The nature of the mechanism underlying arginine vasopressin (AVP) action on intracellular pH (pHi) regulation is not yet defined clearly. In mesangial cells, AVP stimulates the Na+/H+ exchanger1,2 as well as the Na+-dependent or independent Cl- HCO3- exchangers2. In A6 cells (an amphibian distal nephron cell line), however, at either low (10-10 mol/L) or high (10-6 mol/L) concentrations AVP inhibits the basolateral Na+/H+ exchanger activity3. Studies in isolated perfused mouse medullary thick ascending limbs showed that AVP stimulates the basolateral transporters while it simultaneously inhibits the apical Na+/H+ exchanger4.
In addition, most studies have detected AVP activity when applied at the basolateral surface, which is mediated mostly by V2 receptors via the adenylate cyclase/cAMP signaling system5. However, in recent years, V1 receptors have been detected both in apical and basolateral membrane domains, and have been shown to mediate AVP activity via phospholipase C/inositol 3,4,5-triphosphate (IP3)/calcium signaling5,6,7. Previous data from our laboratory have shown that luminal AVP (10-9 mol/L) acts on H+ secretion in both early and late distal tubules of rat kidney via activation of V1 receptors8, whereas peritubular AVP (10-11 and 10-9 mol/L) acts to stimulate bicarbonate reabsorption in both of these segments via activation of V1 receptors, and that V2 receptors have a dose-dependent inhibitor effect, possibly mediated by cAMP (abstract; Musa-Aziz et al, J Am Soc Nephrol 11:7A, 2000). Thus, it is possible that the AVP response of Na+/H+ exchanger may vary with the cell type, cell membrane surface, and hormonal doses being studied.
Studies exploring the mechanisms that control H+ secretion by acid-secreting epithelia have emphasized the importance of cytosolic free calcium concentration ([Ca2+]i) in this process9. Thus, it was shown that AVP increases [Ca2+]i10, while atrial natriuretic peptide (ANP) reduces it11. In addition, ANP has been shown to inhibit cAMP synthesis stimulated by AVP in rat renal papillary collecting tubule cells in culture12. Thus, since AVP stimulates Na+/H+ exchange11, there may be some interaction between AVP and ANP in the regulation of pHi.
The present work investigated the role of AVP (10-12, 10-9, and 10-6 mol/L) in the modulation of pHi, as well as the mechanism of interaction between AVP and ANP (10-6 mol/L) or dimethyl-1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BATPA-AM; 50 
mol/L) in the modulation of pHi and [Ca2+]i. Madin-Darby canine kidney (MDCK) cells, a permanent cell line that is among the best characterized renal epithelial cells, were used. Our previous study demonstrated that these cells possess a basolateral Na+/H+ exchanger accounting for the Na+-dependent pHi recovery13. The present investigation also measured the effect of the V1-receptor specific antagonist [(
-mercapto-, -cyclopentamethylene-propionyl1, O-Me-Tyr2, Arg8) vasopressin (MCMV); 10-5 mol/L] or the V2-receptor specific antagonist [(adamantaneacetyl1, O-Et-D-Tyr2, Val4, aminobutyryl6, Arg8,9) vasopressin; 10-5 mol/L], at either the apical or basolateral membrane surface on Na+-dependent pHi recovery.
Our studies indicate a role of [Ca2+]i in regulating the process of pHi recovery after the acid load induced by NH4Cl, mediated by the basolateral Na+/H+ exchanger and stimulated/impaired by AVP. They are compatible with stimulation of the Na+/H+ exchanger by increases of [Ca2+]i in the lower range (that is, 10-12 or 10-9 mol/L AVP; mediated by basolateral V1 receptors) and inhibition at high [Ca2+]i levels (10-6 mol/L AVP; via activation of basolateral V1 receptors). They also are compatible with inhibition of the Na+/H+ exchanger at high cell cAMP levels (at 10-6 mol/L AVP, mediated by basolateral V2 receptors). ANP and dimethyl-BAPTA-AM, by causing a moderate decrease of [Ca2+]i, do not affect the pHi recovery, but impair the path causing an increase in [Ca2+]i, thus blocking both the stimulatory and inhibitory effects of AVP in this process.
METHODS
Cell culture and fluorescent measurement of pHi and [Ca2+]i were done as we described previously13.
Cell culture
Wild-type MDCK cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were used for all experiments (passages 60 to 66). Serial cultures were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, 100 g/mL streptomycin, and 2 mmol/L L-glutamine. Cells were grown at 37°C, 95% humidified air and 5% carbon dioxide (pH 7.4) in a CO2 incubator (Lab-Line Instruments, Melrose Park, IL, USA). The cells were harvested with trypsin in ethyleneglycol-bis (b-aminoethyl ether)-N, N'-tetraacetic acid (EGTA, 0.02%), and then seeded on sterile glass coverslips and incubated again for 72 hours in the same medium to become confluent.
