0013-722i/92/1~~11-0173~~:~ 00/o Endocrinolo~ Copyright G 1992 by The Endocrine

Vol. 131, No. 1 Printed in U.S A.

Society

Interaction of Ethanol with Signal Transduction Mechanisms Mediating Growth Hormone Release Pituitary Cells in Vitro* PIOTR

A. SOSZYNSKIt

AND

LAWRENCE

by Rat

A. FROHMAN

Division of Endocrinology and Metabolism, Department College of Medicine, Cincinnati, Ohio 45267

of

Internal

Medicine,

University

of

Cincinnati

ABSTRACT The effects of ethanol on signal transduction mechanisms of rat GH (rGH) release were investigated in primary culture of rat anterior pituitary cells. Ethanol (30, 100, and 300 mM) had no significant effect on basal rGH release or cell content after a 4-h incubation or on intracellular CAMP levels at 30 min. Ethanol did not alter rGRH (lo-” M)-stimulated rGH release, but at concentrations of 100 and 300 mM it inhibited rGRH (lo-” M)-stimulated rGH release by 12% (1) < 0.05) and 54% (P < 0.01). In contrast, a dose-dependent stimulatory effect was observed on rGRH-induced CAMP accumulation. Ethanol enhanced the inhibitory effect of SRIH (10-l’ and lo-” M) on rGH release by up to 24% (P < 0.01). Stimulation of rGH release by CAMP derivatives and forskolin was

partially inhibited by ethanol, as was CAMP accumulation after forskolin treatment. Cholera toxin-stimulated rGH release was also inhibited by ethanol, whereas CAMP accumulation was increased. At the higher concentrations, ethanol enhanced rGH release after protein kinase-C activation by phorbol ester and after stimulation of calcium influx with Ca ionophore. No significant ethanol effect was noted on prostaglandin E,-stimulated rGH release, and ethanol did not alter rGH mRNA levels or proliferation of a pituitary somatomammotroph cell line. The results indicate that ethanol exerts multiple effects on systems mediating GH release from the pituitary in uitro. However, the inhibitory influence of ethanol on GH secretion is related primarily to the adenylate cyclase-CAMP pathway, which represents the major signal transducing system in the somatotroph. (Endocrinology 131: 173-180, 1992)

E

signal transduction is derived from studies in brain and liver, in which alterations in the activity of several intracellular pathways [adenylate cyclase-CAMP, protein kinase-C, calcium-calmodulin, and arachidonic acid-prostaglandins (PGs)] have been shown (16-18). Since all of these pathways are involved in GH synthesis and/or release (19, 20), the effects of ethanol may have important physiological consequences. The present study was designed to investigate the effects of ethanol, in concentrations reflecting moderate (30 mM) to severe (100 mM) intoxication in viva and at a higher concentration (300 mM), on rat GH (rGH) release from dispersed anterior pituitary cells in vitro by evaluating the interaction of ethanol with the major signal transduction pathways of GH secretion. In addition, the effects of alcohol were examined on rGH mRNA levels and somatotroph proliferation.

THANOL exerts adverse effects on many physiological processes, with hormone secretion frequently being affected (1, 2). Numerous in viva studies in animals and humans have shown that ethanol decreases circulating GH levels after both acute and chronic administration (3-7). The site of the acute effects of ethanol on GH secretion has not been precisely determined, although most evidence suggests an alteration of hypothalamic function (4, 8, 9) in a manner similar to the perturbation of other endocrine axes, such as GnRH-LH (10) or CRH-ACTH (11). However, ethanol has been reported to directly inhibit basal GH (12) and GnRHstimulated LH release (13), and to stimulate PRL secretion (14) from pituitary cells in vitro, indicating a multiplicity of target sites. The molecular basis of the effects of ethanol has been widely characterized. Ethanol is capable of influencing cell membrane fluidity and membrane phospholipid composition as well as independently altering membrane-associated enzyme activity and signal transduction pathways (1517). However, the magnitude and nature of the effects vary with the type of tissue or cell involved as well as the concentration and duration of ethanol exposure (17). Most of the information regarding ethanol’s effects on molecular

