Biochemical and functional characterization of H+-K+-ATPase in distal amphibian nephron G. PLANELLES,
T. ANAGNOSTOPOULOS,
L. CHEVAL,
AND
A. DOUCET
Laboratoire de Physiologie Renale, Institut National de la Sante et de la Recherche Medicale Unite 323, Centre Hospitalier Universitaire Necker, 75730 Paris Cedex 15; and Laboratoire de Physiologie Cellulaire, Unite de Recherche Associee 219, Centre National de la Recherche Scientifique, College de France, 75231 Paris Cedex 05, France
unlikely (because of unfavorable chemical gradients) and proposed the implication of a primary active transport mediated by electroneutral H+-K+ pump (2). A H+-K+ATPase was primarily described in the gastric mucosa causeproton secretion and K’ reabsorption in the late distal and intestine (9, 11-13). More recently, it was found in tubule of amphibians are active, we evaluated whether these processescould be mediated by an H+-K+-ATPase similar to the distal segments of the rabbit and rat nephron (6,10).
PLANELLES, G., T. ANAGNOSTOPOULOS, L. CHEVAL, AND A. DOUCET. Biochemicaland functional characterization of H+-K+ATPase in distal amphibian nephron. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F806-F812, 1991.-Be-
the gastric H’-K’ pump and to the K+-ATPase previously describedin the terminal segmentsof the mammaliannephron. K+-stimulated ATPase activity was detected in microdissected segmentsof frog and Necturus nephron: its activity was high in the late distal and collecting tubules, whereas it was undetectable in the proximal convoluted tubule and early distal tubule. In frog collecting tubule, K’-ATPase had a high affinity for K’ (K, = 0.30mM), wasinhibited by vanadate, omeprazole, and the imidazopyridine Sch 28080, and was insensitive to ouabain. Furthermore, in vivo administration of Sch 28080 to anesthetizedNecturus induced a significant rise of the steadystate intratubular pH in the late distal tubule, demonstrating that this drug inhibited tubular fluid acidification. It is suggestedthat K+-ATPase present in the terminal segmentsof amphibian nephron is similar to the gastric’ H’-K’ pump and is involved in urinary acidification. frog; Necturus; single-‘nephron segments;Sch 28080; pH-sensitive microelectrodes;proton-potassium pump
This K+-activated
pump, which couples the hydrolysis
of ATP to the transmembrane exchange of K+ and protons against their electrochemical gradients, belongs to the El-E1 class of ATPases, as it displays a phosphorylated intermediate and is inhibited by vanadate (reviewed in Ref. 14). This enzyme was also found to be specifically inhibited by omeprazole (26) and, more recently, by the imidazopyridine Sch 28080 (3,25). The present study was therefore designed to I) determine whether H+-K+-ATPase activities were present in the distal nephron of Necturus, 2) localize this ATPase activity along the Necturus renal tubule, and 3) evaluate whether in vivo inhibition of this pump by Sch 28080 alters distal tubular fluid acidification. To answer the first two questions, we searched for a K+-stimulated, Sch 280804nhibited ATPase activity in single segments of Necturus nephron, obtained by microdissection of collagenase-treated kidneys using the mi-
croassay previously developed for the mammalian nephron (6). However, because microdissection of Necturus THE AMPHIBIAN
DISTAL
TUBULE
is heterogeneous (23),
as it consists of an early distal tubule (EDT) that is functionally similar to the mammalian diluting segment, as well as a late distal tubule (LDT). In Amphiuma, luminal pH (17) and tubule-to-plasma K+ concentration ratio decrease along the LDT (27), indicating that proton secretion and K+ reabsorption occur in that segment. In the LDT of Necturus, measurement of steady-state activities for protons and K+ and potential differences in peritubular blood capillaries, cells, and tubular fluid (2) indicated that K+ must be actively reabsorbed and protons must be actively secreted across the apical membrane (against electrochemical gradients of -20 and 90 mV, respectively). Although the molecular mechanism(s) underlying these active transports across the luminal membrane of Necturus LDT was not formally characterized in that study, its authors concluded that the involvement of Na+K+-2Cl- or K+-Cl- symport and Na+-H+ antiport was F806
0363-6127/91
$1.50
kidney is difficult, preliminary experiments aimed at determining the kinetic and pharmacological properties
of K+-ATPase
were performed
in the kidney of the frog, of samples necessary for such studies can be isolated more easily. By use of the optimal assay conditions determined thereby in the frog nephron, the localization of K+ATPase along the Necturus nephron was analyzed next. To evaluate the involvement of H+-K+-ATPase in distal tubular acidification, the pH of tubular fluid in the distal tubule was determined by pH-sensitive microelectrodes before and after administration of Sch 28080 to anesthetized Necturus. Rana ridibunda, a species in which the large number
METH0DS Animals. Biochemical studies were carried out on two species, R. ridibunda (Couetard, France) and N. maculosus (Nasco). The frogs were anesthetized by
amphibian
Copyright 0 1991 the American Physiological Society
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H’-K’-ATPASE
ALONG
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intraperitoneal injection of 50 mg Inactin (Byk); necturi were anesthetized by immersion into a 0.1% (wt/vol) solution of tricaine methanesulfonate (Sigma) in tap water. Electrophysiological determinations in vivo were performed only in necturi; once the animals were asleep, the head and the branchiae were continuously immersed in the above-mentioned tricaine solution, which was diluted fivefold. Tubule microdissection. A physiological Ringer solution for amphibia was prepared, which contained (in mM) 82 NaCl, 3 KCl, 1.8 CaCIZ, 1.0 MgC&, and 5 tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffered at pH = 7.5 with NaOH. In frogs, the aorta was cannulated from its terminal end, and then it was ligated above the first (upper) renal arteries (16). The effluent was allowed t.o flow into the peritoneal cavity through a large incision of the vena cava. Twenty milliliters of the Ringer solution were perfused to completely remove blood. After the kidneys were rinsed, 10 ml of the same solution supplemented with collagenase (400 U/ml collagenase IA, Sigma) were delivered via the efferent renal vein at a rate of 1 ml/min. The kidneys were excised, cut into thin slices, incubated during 1 h at 28°C into a physiological Ringer solution containing collagenase (4,000 U/ml), and then rinsed with a K’-free Ringer solution. Microdissection was carried out at 4°C in a similar solution. Tubule segments were isolated, photographed at constant magnification, and kept at 4°C until enzymatic determinations. The following four nephron segments were studied: 1) the initial portion of the proximal convoluted tubule (PCT); 2) the EDT or diluting segment, easily recognized by its thin diameter [the thinnest among the major segments of the nephron, the EDT begins after a short transition segment endowed with intraluminal cilia and ends at the juxtaglomerular apparatus (JGA)]; 3) the LDT, which stretches between the JGA and a constriction at the transition with the collecting duct; and 4) collecting tubules, which are recognizable by heterogeneous appearance and branched configuration. The microscopic aspect and intrarenal localization of these nephron segments are illustrated in Fig. 1, which displays a complete frog nephron dissected from acid-treated kidney (see legend to Fig. 1). A few modifications were necessary for dissection of tubules from Necturus because of the presence of thick connective tissue. The aortic cannula was placed via the thoracic aorta, at the upper border of the kidney, and 20 ml of Ringer solution were delivered. Then a rinsing solution (15 ml) supplemented with collagenase (600 U/ ml) was delivered through the caudal vein, after clamping iliac vessels. Kidney fragments were incubated during 60 min in a Ringer solution supplemented with collagenase (8,000 U/ml) at 30°C. Dissection of the segments downstream to the JGA (18) was uncertain because the glomerulus was often lost. Thus the LDT was microdissected upstream, starting from an initial collecting tubule; the boundary between these two segments was assessed from a discernible change in morphology; segments lacking clear-cut boundaries were discarded. Proximal, diluting, and collecting tubules were selected along the criteria defined above, with regard to frog kidney. Determination of K’-ATPose activity. K’-ATPase ac-
AND
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NEPHRON
F807
FIG. 1. A: microscopic appearance of a complete frog nephron dissected from acid-treated kidney. After perfusion with Ringer solution, kidney was removed and incubated for 3 h at 26°C in HCI 18% (vol/ vol). After rinsing in Rqer, it was further incubated for 10 days at 4’C in a 1 CL>(vol/vol) solution of acetic acid. This preparation allowed dissection of entire nephrons. H: schematic localization of glomerulus (G) and segments of nephron used in this study. PCT, proximal convoluted tubule; EDT, early distal tubule; LDT, late distal tubule; CT, collecting Lubule.
