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【摘要】 The effect of arginine vasopressin (AVP) and/or atrial natriuretic peptide (ANP) on the regulation of intracellular pH (pH i ) via H + -ATPase and of cytosolic calcium ([Ca 2+ ] i ) was investigated in Madin-Darby canine kidney (MDCK) cells by the fluorescent probes BCECF-AM and fluo-4-AM, respectively. The pH i recovery rate was examined after intracellular acidification following an NH 4 Cl pulse, in the presence of zero Na + plus Schering 28080 (a specific inhibitor of H + -K + -ATPase). AVP (10 -12 -10 -6 M) increased the rate of pH i recovery and [Ca 2+ ] i in a dose-dependent manner. V 1 - or V 2 -receptor antagonists impaired the effect of AVP on both processes, and DDAVP (10 -12 -10 -6 M; a V 2 -selective agonist) caused a dose-dependent stimulation of them. [Ca 2+ ] i or cAMP (as increased by 10 -5 M thapsigargin or 8-BrcAMP, respectively) alone had no effect on H + -ATPase, but their synergic action was necessary to stimulate H + -ATPase. In agreement with these findings, ANP (10 -6 M) or dimethyl-BAPTA-AM (5 x 10 -5 M), impairing the increase of [Ca 2+ ] i in response to AVP, blocks the stimulatory effect of AVP on H + -ATPase.
【关键词】 atrial natriuretic peptide MadinDarby canine kidney cells
THE EFFECT OF ARGININE VASOPRESSIN (AVP) on H + -ATPase has been subject to some controversy. In a previous in vivo microperfusion study, we demonstrated that in late distal segments of rat kidney luminal AVP (10 -9 M) stimulates H + -ATPase ( 2 ); however, in rabbit cortical collecting duct, a segment that contains several structural components similar to those of late distal tubule, Ando et al. ( 1 ) suggested that luminal AVP (10 -9 M) might impair electrogenic H + secretion. In addition, Borensztein et al. ( 3 ) concluded that in rat medullary thick ascending limb cells, AVP does not directly affect H + -ATPase. On the other hand, most studies have detected AVP action when applied at the basolateral surface mediated mostly by V 2 receptors via the adenylate cyclase/cAMP signaling system ( 14 ). However, in recent years V 1 receptors have been detected in both apical and basolateral membrane domains and have been shown to mediate AVP activity via phospholipase C/inositol 3,4,5-triphosphate (IP 3 )/calcium signaling ( 14, 19, 23 ). Previous in vivo data from our laboratory have shown that luminal AVP (10 -9 M) stimulates bicarbonate reabsorption in both early and late distal tubule of rat kidney via activation of V 1 receptors ( 2 ). In addition, we recently found that peritubular AVP (10 -11 and 10 -9 M) stimulates bicarbonate reabsorption in both these segments via activation of V 1 receptors and that V 2 receptors have a dose-dependent inhibitor effect mediated by cAMP ( 22 ). Thus it is possible that the H + -ATPase response to AVP may vary with the cell type, cell membrane surface, and hormonal doses being studied.
On the other hand, it is known that an increase in cytosolic calcium ([Ca 2+ ] i ) might initiate events that lead to activation of H + -ATPase ( 17 ) and that cell acidification stimulates a calcium-mediated exocytotic insertion of proton pumps, a process important in regulating intracellular pH (pH i ) ( 8, 29, 32 ). Furthermore, we recently found that in Madin-Darby canine kidney (MDCK) cells (a cell line with many morphological and physiological similarities to the mammalian distal nephron) atrial natriuretic peptide (ANP) inhibits [Ca 2+ ] i elevation produced by AVP ( 25 ), suggesting that there may be some interaction between these two vasoactive peptide hormones in the regulation of pH i by H + -ATPase. Besides this finding, ANP has been shown to inhibit cAMP synthesis stimulated by AVP in rat renal papillary collecting tubule cells ( 20 ).
