Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Rat superior cervical ganglion (SCG) neurons possess at least two different types of nucleotide receptors that are both excitatory and thus trigger noradrenaline release (Boehm, 1994; Boehm et al., 1995). One of these receptors is a ligand-gated ion channel that is activated by adenine nucleotides (i.e., a P2X purinoceptor; Boehm, 1999). The other receptor is activated by UTP and UDP and is metabotropic rather than ionotropic (Boehm et al., 1995; Boehm and Huck, 1997a). Similar results have been obtained in neurons from thoracolumbal paravertebral sympathetic ganglia of the rat, but the signaling mechanisms underlying the secretagogue action of uridine nucleotides remained elusive (von Kügelgen et al., 1999). Recently, uridine nucleotide-sensitive P2Y receptors of rat SCG neurons were found to inhibit selectively M-type K⁺ (K_M) channels (Boehm, 1998). The P2 receptors mediating the induction of transmitter release, on the one hand, and the inhibition of K_M channels, on the other hand, both displayed pharmacological characteristics suggestive of a role of P2Y6-like receptors: 1) UDP and UTP were equipotent agonists, even when interconversion was prevented by hexokinase; 2) the receptors were insensitive to the P2 antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) at 10 µM but blocked by reactive blue 2 at this concentration; and 3) the uridine-nucleotide sensitive receptors showed homologous desensitization but no cross-desensitization with ATP (Boehm et al., 1995; Boehm, 1998). We therefore assumed that the inhibition of K_M channels mediated the secretagogue action of uridine nucleotides. Two independent types of results also suggest that an inhibition of K_M channels may stimulate transmitter release from SCG neurons: 1) activation of B₂ bradykinin receptors inhibits K_M channels of SCG neurons via GTP binding proteins (Jones et al., 1995) and triggers tetrodotoxin (TTX)-sensitive noradrenaline release (Boehm and Huck, 1997b), as do UDP and UTP (Boehm et al., 1995); and 2) direct blockade of K_M channels by either Ba²⁺ or linopirdine also elicits TTX-sensitive transmitter release in these neurons (Kristufek et al., 1999).

Apart from uridine nucleotide-sensitive P2Y receptors (Boehm, 1998) and B₂ bradykinin receptors (Jones et al., 1995), M₁ muscarinic receptors cause, on activation, an inhibition of K_M channels (Marrion et al., 1989; Bernheim et al., 1992). However, most recently, B₂ and M₁ receptors were found to use different signaling pathways that finally lead to the closure of K_M channels: The B₂ receptor activates phospholipase C to cause liberation of Ca²⁺ from inositol trisphosphate (IP₃)-sensitive Ca²⁺ stores (Cruzblanca et al., 1998), and cytosolic Ca²⁺ at low micromolar concentrations is known to block K_M channels (Selyanko and Brown, 1996). The M₁ receptor, in contrast, was reported to inhibit K_M channels independent of phospholipase C and IP₃-sensitive Ca²⁺ stores (Cruzblanca et al., 1998; del Rio et al., 1999). The mechanisms by which UTP-sensitive P2Y receptors of SCG neurons cause inhibition of K_M channels are unknown (Boehm, 1998). In heterologous expression systems, all known subtypes of P2Y receptors do couple to phospholipase C and thus cause formation of IP₃ and a resulting increase in intracellular Ca²⁺ (Harden et al., 1995; North and Barnard, 1997; King et al., 1998). Here, we investigate the cellular mechanisms that link the uridine nucleotide-sensitive P2Y receptors to K_M channels, on one hand, and to transmitter release, on the other hand. The experiments focus on the role of the phospholipase C-dependent signaling cascade in the two types of UTP-dependent effects in SCG neurons and suggest that the induction of transmitter release and the inhibition of K_M channels are largely independent phenomena.