Characterization and regulation of H-K ATPase in intercalated cells of rabbit cortical collecting duct

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:443–455 (1997)

© 1997 WILEY-LISS, INC.

JEZ 857

Characterization and Regulation of H-K ATPase inIntercalated Cells of Rabbit Cortical Collecting Duct

RANDI B. SILVER,1* GUSTAVO FRINDT,1 PATRICIA MENNITT,1 ANDLISA M. SATLIN2

1Department of Physiology, Cornell University Medical College, New York,New York, 10021

2Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NewYork 10461

ABSTRACT K-dependent H+ extrusion was investigated using fluorescence techniques in rab-bit cortical collecting tubules (CCTs). Experiments were performed in split-open tubules from nor-mal animals exposed to the intracellular pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein(BCECF). This preparation permitted the study of individual intercalated cells (ICs). In the ICs,partial recovery of pHi was observed in response to an acute acid load upon readdition of 5 mM Kto the superfusate. This recovery was SCH 28080-inhibitable (10–5 M) and ouabain-insensitivesuggesting the process is mediated by a gastric-type H-K ATPase. To see if H-K ATPase plays arole in acid secretion its function was evaluated under chronic metabolic acidosis (CMA) condi-tions. CMA was induced by replacing drinking water with 75 mM NH4Cl in 5% sucrose for 10–14days. The SCH 28080-inhibitable K-dependent pHi recovery rate was three-fold higher in CMAICs compared to controls. To determine the location of the H-K ATPase, CCTs were microperfusedand individual peanut lectin binding (PNA) ICs studied. K-dependent pHi recovery was measuredin response to an NH4Cl pulse. An apical SCH 28080-inhibited K-dependent pHi recovery processwas observed in control and CMA ICs. Taken together these data confirm the existence of a gas-tric-type H-K ATPase in ICs of rabbit CCT. Based on our findings the H-K ATPase is found on theapical side of the cell and is stimulated under conditions of CMA. J. Exp. Zool. 279:443�455,1997. © 1997 Wiley-Liss, Inc.

An H-K ATPase, similar to the acid secretinggastric H-K ATPase, exists in distal nephron seg-ments of rat and rabbit (Doucet and Marsy, ’87;Garg and Narang, ’88; Wingo, ’89). In the stom-ach the H-K ATPase functions to secrete protonsand it is believed there is an associated K+ con-ductance to recycle K from the cell into the lu-men (Cuppoletti and Sachs, ’84; Wolosin andForte, ’81). The renal H-K ATPase is postulatedto function in both K reabsorption (Wingo, ’89)and acid secretion (Garg, ’91).

Renal H-K ATPase enzymatic activity was firstidentified in distal segments of microdissectednephrons of rabbit and rat kidneys (Doucet andMarsy, ’87; Garg and Narang, ’88). The activitywas highest in the connecting tubule, lowest inthe outermedullary collecting tubule, and inter-mediate in the cortical collecting tubule. H-KATPase activity was unaffected by ouabain and in-hibited by omeprazole, vanadate, and the hydropho-bic amine SCH 28080 which suggested that thisATPase behaved similarly to the well characterizedgastric H-K ATPase (Garg, ’91). Furthermore, im-

munoreactivity studies with mouse monoclonal an-tibodies raised against hog gastric H-K ATPase re-vealed diffuse cytoplasmic staining in cells in ratand rabbit cortical collecting tubule (CCTs) dem-onstrating the existence of a related antigen(Wingo et al., ’90).

Several studies demonstrated that K-ATPase ac-tivity could be varied with changes in potassiumintake. For example, enhanced K-ATPase activ-ity was observed in CCTs from K-depleted rats(Doucet and Marsy, ’87) and decreased activitywas reported in CCTs from K-loaded rabbits (Gargand Narang, ’88). An active, omeprazole-sensitiveacidification and K-reabsorption process was alsoshown to exist in isolated perfused outer medul-lary collecting ducts from K-deprived rabbits(Wingo, ’89). Collectively these results provide

*Correspondence to: Dr. Randi B. Silver, Department of Physiol-ogy, Cornell University Medical College, 1300 York Ave., New York,NY 10021. E-mail: [email protected]

444 R.B. SILVER ET AL.

both enzymatic and functional evidence for a roleof the H-K ATPase in the reabsorption of K.

Studies of isolated perfused rabbit CCTs exposedto an ambient in vivo or in vitro acidosis havedemonstrated a reversal in direction of net HCO3transport from that of secretion observed undercontrol conditions, to net absorption (McKinneyand Burg, ’77; Satlin et al., ’92). This change indirection of vectorial HCO3 transport has been pro-posed to be mediated, at least in part, by modifi-cations in function of the predominant subtype ofrabbit CCT intercalated cells (β-IC) (Furuya etal., ’91; Satlin et al., ’92; Silver et al., ’92; Yaso-shima et al., ’92). β-ICs in control rabbit CCTspossess apical Cl/HCO3 exchangers; the vast ma-jority of these cells bind peanut lectin (PNA) totheir apical cell surfaces (Satlin and Schwartz,’89). In response to extracellular acidosis thesecells lose functional apical anion exchange activ-ity, a process that could account for a reductionin net HCO3 secretion (Satlin et al., ’92). The con-tribution of this population of ICs to acidosis-in-duced net H+ secretion has not yet been examined.

