PHYSIOLOGICAL REVIEWS   Vol. 78 No. 1 January 1998, pp. 99-141
Copyright ©1998 by the American Physiological Society

Glial Calcium: Homeostasis and Signaling Function

ALEXEJ VERKHRATSKY, RICHARD K. ORKAND, AND HELMUT KETTENMANN

Department of Cellular Neurosciences, Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; and Institute of Neurobiology, University of Puerto Rico, San Juan, Puerto Rico

I. INTRODUCTION
II. METHODOLOGICAL CONSIDERATIONS
    A. Types of Glial Cells
    B. Experimental Approaches to Measure Intracellular Ca²⁺
III. AN OVERVIEW OF CALCIUM HOMEOSTASIS IN GLIAL CELLS
    A. Resting Intracellular Ca²⁺ in Glial Cells
    B. Ca²⁺-Permeable Channels
    C. Ca²⁺ Storage Organelles, Intracellular Ca²⁺ Release, and Store-Operated Channels
    D. Ca²⁺ Transporters
    E. Intracellular Ca²⁺ Sensors and Effectors
IV. VOLTAGE-GATED CHANNELS AND DEPOLARIZATION-INDUCED CALCIUM SIGNALS
    A. Schwann Cells
    B. Astrocytes
    C. Oligodendrocytes
    D. Mechanisms of Glial Cell Depolarization
V. NEUROTRANSMITTER-INDUCED CALCIUM SIGNALING IN GLIAL CELLS
    A. Glutamate
    B. Purines and Pyrimidines
    C. Monoamines
    D. -Aminobutyric Acid and Glycine
    E. Acetylcholine
    F. Histamine
    G. Substance P
    H. Bradykinin
    I. Endothelins
    J. Other Agonists Linked to Intracellular Ca²⁺ Regulation in Glial Cells
    K. Heterogeneity of Neurotransmitter Receptor Expression in Glial Cells
VI. SPATIOTEMPORAL ORGANIZATION OF CALCIUM SIGNALS
    A. Intracellular Ca²⁺ Oscillations
    B. Intercellular Ca²⁺ Waves
VII. GLIAL CALCIUM SIGNALING AND NEURON-GLIAL INTERACTIONS
VIII. GLIAL CALCIUM AND BRAIN PATHOLOGY
IX. CALCIUM SIGNALS AND GLIAL FUNCTION
X. CONCLUDING REMARKS: CALCIUM SIGNALS ARE A CONSEQUENCE OF GLIAL EXCITABILITY
REFERENCES

	ABSTRACT

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Verkhratsky, Alexej, Richard K. Orkand, and Helmut Kettenmann. Glial Calcium: Homeostasis and Signaling Function. Physiol. Rev. 78: 99-141, 1998.  Glial cells respond to various electrical, mechanical, and chemical stimuli, including neurotransmitters, neuromodulators, and hormones, with an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). The increases exhibit a variety of temporal and spatial patterns. These [Ca²⁺]_i responses result from the coordinated activity of a number of molecular cascades responsible for Ca²⁺ movement into or out of the cytoplasm either by way of the extracellular space or intracellular stores. Transplasmalemmal Ca²⁺ movements may be controlled by several types of voltage- and ligand-gated Ca²⁺-permeable channels as well as Ca²⁺ pumps and a Na⁺/Ca²⁺ exchanger. In addition, glial cells express various metabotropic receptors coupled to intracellular Ca²⁺ stores through the intracellular messenger inositol 1,4,5-trisphosphate. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca²⁺ responses. Such agonist specificity may provide a means for intracellular and intercellular information coding. Calcium signals can traverse gap junctions between glial cells without decrement. These waves can serve as a substrate for integration of glial activity. By controlling gap junction conductance, Ca²⁺ waves may define the limits of functional glial networks. Neuronal activity can trigger [Ca²⁺]_i signals in apposed glial cells, and moreover, there is some evidence that glial [Ca²⁺]_i waves can affect neurons. Glial Ca²⁺ signaling can be regarded as a form of glial excitability.

	I. INTRODUCTION

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Glial intracellular calcium ([Ca²⁺]_i), like that of other eukaryotic cells, is highly regulated; the free [Ca²⁺]_i is four to five orders of magnitude less than that in the narrow system of clefts that constitutes the functional extracellular environment of the nervous system. There is, therefore, a steep electrochemical gradient favoring Ca²⁺ entry; transient cellular activation increasing Ca²⁺ permeability will lead to a transient increase in [Ca²⁺]_i . In addition, there are Ca²⁺ stores within the cell that may release Ca²⁺ in response to specific intracellular chemical messengers, also increasing [Ca²⁺]_i . These transient rises of [Ca²⁺]_i in turn trigger or regulate various intracellular events, including metabolic processes, gene expression, and ion transport systems. Therefore, changes in [Ca²⁺]_i act as an eclectic second messenger system coordinating changes in the external environment with intracellular processes. These observations, in a wide variety of cells, have led to a general appreciation of the specific role of [Ca²⁺]_i in cell signaling (for general references, see Refs. 42, 73, 147, 245, 345). Cells have developed specialized machinery to control the spatial and temporal characteristics of these Ca²⁺ signals. These include transmembrane Ca²⁺ transporters and Ca²⁺-permeable channels, cytoplasmic buffers, and intracellular organelles that are able to accumulate, store, and release Ca²⁺.

View larger version (110K): [in this window] [in a new window]   FIG. 1.   Earliest illustration of all 3 glial elements in central nervous system by Del Rio-Hortega (95). A: protoplasmic astrocytes from gray matter. B: fibrous astrocytes from white matter. C: microglia. D: interfascicular glia, or oligodendrocytes, from white matter. [From Del Rio-Hortega (95).]