Fluorescent measurement of pHi
Intracellular pH was monitored using the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxy-methyl ester (BCECF-AM). Cells grown to confluence on glass coverslips were loaded with the dye by exposure for 20 minutes to 10 mol/L BCECF-AM in solution 1 Table 1. After BCECF-AM entered the cells it was rapidly converted to the anionic free acid form by intracellular esterases. Following the loading period, the glass coverslips were rinsed with the control solution to remove the BCECF-containing solution and placed into a thermo-regulated chamber mounted on an inverted epifluorescence microscope (Nikon, TMD). The measured area under the microscope had a diameter of 260 m 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. Bathing solutions were rapidly exchanged without disturbing the position of the coverslips. All experiments were performed at 37°C. The cells were alternately excited at 455 or 505 nm with a 150 W xenon lamp and the fluorescence emission was monitored at 530 nm by a photomultiplier-based fluorescence system (Georgia Instruments, PMT-400) at time intervals of five seconds. The 505/455 excitation ratio corresponds to a specific pHi. At the end of each experiment, calibration of the BCECF signal was achieved by the high K+-nigericin method14, exposing the cells for 15 minutes to a K+-HEPES buffer solution containing 10 mol/L nigericin (solution 3; Table 1), at pH 6.5, 7.0, or 7.5.
Cell pH recovery
Cell pH recovery was examined following the acidification of pHi with the NH4Cl pulse technique15 after a two-minute exposure to 20 mmol/L NH4Cl (solution 4; Table 1) in the following situations: control (in the presence of external 145 mmol/L Na+, solution 2; Table 1), absence of external Na+ (solution 5; Table 1) or in the presence of AVP (10-12, 10-9, or 10-6 mol/L), V1 and/or V2 receptor antagonists (10-5 mol/L), ANP (10-6 mol/L), or dimethyl-BAPTA-AM (50 mol/L). Since the rate of pH recovery depends on the value of cell pH achieved by the acid load16, we used experiments in which these values were not significantly different between the studied groups Table 2. In all of the experiments, the initial rate of pHi recovery (dpHi/dt, pH units per min) was calculated from the first two minutes of the recovery curve by linear regression analysis.
Table 2 - Summary of pHi responses in MDCK cells to addition of different agents after an acute acid load.
Full table Fluorescent measurement of [Ca2+]i
Changes in [Ca2+]i were monitored fluorometrically using the Ca2+-sensitive probe fluo 4-AM. MDCK cells were grown to confluence on uncoated glass-bottomed microwells (Mat-Tek, Ashland, MA, USA) at a density of 2.5 
105 cells/mL. Twenty-four hours after plating, confluent cultures were loaded with 10 mol/L fluo 4-AM at 37°C for 40 minutes and rinsed in Tyrode solution (solution 6; Table 1) containing 0.2% bovine serum albumin (pH 7.4). Cells were studied at room temperature and fluo 4 fluorescence intensity emitted above 520 nm was imaged by using ultraviolet laser excitation at 488 nm on a Zeiss LSM 510 real-time confocal microscope. The images were continuously acquired before and after the addition of experimental solutions, at time intervals of 10 seconds, for a total of 200 seconds. For each experiment the maximum fluorescent signal for 10 cells was averaged and then used for analysis. Transformation of the fluorescent signal to [Ca2+]i was performed by calibration with ionomycin (30 mol/L; maximum concentration) followed by EGTA (2.5 mmol/L; minimum concentration) according to the Grynkiewicz equation17, using the dissociation constant of 345 nmol/L (according to the Molecular Probes catalog).
Solutions and reagents
The composition of the solutions utilized is described in Table 1. These solutions had an osmolality between 325 and 330 mOsm, which is the value found in the culture medium used for these cells. This osmolality was used to avoid changes 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, USA) and fluo 4-AM, BCECF-AM and dimethyl-BATPA-AM from Molecular Probes (Eugene, OR, USA). AVP (molecular weight 1.084), V1-receptor specific antagonist [anti-V1; (-Mercapto-,-cyclopentamethylene-propionyl1, O-Me-Tyr2,Arg8) vasopressin; (MCMV)], V2-receptor specific antagonist [anti-V2, (adamantaneacetyl1, O-Et-D-Tyr2, Val4, aminobutyryl6, Arg8,9) vasopressin], as well as all other applied chemicals were obtained from Sigma Chemical Company (St. Louis, MO, USA).
Statistics
The results are presented as means 
SEM. N is the number of measurements. Data were analyzed statistically by analysis of variance followed by Bonferroni's contrast test. Differences were considered significant if P < 0.05.
