Kidney International, Vol. 44 (1993), pp. 1235—1241

Changes in pH affect C1 removal and AVP action in collectingtubules

SAN-E ISHIKAWA, Kon OKADA, and TOSHIKAZU SAIT0

Division of Endocrinology and Metabolism, Department of Medicine, Jichi Medical School, Tochigi, Japan

Changes In pH affect C1 removal and AVP action in collecting tubules.We examined what mechanisms are involved in the alteration bychloride (C1) removal of arginine vasopressin (AVP)-induced cellularcAMP production, and cellular free calcium ([Ca211) mobilization in ratrenal papillary collecting tubule cells in culture, using two buffersystems: bicarbonate and non-bicarbonate buffers. The first study wasperformed in the bicarbonate-supplemented buffer. Removal of Cl,which was replaced by methylsulfonate or gluconate, increased cellularpH (pH1) from 7.19 to 7.26. AVP increased cellular cAMP production ina dose-dependent manner; I x l0- and I x l0 M AVP-inducedincreases in cellular cAMP production were significantly enhanced bythe Cl removal. Also, I x l0- a4 AVP-mobilized [Ca21, wasaugmented by the C1 removal (181.3 vs. 224.5 nM, P <0.05). Thesecond study was carried out with the Krebs-Ringer buffered saline(KRB). Removal of C1 lowered pH, from 7.20 to 7.09. AVP-inducedincreases in cellular cAMP production were significantly reduced in theCl-free KRB compared to those in the KRB. The reduction wasobtained with KRB containing less than 25 mri C1. Similar resultswere obtained with 2 x iO M forskolin, a diterpene activator ofadenylate cyclase. I x l0- M AVP-mobilized [Ca2]1 was also dimin-ished by the Cl-free KRB. These results indicate that Cl depletionaffects the cellular response to AVP mediated via the changes in pH1 inrenal papillary collecting tubule cells. The difference in the opposedresponses is based on the activation of the C1/HC03 exchanger in thebicarbonate-supplemented buffer, and may be based on the C1-dependent W-ATPase activation which is probably involved in thenon-bicarbonate buffer system.

Intracellular pH (pH1) as well as extracellular pH (PHe) is animportant factor for regulating the cellular action of argininevasopressin (AVP) in renal collecting tubule cells [1, 2]. Thereare pH1 control systems in renal papillary collecting tubulecells, such as the Na/H exchanger, H-ATPase, Cl/HC03exchanger and others [2—10]. We have recently demonstratedthe involvement of the Na/H exchanger in regulating thecellular action of AVP to increase cellular cAMP production incultured rat renal papillary collecting tubule cells [11, 12]. Also,our preliminary study showed that Cl removal from bicarbon-ate buffer increased the pH, and augmented the cellular re-sponse to AVP, whereas a reduction in the concentration ofHC03 decreased the pH1, resulting in the diminution of thecellular response to AVP [1]. This finding confirms the modu-

Received for publication April 30, 1993and in revised form July 13, 1993Accepted for publication July 15, 1993

© 1993 by the International Society of Nephrology

lation of AVP action by C1/HC03 exchange mediated via thepH1 change in renal papillary collecting tubule cells.

In glomerular mesangial cells Okuda et al [13, 14] suggestedthat Cl is a key anion which acts as a cellular signal transduc-tion produced by AVP or angiotensin II. In their studies all ofthe studies were performed in a bicarbonate-free system, and allof the phenomena, such as cellular free calcium ([Ca2]1)mobilization and inositol trisphosphate production, induced byAVP or angiotensin II was inhibited by the removal of C1 [13,14]. This is probably independent of the modulation by Cl—IHC03 exchanger [15].

The present study was undertaken to determine what mech-anisms are involved in the alteration by C1 removal in theAVP-induced cellular cAMP production and [Ca2}1 mobiliza-tion in rat renal papillary collecting tubule cells, using twobuffer systems, namely, the bicarbonate-supplemented and thenon-bicarbonate buffers.

