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2.
J Pharmacol Exp Ther ; 291(2): 555-61, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10525071

ABSTRACT

The interaction of the cysteine conjugate S-(1-chloro-1,2,2, -trifluoroethyl)-L-cysteine (CTFC) with organic anion and amino acid transport in the basolateral and apical membranes was examined with rabbit renal proximal tubule suspensions and primary cultures of rabbit renal proximal tubule cells. The apparent K(i) for CTFC inhibition of the 1-min uptake of [(3)H]p-aminohippurate in tubule suspensions was 105+/-3 microM and suggests that CTFC interacts with basolateral organic anion transport. Also, the addition of 1 mM CTFC decreased the secretion and intracellular accumulation of fluorescein by approximately 70 to 75%. The addition of 1 mM CTFC to the apical compartment decreased the reabsorption and intracellular accumulation of the amino acid [(3)H]phenylalanine by approximately 60 to 70%. Similar to CTFC, saturating concentrations of the organic anion [(3)H]p-aminohippurate and the amino acid phenylalanine reduced by approximately 75% fluorescein secretion and [(3)H]phenylalanine reabsorption, respectively, by approximately 60 to 70%. Thus, the cysteine conjugate CTFC appears to be a potent inhibitor of basolateral organic anion and apical amino acid transepithelial transport. In contrast to its effects on apical phenylalanine uptake, CTFC had no effect on the basal uptake of [(3)H]phenylalanine by primary cultures. The presence of CTFC in the external bath did trans-stimulate the efflux of fluorescein and [(3)H]phenylalanine across the basal and apical membrane in tubule suspensions or primary cultures, respectively, grown on plastic. Collectively, these data demonstrate that CTFC interacts with, and is transported by, two anatomically and functionally distinct transporters, the basolateral organic anion and apical neutral amino acid pathways, in the rabbit renal proximal tubule cell.


Subject(s)
Amino Acids/pharmacokinetics , Anions/pharmacokinetics , Cell Membrane/drug effects , Cysteine/analogs & derivatives , Kidney Tubules, Proximal/drug effects , Animals , Biological Transport, Active/drug effects , Cells, Cultured , Contrast Media/pharmacokinetics , Cysteine/pharmacology , Epithelium/drug effects , Epithelium/metabolism , Female , Fluorescein/pharmacokinetics , Male , Phenylalanine/pharmacology , Rabbits , Time Factors , p-Aminohippuric Acid/pharmacokinetics
3.
J Pharmacol Exp Ther ; 275(2): 926-32, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7473184

ABSTRACT

To better understand the characteristics of peritubular transport of organic cations (OCs), the uptake of the polyvalent OC dimethylbipyridinium (paraquat) and the structurally similar monovalent OC 1-methyl-4-phenylpyridinium (MPP+) was measured in suspensions of rabbit renal proximal tubules. Compared to the uptake of MPP+, the uptake of paraquat across the peritubular membrane was a low affinity, low capacity carrier-mediated process with a Jmax of 0.52 +/- 0.19 nmol.mg of protein.-1 min-1 and a Km of 162 +/- 25 microM. The uptake of MPP+ was a carrier-mediated process with a measured Jmax and Km of 1.8 +/- 0.09 nmol.mg of protein.-1min-1 and 28 +/- 8 microM, respectively. To determine whether paraquat is a substrate for the monovalent OC pathway, the effect of unlabeled MPP+ and tetraethylammonium (TEA) on paraquat uptake was examined. A 1 mM concentration of the monovalent OC MPP+ and TEA reduced the uptake of [14C]paraquat and [3H]MPP+ by approximately 30 and 90%, respectively, whereas 1 mM paraquat had no effect on [3H]MPP+ or [14C]TEA uptake. Thus, MPP+, but not paraquat, appears to interact with the monovalent OC transporter. On the other hand, the polyvalent OC substrates, including the polyamines putrescine and spermine, the herbicide diquat and the divalent hexamethonium bromidehydrate had no effect on either paraquat or MPP+ uptake. However, the synthetic polyamine methylglyoxal bis(guanyl-hydrazone)dihydrochloride (MGBG; 1 mM) reduced both paraquat and MPP+ uptake (by 60 and 90%, respectively). The ability of MGBG, unlike the other polyvalent substrates, to interact with paraquat transport may be related to structural similarities in the relative location of the two charged nitronium moieties in paraquat and MGBG.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Kidney Tubules, Proximal/metabolism , Paraquat/metabolism , 1-Methyl-4-phenylpyridinium/metabolism , Animals , Cations , In Vitro Techniques , Mitoguazone/metabolism , Rabbits , Tetraethylammonium , Tetraethylammonium Compounds/metabolism
4.
Biochem J ; 284 ( Pt 1): 283-7, 1992 May 15.
Article in English | MEDLINE | ID: mdl-1599407

ABSTRACT

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 Km of 0.63 +/- 0.1 mM. Ciprofibroyl-CoA synthetase was competitively inhibited by 25 and 50 microM-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.