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Cell Culture. Primary cultures of neurons dissociated from SCG of neonatal rats were prepared as previously described in more detail (Boehm, 1994). Briefly, ganglia were dissected from 2- to 6-day-old Sprague-Dawley rat pups, cut into three or four pieces, and incubated in collagenase (no. 9891, 1.5 mg/ml; Sigma Chemical Co., St. Louis, MO) and dispase (no. 165859, 3.0 mg/ml; Boehringer-Mannheim, Indianapolis, IN) for 20 min at 36°C. Subsequently, the ganglia were trypsinized (no. 3703, 0.25% trypsin; Worthington Biochemicals, Freehold, NJ) for 15 min at 36°C, dissociated by trituration, and plated onto 5-mm disks (about 40,000 cells/disk) coated with rat tail collagen (Biomedical Technologies. Cambridge, MA) for superfusion experiments and onto 35-mm culture dishes (no. 153066; Nunc, Naperville, CT) coated with poly(D-lysine) (25 mg/l; Sigma Chemical Co.) for electrophysiological experiments. For the determination of cellular inositol phosphates, cells were plated onto 24 multiwell plates (200,000 cells/well; Nunc) coated with poly(D-lysine) as above. About 50% of the cells in this culture system are neurons; the remainder are provided by non-neural cells, including primarily fibroblasts and glial cells.

Electrophysiology. Electrophysiological experiments were carried out at room temperature (20-24°C) on the somata of isolated neurons using a List EPC-7 amplifier (List Medical, Darmstadt, Germany) and pClamp 6.0 hardware and software (Axon Instruments, Foster City, CA). Unless stated otherwise, currents through K_M channels (I_M) were recorded in the amphotericin perforated-patch configuration (Rae et al., 1991), which prevents rundown of I_M (see Boehm, 1998). Patch pipettes were pulled (Flaming-Brown puller; Sutter Instruments, Novato, CA) from borosilicate glass capillaries (Science Products, Frankfurt/Main, Germany) and front-filled with a solution consisting of 75 mM K₂SO₄, 55 mM KCl, 8 mM MgCl₂, and 10 mM HEPES, adjusted to pH 7.3 with KOH. Then, electrodes were back-filled with the same solution containing 200 µg/ml amphotericin B (in 0.8% DMSO), which yielded tip resistance values of 1 to 3 M. To apply the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to the cytosol of the neurons under investigation, I_M was also recorded in the conventional (open-tip) whole-cell configuration (Hamill et al., 1981) of the patch-clamp technique with an intracellular (pipette) solution containing 120 mM K-aspartate, 30 mM KCl, 3.18 mM CaCl₂, 5 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, and 2 mM Li-GTP, adjusted to pH 6.8 with NaOH. Where indicated, 20 mM KCl was replaced by K₄-BAPTA, which reduces the calculated free Ca²⁺ concentration from 1.4 to 0.02 µM.

Measurement of [³H]Noradrenaline Release. [³H]Noradrenaline uptake and superfusion were performed as described previously (Boehm, 1994). Cultures were labeled with 0.05 µM [³H]noradrenaline (specific activity, 71.7 Ci/mmol) in culture medium supplemented with 1 mM ascorbic acid at 36°C for 1 h. After labeling, culture disks were transferred to small chambers and superfused with a buffer containing (mM) NaCl (120 mM), KCl (6.0 mM), CaCl₂ (2.0 mM), MgCl₂ (2.0 mM), glucose (20 mM), HEPES (10 mM), fumaric acid (0.5 mM), Na-pyruvate (5.0 mM), ascorbic acid (0.57 mM), adjusted to pH 7.4 with NaOH. Superfusion was performed at 25°C at a rate of about 1.0 ml min^-1. Collection of 4-min superfusate fractions was started after a 60-min washout period. When appropriate, thapsigargin (0.3 µM) was included in the superfusion buffer from minute 50 onwards (i.e., 22 min before the application of UTP). [³H] overflow was first induced by inclusion of UTP (10 µM) in the medium from minutes 72 to 74 of superfusion and then by electrical field stimulation (36 monophasic rectangular pulses, 0.5 ms, 0.3 Hz, 50 mA, 50 V cm^-1) from minutes 92 to 94. At the end of experiments, radioactivity remaining in the cultures was extracted by immersion of the disks in 1.2 ml of 2% (v/v) perchloric acid, followed by sonication. Radioactivity in extracts and collected fractions was determined by liquid scintillation counting (Tri-Carb 2100 TR; Packard). Radioactivity released in response to electrical field stimulation from rat sympathetic neurons after labeling with tritiated noradrenaline under conditions similar to those of the present study had previously been shown to consist predominantly of the authentic transmitter and to contain only small amounts (15%) of metabolites (Schwartz and Malik, 1993). Hence, the outflow of tritium measured in this study was assumed to reflect the release of noradrenaline and not that of metabolites.

