Introduction

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Voltage-gated Ca⁺⁺ channels mediate Ca⁺⁺ entry into neurons and regulate neurotransmitter release, firing patterns, gene expression and differentiation (McClesky, 1994; Olivera et al., 1994; Ghosh and Greenberg, 1995). Several types of Ca⁺⁺ channels have been identified with distinct electrophysiological and pharmacological properties (Zhang et al., 1993; Randall and Tsien, 1995). T channels activate at low voltage, inactivate quickly, and are blocked by low concentrations of Ni⁺⁺. L channels are activated by high voltage, inactivate slowly and are blocked by dihydropyridines. N, P and Q channels are activated by high voltage and blocked by the peptide neurotoxins -conotoxin GVIA (N), -agatoxin IVA (P and Q) and -conotoxin MVIIC (N, P and Q). Other channels have also been described that are resistant to organic Ca⁺⁺ channel blockers (G1-G3 and R-type channels) (Forti et al., 1994; Randall and Tsien, 1995).

Several manifestations of ethanol intoxication and dependence appear due to changes in Ca⁺⁺ channel function (Messing and Diamond, 1997). In nerve terminals from rat neurohypophysis and in NGF-differentiated PC12 cells, brief exposure to intoxicating concentrations (10-50 mM) of ethanol inhibits L-type channels by decreasing open channel probability (Wang et al., 1994) and promoting channel inactivation (Mullikin-Kilpatrick and Treistman, 1995). In N1E-115 neuroblastoma and NG108-15 neuroblastoma-glioma cells, high concentrations of ethanol (100-300 mM) reduce T-type currents by 15 to 20% (Twombly et al., 1990), and in rat neurohypophysis, 50 to 100 mM ethanol reduces N-type current by 30 to 40% (Wang et al., 1991). In rat Purkinje neurons P-type currents appear insensitive to ethanol (Hall et al., 1994). The effect of ethanol on other types of Ca⁺⁺ channels is not known.

Protein phosphorylation regulates ion channels (Nestler and Greengard, 1984) and ethanol can alter phosphorylation through actions on signal transduction pathways that regulate protein kinases, particularly cAMP-dependent PKA and PKC (Messing and Diamond, 1997). In some cells (Nagy et al., 1989; Rabin et al., 1993) ethanol stimulates cAMP formation and activates PKA. In rodent hepatocytes (Hoek et al., 1987) and human platelets (Rubin et al., 1988), ethanol activates phospholipase C to generate the PKC activator diacylglycerol (Nishizuka, 1992). PKA activation appears to be required for inhibition of adenosine transporters by ethanol (Coe et al., 1996), whereas PKC activation is important for inhibition of AMPA/kainate receptor currents (Dildy-Mayfield and Harris, 1995) and enhancement of mouse and bovine GABA_A receptor function (Wafford and Whiting, 1992) by ethanol. No studies have identified a role for phosphorylation in the regulation of voltage-gated Ca⁺⁺ channels by ethanol.

In this study, we examined the effect of ethanol on non-L-type Ca⁺⁺ channels, by measuring depolarization-induced rises in [Ca⁺⁺]_i in NGF-differentiated PC12 cells treated with the L channel blocker nifedipine. Our findings indicate that ethanol can inhibit N-type and P/Q-type channels by a mechanism that is antagonized by PKA.

	Methods

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Materials. trk-PC12 6-24 cells were a gift from David Kaplan, (Frederick Cancer Center, Frederick, MD). -agatoxin IVA was generously provided by Michael Adams (University of California, Riverside, CA), Research Biochemicals International as part of the Chemical Synthesis Program of the National Institute of Mental Health (Contract N01 MH3003), and by Pfizer (Groton, CT). Cyclosporin A was supplied by Sandoz Pharmaceuticals (East Hanover, NJ). ¹²⁵I--conotoxin GVIA was purchased from Amersham (Arlington Heights, IL) and unlabeled -conotoxin GVIA was obtained from Peptides International (Louisville, KY). NGF was purchased from Collaborative Biomedical Products (Bedford, MA). Okadaic acid (Na⁺-salt), Sp-cAMPS and deltamethrin were from LC Laboratories (Woburn, MA). Ionomycin and ryanodine were purchased from Calbiochem (San Diego, CA). Ouabain, monensin, poly-L-ornithine and laminin were from Sigma (St. Louis, MO). Fura-2 AM, fura pentapotassium salt and 4-bromo-A23187 were purchased from Molecular Probes (Eugene, OR).

