Neuropharmacologic and behavioral actions of clonidine: interactions with central neurotransmitters.

NEUROPHARMACOLOGIC AND BEHAVIORAL ACTIONS OF CLONIDINE: INTERACTl0NS WITH CENTRAL NEUROTRANSMITTERS Jerry J. Buccafusco Department of Pharmacology and Toxicology Deportment of Psychiatry and Health Behavior Medical College of Georgia Augusta, Georgia 30912 and The Department of Veterans Affairs Medical Center Augusta, Georgia 30912

I. Introduction A. Development of a Novel Antihypertensive Drug B. Current Clinical Indications 11. Receptor Specificity A. Peripheral Sites 8. Central Sites 111. Role of Brain Neurotransmitters in the Antihypertensive Response A. Biogenic Amines 8. Opiates C. Acetylcholine D. Other Neurotransmitters IV. Antiwithdrawal Effects A. Opiate Withdrawal B. A Spinal Cord Model for Opiate Withdrawal C. Other Drugs of Abuse V. Other Pharmacological Actions A. Growth Hormone Secretion B. Inhibition of Cholinesterase Inhibitor Toxicity C. Learning and Memory VI. Summary and Conclusions A. The Diversity of Pharmacological Actions B. Clonidine as a Neuromodulator VII. Future Directions References

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Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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1. Introduction

A. DEVELOPMENT OF A NOVEL ANTIHYPERTENSIVE DRUG T h e impact of the discovery of a novel pharmacological agent on the understanding of a disease process can often be quite dramatic. A case in point is the discovery of the antipsychotic action of chlorpromazine in the early 1950s. In fact, the use of this compound in the treatment of schizophrenia heralded the beginning of modern psychopharmacology. Inquiries into the mechanism of action of this important compound led to the dopamine hypothesis of schizophrenia, a tenet that continues to provide one of the few durable models of brain dysfunction. T h e discovery of chlorpromazine is interesting in another way, that is, in terms of its similarity in certain respects to clonidine. For example, chlorpromazine has amazed researchers over the years regarding its efficacy in a number of psychological and neurological conditions and in the disturbing number of adverse side effects associated with therapy. This pharmacological diversity is a reflection perhaps of the large number of neurotransmitter receptors the drug interacts with. Thus chlorpromazine can inhibit dopamine, a-adrenergic, serotonergic, cholinergic muscarinic, and histaminergic receptors (Baldessarini, 1990). As with chlorpromazine, clonidine began its pharmacological career inauspiciously. The drug was originally synthesized to be employed as a nasal decongestant. Fortunately, the clinical trials initiated to this end resulted in the discovery of the potent hypotensive and sedating actions of the compound (see Kobinger, 1978). T h e unique properties of clonidine unfolded in the mid-1960s and early 1970s and led to an unimaginable wealth of studies. T h e first of these studies, and perhaps the most important up to the present time, concerned the recognition of the role of central adrenergic receptors in cardiovascular regulation and revealed that the brain could be targeted pharmacologiclly to produce an antihypertensive response. In addition to the ground-breaking research that soon followed the discovery of the drug, another feature of similarity with chlorpromazine is clonidine’s pharmacological diversity. As will become evident from the discussion below, clonidine and related drugs have perhaps an unparalleled number of pharmacological and biochemical properties and have found usefulness in a unprecedented number of clinical syndromes. In the case of clonidine, however, this diversity stems not from classical postsynaptic interactions, but most likely through its ability to interact with a number of neurotransmitter sys-

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tems, central and peripheral, through a presynaptic action. For a most excellent earlier review of clonidine and related compounds, the reader is referred to the works of Schmitt (1977) and Kobinger (1978), two individuals who contributed significantly to our current knowledge of the neuropharmacology of central cardiovascular regulation and the mechanism of the antihypertensive action of clonidine. This review will touch upon some of the critical discoveries, but will focus primarily on later accomplishments in the field. Also, the purpose of this review is to summarize the experiments pointing to the mechanism(s) by which clonidine produces its prominent pharmacological actions. B. CURRENT CLINICAL INDICATIONS

In Dr. Schmitt’s review of 1977, the currently recognized pharmacological properties of clonidine included the peripheral actions, hypertension, vasoconstriction, contraction of the nictitating membrane, and hyperglycemia. These actions could be ascribed simply to stimulation of peripheral a-adrenergic receptors and might have been expected in view of the original plan to design a compound with nasal decongestant properties. I n addition, and more importantly, his review underscored the recognition that clonidine possessed a number of pharmacological properties that were due to an action on the central nervous system. These included hypotension, inhibition of sympathetic tone, activation of vagal tone, bradycardia, sedation, antinociception, hypothermia, inhibition of water intake, inhibition of food intake, and aggressive behavior. Direct evidence for the central site of clonidine’s blood pressure-lowering ability was first provided by Kobinger and colleagues in the mid-1960s and by Schmitt and colleagues in the early 1970s. The details of these experiments have been extensively reviewed (Schmitt, 1977; Kobinger, 1978). In retrospect, it is somewhat surprising that a drug associated with this list of pharmacological properties would become a useful adjunct to the antihypertensive armamentarium. It was perhaps prophetic that in the first page of his 1977 review, Dr. Schmitt writes, “I believe we are as yet in a preliminary and rudimentary stage of the pharmacological knowledge of this compound.” A considerable number of pharmacological properties of clonidine have been reported during the past 20 years. Many of these are listed in Table I (also see Fielding and Lal, 1981). Several of these actions demonstrated in experimental animals occur at doses not usually achieved following clinical use. Some of these will be discussed in more detail below.

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TABLE I PHARMACOLOGICAL PROPERTIES OF CLONIDINE Site of a adrenoceptor stimulation Effect

Central

Hypotensiodinhibition of sympathetic tone Bradycardialenhancement of vagal tone Sensitize baroreceptor reflex activity Suppression of renin secretion Inhibition of renal tubule fluid absorption Inhibition of phrenic nerve activity M ydriasis Decreased intraocular pressure Reduced secretions from parasympathetic glands Sedation Antinociceptioli Decreased food intake H yperglycemia Decreased drinking H ypothermia Growth hormone secretion Enhanced learning/memory Antiinflammatory Anticonvulsant action

X

Peripheral

X X

X

X X

X X X

X X X X

X X X X X

X X

The only clinical indication recommended by the manufacturer for clonidine is in the treatment of hypertensive disease. Nevertheless, the drug is prescribed routinely for a number of other conditions. These are listed in Table 11.

TABLE 11 REPORTED CLINICALLY USEFULACTIONSOF CLONIDINE A X D RELATED DRUGS Hypertensiodcardiovascular disease Blockage of withdrawal syndromes (opiate, benzodiazepine, nicotine, alcohol) Schizophrenia Acute mania Anxietylpanic attacks Childhood hyperactivity Alzheimer's disease Head injury

Anakgeskdanesthesia Treatment of short stature Tardive dyskinesia Akathiska Social phobia Korsakoffs psychosis Glaucoma Diabetic diarrhea


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II. Receptor Specificity

A. PERIPHERAL SITES The ability of clonidine to activate a-adrenergic receptors was appreciated in early studies utilizing spinal-transected animals or in isolated tissues removed from central nervous control. Although it is commonly appreciated that clonidine and related drugs exhibit some selectivity for a,-adrenergic receptors, in earlier studies it was common to view this receptor subtype as occurring exclusively presynaptically. That is, that a,-adrenergic receptors existed primarily on noradrenergic (sympathetic) nerve terminals, where as a,-receptors were located on sympathetic end organs, the latter mediating vasoconstriction and the former mediating inhibition of norepinephrine release. In fact, the earlier literature and some texts referred to clonidine as an adrenergic antagonist, rather than an agonist, because of its known sympatholytic action. With the advent of selective antagonists, it was soon determined that both types of receptors were localized postsynaptically and that these postsynaptic a,-adrenergic receptors also mediated vasoconstriction (Langer and Hicks, 1984). Receptor binding analysis has provided evidence that the a, receptor subtype is localized primarily at the synapse, whereas the 01, receptor subtype is primarily extrajunctional (Langer and Shepperson, 1982). The direct vasoconstrictor action of clonidine is not generally exploited clinically except in instances in which it is desired to produce vasoconstriction in patients with severe autonomic failure and significant postural hypotension (Robertson et al., 1983). The a, receptor subtype also exists on nerve terminals of preganglionic and postganglionic autonomic nerve endings. Clonidine has been reported to produce weak ganglionic blockade and direct inhibition of norepinephrine release from sympathetic fibers, but this is usually achieved by high, supraclinical doses or at low, nonphysiological stimulation frequencies (Schmitt, 1977; Haeusler, 1976a). In contrast, the release of acetylcholine from parasympathetic nerve terminals in several tissues is quite sensitive to inhibition by clonidine and other a,adrenergic agonists, including norepinephrine (Deck et al., 1971; Werner et al., 1972; Drew, 1978). This parasympatholytic action of clonidine is often encountered as side effects associated with therapy. For example, patients often complain of dry mouth, constipation, and visual disturbances.

JERRY J. BUCCAFC‘SCO

B. CENTRAL SITES Although clonidine is able to block the release of norepinephrine from peripheral sympathetic fibers under certain conditions of stimulation, the ability of the drug to reduce circulating levels of catecholamines and their metabolites is due primarily to its central action (Haeusler, 1976b). As with its peripheral action, clonidine blocks the release of norepinephrine from central catecholaminergic nerve endings. Also, consistent with its peripheral effects, the earlier literature often referred to clonidine as a central noradrenergic antagonist. It is now well accepted that clonidine produces profound effects on brain catecholaminergic fibers, even under resting conditions. Consistent with this action, clonidine inhibits the turnover rate of brain norepinephrine. Again this response is mediated through stimulation of central a,-adrenergic receptors (Rochette et al., 1974, 1982; Reid, 1974; Svensson et al., 1975; Draper et al., 1977; Jhanwar-Uniyal et al., 1985). Whereas these receptors mediating norepinephrine release are probably located on nerve endings, central a,-adrenergic receptors are found on both pre- and postsynaptic aspects of the central adrenergic neuron (Janowsky and Sulser, 1987). Although this inhibition of brain norepinephrine release, in part, underlies the decrease in plasma 3-methoxy-4-hydroxyphenylglycol (MHPG) levels (Charney et ul., 1981; Siever et al., 1984), the decrease in plasma norepinephrine is due mainly to clonidine’s ability to decrease sympathetic outflow through its central actions (Garty et al., 1990). T h e relationship between clonidine’s central inhibition of norepinephrine release and sympathoinhibition is discussed below. It is interesting that despite the diversity of pharmacological actions, most of clonidine’s effects have been ascribed to stimulation of central o r peripheral a,-adrenergic receptors (Table I). Ligand binding techniques have demonstrated a heterogeneous distribution of these receptors in the brain and spinal cord (U’Prichard et al., 1977) and their localization has been employed to help explain some of the pharmacological properties of clonidine. For example, the high density of binding sites in the hypothalamus and medulla may serve as the substrates for clonidine’s sympatholytic and vagomimetic actions. T h e high density of binding in the cortex and locus coeruleus may underlie clonidine’s sedative, antiwithdrawal, and other psychotropic actions. T h e anatomical centers in the brain that mediate all of the pharmacological properties of clonidine have not been elucidated; however, there is agreement as to the site of clonidine’s antihypertensive effect following parenterdl administration. Although microin-jection studies have demonstrated that clonidine can elicit a decrease in blood pressure after administration to various hypo-


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thalamic, medullary, and even spinal sites (Struyker-Boudier et al., 1974; Connor and Finch, 1981; LoPachin and Rudy, 1981; Sinha et al., 1985; Pitts et al., 1986; Elghozi et al., 1989; Buccafusco and Magri’, 1989; also see Isaac, 1980), microinjection of clonidine receptor-blocking agents directly into a restricted region of the rostra1 ventrolateral medulla (RVL) almost completely inhibits the hypotensive action of systemically administered clonidine (Bousquet and Schwartz, 1983; Punnen et al., 1987). T h e RVL contains the origin of the so-called C1 epinephrinecontaining descending pathway. This pathway ultimately makes synaptic contact with the preganglionic sympathetic cell bodies of the intermediolateral spinal cord, and is considered to provide tonic sympathetic activity to these cells (see Reis et al., 1989). Although clonidine is a potent agonist at central and peripheral a,adrenergic receptors, Bousquet and colleagues ( 1984) reported that the hypotensive action of clonidine may be more related to its chemical structure as an imidazole than to its ability to act as an a, agonist. Subsequently, Reis and colleagues (Ernsberger et al., 1987) determined that norepinephrine was not able to completely displace bound [f~-~H]aminoclonidine from a membrane preparation derived from the ventrolateral medulla. This “imidazo1e”-bindingsite was then characterized and demonstrated to exhibit affinity for clonidine equal to the classical a,-adrenergic receptors in the RVL. Imidazole binding was regionally distributed, with frontal cortex exhibiting classical a,-adrenergic binding but virtually no imidazole binding, and the RVL exhibiting a high degree of both imidazole and ap binding. This imidazole-binding site is distinct from subclasses of histamine receptors, but the imidazole structure of histaminergic ligands may explain earlier contentions that clonidine lowered blood pressure, at least in part, through stimulation of histamine (possibly H,) receptors (Karppanen et al., 1977; FriskHolmberg, 1980). The existence of a new putative receptor almost necessitated the search for an endogenous ligand. To this end Atlas and Burstein (1984) reported the partial isolation from calf brain of a clonidine-displacing substance (CDS). The material, which was not one of the known catecholamines, displaced specifically bound clonidine from rat brain membranes. Almost simultaneously then, Bousquet and Reis reported that CDS microinjected into the RVL produced an alteration in arterial pressure. Unfortunately the responses were in opposite directions. Bousquet and colleagues ( 1986) reported that CDS microinjected into the lateral reticular nucleus of the anesthetized cat regularly produced a hypertensive response. In fact, preinjection of CDS inhibited the subsequent fall in blood pressure produced by clonidine injection. They therefore con-