In the nervous system, Ca²⁺ regulation has been extensively investigated and well characterized in a variety of neurons (17, 134, 290, 387, 430). These investigations demonstrate that neuronal [Ca²⁺]_i participates in the control of important neuronal functions, like electrical excitability, neurotransmitter release, and long-term changes in synaptic efficacy. In parallel, but to a much lesser extent, knowledge has accumulated on the homeostasis and role of Ca²⁺ signaling in glial cells. The perception of the role of glia in brain function has changed dramatically over the last 10 years from that of a supporting glue (Greek glia is glue) with mainly trophic functions to that of a cell with dynamic interactions with neurons actively participating in nervous system function. This change occurred after the development of new techniques, like patch-clamp recording and Ca²⁺ imaging, that revealed that glial cells express a wide variety of ion channels and neurotransmitter receptors that make them able to detect and respond to neuronal activity (429, 432). Changes in glial [Ca²⁺]_i have been measured under a variety of conditions where glial cells are responding to electrical, mechanical, and chemical stimuli. These fluctuations in [Ca²⁺]_i appear to be a consistent response of glial cells to changes in the environment that lead to a change in glial function; they are not passive responses and, therefore, can be considered a form of glial excitability mediated by calcium. This review primarily includes recent insights into the major mechanisms involved in the control of [Ca²⁺]_i and the role of changes in [Ca²⁺]_i in glial signal transduction in response to neuronal activity. In addition, we consider what is known of changes in [Ca²⁺]_i that affect glial function and accompany pathological processes.

	II. METHODOLOGICAL CONSIDERATIONS

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Glial cells (Fig. 1) are found throughout the vertebrate central nervous system (CNS) (for overview on glial cell biology, see Ref. 223). The macroglial cells, astrocytes, and oligodendrocytes are of ectodermal origin, whereas the microglial cells are thought to stem from the mesoderm. Astrocytes are probably the most diverse population of glial cells. One of their hallmarks is the expression of intermediate filament proteins, glial fibrillary acidic protein (GFAP) or S100. There is a battery of commercially available antibodies that can be used as markers to identify astrocytes. However, the expression of GFAP can vary among astrocytes and can change during development, particularly in pathological conditions. The astrocytic response to injury is marked by an increase in GFAP expression, and these cells are termed reactive astrocytes. Astrocytes in culture probably represent reactive astrocytes, since they obviously sense the strange culture environment and prominently express GFAP. Expression of GFAP is, however, not a marker for all astrocytes: Müller cells in the retina do not express GFAP under normal conditions, but only under pathological conditions. What then is the definition of an astrocyte? The answer may be that they characteristically have two contact sites, the neuronal membrane (synaptic regions in the gray matter and axons in the white matter) and the border of the CNS, either the blood system or the ventricular walls. Astrocytes can be subdivided into three major populations: radial astrocytes, fibrous astrocytes, and protoplasmic astrocytes with transition forms between these populations. Bergmann glial cells of the cerebellum are a prominent example of a radial (astrocytic glial cell). Fibrous astrocytes send a large number of processes into all directions, whereas protoplasmic astrocytes, mainly located in the gray matter, have short ramified and crimped processes (for review of astrocyte morphology, see Ref. 354).

Oligodendrocytes are the myelin-producing cells of the CNS (406). They produce myelin proteins such as myelin basic protein, proteolipid protein, myelin-associated glycoprotein, and cyclic nucleotide phosphodiesterase. Antibodies against these proteins can be used as oligodendrocyte markers. In white matter, their function seems to be well defined: they enwrap axons and form the myelin sheath. Oligodendrocytes are prominently found in white matter but can also be found in gray matter. There are also oligodendrocytes that do not myelinate, namely, the perineuronal oligodendrocytes. At present, their functions are not known.

Microglial cells are thought to invade the brain during embryonic and early postnatal period. They stem from the monocytic lineage and thus have many common features with cells of the monocytic lineage. After invasion, they distribute equally in the brain parenchyma, and each cell seems to have a defined territory. Microglial cells are the immunocompetent cells of the CNS and can express the relevant molecules such as the major histocompatibility complex II. Under normal physiological conditions, microglial cells are in a resting state and have a small soma and fine ramified processes. After any disturbance of the nervous system, they can be activated and respond in a defined manner, converting from the resting form ultimately to a cytotoxic, phagocytic cell. This transition is graded and probably, in part, controlled by factors of the immune system, such as complement factors or cytokins (see Refs. 145, 246 for review).

All types of glial cells have been studied under a variety of conditions. There is increasing evidence that the expression of glial properties depends both on the origin of the cells and the precise experimental conditions for study. The variables are numerous and need to be precisely defined in terms of the following: 1) type of cell (including subtype where applicable), e.g., astrocyte, oligodendrocyte, Schwann cell, or microglia; 2) cellular origin, including not only the species and age of the animal but also the brain region; 3) type of preparation, in vivo, acutely prepared slice, or slice and dissociated cells in tissue culture (including time in culture and presence of other cell types), chemical environment during preparation, preservation, and experiment; and 4) experimental approaches, e.g., anatomy, electrophysiology, histochemistry, ion imaging techniques (ion-sensitive dyes, ion probe microscopy), and molecular biology.

Given the multitude of variables, it is hardly surprising that there is little unanimity of opinion or even consistent results regarding the properties of neuroglia. The diversity of results is tending to focus on a few central problems. The control of [Ca²⁺]_i and its variation during glial responses is one of those problems.

Attempts to measure [Ca²⁺]_i in glial cells have paralleled those in other tissues and include the use of radioisotope tracers, Ca²⁺ ion-selective electrodes, electron-probe microscopy, and in more recent time Ca²⁺-sensitive fluorescent dyes (see Refs. 159, 385, 417 for review). Each of the methods has serious limitations that dictate the choice of glial preparation for study. The initial measurements of [Ca²⁺]_i were carried out in steadily growing cultures of glial cell lines (47) and primary glial cultures (283). As indicated above, glial cells change during development. Therefore, it was important to correlate the [Ca²⁺]_i measurement with the developmental stage. Thus Ca²⁺ fluxes and [Ca²⁺]_i recordings were carried out along with immunostaining with stage-specific antibodies (e.g., Refs. 182, 230, 436).

Experiments on cultured cells raised the question of whether the [Ca²⁺]_i handling mechanisms remain unaltered after the cells were removed from their natural environment and maintained under artificial conditions in the absence of neurons. To solve this problem, [Ca²⁺]_i recording techniques were applied first to freshly isolated cells (e.g. Refs. 103, 131) and then to cells in acutely prepared brain slices. Initially, the technique combining patch-clamp electrophysiological recordings with Ca²⁺-sensitive fluorescent dyes was applied to neurons (13, 265); later, it was used in glial cells (235, 237, 301). In these experiments, the patch-clamp whole cell configuration was employed to inject Ca²⁺-sensitive probes into glial cells. This technique confines the [Ca²⁺]_i recording to a single, morphologically identified cell and allows simultaneous electrophysiological recording. However, prolonged intracellular dialysis can significantly disturb [Ca²⁺]_i regulation (235). As an alternative, Ca²⁺-sensitive dye can be injected via microelectrodes into the cell of interest (104), or the slices can be incubated with membrane-permeant forms of fluorescent Ca²⁺ probes (33, 239, 240, 349, 351). A major difficulty with this technique is that the background fluorescence is unknown; this may lead to a miscalculation of [Ca²⁺]_i . This problem can be resolved by combining [Ca²⁺]_i measurements from cells loaded with a permeable form of the dye with subsequent intracellular dialysis. The latter helps to wash the dye from the cell of interest so that an actual background fluorescence value can be determined (240).

