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Biomedical subjects

I S Edelman

Publications and source records attributed to I S Edelman.

At least 55 records · Page 3Linked to original sources
PubMed1983

Critical role played by thyroid hormone in induction of neoplastic transformation by chemical carcinogens in tissue culture.

Incubation of primary cultures of hamster embryo cells (HEC) or mouse fibroblasts (C3H/10T1/2 cells) in media depleted of thyroid hormones does not alter cell growth or survival but renders the cells resistant to neoplastic transformation by benzo[a]pyrene (B[a]P) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), carcinogens which yield transformation rates of 10(-4)-10(-2) in media supplemented with triiodothyronine (T3). In C3H/10T1/2 cells, the times of addition or removal of the hormone indicate that T3 exerts maximum effect when added 12 hr prior to treatment with B[a]P and that the progression of transformation from the time of initiation by the carcinogen to full expression and the appearance of transformed foci was independent of the presence or absence of the hormone in the medium. Dependence of transformation on T3 concentration in the medium was observed over the physiological range of 1 pM to 100 nM in C3H/10T1/2 cells treated with B[a]P. These results were similar to our previous findings on the T3 dose-related induction of radiogenic transformation and of Na+,K+-ATPase activity. The latter effect was used as a measure of T3 induction of protein synthesis. A further indication of the potential involvement of protein synthesis in T3 action is the suppression of T3- and B[a]P-dependent transformation by cycloheximide at concentrations that inhibit protein synthesis by approximately equal to 50% in the C3H/10T1/2 cells. We suggest that thyroid hormone induces the synthesis of a host protein that plays a key role in neoplastic transformation by direct-acting chemical carcinogens and by those requiring metabolic activation. In our previous studies, similar T3-dependent mechanisms were implicated in radiogenic transformations.

Animals↗
PubMed1983

Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder.

Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value. This decrease was accompanied by a proportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations. In contrast, the paracellular resistance was unaffected by trypsinization. Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin. Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin. Trypsinization of the apical membrane was also used to study regulation of Na transport by anti-diuretic hormone (ADH) and aldosterone. Prior exposure of the apical surface to trypsin did not reduce the response to ADH, which indicates that the ADH-induced Na channels were inaccessible to trypsin before addition of the hormone. On the other hand, stimulation of short-circuit current by aldosterone or pyruvate (added to substrate-depleted, aldosterone-repleted bladders) was substantially reduced by prior trypsinization of the apical surface. Thus, the increase in apical Na permeability elicited by aldosterone or substrate involves activation of Na channels that are continuously present in the apical membrane in nonconductive but trypsin-sensitive forms.

Aldosterone↗
PubMed1982

The role of sodium-channel density in the natriferic response of the toad urinary bladder to an antidiuretic hormone.

Urinary bladders of Bufo marinus were depolarized, by raising the serosal K concentration, to facilitate voltage-clamping of the apical membrane. Passive Na transport across the apical membrane was then studied with near-instantaneous current-voltage curves obtained before and after eliciting a natriferic response with oxytocin. Fitting with the constant-field equation showed that the natriferic effect is accounted for by an increase in the apical Na permeability. It is accompanied by a small increase in cellular Na activity. Furthermore, fluctuation analysis of the amiloride-induced shot-noise component of the short-circuit current indicated that the permeability increase is not due to increased Na translocation through those Na channels which were already conducting prior to hormonal stimulation. Rather, the natriferic effects is found to be based on an increase in the population of transporting channels. It appears that, in response to the hormone, Na channels are rapidly "recruited" from a pool of electrically silent channels.

Animals↗
PubMed1982

Aldosterone control of the density of sodium channels in the toad urinary bladder.

Near-instantaneous current-voltage relationships and shot-noise analysis of amiloride-induced current fluctuations were used to estimate apical membrane permeability to Na (PNa), intraepithelial Na activity (Nac), single-channel Na currents (i) and the number of open (conducting) apical Na channels (N0), in the urinary bladder of the toad (Bufo marinus). To facilitate voltage-clamping of the apical membrane, the serosal plasma membranes were depolarized by substitution of a high KCl (85 mM) sucrose (50 mM) medium for the conventional Na-Ringer's solution on the serosal side. Aldosterone (5 X 10(-7) M, serosal side only) elicited proportionate increases in the Na-specific current (INa and in PNa, with no significant change in the dependence of PNa on mucosal Na (Nao). PNa and the control of PNa by aldosterone were substrate-dependent: In substrate-depleted bladders, pretreatment with aldosterone markedly augmented the response to pyruvate (7.5 X 10(-3) M) which evoked coordinate and equivalent increases in INa and PNa. The aldosterone-dependent increase in PNa was a result of an equivalent increase in the area density of conducting apical Na channels. The computed single-channel current did not change. We propose that, following aldosterone-induced protein synthesis, there is a reversible metabolically-dependent recruitment of preexisting Na channels from a reservoir of electrically undetectable channels. The results do not exclude the possibility of a complementary induction of Na-channel synthesis.