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sidered CDS to be an endogenous clonidine antagonist. Reis and colleagues (Meely et al., 1986) reported that CDS was quite potent in inhibiting the binding of [p-3H]aminoclonidine to receptors derived from bovine RVL. In contrast to Bousquet’s findings, microinjection of CDS into the C1 region of the RVL resulted in a dose-dependent decrease in blood pressure. Later this partially purified CDS was demonstrated to bind with greatest affinity to the imidazole receptor, even more so than clonidine (Ernsberger et al., 1988). T h e opposite effects on blood pressure produced by CDS in the two laboratories is problematic, but not completely unexpected in view of the differences in species employed and slight differences in the purification techniques. Furthermore, Bosquet reported no change in heart rate, whereas Reis reported a significant bradycardia. Clearly, the cardiovascular action of Reis’s CDS is more clonidine-like; however, definite conclusions concerning this issue will await complete isolation and identification of the endogenous molecule. Subsequent experiments with a polyclonal antibody to clonidine have suggested that CDS may contain an aminoimidazoline o r guanidine moiety. I t might be pointed out that the importance of the discovery of this new putative receptor and neurotransmitter could extend beyond the CNS because imidazole receptors sensitive to CDS have been characterized in the renal proximal tubule and CDS has been reported to produce contraction of the rat gastric fundus (Felsen Pt al., 1987; Coupry et al., 1989). It will be interesting to determine whether CDS mimics clonidine’s other actions on second-messenger systems o r in inhibiting the release of other neurotransmitters. In addition to the imidazole receptor, evidence also exists for at least two subclasses of a,-adrenergic receptors, aZAand a2Badrenoceptors. Supporting physiological evidence has included the ability of certain antagonists to discriminate between pre- and postsynaptic a,-adrenergic receptors in different peripheral tissues (Bylund, 1978; Ruff010 et al., 1987; Hieble Pt al., 1988). Regarding central a receptors, there is also evidence to suggest a special variant of the receptor in the spinal cord. Stimulation of spinal CY receptors with clonidine does lead to a decrease in blood pressure; however, blockade of this response by selective antagonists has revealed differences in specificity with respect to peripheral a, receptors (Connor and Finch, 1981). Consistent with this concept is the recent finding that intrathecal injection of clonidine or 6-fluoronorepinephrine in unanesthetized rats was less effective than norepinephrine in inhibiting the pressor response to intrathecal injection of the cholinesterase inhibitor neostigmine (see discussion of central adrenergic-cholinergk interactions in cardiovascular regulation below). In-


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stead, clonidine was more effective in this regard when it was administered intracisternally to stimulate medullary 01 receptors (Buccafusco and Magri’, 1989).

111. Role of Brain Neurotransmitters in the Antihypertensive Response

A. BIOGENIC AMINES T h e possibility that the brain, spinal cord, and peripheral sympathetic nerves play a role in the development and/or maintenance of hypertensive disease is no longer in question. Even in animal models of the disease, including mineralocorticoid, salt-sensitive, and renal ischemia models, in which elevated blood pressure was considered to result exclusively from peripheral factors, it is now apparent that the brain and efferent sympathetic activity is an important contributor to the hypertensive process (de Jong, 1984). Some questions yet to be answered concern the degree of CNS contribution to the pathophysiology, the neuronal structures that are involved, and whether neurochemical abnormalities within such structures can be associated with the disease process. Although great strides in these directions have been made in recent years, no one neuropathological entity has emerged as the causative factor. In fact, many neurotransmitter systems, neurohormones, peptides, and several brain areas have been suggested as playing a role. Such diversity is not surprising in view of the complex neuronal interactions that occur in the regulation of a function as vital as blood pressure. However, the discovery of clonidine and its central mechanism in lowering blood pressure and its ability to stimulate adrenergic receptors immediately pointed an accusing finger at the central adrenergic system as a causative factor in hypertension. One of the first studies to implicate a role for central noradrenergic neurons in the development of hypertension in the now classical model for experimental hypertension, the spontaneously hypertensive rat (SHR), was that performed by Haeusler and colleagues (1972). Their approach was to administer the adrenergic neurotoxin, 6-hydroxydopamine, through the lateral cerebral ventricle to young, prehypertensive rats. Under these conditions the development of hypertension was delayed for several weeks and pressure never rose to the highest levels observed for untreated animals. This finding was subsequently reproduced in several laboratories and in other rat models of hypertension (Doba and Reis, 1974; Erinoff et al., 1975; Haeusler, 1976a; van den

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Buuse, et al., 1984). However, although depletion of brain catecholamines inhibited the development of spontaneous hypertension, a permanent reduction in blood pressure was usually not observed in the adult SHR with established hypertension after similar treatment (Haeusler, l976b; Kubo and Misu, 1981; Woodside et al., 1984). Therefore, the concept evolved that central adrenergic neurons participated in the initiation o r triggering of hypertensive disease, but were not involved in maintaining high blood pressure when it had already fully developed. Nevertheless, these findings appeared to be in concert with the centrally mediated antihypertensive properties of clonidine. A role for central adrenergic neurons in hypertension and in the mechanism of the antihypertensive action of clonidine seem to be inexorably linked. This model would predict that elevated blood pressure in hypertensive disease depends upon increased sympathetic activity resulting from the enhanced activity of central adrenergic neurons. In fact, exaggerated central noradrenergic activity has been observed to be associated with experimental hypertension, although this issue is far from resolved. Increases, decreases, or unchanging catecholamine levels and turnover, enzymes, and metabolites have been reported over the years. Such discrepancies are related in part to the different animal models employed, different brain regions examined, and different stages of’ hypertension when measurements were made. A causal relationship between central adrenergic alterations and increased blood pressure has not been unequivocally demonstrated (Versteeg et al., 1984). Nevertheless, the model has heuristic value and goes on to predict that if hypertension is related to enhanced brain catecholaminergic function, then (1) clonidine should lead to a decrease in the release of brain catecholamines and (2) clonidine’s antihypertensive response should be dependent upon intact, functioning brain catecholaminergic neurons. It is clear that the majority of clonidine’s actions are mediated through stimulation of central a,-adrenergic receptors, but a relationship between changes in norddrenergic neuronal activity and clonidine’s cardiovascular actions has not been established. In fact, the preponderance of evidence suggests that this is not the case. Thus, several investigators have demonstrated that the centrally mediated cardiovascular effects of clonidine require neither functioning brain catecholaminergic neurons nor intact stores of catecholamines (Haeusler, 1974; Kobinger and Pichler, 1974, 1975, 1976; Finch et al., 1975; Warnke and Hoefke, 1977; Reynoldson et al., 1979). Similar results have been obtained with respect to other pharmacological actions of clonidine (Table 111). Though decreased catecholamine release may occur as the result of the presynaptic actions of clonidine, the results of an elegant

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TABLE I11 PHARMACOLOGICAL ACTIONS OF CLONIDINE THAT AREUNAFFECTED BY DEPLETION OF BRAINCATECHOLEMINES Pharmacological actions Decrease arterial blood pressure Inhibit sympathetic nerve activity Enhance reflex vagal bradycardia Produce behavioral depression Produce antinociception Produce mydriasis Inhibit phrenic nerve activity Produce sedation Protect against cholinesterase inhibitor toxicity

References Finch et al. (1975); Warnke and Hoefke (1977); Reynoldson et al. (1979) Haeusler (1974); Kobinger and Pichler (1976) Kobling and Pichler (1975) Florio et al. (1975) Paalzow and Paalzow (1976) Koss and Christensen (1979) von Tauberger et al. (1978) Spyraki and Fibiger (1982) Buccafusco et al. (1988b)

neurochemical and neurohistological study by Haeusler and his coworkers (Lorez et al., 1983) have suggested that this direct inhibitory effect on noradrenergic neuronal activity does not appear to be causally related to the drug’s cardiovascular actions. The conclusion may be stated in an alternative fashion: clonidine stimulates central a,-adrenergic receptors, which are located postsynaptically with respect to adrenergic nerve endings. If this is the case, the question arises as to which neurotransmitter system possesses the relevant clonidine-binding site. Any candidate neurotransmitter should (at least for the simplest model) meet the following criteria: 1. The neurotransmitter should be one that is capable of enhancing or mediating increased sympathetic activity. 2. T h e transmitter system might also be involved in the initiation andlor maintenance of experimental hypertension. 3. Inhibition of the transmitter or blockade of its receptors should lead to a decrease in arterial blood pressure. These criteria do not appear to hold, in general, for central adrenergic systems. Clonidine inhibits the firing rate of noradrenergic neurons, and exogenous administration of the neurotransmitter norepinephrine and related compounds, for the most part, also has been demonstrated to evoke a sympathoinhibitory response. Furthermore, depletion of brain catecholamines or a, receptor blockade produces little change in blood pressure. Nevertheless, although the case for a role for central adrenergic systems in the antihypertensive action of clonidine appears weak,

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it is by no means to be totally discounted. For example, Head and coworkers (1983) reported a requirement for intact central noradrenergic and serotonergic neurons in the cardiovascular action of clonidine in the unanesthetized rabbit. Although these investigators employed similar selective neurotoxins as with the above-cited studies, they point to having used longer time intervals between toxin and clonidine administration than had been used in earlier studies. Additionally, the use of an unanesthetized animal model and species specificity are to be considered. It might be pointed out, however, that experiments employing reversible acute depletors of central adrenergic neurons such as reserpine and a-methyltyrosine (see Lorez et al., 1983) provide results more in line with those showing no effect of central catecholamine depletion on clonidine’s cardiovascular actions.

B. OPIATES The possibility that endogenous brain opiates or opioidergic pathways play a role in the cardiovascular changes produced by clonidine was

suggested perhaps with the observation that clonidine itself produces a significant analgesic response. T h e analgesic or antinociceptive response to clonidine has been demonstrated in several animals models using a number of different pain paradigms (Schmitt, 1977; Fielding and Lal, 1981). Although the doses required to produce this type of response have often been greater than those required to evoke a significant antihypertensive response, clonidine has been demonstrated to be more potent than morphine on a molar basis (Paalzow, 1974; Paalzow and Paalzow, 1976; Fielding et al., 1978). This action of clonidine has been exploited therapeutically to enhance the effects of general anesthetics, in postoperative control of pain, and to produce localized analgesia following epidural administration (Eisenach el nl., 1989a,b; Kitahata, 1989; Vercauteren et al., 1990). Despite this apparent close relationship between clonidine and the opiate system, studies performed in animal models have demonstrated that brain or spinal a,-adrenergic receptors mediate the antinociceptive action to clonidine. More controversial, however, is the role endogenous opiate systems play in this property of clonidine (Hynes et al., 1983; Tchakarov et al., 1985; Sherman et al., 1988; Tasker and Melzack, 1989; Mastrianni et al., 1989; Porchet et al., 1990). Apparently endogenous opiate involvement is related to the type of pain test employed, the strain of rat, and the subtype of opiate receptor. When this potential relationship between clonidine and opiate receptors was demonstrated, it was reported to be selective for the p subtype (Mastrianni et al., 1989).

