Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The opioid peptide -endorphin (-EP) is produced in the pituitary, brain, and several other peripheral sites and has been proposed to function as a neurotransmitter or a neuromodulator regulating a variety of physiological functions (O'Donohue and Dorsa, 1982; Wilcox et al., 1986; Amalric et al., 1987; Spanagel et al., 1991). Some of the effects of ethanol intoxication appear to be regulated by the opioid peptide (Gianoulakis et al., 1989; Froehlich and Li, 1994; Wand et al., 1998). We have previously shown that acute exposure of ethanol to hypothalamic cells in primary cultures increases -EP secretion, and chronic exposure of ethanol decreases -EP release and desensitizes the -EP-secreting response to ethanol (Boyadjieva et al., 1997; Boyadjieva and Sarkar, 1997, 1999; De et al., 1999; Simasko et al., 1999). The mechanisms that regulate ethanol-controlled hypothalamic -EP secretion are complex and not well understood.

Previous studies determining the role of the cAMP system and calcium channels revealed that both of these signal transducers contribute significantly to the ethanol-activated -EP secretion from these neurons (Boyadjieva and Sarkar, 1999; De et al., 1999; Simasko et al., 1999). Furthermore, heterologous desensitization of the cAMP system appears to be a mechanism that contributes to the development of ethanol tolerance of -EP neurons following chronic ethanol treatment (Boyadjieva et al., 1997). In addition to cAMP and calcium systems, the protein kinase C (PKC) second messenger system is also involved in neurotransmission of -EP neurons (Shahabi and Sharp, 1993; Suh et al., 1996). Involvement of the PKC system in ethanol-regulated -EP secretion has not been studied.

Protein kinase C, a serine/threonine protein phosphotransferase, represents a family of at least 12 related isoenzymes that differ remarkably in their structure and expression in various tissues, mode of activation, cofactor requirement, and substrate specificity (Nishizuka, 1984; Hug and Sarre, 1993; Newton, 1997). PKC isoenzymes have been categorized in three subclasses by their different mode of activation: conventional PKCs, , I, II, and , which require phosphatidylserine, diacylglycerol, and Ca²⁺; novel PKCs, , , , and , which require phosphatidylserine and diacylglycerol but not Ca²⁺; and the atypical PKCs, , , , and µ, which contain only a phosphatidylserine motif (Newton, 1997). PKC has been shown to participate in the transduction of signals generated by hormones, neurotransmitters, and growth factors (Nishizuka, 1984). PKC activators have various functions including the modulation of cellular growth and differentiation in a variety of cell types (Castagna et al., 1982).

The focus of the present study was to determine the role of the PKC system in ethanol-regulated IR--EP release by determining 1) the effects of acute and chronic treatments with ethanol on the basal and PKC activator-induced -EP release, 2) the action of a general PKC inhibitor on ethanol-induced -EP release, 3) the correlation between the ethanol-induced changes in -EP release and various subclasses of PKC isoenzyme expression, and 4) the effect of isoenzyme-specific PKC inhibitors in ethanol-induced -EP release.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Primary Cultures of Fetal Hypothalamic Cells. Primary cultures of rat fetal hypothalamic cells were prepared according to the method described by us previously (Sarkar and Sakaguchi, 1990). Briefly, mediobasal hypothalamic tissues from the brains of fetuses of 17- to 19-day gestation periods were collected in ice-cold Hanks' balanced salt solution, containing 0.1% bovine serum albumin and 200 µM ascorbic acid (pH 7.4). The tissues were washed two to three times with Hanks' balanced salt solution and once with Hepes-buffered Dulbecco's modified Eagle's medium (HDMEM) and then incubated in HDMEM for 10 to 15 min at 37°C. HDMEM contains 4.5 g/l glucose, 25 mM Hepes, 1% Fungi-bact, 0.1% bovine serum albumin, 200 µM ascorbic acid (pH 7.4). After incubation, tissues were dissociated using 20- and 22-gauge needles fixed to a 20-ml syringe. Then, 4 to 5 × 10⁶ cells/flask were plated in 25-cm² flasks (Corning Glassworks, Corning, NY) previously coated with 100 µg/ml polyornithine in 0.15 M borate buffer (pH 8.4). The cells were grown in HDMEM with 10% heat-inactivated fetal calf serum (Hyclone Laboratories, Logan, UT) in a humidified atmosphere of CO₂:air (7.2:92.8) at 37°C. Two days after plating of cells, the medium was replaced with serum-free HDMEM containing serum supplement (1 µM human transferrin, 5 µg insulin, 20 nM progesterone, 100 µM putrescine, and 60 nM sodium selenite). The medium was replaced every 2 days. The cells were grown 8 to 9 days before treatment.

