Purinergic Signaling in the Regulation of Gout Flare and Resolution.

Gout flares require monosodium urate (MSU) to activate the NLRP3 inflammasome and secrete sufficient IL-1ß. However, MSU alone is not sufficient to cause a flare. This is supported by the evidence that most patients with hyperuricemia do not develop gout throughout their lives. Recent studies have shown that, besides MSU, various purine metabolites, including adenosine triphosphate, adenosine diphosphate, and adenosine bind to different purine receptors for regulating IL-1ß secretion implicated in the pathogenesis of gout flares. Purine metabolites such as adenosine triphosphate mainly activate the NLRP3 inflammasome through P2X ion channel receptors, which stimulates IL-1ß secretion and induces gout flares, while some purine metabolites such as adenosine diphosphate and adenosine mainly act on the G protein-coupled receptors exerting pro-inflammatory or anti-inflammatory effects to regulate the onset and resolution of a gout flare. Given that the purine signaling pathway exerts different regulatory effects on inflammation and that, during the inflammatory process of a gout flare, an altered expression of purine metabolites and their receptors was observed in response to the changes in the internal environment. Thus, the purine signaling pathway is involved in regulating gout flare and resolution. This study was conducted to review and elucidate the role of various purine metabolites and purinergic receptors during the process.

Multimeric Ionotropic Purinoceptor Detection by Protein Cross-Linking.

P2X receptor subunits (P2X1 to P2X7) assemble to form trimeric homomers or heteromers. Here, we describe the use of protein cross-linking to study the composition of P2X receptor complexes. This simple protocol is useful for determining the stoichiometry of P2X heteromeric receptors as well as for assessing the effect of point mutation, truncation, or concatenation on the quaternary architecture of these receptors.

[ROLE OF PURINERGIC RECEPTORS IN IMMUNE RESPONSE].

Purine receptors are located on immune and somatic cells of animal and human organisms. Summation of signals from purine and TOLL-like receptors takes place on the level of inflammasome formation and results in summation of the first and second signals of innate immunity. The first signal--from PAMPs (pathogen associated molecular patterns), the second--from DAMPs (danger associated molecular patterns). Adenosine triphosphate (ATP) is the most studied DAMP. ATP connects with purine receptors which include P2 (P2X7 receptors are the best described), that results in opening of channels of these receptors and transit of ATP into the cell. In parallel exit of K⁺ from cells and entrance of Ca²âº and Na⁺ into the cells is observed, that is associated with activation of the immune competent cell. Damaged cells dying via necrosis or apoptosis are the source of extracellular ATP, as well as activated immunocytes. Signals from P2 and TOLL-like receptors are summarized in effectors of immune response, and activation of P2 receptors in lymphocytes makes a contribution into activation of cells, mediated by T-cell receptor. Negative side of purine receptor activation is a stimulating effect on proliferation and metastasis of malignant cells. The practical output of knowledge on functioning of purine receptors for clinical immunology is the application of agonists and antagonists of purine receptors, as well as explanation of effect of immune modulators from the position of launch of K⁺/Na⁺-pump; resulting in prolonged activation of immune competent cells.

[Adenosine triphosphate stress 99mTc-MIBI single-photon emission computed tomography in the diagnosis of ischemic heart disease].

This review is devoted to possibilities of single-photon emission computed tomography (SPECT) combined with pharmacological test with adenosine triphosphate (ATP) to detect myocardial ischemia in patients with ischemic heart disease (IHD). It contains consideration of contemporary problems and limitations inherent in use of pharmacological stress tests in radionuclide diagnostics; discussion of mechanisms of vasodilating effects of ATP in the context of modern concepts of purine receptors; detailed description of technique of pharmacological testing with ATP, as well as contraindications and possible side effects. Experience of foreign studies with the use of ATP stress testing for verification of presence of ischemia in patients with IHD is also presented.

Non-Edg family LPA receptors: the cutting edge of LPA research.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator with diverse physiological and pathological actions on many types of cells. Originally, LPA was thought to elicit its biological functions through three subtypes of endothelial differentiation gene (Edg) family G protein-coupled receptors (LPA1, LPA2 and LPA3) until our group identified a fourth subtype, LPA4. The discovery of this receptor, which is structurally distinct from the Edg family LPA receptors, led to the identification of two additional LPA receptors, LPA5 and LPA6, homologous to LPA4. These 'non-Edg family' LPA receptors now provide a new framework for understanding the diverse functions of LPA, including vascular development, platelet activation and hair growth. In this review, we summarize the identification, intracellular signalling and biological functions of this novel cluster of LPA receptors.

