Browsing by Author "MORALES, MN"

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    ACTIVATION OF HYPOLIPEMIC DRUGS TO ACYL-COENZYME-A THIOESTERS
    (1986) BRONFMAN, M; AMIGO, L; MORALES, MN
    Compounds possessing the characteristics of CoA thioesters of the hypolipidaemic peroxisome proliferators clofibric acid, nafenopin and ciprofibrate were formed on incubation of the drugs with rat liver microsomal fractions, ATP and CoA. The reactivity of the drugs correlated with their pharmacological potency. It is proposed that the active species of these compounds are their acyl-CoA thioesters.
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    HYPOLIPEMIC DRUGS ARE ACTIVATED TO ACYL-COA ESTERS IN ISOLATED RAT HEPATOCYTES - DETECTION OF DRUG ACTIVATION BY HUMAN LIVER HOMOGENATES AND BY HUMAN PLATELETS
    (1992) BRONFMAN, M; MORALES, MN; AMIGO, L; ORELLANA, A; NUNEZ, L; CARDENAS, L; HIDALGO, PC
    The formation of acyl-CoA esters of the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate and nafenopin was studied in isolated rat hepatocytes. The concentration of ciprofibroyl-CoA in the liver of ciprofibrate-treated rats was in the range of 10-30-mu-M. The three drugs formed acyl-CoA esters when incubated with isolated hepatocytes. Their formation was saturable and reached a plateau after 30 min incubation. Maximal intracellular concentrations of ciprofibroyl-CoA and clofibroyl-CoA (100-mu-M and 55-mu-M respectively) were attained at 0.5 mM of the free drugs in the incubation medium, whereas for nafenopin-CoA, the maximal intracellular concentration (9-mu-M) was reached at 1 mM-nafenopin. At low concentrations of the hypolipidaemic compounds in the incubation medium a significant proportion of the total intracellular drug was present as its acyl-CoA ester (25-35% for ciprofibrate). When isolated hepatocytes were incubated with a ciprofibrate concentration comparable with that observed in the blood of drug-treated rats (0.1 mM), ciprofibroyl-CoA attained an intracellular concentration similar to that previously observed in the liver of treated rats. The formation of ciprofibroyl-CoA by isolated rat hepatocytes was stimulated by the addition of carnitine and partially inhibited by the addition of palmitate. Further, it was shown that human liver homogenates synthesized ciprofibroyl-CoA at a rate similar to that observed for rat liver homogenates. Solubilized human platelets also formed ciprofibroyl-CoA, although at a rate two orders of magnitude lower than that of liver. The results support the view that acyl-CoA esters of hypolipidaemic peroxisome proliferators may be the pharmacologically active species of the drugs.
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    INDUCTION OF PEROXISOMAL FATTY ACYL-COENZYME A-OXIDASE AND TOTAL CARNITINE ACETYL-COENZYME A-TRANSFERASE IN PRIMARY CULTURES OF RAT HEPATOCYTES BY GARLIC EXTRACTS
    (1992) ORELLANA, A; KAWADA, ME; MORALES, MN; VARGAS, L; BRONFMAN, M
    Garlic has been proposed as a natural hypolipidemic substance. Most hypolipidemic compounds induce peroxisomal proliferation and increase enzyme activities associated with peroxisomal beta-oxidation in rat liver. Here we report that garlic methanol-extracts behave as hypolipidemic drugs, increasing the activity of peroxisomal fatty acyl-coenzyme A oxidase and of total carnitine acetyl-coenzyme A transferase in primary cultures of rat hepatocytes. Both enzymes are considered markers associated with increased peroxisomal beta-oxidation. As in the case of hypolipidemic peroxisome proliferators, garlic extracts partially prevented the decrease in fatty acyl-coenzyme A oxidase as the culture aged. No changes were observed in the activity of microsomal NADPH cytochrome c reductase or of mitochondrial glutamate dehydrogenase.
