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Bicarbonate-stimulated ATPase in the renal proximal tubule luminal (brush border) membrane
ARCHIVES
OF
BIOCHEMISTRY
AND
Bicarbonate-Stimulated
BIOPHYSICS
ATPase in the Renal Proximal (Brush Border) Membrane
C. TONY Laboratory
176, 285-297 (1976)
LIANG
of Molecular Aging, Gerontology Institutes of Health, Baltimore
AND
BERTRAM
January
Luminal
SACKTOR
Research Center, National Institute City Hospitals, Baltimore, Maryland
Received
Tubule
on Aging, 21224
National
26, 1976
Brush border membranes of the rabbit renal tubule have an ATPase which was stimulated 60% by 50 mM HCO,-. The K, for HCO,- was 36 mM. Kinetic studies of the “HCO,--ATPase” indicate that HCO,- had no effect on the K, for ATP and ATP did not alter the K, for HCOsm. Several anions, notably SO 32-, also accelerated the rate of dephosphorylation of ATP. The V for “SO 32--ATPase” was fivefold greater than that for “HCO,--ATPase.” The K, for SO,*- was 0.78 mM. Other anions including Cl- and phosphates, did not enhance ATPase activity. Thus, of the anions present in the glomerular filtrate in appreciable concentrations only HCO,- stimulated the luminal membrane enzyme. The anion-stimulated ATPase activity increased sharply from pH 6.1 to 7.1 and moderately with higher pH. The renal ATPase was not inhibited by SCNnor methyl sulfonyl chloride and was relatively insensitive to oligomycin and quercetin. Carbonyl cyanide p-trifluoromethoxy phenylhydrazone increased the basal rate of the membranal ATPase, suggesting that the ATPase activity is limited by transmembrane H+ flux. Carbonic anhydrase significantly increased the HCOs--stimulated ATPase activity. This increment was blocked by Diamox. These findings provide evidence consistent with the hypothesis that the brush border membrane ATPase is involved in the extrusion of H+ from tubular cell to lumen and support suggested interrelationships between HCO,--stimulated ATPase; H+ secretion, and bicarbonate transport in the kidney.
Bicarbonate transport and acid secretion are found in the stomach, salivary gland, and pancreas. It is of interest, therefore, that these organs possess an active HCOs--stimulated ATPase (l-9). Subcellularly, the enzyme is localized in the “microsomal” fraction (3-81, although enhancement of ATPase activity by HCO,and other anions is reported in mitochondrial preparations from liver (10, 11) and heart (121, as well as gastric mucosa (4). In the kidney, 80 to 90% of the filtered bicarbonate is reabsorbed in the proximal tubule (131, and acidification of the tubular fluid is a prominent feature of renal function (14, 15). This focuses attention on the possible role of the proximal tubule luminal (brush border) membrane in these transport processes. Indeed, the presence of a HCO,--stimulated ATPase in a membrane fraction enriched in microvilli has Copyright 0 1976 by Academic All rights
of reproduction
Press, Inc. in any form reserved.
been noted (16). Our previous work with well-characterized proximal tubule brush border membrane preparations as model systems for studying renal transport mechanisms (17-23) prompted us to examine further the HCOa--stimulated ATPase in renal brush border membranes. In this paper we report some kinetic properties of the enzyme and provide evidence relevant to the possible interrelationships between the membrane HCO,--stimulated ATPase, proton secretion, and bicarbonate reabsorption in the renal tubule. EXPERIMENTAL
PROCEDURES
Materials. Nucleotides (sodium salts) were purchased from P-L Biochemicals. Pyruvate kinase, lactate dehydrogenase, carbonic anhydrase, and phosphoenolpyruvate were obtained from Boehringer. Oligomycin and ascorbic acid were from Sigma. Carbonyl cyanide p-trifluoromethoxy phenylhydra285
286
LIANG
AND
zone (FCCP)’ was purchased from Pierce Chemicals. Quercetin was kindly donated by Dr. E. Racker, Cornell University. Other chemicals were reagent grade. Preparation of membranes. Brush border membranes, derived from the proximal tubules of the rabbit kidney cortex, were isolated by a modification (21) of the procedure previously detailed (23). The brush border membranes sedimenting in the final centrifugation were resuspended in a medium comprised of 75 mM Tris chloride, pH 7.4, 6 mM MgCl,, and 160 mM sucrose. The quality of preparations was randomly evaluated by specific enzyme markers
(23). Mitochondria from the rabbit renal cortex were isolated by the procedure of Lardy and Wellman (24) for rat liver mitochondria. The isolation medium contained 210 mM mannitol, 70 mM sucrose, 0.1% bovine serum albumin, and 1 mMN-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), adjusted to pH 7.4 with Tris hydroxide. Determination ofATPase activities. Total ATPase activity was assayed in a 0.2-ml reaction mixture containing 75 mM Tris chloride buffer, pH 7.4, 6 mM MgCl,, 3 mM Na,ATP, 1.6 mM ouabain, and 50 mM NaHC03. Sucrose was added to maintain tonicity at 300 mOSM. The NaHCO, solution was prepared and titrated to pH 7.4 immediately prior to use. Except when indicated, the incubation was carried out for 15 min at 37°C. The reaction was terminated by the addition of 0.1 ml of 12% perchloric acid, followed by placing the tubes in ice. The inorganic phosphate liberated was determined by the method of Chen et al. (25). Mg2+-ATPase activity was measured in the identical manner except that NaHCO, was omitted. The difference between total ATPase activity and Mg*+-ATPase activity was taken to be the “HCO,-ATPase” activity. In determining the effect of pH on ATPase activities, the buffer used was 40 mM Tris plus 40 mM piperazine-iV,N’-bis(2-ethanesulfonic acid) (Pipes) adjusted with HCl to the indicated final pH value. In experiments in which ATPase was measured in the presence of inorganic phosphate or thiosulfate activity was determined by ADP production. The ATPase reaction was carried out and terminated as described above. The mixture was neutralized with KOH and the insoluble potassium perchlorate was removed by centrifugation. Aliquots of the supernatant were assayed spectrophotometrically for ADP in a medium containing 90 pmol of Tris chloride, pH 7.4, 2.5 pmol of MgCI,, 5 pmol of KCl, 0.15 Fmol of phosphoenolpyruvate, 0.15 pmol of NADH, 20 pg of ’ Abbreviations used: FCCP, carbonyl cyanide ptrifluoromethoxy phenylhydrazone; Hepes, N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid; Pipes, piperazine-N,N’-bis(2-ethanesulfonic acid); MSC, methane sulfonyl chloride.
SACKTOR pyruvate kinase, and 8 pg of lactate dehydrogenase in a total volume of 1 ml. Other methods. Succinic dehydrogenase activity was determined polarographically (26). Protein was measured by the procedure of Lowry et al. (27) using bovine serum albumin as the standard. RESULTS
Stimulation of ATPase activity by HCO,-.Total (Mg2+ + HCO,-)-ATPase and Mg2+-ATPAse activities in the renal brush border membranal preparations were linear with protein concentration from 4 to 20 pg per reaction mixture and with time of incubation for at least 20 min. As shown in Table I, the presence of 50 mM HCO,- increased the Mg2+-ATPase activity of isolated brush border membranes from 0.220 to 0.345 pm01 P/min/mg of protein, a stimulation of 57%. The activity in the homogenate of the renal cortex was also enhanced by the anion. Relative to the specific activity in the homogenate, the “HCO,--ATPase” in the brush border membranal preparation was increased 4.3fold, 0.028 compared to 0.118 pmol P/min/ mg of protein. This indicated an enrichment of the activity in these membranes. This increase in relative specific activity, however, was not as great as that found for enzymes localized exclusively in brush border membranes, e.g., trehalase and yglutamyl transpeptidase (28). Rather, the increase was comparable to the enrichment found with enzymes, e.g., cyclic AMP phosphodiesterase (29), that were present in other membranes of the renal cortex in addition to the brush border membrane. In view of the reported presence of a HCO,--stimulated ATPase in mitochondria from various tissues (lo-12), mitochondria from the renal cortex were examined and found to contain an ATPase that was enhanced about 60% by HCO,-. Sonication of the isolated mitochondria increased the Mg2+- and Mg2+ + HCO,-ATPase activities several-fold. In fact, the specific activity of the “HCO,--ATPase” in sonicated mitochondria was greater by approximately 50% than in the brush border membranes. Estimates, based on relative specific activities of “HCO,--ATPase,” succinic dehydrogenase, and y-glutamyl transpeptidase in homogenates and mitochon-
“HCO,--ATPase”
IN BRUSH
BORDER
TABLE LOCALIZATION OF THE HCOJ--S~~~~~~~~~ Preparation Crude homogenate Brush border membrane Mitochondria Intact Sonicated
Total ATPase
287
MEMBRANES
I
ATPase ACTIVITY IN THE RENAL CORTEX” “HCO,--ATPase” Mg2+-ATPase (pm01 Plminlmg of protein)
0.097 + 0.007 (4) 0.345 ‘- 0.020 (16)
0.069 L 0.003 (4) 0.220 ? 0.017 (16)
0.028 + 0.005 (4) 0.118 r 0.008 (16)
0.139 + 0.018 (5) 0.470 k 0.120 (4)
0.085 + 0.009 (5) 0.290 k 0.070 (4)
0.054 t 0.019 (5) 0.190 + 0.060 (4)
(LATPase activities were assayed as described in the text. Total (Mg2+ + HCO,-)- and Mg2+-ATPase represent the activities in the presence and absence of 50 mM NaHCO,, respectively. The difference between total ATPase and Mg*+-ATPase is indicated as the “HCO,--ATPase.” Each datum represents the mean _t SEM for the number of experiments indicated in parentheses.