In this investigation the presence of function-ally active H-K ATPase in the rabbit CCT wastested by measuring the rate of K-dependent H+

efflux in response to an acute acid load in splitrabbit CCTs loaded with the intracellular pH in-dicator, BCECF. The split tubule preparationwhich has the geometry of a flat monolayer per-mits single cell fluorescence measurements. Wemonitored pHi in individual ICs of the rabbit CCT.We demonstrated in ICs a K-dependent intracel-lular alkalinization process in response to an acuteacid load which was unaffected by ouabain andinhibited by SCH 28080.

In order to determine if the H-K ATPase in theICs could play a role in acid secretion, studieswere performed to determine if the H-K ATPasecould be stimulated during metabolic acidosis. H-K ATPase was assayed in individual ICs in split-open CCTs and isolated microperfused CCTs fromrabbits subjected to chronic metabolic acidosis(CMA) in order to determine if the location of thetransporter under CMA conditions is consistent withnet H+ secretion into the tubule lumen.

MATERIALS AND METHODSGeneral

New Zealand white rabbits of either sex (2–3.5kg body weight) maintained on standard chowwere used in this investigation. CMA was inducedin the experimental group by replacement of the

drinking water with a 75 mM NH4Cl solution pre-pared in 5% sucrose for 10–14 days and rationingfood intake to 60–80 gm/daily. In the CMA study,two groups of control animals were studied, onesubject to no dietary manipulation or restriction(untreated animals), and the other provided adrinking water solution containing 75 mM NaClprepared in 5% sucrose for 14 days and food in-take restricted to 60–80 gm/day (NaCl-treated ani-mals). Arterial blood was obtained by cardiacpuncture at 8 days in CMA animals and both bloodand urine were sampled at the time of death incontrols and acid fed animals. All animals werekilled by an intracardiac injection of 100 mg/kgbody weight pentobarbital sodium.

Plasma samples were analyzed for pH using anIL Instrumentation Laboratory blood gas machineand tCO2 with a Natelson microgasometer. Plasmaand urinary K concentrations were determined byflame photometry (Instrumentation Laboratoriesmodel 943). Urine samples taken from the blad-der at the time of death were analyzed for pHwith a Radiometer pH meter. Because urine wassometimes absent in the bladder and bloodsamples were often difficult to obtain, completechemical profiles of plasma and urine are not re-ported for every animal.

Determination of muscle K contentSkeletal muscle K content was measured on

thigh muscle from control and CMA rabbits as pre-viously described (Satlin et al., ’88). Briefly, rightthigh muscle was removed from each animal, dis-sected free of fascia, and minced. The mincedmuscle was divided between four vials and thewet weight of the muscle determined. Aftersamples were dried at 55°C for 48 hours to con-stant weight, 5 ml of anesthesia grade ether wereadded to each vial for 6–48 hours. The ether wasdecanted and the tissue dried in a vacuumdessicator for 12–48 hours to constant weight.These fat-free tissue samples were then extractedfor another 48 hours at room temperature with 3ml of 0.52 N HNO3 and the extract was analyzedfor K content using a flame photometer with a 50mM K standard. Any extracellular fluid source ofcontamination was ignored, assuming all K to beintracellular. K content per 100 g fat free dryweight of muscle was expressed as the mean ofthree to four samples per analyzed animal.

SolutionsThe compositions of the solutions used are given

in Table 1. Hepes buffer was used in all of the

H-K ATPASE IN INTERCALATED CELLS 445

solutions. In studies of isolated perfused CCTs, 1mM BaCl2 was added to all Na-free solutions tominimize exit of K from intercalated cells (Frindtand Palmer, ’89). All chemicals were obtained fromSigma Chemical Co. (St. Louis, MO) unless oth-erwise stated. Nigericin (Molecular Probes, Eu-gene, OR) was added to potassium Ringer ’ssolutions (solutions a and b) from a 10 mM stock(three parts ethanol: one part dimethylformamide)for a final concentration of 10 µM. Individual vials(50 µg) of the acetoxymethyl derivative of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BC-ECF-AM; Molecular Probes) were stored dry at0°C and reconstituted in dimethylsulfoxide (at aconcentration of 10 mM) for each experiment. Thefinal loading concentration of dye, prepared in NaRinger’s solution, was 10 µM for split-open tubulesand 20 µM for isolated perfused tubules. Rho-damine-conjugated peanut agglutinin (PNA) (Vec-tor Labs, Burlingame, CA) was added to NaRsolution (solution 1) at a concentration of 10 µg/ml.

SCH 28080 (2-methyl 8 (phenylmethoxy) im-idazo (1,2a) pyridine-3-acetonitrile), a gift fromDrs. A. Barnett and J. Kaminski at the ScheringPlough Corporation, was dissolved in methanol orDMSO and added to the appropriate solutions toa final concentration of 10 µM to yield a 0.1% (vol-ume to volume) concentration of vehicle.

Split-open tubulesCCTs were dissected free from the outer cortex

and split along the longitudinal axis exposing theluminal surface (Palmer and Frindt, ’86). The splittubule was transferred to a standard glass cover-slip (~0.17 mm thick) prepared with a patch ofCell Tak (Collaborative Research Incorporated,Bedford, MA) to which the basement membraneof the tubule loosely adhered. This attachment al-

lowed both the apical and basolateral sides of thetissue to come in contact with the superfusing so-lution. Mounted preparations were fitted on theunderside of a flow-through Lucite chamber andplaced apical side up. The topside of the chamberwas sealed with a standard glass coverslip.

