0013-7227/92/1315~2077s03.00/0 Endocrinology Copyright G 1992 by The Endocrine

Vol. 131, No. 5 Printed in U.5’ A

Society

The Effect of “Binge” Ethanol Exposure Hormone and Prolactin Gene Expression M. A. EMANUELE, M. R. KELLEY

J. J. TENTLER,

L. KIRSTEINS,

N. V. EMANUELE,

on Growth and Secretion*

A. LAWRENCE,

AND

Departments of Medicine (M.A.E., N.V.E., A.L.) and Molecular and Cellular Biochemistry (M.A.E., J.T.T., A.L., M.R.K.), and the Molecular Biology Program (M.A.E., N.V.E., M.R.K.), Loyola University Stritch School of Medicine, Maywood, Illinois 60153; and the Research and Medical Services, Veterans Administration Hospital (M.A.E., L.K., N. V.E., A.L.), Hines, Illinois 60141 ABSTRACT The effects of ethanol (EtOH) on GH and PRL have been previously explored, and a dicotomy in results noted. While serum GH levels appear to fall after EtOH exposure, PRL levels rise. We have attempted to expand these studies by examining the impact of acute or “binge” EtOH in uiuo on GH and PRL synthesis and secretion. At 0.5, 1.5, and 3.0 h after one dose of ip EtOH, serum GH levels fell significantly compared with those seen in saline-injected controls. This correlated with a fall in GH mRNA levels, but no change in pituitary GH content. Conversely, serum PRL levels rose significantly, while the mRNA for

PRL decreased by approximately 20%. There was no change in pituitary PRL content. Interestingly, the mRNA for pit-l (GHF-1), a transcription factor important to both GH and PRL gene expression, was unchanged at any time point. Despite the fall in GH and PRL mRNA levels, the pituitary CAMP content was markedly elevated at 0.5 h, with no change at any other time point. In summary, acute EtOH exposure in viuo appears to dampen both GH and PRL synthesis, while serum levels behave dissimilarily. Possible explanations for these findings are discussed. (Endocrinology 131: 2077-2082,1992)

T

animals were given either an ip injection ethanol in a dose of 30% (vol/ vol; 1 cc/SO g BW; 3 g/kg) or saline and killed 0.5, 1.5, 3, or 24 h later (n = 5-6/group at each time point). At the time of death, trunk blood was obtained for ETOH, GH, and PRL determinations. The pituitary was rapidly removed, the posterior pituitary was gently lifted away, and the anterior pituitary was frozen in liquid nitrogen until it could be further processed for RNA extraction and GH and PRL RIAs. Blood EtOH determinations were made using a fluorometric kit obtained from Sigma Chemical Co. (St. Louis, MO).

HE SUPPRESSIVE effect of ethanol (EtOH) on GH secretion and its stimulating effect on PRL release have been previously investigated, and both a pituitary (l-5) and hypothalamic/central nervous system locus of EtOH action have been postulated (6). Our goal is to further elucidate the biochemical and molecular mechanismsfor the induced fall in serum GH and concomitant rise in PRL levels. This report deals with the effects of EtOH at the pituitary level on both GH and PRL synthesis and secretion. To determine the effects of EtOH on GH and PRL regulation, we report here the results of time-course studies correlating serum GH and PRL concentrations with pituitary content and steady state levels of GH and PRL mRNA. These results were compared with pituitary CAMP levels, since this is a known modulator in the synthesis and release of GH and PRL (7). The steady state mRNA levels for Pit-l (GHFl), a transcription factor known to regulate high levels of expression of both GH and PRL in somatotroph and lactotroph cells in the pituitary (8), were also assessed. Materials

and Methods

Adult male Sprague-Dawley rats, 60-90 days old and weighing between 250-300 g, were used for these studies. The animals were housed in a 12-h light, 12-h dark environment and given food and water ad libitum. At 0900 h on the morning of the experiments, the

GH RIA The GH RIA was conducted using materials generously contributed by the NIDDK and the National Hormone and Pituitary Program through Dr. A. F. Parlow. Details of the RIA by our laboratory have been previously described (9). The intraassay coefficient of variation was 4%, while the interassay coefficient of variation was 8%.