Measurement of IP₃. Measurement of inositol polyphosphate formation was determined as described previously (Nanoff et al., 1990). Briefly, SCG cultures were prelabeled with 7 µCi/ml of myo-[1,2-³H]inositol in serum-free culture medium (see earlier) for 24 h. At 30 min before the stimulation with various receptor agonists, the cells were incubated in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH 7.4) containing 0.2% BSA and 10 mM LiCl. The cells were stimulated with nucleotides, bradykinin, or oxotremorine M in PBS containing 10 mM LiCl for various periods of time, and the incubations were terminated by replacing the buffer with 0.4 ml of 5% trichloroacetic acid. Extracts were collected, and the trichloroacetic acid was removed by washing twice with 4 volumes of water-saturated diethyl ether. The samples were neutralized with 20 mM Tris base and placed on a Dowex AG 1X8 column. Fractions containing inositol, inositol monophosphate, inositol diphosphates, and inositol trisphosphates, respectively, were sequentially eluted (see Nanoff et al., 1991) and probed for their radioactive contents by liquid scintillation counting. The radioactivity in the IP₃ fraction was expressed as a percentage of the radioactivity in the inositol fraction.

Statistical Analysis. All data are given as arithmetic mean ± S.E. (n = number of cell culture disks in release experiments, of multiwell cultures in inositol trisphosphate experiments, and of single cells in electrophysiological recordings). Differences between single data points were evaluated by the Mann-Whitney test.

Materials. ()-[ring-2,5,6-³H]Noradrenaline and myo-[1,2-³H]inositol were obtained from NEN (Dreieich, Germany). Bradykinin, Na-UTP, amphotericin B, and TTX were purchased from Sigma (Vienna, Austria). Oxotremorine M, 1-[6-[[(17)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), 1-[6-[[(17)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrollidinedione (U73343), and thapsigargin were obtained from Research Biochemicals Inc. (Natick, MA). BAPTA acetoxymethyl ester (BAPTA-AM) was purchased from Molecular Probes (Eugene, OR). Xestospongin C was obtained from Calbiochem (Bad Soden, Germany). U73122, U73343, xestospongin C, and thapsigargin were first dissolved in DMSO and then diluted into buffer to yield DMSO concentrations of 0.1%. At this concentration, DMSO does not affect any of the parameters investigated.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effects of Cholera Toxin and PTX on UTP-Induced [³H]Noradrenaline Release and Inhibition of I_M. To obtain insight into the types of GTP binding proteins involved in the actions of UTP in SCG neurons, cultures were treated with either PTX or cholera toxin (both 100 ng/ml for 24 h). Although PTX prevents the signaling of G_i- and/or G_o-coupled receptors in SCG neurons (e.g., Freissmuth et al., 1996), cholera toxin has been shown to down-regulate G_s in sympathetic neurons (Boehm et al., 1996). After toxin treatment, cultures were loaded with [³H]noradrenaline to determine the outflow of radioactivity as a measure of transmitter release or I_M was recorded (Fig. 1). UTP-evoked tritium overflow was reduced by 65.8 ± 5.5% (n = 12) after PTX treatment compared with nontreated cultures (Fig. 1, A and B). This effect was specific for the secretagogue action of UTP, because electrically evoked tritium overflow was not altered. The cholera toxin treatment, in contrast, did not affect UTP-evoked overflow but reduced electrically induced overflow, as described previously (Boehm et al., 1996).