Cell culture. trk-PC12 cells were maintained in plastic tissue culture flasks at 37°C in a humidified atmosphere of 90% air and 10% CO₂ in complete medium containing DMEM supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 2 mM glutamine, 50 U/ml of penicillin, 50 µg/ml of streptomycin and 200 µg/ml of G418 (geneticin). For Ca⁺⁺ imaging studies, 5 × 10⁵ cells were plated on 22-mm square glass coverslips (Warner Instruments, Hamden, CT) that had been treated for 30 min with 10% HCl in ethanol, washed in PBS, incubated with 0.1 mg/ml of poly-L-ornithine for 30 min, and then coated with laminin (30 µg/ml) overnight at 37°C. The cells were grown for 24 to 48 hr in complete medium supplemented with 50 ng/ml of NGF. After this time, approximately 85 to 90% of the cells had extended neurites more than one cell body in length.

-Conotoxin GVIA binding. Binding of ¹²⁵I--conotoxin GVIA to cells was measured by a modification of a previously described method (Williams et al., 1992). Cells (1.5 × 10⁵) were plated in poly-L-ornithine treated 24-well plates in complete medium. The after day cells were rinsed with buffer A containing 140 mM NaCl, 5 mM KCl, 12 mM glucose, 10 µM CaCl₂, 1 mg/ml of BSA and 10 mM HEPES (pH 7.4). Cells were then incubated in 0.4 ml of buffer A containing 80 pM ¹²⁵I--conotoxin GVIA for 1 hr at 37°C. This concentration was chosen because it approximates the K_d (60 pM) for equilibrium saturation binding of ¹²⁵I--conotoxin GVIA to brain membranes (Wagneret al., 1988). After aspiration of the buffer, the wells were rapidly rinsed four times with 1 ml of buffer B (160 mM choline chloride, 1.5 mM CaCl₂, 1 mg/ml of BSA and 5 mM HEPES, pH 7.4) at 4°C. After aspiration of all liquid, 1 ml of 1N NaOH was added to each well. Plates were incubated at 37°C overnight and radioactivity contained in 0.8 ml samples taken from each well was determined by liquid scintillation counting. Specific binding of ¹²⁵I--conotoxin GVIA was calculated as the difference between binding measured in the absence and the presence of 500 nM -conotoxin GVIA and accounted for 77 ± 2% of total binding. Protein concentrations were measured by the Lowry method with BSA standards (Lowry et al., 1951).

Measurement of intracellular Ca⁺⁺. Cells attached to coverslips were incubated in DMEM containing 25 mM HEPES (pH 7.4) and 5 µM fura-2 AM for 25 min at 37°C. Cells were rinsed twice with 5 mM KCl buffer (85 mM NaCl, 5 mM KCl, 45 mM choline Cl, 2 mM CaCl₂, 5 mM glucose, 25 mM HEPES, pH 7.4). The coverslip was mounted onto a perfusion chamber (model RC-21B, Warner Instruments, Hamden, CT) and perfused with 5 mM KCl buffer. All experiments were conducted at 27°C.

Addition of drugs. Because dihydropyridines preferentially bind to L channels on depolarized cells (Greenberg et al., 1986), we first incubated cells with 50 mM KCl buffer containing 1 µM nifedipine or 10 µM nimodipine. The 50 mM KCl buffer was identical in composition to 5 mM KCl buffer except that KCl was substituted for choline chloride. Cells were then incubated in 5 mM KCl buffer containing 1 µM nifedipine or 10 µM nimodipine for 5 min to allow [Ca⁺⁺]_i to return to resting levels. Cells were then depolarized in 50 mM KCl buffer in the continued presence of nifedipine or nimodipine, and fluorescence images were recorded. This predepolarization step resulted in maximal inhibition of subsequent depolarization-induced [Ca⁺⁺]_i rises by these dihydropyridines.

Analysis of data. Results are expressed as mean ± S.E. In most experiments control and treatment values were measured using the same cells. This was possible because the magnitude of the rise in [Ca⁺⁺]_i in cells treated with nifedipine alone was similar (97 ± 4%) for up to three successive depolarizations, when each was separated by at least 5 min. In contrast, repeated stimulation with bradykinin elicited progressively smaller Ca⁺⁺ rises when separated by 5-min intervals. Therefore, data from bradykinin experiments were derived from different batches of cells in control and ethanol-exposed conditions. Differences between means were analyzed by two-tailed Student's t tests or by ANOVA, and where P < .05, multiple comparisons were evaluated by the Scheffe F-test.