The present study was designed to further investigate the effect of AVP (10 -12, 10 -9, or 10 -6 M) in regulating the pH i recovery mediated by H + -ATPase. To detect which specific AVP receptor regulates this mechanism, we measured the effect of specific V 1 - or V 2 -receptor antagonists and of a V 2 -selective agonist on this process. pH i recovery was monitored in MDCK cells by using the fluorescent probe BCECF. The experiments were done in nominally HCO 3 - /CO 2 -free medium, after an acid load induced by NH 4 Cl, and in the presence of zero Na + plus Schering 28080 (a specific inhibitor of H + -K + -ATPase), an experimental condition in which the H + -ATPase is the only mechanism of pH i recovery in activity ( 11 ). Because the role of vesicle trafficking and exocytosis in the regulation of H + transport in MDCK cells has been documented ( 5, 21 ), we also examined the effect of AVP on [Ca 2+ ] i. In addition, we studied the interaction of AVP plus ANP (10 -6 M) or dimethyl-BAPTA-AM [an intracellular calcium chelator ( 30 )] and the effect of thapsigargin (an intracellular calcium releaser) and/or 8-BrcAMP (a membrane-permeant cAMP analog) on pH i recovery and [Ca 2+ ] i.
Our present results suggest a role of the increase in [Ca 2+ ] i and of cAMP in regulating the dose-dependent stimulatory effect of AVP on H + -ATPase, via V 1 and V 2 receptor-mediated pathways. In agreement with these results, ANP or dimethyl-BAPTA-AM, impairing the increase in [Ca 2+ ] i in response to AVP, blocks this stimulatory effect of AVP. This hormonal interaction we observed in MDCK cells may represent a relevant mechanism of pH i regulation in the intact animal.
MATERIALS AND METHODS
Cell culture. Serial cultures of wild-type MDCK cells ( passages 69-77; American Type Culture Collection, Rockville, MD) were maintained in DMEM (supplemented with 2 mM glutamine, 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin; GIBCO, Grand Island, NY) at 37°C, 95% humidified air-5% CO 2 (pH 7.4), in a CO 2 incubator (Lab-Line Instruments, Melrose Park, IL). The cells were harvested with trypsin/EGTA (0.02%), seeded on sterile glass coverslips, and then incubated again for 72 h in the same medium to become confluent.
Measurement of pH i by fluorescence microscopy. pH i was monitored using the fluorescent probe BCECF ( 26 ). Briefly, cells grown to confluence on glass coverslips were loaded by exposure for 20 min to 10 µM BCECF-AM in the control solution ( solution 1; Table 1 ). After the loading period, the glass coverslips were placed into a thermo-regulated chamber mounted on an inverted epifluorescence microscope (TMD, Nikon). The measured area under the microscope had a diameter of 260 µm and contained on the order of 40 cells. The coverslips remained in a fixed position so that the same cells were studied throughout the experiment. All experiments were performed at 37°C. The cells were alternately excited at 440 or 490 nm with a 150-W xenon lamp, and the fluorescence emission was monitored at 520 nm by a photomultiplier-based fluorescence system (PMT-400, Georgia Instruments) at 5-s time intervals. The 490/440 excitation ratio corresponds to a specific pH i. At the end of each experiment, calibration of the BCECF signal was achieved by exposing the cells for 15 min to a K + -HEPES buffer solution containing 10 µM nigericin ( solution 2; Table 1 ), at pH 6.5, 7.0, or 7.5.