PRL RIA PRL antibody for use in the RIA was contributed by the NIDDK, National Hormone and Pituitary Program, through Dr. A. F. Parlow. These assays have been operative in our laboratory for 5 yr (10). The interassay coefficient of variation was 24%; the intraassay coefficient of variation was 7%.

Pituitary

GH, PRL, and Pit-l

mRNA determinations

Total RNA was extracted from individual rat pituitaries, and levels of GH, PRL, and Pit-l mRNA were determined using Northern blot hybridization analysis. Total RNA was isolated using a modification of an established procedure (11, 12). Each pituitary was homogenized in 4 M guanidinium thiocyanate and extracted with sodium acetate, phenol, and chloroform, and the RNA was pelleted using isopropanol. Northern blots of RNA from each sample were performed, as described previously (12), loading 10 Kg RNA/lane. The RNA was fixed onto Nytran membrane using a Stratagene UV oven. Hybridization was accomplished under stringent conditions in 50% formamide in a Robbins hybridization incubator at 42 C, and the filters were washed in 0.2 X SSC-0.5% SDS

Received April 3, 1992. Address all correspondence and requests for reprints to: Mary Ann Emanuele, M.D., Department of Molecular and Cellular Biochemistry, 2160 South First Avenue, Maywood, Illinois 60153. *This work was supported by NIAAA Grant ROl-AA-06755 (to M.A.E., N.V.E., and M.R.K.), a V.A. Merit Award (to N.V.E.), a Claire and Leonard Tow Foundation grant (to A.L.), and a Schweppe Career Development Award (to M.R.K.).

2077

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EFFECT

2078

OF EtOH

ON GH AND

at 65 C. The filters were exposed to x-ray film from Amersham (Arlington Heights, IL) for 4 h to overnight. Autoradiographs were analyzed using a scanning densitometer. Sometimes filters, after GH, PRL, or Pit-l cDNA hybridization, were analyzed using the Betascope analyzer from Betagen (Waltham, MA). After analysis for GH, PRL, and Pit-l mRNA, the filters were stripped and reprobed with the cDNA for ribosomal protein gene PO to control for loading differences (13). The gene has been previously shown by this lab not to be affected by EtOH (in preparation). Previous studies using the @-actin cDNA clone as a loading control were less than satisfactory (12). After scanning, the data were corrected for loading differences.

PRL GENE EXPRESSION

800 9 0

Endo. Voll31.

1992 No 5

OCONTROL 0s EtOH

700

5 0 I-

600 ~E

z 3’ Ls2, F 0 5 E m

500 400 300 200 100

CAMP levels Briefly, individual pituitaries were homogenized in 200 ~1 ice-cold 10 rnM PBS, with 4 mM EDTA added as a phosphodiesterase inhibitor. Samples were deproteinized by boiling for 3 min and then centrifuged at 12,000 rpm for 10 min. The CAMP content of the supernatant was measured using a RIA kit (Amersham) based on competition with tritium-labeled CAMP. All samples were measured in triplicate. Radioactivity was quantified by liquid scintillation counting.

1.5

3.0

TIME

24

0

(hours)

FIG. 1. Time-course effect of acute EtOH exposure on serum GH levels. Trunk blood was obtained 0.5, 1.5, 3, and 24 h after an ip injection of EtOH or saline. Serum levels were significantly depressed at 0.5, 1.5, and 3 h; while the downward trend was still present at 24 h, this was not significant. n = 5-6/group. Data are expressed as a percentage of the control value (100%). *, P < 0.05; **, P < 0.01.

cDNA clones for PRL, GH, and Pit-l The GH cDNA clone was obtained from Dr. John D. Baxter, University of California (San Francisco), and the PRL cDNA clone was given to us by Dr. Richard Maurer, University of Iowa (Iowa City). The Pit-l cDNA clone was provided by Holly A. Ingraham in Dr. Michael G. Rosenfeld’s laboratory (La Jolla, CA).

Analysis

-ii E

0 CONTROL FZA EtOH

of data and statistics

-TL

The data were analyzed by analysis of variance, with Bon-Ferroni follow-up. Significant differences were reported for P < 0.05.