APPARATUSSplit-open tubules

Solutions were gravity fed into a six-port Hamil-ton valve. The solution leaving the valve went di-rectly into a miniature water-jacketed glass coil(Radnotti Glass Technology, Monrovia, CA) forregulating the solution temperature. The warmedsolution entered the experimental chamber whichwas mounted on the stage of an inverted epifluo-rescence microscope (Zeiss IM 35) equipped withNormarski differential interference contrast op-tics. The temperature of the superfusate in thechamber was maintained at 37°C and was veri-fied with a thermistor. The flow rate through thechamber averaged 2.3 ml/min with a chamber vol-ume of 250 µl.

The microscope was interfaced to an alternat-ing wavelength illumination system (PTI DeltaScan) equipped with a 75 watt xenon lamp. Oncethe excitation beam (490 nm and 440 nm) fromthe fluorimeter entered the microscope it was re-flected off a dichroic mirror up through the 40×objective (Zeiss, N.A. 0.75). Fluorescence emittedfrom the cells passed back through the objectiveand encountered an emission filter (520 nm) be-fore entering the photometer. The output of thephotometer was stored in a computer which alsocontrolled the illumination system. The area ofthe tubule illuminated was controlled by a dia-phragm located between the light source and the

TABLE 1. The compositions of the solutions are expressed in mM/L.

Solution: NaRinger NH4Cl 0 K, 0 Na K Ringer Calibration Calibration1 2 3 4 a b

ComponentNaCl 136.5 126.5 – – – –NH4Cl – 10.0 – – – –K2HPO4 2.5 – – 2.5 – –NMDG Cl – – 140.0 136.5 – –Hepes 5.0 5.0 5.0 5.0 25.0 25.0KCl – – – – 128.0 123.0pH 7.4 7.4 7.4 7.4 6.8 7.81In addition to the above components all solutions contained the following in (mM): 2CaCl2, 1.2 MgSO4, 5.5 dextrose, 6 L�alanine, and either 5Nalactate or 5 lactic acide in the Na-free NMDG (N-methyl�D-glucamine) containing solutions. NaOH was used in all Na containing solutionsand NMDG powder in the K and Na free solutions and KOH in Na-free calibaration solutions to titrate to the appropriate pH. The solutionswere the same for the perfused tubule experiments except for the following: 20 mM NH4Cl was used to acid load the cells with the appropriatedecrease inNaCl and 2.5 mM NMDG Cl was replaced with 2.5 mM NMDG PO4 in solution 3.


microscope. The area from which the emited lightwas gathered was controlled by a sliding view-finder between the objective and photometerwhich allowed us to limit the measurement to aparticular cell type. This area consisted of a singleIC. In our experience the intercalated cells loadedmuch better than the principal cells in the BC-ECF-loaded tubules. In all experiments auto-fluorescence was measured on the predeterminedcell type to be studied prior to loading the tubulewith dye and was automatically subtracted fromall traces.

The intercalated cells were distinguished withNomarski differential contrast optics as previouslydescribed (Silver et al., ’92). Our earlier work hasshown that the majority of the intercalated cellsin this nephron segment stained with fluoresceinisothiocyanate-labeled peanut lectin which hasbeen reported to be specific for the β intercalatedcell (Le Hir et al., ’82; Schwartz et al., ’85).

The tubule was loaded with BCECF from boththe basolateral and luminal sides at room tem-perature for 60 minutes after which it was super-fused with NaRinger’s solution (solution 1) at 37°Cfor at least 15 minutes prior to the start of theexperiment. The predetermined cell area was thenrealigned in the viewfinder leading into the pho-tomultiplier tube. Calibration of the signal emit-ted from each IC was performed at the end of eachexperiment. Extracellular pH was varied from 6.8to 7.8 (solutions a and b) in the presence of the K/H exchanger, nigericin (10 µM) in 145 mM K ac-cording to the method of Thomas et al. (’79). ThispH range is in the linear portion of the calibra-tion curve for this dye as we and others have pre-viously shown (Silver et al.,’92; Chaillet et al., ’85).The experimentally determined fluorescence ra-tios were then transformed to pH by calculatingthe slope and y intercept of the calibration curveas previously described (Silver et al., ’92).

Isolated perfused tubulesTo determine whether the H-K ATPase can

function to extrude H+ ions from the cell to thelumen, luminal K-dependent pHi recovery afteran acute acid load was measured in individualPNA-binding ICs in isolated microperfused CCTsfrom CMA and control animals. The technique ofin vitro microperfusion of isolated tubules hasbeen previously described (Satlin et al., ’92; Satlinand Schwartz, ’89). Briefly, an isolated outer CCTwas transferred immediately to a temperature-controlled specimen chamber, mounted on concen-tric glass pipettes, and equilibrated at 37°C in

symmetrical NaR solutions (solution 1). Measure-ments of pHi and identification of the predomi-nant IC subtype were performed with BCECF andrhodamine-conjugated PNA visualized with aNikon Diaphot inverted epifluorescence micro-scope equipped with a 75 W xenon lamp. The fluo-rescence of the pH sensitive dye BCECF wasobserved with a Nikon B filter cassette (dichroicmirror 510 nm, emission >520 nm) and that ofrhodamine-conjugated PNA with a Nikon G cas-sette (excitation 535–550 nm; dichroic mirror 580nm; emission >580 nm). A Dage SIT camera(Michigan City, IN) was used to obtain fluores-cent images of the nephron segment which wasvisualized on a black-and-white monitor (VMI 220,Hitachi Densi, Woodbury, NY). pHi was estimatedfrom the 490 nm and 450 nm excitation signalsmeasured with a video photometric analyzer(model 240A, Instrumentation for Physiology andMedicine, San Diego, CA) and a two-channel chartrecorder. For each perfused tubule studied onlyone IC was monitored during the course of theexperiment.

