143

J. Physiol. (1975), 250, pp. 143-160 With 5 text-ftgures Printed in Great Britain

THE EFFECTS OF CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE AND OTHER ADENINE NUCLEOTIDES ON BODY TEMPERATURE

BY M. J. DASCOMBE AND A. S. MILTON From the Department of Pharmacology, University Medical Buildings, Foresterhill, Aberdeen, AB9 2ZD.

(Received 20 November 1974) SUIMMARY

1. Adenosine 3',5'-monophosphate (cAMP), its dibutyryl derivative (Db-cAMP) and other adenine nucleotides have been micro-injected into the hypothalamic region of the unanaesthetized cat and the effects on body temperature, and on behavioural and autonomic thermoregulatory activities observed. 2. Db-cAMP and cAMP both produced hypothermia when applied to the pre-optic anterior hypothalamus. With Db-cAMP the hypothermia was shown to be dose dependent between 50 and 500 gug (0-096-0'96 tmole). 3. AMP, ADP and ATP also produced hypothermia when injected into the pre-optic anterior hypothalamus. 4. The order of relative potencies of the adenine nucleotides with respect both to the hypothermia produced and to the autonomic thermoregulatory effects observed were similar. Db-cAMP was most potent and cAMP least. 5. Micro-injection into the pre-optic anterior hypothalamus of many substances including saline produced in most cats a non-specific rise in body temperature apparently the result of tissue damage. Intraperitoneal injection of 4-acetamidophenol (paracetamol 50 mg/kg) reduced or abolished this febrile response. 6. The hypothermic effect of the adenine nucleotides has been compared with the effects produced in these same cats by micro-injections of noradrenaline, 5-hydroxytryptamine, a mixture of acetylcholine and physostigmine (1: 1), EDTA and excess Ca2+ ions. 7. It is concluded that as Db-cAMP and cAMP both produce hypothermia, it is unlikely that endogenous cAMP in the pre-optic anterior hypothalamus mediates the hyperthermic responses to pyrogens and prostaglandins.

144

M. J. DASCOMBE AND A. S. MILTON INTRODUCTION

Noradrenaline, 5-hydroxytryptamine and prostaglandins of the E series are widely accepted as being involved in body temperature regulation or in fever or in both. All these substances have been shown to stimulate cyclic adenosine 3',5'-monophosphate (cAMP) formation in brain tissue (Kakiuchi & Rall, 1968; Klainer, Chi, Freidberg, Rall & Sutherland, 1962; Zor, Kaneko, Schneider, McCann, Lowe, Bloom, Borland & Field, 1969). The action on body temperature of cAMP when injected directly into the region of the anterior hypothalamus has, therefore, been investigated. Varagic & Beleslin (1973) and Clark, Cumby & Davis (1974) have both shown hypothermic responses to the injection of the dibutyryl derivative of cAMP (Db-cAMP) into the lateral ventricle of the unanaesthetized cat. Whether the observed effects were specifically due to the Db-cAMP, or were of a non-specific nature is not clear. In the present experiments not only Db-cAMP, but also cAMP, adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) have been injected directly into the region of the anterior hypothalamus of the unanaesthetized cat and both their short- and long-term effects on body temperature observed. In addition, the autonomic and behavioural effects of the injection of these substances have been documented. METHODS

Female cats weighing between 2-0 and 3-5 kg were used. Under aseptic conditions a Collison type cannula (21-gauge needle tubing) was stereotaxically implanted into the brain such that its tip lay in the region of the hypothalamus. The co-ordinates were taken from the stereotaxic atlas of the cat brain by Snider & Niemer (1961). The animals were allowed to recover for at least a week before being used for experiments. At the completion of the experiments on each cat, the animal was killed with pentobarbitone sodium 130 mg/kg injected i.P. and the brain perfused in situ with a 4 % formaldehyde solution in 0-9 % NaCl. The brain was removed and 40 sem frozen sections cut to determine the position of the cannula tip and the site of injection of the drugs. Animals were individually caged during the experiments without access to food or water, and at an ambient temperature of 22 + 2° C. Experiments were conducted at the same time each day and each animal was used at intervals of not less than 72 hr. Temperature was measured with a thermistor probe (Yellow Springs Instrument series 400) inserted about 10 cm into the rectum and held in position with adhesive tape and was monitored continuously on a Jaquet multi-channel recorder. The control rectal temperature was taken as being the value maintained constant for at least 30 min, after 1 hr allowed for equilibration after the insertion of the probe. Solutions were injected into the brain through an injection cannula (26-gauge needle tubing) connected by a polyethylene tube to a 10ll. syringe. The injection system was flushed and filled with the solution, and the injection cannula passed

