Plasma membrane Na+-H+ antiporter and H+-ATPase in the medullary thick ascending limb of rat kidney MARC FROISSART, PASCALE BORENSZTEIN, PASCAL HOUILLIER, FRANCOISE LEVIEL, JOSIANE POGGIOLI, ERIC MARTY, MAURICE BICHARA, AND MICHEL PAILLARD Laboratoire de Physiologie et Endocrinologie Cellulaire Rkale, Institut National de la SantG et de la Recherche Midicale U. 356, Universiti Pierre et Marie Curie and Hpital Broussais, 75014 Paris, France Froissart, Marc, Pascale Borensztein, Pascal Houilof this segment (10). Also, arguments supporting the lier, Frangoise Leviel, Josiane Poggioli, Eric Marty, presence of both luminal and basolateral Na+-H+ antiMaurice Bichara, and Michel Paillard. Plasma membrane porters have been obtained from intracellular pH (pHi) Na’-H’ antiporter and H’-ATPase in the medullary thick measurements in the rat CTAL (19). Until now, there is ascending limb of rat kidney. Am. J. Pltysiol. 262 (Cell Physiol. no published study that has unequivocally established 31): C963-C970, 1992.-To characterize H’ transport mechathe cellular mechanisms of H+ extrusion from rat mednisms in a fresh suspension of rat medullary thick ascending ullary TAL (MTAL) cells. In another mammalian spelimb (MTAL) tubules, we have monitored intracellular pH cies, the mouse, Na+-H+ antiport activity has been lo(pHi) with use of the fluorescent probe 2’,7’-bis(carboxyethyl)calized to the luminal membrane of MTAL cells (17,18). 5,6-carboxyfluorescein. First, a Na+-H’ antiporter was identified in bicarbonate-free N-2-hydroxyethylpiperazine-N’-2-ethIn addition, Na+-H+ antiport may not be the sole anesulfonic acid (HEPES)-buffered media at 25°C. pHi recovmechanism of proton secretion by the rat TAL. Indeed, ery of Na-depleted acidified cells was dependent on extracelN-ethylmaleimide (NEM) -sensitive proton-adenosinelular sodium concentration, which was inhibited by amiloride triphosphatase (H+-ATPase) activity has been demonin a manner consistent with simple competitive interaction strated in nonmitochondrial membrane fractions of the with one external transport site (amiloride Ki = 1.5-2.1 X 10m5 M); Na-induced pHi recovery of acidified cells was electroneu- rat MTAL and CTAL (1,22). Also, Good and Kurtz (14) form that the cell pH retral since it was not affected by 5 or 100 mM extracellular have reported in preliminary covery from an acute intracellular acidification of rat potassiumin the presenceor absenceof valinomycin. Second, at 37”C, pHi recovery after acute intracellular acidification MTALs isolated and perfused in vitro had a component caused by 40 mM acetate addition to cell suspensionwas that was sodium independent, ATP dependent, and seninhibited 36% by 200-400 nM bafilomycin A1, a macrolide sitive to dicyclohexylcarbodiimide, and thus that was antibiotic that specifically inhibits vacuolar-type H+-ATPase suggestive of a plasma membrane H+-ATPase. at submicromolarconcentrations. In addition, amiloride-insenThe aim of the present work was therefore to detersitive pHi recovery wasinhibited by bafilomycin A1, 10D3M N- mine the various mechanisms of H+ extrusion and to ethylmaleimide, and lo-* M preactivated omeprazole but not quantify their relative importance in the rat MTAL. We by 10e6M vanadate, 10m4M SCH 28080,or removal of extracellular potassium. Also, metabolic inhibition by absenceof monitored pHi with use of the fluorescent probe 2’,7’(BCECF) in substrate, 10e4M KCN, or 5 X 10e4M iodoaceticacid inhibited bis(carboxyethyl)-5,6-carboxyfluorescein fresh suspensions of rat MTAL tubules. The results amiloride-insensitive pHi recovery. The inhibitory effects of absenceof metabolic substrate and iodoacetic acid were re- demonstrate that both Na+-H+ antiporter and H+-ATPmechanisms of moved by reexpoureto glucoseand L-leucine and by exogenous ase of the vacuolar type are important ATP, respectively. Finally, at 37”C, in bicarbonate-free H+ extrusion from these cells. HEPES-buffered medium,pH 7.4, resting pHi was 7.45 t 0.01 and was lowered to 7.40 2 0.01 (P < 0.05) by 2 X 10D3M METHODS amiloride and to 7.41 t 0.02 (P c 0.05) by 200-400 nM Preparation of rat MTAL tubule suspensions.The method bafilomycin A1. We concludethat both Na+-H’ antiporter and plasma membrane H+-ATPase of the vacuolar type mediate used was derived from that previously described by TrinhTrang-Tan et al. (26, 27). Male Sprague-Dawleyrats (200-300 proton extrusion from rat MTAL cells. g body wt) were starved overnight but allowed free accessto intracellular pH; tubule suspension;amiloride; bafilomycin A,; tap water until anesthetization with pentobarbital sodium.For N-ethylmaleimide; omeprazole;vanadate; SCH 28080 each experiment, four or six kidneys of two or three anesthetized rats were rapidly removedwithout any previous manipuTHE RAT THICK ASCENDING LIMB (TAL) has been relation and immediately immersedinto ice-cold dissectingsocently shown to absorb bicarbonate secondary to proton lution, which took only a few seconds,to avoid anoxic damage secretion within the tubular fluid at substantial rates to medullary tissues and improve cell viability; the kidneys (13) that may account for much of the bicarbonate reab- were then cut into thin slicesalong the corticopapillary axis in sorbed from Henle’s loop in vivo, i.e., -15% of the ice-cold Hanks’ solution containing (in mM) 137 NaCl, 5.4 KCl, 4.2 NaHC03, 0.3 Na2HP02, 0.4 KH2P04, 0.4 MgS04, 0.5 bicarbonate-filtered load (4, 5). Yet, the membrane mechanisms of H+ extrusion by rat TAL cells have been MgC12,1.2 CaClz, and 5 glucose,previously bubbled with 95% 02-5% COZ, according to the original method (26). Under a investigated in only a few studies. That bicarbonate microscope,the inner stripe of outer medullaof each absorption by the rat cortical TAL (CTAL) isolated and dissecting slice was excised and cut into uniform small piecesby gently perfused in vitro is inhibited by the absence of sodium forcing it through a piece of stretched medicalgauze.The small from luminal and peritubular solutions and by the pres- tissuepieceswere then serially subjectedto collagenasedigesence of 10m3M luminal amiloride is consistent with the tion (40 mg%) in Hanks’ solution bubbled with 95% 02’5% presence of a Na+-H+ antiporter in the apical membrane COn at 37°C during six to eight lo- to 15-min periods. After 0363-6143/92 $2.00 Copyright 0 1992 the American Physiological Society