Methods

Cell cultureThe experimental procedure was similar to that described

previously [16, 17], modified from the method of Grenier,Rollins and Smith [18]. Eight male Sprague-Dawley rats weigh-ing 150 to 175 g were used. They were decapitated and theabdomen was immediately rinsed with 1% iodine in 70% etha-nol. Under sterile conditions, renal papillary tissues weredissected out and minced with I ml Krebs-Ringer bufferedsaline (KRB), pH 7.4, containing 1 mg/mI collagenase (type II,Worthington Biochemicals, Freehold, New Jersey, USA) usinga sharp razor blade. The minced renal papillary tissues weretransferred into 15 ml culture tubes containing 3 ml collagenase(1 mg/mI) and kept at 37°C for 70 minutes. Distilled water (3 ml)was added to the tubes to produce hypotonic disruption of thered blood cells. The suspension was drawn up and down apipette to break any tissue clumps before the tubes werecentrifuged at 500 g for four minutes at room temperature, andthe supernatants were removed by aspiration. The pellets wereresuspended in 10 ml 10% BSA (Sigma Chemical Co., St.Louis, Missouri, USA), dissolved in KRB, and centrifuged asdescribed above. After aspiration of the supernatants, thepellets were resuspended in Dulbecco's modified Eagle's Min-imal Essential Medium (DMEM; Flow Laboratories, McLean,Virginia, USA) supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100 jzg/ml streptomycin. The dispersed cellsand tubular fragments were plated into a total of 120 wells of

1235

1236 Ishikawa et a!: C! removal and A VP action

Table 1. Composition of the buffers used in the present experiments

Methylsulfonate

Na K Mg2 Ca2 Cl S042 PO4 HC03 Acetate Tns Glucosegluconate orpropionate pH

KRB 140 5 1.2 1 134 1.2 2 10 10 10 7.4C1-free KRB 140 5 1.2 1 1.2 2 16 10 10 128 7.4Bicarbonate buffer 145 5 1,2 1 126 1.2 1 25 10 7.4C1-free

bicarbonate buffer 145 5 1.2 2 1.2 1 25 6 10 120 7.4

All values are mM.

24-well tissue culture clusters (Costar, Cambridge, Massachu-setts, USA) with 0.5 ml DMEM containing 10% fetal bovineserum, penicillin and streptomycin, and kept in a humidifiedincubator under 95% air and 5% CO2. On the second day ofculture, the medium was changed to 1 ml 99% DMEM contain-ing 1% fetal bovine serum, 5 gJml insulin (Sigma), 5 jzg/mltransferrin (Sigma), 5 x 10..'8 M hydrocortisone (Calbiochem-Boehnnger, La Jolla, California, USA), 5 x 10—8 M T3 (Sigma),100 U/ml penicillin and 100 g/ml streptomycin.

For measurement of [Ca24] the cells were cultured on 14 X30 mm thin glass slides (Matsunami Kogyo Co., Osaka, Japan)in 35 x 10 mm petri dishes (Corning Glassworks, Midfield,Massachusetts, USA). Similarly, the cells were grown on thinglass slides (13 mm in diameter; Matsunami) to measure pH1.

The cultured cells were subjected to the following studies onday 5 of culture.