ICs were identified by adding 20 µM BCECF-AM to the luminal perfusate (solution 1) for 20minutes (Weiner and Hamm, ’89; Weiner andHamm, ’90; Satlin and Schwartz, ’89). These cellswere additionally labeled by perfusing the CCTswith 10 µg/ml rhodamine PNA for 3–5 minutes atroom temperature to test for coincident PNA bind-ing (Le Hir et al., ’82). In the rabbit CCT the vastmajority of cells that stain with BCECF and PNApossess apical Cl/HCO3 exchangers and can befunctionally classified as β-type ICs (Satlin et al.,’92; Satlin and Schwartz, ’89). The luminal solu-tion was then switched from NaR to a Na– andK-free solution (solution 3) for 15 minutes. K exitfrom cells was minimized by addition of 1 mMBaCl2 (Frindt and Palmer, ’89) to this and all Na-free solutions. A PNA-binding IC was identifiedand its pHi monitored for the remainder of theprotocol. ICs residing on the edge of the perfusedtubule were chosen to minimize extraneous fluo-rescence originating from cells above and belowthe focal plane. Measurements were obtainedwithin 30 seconds of each solution change andthen at ~3 minute intervals thereafter. This ap-peared to be an adequate time interval for assess-ing K-dependent changes in pHi.

To test the hypothesis that CMA stimulates api-cal H-K ATPase, the effect of selective addition ofK to the luminal perfusate on pHi recovery froman acute acid load was examined. Intracellularacidification was achieved by changing the peri-


tubular NaR solution to one containing 20 mMNH4Cl (solution 2) for 3 minutes. Upon removalof the NH4Cl from the bath, pHi was monitoredin the absence of bath and luminal Na and K (so-lution 3) for at least 10 minutes. K was then addedback to the lumen (solution 4) as pHi was moni-tored either for 10 minutes or until a steady-statepHi was reached. In another set of experiments Kwas added to the lumen in the presence of the H-K ATPase inhibitor SCH 28080 (10 µM).

An intracellular calibration was performed ineach tubule at the conclusion of the experimentusing the nigericin technique as described above.

STATISTICSResults are presented as means ± S.E.M. where

n equals the number of intercalated cells. No morethan one cell was studied per CCT. The meansrepresent the average of individual ICs. Compari-sons were made by paired and unpaired t-testsand by analysis of variance. Significant differenceswere asserted if P < 0.05.

RESULTSK+-dependent alkalinization in

intercalated cellsFunctional identification of H-K ATPase was

defined as K-dependent intracellular alkaliniza-tion observed in response to an imposed acute acidload with a 10 mM NH4

+ pulse. Figure 1 illus-trates a continuous monitoring of pHi on a singleIC as determined from the fluorescence ratio andthe intracellular calibration performed on this cell.Removal of external NH4

+, Na, and K resulted inan intracellular acidification of 1.0 pH unit belowthe starting pHi value. In the absence of externalK and Na (NMDG substitution, solution 3) no pHirecovery was detectable. Reintroduction of K (5mM) to the superfusate (solution 4) resulted inan intracellular alkalinization at a rate of 0.06∆pH/min and partial recovery of pHi to 7.0. In atotal of 29 ICs studied the K-dependent alkalin-ization averaged 0.17 ± 0.03 pH units to a finalpHi of 6.97 ± 0.06. The initial rate of change of

Fig. 1. Effect of 5 mM extracellular K on pHi recoveryafter acute exposure to an NH4Cl acid pulse in a single inter-calated cell. The ordinate represents the pHi based on theintracellular calibration of the dye in this cell. The tubulewas initially superfused with NaRinger’s solution and thenchanged to 10 mM NH4Cl. Acute exposure to NH4Cl resulted

in slight alkalinization followed by acidification after removalof the NH4Cl. Recovery was absent in the 0 K and 0 Na solu-tion. Readdition of 5 mM K resulted in a K-dependent protonefflux as shown by the increase in pHi. Complete recoverywas observed with the reintroduction of extracellular Na.From Silver and Frindt (’93) with kind permission.


pHi for all of the ICs studied under this conditionwas 0.04 ± 0.01 ∆pH/min. For all of the valuesreported in this study the individual slopes werecalculated once recovery had actually started.

As shown in Figure 1 readdition of Na to thesuperfusate caused complete recovery of pHi to aslightly higher pHi than initially observed (pHi 7.7)at a rate of 0.42 ∆pH/min. In all control ICs stud-ied (n = 29) the subsequent readdition of Naalways resulted in further recovery of pHi back tocontrol values with a mean rate of 0.23 ± 0.02 ∆pH/min. This Na-dependent recovery presumably re-flected basolateral Na/H exchange as described byWeiner and Hamm (’89) in β ICs from rabbit CCT.