Purinergic signaling involved in Müller cell function in the mammalian retina.

Purines (in particular, ATP and adenosine) act as neuro- and gliotransmitters in the sensory retina where they are involved in bidirectional neuron-glia signaling. This review summarizes the present knowledge about the expression and functional importance of P1 (adenosine) and P2 (nucleotide) receptors in Müller glial cells of the mammalian retina. Mammalian Müller cells express various subtypes of adenosine receptors and metabotropic P2Y receptors. Human Müller cells also express ionotropic P2X(7) receptors. Müller cells release ATP upon activation of metabotropic glutamate receptors and/or osmotic membrane stretching. The osmotic mechanism is abrogated under conditions associated with ischemia-hypoxia and inflammation, resulting in swelling of the Müller cells when the extracellular milieu is hypoosmotic. However, exogenous glutamate, which induces the release of ATP and adenosine, and thus activates P2Y(1) and A(1) adenosine receptors, respectively, prevents such osmotic swelling under pathological conditions, suggesting unimpaired receptor-induced release of ATP. In addition to the inhibition of swelling, which is implicated in regulating the volume of the extracellular space, purinergic signaling is involved in mediating neurovascular coupling. Furthermore, purinergic signals stimulate the proliferation of retinal precursor cells and Müller cells. In normal retinal information processing, Müller cells regulate the synaptic activity by the release of ATP and adenosine. In retinopathies, abrogation of the osmotic release of ATP, and the upregulation of ecto-apyrase (NTPDase1), may have neuroprotective effects by preventing the overactivation of neuronal P2X receptors that are implicated in apoptotic cell death. Pharmacological modulation of purinergic receptors of Müller cells may have clinical importance, e.g., for the clearance of retinal edema and for the inhibition of dysregulated cell proliferation in proliferative retinopathies.

Expression and contribution of satellite glial cells purinoceptors to pain transmission in sensory ganglia: an update.

The role of adenosine-5'-triphosphate (ATP) and of the ligand-gated P2X3 receptor in neuronal dorsal root ganglia (DRG) pain transmission is relatively well established. Much less is known about the purinergic system in trigeminal ganglia (TG), which are involved in certain types of untreatable neuropathic and inflammatory pain, as well as in migraine. Emerging data suggest that purinergic metabotropic P2Y receptors on both neurons and satellite glial cells (SGCs) may also participate in both physiological and pathological pain development. Here, we provide an updated literature review on the role of purinergic signaling in sensory ganglia, with special emphasis on P2Y receptors on SGCs. We also provide new original data showing a time-dependent downregulation of P2Y2 and P2Y4 receptor expression and function in purified SGCs cultures from TG, in comparison with primary mixed neuron-SGCs cultures. These data highlight the importance of the neuron-glia cross-talk in determining the SGCs phenotype. Finally, we show that, in mixed TG cultures, both adenine and guanosine induce intracellular calcium transients in neurons but not in SGCs, suggesting that also these purinergic-related molecules can participate in pain signaling. These findings may have relevant implications for the development of new therapeutic strategies for chronic pain treatment.

Neuronal soma-satellite glial cell interactions in sensory ganglia and the participation of purinergic receptors.

It has been known for some time that the somata of neurons in sensory ganglia respond to electrical or chemical stimulation and release transmitters in a Ca2+-dependent manner. The function of the somatic release has not been well delineated. A unique characteristic of the ganglia is that each neuronal soma is tightly enwrapped by satellite glial cells (SGCs). The somatic membrane of a sensory neuron rarely makes synaptic contact with another neuron. As a result, the influence of somatic release on the activity of adjacent neurons is likely to be indirect and/or slow. Recent studies of neuron-SGC interactions have demonstrated that ATP released from the somata of dorsal root ganglion neurons activates SGCs. They in turn exert complex excitatory and inhibitory modulation of neuronal activity. Thus, SGCs are actively involved in the processing of afferent information. In this review, we summarize our understanding of bidirectional communication between neuronal somata and SGCs in sensory ganglia and its possible role in afferent signaling under normal and injurious conditions. The participation of purinergic receptors is emphasized because of their dominant roles in the communication.

Purinergic signalling in autonomic control.

