View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Kidney International, Vol. 1 (l972),p. 100—105

Dual action of and on proximal volume absorption in the rat

H. W. RADTKE, G. RUMRICH, EVA KINNE-SAFFRAN and K. J. ULLRICH

Max-Planck-Institut für Biophysik, Frankfurt, Germany

Dual action ofacetazolamide and furosemide on proximal103 et 3) l'action du furosemide ressemble a celle de l'acéta- volume absorption in the rat kidney. Isotonic fluid absorptionzolamide sauf qu'avec chacune des deux solutions de perfusion from the proximal convolution of the rat kidney was meas- le furosemide est un inhibiteur moms puissant. ured, using the Gertz shrinking droplet method, with simul- En conclusion, l'acétazolamide et le furosemide inhibent Ia taneous perfusion of the adjacent peritubular capillaries. The reabsorption isotonique non seulement en inhibant l'anhydrase capillary perfusate consisted of a modified Ringers solution, carbonique mais en interférant, a une dose un peu plus élevée, containing either buffer or the buffer, avec un autre mécanisme de reabsorption indépendant de l'an- glycodiazine. Different amounts of acetazolamide or furosemide hydrase carbonique. Aux doses employees, ni l'acétazolamide ni were added to the perfusate. It was found that: 1) equimolar le furosemide n'avaient un effet sur l'ATPase de transport du concentrations (30 mM) of glycodiazine are as effective as bi- tissue cortical. Une inhibition directe du système de transport carbonate in promoting proximal isotonic reabsorption; 2) de l'ion hydrogéne pourrait donc éxister. acetazolamide, at either 10-5 or 10 M, inhibited absorption to a greater extent when tested in the bicarbonate-containing solu- tion, but the same level of inhibition was attained when the A parallel study in our laboratory has shown that inhibitor was tested in the glycodiazine-buffered solution at a isotonic fluid absorption from the is dose level of 10-3 M; 3) The action of furosemide resembled that decreased when the ambient bicarbonate concentra- of acetazolamide except that, in either buffer, furosemide was a less potent inhibitor of reabsorption. tion is lowered. Furthermore, a linear correlation was It is concluded that both acetazolamide and furosemide observed between the luminal bicarbonate concentra- inhibit isotonic reabsorption by inhibiting carbonic anhydrase; tion and bicarbonate reabsorption, and a saturation- at a slightly higher dose level, these agents also interfere with some other non-carbonic anhydrase-mediated process. Since type relationship was observed between bicarbonate neither acetazolamide nor furosemide in the tested doses exhibit reabsorption and sodium reabsorption [1]. When an effect on cortical Na-K-ATPase, it is suggested that a direct equimolar concentrations of other organic buffers inhibition of the H transporting system may occur. Dualité d'action de l'acétazolamide et du furosemide dans such as glycodiazine, sulfamerazine, and butyrate l'absorpt ion par le tube contourné proximal du rein chez le rat. were used instead of the bicarbonate buffer, the L'absorption de fluide isotonique par le tube contourné proxi- isotonic absorption of Na, Cl and buffer was as large mal du rein a été étudiée chez Ic rat en employant Ia méthode de Ia gouttelette réabsorbée de Gertz avec perfusion simut- as that observed in the presence of bicarbonate buffer. tanée capillaire peritubulaire adjacent. Le liquide de per- Since these organic ions buffer secreted H instanta- fusion capillaire était compose d'une solution de Ringer tam- neously, their action is less complicated than that of ponnée avec du bicarbonate ou avec le sulfamide, glycodiazine. Différentes quantités d'acétazolamide et de furosemide ont bicarbonate buffer and its carbonic anhydrase (CA)- été ajoutées a Ia solution de perfusion. Les observations sui- catalyzed hydration reaction. By comparing the vantes ont été faites: 1) des concentrations équimolaires effects of the CA-inhibitor acetazolamide on proximal (30 mM) de glycodiazine et de bicarbonate sont également efficaces pour favoriser la reabsorption isotonique par le tube isotonic reabsorption in the presence of glycodiazine proximal; 2) une concentration de 10 ou I Q4 M d'acétazol- buffer with those observed in the presence of bicar- amide inhibe l'absorption dans une plus grande mesure en bonate buffer, the extent to which this agent acts via presence du bicarbonate, mais Ic même degré d'inhibition est atteint en presence de Ia glycodiazine avec une concentration de carbonic anhydrase inhibition (as opposed to a dif- ferent type of action) can be evaluated. Shrinking Received for publication August 15, 1971 accepted in revised form October 29, 1971. drop experiments were performed in which proximal © 1972, International Society of Nephrology reabsorption was assessed during the peritubular

