Activation of H+-K+-ATPase by CO2 requires a basolateral Ba2+-sensitive pathway during K restriction

Xiaoming Zhou, I. Jeanette Lynch, Shen-Ling Xia, and Charles S. Wingo

Laboratory of Epithelial Transport, Division of Nephrology, Hypertension, and Transplantation, Department of Medicine, University of Florida, and Nephrology Section, Veterans Affairs Medical Center, Gainesville, Florida 32608-1197


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We studied the activation of H+-K+-ATPase by CO2 in the renal cortical collecting duct (CCD) of K-restricted animals. Exposure of microperfused CCD to 10% CO2 increased net total CO2 flux (Jt CO2) from 4.9 ± 2.1 to 14.7 ± 4 pmol · mm-1 · min-1 (P < 0.05), and this effect was blocked by luminal application of the H+-K+-ATPase inhibitor Sch-28080. In the presence of luminal Ba, a K channel blocker, exposure to CO2 still stimulated Jt CO2 from 6.0 ± 1.0 to 16.8 ± 2.8 pmol · mm-1 · min-1 (P < 0.01), but peritubular application of Ba inhibited the stimulation. CO2 substantially increased 86Rb efflux (a K tracer marker) from 93.1 ± 23.8 to 249 ± 60.2 nm/s (P < 0.05). These observations suggest that during K restriction 1) the enhanced H+-K+-ATPase-mediated acidification after exposure to CO2 is dependent on a basolateral Ba-sensitive mechanism, which is different from the response of rabbits fed a normal-K diet, where activation of the H+-K+-ATPase by exposure to CO2 is dependent on an apical Ba-sensitive pathway; and 2) K/Rb absorption via the apical H+-K+-ATPase exits through a basolateral Ba-sensitive pathway. Together, these data are consistent with the hypothesis of cooperation between H+-K+-ATPase-mediated acidification and K exit pathways in the CCD that regulate K homeostasis.

hydrogen-potassium-adenosine 3',5'-triphosphatase; cortical collecting duct; acidification; Sch-28080


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE APICAL MEMBRANE of the collecting duct possesses an H+-K+-ATPase that is responsible for potassium (K) absorption in exchange for proton (H) secretion (9, 20). This enzyme appears to serve an important physiological role in the regulation of acid-base balance and potassium homeostasis (18). Changes in acid-base balance have a marked effect on potassium transport that is most notable in the distal nephron and collecting duct (11). Several studies have demonstrated that total CO2 flux (Jt CO2) is stimulated in the collecting duct when the ambient CO2 tension (PCO2) is increased (10, 12, 24). During respiratory acidosis induced by 10% CO2, the cortical collecting duct (CCD) from rabbits adapted to a normal-K diet (K repletion) exhibits enhanced luminal acidification (24). The acidification is in part due to a stimulation of H+-K+-ATPase activity in this segment and can be inhibited by luminal Sch-28080, a specific H+-K+-ATPase inhibitor. Under the same K-replete conditions, the activity of H+-K+- ATPase is also dependent on an apical barium (Ba)-sensitive pathway (24). The coupling of an apical H+-K+-ATPase and an apical Ba-sensitive mechanism (presumably a K channel) has been further supported by the observation that, in the presence of luminal Ba, stimulation by 10% CO2 does not increase either apical proton secretion or 86Rb efflux from lumen to bath (24).

To further address the regulatory mechanism of H+-K+-ATPase in the CCD, we examined the activation of the H+-K+-ATPase after exposure to 10% CO2 in K-restricted rabbits. The present studies are consistent with the hypothesis that an increase in peritubular PCO2 profoundly stimulates H+-K+-ATPase activity. In contrast to findings during K-repletion, this stimulation depends on a basolateral Ba-sensitive pathway, and stimulation of H+-K+-ATPase by 10% CO2 increases lumen-to-bath 86Rb efflux. Overall, these data add to our knowledge of the regulation of the H+-K+-ATPase function in the collecting duct in response to changes in potassium intake.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Animal preparations. Female New Zealand White rabbits were maintained on a low-K diet (0.25% K, TD87433, Teklad, Madison, WI) and allowed free access to tap water for at least 4 days before experimental use.