Cellular cAMP production

The experimental procedure was similar to that described inour previous studies [16, 17]. The cells were rinsed with 1 mlKRB twice and preincubated for 20 to 30 minutes at 37°C withmedium containing 0, 10, 25, or 50 ifiM C1, or the vehicle,Cl-depleted media were made by replacing C1 with sodiummethylsulfonate, sodium gluconate or sodium propionate (pH7.4; Table 1). Treatment with the various media describedabove did not affect the viability of the cultured cells, asassessed by Trypan blue staining. The cells were rinsed andthen incubated at 37°C for 10 minutes with the different con-centrations of AVP (Sigma) or forskolin (Nihon Kayaku Co.,Tokyo, Japan) together with 5 x iO M 3-isobutyl-l-methyl-xanthine (Nakarai Biochemicals, Tokyo, Japan) in 1 ml of amedium similar to that used during the preincubation. Forskolinis a diterpene activator of adenylate cyclase. Control experi-ments were performed in the same manner with the vehicle.After the incubation period, the effector solutions were aspi-rated, and the cells were immediately immersed in 0.2 ml 0.1 NHCI to stop the reaction. They were then collected into glasstubes (6 x 50 mm) and boiled for three minutes. After centri-fuging the tubes at 3,000 g for 15 minutes at room temperature,the supernatants were decanted into other glass tubes. Theywere added to 0.05 ml 50 m sodium acetate, and then werekept at —20°C until the assay for cAMP. cAMP was measuredby RIA using Yamasa cAMP kits (Choshi, Japan) [19]. Thepellets were dissolved in 0.2 ml 1% sodium dodecylsulfate andkept at 4°C until the assay for protein. Protein was measured bythe method of Lowry et al [20].

For the study using the bicarbonate-supplemented buffer (pH7.4; Table I), the cells were rinsed with the buffer and preincu-

bated for 20 minutes at 37°C with the modified bicarbonate-supplemented buffer containing 25, 10 or 2.5 mrt NaHCO3 inthe presence or absence of C1. The cells were rinsed and thenincubated at 37°C for 10 minutes with AVP or forskolin in 1 mlof a medium similar to that used during the preincubation.Control experiments were carried out in the same manner withthe vehicle. The studies were carried out in a humidifiedincubator under 95% air and 5% CO2. The following procedureto collect cells and measure cAMP and protein was similar tothat described above.

Measurement of[Ca],The experimental procedure was similar to that used in our

previous studies [21, 22]. The cells were rinsed twice with 3 mlof the buffer containing 1 ifiM CaC12 and loaded with 5 Mfura-2/AM (Dojin Biochemicals, Kumamoto, Japan) in 1 ml ofthe buffer for 60 minutes at 37°C. After aspiration of thefura-2/AM solution, the cells were further preincubated for 20minutes at 37°C with the varying buffers described above. Theglass slides were then placed in a 1 x 1 cm quartz cuvettecontaining 3 ml of the indicated buffer at 37°C in a fluorescencespectrophotometer (Ca Analyzer, CAF-1 10, Japan Spectro-scopic Co., Tokyo, Japan). The dual wavelength excitationmethod for measurement of fura-2 fluorescence was used. Thefluorescence was monitored at 500 nm with excitation wave-lengths of 340 and 380 nm in the ratio mode. The effectors wereadded after a stable fluorescence signal (R) was achieved. Basaland AVP-stimulated peak values were determined. From theratio of fluorescence at 340 and 380 nm the [Ca2'] wasdetermined as described by Grynkiewicz, Poenie and Tsien[23], using the following expression. [Ca2]1 (nM) = Kd X [(R —

Rmin)/(Rmax— R)] x /3, where R is the ratio of fluorescence of

the sample at 340 and 380 nm, and Rmax and Rmin weredetermined as described previously [21]. The term /3 is the ratioof fluorescence of fura-2 at 380 nm in zero and saturating Ca2tKd is the dissociation constant of fura-2 for Ca2, assumed to be224 flM at 37°C [23].

Measurement of pH,

The experimental procedure was similar to that described inour previous reports [11, 12]. The cells were rinsed twice with1 ml of the buffer and then loaded with 2 .&M BCECF/AM(Molecular Probes, Eugene, Oregon, USA) for 60 minutes at37°C. BCECF/AM was dissolved in KRB or the bicarbonate-supplemented buffer. The glass slides were placed in I x 1 cmquartz cuvette with use of a special holder containing 3 ml of theindicated buffers at 37°C in a fluorescence spectrophotometer

