Cellular origin and hormonal regulation of
K+-ATPase activities sensitive to Sch-28080 in rat
collecting duct
Nicolas
Laroche-Joubert,
Sophie
Marsy, and
Alain
Doucet
Laboratoire de Biologie Intégrée des Cellules
Rénales, Service de Biologie Cellulaire, Commissariat à
l'Énergie Atomique, Saclay, Unité de Recherche
Associée 1859, Centre National de la Recherche Scientifique,
91191 Gif-sur-Yvette Cedex, France
 |
ABSTRACT |
Rat collecting ducts exhibit type I or type III
K+-ATPase activities when animals are fed a normal (NK) or
a K+-depleted diet (LK). This study aimed at
determining functionally the cell origin of these two
K+-ATPases. For this purpose, we searched for an effect on
K+-ATPases of hormones that trigger cAMP production in a
cell-specific fashion. The effects of
1-deamino-8-D-arginine vasopressin (dD-AVP), calcitonin,
and isoproterenol in principal cells,
-intercalated cells, and
-intercalated cells of cortical collecting duct (CCD), respectively,
and of dD-AVP and glucagon in principal and
-intercalated cells of
outer medullary collecting duct (OMCD), respectively, were examined. In
CCDs, K+-ATPase was stimulated by calcitonin and
isoproterenol in NK rats (type I K+-ATPase) and by dD-AVP
in LK rats (type III K+-ATPase). In OMCDs, dD-AVP and
glucagon stimulated type III but not type I K+-ATPase.
These hormone effects were mimicked by the cAMP-permeant analog
dibutyryl-cAMP. In conclusion, in NK rats, cAMP stimulates type I
K+-ATPase activity in
- and
-intercalated CCD cells,
whereas in LK rats it stimulates type III K+-ATPase in
principal cells of both CCD and OMCD and in OMCD intercalated cells.
colonic and gastric hydrogen-potassium-adenenosine triphosphatase; adenosine 3',5'-cyclic monophosphate; potassium depletion; vasopressin; glucagon; isoproterenol; calcitonin
 |
INTRODUCTION |
WE HAVE
PREVIOUSLY REPORTED that rat collecting ducts display two types
of Na+-independent K+-ATPases when animals are
fed a normal or a K+-depleted diet (5). The
K+-ATPase activity in cortical (CCD) and outer medullary
(OMCD) collecting ducts from normal rats (NK rats), namely, type I
K+-ATPase, is highly sensitive to the gastric
H+-K+-ATPase inhibitor Sch-28080, is
insensitive to ouabain, and is specific for K+. Conversely,
type III K+-ATPase activity measured in CCD and OMCD of
rats fed a K+-depleted diet for 2 wk (LK rats) displays a
high sensitivity to both Sch-28080 and ouabain and is activated by
either K+ or Na+. Because these two
K+-ATPase activities display different properties, they
likely reflect the activity of distinct proteins. The purpose of this
study was to determine the cell origin of type I and type III
K+-ATPase activities in CCD and OMCD, as it can direct the
search for a potential role of these ATPases in the regulation of
K+ and/or proton balance.
Although initial studies suggested that type I and type III
K+-ATPase activities might reflect the enzymatic activity
of gastric and colonic H+-K+-ATPase,
respectively, this appears unlikely today (reviewed in Refs.
15 and 20). In the absence of molecular characterization, specific molecular probes of type I and III K+-ATPases were
not available, and consequently we used a functional strategy to
determine their cellular origin.
This strategy was based on two types of considerations, a
well-established property of collecting duct cells and an assumption concerning the regulation of K+-ATPase activities. The
assumption was that K+-ATPase activity might be controlled
by hormones triggering the cAMP signaling pathway in rat CCDs, as is
now well established for Na-K+-ATPase activity
(18). The established property is that different hormones
stimulate adenylyl cyclase (AC) of the rat collecting duct in a
cell-specific manner. In CCDs, AC is stimulated by vasopressin in
principal cells, by calcitonin in
-intercalated cells, and by the
-adrenergic agonist isoproterenol in
-intercalated cells, whereas
in OMCDs vasopressin and glucagon stimulate AC in principal cells
and
-intercalated cells, respectively. Thus we evaluated the effect
of these four mediators on type I and III K+-ATPases in the
collecting ducts of NK and LK rats, respectively. Results demonstrate
that type I and type III K+-ATPase not only differ by their
functional properties but also by their cellular origin.
 |
MATERIALS AND METHODS |
Animals.
