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Kidney International, Vol. 4 (1973). p. 245—251

Effect of mersalyl on the thick ascending limb of Henle's loop

MAURICE BURG and NORDICA GREEN

Laboratory of Kidney and Electrolyte Metabolism, National Heart andLung Institute, National Institutes of Health, Bethesda, Maryland

Effect of mersalyl on the thick ascending limb of Henle's loop. de Henle isolée et perfusee peut être identifié a son effet diuretique The cortical portion of the thick ascending limb of Henle's loop in vivo. Le mersalyl dans Ic bain est inefficace, excepté a une was dissected from rabbit kidneys and perfused in vitro. There concentration plus élevée (10—i M)etalors I'effet n'est pas was active chloride transport resulting in net sodium chloride reversible. Nous concluons que l'effet diurétique des mercuriels reabsorption from the tubule lumen, with the electrical potential est lie, au moms en partie, a l'action inhibitrice de ces drogues difference (PD) oriented positive in the lumen. The addition of sur le transport actif de chlore dans Ia partie large de Ia branche mersalyl ( iO M)tothe lumen caused the PD and net Cl flux ascendante de l'anse de Henle. to decrease, indicating inhibition of the active Cl transport. The decrease in PD caused by the drug in the lumen was approxi- mately 50% at concentrations equal to or greater than iO M, Mercurial diuretics were extensively used and studied andthe decrease in CI transport (measured at 3 x l0 M)was until they were supplanted in the past few years by more similar in magnitude. Addition of p-chloromercuribenzoate to effective agents. There are many excellent reviews of the the tubule lumen immediately reversed the effect of mersalyl pharmacology of the mercurials [1—4]. Despite numerous administration. The diuresis caused by mercurials in vivo simi- investigations, however, it had never been possible to larly is reversed by p-chloromercuribenzoate administration. Therefore, the effect of mersalyl on the isolated perfused thick study directly the specific renal action of the drug that is ascending limbs of Henle's loop in vitro can be identified with responsible for the diuresis. Even the site of action within its diuretic effect in vivo. Mersalyl in the bath was inactive ex- the nephron had been uncertain, and most tubule segments cept at a higher concentration (10 M),andthe effect was not have been implicated at one time or another [3]. The most reversible. We conclude that the diuretic effect of mercurials is due, at least in part, to inhibition by these drugs in the tubule recent studies, accomplished by micropuncture in the dog, lumen of active Cl transport in the thick ascending limb of implicate the loop of Henle [5, 6] and it seems likely that, Henle's loop. despite the conflicting evidence, the thick ascending limb of Henle's loop is the major site of action of mercurials [2]. Effet du mersalyl sur Ia partie large de Ia branche ascendante This nephron segment is not accessible to micropuncture, de I'anse de Henle. La portion corticale de Ia partie large de Ia branche ascendante de l'anse de Henle a été disséquée dans des but has recently been studied directly by means of in vitro reins de lapin et perfusée in vitro. II existe un transport actif de perfusion of tubule fragments dissected from rabbit chlore ayant pour consequence une soustraction nette de NaCI kidneys [7, 8]. Its permeability to water is low, There is net a Ia lumière tubulaire et une difference de potentiel telle que Ia sodium chloride reabsorption which results in lowering of lumiCre est positive. Le mersalyl ( lO M)dansIa lumière induit the NaCI concentration in the lumen. The potential dif- une diminution de Ia difference de potentiel et du flux net de chlore, ce qui traduit l'inhibition du transport actif du chlore. ference (PD), which is positive in the lumen, is caused by La diminution de Ia difference de potentiel est approximative- the active transport of chloride out of the tubule lumen. ment de 50% a des concentrations égales ou supérieures a The reabsorption of Na is largely passive along the electrical iO MetIa diminution du transport de chiore, mesurée a gradient. Net NaCI transport and the electrical PD are 3 x iO M,est du mCme ordre de grandeur. L'addition dep-chloro- mercuribenzoate a Ia lumiCre tubulaire annule immédiatement both inhibited by the addition of ouabain to the bath [7] l'effet du mersalyl. La diurCse induite in vivo par les mercuriels and furosemide to the lumen [9]. est, de Ia mCme facon, annulée par Ic p-chloromercuribenzoate. In the present studies the direct effect of the mercurial Ainsi, l'effet du mersalyl sur la partie large ascendante de l'anse diuretic mersalyl on the isolated perfused cortical thick ascending limb of Henle's loop has been studied. Mersalyl Received for publication March 5, 1973; and in revised form June 26, 1973. was chosen since it causes a diuresis in rabbits [10] and © 1973, by the International Society of Nephrology. contains no theophylline, which might complicate the