Intercellular purinergic signalling, which utilizes ATP as a transmitter, is fundamental for the operation of the autonomic nervous system. ATP is released together with 'classical' transmitters from sympathetic and parasympathetic nerves supplying various peripheral targets, modulates neurotransmission in autonomic ganglia, has an important role in local enteric neural control and coordination of intestinal secretion and motility, and acts as a common mediator for several distinct sensory modalities. Recently, the role of ATP-mediated signalling in the central nervous control of autonomic function has been addressed. Emerging data demonstrate that in the brain ATP is involved in the operation of several key cardiorespiratory reflexes, contributes to central processing of viscerosensory information, mediates central CO(2) chemosensory transduction and triggers adaptive changes in breathing, and modulates the activities of the brainstem vagal preganglionic, presympathetic and respiratory neural networks.

Purinergic receptor immunoreactivity in the rostral ventromedial medulla.

The rostral ventromedial medulla (RVM) has long been recognized to play a pivotal role in nociceptive modulation. Pro-nociception within the RVM is associated with a distinct functional class of neurons, ON-cells that begin to discharge immediately before nocifensive reflexes. Anti-nociceptive function within the RVM, including the analgesic response to opiates, is associated with another distinct class, OFF-cells, which pause immediately prior to nocifensive reflexes. A third class of RVM neurons, NEUTRAL-cells, does not alter firing in association with nocifensive reflexes. ON-, OFF- and NEUTRAL-cells show differential responsiveness to various behaviorally relevant neuromodulators, including purinergic ligands. Iontophoresis of semi-selective P2X ligands, which are associated with nociceptive transmission in the spinal cord and dorsal root ganglia, preferentially activate ON-cells. By contrast, P2Y ligands activate OFF-cells and P1 ligands suppress the firing of NEUTRAL cells. The current study investigates the distribution of P2X, P2Y and P1 receptor immunoreactivity in RVM neurons of Sprague-Dawley rats. Co-localization with tryptophan hydroxylase (TPH), a well-established marker for serotonergic neurons was also studied. Immunoreactivity for the four purinergic receptor subtypes examined was abundant in all anatomical subdivisions of the RVM. By contrast, TPH-immunoreactivity was restricted to a relatively small subset of RVM neurons concentrated in the nucleus raphe magnus and pallidus, as expected. There was a significant degree of co-localization of each purinergic receptor subtype with TPH-immunoreactivity. This co-localization was most pronounced for P2Y1 receptor immunoreactivity, although this was the least abundant among the different purinergic receptor subtypes examined. Immunoreactivity for multiple purinergic receptor subtypes was often co-localized in single neurons. These results confirm the physiological finding that purinergic receptors are widely expressed in the RVM. Purinergic neurotransmission in this region may play an important role in nociception and/or nociceptive modulation, as at other levels of the neuraxis.

Purinergic actions on neurons that modulate nociception in the rostral ventromedial medulla.

The rostral ventromedial medulla (RVM) serves as a critical link in bulbo-spinal nociceptive modulation. Within the RVM, 'off-cells' pause and 'on-cells' discharge immediately prior to a nocifensive reflex. These neurons are also activated and inactivated, respectively, by local or systemic application of opioids. Off-cell activation leads to behavioral anti-nociception and on-cell activation to hyperalgesia. Thus, on- and off-cell populations allow bi-directional modulation of nociception by the RVM. A third neuronal population, neutral cells, shows no reflex-related change in discharge. The role of neutral cells in nociception, if any, is unknown. We investigated the responses of on-, off- and neutral cells to the iontophoretic application of purinergic ligands in lightly anesthetized rats. On-cell firing increased rapidly in response to application of ATP and to the P2X-receptor agonist, alpha,beta-methylene ATP. Off-cell firing increased gradually in response to ATP and to the P2Y-receptor agonist, 2-methylthio-ATP. All of these responses were attenuated or reversed by the non-specific P2-receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Activation of off-cells was preferentially antagonized by the relatively selective P2Y antagonist, MRS2179. By contrast with activation of on- and off-cells by ATP, neutral cell firing was depressed by ATP, adenosine and the P1-receptor agonist, 5'-(N-ethylcarboxamido) adenosine (NECA). Neutral cell responses to these agonists were at least partially reversed by the adenosine-receptor antagonist, 8-phenyltheophylline (8PT). These data imply that on-cells preferentially express P2X-receptors, off-cells P2Y-receptors and neutral cells P1-receptors. Immunohistochemical localization of purinergic receptors confirms the presence of some subtypes of P2X, P2Y and A1 receptors on neuronal cell bodies and fibers within the RVM. The differential responses of on-, off- and neutral-cells to purinergic ligands highlight the value of pharmacological signatures in further delineation of the anatomy, connectivity and function of this therapeutically important system.