Experiments were performed in two groups of male Sprague-Dawley rats
weighing 150-200 g. Rats were fed either the LK diet containing
1.5 meq K+/kg (212K; Usine d'Alimentation Rationnelle,
Epinay, France) for 14 days or a similar diet supplemented with KCl
(150 meq/kg), as described previously (5). All animals had
free access to food and were allowed to drink deionized water ad libitum.
Animal preparation and tubule microdissection.
Animals were anesthetized (pentobarbital sodium, 5 mg/100 g body wt
ip), and their left kidney was infused through an aortic catheter with
5 ml of infusion solution (see composition below) containing 12 mg of
collagenase (from Clostridium histolyticum, 0.75 U/mg;
Serva). Next, the kidney was removed and sliced into small pyramids
that were incubated at 30°C for 20 min. CCD and OMCD were dissected
in the dissection solution (see composition below) at 0-4°C
under stereoscopic observation. They were identified by morphological
and topographical criteria. Infusion solution contained (in mM) 120 NaCl, 5 KCl or RbCl (for ATPase and Rb+ flux measurements,
respectively), 1 CaCl2, 1 MgSO4, 0.2 Na2HPO4, 0.15 NaH2PO4,
5 glucose, 2 lactate, 4 essential and nonessential amino acids, and 20 HEPES; pH was 7.40, and the osmolality was adjusted at 500 mosmol/kgH2O by mannitol addition. The dissection solution
was similar except that the concentration of CaCl2 was 0.25 mM and 0.1% BSA was added.
Determination of K+-ATPase activity.
Pools of four to six CCDs or OMCDs were individually transferred with 1 µl of dissection solution in the concavity of sunken bacteriological
slides coated with dried BSA and were stored on ice until the end of
the dissection. After addition of 1 µl of dissection medium without
or with hormone at two times the required final concentration, samples
were incubated at 37°C for 10 min. Thereafter, pools of nephrons were
rinsed two times in 0.3-1.0 ml of ATPase rinsing solution (0.5 mM
CaCl2, 1 mM MgCl2, 0.8 mM MgSO4,
100 mM Tris · HCl, 0.1% BSA, mannitol up to 500 mosmol/kg H2O, pH 7.4) to remove Na+; the pools were
transferred in 0.5 µl of rinsing solution to the BSA-coated wells of
a 96-well flat-bottom plastic microplate and photographed to determine
the total tubular length of each pool. Two microliters of 10 mM
Tris · HCl were added to each well, and the microplate was
submitted to a freezing/thawing step to allow permeabilization of cell
membranes. After addition of 10 µl of ATPase assay solution to each
well, the microplate was incubated at 37°C for 15 min. ATPase assay
solution contained (in mM) 2.5 KCl, 10 MgCl2, 1 EGTA, 25 Tris · HCl, 5 Tris-ATP, and tracer amounts of
[
-32P]ATP (10 Ci/mmol; DuPont de Nemours, Boston MA)
at pH 7.4 in the absence or presence of 300 µM Sch-28080 for the
measurement of total and Sch-28080-insensitive ATPase activities,
respectively. This concentration of Sch-28080 is sufficient to inhibit
type I and type III K+-ATPase activities (5).
Sch-28080 was prepared from a 100 mM solution in methanol. Methanol
(0.3% vol/vol) was added to the Sch-28080-free ATPase assay medium. In
some experiments, 1 mM ouabain was added to the ATPase assay medium to
discriminate between type I (ouabain-resistant) and type III
(ouabain-sensitive) K+-ATPase activity.
Incubation was stopped by adding 300 µl of an ice-cold 15% (wt/vol)
suspension of activated charcoal. The microplate was centrifuged (2,000 rpm for 3 min), and 100 µl of each supernatant were transferred to a
96-well sample microplate (Wallac). The radioactivity in each well was
determined by Cerenkov counting in a microplate counter (1450 microbeta
Trilux; Wallac). Total and Sch-28080-insensitive ATPase activities were
each determined in quadruplicate. K+-ATPase activity was
taken as the difference between the means of total and
Sch-28080-insensitive ATPase activities and was expressed as picomoles
per millimeter per minute. Data represent means and SE from several animals.
