D-2 dopaminergic agonists and adenosine 3',5'-monophosphate directly regulate the synthesis of alpha-melanocyte-stimulating hormone-like peptides by cultured rat melanotrophs

When placed in short term (2-day) tissue culture, the melanotrophs from the intermediate lobe of the rat pituitary gland synthesize a proopiomelanocortin-like material (POMC-LM). Exposure of these cells to bromocriptine (CB 154), an agonist upon their D-2 dopamine receptor, reduces the synthesis of POMC-LM; spiroperidol, an antagonist of the D-2 receptor, prevents this effect of CB 154. Cultured melanotrophs secrete an alpha MSH-like material. The amount of this alpha MSH-like material, either stored intracellularly or secreted into the culture medium, can be quantified in a specific RIA; the material identified in this manner is designated immunoreactive alpha MSH (IR-alpha MSH). CB 154 inhibits the secretion of IR-alpha MSH from these cells. Either spiroperidol or 8-bromo-cAMP prevent this inhibitory effect of CB 154. The capacity of these cells to synthesize alpha MSH-like molecules and release them into the culture medium can be assessed by incubation in the presence of [3H]tyrosine, followed by immunoprecipitation with an antibody directed against alpha MSH. This newly synthesized immunoprecipitable material is designated immunoprecipitable alpha MSH (IP-alpha MSH) and should be distinguished from IR-alpha MSH. Both CB 154 and quinpirole, a selective D-2 agonist (but not SKF 38393, a selective D-1 agonist), inhibit the synthesis and secretion of IP-alpha MSH. YM-09151-2, a selective D-2 antagonist (but not SCH 23390, a selective D-1 antagonist), blocks the inhibitory effects of quinpirole. Several compounds affecting cAMP metabolism (cholera toxin, forskolin, 8-bromo-cAMP, and 3-isobutyl-1-methylxanthine) can also prevent the inhibitory effect of CB 154 on the synthesis of IP-alpha MSH. We conclude the following. The D-2 receptor in the intermediate lobe directly regulates the synthesis and secretion of IP-alpha MSH. cAMP can regulate either the synthesis of POMC-LM or the processing of this substance into alpha MSH-like peptides.     

Differential effects of haloperidol on the rat pituitary: decreased biosynthesis, processing and release of anterior lobe pro-opiomelanocortin

The effects of chronic haloperidol treatment on pro-opiomelanocortin (POMC) synthesis, processing, and release in the anterior (AL) and intermediate (IL) lobes of the rat pituitary were studied. In the IL, 14 days of haloperidol administration promoted an increase in the level of POMC mRNA, and a corresponding elevation of levels of beta-endorphin (beta E), alpha-melanocyte-stimulating hormone (MSH), and gamma 3 MSH. In the AL, a reduction of POMC mRNA as well as immunoreactive beta E, adrenocorticotropin (ACTH), and gamma 3 MSH was observed. Column chromatography revealed that this treatment promoted an apparent alteration of POMC processing in AL: the conversion of larger, precursor-sized peptides to smaller, more-processed forms was relatively inhibited. Circulating levels of both N-acetyl-beta E and corticosterone were elevated following haloperidol challenge in drug-naive animals. Resting plasma levels of both, however, were not changed following chronic haloperidol treatment. Pituitary culture studies demonstrated that chronic haloperidol treatment increased the releasability of IL-derived products, while simultaneously decreasing the releasability of those products from the AL. These results suggest that pituitary POMC biosynthesis, processing and release are under at least partial dopaminergic control in both the IL and the AL of the pituitary, but by different mechanisms; chronic haloperidol treatment upregulates the POMC system in IL, but downregulates it in AL, despite similarities of the responses of both lobes to acute haloperidol challenge.     