tivity was measured using the assay previously developed (6), with slight modifications. Pieces of nephron were individually transferred to the concavity of a sunken bacteriological slide and then photographed to determine their length, which served as reference for enzymatic activities. To increase cell permeability to reactants during the assay, tubule segments were successively submitted to a hypotonic medium and frozen as follows. The medium surrounding each tubule was aspirated and replaced by an equal volume of cold distilled water, and after 5-10 min at 0-4°C this procedure was repeated. Then the droplet surrounding the tubule was again aspirated and replaced by 0.2 ~1 distilled water. When necessary, drugs were added in the 0.2 ~1 distilled water at six times the final concentration required. Samples were then rapidly frozen on dry ice. After thawing, 1 ~1 of incubation medium (see composition below) was added to each sample, which was then incubated for 15 min at 37°C. Incubation was
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H+-K+-ATPASE
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stopped by cooling samples on crushed ice and by adding 5 ~1 of trichloroacetic acid 5% (wt/vol). Samples were then transferred into 2 ml of a suspension of 10% (wt/ vol) activated charcoal. After mixing and centrifugation, the radioactivity of 500 ~1 of supernatant was determined by scintillation counting. Unless indicated otherwise, the final composition of the incubation medium was (in mM) 25 tris(hydroxymethyl)aminomethane hydrochloride (Tris . HCl), 10 MgCl,, 1 ethylene glycol-bis(P-aminoethyl ether)N,N,N’,N’-tetraacetic acid, 1 ouabain, 2.5 sodium azide, and 5 Tris-ATP, as well as tracer amounts (~5 nCi/pl) of [T-~~P]ATP (2-10 Ci/mmol) in the presence of 2.5 mM KC1 (total ATPase) or in its absence (basal ATPage), pH 7.4. Eight to 14 samples of each segment of nephron were distributed randomly into two groups, one each for measuring total and basal ATPase. K+-ATPase was taken as the difference between the means of each group. ATPase activity was expressed as picomoles of inorganic phosphate released per millimeter of tubule length per hour. Steady-state determination of luminal pH in the distal tubule of Necturus in uiuo. Double-barreled (selective vs. conventional) pH microelectrodes have been constructed as in previous studies (19). Briefly, two capillary tubings (1.2 mm OD, 0.7 mm ID) were firmly attached, twisted manually to 360” on a flame, then pulled on a PE-2 Narishige vertical puller. One barrel was exposed to dimethyl trimethylsilylamine (Fluka, 41716) during 7 min, and then baked at 120°C for 2 h. The tip of the silanized barrel was filled with a COz-equilibrated proton cocktail (Fluka). The next day each barrel was backfilled with appropriate electrolyte solutions (2); then the microelectrodes were calibrated in vitro in the pH range 6.0-7.5. Their slope ranged from 50 to 58 mV/pH unit. The slope was checked again before and after each luminal pH determination in vivo by alternately delivering onto the surface of the kidney two artificial solutions buffered at pH 7.5 and 6.5. The animals were divided into two groups, control and experimental. The experimental group received an intravenous bolus of 125 mg/kg Sch 28080, i.e., 0.45 mmol/kg (dissolved in ethanol 2.5 ml). The control group was injected with the vehicle alone. The luminal pH of studied tubules was measured twice in single subcapsular LDT or EDT loops, before and after intravenous injection of vehicle or Sch 28080; each tubule served as its own control. The site of the first impalement was carefully drawn to recover the appropriate loop after 20-40 min of exposure to the inhibitor or to the vehicle. Necturus has a relatively low glomerular filtration rate (0.2 ml* min-’ . kg-‘; see Ref. 20), and this lag period appeared convenient for allowing the drug to reach the apical cell membrane. Statistics. Statistical significance was assessed by Student’s t test or, when necessary, by variance analysis according to the test of Dunnett (7), with P < 0.05 being con sidered significant. RESULTS
Biochemical properties of frog tubular K+-ATPase tiuity. The effect of increasing K+ concentration
ac(by
NEPHRON
addition of KCl) on ATPase activity of frog collecting tubule is shown in Fig. 2. ATPase activity was stimulated in a dose-dependent fashion as K+ concentration was increased between 0 and 2.5 mM. Maximal activity was reached with 0.75 mM K+, and half-maximal stimulation of the K+-dependent moiety of the activity was observed with 0.32 mM K+. Data in Fig. 3 indicate that the ATPase activities determined in the absence or presence of K+ were linearly related to the length or presence of the
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FIG. 2. Stimulation by K+ of ATPase activity in frog collecting tubule. Each point is mean t SE of 5-6 replicate tubules. Maximal stimulation of K’-ATPase activity was reached at K+ concentration of 0.75 mM. Values statistically different from control (K+ = 0) were assessed by variance analysis *P c 0.001. Inset: Lineweaver-Burke plot of K+-dependent component of ATPase activity indicates apparent K, of 0.32 mM and V,,,,, of -400 pmol . mm-‘. h-l.
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3. Relationship between ATPase activity in frog collecting tubule and tubular length. ATPase activity was measured either in presence of 2.5 mM K+ (solid symbols) or in its absence (open symbols). Different symbol shapes correspond to five different frogs. Both total and basal ATPase activities relate linearly to tubular length (r = 0.913 and 0.939, n = 22) in the 0.6-2.0 mm range. FIG.
