The Journal of Neurophysiology Vol. 79 No. 5 May 1998, pp. 2513-2521
Copyright ©1998 by the American Physiological Society

Diverse Signal Transduction Pathways Mediated by Endogenous P₂ Receptors in Cultured Rat Cerebral Cortical Neurons

Tomoyuki Nishizaki and Masahiro Mori

Department of Physiology, Kobe University School of Medicine, Chuo-ku, Kobe 650, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Nishizaki, Tomoyuki and Masahiro Mori. Diverse signal transduction pathways mediated by endogenous P₂ receptors in cultured rat cerebral cortical neurons. J. Neurophysiol. 79: 2513-2521, 1998. The present study was conducted to assess the intracellular signaling pathways mediated by receptors for ATP, uridine triphosphate (UTP), and 2-methylthio ATP (2-MeSATP), by monitoring patch-clamp currents and intracellular calcium mobilization in cultured rat cortical cerebral neurons. All three agonists evoked potassium currents and increased the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), and these effects were inhibited by the broad G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPS) but not by the G_i/_o-protein inhibitor pertussis toxin (PTX). UTP-evoked currents were inhibited by either the phospholipase C inhibitor neomycin or the selective protein kinase C (PKC) inhibitor GF109203X, and the rise in cytosolic Ca²⁺ was inhibited by either neomycin or the inositol 1,4,5-trisphosphate (IP₃) receptor antagonist heparin, indicating that the UTP receptor involved phospholipase C-mediated phosphatidylinositol signaling. In contrast, 2-MeSATP-induced currents and rise in cytosolic Ca²⁺ were not inhibited by either neomycin, or GF109203X, or heparin. 2-MeSATP elicited single-channel currents in the cell-attached patch-clamp configuration and also in excised patches. The G-protein activator GTPS induced single-channel currents in a fashion that mimicked the effect of 2-MeSATP. These data suggest that 2 MeSATP activated potassium channels by a direct action of G-protein subunits and increased [Ca²⁺]_i by a mechanism independent of phospholipase C stimulation and IP₃ production. ATP-evoked currents were partially inhibited by either neomycin or GF109203X, although the rise in cytosolic Ca²⁺ was not affected by these inhibitors. ATP produced single-channel currents with two major classes of the slope conductance (86 and 95 pS) in cell-attached patches, each of which is consistent with that achieved by 2-MeSATP (85 pS) or UTP (96 pS); the currents with the lower conductance were observed in the outside-out patch-clamp configuration. These results indicate that P₂ receptors for UTP and 2-MeSATP are linked to a PTX-insensitive G-protein involving different signal transduction pathways and that ATP responses are mediated by both of these P₂ receptors.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Purinoceptors play a significant role in the regulation of a variety of physiologically important processes in excitable and nonexcitable cells. These receptors were originally classified as P₁ (adenosine > AMP  ADP  ATP) and P₂ purinoceptors (ATP  ADP  AMP  adenosine) depending on their preference for adenosine or adenine nucleotides (Burnstock and Kennedy 1985). The P₂ purinoceptors can be further subdivided into P_2X, P_2Y, P_2Z, P_2T, and P_2U subtypes (Gordon 1986; Lin et al. 1993; O'Connor et al. 1991), although this nomenclature has been superceded by subdivision into two families (Abbracchio and Burnstock 1994). The P_2X purinoceptors, a family of the ligand-gated receptors, have been cloned as P2X_1-6 and P2X₇ (P_2Z) (Bo et al. 1995; Brake et al. 1994; Chen et al. 1995; Collo et al. 1996; Lewis et al. 1995; Soto et al. 1996; Surprenant et al. 1996; Valera et al. 1994), and otherwise, the P_2Y purinoceptors, linked to G-proteins (Barnard et al. 1994), have been cloned as P2Y₁, P2Y₂ (P_2U), and P2Y_3-6 (Chang et al. 1995; Communi et al. 1995; Janssens et al. 1996; Nguyen et al. 1995; Tokuyama et al. 1995; Webb et al. 1993, 1994, 1996b,c). The P_2Y purinoceptors mainly appear to be coupled to the phospholipase C-mediated signaling pathway (O'Connor et al. 1991), and in addition, to the activation of mitogen-activated protein kinase (MAPK) (King et al. 1996; Patel et al. 1996) and G_i protein (Song and Chueh 1996; Webb et al. 1996a). Some of the P_2Y purinoceptors are expressed in the CNS, suggesting an association with synaptic transmission. Little is known, however, about actual function of P_2Y purinoceptors in neurons and the relevant intracellular signaling pathways.