Purinergic signalling--an overview.

A brief account of the early history of extracellular signalling by ATP will be followed by a summary of the current subclassification of receptors for purines and pyrimidines. On the basis of cloning, transduction mechanisms and pharmacology, the P1 (adenosine) receptor family has 4 subtypes, while the P2 (ATP, ADP and UTP) receptor family has been divided into P2X ionotropic receptors (7 subtypes) and P2Y metabotropic G protein-coupled receptors (8 subtypes). The distribution of purinoceptors in both neuronal and non-neuronal cells and the physiology and pathophysiology of purinergic signalling will be reviewed. Examples of fast purinergic signalling include cotransmission and neuromodulation, exocrine and endocrine secretion, platelet aggregation, vascular endothelial cell-mediated vasodilatation and nociceptive mechanosensory transduction. Examples of slow (trophic) purinergic signalling include cell proliferation, differentiation and apoptosis in embryological development, neural regeneration, bone resorption, cell turnover of epithelial cells in skin and visceral organs, inflammation, wound healing and cancer. Finally the purinoceptor subtypes expressed on astrocytes, oligodendrocytes, Schwann cells, microglia, Müller cells and enteric glial cells will be summarized as well as evidence for non-lytic release of ATP from glial cells.

Characterization of the purinergic receptor subtype on guinea-pig suburothelial myofibroblasts.

OBJECTIVE: To identify particular purinoceptor subtypes by immunohistochemical labelling, as a layer of suburothelial myofibroblasts has been identified in the urinary bladder, and these cells respond to exogenous ATP by generating an intracellular Ca2+ transient, but the particular purinoceptor that responds to ATP is unclear. MATERIALS AND METHODS: Tissue sections and isolated cells from the urothelial layer of the guinea-pig bladder were used. Preparations were labelled with primary antibodies to the intermediate-filament protein, vimentin, or the purinoceptors P2X3, P2Y1, P2Y2, P2Y4 and P2Y6. For single-labelling we used a secondary antibody tagged with the fluorescent marker Cy3, and for double-labelling also a secondary antibody tagged with fluorescein isothiocyanate or Cy2. Images were examined using a confocal microscope, with an argon (488 nm) or helium-neon (543 nm) laser. RESULTS: Vimentin-labelling was confined to the suburothelial layer and appeared as discrete signals. Isolated cells labelled with vimentin and strongly for the P2Y6 antibody. There was weaker staining for P2X3, P2Y2 and P2Y4, but none to P2Y1. With frozen sections there was P2Y6 labelling in the urothelial and suburothelial layer. CONCLUSION: The predominant purinoceptor in suburothelial myofibroblasts, from these labelling studies, is the P2Y6 subtype. However, there was weaker labelling to other subtypes, suggesting multiple receptor subtypes or heterogeneity of receptor subunits. The consequences of there being multiple purinoceptor subtypes in the suburothelial space with respect to sensory signalling are discussed.

Purinergic mechanisms of the nucleus of the solitary tract and neural cardiovascular control.

OBJECTIVES: This review addresses the role of central purinergic receptors in the operation of the cardiovascular reflexes. METHODS: Potential physiological role of purinergic receptors operating in the nucleus of the solitary tract (NTS) was assessed via comparison of the regional patterns of hemodynamic and sympathetic responses evoked by selective stimulation/inhibition of NTS purinergic receptor subtypes, with the patterns evoked by stimulation and unloading of arterial baroreceptors, and other known patterns of autonomic responses. The effects of sino-aortic denervation plus vagotomy and ionotropic glutamatergic blockade of NTS mechanisms on the patterns of the responses were also considered. RESULTS: Selective stimulation of NTS A1 receptors with CPA evoked a pattern of regional autonomic responses consistent with inhibition of baroreflex mechanisms and facilitation/ disinhibition of chemoreflex mechanisms. Selective stimulation of NTS A(2a) receptors with CGS 21680-evoked pattern of the responses different than that evoked by stimulation of baroreflex afferents what remains in contrast to previous reports suggesting that NTS A2a receptors facilitate baroreflex transmission. The pattern of the responses was similar to that observed during hypotensive hemorrhage. Preferential, b -adrenergic iliac vasodilation evoked by stimulation of adenosine A2a receptors and preferential activation of sympathetic output to the adrenal medulla by both adenosine A1 and A2a receptors are consistent with contribution of these receptors to the defense response, stress and exercise. These observations support previous findings that NTS A1 receptors contribute to the hypothalamic defense response. The effects of stimulation and blockade of NTS P2x receptors with alpha, beta-methylene ATP and suramin, respectively, suggested that neuronally-released ATP operating via P2x receptors may be a crucial co-transmitter with glutamate in mediating baroreflex responses. DISCUSSION: The above observations strongly suggest that purinergic receptor subtypes operating in NTS circuitry are linked to specific afferent and descending mechanisms primarily integrated in the NTS.