Determination of K+ pump activity in intact cells.
K+ pump activity was determined on intact CCDs and OMCDs by
measuring Sch-28080-sensitive intracellular accumulation of
86Rb+ under conditions of initial rate,
according to the method developed previously in the laboratory
(9). Pools of 10 CCDs or OMCDs were individually
transferred with 0.7 µl of dissection solution to the concavity of
sunken bacteriological slides coated with dried BSA and were stored on
ice until the end of the dissection. After addition of 0.7 µl of
dissection medium without or with hormone and/or Sch-28080 at 2.7-fold
the required final concentrations, samples were incubated at 37°C for
10 min. 86Rb+ uptake was determined after
addition of 0.5 µl of dissection solution added with
86RbCl (~100 nCi/sample; Amersham) and preequilibrated at
37°C. Incubation was stopped after 1 min by adding 20 µl of
Rb+ rinsing solution containing (in mM) 150 choline
chloride, 1.2 MgSO4, 1.2 CaCl2, 2 BaCl2, 10 HEPES, and mannitol up to 500 mosmol/kg H2O, pH 7.4. The 10 tubules of each slide were then rapidly
rinsed in three successive baths of ice-cold Rb+ rinsing
solution and individually transferred with 0.2 µl of the last rinsing
bath to a microscopy coverglass. After being photographed, each sample
was dropped in a counting vial containing 0.5 ml of 1% (wt/vol)
deoxycholic acid, and its radioactivity was measured by liquid scintillation.
K+-ATPase-mediated Rb+ uptake
(pmol · mm
1 · min
1) was
calculated as the difference between the mean values measured in
samples incubated without Sch-28080 and with 10
4 M
Sch-28080. Data for K+-ATPase-mediated Rb+
uptake are means ± SE from several animals within the same
experimental group.
Statistics.
Results are given as means ± SE from different animals.
Statistical comparisons between groups were performed according to Student's t-test for nonpaired data.
 |
RESULTS |
Effect of hormones on K+ pump activity.
Results for CCDs and OMCDs are summarized in Figs.
1 and 2,
respectively. In CCDs of normal rats, preincubation with
10
8 M 1-deamino-8-D-arginine vasopressin
(dD-AVP), a specific agonist of vasopressin V2 receptors,
did not alter Sch-28080-sensitive Rb+ uptake [in
pmol · mm
1 · min
1; control,
1.4 ± 0.2 (SE); dD-AVP, 1.2 ± 0.1; n = 4;
not significant (NS)]. In contrast, Rb+ uptake was
stimulated by 10
8 M salmon calcitonin (control, 1.4 ± 0.1; calcitonin, 3.0 ± 0.2; n = 8;
P < 0.001) and by 10
6 M of the
-adrenergic receptor agonist isoproterenol (control, 1.3 ± 0.1; isoproterenol, 3.0 ± 0.3; n = 8;
P < 0.001). Conversely, in CCDs from LK rats, neither
calcitonin (control, 1.4 ± 0.2; calcitonin, 1.5 ± 0.1;
n = 6; NS) nor isoproterenol (control, 1.3 ± 0.2;
isoproterenol, 1.5 ± 0.2; n = 6; NS) altered
Sch-28080-sensitive Rb+ uptake, whereas dD-AVP induced a
marked stimulation (control, 1.2 ± 0.2; dD-AVP, 2.7 ± 0.3;
n = 6; P < 0.005).

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Fig. 1.
Effect of cAMP-generating mediators on
K+-ATPase-mediated Rb+ uptake in cortical
collecting ducts (CCDs) from normal and K+-depleted rats.
CCDs microdissected from normal rats (NK rats; A) or rats
fed a K+-depleted diet for 2 wk (LK rats; B)
were preincubated at 37°C for 10 min in the absence (Control) or
presence of either 10 8 M
1-deamino-8-D-arginine vasopressin (dD-AVP),
10 8 M salmon calcitonin, or 10 6 M
isoproterenol before measurement of Sch-28080-sensitive Rb+
uptake. Lines join the values determined on the same animal in the
absence or presence of the different agents, and points are means ± SE from different animals. Values statistically different from
controls were determined by Student's t-test for nonpaired
data: *P < 0.005 and **P < 0.001.