C963

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C964

H+ TRANSPORT

each period, the cell- and tubule fragment-containing supernatants were collected after I-min gravity sedimentation and stored into ice-cold Hanks’ solution. After the last digestion, these supernatantswere sievedthrough a 75pm opening nylon mesh that retained the largest fragments of MTAL. These harvested fragments were then collected in Hanks’ solution containing 1 g% bovine serum albumin (BSA), centrifuged 1 min at low speed(-80 g), and resuspendedafter washingin an appropriate volume of the desired medium. BecauseHanks’ solution hasa nonphysiologicalpH of -6.7, someexperiments (in particular those summarized in Fig. 7 in RESULTS) were performed with Hanks’ medium supplementedwith 24 mM bicarbonate, pH 7.38, which did not appreciably change the main resultsof this study. As observedby light microscopy,the final suspensioncontained almost exclusively MTAL fragments (>95%), occasionalthin descendinglimb fragments, and no isolated cells and medullary collecting tubule segments,as observedby others (26). The lengths of the MTAL fragments ranged in generalfrom 75 to 200 pm. That furosemideinhibits the sodiumtransport-dependent oxygen consumption (26) and NH: entry within the cells through the Na+-K’-2Cl-cotransporter (2) in this preparation provides strong evidence that the apical cell membranesare in free functional contact with the extracellular mediumand thus that the lumina of thesetubules are patent in suspension. Loading cells with BCECF and measuring PHi. To load the MTAL cells with the pH-sensitive probe BCECF, tubules suspensionswere incubated for 30-45 min at 37°C in a bicarbonate-containing mediumbubbledwith 95% 02-5% COz (solution A in Table l), containing 10 PM BCECF acetoxymethyl ester [(BCECF/AM), di sso1ved in dimethyl sulfoxide (DMSO) and storedat -2O”C]. The BCECF-loadedtubules werethen washed five times by gentle centrifugation to remove the extracellular dye and resuspendedin the appropriate medium. Aliquots of the MTAL tubule suspensionwere diluted into glasscuvettes containing 3 ml of the experimental medium to reach a final cytocrit of ~1 vo1%0.BCECF fluorescencewas monitored by use of a Perkin-Elmer 204 spectrofluorometer equippedwith a magnetic stirrer for experiments performed at 25°C (ambient temperature), or a Shimadzu RF-5000 spectrofluorometer equippedwith a water-jacketed temperature-controlled cuvette holder and magneticstirrer for experimentsperformed at 37*C. Fluorescence intensity was recorded at one emission wavelength, 530 nm, whereasthe excitation wavelength alternated either manually (Perkin-Elmer spectrofluorometer) or automatically at 2-s intervals (Shimadzu spectrofluorometer) between two wavelengths,500 and 450 nm. Just before each run,

Table

IN RAT MTAL

the MTAL fragment suspensionaliquot to be added into the spectrofluorometer cuvette was washedand centrifuged in the appropriate medium to further remove any residual extracellular dye. The rate of increase of extracellular fluorescence causedby leakageof BCECF out of the cells was low at 1.55t O.l3%/min (pH 7.4, n = 3) and was neglectedbecauseexperiments were performed for short time periods (~4 min). The values of the fluorescenceratio (Fsoo/Frao) were converted into pHi values with use of calibration curves. Calibration curves were establisheddaily by one of two methods. First, the relationship between intracellular BCECF fluorescenceand pHi wasdeterminedon a sampleof the MTAL fragment suspension under conditions during which pHi and extracellular pH may be assumedto be equal, i.e., by placing the cells in a medium containing 115 mM potassiumand 3.3 PM of the K’-H+ ionophore nigericin; in this case, for the calibration and for each experimental run, the fluorescenceof the extracellular medium at both excitation wavelengths was substractedfrom the total fluorescenceobservedafter addition of BCECF-loaded cells to determinethe signalgeneratedby the intracellular dye. Second, after each experimental run, the cells were permeabilizedwith Triton X-100 (0.025 g%), and the relationship between extracellular BCECF fluorescenceand mediumpH was established; this method was used particularly when somematerials (e.g., high dosesof amiloride) suspectedto interfere with the fluorescence were present in the cuvette during experimental runs. Typical calibration curves are shown in Fig. 1; as can be seen, external calibration was not different from that with nigericin under our experimental conditions, which was routinely observed. Materials. CollagenaseCH gradeII wasobtained from Boehringer Mannheim France (Meylan, France), Hanks’ solution from GIBCO (Paisley, Scotland), BCECF/AM from HSC Research Development (Toronto, Canada), and tetramethylammonium (TMA) chloride from Aldrich-Chimie (Strasbourg, France). Amiloride, DMSO, L-leucine, nigericin, NEM, sodium orthovanadate, iodoacetic acid, and all other chemicals were obtained from Sigma Chimie (La Verpilliere, France). Bafilomycin A1 was kindly given by Prof. K. Altendorf, Universitgt 4