der membranes was negligible.2 This condrial and brush border membranal preparations indicate that mitochondria and clusion was deduced from the findings that brush border membranes possessed 43 and renal cortex homogenates had a y-glutamy1 transpeptidase specific activity of 34 57%, respectively, of the total “HC03-ATPase” activity. pmol/h/mg of protein, brush border memSince electron micrographs of brush bor- branal preparations had a specific activity der membranal preparations as well as of 220, whereas mitochondrial preparaprevious measurements of marker en- tions had a value of only 3 ~mol/h/mg of zymes, i.e., cytochrome oxidase, indicated protein. Thus, contamination of the mitoa small, albeit significant, contamination chondrial preparation with brush border of the preparation with some intact but membranes was less than 1.5%. Assuming mostly fragmented mitochondria (231, it that the brush border membranal prepara was important to estimate how much of tion was 10% mitochondrial fragments, the HCO,--stimulated ATPase found in then from the data in Table I it was evibrush border membranal preparations dent that the contributions of mitochoncould be maximally attributed to mitodria to the Mg2+- and HCO,--stimulated chondrial contamination. For this reason ATPases in the brush border preparations succinic dehydrogenase activities were were 0.029 and 0.019 pmol P/min, respecmeasured in preparations of mitochondria tively; i.e., less than 15% of the ATPase This and brush border membranes. activities determined in the brush border marker enzyme was used because purified membranal preparations could be attribF,, the major mitochondrial ATPase, is an uted to mitochondria. inner membrane constituent and was Anion specificity. Stimulation of the shown to be activated by HCO,- (111, al- Mg2+-ATPase in brush border membranes though the presence of some HCO,--stimuby NaHCO, was due to HCO,- not Na+. As lated ATPase activity in the mitochondrial shown in Table II, Tris bicarbonate inouter membrane could not be excluded creased activity to the same magnitude as (30). The specific activity of the dehydrodid NaHCO,. On the other hand, neither genase in sonicated mitochondria (which Tris chloride nor NaCl had a stimulatory was almost twice that in intact mitochoneffect. However, other anions did increase dria) was 0.22 ng-atoms oxygenlminlmg of basal ATPase activity. When tested as its protein. This value compared to 0.022 ng- sodium salt, SO,“- enhanced ATPase acatomslminlmg of protein for the dehydrotivity 200%, a value threefold that found genase in the brush border membranal with HCO,-. The anions, CH,COO- and preparations. These findings suggested S042-, also increased ATPase activity, but that these brush border membranal prepaonly by about 20 to 30%. In contrast, NO,rations were contaminated by about 10% and Cl- were ineffective. In another set of with mitochondria. Contamination of miz Filburn, C., Liang, C. T., and Sacktor, B., untochondrial preparations with brush bor- published observations,
288
LIANG
AND
experiments, in which ATPase activities were assayed by rate of formation of ADP rather than P, it was found that neither phosphate anions nor thiosulfate increased TABLE ANION
II
SPECIFICITY FOR STIMULATION OF THE BRUSH ATPase” BORDER MEMBRANE
Salt
Activity
relative
P formed
to Mg2+-ATPase ADP formed-
(% increase) NaHCO, Tris HCO, Tris Cl NaHCO, NaCl NaNO, NaCH,COO Na*SO, Na,SO, NGW, Na,HPO,
73 + 3 75 + 5 2kO 67 r Sk0 0 21 k 27 k 199 + -
3
4 8 8
48 r 2 174 -c 16 0 0
u ATPase activities were assayed as described in the text, except that the incubation time was 10 instead of 15 min. Fifty millimolar salts were used in all experiments. Mg*+-ATPase activities measured by the formation of P and ADP were 0.274 -r0.013 pmol P/min/mg of protein and 0.220 k 0.010 pmol ADP/min/mg of protein. Each datum represents the mean percentage increase k SEM for 2 to 10 experiments.
I/\TPl ,rnMi
SACKTOR
activity
above the basal level.
Kinetics of the Mg2+ and HCO,--stimulated ATPase. Figure 1 describes the Mg2+- and Mg2+ + HCO,--ATPase activities with respect to ATP concentration. HCO,- enhanced activity at all concentrations of ATP. Double reciprocal plots of both basal and HCO,--stimulated activities were linear. The K, and V for Mg2+ATPase were 0.24 mM and 0.29 pmol PI minlmg of protein. In the presence of HCO,-, both K, and V were increased, and values of 0.37 mM and 0.50 pmol P/ minlmg of protein were estimated. Also plotted in Fig. 1 (dashed lines) is the relationship between “HCO,--ATPase” activity and concentration of ATP. AK,, and V of 0.43 mM and 0.14 pmol P/min/mg of protein were calculated. The effect of varying concentrations of HCO,- on brush border membrane ATPase activity is shown in Fig. 2. The K, for HCO,- was 36 mM and the V was calculated to be 0.26 pmol P/min/mg of protein. To ascertain whether the binding of HCO,affected the binding of ATP, “HCO,--ATPase” activities were determined at different ATP concentrations in the presence of various concentrations of HCO,-. As shown in Fig. 3, HCO,- increased V but had no effect on the K, for ATP. Similarly, the binding of ATP did
l/CATtl
FIG. 1. The Mg*+- and (MgZ+ + HCO,-)-ATPase activities of renal brush border membranes as a function of ATP concentration. (A) and (B) illustrate u versus S and double reciprocal plots of the data, respectively. ATPase activities were determined as described in the text. The concentration of HCO,- was 50 mM. (01, total (Mg *+ + HCO,J-ATPase; (01, Mg2+-ATPase. The difference between total ATPase and MgZ+-ATPase is designated as “HCO,--ATPase” activity (A) and is shown in the dashed line. Each datum is the average of three experiments.
“HCO,--ATPase”
IN BRUSH
BORDER
TABLE NUCLEOTIDE STIMULATED
,/O’ ...” 0 20
,..f 40
60
Dm~-l,mM,
80
”
Nucleotide
../ 0
02
04
06
08
l/IHCO~l
FIG. 2. The effect of concentration of HCO,- on the brush border membrane ATPase activity. In (A) the data for the total (Mg*+ + HCO,-)-ATPase is shown as (0); the “HCO,--ATPase” (01 is shown in the dashed line. (B) illustrates a double reciprocal plot of the “HCO,--ATPase” values. The concentration of ATP was 3 mM. Each datum is the average of three experiments.
IIKPIWm.4-’
lIMC0~-l(mM)-~
FIG. 3. Double reciprocal plots of “HCO,--ATPase” activity as a function of (A) ATP concentration assayed in the presence of the indicated concentrations of HCO,- and (B) HCO,- concentration assayed in the presence of the indicated concentrations of ATP. Each datum is the average of four experiments.
not alter the K, for HCO,-. Nucleotide specificity. The nucleotide specificity of the rabbit renal brush border membrane ATPase is described in Table III. Basal rates of dephosphorylation of the purine nucleotides, ATP, GTP, and ITP, were essentially similar. HCO,- stimulated the hydrolysis of each to about the same degree, 63% for ATP and 56 and 53% for GTP and ITP, respectively. In general, the rate of dephosphorylation of UTP in the presence of Mg*+ was slightly less than those of the purine nucleotides. However, HCO,- stimulated the basal rate of de-
ATP GTP ITP UTP CTP
289
MEMBRANES
0.312 0.358 0.374 0.283 0.227
III
SPECIFICITY OF THE HCO,-ATPase IN BRUSH BORDER MEMBRANE*
Mgz+
MgZ+ + HCO,-
(pm01 Plminlmg
___of protein)
2 k r 2 f
0.020 0.034 0.048 0.031 0.033
(5) (4) (31 (4) (4)
0.509 0.558 0.572 0.375 0.238
-+ 0.025 ? 0.030 k 0.048 2 0.030 2 0.032
(5) (4) (3) (4) (4)
a ATP(nucleoside triphosphatejase activities were determined as described in the text. The nucleotide concentration was 3 mM and the HCO,- concentration was 50 mM. Each datum represents the mean k SEM for the number of experiments indicated in parentheses.
phosphorylation of UTP only 32%. A clear substrate specificity was observed when CTP was tested. With this pyrimidine triphosphate, the basal rate of dephosphorylation was significantly less than that of ATP and it was not stimulated by HCO,-. Stability of the Mg2+- and HCO,--stimuZated ATPase. Mg2+- and Mg2+ + HCO,-ATPase activities of freshly prepared brush border membranes were not increased or decreased by keeping the membranes on ice overnight, by subjecting the membranes to sonication (30 s at 3 A, three times) or to one cycle of freezing and thawing. Preincubation of the membranes with Triton X-100 (30 pglml, equivalent to a detergent:protein ratio of 0.5) for 20 min at 0°C had no apparent effect on the basal or HCO,--stimulated activities. Increasing the Triton X-100:protein ratio to 1.6 caused a 30% loss of the Mg2+-ATPase and an 80% decrease in the anion-stimulated activity. Preincubation of the brush border membranes with deoxycholate (40 to 60 pg/ml, equivalent to a detergent:protein ratio of 0.75) effected a 15 to 20% activation of the Mg2+-ATPase, but was without significant effect on the HCO,--stimulated ATPase. Increases in the deoxycholate:protein ratio to greater than 1.5 resulted in losses of ATPase activities. Kinetics of the S0,2--stimulated ATPuse. In view of the finding that SOZ2- was the most effective activator of the brush border membrane Mg2+-ATPase (Table II),
290
LIANG
AND
the kinetics of the ATPase stimulated by this anion was examined for comparison with that by HCO,-. Figure 4 shows the Mg2+ + S0,2--ATPase activity with respect to concentration of ATP. Enhancement in activity by S0,2- was found at all concentrations of ATP and was greater than that by HCOB- at each nucleotide concentration (Fig. 1). The V for ATPase stimulated by S0,2- was 1.0 pm01 P/min/ mg of protein, a value twice that for Mg2+ + HCO,--ATPase. The presence of SO,‘increased the K, for ATP to 0.47 mM, from 0.29 mM in the absence of the anion. The relationship between “S0,2--ATPase” and ATP concentration is also shown (dashed lines) in Fig. 4. AK, and V of 0.72 mM and 0.70 pm01 P/min/mg of protein, respectively, were calculated. Thus, the V for “S0,2--ATPase” was fivefold greater than that for “HCO,--ATPase” (Fig. 1). The effect of different concentrations of SO,*- on the brush border membrane ATPase activity is described in Fig. 5. The K, for SO,‘- was 0.78 mM, a concentration 50-fold less than that for HCO,- (Fig. 2). Effect of pH on the Mg’+- and anionstimulated ATPase. Examination of the effect of pH on brush border membrane ,HCO,--stimulated ATPase was complicated by the finding that the K, for HCO,was high (36 mM), and by the difficulty in stabilizing the anion at an optimal concen-
FIG. 4. The Mgz+- and (Mg2+ + S0,2-)-ATPase activities of renal brush border membranes as a function of ATP concentration. (Al and (B) illustrate u versus S and double reciprocal plots of the data, respectively. ATPase activities were determined as described in the text, except that the incubation period was 10 min. The concentration of S0,2m was 25 mM. (01, total (Mg*+ + S032V-ATPase; (01, MgZ+-ATPase. The difference between total ATPase and Mg2+-ATPase is designated as “S032--ATPase” activity (A) and is shown in the dashed line. Each datum is the average of three experiments.
