William C. Buss

Effect of calcium channel antagonists on calcium uptake and release by isolated rat cardiac mitochondria

The effects of calcium channel antagonists on Ca2+ uptake and Na+-induced Ca2+ release were studied in isolated rat cardiac mitochondria. Diltiazem, nitrendipine and nimodipine were more effective inhibitors of Na+-induced Ca2+ release (IC50 = 19-100 microM) than of Ca2+ uptake (IC50 = 0.2-1 mM). Nitrendipine and nimodipine had virtually identical IC50 values for inhibiting Ca2+ uptake, but nitrendipine was 3-4 times more potent than nimodipine at inhibiting Na+-induced Ca2+ release. If these calcium channel antagonists achieve intracellular concentrations in the range of 10(-5)-10(-4) M, our results suggest that calcium channel antagonists would preferentially inhibit mitochondrial calcium release more than mitochondrial calcium uptake.

Effects of rifampicin on RNA and protein synthesis in isolated rat liver mitochondria

Abstract Isolated rat liver mitochondria incorporated [ 3 h]utp in an essentially linear fashion for at least 60 min in the presence and absence of exogenous ribonucleoside triphosphates. Preincubation of isolated mitochondria with rifampicin at 0° in the absence of ribonucleoside triphosphates produced a consistent inhibition of [ 3 h]utp incorporation at 30° that ranged up to 75 per cent and was independent of preincubation time with rifampicin or the length of the assay period. Incorporation of [ 3 H]UMP into mitochondria prepared in the presence of the proteolytic enzyme Nagarse was similarly inhibited by rifampicin. When mitochondria prepared in the presence of Nagarse were preincubated with rifampicin, incorporation of [ 3 h]utp was inhibited by approximately 75 per cent within 2 min, while [ 3 H] leucine incorporation continued in a linear fashion for 4–5 min before decreasing with first-order kinetics. These data demonstrate that rifampicin primarily affects RNA synthesis in mitochondria, and leads secondarily to an inhibition of protein synthesis. A 75 per cent inhibition of mitochondrial RNA synthesis resulting in a 45 per cent inhibition of mitochondrial protein synthesis permits an estimate of 3.3 mm as the half-life of mitochondrial RNA serving a messenger function. It is hypothesized that this rapidly turning-over mitochondrial RNA which is closely linked to protein synthesis serves a regulatory role in oxidative phosphorylation.

Association of tissue-specific changes in translation elongation after cyclosporin with changes in elongation factor 2 phosphorylation

In studies of cyclosporin (CsA) toxicity in Sprague-Dawley rats, CsA administered in vivo produced tissue-specific, dose-dependent changes in microsomal translation throughout the bodies of the animals. The most pronounced translation inhibition was in microsomes from the kidney, the organ in which dose-limiting CsA toxicity occurs. In contrast, translation was stimulated in microsomes from the liver. CsA produced changes at the level of translation elongation, which is regulated by the reversible phosphorylation of elongation factor 2 (EF2). Changes in translation elongation after CsA were found to be associated with, and most likely caused by, changes in EF2 phosphorylation. Reduced renal translation elongation was associated with increased EF2 phosphorylation, and increased hepatic elongation with decreased EF2 phosphorylation. EF2 is phosphorylated by Ca2+ calmodulin-dependent protein kinase III (PKIII). Phosphorylated EF2 is a substrate for protein phosphatase 2A (PP2A), but not calcineurin (protein phosphatase 2B or PP2B), the enzyme inhibited by CsA-cyclophilin complexes in T-cells. When CsA or inhibitors of PKIII (EGTA, trifluoperazine) were added in vitro to assays of EF2 phosphorylation in renal or hepatic cytoplasm, or to assays of renal or hepatic microsomal translation elongation, they were without significant effects. Addition in vitro of the PP2A inhibitor okadaic acid increased EF2 phosphorylation in renal and hepatic cytoplasms, but inconsistently produced an inhibition of microsomal translation. However, in less complex rabbit reticulocyte lysates, addition of okadaic acid inhibited PP2A, increased EF2 phosphorylation, and inhibited translation elongation. Furthermore, addition of EGTA and trifluoperazine to rabbit reticulocyte lysates inhibited Ca2+ calmodulin-dependent PKIII activity, decreased EF2 phosphorylation, and stimulated translation elongation. CsA acting alone or as a complex with cyclophilin could alter EF2 phosphorylation by affecting transcriptional regulation or the enzymatic activity of PKIII, PP2A or EF2. Changes in EF2 phosphorylation and translation in body tissues suggest that CsA causes widespread disturbances in phosphorylation and dephosphorylation pathways regulating cellular processes including transcription and translation factor activity. These disturbances may underlie the broad spectrum of toxicities observed during CsA therapy.