Response to the Schering compoundSCH 28080

In order to further characterize and identify themechanism of this K-dependent alkalinization ob-served in IC’s we monitored recovery from theNH4Cl acid load in the presence of the imi-dazopyridine SCH 28080, a specific inhibitor ofthe gastric H-K ATPase (Wallmark et al., ’87). Fig-ure 2 represents a typical experiment where SCH

28080 (10–5 M) was added to the experimental so-lutions upon removal of the NH4Cl. The inhibitorblocked the K-dependent pHi recovery process butdid not inhibit the recovery response to extracel-lular Na. In a total of 10 IC’s studied the K-de-pendent alkalinization response was negligible inthe presence of the Schering compound (0.003 ±0.006 ∆pH/min) with virtually no change in thepHi observed after addition of K (0.02 ± 0.03 ∆pHivs. 0.17 ± 0.03 ∆pHi in the absence of the drug).The rate of pHi recovery with addition of extra-cellular Na was the same in the Schering groupand the control group (0.24 ± 0.06 ∆pH/min,Schering group vs. 0.23 ± 0.02 ∆pH/min, controlgroup). These results demonstrated that H-KATPase was responsible for the K-dependent pHirecovery in response to an acute acid load.

Response to ouabainThe K-dependent pHi recovery protocol was per-

formed in the presence of 100 µM ouabain addedto the solutions after the NH4Cl pulse to demon-strate that this process was due to an H-K ex-changer and not to substitution of H+ for Na+ in

Fig. 2. Effect of SCH 28080 on K-dependent pHi recoveryfrom an acute acid load in a single intercalated cell. The or-dinate corresponds to the pHi based on the intracellular cali-bration of the BCECF in this tubule. After superfusing thetubule with NaRinger’s solution the tubule was exposed to

10 µM NH4Cl. Upon removal of the NH4Cl, SCH 28080 (10µM) was added to all solutions superfusing the tissue. Addi-tion of the blocker prevented the K-dependent H efflux ob-served in Figure 1 but did not effect the Na-dependentrecovery. From Silver and Frindt (’93) with kind permission.


the Na-K ATPase (Polvani and Blostein, ’88). Fig-ure 3 represents the result of such an experiment.The slight delay in the onset of the K-dependentpHi recovery compared to Figure 1 was variable,and there was no consistent difference betweenthe controls and ouabain-treated cells. Ouabaindid not affect the K-dependent intracellular alka-linization or the Na-dependent alkalinization. Inthe four cells treated with ouabain the K-depen-dent pHi recovery rate was similar to that ob-served in the control ICs (0.05 ± 0.02 ∆pH/minvs. 0.04 ± 0.01 ∆pH/min). The K-dependent changein pHi was 0.36 ± 0.12 ∆pH units compared to0.17 ± 0.03 ∆pH units for controls. This differ-ence reflects the more acidotic pHi after the NH4Clload in the ouabain-treated cells compared to thecontrols. The steady-state pHi after K addition wasquite similar between both groups (6.95 ± 0.06,ouabain treated vs. 6.97 ± 0.06 control).

Figure 4 summarizes the rate of K-dependentpHi recovery observed in control, SCH 28080-treated and ouabain-treated ICs. Both the inhibi-tory effect of the Schering compound and the lackof an effect by ouabain on the initial rate of theK-dependent alkalinization suggest that the pro-

cess observed in the intercalated cells in rabbitCCT under conditions of our study is behavinglike gastric H-K ATPase and unlike Na-K ATPase.

In the next group of experiments we studied theeffect of chronic acid-base disturbance, CMA, onfunctional activity of H-K ATPase in rabbits ICs.

Effects of acid feeding on acid-base andK balance

To ensure that the acid-fed rabbits were in astate of CMA both arterial blood and urinesamples were analyzed for evidence of systemicacidosis. The effect of acid feeding on total CO2(tCO2) was apparent by day 8 at which time theTCO2 (22.4 ± 1.1 mM; n = 13) was already signifi-cantly lower than that observed in untreated ani-mals (27.7 ± 1.1 mM n = 10; P < 0.05). The tCO2remained low through 12–14 days of acid feeding(20.9 ± 1.0 mM n = 23; P = NS compared to 8 d).The state of metabolic acidosis was accompaniedby a progressive fall in urinary pH, from 8.4 ±0.1 (n = 4) in untreated rabbits to 7.8 ± 0.2 (n =8) by day 8 and to 7.0 ± 0.3 (n = 20) by days 12–14 of acid feeding. Blood pH did not differ amongthe groups (7.40 ± 0.02, untreated; n = 10; 7.37 ±

Fig. 3. Effect of ouabain on the K-dependent proton ex-trusion in response to an acute acid load in a single interca-lated cell. The y axis represents the corresponding pHi basedon calibration of the dye in this tissue. Addition of 100 µM

ouabain to the solutions superfusing the tubule after the acidpulse (10 mM NH4Cl) had no effect on the K-dependent re-covery of pHi in this cell. From Silver and Frindt (’93) withkind permission.


g/day). The blood pH(pH = 7.44 ± 0.02, n = 6,NaCl-treated vs. pH = 7.40 ± 0.02, n = 10, un-treated) and tCO2 (tCO2 = 29.1 ± 1.8 mM, n = 6,NaCl-treated vs. 27.7 ± 1.1 mM, n = 10, untreated)were similar in NaCl-treated animals and un-treated controls. The urine pH was also the samein the two control groups (pH = 8.30 ± 0.1, n = 6,NaCl; vs. pH = 8.4 ± 0.1, n = 4, untreated). Themuscle K data (45.0 ± 3.1 mM, n = 6, NaCl-treatedvs. 41.9 ± 1.3 mM, n = 6, untreated) and plasmaK data (4.9 ± 0.2 mM, n = 6, NaCl-treated vs. 5.4± 0.2 mM, n = 9, untreated) were also in the twocontrol groups.

