Increased CO2 stimulates K/Rb reabsorption mediated
by H-K-ATPase in CCD of potassium-restricted rabbit
Xiaoming
Zhou,
Suguru
Nakamura,
Shen-Ling
Xia, and
Charles S.
Wingo
Division of Nephrology, Hypertension and Transplantation,
Department of Medicine, College of Medicine, University of Florida,
and Nephrology Section, Veterans Affairs Medical Center,
Gainesville, Florida 32608-1197
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ABSTRACT |
Apical
H-K-ATPase in the cortical collecting duct (CCD) plays an important
role in urinary acidification and K reabsorption. Our previous studies
demonstrated that an H-K-ATPase mediates, in part, Rb reabsorption in
rabbit CCD (Zhou X and Wingo CS. Am J Physiol Renal Fluid
Electrolyte Physiol 263: F1134-F1141, 1992). The purpose of
these experiments was to examine using in vitro microperfused CCD from
K-restricted rabbits 1) whether an acute increase in
PCO2 and, presumably, intracellular acidosis
stimulate K absorptive flux; and 2) whether this stimulation
was dependent on the presence of a functional H-K-ATPase. Rb
reabsorption was significantly increased after exposure to 10%
CO2 in CCD, and this effect was persistent for the entire
10% CO2 period, whereas 10 µM SCH-28080 in the perfusate
totally abolished the stimulation of Rb reabsorption by 10%
CO2. After stimulation of Rb reabsorption by 10%
CO2, subsequent addition of 0.1 mM methazolamide, an
inhibitor of carbonic anhydrase, failed to affect Rb reabsorption.
However, simultaneous exposure to 10% CO2 and
methazolamide prevented the stimulation of Rb reabsorption. Treatment
with the intracellular calcium chelator MAPTAM (0.5 µM) inhibited the
stimulation of Rb reabsorption by 10% CO2. Similar
inhibition was also observed in the presence of either a calmodulin
inhibitor, W-7 (0.5 µM), or colchicine (0.5 mM), an inhibitor of
tubulin polymerization. In time control studies, the perfusion time did
not significantly affect Rb reabsorption. We conclude the following:
1) stimulation of Rb reabsorption on exposure to 10%
CO2 is dependent on the presence of a functional H-K-ATPase
and appears to be regulated in part by the insertion of this enzyme
into the apical plasma membrane by exocytosis; 2) insertion
of H-K-ATPase requires changes in intracellular pH and needs a basal
level of intracellular calcium concentration; and 3)
H-K-ATPase insertion occurs by a microtubule-dependent process.
exocytosis; intracellular pH; calcium; calmodulin; microtubules; cortical collecting duct
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INTRODUCTION |
IN THE RENAL COLLECTING
DUCT, three distinct pathways of K/Rb reabsorption have been
identified: a Ba-sensitive K-conductive pathway due to Ba-sensitive K
channels, an electroneutral pathway due to an H-K-ATPase, and the
paracellular pathway (45). Previous studies have shown
that 10% CO2 stimulates luminal acidification via an
apical SCH-28080-sensitive H-K-ATPase under K-replete conditions (44). K restriction increased an apical H-K-ATPase
activity of the microperfused collecting duct in rabbit (15,
38). Functional studies have demonstrated that 10%
CO2 increases Rb absorption in the colon, which is mediated
by a related K-absorbing pump (27), and an acute increase
in ambient PCO2 in vitro enhances the rate of
luminal acidification in the collecting duct (22). The
role of ambient PCO2 in the control of
exocytosis has been studied extensively in other acid-transporting
epithelia. In particular, the turtle urinary bladder has many transport
characteristics similar to those of the collecting duct
(4). Acetazolamide has been shown to alkalinize
intracellular pH (pHi) in the turtle urinary bladder
(4), and exposure to CO2 also increases H
secretion in this epithelia. This effect of CO2 on luminal
acidification appears dependent on the exocytosis of vesicles
containing proton pumps at the apical membrane. Exocytosis may be an
important mechanism for regulation of both proton pumps and has been
proposed to occur by the following sequence: 1) exposure to
CO2 decreases pHi, and the rate of
pHi reduction is affected by the presence of carbonic anhydrase; 2) rapid alterations in pHi increase
intracellular free Ca2+ concentration
([Ca2+]i), which accelerates the organization
of the cytoskeleton; and 3) cytoskeleton rearrangement is
dependent on calmodulin and results in the translocation of
tubulovesicles from the cytoplasm to the apical membrane (4, 30,
32, 33, 35). These observations suggest that an intracellular
acid-base balance affects the activity of this enzyme and prompted us
to speculate that increased CO2 might also stimulate K
absorption by the cortical collecting duct (CCD) during K-restricted
conditions, which could be attributable to enhanced H-K-ATPase
activity. The purpose of this study was to examine whether increased
CO2 induced by exposure to 10% CO2 affects
H-K-ATPase-dependent K absorption of the CCD in animals adapted to a
low-K diet (K-restricted animals). In addition, we examined possible
intracellular mechanisms that could explain this effect. Rb
reabsorption was used as a qualitative measure of K reabsorption, and
rabbits were adapted to K-restricted conditions to enhance the
component of Rb/K reabsorption mediated by H-K-ATPase.