In earlier studies using cultured rat neurons from different regions of the CNS, we demonstrated that P₂ purinoceptor agonists activate potassium channels via a pertussis toxin (PTX)-insensitive G-protein-regulated receptor, possibly involving a P_2Y purinoceptor (Ikeuchi and Nishizaki 1995a,b, 1996a,b; Ikeuchi et al. 1995, 1996). ATP receptors evoke potassium currents by interacting with a protein kinase C (PKC) pathway in striatal neurons and spinal neurons (Ikeuchi and Nishizaki 1995a,b). In contrast, 2-methylthio ATP (2-MeSATP) and ADP generated currents by a mechanism independent of PKC activation in medullar, cerebellar, and inferior colliculus neurons (Ikeuchi and Nishizaki 1995b, 1996a; Ikeuchi et al. 1995). These findings suggest that the effects of the P₂ purinoceptor agonists on neurons cannot be explained by a single P_2Y purinoceptor and that coupled potassium channels are activated by different mechanisms. This prompted the present study to assess the intracellular signal transduction pathways responsible for ATP, 2-MeSATP, and uridine triphosphate (UTP) by monitoring patch-clamp currents and cytosolic calcium mobilization in cultured rat cerebral cortical neurons. The results of the present study culminated in the finding that either 2-MeSATP or UTP activates potassium channels and regulates intracellular calcium mobilization via distinct P₂ receptors involving independent signal transduction pathways, and that ATP responses are mediated both by the 2-MeSATP and UTP receptor.

	METHODS

Abstract Introduction Methods Results Discussion References

Cell culture

Cerebral cortical neurons from neonatal rat on day 1 were cultured as described previously (Ikeuchi and Nishizaki 1995a). The cerebral cortex was removed from the brain under ether-inducedanesthesia. The tissues were incubated in 0.25% trypsin in Ca²⁺-,Mg²⁺-free saline for a few minutes at room temperature and then mechanically dissociated by triturating with a Pasteur pipette. The dissociated cells were plated on collagen-coated coverslips and grown in Dulbecco's modified Eagle's medium (DMEM, GIBCO) with 15% fetal bovine serum at 37°C in a humidified atmosphere of 95% air and 5% CO₂. To suppress the growth of glial cells, cytosine -D-arabinofuranoside (Ara-C; Sigma, St. Louis, MO; final concentration, 10 µM) was supplemented to the culture medium 1-3 days after plating. Cultured neurons were used 1-2 wk after plating.

View larger version (26K): [in this window] [in a new window]   FIG. 1. Whole cell currents induced by purinoceptor agonists. A: whole cell patches were made to cells using the basic patch electrode-filling solution, and ATP, ADP, AMP, uridine triphosphate (UTP), 2-methylthioATP (2-MeSATP), ,-methylene ATP (,MeATP), ,-methylene ATP (,-MeATP), or adenosine (AD) at a concentration of 10 µM was applied to a single cell for 5 s indicated by the bars. Holding potential was +60 mV. In this and further figures, outward currents correspond to upward deflections. B: whole cell patches were made to cells using the patch electrode-filling solution with (+) and without 1,2bis ( 2 - aminophenoxy ) ethane - N , N , N ' , N ' - tetraaceticacid (BAPTA; ). Cells were held at voltages as indicated and afterward, ATP, UTP, or 2-MeSATP was applied. Currents obtained were normalized by current density (peak current amplitude/cell capacitance), and whole cell current-voltage relations were calculated. Each point represents the average from 7-10 independent experiments, and the SD is indicated by the bars.