7.2

7.1I7.3

:1 mm

7.3 1

Fi/KRBI OmMCI/KRB _______

ishikawa et a!: C1 removal and A VP action 1237

(CAF-l00, Japan Spectroscopic Co.). The complete intracellu-lar hydrolysis of BCECF/AM to BCECF was judged by changesin the excitation and emission spectra. The fluorescence wasmonitored at 530 nm with excitation wavelengths of 450 and 500nm in the ratio mode. The fluorescence signal was calibrated atseveral pH values (6.6, 7.0 and 7.4) in the KC1 solution (140 mMKC1, 4.6 ma't NaC1, 1 m MgC12, 2 m CaC12, 10 m glucoseand 10 m HEPES) containing the K/H ionophore nigencin(10 /hg/ml) [24].

StatisticsAll values of cellular cAMP, [Ca2]1, and pH1 were analyzed

by an analysis of multiple variance and Student's t-test. P <0.05 was considered significant.

Results

Figures 1 and 2 show the pH1 change of cultured rat renalpapillary collecting tubule cells in the bicarbonate-free KRB.The cells were exposed to the Cl-free KRB, in which C1 wasreplaced by methylsulfonate, gluconate or propionate. In allthree conditions the pH1 significantly acidified. The pH,promptly decreased from 7.20 0.02 to 7.09 0.03 in the cellsexposed to the C1-free KRB supplemented with gluconate (P<0.01; Fig. 1).

The change in pH1 of cultured rat renal papillary collectingtubule cells was studied with the bicarbonate-supplementedbuffer (Figs. I and 3). When the buffer containing 25 mMNaHCO3 was used, the pH1 was 7.19 0.01. The pH, wassignificantly reduced to 7.04 0.02 and 6.84 0.02 afterexposing to the buffer containing 10 and 2.5 ifiM NaHCO5,respectively. A similar study was carried out in the C1-freebicarbonate-supplemented buffer, where C1 was replaced bymethylsulfonate. As shown by open circles in Figure 3, the pH1significantly alkalinized. When NaHCO3 was 25 m in the

6.8

Control Methyl- Gluconate Propnonatesulfonate

Fig. 2. Effect of C1 removal from Krebs-Ringer buffered saline (KRB)on cellular pH (pHi) in the cultured rat renal papillary collecting tubulecells. C1 was replaced by methylsulfonate, gluconate or propionate, asdescribed in Table 1. < 0.01 vs. the control. Values are meansSEM, N = 6.

buffer, the Cl— removal elevated the pH, by a mean value of0.07.

AVP- and forskolin-induced cellular cAMP production werestudied with KRB in the presence and absence of Cl—. Asshown in Figure 4, the removal of Cl— markedly reduced the 1x io and 1 X i0 M AVP-induced cellular cAMP produc-tion. A submaximal stimulatory concentration of 2 x 108 Mforskolin increased cellular cAMP production to 604.6 43.5fmol/jsg protein [16]. In contrast, the replacement of Cl bymethylsulfonate or propionate significantly diminished the cel-lular cAMP response to forskolin. Figure 5 shows that theAVP-induced cellular cAMP production depends on the con-centration of Cl— in the buffer (KRB). C1 was replaced bymethylsulfonate in this study. When the medium Cl wasreduced to 50 mi, the AVP-induced increase in cellular cAMPproduction was not affected. The medium concentration of Clless than 25 ifiM significantly diminished the cellular response ofcAMP production to AVP. The greater reduction of mediumCl concentration produced the greater diminution in cellularcAMP production in response to AVP in the cultured rat renalpapillary collecting tubule cells. There was no difference in thebasal cAMP levels among the different concentrations of me-dium Cl-.

