Effects of physiologic concentrations of lactate, pyruvate and ascorbate on glucose metabolism in unstressed and oxidatively stressed human red blood cells

Glucose metabolism was studied in human red blood cells incubated in the presence of physiologic concentrations of ascorbate (0.1 mM) and/or lactate (2 mM) plus pyruvate (0.1 mM). The total flux through glycolysis, as measured by 14C-labeling of glycolytic intermediates, was increased about 15% by ascorbate, 30% by lactate plus pyruvate, and 40% by ascorbate plus lactate plus pyruvate. We found, however, that physiologic concentrations of ascorbate and/or lactate plus pyruvate had no effect on flux of glucose or recycling of pentoses through the hexose monophosphate shunt. Increased formation of lactate accounted for most of the observed increase in glycolysis with little change in pyruvate formation, indicating that the increased flux of reducing equivalents from glucose was stored as lactate rather than being consumed by red cell metabolism. In all experiments, there was a net increase with time in the absolute amount of both lactate and pyruvate in red cell suspensions, indicating that lactate or pyruvate present at zero time did not function as a stoichiometric source or sink for reducing equivalents. There was little effect on steady-state levels of ATP or 2,3-diphosphoglycerate. Equilibration of ascorbate between red cells and the medium was complete before the addition of 14C-labeled glucose to the medium. Glucose metabolism prevented net oxidation of ascorbate in the incubation medium. Physiologic concentrations of ascorbate, lactate and pyruvate appear to increase flux through glycolysis by increasing the turnover of ATP and/or 2,3-diphosphoglycerate. Red cells were exposed to mild oxidative stress by incubation with 0.27 mM 6-hydroxydopamine, 0.27 mM 6-aminodopamine, 0.13 mM 1,4-naphthoquinone-2-sulfonic acid or 0.27 mM phenylhydrazine. The metabolic response to oxidative stress was determined by measuring the formation of methemoglobin, pyruvate, lactate and CO2 in the presence and absence of physiologic concentrations of lactate, pyruvate and ascorbate. Lactate, pyruvate and ascorbate had no effect on the net methemoglobin accumulation but rather on the distribution of the metabolic sources of reducing equivalents and on the flux of reducing equivalents to oxygen. Physiologic lactate and pyruvate allowed increased flow of reducing equivalents from glycolysis to methemoglobin and ultimately oxygen without the necessity of increased flux through glycolysis. This was accomplished by a decrease in the ratio of newly formed lactate to newly formed pyruvate with no increase in total lactate plus pyruvate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Potentiation of CCl4 of hepatotoxicity in rats by a metabolite of 2-butanone: 2,3-butanediol

The role of ketone metabolism in 2-butanone-induced potentiation of carbon tetrachloride (CCl₄) hepatotoxicity was studied in rats. The blood concentrations of 2-butanol, 3-hydroxy-2-butanone and 2,3-butanediol were determined by gas chromatographic analysis after the oral administration of 2-butanone (2.1 ml/kg). The concentrations of 2-butanol, 3-hydroxy-2-butanone and 2,3-butanediol detected 4 h after dosing were 3.2 mg/100 ml, 2.4 mg/100 ml and 8.6 mg/100 ml, respectively. Eighteen hours after 2-butanone, the concentration of 2,3-butanediol rose to 25.6 mg/100 ml, while the concentrations of 2-butanol and 3-hydroxy-2-butanone declined to 0.6 mg/100 ml and 1.4 mg/100 ml, respectively. A 16-h pretreatment with either 2-butanone (2.1 ml/kg, p.o.) or 2,3-butanediol (2.12 ml/kg, p.o.) markedly enhanced the hepototoxic response to CCl₄ (0.1 ml/kg, i.p.), as measured by serum glutamic pyruvic transaminase activity and hepatic triglyceride content. In vivo, limited formation of 3-hydroxy-2-butanone occurred after this dose of 2,3-butanediol. These data suggest that the production of 3-hydroxy-2-butanone and 2,3-butanediol via 2-butanone metabolism may participate in the augmented necrogenic effect of CCl₄ seen after pretreatment with 2-butanone.     