Coordinate regulation of peptide acetyltransferase activity and proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary

Proopiomelanocortin (POMC) is posttranslationally processed in the intermediate lobe of the pituitary to both N-terminally acetylated and nonacetylated forms of alpha MSH and beta-endorphin (beta END). N-Acetylation substantially modifies the physiological responses produced by both peptides, suggesting that the activity of the peptide acetyltransferase, which posttranslationally acetylates beta END and des-acetyl-alpha MSH, may play an important role in defining the biological activity of the secretory products of the intermediate pituitary lobe. The present results demonstrate that peptide acetyltransferase activity is induced by treating rats chronically with the dopamine receptor antagonist haloperidol. Haloperidol administration produced parallel and essentially equivalent increases in acetyltransferase activity, POMC mRNA levels, and the content and secretion of POMC-derived peptides in the neurointermediate pituitary. Time-course and dose-response studies further demonstrated that acetyltransferase activity covaried with POMC mRNA and peptide levels. Chronic treatment with the dopamine receptor agonist bromocriptine had the opposite effects; it lowered acetyltransferase activity, POMC mRNA levels, and alpha MSH and beta END immunoreactivities. Subcellular fractionation showed that acetyltransferase activity was localized in three subcellular compartments corresponding in density to secretory vesicles, rough endoplasmic reticulum and Golgi apparatus, and cytosol. Haloperidol treatment significantly increased the specific activity of the secretory vesicle-associated acetyltransferase without affecting the specific activity of the enzymes present in the endoplasmic reticulum or cytosol. Together, these data indicate that peptide acetyltransferase activity and POMC biosynthesis are coregulated.     

Effect of salt loading on proopiomelanocortin (POMC) messenger ribonucleic acid levels, POMC biosynthesis, and secretion of POMC products in the mouse pituitary gland

The effect of salt loading on POMC-derived peptide secretion, POMC mRNA levels, and POMC biosynthesis in the mouse pituitary gland was investigated. Plasma alpha MSH levels decreased to 56.9% of the control value after 2 days of salt loading. Concomitantly, POMC mRNA levels and POMC biosynthesis in the intermediate lobe decreased to 47.7% and 66.4% of the control value, respectively. After 4 days of salt loading, plasma alpha MSH levels, POMC mRNA, and POMC biosynthesis returned to control levels. ACTH secretion increased to 142.2% of the control value after 2 days of salt loading, but decreased to 73.4% of the control levels after 4 days. After 9 days, ACTH secretion was still low; however, by 12 days, plasma ACTH levels in salt-loaded mice were not significantly different from control levels. POMC mRNA levels in the anterior lobe increased to 205.0% of the control value after 2 days of salt loading, continued to be high after 4 days, but returned to control levels by 9 days. However, POMC biosynthesis was not altered under these conditions during the entire period of salt loading. Thus, POMC mRNA levels and biosynthesis were modulated differently in the intermediate and anterior lobes of the mouse pituitary gland during salt loading.     

Dopaminergic regulation of the biosynthetic activity of individual melanotropes in the rat pituitary intermediate lobe: a morphometric analysis by light and electron microscopy and in situ hybridization

The primary cell type of the intermediate lobe (IL) of the rat pituitary is a polyhedral secretory cell with a smooth ovoid nucleus. The results of this study demonstrate, however, that IL melanotropes are a heterogeneous cell population. Melanotropes differed in the tinctorial properties of their cytoplasm; some cells appeared distinctly darker, others lighter, and cells staining in intermediate shades were also found. Electron microscopical morphometry revealed that darkly staining melanotropes have a denser cytosol and contain a greater amount of rough endoplasmic reticulum, mitochondria, and secretory vesicles than light cells. In addition, in situ hybridization studies, using a POMC probe, showed that POMC mRNA was distributed unevenly among melanotropes in a pattern comparable to the distribution of light and dark cells. These studies further demonstrated that dopaminergic drug treatments, which are known to alter the secretion of POMC-related peptides from the IL, produced parallel changes in both the histological staining properties and the amount of POMC mRNA per cell. Haloperidol treatment dramatically increased the number of dark melanotropes and the amount of POMC mRNA in each cell and eliminated the cellular heterogeneity in both staining properties and the distribution of POMC mRNA. After bromocriptine treatment the number of light melanotropes increased, and each cell contained reduced levels of POMC mRNA. These findings indicate that individual melanotropes maintain different levels of biosynthetic activity and that treatments that alter the secretion of POMC peptides affect both the rate of POMC synthesis in individual melanotropes and the cellular heterogeneity of the IL.     