CCD isolation and microperfusion. Standard in vitro microperfusion methods (2) as modified in this laboratory were used (17). Briefly, rabbits were decapitated, the left kidney was quickly removed, and 1- to 2-mm slices were placed in a chilled petri dish containing the bath solution used in each protocol (Table 1) gassed with 5% CO2-95% O2. All of the dissection solutions and bath solutions contain 5% vol/vol fetal calf serum.

                              
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Table 1.   Composition of the experimental solutions

Dissection proceeded superficially from the corticomedullary junction, and an isolated CCD was transferred to a thermostatically controlled chamber (set as 37°C) where the two ends of the tubule were aspirated into the holding pipettes. The perfusing pipette was advanced 100 µm beyond the holding pipette, and the transepithelial voltage (VT or voltage) was continuously monitored by means of Ag/AgCl electrodes and a FD-223 high-impedance electrometer (World Precision Instruments, Sarasota, FL). The reported voltages represent the time-averaged voltages over the period of sample collections.

Chemicals and solutions. All chemicals were analytic grade or the highest available purity. [3H]inulin and 86Rb were obtained from New England Nuclear (Boston, MA). Sch-28080 (gift of Dr. James Kaninsky, Schering, Bloomfield, NJ) was dissolved in DMSO, and the final concentration of DMSO in perfusion solution did not exceed 0.1% (vol/vol).

The composition of the luminal perfusion and bath solutions for those experiments is listed in Table 1. The bath solutions were gassed with 5% CO2-95% O2 (pH = 7.4 ± 0.04) or 10% CO2-90% O2 (pH = 7.1 ± 0.03) for 60 min, respectively. The solution with 10% CO2 was regassed for 30 min just before use. The bath solutions were continuously exchanged at a rate of 0.64 ml/min. To facilitate comparison of the present results with the observations made from rabbits on a regular diet, the equilibration time between the 5 and 10% CO2 periods was at least 30 min. Our previous studies have shown that exposure to 10% CO2 for 30 min is sufficient to stimulate H+-K+-ATPase activity during K-replete conditions (24). All of the perfusates contained 50 µCi of [methoxy-3H]inulin exhaustively dialyzed according to the method of Schafer et al. (14) and were gassed with 5% CO2-95% O2. Effluent fluid was collected into a constant-volume pipette for measurement of volume flux (Jv), Jt CO2, and 86Rb efflux (KRb). The flow rate of the perfusate was maintained between 3.5 and 6.0 nl/min in the KRb and the Jt CO2 measurements.

Jv measurement. Jv was determined from timed collections of the effluent fluid using the equation
J<SUB>v</SUB><IT>=</IT>[<IT>V</IT><SUB>o</SUB><IT>&cjs0823;  L</IT>(cpm<SUB>o</SUB><IT>&cjs0823;  </IT>cpm<SUB>i</SUB><IT>−1</IT>)]
where Jv is the net volume absorption in nanoliters per millimeter per minute, cpmo and cpmi are the [3H]inulin counts per minute (cpm) per nanoliter in the collected and the perfused fluid, respectively, Vo is the rate of fluid collection in nanoliters per minute, and L is the tubule length in millimeters. In all experiments, the percent recovery of [3H]inulin was >95% (i.e., net volume change of fluid absorption and fluid leak was <5%). Because Jv was negligible under the conditions of these experiments, Jt CO2 was calculated assuming the rate of fluid collection was same as the rate of fluid perfusion as expressed in the following equation.

Jt CO2 measurement. Luminal acidification rate was determined as Jt CO2 (in pmol · mm-1 · min-1) by microcalorimetry and was calculated by the following formula
J<SUB>tCO<SUB>2</SUB></SUB><IT>=V</IT><SUB>o</SUB><IT>&cjs0823;  L</IT>([tCO<SUB>2</SUB>]<SUB>i</SUB><IT>−</IT>[tCO<SUB>2</SUB>]<SUB>o</SUB>)
where Vo and L have the same meanings as previously, and [tCO2]i and [tCO2]o are the perfused and collected fluid total CO2 content (tCO2) concentration (in mmol/l), respectively.

tCO2 was measured by a picapnotherm (GVH-1, World Precision Instruments, Sarasota, FL) and displayed a linear response throughout the range used. The slope of all curves exhibited a sensitivity >100 U/(meq/l) and could readily detect differences in tCO2 of 1.0 mmol/l.