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effects of cholera toxin and PTX on UTP-induced [3H]noradrenaline release and inhibition of IM. A and B, neurons, either untreated or treated with PTX or cholera toxin (ChTX) (each 100 ng/ml for 24 h), were labeled with [3H]noradrenaline, superfused, and exposed to 10 µM UTP or to electrical field stimulation (EFS; each for 2 min). A, time course of [3H] outflow (n = 3). B, results obtained in 12 cultures by showing UTP and electrically evoked overflow as percent of cellular radioactivity. *P < .05 and **P < .01 versus results obtained in untreated cultures. C, IM was measured by the amphotericin B-perforated patch technique in an untreated neuron (top traces) and in a neuron treated with PTX (as above; bottom traces). The current traces shown were obtained by clamping the cells at 30 mV and by applying 1-s hyperpolarizing voltage steps to 55 mV; the recordings were performed before (control), during (UTP), and after (washout) the application of 10 µM UTP. D, inhibitory action of 10 µM UTP on IM determined in five or six neurons, either untreated (open columns) or treated with PTX (filled columns) or cholera toxin (hatched columns) (as above), respectively.

Time Course of UTP-Induced Accumulation of IP₃ and Inhibition of I_M. To obtain further insight into the mechanisms underlying the UTP-induced inhibition of I_M, we first investigated the time course of this effect. I_M relaxation amplitudes were slowly reduced when UTP (10 µM) was present for 10 to 60 s, and the effect reached a maximum after about 30 s (Fig. 2A). The B₂ and M₁ receptor-dependent inhibition of I_M in SCG neurons involves G_q and/or G₁₁ subunits (Jones et al., 1995; Haley et al., 1998), proteins that are commonly linked to phospholipase C (Exton, 1996). Therefore, we investigated whether UTP might cause an accumulation of IP₃ in cultures of SCG neurons and compared the time course of this effect with the time course of I_M inhibition. As shown in Fig. 2B, the UTP-induced IP₃ production showed a delayed onset and reached a maximum after 30 s. Thus, the accumulation of IP₃ and the inhibition of I_M by UTP appeared to occur in parallel.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Time course of the UTP-induced inhibition of IM and accumulation of IP3. A, time dependence of the inhibition of IM relaxation amplitudes (measured as shown in Fig. 1C) by 10 µM UTP applied for the indicated periods of time (n = 4; *P < .05 versus values obtained in the absence of UTP). B, time dependence of the generation of IP3 by 10 µM UTP applied for the indicated periods of time (n = 4); radioactivity in the IP3-fraction is depicted as percentage of the radioactivity in the fraction of inositol (*P < .05 versus values obtained in the absence of UTP).

Accumulation of IP₃ in Presence of Nucleotides: Comparison with Bradykinin and Oxotremorine M. The P2Y receptors of SCG neurons that mediate the inhibition of I_M are activated by UTP, UDP, and ADP but not by ATP (Boehm, 1998). We therefore tested these nucleotides for their capacity to cause an accumulation of IP₃. At concentrations that cause maximal inhibition of I_M (Boehm, 1998), 10 and 100 µM, all of these nucleotides were able to raise the levels of cellular IP₃. However, in comparison with bradykinin (1 µM) and oxotremorine M (10 µM), the nucleotides caused only modest increases in IP₃. As expected, the accumulation of IP₃ in the presence of UTP or oxotremorine M was not altered when cultures had been treated with PTX. However, the UTP- and bradykinin-evoked increases in IP₃ were abolished or largely reduced in the presence of the phospholipase C inhibitor U73122 (1 µM; Jin et al., 1994).