	Results

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Identification of Ca⁺⁺ channels in trk-PC12 cells. PC12 cells express N-type, L-type and P/Q-type channels, and treatment with NGF for several days increases expression of N-type channels (Plummer et al., 1989; Liu et al., 1996). To facilitate our studies of non-L-type channels, we used the PC12 clone trk-PC12 6-24, which overexpresses the tyrosine kinase NGF receptor trkA and undergoes rapid neuronal differentiation after brief NGF treatment (Hempstead et al., 1992). We found that binding of the N channel antagonist ¹²⁵I--conotoxin GVIA (80 pM) to undifferentiated trk-PC12 6-24 cells (13.44 ± 1.44 fmol/mg; n = 6) was 2.6-fold higher than binding to wild-type PC12 cells (5.14 ± 0.45 fmol/mg; n = 9; P < .05). Treatment with 50 ng/ml of NGF for 48 hr increased binding in trk-PC12 cells to 20.29 ± 0.98 fmol/mg (n = 4; P < .05 compared to untreated trk-PC12 cells). This indicates that N channel expression is markedly increased in this PC12 clone especially after treatment with NGF. Therefore we used NGF-differentiated trk-PC12 6-24 cells for our studies.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Multiple Ca++ channel subtypes mediate depolarization-evoked increases in [Ca++]i in trk-PC12 cells. A, Cells were depolarized in 50 mM KCl buffer without () or with () 1 µM nifedipine, () 5 mM EGTA or () 1 µM nifedipine plus 1 µM -conotoxin GVIA. Data are mean ± S.E. [Ca++]i values from 10 (), 60 (, ) or 120 () cells from 14 separate experiments. Perfusion with 50 mM KCl buffer was begun just after images were taken for the "0" time point. B, Cells treated with 1 µM nifedipine were depolarized in the presence 1 µM -conotoxin GVIA (Ctx), 300 nM -agatoxin IVA (Aga), a combination of -conotoxin GVIA and -agatoxin IVA(Ctx+Aga) or a combination of -conotoxin GVIA, -agatoxin IVA and 100 µM nickel (Ctx+Aga+Ni). Data are mean ± S.E. values for maximal increases in [Ca++]i from 68 (Ctx), 65 (Aga), 33 (Ctx+Aga) and 27 (Ctx+Aga+Ni) cells in three to four experiments and are expressed as percent of control values measured in the same cells depolarized without -conotoxin GVIA, -agatoxin IVA, or nickel. *P < .05 compared to Ctx or Aga alone, but not to Ctx+Aga+Ni (ANOVA).

Ethanol inhibits K⁺-evoked [Ca⁺⁺]_i rises. In depolarized, nifedipine-treated cells, ethanol inhibited the peak [Ca⁺⁺]_i rise, but had little effect on the plateau phase of the response (fig. 2A). Inhibition of the peak was concentration-dependent and was maximal with 50 mM ethanol (fig. 2B). The effect of ethanol was not immediate, but required at least 8 min of preincubation to develop completely (fig. 2C). After washout of ethanol, inhibition reversed slowly and only partially after 20 min (fig. 2C).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Inhibition of depolarization-evoked [Ca++]i rises by ethanol. A, Nifedipine-treated cells were preincubated for 8 min in 5 mM KCl buffer in the absence () or presence () of 50 mM ethanol, and then were depolarized in 50 mM KCl buffer in the continued absence or presence of ethanol. Perfusion with 50 mM KCl buffer was begun just after images were taken for the "0" time point. Data are mean ± S.E. [Ca++]i values from 48 cells in four experiments B, Nifedipine-treated cells were depolarized in the presence of 0 to 150 mM ethanol. Data are mean ± S.E. values for maximal increases in [Ca++]i from 26 to 45 cells in three to four experiments. C, Nifedipine-treated cells were incubated with 50 mM ethanol for the indicated times before depolarization with 50 mM KCl (). Some cells treated for 8 min with 50 mM ethanol before depolarization were then incubated for an additional 20 min in two exchanges of ethanol-free 5 mM KCl buffer before being depolarized a second time to measure [Ca++]i rises following washout of ethanol (). Data shown are mean ± S.E. values for maximal increases in [Ca++]i from 18 to 24 cells in three to four experiments. D, Nifedipine-treated cells were incubated with 50 mM ethanol in the absence (None) or presence of 1 µM -conotoxin GVIA (Ctx) or 1 µM -agatoxin IVA (Aga). Data shown are mean ± S.E. values for maximal increases in [Ca++]i from 45 (None), 28 (Ctx) and 21 (Aga) cells in three to six experiments. *P < .05 compared to cells depolarized in the absence of toxins (ANOVA). In B to D data are expressed as percent of control values measured in the same cells depolarized without ethanol, -conotoxin GVIA or -agatoxin IVA.