Dissolution behavior of 17β-estradiol (E2) from povidone coprecipitates. comparison with microcrystalline and macrocrystalline E2

The dissolution rates of macrocrystalline 17β-estradiol (E₂), microcrystalline E₂and E₂-povidone coprecipitates and physical mixtures varying in weight ratio from 1 : 1 to 1:49 were determined at 37°C. E₂ dissolution from the coprecipitates was markedly faster than that from either macro- or microcrystalline forms of the drug and was found to increase with decreasing E₂-to-povidone weight ratio. Based on the results of X-ray diffraction, differential scanning calorimetric. Raman spectroicopic, and thermal gravi metric analysis studies, it is concluded that the formation of a more water soluble, high energy state of E₂ is responsible for the increased dissolution rate of this natural cstrogen from low weight ratio povidone coprecipitates. These findings suggest that the use of E₂-povidone coprecipitates may increase the systemic availability of E₂.     

Further studies of the response of kidney Lysosomes to aminoglycosides and other cations

Rat renal cortical lysosomes were isolated in 0.3 M sucrose containing 1 mM EDTA by differential centrifugation. Lysosomes were incubated in isotonic sucrose or isotonic glycine with various concentrations of endogenous and exogenous compounds at 37° for 1 hr. Lysosomes were resedimented, and the N-acetyl-β-glucosaminidase (NAG) activity was measured in the supernatant fraction and the disrupted pellet and the percentage of total NAG released was calculated. Gentamicin and its C₁ and C₂ components had similar potencies for inhibiting NAG release from lysosomes at low concentrations. The release of alpha-galactosidase and beta-galactosidase from lysosomes was also inhibited by streptomycin and gentamicin. Mepacrine at low concentrations stabilized lysosomes and at high concentrations disrupted lysosomes. This drug also enhanced the effect of low concentrations of gentamicin on lysosomes. Inositol hexaphosphoric acid was a potent antagonist of the effect of low concentrations of gentamicin and mepacrine on lysosomes. Rats were treated with gentamicin at doses of 40, 80 and 160 mg/kg for 1 and 3 days. NAG excretion in gentamicin-treated groups as compared to saline controls was unchanged at day 1. Only the 160 mg/kg treatment group showed a tendency toward elevated renal cortical NAG at day 1 (P < 0.06). All treatment groups had elevated renal cortical NAG at day 3, while the 160 mg/kg group also had elevated NAG excretion. Lysine, arginine, l-canavanine and polymyxin B all affected NAG release from lysosomes in vitro. Lysine enhanced the disruptive effect of high gentamicin concentrations on lysosomes. Ferric and ferrous ions, tested over widely varied concentrations, inhibited NAG release at low concentrations while enhancing NAG release at high concentrations. We therefore conclude that the nephrotoxicity of aminoglycoside and other endogenous and exogenous renally excreted cationic compounds may be produced by their effects on lysosomes in the proximal renal tubule.     

Relationship between liver and kidney levels of glutathione and metallothionein in rats

The purpose of this study was to test if the tissue levels of glutathione and metallothioneins were inter-related. In rats, intraperitoneal administration of diethyl maleate or bromobenzene decreased glutathione levels in both the liver and the kidney before doubling the metallothionein concentration in the liver and increasing that in kidneys by 40% starting from 6 h after intraperitoneal administration. Both Zn and Cd produced an increase in hepatic and renal metallothionein levels. However, unlike the responses to diethyl maleate and bromobenzene, the increase in metathionein caused by the metals was not preceded by any significant changes in glutathione levels. Cd decreased the concentration of glutathione in the liver (at 36 h) and kidneys (at 24 h). In contrast, Zn produced an increase and no change in hepatic and renal glutathione concentrations, respectively. The conclusion is that tissue levels of metallothionein and glutathione are not always interrelated.     