Tetrodotoxin-sensitive Na+ channels and muscarinic and purinergic receptors identified in human erythroid progenitor cells and red blood cell ghosts.

This article concerns the identification of different types of voltage-gated Na(+) channels and of muscarinic and purinergic receptors that are expressed in human erythroid precursor cells and red cell ghosts. We analyzed, by RT-PCR, RNA that was extracted from purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes in 7 to 14 days. These extracts were free of white cell and platelet contamination. Two types of voltage-gated, tetrodotoxin-sensitive Na(+) channels were found. These were Na(v)1.4 and Na(v)1.7, the former known to be present in skeletal muscle and the latter in peripheral nerve. By using a pan Na(+) channel antibody and Western blotting, an immunoreactive channel was detected in ghosts of human red blood cells, consistent with the expression of these two channels. The transcripts for four of the five known subtypes of muscarinic receptors were also identified, including subtypes M2, M3, M4, and M5, whereas subtype M1 was not found. Expression was also detected for the purinergic type receptors P2X(1), P2X(4), P2X(7), and P2Y(1) whereas types P2Y(2), P2Y(4), and P2Y(6) were not found. We also searched for but did not find transcripts for hBNP-1, a type 1b human brain sodium phosphate cotransporter, and cystic fibrosis transmembrane conductance regulator (CFTR). Implications regarding the presence of these different types of channels and receptors in human red blood cells and their functional significance are discussed.

Differential activation of subtype purinergic receptors modulates Ca(2+) mobilization and COX-2 in human microglia.

We have studied modulation of purinergic receptors (P(2Y) and P(2X) subtypes) on changes in intracellular Ca(2+) [Ca(2+)](i) and expression and production of COX-2 in human microglia. Measurements using Ca(2+)-sensitive spectrofluorometry showed adenosine triphosphate (ATP) to cause rapid transient increases in [Ca(2+)](i). Application of ATP plus the P(2X) antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), or treatment with adenosine diphosphate-beta-S (ADP-beta-S), a selective P(2Y) agonist, led to a considerable prolongation in [Ca(2+)](i) responses compared with ATP. The prolonged time courses were consistent with sustained activation of store-operated channels (SOC) since SKF96365, an inhibitor of SOC, blocked this component of the response. RT-PCR data showed that microglia expressed no COX-2 either constitutively or following treatment of cells with ATP (100 microM for 8 h). However, treatment using ATP plus PPADS or with ADP-beta-S led to marked expression of COX-2. The enhanced COX-2 with ATP plus PPADS treatment was absent in the presence of SKF96365 or using Ca(2+)-free solution. Immunocytochemistry, using a specific anti-COX-2 antibody, also revealed a pattern of purinergic modulation whereby lack of P(2X) activation enhanced the production of COX-2 protein. These results suggest that modulation of subtypes of purinergic receptors regulates COX-2 in human microglia with a link involving SOC-mediated influx of Ca(2+).

Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro.

