The effects of antimineralocorticoids on synthesis of aldosterone metabolites in target tissues

[3H]Aldosterone is transformed into several metabolites by subcellular fractions of rat kidney. 80-90% of the metabolites synthesized by nuclei and plasma membranes are 5 alpha-DHAldo and 3 alpha,5 alpha-THAldo in ratios of 1:2 and 1:1 respectively; small quantities of 3 beta,5 alpha-THAldo are also synthesized. In contrast, kidney cytosol metabolizes Aldo principally to 5 beta-reduced products with co-chromatograph with 5 beta-DHAldo and 3 alpha,5 beta-THAldo. Several polar neutral metabolites, as well as sulfate and acidic metabolites are also synthesized by the cytosol fraction. Similar 5 alpha-reduced metabolites, 5 alpha-DHAldo, 3 alpha,5 alpha-THAldo and 3 beta,5 alpha-THAldo are also synthesized when [3H]aldosterone is incubated in vitro with toad urinary bladder for 1 and 5 h. Significant quantities of 5 beta- and 20 beta-reduced products and sulfate and acidic metabolites are also synthesized. The metabolism of [3H]aldosterone in both target tissues is significantly inhibited by aldosterone antagonists. Several of the reduced metabolites of aldosterone synthesized in kidney and toad bladder possess significant mineralocorticoid activity. 5 alpha-DHAldo and 3 alpha,5 alpha-THAldo possess 1/10 and 1/30 and 3 alpha,5 beta possesses 1/80-1/100 of the antinatriuretic activity of Aldo. It is suggested that the metabolism of Aldo in its target tissues may be linked to regulation or expression of the hormone's actions.     

Oxidation of reduced glutathione by subcellular fractions of rat liver

1. A new method was used to diminish the autoxidation of GSH. 2. The oxidation of GSH by liver homogenates was studied with regard to concentration of homogenate, concentration of GSH, time, pH and anaerobiosis. 3. GSH was oxidized by recombinations of the supernatant with microsomes and with mitochondria. Each fraction alone caused little oxidation. 4. Proteins in the supernatant were required to obtain the effect, and low-molecular-weight compounds in the same fraction increased its effect. 5. GSH diminished the formation of malonaldehyde in homogenates. 6. GSH prevented a stimulating effect of the supernatant on the formation of malonaldehyde in microsomes and in mitochondria. 7. The malonaldehyde formation in microsomes together with the supernatant did not start until the concentration of endogenous low-molecular-weight thiols had decreased to a low level. 8. It is suggested that part of the oxidation of GSH in homogenates is coupled to a mechanism that counteracts the peroxidation of membrane lipids.     

Distribution of physostigmine and metabolites in brain subcellular fractions of the rat

The distribution of 3H-physostigmine (Phy) has been studied in the rat brain subcellular fractions at various time intervals following i.v. injection. 3H-Phy or its metabolites rapidly accumulate into the cytoplasm of cells and penetrates the intracellular compartments. Kinetic studies of the subcellular distribution of radioactivity (RA) per gm of rat brain following i.v. injection of 3H-Phy show peak concentrations at 30 min in all subcellular fractions with the exception of mitochondria. In the mitochondrial fraction the RA levels continue to rise from 4682 +/- 875 DPM/gm at 5 min to 27474 +/- 2825 DPM/gm at 60 min (P less than .05). The cytosol contains the highest RA: 223341 +/- 21044 DPM/gm at 30 min which declined to 53475 +/- 3756 DPM/gm at 60 min. RA in synaptosome, microsomes and myelin increases from 5 to 30 min, and declines at 60 min. In vitro studies did not show a greater uptake of RA by the mitochondrial or synaptosomal fractions. The finding of relatively high concentrations of RA in the mitochondrial fraction at 60 min increases the likelihood that Phy or its metabolites could interfere with the physiological function of this organelle.     

The effects of polyuridylic acid on phenylalanine incorporation by subcellular fractions from carbon tetrachloride-poisoned rat liver

1. Ribosomes and microsomes isolated from the livers of rats that had received carbon tetrachloride 1hr. previously had decreased endogenous capacity to incorporate amino acid. 2. The capacity of the isolated structures to respond to a synthetic messenger, polyuridylic acid, and to incorporate phenylalanine was investigated. 3. It was found that ribosomes from carbon tetrachloride-treated animals, prepared with detergent and at high ionic strength, could be restored to the same specific activity as control particles with polyuridylic acid but that these particles required more Mg(2+) in the incubation mixture. 4. Microsomes could also be stimulated to control activities with polyuridylic acid, but had a narrow optimum range of Mg(2+) concentration. 5. Microsomes prepared from poisoned animals could be preprogrammed with polyuridylic acid to a significantly greater degree than could control particles, and this response was greater with increasing Mg(2+) concentrations. These data suggested that in carbon tetrachloride poisoning the messenger-ribosome interaction had been altered. 6. Attempts to deprogramme particles from control and treated animals resulted in decreased endogenous activity of both particles and a decreased capacity for the treated particles to be restored with the synthetic messenger. 7. It is suggested that two effects are present in carbon tetrachloride poisoning, namely an alteration of the messenger-ribosome interaction and an increased lability of the ribosome, as either separate or related events.     

Triacylglycerol synthesis by subcellular fractions of maturing safflower seeds

A subcellular particulate fraction (10³ g, precipitate) prepared from maturing safflower seeds catalysed triacylglycerol synthesis from oleoyl-CoA.     

