Rifampicin inhibition of protein synthesis in mammalian cells.

Rifampicin produces a dose-dependent decrease in protein synthesis in rat thymocytes. At concentrations up to 200 micrograms per milliliter, rifampicin does not alter rat thymic transcription. Rifampicin causes a direct inhibition of protein synthesis in rat thymic and hepatic microsomes, and in cadaveric human hepatic microsomes. Protein synthesis inhibition could explain the toxicity of rifampicin in man.

Dissociation of decreases in renal cellular energetics and recovery of renal microsomal translation during chronic cyclosporine A administration.

Male Sprague-Dawley rats were given oral cyclosporine A or control vehicle, and renal protein synthesis and renal ATP levels were examined. Acute oral cyclosporine A at 5, 10, 25 and 50 mg/kg/day for 6 days reduced [3H]L-leucine incorporation by isolated renal microsomes to 76.2, 56.8, 44.3 and 29.5% of control incorporations, respectively. No significant changes in renal ATP levels were detected by NMR spectroscopy after acute oral cyclosporine A administration at the doses indicated. However, during chronic exposure to cyclosporine A at doses of 5 and 25 mg/kg/day for 30, 60 and 90 days, there was a recovery of renal microsomal protein synthesis by day 30 at 5 mg/kg/day, and by day 45 at 25 mg/kg/day. NMR spectroscopy of the kidneys of these rats demonstrated decreases in renal ATP level by day 60 in animals given cyclosporine A at 25 mg/kg/day. Cyclosporine A administration produced a renal acidosis and up to a 40% decrease in renal ATP level by day 90 in rats fed cyclosporine A at 25 mg/kg. No apparent histologic abnormalities were observed in the ATP-deficient renal tissue by NMR imaging. Reductions in renal ATP level suggest that the recovery of renal microsomal protein synthesis is aberrant in the continued presence of cyclosporine A, or that mitochondria are direct sites of cyclosporine A toxicity.

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.

A new proposal for the mechanism of cyclosporine A nephrotoxicity. Inhibition of renal microsomal protein chain elongation following in vivo cyclosporine A.

In this paper, we report experiments examining the effect of cyclosporine A on "run-off" translation in microsomes isolated from tissues of Sprague-Dawley rats. In microsomes isolated from rat brain, kidney and thymus, cyclosporine A added in vitro in concentrations of up to 100 micrograms/ml did not reduce [3H]L-leucine incorporation relative to controls. A small dose-dependent reduction in [3H]leucine incorporation was observed in microsomes isolated from rat liver when cyclosporine A was added in high concentrations (5 and 6% at 25 and 100 micrograms/ml). However, when cyclosporine A was injected at 50 mg/kg/day for 10 days, [3H]L-leucine incorporation was inhibited 99.9% in microsomes isolated from kidney. The oral administration of cyclosporine A at 50 mg/kg/day for 6-10 days produced a 75% inhibition of incorporation by isolated renal microsomes. These changes were observed in the absence of measurable reductions in "run-off" transcription measured as [3H]UTP incorporation by renal nuclei exposed to cyclosporine A in concentrations of up to 100 micrograms/ml in vitro or isolated from animals given oral cyclosporine A at 50 mg/kg/day for 6 days. Cross-over experiments were performed using microsomes and microsomal supernatant fractions (cell saps) from tissues of animals treated with cyclosporine A and control vehicle. Renal cell sap from cyclosporine A treated animals inhibited [3H]L-leucine incorporation by microsomes isolated from the kidneys or other tissues of animals treated with control vehicle. These experiments demonstrated that a translation inhibitor was present in the cell sap of cyclosporine A treated animals which could directly block translation elongation in microsomes from control animals. When renal cell sap from both control and cyclosporine A treated animals was added to control microsomes, inhibition was still prominent, suggesting the presence of an inhibitor rather than the absence of an elongation factor. Oral administration of cyclosporine A at 50 mg/kg/day for 6 days depressed renal microsomal [3H]L-leucine incorporation equally in male and female rats to 25% of control. The dose-response relationship for microsomal protein synthesis inhibition after 6 days of oral cyclosporine A administration was: 5 mg/kg, 73.7% of control; 10 mg/kg, 64.1% of control; 25 mg/kg, 54.9% of control and 50 mg/kg, 24.1% of control. Renal microsomal protein synthesis following oral cyclosporine A at 50 mg/kg/day was reduced to 54% of control by day 2 and was maximally inhibited at 25-30% of control by day 4.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

EBT, a tryptophan contaminant associated with eosinophilia myalgia syndrome, is incorporated into proteins during translation as an amino acid analog.