RESULTS
pHi
In all experiments, the cell pH recovery was examined following the acidification of pHi with the NH4Cl pulse technique. Figure 1 shows three representative experiments. Cells were first bathed with the control solution Table 1, exhibiting the basal pHi. After a two-minute exposure to 20 mmol/L NH4Cl, during which cell pHi increased transiently, NH4Cl removal caused a rapid acidification of pHi as a result of the NH3 efflux. In the presence of the control solution, the initial fall in pHi was followed by a recovery of pHi towards the basal value Figure 1a. The addition of AVP (10-12 mol/L) to the bath caused a significant increase of the velocity of pHi recovery Figure 1b, whereas in the presence of AVP (10-6 mol/L), the velocity of pHi recovery decreased significantly Figure 1c.
Figure 1.
Intracellular pH (pHi) recovery after cellular acidification with the NH4Cl pulse technique. (A) In the presence of control solution the initial fall in pHi is followed by a recovery of pHi towards the basal value. (B) The addition of 10-12 mol/L AVP to the bath causes a significant increase of the velocity of pHi recovery. (C) The addition of AVP (10-6 mol/L) to the bath causes a significant decrease of the velocity of pHi recovery. B, basal pHi.
Full figure and legend (23K)Table 2 summarizes the main values of pHi responses found in all of the experimental groups. Our results found that MDCK cells in pH 7.4
HCO3-- free solution had a mean baseline pHi of 7.15 0.005 (N = 199).
Figure 2 indicates the effect of the absence of external Na+ on the main pHi recovery rate. In the control situation (that is, in the presence of external Na+ 145 mmol/L), the main pHi recovery rate in the first two minutes was 0.101 0.005 pH units (U)/min (N = 62), and the final pHi was not significantly different from the basal value (7.17 0.01 vs. 7.14 0.04; Table 2). In the absence of external Na+, the pHi recovery rate was reduced to 34% of the control value (and pHi recovery was not complete; Table 2). This effect was reversed with the return of Na+ to the bathing solution (and final pHi was not significantly different from the basal value; Table 2), indicating that the pHi recovery is mostly dependent on Na+/H+ exchange.
Figure 2.
Effect of arginine vasopressin (AVP; 10-12, 10-9, and 10-6 mol/L) on the initial rate of pHi recovery following acute intracellular acidification in Madin-Darby canine kidney (MDCK) cells. The experiments were done in the presence of 145 mmol/L (
) or absence (
) of extracellular Na+. *P < 0.05 vs. control; +P < 0.05 vs. AVP (10-12 mol/L); #P < 0.05 vs. AVP (10-9 mol/L).
Figure 2 also shows that the addition of AVP (10-12 or 10-9 mol/L) to the bath causes a significant increase of the velocity of pHi recovery (77 or 31% of the control value, respectively), and during both situations the final pHi was not significantly different from the basal value Table 2. However, the addition of AVP (10-6 mol/L) significantly decreased the velocity of pHi recovery by 67% of the control value, and during this situation the pHi recovery was not complete Table 2. In the absence of external Na+ both stimulatory effects of AVP were significantly inhibited and pHi recovery was not complete Table 2. With the return of Na+ to the bathing solution both stimulatory effects of AVP subsequently were partly recovered, indicating that they are mostly dependent on Na+/H+ exchange.
We performed a series of experiments in which MDCK cells were grown on permeant filter supports (Transwell 3.0 m pore size, 12 mm diameter; Costar, Cambridge, MA, USA), making it possible to independently measure the effect of the V1 or V2-receptor antagonists on Na+-dependent pHi recovery at either the apical or basolateral membrane surface. Figure 3 summarizes the results found in the presence of the receptor antagonists at the basolateral membrane surface. In the control situation, the pHi recovery rate in the first two minutes was 0.099 0.01 (N = 12), a value not significantly different from 0.101 0.005 pH U/min (N = 62), the control value found when the cells were grown on coverslips. In the presence of V1 and/or V2 receptor antagonists, the pHi recovery rate was not significantly different from the control value. These data indicate that these antagonists have no intrinsic effects on pHi responses. Figure 3 also shows that V1 or V1 plus V2 receptor antagonists return both the stimulatory and inhibitory effects of AVP to control levels. Figure 3 also indicates that the V2 receptor antagonist converts the inhibitory effect of AVP to a stimulatory effect. However, another series of experiments (data not shown) indicates that V1 or V2 receptor antagonists at the apical membrane surface did not affect either the stimulatory or inhibitory effects of AVP, showing that the inhibitors have no effect at the apical side and could not traverse the cells and membranes to the opposite side. Based on these data, we speculate that the V1 and V2 receptors responsible for the Na+-dependent pHi recovery observed in the present study are located on the basolateral membrane surface.
Figure 3.