SACKTOR
0
20
10 NazS03
30
(mM)
FIG. 5. The effect of concentration of Na$SO, on the brush border membrane ATPase activity. Total (Mg*+ + S0,2-)-ATPase is shown as (0); the “SO,‘-ATPase” (0) is shown in the dashed line. The concentration of ATP was 3 mM. Incubation time was 10 min. The insert shows a double reciprocal plot of the “SO,*--ATPase” values. Each datum is the average of two experiments.
tration, considering that the pK’ of H,CO, is approximately 6.3 and the potential loss of CO,. To circumvent this problem, the effect of pH on the ATPase was studied with SOs2- as the activating anion. In this case, the pK1 and pK2 for H&SO3 are approximately 2 and 7.2, respectively, and the K, was found to be only 0.78 mM. Thus, an optimal concentration of activator could be maintained throughout the physiological pH range by using 25 mM Na2S0, in the incubation mixture. Figure 6 describes the pH profile for the brush border membrane Mg2+- and Mg2+ + SO,‘--ATPase activities. Mg2+-ATPase activity gradually increased from 0.14 to 0.35 pmol P/min/mg of protein as the pH was raised from 6.1 to 8.3. In contrast, the S0,2--stimulated activity increased sharply within a narrow pH range, 6.1 to 7.1, and increased moderately at higher pH values. Because brush border membranes were reported to possess alkaline phosphatase and 5’-nucleotidase activities (28), the possible contributions of these phosphatases to the rate of formation of P with increased alkalinity and presence of SOa2- were evaluated. As
“HCO,--ATPase”
IN BRUSH
Or
a#fl Hg . 6.0
70
8.0
PH
FIG. 6. The effect of pH on ATPase activities of brush border membranes. (01, total (MgZ+ + S0,2m)ATPase; (O), Mg2+-ATPase. The “SO,*--ATPase” (A) is shown in the dashed line. The ATP and Na$O, concentrations were 3 and 25 mM, respectively. Values are the averages of three experiments. The dephosphorylation of 3 mM AMP, in the presence (D) or absence (0) of Na2S03, at the different pH values is shown. Each datum represents the average of two experiments.
shown in Fig. 6, low rates of dephosphorylation of AMP were found at the different pH values, in the absence and presence of S0,2-. This indicated that these phosphatases were not activated by SOs2- nor did they contribute significantly to the sharp increase in the rate of dephosphorylation of ATP observed in the presence of S0,2- in the pH range 6.1 to 7.1. The response of the S0,2--stimulated ATPase to pH, illustrated in Fig. 6, raised the question as to whether the increase in activity with higher pH merely reflected an increase in the concentration of the SOs2- species or was, in fact, an intrinsic effect on the enzyme. In the physiological pH range, sulfite exists principally as SOs2- and HSO,-. The concentration of H,SO, is negligible, pK’ being about 2. To answer this question, the Na2S03 concentration in the reaction mixtures was adjusted so that at each pH value the concentration of the SOs2- species was maintained at 9.6 mM, although the concentration of the HSO,- species varied from 38.4
BORDER
291
MEMBRANES
mM at pH 6.4 to 0.4 mM at pH 8.3. The pH profile of the anion-stimulated ATPase activity, at this constant level of S032-, is described in Fig. 7. Comparison of Figs. 7 and 6 revealed the marked similarity in whether concentration of pH profiles, S0,2- was held constant or varied. These results, therefore, suggested that the effect of pH on the stimulated enzyme activity was on the ATPase itself rather than due to an increase in the concentration of activator species. Moreover, the relatively large sulfite-stimulation at pH 8.3, when the concentration of HSO,- was low (0.4 mM), and the relatively small activation when the concentration of HSO,- was high (38.4 mM), strongly supported the view that S0,2- was, indeed, the anion species that activated the ATPase. Effect of inhibitors. In previous studies it was reported that SCN-, which in-
O.l}
0.
7.0
6.0
PH
7. The effect of pH on SO,‘--stimulated ATPase at a constant concentration of SO,*-. The concentration of the S0,2m species was maintained at 9.6 mM at different pH values by the use of 48,25, 22, 12, and 10 mM NalSO, at pH 6.4, 6.8, 7.1, 7.6, and 8.3, respectively. The ATP concentration was 3 mM. CO), total (Mg*+ + SO,*-)-ATPase; (01, Mg2+-ATPase; (A), “S032m-ATPase” in the dashed line. Each datum represents the average of two experiments. FIG.
292
LIANG
AND
hibited acid secretion in gastric mucosa, salivary gland, and pancreas (l-4, 7, 81, was a specific inhibitor of HCO,--stimulated ATPase. In contrast, SCN- was found in the present study to be an ineffective inhibitor of the renal brush border membrane ATPase (Fig. 8). As shown, 1 mM NaSCN had no effect on the rate of dephosphorylation and 10 mM inhibited 30% or less. These same concentrations of SCN- inhibited 60 and lOO%, respectively, of the HCO,--stimulated ATPase in the submandibular gland (7). Moreover, in the brush border membranes SCN- did not distinguish between Mg*+-ATPase activity or the activities stimulated by HCO,- and SO,*-. Interestingly, Ullrich et al. (31) recently reported that H+ secretion in the microperfused proximal tubule was in variance with that in the stomach and pancreas in that in the kidney the process was insensitive to SCN-. The HCO,--stimulated ATPase in the renal brush border membrane also differed from the enzymic activity in the gastric mucosa in its sensitivity to methane sulfonyl chloride. In the renal membrane preparation, as illustrated in Fig. 9, Mg*+ATPase was strongly inhibited by concentrations of the inhibitor (5 to 10 mM) which had no effect on the “HCO,--ATPase.” The
I
0
5 [NaSCNl
10 (mM)
FIG. 8. The inhibition of anion-stimulated ATPase of brush border membranes by NaSCN. ATP concentration was 3 mM. When indicated, 50 mM NaHCO, or 25 rnM Na2S0, was used. (01, Mg2+ATPase; (01, (Mg*+ + HCO,-)-ATPase; (a), (Mg’+ + SO,*-l-ATPase. The 100% activity was, respectively, 0.218, 0.349, or 0.569 pmol P/min/mg protein. Each value represents the average of three experiments.
SACKTOR
“--i;-ll
0 I 1o-4
1o-2
CMSC~(M) FIG. 9. The effect of methane sulfonyl chloride (MSC) on the ATPase activities of brush border membranes. ATPase activities were determined as described in the text. (01, total (Mg”+ + HCO,-lATPase; (0) Mg*+-ATPase; (A,, “HCO,--ATPase” shown in the dashed line. Each value represents the average of three experiments.
relative insensitivity of the anion-stimulated ATPase in brush border membranes to this inhibitor was also evident when S0,2- was used as activator. In these experiments, not illustrated, Mg*+-ATPase activity in the absence and presence of 5 mM methyl sulfonyl chloride was 0.212 and 0.132 pmol Plminlmg of protein, respectively. On the other hand, Mg*+ + SO,*-ATPase activities were 0.543, in the absence, and 0.454, in the presence of inhibitor. Thus, the Mg*+-ATPase was inhibited 38%, whereas the “SO,*--ATPase” activity was unaffected. In contrast, Blum et al. (4) reported that Mg*+ + HCO,--ATPase was inhibited 50% by 1 mM methane sulfonyl chloride whereas Mg2+-ATPase was not inhibited by this concentration of inhibitor. In other experiments, not shown, oligomycin (12.5 g/ml) inhibited the Mg*+- and Mg*+ + HCO,--ATPase activities in the brush border membranes by 23 and 36%, respectively. At this concentration of oligomycin, the renal cortex mitochondrial ATPase was inhibited over 95%. The effect of quercetin, a recently described inhibitor of mitochondrial ATPases (321, was also tested. At concentrations of 50 and 100 pg/ ml, levels greatly in excess of that needed to inhibit mitochondrial ATPase, brush border membrane Mg*+- and Mg2+ + HCO,--ATPase activities were inhibited only 6 and 13%, respectively, at the lower concentration, and 24 and 25%, respec-
“HCO,--ATPase”
IN BRUSH
tively, at the higher concentration of inhibitor. FCCP had little effect on the Mg’+- or Mg2+ + HCO,--ATPase activities in brush border membranes. Since the usual reaction medium for measuring ATPase activity in the present study contained high concentrations of salts, i.e., 50 mM NaHCO, and 75 mM Tris chloride buffer, and it was found previously that high concentrations of salt made the membranes more permeable to various ions and presumably H+ (211, the effect of FCCP was tested additionally using a reaction system in which salt concentrations were minimized, i.e., 2 mM Tris-Hepes, as buffer, and 2 mM Na,SO,, as activator. In this case, as shown in Table IV, the proton conducting agent stimulated basal ATPase activity by 23%, and Mg2+ + S0,2-ATPase activity by little, it at all. Effect of carbonic anhydrase on the HCO,--stimulated ATPase. Added carbonic anhydrase significantly increased HCO,--stimulated ATPase activity (Table V). In contrast, Mg2+-ATPase was not affected. The carbonic anhydrase mediated increment in HCO,--stimulated ATPase activity was completely abolished by Diamox (sodium acetazolamide), a specific inhibitor of carbonic anhydrase (33). Because carbonic anhydrase added exogenously to the reaction mixture would increase the formation of CO*, and the escape of this CO, would tend to make the reaction medium more alkaline, the pH during the course of the incubation was carefully monitored. In these experiments the pH gradually rose from 7.53 to 7.60. As shown in Fig. 6, however, this increase of TABLE
IV
OF FCCP ON THE ANION-STIMULATED ATPase IN BRUSH BORDER MEMBRANES”
EFFECT
MgZ+-ATPase (pm01 P/min/mg Control +FCCP
0.233 0.286
Mg*+ + SO,*--ATPase of protein) 0.530 0.565
n The reaction mixture contained 300 mM sucrose, 2 mM Tris-Hepes, pH 7.5, 3 mM MgCl,, 1.5 mM ouabain, 3 mM tris-ATP, -r- 2 mM Na,SO,, f 10 FM FCCP. The incubation time was 5 min. Each datum is the average of two experiments.
BORDER
293
MEMBRANES TABLE
V
EFFECT OF CARBONIC ANHYDRASE ON THE HCO,-STIMULATED ATPase IN BRUSH BORDER MEMBRANES’” Mg2+-ATPase
Experiment Car-
(pm01 Plminlmg
Diamox
“HCO,-ATPase” of protein)
bonic anhydrase -
-
0.281 0.301 0.296 0.310
+ -
+ +
+
f k f ”
0.024 0.031 0.026 0.029
0.166 0.163 0.208 0.172
t + k f
0.009 0.011 0.016 0.011
” ATPase activities were determined as described in the text. “HCO,--ATPase” represents the difference in activities between total (Mg*+ + HCO,-)ATPase and MgZ+-ATPase. When indicated, 40 units of carbonic anhydrase were used. Each unit is defined as the enzyme activity required to reduce the pH of a CO,-containing solution from 8.7 to 6.7 twice as fast as the pH of the blank, assayed by Boehringer. When indicated the Diamox concentration was 0.5 mM. Each datum represents the mean t SEM for four experiments.