1. Experimental solutions

NaCl 135 67.5 67.5 67.5 TMA-Cl 135 67.5 67.5 67.5 5 KC1 3 3 3 3 25 NaHCOa HEPES 10 10 10 10 10 CaC& 1.8 1.8 1.8 1.8 1.8 1.8 1 1 1 1 1 MgSO, 1 NaH2P04 0.2 0.2 Na2HP04 0.8 0.8 KH2POr 0.2 0.2 0.2 0.2 K2HP0, 0.8 0.8 0.8 0.8 Glucose 5 5 5 5 5 L-Leucine 5 5 5 5 5 Values are given in mM. TMA, tetramethylammonium. Solution A was bubbled with 95% 02-5% Con, pH 7.4; solutions B-F were bubbled with 100% O2 and adjusted to pH 7.4 with tris(hydroxymethyl)aminomethane base.

I

I

I

I

I

J

6.6

6.8

7.0

7.2

7.4

7.6

PH

Fig. 1. Relationships between fluorescence intensity ratio (F&F& of 2’,7’-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and pH. n , BCECF-loaded medullary thick ascending limb (MTAL) cells were washed and suspended in 3 ml (final cytocrit -1 vo1%0) of highpotassium (115 mM) medium containing 3.3 PM nigericin [intracellular pH (pHi) = medium pH]; Fbm/F450ratio was linearly related to pHi over this pH range (y = 2.0x - 11.5; r = 0.99). q , Suspension was then exposed to Triton X-100 (0.025 g%) to obtain release of dye from cells to extracellular medium; a linear relationship between Fm/F4m and medium pH was again observed (y = 1.9x - 10.8, I- = 0.99), which was not significantly different from previous one. Medium pH was measured by glass pH electrode.

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H+ TRANSPORT

Osnabruck, Germany. Omeprazolewas a gift from Astra France Laboratories (omeprazolewas activated at pH c 2 for 30 min at 0°C in the dark before use). (3-Cyanomethyl-2-methyl-& phenylmethoxy)imidazo[ 1,2-culpyridine(SCH 28080)was a gift from Schering-PloughLaboratories. Statistics. Resultsare expressedas meanst SE. Statistical significancebetweenexperimental groupswas assessed by Student’s t test or by one-way analysis of variance, completedby Dunnett’s t test.

IN RAT MTAL r 0.04 c 1, i

0.03

CL .

s,

0.02

z g 0.01

RESULTS

Na+-H+ antiport. To look for the presence of a Na+H+ antiporter in rat MTAL cells, experiments were performed at 25°C (ambient temperature), since possible plasma membrane H+-ATPase activity was expected to be minimal at this temperature, in bicarbonate-free N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-buffered media bubbled with 100% O2 (solution B in Table 1) to avoid interference that would have been caused by bicarbonate transport. Under these conditions, the resting pHi was 7.37 t 0.02 with an extracellular pH of 7.34 t 0.02 (n = 5 in 3 separate experiments). If a Na+-H+ antiporter were present in rat MTAL cells, then pHi recovery after maneuvers that acidify cells should be sodium dependent and amiloride sensitive. The following protocol was used to obtain sodium-depleted acidified cells. First, MTAL fragments were incubated and washed several times during 30 min in a sodium-free TMA medium bubbled with 100% O2 (solution C in Table 1) to nearly completely remove sodium from the cells; we have checked that, under these conditions, cell sodium was undetectable by flame photometry. Then MTAL cells were suddenly exposed to 3.3 PM nigericin, a K+-H+ ionophore that caused exchange of internal K+ for external H+ and thus acidified the cells until a plateau was obtained. After the ionophore was extracted by adding 1 g% BSA to the suspension, the cells were washed and resuspended in sodium-free TMA medium. In 16 MTAL fragment suspensions subjected to these maneuvers, pHi fell to a stable value of 6.87 t 0.04 (extracellular pH 7.39 t 0.01). To initiate a sodium-dependent pHi recovery of sodium-depleted acidified MTAL cells, two protocols were used. First, various amounts of NaCl were added to TMA medium containing acidified cells. Second, sodium-depleted acidified cells were added to an isosmotic sodiumcontaining medium (various amounts of NaCl isosmotitally replacing TMA-Cl). External sodium initiated prompt concentration-dependent pHi recovery in both cases (data not shown). The first 12 s of the time course of sodium-dependent pHi recovery was fitted to a linear equation to calculate the initial rate of pHi recovery (dpHi/dt). Correlation coefficients for these linear fits averaged 0.970 t 0.005. As shown in Fig. 2, dpHi/dt rose as the external sodium concentration was augmented and reached a plateau, which indicated that the sodium dependency of pHi recovery was a saturable process. It is also clear from Fig. 2 that the relationships between the dpHi/dt and external sodium concentration were different in the two protocols; indeed, Lineweaver-Burk plots of the data (not shown) led to calculated values of apparent Michaelis constant for Na [K, (Na)] of 11 vs. 36 mM and of maximal velocity ( Vmax) of 0.015 vs. 0.033

C965

0

Fig. 2. Sodium dependency of pHi recovery at 25°C. Left: acidified Nadepleted MTAL cells were washed and suspended in Na-free tetramethylammonium (TMA) medium and pHi was monitored; at time 0, NaCl was added to cuvette to reach various final external Na concentrations ([Na],). Adding 100 mM TMA-Cl did not cause pHi to recover. Right: acidified Na-depleted MTAL cells were washed and suspended at time 0 in either Na-free TMA medium ([Na], = 0 mM) or in isosmotic Na-containing TMA medium (various amounts of NaCl replacing TMA-Cl). Initial rates of pHi recovery (dpHi/dt, first 12 s) are plotted as functions of [Na],. 0.020