Patch-clamp recording

Whole cell voltage-clamp and single-channel currents were recorded using an Axopatch-200A amplifier (Axon Instruments, Foster, CA). Currents obtained were stored on magneto optical disks (MK128D, Mitsubishi-Kasei, Tokyo, Japan) and analyzed on a microcomputer using pClamp software (Axon Instruments; Version 6). Cells were bathed at room temperature (20-22°C) in a standard extracellular solution containing (in mM) 145 NaCl, 5 KCl, 2.4 CaCl₂, 1.8 glucose, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 0.3 × 10³ tetrodotoxin, pH 7.4. The basic patch electrode-filling solution was (in mM) 150 KCl, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) tetrapotassium salt, hydrate, and 10 HEPES, pH 7.2. In some experiments, BAPTA-free patch electrode-filling solution was used. After formation of whole cell patches, series resistance (R_s) compensation was made up to ~95%. Purinoceptor agonists were applied to cells for 5 s by an air pressure microinjector (PV 830 Pneumatic Picopump, World Precision Instruments, Sarasota, FL). To normalize current amplitudes, cell capacitance was measured and current density (peak current amplitude/cell capacitance) was calculated.

For single-channel current recording, cell-attached patches were made using the patch electrode filled with standard extracellular solution in the absence and presence of ATP, UTP, or 2-MeSATP, which were bath-applied to cells. In outside-out patches, the patch electrode was filled with the same intracellular solution as used in whole cell, patches and agonists were bath-applied during recording. Currents were filtered at 2 kHz and digitized at 1 kHz.

,-Methylene ATP (,-MeATP), ,-methylene ATP (,-MeATP), 2-MeSATP, and UTP were purchased from RBI, PTX from Sigma, guanosine-5'-O-(2-thiodiphosphate) (GDPS) and guanosine-5'-O-(3-thiotriphosphate) (GTPS) from Boehringer Mannheim (Mannheim, Germany), GF109203X, 12-O-tetradecanoyl phorbol-13-acetate (TPA), neomycin, tetrodotoxin, and BAPTA from Wako Pure Chemical (Osaka, Japan).

Assay of intracellular free Ca²⁺ concentrations ([Ca²⁺]_i)

Cells were incubated with 4 µM fura-2/AM (Molecular Probes) at 37°C for 1 h in a standard extracellular solution together with 0.02% pluronic F-127; the cell-permeant AM esters serves as a Ca²⁺-sensitive indicator by deesterification in cells. Fura 2-loaded cells were placed into a recording chamber onto the stage of a Nikon DIAPHOT 300 microscope and bathed in the standard Ca²⁺-containing extracellular solution. Whole cell patches were made from fura 2-loaded cells using a patch electrode filled with the following intracellular solution (in mM): 150 KCl, 10 HEPES, 0.1 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 0.4 µM fura 2/AM, pH 7.2. Cells were viewed using a fluorescence ×20 dry objective lens and a ×20 dry phase-contrast objective. Fura-2 was excited at 340 and 380 nm alternatively switched every 500 ms. The fluorescence signal was filtered through a band-pass filter transmitting 500-511 nm and detected by an intensified charge-coupled device camera (CAM-220, Hamamatsu Photonics, Hamamatsu, Japan). Ratio images were calculated in real time, stored on hard disk, and analyzed using ARGUS-50/CA software (Version 3.0). [Ca²⁺]_i was calculated from the fluorescence ratio/Ca²⁺ concentration calibration curve made before experiments. Simultaneously, whole cell currents were recorded and analyzed as described above. The Ca²⁺-free extracellular solution was (in mM) 145 NaCl, 5 KCl, 2.4 CaCl₂, 1.8 glucose, 10 HEPES, 10 BAPTA, and 0.3 × 10³ tetrodotoxin, pH 7.4.