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    PALMITOYL-COA AND THE ACYL-COA THIOESTER OF THE CARCINOGENIC PEROXISOME-PROLIFERATOR CIPROFIBRATE POTENTIATE DIACYLGLYCEROL-ACTIVATED PROTEIN KINASE-C BY DECREASING THE PHOSPHATIDYLSERINE REQUIREMENT OF THE ENZYME
    (1990) ORELLANA, A; HIDALGO, PC; MORALES, MN; MEZZANO, D; BRONFMAN, M
    To gain insight into the mechanism by which long-chain acyl-CoA thioesters potentiate diacylglycerol-activated protein kinase C, the cofactor dependence of this activating effect was studied with purified rat brain enzyme and histone H1 as substrate. Using two different assay systems, palmitoyl-CoA was found to decrease greatly the amount of phosphatidylserine required to activate the kinase. No relative changes were observed in the dependence of the enzyme for other cofactors (diacylglycerol, ATP, and Ca2+) in the presence of palmitoyl-CoA. The potentiating effect of palmitoyl-CoA and the decrease in phosphatidylserine requirement of the kinase was also demonstrated using the 47-kDa protein of human platelets as substrate and platelet protein kinase C as source of enzyme. The acyl-CoA thioester of the carcinogenic peroxisome-proliferator ciprofibrate was also found to decrease the phosphatidylserine requirement of protein kinase C. The data suggest that acyl-CoAs may play a role in the regulation of protein kinase C activity.
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    POTENTIATION OF DIACYLGLYCEROL-ACTIVATED PROTEIN KINASE-C BY ACYL-COENZYME A THIOESTERS OF HYPOLIPEMIC DRUGS
    (1989) BRONFMAN, M; ORELLANA, A; MORALES, MN; BIERI, F; WAECHTER, F; STAUBLI, W; BENTLEY, P
    Acyl-Coenzyme A thioesters of the hypolipidaemic and cancerinogenic peroxisome proliferators clofibric acid, nafenopin, ciprofibrate, benzafibrate and tibric acid were found to greatly increase the activity of rat brain protein kinase C. Maximal activation required the simultaneous presence of Ca2+, phosphatidylserine and diolein, thus differentiating their action from that of other tumor promoters such as phorbol esters. Under similar conditions the unesterified drugs were comparatively ineffective. Similar results were obtained using the rat liver enzyme. The data suggest that acyl-coenzyme A thioesters of hypolipidaemic drugs, may play a role in the induction of liver tumors by these compounds, through the potentiation of protein kinase C.
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    SUBCELLULAR-DISTRIBUTION AND CHARACTERISTICS OF CIPROFIBROYL-COA SYNTHETASE IN RAT-LIVER - ITS POSSIBLE IDENTITY WITH LONG-CHAIN ACYL-COA SYNTHETASE
    (1992) AMIGO, L; MCELROY, MC; MORALES, MN; BRONFMAN, M
    The subcellular distribution and characteristics of ciprofibroyl-CoA synthetase were studied in rat liver and compared with those of long-chain acyl-CoA synthetase (palmitate as substrate) which, as already known, is distributed among mitochondria, microsomes and peroxisomes. Upon differential centrifugation, the subcellular distribution of ciprofibroyl-CoA synthetase followed closely that of palmitoyl-CoA synthetase and was specifically inactivated in the mitochondrial fraction by freezing and thawing, a behaviour already described for palmitoyl-CoA synthetase. Both enzyme activities were found to co-purify through several steps from rat liver microsomes. By using a partially purified enzyme, the activation of ciprofibrate to its acyl-CoA ester followed Michaelis-Menten kinetics with an apparent K(m) of 0.63 +/- 0.1 mM. Ciprofibroyl-CoA synthetase was competitively inhibited by 25 and 50-mu-M-palmitic acid. Higher concentrations of the fatty acid resulted in a mixed type of inhibition. Conversely, ciprofibrate up to 0.5 mM was found to inhibit competitively palmitoyl-CoA synthetase, whereas higher concentrations also resulted in a mixed inhibition. The highest activity of ciprofibroyl-CoA synthetase was found in fat and liver homogenates. The distribution of the enzyme in different rat tissues was similar to that of palmitoyl-CoA synthetase. The present results suggest that long-chain acyl-CoA synthetase and ciprofibroyl-CoA synthetase activities reside in identical or closely related proteins.