CYCLIC AMP AND BODY TEMPERATURE

145

through the previously implanted guide cannula to a site 1 mm beyond the tip of the outer tube (Myers, 1966). The volume of solution injected over a period of 20-30 see in all experiments was 2-5 plu.; solutions were not washed in. In some animals 2-5 ,d. 5 % Evans Blue solution was injected into the brain of the unanaesthetized cat before it was killed to aid in the determination of the site of the cannula tip and to give some indication of the spread of drugs previously administered. Drug solutions were made up in 0 9 % NaCl and filtered through either 0-22 or 0-025 jsm pore size Millipore filters. The adenine compounds were then sealed in single dose glass ampoules and stored in the dark at -20° C. In several experiments in which the effect of 4-acetamidophenol (Paracetamol, 4-Ac) on the hyperthermia produced by the drugs was examined, paracetamol 50 mg/ kg was injected i.P. The paracetamol was dissolved in 1 ml. of hot ethylene glycol and made up to 5 ml with 0-9 % NaCl to give an injected concentration of 50 mg/ml. Dose related volumes of ethylene glycol/saline containing no paracetamol were used for control injections. All glassware, filters and 0-9 % NaCl used were sterile and pyrogen-free. Clean glassware, 0-025 ,sm Millipore filters and Swinnex filter holders were soaked in 3 N-HC1 for at least 12 hr, rinsed with sterile, pyrogen-free water until neutral to wide range pH indicator paper (BDH) and sealed in aluminium foil. Glassware was heated at 300 'C in a hot air oven for at least 12 hr. Filter units were autoclaved at 121° C and 103-4 kPatm for 15 min. 0-22 ,um Millipore filters were used in the form of MillexTM disposable filter units. The micro-injection system was rinsed wi th sterile, pyrogen-free water and steam sterilized for 30 min immediately before use. In some experiments the micro-injection system was immersed in 1 N-NaOH overnight before being rinsed and steam sterilized. Drug u8ed Adenosine 3',5'-cyclic monophosphoric acid monosodium salt; adenosine (Sigma Chemical Company). N6-2'-O-dibutyryl adenosine 3'5'-cyclic monophosphoric acid monosodium salt (Sigma Chemical Company and Boehringer Mannheim). Adenosine 5'-monophosphate, adenosine 5'-diphosphate and adenosine 5'-triphosphate, disodium salts (Boehringer Mannheim). Ethylenediaminetetra-acetic acid disodium salt, sodium n-butyrate, 5-hydroxytryptamine creatinine sulphate, O-acetylcholine chloride (BDH Chemicals Ltd). Physostigmine sulphate (Maefarlan Smith Ltd). L-noradrenaline bitartrate (Koch Light). 4-Acetamidophenol (Koch Light).

RESULTS

Adenine nucleotides. Db-cAMP was injected into fourteen cats in doses ranging from 0-096 to 0-96 Iamole (50-500,g). At dose levels above 0-096 Itmole all cats responded to the drug within 10 min of injection. Db-cAMP produced a dose related hypothermia in eight animals. In four animals hypothermia in response to Db-cAMP occurred after about 5 min and was observed with all doses of Db-cAMP above 0-096 Itmole (Fig. 1). In these animals injections of 0-9 % NaCl 0-39 ptmole, sodium n-butyrate 0-96 and 1-93 /tmole and NaCl 1-93 Iumole failed to produce a large rise in rectal temperature, the rise in temperature being less than 0.60 C, during the 3 hr period following injection. In one other animal

~M. J. DASCOMBE AND A. S. MILTON injections of 0-9 % NaCI caused a rise (mean ± s.E. of mean of n experiments) in rectal temperature of 1DO + 0-2 (n = 4); sodium n-butyrate 1-93 lzmole, 1*3 +0-3 (n = 2) and NaCi 1*93flmole, 1*1 +0*4' C (n = 3) at 3 hr following injection. These rises in temperature which were continuous from the time of injection and sustained throughout the time of the experiments, were associated with sedation, a crouched posture and ear skin vasoconstriction. Db-cAM1P 0-24 molee caused a rise of 1.0 + 0.30 C (n = 2) at 3 hr, commencing 60 min after injection. Db-cAMP 0-48 molee caused a fall in temperature of 1.1 + 0.10 C (n = 2) below control values beginning about 5 min after the injection. The development of hypo146146