Figure 6 shows the effect of Cl— removal in the bicarbonate-supplemented buffer on AVP-induced increase in cellularcAMP production in rat renal papillary collecting tubule cells inculture. In the control condition which contained 25 msNaHCO3, 1 x l0 and I x iO M AVP significantly increasedcellular cAMP production. Diminution of medium HC03concentration to 10 or 2.5 m dose-dependently reduced theAVP-induced cellular cAMP production. However, the Cl

7.3

7.2

7.1

7.0

I*

6.9

Fig. 1. Alteration in pH, after the removal of C1 in the bicarbonate-supplemented buffer (A) and in the non-bicarbonate buffer, KRB (B).C1 was replaced by gluconate.

800

700

600

500

400

300

: flhIVehicle 1x1O°M AVP 1x1OM AVP 2x108M Forskolin

1238 Ishikawa et a!: C1 removal and AVP action

1.

*+

**

2.5 10 25

7.3

7.2

7.1

I0.

7.0

6.9

6.8

6.7

NaHCO3, mM

Fig. 3. Changes in cellular pH (pHi) after the exposure to the modifiedbicarbonate buffers in the cultured rat renal papillary collecting tubulecells. The horizontal axis shows the reduced concentration of NaHCO3in the buffer, represented by closed circles (•). Open circles (0) showthe removal of C1 from each modified bicarbonate buffer. C1 wasreplaced by methylsulfonate. < 0.01 vs. the control which wasstudies in the presence of 25 m NaHCO3; tP < 0.01 vs. the pH1 in thepresence of Cl—. Values are means SEM, N = 6.

Fig. 4. Reduction by the Ct-free KRB of the A VP- and forskolin-induced cellular cAMP production in the cultured rat renal papillarycollecting tubule cells. Symbols are: (Eli) Control group, and C1 wasreplaced by gluconate (), methylsulfonate () or propionate()• < 0.01 vs. the control group. Values are means SEM, N =6.

removal from such modified buffers markedly enhanced theAVP-induced increase in cellular cAMP production (Fig. 6).There was no alteration in the basal levels of cAMP between thepresence and absence of C1. Similar results were obtainedwith 2 x 10—8 M forskolin, which was a submaximal stimulatory

900

800

700

6000

500

400E

300

200

100

001025 50 140

C1, mM

Fig. 5. Effect of C1 depletion from KRB on the A VP-induced cellularcAMP production in rat renal papillary collecting tubule cells inculture. C1 was replaced by methylsulfonate. Symbols are: (•) vehi-cle; (0) and (•) are the 1 x iO and 1 x iO M AVP groups,respectively. < 0.01 and ** < 0.05 vs. the control group, whichwas studied in the presence of 140 mri CEl. Values are means SEM,N = 6.

concentration. 2 x 108 M forskolin significantly increasedcellular cAMP production from 281.6 17.9 to 781.0 52.4fmolIpg protein. When the medium HCO3 concentration wasreduced to 10 or 2.5 m, the forskolin-induced increase incAMP production was diminished. In contrast, the Cl- removalfrom the modified buffers significantly augmented its response(data not shown).

AVP-mobiized [Ca2'11 in renal papillary collecting tubulecells was examined with the Cl-depleted KRB (Figs. 7 and 8).In the control condition I X 10 M AVP increased [Ca2] from95.7 5.8 to 195.4 9.1 rIM (P <0.01). When medium C1 wasremoved and replaced by either methylsulfonate, gluconate orpropionate, the AVP-induced mobilization of [Ca2]1 was sig-nificantly reduced. The data were shown as the peak [Ca2]1 inresponse to 1 X lO M AVP. The basal level of [Ca2]1 alsodecreased when Cl— was replaced by methylsulfonate.

Figures 7 and 9 show the augmentation by C1 removal of 1x l0— M AVP-mobilized [Ca2]1 with the bicarbonate-supple-mented buffer. In the control state supplemented with 25 mMNaHCO3 1 x lO M AVP increased [Ca2]1 from 100.4 6.5to 181.3 6.6 nM (P < 0.01). After cells were exposed to thebuffer in the absence of Cl—, both the basal and the AVP-mobilized peak [Ca2i significantly elevated. Similarly, theAVP-mobiized peak [Ca2]1 significantly decreased depen-dently on the concentration of 10 and 2.5 mM NaHCO3. TheCl removal augmented the basal and the AVP-rnobilized peak

300

* *

0Basal lXlIr7M AVP

Fig. 8. Inhibition by the C1 removal from KRB of the 1 X 1O MAVP-mobilized [Ca't11 in rat renal papillary collecting tubule cells inculture. Symbols are: Control group (E:J); Cl— was replaced bymethylsulfonate (), gluconate() orpropionate (). < 0.01vs. the control. Values are means SEM, N = 6.