	III. AN OVERVIEW OF CALCIUM HOMEOSTASIS IN GLIAL CELLS

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The general mechanisms of [Ca²⁺]_i homeostasis are common to all eukaryotic cells (see Refs. 73, 245, 345 for review). Intracellular Ca²⁺ is determined by the interaction of membrane Ca²⁺ transporters and cytoplasmic calcium buffers (Fig. 2). The Ca²⁺ transporters are represented by several superfamilies of transmembrane Ca²⁺-permeable channels, ATP-driven calcium pumps, and electrochemically driven Ca²⁺ exchangers. The resulting Ca²⁺ fluxes may either deliver or remove Ca²⁺ from the cytoplasm. Upon entering the cytoplasm, most Ca²⁺ is trapped by Ca²⁺-binding proteins; this determines the Ca²⁺-buffering capacity of the cell. Calcium transporters are localized in the cell membrane (providing Ca²⁺ exchange between the cell interior and exterior) and in the membrane of intracellular organelles [e.g., endoplasmic reticulum (ER), mitochondria, Golgi complex, and nucleus]. The latter forms the intracellular Ca²⁺ storage system (352), which actively accumulates Ca²⁺. Accumulated Ca²⁺ is bound to intraluminal proteins, and it can be rapidly released via intracellular Ca²⁺ channels. This general scheme is applicable to all types of glial cells (429). A peculiar feature of glial cells is their high degree of heterogeneity with respect to the expression of various molecular cascades involved in [Ca²⁺]_i regulation.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   General scheme of molecular cascades involved in intracellular calcium signaling (see discussion in text). VGCC, voltage-gated Ca2+ channels; SOCC, stores-operated Ca2+ channels; PMCA, plasmalemmal Ca2+-ATPase; Ca2+-BP, Ca2+ binding proteins; InsP3R, inositol 1,4,5-trisphosphate receptor/inositol 1,4,5-trisphosphate-gated Ca2+ channel; RyR, ryanodine receptors/Ca2+-gated Ca2+ channel; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase. Intracellular Ca2+ sensors are as follows: CaM, calmodulin; CaM kinase, Ca2+/calmodulin-dependent protein kinase; CaM AC, Ca2+/calmodulin-dependent-adenylate cyclase; CaM-phosphatase, Ca2+/calmodulin-dependent-protein phosphatase; Ras, p21ras guanine nucleotide-binding proteins; Raf, raf protein kinase; MEK, mitogen-activated/extracellular regulated kinase; MAPK, mitogen-activated protein kinase; IEG, immediate early genes.

Free cytoplasmic Ca²⁺ is a minor part (<0.001%) of total calcium in glial cells. Most is associated with intracellular organelles (e.g., ER, mitochondria, and Golgi apparatus). Resting [Ca²⁺]_i in glial cells varies from 30-40 to 200-400 nM (see Table 1). This variation is not only among subtypes of glia, but also within the same population of cells. It may reflect method-induced artifacts or indicate the flexibility of [Ca²⁺]_i homeostasis. Most measurements were made using membrane-permeable forms of calcium indicators; thus all the problems associated with this method (uncertain calibration, dye Ca²⁺ buffering, compartmentalization, and photobleaching) may contribute to the variability. Nevertheless, even in experiments performed on Bergmann glial cells in cerebellar slices (235) with careful intracellular calibration procedures, the resting [Ca²⁺]_i ranged from 30 to 200 nM. This variability did not appear to reflect cell damage, because in all cases the resting potential determined by whole cell recordings remained about normal (75 to 60 mV).

The initial electrophysiological surveys of glial cells of various origin (248, 249, 361; see Ref. 392 for review) did not reveal voltage-sensitive channels. With improved techniques, e.g., voltage clamping and patch clamping, the surprising finding was made that some glial cells exhibit a variety of voltage-gated ion channels that were previously believed to be present only in electrically excitable cells (23, 37, 393). Several populations of both peripheral and central macroglia were shown to express voltage-gated Ca²⁺ channels similar to those found in neurons (392). Later, it was found that Ca²⁺ influx through voltage-gated channels significantly increases [Ca²⁺]_i in astro- and oligodendrocytes as well as in Schwann cells. However, not all glia express Ca²⁺ channels. For example, Bergmann glial cells, microglia, and certain populations of astrogliomas seem to lack voltage-dependent Ca²⁺ channels (192, 239, 429). Nevertheless, Ca²⁺ may enter glial cytoplasm via ligand-gated channels that are abundantly expressed in almost all glial cell subtypes (397). Finally, certain types of glial cells (e.g., retinal Müller cells or cultured astrocytes) express nonspecific cation channels that may also pass Ca²⁺ (227, 356).

Little is known about Ca²⁺ storage organelles in glial cells. Many contain an elaborate ER (143, 354) that presumably serves as a major substrate for rapidly exchanging Ca²⁺ stores. Calcium accumulation by glial ER involves ER pumps that, like other cells, are inhibited by thapsigargin (60, 235, 237) and cyclopiazonic acid (158). Aplysia glial cells were found to have an unusual analog of Ca²⁺ stores, so called "gliagrana" (217, 218), which may retain an enormously high (up to 50-100 mM) Ca²⁺ concentration. The density of these gliagrana varies with fluctuations in extracellular Ca²⁺ ([Ca²⁺]_o). Increases or decreases in glial calcium depending on [Ca²⁺]_o suggest the possible involvement of glia in the regulation of [Ca²⁺]_o . Similar stores have been described in frog ependymal glia and human astrocytes (143).

