GLIA 5~81-94(1992)

Synthesis and Release of Neuroactive Substances by Glial Cells DAVID L. MARTIN Wadsworth Center for Laboratories and Research, New York State Department of Health, and Department of Environmental Health and Toxicology, State University of New York at Albany, Albany, New York 12201-0509

KEY WORDS

Neurotransmitters in glia, Neuropeptides in glia, Release processes by glia

ABSTRACT Glia contain, synthesize,or release more than 20 neuroactive compounds including neuropeptides, amino acid transmitters, eicosanoids, steroids, and growth factors. The stimuli that elicit release differ among compoundsbut include neuropeptides, neurotransmitters, receptor agonists, and elevated external [K+l. The mechanisms of release are poorly understood in most cases. Many of the neuroactive compounds are localized in discrete subpopulations of glia. Thus, glia are equipped to send as well as receive chemical messages and appear to be present as classes of cells with differing abilities to communicate chemically. It is possible that glia are as diverse as neurons in their functional characteristics.

INTRODUCTION Until recent years glial cells were thought of as somewhat passive companions to neurons that performed a variety of essential but almost perfunctory duties. Astrocytes, for example, help to maintain the ionic compositionof the extracellular space,take up and degrade neurotransmitters that overflow from the synapse, and provide glutamatergic and y-aminobutyric acid (GAl3A)ergicneurons with glutamine (Erecinska, 1990; Kimelberg and Katz, 1986; Walz, 1989). This vision of largely static and passive glia is gradually being replaced by a more dynamic picture in which glia play an active role in the development and function of the nervous system. It is, for example, becoming widely accepted that glia are important determinants of development in the CNS. Early in development, neurons migrate along the fibers of radial glia (Hatten, 1990; Hatten and Mason, 1990; Rakic, 1971). During migration the glia appear to serve merely as guides for neuronal movement, but the already established scaffold of radial glia determines the subsequent destination of the neurons. In the lateral geniculate nucleus, this initial neuronal migration is followed by the development of astrocytic laminae and then by the organization of the neurons into their characteristic laminar pattern (Hutchins and Casagrande, 1988).Neurons and 01992 Wiley-Liss, Inc.

glia undoubtedly must engage in complex molecular interactions during these processes. In cell culture, serine proteases appear to be involved in neuronal migration (Krysotek and Seeds, 1981a,b;Moonen et al., 1982), and glial-derived protease inhibitors, such as the nexins, suppress migration and induce neurite outgrowth (Gloor et al., 1986; Gurwitz and Cunningham, 1988; Wagner et al., 1991). Thus, radial glia and astrocytes each appear t o play a central role in developingthe final anatomic organizational patterns that are characteristic of the adult nervous system. In adult animals, neurons and glia have an intimate yet plastic morphological relationship. In parasympathetic ganglia, nerve terminals on neurons are more abundant near glial nuclei than elsewhere on the cell body, and the position and number of glia associated with identified parasympathetic neurons change gradually throughout the life of an animal, indicating that the relationships between neurons and glia are not fixed (Pomeroy and Purves, 1988). Astrocytes and neurons have an even more dynamic morphological relationship in the supraoptic and paraventricular nuclei of the

Received May 2,1991; accepted July 2,1991. Address reprint requests to David L. Martin, Ph.D., Wadsworth Center For Laboratories and Research, P.O. Box 509, Albany, NY 12201-0509.

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hypothalamus and the neurohypophysis (Perlmutter et al., 1985; Smithson et al., 1990; Theodosis and Poulain, 1989;Tweedle and Hatton, 1982).The magnocellular neuropeptidergic neurons are enclosed by glia cells during periods of low secretory activity, but the glial enclosure diminishes during periods of high secretory activity thereby allowing closer juxtaposition of the neuronal membranes. This extensive morphological rearrangement, which also includes changes in synaptic contacts, occurs during parturition, lactation, and dehydration, and can be induced by treatment with oxytocin and p-adrenergic agonists (Smithson et al., 1990; Theodosis et al., 1986). These kinds of interactions imply that neurons and glia engage in reciprocal communication. Astrocytes do have receptors for many neurotransmitters and neuropeptides (Kimelberg, 19881, indicating that they can receive messages from neurons, and there are a few fairly direct observations consistent with neuronal-toglial communication. In the superior cervical ganglion, stimulation of the preganglionic nerve leads to an increase in the cyclic adenosine monophosphate (CAMP) levels in the satellite glia (Ariano et al., 1982; Briggs et al., 1982). In the frog optic nerve action potentials transiently alter the properties of NaC channels in neighboring astrocytes (Marrero et al., 1989), an effect that may be due to the release of a transmitter-like compound from the nerve axon or to changes in the ionic environment in the space between the cells. In cerebellum, glia appear to be among the main target cells of a diffusible, endothelium-derived-relaxing-factor (EDRFI-like substance that is released in response to excitatory amino acids (Garthwaite and Garthwaite, 1987). The idea that neurons send messages to glia suggests that glia also send messages to neurons, as signalling systems in nature are generally characterized by feedback loops. At the present time the ability of glia to synthesize, store, and release neuroactive compounds is the principal line of evidence that glia can and do send signals to neurons. Obtaining clear evidence that the behavior or function of a neuron in situ is directly affected by a compound released by a glial cell remains an important and difficult problem. Bowery et al. (1976) provided evidence that GABA released from satellite glia in the superior cervical ganglion can depolarize ganglion neurons. In these experiments the glia were preloaded with GABA and release was elicited by bathing the ganglia with p-alanine, an alternative substrate for the GABA uptake system in ganglion glia. P-Alanine was thought to induce GABA release by blocking the reuptake of GABA or perhaps by inducing heteroexchange on the GABAIP-alanine transport system. Ganglion glia do not contain high levels of GABA in vivo, and this affect probably does not represent a recognized physiological function in the ganglion. Nevertheless, it does provide evidence that substances released from glia can affect neurons in tissue and supports the idea that glial transport systems can be an important route of release.

