Christina Gianoulakis

Long term ethanol alters the binding of 3H-opiates to brain membranes

In order to examine whether ethanol treatment has selective or differential effects on brain binding sites for opiates, male Sprague Dawley rats were fed for 15 or 21 days with a complete liquid diet containing 6.5% ethanol (v:v) or an isocaloric amount of sucrose. The binding of 3H-DADL-enkephalin, 3H-dihydromorphine and 3H-naloxone to the brain membranes from rats treated with ethanol was increased. However, addition of ethanol directly in the incubation medium decreased the binding of 3H-DADL enkephalin and increased the binding of 3H-dihydromorphine to brain membranes from both control and ethanol treated rats. Direct exposure of brain membranes to ethanol caused no significant change in the binding of 3H-naloxone. Thus chronic ethanol ingestion alters the binding of opiate ligands to brain membranes. Furthermore, the direct effect of ethanol appears to be different for the different classes of opiate binding sites.

Biosynthesis of β-endorphin by the neurointermediate lobes from rats treated with morphine or alcohol

Rats were rendered tolerant to either morphine or alcohol, by 21- day drug treatment. The neurointermediate lobes (NIL) were removed and incubated with [3H]-phenylalanine for 3 hrs. The biosynthesized pro-opiomelanocortin (POMC), β-lipotropin (β-LPH) and β-endorphin- like peptides (β-EPLPs) were purified from the total protein extract of the NIL by immunoprecipitation with an antiserum to β-endorphin (β-EP), and analyzed by sodium dodecyl sulfate polyacrylamide disc gel elecrophoresis. The β-EPLPs were further characterized by extraction from the gel and microsequencing. The homology of rat POMC to authentic bovine POMC was established by extraction from the gel and peptide mapping of its tryptic digestion products. Furthermore, the β-endorphin like immunoreactivity (β-EPLI) was estimated in the incubation medium and in the NIL extract. The morphine treatment induced a decrease in the degree of incorporation of [3H]- phenylalanine into POMC, β-LPH and β-EPLPs, associated with a decrease in the content of β-EPLI in the NIL extract and in the incubation medium. Alcohol induced an increase in the degree of incorporation of [3H]-phenylalanine into POMC, β-LPH and β-EPLPs, and an increase in the β-EPLI content in the incubation medium, but no change in the β-EPLI in the NIL extract. These results indicate an effect of chronic morphine and alcohol treatment on the biosynthesis and release of β-EPLPs by the NIL.

Effect of chronic morphine treatment on β-endorphine biosynthesis by the rat neurointermediate lobe

The effect of chronic morphine treatment on the in vitro biosynthesis of beta-endorphin by rat pars intermedia was investigated. Tolerance and physical dependence were induced in 200 g rats by the subcutaneous implantation of 75 mg morphine pellets for either 3 days or 15 days. Immediately following sacrifice of the animals the neurointermediate lobes were removed and incubated with [3H] phenylalanine. The protein extracts of the lobes were analyzed for the incorporation of the labelled amino acid into total protein, pro-opiomelanocortin, beta-lipotropin (beta-LPH) and beta-endorphin. the biosynthesized products were purified by immunoprecipitation with an antiserum to beta-endorphin. The identity and purity of beta-endorphin were verified by polyacrylamide disc gel electrophoresis with sodium dodecyl sulfate, and microsequencing. The identity of pro-opiomelanocortin (POMC) was verified by peptide mapping of its tryptic digestion products. The results showed that morphine treatment induced a decrease in the incorporation of the radioactive amino acid into total protein, pro-opiomelanocortin, beta-LPH and beta-endorphin. The decrease was more pronounced for the incorporation into beta-LPH and beta-endorphin than into pro-opiomelanocortin and total proteins, suggesting an effect of morphine treatment on the processing of the pro-opiomelanocortin to its final maturation products.

Hyperlipoproteinemia induced by a transplantable pituitary tumor in the rat

The MtT-F4 tumor, a transplantable pituitary tumor of rats, induces significant hyperlipidemia in male Fisher 344 rats. The increasive hypercholesterolemia was accompanied by hypertriglyceridemia only in the first month of tumor implantation. Clofibrate feeding inhibited the development of hypercholesterolemia and maintained normal serum triglyceride levels. In contrast to the changes in lipoprotein cholesterol distribution and profile found in experimental hyperlipidemia induced by high fat and cholesterol feeding, the hypercholesterolemic tumor-bearing rats showed no accumulation of cholesterol in the very low density and intermediate density lipoproteins, and no appearance of a new class of lipoprotein, B-VLDL. An HDLc-like lipoprotein appeared as hypercholesterolemia developed. Increased amounts of cholesterol were deposited in the aorta. The effects are attributed to the lipolytic hormones secreted by the tumor and antagonism to their action by clofibrate.

Endorphins in Fetomaternal Physiology

Following the demonstration of specific opiate receptors in the nervous system of vertebrates and the isolation of a number of endogenous opiate ligands (β-endorphin, enkephalins, dynorphins, and neoendorphins) for these receptors, it was suggested that interactions of endogenous opiate peptides with opiate receptors may be involved in regulating some physiological processes. Initially, the analgesic activity of the endogenous opiates was investigated. However, it was soon noticed that various types of stress stimulate the release of endogenous opiates, suggesting that endogenous opiates may be important in the adaptation of the organism to various stressful situations. Endorphins were also found to modulate the release of prolactin (PRL) and the gonadotropin hormones luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary in humans and experimental animals and to influence the reproductive behavior in animals.

PRO-OPIOMELANOCORTIN: A NEW CONCEPT ISSUED FROM ACTH AND LPH BIOSYNTHETIC STUDIES

We have investigated the biosynthesis of all portions of the precursor molecule related to ACTH/LPH/MSH/ endorphin in order to describe the important cellular events of maturation. Our approach which is based mainly on chemical characterization of the material gives more definitive results than the immunological approach. It has permitted us to demonstrate the presence of the complete N-terminal fragment as a main product of secretion in human, porcine and rat. They were characterized and results indicated (a) that rat N-terminus is a mixture of 2 molecules with one mutation at the N-terminus (Arg/Trp), thus could have two genes; (b) gamma-MSH region of human and porcine is identical to bovine; (c) the sequence of both porcine and human are quite homologous to the bovine species.

EFFECT OF MORPHINE OR ALCOHOL TREATMENT ON THE BIOSYNTHESIS OF β-ENDORPHIN BY THE NEUROINTERMEDIATE LOBE

Rats were rendered tolerant to either morphine or alcohol, by 21 day drug treatment. Morphine treatment induced a decrease in the degree of incorporation of (3H)-phenylalanine into pro-opiomelanocortin (POMC), β-lipotropin (β-LPH) and β-endorphin (β-EP) associated with a decrease in the content of β-endorphin like immunoreactivity (β-EPLI) in the neurointermediate lobe (NIL) extract and in the incubation medium. Alcohol induced an increase in the degree of incorporation of (3H)-phenylalanine into POMC, β-LPH and β-EP and an increase in the β-EPLI content in the incubation medium but not in the NIL extract. These results indicate an effect of chronic morphine and alcohol treatment on the biosynthesis and release of β-EP by the rat NIL.