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T h e cardiovascular depressant effects of morphine and other p agonists are well known and the potential role of the opioidergic system in the development or maintenance of hypertension has been explored in recent years. Perhaps most pertinent to the present discussion is the issue of whether brain opiate receptor blockade can alter the cardiovascular changes induced by clonidine. Several studies from different laboratories have confirmed the observation that naloxone administration blocks or reverses the clonidine-induced decrease in blood pressure in experimental animals. This inhibitory action is observed more prominently in hypertensive animals (Bennett et al., 1982; Ramirez-Gonzalez et al., 1983; Farsang et al., 1984a; Mastrianni and Ingenito, 1987). T h e results in human studies have been less consistent (Bramnert and Hokfelt, 1983; Rodgers and Cubeddu, 1983; Fuenmayor and Cubeddu, 1986), although the naioxone sensitivity of the clonidine response may be selective for a specific subtype of hypertensive patient (Farsang et al., 1984b). This naloxone-sensitive subset was characterized as exhibiting higher cardiac output, stroke index, plasma renin activity, and plasma epinephrine activity than nonresponders. Also, responders were in general more affected by the clonidine treatment itself. The concept that has developed, therefore, is that in certain forms of hypertension, the antihypertensive action of clonidine is mediated through release of an endogenous opioid transmitter. I n consideration of the simple criteria presented above for a neurotransmitter system to be a candidate for the target of interaction with clonidine in cardiovascular regulation, the endogenous opiate system appears to fall short in this regard. The candidate transmitter should, according to the model, mediate an increase in sympathetic activity. Isolated reports have indicated that under certain conditions morphine and other IJ. agonists increase sympathetic tone and blood pressure, but the predominant view is that morphine and endogenous @-endorphinproduce cardiovascular inhibitory actions (see Ramirez-Gonzalez et al., 1983; Versteeg et al., 1984).Although Met-enkephalin-like opiate activity may be sympathoexcitatory (Versteeg et al., 1984; Fuenmayor and Cubeddu, 1986), biochemical studies have demonstrated the ability of clonidine to cause release of @-endorphinsfrom the brain, in vitro (Versteeg et al., 1984; Mastrianni and Ingenito, 1987). In order to support a role for endogenous opiates in hypertension it must be considered that @-endorphinnormally produces inhibitory sympathetic influences, and that in established hypertension such influence is reduced. The possibility that central a,-adrenergic stimulation causes @-endorphin release, which in turn reduces blood pressure, coupled with the suggestion that such opiate-mediated sympathoinhibition could be reduced in hypertension, deserves merit. This scenario does not quite fit with the

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model presented above, but it does suggest an alternative hypothesis that has been advanced before. That is, that sympathoexcitation associated with hypertension is related to a lack of inhibitory transmitter rather than an excess of an excitatory transmitter. Whereas such a possibility has not been tested for &endorphin, administration of naloxone to hypertensive animal models and to humans has not been associated with dramatic cardiovascular changes (Bramnert and Hokfelt, 1983; Farsang et al., 1984a; Ramirez-Conzalez et al., 1983; Fuenmayor and Cubeddu, 1986; Mastrianni and Ingenito, 1987; Florentino et al., 1987). It could be argued that naloxone is a nonselective opiate antagonist and could potentially eliminate both positive and negative influences of endogenous brain opiates on sympathetic outflow and the cardiovascular system. Finally, to be considered is the ability of naloxone to elevate arterial pressure during shock. This action of naloxone has been attributed to blockade of the central and peripheral cardiovascular depressant effects of the P-endorphin that is released during the development of shock (Holaday, 1983). It is possible that naloxone-induced reversal of clonidine’s antihypertensive action in hypertensive animals may be related to this mechanism, particularly because (3-endorphin release has been associated with clonidine treatment. To further support this possibility is the observation that naloxone appears to more readily reverse the effects of clonidine (that is, once clonidine has produced a decrease in blood pressure) than it is able to prevent a clonidine-induced decrease (Bramnert and Hokfelt, 1983; Farsang et al., 1984b; Shropshire and Wendt, 1983; Ramirez-Gonzalez et al., 1983; Fuenmayor and Cubeddu, 1986; Mastrianni and Ingenito, 1987; Florentino et al., 1987). Nevertheless, the participation of central opiate systems in clonidine’s cardiovascular responses must be considered a viable possibility and may play a greater role in certain types of hypertensive disease o r in certain circumstances of clonidine utilization.

C. ACETYLCHOLINE Studies from several laboratories over the past 40 years have pointed to the critical role of central cholinergic neurons in cardiovascular reg-

ulation. Drugs that activate central muscarinic receptors or enhance neuronally released acetylcholine can elicit a reproducible and significant increase in arterial blood pressure in animals and man (for review, see Brezenoff and Giuliano, 1982; Buccafusco and Brezenoff, 1986). Perhaps the earliest study to point to an interaction between clonidine and central cholinergic neurons involved in cardiovascular regulation


69

was reported by Bently and Li (1968).They demonstrated that clonidine pretreatment in rats prevented the centrally mediated hypertensive response to the cholinesterase inhibitor physostigmine. Subsequently, it was demonstrated that pretreatments that increased central cholinergic activity reduced the hypotensive actions of clonidine (Laubie, 1975). Characterization of the clonidine/physostigmine interaction was then carried out by Buccafusco and colleagues, who confirmed the marked inhibitory action of clonidine on the hypertensive response to physostigmine (Buccafusco and Spector, 1980a).They demonstrated that the ability of clonidine to inhibit the pressor response to cholinergic stimulation was selective for indirect-acting agonists such as physostigmine. The pressor response produced by arecoline, a direct muscarinic receptor agonist, was not sensitive to clonidine pretreatment (Buccafusco and Spector, 1980b). This observation was consistent with the finding that clonidine produced a significant reduction in the biosynthesis of acetylcholine in several regions of rat brain, particularly in regions important for cardiovascular regulation, the hypothalamus and medulla (Buccafusco and Spector, 1980c; Buccafusco, 1982, 1984~). The latter action of clonidine was demonstrated to be mediated through stimulation of central a-adrenergic receptors. Also, both clonidine and the related drug a-methyldopa produced decreases in blood pressure and inhibition of brain acetylcholine biosynthesis in hypertensive animals at respective clinically relevant doses (Buccafusco, 1984a,b). Interestingly, blockade of central a-adrenergic receptors with phentolamine reduced the inhibitory effect of clonidine on acetylcholine biosynthesis, but did not itself alter cholinergic dynamics (Buccafusco and Spector, 1980a). In retrospect, it was probably not too surprising to find this ability of clonidine and related drugs to inhibit central cholinergic function, because this relationship was well known for peripheral cholinergic, parasympathetic systems. Thus, stimulation of a,-adrenergic receptors located on parasympathetic nerve endings leads to inhibition of evoked acetylcholine release (see Schmitt, 1977). The central and peripheral “anticholinergic”properties of clonidine may in fact be responsible for many of the side effects reported to occur with therapy (Table IV). Sedation and dry mouth, perhaps the most common side effects, may reflect, respectively, peripheral and central cholinergic blockade. The possibility that inhibition of central cholinergic activity by clonidine leads to its antihypertensive actions as well must be considered. The ability of central muscarinic cholinergic stimulation in several brain regions, including the posterior hypothalamus (Brezenoff and Wirecki, 1970; Brezenoff, 1972; Buccafusco and Brezenoff, 1978, 1979), rostra] medulla (Willette et al., 1984; Giuliano et al., 1989), and spinal

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TABLE IV SIDEEFFECTSTHAT HAVEBEENASSOCIATED WITH CLONIDINE THERAPY OF HYPERI‘ENSIOH

Common

Occasional

Dry mouth Drowsiness Sedation

Constipation Dizziness Headache Fatigue

Rare Gastrointestinal Metabolic CNS

Urinarv retention

cord (Marshall and Buccafusco, 1987; Magri’ and Buccafusco, 1988, 1989; Buccafusco and Magri’, 1990), to produce quite profound increases in blood pressure is consistent with the first of the criteria presented above for a candidate neurotransmitter system. With regard to the second criterion, considerable evidence has been provided in recent years that central cholinergic neurons play an important role in the development and maintenance of experimental hypertension. For example, the pressor response to physostigmine in hypertensive rats has been reported to be significantly greater than similar responses elicited from normotensive controls (Kubo and Tatsumi, 1979; Buccafusco and el al., 1980; Makari et al., 1989; Buccafusco Spector, 1 9 8 0 ~McCaughran ; et al., 1990a). T h e exaggerated pressor response to physostigmine in hypertensive rats suggests that acetylcholine release is enhanced at the site of action of physostigmine, presumably within the rostra1 ventroiateral medulla (Punnen et al., 1986; Giuliano et al., 1989). Although there have been no studies to date that have directly measured brain acetylcholine synthesis or release in hypertensive and normotensive rats, certain markers for the presence of cholinergic neurons have been found to be altered in hypertensive rats. These include the activities of acetylcholinesterase (Yamori, 1976) and choline acetyltransferase (Helke et d., 1980; Edwards et al., 1983) and the density of muscarinic receptors (Edwards et al., 1983; Hershkowitz et al., 1983). The differences, in general, have been positive for hypertensive as compared with normotensive animals. T h e first neurochemical evidence for enhanced brain cholinergic activity in spontaneously hypertensive rats was provided by ‘Trimarchi and Buccafusco ( 1987). They employed the high-affinity uptake of choline by freshly prepared crude synaptosomal fractions as a relative measure of cholinergic activity (Kuhar and Murrin, 1978; Jope, 1979). ‘They reported a significant age-dependent increase in the V,,,, for high-affinity choline uptake into synaptosomal membranes derived


71

from the medulla-pons and hypothalamus (but not the striatum) of the SHR compared with age-matched normotensive Wistar Kyoto (WKY) and compared with normotensive outbred normotensive Wistar rats. Also, a highly significant correlation was found between resting systolic blood pressure and the V,,, for high-affinity choline uptake in both the medulla-pons and hypothalamus. This increase was not a consequence of elevated blood pressure and, in fact, the neurochemical changes occurred prior to the development of significant hypertension. The third component of the model would predict that inhibition of brain cholinergic function should lead to an antihypertensive response. Accordingly, it has been demonstrated that depletion of endogenous stores of brain acetylcholine in the adult SHR results in a marked decrease in blood pressure (Buccafusco and Spector, 1980c; Brezenoff and Caputi, 1980; Giuliano and Brezenoff, 1987). Depletion of brain acetylcholine was accomplished in unanesthetized rats by intracerebroventricular administration of hemicholinium-3 (HC-3), a selective inhibitor of high-affinity choline uptake. Inhibition of choline uptake interferes with the rate-limiting step in acetylcholine synthesis and results in a time-dependent depletion of the transmitter (Gardiner, 1961; Rommelspacher et al., 1974; Finberg et al., 1979). Central administration of HC-3 also prevents the pressor response to physostigmine (Brezenoff and Rusin, 1974; Giuliano et al., 1989). Giuliano and Brezenoff (1987) have extended the findings in the SHR indicated above regarding the ability of central injection of HC-3 to lower resting blood pressure. This antihypertensive response was reported to occur in other rat models of hypertension, including deoxycorticosterone acetate (DOCA) salt hypertension and aortic constriction hypertension. In their study, the antihypertensive effectiveness of HC-3 was not a consequence of the hypertension per se, because it could be reproduced in SHRs whose pressure was lowered to normotensive levels by intravenous infusion of vasodilators. Finally, Buccafusco (1984~)reported that central HC-3 treatment and systemic a-methyldopa both produced antihypertensive responses in the SHR. HC-3 pretreatment greatly enhanced the antihypertensive effectiveness of a-methyldopa, a result that alluded to a common mechanism of action. As with depletion of brain acetylcholine, selective blockade of brain muscarinic receptors results in a marked antihypertensive response in the SHR (Coram and Brezenoff, 1983). More recent studies have demonstrated that the M, muscarinic receptor subtype mediates the cardiovascular consequences of muscarinic stimulation and blockade (Pazos et al., 1986; Xiao and Brezenoff, 1988; Sundaram et al., 1988; Giuliano et al., 1989).

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Although the actual role of brain cholinergic systems in hypertensive disease is far from clear, it does fulfill all three criteria put forth above for a candidate neurotransmitter mediating clonidine’s cardiovascular actions. Interestingly, morphine and other receptor agonists also inhibit the release of acetylcholine from peripheral and central cholinergic neurons. This relationship is discussed in more detail below in considering the antiwithdrawal action of clonidine. However, as indicated above, clonidine does enhance brain P-endorphin release. This clonidineopiate interaction could have relevance here because release of the kopiate agonist could also, in theory, result in inhibition of the function of brain cholinergic pressor neurons. Finally, it should be pointed out that the proposed site for clonidine’s antihypertensive action, the rostral ventrolateral medulla, is also the site that mediates the hypertensive response following systemic injection of physostigmine (Punnen et al., 1986; Giuliano et al., 1989). Moreover, this site is the only brain region demonstrated thus far to mediate a significant decrease in blood pressure in norniotensive rats following acetylcholine depletion or muscarinic blockade (Sundaram and Sapru, 1988; Giuliano et al., 1989). Therefore, the role of brain cholinergic pressor systems in normal cardiovascular regulation, in hypertensive disease, in maintaining tonic sympathetic neuronal activity, and in mediating the antihypertensive action to clonidine and related drugs should continue to be considered a viable possibility.