245 246 Burg / Green

interpretation [11]. The major finding is that mersalyl in Table 1.Effectof mersalyl (3 x l0 M)inthe lumen on the thick the lumen inhibits the net Cl transport and the electrical ascending limb of Henle's loop PD, thus indicating that the drug inhibits active Cl transport Net chloride transport, lumen to bath in this segment. (Eq cm1 sec1 x Tubule Control Mersalyl After control Methods 1 6.2 3.7 4.7 The method employed in these studies has previously 2 12.9 7.3 12.1 been described in detail [7, 9, 12] and is summarized below 3 6.7 2.9 4.8 with additions and modifications. 4 6.8 3.7 5.0 Cortical segments from thick ascending limbs of Henle's 5 9.4 7.2 7.9 6 6.9 5.4 6.1 loop were dissected from rabbit kidney and were perfused 7 8.0 3.4 7.4 at 37° C using concentric glass pipets. The pipet system 8 8.2 3.5 7.1 used includes [13] 1) a means of changing perfusion fluid Mean 8.13 4.64 6.89 with the tubule in place, 2) a means of positioning liquid SEM 0.77 0.62 0.86 dielectric (Sylgard 184) at the outside of the tubule at both ends for insulation and 3) a volumetric constriction pipet aEach result is the mean of three or more collection periods for collection of tubule fluid. The bath solutions were over 15 to 30 mm. During the postcontrol periods, p-chloro- mercuribenzoate (PCMB) was either added to the mersalyl bubbled with 95% 02 + 5% CO2 gas mixture before and already present in the lumen (3 x l05M, tubules 2, 3, and 6) during the experiment. or else PCMB replaced the mersalyl (3 x iO MofPCMB, The tubules were dissected at room temperature in the tubules 1 and 4; l0— M,tubules5, 7 and 8). The drug(s) were saline buffer solution that was used in the bath during continuously present during the postcontrol periods except in perfusion. It contained NaCI, 115 mM; 5 mM; Na tubules 7 and 8 from which the PCMB was removed after being KCI, present for only 2 to 3 mm. The mean paired differences were as Na HCO3, 25 mM; acetate, 10mM; CaCl2, 1 mr.t; MgSO4, follows: control minus mersalyl, 3.51 0.5 5EM(P<0.01); post- 1.2 mM;NaH2PO4,1.2 mM;andglucose, 5 m plus 5% v/v control minus mersalyl, 2.25 0.58 (P< 0.01); and control rabbit serum. The perfusate contained NaCI, 150 mM; minus after control, 1.25 0.17 (P< 0.01). Mean Cl concentra- tions were as follows: 120 mEq/liter1 in the bath, 152 mEq/ K2HPO4,2.5 mM;CaC12,1.0 mM;andMgSO4, 1.2 mM, liters—1 in the perf used fluid, and 108 mEq/liters in the control- adjusted to pH 7.4 (unless otherwise noted) with HCI. collected tubule fluid. Mean tubule length was 1.26 mm; mean Perfusions were carried out by gravity. Perfusion rate was perfusion rate, 1.34 n1/min. Net chloride transport was cal- measured only in the experiments in which Cl transport culated, as previously [8], from the concentrations of Cl in the was determined (Table 1). In the other experiments (in perfused and collected fluid and the perfusion rate (assuming which only PD was measured), the perfusion pressure was no net fluid transport). initially adjusted upwards to achieve a flow rate greater than the slow rates at which the PD is flow-dependent [7]. The tralized with NaOH prior to addition. Cysteine (Calbio- Na and K concentrations were checked in all of the per- chem) solutions were bubbled with N2 gas prior to combina- fusion fluids and baths, using flame photometry (IL), and tion with HgCl2 (Merck, reagent grade). The pH of each osmolality, with the use of a freezing point apparatus solution was measured and was adjusted with HCI to the (Bowman-Aminco) [14]. Chloride concentration was control pH (or other, as noted). No rabbit serum was measured in bulk solutions using a chioridometer (Cotlove- added to any solutions containing the drugs in order to Aminco) [15] and in microsamples of perfused and collected avoid possible protein binding. fluid using a modification [12] of the Ramsay method (16]. Results are given as mean (numberof tubules The transepithelial electrical PD was measured between studied). Statistical significance was estimated using calomel cells connected to the fluids in the bath and perfu- Student's t test. sion pipet with bridges containing 0.16 MNaClagar. The PD was displayed on an oscilloscope (Tektronix 561A) or Results a recorder (Hewlett Packard 680 M) connected by an amplifier (Bak ELSA—3 or 4). The PD was not corrected The mean electrical PD across the epithelium of the for liquid junction potentials since they are small and cortical thick ascending limb of Henle's loop is 6.42.0SD cannot be readily calculated for the complex solutions used my (lumen positive) when the solutions in the lumen and [17] (the liquid junction potential with the solutions just bath contain equal concentrations of Na [7]. The PD listed is less than + 1.5 my, which would be subtracted results from the active transport of Cl across the epithelium from the recorded PD for correction). [7], and drives the Na transport, which is largely passive. Solutions of mersalyl acid (Sterling Winthrop Research Addition of mersalyl to the lumen caused the PD to Institute) lost effectiveness on standing and, therefore, decrease by approximately one-half (Fig. 1 through 4). were freshly prepared during each experiment. P-chloro- Net Cl reabsorption was similarly inhibited (Table 1). mercuribenzoic acid (Nutritional Biochemicals) was neu- Therefore, the addition of mersalyl to the lumen inhibits Mercurial diuretics 247