Materials and Methods Cell culture

and experimental

protocols

Pituitaries for dispersion were obtained from male Sprague-Dawley rats (175-200 e: Harlan, Inc.. Indianauolis. IN). Animals were housed under controllrd envir&mental cond&ons (temperature, 23 t 1 C; constant humidity; lights on, 0600-1800 h), with food and water provided nd libitum. Anterior pituitary glands were removed and enzymatically dispersed, as previously described (21). The dispersed cells (2 X lo5 cells in 25 rl) were plated onto the surface of 24.well culture plates. After a l-h attachment period, the cells were flooded with 1 ml aModified Eagle’s Medium ((UMEM; Sigma Chemical Co., St. Louis, MO) containing 25 pg/ml gentamicin sulfate (Gibco, Grand Island, NY) and 10% horse serum (Flow Laboratories, Inc., McLean, VA) and placed in a humidified incubator at 37 C in a 95% air-5% CO2 atmosphere.

Received January 23, 1992. Address all correspondence and requests for reprints to: Lawrence A. Frohman, M.D., Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, 231 Bethesda Avenue, ML 547, Cincinnati, Ohio 45237-0547. * This work was supported in part by USPHS Grant DK-30667. t Visiting scientist from the Department of Endocrinology, Medical Center for Postgraduate Education, Warsaw, Poland; supported by a research fellowship from the Fogarty International Center.

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ETHANOL

174

AND

SIGNAL

TRANSDUCTION

MECHANISMS

125

125

OF GH RELEASE

Endo. Voll31.

rGRH , 1O-“M

1992 No 1

rGRH, 1O-‘M

0 0

0

125

:

I

100 8

-

0

i?e a-w6

rGRH , 1O-“M

500

0

75

50

E 0 25

0

-

!I I -

0 b g 0 z M

400

300

+= G 200 44 E 2

100

4

0

125

0

125

L

SRIHI lo-“M

SRIH. 1O-gM T I

0

Ethanol, mM

1. Effects of ethanol on basal rGH release (top), rGH cell content (middle), and intracellular CAMP accumulation (bottom) in dispersed anterior pituitary cells. Shown are the mean + SEM of normalized data, expressed as a percentage of the control value (0 mM ethanol) from two to four separate experiments. Basal rGH secretion was 1,677 t 60 ng/4 h/2 x lo5 cells, basal rGH content was 18,138 + 1,617 ng/2 x lo” cells, and basal CAMP content was 0.51 + 0.05 pmol/30 min/2 x lo5 cells in the absence of ethanol.

rGRH, 1OmgM

FIG.

I(

Ethanol, mM

2. Effects of ethanol on GRH-stimulated rGH release (top) and intracellular CAMP accumulation (middle), and SRIH-inhibited rGH release (bottom) in dispersed anterior pituitary cells. Shown are the mean + SEM of normalized data expressed as a percentage of the control value (0 mM ethanol) from two to four separate experiments. Top, Basal rGH secretion was 1,687 f 59 ng/4 h/2 X lo5 cells in the

FIG.

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ETHANOL

AND

SIGNAL

TRANSDUCTION

After a 3-day culture period, medium was removed from the cells and replaced with 1 ml nMEM containing 0.1% BSA for a 60.min preincubation period. Fresh cvMEM with various test substances or vehicle was then added in a total volume of 1 ml. The cells were incubated at 37 C for periods of 30 min for measurements of CAMP or for 4 h for measurement of rGH (and CAMP when cholera toxin was the stimulus) (20). At the end of the incubation period, the medium was removed and stored at -20 C for measurement of rGH. The cells were extracted with 0.01 M NaOH for determination of intracellular rGH content or with 0.1 M HCI in 95% ethanol for assay of intracellular CAMP. Each experimental protocol was repeated at least twice. In experiments designed for rGH mRNA determination, 1.5 x lo6 cells were plated in 12-well plates and cultured in nMEM containing 10% horse serum for 3 days. Cells were then washed with tvMEM containing 0.1% BSA, as described above, and cultured in the same medium in the absence or presence of ethanol for the following 4 days. Medium was changed daily. At the end of the culture period, medium was removed, and total RNA was isolated from cells by a single step acid-guanidinium-phenol-chloroform extraction (22). A previously described rat somatomammotroph cell line (I’(,) was used to evaluate the effects of ethanol on cell proliferation (23). Cells were cultured in control or ethanol-containing aMEM with 10% horse serum under the environmental conditions described above. Cells were plated onto a six-well plate in a density of 5 X 10’ for 6-day culture and 2.5 X 10’ for lo-day culture; medium was supplemented every second day. At the end of the experiment, cells were removed from wells by trypsinization, and the proliferation rate was determined by cell counting.