100 Dual action of acetazolamide and furosemide 101 perfusion ofeitherglycodiazineor bicarbonate- acetate 5 mM, CaCI2 1.5 m, and 15 mrvt NaHCO3 or buffered solutions to which varying amounts of 15 mM Na-glycodiazine respectively. Again, when acetazolamide or I urosemide were added. In low NaHCO3 was used, the solution was equilibrated with concentrations, both inhibited isotonic ab- 2.5% CO2 in 02 so that the pH was again equal to 7.5. sorption in the bicarbonate-buffered system supposedly The composition of the respective "buffer-free solu- via the inhibition of carbonic anhydrase; acetazol- tion" used in some experiments was identical with amide, in concentrations higher than 10 M, and the exception that HCO3 or glycodiazine were replaced furosemide, in concentrations higher than 3 x 10 M, by an equimolar amount of NaCI. also inhibited the bicarbonate-free system supposedly The indicated concentration differences between via direct interference with the H and/or Na capillary perfusate and injected shrinking droplet transporting systems themselves. Because both drugs, fluid were chosen in order to keep the concentration at the cited concentrations, failed to inhibit the Na-K- of the constituents of the droplet constant during the ATPase in a membrane fraction isolated from rat shrinking process. These stationary concentrations kidney cortex, it is suggested that they exert a direct were determined in earlier work' by analyzing the inhibitory effect on the H transport system. Na, Cl, and acetate concentration at different times during the course of the absorption process. Both the droplet fluid and the capillary perfusate Methods contained identical concentrations of acetazolamide The technique for the evaluation of isotonic fluid (or, alternatively, furosemide). absorption in Wistar strain rats was that of the Gertz The activity of the ATP-phosphohydrolase was shrinking droplet [2]. The calculation of reabsorptive determined in isolated plasma membranes using three half-time (t' /2)wasimproved by correcting the tubular different incubation media. All media contained length for the oil-water nieniscus, and by using a pro- 3 x 10 M TRIS-buffer, pH 7.6, 3 x l0 M TRIS-ATP gram which allowed individual regression lines and and 6xl0 2MMgSO4. Media 2 and 3 also contained correlation coefficients to be calculated for each in- 2 x 10-2 M KC1 and 1 x 10' M NaCI. To Medium 3, vestigated tubule. This methodology has been de- 2 x 10 M ouabain was added [6]. scribed in detail by Györy [3]. The rate of isotonic After 30 minutes of incubation at 370 C, the reaction reabsorption per unit tubular length was calculated as was stopped by heating and the samples were centrif- uged. The amount of inorganic phosphate in the O.3472irr2 — supernatant was determined using a modified method ti'2 of Fiske-Subbarow and Bartlett [7, 8]. The mean of Wedid not use the measured radius (r) of the colored the activity difference found in the incubation Media 1 oil column [3]; instead, an estimated r of 15 j.twas and 3 was taken as the Mg tATPase, whereas the used. Simultaneous perfusion of the peritubular capil- difference between incubation Medium 2 and the laries was carried out at a rate of approximately Mg -ATPase was taken to be the sodium and potas- 2 hI/mm, with a technique developed in this labora- sium-stimulated, ouabain-inhibited ATPase (Na-K- tory [4]. ATPase). When furosemide or acetazolamide were The solution used for capillary perfusion contained tested, these inhibitors were added to all three media. the following: NaCI 115 m, Na-acetate 10 mM, The activity of the enzyme is expressed as specific CaCl2 1.5 m, and 30 mrvi NaHCO3 or, alternatively, activity (mol/hr/mg protein). The amount of protein 30 mM Na-glycodiazine. Solutions which contained in the plasma membrane fraction was determined by bicarbonate were equilibrated with 95% oxygen and the method of Lowry et al [9]. 5% CO2 which maintained the pH of the solution at The substances used were of "highest purity": 7.5. Glycodiazine solutions were adjusted to pH 7.5 1) 2-acetylamino- 1 ,3,4-thiadiazol-5-sulfonamid (Dia- with HCI. To be sure that the pH of the solutions did mox®); 2) 4-chlor-N-(2-furfuryl)-5-sulfamoylanthra- not change during perfusion of the blood capillaries, nilacid (Lasix®); 3) 2-benzolsulfonamide-5-methoxy- they were withdrawn several times from the capillaries ethoxypyrimidin-natrium (Redul®); pK =5.7. directly into an indwelling ultramicro glass electrode [5], and their pH was found to be the same as in the Ulirich, K. J., Baldamus, C. A., Frömter, E., Lüer, K., Radtke, H. W., Rumrich, G., and Sauer, F.: Transport para- infused solutions, that is, 7.5. The solution injected in meters for sodium, chloride and bicarbonate in the proximal the tubule lumen contained: NaC1 135 m, Na- tubules of the rat kidney. In preparation. 102 Radtkeet a!.