Aldosterone↗
PubMed1982

Characteristics of antibodies to guinea pig (Na+ + K+)-adenosine triphosphatase and their use in cell-free synthesis studies.

Antibodies have been produced, in three rabbits, to Na/K-ATPase purified from guinea pig renal outer medulla. Each rabbit produced antibodies to both the alpha (catalytic) and the beta (glycoprotein) subunits of Na/K-ATPase. The titers of the anti-alpha and anti-beta antibodies varied with time and between rabbits. None of the antisera inhibited Na/K-ATPase activity under various preincubation conditions. A method is presented for separating small amounts of anti-alpha subunit from anti-beta subunit antibodies. There was no cross-reactivity of antibodies to one subunit with the other subunit. The alpha subunit of the Na/K-ATPase was cleaved into a 41,000-dalton peptide (that contains the ATP phosphorylating site) and a 58,000-dalton hydrophobic peptide as described by Castro and Farley (Castro, J., Farley, R.A., 1979, J. Biol. Chem. 254: 2221-2228). Anti-alpha antibodies from all of the rabbits reacted with both proteolytic fragments. The anti-guinea pig Na/K-ATPase antisera (pooled) cross-reacted with the alpha subunit of Na/K-ATPase from human, cow, dog, rabbit, rat, mouse, turtle, and toad; and with the beta subunit from human, rat, and mouse. The loci of cross-reactivity were investigated using partially purified canine kidney Na/K-ATPase cleaved with trypsin as described above. The anti-sera from rabbits 1 and 2 cross-reacted with the 41,000-dalton peptide from the dog but very little with the 58,000-dalton peptide. No cross-reactivity was observed with antiserum from rabbit 3 to either fragment. Guinea pig kidney RNA was translated in a rabbit reticulocyte lysate system followed by immunoprecipitation with the antisera. The molecular weight of the cell-free synthesized alpha chain was 96,000 daltons. Its identity was established with purified anti-alpha antibodies and by immunocompetition with purified Na/K-ATPase and Ca-ATPase. Translation of the beta subunit was not detected in this system.

Animals↗
PubMed1982

Glucocorticoid stimulation of Na-K-ATPase in superfused distal segments of kidney tubules in vitro.

The ability of glucocorticoids to regulate Na-K-ATPase activity directly was assessed in separated rat kidney tubules derived from the distal nephron. These tubules were superfused under sterile conditions and maintained in a viable condition for at least 24 h in a newly devised apparatus. Viability was assessed by measuring O2 consumption, protein/DNA ratios, and Na-K-ATPase and Mg-ATPase activities. At a concentration of 10(-8) M, dexamethasone elicited a 27% increase in tubular Na-K-ATPase activity in 6 h and a 32% increase in 24 h. In a separate series, assayed at 24 h, the maximal effect was obtained at a dexamethasone concentration of less than 10(-8) M, and by inspection half-maximal stimulation was obtained at approximately 10(-9) M. At a concentration of 10(-7) M, 17 beta-estradiol, testosterone, progesterone, and deoxycorticosterone acetate had no significant effect on tubular Na-K-ATPase activity. These results as well as the time-course and dose-response data imply that the response is mediated by the glucocorticoid receptor pathway. Since the magnitude of response in vitro was similar to the one obtained after injection of dexamethasone in vivo, much if not all of the action appears to be direct and independent of glucocorticoid-induced changes in the filtered Na+ load.

Adrenalectomy↗
PubMed1982

Hormones and the single cell: a relationship prerequisite for transformation.