-

0.015

flmiloride

OL

0

, JAM

\IO-6

IO-5

Amiloride,

IO-4

10-3

M

Fig. 3. Effect of amiloride on sodium-dependent initial rates of pHi recovery (dpHi/dt). Sodium-depleted acidified MTAL cells were exposed to 40 mM sodium by addition of sodium chloride to the suspension. Points are means k SE of at least 3 determinations in 7 separate experiments. Inset: Dixon plot of data (r = 0.998); apparent inhibitory content (Ki) of amiloride, 6.9 x 10s5 M; Ki of amiloride corrected for external sodium concentration, 1.5 X 10m5 M. Extracellular pH was 7.40.

pH/s with NaCl addition and NaCl isosmotically replacing TMA-Cl, respectively. Whether these differences are due to changes in medium osmolality and/or cellular volume or surface was beyond the scope of this work and will not be discussed further. The effects of various concentrations of amiloride on -40 mM sodium-induced pHi recovery are shown in Fig. 3; this sodium concentration was chosen because it is sufficiently higher than the K, (Na) value to induce Na+H+ antiport activity approaching the Vmax (see Fig. 2, left). With 10m3 M amiloride, pHi recovery was almost completely inhibited. The Dixon plot of the data (Fig. 3, inset) yielded a straight line consistent with a single amiloride inhibitory site having an apparent inhibitory constant (Ki) of 6.9 x 10e5 M. When corrected for the value of external sodium concentration (38.7 t 0.3 mM), with the appropriate equation [(Ki = apparent Ki/ (l+Na/Na&)], the value of the Ki for amiloride was 1.5 x 10D5M. To determine the mechanism by which amil-

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C966

H+ TRANSPORT

oride inhibits Na+-induced pHi recovery in MTAL cells, the latter was estimated at various Na+ concentrations in the presence of three different amiloride concentrations (10e6, 2 x 10v6, and low4 M). The results obtained after Lineweaver-Burk linearization of the data are shown in Fig. 4. It is apparent that amiloride did not modify the y-intercept (no effect on Vmax)but altered the slope of the regression line [effect on K, (NJ. Plotting the slope of the regression line against the amiloride concentration (Fig. 4, inset) yielded a straight line from which was derived the value of 2.1 X 10e5M for the Ki of amiloride. Note that plotting the apparent & (Na) values against the corresponding amiloride concentrations (not shown) also yielded a straight line from which were derived the values of 11.3 mM for & (Na)and 2.1 X 10e6M for amiloride Ki. Thus it may be concluded that amiloride inhibited the Na+-induced pHi recovery in acidified MTAL cells by simple competitive interaction with one external Na+-transport site (amiloride Ki = 1.52.1 x lo-' M). The stoichiometry of the Na+-H+ antiporter is one for one as has been established in numerous cell types. The rate of transport should not therefore be dependent on the membrane potential. The contribution of the membrane potential to Na+-induced H+ movements was evaluated by modifying the membrane potential by introduction of the K+ ionophore valinomycin. pHi recovery of acidified MTAL cells in TMA medium after 27 mM NaCl addition was studied with and without 9 PM valinomycin at two different extracellular potassium concentrations (5 and 100 mM). There was no significant difference in the initial pHi, and the initial rate of pHi recovery was similar in the different situations (Table 2), which indicated that sodium-induced H+ movements were not membrane potential sensitive. Finally, we determined the role of the Na+-H+ antiKi =2.1dO-5M

400

o-d 10

/

loo

fimiloride

, pM

1 O4 M amiloride

0

0.02

0.06

0.04

0.08

0.1

1 /[Nab

Fig. 4. Lineweaver-Burke plot of initial rates of pHi recovery of acidified sodium-depleted MTAL cells upon addition of sodium chloride in the absence (control) and presence of amiloride. Each regression line (r 2 0.60, P < 0.05) was obtained from at least 12 determinations. Data points are shown only for the control line. Inset: slope of regression line against corresponding amiloride concentration (r = 0.999). Extracellular pH was 7.40.

IN RAT MTAL

2. Na-dependent PHi recovery from intracellular acidification with or without 9 pM valinomycin Table

Extracellular Concentration,

dpHi/dt,

K mM

Control

pH/min P Value

Valinomycin

100 0.66 =f:0.06 0.60 t 0.06 NS 5 0.48 t 0.06 0.54 t 0.18 NS Values are means t SE of 3 determinations in 2 separate experiments of initial rate of pHi recovery (dpHi/dt) after 27 mM NaCl addition to acidified medullary thick ascending lines cell-containing TMA medium. There was no significnat difference in initial pHi in the various situations. 40mM

acetate

7.5 r 7.4

-

f Control

7.2 z p,

7.1

2x10

-3M

amiloride

7.0 7-3;: 6.9 6.8 -

I

I

I

1

1

2

3 min

Fig. 5. Time course of MTAL pHi after acute cell acidification at 25OC. Cells were incubated in TMA medium containing 67.5 mM sodium in the absence (control) or presence of 2 x low3 M amiloride. Addition of 40 mM potassium acetate induced immediate cell acidification (acetic acid entry) followed by a complete pHi recovery in control and almost no recovery in the presence of amiloride.

