Pharmacological profiles of the murine gastric and colonic H,K-ATPases

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Abstract

Background

The H,K-ATPase, consisting of α and β subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα1 and HKα2 encoded by different genes. The ouabain-resistant gastric HKα1-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα2-H,K-ATPase from different species shows poor primary structure conservation of the HKα2 subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKα1, HKα2, or HKβ genes.

Methods

Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα1, HKα2, or HKβ knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined.

Results

The colonic H,K-ATPase demonstrated slightly greater affinity for K+ than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKα2 KO but was observed in HKβ KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKα1-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα2-H,K-ATPase was not inhibited by these compounds.

Conclusions

These data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride.

General Significance

Characterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.

Introduction

The H,K-ATPase is an integral membrane protein that actively transports protons (H+) and potassium (K+) ions across the plasma membrane and is important for acid–base balance and potassium homeostasis [1]. The H,K-ATPase belongs to the P-type ATPase family and consists of two essential subunits, α and β. The α subunit contains ATP and cation binding sites responsible for ATP hydrolysis and electroneutral exchange of H+ and K+. The highly glycosylated β subunit is involved in the correct assembly, plasma membrane localization, and stabilization of the holoenzyme [2]. There are two isoforms of the α subunit (HKα1 and HKα2) and each exhibits a unique tissue distribution. The HKα1 isoform is expressed in the stomach, kidney, and other tissues [3], [4]. The HKα1 isoform in the gastric parietal cells is the motor of the ion transport system responsible for acid secretion. The gastric H,K-ATPase is sensitive to the P-type ATPase inhibitor orthovanadate, and to the more specific inhibitors Sch28080 and omeprazole, but is insensitive to ouabain, an Na,K-ATPase inhibitor [5]. The HKα2 isoform is expressed in the distal colon, skin, kidney, prostate, and uterus [6], principally expressed in distal colon epithelial cells, and renal collecting duct. Colonic H,K-ATPase was initially characterized as a ouabain-insensitive activity in the rabbit descending colon epithelium membranes [7]. Biochemical studies found that the apical membranes from rat distal colon epithelial cells contain both ouabain-sensitive and ouabain-insensitive K-ATPase activities [8], [9].

HKα2 has been cloned from several species [10], [11], [12], [13]. The mammalian interspecies difference in amino acid composition of the α2 subunit of the colonic H,K-ATPase is about 15%, in contrast to only 1–2% difference in amino acid sequence of gastric H,K-ATPase and Na,K-ATPase from different species. The properties of the colonic H,K-ATPase have been studied in several functional expression systems including Xenopus oocytes [13], [14], [15], [16], [17], [18], HEK 293 cells [19], [20], [21], and baculovirus expression system [22], [23], [24]. These studies yield rather diverse results, especially regarding the pharmacological sensitivity to ouabain and Sch28080. This might be due to the different species and functional expression systems used. Most functional expression studies have been performed with rat colonic H,K-ATPase [15], [16], [21], [22], [23]. Expression of rat HKα2 in Xenopus oocytes resulted in Sch28080-insensitive and poorly ouabain-sensitive K+-activated rubidium uptake and K-ATPase activity, with IC50 values at 0.4–0.6 mM or 1 mM, respectively [15], [16]. Rat HKα2 expressed in HEK293 cells resulted in a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity and rubidium uptake [21]. Similar results were observed with rat HKα2 expressed in baculovirus expression system [23]. However, rat HKα2 without pairing β subunit expressed in insect Sf9 cells displayed a ouabain-resistant, Sch28080-sensitive K-ATPase activity with 18% inhibition at 0.1 mM Sch28080 [22]. Overall, the functionally expressed rat colonic H,K-ATPase is rather insensitive to the inhibition by Sch28080 and ouabain. Bufo marinus bladder H,K-ATPase expressed in Xenopus oocytes displays intermediate sensitivity to ouabain and Sch28080 with Ki of 25 μM and 230 μM, respectively [13]. Expression of human HKα2 in Xenopus oocytes, HEK 293 cells, and baculovirus expression system resulted in similar Sch28080- and ouabain-sensitive rubidium uptake and ATPase activity [18], [20], [24]. However, guinea pig HKα2 expressed in HEK 293 cells shows intermediate ouabain sensitivity (IC50 value of 52 μM) but resistance to Sch28080 [19]. Thus, there is a need to characterize the pharmacological profile of the colonic H,K-ATPase in mouse, given the ability to examine the effect of single gene disruption in the intact animal.

In recent years, mouse models with targeted disruption of the HKα1, HKα2, or HKβ genes [25], [26], [27] have been generated, allowing the contribution of the specific gene to functional activity to be unambiguously deciphered. In the present study, we measured K-ATPase activity in the native tissues of wild-type mice compared to the HKα1, HKα2, or HKβ knockout (KO) mice. Use of the KO mice allows the wild-type enzymatic activities to be specifically linked to each gene in question. Results from our study of HKα1 knockout mice were consistent with the reported properties of the enzyme, thereby validating the assay conditions. Comparison of the K-ATPase activity between wild-type and HKα2 knockout mice demonstrated that the colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, our pharmacological studies suggested that the gastric H,K-ATPase is sensitive to amiloride.

Section snippets

Animals

All animal studies were approved by and performed in accordance with the VAMC IACUC. All mice were housed and bred in the VAMC animal facility with free access to standard laboratory chow (Harlan) and water. Mice between 10 and 20 weeks were used for the experiments. For HKα1 knockout mice [27], heterozygous 129 Black Swiss mice were bred for wild type and knockout, and siblings were used for each experiment. The genotypes of the experimental mice were confirmed by polymerase chain reaction

Results

Since the K+-dependent ATP hydrolysis activity of the gastric enzyme has been well established, wild-type and HKα1 knockout mice were used to validate the K-ATPase assay. As expected, wild-type murine gastric membrane vesicles exhibited ouabain-resistant, Sch28080- and orthovanadate-sensitive K-ATPase activity. The specific activity was 8.8 ± 1.0 μmol/mg/h (n = 6). Gastric membranes from HKα1 knockout animals were devoid of this K-ATPase activity. As well, the K-ATPase activity was measured in the

Discussion

By comparing the colonic K-ATPase activity in the native tissue of the wild-type and knockout mice of HKα2, we demonstrated that HKα2 is responsible for colonic K-ATPase activity and that the mouse colonic K-ATPase activity was ouabain- and Sch28080-insensitive. However, earlier studies obtained from the functional expression systems with HKα2 from different species demonstrate different degrees of ouabain sensitivity, for example, human nongastric H,K-ATPase is sensitive to ouabain inhibition

Note added in proof

Since acceptance of this manuscript we note an additional citation that supports these studies: A.T.P. Skrabanja, P. Asty, A. Soumarmon, J. Joep, H.H.M. de Pont, M. J.M. Lewin, H+ transport by reconstituted gastric (H+ + K+)-ATPase, Biochim. Biophys. Acta 860 (1986), 131–136.

Acknowledgments

The authors wish to thank Alicia Rudin and Jeannette Lynch for their assistance in the animal studies. This work was supported by the Department of Veterans Affairs and the National Institutes of Health Grant DK-49750.

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  • 1

    Current address: Institute of Medical Biology, CAMS, Kunming, Yunnan Province 650118, China.

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