Treatments. Before treatment, cultures were washed with HDMEM containing serum supplement and then treated with different drugs. For secretion studies, the cultures were treated with different doses of a PKC stimulator or a PKC inhibitor in the presence or absence of 50 mM ethanol. We used 4-phorbol-12-myristate-13-acetate (PMA) as a PKC stimulator (Nishizuka, 1984), which activates Ca²⁺-ATPase and potentiates forskolin-induced cAMP formation (Ebeling et al., 1985; Kraft et al., 1987; Hug and Sarre, 1993). We used chelerythrine chloride as a PKC inhibitor, which is a potent, selective, cell-permeable inhibitor that acts on the catalytic domain. It is a competitive inhibitor with respect to phosphate activator and a noncompetitor with respect to ATP (Herbert et al., 1990). Two specific PKC isoenzyme blockers, Rottlerin (6 and 100 µM; Calbiochem, La Jolla, CA) and PKC- (V1-2+; 150 µg/ml; Calbiochem), were used. A scrambled PKC- (V1-2; 150 µg/ml; Calbiochem) was also used as negative control. Both of these peptides were used after permeabilization of cells to better incorporate the peptide into the cells (Johnson et al., 1996a). Rottlerin inhibits protein kinases with specificity for PKC- at a dose range of 3 to 6 µM and for PKC-, -, and - at a dose range of 80 to 100 µM (Gschwendt et al., 1994). V1-2 has been shown to selectively inhibit the translocation of PKC- isozymes (Johnson et al., 1996b). The media samples from these cultures were collected into 12 × 75 mm glass tubes containing 2000 IU of trasylol (Research Plus, Bayonne, NJ) and 100 µg of bacitracin (Sigma, St. Louis, MO). The collected media were then boiled for 5 min and stored at 70°C until use. After treatment, the cells were washed with phosphate-buffered saline (pH 7.4), lysed with 2 ml of 0.1 N NaOH, and used for analysis of cellular DNA contents.

Radioimmunoassay of Immunoreactive -EP. To measure the IR--EP levels in culture media, we used a radioimmunoassay system (Sarkar and Yen, 1985). In this system, antihuman -EP was used as an antiserum (Y-10, supplied by Dr. S. S. C. Yen, La Jolla, CA), ovine -EP as standard (this has the same sequence of amino acid as rat -EP), and ¹²⁵I-ovine -EP. At 1:30,000 dilution the minimum detectable amount of -EP was 3 pg/tube. The antibody we used does not have any cross-reactivity with -, -, and neoendorphin. The cellular DNA contents were measured by the diphenylamine method (Labarca and Paigen, 1980). The units of DNA values are micrograms per milliliter. The amounts of IR--EP were normalized using DNA values of each culture.