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H+-K+-ATPASE
ALONG
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sample, which illustrates the sensitivity and the specificity of the assay. Next, we studied some pharmacological properties of the tubular K’-ATPase activity. K+-ATPase was not sensitive to the Na+-K+-ATPase inhibitor ouabain (~1 mM) and to the mitochondrial Fo-F1 ATPase inhibitor azide (up to 2.5 mM), and these two compounds were routinely added to the incubation medium to lower the basal ATPase activity. Conversely, K+-ATPase activity in the frog collecting tubule was completely inhibited in the presence of 10 PM of the E1-EP ATPase inhibitor vanadate [control, 229 t 33 (SD) pmol . mm-l. h-l, n = 5; vanadate, 13 t 25 pmol mm-l h-‘, n = 5; P < 0.001, variance analysis]. K+-ATPase activity was also completely inhibited by 100 PM of the inhibitor of gastric H+-K+-ATPase omeprazole, after its activation at pH = 2.00 for 30 min at 0°C [control, 229 t 33 (SD) pmol mm-l h-l, n = 5; omeprazole, 0 t 20 pmol. mm-l. h-l, n = 5; P < 0.001, variance analysis]. However, when preactivated in vitro at acid pH, omeprazole is no longer specific for H+-K+-ATPase, as it also inhibits the electrogenic, N-ethylmaleimide (NEM)-sensitive H+-ATPase (unpublished observation). Thus we looked for the action of the specific inhibitor of gastric H+-K+-ATPase, Sch 28080. At a concentration of 100 PM, Sch 28080 completely inhibited K+-ATPase activity [control, 324 t 39 (SD) pmol mm-’ . h-l, n = 4; Sch 28080,O t 37 pmol mm-’ h-‘, n = 5; P < 0.001, unpaired t test]. The effect of increasing concentrations of Sch 28080 on ATPase activity of the frog collecting tubule is shown on Fig. 4. The thresholds of inhibition and half-maximal inhibition of total ATPase activity were observed with 10m7and 10m6M Sch 28080, respectively. This inhibitory action of Sch 28080 was restricted to the K+-stimulated moiety of the ATPase activity, as basal ATPase activity was not modified by this drug up to a concentration of 10m4M (Fig. 4). Finally, since amphibia are poikilotherms, we looked for the temperature dependence of K+-ATPase activity. In frog collecting tubule, K’-ATPase activities were similar when measured either at 25°C or at 37°C [ZSOC, 241 -t 34 (SD) pmol mm-’ *h-l, n = 5; 37”C, 219 t 44 pmol l
l
l
mm-‘. h-l, n = 5; not significant (NS)]. This unexpected finding likely results from opposite actions of thermic activation and denaturation of the enzyme. Localization of K+-ATPase along the frog and Necturus nephron. Figure 5 displays mean K+-ATPase activities
measured in the successive nephron segments of frog (Fig. 5, top) and Necturus (Fig. 5, bottom). In these two species, K+-ATPase activity was exclusively measurable in the LDT and the collecting tubule, whereas K+-ATPase activity was almost undetectable and not statistically different from zero in the PCT and the EDT (P > 0.5). In the terminal segments of nephron, K+-ATPase activity was higher in the LDT than in the collecting tubules of the frog, whereas it was similar in these two nephron segments of the Necturus. Basal ATPase activities measured in the different segments of frog and Necturus nephrons are given in Table 1. Effect of Sch 28080 on tubular fluid acidification. Intratubular pH was measured in LDT under free-flow conditions before and after intravenous administration of either Sch 28080 or vehicle to anesthetized necturi. Results in Fig. 6 clearly indicate that intratubular pH values in LDT were similar before and after administration of vehicle (control period, 6.57 $- 0.06; experimental period, 6.56 t 0.07; n = 15, NS by paired t test). By contrast, 500 -
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FIG. 4. Effect of Sch 28080 concentration on ATPase activity measured in absence (0) or presence of K+ (0) in frog collecting tubule. Each point is mean k SE of 4-5 replicate tubules. Sch 28080 inhibited total ATPase activity (solid line) in a dose-dependent fashion, whereas it did not alter basal ATPase activity (dashed line). Values statistically different from control (no Sch 28080) were determined by variance analvsis. *P < 0.005. **P < 0.001.
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5. Distribution of K’-ATPase activity along frog (top) and nephron (bottom): n. number of animals.
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H+-K+-ATPASE
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1. Basal ATPase activity along the frog and Necturus nephron
TABLE
ATPase Activity, pmol mm-’ l
Frog
l
h-’ Necturus
3,084+241 (5) PCT 916*98 (5) EDT 6791t47 (6) 918,+150(5) 1,473&86(5) LDT 2,044&196 (5) CT 434t,65 (7) 2,125+_189 (5) Values are means k SE; number of animals is in parentheses. Basal ATPase activity was determined in absence of K+ in successive segments of nephron dissected from frog and Necturus kidneys. PCT, proximal convoluted tubule; EDT, early distal tubule; LDT, late distal tubule; CT, collecting tubule. Vehicle
SCH 28080
E CI FIG. 6. Intraluminal pH in the LDT before (C) and after (E) administration of either vehicle (left) or Sch 28080 (right) to anesthetized Necturus. Lines join pH values determined in same nephron; different length of dashes indicate lines corresponding to distinct animals. Points are means Al=SE from individual values. Mean ApH values induced by vehicle and Sch 28080 were -0.01 ,t 0.07 (n = 15) and 0.32 & 0.08 (n = l7), respectively. Statistical analysis was performed by Student’s t test for paired data. *P < 0.005. C-
injection of the K+-ATPase inhibitor induced a significant increase of intratubular pH in LDT (control period, 6.47 t 0.03; experimental period, 6.77 t 0.08; n = 17, P < 0.005 by paired t test). In this nephron segment, the mean ApH (A@ = PHexperimental - pHcontrod induced by vehicle administration [ApH = -0.01 t 0.07 (SE), n = 15 ] was not significantly different from zero (P > 0.9), whereas that induced by Sch 28080 [ApH = 0.32 t 0.08 (SE), n = 171 was significantly different from zero (P C 0.005). To exclude that these effects could be due to some nonspecific action of Sch 28080, in a subset of experiments we evaluated whether Sch 28080 would alter the intraluminal pH in the EDT, a nephron segment that is devoid of K+-ATPase activity (Fig. 5). The mean luminal pH values in seven segments of EDT from three animals were 7.62 t 0.11 and 7.59 -+ 0.08 before and after administration of Sch 28080, respectively. In the same animals, Sch 28080 again induced a statistically significant ApH in the corresponding LDT [0.51 t 0.09 (SE), n = 61. DISCUSSION
The results of this study demonstrate the presence of a K+-ATPase activity in the amphibian kidney. K+ATPase activity in frog kidney displays kinetic and pharmacological properties similar to those previously described in the mammalian kidnev. Indeed, 1) the appar-
NEPHRON