Ethanol does not inhibit Ca⁺⁺-induced Ca⁺⁺ release. Voltage- or receptor-mediated increases in [Ca⁺⁺]_i stimulate Ca⁺⁺ release from intracellular stores by activating ryanodine receptor/Ca⁺⁺ release channels present in endoplasmic reticulum (Meissner, 1994). These channels are activated by caffeine and are inhibited by micromolar concentrations of ryanodine. Because Ca⁺⁺-induced Ca⁺⁺ release contributes to depolarization-induced Ca⁺⁺ rises in PC12 cells (Reber and Reuter, 1991), we examined whether ethanol reduces depolarization-evoked [Ca⁺⁺]_i rises by inhibiting Ca⁺⁺ release channels. Caffeine stimulated a peak rise in [Ca⁺⁺]_i that was approximately 3.5-fold the resting level (fig. 3A). Preincubation with 50 mM ethanol did not alter resting [Ca⁺⁺]_i or inhibit the response to caffeine. Ryanodine reduced depolarization-evoked [Ca⁺⁺]_i rises, but 50 mM ethanol still inhibited the remaining [Ca⁺⁺]_i rise by 53 ± 11% (fig. 3B). This percentage of inhibition by ethanol was similar to that observed in cells depolarized without ryanodine (41 ± 4%; P = .175). These results indicate that ryanodine receptor/Ca⁺⁺ release channels contribute to depolarization-induced [Ca⁺⁺]_i rises in trk-PC12 cells, but these channels are not inhibited by 50 mM ethanol.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Ethanol does not inhibit ryanodine receptor/Ca++ release channels. A, Cells were incubated in 5 mM KCl buffer alone (Con) or in buffer containing 50 mM ethanol (Eth) for 8 min before treatment with 30 mM caffeine in the continued absence (Caf) or presence of ethanol (Caf + Eth). Data shown are mean ± S.E. resting and peak [Ca++]i values from 25 cells in three experiments. B, Nifedipine-treated cells were preincubated without (Con, Ryan) or with (Ryan+Eth) 50 mM ethanol and then depolarized in the absence (Con) or presence (Ryan, Ryan+Eth) of 10 µM ryanodine. Data shown are mean ± S.E. values for maximal increases in [Ca++]i from 20 cells in four experiments. *P < .05 compared to Con and **P < .05 compared to Con and Ryan (ANOVA).

Ethanol does not impair capacitative Ca⁺⁺ entry. Release of Ca⁺⁺ from intracellular stores stimulates influx of extracellular Ca⁺⁺ into the cytoplasm by a process termed capacitative Ca⁺⁺ entry (Berridge, 1995). This Ca⁺⁺ current is carried by Ca⁺⁺ release-activated Ca⁺⁺ channels and sustained elevations in [Ca⁺⁺]_i due to calcium entry through these channels can be stimulated by treating cells with agents that deplete intracellular Ca⁺⁺ stores, such as thapsigargin (Berridge, 1995). In thapsigargin-treated trk-PC12 cells, [Ca⁺⁺]_i rose slowly over 3 min to 316 ± 10 nM and remained elevated for at least 5 min thereafter (fig. 4A). Addition of 50 mM ethanol did not reduce [Ca⁺⁺]_i, suggesting that ethanol does not inhibit Ca⁺⁺ release-activated Ca⁺⁺ channels in trk-PC12 cells.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Ethanol does not inhibit capacitative Ca++ entry or Ca++ removal after Ca++ loading in ionomycin. A, Cells () were treated with 1 µM thapsigargin and images recorded for 8 min. In some cells () 50 mM ethanol was added 180 sec (arrow) after beginning treatment with thapsigargin. Data are mean ± S.E. [Ca++]i values from 21 () and 23 () cells in three experiments. B, Cells were preincubated for 8 min in 2 mM CaCl2 buffer in the absence () or presence () of 50 mM ethanol. 2 mM CaCl2 buffer was identical in composition to 5 mM KCl buffer except that NaCl was replaced by N-methyl-D-methylglucamine to reduce Na+-Ca++ exchange. Cells were then exposed to 5 µM ionomycin and [Ca++]i was allowed to rise to approximately 200 nM over the next 60 sec. Cells were then perfused with low Ca++ buffer that was identical in composition to 2 mM CaCl2 buffer except that the concentration of CaCl2 was lowered to 400 nM and choline chloride was increased by 1.6 mM to maintain osmolarity constant. Images were recorded for another 80 sec as [Ca++]i fell to resting levels. Data are mean ± SE [Ca++]i values for 32 () and 15 () cells from three experiments. Mean values for the rate of decline in [Ca++]i are given in the text.