Table 1. Composition of solutions
Cell pH recovery. After the acidification of pH i by a 2-min exposure to 20 mM NH 4 Cl ( solution 3; Table 1 ) ( 4 ), cell pH recovery was examined in the presence of zero Na + ( solution 4; Table 1 ) plus Schering 28080 (1 x 10 -5 M), in the control situation; or in the presence of the following: AVP (10 -12, 10 -9, or 10 -6 M); V 1 -receptor specific antagonist [ -mercapto-, -cyclopentamethylene-propionyl 1, O -Me-Tyr 2,Arg 8 ]vasopressin (10 -5 M); V 2 -receptor-specific antagonist [adamantaneacetyl 1, O -Et- D -Tyr 2, Val 4,aminobutyryl 6,Arg 8,9 ]vasopressin (10 -5 M); V 2 -selective agonist [deamino-Cys 1, D -Arg 8 ]vasopressin (DDAVP; 10 -12, 10 -9, or 10 -6 M); ANP (10 -6 M); dimethyl-BAPTA-AM (5 x 10 -5 M); the intracellular calcium releaser thapsigargin (10 -5 M); or a membrane-permeant cAMP analog [8-BrcAMP (10 -5 M)]. Because the rate of pH recovery depends on the value of cell pH achieved by the acid load ( 34 ), we used experiments in which these values were not significantly different between the studied groups ( Table 2 ). In all the experiments, no pH change was observed initially after the acid pulse for a period of 1-2 min, and pH recovery started thereafter. We calculated the rate of pH i recovery (dpH i /d t, pH units/min) from the first 2 min after the start of the pH i recovery curve by linear regression analysis (see Fig. 1 ).
Table 2. Summary of pH i responses in MDCK cells to addition of different agents after acute acid load
Fig. 1. Intracellular pH (pH i ) recovery after cellular acidification with the NH 4 Cl pulse technique in MDCK cells. A : in the presence of 145 mM Na + in the bath, the initial fall in pH i is followed by a recovery of pH i toward the basal value (B). B : in the presence of zero Na + external solution plus Schering 28080 (Sch; 1 x 10 -5 M; a specific inhibitor of H + -K + -ATPase), the pH i recovery rate is markedly decreased, and the final pH i was significantly different from the basal value; this effect is subsequently reversed with the return of 145 mM Na + solution to the bath. C : in the presence of zero Na + external solution plus Schering 28080, the addition of AVP (10 -12 M) causes a significant increase in the velocity of pH i recovery, but the pH i recovery was not complete; with the return of 145 mM Na + solution to the bathing medium, the pH i recovery rate increased, and the final pH i was not significantly different from the basal value.
Measurement of [Ca 2+ ] i by fluorescence microscopy. In all experimental groups, changes in [Ca 2+ ] i were monitored fluorometrically by using the calcium-sensitive probe fluo-4-AM ( 26 ) in the presence of zero Na + plus Schering 28080 after the acid load. Briefly, confluent cultures were loaded with 10 µM fluo-4-AM at 37°C for 40 min and rinsed in Tyrode's solution ( solution 5; Table 1 ) containing 0.2% bovine serum albumin (pH 7.4). Fluo-4 fluorescence intensity emitted above 505 nm was imaged in vivo by using laser excitation at 488 nm on a Zeiss LSM 510 confocal microscope. The images were continuously acquired before and after substitution of experimental solutions, at time intervals of 10 s, for a total of 200 s. For each experiment, the maximum fluorescent signal for 10 cells was averaged and then used for analysis. Transformation of the fluorescent signal to [Ca 2+ ] i was performed by calibration with ionomycin (30 µM; maximum Ca 2+ concentration) followed by EGTA (2.5 mM; minimum Ca 2+ concentration) according to the Grynkiewicz equation ( 15 ). The Grynkiewiez method for the determination of [Ca 2+ ] i was originally used with fura-2 fluorescence. However, in previous studies, we have results indicating that the basal and nonbasal levels of [Ca 2+ ] i measured from single-wavelength fluo-4 were similar to those measured from dual-wavelength fura-2. In these studies, we measured the [Ca 2+ ] i in MDCK cells with fluo-4 or fura-2 for a total time of 200 s, and for each experiment the maximum fluorescent signal was used for analysis. Using fura-2, we found that MDCK cells exhibited a baseline [Ca 2+ ] i of 99 ± 10 nM ( n = 10), and in presence of angiotensin II (10 -12, 10 -9, or 10 -7 M) it increased respectively to 132 ± 7, 177 ± 8, and 234 ± 7 nM ( n = 10) ( 26 ). Using fluo-4, we found that MDCK cells exhibited a baseline [Ca 2+ ] i of 100 ± 10 nM ( n = 40), and in the presence of angiotensin II (10 -12, 10 -9, or 10 -7 M) it increased respectively to 145 ± 8, 180 ± 8, and 234 ± 7 nM ( n = 10) ( 24 ).