Glucose metabolism of oxidatively stressed human red blood cells incubated in plasma or medium containing physiologic concentrations of lactate,pyruvate and ascorbate

Red cells suspended in either defined medium or buffered plasma were oxidatively stressed by incubation in the presence of 1, 4-naphthoquinone-2-sulfonate at concentrations which caused less than 50% methemoglobin accumulation, stimulation of the hexose monophosphate shunt to less than 15% of capacity, and about a 30% increase in flux through glycolysis. Normal plasma concentrations of lactate and pyruvate in either defined medium or buffered plasma allowed increased contribution of reducing equivalents from glycolysis in response to oxidative stress. Increased utilization of reducing equivalents by the red cell was observed as increased accumulation of pyruvate, whereas accumulation of lactate represented storage of reducing equivalents. Exogenous lactate or pyruvate did not serve as a net electron source or sink since the total content in red cell suspensions of both lactate and pyruvate was increased during exposure to oxidative stress. If exogenous lactate had been used as a net source of reducing equivalents, the lactate concentration would have decreased during incubation of red cell suspensions. Plasma ascorbate or other constituents did not alter the qualitative response of glycolysis to oxidative stress (decreased lactate accumulation, increased pyruvate accumulation, and increased total flux through glycolysis), but plasma constituents did raise significantly the dose of oxidant agent required to elicit a given quantitative response. At levels of oxidative stress likely to be encountered in vivo, glycolysis and the hexose monophosphate shunt may be equal in importance as aerobic/antioxidant pathways.     

Interaction of capsaicinoids with drug-metabolizing systems. Relationship to toxicity

The interaction of capsaicin with microsomal drug-metabolizing systems was assessed to determine the role that bioactivation of capsaicin may play in the induction of hepatotoxicity and neurotoxicity. Capsaicin produced a type I spectral change in rat hepatic microsomes in a high affinity (K_s = 8 μM) concentration-dependent manner and was approximately equipotent with SKF-525A in inhibiting ethylmorphine demethylation. Capsaicin (10 mg/kg, s.c.) inhibited biotransformation in vivo as measured by prolongation of pentobarbital sleep time. Reactive metabolites of capsaicin were studied using [³H]dihydrocapsaicin. [³H]Dihydrocapsaicin bound irreversiblyto hepatic microsomal protein after in vitro incubation or in vitro administration. No binding was observed in spinal cord or brain. Although the bioactivation and subsequent covalent binding of capsaicin equivalents may initiate events associated with the hepatotoxicity of capsaicin, it appears that capsaicin-induced neuropathy does not involve covalent interactions with neuroproteins in spinal cord or brain.     

Acute tobacco smoke exposure alters the profile of metabolites produced from benzo[a]pyrene by the isolated perfused rabbit lung

The influence of whole tobacco smoke or the gas phase from smoke on the metabolism of $\text{[}{}^{14}\text{C]benzo}\text{[a]}\text{pyrene}$ was e xamined using the isolated perfused rabbit lung model. Fresh whole tobacco smoke mixed with the air ventilating the perfused lung produces an immediate and dose related decrease in the metabolism of $\text{[}{}^{14}\text{C]benzo}\text{[a]}\text{pyrene}$. The metabolites of $\text{[}{}^{14}\text{C]benzo}\text{[a]}\text{pyrene}$, diols, quinones, phenols and polar compounds are generally decreased in quantity. At the lowest level of smoke administered the percentage of BP-7,8-diol produced is increased dramatically. The results indicate that one of the factors contributing to the carcinogenicity of tobacco smoke may be its ability to produce an immediate alteration in the pulmonary metabolism of polycyclic aromatic hydrocarbons.     