This paper is concerned with the synthesis, storage, and release of neuroactive compounds by glia. The term neuroactive compound is used in a broad sense to mean any compound that can elicit a physiological response from a neuron by activating a receptor, whether the response is electrophysiological, metabolic, or developmental in nature. The term glia is also used in a broad sense to include the various kinds of glial cells found throughout the nervous system including astrocytes, oligodendrocytes, satellite cells, and Schwann cells. We will refer to signalling compounds released by glia as gliotransmitters (Martin et al., 19881, a term that reflects origin and function of the compounds. SYNTHESIS AND STORAGE OF NEUROACTIVE SUBSTANCES BY GLIA Glia contain, synthesize, or release at least 20 potentially neuroactive compounds that may serve as gliotransmitters (Tables 1-3). Many of these compounds, such as the enkephalins, somatostatin, and substance P, are well-established hormones or neuropeptides that are thought t o serve only as messengers. Others, such as the prostanoids, EDRF, and steroids, have well-established paracrine or endocrine activity in other tissues. A few are well-characterized neurotransmitters (e.g., glutamate, aspartate), but, because of their metabolic roles, their mere presence in glia does not support the idea that glia use them for signalling. They are listed, however, because they are neuroactive and are released by glia in response to a variety of stimuli (Table 3). The ability of glia to synthesize neuroactive compounds is important, because, in general, cells that use a compound for signalling also synthesize it. In the absence of synthesis, the presence of a compound in a cell may indicate that the compound is merely being accumulated as part of catabolism. A possible example of this is the presence of GABA in astrocytes of mature animals (Blomqvist and Broman, 1988). Astrocytes in culture have very little capacity to synthesize GABA from glutamate (Wu et al., 19791,but astrocytes in vivo do accumulate GABA when GABA degradation is blocked and tissue levels are elevated (Blomqvist and Broman, 1988; Neal et al., 1989; Storm-Mathisen et al., 1986), and they may also accumulate GABA post mortem when GABA catabolism has ceased and tissue levels increase. Glia do, in fact, synthesize many neuroactive compounds, and in some cases synthesis can be stimulated by physiological agonists or drugs (Table 2). Cultured astrocytes synthesize and release several eicosanoids including prostaglandins D,, I,, E,, FZa,and thromboxane A, and B, (Gebicke-Haerter et al., 1988; Ishizaki et al., 1989; Jeremy et al., 1987; Seregi et al., 1987; Wroblewska et al., 1988). Synthesis is stimulated by physiologically relevant agonists, an important indication that eicosanoid synthesis by glia may be significant in vivo. Thus, purinergic (P2) agonists stimulate the

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RELEASE OF NEUROACTIVE COMPOUNDS FROM GLIA

TABLE 1 . Presence of neuroactive compounds in glia Compound

System

Reference

Adenine nucleotides (ATP, CAMP) Angiotensinogen

Satellite glia in superior cervical ganglion; cultured astrocytes Immunocytochemistry in vivo, astrocyte cultures

Enkephalin peptides including proenkephalin GABA

Immunocytochemistry in vivo; by RIA or immunocytochemistry in cultured astrocytes Immunocytochemistry in viva and in slices

Glutamate

Cultured astrocytes, by immunocytochemistry in slices Cultured astrocytes, oligodendrocytes Cultured cerebellar astrocytes Immunocytochemistry in viva Cultured astrocytes; immunocytochemistry in vivo

Ariano et al., 1982; Briggs et al., 1982; Madelian et al., 1985; Shain et al., 1986 Deschepper et al., 1986; Deschepper and Flaxman, 1990; Intebi et al., 1990; Kumar et al., 1988; Thomas and Semia, 1988 Hauser et al.. 1990; Melner et al., 1990 Spruce et al., 1990; Shinoda et al., 1989; Zagon et al.; 1985 Blomqvist and Broman, 1988; Neal et al., 1989; Storm-Mathisen et al., 1986 Erecinska, 1990 (review); Storm-Mathisen et al., 1986

Nerve growth factor Somatostatin Substance P Taurine

Furukawa et al., 1986, 1987; Gonzalez et al., 1990 Shinoda et al., 1989 Kostyk et al., 1989 Michel et al., 1986 Huxtable, 1989 (review); Martin and Shain, 1979; Philibert et al., 1988; Storm-Mathisen and Ottersen, 1986