porter under more physiological conditions, i.e., without previous cell sodium depletion or prolonged acidification. When MTAL cells were incubated in a bicarbonate-free HEPES-buffered medium containing 67.5 mM NaCl bubbled with 100% 02 (solution D in Table l), pHi recovery after acetate-induced cell acidification (rapid intracellular diffusion of acetic acid) was completely suppressed in the presence of 2 x 10V3 M amiloride (Fig. 5). This sodium concentration was used to obtain nearmaximal inhibition of Na+-H+ antiport with 2 X low3 M amiloride because the poor amiloride solubility prevents using a higher concentration of the drug. These results indicate that Na+-H+ antiport was the predominant, if not the sole, mechanism of H+ extrusion at 25°C. In addition, the resting pHi before acetate addition was affected by the inhibition of Na+-H+ antiport, since it was lower in the presence (7.26 t 0.04, n = 4) than in the absence (7.40 t 0.07, n = 6) of amiloride (P < 0.05) (Fig. 5). Plasma membrane H+-ATPase. To look for the presence of a plasma membrane H+-ATPase, we carried out experiments at 37°C to unmask this active H+ transport mechanism. We first determined the extent of inhibition of Na+-H+ antiporter caused by 2 x 10e3 M amiloride with 67.5 mM external sodium at 37°C; MTAL cells were sodium depleted and acidified as described above and, in addition, were preincubated during 45-60 min in the absence of metabolic substrates, i.e., glucose and L-leutine, to minimize any ATP-dependent process such as a proton pump (see Fig. 9 below). As shown in Fig. 6, abrupt exposure to 67.5 mM sodium initiated vigorous Na*-H+ antiport activity, which was suppressed 93% by

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H+ TRANSPORT 63.5

C967

IN RAT MTAL 40 mM acetate

mM NaCl

-o-

7.4

7.3

-

Control

z

7.2

7.3

.

7.2

-

7.1

-

z m

Control Bafilomycin

-

7.01 -40

”

” -20

” 0

’ 20

” 40

Time, 40 mM

0

10

20

30

40

Time

60

60

70

80

Fig. 6. Inhibition of Na+-H’ antiport by 2 x low3 M amiloride at 37OC. Cells were sodium depleted, acidified, and metabolic substrate depleted during 45-60 min; pHi was then recorded before and after addition of 67.5 mM NaCl to the suspension. In the presence of 2 mM amiloride, initial rate of pHi recovery was inhibited 93% (0.09 k 0.05 vs. 1.25 rt 0.07 pH/min). Each point is mean k SE of 7 determinations in 2 separate experiments. x 10m3M amiloride. Thus further experiments were performed in bicarbonate-free HEPES-buffered medium, external pH 7.4, containing 67.5 mM sodium, and bubbled with 100% O2 (solution D in Table 1). Results obtained with bafilomycin A1, a macrolide antibiotic that specifically inhibits vacuolar H+-ATPases (6), are summarized in Fig. 7. Under control conditions, the resting pHi averaged 7.45 t 0.01; after addition of 40 mM potassium acetate to the tubules suspension, pHi immediately fell then recovered toward baseline levels (Fig. 7A). The initial rate of pHi recovery (dpHi/dt), calculated by fitting first-order regression lines to the initial 20 s of the time course, averaged 0.36 t 0.04 pH/ min (n = 13). Bafilomycin A1 (200-400 nM, corresponding to ~0.8-1.6 pM/mg cell protein), added to the suspension aliquots 2-3 min before the pHi measurements, decreased the resting pHi to 7.41 t 0.02 (P C 0.05) and reduced 36% the dpHi/dt after intracellular acidification (0.23 t 0.02 pH/min, n = 11, P c 0.02); there was no difference in the extracellular pH (7.41 t 0.01 vs. 7.40 t 0.01) and in the acidification pHi (7.11 t 0.01 vs. 7.09 t 0.01) between control and bafilomycin A1 conditions. In the presence of 2 x low3 M amiloride (Fig. 7B) added to the medium 2-3 min before the measurements to inhibit Na+-H+ antiport, a significant fall in the resting pHi was observed (7.40 t 0.01, P < 0.05 compared with control); the dpHi/dt after acetate addition was also significantly decreased (0.17 t 0.01 pH/min, n = 6, P < 0.01 compared with control) but was nevertheless substantial. Thus inhibition of Na+-H+ antiport was responsible for only 53% inhibition of the pHi recovery rate instead of 93% inhibition when ATP-dependent processes were not operational (Fig. 6). Bafilomycin A1, added to the suspension with amiloride, further decreased the resting pHi to 7.36 t 0.01 (P < 0.05 compared with amiloride value) and reduced 29% the dpHi/dt after intracellular acidification (0.12 t 0.01 pH/min, n = 8, P < 0.01 compared with amiloride value); here also, there was no difference

*

” 80

’ 100

s

-

90

, t

’ 60

acetate

6.9"""""""""'

2

Al

Amlloride Amiloride Bafilomycin

+ A1

ax 7.2 = 7.1 7.0 6.9 i 6.81 -40

’

’ -20

’

’ 0

’

’ 20

’

Time,

’ 40

’

’ 60

*

’ 80

*

’ 100

s

Fig. 7. Effect of bafilomycin Al on MTAL pHi at 37°C. Cells were incubated in TMA medium containing 67.5 mM sodium; bafilomycin A1 was added 2-3 min before the measurements at 200 or 400 nM. (There was no difference between these 2 concentrations, and the results were pooled.) A: effect on control cells. B: effect on cells treated for 2-3 min with 2 x 10m3M amiloride. Differences in resting pHi and rate of pHi recovery after acidification were significant in A and B conditions (see text).