Results

Blood EtOH levels were 360 mg/lOO ml at 0.5 h, 260 mg/ 100 ml at 1.5 h, 220 mg/lOO ml at 3 h, and unmeasurableat 24 h. Large differences in serum GH levels in the control salineinjected animalsprobably reflect the normal pulsatility noted by others in GH (7, 14-17). Despite this highly variable baseline, serum GH levels were significantly depressed at 0.5, 1.5, and 3 h in the EtOH-treated animals compared to values in saline-injected controls (Fig. 1). At 0.5 h after injection, the control group had values of 119 + 4 rig/ml compared to 22 + 3 rig/ml in the EtOH-treated animals. At 1.5 h, the levels were 311 f 100 rig/ml in the control and 80 + 15 rig/ml in the EtOH-exposed rats, and at 3 h, they were 636 + 111 and 162 f 10 rig/ml in the control and EtOHtreated animals, respectively. There continued to be a downward trend in EtOH-treated animals at 24 h (97 + 21 rig/ml for the saline-injected group compared to 42 + 9 rig/ml for the EtOH-treated group), but this was not statistically significant. Pituitary GH levels were unchanged between the two groups at any time point (Fig. 2). The pituitary GH contents at 0.5 h were 27,500 f 4,300 (control) and 19,700 f 6,000 rig/ml (EtOH); at 1.5 h, the levels were 65,100 + 4,400 and 52,200 + 4,200 rig/ml in the control and EtOH-treated animals, respectively. While a trend was noted toward increasedpituitary GH content at 3 h (30,900 + 7,200 rig/ml

d 0.5

1.5 TIME

3.0

24

0

(hours)

FIG. 2. The effect of acute EtOH

on pituitary GH content. No icant difference was found at any time point, although there trend for GH levels to be higher in EtOH-treated animals at 3 h. n = &g/group. Data are expressed as a percentage of the value.

signifwas a and 24 control

in the control group; 63,800 + 20,500 rig/ml in the EtOH group), this did not achieve statistical significance. At 24 h, there was also a tendency for the GH content to be higher in the EtOH group (29,100 + 16,900) compared to the EtOHexposed animals (59,400 + 29,400 rig/ml), but this was not significant. In contrast, serum PRL values were significantly higher in the ETOH-treated animals at 1.5 h (11 f 2 rig/ml in the control group; 23 f 5 rig/ml in the EtOH group) and 3 h (8 + 2 and 22 k 3 rig/ml in control and EtOH groups; Fig. 3). By 24 h, there was no change between the two groups in serum PRL levels (control, 10 + 1 rig/ml; EtOH, 7 + 1.5 mg/ ml). Similarily, there was no change in serum PRL 0.5 h after EtOH exposure (control, 11.5 f 3.5 rig/ml; EtOH, 12.0 + 2.7 n&4. The pituitary PRL content was not different between the two groups at any time point (Fig. 4). Although there was an upward trend at 1.5 h in the EtOH-exposed animals (58,833

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EFFECT

OF EtOH

ON GH AND

PRL

E r

33;

z F k

25

EXPRESSION

2079

80

35 \

GENE

0 CONTROL FZ3 EtOH /

T

**

0 CONTROL hz9 EtOH

70

*

’

20 I

i

; hid 24

0.5

15

30

TIME (hours)

FIG. 3. Time-course

effect of EtOH injection (ip) on serum PRL levels 0.5, 1.5, 3, and 24 h after EtOH or saline exposure. PRL was significantly elevated after EtOH administration at 1.5 and 3 h, with return to control values by 24 h. n = 5-6/group. The data are expressed as a percentage of the control value. *, P < 0.05.

z b-

30 80

1

0 CONTROL ZBEtOH

i

FIG. 5. CAMP levels in pituitary cells after either EtOH or saline ip injections. The effect of EtOH on pituitary CAMP was significant at 0.5 h (lo-fold increase), with a rapid return to control levels at 1.5 and 3 h. n = 5-6/group. **, P < 0.01.

I

0 CONTROL eZa EtOH

15 15

33

24 0

TIME (hours)

FIG. 4. Pituitary was no statistical n = 5-6/group.