Solutions and reagents. The composition of the solutions utilized is described in Table 1. These solutions had an osmolality of 300 mosmol/kgH 2 O, which is the value found in the culture medium used for these cells. This osmolality was used to avoid changes in volume when the cells were transferred from the culture medium to the experimental solutions. Twenty-eight-amino acid ANP was purchased from Bachem Fine Chemicals (New Haven, CT) and fluo-4-AM and BCECF-AM from Molecular Probes (Eugene, OR). AVP (molecular weight 1.084), as well as all other applied chemicals, was obtained from Sigma (St. Louis, MO).
Statistics. The results are presented as means ± SE; n is the number of experiments. Data were analyzed statistically by analysis of variance followed by the Bonferroni contrast test. Differences were considered significant if P < 0.05.
RESULTS
pH i. Figure 1 A shows a representative experiment in which MDCK cells were first bathed with 145 mM Na + solution, exhibiting the basal pH i. After a 2-min exposure to NH 4 Cl, during which cell pH i increased transiently, the removal of NH 4 Cl caused a rapid acidification of pH i as a result of NH 3 efflux. In the presence of external 145 mM Na +, the initial fall in pH i is followed by a recovery toward the basal value. This behavior has been shown before in MDCK cells, and evidence was presented suggesting that it was due to the activity of the Na + /H + exchanger, H + -K + -ATPase, and H + -ATPase ( 10 ). Figure 1 B indicates that, in the presence of external zero Na + plus Schering 28080, the pH i recovery rate is markedly decreased and the final pH i was significantly different from the basal value; this behavior is due to the inhibition of the activity of the Na + /H + exchanger (by the removal of extracellular Na + ) and of the H + -K + -ATPase (by the specific inhibitor Schering 28080), the H + -ATPase being the only mechanism of pH recovery showing activity ( 10 ). Figure 1 B also shows that this effect is subsequently reversed with the return of the 145 mM Na + solution to the bathing medium. Thus, because the purpose of the present investigation was to study the effect of AVP in regulating the process of pH i recovery mediated by H + -ATPase, in all our experiments the cell pH recovery was examined after the acidification of pH i with the NH 4 Cl pulse technique, in the presence of zero Na + plus Schering 28080. Figure 1 C indicates that the addition of AVP (10 -12 M) to the bath caused a significant increase in the velocity of pH i recovery via H + -ATPase, but during this experimental situation the pH i recovery was not complete. With the return of the 145 mM Na + solution to the bathing medium, the pH i recovery rate increased and the final pH i was not significantly different from the basal value.
Table 2 summarizes the main values of pH i responses found in all the studied experimental groups. Our results indicate that MDCK cells in pH 7.4 HCO 3 - -free solution have a mean baseline pH i of 7.15 ± 0.02 ( n = 167).
Figure 2 indicates that in the control situation the pH i recovery rate was 0.022 ± 0.003 pH units/min ( n = 10) [the final pH i was significantly different from the basal value (6.69 ± 0.04 vs. 7.15 ± 0.03; Table 2 )]. The addition of AVP (10 -12, 10 -9, or 10 -6 M) to the bath caused a dose-dependent significant increase of the velocity of pH i recovery [of 55, 82, and 155% of the control value, respectively; during these situations, the final pH i was still significantly different from the basal value ( Table 2 )].
Fig. 2. Effect of AVP (10 -12, 10 -9, or 10 -6 M) on the initial rate of pH i recovery mediated by H + -ATPase after acute intracellular acidification in Madin-Darby canine kidney (MDCK) cells. The experiments were done in the presence of zero Na + external solution plus Schering 28080. Values are means ± SE; n = no. of experiments. * P < 0.05 vs. control.