In situ hybridization detection of marked differences in pre-proopiomelanocortin messenger ribonucleic acid content of individual corticotropes and melanotropes

Recently, heterogeneity of POMC mRNA content between intermediate lobe melanotropes of the rat pituitary gland was demonstrated by in situ hybridization of tissue sections. In the present study the heterogeneity of POMC mRNA content in dispersed rat pituitary cells has been investigated. Acutely dispersed cells from adult male rat anterior or neurointermediate lobe tissues were adhered to poly-L-lysine-coated coverslips. The cells were fixed and then hybridized with 35S-labeled POMC or 1B15 (cyclophilin) cRNA. Parallel studies measuring constitutively expressed cellular 1B15 mRNA content were undertaken to ensure that the apparent single cell differences in POMC mRNA were not inherent to the in situ hybridization procedure. When classified by image analysis, extensive differences in silver grain densities were seen over POMC mRNA-containing cells from both lobes. To determine if mRNA in polysomal configurations was less accessable for hybridization with probes than naked mRNA, cells were preincubated with pactamycin, a potent inhibitor of ribosomal initiation of protein synthesis. Pactamycin had no effect on these results. Thus, there appears to be large differences in POMC mRNA content between individual pituitary cells expressing the same gene product.     

Immunoreactive proopiomelanocortin (POMC) peptides and POMC-like messenger ribonucleic acid are present in many rat nonpituitary tissues

Immunoreactive (IR) POMC peptides have been found in several rat nonpituitary tissues. We found IR-ACTH, IR-beta-endorphin (beta END), and IR-gamma MSH in extracts from the following eight rat nonpituitary tissues, listed in order of decreasing POMC peptide concentrations: testis, duodenum, kidney, colon, liver, lung, stomach, and spleen, but not in adrenal or muscle extracts. Concentrations were very low and ranged from less than 0.00003% to 0.0005% of pituitary levels. In testis, duodenum, and colon, IR-gamma MSH and IR-beta END concentrations were only 5-37% of IR-ACTH levels. Gel filtration chromatography showed that IR-ACTH and IR-beta END coeluted in a major peak of 15,000 daltons, which is slightly larger than expected for a C-terminal peptide containing rat ACTH and beta-lipotropin. There were also a minor higher mol wt peak of IR-ACTH and IR-beta END and a minor IR-beta END peak that eluted in the position of mature beta END. There was no peak of IR-ACTH that corresponded to the size of mature ACTH. To determine whether these nonpituitary tissues also contained a POMC-like mRNA, which would confirm that the peptides were synthesized locally within the tissues, we examined poly(A) RNA prepared from 10 nonpituitary tissues and total RNA from pituitary by Northern blot hybridization for the presence of a POMC-like mRNA with an exon 3 riboprobe. Pituitary contained a single POMC mRNA species of about 1000 nucleotides. A short POMC-like mRNA of about 800 bases was found in all nonpituitary tissues, except spleen and muscle. Compared to POMC mRNA levels in pituitary, the concentration of POMC-like mRNA was 0.5% in testis and 0.03-0.07% in the other tissues. The ratio of POMC-like mRNA to IR-POMC peptide concentrations in nonpituitary tissues was at least 1000 times greater than that in the pituitary. We conclude that the POMC gene is expressed in many nonpituitary tissues and that either the short POMC-like mRNA is translated much less efficiently or POMC peptides are released or degraded much more rapidly in nonpituitary tissues than in the pituitary.     

Immunocytochemical demonstration of melanin-concentrating hormone and proopiomelanocortin-like products in the brain of the trout and carp

Immunocytochemistry on frozen sections revealed that in both the trout and the carp, parvocellular neurones located in the medial basal hypothalamus (medial nucleus lateralis tuberis) were immunostained by antisera against three molecules known to be derived from the proopiomelanocortin (POMC) molecule, viz: alpha-melanocyte-stimulating hormone (alpha MSH), ACTH, and salmonid NPP--the whole N-terminal sequence preceding ACTH in the POMC precursor. Axons from these neurones extended into various regions of the brain but did not appear to project into the pituitary gland. Antiserum against salmonid melanin-concentrating hormone (MCH) immunostained magnocellular neurones in the lateral basal hypothalamus (lateral nucleus lateralis tuberis). Axons from some of these neurones projected into the brain while other axons extended into the pituitary gland. In the carp, but not in the trout, some MCH neurones were also immunostained by antisera against alpha MSH but not by antisera against the other POMC molecules.     