0.07 units was too small to account for the 25% increase in “HCO,--ATPase” activity that was found in the experiments in which carbonic anhydrase was present (Table V). Figure 10 demonstrates that the effect of carbonic anhydrase on the kinetics of the “HCO,--ATPase” was to increase V without changing the K, for ATP. When the effect of carbonic anhydrase was examined with respect to concentration of HC03-, both V and K, were found to increase. DISCUSSION
The results described in this study show that the specific activity of the “HC03-ATPase” in renal brush border membranal preparations was increased about fourfold relative to that in the renal cortex homogenate. Mitochondrial contamination could account maximally for only 15% of the activity in the brush border preparation. These findings indicate the presence of a HCO,--stimulated ATPase in the proximal tubule luminal membrane. This membrane is the major site of H+ secretion and bicarbonate3 reabsorption in the kidney 3 “Bicarbonate” term
without
reabsorption
specifying
the species transported
is used as a general HCO,- or CO, is across the membrane. whether
294
LIANG
AND
SACKTOR
mM, when the ATPase was activated by SO,z- or HC03-, is in accord with the findings that the K, for ATP also varied with the anion with the isolated F, component of the mitochondrial ATPase (11, 35). An anion-dependent conformational change of l/lATPl imw' l/[HCOO-lhMi-' enzyme subunits which mediates an active/inactive state transition was sugFIG. 10. The effect of carbonic anhydrase on the gested by Moyle and Mitchell (35). kinetics of the HCO,--stimulated ATPase of brush The acceleration of the rates of dephosborder membranes. ATPase activities were deterphorylation of ATP, GTP, and ITP by 50 mined as described in the text and in Table V. (01, of Mg*+-ATPase; (O), MgZ+-ATPase in the presence of mM HCO,- was the same. Stimulation carbonic anhydrase; (A), “HCO --ATPase”; (A), the rate of dephosphorylation of UTP was “HCO,--ATPase” in the presence if carbonic anhy considerably less, and with CTP it was drase. Each value represents the average of three negligible. This nucleotide specificity with experiments. rabbit renal brush border membranes differs from that reported with microvillar membranes of rat kidney (16) in which the rates of dephosphorylation of GTP, ITP, and, if the general concept of the coupling UTP, and CTP were 20% that for ATP. of ATP hydrolysis to cation translocation ATPases in other across membranes (34) is applicable to the The HCO,--stimulated extrusion of H+ from renal tubular cell to preparations also exhibited varied nucleoIn gastric mucosa (4), lumen, then the finding of a brush border tide specificities. HCO,--stimulated ATPase takes on added GTP was hydrolyzed at about 50% the rate for ATP, UTP about lo%, and CTP not at significance. (ll), at a HCO,The renal brush border ATPase was all; in liver mitochondria concentration of 20 mM, the V for ATP and stimulated by several anions. In addition to HC03-, SOX2- was a potent activator of ITP were the same, whereas that for GTP the brush border ATPase. CH,COO- and was half this rate. The present findings that carbonic anS0,2- were less effective than HCOs-, whereas phosphate anions and CL- had no hydrase increased the rate of the brush membrane HCO,--stimulated effect on ATPase activity. Thus, of the border ATPase and this enhancement was anions present in the glomerular filtrate blocked by Diamox may be relevant to proin appreciable concentrations, i.e., HC03-, posed mechanisms of H+ and bicarbonate Cl-, and phosphates, only HCO,- stimulated the luminal membrane ATPase. transport. In the intact dog, 40% of the Moreover, the K, for HCO,-, i.e., 36 mM, filtered bicarbonate was excreted when approximates the physiological concentracarbonic anhydrase was inhibited by Diation of the anion in the glomerular filtrate. mox (36). Diamox prevented acidification The selective stimulation of the renal of the filtrate in the proximal tubule in brush border ATPase by anions, e.g., situ (37), and it inhibited the secretory HCO,- and SOS*-, confirms previous studrate of H+ in perfused tubules (31). As ies on the activation by various anions of illustrated diagrammatically in Fig. 11, crude membranal ATPases from gastric carbonic anhydrase in the proximal tubumucosa (4) and pancreas (9) and of the lar cell has a dual localization; approxihighly purified enzyme from mitochondria mately 90% of the activity is found in the (11, 35). Also consistent with the view that cytosol, the remainder is localized in the HC03- and SOs2- stimulate the same, brush border membrane (38). According to rather than distinct, enzyme is the finding one generally accepted hypothesis for bithat gel electrophoresis of membranes incarbonate transport (14, 15), extrusion of dicated a single ATPase band, sensitive to H+ from cell to lumen favors the converboth HCO,- and S03*- (9). The present sion of HCO,- to H,CO, in the glomerular observation that the K, for ATP was filtrate. The brush border carbonic anhyslightly different, 0.72 compared to 0.43 drase enhances the breakdown of this
“HCO,--ATPase”
+
IN BRUSH
BORDER
MEMBRANES
295
tion of ATP by about 25% is consistent with the presence of a membranal ATPase Na' r + _ whose activity is limited by transmembrane H+ flux. This argues for a role for the enzyme in the transport of H+. In addition, Diamox, which inhibited the carbonic anhydrase enhanced “HCO,--ATPase” in brush border membranes (Table V), also reduced the lumen-positive active transport potential in loops of proximal tubules (42). Strengthening the hypothesis further is the suggestion that the bicarbonate species which is transported across brush borANTILUMINAL LUMINAL BASAL-LATERAL BRUSH BORDER der membrane vesicles is probably CO, MEMBRANE MEMBRANE (22). Lastly, Pitts and Alexander (43) from FIG. 11. A diagrammatic model for bicarbonate clearance measurements in dogs first posreabsorption in the renal proximal tubular cell and tulated an exchange of cellular H+ for filthe postulated role of the HCO,--stimulated ATPtered Na+, and this view was supported by ase. stop-flow microperfusion studies in rats (31). Moreover, studies in which isolated H&O, to CO, and H,O. The COz, thus kidneys were perfused with HCO,--free formed, is transported across the brush media or with media containing HC.O,- + border membrane into the cell, where it is Diamox showed that reabsorption of bicarhydrated by the cytosolic carbonic anhybonate accounts for a small but significant drase. Dissociation of the H&O, generates fraction of the Na+ reabsorbed (44). Thus, intracellular HCO,- and H+. A significant exchange of H+ for Na+ may represent one share of the bicarbonate that is reabsorbed of the mechanisms for maintaining cellufrom the glomerular filtrate has been at- lar electroneutrality. Interestingly, expertributed to H+ secretion by this mechaiments with brush border membrane vesinism (39, 40). It is now further postulated, cles currently in progress in this laboraas diagrammed in Fig. 11, that the brush tory4 indicate a coupling of the transports border ATPase is involved in the extrusion of the two cations. of H+ from cell to lumen and that intracelOn the other hand, there are observalular HCO,- by stimulating this ATPase tions which at this time are seemingly augments translocation of H+ across the drawbacks to the tentatively proposed membrane. Thus, reabsorbed bicarbonate, scheme. As diagrammed in Fig. 11, in the form of HC03-, may act as a positive HCOa--stimulated ATPase is localized on effector promoting the reabsorption of ad- the inner surface of the brush border memditional bicarbonate. brane, thus accessible to intracellular Other evidence is available which lends ATP. In the present in vitro experiments, support for this hypothesis. That a “pump” the nucleotide was added to the medium, mechanism is needed to extrude H+ from external to the membrane vesicle. Alproximal tubular cell to lumen via the though membranes are presumed to be relbrush border membrane is indicated from atively impermeable to ATP, sonication findings that the transmembrane electroand detergents, which disrupt membrane chemical potential of the brush border is integrity, did not enhance rates of dephospositive on the outside and negative on the phorylation. Admittedly, the sidedness of inside (22, 41, 42). In other membrane sys- the population of vesicles is not known. It tems, ATPases are known to couple ATP is also probable that after sonication the hydrolysis to H+ translocation against membranes resealed. Tending to refute electrochemical gradients (34). The pres- this objection further are the findings that ent finding that in brush border membranes the proton-conductor, FCCP, in4 Chernoff, A., and Sacktor, B., unpublished observations. creased the basal rate of dephosphorylaHCOS-
l c-
_
+ _
HCOS-
l _
1
HCOS-
296
LIANG
AND
high concentrations of salts such as used in most of the present experiments increased membrane permeability (21) and that the kidney in contrast to other mammalian tissues is readily permeable to added adenine nucleotides (45). Another apparent inconsistency to the proposed hypothesis relates to the effect of carbonic anhydrase on the kinetics of the HCO,--stimulated ATPase. If the action of carbonic anhydrase is to accelerate the formation of CO, in the medium, then an increase in the HCO,--stimulated ATPase activity is understandable as more CO, is available to diffuse across the brush border membrane. The affinity of HC03- to the ATPase should not be altered. As shown in Fig. 10, however, the effect of carbonic anhydrase was to increase both V and the K, for HC03-. The explanation of this increase in K, remains to be formulated. A second mechanism for the reabsorption of the filtered bicarbonate in the proximal tubule depends on the transport of bicarbonate in its ionic form, rather than as CO, (14, 15). Indeed, a considerable fraction of the total bicarbonate may be transported by this process. This fraction would not be directly affected by H’ extrusion. Nevertheless, if HCO,- is cotransported with Na+ (441, then mechanisms which mediate the eMux of H+ will enhance the influx of Na+ and, indirectly, of HCO,-. Thus, in both mechanisms of bicarbonate transport the suggestion that bicarbonate reabsorption, H+ secretion, and the HC03--stimulated ATPase are interrelated remains a viable hypothesis which merits further testing.
SACKTOR
6.
7. 8. 9. 10. 11. 12. 13.
14.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
REFERENCES 1. KASBEKAR, D. K., AND DURBIN, R. P. (1965) Biochim. Biophys. Actu 105, 472-482. 2. SACHS, G., MITCH, W. E., AND HIRSCHOWITZ, B. I. (1965)Proc. Sot. Exp. Biol. Med. 119: 10231027. 3. WIEBELHAUS, V. D., SUNG, C. P., HELANDER, H. F., SHAH, G., BLUM, A. L., AND SACHS, G. (1971) Biochim. Biophys. Actu 241, 49-56. 4. BLUM, A. L., SHAH, G., ST. PIERRE, T., HELANDER, H. F., SUNG, C. P., WIEBELHAUS, V. D., AND SACHS, G. (1971) Biochim. Biophys. Actu 249, 101-113. 5. SACHS, G., SHAH, G., STRYCH, A., CLINE, G.,
26.
27.
28.
29. 30.
AND HIRSCHOWITZ, B. I. (1972) Biochim. Biophys. Actu 266, 625-638. DE PONT, J. J. H. H. M., HANSEN, T., AND BONTING, S. L. (1972) Biochim. Biophys. Acta 274, 180-200. IZUTSU, K. T., AND SIEGEL, I. A. (1972) Biochim. Biophys. Actu 284, 478-484. SIMON, B., KINNE, R., AND SACHS, G. (1972) Biochim. Biophys. Actu 282, 293-300. SIMON, B., ANDTHOMAS, L. (1972)Biochim. Biophys. Actu 288, 434-442. FANESTIL, D. D., HASTINGS, A. B., AND MAHOWALD, T. A. (1963) J. Biol. Chem. 238, 836-842. EBEL, R. E., AND LARDY, H. A. (1975) J. Biol. Chem. 250, 191-196. RACKER, E. (1962) Fed. Proc. 21, 54. GOTTSCHALK, C. W., LASSITER, W. E., AND MYLLE, M. (1960) Amer. J. Physiol. 198, 581585. RECTOR, F. C., JR. (1973) in Handbook of Physiology, (Orloff, J., and Berliner, R. W., eds.), Section 8, pp. 431-454, Waverly Press, Baltimore. MALNIC, G., AND GIEBISCH, G. (1972) Kidney Internat. 1, 280-296. KINNE-SAFFRAN, E., AND KINNE, R. (19741Proc. Sot. Exp. Biol. Med. 146, 751-753. CHESNEY, R. W., SACKTOR, B., AND ROWEN, R. (1973) J. Biol. Chem. 248, 2182-2191. CHESNEY, R., SACKTOR, B., AND KLEINZELLER, A. (1974)Biochim. Biophys.Actu 332,263-277. ARONSON, P. S., AND SACKTOR, B. (1974) Biochim. Biophys. Actu 356, 231-243. MITCHELL, M. E., ARONSON, P. S., AND SACKTOR, B. (1974) J. Biol. Chem. 249, 6971-6975. ARONSON, P. S., AND SACKTOR, B. (1975) J. Biol. Chem. 250, 6032-6039. BECK, J. C., AND SACKTOR, B. (1975) J. Biol. Chem. 250, 8674-8680. BERGER, S. J., AND SACKTOR, B. (1970) J. Cell Biol. 47, 637-645. LARDY, H. A., AND WELLMAN, H. (1952) J. Biol. Chem. 195, 215-224. CHEN, P. S., JR., TORIBARA, T. Y., AND WARNER, H. (1956) Anal. Chem. 28, 1756-1758. BERNATH, P., AND SINGER, T. P. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, pp. 597-614, Academic Press, New York. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. SACKTOR, B. (1976) in Mammalian Cell Membranes (Jamieson, G. A., and Robinson, D. M., eds.), Vol. 4, Butterworths, London, in press. FILBURN, C., AND SACKTOR, B. (1976)Arch. Biothem. Biophys. 174, 249-261. GRISOLIA, S., AND MENDELSON, J. (1974) Bio-
“HCOsm-ATPase”
IN BRUSH
them. Biophys. Res. Commun. 58, 968-973. 31. ULLRICH, K. J., RUMRICH, G., AND BAUMANN, K. (1975) Pflugers Arch. 357, 149-163. 32. LANG, D. R., AND RACKER, E. (1974) Biochim. Biophys. A& 333, 180-186. 33. MAREN, T. H. (1967) Physiolog. Rev. 47,595-781. 34. MITCHELL, P. (1973) FEB.!? Lett. 33, 267-274. 35. MOYLE, J., AND MITCHELL, P. (1975) FEBS Lett. 56, 55-61. 36. BERLINER, R. W. (1952)Fed. Proc. l&695-700. 37. CLAPP, J. R., WATSON, J. F., AND BERLINER, R. W. (1963) Amer. J. Physiol. 205, 693-696. 38. MAREN, T. H., AND ELLISON, A. C. (1967) Mol. Pharmacol. 3, 503-508.