Microglial cells are the pathologic sensors in the brain. ATP released from damaged cells is a candidate for signalling neural injury to microglia. Moreover, ATP is an extracellular messenger for propagating astrocyte activity in the form of Ca2+ waves. To test for the functional expression of purinoreceptors in microglial cells we employed the patch-clamp technique in acute slices of adult mouse brain. ATP triggered a nonselective cationic and a K+ current. Pharmacological screening with purinergic ligands indicated the presence of P2Y1 and P2Y2/4 receptors linked to the activation of a K+ current and P2X receptors, including P2X7, linked to the activation of a nonselective cationic current. These findings suggest that microglial cells in situ express different purinergic receptors with distinct sensitivity and functional coupling. To test for the involvement of purinoreceptors in microglial activation, we stimulated cultured microglial cells with lipopolysaccharide and measured the release of tumour necrosis factor alpha, interleukin-6, interleukin-12 and macrophage inflammatory protein 1alpha, induction of K+ outward currents and nitric oxide release. All these parameters were reduced in the presence of purinergic ligands, indicating that purinergic receptor activation attenuated indicators of microglial activation.

G protein-coupled receptor genes in the FANTOM2 database.

G protein-coupled receptors (GPCRs) comprise the largest family of receptor proteins in mammals and play important roles in many physiological and pathological processes. Gene expression of GPCRs is temporally and spatially regulated, and many splicing variants are also described. In many instances, different expression profiles of GPCR gene are accountable for the changes of its biological function. Therefore, it is intriguing to assess the complexity of the transcriptome of GPCRs in various mammalian organs. In this study, we took advantage of the FANTOM2 (Functional Annotation Meeting of Mouse cDNA 2) project, which aimed to collect full-length cDNAs inclusively from mouse tissues, and found 410 candidate GPCR cDNAs. Clustering of these clones into transcriptional units (TUs) reduced this number to 213. Out of these, 165 genes were represented within the known 308 GPCRs in the Mouse Genome Informatics (MGI) resource. The remaining 48 genes were new to mouse, and 14 of them had no clear mammalian ortholog. To dissect the detailed characteristics of each transcript, tissue distribution pattern and alternative splicing were also ascertained. We found many splicing variants of GPCRs that may have a relevance to disease occurrence. In addition, the difficulty in cloning tissue-specific and infrequently transcribed GPCRs is discussed further.

Purine-mediated signalling in pain and visceral perception.

Receptor subtypes for purines have been identified in a variety of tissues, increasing interest in the roles of purine-mediated signalling in pathophysiological processes. Growing evidence supports the involvement of one of the purinoceptor subtypes, P2X3, in nociception. In this article, recent studies of purine-mediated nociception and visceral pain will be discussed. Furthermore, a novel hypothesis is proposed for purine-mediated mechanosensory transduction where ATP released during distension from epithelial cells lining tubes (such as ureter and gut) and sacs (such as the bladder) acts on P2X3 receptors on a subepithelial nerve plexus to initiate impulses that are relayed via the spinal cord to pain centres in the brain.

Effects of ATP and derivatives on neuropile glial cells of the leech central nervous system.

We investigated the effects of ATP (adenosine 5'-triphosphate) and derivatives on leech neuropile glial cells, focusing on exposed glial cells. ATP dose-dependently depolarized or hyperpolarized neuropile glial cells in situ as well as exposed neuropile glial cells. These potential shifts varied among cells and repetitive ATP application did not change their amplitude, duration or direction. In exposed neuropile glial cells, ATP most frequently induced a Na(+)-dependent depolarization and decreased the input resistance. The agonist potency ATP > ADP (adenosine 5'-diphosphate) > AMP (adenosine 5'-monophosphate) > adenosine indicates that P2 purinoceptors mediate this depolarization. The P2Y agonist 2-methylthio-ATP mimicked the ATP-induced depolarization, whereas the P2Y antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid) reduced it. P2X agonists were without effect. Because the P1 antagonist 8-SPT (8-(p-sulphophenyl)-theophylline) also depressed ATP-induced depolarizations and some ATP-insensitive glial cells responded to adenosine, we suggest coexpression of metabotropic P2Y and P1 purinoceptors. The ATP-induced depolarization requires activation of Na(+) channels or nonselective cation channels, whereas the ATP-induced hyperpolarization indicates activation of K(+) channels. ATP also increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), that is independent of Ca(2+) influx but reflects intracellular Ca(2+) release possibly triggered by IP(3) formation. ADP and AMP also increased [Ca(2+)](i), but were less efficient than ATP; adenosine and 2-methylthio-ATP did not affect [Ca(2+)](i). In view of the mobilization of intracellular Ca(2+), ATP is clearly different from other leech neurotransmitters, because it enables intracellular Ca(2+) signaling without causing prominent changes in glial membrane potential. Thus disturbance of the extracellular microenvironment and the demand for metabolic energy are minimized.