in the extracellular pH (7.40 t 0.01 vs. 7.39 t 0.01) and in the acidification pHi (6.89 * 0.015 vs. 6.89 t 0.015) between amiloride and amiloride plus bafilomycin A1 conditions. The residual pHi recovery in the presence of amiloride plus bafilomycin A1 was likely explained by slow entry of the acetate anion into the cells with subsequent consumption of H+ and by possible residual Na+H+ exchange activity. Thus, at 37”C, both Na+-Hf antiport and bafilomycin Al-sensitive H+ transport (proton pump) mediated H+ extrusion from rat MTAL cells and were responsible for 53-60% and 36%, respectively, of the pHi recovery from an acute intracellular acidification. lMetaboZic inhibition studies. To determine whether the bafilomycin Al-sensitive pHi recovery after cell acidification (i.e., the proton pump) requires energy from ATP hydrolysis, as expected, we carried out experiments in the presence of 2 x 10m3M amiloride and 67.5 mM extracellular [ Na] under various conditions of inhibition of cellular ATP production. The results are summarized in Fig. 8. When MTAL tubule suspensions were preincubated for 15 min in the presence of 10m4 M KCN, which inhibits the mitochondrial ATP production, the resting pHi fell to 6.97 t 0.03 (n = 11, P < 0.01 VS. amiloride) and the dpHi/dt after cell acidification by acetate was decreased to 0.09 t 0.02 pH/min (P < 0.01 vs. amiloride). In the presence of 5 x lOa M iodoacetic acid (IAA), which inhibits the glycolytic pathway of ATP production, the resting pHi fell to 7.14 t 0.02 pH (n = 11, P < 0.01 vs. amiloride), and the dpHi/dt after cell acidification was also decreased to 0.08 t 0.01 pH/min (n = 11, P < 0.01 vs. amiloride). Finally, when MTAL

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C968

H+ TRANSPORT

40mM

acetate

7.20

6.90

6.70 -15

0

15

30

45

60

Time,s Fig. 6. Effect of inhibition of cellular ATP production on MTAL pHi at 37°C. Cells were incubated in TMA medium containing 67.5 mM sodium in the presence of 2 x 10T3 M amiloride alone (0) and amiloride plus lo-' M KCN (D), 5 x lo-’ M iodoacetic acid (A), or absence of glucose and leucine (A); 90 s later (time 0), cell acidification was induced by 40 mM potassium acetate, and pHi was recorded for 60 s. 0.20 -

B

P < 0.001

v i

-

A

0.15 E

i 4 0.10 ;; .

?e 0.05 0.00 -

3 6lU-

61u+

1eu-

1eu+

P .z 0.05

IAA

IAR+RTP

Fig. 9. Reversibility of the metabolic inhibition of the amiloride-insensitive pH, recovery in MTAL cells at 37°C. A: aliquots of suspensions were incubated in TMA medium containing 67.5 mM Na and free of glucose and leucine for 15 min. During the following 30-min period, substrate-free condition was maintained (Glu-, Leu-) or substrates were reintroduced (Glu+ and Leu+). Then cell acidification was induced by adding 40 mM potassium acetate, and initial rates of pH, recovery (dpH,/dt) were determined. B: aliquots of suspensions were incubated for 30 min with or without 0.25 mM ATP, and iodoacetic acid (IAA) was added for 15 min further in all aliquots. Then cell acidification was induced by 40 mM potassium acetate and dpHi/dt values were determined.

cells were preincubated for 15 min in the absence of the two metabolic substrates, glucose and L-leucine (solution E in Table l), the resting pHi was 7.22 + 0.02 (n = 8), and the dpHi/dt after cell acidification was markedly inhibited (0.04 + 0.01 pH/min, P < 0.01 vs. amiloride). To make sure that the effects described above were actually due to cellular ATP depletion, we determined whether they were reversible by cellular repletion of ATP. First, we tested the effects of reintroducing metabolic substrates (glucose and L-leucine) in MTAL cells initially incubated for 15 min without these substrates (Fig. 9). Aliquots of the suspensions were reexposed to a medium containing 9 mM glucose and 5 mM L-leucine for 30 min, whereas other aliquots were kept substrate

IN RAT MTAL

free for the same time. In the prolonged absence of metabolic substrates, the amiloride-insensitive dpHi/dt after cell acidification was completely suppressed (0.003 + 0.042 pH/min), while the reintroduction of metabolic substrates restored the dpHi/dt to 0.14 rt 0.019 pH/min (n = 9, P < 0.01). In a second series of experiments, exogenous ATP was added to MTAL tubule suspensions incubated in the presence of IAA to augment ATP produced by the mitochondrial metabolism. Indeed, exogenous ATP has been recently shown to increase the ATP content of isolated kidney tubules, which requires ongoing mitochondrial oxidative phosphorylation (28). Aliquots of MTAL tubule suspensions were incubated for 30 min with or without 0.25 mM ATP in the medium and then exposed to 5 x 10m4 M IAA for 15 min. The inhibition by IAA of the amiloride-insensitive dpHi/dt after cell acidification was significantly removed by the presence of exogenous ATP (P < 0.05, Fig. 9). Taken together, these findings are consistent with the presence in rat MTAL cells of a H+ pump that utilizes ATP derived from both glycolysis and mitochondrial sources. Type of H+-ATPase. The experiments described above have shown that the ATP-dependent amiloride-insensitive dpHi/dt after cell acidification was inhibited by bafilomycin Ai, which indicates that a plasma membrane proton pump of the vacuolar type is present in rat MTAL cells. To further characterize this proton pump, we tested the effects of other putative inhibitors and of the external potassium concentration on the amiloride-insensitive dpHi/dt after acetate-induced cell acidification. Results are summarized in Fig. 10. NEM (10m3 M), an inhibitor of nonmitochondrial H+-ATPases (24), significantly reduced the dpHi/dt from 0.17 + 0.03 (2 X 10e3 M amiloride alone) to 0.05 + 0.01 pH/min (n = 17, P < 0.01). Vanadate (10e6 M), a well-known inhibitor of phosphorylating ATPases of the EIEP type such as the Na+-K+ATPase and the H+-K+-ATPase, did not modify the dpHi/dt (0.14 f 0.01 pH units/min, n = 12) (Fig. 10). In contrast, preactivated omeprazole (10m4 M), also known to inhibit the H+-K+-ATPase, was responsible for a marked decrease in the dpHi/dt (0.06 + 0.01 pH units/ min, n = 10, P < 0.01 vs. amiloride alone) (Fig. 10). It should be noted that omeprazole has been recently reported to inactivate the proton channel of vacuolar H+-

Control

NEM

Omep.