Effects of a Phospholipase C Inhibitor on Reduction of I_M by UTP, Bradykinin, and Oxotremorine M. The results shown earlier suggested that the inhibition of I_M by UTP may involve a phospholipase C-mediated generation of IP₃. To corroborate this assumption, neurons were treated with U73122, and the effect of UTP on I_M was tested. Before the application of this phospholipase C inhibitor, the neurons under investigation showed a clear-cut inhibition of I_M by UTP (10 µM; 39.9 ± 7.5% inhibition; n = 6), bradykinin (1 µM; 55.3 ± 12.0% inhibition; n = 5), and oxotremorine M (10 µM; 79.7 ± 7.5% inhibition; n = 6). However, when these neurons had been treated with U73122 (1 µM) for 15 min, the subsequent application of UTP (3.5 ± 3.9% inhibition) and bradykinin (5.5 ± 5.5% inhibition) failed to cause significant alterations in I_M relaxations, whereas oxotremorine M (51.6 ± 7.9% inhibition) still caused a significant reduction (Fig. 3, A and C). Furthermore, the effect of U73122 on the receptor-dependent modulation of I_M was irreversible for up to 60 min (Fig. 3B). To exclude the possibility that U73122 had abolished the effects of UTP and bradykinin on I_M by some unspecific action, an isomer that lacks the inhibitory effect on phospholipase C, U73343, was tested (Jin et al., 1994). This agent failed to alter the inhibition of I_M by any of the agonists used (Fig. 3C).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Accumulation of IP3 in the presence of nucleotides: comparison with bradykinin and oxotremorine M. Generation of IP3 in response to UDP, UTP, ADP, ATP, bradykinin (Bk), and oxotremorine M (OxoM), each applied for 60 s, in untreated cultures (control), in cultures treated with PTX (100 ng/ml for 24 h), and in the presence of 1 µM concentration of the phospholipase C inhibitor U73122. The numbers of cultures investigated are given in the columns. Radioactivity in the IP3 fraction was calculated as a percentage of the radioactivity in the fraction of inositol; values obtained in the presence of agonists are expressed as a percentage of the values obtained in their absence within the same preparation. In untreated and in PTX-treated cultures, all values obtained in the presence of agonists were significantly different from those obtained in their absence (*P < .05 versus the effects obtained with the same agonist in the absence of U73122).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effects of a phospholipase C inhibitor on the reduction of IM by UTP, bradykinin, and oxotre-morine M. A, effect of 10 µM UTP on IM in a SCG neuron was investigated before and after a 15-min application of 1 µM concentration of the phospholipase C inhibitor U73122. The current traces shown were elicited by 1-s hyperpolarizations from 30 to 55 mV and were obtained before (control), during (UTP), and after (washout) the application of 10 µM UTP. B, time course of IM relaxation amplitudes in an additional SCG neuron. Note that 1 µM U73122 irreversibly abolished the inhibitory actions of UTP (10 µM) and bradykinin (Bk, 1 µM). C, summary of the effects of the active phospholipase C inhibitor U73122 and of the less active isomer U73343 (both at 1 µM) on the inhibitory actions of UTP (U, 10 µM), bradykinin (B, 1 µM), and oxotremorine M (O, 10 µM) on IM; experiments were performed as shown in B (n = 5 to 6; *P < .05 between the effects before and after application of 1 µM U73122 for 15 min).