ent Michaelis constant (&J of the frog enzyme for K+ (apparent Km = 0.3 mM; Fig. 2) is similar to that previously reported in the rabbit distal tubule (apparent &, = 0.25-0.40 mM; see Ref. 6); 2) in both frog and rabbit kidney, K+-ATPase is totally inhibited by vanadate and preactivated omeprazole (6); 3) the apparent sensitivity (Ki) to the imidazopyridine Sch 28080 is similar in frog (apparent Ki z 1O-6M; Fig. 4) and rat (apparent Ki = 4 x 10-7; see Ref. 5); and 4) the localizations of the K+ATPase along the nephrons of frog (Fig. 5) and rabbit are similar (6). Based on the similarities between frog and mammalian K+-ATPase on the one hand, and between the distribution patterns of K+-ATPase along the frog and Necturus nephron (Fig. 5) on the other, it can be inferred that the Necturus renal K+-ATPase activity might be similar to that in frog and mammalian nephron. K’-ATPase activity was first described in the gastric mucosa (9, 13), where it functions as an H+-K+ pump, accumulating K+ inside the cell while extruding protons. Although the renal K+-ATPase from either mammalian or amphibian nephron displays the same pharmacological properties as the gastric pump (3, 14, 25), it has not yet been clearly established whether this enzyme was a pump capable of exchanging K+ against protons. Indeed, the only report dealing with K’-ATPase-mediated ion flux in kidney indicates that 100 PM omeprazole completely abolished proton secretion (as measured by net total COZ flux) and K+ reabsorption in outer medullary collecting tubules from K’-depleted rabbits (28). However, the observation that omeprazole completely abolished urinary acidification in the medullary collecting tubule casts doubts on the specificity of this drug. Indeed, it has been firmly established that at least part of the medullary collecting tubule acidification is mediated by an electrogenic, NEM-sensitive H+ pump, distinct from the H+-K+-ATPase (1, 4, 21, 22). Besides, omeprazole was reported to inhibit NEM-sensitive H+ pump in addition to the H+-K+ pump (8). Thus in the present study we chose Sch 28080 as a specific inhibitor of H+-K+ATPase to evaluate whether K+-ATPase is involved in urinary acidification in the LDT of Necturus nephron. The present finding that Sch 28080 inhibited tubular urinary acidification in vivo, as well as other results indicating that in vitro addition of this drug partially inhibited ouabain-insensitive Rb+ uptake in the rat collecting tubule (5), strongly suggest that the renal K+ATPase works as an H+-K+ pump and may therefore be similar to the gastric enzyme. That the distal nephron is a major site of acid secretion in the amphibian nephron is underscored by the observation that 75% of total bicarbonate absorption precisely occurs in the distal portion of the nephron (15, 17, 24, 29). In our in vivo experiments a specific inhibitor of the H’-K+-ATPase, Sch 28080, elicited a rise of the steadystate luminal pH by 0.3 pH units in LDT (Fig. 6). As we lack information on the metabolism of the drug in Necturus in vivo, the amount administered intravenously was 4.5 x 10W4mol/kg body wt, recalling that complete inhibition of the K+-ATPase activity in vitro was obtained at a lower concentration, 10W4M. Despite uncertainties on tissue distribution in vivo, it is reasonable to assume that a substantial fraction of Sch 28080 (though
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H+-K+-ATPASE
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hardly estimated) was filtered. If the filtered load were high, nonspecific effects of the drug upstream could raise EDT luminal pH values and consequently deliver an alkaline fluid into the LDT. However, we checked that intravenous injection of Sch 28080 failed to alter EDT luminal pH, whereas it dissipated, in part, the LDT transepithelial pH gradient. Taken together, these observations rule out nonspecific effects of the drug on the LDT. At least three mechanisms may account for the relatively small change in steady-state luminal pH in the LDT: 1) the time of exposure to Sch 28080 (20-40 min) may have fallen short of achieving its full effect on LDT; 2) the drug may have been heavily metabolized; and 3) luminal acidification could be the result of two complementary processes, the H+-K+ pump and the electrogenic H-ATPase, with the latter sustaining a residual transepithelial H+ gradient after exposure to Sch 28080. Because we measured only steady-state luminal pH values, and not a rate of proton secretion, we cannot assess quantitatively the contribution of the H+-K+ pump to H+ secretion, even with the assumption of full inhibition of the H+-K+ pump by Sch 28080. The presence of H+-K+ pump in the LDT of Necturus has been anticipated on the following observations: K+ absorption in this segment cannot be achieved by passive diffusion or secondary active transport (Na+-K+-2Clor K+-Cl- symport), given the energetic constraints prevailing across the apical membrane (2). A similar analysis ruled out the presence of an apical Na+-H+ antiporter (2). Thus only an H+-K+ pump could fulfill both H+ secretion and K+ uptake, even though net luminal acidification could be the result of both an H+-K+-ATPase and an H+-ATPase. Our study demonstrates the presence of the H’-K+-ATPase in the distal tubule. The fact that the inhibition of this pump by Sch 28080 does not account for part of the acid secretion is consistent with the presence of an H+-ATPase, also. In summary, we demonstrated the presence of a ouabain-insensitive, vanadate-, omeprazole-, and Sch 28080inhibited K+-ATPase activity in the LDT and collecting tubule of amphibian kidney. This enzyme, which is involved in the acidification of the tubular fluid in the distal tubule of Necturus, is similar to the gastric H+K+-ATPase and is likely to originate in luminal cell membranes where it may also participate to K+ reabsorption. We are indebted to Hassle Laboratories (Molndal, Sweden) and to Schering (Bloomfield, NJ) for kindly providing omeprazole and Sch 28080, respectively. We also acknowledge the skillful technical assistance of P. Hulin and the secreterial help of V. Biausque. L. Cheval was supported in part by a grant from the Minis&e de la Recherche et de la Technologie. Address for reprint requests: A. Doucet, Laboratoire de Physiologie Cellulaire, URA CNRS 219, College de France, 11 Place M. Berthelot, 75231 Paris Cedex 5, France. Received 13 August 1990; accepted in final form 10 December 1990.
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AND
pro-
AND
NECTURUS
Fall
NEPHRON
ton pump in the rat kidney: localization along the nephron. J. Biol. Chem. 261: 12526-12534,1986. 2. ANAGNOSTOPOULOS, T., AND
G. PLANELLES. Cell and luminal activities of chloride, potassium, sodium and protons in the late distal tubule of Necturus kidney. J. Physiol. Lond. 393: 73-89,1987. C., B. M. ANDERSSON, P. NORDBERG, AND B. WALL3. BRIVING, MARK. Inhibition of gastric H+/K+-ATPase by substituted imidazo[ 1,2-alpyridines. Biochim. Biophys. Acta 946: 185-192,1988. 4. BROWN, D., S. HIRSCH, AND S. GLUCK. Localization of a protonpumping ATPase in rat kidney. J. Clin. Inuest. 82: 2114-2126, 1988. 5. CHEVAL, L., C. BARLET-BAS, C. KHADOURI, E. FERAILLE, S. MARSY, AND A. DOUCET. K+-ATPase-mediated Rb transport in rat collecting tubule: modulation during K deprivation. Am. J. Physiol. 260 (Renal 6. DOUCET, A., AND
7.
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10. 11.
12. 13.
FZuid Electrolyte
Physiol.
29): F800-F805,1991.
S. MARSY. Characterization of K-ATPase activity in distal nephron: stimulation by potassium depletion. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F418-F423,1987. DUNNETT, C. W. A multiple comparison procedure for comparing several treatments with a control. J. Am. St&. Assoc. 50: 10961121,1955. FROISSART, M., E. MARTY, F. LEVIEL, M. BICHARA, J. POGGIOLI, AND M. PAILLARD. Plasma membrane H’-ATPase and Na+-H+ antiporter in medullary thick ascending limb (MTAL) of rat kidney (Abstract). Kidney Int. 37: 537, 1990. GANSER, A. L., AND J. G. FORTE. K’-stimulated ATPase in purified microsomes of bullfrog oxyntic cells. Biochim. Biophys. Actu 307: 169-180,1973. GARG, L. C., AND N. NARANG. Ouabain-insensitive K-adenosine triphosphatase in distal nephron segments of rabbit. J. CZin. Inuest. 81: 1204-1208,1988. GUSTIN, M. C., AND D. B. P. GOODMAN. Isolation of brush-border membrane from the rabbit descending colon epithelium. Partial characterization of a unique K+-activated ATPase. J. Biol. Chem. 256: 10651-10656,198l. KAUNITZ, J. D., AND G. SACHS. Identification of a vanadatesensitive potassium-dependent proton pump from rabbit colon. J. Biol. Chem. 261: 14005-14010,1986. LEE, J., G. SIMPSON, AND P. SCHOLES. An ATPase from dog gastric mucosa: changes of outer pH in suspension of membrane vesicles accompanying ATP hydrolysis. Biochem. Biophys. Res. Commun.
60: 825-832,1974.
14. LEWIN, M. J. M. Molecular mechanisms of gastric HCl secretion. In: Molecular and Cellular Basis of Digestion, edited by P. Desnuelle, H. Sjostrom, and 0. Noren. Amsterdam: Elsevier, 1986, p. 507-526.