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Fig. 2.
Effect of cAMP-generating mediators on
K+-ATPase-mediated Rb+ uptake in outer
medullary collecting ducts (OMCDs) from NK (A) and LK
(B) rats. OMCDs microdissected from NK rats or LK rats were
preincubated at 37°C for 10 min in the absence (Control) or presence
of either 10 8 M dD-AVP or 10 6 M glucagon
before measurement of Sch-28080-sensitive Rb+ uptake. Lines
join the values determined on the same animal in the absence or
presence of the different agents, and points are means ± SE from
different animals. Values statistically different from controls were
determined by Student's t-test for nonpaired data:
*P < 0.005 and **P < 0.001.
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In OMCDs of normal rats, neither dD-AVP (control, 1.4 ± 0.1;
dD-AVP, 1.6 ± 0.1; n = 4; NS) nor
10
6 M glucagon (control, 1.3 ± 0.1; glucagon,
1.1 ± 0.1; n = 5; NS) stimulated
K+-ATPase-mediated Rb+ uptake. Conversely, in
OMCDs from LK rats, Sch-28080-sensitive Rb+ uptake was
stimulated by dD-AVP (control, 1.1 ± 0.1; dD-AVP, 3.0 ± 0.4; n = 4; P < 0.005) and by glucagon
(control, 1.3 ± 0.2; dD-AVP, 2.8 ± 0.2; n = 6; P < 0.001).
In both CCDs and OMCDs, none of the hormones tested altered
Sch-28080-insensitive Rb+ uptake (Table
1).
Effect of hormones and cAMP on K+-ATPase activity.
Hormone-induced changes in K+-ATPase-mediated
Rb+ uptake may result from a direct stimulation of
K+-ATPase or from indirect stimulation brought about by
hormone-induced alterations of regulatory parameters such as ion
concentrations or gradients. To evaluate whether hormones directly
modulate K+-ATPase, we determined their effect on
K+-ATPase activity measured under maximal velocity
conditions in permeabilized cells. These experiments were limited to
hormones and nephron segments where Rb+ uptake was
modified, i.e., calcitonin and isoproterenol in CCD of NK rats, dD-AVP
in CCD of LK rats, and dD-AVP and glucagon in OMCD of LK rats.
Results summarized in Fig. 3 indicate
that hormones that stimulated Sch-28080-sensitive Rb+
uptake also stimulated K+-ATPase activity. Thus, in CCDs of
NK rats, calcitonin (in
pmol · mm
1 · min
1; control,
0.7 ± 0.3; calcitonin, 3.4 ± 0.4; n = 6;
P < 0.001) and isoproterenol (control, 0.5 ± 0.2; isoproterenol, 3.6 ± 0.6; n = 6;
P < 0.001) stimulated K+-ATPase activity.
In LK rats, K+-ATPase activity was stimulated by dD-AVP in
CCDs (control, 2.8 ± 0.4; dD-AVP, 7.1 ± 1.1;
n = 6; P < 0.005) and by both dD-AVP (control, 1.3 ± 0.3; dD-AVP, 3.8 ± 0.7; n = 9; P < 0.005) and glucagon in OMCDs (control, 1.3 ± 0.3; glucagon, 4.7 ± 1.0; n = 8;
P < 0.01). None of the hormones tested altered
Sch-28080-insensitive ATPase activity (Table
2).

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Fig. 3.
Effect of cAMP-generating mediators on K+-ATPase
activity in CCD and OMCD from NK and LK rats. A: CCD from NK
rats; B: CCD from LK rats; C: OMCD from LK rats.
CCDs and OMCDs from either NK or LK rats were preincubated at 37°C
for 10 min in the absence (filled bars) or presence (shaded bars) of
either 10 6 M isoproterenol (Iso), 10 8 M
salmon calcitonin (Calc), 10 8 M dD-AVP, or
10 6 M glucagon (Glu) before measurement of
Sch-28080-sensitive K+- ATPase activity. Data are
means ± SE for the no. of animals indicated in bars. Values
statistically different from controls were determined by Student's
t-test for nonpaired data: *P < 0.01, **P < 0.005, and ***P < 0.001.
