The short-term effect of one single injection of aldosterone on the renal sodium transport on one hand, and the Na-K-ATPase activity on the other hand, was studied in chronic adrenalectomized rats. Sodium transport was estimated by clearances, and Na-K-ATPase was measured in microdissected fragments of the nephron, according to our microtechnique previously described. Five to eight days after adrenalectomy, only 30% of the initial enzyme activity was recovered in the cortical collecting tubule (CCT). Administration of aldosterone completely restored the ATP-ase activity within three hours. Adrenalectomy also curtailed by 20-45% the activity of other nephron segments but aldosterone had no stimulatory effect on them. Sodium-reabsorption also increased after the hormone injection, following the same time (0.5 less than t1/2 less than 1 h) and dose dependencies (0.8 less than K1/2 less than 0.9 micrograms/kg) as those observed for the enzyme activity in the CCT. It is concluded that the short-term stimulation of Na-K-ATPase in the collecting tubule, after an acute administration of aldosterone, may be responsible for the simultaneous increase in sodium transport.
Both glucocorticoids and mineralcorticoids stimulate the renal Na-K-ATPase. However, the exact site of their respective action is not precisely determined and it is still unknown whether these effects are cumulative or not. We studied the effects of dexamethasone and aldosterone on Na-K-ATPase activity in microdissected nephron segments from adrenalectomized rabbits. In proximal convoluted tubule (PCT) the enzyme activity was altered neither by adrenalectomy nor by any steroid replacement. In the medullary thick ascending limb of the loop of Henle (MAL) and the distal convoluted tubule (DCT), Na-K-ATPase activity decreased by 40% after adrenalectomy, and was restored to control level three hours after administration of dexamethasone (100 micrograms/kg) but not by aldosterone (up to 10 micrograms/kg). In the cortical (CCT) and medullary (MCT) collecting tubule the enzyme activity decreased by 75% after adrenalectomy but in contrast with the MAL and the DCT, these two segments were sensitive to both dexamethasone (100 micrograms/kg) and aldosterone (10 micrograms/kg) and recovered their activities within 3 h after the hormone injection. These effects were not additive. Spironolactone (100 micrograms/kg) abolished the action of each of the two hormones on the CCT and MCT. In contrast, spironolactone did not curtail the effect of dexamethasone on MAL and DCT. These results indicate that whereas glucocorticoid action is localized in MAL, DCT, CCT and MCT, the mineralocorticoid effect is restricted to the CCT and MCT exclusively. They also suggest that, in the CCT and MCT, the two types of hormones share the same receptors.
To determine the number of Na-K-ATPase units and the enzyme's turnover rate along the rabbit nephron, the specific binding of [3H]ouabain and the Na-K-ATPase activity were measured in single nephron segments microdissected from collagenase-treated kidneys. The highest density of Na-K-ATPase (20-30 fmol X mm-1) was found in the distal convoluted tubule and the medullary thick ascending limb. Binding was intermediate (10 fmol X mm-1) in the proximal convoluted tubule and connecting tubule, and it was lowest (2-7 fmol X mm-1) in the pars recta, the cortical thick ascending limb, and the collecting tubule. In the medullary thick ascending limb, Scatchard analysis of the specific [3H]ouabain binding indicated a dissociation constant of 1.8 microM. The pump activity was proportional to the number of catalytic units, indicating that the maximal turnover rate of Na-K-ATPase (2,000 ATP molecules per minute per ouabain binding site) was similar in the various segments of the nephron. The method developed for quantitating [3H]ouabain binding is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine the number of Na-K-ATPase units in each region of the nephron.
Both aldosterone and dexamethasone are known to stimulate renal Na-K-ATPase activity although their action is restricted to specific nephron segments: the collecting tubule, the target site for mineralocorticoids, and the thick ascending limb and distal convoluted tubule, the target sites for glucocorticoids. As this stimulation by corticosteroids is very fast, we attempted to establish whether it occurs through de novo synthesis of new Na-K-ATPase units or by increasing the specific activity of the Na-K-ATPase units already present. For this purpose we studied the effects of aldosterone and dexamethasone on Na-K-ATPase specific activity in microdissected nephron segments from adrenalectomized rabbits. This specific activity was determined by the ratio of ATPase activity over the apparent number of catalytic units, as measured by specific 3H ouabain binding. In the proximal tubule, neither adrenalectomy nor steroid replacement altered Na-K-ATPase activity or the apparent number of catalytic sites. In other nephron segments, adrenalectomy reduced Na-K-ATPase activity and specific 3H ouabain binding concomitantly, and therefore left this enzyme's specific activity unaltered. In the cortical and outer medullary collecting tubules, 10 micrograms/kg aldosterone simultaneously restored both the activity and apparent number of catalytic units of Na-K-ATPase to their control levels, and therefore did not modify the specific activity of the pump. Conversely, 100 micrograms/kg dexamethasone increased Na-K-ATPase activity in the thick ascending limb and distal convoluted tubule without changing the apparent number of catalytic units.(ABSTRACT TRUNCATED AT 250 WORDS)
To test whether sodium availability controls the concentration of renal Na-K-ATPase, we evaluated the effect of chronic alterations in apical membrane sodium permeability in specific nephron segments on the maximal activity of Na-K-ATPase. For this purpose Na-K-ATPase activity was determined in nephron segments microdissected from rats treated continuously for 3-8 days with either furosemide or amiloride, two diuretics known to lower the apical permeability to sodium in the thick ascending limb and the collecting tubule, respectively. Unexpectedly, Na-K-ATPase activity was decreased neither in the thick ascending limb nor in the collecting tubule after administration of either drug. In fact, both diuretics paradoxically increased the pump activity by 60-150% in the collecting tubule. This stimulation of Na-K-ATPase activity was accompanied with an enhancement of the collecting tubule diameter. Stimulation of Na-K-ATPase was identical in the collecting tubule of diuretic-treated rats receiving spironolactone. These results suggest that Na-K-ATPase maximal activity is not controlled by sodium availability or by aldosterone under these conditions and that chronic administration of furosemide or amiloride induces Na-K-ATPase activity in the collecting tubule. This effect appears to be independent of aldosterone.
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