Fig. 7. 1 X io- MAVP-mobilized[CaJ,in the bicarbonate-supplemented buffer (A)and in the non-bicarbonate buffer, KRB (B).Left panel; control, right panel; C1-freecondition. C1 was replaced bymethylsulfonate.

Ishikawa et al: C1 removal and AVP action 1239

,/,,

I

1100

1000

900

800

700

600

500

400

300

200

100

0

--__*_

NaHCO3, mM

DiscussionI I

[Ca2 f] in the buffer containing the reduced concentration ofNaHCO3.

2.5 10 25

We demonstrated by two opposite phenomena that the C1-

Fig. 6. Modulation of the A VP-induced cellular cAMP production bythe modified bicarbonate-supplemented buffer in rat renal papillarycollecting tubule cells. The concentration of bicarbonate was 25, 10 or

free condition modulated the hormonal response of cultured ratrenal papillary collecting tubule cells. The Cl-free condition,based on the bicarbonate-supplemented buffer, alkalinized the

2.5 mai. C1 was removed from these modified buffers, represented by pH, and significantly enhanced the AVP-induced increases inthe open marks (0, 0, ). C1 was replaced by methylsulfonate.Vehicle groups are circles (S, 0), and the 1 x iO and 1 X l0 M AVPgroups are squares (U, 0) and triangles (A, ),respectively. < 0.01vs. the control which was studied in the presence of 25 mM NaHCO3.tP < 0.01 and tP < 0.05 vs. the cAMP levels in the presence of C1.

cellular cAMP production and [Ca2]1 mobilization. In contrast,the CY-free state, based on KRB, acidified the pH1 andmarkedly reduced them. The difference seems likely to dependon the alteration in pH1.

Values are means SEM, N = 6. In a series of our studies [1, 11, 12], the regulation of pH1 is

-400 CVHCO3 buffer

0 m CI/HCO3 buffer

V

50 1 _t_KRB 0 mii CI/KAB

200

100

-1 mm

1240 Ishikawa et a!: C1 removal and A VP action

NaHCO3, mM

Fig. 9. Modulation of the I x 1O MAVP-mobilized [CaJ1 by themod tfled bicarbonate buffer in rat renal papillary collecting tubule cellsin culture. The concentration of bicarbonate was 25, 10 or 2.5 mM,represented by the closed symbols (•, U). C1 was removed from thesemodified buffers, represented by the open symbols (0, 0). C1 wasreplaced by methylsulfonate. The control group is represented bycircles (•, 0), and the 1 x l0— M AVP group by squares (U, 0). *P <0.01 and ** < 0.05 vs. the control which was studied in the presenceof 25 mi NaHCO3. tP < 0.01 and 1P < 0.05 vs. the [Ca2], levels in thepresence of C1. Values are means SEM, N = 6.

involved in the cellular action of AVP in renal collecting tubulecells. The treatment of cells with the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP) dose-dependentlyreduced the pH1 without any change in extracellular pH (PHe).Attenuation by CCCP of AVP-induced cellular cAMP produc-tion and [Ca2]1 mobilization depends on the acidified pH1.Similar results were obtained with forskolin, a diterpene acti-vator of adenylate cyclase. Namely, the greater reduction inpH1 produces the greater diminution in cellular response toAVP. Also, the Na/H exchanger system participates in sucha pH control and indirectly regulates the cellular action of AVPmediated via the pH1 in renal collecting tubule cells [11, 12].

