Studies on rats (1, 2) and dogs ( 1 ) have shown that amiloride ( 3'5-diamino-6chloropyrazinoyl-guanidine induces natriuresis accompanied by a potassium sparing effect. These findings, plus the fact that Baer et al. (1) found an inhibition of sodium reabsorption in the distal samples, using the stop-flow technique, led to the conclusion that the site of action of amiloride is in the distal tubule.Further studies have shown that the drug is a potent inhibitor of sodium transport in the amphibian skin and bladder (3, 4). In view of the latter, we decided to study the effect of amiloride on the proximal tubule of the rat using the microperfusion technique and compare its action to that of other diuretics.Methods. Sprague-Dawley rats weighing 190 2 g were anesthetized with Inactin (100 mg/kg body wt) and then infused through the right jugular vein with Ringer's solution at a rate of 30 ml/hr-kg. Using the microperfusion technique and apparatus of Sonnenberg and Deetjen ( 5 ) , proximal convoluted tubules were perfused at a rate of 20 nl/min with Ringer's solution containing 22Na and 3H-inulin.After attaining a control perfusion and concomitant collection of urine from approximately 1-3 separate proximal tubules in any given rat, each of the rats was injected through the left jugular vein with 1301 either amiloride (2 or 10 mg/kg body wt), furosemide (5 or 50 mg/kg body wt), or ethacrynic acid (5 or 10 mg/kg body wt). In each respective instance the level of diuretic was maintained by a constant infusion of amiloride (2 or 10 mg/hr-kg), furosemide (5 or 50 mg/hr-kg), or ethacrynic acid (5 or 10 mg/hr-kg). This was accomplished by dissolving the respective diuretic in Ringer's solution and maintaining the Ringer infusion rate of 30 ml/hr-kg. This rate was appropriately corrected for changes in urine flow. Under this diuretic condition, several proximal tubules were then perfused and urine samples collected concomitantly. Each of the tubules perfused in the control period and in the diuretic period was injected with a latex solution (6) for later tubule identification and measurement. Concentrations of 3H-inulin and 22Na were determined with a three-channel liquid scintillation counter.Results. Control. Thirty-eight proximal tubules were perfused in rats preceding administration of the diuretic. Figure 1 presents a semilogarithmic plot of collectedfluid to perfused-fluid concentration ratios ( C L (~) / C~) of 3H-inulin and 22Na versus the length of the perfused tubule. Correlation between the logarithm of the concentration ratios and perfused length was statistically significant ( P < 0.05) for both 3H-inulin and 22Na. While the concentration of inulin increased, the concentration of 22Na decreased along the perfused tubule. The slopes were 6.50 X mm-1 and -3 1 .O X mm-1 for "-inulin and 22Na respectively. That is, for each mm of tubule
In a Na-rich bathing system, addition of amiloride to the mucosal fluid of turtle bladders produces decreased in the transepithelial potential difference (PD), short-circuiting current (I-sc), and conductance. Removal of amiloride results in complete reversal of these changes; and this reversibility is incomplete in amiloride-blocked bladders exposed to ouabain. In a Na-free bathing system, step increased in mucosal [Na] evoke rapid initial spikes in PD, Isc, and conductance, the magnitudes of which are independent of prior ouabain treatment. After these spikes, PD and Isc in the ouabain-treated bladder rapidly decay, while conductance remains unchanged and high. This unchanging conductance, plus the fact that ouabain inhibits half the microsomal (Na plus K). ATPase of this tissue within 1 min, suggests that ouabain inhibits Na pumping without changing tissue conductance. The delayed decrease in conductance (beginning 30 min after ouabain addition), a nonspecific and secondary effect of ouabain, is due to a concomitant collapse of the intercellular channels.
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