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Because all of these hormones may be coupled to transduction pathways
other than AC, in a separate experimental series we verified that
stimulation of K+-ATPase activity was mediated by cAMP.
Results in Fig. 4 demonstrate that the
permeant analog of cAMP, dibutyryl-cAMP (DBcAMP, 10
3 M),
stimulated K+-ATPase activity in the CCD of NK (in
pmol · mm
1 · min
1: control,
0 ± 0.1; DBcAMP, 1.8 ± 0.3; n = 6;
P < 0.001) and LK (control, 0.5 ± 0.3; DBcAMP,
2.4 ± 0.3; n = 3; P < 0.005)
rats and in the OMCD of LK rats (control, 1.5 ± 0.8; DBcAMP,
6.6 ± 0.6; n = 4; P < 0.005).
Furthermore, in the same experiments, we evaluated the effect of
ouabain (1 mM) on basal and DBcAMP-stimulated K+-ATPase
activity. In NK rats, ouabain did not alter K+-ATPase
activity, demonstrating that cAMP-stimulated activity was also
accounted for by ouabain-resistant type I K+-ATPase. In
contrast, ouabain abolished both basal and cAMP-stimulated activity in
LK rats (CCD and OMCD), as expected for type III K+-ATPase.

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Fig. 4.
Effect of cAMP on K+-ATPase activity in CCD and OMCD
from NK and LK rats. A: CCD from NK rats; B: CCD
from LK rats; C: OMCD from LK rats. CCDs and OMCDs from
either NK or LK rats were preincubated at 37°C for 10 min in the
absence (filled bars) or presence (shaded bars) of 10 3 M
dibutyryl-cAMP before measurement of Sch-28080-sensitive
K+-ATPase activity. K+-ATPase activity was
determined in either the absence ( Ouab) or presence (+Ouab) of 1 mM
ouabain. Due to seasonal variations, in this experimental series,
K+-ATPase activities in CCDs were lower than those
described in Fig. 3. Data are means ± SE from different animals
(n). In CCD and OMCD of LK rats, basal and cAMP-stimulated
K+-ATPase activities determined in the presence of ouabain
were not statistically different from 0. Values statistically different
from controls were determined by Student's t-test for
nonpaired data: **P < 0.005 and ***P < 0.001.
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DISCUSSION |
Cellular specificity of hormone receptors coupled to AC in rat
collecting duct.
Early studies have shown that AC is stimulated by vasopressin,
isoproterenol, calcitonin, and glucagon in the rat CCD and by
vasopressin and glucagon in the OMCD (27). The cell
specificity of action of these hormones in rat collecting duct has been
established on the basis of 1) their functional effects on
water and ion transport and/or 2) the ability of various
mediators to downregulate cAMP accumulation induced by some hormones
but not by others.
That vasopressin and cAMP control water permeability of the collecting
duct (29, 30) demonstrates that vasopressin V2 receptors are expressed in principal cells of CCD and OMCD. In vitro
microperfusion of rat CCD has shown that calcitonin stimulates the
secretion of proton, whereas isoproterenol stimulates that of
bicarbonate (33), suggesting that these two hormones act on
- and
-intercalated cells, respectively. That vasopressin, isoproterenol, and calcitonin stimulate AC in different cells from rat
CCD is further supported by the finding that PGE2 inhibits cAMP production in response to isoproterenol but not to vasopressin (8), whereas a
-adrenergic agonist inhibits cAMP
accumulation in response to vasopressin but neither to isoproterenol
nor to glucagon (7).
Although the effect of glucagon on ion transport in rat OMCD is not
known, it likely stimulates AC in a different cell type than
vasopressin, i.e., in
-intercalated cells. Indeed,
-adrenergic agonist reduces cAMP accumulation induced by vasopressin but not by
glucagon (7), whereas, conversely, carbachol inhibits cAMP accumulation in response to glucagon but not to vasopressin
(6).
This cellular distribution of hormone action is not altered in LK rats
since 1) in CCDs, K+ depletion does not alter
the stimulation of AC by vasopressin, calcitonin, and isoproterenol and
2) the increased stimulations of AC by vasopressin and
glucagon observed in OMCD from LK rats are entirely accounted for by
the OMCD hypertrophy induced by K+ depletion
(19).