During the period of acid feeding, experimen-tal animals gained weight at a rate of 23 ± 3 g/day (n = 19), a rate similar to that observed inNaCl fed, food restricted rabbits (28 ± 5 g/day, n= 5) and within range of the daily body weightgains reported previously for NZW rabbits of simi-lar age allowed free access to food (Schwartz andEvan, ’93).

K+-dependent alkalinization in ICs fromchronic metabolic acids

Figure 5 is a record obtained from a single ICfrom a 10 day acid-fed animal. Removal of exter-nal NH4

+, Na and K resulted in intracellularacidification of 0.70 pH units below the start-ing pHi value of about 7.20. In the absence ofexternal K and Na (solution 3) no pHi recoverywas detectable. Reintroduction of K (5 mM) tothe superfusate (solution 4) resulted in intrac-ellular alkalinization at a rate of 0.16 ∆pH/minto pHi 7.25.

As shown in Figure 6, for all of the ICs studiedunder this condition the initial rate of change ofpHi was 0.09 ± 0.02 ∆pH/min (n = 15 ICs) a ratesignificantly greater than controls 0.03 ± 0.01∆pH/min (n = 7) (P < 0.01). This difference intransport demonstrates that under conditions ofCMA there is stimulation of H/K exchange.

Readdition of Na to the superfusate resulted inadditional recovery of pHi to a higher pHi valuethan initially observed (pHi 7.55) at a rate of 0.32∆pH/minute.

In order to assess whether the increased H-KATPase activity observed with acid feeding was dueto the CMA itself or a secondary effect of the Clload, additional experiments were performed onsplit-opened tubules from rabbits maintained on 75mM NaCl for 10–12 days. There was no differencein the H-K ATPase activity (+K ∆pH/min 0.04 ±0.02 NaCl treated vs. 0.03 ± 0.01 untreated) be-tween this group and the untreated controls.

Fig. 4. Summary of the initial rate of K-dependent pHirecovery (dpH/dt) in response to an NH4Cl acid load in ICsunder control (n = 29), SCH 28080-treated (n = 10) and oua-bain-treated (n = 4) ICs. Results are expressed as the mean± S.E. From Silver and Frindt (’93) with kind permission.

0.02, 8 d, n = 14; 7.36 ± 0.02, 14 d, n = 21) pre-sumably reflecting the respiratory compensationin response to the CMA.

To determine whether systemic acidosis induceda significant loss of total body K, which could initself stimulate H-K-ATPase activity in the col-lecting duct (Wingo, ’89), we analyzed the effectsof CMA on plasma K concentration. The plasmaK concentration in untreated animals (5.4 ± 0.2mEq/L; n = 9) did not differ from that observed inCMA animals examined at either 8 days (5.0 ±0.2 mEq/L; n = 6) or 12–14 days (5.3 ± 0.2 mEq/L; n = 23) of acid feeding. Because a decrease intotal body K content may not be immediately ap-parent from examination of the extracellular Kconcentration, we estimated intracellular K stores(Stokes, ’81) by measuring skeletal muscle K con-tent in untreated and CMA animals. Thigh muscleK content in CMA animals did not differ from thatobserved in untreated animals (45.0 ± 2.4 mEq/100 g fat free dry weight; n = 19 vs. 41.9 ± 1.3mEq/100 g fat free dry weight; n = 6, P = NS).

In order to be certain that stimulation of H-KATPase in CMA rabbits was due to CMA and nota secondary effect of the distal Cl provided in theNH4Cl fed animals, we studied a group of ani-mals maintained on drinking water containing 75mM NaCl prepared in 5% sucrose with similarfood restriction used in the NH4Cl group (60–80


Response to SCH 28080To test whether the K-dependent pHi recovery

observed in CMA in response to an acute NH4Clacid load was due to H-K-ATPase, pHi recoverywas monitored in the presence of SCH 28080. Fig-ure 7 represents a typical experiment from a 12day acid loaded animal where SCH 28080 (10–5

M) was added to the experimental solutions uponremoval of the NH4Cl. The addition of SCH 28080prevented K-dependent pHi recovery. In a total of6 ICs studied from CMA rabbits, the presence ofSCH 28080 prevented K-dependent alkalinizationof pHi (0.005 ± 0.004 ∆pH/min). These results areconsistent with the view that H-K-ATPase is re-sponsible for the K-dependent pHi recovery uponan acute acid load in chronically acidotic animals.