Effect of V1 or V2 receptor antagonists (10-5 mol/L) alone or plus AVP (10-12, 10-9, or 10-6 mol/L) on the initial rate of pHi recovery following acute intracellular acidification in MDCK cells. These experiments were done in cells growing on permeant filter supports in the presence of the agents at the basolateral membrane surface. Symbols are: () without antagonists; () + anti-V1; (
) + anti-V2; (
) anti-V1 + anti-V2. *P < 0.05 vs. control; +P < 0.05 vs. AVP (10-12 mol/L); #P < 0.05 vs. anti-V2; &P < 0.05 vs AVP (10-6 mol/L); @P < 0.05 vs. AVP (10-6 mol/L) + anti-V1.
Figure 4 gives the effect of addition of ANP (10-6 mol/L) alone or plus AVP (10-12, 10-9, or 10-6 mol/L) to the bath on the rate of pHi recovery, again using MDCK cells on glass coverslips. With ANP alone the pHi recovery rate was not significantly different from the control value, and the final pHi was not significantly different from the basal value Table 2. However, ANP impaired both the stimulatory effect of AVP (at 10-12 and 10-9 mol/L, where during these situations, the final pHi was not significantly different from the basal value; Table 2), as well as inhibited the effects of AVP (10-6 mol/L) on the net rate of pHi recovery (but during this situation pHi recovery was not complete; Table 2). These results indicate that ANP alone does not affect cellular pH recovery, but impairs both the stimulatory and inhibitory effects of AVP.
Figure 4.
Effects of arginine vasopressin (AVP; 10-12, 10-9, or 10-6 mol/L; ) and/or atrial natriuretic peptide (ANP, 10-6 mol/L; ), and/or dimethyl-BAPTA/AM (50
mol/L; ) on the initial rate of pHi recovery following acute intracellular acidification in MDCK cells. *P < 0.05 vs. control; #P < 0.05 vs. AVP (10-12 mol/L); +P < 0.05 vs. AVP (10-9 mol/L); &P < 0.05 vs. AVP (10-6 mol/L).
As some studies have shown the importance of cytosolic free calcium concentration for cellular H+ secretion13,18, we studied the effect of addition of dimethyl-BAPTA-AM (50 mol/L; an intracellular calcium chelator13,19) to the medium on cellular pH recovery. Figure 4 also shows that with dimethyl-BAPTA-AM alone, the pHi recovery rate was not significantly different from the control value (and the final pHi was not significantly different from the basal value; Table 2). Dimethyl-BAPTA-AM impairs both stimulatory effects of AVP (10-12 and 10-9 mol/L) on the rate of pHi recovery, and during both situations the final pHi was not significantly different from the basal value Table 2. Dimethyl-BAPTA-AM also impairs the inhibitory effect of AVP (10-6 mol/L) on the net rate of pHi recovery despite that during this situation the pHi recovery was not complete Table 2. Taken together, these results suggest a role of cytosolic free calcium in regulating the net rate of pHi recovery, mediated by Na+/H+ exchange and stimulated/impaired by AVP.
[Ca2+]i
Figure 5 shows that the addition of AVP (10-12, 10-9, or 10-6 mol/L) to the bath of MDCK cells seeded on glass coverslips caused a significant increase of cell calcium fluorescent signal, in a dose-dependent manner. For each experiment, the maximum fluorescent signal for 10 cells was averaged and then used to calculate [Ca2+]i.
Figure 5.
Cell calcium fluorescent signal tracings during three representative experiments. The images were continuously acquired before and after addition of AVP (10-12, 10-9, or 10-6 mol/L), at time intervals of 10 seconds. The addition of AVP to the bath causes a significant and dose-dependent increase of the fluorescent signal.
Full figure and legend (36K)Figure 6 summarizes the main values of [Ca2+]i found in all of the studied experimental groups. MDCK cells exhibited a mean baseline [Ca2+]i of 100 0.38 nmol/L (N = 386). The subsequent addition of AVP (10-12, 10-9, and 10-6 mol/L) increased [Ca2+]i progressively from control values to 338 1.4 nmol/L (N = 25), in a dose-dependent manner. The addition of ANP (10-6 mol/L) to the bathing solution leads to a rapid and significant decrease in [Ca2+]i from control values to 40.8 0.31 nmol/L (N = 149). In the presence of ANP, the subsequent addition of AVP (10-12, 10-9, and 10-6 mol/L) caused a recovery of [Ca2+]i that reached 89.8 1.8 nmol/L (N = 26), thus without exceeding normal baseline values even at AVP (10-6 mol/L). Figure 6 also shows that the addition of dimethyl-BAPTA-AM to the bathing solution leads to a significant decrease in [Ca2+]i from control values to 50.5 0.68 nmol/L (N = 129). In the presence of dimethyl-BAPTA-AM, the subsequent addition of AVP (10-12, 10-9, and 10-6 mol/L) caused a recovery of [Ca2+]i to 112 1.07 nmol/L (N = 51), 115 0.76 nmol/L (N = 43) and 153 1.87 nmol/L (N = 37), respectively.