86Rb efflux measurement. K absorptive flux was assessed qualitatively as the 86Rb lumen-to-bath efflux coefficient (KRb or 86Rb efflux in nm/s). 86Rb was selected because stimulation of H+-K+-ATPase activity with Rb is similar to that observed with K (3) and because these ions are handled qualitatively similarly by the CCD (16). 86Rb efflux was determined by the disappearance of 86Rb from the perfusate according to the following equation
K<SUB>Rb</SUB><IT>=2</IT>(<IT>V</IT><SUB>o</SUB><IT>&cjs0823;  L</IT>)(Rb<SUP>*</SUP><SUB>i</SUB><IT>−</IT>Rb<SUP>*</SUP><SUB>o</SUB>)<IT>&cjs0823;  </IT>(Rb<SUP>*</SUP><SUB>i</SUB><IT>+</IT>Rb<SUP>*</SUP><SUB>o</SUB>)
where Rb*i and Rb*o are the 86Rb counts per minute (cpm) per nanoliter in the perfused and collected fluid, respectively.

Counts for 3H and 86Rb were measured by a liquid scintillation counter (LS-7800, Beckman Instruments, Irvine, CA). The overlap of 86Rb counts in the 3H channel was corrected as previously described (21). Positive numbers denote net absorption, and negative numbers denote net secretion. At least three collections were obtained for measurement of Jv, Jt CO2, and KRb, respectively.

Data analysis. All the data are expressed as means ± SE. Statistical analyses were performed by paired t-test or ANOVA for repeated measures as appropriate. Post hoc comparisons were made by the Ryan-Einot-Gabriel-Welch F-test. The null hypothesis was rejected at the 0.05 level of significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of 10% CO2 on Jt CO2. Figure 1A shows the measurement of Jt CO2. Six CCD tubules isolated from K-restricted rabbits were perfused with solution A (Table 1) in both perfusate and bath (gassed with 5% CO2 as control). Jt CO2 increased ~200% when 10% CO2 was present in the bath solution (4.9 ± 2.1 pmol · mm-1 · min-1 in the 5% CO2 period; 14.7 ± 3.4 pmol · mm-1 · min-1 in the 10% CO2 period; n = 6; P < 0.01). VT was not significantly affected (2.3 ± 1.4 mV in 5% CO2 period and 1.5 ± 1.7 mV in 10% CO2 period; n = 6; Fig. 1B). The enhancement of Jt CO2 with exposure to 10% CO2 was similar to our previous observations from K-replete rabbits (24), except that the magnitude of the Jt CO2 response in the present studies is greater than that observed under the K-replete condition (see Table 2).


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Fig. 1.   Effect of 10% CO2 on total CO2 flux (Jt CO2) and transepithelial voltage (VT ) in cortical collecting duct of potassium-restricted rabbits. A: stimulation of Jt CO2 from 5 to 10% CO2 was seen in all 6 tubules (P < 0.01). B: VT was shifted to less positive or more negative in 5 of 6 tubules but not significantly (NS).


                              
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Table 2.   Comparison of exposure to 10% CO2 in K-replete and K-restricted rabbits

Effect of luminal Sch-28080 on Jt CO2. To further examine the effect of 10% CO2 ascribed to the H+-K+-ATPase, six tubules were perfused with solution A symmetrically (with the addition of the H+-K+-ATPase inhibitor Sch-28080 to the luminal solution). Figure 2A shows that 10 µM Sch-28080 completely abolished the stimulation of Jt CO2 after exposure to 10% CO2 (5.5 ± 1.0 pmol · mm-1 · min-1 in 5% CO2 period; 4.3 ± 1.5 pmol · mm-1 · min-1 in 10% CO2 period; n = 6). The change in VT was not significantly different during these experiments (5% CO2 period, -1.7 ± 2.0 mV; 10% CO2 period, -1.9 ± 3.1 mV; n = 6; Fig. 2B). These data support the assertion that stimulation of Jt CO2 on exposure to 10% CO2 is mediated by the H+-K+- ATPase.


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Fig. 2.   Stimulation of Jt CO2by 10% CO2 is dependent on an apical H+-K+-ATPase in the cortical collecting duct of K-restricted rabbits. A: stimulation of Jt CO2 by 10% CO2 was blocked by luminal Sch-28080 (10 µM). B: VT was not significantly affected.