Effect of hypoxia on the conversion of angiotensin I to II in the isolated perfused rat lung

Acute hypoxia in the intact animal and in cultured endothelial cells has been shown to be associated with a decrease in conversion of angiotensin I (AI) to angiotensin II (AII). Alterations in capillary surface and in contact time resulting from hemodynamic changes have been shown to influence the rate of pulmonary AI conversion. The dependency of AI conversion on hemodynamics complicates the interpretation of experiments showing changes in AI conversion in intact animals. We studied the effect of acute hypoxia on AI conversion in the isolated rat lung perfused at constant flow without recirculation of perfusate. Three levels of oxygenation were produced by ventilating lungs and equilibrating perfusate with a range of hypoxic gas mixtures. AI (1 μg) was injected into the pulmonary artery, and the effluent was collected for measurement of AI and All. Instead of the expected hypoxic inhibition, percent conversion of AI to All increased slightly but significantly from 69.3 ± 3.1 (mean ± S.E.M.) at normal oxygenation to 74.4 ± 3.0 at moderate hypoxia (P < 0.005, paired t) and to 73.5 ± 3.9 at severe hypoxia (P < 0.01, paired t). Decreasing mean transit time of substrate through the lung (by increasing perfusate flow rate from 5 to 20 ml/min) resulted in a significant decrease in conversion of AI from 88.7 ± 2.9 to 73.4 ± 2.1% (P < 0.001, paired t). These data confirm the effect of contact time on the rate of AI conversion in the lungs. The isolated rat lung preparation does not exhibit the phenomenon of hypoxia-induced inhibition of AI conversion. The authors speculate that hypoxia-induced inhibition of AI conversion in vivo may be secondary to the effects of hypoxia on hemodynamics.     

Irreversible blockade of [3H]spiperone- but not [3H]dopamine-labeled dopamine receptors with phenoxybenzamine

Calf caudate homogenates, preincubated with 10 ⁵M phenoxybenzamine followed by thorough washing, exhibited an essentially complete loss of dopamine-associated high affinity [³H]spiperone specific binding. This effect of phenoxybenzamine was inhibited in a potent and concentration-dependent manner by domperidone, a potent and specific dopamine-receptor blocker. No effect of phenoxybenzamine was observed on [³H]dopamine specific binding. These data indicate that the blockade of [³H]spiperone binding occurred at the dopamine-receptor binding site, or sites, and that the [³H]spiperone binding was molecularly distinct from the [³H]dopamine specific binding. The characteristics of dopamine-associated [³H]spiperone specific binding suggest that there may be two sites labeled by [³H]spiperone.     

Effects of superoxide dismutase and catalase on catalysis of 6-hydroxydopamine and 6-aminodopamine autoxidation by iron and ascorbate

The effects of superoxide dismutase and catalase on autoxidation of 6-hydroxydopamine and 6-aminodopamine in several chemical environments were studied. Inhibition by superoxide dismutase of autoxidation of 6-hydroxydopamine to its p-quinone required the presence of metal chelators, EDTA or diethylenetriamine pentaacetic acid (DETAPAC). A “lag” period in 6-hydroxydopamine autoxidation in the presence of superoxide dismutase couid be prevented by carrying out autoxidation in a mixture of 6-hydroxydopamine and its p-quinone, conditions in which adequate levels of the semiquinone are available for reaction with O₂. Catalase potentiated the inhibitory effect of superoxide dismutase in the presence of EDTA but had no effect in the presence of DETAPAC. Superoxide dismutase and catalase had no effect on the initial rate of 6-aminodopamine autoxidation to the p-quinone imine or on later intracyclization and polymer formation. Iron chelated by EDTA functioned as a catalyst in 6-hydroxydopamine autoxidation, making the reaction independent of superoxide as shown by the lack of effect of superoxide dismutase. the presence of iron-EDTA resulted in bleaching of the p-quinone product and the consumption of the H₂O₂ formed during autoxidation. Catalase had no effect on the rate of 6-hydroxydopamine autoxidation but completely prevented bleaching of the p-quinone, probably by preventing formation of hydroxyl radical by a Fenton reaction between iron-EDTA and H₂0₂. Ethanol, which scavenges hydroxyl radical but not superoxide of H₂O₂, similarly prevented bleaching of the p-quinone with no other effects on autoxidation. Iron-EDTA also catalyzed 6-aminodopamine autoxidation with associated consumption of H₂O₂. Superoxide dismutase, catalase and ethanol had no effect on 6-aminodopamine autoxidation in the presence or absence of iron-EDTA, showing the independence of the kinetics of 6-aminodopamine autoxidation and polymerization from its products, superoxide, H₂0₂ and hydroxyl radical. Chelation by DETAPAC prevented the effects of iron on 6-hydroxydopamine and 6-aminodopamine autoxidation. Autoxidation of 6-hydroxydopamine in the presence of ascorbate exhibited a lag phase followed by a linear phase of autoxidation. Superoxide dismutase, catalase and ethanol had no effect on 6-hydroxydopamine or 6-aminodopamine autoxidation in the presence of ascorbate. Autoxidation of 6-hydroxydopamine or 6-aminodopamine in the combined presence of iron-EDTA and ascorbate showed the full effects of both additions, except that 6-hydroxydopamine autoxidation exhibited no lag phase and the iron-catalyzed autoxidation of ascorbate occurred simultaneously with the 6-hydroxydopamine or 6-aminodopamine reactions.     