Figure 6.
Effects of AVP (10-12, 10-9, or 10-6 mol/L; ) and/or ANP (10-6 mol/L;), and/or dimethyl-BAPTA/AM (50 mol/L; ) on free calcium concentration in the cytosol ([Ca2+]i) of MDCK cells. *P < 0.05 vs. control (C); #P < 0.05 vs. AVP (10-12 mol/L); @P < 0.05 vs. AVP (10-9 mol/L); &P< 0.05 vs. AVP (10-6 mol/L).
DISCUSSION
The purpose of this study was to clarify the mechanism of interaction between AVP and ANP on the initial rate of pHi recovery following acute intracellular acidification in Madin-Darby canine kidney (MDCK) cells, a permanent cell line originated from the renal collecting duct. In the present study, the MDCK cells were from passage 60 to 66, thus from cell strain I according to Richardson, Scalera and Simmons20. Our data demonstrate that MDCK cells in pH 7.4
HCO3--free solution maintain a mean baseline pHi of 7.15 0.005 (N = 199), a value compatible with the MDCK cell subtype C1121. Our data are in accordance with the studies of Wiegmann et al18, who used both fluorometry and video microscopy to show that MDCK cells had a mean pHi of 7.12 0.01 (N = 50). Our present results also agree with the value of 7.17 0.01 (N = 23) found by Fernández and Malnic22 in MDCK cells strain I, and with the value of 7.17 0.04 (N = 173) found in our previous studies performed in MDCK cells13.
Our data show that in the absence of external Na+ the net rate of pHi recovery was reduced to 34% of the control value Figure 2. This effect is partly reversed with the return of Na+ to the bathing solution, indicating that the pHi recovery is mostly dependent on Na+/H+ exchange Table 2. This result is in accordance with Fernández and Malnic22, who found three different mechanisms of pHi recovery in MDCK cells: the Na+/H+ exchanger (the most important), the H+-K+ATPase, and the vacuolar H+ATPase.
Our results indicate, to our knowledge for the first time in MDCK cells, that low concentrations (10-12 or 10-9 mol/L) of AVP stimulate and a high concentration (10-6 mol/L) of AVP inhibits the velocity of pHi recovery Figure 2. In the absence of external Na+, both stimulatory effects of AVP are significantly inhibited, and with the return of Na+ to the bathing solution, they are subsequently partly recovered, indicating that they are mostly dependent on Na+/H+ exchange Table 2.
In our previous studies using permeant filter supports, the Na+/H+ exchanger accounting for the Na+-dependent pHi recovery in MDCK cells was shown to be located on the basolateral membrane13. Our present data indicate that V1 or V2 receptors antagonists at the apical membrane surface do not affect the stimulatory and inhibitory effects of AVP on the velocity of pHi recovery. However, V1 or V1 plus V2 receptor antagonists on the basolateral membrane surface return both the stimulatory and inhibitory effects of AVP to control levels Figure 3. In addition, the V2 receptor antagonist at the basolateral membrane surface converts the inhibitory effect of AVP to a stimulatory effect. Based on these data, it can be concluded that both the stimulatory and inhibitory effects of AVP on the basolateral Na+/H+ exchanger that account for the Na+-dependent pHi recovery are via activation of V1 receptors located on the basolateral membrane surface, and that basolateral V2 receptors have a dose-dependent inhibitor effect. In vivo experiments from our laboratory showed a luminal effect of AVP via V1 receptors, as opposed to the present results. This finding may be due to the use of rat tubules in the previous experiments rather than dog cells, and to differences between in vivo and cultured cells8. On the other hand, capillary perfusion experiments in the rat have shown the presence of both V1 and V2 receptors on the basolateral membrane of distal tubule cells (abstract; Musa-Aziz et al, J Am Soc Nephrol 11:7A, 2000).
In the present experiments on MDCK cells, ANP counteracted both the stimulating and the inhibiting effects of AVP Figure 4. These data are compatible with the identification of ANP receptors in MDCK cells23. Although only few ANP receptors have been found in the cortical distal tubule, such receptors are widely distributed in renal tissue, their mRNA having been detected in cortical and especially in medullary collecting duct cells24. Thus, it is possible that MDCK cells present properties more akin to the medullary collecting duct with respect to these receptors. On the other hand, these results are very similar to those we obtained with Ang II in MDCK cells: ANP (10-6 mol/L) counteracted both the stimulating and the inhibiting effects of Ang II on the net rate of Na+-dependent pHi recovery13. In addition, an interaction between ANP and AVP has been observed in other tissues: ANP inhibits cAMP synthesis stimulated by AVP in rat renal papillary collecting tubule cells in culture12, and AVP stimulated Na+/H+ exchange in vascular smooth muscle cells in culture11.