PRL content after difference between

ip EtOH or saline injection. There the two groups at any time point.

f 8,786 rig/ml) compared to the saline-injected controls (28,500 f. 8,893 rig/ml), this was not statistically significant. At 3 h, the values were 23,167 & 6,765 and 22,000 ?z 9,656 rig/ml in the control and EtOH groups, and at 24 h, they were 15,133 f 6,205 and 13,667 + 726 rig/ml, respectively. Unfortunately, we do not have pituitary content data for 0.5 h after injection. CAMP levels were IO-fold elevated 0.5 h after treatment in the EtOH-treated animals (6.0 + 0.78 US. 58.5 f 7.5 pmol/ pituitary, EtOH VS. control groups), with a rapid return to control values by 1.5 h (control, 18 + 0.54; EtOH, 21 + 0.9) and 3 h (control, 12.6 + 0.6; EtOH, 17.4 + 2.3; Fig. 5). The steady state GH mRNA levels were significantly decreased by EtOH (compared to saline) at 0.5 and 1.5 h (Fig. 6). PRL mRNA levels were decreased at 1.5 and 3.0 h in EtOH-treated animals (Fig. 7). The mRNA levels for Pit-l, however, were not different between the two groups (Fig. 8) at any time point.

30

24.0

TIME (hours)

FIG. 6. The effect of acute EtOH on the steady state level of GH mRNA, as assessed by Northern blot analysis. The mRNA for GH was significantly depressed at 0.5 and 1.5 h. n = 5-6/group. Values were obtained by densitometric scanning of autoradiograms from Northern blot analysis and expressed in arbitrary densitometer units. The data were corrected for loading, as explained in Materials and Methods. Data are expressed as a percentage of the control value. *, P < 0.05.

Discussion The influence of ethanol on the secretion of GH has been previously studied, and an attenuation of basal release noted in both male (1, 2, 5, 6) and female (3, 6) rats. While GH release factor (GRF)-stimulated secretion appears to be unchanged (3), clonidine-stimulated GH release was found to be blunted by ethanol (6), an effect that was overcome by the administration of GRF, thus leading the researchers to conclude that ethanol may reduce GH levels by diminishing oc2-mediated GRF release. Studies examining the effect of EtOH on GH synthesis, however, have not been performed to our knowledge. We confirmed the ETOH-induced decrease in serum GH levels noted by others (1, 3) and found that the effect had abated by 24 h. The marked variability in baseline GH levels in the control saline-injected animals reflects the enormous pulsatility of GH, for which three high amplitude peaks occur every 8 h (7), and basal values any-

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EFFECT

OF EtOH

ON GH AND

:

KI CONTROL FZZ EtOH

i 1.5

30

24

0

T,ME (mum)

FIG. 7. PRL mRNA levels after EtOH exposure. The levels were significantly decreased at 1.5 and 3 h, with a return to the control level by 24 h. Data were corrected for loading, as explained in Materials and Methods, and expressed in arbitrary densitometer units. The results are expressed as a percentage of the control value. n = 5-8/group. **, P < 0.01.

0 700 Y 0 Q

0 600

EII XC

CONTROL EtOH

0 500

2 F

0 400

&

0 300

a 0 200 0 100 0 ooc 15

30

60

TIME (hours:

8. The mRNA levels of Pit-l were unaltered by EtOH exposure. Data are expressed as a percentage of the control value. Loading corrections were performed as described in Materials and Methods. n = 55S/group.

FIG.

where from 1-2 to lo-20 rig/ml have been reported (14-16). Such pulsatility has been documented in studies in which multiple sequential blood sampling was performed in individual animals. In studies with a design similar to ours (i.e. a single trunk blood sample taken at the time of death), the control group variability we see would be anticipated and has, in fact, been noted by others (16, 17). Since the pituitary GH content was unchanged between the EtOH- and salinetreated animals at any time point, this nondepletion suggests a block in the release of GH from the pituitary. The idea that EtOH can impair protein secretion is supported by the finding of Ghosh et al. (18), who showed that altered apoprotein-E glycosylation occurred in the Golgi apparatus of the hepatocyte after EtOH exposure, leading to diminished release, and Tuma et al. (19), who also demonstrated impaired protein trafficking in the liver by EtOH. In other studies we have noted that acute EtOH exposure decreases serum LH levels at the same time that the pituitary LH content actually