TABLE 2. Synthesis of neuroactive comoounds in d i a Compound Adenine nucleotides (ATP, CAMP) Angiotensinogen Eicosanoids (prostaglandins Dz, Ez, Fz,,12; thromboxanes Ap, Bz; hydroxyeicosatetraenoic acids) Endothelium-derived relaxing factor (NO) Enkaphalin peptides including proenkephalin y-Aminobutyric acid (GABA) Glutamate Insulin-like growth factors I and I1 Nerve growth factor Neuropeptide Y Somatostatin Steroids (progesterone and others) Taurine Thyroid hormone (T3)

Svstem

Reference

CAMPin cultured astrocytes, glia in superior cervical ganglion In situ hybridization in viva; presence of mRNA and direct measurement in cultured astrocytes Cultured astrocytes, gliomas; synthetic enzyme (cyclooxygenase) by immunocytochemistry in viva Cerebellar astrocytes in culture Proenkephalin mRNA in viva and in cultured astrocytes From glutamate by cultured astrocytes at very low rates Cultured astrocytes mRNA in cultured astrocytes Production of and mRNA in cultured astrocytes and oligodendrocytes Preproneuropeptide Y mRNA in astrocytes from neonatal rats mRNA in cultured cerebellar astrocytes Synthetic enzyme (cytochrome P4~oscc) in white matter and cultured oligodendrocytes; pregnenolone and progesterone synthesis in oligodendrocytes Synthetic enzyme (cysteine sulfinate decarboxylase); synthesis from cysteine in cultured cerebellar astrocytes Synthetic enzyme (type I1 iodothyronine 5’-deiodinase) in cultured astrocytes

synthesis of prostaglandin D, and thromboxane A, (Gebicke-Haerter et al., 1988; Pearce et al., 1989),while angiotensin I1 and bradykinin stimulate the synthesis of prostaglandin I, (Wroblewska et al., 1988). On the other hand, synthesis of prostaglandin I, is lower in cultured astrocytes than in cultured endothelial or smooth-musclecells (Wroblewska et al., 19881,and glia cells and neurons in vivo both contain cyclooxygenase, the synthetic enzyme for prostanoids (Tsubokura et al., 1991).At present the actual contribution of astrocytes to prostanoid synthesis and release in vivo is unknown. Cultured astrocytes synthesize and release compound(s) with the properties of EDRF when stimulated

Ariano et al., 1982; Briggs et al., 1982; Madelian et al., 1985; Shain et al., 1986 Deschepper and Flaxman, 1990; Intebi et al., 1990; Kumar et al., 1988; Stornetta et al., 1988 Hartung and Toyka, 1987; Jeremy et al., 1987; Gebicke-Haerter et al., 1988; Ishizaki et al., 1989; Pearce et al., 1989; Seregi et al., 1987; Tsubokura et al., 1991; Wroblewska et al., 1988 Murphy et al., 1990 Spruce et al., 1990 Hauser et al., 1990; Melner et al., 1990; Shinoda et a]., 1989; Vilijn et al., 1988 Wu et a]., 1979 Erecinska, 1990 (review) Adamo et al., 1988; Balloti et al., 1987; Rotwein et al., 1988 Furukawa et al., 1986, 1987; Yamakuni et al., 1987; Gonzalez et al., 1990; Lu et al., 1991 Masters et al., 1990 Shinoda et al., 1989 Hu et al., 1987; Jung-Testas et al., 1989; Le Goascogne et al., 1987 Tappaz et al., 1990 Martin et al., unpublished data Courtin et al., 1988; Farwell and Leonard, 1989; Leonard et al., 1990

by bradykinin, quisqualate, or norepinephrine (Murphy et al., 1990). EDRFs were first identified as nonprostanoid vasodilators; their number is unknown, but one has been identified as nitric oxide. Evidence that glia synthesize steroids has been found both in cultured cells and in vivo. Cultured oligodendrocytes contain cytochrome P450scc, one of the enzymes of steroid biosynthesis (Jung-Testas et al., 19891, convert labeled mevalonate to pregnenolone, and convert pregnenolone to progesterone and other steroids (Hu et al., 1987). Cytochrome P450scc also is present in white matter in rat brain, as shown by immunocytochemistry (Jung-Testas et al., 1989).The function of these steroids

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TABLE 3. Release of neuroactive substances from glial cells Compound

, Adenine nucleotides (CAMP) Angiotensinogen

System Astrocytes and astrocytoma cells in culture Cultured astrocytes

Eicosanoids (prostaglandins Dz,Ez, 12; thromboxane Az)

Cultured astrocytes

Endothelium-derived relaxing factor (NO) Enkephalin peptides

Cultured forebrain astrocytes Cultured astrocytes from cortex and cerebellum Glia in superior cervical ganglion Cultured astrocytes, astrocytomas, miiller cells

7-Aminobutyric acid (GABA) Glutamate, L-aspartate, and D-aspartate Insulin-like growth factors Nerve growth factor Taurine

Cultured astrocytes release an unidentified insulinlike factor Cultured astrocytes Cultured astrocytes and glioma cells