Effect of luminal Ba on Jt CO2. Because previous investigations in CCD from K-replete rabbits have suggested that activation of the H+-K+-ATPase on exposure to 10% CO2 is dependent on an apical Ba-sensitive pathway (24), we examined whether a similar response was observed in K-restricted rabbits. Five tubules were perfused with solution A as bath and solution B as perfusate with 2 mM Ba present throughout the entire experiment. As shown in Fig. 3A, exposure to 10% CO2 markedly stimulated the Jt CO2 even in the presence of 2 mM luminal Ba (6.0 ± 1.0 pmol · mm-1 · min-1 in the 5% CO2 period; 16.8 ± 2.8 pmol · mm-1 · min-1 in the 10% CO2 period; n = 5; P < 0.01) without significantly affecting VT (-13.1 ± 8.5 mV in 5% CO2 period and -11.8 ± 7.3 mV in 10% CO2 period; Fig. 3B). The present data also show a tendency for VT in the presence of luminal Ba to be more negative than under similar condition but without luminal Ba during the 5% CO2 period (-13.1 ± 8.5 vs. 2.3 ± 1.4 mV; P = 0.08, not significant by unpaired comparison). This could be due to an inhibitory effect on apical K conductances. The same concentration of luminal Ba has been shown to make VT during the 5% CO2 period become more negative than that without luminal Ba and to inhibit the stimulation of Jt CO2 by 10% CO2 under K-replete conditions (24).


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Fig. 3.   Stimulation of Jt CO2by 10%CO2 is not dependent on an apical Ba-sensitive pathway during K restriction. A: 2 mM luminal Ba did not inhibit 10% CO2-stimulated increase in Jt CO2 (P < 0.01). B: VT was not significantly affected.

These data demonstrate clear differences from K-replete animals and suggest that the stimulation of Jt CO2 during K-restriction conditions is independent of an apical Ba-sensitive pathway. Moreover, the magnitude of Jt CO2 stimulation in the present studies is also significantly greater than that observed under the K-replete condition. We have summarized the present findings as well as the previous observations in Table 2 for comparison.

Effect of bath Ba on Jt CO2. The previous observations raise the possibility that activation of the H+-K+- ATPase by 10% CO2 during K restriction may be dependent on a basolateral Ba-sensitive pathway, because the apical Ba-sensitive pathway seems not be involved in this condition. To test this hypothesis, six tubules were perfused with solution B containing 2 mM Ba in the bath and solution C as perfusate. As shown in Fig. 4A, peritubular application of 2 mM Ba fully inhibited the stimulation of Jt CO2 after exposure to 10% CO2 (3.4 ± 1.2 pmol · mm-1 · min-1 in 5% CO2 period; 1.8 ± 1.0 pmol · mm-1 · min-1 in 10% CO2 period; n = 7). VT was not significantly altered during the experiments (5% CO2 period, 3.5 ± 1.5 mV; 10% CO2 period, 3.3 ± 1.2 mV; n = 7). These data suggest that the effect of 10% CO2 on acidification is dependent on a basolateral Ba-sensitive pathway.


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Fig. 4.   Stimulation of Jt CO2by 10% CO2 was blocked by 2 mM bath Ba (evidence for basolateral Ba-sensitive pathway). A: in 3 tubules Jt CO2 exhibited a small increase and in 4 Jt CO2 decreased. Although the average Jt CO2 decreased, the effect was not significant. B: before and after exposure of 10% CO2, VT was largely unchanged.

86Rb efflux response to 10% CO2 and effect of Sch-28080 on 86Rb efflux. Because 86Rb trace efflux is a qualitative marker of K efflux, we examined whether exposure to 10% CO2 increased KRb from lumen to bath and whether H+-K+-ATPase mediated the effect of 10% CO2 on KRb in CCD of K-restricted rabbits. The tubules were perfused with symmetrical solution A containing either vehicle, 0.1% DMSO, or 10 µM Sch-28080 in the luminal perfusate.

As shown in Fig. 5A, 10% CO2 substantially increased KRb from 93.1 ± 23.8 to 249 ± 60.2 nm/s (n = 7; P < 0.05). The stimulation of KRb by 10% CO2 is consistent with an increase of K efflux from lumen to bath.