Ethanol does not alter Ca⁺⁺ extrusion or Na⁺-Ca⁺⁺ exchange. The level of [Ca⁺⁺]_i is tightly regulated by mechanisms for Ca⁺⁺ extrusion and sequestration that serve to restore the resting level of [Ca⁺⁺]_i after depolarization. These mechanisms include Na⁺-Ca⁺⁺ exchange and active Ca⁺⁺ transport across the plasma membrane and into endoplasmic reticulum through the action of Ca⁺⁺-ATPases (Clapham, 1995). To examine whether ethanol inhibits depolarization-induced rises in [Ca⁺⁺]_i by promoting Na⁺-Ca⁺⁺ exchange we incubated cells in Na⁺-free buffer to prevent Ca⁺⁺ efflux through Na⁺/Ca⁺⁺ exchangers. In Na⁺-free buffer, resting [Ca⁺⁺]_i was higher (172 ± 10 nM, n = 20, P = .0001) than in buffer containing Na⁺ (96 ± 8 nM, n = 48), reflecting inhibition of Ca⁺⁺ efflux through Na⁺-Ca⁺⁺ exchangers in Na⁺-free buffer. However, depolarization increased [Ca⁺⁺]_i to a maximum of 449 ± 32 nM (n = 20) that was similar to that observed in cells depolarized in Na⁺-containing buffer (465 ± 36 nM, N = 48, P = .08). Moreover, in cells depolarized in Na⁺-free buffer, ethanol inhibited the rise in [Ca⁺⁺]_i by 37 ± 6% (n = 20), which is similar to the level of inhibition observed in cells depolarized in Na⁺-containing buffer (41 ± 4%, n = 45, P = .62). These findings indicate that Na⁺-Ca⁺⁺ exchange does not modulate the peak rise in [Ca⁺⁺]_i during depolarization and that ethanol does not reduce depolarization-induced [Ca⁺⁺]_i rises by enhancing Na⁺-Ca⁺⁺ exchange.

Ethanol enhances [Ca⁺⁺]_i rises stimulated by bradykinin. In PC12 cells, bradykinin activates B₂ bradykinin receptors coupled to inositol 1,4,5-trisphosphate production leading to a rapid rise in [Ca⁺⁺]_i (Fasolato et al., 1988). The response to bradykinin is biphasic with an initial peak rise due to IP₃-mediated Ca⁺⁺ release, and a subsequent plateau phase due to Ca⁺⁺ influx through membrane channels that are insensitive to L and N channel antagonists (Fasolato et al., 1988; Sher et al., 1988). If ethanol reduces depolarization-induced [Ca⁺⁺]_i rises by promoting sequestration or extrusion of Ca⁺⁺, then ethanol should also reduce bradykinin-stimulated [Ca⁺⁺]_i rises. However, in contrast to depolarization-evoked [Ca⁺⁺]_i rises, the peak rise induced by bradykinin was potentiated by ethanol (fig. 5). This suggests that inhibition by ethanol is specific for depolarization-evoked [Ca⁺⁺]_i rises mediated by voltage-gated Ca⁺⁺ channels. These results provide additional evidence that ethanol does not promote Ca⁺⁺ sequestration or extrusion, reduce Ca⁺⁺ mobilization, or inhibit other mechanisms for Ca⁺⁺ entry in trk-PC12 cells.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Ethanol enhances bradykinin-induced Ca++ mobilization in trk-PC12 cells. Cells were preincubated in 5 mM KCl buffer without () or with () 50 mM ethanol for 8 min and then 100 nM bradykinin was added ("0" time point) in the continued absence or presence of ethanol. Data are mean ± S.E. [Ca++]i values from 73 cells in three experiments. *P = .023 compared to the mean peak [Ca++]i value recorded in cells treated without ethanol (t test).