RIAs rGH was measured by a specific RIA, as previously described (24). The results were expressed in terms of the NIDDK rGH RP-1 reference standard, with intra- and interassay coefficients of variation of 6.5% and 8.9%, respectively. CAMP levels were determined by RIA (25), with intra- and interassay coefficients of variation of 5.8% and 7.5%, respectively.

Northern

blot hybridization

Equal aliquots of total RNA (2 fig) were electrophoresed in formaldehyde-agarose (1%) gel and transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH). The membrane was baked for 1 h at 80 C and hybridized with a nick-translated V-labeled plasmid, prGH-1, containing the rGH cDNA insert, as described previously (26). After autoradiography of the hybridized membrane, the rGH mRNA signal was evaluated by scanning densitometry.

Statistical

analysis

Data from two to four separate experiments performed with each test agent were normalized by expressing the results as a percentage of the mean control value (i.e. in the absence of ethanol) in each set of incubations and then combined for statistical analysis. Differences between groups were compared by an analysis of variance, followed by Duncan’s multiple range test. The results are expressed as the mean + SEM.

Chemicals

MECHANISMS

acetic acid containing 0.05% BSA, and subsequently diluted with PBS1% BSA. Forskolin (Calbiochem, La Jolla, CA), [email protected] acetate (PMA; Sigma) and prostaglandin E2 (PGE,; Sigma) were dissolved in absolute ethanol and further diluted with PBS-l% BSA. (Bu)~cAMP (Sigma), 8-bromo-CAMP (8-Br-CAMP; Sigma), and cholera toxin (Sigma) were dissolved and diluted in PBS-l% BSA. Calcium ionophore (A23187; Sigma) and I-oleyl-2-acetyl-sn-glycerol (OAG; Sigma) were initially dissolved in dimethylsulfoxide and further diluted with PBS-l% BSA.

Results Effects of ethanol

hormone (rGRH) Inc., Belmont, CA)

and somatostatin (SRIH; Peninwere initially dissolved in 0.05 M

absence of ethanol or GRH. Control values (0 mM ethanol) represent 414% (10-i’ M rGRH) and 760% (lo-’ M rGRH) of the basal value. Middle, Basal CAMP content was 0.51 + 0.05 pmol/30 min/2 x 10” cells in the absence of ethanol or GRH. Control values (0 mM ethanol) represent 1,392% (10-i’ M rGRH) and 10,581% (10e9 M rGRH) of the basal value. Bottom, Basal rGH secretion was 1,776 f 77 ng/4 h/2 x lo5 cells in the absence of ethanol or SRIH. Control values (0 mM ethanol) represent 80% (lo-ii M SRIH) and 12% (10e9 M SRIH) of the basal value. *, P < 0.05; **, P < 0.01 [us. control (0 mM ethanol)].

on basal rGH and CAMP levels

After a 4-h incubation, ethanol (30, 100, and 300 mM) had no significant effect on basal rGH releaseor cell content (Fig 1). Intracellular CAMP levels were also unchanged in the presenceof all ethanol concentrations. Interaction

of

ethanol

with GRH and SRIH

GH secretion in responseto GRH at a concentration (10-l’ previously shown to produce half-maximal stimulation was not significantly altered by ethanol (Fig. 2). However, at concentrations of 100 and 300 mM, ethanol impaired rGH release in response to a maximal stimulatory concentration of GRH (lo-” M) by 12% (P < 0.05) and 56% (P < O.Ol), respectively. In contrast, intracellular CAMP accumulation in response to both GRH concentrations was markedly enhanced by ethanol in a dose-dependent manner (Fig. 2). CAMP responses to GRH (lo-” M) were significantly increasedin the presence of 100 and 300 mM ethanol by 66% (P < 0.05) and 340% (P < O.Ol), respectively. Ethanol produced a dose-dependent enhancement of GRH-stimulated (lo-” M) CAMP accumulation, with increases of 32% (P < 0.05), 63% (P < O.Ol), and 135% (P < 0.01) at 30, 100, and 300 mh4,respectively. The effect of a half-maximal inhibitory concentration of SRIH (lo-” M) on rGH releasewas enhanced at 100 and 300 mM ethanol by 11% (P < 0.05) and 24% (P < O.Ol), respectively (Fig. 2). The suppressionof rGH secretion by a maximal inhibitory concentration of SRIH (10m9M) was also enhanced by 14% (P < 0.05), 20% (P < O.Ol), and 19% (P < 0.01) in the presence of 30, 100, and 300 mM ethanol, respectively. Intracellular CAMP levels were markedly suppressedby SRIH (10-l’ M and lo-’ M) and were not significantly altered by the presence of ethanol at any of the concentrations (data not shown). M)