The direct oncogenic potential of radiation and chemicals is demonstrable in cell cultures, where host-mediated influences do not prevail. These systems afford the opportunity of investigating factors that modulate neoplastic transformation. Agents such as retinoids modulate late events and inhibit the expression of transformation and the promotional effects of 12-O-tetradecanoylphorbol-13-acetate. Thyroid hormones play a role in early events, in initiation of transformation. Interaction of triiodothyronine (T3) in a culture medium with single cells is a prerequisite for the initiation of transformation following exposure to X-rays and to benzo[a]pyrene. When cloned hamster embryo cells and mouse 10T 1/2 cells are maintained in a medium containing serum without thyroid hormones (hypothyroid conditions) and exposed to 3 or 4 Gy X-rays, no transformation is observed, although cell survival and cell growth are unaffected by thyroid hormone level. Supplementation of the medium with 10-12M to 10-7M T3 12 h before exposure to X-rays results in a transformation frequency related to the dose of T3, with a peak at 10-10M. The curve is similar to that induced by Na/K ATPase. Addition of T3 at the time of radiation results in a lower transformation frequency; if it is added after radiation no transformation is observed. The effect of T3 in the process of initiation is not mimicked by reverse T3 and is abolished by addition of 100 ng cyclohexamide. The data suggest that the effect of T3 in rendering the cell competent to transform involves synthesis of a cellular 'transforming protein'. Ongoing experiments indicate that exposure to 1 microgram/ml benzo[a]-pyrene results in transformation only in the presence of T3. Under hypothyroid conditions, no transformation is observed.

Animals↗
PubMed1981

31P NMR studies of intact anchorage-dependent mouse embryo fibroblasts.

31P NMR spectra of "normal" and x-ray-transformed anchorage-dependent mouse embryo fibroblasts (C3H/10T 1/2) at 145.7 MHz and 37 degrees C were obtained by using a solenoid coil probe with a sensitivity approximately 2.6 times higher than that of the standard Helmholtz coil probe. The cells were grown as monolayers on the surface of microcarrier beads. Results show that these cells have low levels of NMR-detectable ADP, are highly impermeable to protons, are capable of maintaining high ATP levels in the absence of respiration, and, when ATP generation is blocked, ultimately hydrolyze their existing ATP to a nonphosphorylated product and Pi. Only small differences were observed between the spectra of "normal" and transformed cells. However, the latter accumulated 2-deoxyglucose 6-phosphate at a faster rate.

Adenine Nucleotides↗
PubMed1981

Crucial role of thyroid hormone in x-ray-induced neoplastic transformation in cell culture.

Incubation of mouse embryo fibroblasts (C3H/10T1/2) in media depleted of thyroid hormone for 1 week rendered the cells completely resistant to the transforming action of an x-ray dose, 4 grays, that yields transformation frequencies (no. foci per surviving cells) of approximately 10(-3) in media supplemented with triiodothyronine (T3) (1 nM). Studies on the timing of the additions or removal of the hormone indicate that T3 was maximally effective when added 12 hr before irradiation and that progression from the time of irradiation to the appearance of foci (6 weeks) was independent of the presence or absence of the hormone. The dependence of x-ray-induced transformation on the concentration of T3 in the medium was virtually the same as that for augmentation of Na+,K+-ATPase activity. The latter effect was used as a measure of T3 induction of protein synthesis. A further indication of the involvement of protein synthesis in the process is the abolition of T3- and x-ray-dependent transformation by cycloheximide at a concentration (100 ng/ml) that inhibits 50% of protein synthesis. We propose that thyroid hormone induces the synthesis of a host protein that is an obligatory participant in x-ray-mediated transformation.

Animals↗
PubMed1981

Nuclear binding of T3 and effects of QO2, Na-K-ATPase, and alpha-GPDH in liver and kidney.

Thyroid status was altered by use of a low-iodine-perchlorate (PC) regimen and either reversal with NaI or injections of L-3,5,3'-triiodothyronine (T3). The PC regimen decreased renal and hepatic oxygen consumption (QO2), alpha-glycerophosphate dehydrogenase (alpha-GPDH), and Na+-K+-dependent adenosine triphosphatase (Na-K-ATPase) to comparable extents (25 vs. 23%, 26 vs. 39%, and 41 vs. 51%, respectively). Administration of T3 to hypothyroid rats elicited dose-dependent increases in hepatic and renal cortical QO2, ouabain-sensitive oxygen consumption (QO2(t)), alpha-GPDH, and Na-K-ATPase activities. The half-maximal increases in all of the response parameters in both kidney and liver were obtained at dosages of 6-32 micrograms T3/100 g body wt. The equivalences in the renal cortical vs. hepatic responses were indicated by correlation coefficients of approximately 0.97. Kidney and liver nuclei also showed similar high-affinity binding of 125I-T3-K1/2 = 29 vs. 18 micrograms T3/100 g body wt, and Nmax = 1.8 vs. 2.1 ng T3/mg DNA. The patterns of the responses plotted as a function of T3 occupancy of the high-affinity nuclear binding sites were indistinguishable in kidney and liver. These results imply similar modes of action of T3, probably initiated at the nuclear level, in both kidney and liver.