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Fig. 1. Rectal temperature records of an unanaesthetized cat. At the arrow 2-5 #Il. drug solution injected into the pre-optic anterior hypothalamus. 0.9 % NaCi, ( x); Db-cAMP, 0-096,umole (M), 0-24,smole ([:]), 0-48flmole (@) and 0-96,smole (0).

thermia in all five animals was associated with autonomic heat loss activity, such as ear skin vasodilation, polypnoea, panting and occasionally sweating from the paw pads, and lasted 60-90 min. After this time temperature returned to control levels within 2-3 hr, accompanied by vasoconstriction and shivering. The injection sites in four of these animals were shown post mortem to be in the pre-optic anterior hypothalamus and in one cat the site was in the optic chiasma 1 mm ventral to the pre-optic anterior

hypothalamus. In a further three animals, control injections of 0-9 % NaCl, sodium n-butyrate and NaCi produced hyperthermic responses continuously from the time of the injection and of more than 6 hr duration. In these animals, Db-cAMP 0-24 and 0-48 ptmole produced an initial rise in rectal temperature of 0-15-0. 850 C. This initial rise was followed, after some 20 min, by a period of active heat loss accompanied by a fall in body temperature lasting

147 CYCLIC AMP AND BODY TEMPERATURE 60-70 min (Fig. 2) which in two animals was 015-0 3° C below control values. A sustained rise in temperature which followed, lasted throughout the remaining time of the experiments. The magnitude of the initial hyperthermia after an injection of Db-cAMP was greatest in that animal which developed fever most rapidly and achieved the highest level of rectal temperature after a control injection. The injection site in this animal was in the columna fornicis of the anterior hypothalamus. Db-cAMP 0-96 ,mole in two of these animals produced falls in body temperature of as much as

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1-10 C below control values (Fig. 2). In the third animal Db-cAMP 0-96 Itmole produced extreme hyperactivity which although associated with peripheral vasodilatation, panting and intermittent stretching out, produced a rise in rectal temperature. The injection sites were determined to be in the dorsal region of the pre-optic anterior hypothalamus in two animals and in the anterior commissure in the third cat. Injection of Db-cAMP 0-24 and 0-48 /tmole into the region rostral to the pre-optic area was without effect on body temperature. Db-cAMP 0-96 /tmole into this site caused a fall in temperature starting 20 min after injection and reaching 1.10 C below control value. Db-cAMP 0-96 pmole in

148148

~M. J. DASCOMBE AND A. S. MILTON this animal also produced intermittent convulsions that were associated with transient increases in temperature during the established hypothermia. In two animals which received the highest dose of 1Db -cAMP (O- 9 6fmole) a rise in body temperature was observed which paralleled the increased motor activity. The increased temperature was not sustained and fell as the motor effects of the drug wore off. One of these animals was shown to have its injection site in the posterior hypothalamus. In the second cat, although the cannula was in the pre-optic anterior hypothalamus area, the drug solution had access to the III ventricle as evidenced by the observed

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presence of c.s.f. in the guide cannula du-ring experiments and the intraventricular distribution of dye injected prior to the histological study. The increase in both motor activity and temperature produced by 1Db-cAMfP 0-96 gtmole was greater and quicker in onset when administered into the posterior hypothalamus rather than into the pre-optic anterior hypothalamus (Fig. 3). In the animal with the injection site in the pre-optic anterior hypothalamus Db-cAMP 0-48 /tmole produced falls in temperature after about 10 min accompanied by ear skin vasodilatation and panting, proceeded by an initial hyperthermia that was always associated with increased motor activity and agitation, Db-cAMP 0-24 jt-mole had no effect on behaviour, autonomic activity or temperature. The same doses into the

149 CYCLIC AMP AND BODY TEMPERATURE posterior hypothalamus, although producing defaecation, vocalization and ear skin vasodilatation, had no effect on rectal temperature. Injection of Db-cAMP (0-24 and 0-48 tmole) into the anterior hypothalamus of one animal produced slight hypothermia in response to the higher dose of the compound (0-48 mole) 5-10 min after injection, the lower dose being without effect. Administration of these doses (0-24 and 0*48 Itmole) Db-cAMP to the anterior hypothalamus of a second animal and to the region dorsal to the anterior hypothalamus in another animal was without effect. Db-cAMP 0 96 Etmole caused an increase in body temperature associated with increased motor activity in all three animals. Fig. 4 summarizes the response of these animals to Db-cAMP 0-48 Emole.