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MATERIALS AND METHODS |
In vivo conditions.
Female New Zealand White rabbits were maintained on a K-restricted diet
(0.25% K, TD 87433, Teklad, Madison, WI) for at least 4 days before
experimentation and allowed free access to tap water.
In vitro methods.
Standard in vitro microperfusion methods (2) as modified
in this laboratory were used (38, 43). Briefly, rabbits
were decapitated, the left kidney was quickly removed, and 1- to 2-mm slices were placed in a chilled petri dish containing Ringer
bicarbonate solution [(in mM) 145 Na, 5 K, 112 Cl, 25 HCO3, 1.8 Ca, 2.3 PO4, 1.0 Mg, 1.0 SO4, 10 acetate, 8 glucose, and 5 alanine] gassed with 5%
CO2-95% O2 with 5% vol/vol fetal calf serum.
Dissection proceeded superficially from the corticomedullary junction.
Tubules were transferred to a thermostatically controlled chamber
(37°C), and the two ends of the tubule were aspirated into the
holding pipettes. The perfusing pipette was advanced 100 µm beyond
the holding pipette. The composition of the bath solution was identical
to that of dissection solution and was gassed with 5%
CO2-95% O2 (pH = 7.4 ± 0.04) or
10% CO2-90% O2 (pH = 7.1 ± 0.03)
as appropriate. The bath solution was continuously exchanged at a rate
of 0.64 ml/min. The equilibration time between two periods was 30 min
unless otherwise indicated. The electrolyte composition of the
perfusate was identical to that of the bath and was gassed with 5%
CO2-95% O2. The perfusate contained 50 µCi
of [methoxy-3H]inulin exhaustively dialyzed according to
the method of Schafer et al. (28). The flow rate of the
perfusate was maintained between 4 and 6 nl/min. Effluent fluid was
collected into a constant-volume pipette for measurement of volume and
isotopic flux.
Volume flux was determined from timed collections of the effluent fluid
by using the equation Jv = (cpmo/cpmi
1) · Vo/L, where
Jv is the net volume absorption in nanoliters
per millimeter per minute; cpmo and cpmi are
the [3H]inulin counts per min 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% or the experimental
result was discarded.
K absorptive flux was assessed as the 86Rb lumen-to-bath
efflux coefficient (KRb or Rb reabsorption).
Isotopic 86Rb was used as a tracer marker for measurement
of K reabsorption because stimulation of K-ATPase activity with Rb is
similar to that observed with K (5), and there is no
evidence that the two ions are transported by different pathways
(37). 86Rb reabsorption was determined by the
disappearance of 86Rb from the perfusate according to the
following equation
where Rb
and Rb
are the
86Rb counts per minute per nanoliter in the perfused and
collected fluid, respectively. At least three and generally four
collections were obtained for measurement of Rb reabsorption. Counts
for 3H and 86Rb were measured by an LS-7800
liquid scintillation counter (Beckman Instruments, Irvine, CA). The
overlap of 86Rb counts in the 3H channel was
corrected as previously described (43).