To obtain information on the mechanism of the interaction of these hormones on pHi regulation, we studied their effects on the regulation of [Ca2+]i. Our results indicate that MDCK cells exhibited a mean baseline [Ca2+]i of 100 0.38 nmol/L (N = 386). These data agree with the value of 120 29 nmol/L (N = 6) found by Borle and Bender25 or of 125 7 nmol/L (N = 50) found by Weigmann et al in MDCK cells18. This value also is not significantly different from the basal value of [Ca2+]i monitored with the fluorescent probe Fura-2 in these cells while in suspension, as described in our previous study (99.0 10 nmol/L, N = 20)13.
Our data show that [Ca2+]i increases progressively as AVP concentrations increase from 10-12 to 10-6 mol/L Figures 5 and 6. These results are in accordance with data from the literature. It has been proposed that V1 receptors mediate AVP action mostly via a Gq11 protein-phospholipase C-IP3-protein kinase C-Ca2+ pathway6,26,27,28. Besides, it is known that protein kinase C, via phosphorylation, may stimulate the Na+/H+ exchanger29. This behavior is compatible with our data showing that low concentrations of AVP stimulate the rate of Na+-dependent pHi recovery via V1 receptors Figure 3.
At high concentrations, AVP is known to interact with V1 receptors, causing the liberation of arachidonic acid, which is part of a path that elevates cell calcium by activating voltage-sensitive calcium channels of the plasma membrane27. At high cytosolic concentrations, calcium may inhibit Na+/H+ exchange by activating Na+/Ca2+ exchange at the cell membrane, thereby increasing cell sodium, which decreases the gradient responsible for H+ extrusion by the exchanger29,30. This behavior is compatible with our data showing that (1) the effect on the rate of Na+-dependent pHi recovery with AVP (10-12 mol/L) is higher than with AVP (10-9 mol/L), but in the presence of V1 antagonist is similar for both doses, and (2) AVP (10-6 mol/L) inhibits the rate of Na+-dependent pHi recovery via V1 receptors Figure 3.
On the other hand, it is well known that V2 receptors are present mostly at the basolateral membrane, where they mediate the hydro-osmotic effect of AVP at picomolar concentrations. This mechanism is known to involve a dose-dependent adenylate cyclase, cAMP, protein kinase A pathway that, at high AVP concentrations, is expected to inhibit the Na+/H+ exchanger31. This behavior also is compatible with our present data, since in the presence of a V2 receptor antagonist on the basolateral membrane surface, the inhibitory effect of 10-6 mol/L AVP on the rate of pHi recovery is converted to a stimulatory effect that is significantly higher than the control or 10-6 mol/L AVP plus V1 receptor antagonist values Figure 3.
Our results show that when 10-6 mol/L ANP is added to the bathing solution, [Ca2+]i decreases to approximately 41% of the control value. In the presence of ANP, the subsequent addition of AVP (from 10-12 to 10-6 mol/L) caused a recovery of [Ca2+]i without exceeding normal baseline values even at AVP 10-6 mol/L Figure 6. Our results also show that ANP alone does not affect the rate of Na+-dependent pHi recovery, but ANP impairs both the stimulatory and inhibitory effects of AVP in this process Figure 4. These data are compatible with our previous results in MDCK cells showing that—in contrast to EGTA—ANP alone does not affect the rate of Na+-dependent pHi recovery, since it causes only a moderate decrease of cytosolic free calcium as compared to the minimal [Ca2+]i values found in presence of EGTA (to 35 and 15% of the control value, respectively)13. On the other hand, ANP impairs both stimulatory and inhibitory effects of AVP on the rate of Na+-dependent pHi recovery because it impairs the increase of [Ca2+]i in response to AVP, thus modulating the cellular action of AVP. It is possible that this is the mechanism by which ANP inhibits the AVP-stimulated Na+/H+ exchange in vascular smooth muscle cells in culture11, and impairs both the stimulatory and inhibitory effects of Ang II on the rate of Na+-dependent pHi recovery in MDCK cells13.
This behavior is also in agreement with the results concerning the effect of dimethyl-BAPTA-AM on the rate of pHi recovery. Similar to ANP, dimetyl-BAPTA/AM alone does not affect the rate of pHi recovery since, like ANP, it causes only a moderate decrease (to about 51%) of cytosolic free calcium Figure 6. On the other hand, like ANP, dimethyl-BAPTA-AM impairs both stimulatory and inhibitory effects of AVP on the rate of pHi recovery since, like ANP, it impairs the increase of [Ca2+]i in response to AVP. In addition, with dimethyl-BAPTA-AM plus AVP (10-6 mol/L), the [Ca2+]i values are not significantly different from AVP (10-12 mol/L) values [153 1.87 (N = 37) and 160 1.45 (N = 53) nmol/L, respectively], but the pHi recovery values measured in the presence of dimethyl-BAPTA-AM plus AVP 10-6 mol/L are significantly smaller than the values found in presence of AVP 10-12 mol/L Figure 4, because in the presence of high doses of AVP an inhibitory effect occurs on the Na+/H+ exchange mediated by cAMP-protein kinase A, via V2 receptors.
