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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha -intercalated cells, and beta -intercalated cells of cortical collecting duct (CCD), respectively, and of dD-AVP and glucagon in principal and alpha -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 alpha - and beta -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -intercalated cells, and by the beta -adrenergic agonist isoproterenol in beta -intercalated cells, whereas in OMCDs vasopressin and glucagon stimulate AC in principal cells and alpha -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 [gamma -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.

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).

                              
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Table 1.   Sch-28080-insensitive rubidium uptake in collecting duct of normal and K+-depleted rats

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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Table 2.   Sch-28080-insensitive ATPase activity in collecting duct of normal and K+-depleted rats

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha - and beta -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 alpha -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 alpha -intercalated cells. Indeed, alpha -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 alpha - and beta -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 alpha - and beta -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 alpha -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 alpha -and beta -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 alpha -intercalated cells is missing in OMCD alpha -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 alpha -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 alpha -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 alpha -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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