The [Ca2+]i of CCD cells was measured with
fluo-4 and Fura Red using a confocal fluorescence system (Laser
Scanning Module; LSM 510, Zeiss, Thornwood, NY). Tubules were loaded
with Ringer solution containing 10 µM fluo-4 acetoxymethyl ester
(Molecular Probes, Eugene, OR) by 60-min incubation (either loaded from
lumen or from bath), then washed at least 15 min by the Ringer
solution. To prevent possible artifacts by the use of single-wavelength fluorescent studies, we used the fluorescent indicator Fura Red (15 µM) to coload cells at the same time, so that a ratio signal could be
measured (16). The dyes were dissolved in DMSO, and the
final concentration of DMSO in the loading solution was always <0.25%
(vol/vol).
Fluorescence measurements were carried out with the fluar ×20
(numerical aperture = 0.75) and plan-neo ×40 (numerical
aperture = 0.75) objectives with an inverted microscope (Axiovert
100M, Zeiss). Images were obtained at a rate of 1 image/10 s and stored onto a hard disk as 515 × 512 pixels for further analysis.
Windows NT-based Zeiss LSM image-analysis software was used to
calculate the fluorescent intensities and plot the time courses.
All chemicals were analytical grade or of the highest available purity.
[3H]inulin and 86Rb were obtained from New
England Nuclear (Boston, MA). SCH-28080 (graciously supplied by Dr.
James J. Kaminski, Schering, Bloomfield, NJ) was dissolved in DMSO and
applied to the perfusate with a final concentration of 10 µM. The
final concentration of DMSO was 0.1% (vol/vol), and this concentration
has been previously demonstrated not to affect Rb efflux
(44). Methazolamide (American Cyanamide, Pearl River, WY),
bis-(2-amino-5-methyl-phenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM; Sigma, St. Louis, MO), and
N-(6-aminohexyl)-5-choloro-L-naphthalene-sulfonamide
(W-7, Sigma) were also dissolved in DMSO and applied to the bath with the final concentration of 0.1 mM, 0.5 µM, and 0.5 µM,
respectively. Colchicine (Sigma) was dissolved in a 154 mM NaCl
solution and applied to the bath with a final concentration of 0.5 mM.
Data are expressed as means ± SE. Statistical analyses were
performed by ANOVA for repeated measures or paired t-test 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.
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RESULTS |
To examine whether an acute increase in
PCO2 stimulates K absorptive flux, we perfused
the CCD with symmetrical Ringer bicarbonate solution gassed with 5%
CO2 (5% CO2 period) or 10% CO2
(10% CO2 period). After measurement of the basal rate of
Rb reabsorption (5% CO2 period), effluent fluid was
collected from 30 to 120 min after exposure to 10% CO2.
Exposure to 10% CO2 decreases pHi (29, 35), which could provide a greater concentration of protons (or,
more precisely, hydronium ions) as a substrate for the H-K-ATPase. The
rate of reduction of pHi is dependent on the activity of
carbonic anhydrase that catalyzes the hydration of CO2 to
carbonic acid (8, 10, 20). To evaluate the role of
carbonic anhydrase on the stimulation of Rb reabsorption by 10%
CO2, 0.1 mM methazolamide, an inhibitor of this enzyme, was
added to the bath from 120 to 180 min after exposure to 10%
CO2. As shown in Fig. 1,
exposure to 10% CO2 significantly stimulated Rb efflux,
and this effect was persistent for the entire 10% CO2
period. The subsequent addition of methazolamide did not significantly
inhibit KRb (72.4 ± 11.8, 5%
CO2 period; 121 ± 29.9, 135 ± 34.3, and
135 ± 41.3 nm/s at 30-60, 60-90, and 90-120 min
after exposure to 10% CO2, respectively; 133 ± 29.0 nm/s, methazolamide period, n = 6). To examine whether the H-K-ATPase contributes to the effect of 10% CO2 on Rb
reabsorption, we repeated the experiments under conditions identical to
those described above (Fig. 1) except for the presence of luminal 10 µM SCH-28080, a specific inhibitor of H-K-ATPase. SCH-28080 totally blocked the stimulation of Rb reabsorption in response to 10% CO2, and methazolamide had no significant effect on Rb
reabsorption under these conditions (90.8 ± 16.5, 5%
CO2 period; 80.5 ± 12.3, 83.9 ± 14.3, and
78.7 ± 10.9 nm/s at 30-60, 60-90, and 90-120 min
after exposure to 10% CO2, respectively; 81.9 ± 13.5 nm/s, methazolamide period, n = 7, Fig.