Reference Release stimulated by Martin et al., 1990; unpublished data; Penit p-Adrenergic agonists (CAMP) et al., 1974 Deschepper and Flaxman, 1990; Intebi et al., Dexamethasone 1990 P2-purinergic agonists, Gebicke-Haerter et al., 1988; Hartung and Toyka, 1987; Jeremy et al., 1987; Wroblewska angiotensin 11, et al., 1988 bradykinin, phorbol esters, A23187 Murphy et al., 1990 Bradykinin, quisqualate, norepinephrine Melner et al., 1990; Shinoda et al., 1989 Forskolin, p-adrenergic agonists, cpt-CAMP Bowery et al., 1976 p-alanine, p-aminobutyric acid High [K'],, swelling, Holopainen and Kontro, 1990; kainate, quisqualate, Kimelberg et al., 1990; Lehmann and veratridine Hansson, 1988; Martin et al., 1990a; Szatkowski et al., 1990 Kadle et al., 1988 Greater in growing cells p-Adrenergic agonists, 5-hydroxytryptamine, adenosine, U50-488 (K-Opioid),ethanol, , elevated [K'],, reduced osmolarity, elevated temmrature

is unknown, but it is interesting that progesterone sulfate behaves as a G B A A antagonist and increases the firing rate of neurons when applied directly (see refs. in Hu et al., 1987). Astrocytes appear capable of activating thyroxin, a hormone that is extremely important in normal brain development.A large majority of 3,5,3' triiodothyronine (T3,the most active form of the hormone in brain) is produced intracerebrally from thyroxin (TJ; the enzyme responsible for this conversion (type I1 iodothyronine deiodinase) is present in cultured astrocytes (Courtin et al., 1988; Leonard et al., 1990). This enzyme is highly regulated, as its activity is increased 10-15-fold when cells are grown in the absence of thyroid hormone or in the presence of dibutyryl CAMP (dbcAMP). Its activity in the presence of dbcAMP can be increased further by hydrocortisone and insulin. The target of astrocytically produced T, is unclear; it may be the astrocyte itself, or it may be surrounding cells, including neurons. Astrocytes from certain brain regions synthesize and process proenkephalin and release enkephalin peptides (Melner et al., 1990; Shinoda et al., 1988; Spruce et al., 1990). Immunocytochemical and in situ hybridization experiments have shown that proenkephalin and its mRNA are present in a subpopulation of cerebellar astrocytes in gray and white matter but not in Bergmann glia of young rats (Hauser et al., 1990; Spruce et al., 1990). Proenkephalin mRNA and enkephalin peptides also are expressed by cultured astrocytes from cerebellum, cerebral cortex, striatum, and hypothalamus (Shinoda et al., 1989;Vilijn et al., 1988).The level of proenkephalin mRNA in hypothalamic astrocytes is

Furukawa et al., 1986, 1987 Huang et al., 1990; Madelian et al., 1988; Martin et al., 1988 (review); Pate1 et al., 1974; Perrone et al., 1986; Philibert et al., 1988; Ryan et al., 1975; Shain and Martin, 1984; Shain et al., 1987; Tigges et al., 1990

comparable to the level found in cultured neurons from hypothalamus and is greater than the level in astrocytes from other regions (Vilijn et al., 1988).Not all glial cells in culture express proenkephalin mRNA, as it is observed only in type 1 astrocytes (Melner et al., 1990; Spruce et al., 1990)and not in oligodendrocytesor mixed type 2 astrocyte-oligodendrocyte cultures (Melner et al., 1990).The expression of proenkephalin mRNA by cultured astrocytes is substantially increased by the p-adrenergic agonist isoproterenol (IPR) and by a permeant CAMPderivative (Melner et al., 1990). In contrast to proenkephalin, somatostatin mRNA and peptides have been found in cerebellar astrocytes but not in astrocytes from cerebral cortex or striatum (Shinoda et al., 1989). Angiotensinogen, the precursor to the hormone angiotensin 11, is produced in large amounts in brain, principally by glial cells. Immunocytochemical and in situ hybridization studies have shown that angiotensinogen and its mRNA are present in glial fibrillary acidic protein (GFAF')-positive cells, particularly in subcortical regions such as the hypothalamic and preoptic areas, the midbrain, and the medulla (Deschepperet al., 1986; Intebi et al., 1990; Stornetta et al., 1988; Thomas and Sernia, 1988). Cultured astrocytes also produce angiotensinogen and express its mRNA, and hypothalamic and thalamic astrocytes express greater amounts of each than do cerebral cortical astrocytes (Intebi et al., 1990).Angiotensinogen production and its mRNA levels in cultured hypothalamic astrocytes are regulated by glucocorticoids (Deschepper and Flaxman, 1990), but are unaffected by forskolin, which elevates intracellular CAMP levels (Intebi et al., 1990). In the periphery,