PRL

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EXPRESSION

Endo. 1992 Vol 131. No 5

increases, again suggesting a blockage of release (12). However, the results observed with PRL release (see below) lead us to conclude that there is not a global effect of EtOH impeding hormone release from the pituitary. The rise in serum PRL after acute EtOH exposure reported here has been demonstrated by this laboratory in in vitro studies of male rat pituitary cells (20) and by others in male (21) and female rats (22, 23) and has been attributed to the stress effect of drinking on this stress-responsive hormone. Similarily, the ability of ethanol to increase the activity of the hypothalamic-pituitary-adrenal axis, another stress-responsive hormonal system, has been documented in both acute and chronic studies (24). An EtOH-induced increased rate of CRF biosynthesis was noted (25) in addition to an altered ability of CRF or stress to stimulate ACTH secretion. The mechanisms through which these stress changes take place, however, remain speculative. Numerous changes in the brain levels and/or rate of turnover of neurotransmitters, such as dopamine, acetylcholine, and norepinephrine have been reported after EtOH exposure (26-28), in addition to effects on TRH (29), which is known to regulate PRL release, and arginine vasopressin (30). The fact that ethanol alters the secretion of a brain neurotransmitter, however, suggests a potential role for this mediator in EtOH-induced hormone changes, without explaining the mechanisms involved in this change. The EtOH-induced rise in serum PRL is not accompanied by any change in pituitary PRL content, a finding reported by others (21). This may be due to one of several reasons. First, it may well be that the actual amount of extra secreted PRL is small compared to the total pituitary PRL pool, and thus, any pituitary differences might be so low as not to be easily detected. Indeed, the average rise in serum PRL between EtOH and control groups of 12-14 rig/ml reflects an increased total secretion of only 120-140 ng, assuming distribution through a lo-ml volume of rat blood. Such an amount would be small compared to the approximately 25,000 ng total content of pituitary PRL seen in these studies. Another possible explanation for the associated increase in serum PRL with no change in pituitary PRL content and decrease in PRL mRNA (see below) might be that EtOH and/ or its metabolites somehow induce increased translational efficiency, such that more PRL protein is translated from lesser amounts of PRL mRNA. The EtOH-induced suppression of GH mRNA suggests that EtOH impacts not only on GH secretion, but on GH synthesis as well. Our results also have shown a decrease in steady state levels of PRL mRNA, but not in the levels of the transcription factor Pit-l (GHF-1). Pit-l (GHF-1) is a POU/ homeodomain transcription factor thought to be important in the regulation of both GH and PRL (as well as TSH) transcription (8). These findings of changes in GH and PRL mRNA, but not in Pit-l (GHF-1) message, indicate that the effects of EtOH on pituitary hormone mRNAs are not global. This is in agreement with our recent reports that ip EtOH administered to castrated male rats resulted in a dramatic 80% reduction in LH/3 mRNA, but no change in FSHP or common (Y mRNA (12, 31). This change was coupled with