15. MONTGOMERY, H., AND J. A. PIERCE. The site of acidification of the urine within the renal tubule in amphibia. Am. J. Physiol. 118: 152-155,1937. 16. OBERLEITHNER, H., F. LANG, G. MESSNER, AND W. WANG. Mechanism of hydrogen ion transport in the diluting segment of frog kidney. Pfluegers Arch. 402: 272-280, 1984. 17. PERSSON, B. E., AND A. E. G. PERSSON. Acidification of the distal tubule of Amphiuma kidney. Acta Physiol. Stand. 117: 343-349, 1983.
18. PERSSON, B. E., T. SAKAI, AND D. J. MARSH. Juxtaglomerular interstitial hypertonicity in Amphiuma: tubular origin. TGF signal. Am. J. Physiol. 254 (Renal Fluid Electrolyte Physiol. 23): F445F449,1988. 19. PLANELLES, G., A. KURKDJIAN, AND T. ANAGNOSTOPOULOS. Cell and luminal pH in the proximal tubule of Necturus kidney. Am. J. PhysioZ. 247 (Renal Fluid Electrolyte Physiol. 16): F932-F938,1984. 20. RENKIN, E. M., AND J. P. GILMORE. Glomerular filtration. In: Handbook of Physiology. Renal Physiology. Washington, DC: Am. Physiol. Sot., 1973, sect. 8, chapt. 9, p. 185-248. 21. SCHWARTZ, G. J., AND Q. AL-AWQATI. Carbon dioxide causes exocytosis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules. J. Clin. Invest. 75: 1638-1644, 1985. 22. STONE, D. K., D. W. SELDIN, J. P. KOKKO, AND H. R. JACOBSON. Anion dependence of rabbit medullary collecting duct acidification. J. Clin. Inuest. 71: 1505-1508, 1983. L. C. Isolated, perfused amphibian renal tubules: the 23. STONER, diluting segment. Am. J. Physiol. 233 (Renal Fluid Electrolyte Physiol. 2): F438-F444, 1977.
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F812 24. WALKER, 25.
H+-K+-ATPASE
ALONG
FROG
A. M. Ammonia formation in the amphibian kidney. Am.
J. Physiol. 131: 18’7-194, 1940. WALLMARK, B., C. BRIVING, J. FRYKLUND, K. MUNSON, SON, J. MENDLEIN, E. RABON, AND G. SACHS. Inhibition
R. JACKof gastric H+, K+-ATPase and acid secretion by SCH 28080, a substituted pyridyl( 1,2a)imidazole. J. Biol. Chem. 262: 2077-2084, 1987. 26. WALLMARK, B., B. M. JARESTEN, H. LARSSON, B. RYBERG, A. BRANDSTROM, AND E. FELLENIUS. Differentiation among inhibitory actions of omeprazole, cimetidine, and SCN- on gastric acid secretion. Am. J. Physiol. 245 (Gastrointest. Liver Physiol. 8): G64G71,1983.
AND
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27. WIEDERHOLT, M., W. J. SULLIVAN, AND G. GIEBISCH. Potassium and sodium transport across single distal tubules of Amphiuma. J. Gen. Physiol. 57: 495-525, 1971. 28. WINGO, C. S. Active proton secretion and potassium absorption in the rabbit outer medullary collecting duct. Functional evidence for proton-potassium-activated adenosine triphosphatase. J. Clin. Invest. 84: 361-365, 1989. 29. YUCHA, C. B., AND L. C. STONER. Bicarbonate transport by amphibian nephron. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol.
20): F865-F872,
1986.
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T. ANAGNOSTOPOULOS,
L. CHEVAL,
AND
A. DOUCET
Laboratoire de Physiologie Renale, Institut National de la Sante et de la Recherche Medicale Unite 323, Centre Hospitalier Universitaire Necker, 75730 Paris Cedex 15; and Laboratoire de Physiologie Cellulaire, Unite de Recherche Associee 219, Centre National de la Recherche Scientifique, College de France, 75231 Paris Cedex 05, France
unlikely (because of unfavorable chemical gradients) and proposed the implication of a primary active transport mediated by electroneutral H+-K+ pump (2). A H+-K+ATPase was primarily described in the gastric mucosa causeproton secretion and K’ reabsorption in the late distal and intestine (9, 11-13). More recently, it was found in tubule of amphibians are active, we evaluated whether these processescould be mediated by an H+-K+-ATPase similar to the distal segments of the rabbit and rat nephron (6,10).
PLANELLES, G., T. ANAGNOSTOPOULOS, L. CHEVAL, AND A. DOUCET. Biochemicaland functional characterization of H+-K+ATPase in distal amphibian nephron. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F806-F812, 1991.-Be-
the gastric H’-K’ pump and to the K+-ATPase previously describedin the terminal segmentsof the mammaliannephron. K+-stimulated ATPase activity was detected in microdissected segmentsof frog and Necturus nephron: its activity was high in the late distal and collecting tubules, whereas it was undetectable in the proximal convoluted tubule and early distal tubule. In frog collecting tubule, K’-ATPase had a high affinity for K’ (K, = 0.30mM), wasinhibited by vanadate, omeprazole, and the imidazopyridine Sch 28080, and was insensitive to ouabain. Furthermore, in vivo administration of Sch 28080 to anesthetizedNecturus induced a significant rise of the steadystate intratubular pH in the late distal tubule, demonstrating that this drug inhibited tubular fluid acidification. It is suggestedthat K+-ATPase present in the terminal segmentsof amphibian nephron is similar to the gastric’ H’-K’ pump and is involved in urinary acidification. frog; Necturus; single-‘nephron segments;Sch 28080; pH-sensitive microelectrodes;proton-potassium pump
This K+-activated
pump, which couples the hydrolysis
of ATP to the transmembrane exchange of K+ and protons against their electrochemical gradients, belongs to the El-E1 class of ATPases, as it displays a phosphorylated intermediate and is inhibited by vanadate (reviewed in Ref. 14). This enzyme was also found to be specifically inhibited by omeprazole (26) and, more recently, by the imidazopyridine Sch 28080 (3,25). The present study was therefore designed to I) determine whether H+-K+-ATPase activities were present in the distal nephron of Necturus, 2) localize this ATPase activity along the Necturus renal tubule, and 3) evaluate whether in vivo inhibition of this pump by Sch 28080 alters distal tubular fluid acidification. To answer the first two questions, we searched for a K+-stimulated, Sch 280804nhibited ATPase activity in single segments of Necturus nephron, obtained by microdissection of collagenase-treated kidneys using the mi-
croassay previously developed for the mammalian nephron (6). However, because microdissection of Necturus THE AMPHIBIAN
DISTAL
TUBULE
is heterogeneous (23),
as it consists of an early distal tubule (EDT) that is functionally similar to the mammalian diluting segment, as well as a late distal tubule (LDT). In Amphiuma, luminal pH (17) and tubule-to-plasma K+ concentration ratio decrease along the LDT (27), indicating that proton secretion and K+ reabsorption occur in that segment. In the LDT of Necturus, measurement of steady-state activities for protons and K+ and potential differences in peritubular blood capillaries, cells, and tubular fluid (2) indicated that K+ must be actively reabsorbed and protons must be actively secreted across the apical membrane (against electrochemical gradients of -20 and 90 mV, respectively). Although the molecular mechanism(s) underlying these active transports across the luminal membrane of Necturus LDT was not formally characterized in that study, its authors concluded that the involvement of Na+K+-2Cl- or K+-Cl- symport and Na+-H+ antiport was F806
0363-6127/91
$1.50
kidney is difficult, preliminary experiments aimed at determining the kinetic and pharmacological properties
of K+-ATPase
were performed
in the kidney of the frog, of samples necessary for such studies can be isolated more easily. By use of the optimal assay conditions determined thereby in the frog nephron, the localization of K+ATPase along the Necturus nephron was analyzed next. To evaluate the involvement of H+-K+-ATPase in distal tubular acidification, the pH of tubular fluid in the distal tubule was determined by pH-sensitive microelectrodes before and after administration of Sch 28080 to anesthetized Necturus. Rana ridibunda, a species in which the large number
METH0DS Animals. Biochemical studies were carried out on two species, R. ridibunda (Couetard, France) and N. maculosus (Nasco). The frogs were anesthetized by
amphibian
Copyright 0 1991 the American Physiological Society
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H’-K’-ATPASE
ALONG
FROG