The cell specificity of action of vasopressin, calcitonin, and
isoproterenol in CCD and of vasopressin and glucagon in OMCD was used
to localize functionally type I and type III K+-ATPases in
collecting ducts of NK and LK rats, respectively.
Cellular origin of type I K+-ATPase.
In CCD from NK rats, cAMP stimulated ouabain-insensitive
K+- ATPase, i.e., type I K+-ATPase (Fig. 4).
Stimulation was also induced by calcitonin and isoproterenol but not by
a specific vasopressin V2 agonist (Fig. 1). These findings
indicate that type I K+-ATPase is present in both
- and
-intercalated cells of rat CCD, although it does not exclude its
presence in principal cells (where it could be unresponsive to AC
stimulation). Expression of type I K+-ATPase activity in
CCD intercalated cells is consistent with the finding of Silver et al.
(34) who reported that K+-dependent proton
extrusion in intercalated cells of rat CCD is sensitive to Sch-28080
and insensitive to ouabain, like type I K+-ATPase.
Localization of type I K+-ATPase in both
- and
-intercalated cells may also account for the lack of effect of
Sch-28080 on bicarbonate transport by in vitro microperfused rat CCD
(17). Assuming that K+-ATPase from both cell
types contributes equally to bicarbonate transport (but in reverse
direction), their inhibition should reduce unidirectional fluxes
without detectable alteration of the transepithelial net flux. This
hypothesis is supported by the fact that, under acute alkalosis, a
condition that stimulates bicarbonate secretion over its reabsorption,
Sch-28080 reduces bicarbonate secretion in in vitro microperfused CCDs
(17).
Despite pharmacological similarities (5, 16, 25), it
remains uncertain whether type I K+-ATPase and gastric
H+-K+-ATPase are a same molecular entity.
Indeed, 1) expression of gastric
H+-K+-ATPase
-subunit mRNAs in the rat
collecting duct was reported in some (1, 3) but not all
studies (10, 14), and 2) expression of gastric
H+-K+-ATPase in rat kidney was revealed by some
(4, 35) but not all anti-gastric
H+-K+-ATPase antibodies (35).
Nevertheless, the functional localization of type I
K+-ATPase in
-and
-intercalated cells of CCD from NK
rats is consistent with the localization of gastric
H+-K+-ATPase in studies where expression of
this pump was observed. By in situ hybridization, Ahn et al. (1,
3) reported a much higher labeling in intercalated cells than in
principal cells. The first immunolocalization of gastric
H+-K+-ATPase in the kidney revealed that only
some cells of the rat CCD were labeled (35). Later,
Bastani (4) demonstrated that these were intercalated
cells because gastric H+-K+-ATPase colocalized
with H+-ATPase in the rat CCD. Altogether, these results
suggest that type I K+- ATPase is the enzymatic
counterpart of the gastric, or a gastric-like, H+-K+-ATPase in the rat CCD.
Although type I K+-ATPase activity was detected repeatedly
in rat OMCD, it was not altered by either dD-AVP or glucagon (Fig. 2),
precluding the determination of its cellular origin. The demonstration of Sch-28080-sensitive, ouabain-insensitive K+-dependent
bicarbonate reabsorption by in vitro microperfused OMCD from normal
rats (17, 28) suggests that type I K+-ATPase
originates in intercalated cells in OMCD also. This would also be
consistent with the localization of gastric
H+-K+-ATPase immunoreactivity in OMCD
intercalated cells (4). The lack of effect of glucagon on
type I K+-ATPase activity in OMCD suggests that some
intermediate of the signaling pathway that mediates calcitonin
stimulation of type I ATPase in CCD
-intercalated cells is missing
in OMCD
-intercalated cells.
Cellular origin of type III K+-ATPase.
We have previously reported that ouabain-insensitive type I
K+-ATPase activity disappears from the rat CCD and OMCD
during dietary K+ depletion and is replaced by
ouabain-sensitive type III K+-ATPase (5). The
present results confirm this finding, since 1) ouabain
abolished both basal and cAMP-stimulated K+-ATPase in CCD
and OMCD of LK rats (Fig. 4) and 2) calcitonin and
isoproterenol no longer stimulated K+-ATPase in CCD from LK
rats (Fig. 1). In addition, they show that type III
K+-ATPase was stimulated by dD-AVP in CCD and by both
dD-AVP and glucagon in OMCD (Figs. 1 and 2), demonstrating that it is
present in principal cells of CCD and OMCD and in OMCD
-intercalated cells. Thus K+ depletion not only modifies the type of
K+-ATPase expressed in the collecting duct but also the
cellular origin of K+-ATPase.