RELEASE O F NEUROACTIVE COMPOUNDS FROM GLIA

angiotensinogen serves as the precursor for angiotensin 11, a hormone that is involved in the maintenance of blood pressure and electrolyte homeostasis, but the function of these peptides in the nervous system is poorly understood. The entire angiotensinoged angiotensin system is present in brain, suggesting that angiotensinogen may function as an extracellular reservoir of angiotensin peptides as it does in the periphery. On the other hand, angiotensinogen also is related structurally to the serpin family of protease inhibitors (Carrel and Travis, 1985) and may have an additional function unrelated to its precursor role. As noted above, astrocytes produce protease inhibitors that affect neuronal migration and neurite outgrowth. Finally, glial cells have been reported to synthesize and secrete a number of growth factors including p-nerve growth factor (NGF), insulin, and insulin-likegrowth factors (IGF) I and 11. Cultured astrocytes express the mRNA for NGF and synthesize and secrete the protein (Furukawa et al., 1986, 1987; Gonzalez et al., 1990;Lu et al., 1991;Yamakuni et al., 1987).The mRNA for NGF is expressed at similar levels by cultured type I astrocytes, oligodendrocyte progenitor cells, and neurons, while NGF-like immunoreactivity is present in oligodendrocytes and a subpopulation of neurons (Gonzalez et al., 1990). The expression and secretion of NGF by astrocytes appears to be strongly regulated and may be related to cell growth. In mouse astrocytes, NGF secretion declines as the cells become confluent or when the cells are fed serum-free medium, which slows growth and induces shape change (Furukawa et al., 1987). Immunocytochemical experiments indicate that rat cortical astrocytes do not contain detectable levels of NGF except when grown in serum-free medium when a subpopulation strongly expresses the protein (Gonzalez et al., 1990). In contrast, NGF mRNA is reported to be lower in rat hippocampal astrocytes grown in serumfree medium and greater when glial growth is induced, as in transected optic nerve (Lu et al., 1991). The discordance between the decline of mRNA levels and secretion on the one hand, and the increase of immunoreactivity on the other requires clarification. Assuming that the discordance does not have a trivial explanation (i.e., methodologic problems, tissue differences), the increase in cellular NGF content may result from a change in translation of the message, a change in processing of the NGF precursor, or a reduction in the secretion of NGF. The latter possibility is attractive in light of the reduction in NGF secretion when astrocytes were transferred to serum-free medium. Cultured astrocytes contain the mRNA for IGF-I (Balloti et al., 1987; Rotwein et al., 1988) at levels similar to that found in cultured neurons (Rotwein et al., 1988), and the mRNA levels are down-regulated by glucocorticoids (dexamethasone) (Adamo et al., 1988). In contrast IGF-I1 mRNA is present only in astrocyte cultures and not neuronal cultures. Although the presence of IGF proteins has not been demonstrated in astrocytes, these cells do secrete compounds with biological and immunological properties similar to insu-

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lin (Kadle et al., 1988). In rats IGF mRNA is expressed most strongly at embryonic day 14 and declines by 60-75% to roughly adult levels by birth (Rotwein et al., 1988). Thus, it may continue to play a functional role beyond the period of brain development. Astrocytes also synthesize and contain taurine, which has inhibitory effects on neurons (reviewed by Huxtable, 1989). Immunocytochemical studies have shown that taurine is preferentially localized in astrocytes in brain stem, thalamus, and hypothalamus but is preferentially localized in neurons in the cerebral cortex, hippocampus, striatum, and cerebellum (StormMathisen and Ottersen, 1986). Astrocytes in culture contain high concentrations of taurine (>20 mM). Much of this taurine is accumulated from the serum-containing growth medium (which contains -15 pM taurine), but a substantial part appears to be synthesized by the astrocytes themselves, as they cannot be depleted completely of taurine by growing them in taurine-free medium (R.A. Waniewski, personal communication). Astrocytes do contain one of the enzymes of taurine biosynthesis, cysteine sulfinate decarboxylase (Tappaz et al., 1990), and are also able to convert labeled cystine to taurine (Martin and Battaglioli, unpublished data). FUNCTIONAL DIVERSITY OF ASTROCYTES The possibility that astrocytes are functionally diverse and specialized is a long-standing and intriguing problem. The ability of astrocytes to accumulate and degrade transmitters, for example, suggests that individual astrocytes may be specialized to metabolize the transmitters released by neighboring neurons, but aside from some differences among astrocytes from different brain regions, little evidence of a simple relationship of this kind has emerged as yet. The specific localization of neuroactive substances in subpopulations of astrocytes provides strong evidence that astrocytes differ in their abilities to send messages. Angiotensinogen, for example, is produced by a large number of astrocytes in certain nuclei of the hypothalamus, midbrain, and brain stem but is not produced by astrocytes in other brain regions (Stornetta et al., 1988). Substance-P immunoreactivity is found in a very small minority of astrocytes immediately adjacent to blood vessels in striatum and in white matter (Kostyk et al., 1989; Michel et al., 1986). Proenkephalin mRNA is present in a subpopulation of astrocytes in cerebellum but is not present in Bergmann glia (Spruce et al., 1990). Somatostatin is produced by astrocytes cultured from cerebellum but not by astrocytes from striatum or cerebral cortex (Shinoda et al., 1989). Taurine is present predominantly in astrocytes in brain stem, hypothalamus, and thalamus, but is present predominantly in neurons in cerebellum, caudate, and cortex (StormMathisen and Ottersen, 1986). It is likely that similar studies of other compounds will produce additional evidence of functional diversity and specialization among astrocytes. Thus, astrocytes appear to be much