BORDER
MEMBRANES
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39. RECTOR, F. C., JR., CARTER, N. W., AND SELDIN, D. W. (1965) J. Clin. Invest. 44, 278-290. 40. VIEIRA, F. L., AND MALNIC, G. (1968) Amer. J. Physiol. 214, 710-718. 41. MARUYAMA, T., AND JOSHI, T. (1972) Biochim. Biophys. Acta 282, 214-225. 42. FROMTER, E., AND GESSNER, K. (1975) Pflugers Arch. 357, 209-224. 43. PITTS, R. F., AND ALEXANDER, R. S. (1945)Amer. J. Physiol. 144, 239-254. 44. BESARAB, A., SILVA, P., Ross, B., AND EPSTEIN, F. H. (1975) Amer. J. Physiol. 228, 1525-1530. 45. WEIDEMANN, M. J., HEMS, D. A., AND KREBS, H. A. (1969) Biochem. J. 115, l-10.
OF
BIOCHEMISTRY
AND
Bicarbonate-Stimulated
BIOPHYSICS
ATPase in the Renal Proximal (Brush Border) Membrane
C. TONY Laboratory
176, 285-297 (1976)
LIANG
of Molecular Aging, Gerontology Institutes of Health, Baltimore
AND
BERTRAM
January
Luminal
SACKTOR
Research Center, National Institute City Hospitals, Baltimore, Maryland
Received
Tubule
on Aging, 21224
National
26, 1976
Brush border membranes of the rabbit renal tubule have an ATPase which was stimulated 60% by 50 mM HCO,-. The K, for HCO,- was 36 mM. Kinetic studies of the “HCO,--ATPase” indicate that HCO,- had no effect on the K, for ATP and ATP did not alter the K, for HCOsm. Several anions, notably SO 32-, also accelerated the rate of dephosphorylation of ATP. The V for “SO 32--ATPase” was fivefold greater than that for “HCO,--ATPase.” The K, for SO,*- was 0.78 mM. Other anions including Cl- and phosphates, did not enhance ATPase activity. Thus, of the anions present in the glomerular filtrate in appreciable concentrations only HCO,- stimulated the luminal membrane enzyme. The anion-stimulated ATPase activity increased sharply from pH 6.1 to 7.1 and moderately with higher pH. The renal ATPase was not inhibited by SCNnor methyl sulfonyl chloride and was relatively insensitive to oligomycin and quercetin. Carbonyl cyanide p-trifluoromethoxy phenylhydrazone increased the basal rate of the membranal ATPase, suggesting that the ATPase activity is limited by transmembrane H+ flux. Carbonic anhydrase significantly increased the HCOs--stimulated ATPase activity. This increment was blocked by Diamox. These findings provide evidence consistent with the hypothesis that the brush border membrane ATPase is involved in the extrusion of H+ from tubular cell to lumen and support suggested interrelationships between HCO,--stimulated ATPase; H+ secretion, and bicarbonate transport in the kidney.
Bicarbonate transport and acid secretion are found in the stomach, salivary gland, and pancreas. It is of interest, therefore, that these organs possess an active HCOs--stimulated ATPase (l-9). Subcellularly, the enzyme is localized in the “microsomal” fraction (3-81, although enhancement of ATPase activity by HCO,and other anions is reported in mitochondrial preparations from liver (10, 11) and heart (121, as well as gastric mucosa (4). In the kidney, 80 to 90% of the filtered bicarbonate is reabsorbed in the proximal tubule (131, and acidification of the tubular fluid is a prominent feature of renal function (14, 15). This focuses attention on the possible role of the proximal tubule luminal (brush border) membrane in these transport processes. Indeed, the presence of a HCO,--stimulated ATPase in a membrane fraction enriched in microvilli has Copyright 0 1976 by Academic All rights
of reproduction
Press, Inc. in any form reserved.
been noted (16). Our previous work with well-characterized proximal tubule brush border membrane preparations as model systems for studying renal transport mechanisms (17-23) prompted us to examine further the HCOa--stimulated ATPase in renal brush border membranes. In this paper we report some kinetic properties of the enzyme and provide evidence relevant to the possible interrelationships between the membrane HCO,--stimulated ATPase, proton secretion, and bicarbonate reabsorption in the renal tubule. EXPERIMENTAL
PROCEDURES
Materials. Nucleotides (sodium salts) were purchased from P-L Biochemicals. Pyruvate kinase, lactate dehydrogenase, carbonic anhydrase, and phosphoenolpyruvate were obtained from Boehringer. Oligomycin and ascorbic acid were from Sigma. Carbonyl cyanide p-trifluoromethoxy phenylhydra285
286
LIANG
AND
zone (FCCP)’ was purchased from Pierce Chemicals. Quercetin was kindly donated by Dr. E. Racker, Cornell University. Other chemicals were reagent grade. Preparation of membranes. Brush border membranes, derived from the proximal tubules of the rabbit kidney cortex, were isolated by a modification (21) of the procedure previously detailed (23). The brush border membranes sedimenting in the final centrifugation were resuspended in a medium comprised of 75 mM Tris chloride, pH 7.4, 6 mM MgCl,, and 160 mM sucrose. The quality of preparations was randomly evaluated by specific enzyme markers
(23). Mitochondria from the rabbit renal cortex were isolated by the procedure of Lardy and Wellman (24) for rat liver mitochondria. The isolation medium contained 210 mM mannitol, 70 mM sucrose, 0.1% bovine serum albumin, and 1 mMN-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes), adjusted to pH 7.4 with Tris hydroxide. Determination ofATPase activities. Total ATPase activity was assayed in a 0.2-ml reaction mixture containing 75 mM Tris chloride buffer, pH 7.4, 6 mM MgCl,, 3 mM Na,ATP, 1.6 mM ouabain, and 50 mM NaHC03. Sucrose was added to maintain tonicity at 300 mOSM. The NaHCO, solution was prepared and titrated to pH 7.4 immediately prior to use. Except when indicated, the incubation was carried out for 15 min at 37°C. The reaction was terminated by the addition of 0.1 ml of 12% perchloric acid, followed by placing the tubes in ice. The inorganic phosphate liberated was determined by the method of Chen et al. (25). Mg2+-ATPase activity was measured in the identical manner except that NaHCO, was omitted. The difference between total ATPase activity and Mg*+-ATPase activity was taken to be the “HCO,-ATPase” activity. In determining the effect of pH on ATPase activities, the buffer used was 40 mM Tris plus 40 mM piperazine-iV,N’-bis(2-ethanesulfonic acid) (Pipes) adjusted with HCl to the indicated final pH value. In experiments in which ATPase was measured in the presence of inorganic phosphate or thiosulfate activity was determined by ADP production. The ATPase reaction was carried out and terminated as described above. The mixture was neutralized with KOH and the insoluble potassium perchlorate was removed by centrifugation. Aliquots of the supernatant were assayed spectrophotometrically for ADP in a medium containing 90 pmol of Tris chloride, pH 7.4, 2.5 pmol of MgCI,, 5 pmol of KCl, 0.15 Fmol of phosphoenolpyruvate, 0.15 pmol of NADH, 20 pg of ’ Abbreviations used: FCCP, carbonyl cyanide ptrifluoromethoxy phenylhydrazone; Hepes, N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid; Pipes, piperazine-N,N’-bis(2-ethanesulfonic acid); MSC, methane sulfonyl chloride.
SACKTOR pyruvate kinase, and 8 pg of lactate dehydrogenase in a total volume of 1 ml. Other methods. Succinic dehydrogenase activity was determined polarographically (26). Protein was measured by the procedure of Lowry et al. (27) using bovine serum albumin as the standard. RESULTS
Stimulation of ATPase activity by HCO,-.Total (Mg2+ + HCO,-)-ATPase and Mg2+-ATPAse activities in the renal brush border membranal preparations were linear with protein concentration from 4 to 20 pg per reaction mixture and with time of incubation for at least 20 min. As shown in Table I, the presence of 50 mM HCO,- increased the Mg2+-ATPase activity of isolated brush border membranes from 0.220 to 0.345 pm01 P/min/mg of protein, a stimulation of 57%. The activity in the homogenate of the renal cortex was also enhanced by the anion. Relative to the specific activity in the homogenate, the “HCO,--ATPase” in the brush border membranal preparation was increased 4.3fold, 0.028 compared to 0.118 pmol P/min/ mg of protein. This indicated an enrichment of the activity in these membranes. This increase in relative specific activity, however, was not as great as that found for enzymes localized exclusively in brush border membranes, e.g., trehalase and yglutamyl transpeptidase (28). Rather, the increase was comparable to the enrichment found with enzymes, e.g., cyclic AMP phosphodiesterase (29), that were present in other membranes of the renal cortex in addition to the brush border membrane. In view of the reported presence of a HCO,--stimulated ATPase in mitochondria from various tissues (lo-12), mitochondria from the renal cortex were examined and found to contain an ATPase that was enhanced about 60% by HCO,-. Sonication of the isolated mitochondria increased the Mg2+- and Mg2+ + HCO,-ATPase activities several-fold. In fact, the specific activity of the “HCO,--ATPase” in sonicated mitochondria was greater by approximately 50% than in the brush border membranes. Estimates, based on relative specific activities of “HCO,--ATPase,” succinic dehydrogenase, and y-glutamyl transpeptidase in homogenates and mitochon-
“HCO,--ATPase”
IN BRUSH
BORDER
TABLE LOCALIZATION OF THE HCOJ--S~~~~~~~~~ Preparation Crude homogenate Brush border membrane Mitochondria Intact Sonicated
Total ATPase
287
MEMBRANES
I
ATPase ACTIVITY IN THE RENAL CORTEX” “HCO,--ATPase” Mg2+-ATPase (pm01 Plminlmg of protein)
0.097 + 0.007 (4) 0.345 ‘- 0.020 (16)
0.069 L 0.003 (4) 0.220 ? 0.017 (16)
0.028 + 0.005 (4) 0.118 r 0.008 (16)
0.139 + 0.018 (5) 0.470 k 0.120 (4)
0.085 + 0.009 (5) 0.290 k 0.070 (4)
0.054 t 0.019 (5) 0.190 + 0.060 (4)
(LATPase activities were assayed as described in the text. Total (Mg2+ + HCO,-)- and Mg2+-ATPase represent the activities in the presence and absence of 50 mM NaHCO,, respectively. The difference between total ATPase and Mg*+-ATPase is indicated as the “HCO,--ATPase.” Each datum represents the mean _t SEM for the number of experiments indicated in parentheses.