	RESULTS

Abstract Introduction Methods Results Discussion References

Whole cell currents evoked by ATP, UTP, and 2-MeSATP

P₂ purinoceptor agonists (ATP, UTP, and 2-MeSATP) produced outward whole cell currents at a holding potential of +60 mV in cultured cerebral cortical neurons (Fig. 1A). In addition, ADP, AMP, and ,-MeATP, or the P₁ purinoceptor agonist, adenosine also evoked currents, although no response was induced by ,-MeATP (Fig. 1A). The currents were blocked either by replacement of KCl with CsCl in the patch electrode-filling solution (Fig. 2) or by treatment with the nonselective K⁺ channel blocker, tetraethylammonium (1 mM; data not shown). The current-voltage relations obtained with voltage steps from 120 to +120 mV exhibited a reversal potential of about 80 mV, consistent with the expected equilibrium potential for K⁺ (Fig. 1B). These results indicate that these agonists activated potassium channels.

View larger version (34K): [in this window] [in a new window]   FIG. 2. Regulation of ATP-, UTP-, and 2-MeSATP-induced currents. KCl (150 mM) in the patch electrode-filling solution was replaced with CsCl (150 mM). Guanosine-5'-O-(2-thiodiphosphate) (GDPS; 100 µM) was added to the patch electrode-filling solution 5 min before application of the agonists. Cells were treated with PTX (0.1 µg/ml) for 24 h before experiments. In other cases, ATP, UTP, or 2-MeSATP at a concentration of 10 µM was applied to cells in the presence and absence of neomycin (500 µM) or GF109203X (500 nM). Each set of experiment was carried out in a single cell. The holding potential was +60 mV. In the accompanying graph, each value (±SD) represents the mean from 8-10 cells. *P < 0.1, **P < 0.01, unpaired t-test.

The currents evoked by ATP, UTP, or 2-MeSATP were inhibited by the broad G-protein inhibitor, GDPS (100 µM), whereas they were not affected by the G_i/G_o-protein inhibitor, PTX (0.1 µg/ml; Fig. 2). This indicates that the receptors for ATP, UTP, and 2-MeSATP were linked to a PTX-insensitive G-protein. The patch electrode-filling solution used here did not contain Mg²⁺, a necessary factor for activating G-proteins, suggesting that G-proteins could be activated by intrinsic Mg²⁺ even if it is diluted. ATP-evoked currents were inhibited by 21 ± 6% and 44 ± 8%(mean ± SD) by the phospholipase C inhibitor, neomycin (500 µM), and by the selective PKC inhibitor, GF109203X (500 nM), respectively (Fig. 2). UTP-evoked currents were fully inhibited by neomycin and GF109203X, whereas these inhibitors had no effect on 2-MeSATP-evoked currents (Fig. 2). These results suggest that UTP activated K⁺ channels by phospholipase C-mediated PKC activation and that the ATP-sensitive channel was only regulated in part by PKC activation. Otherwise, the 2-MeSATP-sensitive channel was regulated by a mechanism independent of PKC activation.

Intracellular Ca²⁺ mobilization induced by ATP, UTP, and 2-MeSATP

ATP, UTP, and 2-MeSATP increased [Ca²⁺]_i in Ca²⁺-containing extracellular solution (Fig. 3C). Increases were also observed in Ca²⁺-free extracellular solution (Fig. 3A), suggesting that these agonists stimulated Ca²⁺ release from intracellular calcium stores. Notably, there was no relation between current amplitude and rise in intracellular Ca²⁺ induced by these agonists (Fig. 3C). When monitored with the patch electrode-filling solution containing 10 mM BAPTA, neither of these agonists increased [Ca²⁺]_i, while currents were evoked (Fig. 3B). These findings, together with the finding that there was no significant difference in the whole cell current-voltage relations between in the presence and absence of BAPTA in the patch pipette (Fig. 1B), indicate that intracellular Ca²⁺ did not activate K⁺ channels.