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Fig. 4. Positions of injection sites in fourteen cats. All sites were 1-3 mm left of mid line and have been superimposed on a sagittal section of the cat brain. Db-cAMP 0 48 molee injected at these sites caused: (0) a fall in body temperature (0.81-50 C below control values) within 10 min of injection; (0) a fall in body temperature (less than 0.40 C below control values), with one exception, 20 min after injection, and (0) no effect on body temperature.

Db-cAMP (0-24-0*96 gimole) in all fourteen animals commonly caused vocalization, defaecation and micturition, followed by mydriasis and salivation. Non-thermoregulatory behavioural effects of Db-cAMP occasionally seen in addition to increased motor activity, were contraversive circling, grooming activity and scratching of the head and neck regions. Eight out of nine cats which received 0-96 Emole Db-cAMP also showed a strong desire to escape the confines of their cages. In addition, five of these animals became extremely agitated and hyperactive, with two convulsing when subjected to external tactile or auditory stimuli. The injection sites in both these two animals was shown post mortem to be in the rostral region of the pre-optic area. The lowest dose tested, 0-096 #mole,

M. J. DASCOMBE AND A. S. MILTON 150 produced no effects on body temperature, behaviour or autonomic activity. In eleven of these cats the responses to injection of other adenine nucleotides were studied. In four animals with injection sites in the pre-optic anterior hypothalamus and in one animal with its injection site 1 mm ventral to the

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Time (h) Fig. 5. Rectal temperature records of an unanaesthetized cat. At the arrow 2*5 #sl. drug solution injected into the optic chiasma, 1 mm ventral to the pre-optic anterior hypothalamus (a) 0-9% NaCl (Fl); cAMP 0-48 mole (x); AMP 0-48 #mole (0); (b) ADP 0-48 molee (-); ATP 0-48 mole (U) and Db-cAMP 0-48 #mole (A).

pre-optic anterior hypothalamus, in the optic chiasma, ATP, ADP, AMP and cAMP (0.48 and 0-96Qamole) all produced ear skin vasodilatation, which in four cats was associated with falls in rectal temperature below control values (Fig. 5). Hypothermia in response to ATP (0-48 and 0-96 #mole) was also occasionally associated with polypnoea and in one animal, where

151 CYCLIC AMP AND BODY TEMPERATURE the cannula tip was shown to be in the pre-optic anterior hypothalamus, with panting and stretching out. ATP was more potent when injected into the pre-optic anterior hypothalamus than either ADP or AMP, which were approximately equipotent, whilst cAMP was the least active of the nucleotides. Injection of the adenine nucleotides (0.48 and 0 96 ,mole) into the anterior hypothalamus of two cats produced heat loss activity associated with a fall in temperature in only one animal, in response to high dose (0.96 tmole) ATP and AMP. ATP 0-96 Fmole into the fasciculus mamillothalamicus dorsal to the anterior hypothalamus of one other animal caused a slight fall in temperature associated with heat loss activity. All other adenine nucleotides at this latter site were without effect. The hypothermic responses to the nucleotides were more marked and more reproducible in the pre-opticus than in the anterior hypothalamus. The latency and the rates of fall in body temperature produced by these nucleotides were similar to those produced by Db-cAMP (Fig. 5). The magnitude of the response, however, was less than that produced by Db-cAMP lasting only 30-40 min before the onset of ear skin vasoconstriction and shivering. Six of the animals produced hyperthermic responses to injections of 0 9 % NaCl, sodium n-butyrate 0-96 and 1*93 mole and NaCI 1-93 gmole and the onset of hyperthermia following central injection tended to mask the hypothermic responses, both to Db-cAMP and the other nucleotides. In all eight of these animals ATP (0.48 and 0*96 ,mole) produced vocalization, defaecation and micturition, whilst salivation was occasionally seen. ADP and AMP (0-48 and 0-96 #mole) produced ear skin vasodilatation in these animals; however, with the exception of occasional vocalization, no other response to these compounds was seen. Administration of cAMP (0.48 and 0*96 tmole) produced only slight ear skin vasodilatation. The extreme hyperactivity and agitation produced by Db-cAMP were not observed in response to these nucleotides. In three of the eleven animals tested the adenine nucleotides produced no effect on behaviour, autonomic activity or body temperature. The injection sites in these animals were in the region rostral to the pre-optic area, the posterior hypothalamus and the anterior commissure. The dose of adenosine micro-injected in these experiments was limited to 0-047 gimole (12.5 /tg) due to the poor solubility of this nucleoside in 0 9 % NaCl in comparison to that of the sodium salts of the nucleotides. Administration of this nucleoside in this dose into the anterior hypothalamus, the anterior commissure, the fasciculus mamillothalamicus or the preoptic anterior hypothalamus was without effect on body temperature or behavioural activity. With the exception of ear skin vasodilatation when