intraperitoneal injection of 50 mg Inactin (Byk); necturi were anesthetized by immersion into a 0.1% (wt/vol) solution of tricaine methanesulfonate (Sigma) in tap water. Electrophysiological determinations in vivo were performed only in necturi; once the animals were asleep, the head and the branchiae were continuously immersed in the above-mentioned tricaine solution, which was diluted fivefold. Tubule microdissection. A physiological Ringer solution for amphibia was prepared, which contained (in mM) 82 NaCl, 3 KCl, 1.8 CaCIZ, 1.0 MgC&, and 5 tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffered at pH = 7.5 with NaOH. In frogs, the aorta was cannulated from its terminal end, and then it was ligated above the first (upper) renal arteries (16). The effluent was allowed t.o flow into the peritoneal cavity through a large incision of the vena cava. Twenty milliliters of the Ringer solution were perfused to completely remove blood. After the kidneys were rinsed, 10 ml of the same solution supplemented with collagenase (400 U/ml collagenase IA, Sigma) were delivered via the efferent renal vein at a rate of 1 ml/min. The kidneys were excised, cut into thin slices, incubated during 1 h at 28°C into a physiological Ringer solution containing collagenase (4,000 U/ml), and then rinsed with a K’-free Ringer solution. Microdissection was carried out at 4°C in a similar solution. Tubule segments were isolated, photographed at constant magnification, and kept at 4°C until enzymatic determinations. The following four nephron segments were studied: 1) the initial portion of the proximal convoluted tubule (PCT); 2) the EDT or diluting segment, easily recognized by its thin diameter [the thinnest among the major segments of the nephron, the EDT begins after a short transition segment endowed with intraluminal cilia and ends at the juxtaglomerular apparatus (JGA)]; 3) the LDT, which stretches between the JGA and a constriction at the transition with the collecting duct; and 4) collecting tubules, which are recognizable by heterogeneous appearance and branched configuration. The microscopic aspect and intrarenal localization of these nephron segments are illustrated in Fig. 1, which displays a complete frog nephron dissected from acid-treated kidney (see legend to Fig. 1). A few modifications were necessary for dissection of tubules from Necturus because of the presence of thick connective tissue. The aortic cannula was placed via the thoracic aorta, at the upper border of the kidney, and 20 ml of Ringer solution were delivered. Then a rinsing solution (15 ml) supplemented with collagenase (600 U/ ml) was delivered through the caudal vein, after clamping iliac vessels. Kidney fragments were incubated during 60 min in a Ringer solution supplemented with collagenase (8,000 U/ml) at 30°C. Dissection of the segments downstream to the JGA (18) was uncertain because the glomerulus was often lost. Thus the LDT was microdissected upstream, starting from an initial collecting tubule; the boundary between these two segments was assessed from a discernible change in morphology; segments lacking clear-cut boundaries were discarded. Proximal, diluting, and collecting tubules were selected along the criteria defined above, with regard to frog kidney. Determination of K’-ATPose activity. K’-ATPase ac-
AND
Nh’:C’I’I:RILS
NEPHRON
F807
FIG. 1. A: microscopic appearance of a complete frog nephron dissected from acid-treated kidney. After perfusion with Ringer solution, kidney was removed and incubated for 3 h at 26°C in HCI 18% (vol/ vol). After rinsing in Rqer, it was further incubated for 10 days at 4’C in a 1 CL>(vol/vol) solution of acetic acid. This preparation allowed dissection of entire nephrons. H: schematic localization of glomerulus (G) and segments of nephron used in this study. PCT, proximal convoluted tubule; EDT, early distal tubule; LDT, late distal tubule; CT, collecting Lubule.
tivity was measured using the assay previously developed (6), with slight modifications. Pieces of nephron were individually transferred to the concavity of a sunken bacteriological slide and then photographed to determine their length, which served as reference for enzymatic activities. To increase cell permeability to reactants during the assay, tubule segments were successively submitted to a hypotonic medium and frozen as follows. The medium surrounding each tubule was aspirated and replaced by an equal volume of cold distilled water, and after 5-10 min at 0-4°C this procedure was repeated. Then the droplet surrounding the tubule was again aspirated and replaced by 0.2 ~1 distilled water. When necessary, drugs were added in the 0.2 ~1 distilled water at six times the final concentration required. Samples were then rapidly frozen on dry ice. After thawing, 1 ~1 of incubation medium (see composition below) was added to each sample, which was then incubated for 15 min at 37°C. Incubation was
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H+-K+-ATPASE
ALONG
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stopped by cooling samples on crushed ice and by adding 5 ~1 of trichloroacetic acid 5% (wt/vol). Samples were then transferred into 2 ml of a suspension of 10% (wt/ vol) activated charcoal. After mixing and centrifugation, the radioactivity of 500 ~1 of supernatant was determined by scintillation counting. Unless indicated otherwise, the final composition of the incubation medium was (in mM) 25 tris(hydroxymethyl)aminomethane hydrochloride (Tris . HCl), 10 MgCl,, 1 ethylene glycol-bis(P-aminoethyl ether)N,N,N’,N’-tetraacetic acid, 1 ouabain, 2.5 sodium azide, and 5 Tris-ATP, as well as tracer amounts (~5 nCi/pl) of [T-~~P]ATP (2-10 Ci/mmol) in the presence of 2.5 mM KC1 (total ATPase) or in its absence (basal ATPage), pH 7.4. Eight to 14 samples of each segment of nephron were distributed randomly into two groups, one each for measuring total and basal ATPase. K+-ATPase was taken as the difference between the means of each group. ATPase activity was expressed as picomoles of inorganic phosphate released per millimeter of tubule length per hour. Steady-state determination of luminal pH in the distal tubule of Necturus in uiuo. Double-barreled (selective vs. conventional) pH microelectrodes have been constructed as in previous studies (19). Briefly, two capillary tubings (1.2 mm OD, 0.7 mm ID) were firmly attached, twisted manually to 360” on a flame, then pulled on a PE-2 Narishige vertical puller. One barrel was exposed to dimethyl trimethylsilylamine (Fluka, 41716) during 7 min, and then baked at 120°C for 2 h. The tip of the silanized barrel was filled with a COz-equilibrated proton cocktail (Fluka). The next day each barrel was backfilled with appropriate electrolyte solutions (2); then the microelectrodes were calibrated in vitro in the pH range 6.0-7.5. Their slope ranged from 50 to 58 mV/pH unit. The slope was checked again before and after each luminal pH determination in vivo by alternately delivering onto the surface of the kidney two artificial solutions buffered at pH 7.5 and 6.5. The animals were divided into two groups, control and experimental. The experimental group received an intravenous bolus of 125 mg/kg Sch 28080, i.e., 0.45 mmol/kg (dissolved in ethanol 2.5 ml). The control group was injected with the vehicle alone. The luminal pH of studied tubules was measured twice in single subcapsular LDT or EDT loops, before and after intravenous injection of vehicle or Sch 28080; each tubule served as its own control. The site of the first impalement was carefully drawn to recover the appropriate loop after 20-40 min of exposure to the inhibitor or to the vehicle. Necturus has a relatively low glomerular filtration rate (0.2 ml* min-’ . kg-‘; see Ref. 20), and this lag period appeared convenient for allowing the drug to reach the apical cell membrane. Statistics. Statistical significance was assessed by Student’s t test or, when necessary, by variance analysis according to the test of Dunnett (7), with P < 0.05 being con sidered significant. RESULTS
Biochemical properties of frog tubular K+-ATPase tiuity. The effect of increasing K+ concentration
ac(by
NEPHRON
addition of KCl) on ATPase activity of frog collecting tubule is shown in Fig. 2. ATPase activity was stimulated in a dose-dependent fashion as K+ concentration was increased between 0 and 2.5 mM. Maximal activity was reached with 0.75 mM K+, and half-maximal stimulation of the K+-dependent moiety of the activity was observed with 0.32 mM K+. Data in Fig. 3 indicate that the ATPase activities determined in the absence or presence of K+ were linearly related to the length or presence of the
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FIG. 2. Stimulation by K+ of ATPase activity in frog collecting tubule. Each point is mean t SE of 5-6 replicate tubules. Maximal stimulation of K’-ATPase activity was reached at K+ concentration of 0.75 mM. Values statistically different from control (K+ = 0) were assessed by variance analysis *P c 0.001. Inset: Lineweaver-Burke plot of K+-dependent component of ATPase activity indicates apparent K, of 0.32 mM and V,,,,, of -400 pmol . mm-‘. h-l.