The major mechanism for Ca²⁺ release from internal stores involves activation of inositol 1,4,5-trisphosphate (InsP₃)-gated Ca²⁺ release channels (InsP₃ receptors, Refs. 34, 139). The production of InsP₃ , in turn, is achieved by the activation of phospholipase C (PLC) coupled via G proteins with numerous "metabotropic" plasmalemmal receptors. The nature of the InsP₃ receptors subtypes in different glial cells is not known in detail. In rat cortical astrocytes and cerebellar Bergmann glial cells, only type 3 but not type 1 and 2 InsP₃ receptors have been immunolocalized (453). Oligodendrocytes were reported to transiently express type 1 InsP₃ receptors in a short period during the onset of myelination (98). The direct activation of InsP₃ receptors by photorelease of InsP₃ from caged compound was shown in cultured astrocytes (224, 382). Astrocytic InsP₃ receptors appear to be substantially more sensitive to InsP₃ than InsP₃ receptors in Purkinje neurons; the threshold InsP₃ concentration for activation of the InsP₃-gated channel in astrocytes was 0.2-0.5 µM, whereas in Purkinje neurons, it was 9 µM (224). Inositol 1,4,5-trisphosphate-induced Ca²⁺ release is involved in the majority of glial responses to neurotransmitters and neurohormones (see sect. V). The glial expression of another type of intracellular Ca²⁺ release channel, the Ca²⁺-gated channel [or ryanodine receptor (RyR); Refs. 138, 287] is still debatable. Functional Ca²⁺-induced Ca²⁺ release (CICR), sensitive to the classical modulators ryanodine and caffeine and activated under physiological conditions, has been demonstrated only for periaxonal Schwann cells (258) and for freshly isolated Müller glial cells from salamander retina (219). In astrocytes, data on CICR are controversial; there is the one report that caffeine triggered a [Ca²⁺]_i increase in cultured embryonic cortical astrocytes (158). In contrast, several observations in cultured and freshly isolated astrocytes failed to detect an obvious caffeine-triggered [Ca²⁺]_i effect (60, 103). However, ryanodine and dantrolene, believed to be CICR antagonists, modulated [Ca²⁺]_i responses in astrocytes (60, 253). Finally, in Bergmann glial cells, studied in cerebellar slices, caffeine and ryanodine triggered a moderate [Ca²⁺]_i elevation and attenuated [Ca²⁺]_i transients evoked by kainate (S. Kirischuk and A. Verkhratsky, unpublished observations). In oligodendrocytes, caffeine and ryanodine did not affect [Ca²⁺]_i (238). In general (with several exceptions), glial [Ca²⁺]_i appears to be insensitive to caffeine, reflecting either an absence of ryanodine receptors in glial cells or the specific expression of "brain" ryanodine receptor isoform (RyR3), which is not modulated by caffeine (149, 395). This particular ryanodine receptor subtype could be activated by a newly discovered second messenger, cyclic ADP ribose (cADPR) (141); the possible effect of cADPR on glial [Ca²⁺]_i has not been investigated.

The amount of Ca²⁺ bound to internal stores may also regulate a distinct type of plasmalemmal Ca²⁺ permeability: it is widely recognized that the depletion of [Ca²⁺]_i pools activates a capacitative Ca²⁺ influx. This influx is associated with the activation of specific, store-operated plasmalemmal Ca²⁺ channels (75, 188). The existence of these channels in glial cells has not been clearly shown, although there are a number of suggestions that they might be important for Ca²⁺ homeostasis in gliomas (175, 362), astrocytes (418), and microglial cells (292, 293), although at least one group (347) reported that their attempts to find the capacitative Ca²⁺ entry in cultured astrocytes failed. In microglial cells, the long-lasting activation of capacitative Ca²⁺ entry after the maximal depletion of intracellular Ca²⁺ stores has been described recently (416). Once activated, the capacitative Ca²⁺ entry pathway in microglial cells remained operative for tens of minutes, creating a steady-state [Ca²⁺]_i elevation that dramatically outlasted the period of agonist action.

Mitochondria are another capacious Ca²⁺ storage site. However, their role in [Ca²⁺]_i homeostasis in glial cells is little understood. The dissipation of the mitochondrial electrochemical gradient by protonophores [carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone] triggered Ca²⁺ release in oligodendrocytes (236, 238). However, CCCP treatment did not influence the kinetic parameters of the depolarization-triggered [Ca²⁺]_i transients (238). This suggests that mitochondrial Ca²⁺ accumulation does not play an important role in calcium signal termination under physiological conditions. Under pathological conditions, which may substantially disturb mitochondria, glial [Ca²⁺]_i homeostasis might be markedly affected. Alternatively, mitochondria could play an active role in Ca²⁺ signaling also under physiological conditions: in cultured oligodendrocytes, mitochondrial Ca²⁺ release/accumulation actively shaped intracellular Ca²⁺ waves and [Ca²⁺]_i oscillations originated from InsP₃-sensitive Ca²⁺ stores (388). In cultured astrocytes, histamine-evoked [Ca²⁺]_i oscillations were accompanied by oscillations in intramitochondrial free Ca²⁺ (209), also suggesting that mitochondrial Ca²⁺ store may play an active role in [Ca²⁺]_i homeostasis.

The intracellular distribution of active mitochondria is different in oligodendrocyte progenitors and mature oligodendrocytes. In the former, both rhodamine-123 staining and CCCP-induced Ca²⁺ release were confined to the tips of cellular processes, suggesting that active mitochondria were concentrated in these particular areas. Conversely, in mature oligodendrocytes, mitochondria were evenly distributed (236). Presumably, the preferential localization of active mitochondria in the processes of oligodendrocytic progenitors might be important to supply energy for protein synthesis during cellular growth; it could also be important for [Ca²⁺]_i handling in this subcellular compartment.