In conclusion, the results obtained in our studies suggest that [Ca2+]i has a role in regulating the process of pHi recovery after the acid load induced by NH4Cl, which is mediated by the basolateral Na+/H+ exchanger and stimulated/impaired by AVP. The data are compatible with stimulation of the Na+/H+ exchanger by increases of [Ca2+]i in the lower range (at 10-12 or 10-9 mol/L AVP; mediated by basolateral V1 receptors via a Gq11 protein phospholipase C IP3 protein kinase C–Ca2+ pathway) and inhibition at high [Ca2+]i levels (at 10-6 mol/L AVP; via activation of basolateral V1 receptors causing the liberation of arachidonic acid). They are also compatible with inhibition of the Na+/H+ exchanger at high cell cAMP levels (at 10-6 mol/L AVP, mediated by basolateral V2 receptors). While ANP and dimethyl-BAPTA/AM cause a moderate decrease of [Ca2+]i, this does not affect the pHi recovery, but, by impairing the path causing the increase in [Ca2+]i, they block both the stimulatory and inhibitory effects of AVP during this process. The question of whether [Ca2+]i modification represents an important direct mechanism for exchanger activation or is a side effect of other signaling pathways must await additional studies.
References
| 1. | Ganz MB, Boyarsky G, Sterzel RB & Boron WF. Arginine vasopressin enhances pHi regulation in the presence of HCO3 by stimulating three acid-base transport systems. Nature 1989; 337: 648−651. | Article | PubMed | ISI | ChemPort | |
| 2. | Ganz MB, Boyarsky G, Boron WF & Sterzel RB. Effect of angiotensin II and vasopressin on intracellular pH of glomerular mesangial cells. Am J Physiol 1988; 254: F787−F794. | PubMed | ISI | ChemPort | |
| 3. | Casavola V, Guerra L & Helmle-Kolb C et al. Na+/H+ exchange in A6 cells: Polarity and vasopressin regulation. J Membr Biol 1992; 130: 105−114. | PubMed | ISI | ChemPort | |
| 4. | Sun AM, Kikeri D & Hebert SC. Vasopressin regulates apical and basolateral Na+-H+ antiporters in mouse medullary thick ascending limbs. Am J Physiol 1992; 262: F241−F247. | PubMed | ISI | ChemPort | |
| 5. | Grider J, Falcone J & Kilpatrick E et al. Effect of luminal vasopressin on NaCl transport in the medullary thick ascending limb of the rat. Eur J Pharmacol 1996; 313: 115−118 10.1016/0014-2999(96)00620-6. | Article | PubMed | ISI | ChemPort | |
| 6. | Ikeda M, Yoshitomi K & Imai M et al. Cell Ca2+ response to luminal vasopressin in cortical collecting tubule principal cells. Kidney Int 1994; 45: 811−816. | PubMed | ISI | ChemPort | |
| 7. | Nonoguchi H, Takayama M & Owada A et al. Role of urinary arginine vasopressin in the sodium excretion in patients with chronic renal failure. Am J Med Sci 1996; 312: 195−201. | PubMed | ISI | ChemPort | |
| 8. | Barreto-Chaves MLM & Mello-Aires M. Luminal arginine vasopressin stimulates Na+-H+ exchange and H+-ATPase in cortical distal tubule via V1 receptor. Kidney Int 1997; 52: 1035−1041. | PubMed | ChemPort | |
| 9. | Slotki I, Schwartz JH & Alexander EA. Interrelationship between cell pH and cell calcium in rat inner medullary collecting duct cells. Am J Physiol 1993; 265: C432−C438. | PubMed | ISI | ChemPort | |
| 10. | Burgess WJ, Balment RJ & Beck JS. Effects of luminal vasopressin on intracellular calcium in microperfused rat medullary thick ascending limb. Renal Physiol Biochem 1994; 17: 1−9. | PubMed | ISI | ChemPort | |
| 11. | Caramelo C, Lopez-Farré A & Riesco A et al. Atrial natriuretic peptide and cGMP inhibit Na+/H+ antiporter in vascular smooth muscle cells in culture. Kidney Int 1994; 45: 66−75. | PubMed | ISI | ChemPort | |
| 12. | Ishikawa S, Saito T & Okada K et al. Atrial natriuretic factor increase cyclic GMP and inhibits cyclic AMP in rat renal papillary collecting tubules cells in culture. Biochem Biophys Res Commun 1985; 130: 1147−1153. | Article | PubMed | ISI | ChemPort | |
| 13. | Oliveira-Souza M & Mello-Aires M. Interaction of angiotensin II and atrial natriuretic peptide on pHi regulation in MDCK cells. Am J Physiol 2000; 279: F944−F953. | ISI | ChemPort | |