Figure 3 shows that in the presence of V 1 - or V 2 -receptor antagonists, the pH i recovery rate was not significantly different from the control value. These data indicate that these antagonists have no intrinsic effects on pH i responses. Figure 3 also indicates that V 1 - or V 2 -receptor antagonists returned both stimulatory effects of AVP (10 -12 or 10 -6 M) to control levels, suggesting that the stimulation of H + -ATPase by AVP is via V 1 and V 2 receptor-mediated pathways.
Fig. 3. Effect of vasopressin V 1 - or V 2 -receptor antagonists (10 -5 M) plus AVP (10 -12 or 10 -6 M) on the initial rate of pH i recovery mediated by H + -ATPase after acute intracellular acidification in MDCK cells. The experiments were done in the presence of zero Na + external solution plus Schering 28080. Values are means ± SE; n = no. of experiments. * P < 0.05 vs. control. # P < 0.05 vs. AVP (10 -12 M). + P < 0.005 vs. AVP (10 -6 M).
Figure 4 indicates that DDAVP (a V 2 -selective agonist) caused a dose-dependent significant increase of the rate of pH i recovery, confirming that V 2 receptors have a stimulatory effect on H + -ATPase.
Fig. 4. Effect of the V 2 -selective agonist (DDAVP; 10 -12, 10 -9, or 10 -6 M) on the initial rate of pH i recovery mediated by H + -ATPase after acute intracellular acidification in MDCK cells. The experiments were done in the presence of zero Na + external solution plus Schering 28080. Values are means ± SE; n = no. of experiments. * P < 0.05 vs. control.
Figure 5 shows that with ANP alone the pH i recovery rate was not significantly different from the control value; however, ANP impaired the stimulatory effects of AVP (10 -12 or 10 -6 M). Figure 5 also indicates that, similar to ANP, dimethyl-BAPTA-AM alone did not affect the pH i recovery rate but impaired the stimulatory effects of AVP (10 -12 or 10 -6 M).
Fig. 5. Effect of atrial natriuertic peptide (ANP; 10 -6 M) or dimethyl-BAPTA-AM (5 x 10 -5 M) alone or plus AVP (10 -12 or 10 -6 M) on the initial rate of pH i recovery mediated by H + -ATPase after acute intracellular acidification in MDCK cells. The experiments were done in the presence of zero Na + external solution plus Schering 28080. Values are means ± SE; n = no. of experiments. * P < 0.05 vs. control. # P < 0.05 vs. AVP (10 -12 M). + P < 0.05 vs. AVP (10 -6 M).
Figure 6 indicates that with thapsigargin alone or 8-BrcAMP alone the pH i recovery rate was not significantly different from the control value; however, thapsigargin plus 8-BrcAMP caused a dose-dependent significant increase in the velocity of pH i recovery (of 114% of the control value).
Fig. 6. Effect of thapsigargin (an intracellular calcium releaser; 10 -5 M) and/or 8-BrcAMP (a membrane-permeant cAMP analog; 10 -5 M) on the initial rate of pH i recovery mediated by H + -ATPase after acute intracellular acidification in MDCK cells. The experiments were done in the presence of zero Na + external solution plus Schering 28080. Values are means ± SE; n = no. of experiments. * P < 0.05 vs. control, thapsigargin, or 8-BrcAMP.
[Ca 2+ ] i. Table 3 shows that MDCK cells exhibited a mean baseline [Ca 2+ ] i of 104 ± 4 nM ( n = 122). AVP (10 -12 or 10 -6 M) increased [Ca 2+ ] i in a dose-dependent manner (by 141 or 281% of the control value, respectively). This stimulatory effect of AVP was significantly reduced by V 1 - or V 2 -receptor antagonists. DDAVP (10 -12, 10 -9, or 10 -6 M) increased [Ca 2+ ] i in a dose-dependent manner (by 23, 53, or 78% of the control value, respectively). ANP or dimethyl-BAPTA-AM led to a significant decrease in [Ca 2+ ] i (of 55 or 50% of the control value, respectively). In the presence of ANP or dimethyl-BAPTA-AM, AVP caused a recovery of [Ca 2+ ] i without exceeding normal baseline values even at AVP (10 -6 M). Thapsigargin (10 -5 M) alone or plus 8-BrcAMP (10 -5 M) increased [Ca 2+ ] i (by 250 or 241% of the control value, respectively); however, with 8-BrcAMP (10 -5 M) alone, [Ca 2+ ] i was not significantly different from the control value.