A low [Ca²⁺]_i and recovery from increases in [Ca²⁺]_i produced by receptors/channels activation is provided by plasmalemmal Ca²⁺ pumps (57) and an electrochemically driven Na⁺/Ca²⁺ exchanger (40). There is little information on the properties of glial Ca²⁺ pumps. However, it has been shown in oligodendrocytes that La³⁺-sensitive Ca²⁺-ATPases are primarily responsible for the restoration of [Ca²⁺]_i after a depolarization-triggered [Ca²⁺]_i increase (238). In contrast, substantially more information is available on the expression of a Na⁺/Ca²⁺ exchanger. Initial evidences concerning the existence of functional Na⁺/Ca²⁺ exchange in glial cells derived from radiotracer experiments demonstrating that transmembrane fluxes of ⁴⁵Ca²⁺ in glial cells are controlled by extracellular Na⁺ concentration ([Na⁺]_o) (254). In several astrocytic preparations, reduction of the Na⁺ gradient, by lowering [Na⁺]_o , increased [Ca²⁺]_i (96, 156, 158), reduced the Ca²⁺ efflux rate (178, 410), and affected the kinetics of the stimulus-evoked [Ca²⁺]_i (84, 203). Biochemical studies (both the presence of protein and specific mRNA) revealed the expression of a heart isoform of the Na⁺/Ca²⁺ exchanger in astroglial cells (156, 410). Astrocytic Na⁺/Ca²⁺ exchanger was inhibited by 30-min incubation with 0.1-1 mM ascorbic acid (409) and was stimulated by sodium nitroprusside and 8-bromoguanosine 3',5'-cyclic monophosphate (11, 411), suggesting that Na⁺-dependent Ca²⁺ transport in glial cells may be the target for nitric oxide. However, the relative importance of Na⁺/Ca²⁺ exchanger in regulation of [Ca²⁺]_i was questioned in several reports that showed only a minor effect of low [Na⁺]_o on [Ca²⁺]_i (103, 203). Recently, it was demonstrated (156) that a decrease in [Na⁺]_o , by itself, is not sufficient to increase Ca²⁺ influx via the Na⁺/Ca²⁺ exchanger in astrocytes. Nevertheless, under conditions of elevated intracellular Na⁺ concentration ([Na⁺]_i), in ouabain-treated cells, lowering [Na⁺]_o produced a dramatic increase in [Ca²⁺]_i . Interestingly, a ouabainlike compound has been proposed to act as a vertebrate adrenocortical hormone (163). Thus it is possible that the physiological effect of this compound might be to increase Ca²⁺ influx via an increase in [Na⁺]_i .

In Bergmann glial cells in situ, Na⁺/Ca²⁺ exchanger also seems to play a relatively minor role in regulating resting [Ca²⁺]_i . However, Ca²⁺ flux through the exchanger became significant under conditions of elevated [Na⁺]_i . Stimulation of Bergmann glial cells with kainate increased [Na⁺]_i to >30 mM, which turned the exchanger in the reverse mode, providing thus the additional pathway for Ca²⁺ influx. This Ca²⁺ influx significantly altered both the amplitude and kinetics of the kainate-triggered [Ca²⁺]_i signals (233).

An astrocytic Na⁺/Ca²⁺ exchanger may be an important means for glia to regulate the ionic content in the interstitium. Neurons, when being electrically excited, can decrease both [Ca²⁺]_o and [Na⁺]_o in the intercellular clefts (30). Under conditions of lowered [Na⁺]_o , the Na⁺/Ca²⁺ exchanger could reverse and supply the interstitium with Ca²⁺ by expelling it from adjacent astrocytes.

Finally, Na⁺/Ca²⁺ exchanger may be involved in mediating Ca²⁺ excitotoxicity under pathological conditions. In particular, reversal of Na⁺/Ca²⁺ exchanger was found to play an important role in the astrocytic injury due to Ca²⁺ reperfusion after periods of Ca²⁺ depletion (a phenomenon similar to the"Ca²⁺ paradox" well described in cardiac muscle). The reperfusion-induced Ca²⁺ excitotoxicity was significantly decreased in astrocytes in which expression of Na⁺/Ca²⁺ exchanger was inhibited by treatment with antisense oligodeoxynucleotides to the exchanger (282).

After entering the cytoplasm, Ca²⁺ binds to a number of proteins that trigger various intracellular signal transduction pathways (Fig. 2, see Ref. 147 for review). Probably the best known cytoplasmic Ca²⁺ sensor is calmodulin (CaM), which regulates the functional activity of at least three broad classes of enzymes, namely, CaM-dependent protein kinases, protein phosphatases, and adenylate cyclases. The latter either interact with cytoplasmic enzymes or transfer the signal further down to the nucleus, initiating other pathways responsible for gene expression. An alternative way to connect cytoplasmic Ca²⁺ signals and gene expression is associated with Ras proteins (small guanine nucleotide-binding proteins) which after being activated by Ca²⁺ trigger a cascade of phosphorylation events that lead to a modulation of gene expression (127). Finally, cytoplasmic Ca²⁺ signals may propagate to the nucleus, where they directly stimulate the synthesis of immediate early genes as well as structural genes. Unfortunately, little is known of the expression and role of these systems in glial cells; their characterization in glia is an important problem awaiting an experimental solution.

	IV. VOLTAGE-GATED CHANNELS AND DEPOLARIZATION-INDUCED CALCIUM SIGNALS

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Voltage-gated Ca²⁺ channels form an important pathway for Ca²⁺ entry in excitable cells; the latter have been found to express a variety of Ca²⁺ channels, differing in their voltage dependence, kinetics, and pharmacological properties (177, 190). Calcium channels are integral membrane proteins composed of five subunits, each playing a distinct role in channel function. The Ca²⁺ channel subunits are encoded by several gene families. The functional heterogeneity of Ca²⁺ channels arises mainly from differences in ₁-subunit proteins; at least six major subtypes of ₁-subunit have been cloned and characterized (177). On the basis of physiological properties and pharmacological profile, Ca²⁺ channels are classified as low-voltage-activated or T-type channels and several types of high-voltage-activated channels (code named as L, N, P, Q, and R types). Molecular classification based on the diversity of ₁-subunit distinguishes six types of Ca²⁺ channels (CaCh1-6). Glial cells, although being inexcitable from the classical point of view (they are not able to generate action potentials) are capable of expressing voltage-gated Ca²⁺ channels. These have been found in several populations of macroglial cells but so far not in microglia.

Several early attempts to identify Ca²⁺ currents (I_Ca) in cultured Schwann cells (see Ref. 392 for review) were unsuccessful. In 1991, Amedee et al. (5) discovered that Schwann cells are able to express Ca²⁺ channels only when cocultured with neurons. Later, it was shown that expression of Ca²⁺ channels in Schwann cells could be also induced by addition of a nonhydrolyzable analog of adenosine 3',5'-cyclic monophosphate (cAMP) to the culture media (28). Whole cell patch-clamp studies of Schwann cells in organotypic cultures of mouse dorsal root ganglia (DRG) revealed voltage-dependent Ca²⁺ currents. At 10 mM Ca²⁺ outside, the I_Ca had an activation threshold at 45 mV, current-voltage curve maximum at 10 mV, and was rapidly inactivated (complete decay of current took ~150-200 ms). The Ca²⁺ currents in cultured Schwann cells were insensitive to L-type Ca²⁺ channel modulators (nifedipine and BAY K 8644) but were blocked by 5 mM Co²⁺. In a minor cell subpopulation, a slowly decaying, nifedipine-sensitive current component was observed when using 89 mM Ba²⁺ as a charge carrier. The expression of voltage-gated Ca²⁺ channels should provide a means for generating [Ca²⁺]_i transients upon Schwann cell depolarization. However, a direct attempt to measure [Ca²⁺]_i elevation in Schwann cells in a similar DRG coculture (267) failed to detect any measurable [Ca²⁺]_i elevation in response to depolarization by 50 mM KCl.

