In pancreas, the activities of several sex steroid-transforming enzymes have been reported. Data have been obtained in perfused organs, total tissue homogenates, and subcellular organelles. These data, concurrent with the description of the presence of ligand-regulated steroid receptors, as well as the sexually dimorphic behavior of some pancreatic tumors, are clear evidence in support of the participation of steroid hormones in the pancreatic function. In this study, the steroidogenic ability of the pancreas was demonstrated by two different methods: (a) in tissue homogenates, by the identification of cytochrome P-450scc gene (CYP11A) transcripts after reverse transcription-polymerase chain reaction amplification (RT-PCR); and (b) in isolated mitochondria by the glutethimide-dependent inhibition of cholesterol-pregnenolone biotransformation. The results obtained in a series of independent experiments showed that (a) the pancreatic tissue possessed transcriptional activity of the CYP11A gene, although to a lesser extent than the typical steroidogenic tissues, and (b) isolated mitochondria obtained from the pancreas were able consistently to synthesize pregnenolone; furthermore, the addition of the specific inhibitor aminoglutethimide (AMG) blocked its synthesis. On the whole, these findings are interpreted as clear evidences of the activity of the cytochrome P-450scc enzymatic complex (P450scc), responsible for the transformation of cholesterol into pregnenolone and considered the first and limiting step in steroid biosynthesis.

The effect of 6-ketocholestanol was studied on CCCP-induced uncoupling in liver mitochondria, submitochondrial particles and cytochrome oxidase proteoliposomes. It was found that 6-ketocholestanol prevents and reverses uncoupling induced by nM concentrations of CCCP on the three systems assayed. As it was reported on kidney mitochondrial membranes [Chavez et al. (1996) FEBS Lett. 379, 305-308], the recoupling effect caused by 6-ketocholestanol on submitochondrial particles and proteoliposomes could be due to a diminution of membrane fluidity.

This study shows that the nonsteroidal antiinflammatory drug, ketorolac, protects against myocardial damage induced by reperfusion. This effect was analyzed after 5 min of coronary occlusion in rat hearts. The results indicate that ketorolac, at a dose of 1 mg/kg, effectively protects the heart against reperfusion arrhythmias. Furthermore, it protects from the release of lactate dehydrogenase and creatine kinase to the plasma. We propose that the protective effect of the drug might he due to its chelating action on calcium ions, thus preventing the overload of such cation in myocardial cells.

This work shows that 6-ketocholestanol (kCh) inhibits the effect of carbonyl cyanide-m-chlorophenyl hydrazone (CCP) on mitochondrial Ca2+ efflux. Such an effect proved to be caused by diminution of membrane fluidity, therefore, it is affected by the incubation temperature. Furthermore, kCh reversed CCP-induced Ca2+ efflux depending on the accumulation of phosphate. It is also shown that kCh enhances the effect of carboxyatractyloside on membrane permeability transition.

1. In order to explore the mechanism of inhibition of hydroxylases involved in steroidogenesis, by gossypol, we studied the effect of this drug on adrenal cortex mitochondria, and compared it with those on kidney and heart.2. The uncoupler effect of gossypol (collapse of DELTApsi and Ca2+ efflux) was found to be lower in adrenal cortex mitochondria than in kidney and heart mitochondria.3. Gossypol produced more extensive changes on the membrane lipidic matrix (increase in the order parameter for 5-doxylstearic acid) in adrenal cortex mitochondria than in the other mitochondria studied.4. The results described above indicate that the mechanism of inhibition of gossypol of steroidogenic adrenal enzymes could be attributed to an alteration of the lipidic matrix which, in turn, modifies protein function.

Cholesterol side-chain cleavage and 11 beta-hydroxylation were assessed in isolated adrenal cortex mitochondria by formation of pregnenolone and corticosterone, respectively, in the presence and absence of gossypol. Pregnenolone biosynthesis was inhibited when increasing concentrations of gossypol were added. The control value of 4 nmol min-1 mg-1 dropped to 2 nmol min-1 mg-1 with 30 microM of the drug in the incubation medium. A more pronounced inhibitory effect was observed upon 11 beta-hydroxylation of steroids; I50 was 11 microM. Seventy-five percent of corticosterone production was impaired when 30 microM of gossypol were present. Bovine serum albumin prevented and reversed the inhibitory action of the drug. Kinetic studies showed a linear mixed type inhibition, suggesting a direct action of the drug upon the enzymatic complex. This study demonstrates a direct inhibitory effect of gossypol upon the steroidogenic enzymes located in the inner mitochondrial membrane of the adrenal cortex.

Ca2+ energy-coupled transport was analized in adrenal cortex mitochondria using the sesquiterpenic drug perezone. Perezone promotes Ca2+ efflux by inducing collapse of the membrane potential and oxidation of pyridine nucleotides. The effect of perezone on mitochondrial Ca2+ release follows a dose-response relationship and is dependent of the reduction of the drug. These data suggest that perezone may produce a cytotoxic effect through an impairment in Ca2+ homeostasis.

Adrenal cortex mitochondria isolated from mongrel dogs were incubated with cholesterol in the presence and absence of adriamycin (ADM). The capacity for cholesterol side chain cleavage was assessed by determining the pregnenolone yield. ADM behaves as an inhibitor of pregnenolone biosynthesis with a calculated IC50 of 110 microM. The inhibitory effect follows a dose-response relationship depending upon ADM concentration.

Inhibition of 11β-hydroxylase activity was observed to be due to the interaction of adriamycin with adrenal cortex mitochondria. The inhibition of the enzyme was uncompetitive, with an apparent Ki of 100 μm, and was dependent upon the concentration of the drug and the time of incubation. Adriamycin increased the oxygen consumption of these mitochondria. EPR studies showed that adriamycin was reduced to a free radical semiquinone which served to shuttle electrons to oxygen, leading to an impairment in the reduction of cytochrome P450. It is suggested this may be the mechanism for the inhibitory effect of the drug on 11β-hydroxylase activity.

The present paper describes experiments that show an interaction between ATP and the ATPase inhibitor quercetin which induce an increase in the fluorescence intensity. The experiments show also that the fluorescence intensity is higher with ATP than that observed with ADP and other purine or pyrimidine nucleotides. This fluorescence developed by the interaction ATP-quercetin can be used to monitor ATP- dependent enzymatic reactions as well as for determination of ATP.

The effect of the local anesthetic tetracaine on some functions of rat heart mitochondria was studied, and the results show that this tertiary amine inhibits the oxidation of NAD-dependent substrates by affecting the mitochondrial membrane in a site located between the NADH dehydrogenase and Co Q span through a process which is not affected by K+, Ca2+, or Mg2+. Also the results show that tetracaine induces sodium uptake probably by a H+/Na+ exchange reaction.