der membranes was negligible.2 This condrial and brush border membranal preparations indicate that mitochondria and clusion was deduced from the findings that brush border membranes possessed 43 and renal cortex homogenates had a y-glutamy1 transpeptidase specific activity of 34 57%, respectively, of the total “HC03-ATPase” activity. pmol/h/mg of protein, brush border memSince electron micrographs of brush bor- branal preparations had a specific activity der membranal preparations as well as of 220, whereas mitochondrial preparaprevious measurements of marker en- tions had a value of only 3 ~mol/h/mg of zymes, i.e., cytochrome oxidase, indicated protein. Thus, contamination of the mitoa small, albeit significant, contamination chondrial preparation with brush border of the preparation with some intact but membranes was less than 1.5%. Assuming mostly fragmented mitochondria (231, it that the brush border membranal prepara was important to estimate how much of tion was 10% mitochondrial fragments, the HCO,--stimulated ATPase found in then from the data in Table I it was evibrush border membranal preparations dent that the contributions of mitochoncould be maximally attributed to mitodria to the Mg2+- and HCO,--stimulated chondrial contamination. For this reason ATPases in the brush border preparations succinic dehydrogenase activities were were 0.029 and 0.019 pmol P/min, respecmeasured in preparations of mitochondria tively; i.e., less than 15% of the ATPase This and brush border membranes. activities determined in the brush border marker enzyme was used because purified membranal preparations could be attribF,, the major mitochondrial ATPase, is an uted to mitochondria. inner membrane constituent and was Anion specificity. Stimulation of the shown to be activated by HCO,- (111, al- Mg2+-ATPase in brush border membranes though the presence of some HCO,--stimuby NaHCO, was due to HCO,- not Na+. As lated ATPase activity in the mitochondrial shown in Table II, Tris bicarbonate inouter membrane could not be excluded creased activity to the same magnitude as (30). The specific activity of the dehydrodid NaHCO,. On the other hand, neither genase in sonicated mitochondria (which Tris chloride nor NaCl had a stimulatory was almost twice that in intact mitochoneffect. However, other anions did increase dria) was 0.22 ng-atoms oxygenlminlmg of basal ATPase activity. When tested as its protein. This value compared to 0.022 ng- sodium salt, SO,“- enhanced ATPase acatomslminlmg of protein for the dehydrotivity 200%, a value threefold that found genase in the brush border membranal with HCO,-. The anions, CH,COO- and preparations. These findings suggested S042-, also increased ATPase activity, but that these brush border membranal prepaonly by about 20 to 30%. In contrast, NO,rations were contaminated by about 10% and Cl- were ineffective. In another set of with mitochondria. Contamination of miz Filburn, C., Liang, C. T., and Sacktor, B., untochondrial preparations with brush bor- published observations,
288
LIANG
AND
experiments, in which ATPase activities were assayed by rate of formation of ADP rather than P, it was found that neither phosphate anions nor thiosulfate increased TABLE ANION
II
SPECIFICITY FOR STIMULATION OF THE BRUSH ATPase” BORDER MEMBRANE
Salt
Activity
relative
P formed
to Mg2+-ATPase ADP formed-
(% increase) NaHCO, Tris HCO, Tris Cl NaHCO, NaCl NaNO, NaCH,COO Na*SO, Na,SO, NGW, Na,HPO,
73 + 3 75 + 5 2kO 67 r Sk0 0 21 k 27 k 199 + -
3
4 8 8
48 r 2 174 -c 16 0 0
u ATPase activities were assayed as described in the text, except that the incubation time was 10 instead of 15 min. Fifty millimolar salts were used in all experiments. Mg*+-ATPase activities measured by the formation of P and ADP were 0.274 -r0.013 pmol P/min/mg of protein and 0.220 k 0.010 pmol ADP/min/mg of protein. Each datum represents the mean percentage increase k SEM for 2 to 10 experiments.
I/\TPl ,rnMi
SACKTOR
activity
above the basal level.
Kinetics of the Mg2+ and HCO,--stimulated ATPase. Figure 1 describes the Mg2+- and Mg2+ + HCO,--ATPase activities with respect to ATP concentration. HCO,- enhanced activity at all concentrations of ATP. Double reciprocal plots of both basal and HCO,--stimulated activities were linear. The K, and V for Mg2+ATPase were 0.24 mM and 0.29 pmol PI minlmg of protein. In the presence of HCO,-, both K, and V were increased, and values of 0.37 mM and 0.50 pmol P/ minlmg of protein were estimated. Also plotted in Fig. 1 (dashed lines) is the relationship between “HCO,--ATPase” activity and concentration of ATP. AK,, and V of 0.43 mM and 0.14 pmol P/min/mg of protein were calculated. The effect of varying concentrations of HCO,- on brush border membrane ATPase activity is shown in Fig. 2. The K, for HCO,- was 36 mM and the V was calculated to be 0.26 pmol P/min/mg of protein. To ascertain whether the binding of HCO,affected the binding of ATP, “HCO,--ATPase” activities were determined at different ATP concentrations in the presence of various concentrations of HCO,-. As shown in Fig. 3, HCO,- increased V but had no effect on the K, for ATP. Similarly, the binding of ATP did
l/CATtl
FIG. 1. The Mg*+- and (MgZ+ + HCO,-)-ATPase activities of renal brush border membranes as a function of ATP concentration. (A) and (B) illustrate u versus S and double reciprocal plots of the data, respectively. ATPase activities were determined as described in the text. The concentration of HCO,- was 50 mM. (01, total (Mg *+ + HCO,J-ATPase; (01, Mg2+-ATPase. The difference between total ATPase and MgZ+-ATPase is designated as “HCO,--ATPase” activity (A) and is shown in the dashed line. Each datum is the average of three experiments.
“HCO,--ATPase”
IN BRUSH
BORDER
TABLE NUCLEOTIDE STIMULATED
,/O’ ...” 0 20
,..f 40
60
Dm~-l,mM,
80
”
Nucleotide
../ 0
02
04
06
08
l/IHCO~l
FIG. 2. The effect of concentration of HCO,- on the brush border membrane ATPase activity. In (A) the data for the total (Mg*+ + HCO,-)-ATPase is shown as (0); the “HCO,--ATPase” (01 is shown in the dashed line. (B) illustrates a double reciprocal plot of the “HCO,--ATPase” values. The concentration of ATP was 3 mM. Each datum is the average of three experiments.
IIKPIWm.4-’
lIMC0~-l(mM)-~
FIG. 3. Double reciprocal plots of “HCO,--ATPase” activity as a function of (A) ATP concentration assayed in the presence of the indicated concentrations of HCO,- and (B) HCO,- concentration assayed in the presence of the indicated concentrations of ATP. Each datum is the average of four experiments.
not alter the K, for HCO,-. Nucleotide specificity. The nucleotide specificity of the rabbit renal brush border membrane ATPase is described in Table III. Basal rates of dephosphorylation of the purine nucleotides, ATP, GTP, and ITP, were essentially similar. HCO,- stimulated the hydrolysis of each to about the same degree, 63% for ATP and 56 and 53% for GTP and ITP, respectively. In general, the rate of dephosphorylation of UTP in the presence of Mg*+ was slightly less than those of the purine nucleotides. However, HCO,- stimulated the basal rate of de-
ATP GTP ITP UTP CTP
289
MEMBRANES
0.312 0.358 0.374 0.283 0.227
III
SPECIFICITY OF THE HCO,-ATPase IN BRUSH BORDER MEMBRANE*
Mgz+
MgZ+ + HCO,-
(pm01 Plminlmg
___of protein)
2 k r 2 f
0.020 0.034 0.048 0.031 0.033
(5) (4) (31 (4) (4)
0.509 0.558 0.572 0.375 0.238
-+ 0.025 ? 0.030 k 0.048 2 0.030 2 0.032
(5) (4) (3) (4) (4)
a ATP(nucleoside triphosphatejase activities were determined as described in the text. The nucleotide concentration was 3 mM and the HCO,- concentration was 50 mM. Each datum represents the mean k SEM for the number of experiments indicated in parentheses.
phosphorylation of UTP only 32%. A clear substrate specificity was observed when CTP was tested. With this pyrimidine triphosphate, the basal rate of dephosphorylation was significantly less than that of ATP and it was not stimulated by HCO,-. Stability of the Mg2+- and HCO,--stimuZated ATPase. Mg2+- and Mg2+ + HCO,-ATPase activities of freshly prepared brush border membranes were not increased or decreased by keeping the membranes on ice overnight, by subjecting the membranes to sonication (30 s at 3 A, three times) or to one cycle of freezing and thawing. Preincubation of the membranes with Triton X-100 (30 pglml, equivalent to a detergent:protein ratio of 0.5) for 20 min at 0°C had no apparent effect on the basal or HCO,--stimulated activities. Increasing the Triton X-100:protein ratio to 1.6 caused a 30% loss of the Mg2+-ATPase and an 80% decrease in the anion-stimulated activity. Preincubation of the brush border membranes with deoxycholate (40 to 60 pg/ml, equivalent to a detergent:protein ratio of 0.75) effected a 15 to 20% activation of the Mg2+-ATPase, but was without significant effect on the HCO,--stimulated ATPase. Increases in the deoxycholate:protein ratio to greater than 1.5 resulted in losses of ATPase activities. Kinetics of the S0,2--stimulated ATPuse. In view of the finding that SOZ2- was the most effective activator of the brush border membrane Mg2+-ATPase (Table II),
290
LIANG
AND
the kinetics of the ATPase stimulated by this anion was examined for comparison with that by HCO,-. Figure 4 shows the Mg2+ + S0,2--ATPase activity with respect to concentration of ATP. Enhancement in activity by S0,2- was found at all concentrations of ATP and was greater than that by HCOB- at each nucleotide concentration (Fig. 1). The V for ATPase stimulated by S0,2- was 1.0 pm01 P/min/ mg of protein, a value twice that for Mg2+ + HCO,--ATPase. The presence of SO,‘increased the K, for ATP to 0.47 mM, from 0.29 mM in the absence of the anion. The relationship between “S0,2--ATPase” and ATP concentration is also shown (dashed lines) in Fig. 4. AK, and V of 0.72 mM and 0.70 pm01 P/min/mg of protein, respectively, were calculated. Thus, the V for “S0,2--ATPase” was fivefold greater than that for “HCO,--ATPase” (Fig. 1). The effect of different concentrations of SO,*- on the brush border membrane ATPase activity is described in Fig. 5. The K, for SO,‘- was 0.78 mM, a concentration 50-fold less than that for HCO,- (Fig. 2). Effect of pH on the Mg’+- and anionstimulated ATPase. Examination of the effect of pH on brush border membrane ,HCO,--stimulated ATPase was complicated by the finding that the K, for HCO,was high (36 mM), and by the difficulty in stabilizing the anion at an optimal concen-
FIG. 4. The Mgz+- and (Mg2+ + S0,2-)-ATPase activities of renal brush border membranes as a function of ATP concentration. (Al and (B) illustrate u versus S and double reciprocal plots of the data, respectively. ATPase activities were determined as described in the text, except that the incubation period was 10 min. The concentration of S0,2m was 25 mM. (01, total (Mg*+ + S032V-ATPase; (01, MgZ+-ATPase. The difference between total ATPase and Mg2+-ATPase is designated as “S032--ATPase” activity (A) and is shown in the dashed line. Each datum is the average of three experiments.