Coordinate increases and decreases in mitochondrial RNA and ATP syntheses produced by propranolol and rifampicin

A variety of compounds were examined for their capacity to alter RNA synthesis in isolated rat cardiac and hepatic mitochondria. The beta-adrenergic blocking agents propranolol and butoxamine, and the antiarrhythmic agent quinidine, produced a concentration-dependent stimulation of RNA synthesis in cardiac and hepatic mitochondria. In contrast, the antitubercular antibiotic rifampicin produced a concentration-dependent inhibition of RNA synthesis in cardiac and hepatic mitochondria. Propranolol, as a representative compound which stimulated RNA synthesis, was also found to stimulate ATP synthesis in isolated mitochondria, whereas rifampicin inhibited ATP synthesis. Coordinate increases and decreases in RNA and ATP syntheses suggest that agents which stimulate or inhibit RNA synthesis may rapidly alter ATP synthesis. This finding is consistent with the rapid turn-over of mitochondrial RNA with a messenger function (1.4 and 3.3 min in isolated rat cardiac and hepatic mitochondria), and it suggests that mitochondrial RNA must continue to be synthesized to maintain inner membrane systems required for ATP synthesis. Stimulation of RNA and ATP syntheses by propranolol through membrane stabilization or other actions represents a heretofore unrecognized action of propranolol which may contribute to its beneficial therapeutic effects.

The dose-dependent inhibition of rat renal translation elongation seen after in vivo cyclosporin A is not caused by cyclosporin metabolites.

Cyclosporin A (CsA) given to Sprague-Dawley rats in vivo produced a tissue-specific, dose-dependent inhibition of translation elongation in renal microsomes. CsA at an oral dose of 50 mg/kg/day for 6 days reduced renal microsomal translation by 70.5%. Renal cytoplasm from rats treated in vivo with CsA inhibited translation by 55% when added to renal microsomes isolated from tissues of control animals. In contrast, CsA added to renal microsomes in vitro did not inhibit translation. Renal cytoplasm from CsA-treated rats containing translation inhibitory factor was found by HPLC to contain CsA and CsA metabolites M1 and M17. CsA metabolites M1, M17, M18 and M21 were isolated from human bile and tested in vitro for translation elongation inhibitory activity in renal microsomes. CsA, M18 and M21 did not inhibit translation elongation at concentrations of up to 2500 ng/ml. M17 inhibited translation elongation, but only by 8.4% at the highest concentration tested (2500 ng/ml), a level 20-fold higher than that measured in renal cytoplasm (125 ng/ml). M1 produced a concentration-dependent inhibition of translation elongation, beginning at 500 ng/ml, or approximately 2-fold higher than that found in renal cytoplasm (260 ng/ml). M1 at 2500 ng/ml or approximately 10-fold higher than the concentration measured in renal cytoplasm, inhibited translation elongation by 23.8%, only 1/3 that observed upon addition of renal cytoplasm containing translation inhibitory factor. We conclude from these findings that the dose-dependent inhibition of renal translation elongation following in vivo CsA cannot be explained by the renal formation or uptake of known CsA metabolites.

Studies on the mechanism of glucocorticoid hormone induced alterations in rat thymic transcription—II. Partial purification and characterization of RNA polymerases II from hydrocortisone and control vehicle treated animals

In experiments designed to study the mechanism of glucocorticoid hormone induced reductions in rat thymic transcription, adrenalectomized rats were injected with hydrocortisone (50 mg/kg) or control vehicle 12 h prior to sacrifice. Thymic nuclei were used to prepare soluble nuclear extracts containing RNA polymerase II. Nuclear extract RNA polymerases II were then partially purified (600-fold) on DEAE-Sephadex columns and characterized. The responses of partially purified thymic RNA polymerases II from rats treated in vivo with hydrocortisone or vehicle were similar to: pH, temperature, ionic strength, trypsin proteolysis, and inhibition by alpha-amanitin; however, RNA polymerase II from hydrocortisone treated animals was consistently reduced in activity compared to control RNA polymerase II. Determination of the apparent specific activities of peak RNA polymerase II fractions from DEAE-Sephadex columns suggested that the specific activity of RNA polymerase II from hydrocortisone treated animals was reduced compared to RNA polymerase II activity from control animals. The fact that both nuclear extract and partially purified RNA polymerases II from hydrocortisone treated rats were reduced in activity when assayed in reconstituted transcriptive systems suggests a denatured, defective or modified RNA polymerase II molecule acting as a transcription inhibitor. Thermally denatured nucleoplasmic RNA polymerase II fractions were shown to interfere with transcription by native nucleoplasmic RNA polymerase II in vitro, but did not appear to inhibit transcription to he degree observed in vitro following in vivo hydrocortisone administration.