The tryptophan dimer 1,1'-ethylidenebis[L-tryptophan] was identified as a contaminant of tryptophan preparations associated with Eosinophilia-Myalgia Syndrome. In this paper, we describe experiments examining the hypothesis that 1,1'-ethylidenebis[L-tryptophan] acts as an amino acid analog replacing L-tryptophan during the synthesis of proteins. We propose further that proteins containing 1,1'-ethylidenebis[L-tryptophan] are rejected in an autoimmune process identified clinically as Eosinophilia-Myalgia Syndrome. Rabbit reticulocyte lysates containing an estimated 1 microM L-tryptophan were used to assay the ability of 1,1'-ethylidenebis[L-tryptophan] to compete with 3H-L-tryptophan for incorporation into proteins translated from BMV RNA. 1,1'-Ethylidenebis[L-tryptophan] in concentrations of 40, 80 and 110 microM reduced lysate 3H-L-tryptophan incorporation to 81%, 76% and 75% of control incorporation obtained in the absence of 1,1'-ethylidenebis[L-tryptophan]. In the presence of 20 microM L-tryptophan, 110 microM 1,1'-ethylidenebis[L-tryptophan] reduced 3H-L-tryptophan incorporation to 56% of control incorporation. In contrast, ethyl-L-tryptophan did not significantly reduce 3H-L-tryptophan incorporation. In the presence of 110 microM 1,1'-ethylidenebis[L-tryptophan] and 20 microM L-tryptophan, 3H-L-leucine incorporation was not significantly reduced compared to incorporation in the absence of 1,1'-ethylidenebis[L-tryptophan], demonstrating that proteins were translated to full length during elongation. These findings suggest that 1,1'-ethylidenebis[L-tryptophan], but not ethyl-L-tryptophan, reduced 3H-L-tryptophan incorporation into proteins by substituting for L-tryptophan rather than by causing premature termination or significant slowing of nascent protein chains.

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.

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.

The 105mm camera as a supplement to nuclear venography.

Nuclear venograms were supplemented by contrast venograms with use of a 105mm spot film camera under direct visualization. This technique is a simple effective way of resolving questionable areas observed on nuclear venograms.

Characterization of the inhibition of renal translation in the Sprague-Dawley rat following in vivo cyclosporin A.

"Run-off" translation assays in microsomes isolated from Sprague-Dawley rats were used to examine the capacity of cyclosporin A (CsA) to alter translation elongation. CsA added in vitro in concentrations of up to 100 micrograms/ml did not reduce "run-off" translation measured as 3H-L-leucine incorporation in microsomes isolated from the rat kidney or thymus. In contrast, the oral administration of CsA at 50 mg/kg/day for 6 days resulted in reductions in 3H-L-leucine incorporation by microsomes isolated from rat kidneys and unstimulated thymus to 21.6 and 83.0% of control values, respectively. In cross-over experiments between renal microsomal and cytoplasmic fractions, 3H-L-leucine incorporation was inhibited following the addition of renal cytoplasm from CsA treated rats to renal microsomal fractions from control vehicle treated rats. Translation inhibition was still observed when renal cytoplasm from CsA treated rats was added along with renal cytoplasm from control rats to renal microsomes isolated from rats treated with control vehicle. Reductions in renal 3H-L-leucine incorporation were not due to a stimulation of renal protease activity. Experiments varying the concentration of individual components of the microsomal translation assays suggested that renal microsomes from CsA treated animals were saturated with substrate or cofactor at lower concentrations than control microsomes. Time course experiments showed a marked reduction in the duration and extent of 3H-L-leucine incorporation by renal microsomes from CsA treated animals compared to controls. Sucrose density gradient analysis of microsomes from CsA treated animals confirmed that elongation was inhibited. These findings suggest that the in vivo administration of CsA results in the formation of a direct-acting inhibitor of renal translation rather than reducing translation by producing changes in renal transcription. The observation that renal translation is inhibited only after in vivo CsA suggests that a CsA metabolite formed in, or taken up by, the kidney produces translation inhibition, or that a cellular product involved in translation or translation regulation is formed or induced. We propose that the CsA induced inhibition of renal translation elongation accounts, at least in part, for CsA-induced nephrotoxicity.

Tissue specificity of translation inhibition in Sprague-Dawley rats following in vivo cyclosporin A.