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effects of an IP3 antagonist on the reduction of IM by UTP, bradykinin, and oxotremorine M. A, effects of 10 µM UTP and 10 µM oxotremorine M (OxoM) on IM in a SCG neuron were investigated before and after a 10-min application of 10 µM concentration of the IP3 antagonist xestospongin C; the graph shows the time course of IM relaxation amplitudes. B, effect of 10 µM UTP on IM was investigated in a SCG neuron incubated for 30 min in 0.1% DMSO (top) and then in a neuron in the same culture dish incubated for 30 min in 10 µM xestospongin C (bottom). The current traces shown were elicited by 1-s hyperpolarizations from 30 to 55 mV and were obtained before (control), during (UTP), and after (washout) the application of 10 µM UTP. C, summary of the effects of incubation of neurons in either 10 µM xestospongin C or 0.1% DMSO, both for 30 min, on the inhibitory actions of UTP (10 µM), bradykinin (Bk, 1 µM), and oxotremorine M (OxoM, 10 µM) on IM; experiments were performed as shown in B (n = 4 or 5; ** P < .01 versus the inhibition of IM in neurons treated with DMSO).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effects of Ca2+ chelators and Ca2+-ATPase inhibition on the reduction of IM by UTP. A, effects of 10 µM UTP and 1 µM bradykinin (Bk) on IM in a SCG neuron were investigated before and after a 15-min application of 1 µM concentration of the Ca2+-ATPase inhibitor thapsigargin. The current traces shown were elicited by 1-s hyperpolarizations from 30 to 55 mV and were obtained before (control), during (UTP, Bk), and after (washout) the application of 10 µM UTP and 1 µM bradykinin, respectively. B, effects of thapsigargin (1 µM) on the inhibitory actions of UTP (10 µM), bradykinin (Bk, 1 µM), and oxotremorine M (OxoM, 10 µM) on IM; the effects of receptor agonists were tested before and after a 15-min application of 1 µM thapsigargin (n = 5 or 6; *P < .05 versus the inhibition of IM before the application of thapsigargin). C, effects of UTP and oxotremorine M (OxoM; both at 10 µM) were investigated in neurons incubated in 3 µM BAPTA-AM for 30 min, followed by a 30-min incubation in regular buffer, and were compared with those obtained in control neurons (n = 4 to 6; *P < .05 versus the inhibition of IM in neurons not treated with BAPTA-AM). D, effects of UTP (10 µM) on IM were investigated in the perforated-patch configuration in the absence and presence extracellular Ca2+ (extra) and in the conventional whole-cell configuration with 20 mM KCl or 20 mM BAPTA added to the pipette solution (intra; n = 4; *P < .05 versus the inhibition of IM in neurons dialyzed with pipette solution containing KCl instead of BAPTA).

Effects of an IP₃ Antagonist on Reduction of I_M by UTP, Bradykinin, and Oxotremorine M. IP₃ elicits cellular effects by activating IP₃ receptors located at the endoplasmic reticulum (Wilcox et al., 1998). Xestospongins are cell-permeable antagonists at these receptors, with xestospongin C being the most potent isomer (Gafni et al., 1997). When a neuron that displayed a 54% reduction of I_M relaxations in the presence of UTP (10 µM) was superfused with 10 µM xestospongin C for 10 min, the inhibition of I_M by UTP was reduced to 35%. In contrast, the inhibition of I_M by oxotremorine M amounted to 69% before and to 78% after the application of xestospongin C (Fig. 5A). Xestospongin C has been reported to block IP₃-mediated responses in intact PC12 cells more efficiently after prolonged periods of application (Gafni et al., 1997). Therefore, SCG neurons were first incubated in vehicle (0.1% DMSO) for 30 min, and the effects of UTP (10 µM), bradykinin (1 µM), and oxotremorine M (10 µM) on I_M were investigated. Thereafter, neurons in the very same culture dish were exposed to 10 µM xestospongin C again for 30 min, and the three receptor agonists were applied. When the results obtained in neurons treated with xestospongin C were compared with those obtained in neurons treated with vehicle, the inhibitory actions of UTP and bradykinin were reduced from 44.2 ± 7.4 to 8.9 ± 2.7% and from 58.3 ± 12.9 to 9.7 ± 5.8% inhibition, respectively. In contrast, the effect of oxotremorine M was not altered (Fig. 5, B and C). Hence, the IP₃ antagonist selectively counteracted the inhibition of I_M by UTP and bradykinin.