Ontogenesis of proopiomelanocortin gene expression and regulation in the rat pituitary intermediate lobe

During ontogenesis, proopiomelanocortin (POMC) mRNA appears in the pituitary intermediate lobe (IL) at embryonic day 16 (E16), rather later than in the hypothalamic arcuate nucleus (E13) or the pituitary anterior lobe (E15). POMC mRNA onset in the IL correlates with the appearance of POMC-derived peptides detected by immunocytochemistry (ICC), indicating that there is probably no time lag between POMC mRNA translation. Subsequently, while the IL lobular organization developed progressively, the number of in situ hybridization-(ISH) and ICC-positive cells increased until after birth. During postnatal development, coinciding with innervation of the IL, the POMC mRNA level in the lobe, measured by quantitative ISH, increased about 4-fold to reach the adult value at weaning. The effects of acute or chronic postnatal treatment with a dopamine antagonist (haloperidol) or a dopamine agonist (bromocriptine) show that the physiological dopaminergic inhibitory control of POMC gene expression operates as early as postnatal day 5. The subsequent increase in mRNA levels despite the inhibitory innervation raises the question of the existence of some unknown positive regulation active during postnatal development.     

Concomitant dopaminergic and glucocorticoid control of pituitary proopiomelanocortin messenger ribonucleic acid and beta-endorphin levels

Synthesis and secretion of POMC-derived peptides appear to be differentially regulated in the anterior pituitary (AP) and neurointermediate lobe (NIL). In the AP, glucocorticoids inhibit, and CRF and arginine vasopressin stimulate, synthesis of POMC and release of immunoreactive (ir)-beta-endorphin (beta EP); in the NIL, synthesis and release of POMC and its derivatives are under tonic inhibitory dopaminergic control. There is, however, evidence for some overlap of these control mechanisms under certain circumstances. In the present study we have used specific RIA and Northern blot analysis to examine the effects of chronic treatment with dopaminergic agents and dexamethasone (DM) (both alone and in combination) on AP and NIL content of ir-beta EP and POMC messenger RNA (mRNA), and/or hypothalamic ir-arginine vasopressin and ir-CRF content. In the NIL, the dopamine agonist bromocriptine reduced and the antagonist haloperidol raised both POMC mRNA and ir-beta EP content. Long term DM treatment did not alter NIL ir-beta EP content in the intact rat, but increased levels of POMC mRNA. DM abolished the haloperidol-induced increase in NIL ir-beta EP content but further increased the haloperidol-induced rise in POMC mRNA. DM treatment lowered both ir-beta EP and POMC mRNA in the AP as well as lowering levels of hypothalamic ir-CRF. In DM-treated rats, haloperidol partially restored AP ir-beta EP and POMC mRNA to control untreated levels. These findings further support the proposition that both dopaminergic agents and glucocorticoids can modulate POMC mRNA levels and/or tissue content of ir-beta EP in both the NIL and AP of the rat. The effects of DM on the NIL, both alone or with haloperidol, suggest that glucocorticoids may have both direct and indirect effects on POMC gene expression in this tissue.     

Development of pituitary pro-opiomelanocortin gene and peptide expression: characterization and effect of repeated intermittent maternal isolation.

The dynamics of pro-opiomelanocortin (POMC) biosynthesis in the adult rat are altered by demands imposed on the system, such that acute stress increases in the efficiency of anterior pituitary (AP) posttranslational events, while repeated stress increases pretranslational events. In contrast, the developing animal has a limited adrenocortical response to acute stress during the first 2 weeks of life (stress nonresponsive period). In this study, we investigated how the maturing AP and intermediate lobe (IL) POMC cells respond to repeated demand. Measurements of AP and IL POMC mRNA and POMC peptides were performed using Northern gels and radioimmunoassay, respectively. Plasma ACTH and corticosterone measurements were also performed. Maternal isolation, for 1 h on 3 consecutive days, was used as a repeated stress stimulus. The developing AP and IL exhibit an age-related increase in POMC mRNA and peptide levels. On the other hand, AP and IL do not respond to repeated intermittent maternal isolation during the first 2 weeks of life. However, a significant corticosterone release is seen in the 14 and 21-day-old animals. A change in POMC mRNA level is only detected in the 21-day-old AP where levels decrease. Therefore, an adrenocortical response to repeated intermittent maternal isolation predates the appearance of glucocorticoid inhibition of POMC expression in the 21-day-old animal. We propose that an immature neuronal inhibitory circuit during the 3rd week of life causes a sustained corticosteroid response which may in turn trigger AP-delayed feedback.     