Table 3. Cytosolic free calcium concentration in MDCK cells subjected to different agents
DISCUSSION
The purpose of this study was to clarify the effect of AVP (10 -12, 10 -9, or 10 -6 M) in regulating the process of pH i recovery mediated by H + -ATPase and to elucidate the interaction between ANP and AVP in this process. The experiments were done in MDCK cells, a permanent cell line originated from the renal collecting duct. The cells were from passages 66-75 and exhibited the resistance of 320 ·cm 2, thus from cell strain I according to Richardson et al. ( 27 ).
The heterogeneity of strain I was confirmed by Gekle et al. ( 12 ), who cloned two MDCK cell subtypes, C7 and C11, with different morphologies and functions. The C7 subtype resembles principal cells of the renal collecting duct and exhibits a pH i of 7.39. The C11 subtype establishes a transepithelial Cl - and pH gradient, two-thirds of them exhibited peanut lectin binding capacity (thus resembling intercalated cells of the renal collecting duct), and they maintain a pH i at 7.16. Our present data demonstrate that MDCK cells, in a pH 7.4 HCO 3 - -free solution, maintain a mean baseline pH i of 7.15 ± 0.02 ( n = 167), a value compatible with the MDCK cell subtype C11. However, we did not distinguish between the two cell subtypes present in our preparation.
According to Fernández and Malnic ( 10 ), the more important of the mechanisms of pH i recovery after an acid load in strain I MDCK cells is the Na + /H + exchanger; however, the reduction of extracellular K + or the addition of Schering 28080 caused a significant reduction of Na + -independent pH i recovery, confirming the presence of a H + -K + -ATPase in these cells. In addition, when a low-K + extracellular solution plus concanamycin (a specific inhibitor of the vacuolar H + -ATPase) was used, the Na + -independent pH i recovery was almost completely inhibited, showing the presence of a vacuolar H + -ATPase in these cells. This H + -ATPase was also found in clone C11 of MDCK cells ( 11 ). The two Na + -independent proton secretion mechanisms, the H + /K + -ATPase and the vacuolar H + -ATPase, are mechanisms similar to those found in the intercalated cells of mammalian collecting duct.
AVP. We studied the effect of AVP in regulating the process of pH i recovery in the presence of zero Na + plus Schering 28080, a situation in which the H + -ATPase is the only mechanism of pH i recovery showing activity, according to the aforementioned authors. Our results indicate, to our knowledge for the first time in MDCK cells, a dose-dependent stimulatory effect of AVP on H + -ATPase ( Fig. 2 ). This hormonal effect is via V 1 and V 2 receptors, because we demonstrated that V 1 - or V 2 -receptor antagonists prevent the stimulatory effect of AVP on the net rate of pH i recovery ( Fig. 3 ).
Our results show that [Ca 2+ ] i increases progressively as AVP concentrations increase from 10 -12 to 10 -6 M and that the V 1 -receptor antagonist reduces this stimulatory effect of AVP ( Table 3 ). These results are in accordance with data from the literature. It has been proposed that V 1 receptors mediate AVP action mostly via a G q11 protein-phospholipase C-IP 3 -protein kinase C-Ca 2+ pathway ( 6, 7, 19, 35 ). On the other hand, at high concentrations, AVP is known to interact with V 1 receptors, causing the liberation of arachidonic acid, which is part of a path that elevates cell calcium ( 9 ). The rise in [Ca 2+ ] i may enhance active proton transport by an increased exocytotic insertion of proton pumps into the apical membrane (as occurs in the turtle urinary bladder, proximal tubule, and cortical collecting duct) ( 13, 28 ). This behavior is compatible with our data showing that a V 1 -receptor antagonist impairs both stimulatory effects of AVP (10 -12 or 10 -6 M) on pH i recovery mediated by H + -ATPase ( Fig. 3 ) and on [Ca 2+ ] i ( Table 3 ). In addition, our results are in accordance with studies suggesting that a rise of [Ca 2+ ] i might represent part of a physiological mechanism to stimulate H + -ATPase-mediated protein export after cell acidification ( 17 ).