Effects of a Ca²⁺ ATPase Inhibitor on UTP-Induced [³H]Noradrenaline Release and Inhibition of I_M. Activation of IP₃ receptors results in liberation of Ca²⁺ from the endoplasmic reticulum into the cytosol (Wilcox et al., 1998). To find out whether intracellular Ca²⁺ stores are required for the inhibition of I_M by UTP, neurons were treated with the Ca²⁺-ATPase inhibitor thapsigargin, which had been shown previously to entirely deplete Ca²⁺ stores in sympathetic neurons at a concentration of 0.1 µM (Foucart et al., 1995). Before the application of thapsigargin, I_M was reduced by UTP (10 µM), bradykinin (1 µM), and oxotremorine M (10 µM) by 36.1 ± 6.5% (n = 5), 67.7 ± 7.7% (n = 6), and 82.9 ± 6.4% (n = 5), respectively. When neurons had been treated with thapsigargin (1 µM for 15 min) and the receptor agonists were applied again in the continuing presence of the Ca²⁺-ATPase inhibitor, the inhibitory actions of UTP (9.8 ± 3.0% inhibition) and bradykinin (6.7 ± 2.8% inhibition) were reduced, whereas the inhibition by oxotremorine M (61.1 ± 8.1%) was not significantly altered (Fig. 6, A and B).

Effects of Ca²⁺ Chelators on Reduction of I_M by UTP and Oxotremorine M. To reveal whether increases in cytosolic Ca²⁺ concentrations are required for the inhibition of I_M by UTP, neurons were incubated in 3 µM concentration of the cell-permeable Ca²⁺ chelator BAPTA-AM for 30 min, followed by an incubation in regular bathing solution. In these neurons, UTP (10 µM) did not affect I_M relaxations (2.2 ± 4.0% inhibition, n = 6), whereas in sister cultures not treated with BAPTA-AM the inhibition by UTP amounted to 31.5 ± 5.4% (n = 6; P < .05). In contrast, oxotremorine M (10 µM) reduced I_M in neurons treated with BAPTA-AM (85.4 ± 3.0% inhibition; n = 4) to the same extent as in nontreated neurons (88.7 ± 2.7% inhibition; n = 5; Fig. 6C). To corroborate a role of increases in intracellular Ca²⁺ in the UTP-dependent inhibition of I_M, currents were determined in the open-tip whole-cell configuration of the patch-clamp technique with either 20 mM KCl or 20 mM K-BAPTA added to the pipette solution. With BAPTA in the recording electrode, UTP (10 µM) reduced I_M relaxations by 6.4 ± 9.3% (n = 4), with KCl instead of BAPTA, the inhibition amounted to 44.8 ± 11.4% (n = 4; P < .05; Fig. 6D).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In heterologous expression systems, all types of P2Y receptors couple to phospholipase C to generate inositol polyphosphates, which then liberate Ca²⁺ from intracellular stores. However, the signaling cascades of native P2Y receptors, in particular of those expressed in neurons, are less well characterized (Harden et al., 1995; North and Barnard, 1997; King et al., 1998). In rat SCG neurons, a P2Y6-like G protein-coupled nucleotide receptor mediates UTP-evoked transmitter release, on one hand, and UTP-dependent inhibition of K_M channels, on the other hand (Boehm et al., 1995; Boehm, 1998). The present results demonstrate that these two effects involve different signaling cascades, and only the latter action, the inhibition of K_M channels, involves an accumulation of IP₃ with resulting increases in cytosolic Ca²⁺.