Western Blot Analysis. Treated cultures were washed with cold phosphate-buffered saline (pH 7.4) and lysed in lysis buffer (20 mM Tris, 50 mM -mercaptoethanol, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 10 mM aprotinin), sonicated, and then centrifuged at 100,000g at 4°C for 1 h. Supernatant was collected and used as cytosolic fraction. To extract the membrane-bound protein, the pellet was dissolved in 1% Nonidet P-40 and 10 mM EGTA. The protein concentrations of both cytosolic and membrane fractions were measured by the BCA protein assay method (Pierce, Rockford, IL). Each sample containing 100 µg of total protein was analyzed in 12% sodium dodecyl sulfate-polyacrylamide gel at a constant current. The protein was transferred to nitrocellulose membrane (0.45-µm pore size; Amersham Biosciences, Arlington Heights, IL), and then the membrane was blocked with 5% milk. The membrane was incubated with 1 µg/ml anti-PKC-, -I, -II, -, -, -, or - (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at room temperature. After washing with Tris-NaCl buffer, the membrane was incubated with antirabbit IgG (Amersham Biosciences) at room temperature for 2 h. Immunoreactivity was detected using an enhanced chemiluminescence Western blotting system (Amersham Biosciences). After transfer, the membrane was stained with ponceau S solution. The ponceau-stained bands were scanned using a laser scanner (Personal Densitometer; Molecular Dynamics, Sunnyvale, CA). After immunodetection, the PKC-positive bands were scanned with the laser scanner, and the percentage of the ratio of this value and the total protein value from ponceau S stained was calculated to determine the arbitrary unit of PKC isoenzymes.

Statistics. The data shown in figures and text are mean ± S.E. Data were analyzed using one-way analysis of variance. A post-hoc test was conducted using the Student-Newman-Keuls test. A value of p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effects of Ethanol on Basal and PMA-Induced IR--EP Release. In the first study, the effect of the PKC-stimulating agent PMA on -EP release in primary cultures of hypothalamic cells was studied. The cultures were treated with 0, 0.1, and 1 µM doses of PMA for 1, 3, 6, 12, and 24 h, and culture media samples were collected and assayed for IR--EP. PMA increased the release of IR--EP from primary cultures of fetal hypothalamic cells between 1 and 12 h (Fig. 1A). The release of IR--EP was significantly increased at the 0.1 µM dose of PMA at 3 h. The 1 µM dose of PMA significantly increased IR--EP release between 1 and 24 h.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Dose- and time-dependent effects of the protein kinase C activator PMA on basal (A) and ethanol-regulated IR--EP secretion from hypothalamic neurons in primary cultures (B). A, hypothalamic cells were grown in cultures for 9 days and treated with PMA (0, 0.1, and 1.0 µM) for various periods of time followed by measurement of media levels of IR--EP. Data are means ± S. E. of six to nine cultures. *, p < 0.05 compared with control group (0 dose). B, after growing in culture for 9 days, hypothalamic cells were treated with vehicle, 1 µM PMA, 50 mM ethanol, or 1 µM PMA and 50 mM ethanol for 3 h (acute treatment), or treated first with 50 mM ethanol for 21 h and then with either 1 µM PMA or 1 µM PMA and 50 mM ethanol for an additional 3 h (chronic treatment). Media samples were collected and assayed for IR--EP levels. Data are means ± S.E. of six to eight cultures. a, p < 0.05, compared with control. b, p < 0.05, compared with PMA alone.