SACKTOR
0
20
10 NazS03
30
(mM)
FIG. 5. The effect of concentration of Na$SO, on the brush border membrane ATPase activity. Total (Mg*+ + S0,2-)-ATPase is shown as (0); the “SO,‘-ATPase” (0) is shown in the dashed line. The concentration of ATP was 3 mM. Incubation time was 10 min. The insert shows a double reciprocal plot of the “SO,*--ATPase” values. Each datum is the average of two experiments.
tration, considering that the pK’ of H,CO, is approximately 6.3 and the potential loss of CO,. To circumvent this problem, the effect of pH on the ATPase was studied with SOs2- as the activating anion. In this case, the pK1 and pK2 for H&SO3 are approximately 2 and 7.2, respectively, and the K, was found to be only 0.78 mM. Thus, an optimal concentration of activator could be maintained throughout the physiological pH range by using 25 mM Na2S0, in the incubation mixture. Figure 6 describes the pH profile for the brush border membrane Mg2+- and Mg2+ + SO,‘--ATPase activities. Mg2+-ATPase activity gradually increased from 0.14 to 0.35 pmol P/min/mg of protein as the pH was raised from 6.1 to 8.3. In contrast, the S0,2--stimulated activity increased sharply within a narrow pH range, 6.1 to 7.1, and increased moderately at higher pH values. Because brush border membranes were reported to possess alkaline phosphatase and 5’-nucleotidase activities (28), the possible contributions of these phosphatases to the rate of formation of P with increased alkalinity and presence of SOa2- were evaluated. As
“HCO,--ATPase”
IN BRUSH
Or
a#fl Hg . 6.0
70
8.0
PH
FIG. 6. The effect of pH on ATPase activities of brush border membranes. (01, total (MgZ+ + S0,2m)ATPase; (O), Mg2+-ATPase. The “SO,*--ATPase” (A) is shown in the dashed line. The ATP and Na$O, concentrations were 3 and 25 mM, respectively. Values are the averages of three experiments. The dephosphorylation of 3 mM AMP, in the presence (D) or absence (0) of Na2S03, at the different pH values is shown. Each datum represents the average of two experiments.
shown in Fig. 6, low rates of dephosphorylation of AMP were found at the different pH values, in the absence and presence of S0,2-. This indicated that these phosphatases were not activated by SOs2- nor did they contribute significantly to the sharp increase in the rate of dephosphorylation of ATP observed in the presence of S0,2- in the pH range 6.1 to 7.1. The response of the S0,2--stimulated ATPase to pH, illustrated in Fig. 6, raised the question as to whether the increase in activity with higher pH merely reflected an increase in the concentration of the SOs2- species or was, in fact, an intrinsic effect on the enzyme. In the physiological pH range, sulfite exists principally as SOs2- and HSO,-. The concentration of H,SO, is negligible, pK’ being about 2. To answer this question, the Na2S03 concentration in the reaction mixtures was adjusted so that at each pH value the concentration of the SOs2- species was maintained at 9.6 mM, although the concentration of the HSO,- species varied from 38.4
BORDER
291
MEMBRANES
mM at pH 6.4 to 0.4 mM at pH 8.3. The pH profile of the anion-stimulated ATPase activity, at this constant level of S032-, is described in Fig. 7. Comparison of Figs. 7 and 6 revealed the marked similarity in whether concentration of pH profiles, S0,2- was held constant or varied. These results, therefore, suggested that the effect of pH on the stimulated enzyme activity was on the ATPase itself rather than due to an increase in the concentration of activator species. Moreover, the relatively large sulfite-stimulation at pH 8.3, when the concentration of HSO,- was low (0.4 mM), and the relatively small activation when the concentration of HSO,- was high (38.4 mM), strongly supported the view that S0,2- was, indeed, the anion species that activated the ATPase. Effect of inhibitors. In previous studies it was reported that SCN-, which in-
O.l}
0.
7.0
6.0
PH
7. The effect of pH on SO,‘--stimulated ATPase at a constant concentration of SO,*-. The concentration of the S0,2m species was maintained at 9.6 mM at different pH values by the use of 48,25, 22, 12, and 10 mM NalSO, at pH 6.4, 6.8, 7.1, 7.6, and 8.3, respectively. The ATP concentration was 3 mM. CO), total (Mg*+ + SO,*-)-ATPase; (01, Mg2+-ATPase; (A), “S032m-ATPase” in the dashed line. Each datum represents the average of two experiments. FIG.
292
LIANG
AND
hibited acid secretion in gastric mucosa, salivary gland, and pancreas (l-4, 7, 81, was a specific inhibitor of HCO,--stimulated ATPase. In contrast, SCN- was found in the present study to be an ineffective inhibitor of the renal brush border membrane ATPase (Fig. 8). As shown, 1 mM NaSCN had no effect on the rate of dephosphorylation and 10 mM inhibited 30% or less. These same concentrations of SCN- inhibited 60 and lOO%, respectively, of the HCO,--stimulated ATPase in the submandibular gland (7). Moreover, in the brush border membranes SCN- did not distinguish between Mg*+-ATPase activity or the activities stimulated by HCO,- and SO,*-. Interestingly, Ullrich et al. (31) recently reported that H+ secretion in the microperfused proximal tubule was in variance with that in the stomach and pancreas in that in the kidney the process was insensitive to SCN-. The HCO,--stimulated ATPase in the renal brush border membrane also differed from the enzymic activity in the gastric mucosa in its sensitivity to methane sulfonyl chloride. In the renal membrane preparation, as illustrated in Fig. 9, Mg*+ATPase was strongly inhibited by concentrations of the inhibitor (5 to 10 mM) which had no effect on the “HCO,--ATPase.” The
I
0
5 [NaSCNl
10 (mM)
FIG. 8. The inhibition of anion-stimulated ATPase of brush border membranes by NaSCN. ATP concentration was 3 mM. When indicated, 50 mM NaHCO, or 25 rnM Na2S0, was used. (01, Mg2+ATPase; (01, (Mg*+ + HCO,-)-ATPase; (a), (Mg’+ + SO,*-l-ATPase. The 100% activity was, respectively, 0.218, 0.349, or 0.569 pmol P/min/mg protein. Each value represents the average of three experiments.
SACKTOR
“--i;-ll
0 I 1o-4
1o-2
CMSC~(M) FIG. 9. The effect of methane sulfonyl chloride (MSC) on the ATPase activities of brush border membranes. ATPase activities were determined as described in the text. (01, total (Mg”+ + HCO,-lATPase; (0) Mg*+-ATPase; (A,, “HCO,--ATPase” shown in the dashed line. Each value represents the average of three experiments.
relative insensitivity of the anion-stimulated ATPase in brush border membranes to this inhibitor was also evident when S0,2- was used as activator. In these experiments, not illustrated, Mg*+-ATPase activity in the absence and presence of 5 mM methyl sulfonyl chloride was 0.212 and 0.132 pmol Plminlmg of protein, respectively. On the other hand, Mg*+ + SO,*-ATPase activities were 0.543, in the absence, and 0.454, in the presence of inhibitor. Thus, the Mg*+-ATPase was inhibited 38%, whereas the “SO,*--ATPase” activity was unaffected. In contrast, Blum et al. (4) reported that Mg*+ + HCO,--ATPase was inhibited 50% by 1 mM methane sulfonyl chloride whereas Mg2+-ATPase was not inhibited by this concentration of inhibitor. In other experiments, not shown, oligomycin (12.5 g/ml) inhibited the Mg*+- and Mg*+ + HCO,--ATPase activities in the brush border membranes by 23 and 36%, respectively. At this concentration of oligomycin, the renal cortex mitochondrial ATPase was inhibited over 95%. The effect of quercetin, a recently described inhibitor of mitochondrial ATPases (321, was also tested. At concentrations of 50 and 100 pg/ ml, levels greatly in excess of that needed to inhibit mitochondrial ATPase, brush border membrane Mg*+- and Mg2+ + HCO,--ATPase activities were inhibited only 6 and 13%, respectively, at the lower concentration, and 24 and 25%, respec-
“HCO,--ATPase”
IN BRUSH
tively, at the higher concentration of inhibitor. FCCP had little effect on the Mg’+- or Mg2+ + HCO,--ATPase activities in brush border membranes. Since the usual reaction medium for measuring ATPase activity in the present study contained high concentrations of salts, i.e., 50 mM NaHCO, and 75 mM Tris chloride buffer, and it was found previously that high concentrations of salt made the membranes more permeable to various ions and presumably H+ (211, the effect of FCCP was tested additionally using a reaction system in which salt concentrations were minimized, i.e., 2 mM Tris-Hepes, as buffer, and 2 mM Na,SO,, as activator. In this case, as shown in Table IV, the proton conducting agent stimulated basal ATPase activity by 23%, and Mg2+ + S0,2-ATPase activity by little, it at all. Effect of carbonic anhydrase on the HCO,--stimulated ATPase. Added carbonic anhydrase significantly increased HCO,--stimulated ATPase activity (Table V). In contrast, Mg2+-ATPase was not affected. The carbonic anhydrase mediated increment in HCO,--stimulated ATPase activity was completely abolished by Diamox (sodium acetazolamide), a specific inhibitor of carbonic anhydrase (33). Because carbonic anhydrase added exogenously to the reaction mixture would increase the formation of CO*, and the escape of this CO, would tend to make the reaction medium more alkaline, the pH during the course of the incubation was carefully monitored. In these experiments the pH gradually rose from 7.53 to 7.60. As shown in Fig. 6, however, this increase of TABLE
IV
OF FCCP ON THE ANION-STIMULATED ATPase IN BRUSH BORDER MEMBRANES”
EFFECT
MgZ+-ATPase (pm01 P/min/mg Control +FCCP
0.233 0.286
Mg*+ + SO,*--ATPase of protein) 0.530 0.565
n The reaction mixture contained 300 mM sucrose, 2 mM Tris-Hepes, pH 7.5, 3 mM MgCl,, 1.5 mM ouabain, 3 mM tris-ATP, -r- 2 mM Na,SO,, f 10 FM FCCP. The incubation time was 5 min. Each datum is the average of two experiments.