Mechanisms of UTP-Dependent Inhibition of K_M Channels. Our results demonstrate that UTP inhibits K_M channels of SCG neurons via the signal transduction cascade: P2Y receptors  G_q/11  phospholipase C  IP₃  IP₃ receptor  Ca²⁺ release  K_M channel blockade. This conclusion is based on the following results: 1) the UTP-dependent inhibition of I_M was not altered by either PTX or cholera toxin. Hence, members of the toxin-insensitive family of G proteins (Fields and Casey, 1997) must have mediated the inhibition of I_M by UTP. Previously, q and/or 11 G protein subunits have been shown to be involved in receptor-dependent inhibition of I_M (Caulfield et al., 1994; Jones et al., 1995; Haley et al., 1998). 2) UTP as well as the other nucleotides tested induced the formation of IP₃, and this effect was not altered by PTX but abolished by the phospholipase C inhibitor U73122. The UTP-dependent generation of IP₃ and inhibition of I_M occurred in parallel. Furthermore, U73122 abolished the inhibition of I_M by UTP. This effect appeared specific for phospholipase C, because an isomer (U73343) that fails to block this enzyme (Jin et al., 1994) did not mimic the action of U73122. 3) Xestospongin C, a noncompetitive antagonist of IP₃ receptors (Gafni et al., 1997), largely reduced the inhibition of I_M by UTP, whereas the inhibition by oxotremorine M remained unaltered. This observation verifies that xestospongin C did not interfere with the receptor-mediated modulation of I_M by some unspecific effect. 4) Thapsigargin, which inhibits the endoplasmic Ca²⁺-ATPase (Thastrup et al., 1990) and thereby depletes intracellular Ca²⁺ stores in SCG neurons (Foucart et al., 1995), significantly attenuated the inhibitory actions of UTP on I_M. 5) Finally, intracellular application of the Ca²⁺ chelator BAPTA prevented the inhibition of I_M by UTP, which, however, was not altered by the removal of extracellular Ca²⁺. Hence, release of Ca²⁺ from intracellular stores into the cytosol, but not transmembrane Ca²⁺ entry, was involved in the UTP-induced inhibition of I_M. Cytosolic Ca²⁺ concentrations in the submicromolar to low micromolar range have been shown before to directly block K_M channels (Selyanko and Brown, 1996).

Mechanisms of UTP-Evoked Transmitter Release. The PTX treatment, which did not alter the UTP-induced inhibition of I_M or the accumulation of IP₃, clearly reduced the secretagogue action of UTP. Conversely, thapsigargin attenuated the UTP-induced inhibition of I_M but not UTP-evoked noradrenaline release. These results permit several conclusions: 1) UTP-evoked transmitter release involves G proteins other than those involved in the UTP-dependent inhibition of I_M, namely G_i and/or G_o; 2) intact intracellular Ca²⁺ stores are required for the inhibition of I_M, but not for the stimulation of noradrenaline release; and 3) as a consequence, the inhibition of I_M cannot be the major mechanism by which UTP depolarizes SCG neurons to finally evoke transmitter release. The reduction of UTP-evoked noradrenaline release by PTX was not complete but amounted to only 66%. Thus, PTX-insensitive G proteins are also involved in the secretagogue action of the uridine nucleotide. These G proteins cannot include G_s, because down-regulation of G_s by cholera toxin (Boehm et al., 1996) failed to affect the induction of transmitter release by UTP. Therefore, it appears likely that the toxin-insensitive G proteins that mediated the formation of IP₃ and the inhibition of I_M (i.e., G_q and/or G₁₁) also contributed to the secretagogue action of UTP. Direct K_M channel blockade by either Ba²⁺ or linopirdine has been found to trigger noradrenaline release from SCG neurons, although the secretagogue actions of these agents are much weaker than those of UTP (Kristufek et al., 1999). However, the lack of effect of thapsigargin on UTP-evoked noradrenaline release argues against an unequivocal role of K_M channel inhibition in the secretagogue action of the nucleotide. Therefore, additional G_q/G₁₁-mediated effects must be involved in the transmitter release stimulated by the activation of P2Y receptors. In accordance with this hypothesis, we found that the protein kinase C inhibitors bisindolylmaleimide I and staurosporine reduced the secretagogue action of UTP by 71 and 91%, respectively (Moskvina et al., 1999).