Alcohol induced alterations in polyunsaturated fatty acid composition and metabolism

Alcohol induced alterations in polyunsaturated fatty acid composition and metabolism

Concurrent Sessions Tuesday 22 July EFA & Eicosanoids 1997 - Edinburgh T12 T13 Identification of Residues Determining Ligand Selectivity For EP 2 ...

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Concurrent Sessions Tuesday 22 July

EFA & Eicosanoids 1997 - Edinburgh

T12

T13

Identification of Residues Determining Ligand Selectivity For EP 2 Prostanoid Receptor. D W Gil, KM

Alcohol Induced Alterations in Polyunsaturated Fatty Acid Composition and Metabolism. Norman

Kedzie, JE Donello, HA Krauss, DF Woodward, JW Regan. Allergan, Irvine, CA and Univ. of Arizona, Tucson, AZ, USA.

Salem, Jr., Nils U. Olsson, Joseph R. Hibbeln, Robert J. Pawlosky. LMBB, NIAAA, NIH, Rockville, MD, USA.

A high degree of homology between the four Gs-coupled prostaglandin receptors (EP=, EP 4, IP, DP) and the four Gq/Gicoupled receptors (EP~, EP 3, FP, TP) suggests that PG receptors evolved functionally from an ancestral EP receptor before the development of distinct binding epitopes. If so, ligand selectivity should be determined by a limited number of amino acids. EP 2 receptor transmembrane domain residues that are similar in the EP 4 receptor, but are not homologous in the IP receptor, were mutated to the corresponding IP receptor residue. Activation of the mutant receptors by PGE 2 (EP 2 ligand), iloprost (IP ligand), and PGE~ (EPJlP ligand) was determined using a cyclic AMP-dependent reporter gene assay. Changing a single amino acid in the 7th transmembrane domain (TM7) of the EP 2 receptor enhanced iloprost potency approximately 100-fold so that the receptor was activated by all three drugs. An amino acid change in the third transmembrane domain had no effect on drug potency but improved the response of the TM7 mutant to the three drugs. The potency of PGFz~ and P G D 2 was not enhanced by these mutations. The TM7 arginine residue has been implicated as a counter ion for the prostaglandin carboxylic acid and consequently mutations of this residue were tested. The potency of all prostaglandins was reduced 10-100 fold by changes in this residue. Thus, we have identified a single amino acid change that is important for iloprost activity and may have been critical in the evolution of the prostacyclin binding epitope.

Alcohol is a complex drug that appears to act through multiple mechanisms. One of the impol~ant mechanisms for its action involves the alteration in essential fatty acid (EFA) metabolism and the resulting losses in long chain polyunsaturates like arachidonic (AA) or docosahexaenoic (DHA) acids in biological tissues. Several studies involving various mammalian species will be p~esented which demonstrates the losses of EFAs after alcohol exposure. Animal models of alcoholism using a diet low, but not inadequate in EFAs and some vitamins have been used to better simulate the human condition. Alcohol-induced losses in liver DHA, for example, will be s h o w n for felines, rhesus and human subjects. Losses of DHA in the central nervous system have been demonstrated in rhesus and domestic cats after alcohol exposure as well. Ad libitum drinking in rhesus monkeys for 5 years leads to the development of liver fibrosis and alteled electroretinograms. Assessment of bz vivo deuterium accumulation in AA and DHA fi'om the deuterated 18-carbon EFA precursors does not suppo~ the view that alcohol inhibits desaturase enzymes thereby slowing AA and DHA formation. Related in vivo human studies of volunteers, smokers and alcoholic-smokers on withdrawal will be presented. Our studies indicate a marked increase in alcohol-induced EFA degradation as indicated by aldehydes, isoprostanes and other markers. It is hypothesized that the prooxidative challenge posed by alcohol together with a diet with often lower levels of both EFAs and antioxidant nutrients lead to diminished levels of tissue long chain polyunsaturates such as DHA. This then contributes to the multiple organ system pathologies obsetwed in alcoholics.

T14

T15

Effect of ethanol exposure on fetal guinea-pig brain essential fatty acid content GC Burdge and AD Postle. Child Health, University of Southampton, Southampton, UK

ETHANOL BINDS TO LIPID CARRIERPROTEINS W. G. Wood, S. V. Chochina, U. Igbavboa, F. Schroeder, and N. A. Avdulov. VA Medical Center, GRECC and Univ. Minn. Depart. Pharmacol., Minneapolis, Minnesota, US, 55417

Chronic maternal ethanol consumption during pregnancy may result in severe impairment of both fetal brain growth and function: fetal alcohol syndrome (FAS). The mechanisms by which ethanol exposure alters brain development are not known. Adequate accumulation of docosahexaenoic acid (22:6033, DHA) into phospholipids in the developing central nervous system is critical for subsequent optimal function. We have used a guineapig model to test the hypothesis that ethanol modifies DHA assimilation into developing brain as a possible pathological mechanism in FAS. Adult female guinea-pigs were fed either chow or chow+6g/kg ethanol/day both before (14 days) and throughout pregnancy. Fetuses were delivered by Caesarian section at term (68 days), and fetal brain phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species compositions determined by HPLC. Ethanol exposure resulted in increased total PC (1.3 fold) and decreased total PE (1.6 fold) contents. Concentrations of PUFA-containing PC species, including DHA, were significantly lower in ethanol-exposed fetal brain while disaturated and monounsaturated species were increased. Ethanol exposure resulted in differential decreases in DHA-containing PE species and increases in arachidonate and oleate species. These biochemical changes were accompanied by impaired motor function. Feeding maternal guinea-pigs a combination of ethanol and tuna oil (0.5/gday containing 26% DHA) both before and throughout pregnancy increased the DHA content of both fetal brain PC (1.4 fold) and PE (2. I fold) above control levels. This was associated with a subiective decrease in the severity of impaired neonatal motor function. There was no significant change in the DHA content of fetal brains from mothers fed tuna oil alone. Together these results suggest that impaired DHA assimilation may contribute significantly to the pathogenesis of FAS and that maternal dietary supplementation with DHA might potentially be useful in reducing the severity of ethanol-induced brain damage.

Ethanol alters the biological activity of various proteins. The molecular mechanism of how ethanol affects activity of proteins is not well-understood. One hypothesis that has been previously proposed is that ethanol as well as general anesthetics directly bind to certain proteins and such binding alters protein function. However, much of the evidence in support of that hypothesis is based on changes in protein activity and/or an interaction with lipids and proteins. We have been examining the interaction of ethanol with different lipid carrier proteins using fluorescence spectroscopy. Ethanol (25-200 retool) competitively inhibited 1,8-ANS binding to BSA. Binding of cis- parinaric acid to BSA was decreased by ethanol and we found that ethanol was acting on three of the five fatty acid-binding sites on BSA. Sterol carrier protein-2 (SCP-2) binds fatty acids, phospholipids, cholesterol and expression of SCP-Z was increased in brain of chronic ethanol-treated mice. Ethanol inhibited binding of NBDstearic acid, phosphatidylcholine, and cholesterol to SCP2. Liver-fatty acid binding protein, a protein that binds both fatty acids and cholesterol was unaffected by ethanol in vitro. Ethanol-induced changes in activity of certain proteins may result from direct binding of ethanol to specific hydrophobic binding sites and/or displacement of endogenous ligands from those sites. Supported in part by NIH grants AA 10806, AG 11056 and the Department of Veterans Affairs.

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