2). Thus H-K-ATPase appears to mediate
the stimulatory effect of 10% CO2 on
KRb. The lack of inhibitory effect of
methazolamide on KRb after exposure to 10%
CO2 implies that carbonic anhydrase is not necessary to
maintain the activation of H-K-ATPase by 10% CO2. To
examine whether carbonic anhydrase was required for initiating the
stimulation of KRb by 10% CO2, we
simultaneously exposed the CCD to methazolamide and 10%
CO2 after measurement of the basal rate of
KRb. Under these conditions, 10%
CO2 did not stimulate Rb reabsorption (98.6 ± 14.1 vs. 86.2 ± 16.5 nm/s, n = 6, Fig.
3). Time control experiments demonstrated
that perfusion time did not significantly affect KRb (Table 1).
These data suggest that carbonic anhydrase is necessary for initiating
the activation of H-K-ATPase on exposure to 10% CO2. To
examine whether methazolamide inhibited the basal rate of Rb
reabsorption, we perfused the CCD in the presence of 5%
CO2 throughout the experiment and added methazolamide
during the second period. As shown in Fig.
4, methazolamide did not significantly inhibit KRb (60.4 ± 10.1 vs. 59.1 ± 10.6 nm/s, n = 6). Because our previous studies
demonstrated that H-K-ATPase contributed to the Rb reabsorption when
the CCD was perfused in the presence of 5% CO2
(43), it appears that methazolamide has no significant effect on basal H-K-ATPase activity.

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Fig. 1.
Effect of 10% CO2 and 0.1 mM methazolamide in the
absence of SCH-28080 on the 86Rb lumen-to-bath efflux
coefficient (KRb; n = 6).
*P < 0.05 compared with the 5% CO2 period
(ANOVA).
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Fig. 2.
Effect of 10% CO2 and 0.1 mM methazolamide in the
presence of SCH-28080 on KRb (n = 7; ANOVA). None of the means of the 10% CO2 periods were
significantly different from those of the 5% CO2 period.
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Fig. 3.
Effect of simultaneous exposure to 10% CO2
and 0.1 mM methazolamide on KRb [n = 6;
not significant (NS) by paired t-test].
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Fig. 4.
Effect of 0.1 mM methazolamide on
KRb. The entire experiment was conducted in the
presence of 5% CO2 (n = 6; NS by paired
t-test).
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The time frame for the stimulation of Rb efflux on exposure to 10%
CO2 suggests that existing H-K- ATPase pump units
contribute to this effect. This could involve activation of previously
inactive H-K-ATPase located at the apical plasma membrane or
translocation of H-K-ATPase by the insertion of the enzyme to the
apical membrane of the CCD via exocytosis, or a combination of both
mechanisms. To further test the insertion hypothesis, we examined the
effects of several agents known to block the insertion cascade on
Rb reabsorption after exposure to 10% CO2. First,
the CCD was perfused in the presence of 0.5 µM MAPTAM, a
cell-permeable intracellular Ca2+ chelator, throughout the
experiment. In this case, 10% CO2 failed to stimulate Rb
reabsorption (89.5 ± 19.5 vs. 85.6 ± 23.6 nm/s, n = 6, Fig. 5). To assess
whether 10% CO2 increased
[Ca2+]i, we perfused the CCD and measured
[Ca2+]i by using a confocal fluorescence
system with fluo-4 and Fura Red. [Ca2+]i
failed to increase during the exposure to 10% CO2
(5-15 min; n = 44 cells/3 tubules). However, a
decrease in [Ca2+]i was observed by adding 5 mM EGTA to the bath solution containing 1.5 mM Ca2+. These
observations suggest that basal-level [Ca2+]i
is critical for 10% CO2 to stimulate
KRb. Second, to evaluate the role of calmodulin
in the stimulation of KRb by 10%
CO2, the CCD was perfused in the presence of 0.5 µM W-7,
an inhibitor of calmodulin, throughout the experiment. W-7 completely
abolished the stimulation of 10% CO2 on Rb reabsorption.
In fact, Rb reabsorption was significantly reduced during the 10%
CO2 period (89.3 ± 14.0 vs. 65.1 ± 13.0 nm/s,
P < 0.05, n = 6, Fig.