Interaction

Rat GH-releasing sula Laboratories,

175

OF GH RELEASE

of

ethanol

with CAMP, forskolin,

and cholera toxin

Ethanol significantly impaired rGH releasein responseto (Bu)*cAMP (5 X 10m3M) by 11% (P < 0.05) at 30 mM, by 14% (P < 0.01) at 100 mM, and by 46% (P < 0.01) at 300 mM (Fig. 3). Similar effects were observed with 8-Br-CAMP (data not shown). Forskolin (lo-” M)- and cholera toxin (lo-” M)-stimulated rGH release was inhibited by ethanol (Fig. 3). The impairment of the effects of forskolin was observed at all concentrations, with 12%, 21%, and 42% (P < 0.01 each) attenuation in the presenceof 30, 100, and 300 mM ethanol, respec-

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ETHANOL

176 1251

:

5

(B&.cAMP,

loo-

AND SIGNAL

TRANSDUCTION

**

l *

lli z

Endo. 1992 Vol 131 -No 1

Forskolin-stimulated CAMP accumulation was markedly inhibited at the higher ethanol concentrations, with 25% and 46% decreases (P < 0.01 each) at 100 and 300 mM, respectively. In contrast, ethanol enhanced CAMP accumulation in response to cholera toxin by 16%, 23%, and 83% (P < 0.01 each) at 30, 100, and 300 mM, respectively (Fig. 3).

8

b x

OF GH RELEASE

tively. Impairment of cholera toxin-stimulated rGH release was observed at the higher ethanol concentrations, with 12% and 22% decreases (P < 0.01 each) at 100 and 300 mM, respectively.

10B3M

x

MECHANISMS

75-

50 -

0

I!

Interaction PGE,

25 F O0 125

1

0

30

Forskolin,

100

300

1O’%

Ch. Toxin,

+*

7

I 1

30

F orskolin,

lo-“M

100

300

1 OB6M

30

100

I. Toxin , lo-“M

z 150

**

300

+* T

OAG, calcium

ionophore,

and

on rGH mRNA

levels

Four days of culture in medium containing the three ethanol concentrations (30, 100, and 300 mM) had no significant effect on the level of rGH mRNA in dispersed rat anterior pituitary cells, as revealed by Northern blot analysis (Fig. 5). I ’ Effect of ethanol

on cell proliferation

Pituitary somatomammotroph (PO) cell line proliferation was not affected by any of the ethanol concentrations used (Fig. 6). A slight inhibition of I’,, cell growth, observed after 6 and 10 days of culture at a highest ethanol concentration (300 mh4), was not statistically significant.

**

z x ; 5 E 0 0

with PMA,

Phorbol ester (PMA; lo-” M)-stimulated rGH release was enhanced by 23% (P < 0.01) at 300 mM ethanol. Ethanol did not significantly modify the rGH response to OAG (10m4 M) (Fig. 4). However, OAG-stimulated rGH release was of a relatively small magnitude (18% above basal; P < 0.05). Calcium ionophore (A23187; 10mhM)-stimulated rGH secretion was significantly enhanced by ethanol, with 19% (P < 0.05) and 45% (P < 0.01) increases at 100 and 300 mM ethanol, respectively (Fig. 4). Ethanol exhibited no significant effect on PGE, (10m7 M)-stimulated rGH release, despite its marked enhancement of the CAMP response (data not shown) at 100 mM (107%) and 300 mM (281%; P < 0.01 each). Effect of ethanol