Ethanol exposure decreases pituitary corticotropin-releasing factor binding, adenylate cyclase activity, proopiomelanocortin biosynthesis, and plasma beta-endorphin levels in the rat

Animals exposed continuously for 14 days to ethanol vapor in an inhalation chamber at sufficient ethanol vapor concentration to maintain blood ethanol levels from 100-250 mg/100 ml exhibited approximately 36% lower corticotropin-releasing factor binding and 24% lower adenylate cyclase activity in anterior (AL) and neurointermediate lobe (NIL) membranes of the pituitary gland compared to controls not treated with ethanol. To determine the effect of chronic ethanol exposure on proopiomelanocortin (POMC) biosynthesis, the levels of POMC mRNA in the AL and NIL were quantified by Northern blot and slot blot techniques. Ethanol treatment for 1, 7, or 14 days produced a time-related decrease in POMC mRNA levels, relative to total RNA levels, in both the AL and NIL. Ethanol treatment caused a greater reduction in NIL POMC mRNA than in AL POMC mRNA. Exposure to ethanol vapors for 14 days decreased immunoreactive beta-endorphin in plasma by approximately 82%. The observed reduction of immunoreactive beta-endorphin in plasma after long term exposure of rats to ethanol may be related to the alcohol-mediated decrease in corticotropin-releasing factor binding and adenylate cyclase activity, which, in turn, leads to decreased intracellular POMC levels through reduced production of POMC mRNA in the AL and NIL of the rat pituitary gland.     

Dopaminergic control of prolactin mRNA accumulation in the pituitary of the male rat

Dopaminergic control of the expression of the prolactin gene was investigated by administration of biomoergocryptine (CB154) to male rats. The effects of the drug on the following parameters were measured: (i) circulating levels of GH and PRL; (ii) synthesis of GH and PRL measured by pulse labeling pituitary fragments in vitro; (iii) GH and PRL mRNA activities; and (iv) content of PRL mRNA. After 1 day of CB154 administration, serum PRL fell to undetectable levels whereas it took 3 days to observe a 50% reduction in PRL synthesis. This effect was accounted for by a parallel decrease in PRL mRNA activity and content. GH synthesis and GH mRNA were not affected by the treatment. Our results show that the dopaminergic inhibition of PRL production involves regulation at a pre-translational level.     

Regulation of the pro-opiomelanocortin mRNA levels in rat pituitary by dopaminergic compounds.

Dopamine-containing neurons directly innervate the intermediate lobe of the pituitary and dopaminergic compounds exert inhibitory effects on the secretion and the content of alpha-melanocyte-stimulating hormone and beta-endorphin in this tissue. In this study, we have investigated the effects of dopamine receptor agonists and antagonists on the level of pro-opiomelanocortin (POMC) mRNA in rat pituitary. RNAs isolated from neurointermediate pituitary (NIP) or anterior pituitary were spotted on nitrocellulose filters and the levels of POMC mRNA were quantified by hybridization to a POMC-specific complementary DNA probe coupled with autoradiography and densitometry. Administration of a dopamine receptor antagonist, haloperidol (2 mg/kg per day), to adult female rats resulted in a 3- to 5-fold increase in POMC mRNA level in the NIP. Treatment with the dopamine agonist 2-Br-alpha-ergocryptine (1 mg/kg per day) decreased significantly the content of POMC mRNA in the NIP. These drugs had no apparent effect on the POMC mRNA levels in the anterior pituitary. The effect of haloperidol and ergocryptine on POMC mRNA in the NIP is time- and dose-dependent. The elevation of POMC mRNA content in the NIP by haloperidol can be observed as early as 6 hr after treatment. These effects of dopaminergic compounds can also be demonstrated in adult male and ovariectomized female rats. The beta-endorphin content of the NIP, as measured by radioimmunoassay, and the de novo synthesis of POMC, as determined by radioactive amino acid labeling and two-dimensional gel electrophoresis analysis, also show negative regulation by dopaminergic compounds.     