Uanadate

Fig. 10. Effect of various inhibitors on the amiloride-insensitive MTAL pH, recovery after cell acidification induced by 40 mM potassium acetate in TMA medium containing 67.5 mM sodium at 37°C at control or with lo-’ M N-ethylmaleimide (NEM), 10e4 M omeprazole (omep.), and 10m5M vanadate.

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H+ TRANSPORT

ATPases (29). Also, 10D4M SCH 28080, an imidazopyridine compound that inhibits H+-K+-ATPases by competition on the external K+-uptake site of the EP conformation of the pump (7), did not affect the cell pH recovery after intracellular acidification caused by addition of 40 mM sodium acetate to the suspension (0.14 t 0.02 vs. 0.16 t 0.01 pH/min, P = NS). Finally, we tested whether the H+-ATPase was dependent or not on the extracellular potassium concentration. After cell acidification with 40 mM sodium acetate, the amiloride-insensitive dpHi/dt was not modified by removal of external potassium (solution F in Table 1) (0.11 t 0.02 vs. 0.06 t 0.01 pH/min, P = NS). Note that this negative result was obtained after the BCECF-loaded cells were washed and centrifuged six or seven times in the K+-free medium to completely remove potassium from the extracellular medium. Taken together, these results are consistent with the presence of a vacuolar-type H+-ATPase and not a H+-K+-ATPase in the plasma membrane of rat MTAL cells. DISCUSSION

This study demonstrates that cells of rat MTALs extrude protons to recover from an intracellular acidification by two mechanisms: 1) by a saturable sodiumdependent, amiloride-sensitive, and electroneutral transport mechanism (these characteristics identify this transport mechanism as a plasma membrane Na+-H+ antiporter); and 2) by a bafilomycin Al-sensitive transport mechanism requiring energy from ATP. This H+ pump is also sensitive to NEM and preactivated omeprazole, insensitive to vanadate and SCH 28080, and not dependent on extracellular potassium; these characteristics are those of a vacuolar-type H+-ATPase (6, 9, 24, 25). Na+-H+ antiport and plasma membrane H+-ATPase were responsible for 53-60% and 36%, respectively, of the pHi recovery after cell acidification. It is important to note that the latter results were obtained under defined experimental conditions (in the absence of exogenous bicarbonate for instance) and from kidneys removed from starved rats. The sodium dependency of tubular fluid acidification in the isolated perfused rat CTAL has been demonstrated by Good (lo), who found that removing sodium from perfusate and bath, or inhibiting sodium transport by removing bath potassium inhibited transepithelial bicarbonate transport; in addition, bicarbonate absorption was inhibited 67% by 10D3M luminal amiloride (10). These results are consistent with the presence of an apical Na+-H+ antiporter mediating a large part of the luminal proton secretion by the rat CTAL. Krapf (19), by monitoring pHi transients in the rat CTAL, has confirmed the presence of an apical Na+-H+ antiporter, but has also suggested the presence of a basolateral Na+H+ antiporter involved in the cell pH response to a NH:-induced acid load; however, it is worth noting that, in the latter work (19), 1 mM bath amiloride did not affect the resting pHi in the presence or absence of CO,/ HCO:, which suggests that the basolateral Na+-H+ antiporter in this nephron segment may be inactive under basal conditions. The present study is the first, to our knowledge, to fully characterize a plasma membrane

IN RAT MTAL

C969

Na+-H+ antiporter in rat MTAL cells; this transporter is active under basal conditions since its inhibition by amiloride led to significant intracellular acidification. In addition, the data summarized in Figs. 3 and 4 are consistent with simple competitive inhibition by amiloride binding to only one external site of the Na+-H+ exchanger of the rat MTAL with an apparent amiloride Ki of 1.5-2.1 x 10B5M at 25OC. Quite similar findings (apparent amiloride Ki of 1.06 X 10m5M with 1 binding site at 37°C) have been obtained in the mouse MTAL (17) in which Na+-H+ exchange has been localized to the luminal, but not basolateral, membrane (17, 18). Although the luminal or basolateral localization cannot be assessedin a suspension like ours, these features taken together make it very likely that the Na+-H+ antiporter of the rat MTAL is located in the luminal membrane and is therefore responsible for proton secretion in this segment. It may be noted that an antidiuretic hormone (ADH) -induced basolateral Na+-H+ antiporter involved in cell volume regulation has been described in the mouse MTAL (15,16). Whether such an ADH-induced basolatera1 transporter may also support cell volume regulation in the rat MTAL is at present unknown. Active ATP-dependent H+ extrusion from rat MTAL cells by a plasma membrane H+ pump is fully documented and characterized in the present study, which is in agreement with a preliminary report in rat MTALs isolated and perfused in vitro (14). Sodium-independent and amiloride-insensitive pHi recoveries after acute cell acidification have also been observed but not characterized in mouse MTAL cells (17, 18). As outlined above, it can be accepted that the H+-ATPase identified in the present work is of the vacuolar type. Indeed, bafilomycin A1 was shown to specifically inhibit vacuolar H+ATPases of various origins at submicromolar concentrations (6) and is demonstrated in this study to inhibit, like NEM, the plasma membrane H+-ATPase of intact MTAL cells. The sensitivity of this H+-ATPase to preactivated omeprazole, which is a sulfhydryl reagent, is of no surprise since this drug has been reported to inhibit vacuolar H+-ATPases purified from the kidney (29). In addition, the present inhibitory effect of omeprazole provides further evidence for the location of the H+ATPase within the plasma membrane of rat MTAL cells since this drug reacts exclusively from the external surface of intact cells (23, 29). Our data are consistent with a previous immunocytochemical study, in which affinitypurified antibodies against subunits of the bovine medullary H+-ATPase were used, that has localized vacuolar H+-ATPases to the apical pole of rat MTALs and CTALs (8). Thus proton pumps other than the H+-K+-ATPase are sensitive to preactivated omeprazole; this drug therefore cannot be used to define the type of a proton pump but may be useful to localize it to the plasma membrane in intact cell preparations. From our data, we can speculate on the potential role of the Na+-H+ antiporter and H+-ATPase in proton secretion by the rat MTAL, since both transporters are likely located in the luminal membrane of this segment, as discussed above. Separate inhibitions of Na+-H+ antiport by amiloride and of H+-ATPase by bafilomycin A1 were responsible for similar falls in resting pHi and inhibitions of pHi recovery after cell acidification. Thus