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EFFECT

OF EtOH

ON GH AND

significant decreases in serum levels of both LH and FSH. Given the relatively long half-life of the PLH mRNA of greater than 24 h (32), the marked fall in steady state levels must be due at least in large part to diminished mRNA stability. Estimates of the half-life of GH mRNA vary from approximately 24-60 h (33,34). Thus, the more modest 30% decrease in GH and PRL messages might be due to either decreased transcription or increased GH and PRL mRNA degradation. Analysis of the effect of EtOH on GH and PRL transcription rates and half-life studies are currently in progress to delineate at what level EtOH is exerting its effect on the steady state mRNA levels. The sharp lo-fold rise in pituitary CAMP levels is intriquing. Our data reflect total pituitary CAMP, and thus, we cannot say whether the effect of EtOH is seen in all of the different cell populations of pituitary (e.g. lactotropes, gonadotropes, etc.) or is restricted to a few. The former possibility is more likely, since data from several other laboratories have consistently demonstrated that EtOH acutely causes enhanced CAMP accumulation in a diverse variety of cells, including hepatocytes (35), NIE-115 neuroblastoma cells (36), PC12 cells (37), and platelets (38). Thus, it seems reasonable to suppose that the CAMP response to EtOH in pituitary cells is a generalized cellular phenomenon, and our findings of increased total pituitary CAMP reflect rises in each of the cellular subtypes. It is relevant that CAMP has been shown to stimulate transcription and steady state levels of the Pit-l, GH, and PRL genes as well as secretion of both of these hormones (7). The elevated CAMP levels seen in our EtOH-treated rats compared to control rats might then be expected to result in substantial increases in steady state levels of GH and PRL mRNAs. Thus, the 30% decrement in GH and PRL messages in EtOH-treated rats and the lack of apparent change in Pit-l (GHF-1) message might be inappropriate in light of the sharp CAMP rise. It may be that EtOH caused a marked reduction in CAMP-mediated GH and PRL gene transcription and/or diminished stability of these messages. An alternative explanation for the decrease in GH mRNA steady state levels in the presence of a markedly elevated CAMP level could be a block in transcription by EtOH begore Pit-l involvement. Alternatively, EtOH could be inducing GRF receptor down-regulation via CAMP elevation. In other cell systems CAMP is known to mediate receptor down-regulation via phosphorylation after CAMP and protein kinase-A have been activated (39). In our system, the decrease in GH mRNA levels might be attributed to diminished GRF receptor number in pituitary cells secondary to EtOH-induced CAMP elevation. A final potential explanation for the elevated CAMP levels could be the known increase in membrane bulk fluidity that occurs with EtOH (40). Most investigators, however, agree that bulk membrane lipid pertubations do not totally account for the deleterious actions of EtOH, and there is unanimity of opinion that specific targets within the cell membrane exist for EtOH (40). As there are no studies examining the effect of EtOH on pituitary cell mechanisms, it is not possible to definitely conclude that the CAMP rise is based on an alteration in membrane fluidity, although this may be a mechanism.

PRL

GENE

EXPRESSION

2081

Previous data from our laboratory have shown that EtOH can directly suppress GH release and stimulate PRL release in primary cultures of anterior pituitary cells (4, 20). Thus, at least part of the effect we have reported in vim is mediated at the pituitary level. Our data do not, of course, exclude the possibility of a hypothalamic effect as well. In conclusion, our in vivo results confirm that acute EtOH administration can cause substantial disruption of two neuroendocrine hormones crucial to homeostasis of the organism and begin to unravel the molecular mechanisms for these changes. Work on detailed analysis of the effect of EtOH on the CAMP transcription cascade and on GH and PRL gene transcription rates and mRNA stability is currently in progress in our laboratory, as well as studies on the effect of EtOH on hypothalamic GRF and somatostatin, the primary brain hormones that control GH synthesis and release.

Acknowledgment We would ance.

