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Biomedical subjects

D Marver

Publications and source records attributed to D Marver.

33 records · Page 2Linked to original sources
PubMed1980

Identification of mineralocorticoid target sites in the isolated rabbit cortical nephron.

Previous evidence suggests that the activity of the mitochondrial enzyme citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH(2)COO --> acetyl-CoA), EC 4.1.3.7] is increased in target tissues upon acute administration of aldosterone. Therefore, an ultramicro assay was established to determine citrate synthase levels in isolated rabbit nephron segments as a means of localizing mineralocorticoid-responsive sites within the renal cortex. The relative citrate synthase activities in normal rabbit segments (per kg of dry tissue) correlated with the metabolic activity of the segments. The order was: distal convoluted tubule > proximal convoluted tubule > cortical thick ascending limb of Henle > cortical collecting duct > pars recta. When these segments were isolated from adrenalectomized rabbits, only the citrate synthase activity in the cortical collecting duct was significantly decreased compared to normal values (3.2 mol of citrate/kg dry wt per hr compared to 7.1; P < 0.001). Furthermore, enzyme activities in segments isolated from adrenalectomized rabbits 90 min after intravenous injection of aldosterone (10 mug/kg) were unchanged from normal or adrenalectomized rabbit tubule values for all segments except the cortical collecting duct. In this segment, aldosterone significantly increased citrate synthase activity compared to adrenalectomized rabbit values (8.1 mol/kg per hr compared to 3.2; P < 0.001), in contrast to the effect of dexamethasone at 10 mug/kg (4.4 mol/kg per hr compared to 3.2; P, NS). Spirolactone SC 26304 administered 30 min prior to injection of aldosterone inhibited the increase in collecting duct citrate synthase activity seen with aldosterone alone (3.4 mol/kg per hr compared to 8.1; P < 0.001). These findings suggest that the collecting duct is the primary target for aldosterone in the renal cortex.

Adrenalectomy↗
PubMed1979

The mineralocorticoid antagonist activity of an 11 beta,18-oxidopregnane.

Removal of the 18-hydroxy group of the hemiacetal form of aldosterone transforms its activity from that of pure agonist to predominant antagonist. The 18-deoxy derivative possesses one third of the binding affinity of aldosterone for the cytoplasmic mineralocorticoid receptor of rat kidney and exhibits an approximate 2:1 antagonist to agonist ratio in both toad bladder and adrenalectomized rat bioassay systems. The promising properties of the 11 beta,18-oxidopregnane tested included very low androgen receptor affinity and approximately equal effectiveness in vitro and in vivo in displacing aldosterone from mineralocorticoid-binding sites in the rat.

Aldosterone↗
PubMed1976

Photoaffinity site-specific covalent labeling of human corticosteroid-binding globulin.

A method was developed for the synthesis of high-specific-activity 21-diazo-21-[6,7-(3)H]deoxycorticosterone, an analog of corticosterone. This analog was used as a photoaffinity label of a high affinity steroid-binding protein, human corticosteroid-binding globulin. Based on direct binding studies and crosscompetition experiments, this diazo derivative exhibited the requisite affinity (within a factor of 1.5 times that of corticosterone) and site specificity to qualify as an affinity labeling legand. Irradiation of corticosteroid-binding globulin with the 21-diazo derivative resulted in irreversible binding to corticosteroid-binding globulin, identified by polyacrylamide gel electrophoresis. Specificity of covalent binding to corticosteroid-binding globulin was established by competition analysis with various steroids. Irreversibility of photodependent binding was shown by persistence of the complex on electrophoresis (in contrast to the noncovalently linked complex), and resistance to exchange with corticosterone or pregnanediol and to solvent extraction. Site specificity of covalent binding was inferred from the effects of a scavenger, Tris-HC1, and fluorescence quenching of a neighboring tryptophan.

Affinity Labels↗
PubMed1975

Autoradiographic localization of corticosterone receptors (type III) to the collecting tubule of the rat kidney.