8

7 o Mersalyl10— 5M 8 •Control 6 7 5 • 6

3

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o Mersalyl3xl05M PCMB3x105 PCMB 105M • Control I I I I I — J —10 10 20 30 40 50 60 Minutes 0 10 20 30 40 50 60 Fig. 1. Addition of mersalyl to the lumen of the thick ascending limb of Henle's loop caused the transepithelial potential differ- Minutes ence (PD) to decrease reversibly. Fig. 3. Effect of mersalyl administration on the PD was reversed immediately when p-chloromercuribenzoate (PCMB) was sub- tuti stituted for the mersalyl. The reversal was more rapid with PCMB than spontaneously (Fig. 1). 90 -

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10- 1 I I Control Mersalyl PCMB a or 106 io— io— io— PCMB + mersalyl Mersalyl concentration, M Fig. 4. Inhibition of the PD across the thick ascending limb of Fig. 2. The maxima! inhibition of the PD by mersalyl in the Henle's loop by mersalyl (3 X 10 Minthe lumen) was reversed lumen of the thick ascending limb of Henle's loop was approxi- by the addition of p-chloromercuribenzoate (PCMB, 10—s M, mately 50% and was reached at a concentration of 10 Mmer- 3tubules; 3 x io— M,8tubules) whether the PCMB replaced salyl in the lumen. The PD indicated is the stable value reached mersalyl (A) or was added to it (s). The PD indicated with 5 to 10 mm after the drug was added. The pH of the perfusate mersalyl is the stable value reached 5 to 10 mm after addition containing mersalyl was 7.4 (.)or6.0 to 6.5 ()(resultsof of the drug. The PD with PCMB or PCMB+ mersalyl is maxi- 32 measurements in 27 tubules). mum value reached immediately after adding the PCMB. 248 Burg /Green active Cl transport across the cortical thick ascending limb of Henle's loop and thus decreases net NaC1 reabsorption. Mersalyl ( 10M)added to the lumen caused the PD to decrease to a mean value of 48 2% of the control 6 PCMB 3x 105M o HgCl2 3 x 106M (30 measurements in 27 tubules). The effect did not differ • HgCl2 106M appreciably over the concentration range from 10 to 5 •Control 3 X 10 M (Fig. 2). In contrast, furosemide or ethacrynic acid inhibit to a greater extent. Addition of 10 M of the latter drugs caused a greater decrease in the PD (and Cl transport) than did mersalyl [9, 181. This difference is consistent with the observation that furosemide and 3 ethacrynic acid produce a greater maximum diuresis in vivo than do the mercurial diuretics [2]. 2 Mercurials have a wide range of biological actions be- cause of the toxic heavy metal which they contain. Therefore, inhibition of a transport process in kidney tissue in vitro is not necessarily related to the diuresis observed in vivo. In order to investigate whether the action of mersalyl just described is responsible for diuresis, the additional effect —10 10 20 30 40 50 of the nondiuretic mercurial p-chloromercuribenzoate was Minutes tested. Both the mercurial diuretics and p-chloromercuri- Fig. 5. The additionofmercuric chloride to the lumencaused benzoate previously were found to inhibit electrolyte inhibition ofthe PD acrossthethick ascending limb of Henle's transport in kidney slices [19, 20] and tubule suspensions loop at a concentration of3x 1O M.Theinhibition, however, did not reverse when p-chloromercuribenzoate (PCMB) was (Burg M. B., unpublished observations) and to inhibit the substituted for the HgCl2. renal Na and K-activated ATPase [21]. In contrast, the diuretic action of mercurials in vivo is reversed by addition of p-chloromercuribenzoate [22,23]. In the present studies, The mean PD 18 to 30 minutes after HgCI2 was placed in when p-chloromercuribenzoate (1 to 3 x l0 M) replaced the lumen was 2.3 my (four tubules studied) mersalyl in the lumen, the PD returned immediately to the compared to 7.31.7 my, control PD in the same tubules. control value (Fig. 3). The reversal was much more rapid In contrast to its reversal of the mersalyl effect, addition of than when mersalyl was simply removed (Fig. 1). Addition p-chloromercuribenzoate to the lumen (l0 to 3 x 10 M) of p-chloromercuribenzoate also reversed the mersalyl did not reverse the effect of HgCl2 in the lumen. The effect despite the continued presence of mersalyl in the maximum PD during 2 to 5 minutes following substitution lumen (Fig. 4). Therefore, p-chloromercuribenzoate in the of p-chloromercuribenzoate was only 0.3 0.3 my [3]. lumen of the cortical thick ascending limb of Henle's loop Therefore, release of mercuric ion into the tubule fluid in vitro reverses the inhibitory effect of mersalyl, exactly cannot account for the action of the mercurial diuretics. analogous to the result in vivo, providing evidence that the Either mercuric ion is released within the tissue or, more in vitro effect is the cause of at least part of the diuresis likely, the intact molecule of the organic mercurial itself is observed in vivo. the effective form of the drug, rather than the mercuric ion. The action of mercurial diuretics is enhanced by prior Mercuric cysteine is a potent diuretic [26, 27]. In the administration of acidifying salts such as NH4CI [24]. present experiments, however, even a very high concentra- Consequently, it was postulated that the diuretic action tion of mercuric cysteine (l0 M of Hg mixed with of mercurials requires an acid pH in the tubule lumen [1]. 2.2 x 10 M cysteine, pH 6.5 to 7.4) in the tubule lumen had In the present experiments, mersalyl was as effective at little effect on the PD. In 4 experiments the mean PD was pH 7.4 in the perfusate as at pH of 6.0 to 6.5 (Fig. 2), which 6.8 my 1.25 SEM in the control periods, 6.4 0.9 my is contrary to this theory (in the absence of the drug there during 10 to 15 minutes of perfusion with the mercuric was no difference in the steady state PD with variation of cysteine, and 6.9 0.9 my in the postcontrol periods. The lumen pH between 6.0 and 8.0). discrepancy between the in vivo and in vitro results could be It has also been proposed [1, 25] that mercuric ions are explained if mercuric cysteine acted at a site other than the released from the organic mercurial diuretics and that the lumen of the thick ascending limb, or was altered in the mercuric ion is the actual effective form of the drug. In the body to a different chemical form. present experiments, addition of mercuric chloride to the Since the principal excretory product of mersalyl in the lumen inhibited the PD across the cortical thick ascending urine is the cysteine complex of mersalyl [25], it is possible limb of Henle's loop. The concentration