Chloroquine- and primaquine-induced alterations of glucose metabolism in the uninfected red cell

The effects of chloroquine and primaquine on glucose metabolism in uninfected red cells were studied. The flux of glucose through the hexose monophosphate shunt was decreased by chloroquine and increased by primaquine; the flux through glycolysis was not altered significantly by either drug. Since the hexose monophosphate shunt was the metabolic pathway affected by chloroquine and primaquine, measurements were made of the intracellular concentration of NADPH, a major product of the hexose monophosphate shunt in red cells. After incubation with either drug in the absence of glucose, the concentration of NADPH was lower than in control red cells; in the presence of glucose, higher concentrations of NADPH were maintained. Primaquine was more potent than chloroquine in lowering the NADPH concentration. Neither chloroquine nor primaquine inhibited the capacity of the red cell to increase flux through the hexose monophosphate shunt in response to gradual infusion of hydrogen peroxide. No qualitative differences in the effects of chloroquine or primaquine on glucose metabolism were observed when experiments were carried out with red cells containing methemoglobin or carbonmonoxyhemoglobin. This observation leads to the reassessment of the role of oxyhemoglobin in the mechanism of action of primaquine. Since incubation with chloroquine significantly decreased glucose flux through the hexose monophosphate shunt but resulted in only slightly lower NADPH levels, and the turnover of NADPH in control red cells in 1 hr was about ten times the total NADPH content, it follows that chloroquine both decreases utilization of NADPH and inhibits flux through the hexose monophosphate shunt. The effects of primaquine, a significant increase in the flux through the hexose monophosphate shunt with significantly lower NADPH concentrations, can be explained by the capacity of primaquine to undergo oxidation-reduction reactions which result in increased NADPH utilization and, therefore, increased flux through the hexose monophosphate shunt. The observed alterations in metabolism of uninfected red cells may be relevant to understanding the mechanisms of prophylactic and therapeutic effects of antimalarial agents.     

Role of red cell membrane lipid peroxidation in hemolysis due to phenylhydrazine

Although red cell membrane lipid peroxidation has been identified as a consequence of certain oxidizing hemolytic drugs, the relative contribution of lipid peroxidation to red cell damage leading to hemolysis is unclear. This has been evaluated by studying the response to phenylhydrazine of vitamin E-deficient rats as compared to vitamin E-supplemented rats. Following repetitive phenylhydrazinc injections, a lower hematocrit was observed in the vitamin E-deficient group which was associated with higher levels of lipid peroxidation, as indicated by the fluorescence of lipid-containing red cell extracts. However, no significant difference in the initial extent of hemolysis following phenyl-hydrazine injection was observed. Evidence was also obtained suggesting that malonaldehyde, a decomposition product of polyunsaturated fatty acids, is capable of cross-linking hemoglobin to the red cell membrane. These findings suggest that red cell membrane lipid peroxidation is of relatively minor consequence in the acute response to phenylhydrazine but may be of importance in chronic hemolysis due to this oxidizing drug.     