Animals↗
PubMed1981

Glucocorticoid receptors in rat kidney cortical tubules enriched in proximal and distal segments.

Cytoplasmic and nuclear binding of [3H]triamcinolone acetonide was assessed in isolated rat kidney cortical tubules, enriched in distal (fraction A) or in proximal segments (fraction B). The concentration dependence of specific [3H]triamcinolone acetonide binding in cytoplasm was determined (range = 4.4 X 10(-10) to 2.1 X 10(-7) M) and analyzed by a least-squares curve-fitting method. A single, high-affinity binding class with a dissociation constant of 1 X 10(-8) M (25 degrees C) was obtained in both fractions A and B. Based on competition for the [3H]triamcinolone acetonide sites, the following sequence of affinities was obtained: triamcinolone acetonide = dexamethasone > progesterone = corticosterone > d-aldosterone > 17 beta-estradiol. These specificities imply that these sites are glucocorticoid receptors. Fraction B contained 1.6 times more cytosol sites for [3H]triamcinolone acetonide than fraction A (5.0 +/- 0.5 X 10(-13) vs. 3.0 +/- 0.5 X 10(-13) mol/mg protein). In the presence of a onefold excess of d-aldosterone specific cytoplasmic binding of [3H]triamcinolone acetonide was 1.4-fold greater in fraction B than in fraction A, and specific nuclear binding was 1.3-fold greater in fraction B than in fraction A (5.1 +/- 0.6 X 10(-13) vs 4.0 +/- 0.5 X 10(-13) mol/mg DNA). These results and the measured lengths of proximal and distal tubules yielded estimates of a higher proximal content (three- to sixfold) compared to distal content of glucocorticoid receptors.

Animals↗
PubMed1981

Receptors and effectors in hormone action on the kidney.

An analysis is presented of the similarities and differences in receptor-effector relationships in the actions of aldosterone and triiodothyronine on the kidney. Both agents augment tubular reabsorption of sodium at different sites in the nephron. Aldosterone acts primarily distally and triiodothyronine, primarily proximally. In both cases, the respective hormone-receptor complexes associate with target cell chromatin and modulate the abundance of specific coding by mRNAs for regulatory proteins. The quantitative relationships between nuclear-receptor occupancy and responses were analyzed by fractional plots in a bounded domain. When applied to the toad bladder, this analysis revealed a parabolic dependence of the increase in transepithelial Na+ transport on the abundance of nuclear of the increase in transepithelial Na+ transport on the abundance of nuclear aldosterone-receptor complexes. In contrast, four independent response parameters (QO2, QO2(t), Na-K-ATPase, alpha-glycerophosphate dehydrogenase) exhibited a hyperbolic dependence on nuclear abundance of T3 in the rat kidney. The observed cosaturation of nuclear occupancy and responses in both systems, for both hormones, implies that the respective high-affinity binding sites are authentic receptors. Further information is needed on the molecular bases of the nonlinear response-occupancy relationships in hormone action on the kidney.

Aldosterone↗
PubMed1980

Current-voltage analysis of apical sodium transport in toad urinary bladder: effects of inhibitors of transport and metabolism.

The basal-lateral surface of the epithelium of the urinary bladder of the toad (Bufo marinus) was depolarized by exposure of the serosal surface to 85 mM KCL and 50 mM sucrose. The extent of depolarization appeared to be virtually complete, as evaluated by the invariance in the transepithelial electrical potential difference and conductance on addition of nystatin (a monovalent cation ionophore) to the serosal medium. The Na-specific current (INa) was defined as the current sensitive to the removal of Na from the mucosal medium or inhibitable by addition of amiloride to this medium. In the presence of the high K-sucrose serosal medium, rapid, serial, stepwise clamping of the transepithelial voltage (V) yielded a curvilinear dependence of INa on V; which is taken to represent the I--V curve of the apical Na channels. The constant field equation (Goldman, D.E. 1943; J. Gen. Physiol. 27:37) fits the I--V data points closely, allowing estimates to be made of the permeability to Na of the apical membrane (PNa) and of the intracellular Na activity (Nac). Exposure of the apical surface to amiloride (5 X 10(-7) M) decreased PNa in proportion to the decrease in INa (i.e., approximately 70%) but decreased Nac only 25%. In contrast, an equivalent reduction in INa elicited by exposure of the basal-lateral surface to ouabain was accompanied by only a 20% decrease in PNa and a sixfold increase in Nac. The effects of amiloride on PNa and ouabain on Nac are consistent with the primary pharmacological actions of these drugs. In addition, PNa appears to be under metabolic control, in that 2-deoxyglucose, a specific inhibitor of glycolysis, decreased INa and PNa proportionately, and lowered Nac marginally, effects indistinguishable from those obtained with amiloride.

