Vol. zyx 23, zy No. zy 1 January 1999 Effects of Ethanol on a-Adrenergic and P-Adrenergic Agonist-Stimulated P-Endorphin Release and cAR/IP Production in Hypothalamic Cells in Primary Cultures Alok De, Nadka Iv. Boyadjieva, and Dipak K. Sarkar We have previously shown that low concentrationsof ethanol rapidly stimulate p-endorphin (p-EP) releasefrom hypothalamic neurons in primarycultures and that chronic exposuresto these concentrations of ethanol desensitize p-EP neurons to ethanol challenges. We have also shown that chronic ethanol desensitizes dibutyryl CAMP-, adenosine-, and prostaglandin E, -stimulated p-EP release and the cAMP content in hypothalamic neurons. In this study, we determined the effects of ethanol zyxwvutsr (50 mM) on p-adrenergic agonist (isoprotere- nol) or a-adrenergic agonist zyxwvutsrq (I-pheny1ephrine)-induced p-EP release and cellular contents of cAMP to identify whether ethanol causes heterologous desensitization of the adenylate cyclase system in this neuronal cell population. Both isoproterenoland I-phenylephrine in- creased p-EP levels in culture media and elevatedthe cAMP content in cell extracts in a concentration (0.1 and 10 pM)-dependent fashion between zyxwvutsrq 3 to 6 hr. A 50 mM dose of ethanol increased p-EP and cAMP levels at 3 hr, but it did not elevate p-EP and cAMP levels after 48 hr of exposure. Acute exposure (3 hr) of these cells to eth- anol moderately enhanced the isoproterenol-stimulated and I-phenylephrine-stimulated levels of media p-EP and intracellular levels of CAMP. However, chronic exposure (48 hr) to ethanol re- duced the magnitude of both a- and p-adrenergic receptor agonist- stimulated p-EP release and cAMP production. These results con- firm our previous findings that the ethanol action on p-EP secretion is mediated by the cAMP system and further suggest that chronic ethanol causes heterologous desensitization of the adenylate cy- clase system in the p-EP neuronal cell population. Key Words: Acute Ethanol, Chronic Ethanol, I-Phenylephrine, Isoproterenol, Hypothalamic p-Endorphin Neurons. HE OPIOID peptide P-endorphin (P-EP) is produced T in the hypothalamus and regulates a variety of brain functions related to drug abuse, including psychomotor stimulation, positive reinforcement, and mood.’-4 This peptide also mediates prenatal ethanol-induced growth re- tardation,s behavioral abnormalities, central nervous system damage, undernutrition, hypoxia, and acidosis,637 defects in learning and memory,6 neuroendocrine abnor- nialitie~,”~~~ and increased infant mortality.’ Ethanol con- trols P-EP neurotransmission by modulating the release zyxwvu From the Department of Veterinary and Comparative Anatomy, Pharma- cology. and Physiology. Wushington State University, Pullman, Washingon. Received zyxwvutsrqpon for publication May zyxwvutsrqp 14, 1998; Accepted October 14, 1998 This investigution was supported by the National Institutes of Health Grants AA08757 and AA00220. Reprint reqirests: Dipak K. Sarkar, Ph.D., Department of Veterina y and Comparative Anatomy, Pharmacology. and Physiology. Washington State University, Piillman, WA 99164-6520. FAX # 509-335-4650, eniail- sarkur@vetmed. wsii. edir Copyright zyxwvutsrqp 0 1999 by The Research Society on Alcoholism. 46 and synthesis of the peptide both in vivo and in vitro.”-” In primary cultures of fetal hypothalamic cells, low concen- trations of ethanol rapidly stimulated basal and secretogogue-induced P-EP release,’3y14 whereas constant exposure to ethanol desensitized these cultured neurons, suggesting the development of a tolerance to constant eth- Recent studies have shown that adaptive responses to ethanol, such as tolerance and physical dependence, may be mediated in part by changes in CAMP signal transduc- tion. 17-24 Ethanol acutely increases hormone- and neurotransmitter-stimulated CAMP levels in brain tissue membranes and clonal cell line^^^^''^'^ through adenylate cyclase a c t i ~ i t y . ” ~ ’ ~ , ~ ~ - ~ ~ In brain tissue membranes or in clonal cell lines, constant exposure to ethanol decreases receptor-stimulated intracellular CAMP level^.^^.^^-^' Re- cently, it has been shown in primary cultures of hypotha- lamic cells that the CAMPsystem may be involved in con- trolling P-EP secretion, as well as in regulating the stimulatory and adaptive responses of P-EP neurons to ethanol. In primary cultures of hypothalamic cells, acute treatment with ethanol stimulates and chronic ethanol de- sensitizes various secretogogue-induced P-EP releases and cAMP content^.^*-^^ However, ethanol effects on noradrenergic-regulated P-EP release in primary cultures of hypothalamic neurons have not been determined. Pharmacological studies have shown that the noradren- ergic system is involved in a variety of physiological func- tions in the central nervous system, including antinocicep- ti~n,’~ anxiety?7 a t t e n t i ~ n , ~ ~ blood pressure regulation?’ and cognitive function^.^"-^^ The a-adrenoreceptors are involved in the control of neuronal e ~ c i t a t i o n . ~ ~ , ~ ~ - ~ ~ In pinealocytes, the activation of the a-adrenergic system in- creases CAMP levels.47 P-Adrenoreceptors stimulate the secretion of pro-opiomelanocortin (P0MC)-derived pep- tides, such as P-EP via CAMP and Ca’+-dependent path- ways in the anterior pituitary gland.48 Hence, the possibil- ities arise that the secretion of hypothalamic P-EP is controlled by a-adrenoreceptor and P-adrenoreceptor reg- ulators, and that ethanol may control the noradrenergic agent-regulated P-EP release. In the study presented herein, we determined the effects of ethanol on the P-adrenergic agonist isoproterenol- and a-adrenergic ago- nist I-phenylephrine-induced P-EP release and cellular lev- an01.’~J~ Alcohol Clin Exp Res, Vol 23, No 1, 1999: pp 46-51