On the other hand, it is well known that V 2 receptors mediate a dose-dependent adenylate cyclase-cAMP-protein kinase A pathway that, in endocytotic vesicles, is observed to inhibit the H + -ATPase ( 16, 31 ). This finding is not compatible with our present results, which show that, in MDCK cells, a V 2 -receptor antagonist impairs the stimulatory effects of AVP (10 -12 or 10 -6 M) on pH i recovery mediated by H + -ATPase ( Fig. 3 ). Thus it is possible that the effect of AVP on H + -ATPase may vary with the cell type, membrane surface, and/or the experimental procedure used. To confirm our results, we did experiments with DDAVP (an AVP analog that specifically binds to adenylyl cyclase-coupled V 2 receptors). These experiments confirmed that, in MDCK cells, action via V 2 receptors has a dose-dependent stimulatory effect on H + -ATPase ( Fig. 4 ) and on [Ca 2+ ] i ( Table 3 ). These data are in accordance with the studies demonstrating that AVP causes a [Ca 2+ ] i increase, mediated by V2, through a cAMP-dependent PKA pathway in rat cortical thick ascending limb ( 18 ) and in primary cultures of rabbit cortical collecting system cells ( 33 ). These findings are also compatible with our present data showing that, in the presence of anti-V 1, AVP (10 -12 M or 10 -6 M) had a stimulatory effect on [Ca 2+ ] i ( Table 3 ).
The relationship between an increase in [Ca 2+ ] i and H + -ATPase activation is not present in all experimental situations we have studied. Thus we have shown that although AVP (10 -12 M) increased [Ca 2+ ] i and the velocity of pH i recovery (by 141 and 55% of the control value, respectively), AVP (10 -6 M) plus anti-V 1 or anti-V 2 increased [Ca 2+ ] i (by 140 and 198% of the control value, respectively) but did not affect the velocity of pH i recovery ( Table 3 and Fig. 3 ). Because the increase in [Ca 2+ ] i is not always related to activation of H + -ATPase, it is possible that an additional signaling mechanism(s) is responsible for the stimulation of H + -ATPase by AVP. An additional possibility would be that the relationship between [Ca 2+ ] i and H + -ATPase is not causal, but indirect, the main activating path being, for example, PLC-IP 3 -PKC and/or via arachidonic acid and/or cAMP-PKA, and [Ca 2+ ] i being a consequence of the activation of these pathways but not a direct cause for H + -ATPase activation.
To investigate whether blocking the increase in [Ca 2+ ] i would affect its stimulatory effect on H + -ATPase, we performed experiments in which ANP or dimethyl-BAPTA were added to AVP in our preparation. These agents are known to markedly decrease cell Ca 2+ in many tissues.
AVP plus ANP or dimethyl-BAPTA-AM. We found that, during the addition of 10 -6 M ANP alone, the pH i recovery rate was not significantly different from the control value; however, ANP impairs the stimulatory effect of AVP (10 -12 and 10 -6 M) on the velocity of pH i recovery ( Fig. 5 ). These data are compatible with a role of the increase in [Ca 2+ ] i in regulating the stimulatory effect of AVP on H + -ATPase. ANP alone does not affect the velocity of pH i recovery because it causes a decrease in cytosolic free calcium that by itself does not impair cellular H + secretion. However, ANP impairs the effect of AVP on the velocity of pH i recovery because it impairs the increase in [Ca 2+ ] i in response to AVP, thus modulating the cellular action of AVP ( Table 3 ). In addition, our data are in accordance with studies showing that ANP inhibits cAMP synthesis stimulated by AVP ( 20 ). Similar to ANP, dimethyl-BAPTA-AM alone does not affect the rate of pH i recovery although it causes, like ANP, a decrease in cytosolic free calcium. On the other hand, like ANP, dimethyl-BAPTA-AM impairs the stimulatory effect of AVP on the velocity of pH i recovery because it blocks, like ANP, the increase in [Ca 2+ ] i in response to AVP ( Fig. 5 and Table 3 ).