D. OTHERNEUROTRANSMITTERS .4lmost every putative neurotransmitter or modulator discovered in the mammalian central nervous system, when centrally administered, has been demonstrated to alter resting hemodynamics. Perhaps it is not too surprising in view of the importance of the regulation of the cardiovascular system to virtually every physiological function. Less is known, however, regarding the role of these substances in mediating the antihypertensive action of clonidine. However, using the rostral ventrolateral medulla as an example, it is at this important regulatory site that receptors for catecholamines, excitatory and inhibitory amino acids, acetylcholine, opiates, and several other neuropeptides are known to be linked to cardiovascular pathways entering or leaving the region (see Reis et ui., 1989). Because this region may also be the primary site of action for systemically administered clonidine, a potential interaction of this imidazoline with each of these neurotransmitter systems is conceivable.


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IV. Antiwithdrawal Effects

A. OPIATEWITHDRAWAL Despite the relative explosion in research in the area of narcotic analgesics and the opioidergic nervous system during the last decade, very little progress has been made with regard to the pharmacological treatment of opiate addiction. Methadone maintenance continues to provide the mainstay treatment in most clinical settings. However, within the best inpatient environment, rates of retention vary from about 50 to 85% and only 12-28% are likely to remain abstinent for 1-3 years after detoxification Uaffe, 1987). In addition, long-term treatment with methadone can be associated with significant side effects, such as constipation, excessive sweating, decreased sexual function, hormonal abnormalities, and sleep disturbances, effects to which tolerance may not develop. Again, despite the significant research effort in this area, attempts to separate the psychic and dependence properties from the analgesic property of opiates have not led to a clinically useful drug. Furthermore, the endogenous opiate peptides are not devoid of the problems of tolerance or dependence. Also, the earlier suggestion that opiate receptor changes might underlie dependence has not been realized (Redmond and Krystal, 1984). However, during the last few years an alternate pharmacological approach to opiate detoxification has been developed and used quite successfully. Clonidine has been found in several clinical studies to relieve primarily the autonomic components of withdrawal, whereas many of the subjective o r behavioral effects, such as craving, may not be fully reduced in magnitude. Although it is recognized that clonidine and related drugs cannot fully substitute for morphine, and it is not clear whether clonidine can alter relapse rates, clonidine treatment has been employed successfully to facilitate the initiation of opiate antagonist (naltrexone) therapy in heroin addicts (Charney et al., 1982). Perhaps even more importantly, clonidine has provided an important research tool with which to investigate the processes of dependence and withdrawal. Noradrenergic neurons are found in greatest concentration within the pontine locus coeruleus, where they coexist with a high density of opiate receptors (Kosterlitz and Hughes, 1975; Bird and Kuhar, 1977). Both morphine and clonidine can suppress the firing rate of locus coeruleus neurons, although the response to each agonist is mediated through respective opiate and a,-adrenergic receptors (Svensson et al., 1975; Cedarbaum and Aghajanian, 1977), and the apparent firing rate

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of the catecholamine neurons in this brain region increases during morphine withdrawal (Aghajanian, 1978). These findings provide the basis for the noradrenergic hypothesis of narcotic withdrawal. In brief, these data have encouraged the hypothesis that noradrenergic hyperactivity originating within the locus coeruleus is responsible for the behavioral and sympathetic responses associated with narcotic withdrawal (Gold et nl., 1978a,b, 1979, 1980). Although this may be true for the behavioral effects of withdrawal, this hypothesis does not appear to accommodate all the facts concerning the pharmacological properties of clonidine. For example, as discussed above, enhanced central noradrenergic activity is usually associated with sympatho-inhibition, not sympatho-excitation (Schmitt and Fenard, 1971; Heise and Kroneberg, 1973; de Jong et al., 1975; Buccafusco and Brezenoff, 1977). Also, the possibility that clonidine acts indirectly to inhibit norepinephrine release to produce its sympatho-inhibitory actions is contradicted by the fact that depletion of norepinephrine or destruction of norepinephrine nerve terminals does not impair the sympatho-inhibitory actions of clonidine (Haeusler, 1974; Kobinger and Pichler, 1974, 1975, 1976; Finch et al., 1975; Warnke and Hoefke, 1977; Reynoldson et a!., 1979; Lorez et nl., 1983; Buccafusco et a/., 198813). These arguments correspond to those presented above for clonidine’s antihypertensive action and so will not be elaborated here. Finally, studies from several laboratories have demonstrated that significant morphine withdrawal responses could be expressed from brain regions other than the locus coeruleus, including the spinal cord (Wikler and Frank, 1948; Martin and Eades, 1964; Martin et al., 1976; Gilbert and Martin, 1976; Davies, 1976; Yaksh et al., 1977; Delander and Takemori, 1983). The fact that enhanced withdrawal-associated behaviors and sympathetic activity (as measured by postwithdrawal blood pressure increase) can be elicited from cardiovascular centers at different levels of the neuraxis (Marshall and Buccafusco, 1985a) suggests that a redundancy in the mechanism of dependencelwithdrawal may exist. Clonidine can interact with a host of potential neurotransmitters and modulators; however, significant evidence exists to support the concept that cholinergic hyperexcitability underlies the sympatho-excitation associated with narcotic withdrawal. In addition to the fact that certain central cholinergic pathways mediate sympatho-excitation, previous neurochemical and pharmacological data demonstrate that opiates inhibit the function of cholinergic neurons and that during withdrawal an enhanced cholinergic activity is expressed (Redmond and Krystal, 1984; Crossland, 1971; Domino and Wilson, 1973; Pinsky et al., 1973; Vasko and Domino, 1978; Casamenti et al., 1980; Crossland and Ahmed, 1984).


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This enhanced cholinergic activity may occur over pathways similar to those involved in central cardiovascular regulation and hypertension (as discussed above). Another feature linking clonidine and morphine with cholinergic neurons is their ability to inhibit the electrically induced release of acetylcholine from parasympathetic nerve endings (for review, see Kosterlitz et al., 1972; Schmitt, 1977).This shared ability is mediated through stimulation of respective presynaptic opiate and a,-adrenergic receptors. Presynaptic p-opiate and a, receptors both function to inhibit acetykholine release from the superior cervical ganglion by reducing calcium influx during preganglionic nerve stimulation (Araujo and Collier, 1987). Also, the withdrawal syndrome evoked after prolonged exposure of the isolated guinea pig ileum to opiates is inhibited by clonidine. This antiwithdrawal effect of clonidine is mediated through stimulation of presynaptic a, receptors. Likewise, the withdrawal syndrome evoked after prolonged exposure to clonidine in this preparation is inhibited by opiate agonists, mediated through opiate receptors (Chahl, 1985; Araujo and Collier, 1987). Thus clonidine’s antiwithdrawal action in humans may be due at least in part, through its common ability with morphine, to inhibit the function of central as well as peripheral cholinergic neurons. In support of this concept are the findings that the sympatho-excitatory response associated with precipitated withdrawal in the rat is inhibited both by clonidine and by hemicholinium-3, a drug that depletes endogenous stores of brain acetylcholine (Buccafusco, 1983; Marshall and Buccafusco, 1987). Therefore, although several brain neurotransmitter systems have been implicated in mediating the symptoms of narcotic withdrawal (for review, see Redmond and Krystal, 1984),evidence appears most compelling for the role of cholinergic neurons in mediating the autonomic symptoms associated with abstinence. If cholinergic neurons do provide the substrate for certain components of the withdrawal syndrome and the antiwithdrawal effects of clonidine, it would follow that anticholinergic agents should be equally effective as antiwithdrawal agents (as indicated above for the experimental drug hemicholinium-3). In fact, the effectiveness of anticholinergic drugs in this regard is controversial (Redmond and Krystal, 1984).Such inconsistencies may be related to the fact that cholinergic neurons do not participate in mediating all withdrawal signs. Furthermore, drugs such as atropine are nonspecific with regard to muscarinic receptor blockade. For example, the sympatho-excitatory effects of central cholinergic stimulation appear to be mediated through M, rather than M, receptors (Pazos et al., 1986; Xiao and Brezenoff, 1988). In concert with this cardiovascular data, Buccafusco ( 1991) has recently demonstrated that the

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selective M , antagonist pirenzepine did not alter the autonomic or behavioral components of naloxone-precipitated withdrawal in morphinedependent rats. In contrast, the MJM, selective antagonist 4-DAMP was effective in almost completely eliminating the cardiovascular (sympathoexcitatory) signs of withdrawal, and was more effective than clonidine under similar conditions in inhibiting the behavioral signs. B. A SPINAL C O R D MODELFOR OPIATE WITHDRAWAL

'That the spinal cord could provide a useful model system in which to study the neuronal interaction associated with morphine withdrawal was appreciated more than 30 years ago. For example, Martin and colleagues demonstrated that morphine-dependent spinal-transected dogs exhibit many of the autonomic and somatic reflex signs of withdrawal from spinal cord segments distal to the level of transection (Martin and Eades, 1964; Martin et al., 1976). T h e localized intrathecal, subarachnoid administration of morphine and opiate antagonists in dependent rats also indicates that spinal cord neurons participate in the expression of several characteristic behavioral signs and symptoms of the withdrawal response (Yaksh et al., 1977; Delander and Takemori, 1983). In fact, withdrawal-enhanced activity of sympathetic autonomic neurons originating within the spinal cord has led to identification of these neurons as a possible site for the antiwithdrawal effect of clonidine (Franz et al., 1982). High concentrations of opiate peptides and receptors are localized in spinal laminae 1-111 (H8kfelt et al., 1977; Atweh and Kuhar, 1977). This region also contains a high density of small-diameter (C and A6) primary afferent fibers known to transmit thermal and nociceptive information capable of producing reflex autonomic changes (see Martin, 1982; Carew, 1982). Opiate receptors exist on primary afferent fibers (LaMotte et a/.,1976; Jessell et al., 1979), which, when occupied by morphine, results in a decreased excitability of the terminals (Sastry, 1978; Carstens el ut., 1979). These afferent fibers are generally believed to employ substance P as their neurotransmitter and carry sensory pain information and perhaps information related to local autonomic reflex activity (for review, see Neale and Barker, 1983). However, this concept has been challenged (Bossut et al., 1988).Another possible candidate for carrying afferent nociceptive information is glutamate. The dorsal root and dorsal horn are selectively rich in glutamate, and it has been demonstrated that a subset of spinal neurons directly excited by dorsal root fibers has excitatory membrane receptors activated by L-glutamate (Puil, 1983;


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Hunt, 1983; Blumenkopf, 1988). Irrespective of the nature of the excitatory primary afferent transmitter, other studies support the concept that spinal opiate withdrawal may be mediated over afferent cardiovascular reflex pathways (Marshall and Buccafusco, 1987). An established role for spinal cholinergic neurons has been limited primarily to preganglionic and motor neurons and Renshaw cells. Application of muscarinic agents to spinal motor neurons or sympathetic preganglionic neurons does not elicit excitation (Puil, 1983). If spinal motor neurons are cholinoceptive, the cholinergic receptors activated usually produce inhibitory responses. Collaterals from these neurons activate Renshaw cells (classic recurrent inhibition) that are dually innervated by nicotinic as well as muscarinic receptors (Puil, 1983). Nevertheless, stimulation of spinal muscarinic receptors results in a sympathoexcitatory response (Marshall and Buccafusco, 1987; Magri’ and Buccafusco, 1988). This duality of spinal cholinergic function is interesting because, when morphine withdrawal is precipitated directly from the spinal cord, cholinergic neurons play an inhibitory role. On the other hand, if withdrawal involves higher centers, spinal cholinergic neurons play an excitatory role (Marshall and Buccafusco, 1987). The results of these recent studies suggest the presence of a local spinal cholinergic inhibitory circuit as well as an ascending excitatory pathway (Marshall and Buccafusco, 1987; Magri’ and Buccafusco, 1988). The latter is a novel concept and may serve to explain why excitatory spinal muscarinic synapses have been difficult to identify. Direct evidence for the presence of an ascending vasomotor pathway is not yet available. However, transection of the spinal cord was reported not to significantly alter the level of endogenous spinal acetylcholine below the point of the transection, but did reduce levels above the transection, a finding that is consistent with the presence of primarily ascending spinal cholinergic neurons (Potter and Neff, 1984). For the local inhibitory pathway, it is also interesting that high levels of muscarinic receptors exist in the dorsal horn. After dorsal rhizotomy, the level of choline acetyltransferase (the acetylcholine synthetic enzyme) and the density of muscarinic and opiate receptor binding sites were decreased, suggesting that primary afferent fibers may receive a cholinergic as well as opiate innervation (Gillberg and Wiksten, 1986). These results may help to explain the finding that intrathecal injection of muscarinic agonists produces antinociception and a reduction in substance P levels (Smith et al., 1989). T h e use of postwithdrawal arterial blood pressure increase as an index of the sympathetic component of the narcotic abstinence syndrome dates back to the original studies of Himmelsbach in the 1930s (Himmelsbach, 1937, 1939). Perhaps due to the complexities of main-