6). These observations indicate a
critical role for calmodulin in the activation of
KRb by 10% CO2. Third, to examine
whether microtubules were involved in the effect of 10%
CO2, the CCD was simultaneously exposed to 10%
CO2 and 0.5 mM colchicine, an inhibitor of tubulin
polymerization. Colchicine totally blocked the stimulatory effect of
10% CO2 on Rb reabsorption (85.0 ± 15.0 vs.
81.3 ± 13.6 nm/s, n = 6, Fig. 7). These data indicate a requirement for
an intact cytoskeletal system in the activation of
KRb by 10% CO2. In contrast, after stimulation of KRb by 10% CO2,
subsequent addition of colchicine had no inhibitory effect on Rb
reabsorption (72.5 ± 7.9 nm/s, 5% CO2 period;
97.3 ± 10.1 nm/s, 10% CO2 period; and 110 ± 13.2 nm/s, 10% CO2+colchicine period, n = 6, Fig. 8).

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Fig. 5.
Effect of 10% CO2 in the presence of 0.5 µM
bis-(2-amino-5-methyl-phenoxy)-ethane-N,N,N',N'-tetraacetic
acid tetraacetoxymethyl ester (MAPTAM) throughout the experiment on
KRb (NS by paired t-test).
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Fig. 6.
Effect of 10% CO2 on
KRb in the presence of 0.5 µM
N-(6-aminohexyl)-5-choloro-L-naphthalene-sulfonamide
(W-7) throughout the experiment (paired t-test).
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Fig. 8.
Effect of 0.5 mM colchicine on KRb
after prior stimulation of KRb by 10%
CO2. *P < 0.05 compared with the 5%
CO2 period (ANOVA).
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DISCUSSION |
In renal collecting ducts, K reabsorption occurs in the
intercalated cells of CCD and outer medullary collecting duct (OMCD) and is mediated by H-K- ATPase pumps in the luminal membrane that reabsorb K and secrete H. The activity of these pumps is increased by
hypokalemia (15, 25, 38). The renal response to K
depletion (KD) is sufficiently effective that a low-K diet will not
lead to significant K losses. Microperfusion studies have demonstrated a K-dependent, SCH-28080-sensitive, acid-secreting pathway in the
collecting ducts of normal and KD animals (15, 23-25,
36). Previous functional studies have demonstrated H-K-ATPase
sensitivity to SCH-28080 in KD rats and rabbits (1, 15, 23,
25). On the basis of the inhibitory profile of H-K-ATPase
activity in KD animals by SCH-28080, it has been presumed that the
gastric H-K-ATPase is the isoform responsible for increased activity
(15, 41). However, colonic H-K-ATPase (HK
2)
is induced and mediates increased bicarbonate reabsorption in OMCD and
inner medullary collecting duct (IMCD) of KD rats (23,
25). Recent studies show that, in KD, an H-K-ATPase activity (as
assayed by ATP hydrolysis) is induced in OMCD and CCD and is sensitive
to both ouabain and SCH-28080 (1). HK
2 is
sensitive to both ouabain and SCH-28080 in KD (1, 25) and
is likely responsible for increased bicarbonate and K reabsorption in
collecting ducts. As such, this isoform appears to play an important
role in the maintenance of metabolic alkalosis in KD. Our results
provide direct evidence that increased CO2 stimulates
K-absorptive flux, assessed as the KRb or Rb
efflux, in the CCD of rabbits under K-restricted conditions. Moreover, SCH-28080 inhibited the stimulation of Rb efflux by 10%
CO2, suggesting that this enhanced
KRb is mediated by an H-K-ATPase. The rapid response to 10% CO2 and the effects of various maneuvers
designed to inhibit exocytosis suggest that the stimulus increases the number of functional H-K-ATPase units in the apical membrane. In the
gastric gland, exocytosis of H-K-ATPase to the canalicular (apical)
membrane occurs in response to several stimuli (11, 32).