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3. Relationship between ATPase activity in frog collecting tubule and tubular length. ATPase activity was measured either in presence of 2.5 mM K+ (solid symbols) or in its absence (open symbols). Different symbol shapes correspond to five different frogs. Both total and basal ATPase activities relate linearly to tubular length (r = 0.913 and 0.939, n = 22) in the 0.6-2.0 mm range. FIG.
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H+-K+-ATPASE
ALONG
FROG AND NECTURUS
sample, which illustrates the sensitivity and the specificity of the assay. Next, we studied some pharmacological properties of the tubular K’-ATPase activity. K+-ATPase was not sensitive to the Na+-K+-ATPase inhibitor ouabain (~1 mM) and to the mitochondrial Fo-F1 ATPase inhibitor azide (up to 2.5 mM), and these two compounds were routinely added to the incubation medium to lower the basal ATPase activity. Conversely, K+-ATPase activity in the frog collecting tubule was completely inhibited in the presence of 10 PM of the E1-EP ATPase inhibitor vanadate [control, 229 t 33 (SD) pmol . mm-l. h-l, n = 5; vanadate, 13 t 25 pmol mm-l h-‘, n = 5; P < 0.001, variance analysis]. K+-ATPase activity was also completely inhibited by 100 PM of the inhibitor of gastric H+-K+-ATPase omeprazole, after its activation at pH = 2.00 for 30 min at 0°C [control, 229 t 33 (SD) pmol mm-l h-l, n = 5; omeprazole, 0 t 20 pmol. mm-l. h-l, n = 5; P < 0.001, variance analysis]. However, when preactivated in vitro at acid pH, omeprazole is no longer specific for H+-K+-ATPase, as it also inhibits the electrogenic, N-ethylmaleimide (NEM)-sensitive H+-ATPase (unpublished observation). Thus we looked for the action of the specific inhibitor of gastric H+-K+-ATPase, Sch 28080. At a concentration of 100 PM, Sch 28080 completely inhibited K+-ATPase activity [control, 324 t 39 (SD) pmol mm-’ . h-l, n = 4; Sch 28080,O t 37 pmol mm-’ h-‘, n = 5; P < 0.001, unpaired t test]. The effect of increasing concentrations of Sch 28080 on ATPase activity of the frog collecting tubule is shown on Fig. 4. The thresholds of inhibition and half-maximal inhibition of total ATPase activity were observed with 10m7and 10m6M Sch 28080, respectively. This inhibitory action of Sch 28080 was restricted to the K+-stimulated moiety of the ATPase activity, as basal ATPase activity was not modified by this drug up to a concentration of 10m4M (Fig. 4). Finally, since amphibia are poikilotherms, we looked for the temperature dependence of K+-ATPase activity. In frog collecting tubule, K’-ATPase activities were similar when measured either at 25°C or at 37°C [ZSOC, 241 -t 34 (SD) pmol mm-’ *h-l, n = 5; 37”C, 219 t 44 pmol l
l
l
mm-‘. h-l, n = 5; not significant (NS)]. This unexpected finding likely results from opposite actions of thermic activation and denaturation of the enzyme. Localization of K+-ATPase along the frog and Necturus nephron. Figure 5 displays mean K+-ATPase activities
measured in the successive nephron segments of frog (Fig. 5, top) and Necturus (Fig. 5, bottom). In these two species, K+-ATPase activity was exclusively measurable in the LDT and the collecting tubule, whereas K+-ATPase activity was almost undetectable and not statistically different from zero in the PCT and the EDT (P > 0.5). In the terminal segments of nephron, K+-ATPase activity was higher in the LDT than in the collecting tubules of the frog, whereas it was similar in these two nephron segments of the Necturus. Basal ATPase activities measured in the different segments of frog and Necturus nephrons are given in Table 1. Effect of Sch 28080 on tubular fluid acidification. Intratubular pH was measured in LDT under free-flow conditions before and after intravenous administration of either Sch 28080 or vehicle to anesthetized necturi. Results in Fig. 6 clearly indicate that intratubular pH values in LDT were similar before and after administration of vehicle (control period, 6.57 $- 0.06; experimental period, 6.56 t 0.07; n = 15, NS by paired t test). By contrast, 500 -
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NEPHRON
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FIG. 4. Effect of Sch 28080 concentration on ATPase activity measured in absence (0) or presence of K+ (0) in frog collecting tubule. Each point is mean k SE of 4-5 replicate tubules. Sch 28080 inhibited total ATPase activity (solid line) in a dose-dependent fashion, whereas it did not alter basal ATPase activity (dashed line). Values statistically different from control (no Sch 28080) were determined by variance analvsis. *P < 0.005. **P < 0.001.