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taining patent arterial catheters in animals, the models developed since that time depended primarily on measurement of a spectrum of associated behavioral symptoms as well as changes in body temperature and weight loss. Some of these behavioral changes such as “wet dog shakes” and escape or jumping behavior were characteristic of the opiate withdrawal syndrome in rodents. Studies over the last several years have established that, as in clinical subjects, the withdrawal-associated increase in mean arterial pressure (MAP) can provide a reliable and sensitive index of both the intensity of the sympathetic component of abstinence as well as the degree of physical dependence produced as a result of chronic morphine administration in rats (Buccafusco, 1983; Buccafusco et nf., 1984; Buccafusco and Marshall, 1985; Marshall and Buccafusco, 1985a,b,c, 1987). Furthermore, this autonomic symptom of withdrawal has been employed to predict the antiwithdrawal potential of clonidine and the related drug guanfacine (Buccafusco et al., 1984). Because these experiments can be carried out in unanesthetized, freely moving animals, the classical behavioral signs of morphine withdrawal can also be recorded simuitaneously. Therefore, by combining measures of the cardiovascular and behavioral changes associated with withdrawal, a composite picture of withdrawal can be produced in the rat that is similar in many ways to that originally employed by Himmeslbach in patients. ’The pressor response and behavioral changes associated with morphine withdrawal in dependent rats are quite marked following systemic injection of the narcotic antagonist naloxone (there is no effect on blood pressure and no obvious changes in behavior following similar injection of naloxone in nondependent, naive animals). Behavioral and autonomic signs of withdrawal can be elicited following the intraarterial injection (Marshall and Buccafusco, 1985b,c) of naloxone as well as following the injection of the antagonist into localized areas of the CNS (Marshall and Buccafusco, 1985a). In fact, marked behavioral and autonomic withdrawal symptoms can be obtained following intrathecal (i.t.) injection at the level of the spinal sympathetic (thoracic) outflow (Buccafusco and Marshall, 1985; Marshall and Buccafusco, 1985a, 1987). T h e importance of the spinal cord in mediating autonomic symptoms of withdrawal was underscored by the observation that two consecutive withdrawal responses of equal magnitude could be elicited when the first injection of naloxone was made into the lateral cerebral ventricle and the second was into the intrathecal space. Conversely, once the spinal cord was withdrawn (through i.t. injection of naloxone), a second withdrawal response could not be produced by injection of naloxone via any other route (Marshall and Buccafusco, 1985a). Also, the spinal cord in isolation was capable of mediating a withdrawal response, i.e., a marked

CLONIDINEINEUROTRANSMITTER INTERACTIONS

\

-

79

? ....*..*.,............................ ...........-..........-.--*-. 3

#I)-

_ , ~ ~ ~ ~ _ ~ _ ~ ~ ~ o ~ ~ o - o - - - - ~ = ~ - ~ - -

SPINO-MEDULLARY REFLEX DESCENDING FROM HIGHER CENTERS

FIG. 1. Model of spinal and medullary interactions proposed for cholinergic, opioidergic, and adrenergic receptors in the morphine Withdrawal syndrome; ACh, cholinergic neuron; ENK, enkephalinergic interneuron; a,a-adrenergic (presynaptic)receptors; +, excitatory influence; -, inhibitory influence; ?, unknown neurotransmitter.

pressor response was observed in spinal-transected (Cl), morphine-dependent rats (but not in spinal-transected, nondependent animals) (Marshall and Buccafusco, 1985~). The schematic model of putative neuronal interactions illustrated in Fig. 1 is not meant to indicate actual anatomical connections. Nevertheless, pharmacological manipulations of these potential pathways have provided significant insight into the nature of spinal cord pathways involved in autonomic regulation, in the local generation of opiate withdrawal symptoms, and in the antimorphine withdrawal action of clonidine. In spinal-transected rats (control or morphine dependent), local cardiovascular reflex pathways are intact, because a tail or foot pinch reliably elicits an increase in blood pressure (Marshall and Buccafusco, 1987).

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The fact that such a reflex pathway (see the spinal reflex pathway depicted in Fig. 1) is involved in opiate withdrawal is underscored by the observation that dorsal root deafferentation completely abolished the pressor response to naloxone in morphine-dependent, spinal-transected rats (Buccafusco and Marshall, 1985). T h e interactions proposed for the spinal reflex pathway are also based upon the facts that primary afferent fibers d o not directly innervate the cell bodies of the preganglionic neurons (Puil, 1983) and that enkephalin-containing interneurons can produce pre- and postsynaptic inhibitory influences on primary afferent tertninals in the dorsal horn (Sastry, 1978; Carstens et al., 1979). Chronic stimulation of opiate (enkephalin) receptors could lead to inhibition of the reflex pathway, which in turn would result in the activation of compensator): mechanisms. During withdrawal such compensatory mechanisms would be suddenly unopposed, leading to the withdrawal pressor response. ‘The inhibitory cholinergic influence is suggested by the following findings: (1) Intrathecal injection of naloxone to morphine-dependent rats produces a withdrawal-associated increase in blood pressure (Buccafusco, 1983; Marshall and Buccafusco, 1985b). (2) Intrathecal injection of atropine or hemicholinium-3 enhanced the pressor response to i.t. injection of naloxone in morphine-dependent rats (Marshall and Buccafusco, 1987). Because i.t. injections of cholinergic antagonists d o not alter resting levels of blood pressure, the cholinergic inhibitory system proposed must be activated during withdrawal (or during activation of the reflex pathway). It is also possible that this cholinergic influence may be provided by recurrent collaterals from the preganglionic fibers themselves. If this inhibitory influence is mediated through Renshaw cells, both nicotinic and muscarinic receptors could be involved. (3) The a2 agonist clonidine can produce marked inhibition of central cholinergic function and inhibits the sympathetic component of morphine withdrawal (see above); however, in dependent rats, if naloxone is injected by the intrathecal route, local clonidine pretreatment does not affect the pressor response associated with withdrawal (Buccafusco, 1990). (4) Activation of spinal muscarinic receptors results in an antinociceptive response in the rat (Yaksh et al., 1985; Smith et al., 1989). Spinal antinociception was also associated with a selective decrease in the levels of substance P in the dorsal segment of the cord. Because dorsal root afferents have muscarinic receptors, spinal cholinergic (muscarinic) inhibitory pathways may directly inhibit dorsal root afferent terminals, an action that could account both for the antinociceptive (Smith et al., 1989) and withdrawal-potentiating (Marshall and Buccafusco, 1987) actions of local spinal cholinergic neurons. Finally, as depicted in Fig. 1, dorsal afferent fibers involved in cardiovascular re-

CLONIDINE/NEUROTRANSMITTERINTERACTIONS

81

flexes may activate local or ascending sympatho-excitatory pathways, analogous to stimulation of ascending nociceptive pathways. To support a role for primary afferents mediating a sympathetic component of withdrawal is the study by Sharpe and Jaffe (1986), in which neonatal capsaicin treatment was employed to produce permanent degeneration of primary afferent fibers in the adult rat. Capsaicin treatment significantly attenuated naloxone-precipitated autonomic abstinence symptoms such as salivation, lacrimation, and rhinorrhea in rats implanted with morphine pellets several days earlier. In addition to the local reflex circuit, cholinergic neurons participate in sympatho-excitatory responses in the spinal cord. Buccafusco and coworkers were the first to demonstrate that intrathecal injection of cholinergic agonists in freely moving rats produces a marked pressor response (Magri’ and Buccafusco, 1988). This response is greatest when the injection is made at the lower thoracic level (T10-T 13).The pressor response can be elicited following i.t. injection of low microgram doses of carbachol or neostigmine. The effect of the latter drug is mediated through the release of spinal acetylcholine because it is blocked by both atropine and by local depletion of acetylcholine. As with the local spinal circuit, these cholinergic neurons may not participate in mediating tonic sympathetic activity because spinal treatment with muscarinic-blocking drugs or depletion of spinal acetylcholine does not affect resting blood pressure (Magri’ and Buccafusco, 1988). The trajectory of this pathway is unknown but may involve local spinal interaction or a pathway ascending to higher centers, because (1) spinal section at C 1 blocks the pressor response to i.t. injection of both neostigmine (indirect receptor agonist) and carbachol (direct receptor agonist) (Marshall and Buccafusco, 1987); (2)pretreatment with intracisternal injection of hemicholinium-3 to deplete selectively medullary levels of acetylcholine blocks the pressor response to intrathecal injection of carbachol (Magri’ and Buccafusco, 1989) (note i.t. injection of hemicholinium-3has no effect on the pressor response to carbachol) (Magri’ and Buccafusco, 1988); and (3) intracisternal injection of clonidine (which inhibits the function of central cholinergic neurons) (Buccafusco and Brezenoff, 1977; Buccafusco and Spector, 1980c; Buccafusco, 1984a,b,c; Magri’ et al., 1988) blocks the pressor response to intrathecal injection of neostigmine (Magri’ and Buccafusco, 1989). Therefore, the cholinergic excitatory system is cholinoceptive, but may also be cholinergic (see the spino-medullary reflex pathway, Fig. 1). As with certain cholinergic neurons of higher centers, spinal cholinergic excitatory neurons are inhibited by pretreatment with i.t. injection of a-adrenergic agonists. If this cholinergic system participates in the pressor response to systemic injection of naloxone in mor-

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phine-dependent rats, this pathway could be the substrate underlying the antiwithdrawal effects of intrathecal injection of clonidine (Magri’ and Buccafusco, 1989). T h e nature of the interactions of the central components of this pathway is not understood. T h e pressor response to neostigmine is not altered in the decerebrate rat preparation (Takahashi and Buccafusco, 1989);therefore, the rostral portion of the circuit must be completed within the medullary-pontine region. Although it is not possible to rule out any particular cell group in integrating this portion of the pathway, several sites in the medulla have been implicated in directly modifying sympathetic vasoconstrictor tone. In recent years it has been demonstrated that narrowly defined anatomical regions and cell groups play a direct role in this process. Of particular interest is the rostral ventroiateral pressor region of the medulla. As discussed previously with respect to clonidine’s antihypertensive action, this area projects directly to spinal sympathetic preganglionic neurons and has been demonstrated, using anatomic, physiologic, and pharmacological techniques, to provide tonic descending vasomotor activity (for review, see Calaresu and Yardiey, i988). Although the descending pathway is not cholinergic, the medullary neurons are cholinoceptive, and cholinergic stimulation increases blood pressure, whereas cholinergic inhibition lowers pressure (Sundaram and Sapru, 1988). T h e RVL is also the origin of the C 1 adrenergic descending neurons. Other regions of importance in cardiovascular regulation include the caudal ventrolateral medulla, the origin of A1 noradrenergic fibers, stimulation of which produces sympatho-inhibition. Also, regions in the dorsomedial medulla may have a sympatho-excitatory role (Calaresu and Yardley, 1988). T h e nucleus tractus solitarius (IVTS), site of termination of baroreceptor and chemoreceptor afferents and origin of the A2 noradrenergic cell group, is also a potential site for cholinergic influences on sympathetic control. This region also contains a relatively high density of cholinergic markers (Simon et al., 1985). Finally, the bulbo-spinal (descending) component of the spinomedullary reflex pathway is not cholinergic because the pressor response to intracisternal (medullary) injection of neostigmine is not blocked following intrathecal injection of atropine (note: i.t. injection of neostigmine is blocked by i.t. pretreatment with atropine). Briefly stated, a sympatho-excitatory cholinoceptive pathway exists in the spinal cord, which subsequently activates a locally active or ascending cholinergic system that interacts with a second cholinergic pressor system, probably localized within the medulla. Both the spinal and medullary cholinergic components of this pathway can be inhibited by a,-adrenergic agonists such as clonidine.