PKA regulates inhibition of [Ca⁺⁺]_i rises by ethanol. Because protein phosphorylation regulates ion channel function (Nestler and Greengard, 1984) and, in some cells, ethanol stimulates second messenger cascades that lead to activation of PKA (Nagy et al., 1989; Rabin et al., 1993) or PKC (Hoek et al., 1987; Rubin et al., 1988) we examined whether ethanol inhibits depolarization-induced [Ca⁺⁺]_i rises by activating these kinases. If ethanol acts by stimulating PKA, then treatment with a PKA agonist should also inhibit depolarization-induced [Ca⁺⁺]_i rises. However, exposure to Sp-cAMPS at a concentration (30 µM) that produces near maximal stimulation of PKA-mediated glycogenolysis in rat hepatocytes (Rothermel et al., 1983) enhanced depolarization-induced [Ca⁺⁺]_i rises (fig. 6A). In addition, inhibition by ethanol was not prevented by treating cells with the PKA antagonist Rp-cAMPS at a concentration (30 µM) that maximally inhibits PKA activation by glucagon in hepatocytes (Rothermel et al., 1984). The PKC activator PMA (Nishizuka, 1992), like ethanol, inhibited depolarization-evoked rises in [Ca⁺⁺]_i (fig. 6A). However, treatment with a maximally effective-concentration of PMA did not prevent further inhibition by ethanol, indicating that PMA and ethanol have different mechanisms of action. Therefore ethanol does not appear to inhibit depolarization-induced [Ca⁺⁺]_i rises in trk-PC12 cells by activating PKA or PKC.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Phosphorylation modulates inhibition of [Ca++]i rises by ethanol. A, Nifedipine-treated cells were first depolarized in 50 mM KCl and control responses were recorded. Cells were then preincubated for 10 min and then depolarized without (open bars) or with (shaded bars) 50 mM ethanol and no added drug (Con), 1 µM PMA (Ph), 30 µM Sp-cAMPS (Sp), 30 µM Rp-cAMPS (Rp), 30 nM okadaic acid (Ok) or 1 µM cyclosporin A (Cy). Data shown are mean ± S.E. values for maximal increases in [Ca++]i from 20 to 92 cells evaluated in three to seven experiments and are expressed as the percent of values measured in the same cells depolarized in the presence of nifedipine alone. *P < .05 compared to depolarization with nifedipine alone. **P < .05 compared to cells depolarized with the same drugs but without ethanol (t test). B, Nifedipine-treated cells were first depolarized in 50 mM KCl and control responses were recorded. Cells were preincubated with ethanol and the indicated concentration of okadaic acid for 10 min and then depolarized in the continued presence of ethanol and okadaic acid. Data are mean ± S.E. values for maximal increases in [Ca++]i from 19 to 48 cells for each concentration of okadaic acid and are expressed as the percent of values measured in the same cells depolarized with nifedipine alone.

	Discussion

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The major findings of this study are that 1) NGF-differentiated trk-PC12 cells express L-type, N-type and P/Q-type channels as determined by responses to selective Ca⁺⁺ channel antagonists, 2) ethanol can inhibit N-type and P/Q-type channels in these cells and 3) inhibition by ethanol is antagonized by PKA. Using NGF-differentiated trk-PC12 cells as a model system to study dihydropyridine-insensitive Ca⁺⁺ channels, we found that K⁺ depolarization evoked biphasic [Ca⁺⁺]_i rises in these cells, with a peak phase mediated mainly by N-type channels sensitive to -conotoxin GVIA and P/Q-type channels sensitive to -agatoxin IVA. The effect of -agatoxin IVA was seen mainly at high concentrations (>200 nM), indicating that these channels are probably Q-type (Randall and Tsien, 1995). Ethanol inhibited the peak [Ca⁺⁺]_i rise with maximal inhibition of approximately 45% at 50 mM ethanol. Ethanol did not act by inhibiting ryanodine receptor/Ca⁺⁺ release channels or capacitative Ca⁺⁺ entry, or by enhancing Ca⁺⁺ sequestration or extrusion. [Ca⁺⁺]_i rises induced by bradykinin, which stimulates IP₃ mediated Ca⁺⁺ mobilization and receptor-gated Ca⁺⁺ influx (Fasolato et al., 1988), were increased by ethanol, indicating that inhibition of [Ca⁺⁺]_i rises by ethanol is specific for depolarization-evoked Ca⁺⁺ entry through voltage-gated channels. Both -conotoxin GVIA and -agatoxin IVA reduced inhibition by ethanol, demonstrating that ethanol inhibited [Ca⁺⁺]_i rises mediated by N-type and P/Q-type channels.