Amiloride↗
PubMed1979

Radio-iodination of plasma membranes of toad bladder epithelium.

The present report describes high yield enzymatic radio-iodination of the apical and basal-lateral plasma membranes of toad bladder epithelium, by a procedure that does not breach the functional integrity of the epithelium, as assessed by the basal and vasopressin-sensitive short-circuit current (SCC). Restriction of the label to the membrane surface, was ascertained by light and electron-microscopic autoradiographs. On the apical surface, the grains were over the glycocalyx and the plasma membrane. Analysis of the labeled glycocalyx by agarose gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as enzymatic and pH-dependent hydrolysis indicated that the glycocalyx is a trichloro-acetic acid-soluble macromolecular complex of high molecular weight composed of a peptide moiety attached to large prosthetic groups (presumably carbohydrates) by O-glycosidic bonds. Analysis of the labeled apical plasma membrane components by agarose gel filtration and SDS-PAGE disclosed the presence of six major species of apparent molecular weights: 23,000, 28,000, 37,000, 44,000, 68,000, and 95,000. More than half of the membrane-associated radio-iodine was in two bands of molecular weights 37,000 and 44,000. Concentrations of vasopressin and cyclic AMP sufficient to increase the SCC significantly did not modify the extent of membrane labeling or the distribution of the label among the apical membrane components (presumably proteins) as assessed by SDS-PAGE. Iodination in the presence of amiloride inhibited incorporation but did not change the pattern of the distribution of the label among the components resolved by SDS-PAGE. Iodination of basal-lateral plasma membranes, at a yield comparable to that obtained with apical labeling, was attained after about 30 min of exposure of the intact bladder to the labeling solutions. Approximately 25% of the basal-lateral labeling was lost when the epithelial cells were harvested after collagenase treatment, implying that some iodination of the basement membrane had taken place. Less than 10% of iodination of the apical or basal-lateral surfaces was accounted for by lipid-labeling. Analysis of the labeled apical and basal-lateral species by enzymatic digestion and thin layer chromatography disclosed that virtually all the radioactivity was present as mono-iodotyrosine (MIT).

Animals↗
PubMed1979

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium.

The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Normarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with 125I. The basal-lateral components yielded a hetero-disperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochrome c reductase activities, were separated from the radio-iodine labeled by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 x g x 1 hr after removal of the mitochrondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.

Animals↗
PubMed1979

Relationship between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in the action of thyroid hormone on rat jejunal mucosa.

Administration of three successive doses of triiodothyronine (T3) (50 micrograms/100 g body wt), given on alternate days to thyroidectomized and euthyroid rats, stimulated oxygen consumption (QO2) and Na+ transport-dependent respiration (QO2 [5]) in the stripped jejunal mucosa, a preparation that consisted mostly of epithelial cells. The increase in QO2(t) accounted for 57% of the increment in QO2 in the transition from the hypothyroid to the euthyroid state and for 29% of the increment in the transition from the euthyroid to the hyperthyroid state. Administration of T3 to hypothyroid rats also increased the yield of epithelial cells. Injection of T3 into thyroidectomized and euthyroid rats increased the specific activity (at Vmax) of the (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) in jejunal crude membrane preparations. No significant change was recorded in the activity of Mg-ATPase in the same preparation. The ratio of QO2/NaK-ATPase and QO2(t)/NaK-ATPase in the various thyroid states remained constant, indicating proportionate increased in the respiratory and enzymatic indices. The effect of administration of T3 to thyroidectomized rats on the number of NaK-ATPase units (recovered in the crude membrane preparation) was estimated by: (a) Na+ + Mg++ + ATP-dependent binding of [3H]-ouabain to crude membrane fractions, and (b) the amount of the phosphorylated intermediate formed in the NaK-ATPase reaction from AT32P(gamma). Estimates were obtained of the maximal number of [3H]ouabain binding sites (Nm) and dissociation constants (Kd). Nm for [3H]ouabain and Nak-ATPase specific activity increased to about the same extent after T3 administration to thyroidectomized rats, with no change in the apparent Kd values. The amount of phosphorylated intermediate formed in jejunal crude membrane preparations also increased significantly. Thus, thyroid hormone administration may increase the number of active Na+pump sites in the plasma membrane. The apparent increase in the number of Na+ pump sites also correlated with the hormone dependent increases in QO2 and QO2(t).

Animals↗