83

Sympatho-excitatory pathways descending from higher centers (Fig. 1) may also play a role in the morphine withdrawal syndrome. Naloxone-induced blood pressure increases were obtained following injection of the antagonist into the lateral cerebral, or fourth ventricle, in dependent rats (Marshall and Buccafusco, 1985a). The possibility exists that naloxone administered via the cerebrospinal fluid could be potentially distributed to all brain areas. However, two consecutive full withdrawal responses could be obtained in the same animal when the first injection of naloxone was made into the lateral ventricle and the second was made either intrathecally or systemically. Once the spinal cord was withdrawn, a second withdrawal response could not be obtained from any other route of naloxone injection (Marshall and Buccafusco, 1985a). Thus, at least two (supraspinal and spinal) distinct sympatho-excitatory pathways associated with opiate withdrawal exist. The descending pathways may interact with the spinal sympatho-excitatory (spino-medullary) cholinergic pathway (Fig. 1). This possibility is based upon the finding that i.t. injection of anticholinergic drugs inhibits the pressor response to naloxone following systemic injection in dependent rats (Marshall and Buccafusco, 1987). As further evidence for this possibility, i.t. pretreatment with clonidine also inhibits the pressor response to systemic administration of naloxone in dependent rats (Buccafusco, 1990). Therefore, withdrawal sympathetic pathways descending from higher centers must interact with spinal cholinergic pressor pathways susceptible to clonidine inhibition. A direct (noncholinergic) pathway may exist, because cholinergic antagonists do not completely eliminate the withdrawal response to systemic injection of naloxone in dependent rats (Marshall and Buccafusco, 1987). This noncholinergic descending withdrawal pathway may occur independently of the spino-medullary reflex pathway proposed, or it may activate the noncholinergic descending limb (Fig. 1) of the circuit. Although the above data are consistent with the proposed interactions illustrated in the model, many other interpretations are possible. Underscored here are the interactions between cholinergic, adrenergic, and perhaps peptidergic or glutaminergic systems; however, it is most likely that other biogenic amines and opioid peptides play a role in the expression of the withdrawal syndrome. It appears that at least some of these interactions take place in higher brain centers. For example, in several preliminary experiments we have observed that pretreatment with cerebroventricular injection of cholinergic antagonists inhibits the pressor response to systemic administration of naloxone in dependent rats (Buccafusco, 1991). As with the spinal cord data, however, if naloxone is administered by cerebroventricular injection to precipitate with-

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drawal (rather than systemically), pretreatment with cerebroventricular injection cholinergic antagonists enhances the pressor response to naloxone. This feature of cholinergic neurons, inhibiting withdrawal evoked locally and facilitating withdrawal evoked systemically, suggests a redundancy in such interactions and supports the use of the spinal cord as a model in examining the autonomic mechanisms of the opiate withdrawal syndrome. I n summary, current findings in this area are generally supportive of the hypothesis that the spinal cord may provide a good model for the autonomic component of the narcotic withdrawal syndrome, that cholinergic pathways within the spinal cord modify the withdrawal response, and that clonidine may evoke its antiwithdrawal actions at the level of the spinal cord through inhibition with the sympatho-excitatory cholinergic pathway. C. OTHER DRUGSOF ABUSE

As indicated in Table 111, clonidine has been examined clinically for the treatment of withdrawal signs associated with abstinence following continued use of other drugs with abuse liability. Much less is known about the mechanism of clonidine’s actions in these situations than is known about the opiate withdrawal syndrome. For example, it is not clear whether clonidine’s antianxiety properties or its sedative/CNS depressive properties (see Fielding and Lal, 1981) could account for the reported benefit of clonidine in alcohol (Wartenburg, 1983), benzodiazepine (Kunchandy and Kulkarni, 1986), and nicotine withdrawal (Glassman and Covey, 1990). Regarding alcohol abuse, however, clonidine has been reported to interfere with the expression of ethanol intoxication in a mouse model. The inhibitory effect was reported to be comparable to that produced by the imidazodiazepine RO15-45 13, which is known to inhibit ethanol’s effects by a central action (Lister et al., 1989). Clonidine has also been found to be more effective than conventional therapy in treating states of acute alcohol withdrawal, including delirium tremens (Cushman, 1987). Here the effect, as with morphine withdrawal, may be due in part to its central sympatholytic action. Clonidine has a clear antiwithdrawal effect in animal models and, moreover, has been demonstrated to reduce voluntary ethanol consumption in rats (Opitz, 1990). T h e effect is most likely mediated by central a2adrenergic receptors because this latter action of clonidine was mimicked by the related drugs guanfacine and tiamenidine. T h e interaction


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with central a,-adrenergic receptors is also consistent with the ability of the selective antagonist yohimbine to block clonidine’s antiwithdrawal action. Thus, in alcohol abuse clonidine may be a therapeutic adjunct useful on several fronts, including alcohol withdrawal and chronic alcoholism. Nicotine as a drug of abuse is not known for the severity of its withdrawal syndrome. However, the drug is a powerful reinforcer in both animals and man. Clonidine has been shown to be effective in reducing craving in smokers abstinent from tobacco for 24 hr, although a significant effect was obtained in women only, regarding smoking cessation. Quit rates, however, have been reported to be higher than placebo for both genders when clonidine therapy was combined with counseling (see Glassman et al., 1988; Glassman and Covey, 1990). In view of the side effects commonly associated with clonidine therapy, it is not clear how this approach will be continued to be viewed in the future. Nevertheless, the ability of clonidine to produce significant beneficial actions in so many different abstinence syndromes produced in the laboratory and observed in the treatment of addicts is unprecedented for a pharmacological agent and most likely is a reflection of the drug’s ability to interact centrally with so many important neuronal systems.

V. Other Pharmacological Actions

A. GROWTH HORMONE SECRETION Not only is the ability of clonidine to elicit secretion of growth hormone (GH) employed currently in the treatment of short stature (Pintor et al., 1987) and as a test of GH secretory reserve (Gil-Ad et al., 1979), but the level of plasma GH produced by clonidine administration is being used routinely in research protocols to assess the status of central a2adrenergic receptors (Siever et al., 1982; Eriksson et al., 1986). In clinical studies this approach has been applied to investigation of the status of central a,-adrenergic receptors in childhood affective disorders, depression, and schizophrenia. As with its antihypertensive response, clonidine’s GH stimulatory action is mediated through postsynaptic a,adrenergic receptors, that is, central stores of brain catecholamines are not required for expression of this action (Conway et al., 1990). That the a,-adrenergic receptors mediating GH secretion are part of a physiologically relevant pathway is indicated by the ability of central a,-adrenergic

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receptor blockade with yohimbine to inhibit the GH response to insulininduced hypoglycemia (Suri et al., 1990). T h e hypothalamus is a rich source of neurotransmitters, modulators, and peptide hormones. Although the site of the a,-adrenergic receptor innervation involved in this response is not known, clonidine does not appear to act entirely through stimulation of growth hormone-releasing hormone (Suri et al., 1990). In a recent study by Conway and co-workers (1990),the GH response to clonidine in rats was significantly inhibited by metergoline and cyproheptadine, serotonin [5-hydroxytryptamine (5HT,) receptor] antagonists. Depletion of brain serotonin levels using the tryptophan hydroxylase inhibitor parachlorophenylalanine (PCPA) also blocked the GH surge to subsequent injection of clonidine. The site of potential interaction between clonidine and serotonin was identified through experiments in which the selective serotonin neurotoxin, 5,7dihydroxytryptamine, was microinjected into selected hypothalamic nuclei. Selective destruction of serotonergic cells in the arcuate nucleus effectively abolished the GH secretion to clonidine. Despite these suggestive experiments, the authors were reluctant to affirm that the relevant a,-adrenergic receptors were located on arcuate serotonergic neurons. Their trepidation was due in part to the counterargument that clonidine-induced activation of a,-adrenergic neurons generally leads to a reduction in serotonergic neuronal activity. Therefore, if serotonin neurons were interjected in the GH pathway, clonidine should inhibit GH release. Nevertheless, the data are strong for an important interaction between central adrenergic and serotonergic interactions in GH secretion. An alternate scenario was presented in an earlier study by Casanueva and co-workers (1984). These investigators reported that in their clinical studies administration of atropine completely blocked the GH secretory peak following either an arginine o r clonidine stimulus. Atropine treatment also blocked the GH peak following physical exercise. Although the authors did not comment upon a possible adrenergic-cholinergic interaction in GH secretion, they did suggest that a cholinergic synapse may be the final common pathway for a variety of GH stimulants. However, as with the serotonin situation, cholinergic agonists also stimulate GH secretion. For the serotonergic or cholinergic scenarios to be correct, clonidine would have to have an excitatory action on the respective systems. Although clonidine does in general inhibit these systems, this possibility cannot be ruled out. Further studies should be quite fruitful in helping to elucidate this interesting property of clonidine and its relationship with these hypothalamic neurotransmitters.


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B. INHIBITION OF CHOLINESTERASE INHIBITOR TOXICITY In developing antidotes to poisoning by cholinesterase inhibitors, several potential target sites at the cholinergic synapse have been studied, including the postsynaptic receptor and acetylcholinesterase itself. Muscarinic receptor-blocking agents such as atropine have been, and continue to be, the primary means for pharmacological intervention in cases of anticholinesterase poisoning. Oxime reactivators may prove useful when the enzyme is inhibited by an organophosphorus agent. One site that has received much less attention is the presynaptic site, the cholinergic nerve terminal. It is reasonable to expect that reducing acetylcholine release would decrease the toxicity of cholinesterase inhibitors. In fact, inhibitory mechanisms are in place to reduce cholinergic neuronal function in situations of postsynaptic overstimulation. These mechanisms include down-regulation or decreased postsynaptic receptor numbers and decreased release of transmitter from the cholinergic nerve terminal. In cases in which poisoning is slow enough, such adaptive changes allow for significant degrees of cholinesterase inhibition without toxicity and even without overt symptoms. In cases of acute, severe poisoning, such adaptive mechanisms are too slow to prevent the development of toxicity. Acceleration of presynaptic down-regulation by pharmacological agents, therefore, may be of use under such circumstances. The examination of this approach to protection has been limited, perhaps due to a paucity of presynaptic cholinergic blocking agents, or from the fear that such agents might prove highly toxic. Complete blockade of acetylcholine release with botulinum toxin underscores this concern. However, an agent that merely accelerates presynaptic down-regulation without completely inhibiting transmitter release might be of value. In support of this possibility, Buccafusco (1982) first demonstrated a marked protection by clonidine against the manifestations of physostigmine toxicity. In the mouse, clonidine’s protective actions were associated with significant inhibition of the increase in brain acetylcholine induced by the reversible cholinesterase inhibitor. That the mechanism of protection was primarily through central cholinergic and peripheral muscarinic pathways was indicated by the lack of protection afforded by clonidine against the toxic effects of the selective, peripherally acting cholinesterase inhibitor neostigmine. More recent studies employing organophosphate cholinesterase inhibitors (soman and echothiophate) substantiated the physostigmine studies (Aronstam et al., 1986; Buccafusco and Aronstam, 1986, 1987). Moreover, the combined use of

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atropine and clonidine in the pretreatment regimen was found to enhance survival following soman administration. During these experiments it was consistently noted that clonidine-pretreated mice that survived LD,, doses of soman had fewer behavioral side effects than mice that did not receive clonidine. This observation was confirmed in a rat model in which the toxic behavioral effects induced by soman administration were quantitated (Buccafusco et al., 1988a). Again clonidine offered protection against the lethal as well as the toxic behavioral effects of soman. This behavioral toxicity included the development of tremors, hind limb extension, convulsions and jerking motions, chewing, and excessive salivation. Soman also decreased the expression of normal ongoing behaviors such as sniffing, rearing, and general locomotor activity. 'The ability of clonidine to inhibit soman-induced convulsive behavior (Buccafusco et al., l988a, 1989) is consistent with anticonvulsive activity in other animal models (see Baran et al., 1989),a feature of its protection that might help to limit the development of more permanent toxic manifestations. T h e protective effects of clonidine and atropine were usually synergistic, even though clonidine antagonized some of the stereotyped behaviors elicited by protective doses of atropine (Molloy et al., 1986). 'Thus, while enhancing the protective actions of atropine, clonidine also may reduce atropine-induced side effects. The mechanism for this latter effect is yet to be identified. The mechanism of the protective actions of clonidine has been investigated and appears to be more complex than simply inhibition of acetylcholine release. That is, whereas clonidine does produce a marked inhibition of acetylcholine synthesis and release at peripheral and central muscarinic synapses, its other actions on the cholinergic system include a reversible inhibition of acetylcholinesterase and a reversible inhibition of muscarinic receptors (Aronstam et al., 1986; Buccafusco and Aronstam, 1986, 1987). This interaction with the enzyme was observed in both in vivo and in vitru preparations, and in both cases the permanent inhibition of enzyme activity produced by soman was reduced by clonidine treatment. This mode of protection of the enzyme may be similar to that produced by reversible carbamate cholinesterase inhibitors, such as pyridostigmine. Reversible inhibition of cholinesterase essentially protects the enzyme from permanent inactivation by irreversible agents such as soman. Clonidine and many of the tested analogs were also found to interact directly with muscarinic receptors, in an atropine-like manner. Therefore, clonidine and several analogs afford protection against soman poisoning by at least three mechanisms: (1) a reduction in the release of acetylcholine in brain and peripheral muscarinic sites, (2) reversible inhibition of cholinesterase, and (3) blockade of central mus-