200

E z

+*

of ethanol

100

Discussion 50

4 0 0 0

30

100

300

Ethanol,

0

30

100

300

mM

FIG. 3. Effects of ethanol on (Bu)zcAMP-stimulated rGH release (top), forskolin- and cholera toxin (Ch. Toxin)-stimulated rGH release (middle), and intracellular CAMP accumulation (bottom) in dispersed anterior pituitary cells. Shown are the mean f SEM of normalized data, expressed as a percentage of the control value (0 mM ethanol) from two separate experiments. Top, Basal rGH secretion was 1513 & 48 ng/ 4 h/2 x lo5 cells in the absence of ethanol or (Bu)+AMP. The control

The results of the present study indicate that ethanol, in concentrations comparable to those observed during intoxication in viva, exerts no major effects on basal rGH release or CAMP levels in dispersed pituitary cells. However, it modifies some of the signal transduction pathways mediating value (0 mM ethanol) represents 630% of the basal value. Middle, Basal rGH secretion was 1344 f 131 ng/4 h/2 x lo” cells in the absence of ethanol, forskolin, or cholera toxin. The control values (0 mM ethanol) represent 892% (1OmfiM forskolin) and 578% (lo-” M cholera toxin) of the basal values. Bottom, Basal CAMP content was 0.44 t 0.05 pmol/2 x lo5 cells in the absence of ethanol, forskolin, or cholera toxin. Control values (0 mM ethanol) represent 5446% (lo-’ M forskolin) and 2995% (10-l’ M cholera toxin) of the basal values. *, P < 0.05; **, P < 0.01 [US. control (0 mM ethanol)].

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ETHANOL

AND

PMA, 10-sM

150

SIGNAL

TRANSDUCTION

MECHANISMS

OF GH RELEASE

OAG, 10-4M

Control

GRH, 1O-8 M

1

30 0 30

175 02

150

z o

125

z M

100

2 a.%-

75

Q) F

50

1

100

300

PGE2, 10-‘M

A231 87, lo-‘M

177

I

100 300 Ethanol, 10” M

FIG. 5. Northern blot analysis of RNA from dispersed pituitary cells, demonstrating rGH mRNA levels in cells cultured for 4 days at three concentrations of ethanol. Shown are duplicate lanes of each experimental group. Each lane contains 2 bg total RNA from individual cultures of 1.5 X lo6 cells. 10.0

6 days 90 F

IO days

7.5

X

= g \ co = E a”

5.0

2.5

0 0

30

100

300

30

100

300

Ethanol, mM FIG. 4. Effect of ethanol on PMA- and OAG-stimulated (top) and A23187- and PGE&imulated (bottom) rGH release in dispersed anterior pituitary cells. Shown are the mean f SEM of normalized data, expressed as a percentage of the control value (0 mM ethanol) from two separate experiments. Top, Basal rGH secretion was 1985 f 277 ng/4 h/2 x 10’ cells in the absence of ethanol, PMA, or OAG. Control values (0 mM ethanol) represent 299% (10m9 M PMA) and 118% (lo-* M OAG) of the basal values. Bottom. Basal rGH secretion was 1392 * 66 ng/4 h/2 x lo5 cells in the absence of ethanol, A23187, or PGE,. Control values (0 mM ethanol) represent 128% (10m6 M A23187) and 501% (W7 M PGE2) of the basal values. *, P < 0.05; **, P < 0.01[us. control (0 mM ethanol)]. rGH secretion in response to stimulation. Other parameters of somatotroph function, such as rGH mRNA expression or cell proliferation, were not significantly affected by ethanol. The lack of an effect of ethanol on rGH cell content or release under basal conditions in the present study is in contrast to the report of Emanuele et al. (12), which described short term and prolonged dose-dependent ethanol-induced inhibition of rGH release from pituitary cells in vitro. The explanation for this discrepancy is not readily apparent, since both experiments used similar concentrations of ethanol and duration of incubation. The suppression of rGH release by more than 90% after 4-h incubation and the 50% decrease