M. J. DASCOMBE AND A. S. MILTON injected into the optic chiasma 1 mm ventral to the pre-optic anterior hypothalamus of one cat, adenosine (0.047 mole) had no effect on autonomic activity. Neurotranmmitters. Noradrenaline, 5-hydroxytryptamine (5-HT) and a mixture of acetylcholine and physostigmine (ACh/phys) in equal parts by weight were administered in the dose range 25-150 n-mole to five animals. Noradrenaline produced a dose dependent fall in body temperature in four cats. Falls in body temperature were observed within 3 min following injection of noradrenaline and were associated with ear skin vasodilatation, panting and stretching out. Defaecation and salivation were also seen. The injection sites in the animals responding to noradrenaline were shown to be in the pre-optic anterior hypothalamus, in the anterior hypothalamus and in anterior commissure. Noradrenaline had no effect on body temperature when injected into the fasciculus mamillothalamicus dorsal to the anterior hypothalamus in one cat. In all five animals, 5-hydroxytryptamine (25-150 n-mole) produced a crouched posture and shivering together with ear skin vasodilatation. In three animals, with injection sites in the anterior hypothalamus and the anterior commissure, 5-hydroxytryptamine caused a rise in body temperature observed 2-3 min after injection and lasting 40-80 min. 5-hydroxytryptamine injected into the pre-optic anterior hypothalamus of one animal, in which its drug solutions had access to the III ventricle from this site, produced a fall in body temperature lasting 2-3 hr. Administration of 5-hydroxytryptamine into the pre-optic anterior hypothalamus of another animal at a site 1 mm caudal to the site in the latter animal produced either a fall or a rise in body temperature in different experiments. Four out of these five animals responded to ACh/physostigmine (25-150 n-mole) with a rise in body temperature within 5 min that lasted 1-2 hr. The rise in temperature was associated with both a crouched posture and ear skin vasodilatation. The largest response to ACh/physostigmine was observed following injection into the anterior hypothalamus, the most caudal site tested at the level of the hypothalamus. The injection site in the animal not responding to ACh/physostigmine was in the pre-optic anterior hypothalamus. Ca2+ ions. Ethylenediamine tetra-acetic acid (EDTA, 0-17-0-67 Mmole) in eight out of ten animals produced a rise in body temperature within 2-5 min, which was maximal in about 20 min, and was accompanied by peripheral vasoconstriction and shivering. Body temperature fell to control level in 60-80 min accompanied by ear skin vasodilatation, polypnoea and panting. Two animals responded to EDTA with a fall in body temperature associated with ear skin vasodilatation and polypnoea. EDTA in all ten animals tested caused vocalization, micturition and mydriasis. In one 152