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carinic receptors. All of these effects were achieved following administration of protective doses of clonidine. Furthermore, the muscarinic receptor down-regulation that occurs in response to elevated transmitter levels following soman administration is prevented in mice protected with clonidine (Aronstam et al., 1987). This may simply be a reflection of clonidine’s ability to limit acetylcholine release and postsynaptic receptor stimulation. It is not clear to what degree each of these three mechanisms contributes to the ability of clonidine to produce protection against the acute lethal actions of soman. However, several centrally acting a-adrenergic agonists of different chemical structures share this ability with clonidine, and its relative potency as a protective agent was related to its affinity for a-adrenergic binding sites labeled with [3H]clonidine (Buccafusco and Aronstam, 1987). Also, the ability of clonidine to inhibit the biosynthesis of brain acetylcholine is mediated through a-adrenergic receptors (Buccafusco, 1982). It is this action of clonidine, therefore, that appears to predominate in its ability to protect against the acutely toxic actions of soman. It is possible that clonidineinduced protection of cholinesterase from irreversible inactivation by soman may provide a more chronic form of protection, that is, protection long after the clonidine is metabolized or excreted. Along these lines, animals pretreated with clonidine that survive the soman challenge for several days appear behaviorally normal as compared with atropine-pretreated animals or saline-pretreated animals that survive an LD,, dose of soman (Buccafusco et al., 1989,1990b).This apparent difference was observed even though protected animals may have received a higher dose of soman. Initially this finding might not seem noteworthy, because protected animals might be expected to have a better prognosis than nonprotected animals. However, soman is an irreversible inhibitor of acetylcholinesterase, and clonidine is a very shortacting drug, particularly in rodents (Jarrot and Spector, 1978). In fact, animals protected to the same extent as clonidine with high doses (25 mg/kg) of atropine did not appear as behaviorally normal as the clonidine-pretreated animals. In rats (Buccafusco et al., 1988a), 0.5 mg/kg of clonidine produced a degree of protection equivalent to 6 mg/kg of atropine against lethal and soman-induced behavioral effects. T h e ability of a single dose of soman to induce behavioral abnormalities several days later has been reported (Haggerty et al., 1986). In fact, the decrease in spontaneous motor activity induced by an LD,, dose of soman in the rat was observed over 21 days. Such chronic toxic behavioral effects have also been observed following exposure to other organophosphate cholinesterase inhibitors in animals, and in humans following accidental intoxication (for review, see Karczmar, 1984). The

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mechanism for this delayed toxicity is not clear, but it has been reported that significant brain pathology can occur as early as 24 hr following soman administration (Churchill et al., 1985; Pazdernik et al., 1985). It has been suggested that the pathology may result from the severe convulsive activity present soon after soman administration (Samson et al., 1985). Atropine pretreatment is only partially effective in reducing soman-induced convulsive activity and hence delayed brain pathology (Pazdernik et al., 1986). Clonidine pretreatment, however, was more effective than atropine in preventing the occurrence of soman-induced convulsive behavior, and survivors in the clonidine group were less behaviorally impaired than the atropine group (Buccafusco et al., 1989). High doses of atropine do not offer a substantial degree of protection against chronic toxicity, thus of the three mechanisms of clonidine protection stated above, direct muscarinic receptor blockade is probably of minor importance. Because posttreatment with clonidine is not as effective as pretreatment (J. J. Buccafusco, unpublished observation), the ability of clonidine to reduce acetylcholine release is an important contribution to its acute protective actions. The ability to protect cholinesterase from irreversible inactivation (Aronstam et al., 1986; Buccafusco and Aronstam, 1986) may be more important for protection against chronic soman toxicity. The ability of clonidine to produce its antihypertensive response as well as several of its other pharmacological actions is not reduced in animals whose central stores of catecholamines are reduced (see above). Thus, although central a,-adrenergic receptors are required for clonidine’s actions, these receptors are not located on central catecholaminergic nerves. That central cholinergic neurons are endowed with inhibitory clonidine-binding sites is suggested by clonidine’s ability to inhibit brain acetylcholine turnover rates (see above) as well as its ability to offer protection against cholinesterase inhibitor intoxication. It is not yet clear, however, whether central catecholaminergic systems are implicated in the toxic and lethal manifestations of soman intoxication or whether the protection afforded by clonidine involves such pathways. In one study, catecholamine depletion with reserpine or a-methyl-ptyrosine enhanced the toxicity of 2-sec-butylphenyl-N-methylcarbamate and malathion, whereas the monoamine oxidase inhibitor pargyline reduced their toxicities (Takahashi et al., 1987). The implication is that endogenous brain catecholamines provided a tonically active protective mechanism against cholinesterase inhibitor toxicity. Conflicting results were obtained in a subsequent study regarding the role of brain adrenergic systems in clonidine-induced protection. Depletion of brain catecholamines, using effective doses of either reserpine o r a-methyl-p-


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tyrosine as the depleting agent, did not alter soman-induced death or behavioral toxicity (Buccafusco et al., 1988b). This finding was consistent with their earlier report that the centrally acting a-adrenergic blocking drug yohimbine had no effect on physostigmine-induced toxicity (Buccafusco, 1982). Therefore, the findings by Buccafusco and co-workers are consistent with the possibility that either that these a receptors (clonidine-binding sites) are not functionally innervated, or, if they are, that there exists no significant noradrenergic inhibitory tone. It is difficult to reconcile the opposite results obtained by the above two studies; however, it is possible that N-methylcarbamate and malathion have a toxicological profile that differs from that of soman and/or different sites of action. For example, acute intoxication by soman is mediated almost entirely through central mechanisms, whereas the former agents are not as lipid soluble and may include peripheral mechanisms in producing acute intoxication. The results of the two studies are in concert, however, with regard to the finding that elevation of brain catecholamines using pargyline results in significant protection against cholinesterase inhibitor toxicity. In fact, in the study by Buccafusco and co-workers (1988b), pargyline and clonidine were additive in their protective actions. The fact that pargyline-induced protection was time dependent suggests that its mechanism is related to brain catecholamine accumulation. T h e concept that elevation of brain catecholamine levels is inhibitory to cholinergic activity is consistent with the opposing nature of cholinergic and adrenergic functions throughout the nervous system. T h e experiment employing the amine-depleting agents indicated that adrenergic inhibitory tone to central cholinergic systems sensitive to soman is minimal (at least with regard to the cholinergic systems involved in respiratory function and certain behavioral activity), but the results from the pargyline experiments indicate that endogenous catecholamines under the proper circumstances can provide protection against cholinergic overstimulation. It has yet to be determined whether cholinergic neurons sensitive to soman receive inhibitory innervation from adrenergic systems, or whether cholinergic neurons are endowed with inhibitory regulatory receptors sensitive to a2agonists. In the latter case, elevation of catecholamine levels in the extracellular fluid following pargyline administration could potentially activate such receptors. Thus, clonidine administration offers significant protection against soman lethality as well as several toxic manifestations of soman administration, including hypertension, excessive salivation, convulsive behavior, locomotor depression, and a wide profile of stereotyped activity. Clonidine’s protective actions are in many ways similar to those of atropine, but in other ways surpass them. Clonidine was more effective than atropine at reduc-

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ing soman-induced convulsive behavior and at reducing the expression of chronic soman toxicity, possibly because the two are casually related. The mechanism by which clonidine offers protection in the rat was indicated by experiments demonstrating clonidine’s ability to inhibit somaninduced brain acetylcholine accumulation and soman-induced muscarinic receptor down-regulation. Finally, clonidine’s protective actions appear to be independent of central adrenergic neurons; however, elevation of brain catecholamines, like clonidine, offered significant protection against soman toxicity. This finding may be interpreted to indicate that endogenous catecholamine systems may modulate cholinergic neurons under certain conditions and may therefore serve as a protective mechanism against cholinergic overstimulation. Utilization of direct receptor agonists such as clonidine to limit the expression of cholinergic activity is one way to induce this effect. However, it is possible that other, as yet unknown, protectants against cholinesterase inhibitor intoxication could be developed.

C. LEARNING AND MEMORY Alzheimer’s disease is one of the greatest clinical challenges of this century. T h e disease itself, responsible for the institutionalization of a large proportion of the more than 1,000,000 individuals in nursing homes in this country, is characterized by a rapid decline in cognitive and memory abilities, usually in the later years. There is currently no conclusive diagnostic test save autopsy, but post mortem studies have indicated ultrastructural pathological changes characterized by neuritic plaques, paired helical filaments, and granulovacuolar bodies. One of the most consistent abnormalities associated with Alzheimer’s disease is that of brain neurochemistry. Several laboratories throughout the world have reported a significant loss of markers specific for brain cholinergic neurons, including the synthetic enzyme choline acetyltransferase and the degradative enzyme acetylcholinesterase (Bowen et al., 1976; Davies, 1979; Chyle et al., 1983; Younkin et al., 1986; for review, see also Perry arid Perry, 1983; Bartus et al., 1985). T h e degeneration of cholinergic neurons is not global, but there appears to be a rather selective loss of cholinergic fibers originating in the diencephalon, the so-called nucleus basalis of Meynert (Whitehouse et al., 1981; Perry and Perry, 1983; Wilcock et al., 1983; Nagai et al., 1983; Younkin et al., 1986). Although the cholinergic hypothesis of Alzheimer’s disease has been challenged (Palmer et al.. 1986, for example) and it is now generally accepted that several other neurotransmitters o r neurohormones may also play a role


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(Gottfries et al., 1986; Kragh-Sorensen et al., 1986), pharmacological treatments that enhance central cholinergic neurotransmission have generally provided the most reproducible beneficial effects. These have included direct-acting muscarinic receptor agonists such as arecoline, and centrally acting cholinesterase inhibitors such as physostigmine (Beller et al., 1985). Acute beneficial effects have been observed for both classes of drugs, although both have serious limitations with regard to long-term therapy. These include steep, inverted-U-type dose-response curves, tolerance to the beneficial effects, severe side effects, and unpredictable patient response to standard dosing regimens. Recent clinical trials show that a long-acting cholinesterase inhibitor, tetrahydroaminoacridine (THA), produces significant clinical improvement in Alzheimer’s patients, an effect that may continue during long-term treatment (Summers et al., 1986). It is interesting that the class of agent that seems to be of greatest benefit in Alzheimer’s disease is an indirect-acting cholinergic agonist. Thus, even though there exists significant degeneration of cholinergic fibers to cortical limbic structures, inhibition of the degradative enzyme is still able to enhance the effectiveness of neurotransmitter released from the remaining, intact cholinergic nerve endings. Another feature of the Alzheimer’s brain is that, for the most part, the density and affinity of muscarinic receptors are intact (Caufield et al., 1982; Whitehouse and Au, 1986), although the results of recent studies in which specific receptor subtypes (M, o r M,) were examined suggest a selective loss of the M, subtype (Whitehouse and Au, 1986; Quirion et al., 1986). It has been suggested that the M, subtype may be located presynaptically, hence, the loss during cholinergic degeneration (Iversen, 1986). The presynaptic muscarinic receptor, however, generally functions as a negative-feedback receptor, which upon activation inhibits the further release of acetylcholine. It is this presynaptic down-regulation that may be responsible for the tolerance that can develop to prolonged treatment with physostigmine (Becker and Giacobini, 1988). This and the often severe side effects to physostigmine and other cholinergic agonists provide a great clinical challenge. I n addition to the well-known alterations in central cholinergic function, Alzheimer’s disease has also been associated, post mortem, with a decline in brain catecholamines related to neuronal degeneration. Two recent studies typify such findings (Palmer et al., 1987a,b), with the latter study revealing an approximately 50% reduction in markers for norepinephrine innervation of the temporal cortex. Although the ability of centrally acting cholinergic agonists to enhance learning and memory performance in animals and humans has been appreciated for many

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years, it is more recently that central catecholaminergic pathways have been implicated in these processes (Mair et al., 1985; Robbins et al., 1985). Such findings have been strengthened by recent reports that stimulation of central a,-adrenergic receptors by clonidine results in enhanced memory performance in rat (Freedman et d., 1979; Sara et d., 1987) and nonhuman primate (Arnsten and Goldman-Rakic, 1985, 1987) models, and in human patients with Korsakoffs psychosis (McEntee and Mair, 1980; Mair and McEntee, 1986). T h e mechanism by which clonidine elicits these changes is not known, although it is clear that in Korsakoffs patients, clonidine is acting through central pathways, and that the beneficial action of clonidine was greatest in patients exhibiting the greatest loss of CSF markers for norepinephrine (Mair and McEntee, 1986). Perhaps the neural substrate for this effect of clonidine is the significant multiple high-affinity a, receptor binding sites that have been measured in human prefrontal cortex (Carlson and Andorn, 1986). Despite these findings in support of the possibility that clonidine and related drugs could provide symptomatic improvement in human dementias, the results reported in the aged nonhuman primate have been inconsistent. T h e aged monkey has been employed as one of the most relevant models for human dementia, particularly Alzheimer's disease. These animals not only exhibit behavioral deficits in standard delayedresponse paradigms, but neurochemical and morphological changes similar to those seen on autopsy of Alzheimer's disease brain are often observed (for review see Bartus and Dean, 1985). In one series of studies (Arnsten and Goldman-Rakic, 1985, 1987),clonidine improved delayedresponse performance in aged monkeys, an effect that was linked to a,adrenergic receptor stimulation within the prefrontal cortex. However, such beneficial actions were not reproduced in another laboratory (Davis et ul., 1988), from which it was reported that clonidine disrupted performance. T h e latter group speculated that these opposite findings might be related to differences in the testing environment o r paradigm. The earlier work (Arnsten and Goldman-Rakic, 1985, 1987) had employed the presentation of three-dimensional stimuli though the use of a Wisconsin General Test Apparatus (WGTA), whereas the latter study (Davis et al., 1988) had employed presentation of two-dimensional, spatially linked stimuli with an automated apparatus. That the effect of clonidine might be task specific was later demonstrated by Arnsten and Goldman-Rakic (1990), who could not obtain the same magnitude of beneficial effect of clonidine in a delayed nonmatch-to-sample task as they had earlier obtained with the delayed-response task. Recent studies in this laboratory using a completely automated sys-