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more specialized than previously imagined, and anatomical or developmental classifications of glia are likely to become unsatisfactory as their functions become clearer. Just as a neuron is identified by the transmitter it releases and its role in circuitry, glial cells may be identified eventually by similar functional criteria. RELEASE OF NEUROACTIVE SUBSTANCES Most of the neuroactive compounds listed in Tables 1 and 2 are released by astrocytes either at a basal rate or in response to stimulation by neurotransmitters, receptor agonists, hormones, or elevated [K+], (Table 3). Taurine, for example, is released when astrocytes are stimulated with P-adrenergic agonists, adenosine, 5-hydroxytryptamine, K-opiates (Madelian et al., 1985, 1988; Martin et al., 1988; Shain et al., 1986; Shain and Martin, 19841,or elevated [K+l, (Martin et al., 1990a,b; Pasantes-Morales et al., 1990a; Philibert et al., 1988; Tigges et al., 1990), while substance P inhibits P-agonist-stimulated release (Perrone et al., 1986). Release in response to p-agonists and adenosine is mediated by CAMP (Madelian et al., 1985, 1988). Other second messengers also appear to be involved, but have not been identified. Ca2+does not appear to be involved (see below). A number of other compounds listed also are released by a variety of agonists. The diversity of the released compounds and the varied nature of the secretagogues suggest that a number of release mechanisms are involved. Aside from nitric oxide, the prostaglandins, and thromboxanes, which appear to be sufficiently lipophilic to leave the cell by diffusion, the mechanisms of release are poorly understood. Mechanisms of Peptide Release The possibility that glial cells can release neuroactive compounds by vesicular mechanisms has received relatively little attention. The fact that glial cells are not known to contain local regions of highly concentrated vesicles or morphologically recognizable release zones does not eliminate the possibility that they can carry out vesicular release. Eukaryotic cells are known to possess two types of vesicular mechanisms for protein release-regulated release, in which newly synthesized proteins are packaged in storage vesicles to be released when the cell is stimulated with a secretagogue, and constitutive release, in which newly synthesized proteins are packaged in vesicles and released from the cell shortly after synthesis (Burgess and Kelly, 1987; Kelly, 1985). Proteins secreted by the regulated system are released in large amounts during a relatively short period after stimulation by an agonist, and the rate of release can exceed the rate of synthesis, at least for a short time. Examples of peptides secreted by the regulated system

are hormones such as oxytocin and vasopressin. The vesicles used for regulated release contain high concentrations of peptides and appear as dense-cored vesicles in electron micrographs. In contrast, proteins secreted by the constitutive system are released as they are synthesized and changes in the rate of release are accomplished by changing the rate of protein synthesis. Examples of proteins secreted by the constitutive system are angiotensinogen and serum albumin, which are secreted by liver cells, and immunoglobins, which are secreted by lymphocytes. The vesicles used for constitutive release do not contain high concentrations of protein and have no specialized appearance in electron micrographs. The regulated system is better adapted for an immediate response and to send messages with short-term effects, while the constitutive system, if it is used for messenger molecules at all, is adapted better for slow, long-term responses. The ability of astrocytes to synthesize and secrete peptides such as angiotensinogen and enkephalins suggests that these cells should possess one or both types of vesicular release mechanism, but this point has received very little attention. Intebi et al. (1990) have suggested that angiotensinogen is released by the constitutive pathway and not the regulated pathway, because its synthesis and release is enhanced by dexamethasone and not by forskolin, an agonist that can increase release by the regulated pathway (Deschepper and Flaxman, 1990; Intebi et al., 1990). On the other hand, the synthesis and release of enkephalin peptides is increased by forskolin (Shinoda et al., 19891, but forskolin also increased the levels of proenkephalin mRNA, suggesting that enhanced enkephalin release results from increased synthesis and may occur by the constitutive pathway. It is not clear, in fact, whether astrocytes even possess the regulated release system, although there is sparse evidence that glia do contain secretory vesicles or their components. Dense-cored vesicles have been observed in astrocytes in the marginal nuclei of the avian spinal cord (Bodega et al., 1989),and dense vesicles have been observed in perivascular satellite cells in the goldfish preoptic area (Gregory et al., 19881, although the latter authors carefully noted that these vesicles could be either secretory or lysosomal. Cerebellar Bergmann glia and some cerebellar astrocytes contain chromogranin-A-like immunoreactivity (McAuliffe and Hess, 1990). Chromogranin A is located in secretory vesicles in endocrine cells and is cosecreted with hormones; in neurons it is found in dense-cored vesicles or the Golgi complex. The function of chromogranin A is unclear; it may have a regulatory function or be a precursor of active chromograninderived peptides such as pancreastatin (McAuliffe and Hess, 1990). The observation of neuropeptide release does not necessarily imply that glia release them by the regulated pathway, since cells will use the constitutive pathway to release “regulated-pathway” proteins when the regulated pathway is blocked (Burgess and Kelly, 1987; Kelly, 1985).

RELEASE OF NEUROACTIVE COMPOUNDS FROM GLIA

Release of Small Molecules Much more attention has been given to the mechanism(s) by which astrocytes release small molecules such as the neuroactive amino acids than to mechanisms of peptide release. In these studies, the term release is used operationally to designate the overflow of a compound from an experimental preparation that may occur by one (or more) of several mechanisms including reversal of an uptake system, vesicular release, and "transmembrane l e a k (a poorly defined phenomenon sometimes invoked to explain basal release). As discussed below, recent studies have provided evidence for another, possibly distinct mechanism that we term tension-controlled release. A major problem in studying release from glia is distinguishing among the mechanistic alternatives.