α1-acid glycoprotein involvement in high affinity binding of tricyclic antidepressants to human plasma

The binding of the tricyclic antidepressants imipramine (IMI) and desmethylimipramine (DMI) to human plasma and individual proteins was studied by equilibrium dialysis. Both drugs bound extensively to plasma, albumin, and α₁-acid glycoprotein, while there was very little binding to the γglobulin fraction. The binding of both IMI and DMI to α₁-acid glycoprotein was high affinity (association constant K, 9.2 × 10⁴/M and 4.7 × 10⁴/M respectively) and low capacity (number of binding sites, n = 1 for both IMI and DMI), whereas the binding to albumin was low affinity (K for IMI, 2.3 × 107²/M and for DMI, 3 × 10²/M) and high capacity (n = 7). The binding of IMI to a mixture of human serum albumin and α₁-acid glycoprotein revealed two sets of binding sites; a high affinity binding site corresponding to α₁-acid glycoprotein and a low affinity binding site corresponding to albumin. The binding affinity and/or number of binding sites for IMI binding to albumin decreased with increasing albumin concentrations. The free fraction in plasma of nineteen normal, male controls was significantly correlated with the concentration of α-acid glycoprotein (r = 0.601, P < 0.01), although there was no correlation with albumin or free fatty acid concentrations in plasma.     

Induction of metallothioneiin by adrrenocortical steroids

Metallothionein concentration in the liver of hamsters was increased by the administration of hydrocortisone and dexamethasone. However, the increase was of a low order of magnitude (40–80%) in comparison to the large increase seen after zinc and cadmium administration (700–2000%). These data suggest that the small increase in metallothionein seen after various forms of stress could be mediated by the endogenous release of adrenocortical steroids and that the large increase after exposure to cadmium or zinc is unlikely to be mediated via adrenocortical steroid hormones.     

The isolated perfused rat brain preparation in the study of monoamine oxidase and benzylamine oxidase: Lack of selective brain perfusion

Monoamine oxidase (MAO) is present in brain blood vessels, and a different amine oxidase, benzylamine oxidase (BzAO), is claimed to exist in porcine cerebral vessels. The object of the present investigation was to evaluate these deaminating activities in the structurally intact brain, utilizing the isolated perfused rat brain preparation (IPRB). [¹⁴C]Benzylamine (10 μM), a substrate for both BzAO and MAO, was perfused via the internal carotid arteries for 5 min, and deaminated metabolites were measured. BzAO and MAO activities were distinguished by the use of the selective inhibitors semi-carbazide and pargyline. Both BzAO and MAO deaminated benzylamine (10 μm), but over 90 per cent of the total deaminated products resulted from BzAO. This was due to the much lower K_m, value of benzylamine for BzAO (2.8μM) than for MAO (169 μM). In vitro assays, however, revealed that the brain contained no measurable BzAO activity, whereas all other head structures (skull, mandible, skin, skeletal muscle, eyes, periocular tissues and tongue) contained BzAO activity. MAO was present both within and outside the brain. These results suggest that the IPRB preparation is not specific for brain perfusion. Experiments with technetium-labeled microspheres showed that, although the brain is preferentially perfused on a per gram basis, 52 per cent of the total perfusate passed through the skull and 13 per cent through extracranial structures. Only 35 per cent was specific for the brain. Other experiments showed that perfusion of the intact rat head produced greater deamination of benzylamine than when the skin and 70 per cent of the muscle were removed. Additionally, perfusion via the pterygopalatine arteries, to bypass the brain, resulted in increased deamination. It is concluded that the IPRB preparation is not specific for brain perfusion and that BzAO activity is present in all head structures other than the brain. The presence of BzAO in bone, skin, and muscle is consistent with suggestions for a physiological function of the enzyme in connective tissue.     