0

1 0

30

100

i 300

0

30

100

300

Ethanol, mM

FIG. 6. Effect of ethanol on rat pituitary somatomammotroph cell line (P,) proliferation. Values are expressed as the mean + SEM of a single experiment (n = 6). Cells were plated at a density of 5 X 10’ for the 6day and 2.5 x 10’ for the lo-day cultures. in rGH cell content after 48-h exposure to ethanol in the latter study suggest severe cellular toxicity of alcohol, which was not seen in our experiments or in other studies conducted under similar conditions (13, 14, 27). GRH and SRIH constitute the two major physiological modulators of GH secretion (19, 28). The interplay between these neurohormones regulates the normal pattern of GH secretion (28). Thus, the potential interaction of ethanol with GRH or SRIH at the pituitary level could alter GH release. In the present study ethanol had no significant effect on the rGH response to moderate stimulation by rGRH (10-l’ M), but substantially decreased the rGH response to maximal stimulation by rGRH (1O-9 M). In contrast to its effects on rGH release, ethanol markedly enhanced intracellular CAMP accumulation in response to GRH. An increase in CAMP production after alcohol administration has been previously reported (16). Most evidence indicates that this effect is exerted by activation of the stimulatory subunit of the G-

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ETHANOL

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protein (G,cu). This action is most pronounced when G,oc is concomitantly activated by specific stimulation, related to occupancy of a linked membrane receptor (29). Our findings, of minimal effects of ethanol on basal CAMP levels but substantial enhancement of stimulated CAMP responses, are consistent with the actions of alcohol observed in other studies using pineal (30), neural (31), and hepatic (32) tissue. Although dispersed pituitary preparations include a mixture of different cell populations, the enhancement of GRHstimulated CAMP accumulation by ethanol indicates that this effect is specific to somatotrophs. The discrepancy between the rGH and CAMP responses in our study may be explained by down-regulation or desensitization of the CAMP-dependent pathway of intracellular signalling by high CAMP levels (33). The rGH release-inhibiting activity of SRIH was enhanced by ethanol in the present study. While inhibition of GH secretion by SRIH may result from several potential actions, suppression of CAMP production by stimulation of the inhibitory G-protein (G,) plays an essential role (34). Since the G, and G, proteins are similar in structure and mechanism of action, ethanol could potentially enhance G, a-subunit function in response to specific stimulation. However, this effect may be normally overbalanced by the marked enhancement of G, function (16). Ethanol administration has also been reported to enhance other actions of SRIH in the central nervous system (35). While these findings suggest that the impairment of GH secretion in vivo is caused by a direct pituitary action, most of the effects are of small magnitude and become significant only at an ethanol concentration of 100 mM, which is comparable to levels observed during severe intoxication (18). Moreover, intense stimulation by GRH is required to show ethanol inhibition of GH release. Although there are very limited data on GRH levels in rat portal blood, peak values as high as 2 X lo-“’ M have been reported (19), which is nearly comparable to the highest concentration of GRH used in our study. Acute (3) and prolonged (Soszynski, P. A., and L. A. Frohman, manuscript in preparation) ethanol administrations severely inhibit pulsatile rGH secretion, which is predominantly GRH mediated (36). Since the suppression of GH release by ethanol is only partial, the effects of ethanol at the level of the pituitary in vivo are probably less important than those occurring within the central nervous system (4, 8). To clarify the mechanisms by which ethanol interferes with rGH release, other factors acting via the G-proteinadenylate cyclase-CAMP pathway were tested. Cholera toxin catalyzes ADP ribosylation of the G, a-subunit of the Gprotein, causing its activation (37), thereby mimicking the effects of GRH binding to the somatotroph receptor. Cholera toxin-induced rGH release was decreased, and CAMP accumulation was increased by ethanol in a pattern similar to that found after GRH stimulation. These data confirm that G, is one of the primary targets of ethanol action in somatotrophs. Enhancement of CAMP responses to cholera toxin by ethanol has also been described in striatal membranes (38). Forskolin is a plant diterpene that binds to the G,adenylate cyclase complex and directly stimulates the cata-