Are Small Molecules Released From Glia by a Vesicular Mechanism? Although the idea that glial cells release small molecules by a vesicular mechanism may seem improbable, it cannot be dismissed out of hand simply because glia do not contain collections of vesicles or recognizable release zones. Currently available morphological data do not eliminate the possibility that glia contain a diffusely organized vesicular release mechanism for small molecules. However, current biochemical evidence is largely inconsistent with the idea that glia release small molecules by a vesicular mechanism. Investigation of the Ca2+dependence of release is one of the few ways that is available to distinguish between vesicular release and other mechanisms such as the reversal of a transport process. Because of the complexity of Ca2+ compartmentation and homeostasis in the cell, the exact role of Ca2+in a release process is difficult to establish. Determining Ca2+ dependence by manipulating [Ca2+l,can be difficult experimentally, and the results, even when clear, can be hard to interpret. Stringently reducing [Ca2+l, by omitting Ca2+ salts from the medium and adding a high concentration of ethylene glycol bis(P-aminoethylether)N,N'-tetraacetic acid (EGTA)or ethylenediaminetetraacetate (EDTA)(- 1mM) can lead to a large increase in the release of some compounds, and it may be difficult to measure evoked release accurately on top of the greatly elevated baseline release that occurs in Ca2+-freemedium. For example, astroglial taurine release is greatly elevated in Ca2+-free medium containing 1 mM EGTA (Holopainen et al., 1985; Martin et al., 1989; Philibert et al., 1988; Tigges et al., 19901, an effect that prevented reliable quantitation of IPR-stimulated release (Martin et al., 1989). The release of labeled D-aspartate also increases when cells are perfused with Ca2+-freemedium containing 1 mM EGTA (Holopainen and Kontro, 1990). Two other approaches to manipulating external Ca2+appear preferable. One is to use a much lower concentration of EGTA

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(-10 kM) to buffer the residual [Ca"], in a medium prepared without added Ca2+salts (Martin et al., 1989; Shain et al., 1989).The internal [Ca2+1in astroglial cells incubated in this medium (46 ? 10 nM) is much lower than in cells incubated in control medium (150 95 nM),indicating that the driving force for Ca2+entry has been reduced substantially. Baseline taurine release was also stable in this medium, indicating that the nonspecific effects of 1 mM EGTA Ca2+-free medium were reduced if not eliminated (Shain et al., 1989). A second approach is to replace external Ca2+with other divalent anions, (e.g.,M$+, Mn2+,Co"). In such experiments, the osmolarity should be kept constant because some release processes, such as taurine release, are very sensitive to small changes in osmotic pressure and can 'be suppressed almost completely when the osmolarity of the medium is increased by 10% (Martin et al., 1989). IPR-stimulated taurine release is unaffected by reducing [Ca2+l, with the Ca2+-freemedium containing 10 FM EGTA and is not blocked by Mn2+,Cd2+,or Co2+ or by the Ca2+channel blockers nifedipine, diltiazem, or verapamil, indicating that the entry of external Ca2+is not required for release (Martin et al., 1989). Unfortunately, the interpretation of simple experiments on the role of external Ca2+is inevitably ambiguous. Ca2+may, of course, be essential for a nonvesicular release process if it is involved as a second messenger or is essential for some other step such as the initial response to the agonist that evokes release. If the stimulus is coupled to the release mechanism via a Ca2+-regulatedprotein kinase, for example, release will be Ca2+ dependent even though vesicles are not involved. On the other hand, a Ca2+-dependent release process may be independent of external Ca2+ if the stimulus causes release of Ca2+ from internal stores. Thus, failure t o observe dependence on external Ca2+ does not necessarily indicate that release is Ca2+independent or that it is nonvesicular. Measuring intracellular [Ca2+1with FURA-2 or similar dyes is a more direct approach to determining the Ca2+dependency of a release process. If release is Ca2+ dependent there should be a close correlation between changes of intracellular [Ca"] and changes in release. Thus, a stimulus that brings about release also should elevate intracellular [Ca2*l and Ca2+ionophores, such as ionomycin, should elevate intracellular [Ca2+land stimulate release, while reducing internal [Ca2+lby, for example, lowering [Ca2+1, should reduce release. No glial release process has been shown to be Ca2+dependent by these or similar strict criteria, but Ca2+ independence has been demonstrated in the case of taurine release (Martin et al., 1989; Shain et al., 1989). IPR stimulates taurine release from astroglia but does not elevate intracellular [Ca2+],as measured with FURA-2. Furthermore, IPR-stimulated taurine release is unaffected by reducing internal [Ca2+l(Martin et al., 1989; Shain et al., 19891, and ionomycin, which increases intracellular [Ca"] to very high levels, does not stimulate taurine release (Shain et al., 1989). The failure of ionomycin to stimulate release even though it elevates

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MARTIN

intracellular [Ca2'] indicates that changes in intracellular [Ca2+] are not involved in the taurine release process per se, and suggest that Ca2+must be involved in some other way in the case of K+-stimulated taurine release, which appears to depend on extracellular Ca2+ (Philibert et al., 1988; Tigges et al., 1990). Biochemical studies of compartmentation are also useful for distinguishing between vesicular and nonvesicular release. Although there are numerous studies indicating that transmitters and similar molecules are stored in and released from a distinct subcellular compartment in neurons, there appears to have been only one such study with glial cells (Waniewski et al., 19911, and the results were inconsistent with vesicular release. Thus, subcellular fractionation experiments showed that taurine is present in a cytoplasmic pool or in a compartment that exchanges taurine freely with the cytoplasm. Furthermore, the specific radioactivity of released taurine was the same as that of cellular taurine, a result consistent with release from a cytoplasmic compartment, not vesicles, and differing from that observed for the release of many transmitters from neurons. Thus, there is at present no evidence that glial cells can release small molecules by a vesicular mechanism.