Effects of Chelerythrine Chloride on Basal and Ethanol-Induced -EP Release. The role of the PKC signaling system in the control of IR--EP release from hypothalamic neurons was further investigated by using the PKC inhibitor chelerythrine chloride. Cultures were pretreated with different doses of chelerythrine chloride (0, 1, 2.5, and 5 µM) for 1 h and then incubated with ethanol for 3 h. As shown in Fig. 2, this inhibitor blocked ethanol-induced IR--EP release in a dose-dependent manner. The minimum dose required to block ethanol-induced IR--EP release is 2.5 µM chelerythrine. Because chronic ethanol markedly reduced -EP release, it is very hard to detect further reduction of the release by the hormone assay we used. A preliminary study determining the effects of PKC blocker Rottlerin showed very low levels of IR--EP in media samples of cultures chronically treated with ethanol (data not shown). We anticipate that after chronic ethanol exposure, chelerythrine treatment will cause further decrease in IR--EP release to make the hormone levels undetectable. Therefore, the effect of chelerythrine on chronic ethanol-regulated IR--EP release was not determined.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Effects of a protein kinase inhibitor, chelerythrine chloride, on ethanol-stimulated IR--EP secretion from hypothalamic neurons in primary cultures. Hypothalamic cells were grown in cultures for 9 days and pretreated with chelerythrine chloride (0, 1.0, 2.5, and 5 µM) for 1 h. The cultures were washed with fresh media and then incubated with media containing 50 mM ethanol or no ethanol (control) for 3 h. The media levels of IR--EP were measured by radioimmunoassay. Data are means ± S.E. of six cultures. a, p < 0.05, significantly different from 0 dose of ethanol with the same dose of chelerythrine. b, p < 0.05, significantly different from 50 mM dose of ethanol alone.

Effects of Ethanol and PMA on Cytosolic and Cell Membrane Levels of Different PKC Isoenzymes. The effect of ethanol and PMA on cytosolic and cell membrane levels of PKC isoenzymes were studied using seven different isoenzymes (PKC-, -I, -II, -, -, -, and -). Cytosolic and cell membrane levels of various PKC isoenzyme proteins were determined by Western blot after acute or chronic ethanol treatments. All the PKC isoenzymes were detectable in both cytosolic and cell membrane preparations irrespective of treatments (Figs. 3-9). Although we have studied seven different PKC isoenzymes, only two PKC isoenzymes,  and , showed higher levels in the membrane fraction than in the cytoplasmic fraction.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Changes in PKC- isoenzyme levels in the cytosolic fraction (panels A and B) and in the membrane fraction (panels C and D) of hypothalamic cells after being treated with ethanol for 3 h (acute) or 24 h (chronic), with or without PMA. A, acute ethanol (50 mM); B, control; C, PMA (1 µM) and ethanol (50 mM); D, PMA (1 µM); E, PMA (1 µM) and ethanol (50 mM); F, chronic ethanol (50 mM). Representative Western blot data for cytosolic and membrane fractions are shown on the top. Mean ± S. E. values of PKC- isoenzyme level in cytosolic and membrane fractions are shown on the bottom. The Western blot membranes were first stained with ponceau S solution prior to immunostain with PKC- antibody. The ponceau-stained bands were scanned using a laser scanner, and the stain intensity was used as the arbitrary value of total protein. After immunodetection, the PKC-positive bands were scanned with the laser scanner, and the percentage of the ratio of this value and the total protein value was calculated to determine the arbitrary amount of PKC- isoenzyme. The values are means ± S.E. of four to six experiments. a, p < 0.05, as compared with control group.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Changes in PKC-I isoenzyme levels in the cytosolic fraction (panels A and B) and in the membrane fraction (panels C and D) of hypothalamic cells after treatment with ethanol for 3 h (acute) or 24 h (chronic), with or without PMA. A, acute ethanol (50 mM); B, control; C, PMA (1 µM) and ethanol (50 mM); D, PMA (1 µM); E, PMA (1 µM) and ethanol (50 mM); F, chronic ethanol (50 mM). Representative Western blot data for cytosolic and membrane fractions are shown on the top. Mean ± S.E. values of PKC-I isoenzyme level in cytosolic and membrane fractions are shown on the bottom. The values are means ± S.E. of four to six experiments.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Changes in PKC-II isoenzyme levels in the cytosolic fraction (panels A and B) and in the membrane fraction (panels C and D) of hypothalamic cells after treatment with ethanol for 3 h (acute) or 24 h (chronic), with or without PMA. A, acute ethanol (50 mM); B, control; C, PMA (1 µM) and ethanol (50 mM); D, PMA (1 µM); E, PMA (1 µM) and ethanol (50 mM); F, chronic ethanol (50 mM). Representative Western blot data for cytosolic and membrane fractions are shown on the top. Mean ± S.E. values of PKC-II isoenzyme level in cytosolic and membrane fractions are shown on the bottom. The values are mean ± S.E. of four to six experiments. a, p < 0.05, as compared with control group.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Changes in PKC- isoenzyme levels in the cytosolic fraction (panels A and B) and in the membrane fraction (panels C and D) of hypothalamic cells after treatment with ethanol for 3 h (acute) or 24 h (chronic) with or without PMA. A, acute ethanol (50 mM); B, control; C, PMA (1 µM) and ethanol (50 mM); D, PMA (1 µM); E, PMA (1 µM) and ethanol (50 mM); F, chronic ethanol (50 mM). Representative Western blot data for cytosolic and membrane fractions are shown on the top. Mean ± S.E. values of PKC- isoenzyme level in cytosolic and membrane fractions are shown on the bottom. The values are means ± S.E. of four to six experiments.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Changes in PKC- isoenzyme levels in the cytosolic fraction (panels A and B) and in the membrane fraction (panels C and D) of hypothalamic cells after treatment with ethanol for 3 h (acute) or 24 h (chronic), with or without PMA. A, acute ethanol (50 mM); B, control; C, PMA (1 µM) and ethanol (50 mM); D, PMA (1 µM); E, PMA (1 µM) and ethanol (50 mM); F, chronic ethanol (50 mM). Representative Western blot data for cytosolic and membrane fractions are shown on the top. Mean ± S.E. values of PKC- isoenzyme level in cytosolic and membrane fractions are shown on the bottom. The values are means ± S.E. of four to six experiments. a, p < 0.05, as compared with control group.