View larger version (25K): [in this window] [in a new window]   View larger version (43K): [in this window] [in a new window]   FIG. 3. Assay of [Ca2+]i. Whole cell patches were made in fura 2-loaded cells using the patch electrode-filling solution containing 0.1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; A and C) or 10 mM BAPTA (B). The currents (I) and/or intracellular Ca2+ waves ([Ca2+]i; Conc., calibrated [Ca2+]i; Ratio, fluorescence ratio F340/380) were monitored in Ca2+-free [extCa2+(); A] and Ca2+-containing extracellular solution [extCa2+(+); B and C]. GDPS (100 µM) or heparin (1 mg/ml) was perfused in the patch electrode-filling solution for 5 min before application of ATP, UTP, or 2-MeSATP at a concentration of 10 µM. Cells were treated with neomycin (500 µM) for 15 min before and during application of the drugs. The holding potential here was +30 mV, because fura 2-loaded cells were not stabilized at a holding potential of +60 mV. In the accompanying graph (D), each value (±SD) represents the mean from 7-10 independent experiments. **P < 0.01, unpaired t-test.

GDPS (100 µM) inhibited the rise in intracellular Ca²⁺ induced by all the agonists (Fig. 3, B and D), suggesting that intracellular Ca²⁺ mobilization was regulated by activation of a G-protein. The increase in [Ca²⁺]_i induced by UTP was blocked either by neomycin (500 µM) or by the inositol 1,4,5-trisphosphate (IP₃) receptor antagonist, heparin (1 mg/ml), indicating that the receptor for UTP was linked to a G-protein involving phospholipase C stimulation; hydrolysis of phosphatidylinositol produces IP₃, to cause cytosolic Ca²⁺ release, and diacylglycerol, to activate PKC. In contrast, intracellular Ca²⁺ rise induced by ATP and 2-MeSATP was not affected by neomycin or heparin (Fig. 3, B and D), suggesting that ATP and 2-MeSATP elevated [Ca²⁺]_i by a mechanism independent of phospholipase C stimulation and IP₃ production.

Single-channel currents elicited by ATP, UTP, and 2-MeSATP

ATP, UTP, and 2-MeSATP, when added to the patch electrode-filling solution, elicited single-channel K⁺ currents in the cell-attached patch-clamp configuration (data not shown). Likewise, bath application of these agonists outside the patch pipette also induced single-channel currents (Fig. 4A). No current was evoked in the presence of tetraethylammonium (1 mM) as observed in whole cell currents (data not shown). UTP-induced currents showed a larger slope conductance (96 ± 9 pS), whereas 2-MeSATP evoked currents with the lower slope conductance (85 ± 7 pS; Fig. 4A). ATP-induced currents had two major classes of the slope conductance (86 ± 8 pS and 95 ± 9 pS; Fig. 4A), each of which was consistent with slope conductances for 2-MeSATP- or UTP-induced currents, respectively.

View larger version (26K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   FIG. 4. Single-channel recordings. Cell-attached patches were made to cells using the patch electrode-filling solution without ATP, UTP, or 2-MeSATP. Each of the agonists (10 µM) or 12-O-tetradecanoyl phorbol-13-acetate (TPA; 50 nM) was applied to a single neuron outside the patch pipette with 5-min intervals, in the presence and absence of GF109203X (500 nM; A). The pipette potential, which represents the voltage loaded on the inside membrane, was +100 mV. a and b, single-channel currents elicited by ATP in the absence; and presence of GF109203X (c); d, single-channel currents elicited by UTP; e, single-channel currents elicited by 2-MeSATP in the absence; and presence of GF109203X (f); g, single-channel currents elicited by TPA. B: ATP, UTP, or 2-MeSATP at a concentration of 10 µM was applied to a single patched cell in the outside-out patch-clamp configuration. GDPS (100 µM) or GTPS (500 µM) was perfused in the patch electrode-filling solution. Holding potential was +60 mV. The single-channel current-voltage (I-V) relations were plotted, and the slope conductances were measured by linear regression fitted to the I-V relation (n = 8-10).