153 CYCLIC AMP AND BODY TEMPERATURE animal where the injection site was shown, post mortem, to be in the posterior hypothalamus, EDTA 0-67 Itmole caused extreme aggression which included spitting and attack, accompanied by pilo-erection and excessive salivation. The cannula sites in all ten animals were shown to be in the hypothalamic region, the two animals responding with hypothermia being both in the columna fornicis of the anterior hypothalamus. Calcium chloride 0-96 lzmole micro-injected into the region of the hypothalamus in six animals caused a fall in body temperature associated with ear skin vasodilatation, within 2-3 min of injection that was maximal in 20-30 min. Return to control level temperature in 60-80 min was accompanied by ear skin vasoconstriction. Calcium chloride 0-96 molee injected into the columna fornicis of one other cat produced a rise in temperature associated with ear skin vasoconstriction in 2-3 min that was maximal in about 30 min. After this time body temperature fell before the onset of a long lasting secondary rise 60-90 min after injection that was associated with a crouched posture and sedation. Control injections of 0*9 % NaCl produced ear skin vasoconstriction, a crouched posture, sedation and a long lasting rise in temperature in this animal 60-70 min after injection. Non-specific febrile response. In ten animals there was a sustained rise in body temperature associated with sedation, ear skin vasoconstriction and crouched posture, following injections of drugs or saline into the hypothalamic region. The hyperthermic response in six animals was accentuated, that is the latency of onset of the response was shorter and the rate of rise in body temperature greater, when drug solutions rather than 0*9 % NaCl were administered (Fig. 2). This effect appeared to be irrespective of drug or concentration of drug solution used in these experiments. In the other four animals, the responses to 0.9 % NaCl and drug solutions were similar. The latency of the rise depended upon the exact placement of the cannula, being shorter at sides nearer the pre-optic anterior hypothalamus, but up to 2-3 hr at sites more distant from this region. In some animals in which this response was absent upon initial use following cannula implantation, hyperthermia following micro-injection was observed to gradually develop as the experimental life of the animal increased. Histological studies of the brains of animals in which this febrile response was prominent commonly showed cell proliferation and/or tissue damage at the site of injection. 4-Acetamidophenol (paracetamol, 4-Ac) 50 mg/kg, injected i.P. after the onset of this temperature rise caused a reduction in this 'fever', irrespective of the drug originally administered. Similarly, pre-treatment of the animals with paracetamol some 30 min before central injection prevented the temperature rise in response to the adenine nucleotides.

154

M. J. DASCOMBE AND A. S. MILTON DISCUSSION

The balance of noradrenaline and 5-hydroxytryptamine in the anterior hypothalamic region has been proposed as a mechanism whereby the body temperature of the cat is regulated (Feldberg & Myers, 1964). Prostaglandins of the E series have been shown to exert a powerful hyperthermic effect in the cat and rabbit (Milton & Wendlandt, 1970, 1971; Feldberg & Saxena, 1971) and sheep (Bligh & Milton, 1972) and have been proposed as mediators of fever (Feldberg & Gupta, 1973). Feldberg, Gupta, Milton & Wendlandt, 1973). Since noradrenaline, 5-hydroxytryptamine and prostaglandins have been shown to stimulate the formation of cyclic adenosine 3',5'-monophosphate (cAMP) in brain tissue (Kakiuchi & Rall, 1968; Klainer et al. 1962; Zor et al. 1969) it is conceivable that their temperature effects may be mediated through the adenyl cyclase system. If this were to be so, then cAMP itself could affect body temperature and indeed have an essential role in thermoregulation and fever. It is generally accepted that cAMP does not readily penetrate cell membranes and to mimic the action of endogenous cAMP it is necessary to administer a derivative of the parent compound which more readily penetrates cell membranes (Robison, Butcher & Sutherland, 1971 a). The N6-2'-O-dibutyryl derivative (Db-cAMP) is commonly used to produce a more potent effect than cAMP itself, possibly owing to its greater lipid solubility and its resistance to hydrolysis by cAMP phosphodiesterase (Posternak, Sutherland & Henion, 1962; Cheung, 1970). Some exceptions have been noted. Kim, Shulman & Levine (1968) reported that Db-cAMP was less effective than cAMP in causing relaxation of the isolated rabbit ileum, and Ryan & Heidrick (1968) showed that Db-cAMP was less effective as an inhibitor of cell growth in tissue culture. There are, furthermore, several reports of Db-cAMP producing responses different from those of the parent compound on various preparations including skeletal muscle (Chambaut, Ebou6-Bonis, Hanoune & Clauser, 1969) melanophores (Hadley & Goldman, 1969), isolated fat cells (Solomon, Brush & Kitabehi, 1970), HeLa cells (Hilz & Tarnowski, 1970) and intestinal smooth muscle (Bowman & Hall, 1070). In the experiments reported here the actions of cAMP with Db-cAMP when injected into the hypothalamic region of the cat brain are compared. Adenosine and its nucleotides are pharmacologically potent and to determine whether the observed responses to cAMP and Db-cAMP were related to the cyclic structure of these compounds we also investigated the effects of adenosine and the nucleotides, ATP, ADP and AMP. Since thermoregulation involves both behavioural and autonomic activities,