This study is the first demonstration of inhibition of an -agatoxin IVA-sensitive channel by ethanol. Using nerve terminals from rat neurohypophysis, Wang et al. (1991) demonstrated inhibition of -conotoxin VIA-sensitive N-type channels by ethanol. However, investigators from the same laboratory found that N-type channels in NGF-differentiated PC12 cells are not inhibited by 50 mM ethanol (Mullikin-Kilpatrick and Treistman, 1995). The discrepancy between their findings and ours may reflect the use of different cell lines and techniques. One key difference may lie in the use of Ba⁺⁺ rather than Ca⁺⁺ as the charge carrier. In the earlier study of N-type channels in neurohypophyseal terminals, Ca⁺⁺ was the charge carrier (Wang et al., 1991), whereas in the study using PC12 cells, Ba⁺⁺ was used (Mullikin-Kilpatrick and Treistman, 1995). Although ethanol inhibits both Ca⁺⁺ and Ba⁺⁺ influx through L-type channels in PC12 cells (Mullikin-Kilpatrick and Treistman, 1994), inhibition of N-type channels by ethanol could be specific for Ca⁺⁺. A similar result was observed with AMPA/kainate receptors expressed in Xenopus oocytes, where inhibition by ethanol was reduced when Ba⁺⁺ was substituted for extracellular Ca⁺⁺ (Dildy-Mayfield and Harris, 1995). If true, this would suggest that inhibition of dihydropyridine-insensitive channels by ethanol involves Ca⁺⁺-activated processes such as phosphorylation by Ca⁺⁺-sensitive kinases or Ca⁺⁺-mediated channel inactivation.

We found that inhibition of depolarization-induced [Ca⁺⁺]_i rises by ethanol required several min of ethanol exposure to become maximal and was long-lasting, only partly reversing 20 min after washout of ethanol. The slow onset and persistence of the effect may reflect the action of metabolic events such as phosphorylation that regulate channel function. Subunits of N and P/Q channels are phosphorylated by PKA and PKC (Hell et al., 1994; Sakurai et al., 1995). PKA activation stimulates N channels in rat nodose ganglion cells (Gross et al., 1990) but inhibits N channels in mouse dorsal root ganglion cells (Gross and MacDonald, 1989) and rat neostriatal neurons (Surmeier et al., 1995). Similarly, PKC activation increases N channel activity in superior cervical ganglion cells (Bernheim et al., 1991), rat hippocampal CA3 and cortical pyramidal cells (Swartz, 1993; Swartz et al., 1993) and several peripheral neurons (Swartz, 1993), but decreases N channel function in freshly dissociated hippocampal neurons (Doerner et al., 1990). PKC potentiates Q channel-mediated synaptic transmission at CA3-CA1 synapses in rodent hippocampus (Wheeler et al., 1994) and activation of PKC or PKA enhances Q-type currents (Randall and Tsien, 1995) expressed by Xenopus oocytes injected with mRNA from cerebellar granule cells (Fournier et al., 1993). However, in intact cerebellar granule cells, activation of PKA slightly decreases non-L- and non-N-type current, half of which is mediated by Q-type channels (Randall and Tsien, 1995). Therefore, phosphorylation appears to regulate N-type and P/Q-type channels, but whether it increases or decreases function may depend on cell-specific splice variants of channel subunits or other proteins that modulate channel function.

Because phosphorylation regulates Ca⁺⁺ channels, we considered whether ethanol inhibits channel function by altering PKC or PKA activity. We found that treatment with the cAMP analog Sp-cAMPS enhanced depolarization-induced [Ca⁺⁺]_i rises whereas the PKA inhibitor Rp-cAMPS had no effect, indicating that ethanol could not inhibit [Ca⁺⁺]_i rises by altering PKA activity. Moreover, although activation of PKC with PMA inhibited depolarization-evoked rises in [Ca⁺⁺]_i, the actions of PMA and ethanol were additive, indicating different mechanisms of action. These findings demonstrate that ethanol does not inhibit depolarization-induced [Ca⁺⁺]_i rises by modulating PKA or PKC activity. Whether ethanol acts by regulating other kinases requires further investigation.