Subject(s)
Acyl Coenzyme A/metabolism , Clofibric Acid/analogs & derivatives , Coenzyme A Ligases/metabolism , Liver/enzymology , Repressor Proteins , Saccharomyces cerevisiae Proteins , Animals , Binding, Competitive , Clofibric Acid/metabolism , Coenzyme A Ligases/isolation & purification , Kinetics , Rats , Rats, Inbred Strains , Subcellular Fractions/enzymology , Tissue Distribution
5.
Biochem J ; 284 ( Pt 1): 289-95, 1992 May 15.
Article in English | MEDLINE | ID: mdl-1599408

ABSTRACT

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 microM. 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 microM and 55 microM respectively) were attained at 0.5 mM of the free drugs in the incubation medium, whereas for nafenopin-CoA, the maximal intracellular concentration (9 microM) 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.


Subject(s)
Blood Platelets/metabolism , Clofibric Acid/analogs & derivatives , Clofibric Acid/pharmacokinetics , Lipids/blood , Liver/metabolism , Nafenopin/pharmacokinetics , Acyl Coenzyme A/metabolism , Animals , Biotransformation , Carnitine/pharmacology , Cells, Cultured , Clofibric Acid/metabolism , Clofibric Acid/pharmacology , Fibric Acids , Humans , Kinetics , Liver/cytology , Nafenopin/analogs & derivatives , Nafenopin/metabolism , Octoxynol , Palmitic Acid , Palmitic Acids/pharmacology , Polyethylene Glycols/pharmacology , Rats , Rats, Inbred Strains
6.
Toxicol Lett ; 60(1): 11-7, 1992 Jan.
Article in English | MEDLINE | ID: mdl-1539178

ABSTRACT

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.


Subject(s)
Carnitine O-Acetyltransferase/biosynthesis , Garlic , Liver/drug effects , Microbodies/drug effects , Oxidoreductases/biosynthesis , Plants, Medicinal , Acyl-CoA Oxidase , Animals , Cells, Cultured , Enzyme Induction/drug effects , Liver/enzymology , Male , Microbodies/enzymology , Plant Extracts/pharmacology , Rats
7.
Eur J Biochem ; 190(1): 57-61, 1990 May 31.
Article in English | MEDLINE | ID: mdl-2364949

ABSTRACT

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.


Subject(s)
Acyl Coenzyme A/pharmacology , Clofibrate/analogs & derivatives , Clofibric Acid/analogs & derivatives , Diglycerides/pharmacology , Glycerides/pharmacology , Palmitoyl Coenzyme A/pharmacology , Phosphatidylserines/metabolism , Protein Kinase C/metabolism , Animals , Blood Platelets/enzymology , Brain/enzymology , Calcium/pharmacology , Clofibric Acid/pharmacology , Cocarcinogenesis , Drug Synergism , Enzyme Activation/drug effects , Fibric Acids , Histones/metabolism , Humans , Microbodies/drug effects , Phosphorylation , Rats
8.
Biochem Biophys Res Commun ; 159(3): 1026-31, 1989 Mar 31.
Article in English | MEDLINE | ID: mdl-2930549

ABSTRACT

Acyl-Coenzyme A thioesters of the hypolipidaemic and cancerinogenic peroxisome proliferators clofibric acid, nafenopin, ciprofibrate, bezafibrate and tibric acid were found to greatly increase the activity of rat brain protein kinase C. Maximal activation required the simultaneous presence of Ca+2, 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 acylcoenzyme 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.


Subject(s)
Brain/enzymology , Hypolipidemic Agents/pharmacology , Protein Kinase C/metabolism , Acyl Coenzyme A/pharmacology , Animals , Diglycerides/pharmacology , Kinetics , Rats
9.
Biochem Biophys Res Commun ; 152(3): 987-92, 1988 May 16.
Article in English | MEDLINE | ID: mdl-3377782

ABSTRACT

The activity of rat brain protein kinase C, measured in the presence of diacylglycerol, phosphatidylserine and Ca+2, was found to be greatly increased by micromolar amounts of long chain acyl-CoAs, using two different assay systems (lipids added as sonicated dispersion or as mixed micelles with Triton X-100). The potentiation phenomenon required the presence of both diacylglycerol and phosphatidylserine; it was observed at low and saturating concentrations of these effectors, and it was inhibited at high, non physiological Ca+2 concentrations. Under similar conditions, fatty acids alone or coenzyme A were ineffective. The data strongly suggest that acyl-CoAs at the intracellular concentration levels, are important in the modulation of protein kinase C, after activation of the enzyme by the phospholipase C/phosphatidylinositol pathway.


Subject(s)
Acyl Coenzyme A/pharmacology , Diglycerides/pharmacology , Glycerides/pharmacology , Protein Kinase C/metabolism , Animals , Brain/enzymology , Calcium/pharmacology , Drug Synergism , Enzyme Activation , Octoxynol , Phosphatidylserines/pharmacology , Polyethylene Glycols , Rats , Structure-Activity Relationship
10.
Biochem J ; 239(3): 781-4, 1986 Nov 01.
Article in English | MEDLINE | ID: mdl-3827829

ABSTRACT

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.


Subject(s)
Acyl Coenzyme A/metabolism , Clofibrate/analogs & derivatives , Clofibrate/metabolism , Clofibric Acid/analogs & derivatives , Microsomes, Liver/metabolism , Nafenopin/metabolism , Propionates/metabolism , Repressor Proteins , Saccharomyces cerevisiae Proteins , Animals , Chromatography, High Pressure Liquid , Clofibric Acid/metabolism , Coenzyme A Ligases/metabolism , Fibric Acids , Hydrolysis , Male , Nafenopin/analogs & derivatives , Rats , Rats, Inbred Strains , Spectrophotometry, Ultraviolet
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