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c970

H+ TRANSPORT

the two mechanisms appear important in H+ extrusion from these cells and therefore may be involved in bicarbonate absorption by rat MTAL cells since both are likely located in the luminal membrane as discussed above. This segment was recently shown to increase its ability to absorb bicarbonate (11) and its NEM-sensitive H+-ATPase activity (22) in response to metabolic acidosis. In addition, peptide hormones such as ADH and glucagon that exert major effects on tubular fluid and final urine acidification (21) have been shown, both in vivo (3, 20) and in vitro (l2), to modulate bicarbonate absorption by the rat TAL. The potential respective roles of the Na+-H+ antiporter and H+-ATPase in the hormonal control of the rat MTAL and in its adaptation to the acid-base status remain to be determined. We are greatly indebted to Drs. M.-M. Trinh-Trang-Tan and L. Bankir for having kindly shown us how to prepare the MTAL tubule suspension. We also thank Dr. R. Costalat for advice about fluorescence techniques and Chantal Nicolas for secretarial assistance. This study was supported by grants from the Institute National de la Sante et de la Recherche Medicale (CJF 8807), the Universites Paris 7 and Paris 6, the Fondation pour la Recherche Medicale FranCaise, and the Fondation de France. Parts of this work were presented at the XIth International Congress of Nephrology, Tokyo and Nara, July 1990, and were published in abstract form (Kidney Int. 35: 458,1989; and Kidney Int. 37: 537,199O) and as Congress proceedings (Kidney Int. 40, Suppl.: S43-S46,1991). Address for reprint requests: M. Bichara, Laboratoire de Physiologie, Hopital Broussais, 96 rue Didot, 75014 Paris, France. Received 10 July 1991; accepted in final form 22 November 1991. REFERENCES Ait-Mohamed, A. K., S. Marsy, C. Barlet, C. Khadouri, and A. Doucet. Characterization of N-ethylmaleimide-sensitive proton pump in the rat kidney. J. Biol. Chem. 261: 12526-12533,1986. 2. Bichara, M., E. Marty, P. Borensztein, and M. Paillard. NH: transport by cells of rat medullary thick ascending limb: electrogenic amiloride-sensitive pathway (Abstract). J. Am. Sot. 1.

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Bichara, M., 0. Mercier, P. Houillier, M. Paillard, and F. Leviel. Effects of antidiuretic hormone on urinary acidification and on tubular handling of bicarbonate in the rat. J. Clin. Inuest.

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Bichara, M., 0. Mercier, M. Paillard, and F. Leviel. Effects of parathyroid hormone on urinary acidification. Am. J. Physiol.

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Bowman, E. J., A. Siebers, and K. Altendorf. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. NatZ. Acad. Sci. USA 85: 7972-

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16. Hebert, S. C. Hypertonic cell volume regulation in mouse thick limbs. II. Na+-H+ and Cl-HCO; exchange in basolateral membranes. Am. J. Physiol. 250 (Cell Physiol. 19): C920-C931, 1986. 17. Kikeri, D., S. Azar, A. Sun, M. L. Zeidel, and S. C. Hebert. Na+-H+ antiporter and Na’-(HCO;), symporter regulate intracellular pH in mouse medullary thick limbs of Henle. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F445-F456, 1990. D., A. Sun, M. L. Zeidel, and S. C. Hebert. Cell 18. Kikeri, membranes impermeable to NH3. Nature Land. 339: 478-480,1989. 19 Krapf, R. Basolateral membrane H/OH/HCOs transport in the rat cortical thick ascending limb. Evidence for an electrogenic Na/ HC03 cotransporter in parallel with a Na/H antiporter. J. CZin. l

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Mercier, O., M. Bichara, M. Delahousse, A. Prigent, F. Leviel, and M. Paillard. Effects of glucagon on H’-HCO; transport in Henle’s loop, distal tubule, and collecting ducts in the rat. Am. J. Physiol. 257 (Renal Fluid Electrolyte PhysioZ. 26): Fl003F1014,1989. 21* Paillard, M., and M. Bichara. Peptide hormone effects on urine acidification and acid-base balance: PTH, ADH, and glucagon. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F973-F985, 1989. NEM-sensi22* Sabatini, S., M. E. Laski, and N. A. Kurtzman. tive ATPase activity in rat nephron: effect of metabolic acidosis and alkalosis. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol.

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Characterization of inside25. Simon, B. J., and G. Burckhardt. out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles. J. Membr. BioZ. 117: 141-151, 1990. 26. Trinh-Trang-Tan, M.-M., N. Bouby, C. Coutaud, and L. Bankir. Quick isolation of rat medullary thick ascending limbs. Enzymatic and metabolic characterization. Pfluegers Arch. 407: 228-234,1986.

Trinh-Trang-Tan, M.-M., 0. Levillain, and L. Bankir. Contribution of leucine to oxidative metabolism of the rat medullary thick ascending limb. PfZuegers Arch. 411: 676-680,1988. J. M., J. A. Davis, A. Lawton, and M. Abarzua. 28. Weinberg, Modulation of cell nucleotide levels of isolated kidney tubules. Am. 27.

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