CYCLIC AMP AND BODY TEMPERATURE 1550 the recorded effects of the compounds on unanaesthetized cats included the behavioural and autonomic responses as well as changes in body temperature. Breckenridge & Lisk (1969) observed that anterior hypothalamic implantation of both cAMP and Db-cAMP caused hyperthermia associated with hyperactivity and aggression in rats. Sustained hyperthermia in response to intraventricular administration of Db-cAMP has been found in the cat, preceded on some occasions by an initial hypothermia lasting 1-3 hr or a transient hyperthermia (Varagic & Beleslin, 1973; Clark et al. 1974). Similar responses are reported here following micro-injection of Db-cAMP into the hypothalamic area of most animals. In our experiments, however, all solutions including 0-9 % NaCl, sodium n-butyrate and a hyperosmotic solution of sodium ions injected into the hypothalamic area, caused in most animals similar sustained rises in body temperature. We must conclude, therefore, that central micro-injection may produce a non-specific, long-lasting rise in body temperature in the cat. A similar response has been observed by Feldberg, Myers & Veale (1970) and by Feldberg & Saxena (1970) following ventricular perfusion and by Varagic & Beleslin (1973) in response to intraventricular injection of Db-cAMP, of ATP and of butyrate; these authors also proposed the rise to be a nonspecific effect. From our observations the hyperthermia appears to be initiated in the pre-optic anterior hypothalamus, the region between the anterior commissure and the optic chiasma known to be involved with thermoregulation and shown to be sensitive in the cat, as well as other species, to bacterial pyrogens (Villablanca & Myers, 1965), leucocytic pyrogen (Jackson, 1967), monoamines (Feldberg & Myers, 1965) and prostaglandins (Milton & Wendlandt, 1970). Although precautions were taken to prevent pyrogenic contamination of the injected solutions, the possibility of such contamination remained. The gradual development of pyrexia of unknown aetiology in some animals in which the response was initially absent, and the tissue disruption commonly evident at the site of injection on histological study both indicate that the hyperthermia may be due to tissue damage. This could presumably be a result of cranial implantation or repeated micro-injections at a single site or both in any individual animal. Paracetamol and indomethacin, antipyretics which inhibit synthesis of prostaglandins (Vane, 1971; Flower & Vane, 1972), have been shown to reduce this fever (Clark et al. 1974; Dey, Feldberg, Gupta, Milton & Wendlandt, 1974). In addition, Dey et al. (1974) have shown that the associated increase in prostaglandin-like activity in cisternal c.s.f. is also reduced and these authors propose that the fever of unknown aetiology in cats is due to increased synthesis and release of prostaglandins, stimulated

M. J. DASCOMBE AND A. S. MILTON possibly by tissue damage. The hyperthermic responses observed in our experiments were reduced by treatment with paracetamol. In addition, administration of paracetamol prior to central micro-injection prevented hyperthermia in response to the adenosine nucleotides. These observations suggest a similar interpretation. It is not possible to determine from these experiments to what extent increased synthesis and release of prostaglandins may be a direct effect of the substances injected. The hypothermic responses to Db-cAMP, ATP, ADP, AMP and cAMP in equimolar doses were similar in both latency and rate of fall of temperature suggesting a common mode of action. Differences in potency of these compounds were apparent in the degree and the duration of the hypothermia developed. The order of potency with respect to the hypothermia induced paralleled the nucleotides ability to produce autonomic effects. Db-cAMiP was most potent, and produced hypothermia in some experiments despite increased motor activity that would have otherwise increased rectal temperature, cAMP was least active. Hypothermia in response to Db-cAMP in some animals was observed without the subsequent development of a sustained hyperthermia during the time of the experiments. These animals did not develop hyperthermia following the injection of 0 9 % NaCl. The transient hyperthermia seen initially after administration of DbcAMP may be due in part to general C.N.S. stimulation made manifest as increased motor activity as suggested by Clark et al. (1974). But in some of the experiments reported here this effect is attributable to the rapid development of pyrexia following micro-injection into brain tissue, before the onset of drug-induced hypothermia. The longer latency of the DbcAMIP effect in these animals possibly represents the time for diffusion of the drug to the pre-optic anterior hypothalamus from injection sites remote from this region (Myers, 1966). The response to the drug then appears as an initial transient hyperthermia followed by a sustained rise in body temperature when the hypothermic effect of Db-cAMP wears off. Falls in body temperature below control values in response to higher doses of the drug support this proposal. The behavioural and autonomic effects observed in response to DbcAMP were qualitatively the same as described by Gessa, Krishna, Forn, Tagliamonte & Brodie (1970). Our results differed, however, from those of these authors who reported that the effects of Db-cAMP lasted many hours. In our experiments, the effects of Db-cAMP lasted less than 3 hr. Furthermore, the adenine nucleotides ATP, AMP and cAMP have been reported as being inactive when centrally injected into the unanaesthetized cat (Gessa et al. 1970; Varagic & Beleslin, 1973), in contrast the results reported here show that localized application of ATP, ADP, AMP and cAMP into the pre-optic anterior hypothalamus in quantities equimolar to the doses 156