of HgCI2 that this complex, rather than mersalyl itself, is present in (3 x 10—6 M) was lower than that of mersalyl, but the the lumen of the thick ascending limb of Henle's loop HgCl2 took longer to act (compare Fig. 5 to Figs. I and 3). in vivo. When the mersalyl-cysteine complex was perfused Mercurial diuretics 249 into the tubule lumen in vitro (mersalyl 10' to i0 Mplus Table 2.Effect of mersalyl and NaCI concentration in the lumen 10% molar excess of cysteine), it caused the PD to decrease on the thick ascending limb of Henle's loopa to a mean value of 66 6% of the control value (E/C, 150 50 150 50 13 measurements in 9 tubules). Therefore, both mersalyl Perfusate [NaCI], mEq/liter' Perfusate [Mersalyl], M 0 0 3 X iO3 X and its cysteine complex can inhibit the PD in the cortical l0 thick ascending limb of Henle's loop in vitro, and either PD,mv might be the effective form in vivo. Mersalyl itself, however, Tubule 1 7.5 22.3 3.8 22.8 is somewhat more potent (as just shown). There was no Tubule 2 8.3 26.5 4.5 23.4 effect of varying pH in the lumen on the action of the Tubule 3 8.0 30.0 4.3 22.8 23.0 2.4 15.0 mersalyl cysteine complex (E/C =0.660.11 [5] at pH 6.5; Tubule 4 6.9 0.65 [8] at pH 7.4 to 7.6). Mean 7.7 25.5 3.8 21.0 Mersalyl in the bath also inhibited the PD, but the effect Difference 17.8 17.3 was different from that in the lumen. Even with no protein a Eachvalue of the PD is the mean of two readings taken one present in the bath, the minimum effective concentration of minute apart. The perfusate with 50 mEq/liter of NaCl was mersalyl was higher (10 M,Fig.6) than in the lumen otherwise identical to that containing 150 mEq/liter'. (3 x 10—6 to 10', Fig. 2) and the rate of decrease of the PD was slower (compare Fig. 6 to Figs. 1 and 3). Further, the effect of mersalyl in the bath did not reverse spontaneously resistance were measured. When the thick ascending limb nor with the addition of p-chloromercuribenzoate to the is perfused with a solution which contains a lower NaCI lumen or bath (Fig. 6). concentration than in the bath, the electrical PD increases. In order to test whether the effect of the addition of This is a consequence of a diffusion potential across the mersalyl to the lumen of the cortical thick ascending limb epithelium, resulting from the imposed chemical concentra- of Henle's loop is accompanied by a major change in Na or tion gradient (Na conductance exceeds Cl conductance) CI permeability, the NaCI diffusion potential and electrical [7]. All other things being equal, the magnitude of the diffusion potential depends on the ratio of Na to Cl conductance. In the experiments in Table 2, tubules were perfused with solutions containing both a normal (150 mM) anda low (50 mM) NaCI concentration. The mean difference in PD with the two solutions was approximately the same whether mersalyl was present (17.3 my difference) or absent 8 (17.8 my difference) indicating little change in the ratio of Na to Cl permeabilities with the drug. The addition of 7 mersalyl (3 x 10 M in the lumen) also caused no definite change in the transepithelial electrical resistance. In 6 5 experiments the mean resistance was 3620 ohm cm in control periods (C), 3860 with mersalyl in the lumen (E),