We have shown that following the in vivo administration of cyclosporin A (CsA) to either male or female Sprague-Dawley rats, there is a time- and dose-dependent inhibition of translation elongation in renal microsomes. Experiments reported in this paper explore the tissue specificity of translation inhibition. Following 6 days of oral 50 mg/kg/day CsA or control vehicle, animals were sacrificed and renal, hepatic and cardiac microsomal and cytoplasmic fractions prepared for "run-off" translation assays. These assays demonstrated that in vivo CsA resulted in a reduction in renal microsomal 3H-L-leucine incorporation to 25% of control values, a reduction of cardiac microsomal incorporation to 60% of controls and a stimulation of hepatic microsomal incorporation to 140% of controls. Cross-over experiments involving the addition of renal cytoplasmic fractions from CsA-treated animals to renal microsomal fractions from control-vehicle-treated animals depressed 3H-L-leucine incorporation to approximately 50% of control values. When the renal cytoplasmic fraction from CsA-treated animals was added to renal, hepatic or cardiac microsomal fractions from control-vehicle-treated animals, 3H-L-leucine incorporation values were consistently reduced to approximately 50% of controls. We have reported that translation elongation is inhibited by renal cytoplasm from CsA-treated rats in the presence of control renal cytoplasm. These data suggest that inhibition arises from the presence of an inhibitor rather than from a deficiency in elongation factors. The data reported in this paper demonstrate that microsomal fractions from various tissues have equal sensitivity to CsA-mediated renal cytoplasmic translation inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Gentamicin administered in vivo reduces protein synthesis in microsomes subsequently isolated from rat kidneys but not from rat brains.

Gentamicin injected in vivo, in doses equivalent to those used therapeutically in humans, produced a decrease in the protein synthesizing capacity of microsomes subsequently isolated from rat kidneys, but not of microsomes isolated from rat whole brains. The decreases in eukaryotic cell protein synthesis observed following in-vivo administration of gentamicin were similar to those observed following the addition of gentamicin to microsomes in vitro.

Metal ion catalyzed oxidation of the antibiotic rifampicin.

The metal ions Cu++, Mn++ and Co+++, but not Ca++, Fe+++, K+, La+++, Mg++, Na+, Sr++ or Zn++ catalyzed the oxidation of rifampicin from the naphthohydroquinone to the naphthoquinone form. This reaction was pH dependent, and occurred at neutral or basic pH more rapidly than at acidic pH. Mn++ catalyzed the most rapid oxidation, followed by Cu++ and then Co+++. Rifampicin oxidation was metal ion dependent and complete oxidation occurred at metal ion concentrations below stoichiometric values. Initial rate studies suggest that the oxidation mechanism is complex.

Inhibitory effects of gentamicin and ethacrynic acid on mammalian microsomal protein synthesis.

The ototoxic antibiotic gentamicin, and the ototoxic diuretic ethacrynic acid, both produce inhibitory effects on protein synthesis in microsomes isolated from rat brain. Inhibitory effects appear to be essentially independent of each other. The inhibitory dose-response curve for gentamicin is logarithmic, while that for ethacrynic acid is linear. The dose-response curves make gentamicin the predominant inhibitor when the drugs are combined at low concentrations and ethacrynic acid the predominant inhibitor when the drugs are combined at high concentrations. Accumulation of aminoglycoside by tissues of the inner ear may result in inhibition of protein synthesis in spite of low and transient plasma levels of the drug. Further inhibition of translation by ethacrynic acid could account, in part, for the ototoxic interaction of aminoglycosides and high ceiling diuretics.

Cyclosporine-induced renal dysfunction: correlations between cellular events and whole kidney function.

The main adverse reaction to the immunosuppressive drug cyclosporine is dose-dependent renal dysfunction. Although renal vasoconstriction without major tubular dysfunction is usually noted, recent studies have demonstrated an inhibition of renal cortical microsomal protein synthesis. Sprague-Dawley rats and appropriate pair-fed controls were given cyclosporine orally in doses of 5, 10, 25, and 50 mg/kg/day for periods up to 10 days. A dose-dependent decline in glomerular filtration rate and effective renal plasma flow was maximal by day 3 and did not worsen despite continued dosing. Microsomal protein synthesis as measured by [3H]leucine incorporation was also depressed in a dose-dependent fashion; however, inhibition did not reach the nadir until day 4, 1 day after renal dysfunction was established. When cyclosporine was discontinued, microsomal protein synthesis was normalized by 4 days after drug withdrawal; in contrast, the return of glomerular filtration rate and effective renal plasma flow to normal required 8 days after drug discontinuation. Tubular function as measured by fractional excretion of lithium, enzymuria, and urinary osmolality was well maintained despite the depression of renal hemodynamics. There was no evidence of tubular necrosis by light or electron microscopy. Although cyclosporine produces reductions in renal microsomal protein synthesis, measured by "run-off" translation assays, these effects appear unlikely to be the direct cause of acute renal dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of mammalian microsomal protein synthesis by aminoglycoside antibiotics.

The aminoglycoside antibiotics gentamicin, kanamycin and netilmicin produce a dose-dependent inhibition of amino acid incorporation in microsomes isolated from human liver and rat brain, kidney and liver. Inhibitory effects on microsomal protein synthesis occur at concentrations that have been shown to accumulate in rodent and human renal cortex and perilymph following therapeutic administration. Inhibition of translation in those tissues which specifically accumulate aminoglycoside antibiotics may, in part, explain toxicities observed following exposure to aminoglycosides.