Recently, voltage-gated Ca²⁺ channels were detected in perisynaptic Schwann cells at the frog neuromuscular junction (368). Calcium channel expression in these cells was visualized using either labeling with monoclonal antibodies against ₂/-subunit (monoclonal antibody 3007; Ref. 424) or fluorescent phenylalkylamine (242). Both markers clearly stained the Schwann cell membrane primarily on the processes close to transmitter release sites. The morphological observations were substantiated by confocal video imaging of [Ca²⁺]_i that demonstrated that perisynaptic Schwann cells respond to high-KCl depolarization with [Ca²⁺]_i transients sensitive to nimodipine (368). Thus it appears the perisynaptic peripheral glia express functional voltage-gated Ca²⁺ channels.

MacVicar (271) first demonstrated Ca²⁺ action potentials in cAMP-treated cultured cortical astrocytes when the K⁺ conductance was blocked and 10 mM Ba²⁺ was added. Subsequently, similar Ca²⁺ action potentials were recorded from Müller glial cells in freshly prepared retinal slices (311), and voltage-clamp experiments on enzymatically dissociated Müller cells revealed currents carried by Ca²⁺ (311).

There are essentially two techniques to detect the presence of voltage-gated Ca²⁺ channels, either by characterizing membrane currents using electrophysiological techniques or by recording [Ca²⁺]_i while activating Ca²⁺ channels with depolarization [commonly by elevating extracellular K⁺ concentration ([K⁺]_o)]. Calcium currents were characterized in detail in cultured cortical astrocytes (24, 71, 85, 273) and type 2 astrocytes from optic nerve (23, 25). A special treatment of cortical astrocytic cultures was necessary to record Ca²⁺ currents, namely, the addition to the culture medium of agents that increase intracellular cAMP. These include treatment with dibutyryl cAMP (71, 236, 273), forskolin, isoprotereneol, or certain types of sera (24, 156). Coculturing with neurons had the same effect (85). In untreated cortical astrocytes, Ca²⁺ currents were usually undetectable. In contrast, in both freshly isolated and cultured astrocytes from the optic nerve, Ca²⁺ currents could be recorded without such pretreatment (23, 25). These results suggest that certain intracellular metabolic processes (i.e., phosphorylation) are necessary to transfer Ca²⁺ channels between "silent" and functional pools and furthermore that neurons may regulate the availability of Ca²⁺ channels in certain types of astrocytes. This also indicates that astrocytes are heterogeneous with respect to Ca²⁺ channel expression.

The parameters of astrocytic Ca²⁺ currents and the types of Ca²⁺ channels expressed vary in cells of different origin. Using a double-microelectrode voltage clamp in cultured cortical astrocytes, MacVicar and Tse (273) recorded a Ca²⁺ current that closely resembled L-type currents described in neurons. This current inactivated slowly, had a typical voltage dependence (threshold at 20 mV and a maximum of the current-voltage curve at +10 mV while using 10 mM Ba²⁺ as a charge carrier), was completely blocked by 1 µM nifedipine, and was potentiated by -adrenergic agonists via increased intracellular cAMP. The amplitude of I_Ca in this preparation was quite substantial, reaching 4-6 nA at 5 mM extracellular Ba²⁺ and 10 nA at 10 mM extracellular Ba²⁺. In contrast, currents recorded from optic nerve astrocytes were much smaller, 200-400 pA, with 10 mM Ba²⁺ as a charge carrier (23). Furthermore, two components of I_Ca were recorded from optic nerve astrocytes (23, 25): inactivating (which was defined as a T-type I_Ca based on its kinetic and voltage dependence) and sustained, sharing properties of an L-type I_Ca (slow inactivation, sensitivity to low concentrations of Cd²⁺, and voltage dependence).

Similarly, small Ca²⁺ currents (<250 pA at 5 mM Ca²⁺ outside) were recorded recently from immature astrocytes in acutely prepared hippocampal slices (Fig. 3, Ref. 2). Hippocampal astrocytes in situ appear to express several types of Ca²⁺ channels as revealed by their sensitivity to various antagonists. The currents observed at voltages between 50 and 20 mV had parameters typical for T-type I_Ca (fast inactivation and sensitivity to amiloride). The I_Ca recorded at higher potentials were partially sensitive to nimodipine, verapamil, and -conotoxin, suggesting the coexpression of L- and N-type Ca²⁺ channels.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Ca2+ currents in glial cells. Left: low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca2+ currents recorded from astrocytes in stratum radiatum of CA1 region of a hippocampal slice. Slices were prepared from 9- to 12-day-old mice. Ca2+ currents were recorded in Na+- and K+-free external solutions supplemented with 1 µM tetrodotoxin; intrapipette solution contained N-methyl-D-glucamine and tetraethylammonium as major cations. Left trace shows family of currents evoked by different depolarizing pulses after a conditioning hyperpolarization to 110 mV for 1.5 s. Current apparently represents superposition of both LVA and HVA Ca2+ currents. Middle trace shows HVA current in a pure form. To isolate HVA component, cells were held at 50 mV for 1.5 s before test depolarizations. Right trace shows LVA current obtained as a result of subtracting HVA component from total current. Corresponding current (I)-voltage (V) curves are shown at bottom. [From Akopian et al. (2). Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.] Right: 2 types of Ca2+ currents recorded from cultured oligodendrocyte. For Ca2+ current isolation, cells were perfused with Cs+, tetraethylammonium, and 4-aminopyridine solution while bathing in Na+-free media; 20 mM Ba2+ was used as a current carrier. Currents shown on left were evoked by test depolarizations to different voltages (indicated near traces) from holding potential of 75 mV. On right, I-V curves for total (LVA + HVA) Ca2+ current and net HVA current are presented. To separate HVA current, cells were held at a holding potential of 40 mV. [From Von Blankenfeld et al. (436) by permission of Oxford University Press.]