Table 1 cm [.10—7cm sec Effect of acetazolamide and furosemide on isotonic 3.Oj reabsorption (x 10 cm3 cm sec') from the proxi-

2.5 mal convolution

2.0 \ b Buffer free Bicarbonate Glyco- 1.5 diazine

1.0 Control 0.69± 0.04 2.75± 0.07 2.74± 0.07 0.5 N=31 (4) N=82 (13) N=72 (9) Acetazolamide M Acetazolamide 1 x 1O 1.46± 0.06 2.24± 0.09 'm3 N= 33 (3) N— 35 (2) cmsecj 3.0 lx 10 0.36±0.03 0.70±0.04 1.95±0.11 N= 48 (3) N— 45 (4) N— 30 (2) 1x103 1.00±0.06 N=26(2) :' Furosemide lxlO—4 1,58±0.07 2.51±0.10 N= 45 (4) N= 47 (3) Furosemide 3x 10 0.89±0.04 1.04±0.06 0—?::' ioI iOI i0I 10 M N— 22 (2) N= 30 (2) Mean values SE; N =numberof measurements; number of Fig. 1. Effect of acetazolamide (upper panel) and furosemide animals in brackets. (lower panel) on proximal isotonic reabsorption (1.0 x l0- cm3 x cm1 x secl): a) 30 m bicarbonate buffer in capillary perfusate, b) 30 mat glycodiazine buffer in capillary perfusate. Table 2 Effect of acetazolamide and furosemide on the Mg+ + ATPase and the Na-K-ATPase activity of plasma Results membranes from rat kidney cortex The effects of increasing perfusate concentrations of acetazolamide and furosemide on isotonic reab- Mg ATPase Na-K sorption are summarized in Table I and Fig. 1. ATPase In the absence of inhibitor drugs, the rate of proxi- mal volume reabsorption was identical irrespective Control 26.83±1.12 23.06±1.40 of the use of 30 m bicarbonate or 30 mivi glycodiazine buffer in the capillary perfusate. These data also show Acetazolamide that reabsorption was increasingly inhibited with l0 26.13±1.64 23.26±1.40 rising concentrations of acetazolamide, but that the 10 26.21±1.03 25.23±0,81 24.93±0.42 25.38±0.76 effect was always greater in the presence of bicarbonate 10 102 25.97± 0.73 22.62± 0.65 buffer than in the presence of glycodiazine. With furosemide, the inhibition of reabsorption was also Furosemide greater in the presence of bicarbonate buffer. l0— 25.44± 1.66 23.28±0.85 Table 2 shows the influence of acetazolamide and 10' 25.80±0.19 22.24± 1.70 furosemide on the Mg -ATPase and Na-K- iO 22.40± 1.46 24.26±0.36 ATPase activity of a plasma membrane fraction 102 14.33±0.69 24.21±0.10 isolated from rat kidney cortex. Acetazolamide, at Mean values SE of 3 experiments are given. Each membrane concentrations ranging from 10 to 10-2 M, had no fraction was prepared from 20 animals. The enzyme activity is effect on either the Na-K-ATPase or the Mg - expressed as specific activity in MM/hr/mg protein. Dual action of acetazolamide and furosemide 103