The presence of type III K+-ATPase in
-intercalated
cells of LK rat OMCD is consistent with the in vitro microperfusion
study of Nakamura et al. (28). These authors reported a
marked inhibition of bicarbonate reabsorption by OMCD of LK rats either
by luminal addition of Sch-28080 or ouabain or by removal of luminal
K+. In CCD and OMCD principal cells of LK rats, the
possible contribution of a Sch-28080- and ouabain-sensitive process to
proton and/or K+ transport has not been evaluated to date.
Our hypothesis is that the main contribution of type III
K+-ATPase in principal cells of LK rat collecting duct is
to energize K+ reabsorption and thereby reduce its urinary loss.
Type III K+-ATPase was thought initially to reflect the
activity of colonic H+-K+-ATPase because
1) it displays the pharmacological profile of the amphibian
ortholog of rat colonic H+-K+-ATPase
(22) and 2) expression of colonic
H+-K+-ATPase
-subunit mRNAs is markedly
induced by K+ depletion in rat collecting ducts
(26). Despite these preliminary conclusions, the identity
of type III K+-ATPase and colonic
H+-K+-ATPase is now questioned because
1) rat colonic H+-K+-ATPase
expressed in recombinant cells is poorly sensitive (or not sensitive)
to ouabain and Sch-28080 (11, 13, 23, 24, 32) and
2) colonic H+-K+-ATPase expressed in
Xenopus oocytes is able to transport Na+ instead
of proton (12), whereas Na+ substitutes for
K+ for activation of type III K+-ATPase
activity (5). The cellular origin of type III
K+-ATPase in OMCD of LK rats is consistent with that of the
distribution of colonic H+-K+-ATPase in
intercalated (2) and principal cells (21,
31). However, with the same molecular probes, these authors
failed to detect colonic H+-K+-ATPase mRNAs and
protein in CCD of LK rats. Altogether, these results suggest that type
III K+-ATPase may not reflect expression of a unique
molecular form of colonic H+-K+-ATPase in the
collecting duct of LK rats.
In conclusion, type I and type III K+-ATPase activities
previously described in the collecting duct of normal and
K+-depleted rats, respectively, not only differ by their
pharmacological properties but also by their cellular origin.
Ouabain-insensitive type I K+-ATPase originates in
intercalated cells, where it likely contributes to proton/bicarbonate
transport, whereas ouabain-sensitive type III K+-ATPase
originates in principal cells, where it may contribute to
K+ reabsorption, and intercalated cells from OMCD. The
pharmacological properties, the function, and the localization of type
I K+-ATPase are consistent with those of a gastric type of
H+-K+-ATPase. In contrast, pharmacological
properties and tubular localization of type III K+-ATPase
are incompatible with that of colonic
H+-K+-ATPase and therefore call for the search
of new molecular forms of H+-K+-ATPase in the
kidney of LK rats. Finally, this study demonstates that
K+-ATPase activity, and by inference
H+-K+-ATPase, can be stimulated acutely by
hormones that trigger the AC/cAMP cascade in the collecting duct, which
opens a new field of investigation.
 |
ACKNOWLEDGEMENTS |
We thank M. Imbert-Teboul for critical reading of the manuscript.
 |
FOOTNOTES |
This work was supported by grants from the Commissariat à
l'Energie Atomique and the Centre National de la Recherche
Scientifique to the Unité de Recherche Associée 1859. N. Laroche-Joubert was supported by a grant from the Ministère de
l'Education Nationale, de la Recherche et de la Technologie.
Address for reprint requests and other correspondence: A. Doucet, URA 1859, Bâtiment 520, Centre d'Etudes de Saclay, 91191 Gif sur Yvette cedex, France (E-mail:
doucet{at}dsvidf.cea.fr).
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 9 February 2000; accepted in final form 2 August 2000.
 |
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