We propose that the stimulation of Rb reabsorption on exposure to 10%
CO2 is dependent on the exocytotic insertion of H-K-ATPase into the apical membrane of the collecting duct. Three different lines
of evidence are consistent with this hypothesis. First, the present
studies show that maneuvers designed to prevent exocytosis also block
the stimulatory effect of 10% CO2 on Rb reabsorption (see
Figs. 5-7). Second, H-K-ATPase has been localized by
immunocytochemistry to the intercalated cell in the CCD and OMCD
(40), and inhibition of this enzyme by SCH-28080 blocks
the effect of 10% CO2 on KRb (42). Third, alterations in ambient
PCO2 stimulate the translocation of
tubulovesicles to the luminal membrane in the CCD (22,
29). Respiratory acidosis increases the surface density of the
apical membrane of intercalated cells concomitantly with a decrease in the number of tubulovesicular profiles in the apical region, but no
such finding occurred in the principal cell (18). These
findings have been taken as evidence that increased CO2
stimulates the fusion of tubulovesicular profiles to the apical
membrane (18). In addition, experiments using fluorescent
dextran demonstrate that CO2 stimulated the exocytotic
fusion of vesicles with the apical membrane in the intercalated cell of
rabbit collecting duct (29). Taken together, these studies
indicate that 10% CO2 stimulates exocytosis both in vivo
and in vitro.
Functional studies have demonstrated that an increase in
CO2 causes a decrease in pHi in ventricular
myocytes (14) and in the perfused CCD (I. D. Weiner,
personal communication). The decrease in pHi stimulates the
exocytotic insertion of H pumps into the apical membrane of the turtle
urinary bladder tissue (4, 30, 35). Consistent with the
present observations, simultaneous exposure to 10% CO2 and
methazolamide inhibited the stimulatory effect of 10% CO2
on KRb (Fig. 3), whereas after 10%
CO2 had increased Rb reabsorption, the subsequent addition
of methazolamide had no detectable effect on KRb
(Fig. 1). Previous studies have demonstrated that methazolamide
dramatically reduced the initial rate of acidification produced by the
carbonic anhydrase-catalyzed hydration of CO2 (26). This may explain the lack of the late effect on Rb
efflux by the addition of methazolamide after the CCD was acidified. In
addition, our previous studies suggest that the H-K-ATPase contributes
to Rb reabsorption in the basal (5% CO2) state
(43), and our present studies demonstrate that
methazolamide does not significantly affect KRb
when the CCD is perfused in the presence of 5% CO2
throughout the experiment (Fig. 4). These data support the hypothesis
that, after H-K-ATPase is inserted into the apical membrane,
methazolamide does not significantly inhibit the activity of this
enzyme. Therefore, methazolamide inhibition of H-K-ATPase appears to be
indirect, and this effect is mediated by inhibition of exocytosis,
which is pH dependent. The proton or hydronium ion source for the
H-K-ATPase after the administration of methazolamide is an
additional issue that is not addressed by these studies. The hydrolysis
of ATP may supply protons for the pump, as suggested for the gastric
H-K-ATPase (17). Alternatively, the noncatalyzed hydration
of CO2 (20) may in part provide protons for
apical secretion.
[Ca2+]i concentration is responsible for a
variety of cellular events. Many Ca2+ responses depend on
protein phosphorylation or dephosphorylation through the reactions with
calmodulin. For example, calmodulin regulates
Ca2+-dependent microtubule assembly and disassembly
(19). Exposure to CO2 in the turtle urinary
bladder appears to stimulate the insertion of proton pumps into the
apical membrane (4). When alterations in
[Ca2+]i are minimized (4, 22,
35) or the function of calmodulin is antagonized
(22), exocytosis and the enhancement of proton secretion
are mitigated. Compatible with these observations, two maneuvers that
should affect [Ca2+]i or calmodulin
activity also inhibited the enhancement of H-K-ATPase-dependent Rb
reabsorption on exposure to 10% CO2 (Figs. 5 and 6). In
the present studies, we observed that MAPTAM prevented an adaptive increase in Rb efflux in response to 10% CO2.
Microperfusion experiments in the OMCD have demonstrated that
1,2-bis(2-aminophenoxy)
ethane-N,N,N',N'-tetraacetic acid (BAPTA; analogous to MAPTAM, an intracellular Ca2+
chelator) substantially attenuated an adaptive increase in
H+ secretion in response to in vitro acidosis
(34). On the basis of our results that
[Ca2+]i failed to increase under 10%
CO2, we speculate that the inhibitory effect of MAPTAM is
related to a decrease in basal intracellular calcium. However, the
failure to detect the increase of [Ca2+]i
under the 10% CO2 condition is consistent with the
functional studies in rat ventricular myocytes (14).