ATPase. The Na-K-ATPase was not inhibited by agreement with those of Schmidt and Dubach [18] who furosemide even at a concentration of 10-2Malthough reported similar results using a Na + K + -ATPase this concentration led to a considerable reduction of preparation from the proximal convolutions of rat the Mg -ATPase activity. kidneys. In addition, Ebel et al [20] found no inhibitory effect in a plasma membrane fraction from total rat Diskussion kidneys using a furosemide dose of 1 m. On the Acetazolamide is the classic inhibitor of carbonic other hand, we observed an inhibitory effect of 10 mM anhydrase [10, 11]; furosemide also has an in vitro furosemide on cortical Mg-ATPase activity, and we carbonic anhydrase inhibitor potency of the same order cannot exclude the possibility that furosemide accu- mulates at the basal infoldings of the proximal tubular of magnitude as that exhibited by sulfanilamide [12]. The effect of acetazolamide in vivo, in the kidney, cells in vivo and thereby inhibits the NatKATPase is generally attributed to its carbonic anhydrase located in these membranes. Nevertheless, we are not inclined to view the action of furosemide as being inhibitory activity, thereby affecting the H-Na exchange process at the luminal cell membrane [13]. localized to the contraluminal Na-K-ATPase, but As shown in this study, acetazolamide has no action rather as being centered on a transport step at the lumi- on renal Na-K-ATPase. This is in agreement with the nal membrane, the nature of which is discussed below. findings of others [14] who have also failed to observe As originally suggested by Pitts and Alexander [13], any effect on renal NaKtATPase preparations by the luminal H-Na exchange in the proximal con- acetazolamide and other related substances (hydro- volution could lead to the reabsorption of luminal bicarbonate in the following sequence: secreted H , tri-chlormethazine and cyclopenthi- azide). An effect of acetazolamide on the energy- reacts with luminal HCO3 to form H2C03 which is producing system, which could be responsible for the then dehydrated to H20 and CO2. Both H20 and CO2 inhibition of proximal salt and water transport, has enter the cell from the lumen by diffusion. Within thus far not been identified. We are therefore inclined the cell the CO2 is rehydrated and could then be used to view the action of acetazolamide as one that is as the source of H + to be secreted. The resulting confined exclusively to a luminal H+Na+ exchange HCO3 should diffuse out of the cell into the contra- system which is somehow linked to a carbonic an- luminal interstitium. This hypothesis is supported by three facts gained by micropuncture: 1) an acid dis- hydrase-catalyzed reaction step. The situation is somewhat different with furosemide. equilibrium pH is observed when acetazolamide is The altered renal ion pattern after the given [21]; this indicates that the H + secreted into the administration of furosemide to the whole animal lumen is not rapidly buffered when the activity of (especially the low or absent urinary excretion of carbonic anhydrase is blocked. Furthermore, the data bicarbonate) suggests a mechanism of action different indicate that the location of the CA must be on the from that of acetazolamide and the other saluretic surface of the brush border. 2) The permeability of the sulfonamides [15]. Thus far, effects on the energy- contraluminal cell site is comparatively high for HCO3 as shown by the electrical measurements of Frömter, producing system in rat kidney cortex [16] or the pas- sive ion permeability of rat proximal convolutions Muller, and Wick [22], which thus allows for intra- have not been detected [17]. A NatKATPase cellular HCO3 to diffuse primarily into blood instead inhibitory effect of furosemide has been described in of returning to the lumen. 3) A stoichiometric relation erythrocytes2 and the distal nephron segments of exists between H secretion and Na absorption [1]. rats [18]. Similarly, inhibition of a microsomal cortical In a parallel study in which the ambient bicarbonate ATPase preparation from rabbit kidney has also been concentration was changed, we observed a linear observed by Murphy and Bader [19] with a half- relation between bicarbonate transport (which indi- maximal concentration of 12 m. However, such cated H secretion) and net sodium transport with a inhibition was not observed in our own NaKt slope of 1:6 in the range of up to 20 mrvi luminal ATPase preparation from rat kidney, at least in the HCO3 concentration. In these experiments approxi- presence of doses up to 10 mi, a concentration which mately one-half of the net sodium transport is active.3 showed a maximal inhibitory effect on proximal 3 U!lrich, J. J.,Baldamus, C. A., Frömter, E.,Luer, K., isotonic reabsorption. Our results are therefore in Radtke, H.W., Rumrich, G., and Sauer, F.: Transport para- meters for sodium, chloride and bicarbonate in the proximal 2 Hoffman, J.P.: Personal communication. tubules of the rat kidney. In preparation. 104 Radtkeet a!.