The pH1 dependence of the cellular action of AVP was alsofound in the modified bicarbonate buffer. There is a Cl—/HC03exchanger in renal collecting tubule [7, 9, 10]. The reducedconcentration of bicarbonate acidified the pH1 and diminishedboth AVP-induced cAMP production and [Ca2]1 mobilization.In the present study we showed additional evidence that theremoval of chloride enhanced the cellular responses of cAMPproduction and [Ca2]1 mobilization to AVP under varying

concentrations of bicarbonate (25, 10 or 2.5 mM). The removalof Cl elevated the pH1 by approximately 0.07 and augmentedthe cAMP response of 1 x l0 M AVP to the similar magnitudeas that of 1 x l0 M AVP in the intact cells. Similar resultswere obtained with 2 x 10—8 M forskolin.

The C1 depletion study using KRB resulted in an oppositeeffect on the cellular action of AVP in renal papillary collectingtubule cells. The C1-free state markedly reduced the AVP-induced increases in cellular cAMP production and [Ca2]1mobilization. Such an inhibition was obtained with less than 25mM C1 [25, 26]. The diminution of the action of AVP dependson the lowered pH1 after the Cl removal. Whether Cl— wasremoved from the bicarbonate-supplemented buffer or KRB,the modulation of the cellular action of AVP by Cl removalwas to an opposite direction. The difference was the alterationin pH1 after the removal of C1. We know that there is aC1/HC03 exchanger. When the study was carried out in thebicarbonate-supplemented buffer, the Cl removal blocked theexchange of C1 and HC03 across plasma membrane, result-ing in an increase in pH1. On the other hand, the experiment wasperformed in KRB in the absence of bicarbonate, and theactivity of C1/HCO3— exchanger weakened. H-ATPase ispresent in apical membrane of renal papillary collecting tubulecells [3, 4, 27, 28]. Several reports suggested that C1 isnecessary for the activation of H-ATPase in nephron seg-ments including proximal tubule, ascending limb of Henle'sloop and cortical and medullary collecting duct [25, 26, 29—31],and osteoclast cells [32]. Taken together, the present resultsindicate that the reduction in pH, after the removal of C1 fromKRB is based on the attenuation in H-ATPase activity. Weknow that 1 x iO M N-ethylmaleimide, an inhibitor ofW-ATPase, decreased the pH, by 0.17, and significantly re-duced the AVP-induced increases in cellular cAMP productionand [Ca2] mobilization in cultured rat renal papillary collect-ing tubule cells [33]. Further study will be needed to elucidatethe interaction of C1 and H-ATPase activity.

In summary, we demonstrated two opposite phenomena thatC1 depletion modulates the hormonal response of the culturedrat renal papillary collecting tubule cells, using bicarbonate-supplemented and non-bicarbonate buffers. The differenceprobably depends on the alteration in pH1 after the Cl— re-moval. The increase in pH, after the Cl— removal from thebicarbonate buffer is based on the C1/HC03 exchanger. Incontrast, the reduction in pH1 after the C1 removal from KRBis probably based on the Cl-dependent H-ATPase activity.These results indicate that Cl— ion is an important anion tocontrol the cellular pH of renal papillary collecting tubule cells,mediated through both the Cl—/HC03 exchanger and theH-ATPase, and that such an alteration in pH1 is involved in thecellular action of AVP in renal papillary collecting tubule cells.

Acknowledgments

The present study was supported by a grant from the Ministry ofEducation, Science and Culture of Japan. This work was, in part,presented at 74th Annual Meeting of The Endocrine Society at SanAntonio, Texas, June, 1992.

— —— —

t

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0---it

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Reprint requests to San-e Ishikawa, M.D., Division of Endocrinologyand Metabolism, Department of Medicine, Jichi Medical School,331/-i Yakush/i Minamikawachi-machi, Tochigi 329—04, Japan.

Ishikawa et a!: C1 removal and AVP action 1241

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