Finally, with the employment of whole cell and perforated patch-clamp recordings, L-type Ca²⁺ currents were recorded in cultured human Müller cells (357). These currents were inhibited by dihydropyridines, but insensitive to -conotoxins. The reverse transcription (RT)-polymerase chain reaction (PCR) examination of total RNA derived from cultured Müller cells revealed expression of mRNAs specific for _1D-, ₂-, and ₃-channel subunits, where ₂-subunit was represented by a splice variant distinct from skeletal muscle _2S- and brain _2B-isoforms.

An important question was to find out whether Ca²⁺ influx via voltage-gated channels alters [Ca²⁺]_i . The initial attempt to resolve this problem employed fura 2 and indo 1 [Ca²⁺]_i recordings from cultured embryonic (272) and neonatal (121) cortical astrocytes. In neonatal astrocytes, membrane depolarization with 25-100 mM KCl resulted in large increases in [Ca²⁺]_i (up to 1 µM) that were inhibited by nimodipine and D-600 (121). In embryonic astrocytes, maintained in confluent culture for 4-6 wk, application of 50 mM KCl generated [Ca²⁺]_i transients with an amplitude of 300-400 nM (272). The KCl-triggered [Ca²⁺]_i elevation was inhibited by nifedipine and significantly potentiated by BAY K 8644, suggesting the influx was mediated by L-type Ca²⁺ channels. In astrocytes kept in a culture for only 1-2 wk, 50 mM KCl did not raise (but rather lowered) [Ca²⁺]_i unless KCl was applied together with BAY K 8644. These results imply that astrocytes at early stages in culture have silent Ca²⁺ channels (272).

The variability of [Ca²⁺]_i channels in cultured cells raised questions as to the presence and function of voltage-gated Ca²⁺ entry in vivo. Freshly isolated mature hippocampal astrocytes (103) promptly respond to KCl application with [Ca²⁺]_i transients (400-800 nM in amplitude in response to 50 mM KCl). Potassium chloride-induced [Ca²⁺]_i transients were blocked by Co²⁺ and verapamil, but, in contrast to cultured astrocytes, they were resistant to dihydropyridines; depolarization-induced [Ca²⁺]_i transients in freshly isolated astrocytes were not affected by dibutyryl cAMP. The importance of voltage-gated Ca²⁺ channels for [Ca²⁺]_i regulation was further illustrated by recording [Ca²⁺]_i elevation evoked by depolarizing steps in voltage-clamped fura 2-loaded astrocytes from hippocampal slices (2). Thus the data available suggest that astrocytes in vivo express voltage-gated Ca²⁺ channels and that Ca²⁺ influx through these channels substantially affects [Ca²⁺]_i .

Oligodendrocytes are heterogeneous with respect to the expression of voltage-gated Ca²⁺ channels. In cultures from cortex, oligodendrocytes expressed both low-voltage (T type) and high-voltage (presumably L type) Ca²⁺ currents (431, 436). The amplitudes of Ca²⁺ currents in mature oligodendrocytes were up to about 200 pA with 20 mM Ca²⁺ as a charge carrier (Fig. 3). In contrast, voltage-clamp analysis of membrane currents in cultured oligodendrocytes isolated from rat optic nerve did not reveal Ca²⁺ currents (23). In situ recordings from oligodendrocytes in a white matter preparation also failed to detect voltage-gated Ca²⁺ currents (31). It could, however, not be excluded that such channels are present in membrane patches remote from the soma such as in the paranodal loops. It is unlikely that even large current injections into the soma will lead to significant membrane depolarization at such distant regions. In support of such an uneven distribution of voltage-gated channels is an observation by Waxman and colleagues (393), who found that voltage-gated Na⁺ channels in Schwann cells are concentrated in the membrane of the paranodal loops.

The expression pattern of Ca²⁺ channels undergoes considerable changes during development. Oligodendrocytic precursors from cortical cultures exhibited both T- and L-type Ca²⁺ currents. (431). Channel density was very low, and whole cell I_Ca were barely detectable (peak amplitudes <100 pA) even when Ba²⁺ was used to carry current. Cultured perinatal and adult oligodendrocyte progenitors from rat optic nerve (45) had only one component of Ca²⁺ current resembling the L type. In the cortical cultures, Ca²⁺ currents were substantially smaller in immature oligodendrocytes/late precursors and could not be detected in young oligodendrocytes. They were readily recorded from mature cells with complex morphology. Although it was not yet possible to detect Ca²⁺ channels in oligodendrocytes in situ, they were found in precursors from slices of mouse corpus callosum (31).

Despite the small amplitude of Ca²⁺ currents in oligodendrocytes, Ca²⁺ influx through voltage-gated channels was found to significantly increase [Ca²⁺]_i . Depolarization of cultured oligodendrocyte precursors and mature oligodendrocytes with KCl revealed substantial [Ca²⁺]_i increases that were sensitive to removal of [Ca²⁺]_o , inhibited by verapamil, and potentiated by BAY K 8644 (44, 45, 230, 238).

The depolarization-induced [Ca²⁺]_i transients in cultured oligodendrocytes were spatially heterogeneous, being in general more pronounced in oligodendrocytic processes (238). Furthermore, at the early developmental stages, T- and L-type Ca²⁺ channels were unevenly distributed over the cell membrane (Fig. 4). A moderate depolarization of the oligodendrocyte precursor by 20 mM K⁺ led to an increase of [Ca²⁺]_i in the processes only, whereas [Ca²⁺]_i levels in the soma remained unaffected. A further increase in [K⁺]_o resulted in a progressive fall in the amplitude of [Ca²⁺]_i elevations in processes, whereas in the soma, [Ca²⁺]_i transients became larger. Moreover, [Ca²⁺]_i signals in processes and in the soma of oligodendrocyte precursors can be dissected pharmacologically; Ni²⁺ (antagonist of low-voltage-activated Ca²⁺ channels) inhibited the depolarization-induced [Ca²⁺]_i transients only in the processes, whereas dihydropyridines preferentially affected somatic depolarization-triggered [Ca²⁺]_i responses (238).