5 and 3570 afterp-chloromercuribenzoate (10 M) was added and the PD had returned to the control value (C'). The C± C difference E— was + 267±106, p >0.05. Thus, it is unlikely that the drug causes any major change in Na or 3 Cl permeability. The addition of furosemide to the lumen was previously noted to cause a small decrease in Na and 2 Cl permeability [9]. A similar, but lesser, decrease in per- meability from mersalyl is not excluded by the present results.

Discussion 30 Minutes On the basis of the present results, mersalyl inhibits active Cl transport in the cortical thick ascending limb of Fig. 6.Effectof mersalyl in the bath on the thick ascending limb of Henle's loop. P-chloromercunbenzoate (PCMB) added to the Henle's loop. Schwartz [28] and Axelrod and Pitts [29] lumen or bath did not reverse the effect of mersalyl in the bath, previously called attention to the disproportionately large contrary to the result when mersalyl was perfused (Fig. 3). A amount of Cl which appears in the urine following ad- third experiment in which the results were otherwise identical ministration of mercurial diuretics. They proposed that the is not shown since the timing was different. The bath and perfusate were artificial saline buffers free of added protein mercurials primarily inhibited Cl transport. At that time, (see Methods). however, the importance of active Cl transport in the 250 Burg! Green nephron was not recognized, and, since the result could tubule cells is a more likely pathway, but there are impor- be rationalized in another manner [4], their idea was not tant differences between species and between the tubular generally accepted. The inhibition of Cl transport, which handling of the various mercurial diuretics, and the exact they proposed and which has been directly demonstrated mechanism remains uncertain [1, 3]. in the present studies to occur in the cortical thick ascending limb of Henle's loop, is a renal action of mercurials which accounts, at least in part, for their diuretic effect. Hopefully, Acknowledgment future direct examination of the effect of mercurials on the Dr. Jack Orloff provided many helpful suggestions during other parts of the kidney tubule will determine whether the course of this study. Mersalyl acid was provided by inhibition of the other segments is also important in the Sterling Winthrop Research Institute. diuresis. P-chloromercuribenzoate antagonizes the diuretic action of the mercurials both in vivo [22, 23] and in vitro (present Reprint requests to Dr. Maurice Burg, National Heart and experiments). Miller and Farah [22, 231 accepted, following Lung Institute, National Institutes of Health, Building 10, Weiner et al. [1, 25], that mercuric ions released from Room 6N307, Bethesda, Maryland 20014, U.S.A. organic mercurials are the effective diuretic form, and they proposed that p-chloromercuribenzoate displaces the References mercuric ions from a renal receptor, but is itself non- diuretic, owing to a different form of binding to the 1. WEINER I, FARAH A: Pharmacology of mercurial diuretics, receptor. In the present studies, the effects of mercuric in Proceedings of the Third International Pharmacology chloride in the lumen differed significantly from those with Meeting. New York, Pergamon Press, 1968, vol. 8 (Salt and Water Balance), pp 15—36 mersalyl. One possible explanation for this difference is 2. GOLDBERG M: The renal physiology of diuretics in Hand- that the organic mercurial molecule must be intact to exert book of Physiology, in press its characteristic effect [30]. In that case, it is necessary to 3. CALFRUNY EJ: The site and mechanism of action of mer- assume that p-chloromercuribenzoate displaces the organic cunal diuretics. Pharm Rev 20:89—116, 1968 mercurial molecule itself from the receptor site, rather than 4. ORLOFF J, BERLINER RW: Renal pharmacology. Ann Rev the mercuric ion. PharmacolI :287—314, 1961 These are a number of possible explanations as to why 5.EVANSON RL, LOCKHART EA, DIRKS JH: Effect of mer- p-chloromercuribenzoate reversed the effect of mersalyl in curial diuretics on the tubular sodium and potassium trans- the lumen, but not of HgCl2 in the lumen nor of mersalyl port in dogs. Am J Physiol 222:282—289, 1972 6. CLAPP JR, ROBINSON RR: Distal sites of action of diuretic in the bath. The one which we favor is that the mercurials drugs in the dog nephron. Am J Physiol 215:228—235, 1968 might have more than one effect on the thick ascending limb 7. BURG M, GREEN N: Function of the thick ascending