Taken together, our findings support the view that the increase in [Ca 2+ ] i, although not exclusive, has a role in regulating the stimulatory effect of AVP on H + -ATPase. However, whether [Ca 2+ ] i elevation represents an important direct mechanism for H + -ATPase activation by AVP or a side effect of other signaling pathways must await additional studies.
Another observation that needs an additional explanation is the inhibition of H + -ATPase activation by receptor antagonists shown in Fig. 3 and Table 2. The inhibition of H + -ATPase activation by AVP by either V 1 - or V 2 -receptor blockers is complete, whereas the addition of the effect of these agents would be expected; i.e., the addition of their individual effects should lead to return of the rate of H + extrusion to the control (pre-AVP) level. To investigate further the role of Ca 2+ and cAMP in determining the AVP effect on H + -ATPase function, we performed measurements using thapsigargin (an intracellular calcium releaser) and 8-BrcAMP (a membrane-permeant cAMP analog).
Thapsigargin and 8-BrcAMP. We found that thapsigargin alone increases [Ca 2+ ] i but not dpH i /min ( Fig. 6 and Table 3 ). On the other hand, 8-BrcAMP neither increases [Ca 2+ ] i nor affects dpH i /min. However, when both agents are given together, a significant increase in dpH i /min was seen (114% of the control value). This suggests that for an effect of AVP on H + -ATPase both of these agents are necessary, one being permissive of the other. This would also explain the role of both V 1 and V 2 receptors in AVP action on H + -ATPase, both being necessary for AVP action and the inhibition of one receptor only abolishing AVP action entirely. In other words, the block of V 1 would abolish the entire effect of AVP because [Ca 2+ ] i is a permissive factor, the same occurring with V 2 and cAMP.
Our present data show that the joint action of two mediators (e.g., PKA plus PKC or Ca 2+ with 1 of them) might be necessary for the stimulation of H + -ATPase. In the presence of DDAVP, although only the V 2 receptor path is activated, the V 1 path is not blocked by an antagonist. Of course, this behavior could not occur in preparations where only one receptor path is active, as occurs with the hydrosmotic effect of AVP (V 2 ) or the luminal effect of AVP on Na + /H + exchange in the distal tubule (V 1 ) ( 2 ). In the present case, we have one effector (H + -ATPase) activated by way of both V 1 and V 2 receptor signaling paths, where the interaction of these pathways would be essential for the final effect. The more detailed nature of this mechanism will demand further investigation.
In conclusion, our present results are compatible with a role of the increase in [Ca 2+ ] i but also of cAMP in regulating the dose-dependent stimulatory effect of AVP on H + -ATPase, via V 1 and V 2 receptor-mediated pathways. This stimulation is also observed in a dose-dependent way when the V 2 -receptor agonist is added to the preparation. In agreement with these results, ANP or dimethyl-BAPTA-AM, impairing the increase in [Ca 2+ ] i in response to AVP, blocks this stimulatory effect of AVP. On the other hand, Ca 2+ alone or cAMP alone is not sufficient to cause stimulation of H + -ATPase action, but their joint action is necessary to obtain this effect. This hormonal interaction we observed in MDCK cells (a cell line with many morphological and physiological similarities with the mammalian collecting duct) may represent a relevant mechanism of pH i regulation and of transepithelial H + secretion modulation in the intact animal.
GRANTS
This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo, Programa de Apoio a Núcleos de Excelência (no. 66.1029/1998-0), and Conselho Nacional de Pesquisas.
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作者单位：Department of Physiology and Biophysics, Instituto de Ciências Biomédicas, University of São Paulo, São Paulo 05508-90 Brazil