Histological evaluation of the dopaminergic regulation of proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary, involving in situ hybridization and [3H]thymidine uptake measurement

The melanotroph, a polyhedral secretory cell with an ovoid smooth nucleus, is the primary cell type of the intermediate lobe (IL) of the rat pituitary. The melanotrophs are not uniform, but differ in the tinctorial properties of their cytoplasm; some cells appear distinctly darker, others lighter, and cells staining in intermediate shades are also found. In addition, in situ hybridization using proopiomelanocortin (POMC) probes shows an uneven distribution of POMC mRNA among melanotrophs, indicating that different cells maintain different levels of biosynthetic activity. Dopaminergic drugs known to alter the secretion of POMC-related peptides from the IL produced parallel changes in histological staining properties and the amount of POMC mRNA per cell, as determined by in situ hybridization. Acute bromocriptine treatment (6 h) produced a dramatic reduction in grain counts over melanotroph cytoplasm (to 10% of the control levels). A similar reduction persisted after chronic treatment. Six hours after a single haloperidol injection, the grain counts were 180% of control levels. After chronic haloperidol treatment, they were further elevated to 300% of control levels. Chronic bromocriptine and haloperidol treatment also changed the thickness of the IL. Bromocriptine reduced and haloperidol treatment increased the number of cell layers in the IL by changing the rate of cell proliferation. Thus, haloperidol treatment significantly increased and bromocriptine treatment significantly decreased the number of melanotrophs labeled by [3H]thymidine. The mitotic index followed the same trend. These results suggest that dopamine regulation of the IL acts by two different mechanisms: POMC gene expression and cellular proliferation. The change in POMC gene expression is the cell's first rapid response. The influence on the cell cycle appears after subchronic and chronic treatment.     

Proopiomelanocortin-derived peptides in rat cerebrospinal fluid and hypothalamic extracts: evidence that secretion is regulated with respect to energy balance

Regulation of proopiomelanocortin (POMC) is an important means of controlling the central melanocortin system. It has never been established whether the spectrum of POMC-derived peptides synthesized and secreted from the hypothalamus is altered in response to changes in energy homeostasis in vivo. To monitor secretion, we analyzed peptide content of rat cerebrospinal fluid. Strikingly, both the POMC precursor and ACTH were readily detected. Moreover, levels of both were lower in samples from obese Zucker rats (fa/fa) vs. lean Zucker rats (+/+, fa/+) and from fasted vs. fed rats, whereas alpha MSH could not be detected. POMC levels were also decreased in hypothalamic extracts from obese and fasted animals. In contrast, despite being the most predominant peptide in extracts, alpha MSH levels were not significantly changed in any of the rat models. The ratio of precursor to derived peptides in cerebrospinal fluid was significantly higher in obese vs. lean and fed vs. fasted rats, indicating that secretion of POMC-derived peptides is differentially down-regulated during negative energy balance. In contrast to peptide analysis, we found that POMC gene expression was not significantly decreased in fasted rat hypothalami. We conclude that regulation of peptide secretion is an important mechanism by which the POMC system is controlled.     

Differences in the Brain and Pituitary β-Endorphin System between the Alcohol-Preferring AA and Alcohol-Avoiding ANA Rats

The content of pro-opiomelanocortin (POMC) mRNA was determined in the hypothalamus, as well as in the anterior and intermediate lobes of the pituitary gland of the alcohol-preferring AA and alcohol-avoiding ANA rats under basal conditions. In addition the content of beta-endorphin-like immunoreactivity (beta-EPLIR) was measured in nine brain regions, the anterior and intermediate lobes of the pituitary gland and the serum. The content of beta-EPLIR was significantly higher in the septum and significantly lower in the amygdala, and periaqueductal gray matter of the AA rats, while there was no significant difference between the AA and ANA rats in the arcuate nucleus plus median eminence, nucleus accumbens, caudate, hippocampus, and cortex. HPLC analysis indicated no significant differences in the relative proportions of non-acetyl and acetyl forms of beta-endorphin peptides in the hypothalamus, distinct brain regions, and anterior and neurointermediate lobes of the pituitary gland, between the AA and ANA rats. The content of POMC mRNA but not of beta-EPLIR was significantly higher in the hypothalamus and neurointermediate lobe of the AA rats, while the content of both beta-EPLIR and POMC mRNA were significantly higher in the anterior pituitary of the AA than of the ANA rats. Thus, there are genetically determined differences in the pituitary and brain beta-endorphin system between the AA and ANA rats, which may be important in controlling the differences in the voluntary ethanol consumption exhibited by these animals.