Since it is very likely that these sodium ions, which concomitantly also the pH) does not influence the are transported actively by the contraluminally located proximal reabsorption process [I]. It is rather only + Na +-KATPase,first have to enter the cell from the the availability of the anions bicarbonate or A, lumen, a ratio of one H secreted per three Na which is responsible for the secretory rate of H, and reabsorbed by the transcellular route may be estimated. secondarily for the reabsorption of Nat Considering The sodium ions entering the cell in this way increase all of the available arguments, we are inclined to the intracellular Na concentration, which in turn postulate a direct effect on the H transport system could govern the rate of the sodium pump. as a second action of the CA-inhibitors, acetazolamide A blockade of carbonic anhydrase activity could and furosemide. therefore cause the following sequence of events to Schwartz, Rosen, and Steinmetz [24], have reached occur: 1) direct inhibition of the CA reaction; H the same conclusion, namely, that acetazolamide production inside the tubular cells is therefore blocked; influences sodium reabsorption via two mechanisms. in addition, H ions would accumulate in the lumen They observed that acetazolamide diminished the H since the formation of , and subsequently secretion in the turtle urinary bladder. Carbon dioxide H20 and C02, would be depressed; 2) an indirect restored the secretory rate to normal, but only at an inhibition of H secretion would result because of the acetazolamide concentration of 5 x l0 M. At higher less favorable gradient; 3) diminution of the H-Na concentrations (5 x l0 M), this was not the case. exchange; 4) a decrease of active Na transport, and In the reaction mechanism of CA, two binding-sites 5) a decrease of passive NaC1 and water transport. seem to be involved [251: 1) a Zn atom serves as the The above sequence could explain the action of binding site for HC0, 0H, and H2O, and also for acetazolamide and furosemide on isotonic proximal competitive inhibitors such as the unsubstituted salt and water reabsorption when the system contains sulfonamide anions and thiocyanate; 2) an adjacent bicarbonate. But how can one explain the action of histidine molecule serves as a translocator in these agents when the system contains no added the hydration-dehydration reaction. On the other hand, bicarbonate? Could perhaps the CO2 created by the probability is very great that one or several histidine metabolism be involved? Unfortunately, data for C02- molecules may also be involved in the mechanism of production by the rat under our experimental condi- transmembrane proton translocation. If the histidine tions are not available. But using data from the dog molecule in CA and the histidine molecule in the [23], the molar ratio of total 02 consumed to net translocation mechanism are the same, or if the sodium reabsorbed (1:22) can be estimated. With the histidine molecule of the translocation mechanism is respiratory ratio of 0.7 which prevails in the kidney, very near the inhibitor-binding Zn molecule, then it the ratio of CO2 formed per net sodium reabsorbed is is conceivable that a sulfonamide molecule bound to approximately 1:30, and 1: 15 per active sodium CA-Zn serves to inhibit CA, and thus influences transport. With a Na-H exchange ratio of 3.0 as proton translocation as well. stated above, all of the CO2 that is produced and converted to H could only account for one-fifth of Acknowledgements the postulated Na-H exchange. But one may, Reprint requests to Prof. Dr. K. J. UI/rich, Max-Planck- however, infer that one CO2 molecule may recycle institut für Biophysik, Kennedyallee 70, 6 Frankfurt/M.-70, five times. This, as well as the assumption that all Germany. endogeneously produced CO2 would serve as the source References of H, is very unlikely. Contrarily, the probability is much higher that in a system with an organic buffer 1. Ullrich, K. J., Radtke, H. W., and Rumrich, G.: The such as glycodiazine, bicarbonate is not necessary at role of bicarbonate and other buffers on isotonic fluid absorption in the proximal convolution of the rat all to promote proximal reabsorption. These organic buffers share two things in common with bicarbonate: kidney. PflugersArch. ges. Physiol. 330: 149—161,1971. 2. Gertz, K. H.: Transtubuläre Natriumchloridflüsse und 1) their undissociated form is very lipid soluble and it Permeabilitat für Nichtelektrolyte im proximalen und can easily penetrate the cell membranes by nonionic distalen Konvolut der Rattenniere. Pflügers Arch. ges. diffusion, and 2) their anionic form, like HCO3, is Physiol. 276: 336—356, 1963. probably relatively impermeable. Considering the 3. Györy, A. Z.: Reexamination of the split oil droplet very rapid equilibration of the undissociated form, it method as applied to kidney tubules. Pflugers Arch. is not surprising that the level of CO2 or AH (and ges. Physiol. 324: 328—343, 1971. Dual action of acetazolamide and furosemide 105