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Fig. 5.   Effect of 10% CO2 on stimulation of 86Rb tracer efflux [86Rb lumen-to-bath efflux coefficient (KRb)]. 86Rb was used as a qualitative marker for measurement of K ion flux from lumen to bath. A: in all 7 tubules KRb increased after exposure to 10% CO2 (P < 0.05). B: VT was shifted to less negative (P < 0.05). C: comparison of effect of VT change on KRb vs. the measurement of KRb.

Concomitantly, the VT became less negative, from -2.4 ± 1.3 mV in 5% CO2 period to -1.2 ± 0.9 mV in 10% CO2 period (n = 7; P < 0.05; Fig. 5B). Although the effect on VT was small, the less negative or lumen-positive voltage could enhance passive 86Rb efflux through the paracellular pathway. To examine whether the changes in VT accounted for the effect of 10% CO2 on 86Rb efflux, we calculated the increase in 86Rb efflux resulting from the change in VT using the Goldman flux equation. The increased KRb as predicted by the change in VT was only 10.6 ± 3.7 nm/s, whereas an increment of 156 ± 58.4 nm/s after 10% CO2 was observed (Fig. 5C). Thus changes in VT had a very small effect on the increase in KRb and could not account for the enhancement of KRb after exposure to 10% CO2 observed in the experiments.

Figure 6A shows that luminal Sch-28080 abolished the stimulation of 10% CO2 on KRb (76.4 ± 15.1 nm/s in 5% CO2 period; 76.8 ± 13.3 nm/s in 10% CO2 period; n = 5) but did not abolish the voltage response to 10% CO2 (-14.6 ± 9.6 mV in 5% CO2 period; -10.7 ± 9.1 mV in 10% CO2 period; n = 5; P < 0.01; Fig. 6B). The effect of Sch-28080 to block KRb stimulation suggests that an H+-K+-ATPase contributes to the enhancement of Rb efflux by 10% CO2 and is in agreement with the effect of Sch-28080 on Jt CO2 (see Fig. 2). The basal VT in this group of experiments was more negative, but not significantly, than that in other groups and it was due to the variance among tubules in this group.


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Fig. 6.   Stimulation of KRb by 10% CO2 was inhibited by Sch-28080. A: the measurement was under the same conditions as in the Fig. 5 except 10 µM Sch-28080 was present in the lumen. The change in KRb is not significant. This is further evidence for activation of luminal H+-K+-ATPase in the stimulation of 10% CO2 and is in agreement with Fig. 2A. B: VT was shifted to less negative (P < 0.01).

Effect of bath Ba on 86Rb efflux. The purpose of the next experiment was to examine whether a basolateral Ba-sensitive pathway of CCD mediates the stimulation of KRb after exposure to 10% CO2 in K-restricted rabbits. In six experiments the CCD was perfused with solution B containing 3 mM Ba as the bath and solution C as the perfusate. Figure 7A shows that Ba totally abolished the stimulation of KRb by 10% CO2 (73.0 ± 8.2 nm/s in 5% CO2 period; 70.9 ± 8.3 nm/s in 5% CO2 period; n = 6). The present observations indicate that the effect of 10% CO2 on KRb by the CCD from K-restricted rabbits is different from that observed in the CCD of rabbits exposed to a normal diet (24) and is in agreement with the data on Jt CO2 presented previously (see Figs. 1 and 4). Thus a Ba-sensitive pathway appears to be present in the basolateral membrane of the rabbit CCD during K restriction, and this pathway mediates the stimulatory effect of 10% CO2 on KRb. The VT became slightly less lumen-negative, but the effect was not significant (from -2.2 ± 3.0 mV in 5% CO2 period to -0.0 ± 1.7 mV in 10% CO2 period) (n = 6; P = not significant; Fig. 7B).


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Fig. 7.   Stimulation of KRb by 10% CO2 was blocked by bath Ba. A: 3 mM bath Ba prevented the increase of KRb in 5 of 6 tubules. This is further evidence for the participation of a basolateral Ba-sensitive pathway in the stimulation by 10% CO2 and is in agreement with Fig. 4A. B: VT was shifted to less negative values, but the effect was not significant.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

H+-K+-ATPase appears to be primarily responsible for enhanced luminal acidification after exposure to 10% CO2 under normal conditions (24). Apical K absorption by H+-K+-ATPase recycles at the luminal membrane, but during K restriction this enzyme is responsible for both H secretion and K absorption, as demonstrated by our previous (19, 22, 23) and present studies.