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Changes of PKC- isoenzyme levels in the cytosolic fraction (panels A and B) and in the membrane fraction (panels C and D) of hypothalamic cells after treatment with ethanol for 3 h (acute) or 24 h (chronic), with or without PMA. A, acute ethanol (50 mM); B, control; C, PMA (1 µM) and ethanol (50 mM); D, PMA (1 µM); E, PMA (1 µM) and ethanol (50 mM); F, chronic ethanol (50 mM). Representative Western blot data for cytosolic and membrane fractions are shown on the top. Mean ± S.E. values of PKC- isoenzyme level in cytosolic and membrane fractions are shown on the bottom. The values are means ± S.E. of four to six experiments. a, p < 0.05, compared with control group. b, p < 0.05, compared with ethanol + PMA group.

View larger version (32K): [in this window] [in a new window]   Fig. 9.   Changes of PKC- isoenzyme levels in the cytosolic fraction (panels A and B) and in the membrane fraction (panels C and D) of hypothalamic cells after treatment with ethanol for 3 h (acute) or 24 h (chronic), with or without PMA. A, acute ethanol (50 mM); B, control; C, PMA (1 µM) and ethanol (50 mM); D, PMA (1 µM); E, PMA (1 µM) and ethanol (50 mM); F, chronic ethanol (50 mM). Representative Western blot data for cytosolic and membrane fractions are shown on the top. Mean ± S.E. values of PKC- isoenzyme level in cytosolic and membrane fractions are shown on the bottom. The values are means ± S.E. of four to six experiments. a, p < 0.05, compared with control group. b, p < 0.05, compared with ethanol + PMA group.