BORDER
293
MEMBRANES TABLE
V
EFFECT OF CARBONIC ANHYDRASE ON THE HCO,-STIMULATED ATPase IN BRUSH BORDER MEMBRANES’” Mg2+-ATPase
Experiment Car-
(pm01 Plminlmg
Diamox
“HCO,-ATPase” of protein)
bonic anhydrase -
-
0.281 0.301 0.296 0.310
+ -
+ +
+
f k f ”
0.024 0.031 0.026 0.029
0.166 0.163 0.208 0.172
t + k f
0.009 0.011 0.016 0.011
” ATPase activities were determined as described in the text. “HCO,--ATPase” represents the difference in activities between total (Mg*+ + HCO,-)ATPase and MgZ+-ATPase. When indicated, 40 units of carbonic anhydrase were used. Each unit is defined as the enzyme activity required to reduce the pH of a CO,-containing solution from 8.7 to 6.7 twice as fast as the pH of the blank, assayed by Boehringer. When indicated the Diamox concentration was 0.5 mM. Each datum represents the mean t SEM for four experiments.
0.07 units was too small to account for the 25% increase in “HCO,--ATPase” activity that was found in the experiments in which carbonic anhydrase was present (Table V). Figure 10 demonstrates that the effect of carbonic anhydrase on the kinetics of the “HCO,--ATPase” was to increase V without changing the K, for ATP. When the effect of carbonic anhydrase was examined with respect to concentration of HC03-, both V and K, were found to increase. DISCUSSION
The results described in this study show that the specific activity of the “HC03-ATPase” in renal brush border membranal preparations was increased about fourfold relative to that in the renal cortex homogenate. Mitochondrial contamination could account maximally for only 15% of the activity in the brush border preparation. These findings indicate the presence of a HCO,--stimulated ATPase in the proximal tubule luminal membrane. This membrane is the major site of H+ secretion and bicarbonate3 reabsorption in the kidney 3 “Bicarbonate” term
without
reabsorption
specifying
the species transported
is used as a general HCO,- or CO, is across the membrane. whether
294
LIANG
AND
SACKTOR
mM, when the ATPase was activated by SO,z- or HC03-, is in accord with the findings that the K, for ATP also varied with the anion with the isolated F, component of the mitochondrial ATPase (11, 35). An anion-dependent conformational change of l/lATPl imw' l/[HCOO-lhMi-' enzyme subunits which mediates an active/inactive state transition was sugFIG. 10. The effect of carbonic anhydrase on the gested by Moyle and Mitchell (35). kinetics of the HCO,--stimulated ATPase of brush The acceleration of the rates of dephosborder membranes. ATPase activities were deterphorylation of ATP, GTP, and ITP by 50 mined as described in the text and in Table V. (01, of Mg*+-ATPase; (O), MgZ+-ATPase in the presence of mM HCO,- was the same. Stimulation carbonic anhydrase; (A), “HCO --ATPase”; (A), the rate of dephosphorylation of UTP was “HCO,--ATPase” in the presence if carbonic anhy considerably less, and with CTP it was drase. Each value represents the average of three negligible. This nucleotide specificity with experiments. rabbit renal brush border membranes differs from that reported with microvillar membranes of rat kidney (16) in which the rates of dephosphorylation of GTP, ITP, and, if the general concept of the coupling UTP, and CTP were 20% that for ATP. of ATP hydrolysis to cation translocation ATPases in other across membranes (34) is applicable to the The HCO,--stimulated extrusion of H+ from renal tubular cell to preparations also exhibited varied nucleoIn gastric mucosa (4), lumen, then the finding of a brush border tide specificities. HCO,--stimulated ATPase takes on added GTP was hydrolyzed at about 50% the rate for ATP, UTP about lo%, and CTP not at significance. (ll), at a HCO,The renal brush border ATPase was all; in liver mitochondria concentration of 20 mM, the V for ATP and stimulated by several anions. In addition to HC03-, SOX2- was a potent activator of ITP were the same, whereas that for GTP the brush border ATPase. CH,COO- and was half this rate. The present findings that carbonic anS0,2- were less effective than HCOs-, whereas phosphate anions and CL- had no hydrase increased the rate of the brush membrane HCO,--stimulated effect on ATPase activity. Thus, of the border ATPase and this enhancement was anions present in the glomerular filtrate blocked by Diamox may be relevant to proin appreciable concentrations, i.e., HC03-, posed mechanisms of H+ and bicarbonate Cl-, and phosphates, only HCO,- stimulated the luminal membrane ATPase. transport. In the intact dog, 40% of the Moreover, the K, for HCO,-, i.e., 36 mM, filtered bicarbonate was excreted when approximates the physiological concentracarbonic anhydrase was inhibited by Diation of the anion in the glomerular filtrate. mox (36). Diamox prevented acidification The selective stimulation of the renal of the filtrate in the proximal tubule in brush border ATPase by anions, e.g., situ (37), and it inhibited the secretory HCO,- and SOS*-, confirms previous studrate of H+ in perfused tubules (31). As ies on the activation by various anions of illustrated diagrammatically in Fig. 11, crude membranal ATPases from gastric carbonic anhydrase in the proximal tubumucosa (4) and pancreas (9) and of the lar cell has a dual localization; approxihighly purified enzyme from mitochondria mately 90% of the activity is found in the (11, 35). Also consistent with the view that cytosol, the remainder is localized in the HC03- and SOs2- stimulate the same, brush border membrane (38). According to rather than distinct, enzyme is the finding one generally accepted hypothesis for bithat gel electrophoresis of membranes incarbonate transport (14, 15), extrusion of dicated a single ATPase band, sensitive to H+ from cell to lumen favors the converboth HCO,- and S03*- (9). The present sion of HCO,- to H,CO, in the glomerular observation that the K, for ATP was filtrate. The brush border carbonic anhyslightly different, 0.72 compared to 0.43 drase enhances the breakdown of this
“HCO,--ATPase”
+
IN BRUSH
BORDER
MEMBRANES
295
tion of ATP by about 25% is consistent with the presence of a membranal ATPase Na' r + _ whose activity is limited by transmembrane H+ flux. This argues for a role for the enzyme in the transport of H+. In addition, Diamox, which inhibited the carbonic anhydrase enhanced “HCO,--ATPase” in brush border membranes (Table V), also reduced the lumen-positive active transport potential in loops of proximal tubules (42). Strengthening the hypothesis further is the suggestion that the bicarbonate species which is transported across brush borANTILUMINAL LUMINAL BASAL-LATERAL BRUSH BORDER der membrane vesicles is probably CO, MEMBRANE MEMBRANE (22). Lastly, Pitts and Alexander (43) from FIG. 11. A diagrammatic model for bicarbonate clearance measurements in dogs first posreabsorption in the renal proximal tubular cell and tulated an exchange of cellular H+ for filthe postulated role of the HCO,--stimulated ATPtered Na+, and this view was supported by ase. stop-flow microperfusion studies in rats (31). Moreover, studies in which isolated H&O, to CO, and H,O. The COz, thus kidneys were perfused with HCO,--free formed, is transported across the brush media or with media containing HC.O,- + border membrane into the cell, where it is Diamox showed that reabsorption of bicarhydrated by the cytosolic carbonic anhybonate accounts for a small but significant drase. Dissociation of the H&O, generates fraction of the Na+ reabsorbed (44). Thus, intracellular HCO,- and H+. A significant exchange of H+ for Na+ may represent one share of the bicarbonate that is reabsorbed of the mechanisms for maintaining cellufrom the glomerular filtrate has been at- lar electroneutrality. Interestingly, expertributed to H+ secretion by this mechaiments with brush border membrane vesinism (39, 40). It is now further postulated, cles currently in progress in this laboraas diagrammed in Fig. 11, that the brush tory4 indicate a coupling of the transports border ATPase is involved in the extrusion of the two cations. of H+ from cell to lumen and that intracelOn the other hand, there are observalular HCO,- by stimulating this ATPase tions which at this time are seemingly augments translocation of H+ across the drawbacks to the tentatively proposed membrane. Thus, reabsorbed bicarbonate, scheme. As diagrammed in Fig. 11, in the form of HC03-, may act as a positive HCOa--stimulated ATPase is localized on effector promoting the reabsorption of ad- the inner surface of the brush border memditional bicarbonate. brane, thus accessible to intracellular Other evidence is available which lends ATP. In the present in vitro experiments, support for this hypothesis. That a “pump” the nucleotide was added to the medium, mechanism is needed to extrude H+ from external to the membrane vesicle. Alproximal tubular cell to lumen via the though membranes are presumed to be relbrush border membrane is indicated from atively impermeable to ATP, sonication findings that the transmembrane electroand detergents, which disrupt membrane chemical potential of the brush border is integrity, did not enhance rates of dephospositive on the outside and negative on the phorylation. Admittedly, the sidedness of inside (22, 41, 42). In other membrane sys- the population of vesicles is not known. It tems, ATPases are known to couple ATP is also probable that after sonication the hydrolysis to H+ translocation against membranes resealed. Tending to refute electrochemical gradients (34). The pres- this objection further are the findings that ent finding that in brush border membranes the proton-conductor, FCCP, in4 Chernoff, A., and Sacktor, B., unpublished observations. creased the basal rate of dephosphorylaHCOS-
l c-
_
+ _
HCOS-
l _
1
HCOS-
296
LIANG
AND
high concentrations of salts such as used in most of the present experiments increased membrane permeability (21) and that the kidney in contrast to other mammalian tissues is readily permeable to added adenine nucleotides (45). Another apparent inconsistency to the proposed hypothesis relates to the effect of carbonic anhydrase on the kinetics of the HCO,--stimulated ATPase. If the action of carbonic anhydrase is to accelerate the formation of CO, in the medium, then an increase in the HCO,--stimulated ATPase activity is understandable as more CO, is available to diffuse across the brush border membrane. The affinity of HC03- to the ATPase should not be altered. As shown in Fig. 10, however, the effect of carbonic anhydrase was to increase both V and the K, for HC03-. The explanation of this increase in K, remains to be formulated. A second mechanism for the reabsorption of the filtered bicarbonate in the proximal tubule depends on the transport of bicarbonate in its ionic form, rather than as CO, (14, 15). Indeed, a considerable fraction of the total bicarbonate may be transported by this process. This fraction would not be directly affected by H’ extrusion. Nevertheless, if HCO,- is cotransported with Na+ (441, then mechanisms which mediate the eMux of H+ will enhance the influx of Na+ and, indirectly, of HCO,-. Thus, in both mechanisms of bicarbonate transport the suggestion that bicarbonate reabsorption, H+ secretion, and the HC03--stimulated ATPase are interrelated remains a viable hypothesis which merits further testing.
SACKTOR
6.
7. 8. 9. 10. 11. 12. 13.
14.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
REFERENCES 1. KASBEKAR, D. K., AND DURBIN, R. P. (1965) Biochim. Biophys. Actu 105, 472-482. 2. SACHS, G., MITCH, W. E., AND HIRSCHOWITZ, B. I. (1965)Proc. Sot. Exp. Biol. Med. 119: 10231027. 3. WIEBELHAUS, V. D., SUNG, C. P., HELANDER, H. F., SHAH, G., BLUM, A. L., AND SACHS, G. (1971) Biochim. Biophys. Actu 241, 49-56. 4. BLUM, A. L., SHAH, G., ST. PIERRE, T., HELANDER, H. F., SUNG, C. P., WIEBELHAUS, V. D., AND SACHS, G. (1971) Biochim. Biophys. Actu 249, 101-113. 5. SACHS, G., SHAH, G., STRYCH, A., CLINE, G.,
26.
27.
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