GF109203X (500 nM) fully inhibited UTP-evoked currents (Fig. 4A), suggesting that UTP regulated the potassium channels as a result of PKC activation. In contrast, 2-MeSATP-evoked currents were not affected by GF109203X (Fig. 4A), suggesting that 2-MeSATP elicited the currents by a mechanism independent of PKC activation. Of ATP-induced currents, only the currents with the higher slope conductance were blocked by GF109203X (Fig. 4A), suggesting that ATP evoked K⁺ current in part by PKC activation. The potent PKC activator, TPA (50 nM) induced single-channel currents with the same level of the slope conductance (95 ± 7 pS) as that achieved by UTP, ~1 min after application outside the patch pipette, and the current was blocked by GF109203X (Fig. 4A), further supporting the idea that the UTP receptor regulates potassium channels by interacting with a PKC pathway.

ATP and 2-MeSATP elicited single-channel currents with a slope conductance of 87 ± 6 pS and 88 ± 9 pS, respectively, without latency in the outside-out patch-clamp configuration, although no current was induced by UTP (Fig. 4B). The currents induced by ATP or 2-MeSATP were completely inhibited by GDPS (100 µM; Fig. 4B) and otherwise, the G-protein activator, GTPS (500 µM), produced single-channel currents with the same level of the slope conductance (88 ± 5 pS) as that achieved by ATP or 2-MeSATP (Fig. 4B). These results suggest that 2-MeSATP activates potassium channels directly by G-proteins and that ATP evokes potassium currents both by the direct action of G-protein subunits and by PKC activation.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

In the present study, the P₂ purinoceptor agonists, ATP, UTP, and 2-MeSATP, evoked potassium currents and increased intracellular calcium concentration in rat cerebral cortical neurons. One assumes that the potassium channels might be activated by calcium. Two kinds of Ca²⁺-dependent potassium channels, such as voltage-dependent BK with high single-channel conductance of 100-250 pS and voltage-independent SK channels with low single-channel conductance of 4-14 pS, have been found in neurons (Hille 1992). The findings that the currents by ATP, UTP, and 2-MeSATP were sensitive to tetraethylammonium and that the channels had conductances of 85-95 pS suggest that the relevant channel was a BK type. However, this is unlikely, because these agonists produced potassium currents under conditions of zero intracellular Ca²⁺; the currents were not affected by intracellular calcium concentrations; and the channels showed no voltage dependency. If depolarizing pulses were applied to cells, then the Ca²⁺- and voltage-dependent potassium currents might be induced also here, as observed in cat cortical neurons (Schwindt et al. 1992).

Receptors for ATP, UTP, and 2-MeSATP were linked to PTX-insensitive G-proteins; however, the responses could not be explained by activation of a single receptor. UTP did not activate potassium channels in excised patches, indicating that the UTP responses require intracellular second messengers. The finding that UTP-induced currents were blocked by neomycin or GF109203X supports this idea; the UTP receptor is linked to a G-protein involving phospholipase C-mediated PKC activation. 2-MeSATP-induced currents otherwise were not affected by neomycin or GF109203X, suggesting that the 2-MeSATP receptor is not coupled to a phospholipase C pathway. 2-MeSATP elicited single-channel currents in excised patches; the currents were blocked by GDPS, whereas GTPS produced currents in a fashion that mimics the effect of 2-MeSATP, indicating that 2-MeSATP-operated potassium channels are activated by a G-protein alone. The K_ACh channel (GIRK1) (Breitwieser and Szabo 1985; Clapham and Neer 1993; Ito et al. 1992; Kubo et al. 1993; Pfaffinger et al. 1985; Takao et al. 1994), K_ATP channel (ROMK1) (Ho et al. 1993; Kirsch et al. 1990), L-type Ca²⁺ channel (Yatani and Brown 1989), and I_f channel (Yatani et al. 1990) are recognized to be activated directly by the -subunits of G-proteins. Taken together, 2-MeSATP may activate potassium channels by a direct action of G-protein dimer. ATP-evoked currents were partially inhibited by either neomycin or GF109203X. ATP elicited single-channel currents in cell-attached patches with two major classes of the slope conductance, and GF109203X blocked currents with the higher slope conductance, which is in good agreement with that achieved by UTP. In addition, ATP induced currents with the same slope conductance as 2-MeSATP induced in outside-out patches. These findings suggest that the ATP responses are mediated by both of the UTP and 2-MeSATP receptor.

