The Mechanism of the Calorigenic Action of Thyroid Hormone : Stimulation of Na+ + K+-activated adenosinetriphosphatase activity

In an earlier study, we proposed that thyroid hormone stimulation of energy utilization by the Na⁺ pump mediates the calorigenic response. In this study, the effects of triiodothyronine (T₃) on total oxygen consumption (Q_OO2), the ouabain-sensitive oxygen consumption [Q_OO2(t)], and NaK-ATPase in liver, kidney, and cerebrum were measured. In liver, ~90% of the increase in Q_OO2 produced by T₃ in either thyroidectomized or euthyroid rats was attributable to the increase in Q_OO2(t). In kidney, the increase in Q_OO2(t) accounted for 29% of the increase in Q_OO2 in thyroidectomized and 46% of the increase in Q_OO2 in euthyroid rats. There was no demonstrable effect of T₃ in euthyroid rats on Q_OO2 or Q_OO2(t) of cerebral slices. The effects of T₃ on NaK-ATPase activity in homogenates were as follows: In liver +81% from euthyroid rats and +54% from hypothyroid rats. In kidney, +21% from euthyroid rats and +69% from hypothyroid rats. T₃ in euthyroid rats produced no significant changes in NaK-ATPase or Mg-ATPase activity of cerebral homogenates. Liver plasma membrane fractions showed a 69% increase in NaK-ATPase and no significant changes in either Mg-ATPase or 5'-nucleotidase activities after T₃ injection. These results indicate that thyroid hormones stimulate NaK-ATPase activity differentially. This effect may account, at least in part, for the calorigenic effects of these hormones.     

Cellulase from Ruminococcus albus and Mixed Rumen Microorganisms

Cellulase in the cultural filtrates of Ruminococcus albus and cellulase extracted from mixed rumen microorganisms were investigated with acid-swollen cellulose and carboxymethylcellulose as substrates. Maximal activity occurred at approximately pH 5.8 and 47 C. Apparent Michaelis constants (K_m) varied between 0.53 and 0.02% carboxymethylcellulose, depending on the level of activity and the method of assay. R. albus cellulase has a lower K_m value than the enzyme extracted from mixed rumen microorganisms. Antisera from rabbits immunized with a cellulase preparation from R. albus inhibited the cellulolytic activity of both systems. Based on the relative degree of inhibition, approximately 20% of the cellulase of the mixed rumen microorganisms was immunologically similar to R. albus cellulase. Ratios of activity in different assay techniques showed the two sources of activity to be similar in the mechanisms of degradation. However, glucose is the main product of cellulose degradation by mixed rumen microorganisms, and cellobiose is the product of degradation by R. albus.     

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

Summary Urinary bladders ofBufo 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.     

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

Summary 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 Nomarski 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 with¹²⁵I. The basal-lateral components yielded a heterodisperse 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 cytochromec reductase activities, were separated from the radio-iodine labeled components 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 × g × 1 hr after removal of the mitochondria 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.     

Effect of aldosterone on incorporation of amino acids into renal medullary proteins

Summary Studies on the effects of pretreatment with aldosterone on the incorporation of³H leucine or³H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 g/100 g body wt) to adrenalectomized rats increased³H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the³H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in³H-methionine incorporation into proteins of renal medullary slices (0.45 g of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K⁺/Na⁺ ratio (0.35 g of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 g/100 g body wt did not augment³H-leucine incorporation into renal medullary proteins but was effective at 8 g/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 gvs. 0.8 g (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70–80 g/100 g body wt) erased the effect of aldosterone (0.8 g/100 g body wt) on amino acid incorporation into medullary proteins.In paired experiments with³H and³⁵S methionine, aldosterone (0.8 g/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of 31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70–80 g/100 g body wt or SC-26304 (80 g/100 g body wt). Dexamethasone (0.8 g/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.     

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.     

Effects of aldosterone on Na transport in the toad bladder II. The anaerobic response

The action of aldosterone on active Na⁺ transport was assessed under aerobic and anaerobic conditions in the isolated urinary bladder of the toad, BUfo marinus. Aldesterone augmented the short-circuit current (I_sc) under rigorous anaerobiosis. Four lines of evidence indicate that the increase in anaerobic I_sc does not represent an equivalent increase in active Na⁺ transport: 1. Net Na⁺ transport, determined by isotopic fluxes, was the same in the aldosterone-treated and control quarter-bladders, and significantly greater than the simultaneously measured I_sc. 2. Amiloride, an inhibitor of the apiral entry of Na⁺, did not reduce the steroid-dependent increase in the anaerobic I_sc. 3. Substitution of choline for Na⁺ in the mucosal medium reduced the magnitude of the anaerobic I_sc values did not eliminate the effect of aldosterone. 4. Addition of ouabain, a potent inhibitor of the Na⁺ pump, partially inhibited the effect of aldosterone on the anerobic I_sc but a significant hormonal increment remained. The source of the anaerobic I_sc was not identified; an effort was made, however, to determine the dependence of this current on glycolysis. During anaerobics, aldosterone increased the integral I_sc by 42% but did not alter lactate production. These results suggest that the steroid-dependent increase in the anaerobic I_sc may involve effects on permeability properties of the epithelium rather than on active tranport systems.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.