Studies in vascular smooth muscle cells have shown that intracellular
acidification is associated with changes in free cytosolic
Ca2+ via a Ca2+-ATPase (7). These
observations suggest that increased Ca2+ extrusion from the
cell may be another reason for the lack of change in
[Ca2+]i when PCO2 is
increased. A significant decrease in Rb reabsorption was observed
during the 10% CO2 period when the CCD was perfused in the
presence of W-7. We do not exclude the possibility that the effect of
W-7 may be due to an action on the basolateral K exit pathway or due to
inhibition of the apical H-K-ATPase. The mechanism(s) for the decrease
in KRb remains to be elucidated.
Exocytosis is, in large part, dependent on the organization of the
cytoskelton, which includes microtubules (3, 11, 22). Therefore, we examined conditions that were expected to inhibit microtubule function at two different stages. First, we simultaneously exposed the CCD to 10% CO2 and colchicine. Under these
conditions, we observed no stimulation of Rb reabsorption (Fig. 7).
Second, we further pursued this question by stimulating Rb reabsorption with 10% CO2 and subsequently examining the effect of
colchicine. In this case, colchicine did not reduce Rb reabsorption
(Fig. 8). These results suggest that exocytosis of the H-K-ATPase
is dependent on the intact function of microtubules. After the
H-K-ATPase inserts into the membrane, the present data suggest that
colchicine does not inhibit the enzyme. Alternatively, exocytosis may
be necessary for the insertion into the apical membrane of a protein that is essential for the functional activity of the H-K-ATPase.
Exposure to 10% CO2, which stimulates exocytosis of
H-K-ATPase, may not be a phenomenon unique to animals adapted to a
K-restricted diet. McKinney and Davidson (22) have
previously shown that the enhancement of acidification by the
collecting duct on exposure to 15% CO2 involves the intact
function of the cell cytoskeleton in normal (K-replete) animals. In CCD
of K-replete animals, the principal cells possess pH-sensitive K
conductance at both the apical and basolateral membrane, whereas
intercalated cells possess H-K-ATPase but small K conductance.
CO2 (10%) could be predicted to decrease Rb reabsorption
via K conductance but increase Rb reabsorption that is mediated by an
H-K-ATPase. Our previous findings that 10% CO2 failed to
increase Rb reabsorption during K repletion suggest that an apical K
exit mechanism is the predominant pathway for K flux under these
conditions and that 10% CO2 stimulates luminal
acidification via an H-K-ATPase-dependent process in K-replete animals
(44). Clearly, the response to 10% CO2 during
K repletion differs from that during K restriction.
In summary, these studies demonstrate that 10% CO2
stimulates Rb reabsorption, and this stimulation was totally abolished by SCH-28080. The rapid response to 10% CO2 implies that
this maneuver increases the functional units of H-K-ATPase at the
apical membrane or the activity of existing pump units. The subsequent addition of methazolamide after exposure to 10% CO2 had no
significant effect on KRb, whereas simultaneous
exposure of the tubules to 10% CO2 and methazolamide
prevented the enhancement of Rb reabsorption by 10% CO2,
suggesting that carbonic anhydrase is not necessary for maintaining
activation of H-K-ATPase by 10% CO2 but is necessary for
initiating activation of this enzyme. MAPTAM, W-7, and colchicine inhibited the stimulation of Rb reabsorption after exposure to 10%
CO2, suggesting that the effect of 10% CO2 on
Rb reabsorption requires at least a basal level of
[Ca2+]i as well as the intact function of
calmodulin and microtubules. These studies suggest that increased
CO2 activates an SCH-28080- sensitive H-K-ATPase in the CCD
of K-restricted rabbits.
 |
ACKNOWLEDGEMENTS |
These studies were supported by the Medical Research Service of the
Department of Veterans Affairs and National Institute of Diabetes and
Digestive and Kidney Diseases Grant R01-DK-49750.
 |
FOOTNOTES |
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 Section, Dept. of Veterans Affairs Medical Ctr.,
Gainesville, FL 32608-1197 (E-mail: wingocs{at}ufl.edu).
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. Section 1734 solely to indicate this fact.
Received 25 October 1999; accepted in final form 15 March 2001.
 |
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