MECHANISMS

OF GH RELEASE

Endo * 1992 Vol 131. No 1

lytic unit of adenylate cyclase (39). It has been previously shown that forskolin-activated CAMP synthesis is inhibited by ethanol in neural (40) and mesenchymal(41) cells in vitro. The mechanism of this effect of ethanol, which is in contrast to its enhancement of CAMP accumulation in response to other stimuli acting via the receptor-G,-protein complex, is not clear, although ethanol has been reported to inhibit forskolin binding (42). In the present study the impairment of forskolin-stimulated rGH release was accompanied by an inhibition of intracellular CAMP accumulation. Further evidence for ethanol impairment of signal transduction in somatotrophs was provided by examining its effects on the stimulation of rGH release by CAMP derivatives. A dose-dependent impairment of CAMP-stimulated rGH secretion was observed, but marked inhibition was found only at the highest ethanol concentration (300 mM). There are limited data available on the action of alcohol on post-CAMP pathways, although ethanol has been reported to inhibit CAMP binding to the regulatory unit of protein kinase-A as well as decrease the activity of this enzyme in vivo (43, 44). This may indicate that ethanol inhibition of the stimulatory effects on rGH release of factors acting via the CAMP pathway also results from a direct impairment of protein kinase-A activation. Thus, while the effects of ethanol on CAMP responses were dependent on the site of stimulatory factor action, the final biological result was the same, i.e. suppression of rGH release. The role of other signal transduction pathways in GH secretion has been less extensively defined, although rGH release is stimulated by protein kinase-C activators, increases in calcium influx, and PG derivatives (19, 20). All of these pathways are potential targets of ethanol action (16-18). In the present study ethanol exhibited a relatively small influence on rGH release in response to the simulation of protein kinase-C by PMA or OAG. It has been previously shown that inositol phosphate production and subsequent protein kinase-C activation are transiently stimulated by ethanol, followed by rapid desensitization and inhibition of enzyme activity (45). Thus, the increase in rGH release after PMA in the presence of a high concentration of ethanol may reflect the initial stimulatory effect of both agents on this pathway. Ethanol has also been shown to enhance nonvoltage-gated calcium influx and calcium-mediated responses (17, 46). The enhancement by ethanol of rGH release after A23187 treatment in the present report is consistent with the above observations. PGE*-stimulated rGH secretion was not significantly affected by ethanol despite the enhancement of CAMP accumulation. The latter observation confirms a previous study, which showed that ethanol potentiates PGE,-mediated CAMP formation (47). Overall, it appears that rGH release mediated by signal transduction mechanisms other than the CAMP-mediated pathway is influenced by ethanol to a lesser extent. The present study has demonstrated that ethanol may alter several intracellular mechanisms modulating rGH release. As in other in vitro systems, ethanol markedly enhanced CAMP accumulation, although only in conjunction with G, protein stimulation. However, the increase in CAMP

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ETHANOL

AND

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TRANSDUCTION

levels was not accompanied by an increase in CAMP-mediated responses. In fact, an impairment of the rGH secretory response has been shown despite activation of the CAMPassociated pathway of signal transduction using stimuli acting at the membrane receptor, the G, protein, adenylate cyclase, and post-CAMP sites. When stimuli acting at steps beyond the G, protein were used (forskolin and CAMP analogs), the inhibitory effects of ethanol were demonstrated even at the lowest concentration (30 mM), which is comparable to circulating ethanol levels in mild intoxication in vim (18). GH release in response to agents acting via other pathways, such as the calcium channel (A23187) or protein kinase-C (PMA or OAG), was unchanged or even enhanced by ethanol, suggesting that its inhibitory effect on hormone secretion may be limited to CAMP-mediated mechanisms. Since the net effect of ethanol on GH secretion is inhibitory, the predominant intracellular action of this agent most likely affects post-CAMP mechanisms. The effects of ethanol reported here may only be of an acute nature, since desensitization and tolerance have been described after prolonged exposure (48). The pituitary may be less sensitive to such effects, since exposure of cells to 100 mM ethanol for 48 h produced effects on the rGH and CAMP responses to rGRH or forskolin stimulation comparable to those occurring at 4 h (our unpublished observation). In conclusion, our results indicate that ethanol exerts multiple effects on systems mediating GH release from the pituitary in vitro. However, the inhibitory influence of ethanol on GH secretion is related primarily to the adenylate cyclase-CAMP pathway, which represents the major signal transducing system in the somatotroph. Acknowledgments The authors with to thank Tom Downs for his advice and suggestions. Rat GH was provided by the NIDDK National Hormone and Pituitary Program.

References

5.

6.

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