4. Frömter, E., Muller, C. W., and Knauf, H.: Fixe nega- ken, H., Springer-Verlag, Berlin, Heidelberg, New tive Wandladungen im proximalen Konvolut der York, 1969, p. 386—405. Rattenniere und ihre Beeinflussung durch Ca-lonen in 16. Janata, V., Turek, J., and Radikovsky, P.: Metabolism VI. Symposium Gesellschaft für Nephrologie, edited in the rat kidney after , furosemide and by Watschinger, B., Wien, 1969, p. 61—64. ethacrynic acid. II. Changes in some enzyme activities 5. Uhlich, F., Baldamus, C. A., and Ullrich, K. J.: Ver- in the renal cortex and medulla mitochondria. Cesk. halten von C02-Druck und Bicarbonat im Gegenstrom- Farm. 19: 53—56, 1970. system des Nierenmarks. Pflugers Arch. ges. Physiol. 17. Ullrich, K. J., Baumann, K., Loeschke, K., Rumrich, 303: 31—48, 1968. G., and Stolte, H.: Micropuncture experiments with 6. Gyory, A. Z., and Kinne, R.: Energy source for trans- saluretic sulfonamides. Ann. N.Y. Aca. Sci. 139: epithelial sodium transport in rat renal proximal 416—423, 1966. tubules. Pflugers Arch. ges. Physiol. 327: 234—260, 18. Schmidt, U., and Dubach, U. C.: Quantitative Histo- 1971. chemie am Nephron in Progress in Histochemistry 7. Fiske, C. H., and Subbarow, Y.: The colorimetric deter- and Cytochemistry, Gustav Fischer-Verlag, Stutt- mination of phosphorus. J. Biol. Chem. 66: 375—400, gart-Portland, Vol. 2, 1971, p. 185—298. 1925. 19. Murphy, J. C., and Bader, H.: The effect of diuretics 8. Bartlett, G. R.: Phosphorus assay in column chromato- on membrane ATPase of rabbit kidney cortex. graphy. J. Biol. Chem. 234: 466—468, 1959. Pharmacol. 10: 162, 1968. 9. Lowry, 0. H., Rosebrough, H. J., Farr, A. L., and Ran- 20. Ebel, H., Ehrich, J., and De Santo, N. G.: Die Wir- dall, R. J.: Protein measurement with the folin phenol kung von Diuretica auf die ATPase-Aktivität einer reagent. J. Biol. Chem. 193: 265—275, 1951. plasmazelimembranreichen Fraktion (PMF) der Rat- 10. Berliner, R. W., and Orloff, J.: Carbonic anhydrase tenniere. Pflugers Arch. ges. Physiol. 307: R 77, 1969. inhibitors. Pharmacol. Rev. 8: 137—174, 1956. 21. Rector, F. C.,Jr., Carter, N. W., and Seldin, D. W.: 11. Maren, T. H., Mayer, F., and Wadsworth, B. C.: Car- The mechanism of bicarbonate reabsorption in the bonic anhydrase inhibition. I. The pharmacology of proximal and distal tubules of the kidney. J. Clin. Diamox (2- acetylamino -1,3,4 -thiadiazole-5- sulfon- Invest. 44: 278—290, 1965. amide). Bull. Johns Hopkins Hosp. 95: 199—243, 1954. 22. Frömter, E., Muller, C. W., and Wick, T.: Permeability 12. Baer, J. E., and Beyer, K. H.: Renal pharmacology. properties of the proximal tubular epithelium of the rat Ann. Rev. Pharmacol. 6: 261—292, 1966. kidney studied with electrophysiological methods in 13. Pitts, R. F., and Alexander, R. S.: The nature of the Electrophysiology of Epithelia, F. K. Schattauer-Ver- renal tubular mechanism for acidifying th eurine. Am. lag, Stuttgart, New York, 1971, p. 119—146. J. Physiol. 144: 239—254, 1945. 23. Deetjen, P., and Kramer, K.: Die Abhangigkeit des 14. Williamson, H. E.: Relationship between ATPase activ- 02-Verbrauchs der Niere von der Na-Ruckresorption. ity and sodium transport and action of diuretics. Pflugers Arch. ges. Physiol. 273: 636—650, 1961. Proc. 4th mt. Congr. Nephrol., Stockholm, edited by 24. Schwartz, J. H., Rosen, S., and Steinmetz, P. R.: Dual AIwall, N., Bergiund, F., and Josephson, B.; S. Kar- effect of acetazolamide on H +secretionby the turtle ger, Basel, Munchen, New York. Vol. 2, p. 144—152, bladder. Fed. Proc. 30: 421, 1971. 1970. 25. Wang, J. H.: Directional character of proton transfer 15. Peters, G., and Roch-Ramel, F.: Furosemide in Hand- in enzyme catalysis. Proc. Nat. Aca. Sci. 66: 874—881, book of Experimental Pharmacology, edited by Her- 1970.