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Spatial heterogeneity of LVA and HVA Ca2+ channels in oligodenroglial precursors. A: differential effect of increasing extracellular K+ on intracellular Ca2+ concentration ([Ca2+]i) in an oligodenroglial precursor cell. Top: pseudocolor images that reflect [Ca2+]i distribution within cell in response to external application of solutions with increasing K+ concentrations. Bottom: changes in fluorescence ratio for fluo 3 (which is a function of [Ca2+]i) were simultaneously measured in soma and in processes. Stars correspond to images shown above. B: distinct pharmacological properties of [Ca2+]i transients in soma (a) and in processes (b) of an oligodendrocyte precursor. Application of 50 mM K+ triggered [Ca2+]i elevation in both regions. However, K+-induced [Ca2+]i elevation was blocked by 50 µM Ni2+ in processes but not in soma. Because Ni2+ preferentially blocks LVA Ca2+ channels, result indicates that these channels are present to a much greater extent in processes than in soma. [From Kirishuck et al. (238). Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.]

An uneven distribution of Ca²⁺ channels was also observed in mature oligodendrocytes; a depolarization-induced [Ca²⁺]_i increase was mainly confined to the processes, whereas [Ca²⁺]_i in the soma increased to a much smaller extent (238).

The opening of voltage-gated Ca²⁺ channels requires depolarization. This depolarization might normally result from local changes in K⁺ concentration that accompany neuronal activity; the [K⁺]_o can increase to 15 mM with intense neuronal firing (405). Such an increase in [K⁺]_o was found to trigger Ca²⁺ influx via voltage-gated channels in cultured oligodendrocytes (238). Much higher levels of depolarization can be achieved under pathological conditions (e.g., spreading depression), when interstitial K⁺ may rise up to 80 mM (313). Alternatively, glial cells can be depolarized by the opening of ligand-gated cationic channels (see sect. V).

	V. NEUROTRANSMITTER-INDUCED CALCIUM SIGNALING IN GLIAL CELLS

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One of the most surprising developments in glial research over the last 25 years has been the discovery that various glial cells express a heterogeneous pattern of functional receptors to a variety of chemicals previously known to affect neurons. These include not only the classical neurotransmitters but also neuromodulators and neurohormones. Table 2 summarizes many of the experimental results that demonstrated an effect of these substances to increase [Ca²⁺]_i . Their effect results from activation of various receptors linked via several pathways to [Ca²⁺]_i regulating molecular cascades.

Glutamate is the major excitatory neurotransmitter in the CNS of mammals, and its action is conducted via activation of highly diversified families of ionotropic and metabotropic receptors. The ionotropic glutamate receptors (GluRs) are ligand-gated cationic channels assembled from five subunits. There are three groups of ionotropic GluRs (according to their pharmacological properties), -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) (180). The recent advances in recombinant DNA techniques have given a precise characterization of the GluRs subunit structure and revealed the molecular determinants of GluRs permeability, gating mechanisms, and agonist specificity (180, 207). The GluR subunits A, B, C, and D (or 1-4) form AMPA-sensitive receptors, GluR5, -6, and -7, and subunits denoted as KA1 and KA2 assembled to form kainate-preferable GluRs. Finally, the NMDA-sensitive GluR subfamily is formed by NMDA R1 and NMDA R2A-D subunits (295). Various GluRs subunits can be differentially assembled forming homo- or heteromeric channels that bear different functional properties. The subunit structure of the GluRs determines their Ca²⁺ permeability, with a crucial role for the GluR B subunit; channels containing GluR B subunit are almost impermeable to Ca²⁺, and those lacking this subunit in the channel pentamer are highly Ca²⁺ permeable (52, 146).

Metabotropic glutamate receptors (mGluRs) also comprise a distinct gene family of at least eight members (mGluRs 1-8); the mGluRs belong to the so-called seven-membrane spanning domains receptors (307, 348). The mGluR1 and -5 are coupled (via G proteins) with PLC, being thus the activators of the InsP₃-mediated intracellular signaling pathway; other mGluRs are connected with adenylate cyclases.

View larger version (29K): [in this window] [in a new window]   FIG. 5.   Stimulation of ionotropic glutamate receptors (GluRs) in Bergmann glial cell in cerebellar slices triggers Ca2+ influx. A: pseudocolor image of a fura 2-loaded Bergmann glial cell (scheme of experiment is shown in inset on left) in control conditions and upon extracellular application of 100 µM kainate. Note preferential increase in [Ca2+]i in cell processes. B and C: [Ca2+]i and membrane current traces recorded from same cell. Both removal of extracellular [Ca2+]i (B) and blockade of ionotropic GluRs by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) inhibited kainate-induced currents and [Ca2+]i elevation, suggesting a key role of Ca2+ influx. F340/380 , fluorescence ratio at 340/380 nm. (From T. Müller and H. Kettenmann, unpublished data.)

Adenosine 5'-triphosphate, adenosine, and related substances control a number of important physiological reactions and act as neurotransmitters in the peripheral nervous system and CNS (54, 102, 133). In recent years, clear evidence that ATP acts as an excitatory neurotransmitter in the CNS has been obtained (108, 109), and pharmacological and molecular characterization of ATP and adenosine receptors (named purinoreceptors) was achieved. Purinoreceptors are represented by a broad family of proteins classified into two major groups (88, 133): 1) adenosine receptors (or P₁ purinoreceptors codenamed also as A₁-A₄ receptors) coupled mainly with adenylate cyclases as well as with PLC (A₁ receptors) and 2) receptors for ATP and related nucleotides known as P₂ purinoreceptors. On the basis of pharmacological properties, the P₂ purinoreceptor family is subclassified into two groups: ionotropic receptors (P_2x and P_2z) and metabotropic receptors (P_2y , P_2u , P_2t and P_2d). Advances in molecular cloning extended this classification by showing that purinoreceptors are encoded by two distinct gene families (55). The family of P_2x receptors comprise several subtypes (labeled P_2x1-7) of ligand-gated ionic channels with a unique two transmembrane domain topology; the members of P_2x family differ in their ion selectivity and gating properties. Cloned P_2z receptors also belong to the P_2x gene family, and they are codenamed as P_2x7 subtype; P_2x7(z) receptors are large transmembrane pores that are activated in fact by a tetraanionic form of ATP (ATP⁴) and may pass molecules with a molecular mass up to 1 kDa. Cloned metabotropic receptors are classified as P_2y family, being represented by seven members (P_2y1-P_2y7). They all are similar to other G protein-linked metabotropic receptors by their seven-transmembrane domain structure and are often associated with PLC and hence InsP₃ turnover. The P_2y1 receptor pharmacologically matches P_2y subtype, P_2y2-P_2u , P_2y3 probably corresponds to P_2t receptor; P_2y4-7 are