Isoproterenol and nucleotide induced stimulation of Ca2+ uptake by microsomal fractions from kidney and isolated glomeruli.

Renal cortical microsomal vesicles possess an ATP-dependent Ca²⁺ uptake system which is two to three times more active in accumulating Ca²⁺ than are the microsomes prepared from the outer medulla or papilla of the cat kidney. The microsomal Ca²⁺ uptake system was unaffected by sodium azide, and electron microscopy confirmed the absence of intact mitochondria. Ca²⁺ accumulating activity was significantly increased by 13 per cent in cortical microsomes prepared from kidneys which had been perfused with isoproterenol (2 × 10^?7 M), whereas medullary or papillary microsomal Ca²⁺ accumulation was unaffected. Perfusate containing a higher isoproterenol concentration (2 × 10^?6 M) stimulated cortical as well as papillary microsomal Ca²⁺ uptake by 13 and 31 per cent respectively, but had no effect on medullary microsomal Ca²⁺ accumulation. A lower isoproterenol concentration (2 × 10^?8 M) did not change the Ca²⁺ uptake activity of microsomes isolated from either region of the cat kidney. The isoproterenol concentrations (2 × 10^?7 and 2 × 10^?6 M) which activated Ca²⁺ uptake in microsomes produced graded increases in renin secretion, whereas 2 × 10^?8 M isoproterenol was relatively inactive in eliciting renin secretion. Renal cortical tissue exposed to cyclic AMP (cAMP) and 5'-AMP during subcellular fractionation showed significant increases in microsomal Ca²⁺ uptake. However, microsomes exposed to cAMP or 5'-AMP in the Ca²⁺ uptake medium were not stimulated. Isoproterenol also activated Ca²⁺ uptake by microsomes prepared from isolated glomeruli, and this stimulation was blocked by propranolol. We conclude that the cat renal cortex possesses specific receptors for isoproterenol which activate Ca²⁺ transport through a nucleotide mediated mechanism.     

Effect of aldosterone on ribonucleic acid polymerase activity in rat kidney cortical and medullary mitochondria

The involvement of mitochondria in the mechanism of action of aldosterone is suggested by the ability of this hormone to alter the activity of certain mitochondrial enzymes. Since the mitochondrion is able to synthesize some of its own macromolecules, we investigated whether the mineralocorticoid effects mitochondrial RNA synthesis in isolated kidney mitochondria. Rat kidney mitochondria were purified and then separated into two populations by sucrose density gradient centrifugation; mitochondria sedimenting at 1.2 M sucrose were predominantly of medullary origin, while mitochondria sedimenting at 1.4 M sucrose were mainly from the cortex. RNA polymerase activity in medullary mitochondria was almost twice that of cortical mitochondria. The activity was characterized as α-amanitin resistant and rifampicin sensitive DNA-dependent RNA polymerase. Adrenalectomy resulted in a 40 per cent decrease in the RNA polymerase activity of cortical mitochondria 5 days later (P < 0.001), but no decrease occurred in medullary mitochondrial activity, compared to sham-operated controls. Three hr after aldosterone administration ( 2 μmg/100 g body wt, i.p.) the RNA polymerase activities of medullary and cortical mitochondria were increased 20 and 50 per cent, respectively, above the adrenalectomized values (P < 0.05, P < 0.001). Spironolactone, an antagonist of aldosterone (7 mg/100 g body wt, i.p., 30 min prior to aldosterone administration), blocked the stimulation of RNA polymerase by aldosterone (P < 0.05). Steroid hormones other than aldosterone did not produce significant enhancement of mitochondrial RNA polymerase activity. No change in RNA polymerase activity occurred after direct addition of aldosterone or spironolactone to the assay medium. The results suggest that the mechanism of mineralocorticoid action is at least in part mediated through its effects on mitochondrially synthesized proteins.