Effects of PKC- and - Isoenzyme Blockers on Ethanol-Induced -EP Release. Because acute ethanol increased PKC- and - isoenzymes levels, the role of these PKC isoenzymes on ethanol-induced -endorphin release was determined. We have used a 6 µM dose of Rottlerin to block the PKC- isoenzyme and a 100 µM dose to block the PKC- isoenzyme (Gschwendt et al., 1994). PKC- isoenzyme translocation-inhibitory peptide (V1-2+) and its negative peptide (V1-2; Johnson et al., 1996b) were also used. Both doses of Rottlerin significantly reduced acute ethanol-induced IR--EP release (Fig. 10), suggesting the possibility of involvement of both PKC- and - isoenzymes in the ethanol action. PKC- isoenzyme translocation-inhibitory peptide V1-2+ also reduced ethanol-induced IR--EP release as compared with ethanol alone-treated groups. The scrambled peptide V1-2, used as control, by itself moderately inhibited ethanol-induced IR--EP release, indicating that the permeabilization procedure used for the treatment of PKC- peptides may have some adverse effect on IR--EP release. However, IR--EP levels of cultures treated with V1-2+ were significantly lower than the V1-2-treated controls, suggesting that inhibition of PKC- isoenzyme translocation reduces the ability of ethanol to induce IR--EP release. A preliminary study determining the effect of PKC blocker Rottlerin (6 and 100 µM) on chronic ethanol-treated IR--EP release revealed very low or undetectable levels of IR--EP in media (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 10.   Effect of PKC blockers on ethanol-stimulated IR--EP secretion from hypothalamic neurons in primary cultures. A group of hypothalamic cell cultures grown for 9 days was pretreated with Rottlerin (6 µM for PKC-) or Rottlerin (100 µM for PKC-) for 1 h. The cultures were washed with fresh media and then incubated with media containing 50 mM ethanol or no ethanol (control) for 3 h. Another group of cultures was transiently permeabilized with saponin (50 µg/ml) with 150 µg/ml PKC- (V1-2+) or scrambled PKC- (V1-2) for 10 min and then incubated with media containing 50 mM ethanol or no ethanol (control) for 3 h. The media levels of IR--EP were measured by radioimmunoassay. Data are mean ± S.E. of six to nine cultures. a, p < 0.01, significantly different from control; b, p < 0.05, significantly different from ethanol alone; c, p < 0.05, significantly different from V1-2-treated group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present data for the first time demonstrate that the PKC-activating agent PMA stimulates ethanol-induced IR--EP release, and PKC-inhibiting agents cause inhibition of ethanol-induced IR--EP release. Furthermore, the data suggest that  and  PKC isoenzymes may be involved in ethanol-modulated IR--EP secretion from hypothalamic cells in primary culture.

The data presented here show that exposure of hypothalamic cells to ethanol or PMA causes stimulation of IR--EP secretion. These results corroborate our previous studies (Boyadjieva et al., 1997; Boyadjieva and Sarkar, 1997) and the findings of Kapcala et al. (1992). The PKC stimulator PMA also has been shown to increase pro-opiomelanocortin fragment release in static cultures of anterior pituitary cells (Abou-Samra et al., 1986). Hence, it appears that the PKC signaling pathway may be important in the regulation of -EP release in both the hypothalamus and the pituitary. In the present study, we found that ethanol-induced IR--EP release was potentiated by PMA. Furthermore, ethanol-induced IR--EP release was significantly reduced by the PKC inhibitor chelerythrine chloride. These results suggest that acute ethanol-regulated IR--EP release may involve the PKC system.

The multigene family of PKC enzymes is involved in the control of many biological events (Borgatti et al., 1996). Depending upon the cell types, the kind of agonist employed, and the length of stimulation that induces PKC activation, the cellular responses vary (Nishizuka, 1992; Dekker and Parker, 1994). Recent studies suggest that in neural cell lines, alcohol increases PKC activity (Messing et al., 1991). In addition, ethanol exposure causes an increase in PKC activity in platelets (Rabbani et al., 1999), human epidermal keratocytes (Kharbanda et al., 1993), macrophages (Rovera et al., 1979), human lymphocytes (Depetrillo and Liou, 1993), PC 12 cells (Messing et al., 1991) and NG 108-15 neuroblastoma-glioma cells (Gordon et al., 1997). Ethanol stimulates or inhibits various PKC isoenzymes in various cell types (Stubbs and Slater, 1999). It has been proposed that ethanol activates PKC by increasing levels of specific PKC isoenzymes (Messing et al., 1991; Gordon et al., 1997). We show herein that acute ethanol increases primarily  and  PKC isoenzymes in hypothalamic cells. The effect of ethanol on other PKC isoenzymes is very modest, if any.