| 14. | Thomas J, Buchsbaum R, Zimniak A & Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 1979; 18: 2210−2218. | Article | PubMed | ISI | ChemPort | |
| 15. | Boron WF & De Weer P. Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J Gen Physiol 1976; 67: 91−112. | Article | PubMed | ISI | ChemPort | |
| 16. | Weintraub WH & Machen TE. pH regulation in hepatoma cells: Roles for Na-H exchange, Cl-HCO3 exchange, and Na-HCO3 cotransport. Am J Physiol 1989; 257: G317−G327. | PubMed | ISI | ChemPort | |
| 17. | Grynkiewicz G, Poenie M & Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440−3450. | PubMed | ISI | ChemPort | |
| 18. | Wiegmann TB, Welling LW & Beatty DM et al. Simultaneous imaging of intracellular [Ca2+] and pH in single MDCK and glomerular epithelial cells. Am J Physiol 1993; 265: C1184−C1190. | PubMed | ISI | ChemPort | |
| 19. | Stuart RO, Sun A & Panichas M et al. Critical role for intracellular calcium in tight junction biogenesis. J Cell Physiol 1994; 159: 423−433. | Article | PubMed | ISI | ChemPort | |
| 20. | Richardson J, Scalera V & Simmons NL. Identification of two strains of MDCK cells which resemble separate nephron tubule segments. Biochim Biophys Acta 1981; 673: 26−36. | PubMed | ISI | ChemPort | |
| 21. | Gekle M, Wünsch S, Oberleithner H & Silbernagl S. Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties. Pflügers Arch 1994; 428: 157−162. | Article | PubMed | ChemPort | |
| 22. | Fernández R & Malnic G. H+ ATPase and Cl- interaction in regulation of MDCK cell pH. J Membr Biol 1998; 163: 137−145 10.1007/s002329900378. | Article | PubMed | ChemPort | |
| 23. | Pandey KN, Inagami T & Misono KS. Three distinct forms of atrial natriuretic factor receptors: Kidney tubular epithelium cells and vascular smooth muscle cells contain different types of receptors. Biochem Biophys Res Commun 1987; 147: 1146−1152. | Article | PubMed | ISI | ChemPort | |
| 24. | Terada Y, Tomita K & Nonoguchi H et al. PCR localization of C-type natriuretic peptide and B-type receptor mRNAs in rat nephron segments. Am J Physiol 1994; 267: F215−F222. | PubMed | ISI | ChemPort | |
| 25. | Borle AB & Bender C. Effects of pH on Ca2+i, Na+i, and pHi of MDCK cells: Na+-Ca2+ and Na+-H+ antiporter interactions. Am J Physiol 1991; 261: C482−C489. | PubMed | ISI | ChemPort | |
| 26. | Yamada H, Seki G & Taniguchi S et al. Roles of Ca2+ and PKC in regulation of acid/base transport in isolated proximal tubules. Am J Physiol 1996; 271: F1068−F1076. | PubMed | ISI | ChemPort | |
| 27. | Burgess WJ, Balment RJ & Beck JS. Effects of luminal vasopressin on intracellular calcium in microperfused rat medullary thick ascending limb. Renal Physiol Biochem 1994; 17: 1−9. | PubMed | ISI | ChemPort | |
| 28. | Bunatowska-Hledin M & Spielman WS. Vasopressin V1 receptors on the principal cells of the rabbit cortical collecting tubule: Stimulation of cytosolic free calcium and inositol phosphate production via coupling to a pertussis toxin substrate. J Clin Invest 1989; 83: 84−89. | PubMed | ChemPort | |
| 29. | Douglas JG & Hopfer U. Novel aspect of angiotensin receptors and signal transduction in the kidney. Annu Rev Physiol 1994; 56: 649−669. | Article | PubMed | ISI | ChemPort | |
| 30. | Good DW, George T & Wang DH. Angiotensin II inhibits HCO3- absorption via a cytochrome P-450-dependent pathway in MTAL. Am J Physiol 1999; 276: F726−F736. | PubMed | ISI | ChemPort | |
| 31. | Borensztein P, Juvin P & Vernimmen C et al. cAMP-dependent control of Na+/H+ antiport by AVP, PTH, and PGE2 in rat medullary thick ascending limb cells. Am J Physiol 1993; 264: F354−F364. | PubMed | ISI | ChemPort | |
Acknowledgments
This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Pesquisas (CNPq). The authors thank Dr. Gerhard Malnic for careful reading of the manuscript. We also thank Dr. David C. Spray for help with the measurement of cytosolic-free calcium technique using a real-time confocal microscope.