like to thank

Janet Flores

for her expert

secretarial

assist-

References 1. Redmond GP 1980 Effect of ethanol on endogenous rhythms of growth hormone action. Alcohol Clin Exp Res 4:50-56 2. Mannisto PT, Vedernekona NN, Borisenko SA, Touminer RK, Kuanmaa K, Burov YZ 1987 Effect of chronic ethanol administration and abstinence on serum thyroid stimulating hormone, prolactin and growth hormone concentrations in rats with high and low ethanol intake. Alcohol 22:251-256 3. Dees WL, Skelley CW, Rettori V, Kentroti MS, McCann SM 1988 Influence of ethanol on growth hormone secretion in adult and prepubertal rats. Neuroendocrinology 48:495-497 4. Emanuele MA, Kirsteins L, Reda D, Emanuele NV, Lawrence AM 1989 The effect of in vitro ethanol exposure on basal growth hormone secretion. Endocr Res 14:283-291 5. Sontag WE, Boyd RL 1989 Diminished insulin-like growth factor1 levels after chronic ethanol: relationship to pulsatile growth hormone release. Alcohol Clin Exp Res 13:3-7 6. Conway D, Mauceric H 1991 The influence of acute ethanol exposure on growth hormone release in female rats. Alcohol 8:159164 7. Frohman LA, Jamsson JO 1986 Growth hormone releasing hormone. Endocr Revi 7:223-253 8. Mangalam H, Albert V, Ingraham H, Kapiloff M, Wilson L, Nelson C, Elsholtz H, Rosenfeld MG 1989 A pituitary POU domain, Pit-l, activates both GH and Prl promoter transcription. Genes Dev 3:946-958 9. Hojvat S, Emanuele N, Baker G, Connick E, Kirsteins L, Lawrence AM 1982 Growth hormone (GH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH)-like peptides in the rodent brain: non-parallel ontogenetic development with pituitary counterparts. Dev Brain Res 4:427-434 10. Emanuele NV, Metcalfe L, Wallock L, Tentler J, Hagen TC, Beer CT, Martinson D, Gout PW, Kirsteins L, Lawrence AM 1986 Hypothalamic prolactin: characterization by radioimmunoassay and bioassay and response to hypophysectomy and restraint stress, Neuroendocrinology 44:217-221 11. Chromczynski I’, Sacchi N 1987 Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 12. Emanuele MA, Tentler J, Emanuele NV, Kelley MR 1991 In viva effects of acute ETOH on rat (Y and /3 luteininizing hormone gene expression. Alcohol 8:345-348 13. Rich BE, Steitz JA 1987 Human acidic ribosomal phosphoprotein

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14. 15.

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EFFECT

OF EtOH

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GH AND

PO, I’1 and P2: analysis of cDNA clones, in vitro synthesis and assembly. Mol Cell Biol 7:4065-4074 Eden S 1979 Age and sex-related differences in episodic GH secretion in the rat. Endocrinology 105:555-560 Millard WJ, Fox TO, Badger TM, Martin JB 1987 Gonadal steroid modulation of GH secretory patterns in the rat. In: Robbins J, Melmed S (eds) Acromegaly. Plenum Press, New York, pp 139-147 Jansson JO, Ekberg S, Isaksson OGP, Eden S 1984 Influence of gonadal steroids on age- and sex-related secretory patterns of growth hormone in the rat. Endocrinology 114:1287-1295 Tannenbaum GS, Martin JB 1976 Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98:562-570 Ghosh P, Chrtel S, Lakshman MR 1991 Effect of chronic ethanol on apolipoprotein (APO) E synthesis and glycosylation in rats. Alcoholism Clin Exp Res 15:725-729 Tuma DJ, Casey CA, Sorrel1 MF 1991 Effects of alcohol on hepatic protein metabolism and trafficking. Alcohol Alcoholism 51:297-303 Emanuele MA, Metcalfe L, Sonru I, Reda D, Kirsteins L, Emanuele NV, Lawrence AM 1987 The effect of ethanol on prolactin release from pituitary cells in vitro. Alcohol 4:429-432 Seilicovich A. Rettori V. Duvilonski B. Diaz MC. Debeirik L 1982 The effect of acute and chronic ethanol administration in male rats. J Alcohol 3:344-348 Esquifino A, Sanchis R, Guerri C 1986 Effect of prenatal alcohol exposure in sexual maturation of female rat offspring. Neuroendocrinology 44~483-487 Dees W, Kozlowski GP 1984 Differential effects of ethanol on LH, FSH, and prolactin secretion in the female rat. Alcohol 1:429-433 Rivier C, Bruhn T, Vale W 1984 Effect of ethanol in the hypothalmic-pituitary-adrenal axis in the rat: role of CRF. J Pharmacol Exp Ther 229:127-131 Rivier C, Imaki T, Vale W 1990 Prolonged exposure to alcohol: effect on CRF mRNA levels and CRF and stress induced ACTH secretion in the rat. Brain Res 520:1-5 Hunt WA, Maychrowicz E, Dalton TK, Swartzevelden HS, Wixon H 1979 Alterations in neurotransmitter activity after acute and chronic ethanol treatment: studies of transmitter interactions. Alcoholic Clin Exp Res 3:359-363 Pohorecky LA 1974 Effects of ethanol on central and peripheral noradrenergic neurons. J Pharmacol Exp Ther 189:380-391

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Endo. 1992 Vol131. No 5

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