Our previous studies (24) from K-replete rabbits have shown that exposure to 10% CO2 profoundly enhanced total CO2 flux of the CCD, and this enhancement was totally blocked by luminal application of either Sch-28080 or Ba. In contrast, exposure to 10% CO2 failed to stimulate Rb efflux by the CCD. These data suggest that stimulation of total CO2 flux on exposure to 10% CO2 is dependent on both the apical H+-K+-ATPase and the apical Ba-sensitive pathway, which leads to our proposal that apical K absorption via the H+-K+-ATPase recycles at the apical membrane via a Ba-sensitive exit mechanism (24). However, the results are quite different in the present studies performed in this segment from rabbits maintained on a K-restricted diet.

First, the magnitude of stimulation of total CO2 flux by exposure to 10% CO2 under K-restricted conditions is significantly greater than that observed under normal conditions (9.8 ± 1.7 vs. 3.5 ± 0.6 pmol · mm-1 · min-1) (see Table 2). This observation is consistent with the enzymatic data that demonstrate increased K-dependent ATP hydrolysis in the permeablized CCD after exposure to a K-deprived diet (3, 4).

Second, although exposure to 10% CO2 dramatically augmented the Sch-28080-inhibitable total CO2 flux (Figs. 1A and 2A), this effect does not rely on the apical Ba-sensitive pathway because luminal Ba failed to alter the response of the CCD to 10% CO2 (Fig. 3A). On the other hand, this Jt CO2does depend on the basolateral Ba-sensitive mechanism, because peritubular Ba completely abolished the stimulation of total CO2 after exposure to 10% CO2 (Fig. 4A).

Third, exposure to 10% CO2 substantially increased the Sch-28080-sensitive 86Rb efflux (Figs. 5A and 6A), and this effect was completely inhibited by peritubular application of Ba (Fig. 7A). However, our previous investigations of K-restricted animals showed that in the presence of luminal Ba, Sch-28080-sensitive 86Rb efflux could still be enhanced (23). These data are consistent with a model in which Rb/K that enters the cell via the apical H+-K+-ATPase under 10% CO2 stimulation exits the cell via a basolateral Ba-sensitive pathway.

Fourth, in our present study, VT did not significantly change from the 5% CO2 period to the 10% CO2 period in the K-restricted rabbits (Fig. 1B). In contrast, CCD in the normal-K-diet rabbits became significantly less lumen negative in the 10% CO2 period (see Table 2). If the differences in K-restricted rabbits and in normal-K-diet rabbits suggest a K-conserving role for the former and a K-recycling role for the later, we should see a more negative VT during 10% CO2 stimulation in the K-restricted rabbits. It is possible that a parallel Cl current was also activated in the basolateral membrane during the 10% CO2 period, which could mitigate the change in VT. The VT in the 5% CO2 period in the presence of luminal Ba was more negative than that without luminal Ba (see Fig. 3B vs. 1B). In our previous experiments with animals in K repletion, we observed a similar effect of luminal Ba on VT (Table 2), although the magnitude of the effect appeared greater, consistent with a greater apical K conductance in K-replete animals.

In the experiments that examined the effect of 10% CO2 on 86Rb efflux, VT became more lumen positive (or less negative) regardless of the absence or presence of luminal Sch-28080 (see Figs. 5B and 6B). The exact reason for VT changes in these experiments is not known. This may reflect differences in the electrogenic Na absorption among the tubules and the sensitivity of this process to 10% CO2, but the present study does not separate these effects. The theoretical estimation of the effect of the VT change on 86Rb efflux is <10% of the total measured enhancement (Fig. 5C). In addition, Sch-28080 completely inhibited the effect of 10% CO2 on KRb (Fig. 6A), which also provides evidence that the H+-K+-ATPase, not the change in voltage, mediates enhancement of 86Rb efflux on exposure to 10% CO2.