Enzymatic synthesis of 3H-labeled Ring-A reduced metabolites of aldosterone and their separation by high pressure liquid chromatography

Specific methods are described for the enzymatic synthesis of each of the six possible 3H-labeled Ring-A reduced metabolites of aldosterone (5 alpha- and 5 beta-DHAldo; 3 alpha,5 alpha-THAldo; 3 beta,5 alpha-THAldo; 3 alpha,5 beta-THAldo; and 3 beta,5 beta-THAldo; see footnote 1 for full names). Use of heated jacketed columns (C8-reverse phase) and two HPLC solvent systems, with isocratic aqueous methanol or acetonitrile, respectively, have been developed which resolve all six Ring-A reduced metabolites of aldosterone. The relative retention times and elution order of each reduced metabolite are different with each solvent system and hence help confirm the identities of Ring-A reduced metabolites made in vivo from physiological quantities of [3H]aldosterone. The use of an on-line beta-radioactivity detector (Berthold LB-504) enhanced the sensitivity of detection and markedly improved the resolution of these metabolites, compared with that obtained by off-line scintillation counting. Thus, the use of increased temperature with these two solvent systems, together with an on-line radioactivity detector, provide a useful and efficient analytical tool for the separation and identification of each reduced metabolite of aldosterone.     

Protein synthesis rates in rat brain regions and subcellular fractions during aging

In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.A preliminary report of these results was previously presented at: WFN-ESN Joint Meeting on: Cerebral Metabolism in Aging and Neurological Disorders, Baden, August 28–31, 1986.     

The role of cytochrome P-450 in the synthesis of polar metabolites of aldosterone by microsomes of male rat liver

Among the tissues of the male rat studied, the largest quantities of the neutral polar metabolites of aldosterone were synthesized by the hepatic microsomal fraction. The polar metabolites of aldosterone were separated by HPLC into six peaks. Three peaks of non-polar (reduced) metabolites were also synthesized. Synthesis of at least four of the neutral polar metabolites was induced by phenobarbital and inhibited by both CO and SKF-525A. The rates of synthesis of these metabolites, which were linear up to 5 minutes, correlated well with the concentration of cytochrome P-450 in the liver microsomes. Addition of aldosterone to the microsomal fraction caused a pronounced type 1 change in the cytochrome P-450 spectrum. The half maximal spectral change (K_s) for aldosterone was calculated to be 8 μM. These experiments indicate that the neutral polar metabolites of aldosterone are produced by cytochrome P-450 dependent hydroxy lations.     

Glycosaminoglycan synthesis by rat fibroblast monolayers: effects of serum and solubilized bone matrix fractions

Glycosaminoglycan synthesis by fibroblasts, from skeletal muscle of the neonatal rat, is stimulated by a bone matrix preparation, soluble in isotonic medium, obtained by extracting decalcified rat bone with 4 M guanidine HCl. The stimulation in glycosaminoglycan synthesis is dependent on the presence of serum in the culture, but the stimulatory effect can be clearly distinguished from that of serum. The stimulatory activity in the bone matrix has been fractionated by ion-exchange chromatography and coelutes largely with anionic, non-collagenous matrix glycoproteins.     

The association of calmodulin with subcellular fractions isolated from rat liver

Calmodulin associated with rat liver mitochondria has been found to belong to a contaminant membranous fraction which contains different subcellular membranes. The concentration of calmodulin in this fraction is relatively high, about 1.6 micrograms/mg protein, and can not be decreased with EGTA. The calmodulin-rich membranous fraction seems to contain cytoskeletal proteins which could be responsible for the binding of calmodulin.     

Induction of citrate synthase by aldosterone in the rat kidney

Summary The possible induction of renal citrate synthase (E.C. 4.1.3.7), by aldosterone was evaluated in the adrenalectomized rat. Three hours after administration of aldosterone (0.8 g/100 g body wt), renal cortical and medullary citrate synthase activity was significantly increased as reported previously by Kinne and Kirsten (Kinne, R., Kirsten, R. 1968.Pfleugers Arch. 300:244). In contrast, no change in this activity was detected in the renal papilla or the liver, under the same conditions. Kinetic analysis revealed that injection of aldosterone had no effect on theK _m s for acetyl-CoA and oxalacetate but augmentedV _max of renal medullary citrate synthase activity by 40%. The aldosterone-dependent increase in medullary citrate synthase activity was proportionate to the associated increase in the quantity of antiserum (specific for citrate synthase) required for half-maximal immuno-precipitation.The possibility that aldosterone induced the synthesis of citrate synthase was evaluated in two sets of experiments. In the first set, adrenalectomized rats were injected intraperitoneally with either aldosterone (0.8 g/100 g body wt) or the diluent, and simultaneously with³H or³⁵S methionine (500 Ci/rat). The isotopes were reversed in about half of the experiments. Three hours after the injection, renal citrate synthase was isolated by ATP-sepharose column chromatography and immuno-precipitation with the specific antiserum. Aldosterone augmented methionine incorporation into renal citrate synthase by 55% but had no effect on incorporation into the hepatic enzyme. In the second set, adrenalectomized rats were injected with either aldosterone (0.8 g/100 g body wt) or the diluent, the kidneys were removed 1 hr later and medullary slices were incubated in either³H-or³⁵S-methionine at 20° for 2 hr. Mitochondrial citrate synthase was isolated either by ATP-sepharose column chromatography and immuno-precipitation, or by polyacrylamide gel electrophoresis. Aldosterone increased methionine incorporation into the immuno-precipitates by 30% and into the enzyme peak resolved by polyacrylamide gel electrophoresis by 43%. The latter increase was eliminated by prior administration of either actinomycin D (70–80 g/100 g body wt) or spirolactone (SC-26304) (80 g/100 g body wt). An equimolar dose of dexamethasone (0.8 g/100 g body wt) had no effect on the isotope ratio associated with citrate synthase activity in the polyacrylamide gels.