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Release by Reversal of Membrane Transport Fig. 1. A: Effect of membrane potential and reaction stoichiometry It has been recognized for many years that the memon the maximum transmembrane glutamate-concentration gradient brane transport systems for small molecules are revers- that can be developed by the transport system described by reaction 2. ible and can catalyze a net outward flux as well as net B: Effect of [K+], on the transmembrane concentration gradient of at two reaction stoichiometries. The glutamate gradient at 3 inward movement of the substrate [Martin (1976) and glutamate mM [K+], was taken as 100%.Transport is electroneutral when n = 2 others], and the possibility that astrocytes can release and k = 1; under these circumstances membrane potential has no compounds by this route is genuine. These transport effect on the glu gradient that can be generated by the transport system and the effect of [K+l, is entirely attributable to changes in the [K'I systems generally operate by coupling the movement of gradient. In both panels the transmembrane gradients were calculated the substrate with the movement of one or more species by of ions, and are, therefore, strongly affected by the transmembrane ion gradients. Because the ions and ~[Gluli [Nat]z[Kt]: (1 + k n) RT v, (Equation 3) many of the substrates are charged, these systems often [Glu], [Nat]:[Kt]t are electrogenic, that is, the transport reaction results in the net movement of charge across the membrane. In addition t o being affected by the ion gradients, electroIonic requirements differ among transport systems. genic transport systems also are affected by the memSynaptosomal taurine transport, for example, requires brane potential. The direction that a system moves its substrate is, of both Nat and C1- ions. Studies in various systems course, a matter of energetics. If the total energy avail- (reviewed by Shain and Martin, 1990) suggest that the able from ion cotransport exceeds the energy required to reaction is move the substrate into the cell, the system will transport the substrate into the cell, but ifthe energy from ion Tau, + nNa: + cC1, =Taui + nNaT + cCli (Reaction 1) cotransport becomes too low to support the existing substrate gradient, the system will run in reverse and In contrast, the glutamate transport system in glial move the substrate from inside to outside. For cotrans- cells appears to involve the cotransport of Nat and K' port systems, the stoichiometry of the transport reac- (Szatkowski et al., 1990). There is also a separate tion determines the substrate concentration gradient C1--dependent glutamate transport system in glia that that the system can generate and the sensitivity of the carries out a rapid exchange reaction (Bridges et al., system to changes in the membrane potential and ion 1987; Erecinska, 1990; Waniewski and Martin, 1984). concentrations. Thus, understanding the nature of a Considering only Na+ and K+ cotransport the reaction transport reaction and its stoichiometry are useful for would be understanding whether it will run in reverse under a + Glu; + nNa1 + k&+ (Reaction 2 ) Glu; + nNa: + particular set of experimental conditions. ~

m+

RELEASE OF NEUROACTIVE COMPOUNDS FROM GLIA

Under normal physiological conditions, the C1- and K+ gradients are closer to their respective equilibrium potentials than Na+ is to its equilibrium potential, so K+ and C1- contribute only a small fraction of the energy contributed by Nat to the transport reaction. Thus, in each case, the principal source of energy for transport in the resting cell is the electrochemical potential gradient of Na+. Nevertheless, changes in K' and Cl- gradients can strongly affect transport either directly or else indirectly through effects on the membrane potential. In glial cells in culture, the taurine transport system generates and maintains a ITauli/[Taul, of about 10,000 and, given the usual membrane potential and intracellular and extracellular "a+], at least two Na+ ions must be cotransported (i.e., n = 2 in reaction 1) to geqerate and maintain the observed taurine gradient (Shain and Martin, 1990). If only one C1- is cotransported (c = l),the system will be electrogenic, and if the cells are depolarized sufficiently, the ion gradients will not supply enough energy to maintain the existing taurine gradient. Under these circumstances, the taurine transport system will run in reverse, moving taurine from inside to outside. Thus, high-K+ medium or other depolarizing conditions could, in principle, lead to taurine efflux by reversing transport. Glial cells do, in fact, release taurine when exposed to high K' medium (Martin et al., 1990a,b; Pasantes-Morales et al., 1990a; Philibert et al., 1988; Tigges et al., 1990), but it is unclear whether this effect is attributable to reversal of transport (Martinet al., 1990a;Pasantes-Morales et al., 1990a). In primary astrocyte cultures and LRM55 cells K' stimulates release only if [K+], is elevated isosmotically by replacing NaCl with KC1. Even strongly depolarizing [K+], does not stimulate release if [K'l is elevated hyperosmotically by adding KC1to the medium (Martinet al., 1990a). K+-stimulatedrelease also can be suppressed by raising the osmolarity with sucrose. In LRM55 cells, elevated [K'], stimulates release at concentrations (