Our previous investigations of K-restricted animals showed that removal of luminal Na could enhance 86Rb efflux, and the enhancement was inhibited by the luminal application of the H+-K+-ATPase inhibitor Sch-28080, but not by luminal Ba (23). This indicates that luminal Ba does not directly inhibit the action of the H+-K+-ATPase. Our previous studies from K-replete animals showed that 86Rb efflux was actually decreased in response to 10% CO2 and this decrease was abolished when apical Ba was present (24). Our present data from K-restricted animals suggest that under 10% CO2, a Ba-sensitive K exit pathway is present in the basolateral membrane of the CCD, and this pathway participates in Rb/K absorption. Thus the basolateral K exit mechanisms appear to play a critical role in differentiating the response to 10% CO2 under K-restricted conditions from that observed under K-replete conditions.

Several studies have shown that the basolateral membrane of rat CCD has Ba-sensitive K conductances (6, 15). Similar Ba-sensitive K conductances were also revealed in the basolateral membrane of rabbit CCD (8, 13). It is possible that blockade of K channels increases the intracellular K activity, which secondarily inhibits the H+-K+-ATPase and thus Jt CO2 (see Fig. 4A) (7, 21). However, Ba may not only affect K channels. For example, Ba also inhibits a K-Cl cotransporter as demonstrated by Greger and Schlatter (5) in the thick ascending limb of Henle's loop. Therefore, future investigation is needed to understand the exact mechanisms of basolateral K exit under stimulation of 10% CO2.

Although the present study has focused on the H+-K+-ATPase stimulation by 10% CO2, our data show that basolateral Ba had a tendency to inhibit Rb efflux, but not significantly with 5% CO2 (unpaired comparison of Figs. 5A and 7A). Because we have not formally examined the quantitative contribution of this basolateral Ba-sensitive mechanism to transepithelial 86Rb efflux, we cannot exclude a significant effect in paired studies of Ba on KRb with 5% CO2.

Our data suggest that apical K absorption via an apical H+-K+-ATPase may exit the cell through either an apical K secretory mechanism or a basolateral K- absorptive mechanism, depending on total body K homeostasis. We propose a cell model showing the coupling of an apical membrane H+-K+-ATPase and a K exit during cellular acidification in K-replete and K-restricted conditions (Fig. 8). This model is based primarily on the differences observed from previous K-replete (24) and present K-restricted rabbits with respect to exposure to 10% CO2. Neither the present study nor evidence in the literature allows unambiguous distinction of the cell types involved in the response to 10% CO2. Some recent data suggest that H+-K+-ATPase alpha 1-subunit is expressed in principal cells (alpha 1 mRNA) (1), although antibody studies localize the H+-K+-ATPase alpha 1-subunit to the apical membrane of intercalated cells (19). The possibility that both cell types respond cannot be excluded.


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Fig. 8.   A cell model of cooperation between H+-K+-ATPase-mediated acidification and Ba-sensitive K exit pathways in the cortical collecting duct (CCD). Proposed model of regulating K homeostasis is based on the observations from microperfusion experiments in normal-K-diet (K repletion) and in low-K-diet (K restriction) rabbits during 10% CO2 stimulation. Only H+-K+-ATPase-mediated processes measured in previous studies (23, 24) and in this paper are illustrated.

In summary, we have shown that during K restriction exposure to 10% CO2 substantially increased the Sch-28080-sensitive JtCO2 even in the presence of luminal Ba and this effect was abolished by peritubular application of Ba. In addition, exposure to 10% CO2 profoundly stimulated 86Rb efflux by the CCD from the K-restricted rabbit, and this stimulation was inhibited not only by luminal application of Sch-28080 but also by peritubular application of Ba. These studies demonstrate that enhancement of H+-K+-ATPase-mediated acidification by 10% CO2 during K restriction is not dependent on an apical Ba-sensitive pathway but rather dependent on a basolateral Ba-sensitive pathway.


    ACKNOWLEDGEMENTS

The authors thank Wei L. Ueberschaer for technical assistance in some experiments reported in this manuscript.


    FOOTNOTES

These studies were supported by funds from the Medical Research Service of the Department of Veterans Affairs and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49750.

Present address of X. Zhou: Dept. of Medicine, Uniformed Services Univ. of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799.

Address for reprint requests and other correspondence: C. S. Wingo, Nephrology and Hypertension (111G), Veterans Affairs Medical Center, Gainesville, FL 32608-1197.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 8 June 1999; accepted in final form 29 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 279(1):F153-F160
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