In the present study, we have shown that ethanol causes an increase in the levels of  and  PKC isoenzymes in both cytosolic and membrane fractions. These two isoenzymes have been found to translocate to different cellular sites due to ethanol treatment in NG 108-15 cells (Gordon et al., 1998) and in human epidermal keratocytes (Kharbanda et al., 1993). Although ethanol-induced changes of PKC isoenzymes in the membrane fraction were not larger than those in the cytosolic fraction, ethanol increased the levels of  and  PKC isoenzymes in both cytosolic fraction and the membrane fraction. Furthermore, isoenzyme-specific blockers Rottlerin and V1-2+ inhibited ethanol-induced -EP release. Hence, these data support the idea that  and  PKC isoenzymes regulate ethanol-induced -EP release.

The basal and PMA-induced release of IR--EP was reduced after chronic ethanol treatment. This suggests that chronic ethanol down-regulates the PKC signaling system. The mechanism by which ethanol down-regulates the PKC system is not apparent from this study. Chronic ethanol consumption decreases phorbol ester binding and PKC activity in the cerebral cortex and hippocampus (Pandey et al., 1963; Battaini et al., 1989). These studies proposed two different mechanisms of ethanol down-regulation of the PKC system. Chronic ethanol may take part in phospholipid metabolism, which leads to the formation of phosphatidylethanol by the action of phospholipase D. This phosphatidylethanol may contribute to the down-regulation of PKC. An alternate mechanism is that chronic ethanol induces heterologous desensitization of membrane receptors leading to a decrease in PKC activity. Whether or not such mechanisms operate in -EP-secreting neurons following chronic ethanol needs to be studied.

The data showing desensitization of the PKC system following chronic ethanol are in contrast to those shown for PC 12 cells, where ethanol stimulates both  and  PKC isoenzymes. In the PC 12 cells, chronic ethanol stimulates both P- and N-type calcium channels (McMahon et al., 2000; Walter et al., 2000). Furthermore, ethanol-regulated expression of P- and N-type calcium channels involves  and  PKC isoenzymes in PC 12 cells (McMahon et al., 2000; Walter et al., 2000). Recently it has been shown that PKC is involved in the effect of acute ethanol on several ion channels. Chronic exposure to ethanol causes an increase in L-type voltage-gated calcium channels in PC 12 cells (Walter et al., 2000). Chronic ethanol has also been shown to increase the density and function of L-type voltage-gated channels in PC 12 cells via a PKC--dependent mechanism. Exposure to ethanol for a short period of time causes inhibition of L-type and N-type voltage-gated calcium channels (McMahon et al., 2000). These effects appear to be dependent on phosphorylation by PKC or cyclic AMP-dependent kinases or other kinases or proteins or neurotransmitters (Stubbs and Slater, 1999). We have previously shown that, in the hypothalamic -EP neurons, ethanol-activated -EP release involves P- and N-type calcium channels (De et al., 1999). Hence, it is possible that ethanol may increase -EP release by enhancing translocation of  and  PKC isoenzymes, leading to activation of P- and N-type calcium channels and neurosecretion. The down-regulation of  and  PKC isoenzyme expression following chronic ethanol may also be responsible for the decreased neurosecretion and development of ethanol tolerance in the -EP neurons. We have also shown that the cAMP system plays a significant role in both ethanol activation and desensitization of -EP neurons. Because both  and  PKC systems have been shown to be regulated by the cAMP signaling in the nervous system, it can be hypothesized that ethanol-activated neurotransmission in -EP neurons is partly mediated by the cross-talk between the cAMP-PKC-Ca²⁺ systems.