Brief exposure to 100 to 150 mM ethanol for 10 min has been reported to increase cAMP levels by 9 pmol/10⁶ cells in NG108-15 cells (Nagy et al., 1989) and by 3 pmol/mg protein in PC12 cells (Rabin et al., 1993). Because ethanol increases cAMP levels and Sp-cAMPS prevented inhibition of [Ca⁺⁺]_i rises by ethanol, it seems paradoxical that ethanol inhibited the response to depolarization. One might expect that ethanol-induced increases in cAMP would antagonize the inhibitory effect of ethanol on depolarization-induced [Ca⁺⁺]_i rises. However, ethanol-induced increases in cAMP are extremely small and are only about 1% of increases stimulated by forskolin (10 µM) or the adenosine agonist 2-chloroadenosine (1 µM) in PC12 cells (Rabin et al., 1993). Moreover, ethanol does not increase cAMP levels in N1E-115 neuroblastoma cells (Stenstrom and Richelson, 1982), some clones of PC12 cells (Rabe et al., 1990), or in all studies involving NG108-15 cells (Gordon et al., 1986). Because Rp-cAMPS had no effect on [Ca⁺⁺]_i rises in ethanol-treated trk-PC12 cells (fig. 6), ethanol may not increase cAMP levels in these cells, or if it does, the increase is too small to alter the response to depolarization or prevent inhibition of [Ca⁺⁺]_i rises by ethanol

In addition to regulating ion channel function, phosphorylation can alter the sensitivity of certain membrane proteins to ethanol. For example, in NG108-15 cells, ethanol-sensitive adenosine transporters require PKA activation to be inhibited by ethanol (Coe et al., 1996), whereas mouse and bovine GABA_A receptors appear to require PKC phosphorylation of a splice-variant long form of the 2 subunit for enhancement of receptor function by ethanol (Wafford and Whiting, 1992). Our findings suggest that the sensitivity of non-L-type Ca⁺⁺ channels to ethanol may also be modulated by phosphorylation since we found that activation of PKA with the selective agonist Sp-cAMPS completely prevented inhibition of depolarization-evoked [Ca⁺⁺]_i rises by ethanol.

The phosphatase inhibitor okadaic acid also prevented inhibition by ethanol and this was reversed by treatment with the specific PKA antagonist Rp-cAMPS, indicating that the effect of okadaic acid was due to preservation of PKA-phosphorylated sites on proteins that regulate channel function. Unlike okadaic acid, which inhibits PP-1 and PP-2A, inhibitors of PP-2B (calcineurin), did not alter inhibition by ethanol. Okadaic acid reduced the effect of ethanol with an IC₅₀ value of approximately 3 nM, close to its IC₅₀ value for inhibition of PP2-A (1 nM) (Bialojan and Takai, 1988). These findings suggest that inhibition of non-L-type channels by ethanol is regulated by a phosphoprotein that is a substrate for PKA and PP-2A. Further work is needed to identify the phosphoproteins that interact with ethanol to regulate channel function.

N-type and P/Q-type channels are expressed in dendrites and presynaptic terminals (Westenbroek et al., 1992, 1995) and regulate synaptic transmission at several synapses (Takahashi and Momiyama, 1993; Wheeler et al., 1994). Thus, inhibition of N-type and P/Q-type channels by intoxicating concentrations of ethanol could depress neurotransmitter release in several brain regions. Such inhibition could underlie inhibition of electrically stimulated release of acetylcholine and other neurotransmitters by ethanol in rat neocortex (Carmichael and Israel, 1975) and contribute to sedation and cognitive impairment observed during ethanol intoxication (Messing and Diamond, 1997). In addition, mutations in the gene encoding the _1A subunit of P/Q-type channels are linked to the tottering and leaner phenotypes in mice (Fletcher et al., 1996) and to familial hemiplegic migraine, episodic ataxia type 2 and spinocerebellar ataxia 6 in humans (Ophoff et al., 1996; Zhuchenko et al., 1997). Ataxia is a common feature of these disorders, supporting a role for ethanol-induced inhibition of P/Q-type channels in the induction of ataxia. Further studies of brain N-type and P/Q-type channels will be required to establish whether ethanol inhibits these channels in multiple regions of the mammalian central nervous system.