limb of Henle's loop. The first effect is that associated with the of Henle's loop. Am J Physiol 224:659—668, 1973 in vivo diuresis. It is caused by diuretic mercurials in the 8. ROCHA AS, KOKKO JP: Sodium and water transport in the tubule lumen and reversed byp-chloromercuribenzoate. The medullary thick ascending limb of Henle: Evidence for second is a nonspecific effect caused by HgC12 and by high active chloride transport. J C/in Invest 52:612—623, 1973 concentrations of mersalyl in the bath. The two effects might 9. BURG M, STONER L, CARDINAL J, GREEN N: Furosemide correspond to two different sites of action. The first site effect on isolated perfused tubules. AmfPhysio/ 225:119—124, could be on the lumen surface and could explain why the 1973 diuretic effect is so rapidly reversed by p-chloromercuri- 10. FARAH A, MARISH G: The influence of sulfhydryl compounds on diuresis and renal and cardiac circulatory changes benzoate. The second site could be within the tubule cells. caused by mersalyl. J Pharm Exp Therap 92:73—82, 1948 The relatively slow but irreversible inhibitory action of the 11. PORUSH JG, GOLDSTEIN MH, EISNER GM, LEvIrr MF: Ef- mercurials could result from gradual entry of these com- fect of organomercurials on the renal concentrating opera- pounds into the cells and subsequent toxic inhibition of the tion in hydropenic man: Comments on site action. J C/in cellular metabolic processes. It is possible that in vivo the Invest40:1475—1484,1961 cells could be protected from this toxic effect by binding of 12. Buxo M, GRANTHAM J, ABRAMOW M, Oiu.orr J: Prepara- mercurials by proteins in the plasma or complexing of tion and study of fragments of single rabbit nephrons. Am mercuric ions with amino acids such as cysteine in the J Physiol 210:1293—1298,1966 tubule fluid. 13.BURG MB: Perfusion of isolated renal tubules. Yale J Biol The probability that the mercurial diuretics act from Med 45:321—326, 1972 14.BOWMAN RL, TRANTHAM HY, CAULFIELD PA: An instru- inside the lumen has been recognized for some time [1, 3]. ment and method for rapid, dependable determination of The question of the pathway by which they reach the tubule freezing point depression. fLab Clin Med 43:310—315, 1954 fluid, however, is unresolved. Binding of the mercurials to 15. 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16. RAMSAYJA,BROWNHJ,CROGHAM PC: Electrometric titra- 24. KIETH NM,WHELANM: A study of the action of ammonium tion of chloride in small volumes. J Exptl Biol 32:822—829, chloride and organic mercurial compounds. J C/in Invest 1955 3:149—202, 1926 17.GRANTHAM BURG JJ, MB, ORLOFFJ: The nature of trans- 25. WEINER IM, LEVY RI,MUDGEOH: Studies on mercurial tubular Na and K transport in isolated rabbit renal collect- diuresis: Renal excretion, acid stability, and structure ing tubules. J C/in Invest49:1815—1826,1970 activity relationships of organic mercurials. J Pharm Exp 18. BURG MB, GREEN N: Effect of ethacrynic acid and other Therap138:96—112,1962 diuretics on the thick ascending limb. Kidneymt.in press 19. MUDGE GH: Electrolyte and water metabolism of rabbit 26. NIGI0VIC V, CH0 KC, CAFRUNY EJ: Diuretic response to kidney slices. Am J Physiol 167: 206—223, 1951 mercuric cysteine: Dependency on urinary pH. J Pharm 20. MACKNIGHT ADC: Water and electrolyte contents of rat Exp Therap 175:741—748, 1970 renal cortical slices incubated in medium containing 27. LEVY RI,WEINERIM,MUDGEOH:The effects of acid-base p-chloromercuribenzoic acid or p-chloromercuribenzoic balance on the diuresis producedby organic and inorganic acid and ouabain. BBA 163: 500—505, 1968 mercurials. J C/in Invest37:1016-1023,1958 21. NECHAY BR,PALMERRF, CHINORYDA,POSEY VA: The 28. SCHWARTZ WB,WALLACEWM:Electrolyte equilibrium problem of Na+ + K+ adenosine triphosphatase as the mercurial diuresis. J C/in Invest 1951 receptor for diuretic action of mercurials and ethacrynic during 30:1089—1104, acid. J Pharm Exp Therap 157:599—617, 1967 29. AXELROD DR. Plrrs RF: The relationship of plasma pH 22. MILLER TB, FARAH AE: Inhibition of mercurial diuresis by and anion pattern to mercurial diuresis. J Clin Invest 31: nondiuretic mercurials. J Pharm Exp Therap 135:102—111, 171—178, 1952 1962 30.KESSLERRH,LOZANOR, Plrrs RF:A comparisonofthe 23. MILLER TB, FARAH AE: On the mechanism of the inhibition pharmacologicalbehavior of chlormerodrin, meralluride, of mercurial diuresis by p-chloromercuribenzoate. J Pharm mersalyl, and mercuric chloride in the dog. J Pharm Exp ExpTherap 136:10—19, 1962 Therap121:432—442,1957