UTP increased [Ca²⁺]_i, which originated from intracellular calcium stores, and the increase was blocked by either neomycin or heparin, further supporting the idea that the UTP receptor is linked to a G-protein involving phospholipase C activation. G_q- and G_i/G_o-proteins are known to be coupled to phospholipase C₂ (Wu et al. 1993) and phospholipase A₂ (Leurs et al. 1994), respectively. The finding that the UTP response was blocked by the broad G-protein inhibitor GDPS but not by the G_i/G_o protein inhibitor PTX strongly suggests that the corresponding G-protein belongs to the class of G_q proteins.

The increases in [Ca²⁺]_i induced by 2-MeSATP were not affected by either neomycin or heparin, suggesting that 2-MeSATP elevates [Ca²⁺]_i by a mechanism independent of phospholipase C activation and IP₃ production. In addition to IP₃ receptors, the ryanodine receptors are major intracellular Ca²⁺ channels (Berridge 1993). Moreover, recent studies suggest that cyclic ADP-ribose releases cytosolic Ca²⁺ by a mechanism independent of IP₃ receptors (Berridge 1993; Galione 1993). It is presently unknown whether the 2-MeSATP-induced intracellular Ca²⁺ rise is due to activation of ryanodine receptors or a third intracellular Ca²⁺ pool related to cADP-ribose. Alternatively, another possible explanation for intracellular calcium mobilization via the 2-MeSATP receptor involves a direct activation by the -subunits of a G-protein. To address this question, we are currently carrying out further examinations.

The rise in intracellular Ca²⁺ induced by ATP was not affected by either neomycin or heparin, whereas the currents were partially inhibited by neomycin or GF109203X. This suggests that, whereas PKC is activated by ATP enough to produce K⁺ currents, IP₃ is not produced enough to release stored calcium and that, therefore, ATP-induced cytosolic Ca²⁺ release is mainly mediated by the 2-MeSATP receptor.

In conclusion, the results presented here demonstrate that at least two different types of the P_2Y purinoceptors, a UTP and 2-MeSATP receptor, linked to a PTX-insensitive G-protein, are expressed in rat cerebral cortical neurons. The UTP receptor is involved in stimulation of phospholipase C, leading to activation of the potassium channels and increase in [Ca²⁺]_i (Fig. 5). The 2-MeSATP receptor exerts activation of the potassium channels, possibly by a direct action by the -subunits of a G-protein, whereas enhancement in [Ca²⁺]_i occurs by a mechanism independent of phospholipase C stimulation (Fig. 5). The ATP responses may be mediated by both receptors for UTP and 2-MeSATP (Fig. 5).

View larger version (23K): [in this window] [in a new window]   FIG. 5. Schematic diagram of intracellular signal transduction pathways mediated by the UTP and 2-MeSATP receptor. 2-MeSATPR, 2-MeSATP receptor; UTPR, UTP receptor; PLC, phospholipase C; (), inhibition.

	FOOTNOTES

  Address for reprint requests: T. Nishizaki, Dept. of Physiology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

  Received 18 September 1997; accepted in final form 6 February 1998.

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