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tem, but one in which environmental distractors were not completely eliminated (other animals in the same testing room), have confirmed the ability of clonidine to produce a modest but significant improvement in performance of a delayed matching-to-sample task in both young and aged monkeys (Jackson and Buccafusco, 1991). In the presence of low, clinically relevant doses of clonidine, an improvement averaging approximately 10% of baseline performance was obtained with the most effective dose determined for each animal (0.5-10.0 pglkg, i.m). This improvement continued for at least 24 hr following the single administration, much longer than would have been predicted from the known half-life of the drug. By 3 days after administration, responding was again at baseline levels. T h e reason for this chronic improvement is not known, but it could be a consequence of enhanced learning rather than just improved recall or attention. Sedative effects were observed at higher doses ( > l o &kg) in young monkeys, but the aged animals were more sensitive in this regard, with the lO-pg/kg dose producing marked sedation and impairment in performance. The sedative action of clonidine itself does not appear to be the cause of the enhanced performance (Arnsten et al., 1988). Again, despite these promising results in nonhuman primates, clinical trials using clonidine and the related drug guanfacine did not improve the neuropsychological rating of intellectual and memory function in Alzheimer’s patients (Schlegel et al., 1989). This disparity in results regarding Korsakoffs patients indicated above may reflect a more consistent or dramatic loss of cortical noradrenergic innervation associated with the latter disease. It is possible that certain subpopulations of dementia patients, including Alzheimer’s dementia, might still benefit from therapy with central a,-adrenergic agonists. Also, it is generally recognized that Alzheimer’s disease involves alterations in several neurotransmitter systems and it may be necessary to address pharmacologically these multiple deficits.

VI. Summary and Conclusions

A. THEDIVERSITY OF PHARMACOLOGICAL ACTIONS T h e considerable number of pharmacological actions reported for clonidine as indicated in Table I and the substantial number of potential and actual clinical uses (Table 11) are probably unprecedented for a single pharmacological entity. Such a diverse pharmacological profile is undoubtedly a reflection of a diverse mechanism of action. Clonidine

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and related drugs have been demonstrated to interact with classical neurotransmitter systems, the catecholamines, indolamines, cholinergic, opioidergic, and amino acid transmitter systems. For each action of clonidine no single mechanism has been clearly identified as mediating the pharmacological effect. Perhaps the only common link is that most, if riot all, of its actions are mediated through stimulation of a,-adrenergic receptors (Table I). However, even this direct mechanism may be complicated. T h e emergence of multiple clonidine-binding sites as well as the discovery of a novel (nonadrenergic) iniidazole receptor allow for an even greater diversity in its mechanism of action. Also to be considered is the strategic location of clonidine-binding sites. The location of such a site on the neuron soma could have a completely different effect in terms of excitability of the cell if the location of the receptors is the nerve terminals. T h e role of clonidine’s presynaptic actions, particularly regarding central catecholamine systems, has been addressed several times. In many of clonidine’s actions such an inhibitory effect on catecholamine release has not appeared to play an important role. Nevertheless, inhibition of catecholamine release cannot be discounted as an important contributor to clonidine’s long-term actions. It is not yet clear whether “postsynaptic” clonidine-binding sites are innervated by catecholamine nerve terminals. For example, although clonidine’s inhibitory action on central cholinergic transmission is inhibited by a,-adrenergic antagonists, blockade of these receptors in the absence of clonidine does not lead to increased acetylcholine release (Buccafusco and Spector, 1980a). Therefore, either the clonidine receptors on cholinergic neurons are not innervated or the putative noradrenergic tone is quiescent under normal circumstances. In either case, it is possible that such receptors, located on cholinergic or on other systems, continually sample and respond to changes in catecholamine levels in the cerebrospinal fluid. The mutually antagonistic action of norepinephrine and acetylcholine in dually innervated autonomic effector organs is enhanced through presynaptic modulation. That is, norepinephrine overflow during periods of intense sympathetic stimulation can reduce the release of acetylcholine from parasympathetic nerve endings. This relationship between adrenergic and cholinergic neurons may be further amplified in the CNS, which is perhaps more of a closed system than the peripheral circulation. This concept of neurotransmission via the brain extracellular fluid has more recently been termed volume transmission (Fuxe and Agnati, 1991). Volume transmission was originally invoked in part to explain the presence of neurotransmitter receptors at extrasynaptic sites, but the concept may also help to explain the lack of effect of ag-

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adrenergic blocking drugs on cholinergic function indicated above. For example, if central cholinergic activity is indeed modulated by extracellular levels of catecholamines, it is possible that after chronic treatment with clonidine, decreased levels of catecholamines in the cerebrospinal fluid could have an impact on cholinergic neurotransmission. Another factor contributing to the diversity of clonidine’s actions is the nature of the signal transduction processes reported to be activated by the drug. Consistent actions on neuronal cAMP and related systems have been difficult to obtain. Clonidine’s effect on brain cAMP have been demonstrated to be dependent upon the brain region examined and other factors related to specific preparations (see Janowsky and Sulser, 1987; Nakamichi et al., 1987). Part of the problem may also reside in the ability of clonidine to increase intracellular pH (see Marx, 1987). T h e change in pH has been related to clonidine’s ability to enhance Na+ / H exchange as demonstrated in platelets following stimulation of a,-adrenergic receptors. The increase in intracellular pH may trigger the decrease in cAMP often observed following a,-adrenergic receptor simulation in these cells. +

B. CLONIDINE AS A NEUROMODULATOR Despite this diversity of action, clonidine has been employed clinically for several years quite successfully. Although newer related drugs have purported to be associated with less severe side effects than clonidine, in fact, the drug is well tolerated by a large proportion of patients. Perhaps this selectivity is related to the drug’s marked potency for central autonomic pathways. This action is important for its antihypertensive and perhaps its antiwithdrawal properties, the current main clinical applications. In this respect, it is interesting that clonidine is a better antihypertensive agent than it is a hypotensive agent. Thus, in the presence of disease the drug’s actions are more apparent. The inhibitory action of clonidine on neurotransmitter systems is generally modulatory, its effectiveness in inhibiting transmitter release frequency dependent. Unlike direct receptor-blocking agents, clonidine’s modulatory ability could allow for a more subtle degree of regulation. Even in high doses, for example, clonidine does not usually alter the steady-state levels of neurotransmitter and does not completely inhibit the release or synthesis process. Finally, part of clonidine’s selectivity may be related to its singular effectiveness and potency in inhibiting central and peripheral cholinergic muscarinic activity. If this possibility has merit, then

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the role of central cholinergic neurons in mediating many of clonidine’s phartnacological properties as well as in the disease process itself deserves further investigation.

VII. Future Directions

There is no doubt that clonidine will continue to be employed as the experimental drug of choice for investigations examining the effect of central a,-adrenergic receptor stimulation. Some of the clinical indications for the drug will disappear, possibly its use as an antipsychotic or antianxiety agent. However, the use of the drug in withdrawal syndromes will continue to be examined. Clonidine’s analgesic actions will continue to be exploited and may actually surpass the use of opiates in the production of localized spinal analgesia and as a supplement to general anesthetics, before, during, and after surgical procedures. T h e advantage of clonidine and related drugs in this regard is the reduced capacity for producing central respiratory depression and an almost nonexistent abuse liability. Although the use of clonidine as a first-line antihypertensive agent has been supplanted by newer agents targeting peripheral nerves or blood vessels, a reexamination of clonidine’s property as a central sympatholytic agent may be in order. It is generally appreciated that lowering of blood pressure in hypertensive disease per se does always result in protection from secondary cardiovascular complications such as coronary artery disease, left ventricular hypertrophy, vascular damage to the eyes, kidney, and brain, and the production of ventricular arrhythmias (Rosenman, 1989). This kind of toxicity has been ascribed to excessive catecholamine excretion subsequent to enhanced sympathetic activity often associated with essential hypertension. In fact, the use of certain classes of peripherally acting antihypertensive agents may actually enhance sympatho-adrenal outflow though activation of cardiovascular reflex activity o r through plasma sodium and fluid loss (1220, 1989). To date there have been no large or multicenter studies regarding the incidence of cardiovascular-related morbidity or lethality following longterm treatment with a central versus peripheral antihypertensive treatment regimen. If such a study confirms the cardiovascular protective action of clonidine and related drugs, a resurgence in the utilization of this class of antihypertensive agent would be expected. It might be point-


99

ed out that clonidine has been demonstrated to provide benefit in chronic heart failure and ischemic heart disease (Giles et al., 1985) and that in hypertensive rats, clonidine significantly reduced blood pressure and heart rate fluctuations, reflecting an enhanced baroreceptor control (Grichois et al., 1990). Finally, clonidine and related drugs may continue to be examined for potential antidementia effects. Despite the disappointing initial results in Alzheimer’s patients mentioned above, clonidine may exhibit beneficial actions in certain categories of human dementia. It might also be borne in mind that central cholinergic agonists such as physostigmine have produced limited benefit in Alzheimer’s patients. The possibility that combined treatment with clonidine and physostigmine may produce an added benefit fits with the observation of multiple neurotransmitter alterations in Alzheimer’s disease. Several studies (discussed above) indicate that clonidine can inhibit many of the pharmacological effects produced by cholinesterase inhibitors, but this does not necessarily imply incompatibility for their use in dementia. The reverse may actually be the case. Although clonidine does inhibit central cholinergic function, studies in our laboratory have demonstrated that this action is selective for certain brain regions. For example, the striatum and hippocampus, which exhibit a high degree of cholinergic innervation, are essentially spared from the inhibitory action of clonidine on their cholinergic neurons (Buccafusco and Spector, 1980a; Buccafusco, 1984a). One hypothesis that is under current investigation in this laboratory is that combined treatment with clonidine and physostigmine results in a greater than additive effect in a delayed matching paradigm in young and aged monkeys. In this situation, it is envisioned that clonidine may have some palliative action of its own on memory and/or learning processes, but may also obviate some of the autonomic side effects associated with physostigmine administration. This selective protective action of clonidine may be due to its inability to interfere with cholinergic function in the hippocampus, a crucial site for memory formation. Along these lines, it has been demonstrated that in rats having lesions of the ascending noradrenergic bundle, the memory-enhancing effect of physostigmine was blocked. A combination of physostigmine and clonidine was required to restore the beneficial effects of central cholinergic stimulation in the lesioned animals (Haroutunian et al., 1990). Thus, clonidine may widen the therapeutic window for physostigmine’s beneficial actions by reducing interfering side effects and by contributing to the relief of noradrenergic deficits associated with aging or the disease process.

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Morphine and enkephalin interactions with putative neurotransmitters in rat hippocampus.

Comparison of the actions of central and peripheral administration of clonidine and BS 100-141 in the rabbit [proceedings].

Central nervous system depressant actions of clonidine and UK-14,304: partial dissociation of EEG and behavioural effects.

[Central neurotransmitters and regulation of gastric acid secretion].

Central actions of valproate sodium.

Actions and interactions of antithrombin and heparin.

Central vasopressor actions of angiotensin.

Brain mechanisms associated with therapeutic actions of benzodiazepines: focus on neurotransmitters.

Molecular biology of phenobarbital actions and interactions.

Trace amines and alternative neurotransmitters in the central nervous system.

Clonidine and related imidazolidines: structure-activity relationship with respect to various cardiovascular actions.

Neurochemical and behavioral effects of clonidine and related imidazolines: interaction with alpha-adrenoceptors.

Cardiovascular actions of N-allyl-clonidine (ST 567), a substance with specific bradycardic action.

The role of serotonin in memory: interactions with neurotransmitters and downstream signaling.

Neuropharmacologic control of neuroendocrine function in man.

Neuropharmacologic Approaches to Cognitive Rehabilitation.

On the mechanism of central hypotensive action of clonidine.

Central actions of histamine: microelectrophoretic studies.

Electrophysiology of oxytocin actions on central neurons.

Endocrine, cardiovascular, and behavioral responses to clonidine in patients with panic disorder.

Somatostatin: central nervous system actions on glucoregulation.

Topical clonidine produces mydriasis by a central nervous system action.

Central antiemetic actions of pimozide and haloperidol in the dog.

Prejunctional clonidine-adenosine interactions in the rat vas deferens [proceedings].