Apical H-K-ATPase activityThe demonstration that CMA stimulates H-K

ATPase activity in split-open tubules led us to testthe hypothesis that under conditions of CMA, theH-K ATPase could function to extrude acid fromthe cell into the tubule lumen. Experiments wereperformed on PNA ICs identified in perfused tu-

bules isolated from CMA and control animals totest whether the location of the H-K-ATPase un-der CMA conditions is apical, consistent with netH+ secretion. H-K-ATPase activity was assayed asthe rate of K-dependent intracellular alkaliniza-tion in response to an acute acid load. CCTs werefirst perfused with BCECF and then rhodamineconjugated PNA, to identify a β-type IC. The lu-men was then perfused with the Na- and K-freesolution. An example of the protocol is shown foran IC from a CMA rabbit in Figure 8. Transientexposure of the IC to 20 mM NH4

+ with subse-quent removal in 0 K, 0 Na (solution 3) led to anintracellular acidification to pHi 6.5 from an ini-tial pHi of 7.25. The mean pHi measured after re-moval of the NH4Cl in CMA ICs was 6.64 ± 0.09(n = 7) from a resting pHi of 7.40 ± 0.07. Therewas a similar change in pHi observed in controlICs (7.52 ± 0.14 to 6.58 ± 0.09; n = 5). As shownin the trace, no change in pHi was observed inthe complete absence of Na and K (bath and lu-men). There was no recovery of pHi in any of theICs studied under this condition as shown by thelack of pHi recovery rates (∆pH/min –0.005 ±

Fig. 5. Effect of 5 mM extracellular K on pHi recoveryafter acute exposure to an NH4Cl acid pulse in a single ICfrom a 10-day acid loaded animal. The y-axis is the pHi asdetermined from the intracellular calibration of the dye inthis cell. Removal of external NH4, Na, and K resulted inintracellular acidification of 0.07 pH units below the starting

pHi value of 7.20. In the absence of external K and Na (NMDGsubstitution, solution 3) no pHi recovery was detectable. Re-introduction of K to the superfusate resulted in an intracellu-lar alkalinization at a rate of 0.162 ∆pH/min to an pHi of7.25. From Silver et al. (’96) with kind permission.


Fig. 6. Comparison of the functional activity of H-KATPase in control and CMA ICs. The functional activity wasassessed as the rate of K-dependent intracellular pH recov-ery from an imposed acid load. The functional activity is aboutthree times higher in the CMA ICs compared to controls (P <0.01). Values are means ± S.E. From Silver et al. (’96) withkind permission.

0.014, n = 6, CMA vs. ∆pH/min –0.017 ± 0.007, n= 7, control). Addition of 5 mM K to the lumenresulted in a rate of pHi recovery of 0.06 ∆pH/min. This apical K-dependent pHi recovery ratewas similar for all of the CMA ICs studied (0.06± 0.01 ∆pH/min, n = 6) and was significantlygreater (P < 0.05) than that observed in controlICs (0.02 ± 0.01, n = 7).

In order to examine whether the intracellularalkalinization observed with readdition of K to thelumen was due to an apical H-K-ATPase, K wasadded to the luminal perfusate in the presence ofSCH 28080 (10–5 M). The luminal K-dependentrecovery after an NH4Cl pulse was abolished inthe presence of the inhibitor (0.005 ± 0.013, n =4). Taken together these data demonstrate thatan apically located H-K ATPase in PNA-positiveICs can be stimulated with CMA.

DISCUSSIONThe BCECF-loaded, split-tubule preparation

was used in this study in order to functionallyidentify a SCH 28080-inhibitable H-K ATPase inindividual ICs of rabbit CCT. Our findings indi-cate that ICs possess an H-K ATPase which is

Fig. 7. Effect of SCH 28080 on K-dependent pHi re-covery from an acute acid load in a single CMA IC. Y-axis, pHi as determined from the intracellular calibrationof the dye in this cell. The solution changes are the sameas Figures 1 and 2. On removal of the NH4Cl, SCH 28080

(10 µM) was added to all solutions superfusing the tis-sue. Addition of the blocker prevented the K-dependentH efflux but did not affect the Na-dependent recovery.From Silver et al. (’96) with kind permission.


activated during acidosis and contributes to therecovery of pHi. This K-dependent H+ effluxmechanism was inhibited by the hydrophobicamine SCH 28080, a specific inhibitor of the gas-tric H-K ATPase (Figs. 2 and 7) but was insensi-tive to ouabain (Fig. 3). The insensitivity toouabain indicates that this transport mechanismis distinctly separate from Na-K ATPase where itis known that protons can substitute for cytoplas-mic Na (Polvani and Blostein, ’88).

The functional identification of the K-dependentH+ transport process in the ICs of rabbit CCTs is

consistent with the findings from enzymatic andimmuno-cytochemical studies from other labora-tories which have also provided evidence for theexistence of an H-K ATPase in the ICs of the CCT(Doucet and Marsy, ’87; Wingo et al., ’90). Previ-ous work done in our laboratory on rabbit CCTsderived from animals maintained on a normal diethas shown that 90% of the cells visually identi-fied as ICs stained positive for FITC-peanut lec-tin (Silver et al., ’92) which indicated that themajority of ICs present in rabbit CCT were β ICs(Le Hir et al., ’82; Schwartz et al., ’85). Other

Fig. 8. Representative tracing of pHi changes recorded ina single PNA-positive IC in a perfused tubule isolated from aCMA rabbit. In the absence of luminal K and Na withdrawalof a 20 mM NH4 pulse led to an intracellular acidification. Inthe absence of luminal and bath K, no recovery of pHi is ob-

served. Restoration of luminal K resulted in an intracellularalkalinization from 6.53 to an pHi of 7.00 at a rate of 0.06∆pH/minute. Addition of K back to the bath did not changethe steady-state pHi. Readdition of bath Na led to full recov-ery of pHi. From Silver et al. (’96) with kind permission.


groups have also reported this high proportion ofβ intercalated cells in rabbit CCT (Schwartz etal., ’85; Furuya et al., ’91). In the present study,we did not confirm the identity of the IC subtypeused for the split-opened tubule experiments butbased on the high percentage of β cells known toexist in rabbit CCT it is likely they express func-tional H-K ATPase.

