Amiloride, a pyrazine diuretic, has been shown to decrease the secretion of K+ by the renal tubule (1-3). Although this effect has been explained as a result of the inhibitory aation of the drug on Na+ reabsorption, the possibility exists that amiloride might directly affect K+ permeability. This thought led to the speculation that amiloride might likewise affect the K+ permeability of the frog gastric mucosa which lends itself well to such an investigation. From the literature (4, S ) , it appears that the K+ resistance of the nutrient membrane of frog gastric mucosa is about half the C1-resistance and that other ionic resistances are very high relative to the K+ and C1-resistances.Since K + has been found to be essential for HCl secretion (6), perhaps if amiloride affected the K+ permeability it might also decrease the H+ secretory rate. Experiments were, therefore, designed to test the effect of arniloride on the transmembrane resistance, the transmembrane potential difference (PD), and the H-+ secretory rate in high and low nutrient K + -solutions.Methods. The experiments were performed on gastric mucosae of Rana pipiens with an in vitro method described in detail elsewhere ( 7 ) . In this method each mucosa was mounted between cylindrical chambers. Two pairs of electmodes were used, one for sending current across the mucosla and the other for measuring the PD. The resistance was determined as the change in PD per unit of applied current. The nutrient bathing solution contained ( m u ) : Na+, 101; K+, 4; Ca2+, 1; Mg2+, 0.8; C1-, 81; HC03-, 25; phosphate, 1.0; and glucose, 10; and the secretory bathing solution: Na+, 102; K+, 4; C1-, 106. In high K+ nutrient solutions, K+ replaced Na+, B~t h sides of the mucosa were gassed with 95% O2 and 5% C02.The pH of the secretory solution was maintained at 4.90. After the control part of the experiment, amiloride was added in the nutrient solution to a 0.5 or 2.0 mM concentra tion.Results. Figure 1 shows the effects of adding amiloride to the nutrient solution. At the time indicated by the arrow, amiloride was added to a concentration of 0.5 mM in the nutrient solution. A comparison was generally made of the changes in the parameters in about 30 min following the addition of amilorirde in the nutrient solution to the control values just before the addition. As shown in Fig. 1, the resistance inlcreased about SO%, the PD decreased about 15%, and the H+ rate decreased about 30%. Washing both sides of the mucosa with regular solutions produced only a partial recovery of the parameters towards control values. In about 40% of the experiments after washing with regular solutions, the resistance decreased to some extent towards control values while the H+ rate persisted at about the same level as with amiloride in the nutrient solution. Table I sulmmarizes the main results. Columns 111, V and VII give, respectively, the average resistances, PD and H+ rates of mucosae bathed in regular solutions without amiloride. Columns IV, VI and VIII give, respectively, the average percentag...
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
Amiloride (3,5-diamino-6-chloropyrazinoylguanidine) was found to inhibit the unidirectional flux of 22Na in perfused proximal tubules of rats when the drug was present in the blood (1). The drug did not inhibit the net water reabsorption, therefore, it apparently did not have an affect on the active reabsorption of sodium.Since amiloride has been found to be a potent inhibitor of sodium reabsorption in the amphibian bladder and skin (2, 3) and since it has been found by the same authors that the inhibition of sodium transport is about one thousand times higher when the drug is on the source than when it is on the sink site of the sodium transport, experiments were designed to test the effect of the drug when present in the luminal and not in the interstitial site of the tubule cell.Methods. Sprague-Dawley rats weighing about 200 g were anesthetized with Inactin (100 mg/kg body wt.) and then infused through the right jugular vein with Ringer's solution a t a rate of 30 ml/hr-kg. Using a modification of the microperfusion technique and apparatus of Sonnenberg and Deetjen (4), proximal tubules were perfused at a rate of 20 nanoliters per min.Two pumps were used, one contained a control solution (Ringer's with 22Na and 14Cinulin) and the other contained the same solution plus 1 P M amiloride. A selected proximal tubule was first perfused with the control solution for 30-40 rnin, and 2-4 samples were obtained during this period. Then the control pump was withdrawn and substituted by the "amiloride" pump. Perfusion was restarted at exactly the same location. The amiloride perfusion was administered for 45-60 min during which time 2-4 samples were obtained. The use of the 2 pumps was randomized with ref-erence to their use for control or for amiloride perfusions.Each of the tubules perfused in the control period and in the diuretic period was injected with a latex solution ( 5 ) for later tubule identification and measurement. Concentrations of l4C-inulin and 22Na were determined with a three channel liquid scintillation counter.Results. Figure 1, presents a plot of the concentration ratio of collected over perfused fluid [ (TF)/(IF)] of 14C-inulin and 22Na, versus time. In this selected experiment three perfusion periods are presented: control, amiloride, and recovery or return to control. The (TF) / (IF) values for inulin were definitely reduced by amiloride and increased slightly when the amiloride perfusate was substituted by the control. The (TF)/(IF) values for sodium were definitely increased by amiloride returning to low levels when a control perfusate was reinstated. Table I, presents compiled data from eight perfusions (8 rats). The values presented for 0.2 / \ r -Control Amiloride Control w -Ic------+l 1 I I I I o 20 40 60 80 loo Time [minutes) FIG. 1. Effect of amiloride on proximal tubular reabsorption of water and sodium. Open circles are the collected fluid to perfusate ratios ( [ TF]/ [IF] ) of inulin concentration; dots are the same ratios for Na. Zero time refers to the beginning of the tubular perfusi...
The addition of ethacrynic acid to a concentration of 1 mM in the nutrient solution bathing the frog gastric mucosa in vitro produced an immediate decrease in resistance followed by an increase in resistance and a decrease in H+ secretory rate. The latter effect was irreversible and the former reversible. During the initial phase for nutrient solutions of 4 mM K+ and 79 mM K+ containing 1 mM ethacrynic acid, the decrease in resistance was about 30% and the decrease in the transmembrane potential difference (P.D.) was about 1 mV in the low K+ case and 2 mV in the high K+ case. The addition of ethacrynic acid to a 4 mM K+ nutrient solution containing 1 mM Ba2+ produced initially a 19% decrease in resistance and both positive and negative changes in P.D. In the absence of Ba2+, these results suggest strongly a marked increase of K+ permeability with the possibility of some increase of Cl− permeability. In the presence of Ba2+, as a result due to the increased K+ resistance of the nutrient membrane, ethacrynic acid may affect predominantly either K+ or Cl− permeability.
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