On
5
PCT FIG. Necturus
EDT
5. Distribution of K’-ATPase activity along frog (top) and nephron (bottom): n. number of animals.
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F810
H+-K+-ATPASE
ALONG
FROG AND NECTURUS
1. Basal ATPase activity along the frog and Necturus nephron
TABLE
ATPase Activity, pmol mm-’ l
Frog
l
h-’ Necturus
3,084+241 (5) PCT 916*98 (5) EDT 6791t47 (6) 918,+150(5) 1,473&86(5) LDT 2,044&196 (5) CT 434t,65 (7) 2,125+_189 (5) Values are means k SE; number of animals is in parentheses. Basal ATPase activity was determined in absence of K+ in successive segments of nephron dissected from frog and Necturus kidneys. PCT, proximal convoluted tubule; EDT, early distal tubule; LDT, late distal tubule; CT, collecting tubule. Vehicle
SCH 28080
E CI FIG. 6. Intraluminal pH in the LDT before (C) and after (E) administration of either vehicle (left) or Sch 28080 (right) to anesthetized Necturus. Lines join pH values determined in same nephron; different length of dashes indicate lines corresponding to distinct animals. Points are means Al=SE from individual values. Mean ApH values induced by vehicle and Sch 28080 were -0.01 ,t 0.07 (n = 15) and 0.32 & 0.08 (n = l7), respectively. Statistical analysis was performed by Student’s t test for paired data. *P < 0.005. C-
injection of the K+-ATPase inhibitor induced a significant increase of intratubular pH in LDT (control period, 6.47 t 0.03; experimental period, 6.77 t 0.08; n = 17, P < 0.005 by paired t test). In this nephron segment, the mean ApH (A@ = PHexperimental - pHcontrod induced by vehicle administration [ApH = -0.01 t 0.07 (SE), n = 15 ] was not significantly different from zero (P > 0.9), whereas that induced by Sch 28080 [ApH = 0.32 t 0.08 (SE), n = 171 was significantly different from zero (P C 0.005). To exclude that these effects could be due to some nonspecific action of Sch 28080, in a subset of experiments we evaluated whether Sch 28080 would alter the intraluminal pH in the EDT, a nephron segment that is devoid of K+-ATPase activity (Fig. 5). The mean luminal pH values in seven segments of EDT from three animals were 7.62 t 0.11 and 7.59 -+ 0.08 before and after administration of Sch 28080, respectively. In the same animals, Sch 28080 again induced a statistically significant ApH in the corresponding LDT [0.51 t 0.09 (SE), n = 61. DISCUSSION
The results of this study demonstrate the presence of a K+-ATPase activity in the amphibian kidney. K+ATPase activity in frog kidney displays kinetic and pharmacological properties similar to those previously described in the mammalian kidnev. Indeed, 1) the appar-
NEPHRON
ent Michaelis constant (&J of the frog enzyme for K+ (apparent Km = 0.3 mM; Fig. 2) is similar to that previously reported in the rabbit distal tubule (apparent &, = 0.25-0.40 mM; see Ref. 6); 2) in both frog and rabbit kidney, K+-ATPase is totally inhibited by vanadate and preactivated omeprazole (6); 3) the apparent sensitivity (Ki) to the imidazopyridine Sch 28080 is similar in frog (apparent Ki z 1O-6M; Fig. 4) and rat (apparent Ki = 4 x 10-7; see Ref. 5); and 4) the localizations of the K+ATPase along the nephrons of frog (Fig. 5) and rabbit are similar (6). Based on the similarities between frog and mammalian K+-ATPase on the one hand, and between the distribution patterns of K+-ATPase along the frog and Necturus nephron (Fig. 5) on the other, it can be inferred that the Necturus renal K+-ATPase activity might be similar to that in frog and mammalian nephron. K’-ATPase activity was first described in the gastric mucosa (9, 13), where it functions as an H+-K+ pump, accumulating K+ inside the cell while extruding protons. Although the renal K+-ATPase from either mammalian or amphibian nephron displays the same pharmacological properties as the gastric pump (3, 14, 25), it has not yet been clearly established whether this enzyme was a pump capable of exchanging K+ against protons. Indeed, the only report dealing with K’-ATPase-mediated ion flux in kidney indicates that 100 PM omeprazole completely abolished proton secretion (as measured by net total COZ flux) and K+ reabsorption in outer medullary collecting tubules from K’-depleted rabbits (28). However, the observation that omeprazole completely abolished urinary acidification in the medullary collecting tubule casts doubts on the specificity of this drug. Indeed, it has been firmly established that at least part of the medullary collecting tubule acidification is mediated by an electrogenic, NEM-sensitive H+ pump, distinct from the H+-K+-ATPase (1, 4, 21, 22). Besides, omeprazole was reported to inhibit NEM-sensitive H+ pump in addition to the H+-K+ pump (8). Thus in the present study we chose Sch 28080 as a specific inhibitor of H+-K+ATPase to evaluate whether K+-ATPase is involved in urinary acidification in the LDT of Necturus nephron. The present finding that Sch 28080 inhibited tubular urinary acidification in vivo, as well as other results indicating that in vitro addition of this drug partially inhibited ouabain-insensitive Rb+ uptake in the rat collecting tubule (5), strongly suggest that the renal K+ATPase works as an H+-K+ pump and may therefore be similar to the gastric enzyme. That the distal nephron is a major site of acid secretion in the amphibian nephron is underscored by the observation that 75% of total bicarbonate absorption precisely occurs in the distal portion of the nephron (15, 17, 24, 29). In our in vivo experiments a specific inhibitor of the H’-K+-ATPase, Sch 28080, elicited a rise of the steadystate luminal pH by 0.3 pH units in LDT (Fig. 6). As we lack information on the metabolism of the drug in Necturus in vivo, the amount administered intravenously was 4.5 x 10W4mol/kg body wt, recalling that complete inhibition of the K+-ATPase activity in vitro was obtained at a lower concentration, 10W4M. Despite uncertainties on tissue distribution in vivo, it is reasonable to assume that a substantial fraction of Sch 28080 (though
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H+-K+-ATPASE
ALONG
FROG
hardly estimated) was filtered. If the filtered load were high, nonspecific effects of the drug upstream could raise EDT luminal pH values and consequently deliver an alkaline fluid into the LDT. However, we checked that intravenous injection of Sch 28080 failed to alter EDT luminal pH, whereas it dissipated, in part, the LDT transepithelial pH gradient. Taken together, these observations rule out nonspecific effects of the drug on the LDT. At least three mechanisms may account for the relatively small change in steady-state luminal pH in the LDT: 1) the time of exposure to Sch 28080 (20-40 min) may have fallen short of achieving its full effect on LDT; 2) the drug may have been heavily metabolized; and 3) luminal acidification could be the result of two complementary processes, the H+-K+ pump and the electrogenic H-ATPase, with the latter sustaining a residual transepithelial H+ gradient after exposure to Sch 28080. Because we measured only steady-state luminal pH values, and not a rate of proton secretion, we cannot assess quantitatively the contribution of the H+-K+ pump to H+ secretion, even with the assumption of full inhibition of the H+-K+ pump by Sch 28080. The presence of H+-K+ pump in the LDT of Necturus has been anticipated on the following observations: K+ absorption in this segment cannot be achieved by passive diffusion or secondary active transport (Na+-K+-2Clor K+-Cl- symport), given the energetic constraints prevailing across the apical membrane (2). A similar analysis ruled out the presence of an apical Na+-H+ antiporter (2). Thus only an H+-K+ pump could fulfill both H+ secretion and K+ uptake, even though net luminal acidification could be the result of both an H+-K+-ATPase and an H+-ATPase. Our study demonstrates the presence of the H’-K+-ATPase in the distal tubule. The fact that the inhibition of this pump by Sch 28080 does not account for part of the acid secretion is consistent with the presence of an H+-ATPase, also. In summary, we demonstrated the presence of a ouabain-insensitive, vanadate-, omeprazole-, and Sch 28080inhibited K+-ATPase activity in the LDT and collecting tubule of amphibian kidney. This enzyme, which is involved in the acidification of the tubular fluid in the distal tubule of Necturus, is similar to the gastric H+K+-ATPase and is likely to originate in luminal cell membranes where it may also participate to K+ reabsorption. We are indebted to Hassle Laboratories (Molndal, Sweden) and to Schering (Bloomfield, NJ) for kindly providing omeprazole and Sch 28080, respectively. We also acknowledge the skillful technical assistance of P. Hulin and the secreterial help of V. Biausque. L. Cheval was supported in part by a grant from the Minis&e de la Recherche et de la Technologie. Address for reprint requests: A. Doucet, Laboratoire de Physiologie Cellulaire, URA CNRS 219, College de France, 11 Place M. Berthelot, 75231 Paris Cedex 5, France. Received 13 August 1990; accepted in final form 10 December 1990.
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