CYCLIC AMP AND BODY TEMPERATURE 157 of Db-cAMP employed by these authors and ourselves, produced ear skin vasodilatation in the unanaesthetized cat. In addition, ATP produced vocalization, defaecation, micturition, polypnoea and salivation. The responses to micro-injection of noradrenaline and 5-hydroxytryptamine are in agreement with the central effects documented for these monoamines in the cat (Feldberg & Myers, 1965; Kulkarni, 1967). Hyperthermia was produced by micro-injection of a mixture of acetylcholine and physostigmine into the region of the anterior hypothalamus of the unanaesthetized cat. This observation is in keeping with the work of Myers & Yaksh (1969), who concluded from their work in the monkey, the existence of a cholinergic heat production pathway projecting from the anterior hypothalamus to the posterior hypothalamus. EDTA which is a divalent ion chelating agent has previously been reported to produce a rapid peak hyperthermia when injected into the ventricular system of the cat (Clark, 1971). A similar rapid peak hyperthermia was seen in eight out of ten cats when the chelating agent was locally applied to the hypothalamic region. In two animals, however, where both the cannula tips were in the columna fornicis of the anterior hypothalamus, EDTA produced hypothermia associated with ear skin vasodilatation. Ca2+ ions have been reported to produce hypothermia, associated with ataxia and sedation when perfused through the ventricles of the rabbit (Feldberg & Saxena, 1970) and the cat (Myers & Veale, 1970). A similar effect on body temperature is reported here in the absence of sedation and ataxia when Ca2+ ions were micro-injected into the anterior hypothalamic region in six out of seven cats. Injections of Ca2+ ions into the columna fornicis of one cat, however, produced a rise in body temperature. In all animals, Ca2+ ions had, as would be expected, the opposite effect on body temperature to that of EDTA which is capable of binding Ca2+ ions. The hypothermic response to both cAMP and to noradrenaline in these experiments in the cat is consistent with the proposal that cAMP mediates many adrenergic responses (Robison, Butcher & Sutherland, 1971 b). Both intrahypothalamic noradrenaline (Cooper, Cranston & Honour, 1965) and Db-cAMP (Laburn, Rosendorff, Willies& Woolf, 1974) cause hyperthermia in the rabbit. If it is shown that the response to Db-cAMP in the rabbit is not a non-specific effect, the apparent species difference in the responses to noradrenaline and Db-cAMP in the cat and the rabbit would support this proposal of Robison et al. (1971 b). Hypothermia and associated heat loss activity in response to equimolar quantities of Db-cAMP, cAMP, ATP, ADP and AMP, which differ only in their relative potency, indicates that this effect is a common property of these compounds. The effects of the adenine nucleotides on body

M. J. DASCOMBE AND A. S. MILTON temperature appear to be independent of the cyclic structure of cAMP, although it is not possible to determine from these experiments whether ATP, ADP and/or AMP modifyendogenous levels of cAMP. Adenine-ribose compounds have been reported as increasing cAMP levels in tissue from all regions of the brain of all species thus far tested (Rall & Sattin, 1970). The hypothermic response to adenine nucleotides could be due to a localized increase in free Ca2+ since Duffy & Schwarz (1974) have reported the displacement of Ca2+ ions from isolated erythrocyte membranes in response to cAMP and ATP. This hypothesis is strengthened by the observation that Ca2+ ions when injected in equimolar quantities produced hypothermia. That increased availability of Ca2+ ions in the columna fornicis causes an increase in body temperature does not argue against this proposal, as hypothermia caused by Db-cAMP injected into this area was preceded by transient hyperthermia, which may be due, in part, to the effect of Ca2+ ions in this region. That cAMP and Db-cAMP produce hypothermia when locally applied to the pre-optic anterior hypothalamus of the conscious cat compels the conclusion that it is unlikely that endogenous cAMP in this region mediates the hyperthermic response to pyrogens or to prostaglandins. 158

This work was supported by a grant from the Medical Research Council. M. J. Dascombe is in receipt of a Medical Research Council scholarship.

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