The K-dependent intracellular pH recovery ratemeasured in the ICs averaged 0.04 ± 0.01 ∆pH/min. Kleinman et al. (’93) reported a similar rateof K-dependent H+ efflux in response to an acuteacid load in cultured inner medullary collectingduct cells. To compare this rate with that observedfor transepithelial K transport (Wingo, ’89) we cal-culated the K+ (or H+) flux (pmol mm–1 min–1) in-duced by the addition of K using the equation:

JK(H) = (dpHi/dt) (VIC) (#ICs/mm tubule) (BT)

where VIC is the volume of cell water in an ICand BT is the buffer capacity. The cell volume wascalculated to be 7.0 × 10–13 L based on measure-ments of luminal surface area and height of anindividual IC (O’Neil and Hayhurst, ’85). They alsoreported a value of 127 ICs/mm tubule for the rab-bit which was used in this calculation. Using ourmean rate of recovery (0.04 ∆pH/min) and an in-tracellular buffering capacity of 11 mM/pH unitas determined by Boron (’89), the H+ efflux, andby inference the K+ influx, is 0.04 pmole/min mm.This value is significantly lower than the reportedrate of transepithelial net K absorption of 5.0 ±1.0 pmol/min mm measured in isolated and per-fused outer medullary collecting duct from K-de-prived rabbits (Wingo, ’89). This great differencesuggests that H-K ATPase may be more impor-tant under conditions of chronic K-deprivationand/or acid-base disturbances than under basalconditions or relatively acute perturbations.

Our results demonstrate that during acute aci-dosis H-K-ATPase functions to extrude H+ fromthe cell. If one of the primary functions of the ex-changer is to secrete H+ then this would necessi-tate that the exchanger be located on the apicalmembrane of the ICs. If this were so then H-KATPase would be placed on the same membraneas the Cl/HCO3 exchanger known to exist in theβ type ICs of rabbit CCT. Theoretically a cell se-creting HCO-3 via the apical HCO3/Cl exchangerand secreting H+ through an H-K ATPase wouldnot be expected to accomplish transepithelial netHCO-3 transport. However, this assumes that both

transporters are functioning at equal rates. Whilewe have not determined the transcellular HCO3

–

flux in the cells we studied we would like to specu-late that these cells may achieve net acid se-cretion through the H-K ATPase under someconditions with the concomitant down regula-tion of the Cl/HCO3 exchanger. Within the con-text of our proposed model H-K ATPase may beactivated during chronic acid-base and or kalemicperturbations and thus be quiescent under nor-mal conditions in the adult animal, allowing nettransepithelial HCO3

– transport to occur.In order to investigate the possibility that H-K

ATPase is stimulated with chronic acidosis andthereby contributing to net acid secretion, experi-ments were performed on ICs from CMA rabbits.Our results demonstrated a stimulation of H-KATPase activity in ICs from CMA rabbits (Fig. 6)and microperfused tubules (Fig. 8). The perfusedtubule data indicate CMA enhances the activityof an apically located H-K ATPase in PNA bind-ing β-type ICs. The absence of changes in skel-etal muscle cell K content in the acid fed animalssuggests that stimulation of H-K ATPase in thisinvestigation is probably not due to acidosis-in-duced total-body K depletion but to either a di-rect or indirect effect of systemic acidosis.

The mechanism responsible for the stimulationof H-K ATPase remains to be determined but maybe the result of insertion of either new or pre-exist-ing pumps in the membrane or a greater flux acrossthe pumps already residing in the membrane. Inboth control and CMA conditions the K-dependentrecovery of pHi from an acid load was only partial(Figs. 1 and 5). This may be due to a specific pHirange within which this transporter operates.

In conclusion the data demonstrate a SCH28080 sensitive K-dependent H+ extrusion mecha-nism in ICs from rabbit cortical collecting tubuleconsistent with the gastric isoform of H-K ATPase.The H-K ATPase activity was increased under con-ditions of chronic metabolic acidosis. Taken to-gether with the data on perfused tubules, thissuggests an acid-secretory role for the H-KATPase that is coupled to K movement.

ACKNOWLEDGMENTSR.B. Silver thanks Dr. Zadunaisky for inviting

her to the symposium honoring Dr. Kleinzeller. Itis a privilege to be included among those who areinspired by him.

Funding was provided by an Investigatorshipfrom the American Heart Association, New YorkCity Affiliate, the American Heart Association


Grant-in-Aid and NIH DK45828 to R.B.S. L.M.S.was supported by NIH DK38470 and a Grant-in-Aid from the American Heart Association.

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Characterization and regulation of H-K ATPase in intercalated cells of rabbit cortical collecting duct