Recently, a class of receptors exhibiting high affinity for corticosterone was described in rat kidney (Feldman, D. et al., Endocrinology 92: 1429, 1973). These receptor sites exhibited negligible affinity for dexamethasone and aldosterone and were designated Type III to distinguish them from sites having high affinity for aldosterone (Type I), and sites with high affinity for dexamethasone and corticosterone (Type II). To visually localize Type III sites in the kidney and demonstrate whether or not they represent intracellular steroid receptors, we used an autoradiographic procedure for diffusible substances. Male adrenalectomized rats were injected intravenously with the following combination of steroids per 100 g body weight: 4 x 10(-9) mol [3H]corticosterone, 4 x 10(-9) mol unlabeled aldosterone, and 4 x 10(-9) mol unlabeled dexamethasone. To differentiate "nonspecific" binding, each experimental animal was paired with a control animal that received the same steroids plus 250-fold unlabeled corticosterone. At 3 min, 10 min, and 30 min, kidneys were removed, cut into quadrants, and frozen in isopentane cooled by liquid nitrogen. For autoradiography, 4 mum frozen sections were cut, pressed into contact with emulsion precoated slides at -30 C, melted and simultaneously dried under a jet of dry nitrogen gas, and exposed at 4 C for 2 to 6 weeks. At all three time intervals, silver grains representing [3H]corticosterone binding sites, were concentrated over collecting tubules, only in the outer medulla and cortex (those in the inner medulla and papilla were not labeled). In the labeled segments of the nephron, some of the cells showed an apparent high ratio of cytoplasmic to nuclear grains and in others nuclear labeling was more prominent. A small population of cells within labeled collecting tubules (possibly dark cells) were not labeled. Although no function can yet be ascribed to Type III receptors in the kidney, they may represent an important steroid-mediated renal mechanism.

Animals↗
PubMed1974

Renal aldosterone receptors: studies with (3H)aldosterone and the anti-mineralocorticoid (3H)spirolactone (SC-26304).

In vivo, a spirolactone (SC-26304) inhibited the effects of aldosterone on urinary K(+):Na(+) ratios and the binding of [(3)H]aldosterone to renal cytoplasmic and nuclear receptors. Cytoplasmic binding of [(3)H]aldosterone and [(3)H]spirolactone (SC-26304) was similar in magnitude and involved the same set of sites. Under three sets of conditions-(i) in the intact rat, (ii) in kidney slices, and (iii) in reconstitution studies (mixing prelabeled cytoplasm with either purified renal nuclei or chromatin), [(3)H]spirolactone (SC-26304) did not yield specific nuclear complexes in contrast to the reproducible generation of these complexes with [(3)H]aldosterone. In glycerol density gradients, cytoplasmic [(3)H]aldosterone receptor complexes sedimented at 8.5 S and 4 S in low concentrations of salt and at 4.5 S in high concentrations of salt. Cytoplasmic [(3)H]spirolactone (SC-26304) receptor complexes sedimented at 3 S in low concentrations of salt and 4 S in high concentrations of salt. These results are discussed in terms of an allosteric model of the receptor system.

Adrenalectomy↗
PubMed1966

Correlation between location and time of expression for genes in a single operon.

When the histidine Operon in Salmonella typhimurium becomes derepressed in the presence of 4-amino-5-imidazole carboxamide ribonucleoside, derepression of the enzymes for histidine biosynthesis Occurs in a temporal sequence which corresponds with the positional sequence of the genes in the histidine Operon, approximately 20 minutes intervening between the derepression of the first and that of the last enzyme studied. In the case of the deletion mutant, hisH,B22, the interval between the derepression of the first and that of the last enzyme was reduced from 20 minutes to 10 minutes. This reduction was due exclusively to the fact that the interval between the derepressions of the two enzymes, the structural genes for which are located on either side of the deletion, was almost completely eliminated. The results support the genetic evidence for the physical absence of genetic material situated between these two genes.

Chromosome Mapping↗