Molecular identification of the renal H+,K+-ATPases.

The pharmacological properties of H+,K+-ATPase activity described in the kidney were not necessarily consistent with the properties of the well-characterized gastric H+,K+-ATPase. Recent molecular biology experiments suggest that renal H+,K+-ATPase activity may be the product of several closely related P-type ATPases. At least 3 different pumps containing the HKalpha1, HKalpha2a, and HKalpha2c subunits have been detected in rabbit kidney. The current view is that these HKalpha subunits arose through gene duplication early in evolution and the proteins evolved their differing activities over time. The HKbeta protein associates with HKalpha1 in gastric tissues and is the likely mate for the HKalpha1 subunit in renal tissues. Three distinct beta subunits have been implicated as possible partners for the HKalpha2 subunits, but it remains to be determined which beta subunit predominantly associates with the HKalpha2 subunits in vivo. Sequence analysis suggests the beta subunit was constrained by size and shape of the protein rather than specific amino acid content during the course of evolution. Multiple H+,K+-ATPases in the kidney may be an important adaptation providing redundancy for the essential physiological function of maintaining ionic balance.

H-K-ATPase in the RCCT-28A rabbit cortical collecting duct cell line.

In the present study, we demonstrate that the rabbit cortical collecting duct cell line RCCT-28A possesses three distinct H-K-ATPase catalytic subunits (HKalpha). Intracellular measurements of RCCT-28A cells using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) indicated that the mechanism accounting for recovery from an acid load exhibited both K+ dependence and sensitivity to Sch-28080 characteristic of H-K-ATPases. Recovery rates were 0.022 +/- 0.005 pH units/min in the presence of K+, 0.004 +/- 0.002 in the absence of K+, and 0.002 +/- 0.002 in the presence of Sch-28080. The mRNAs encoding the HKalpha1 subunit and the H-K-ATPase beta-subunit (HKbeta) were detected by RT-PCR. In addition, two HKalpha2 species were found by RT-PCR and 5' rapid amplification of cDNA ends (5'-RACE) in the rabbit renal cortex. One was homologous to HKalpha2 cDNAs generated from other species, and the second was novel. The latter, referred to as HKalpha2c, encoded an apparent 61-residue amino-terminal extension that bore no homology to reported sequences. Antipeptide antibodies were designed on the basis of this extension, and these antibodies recognized a protein of the appropriate mass in both rabbit renal tissue samples and RCCT-28A cells. Such findings constitute very strong evidence for expression of the HKalpha2c subunit in vivo. The results suggest that the rabbit kidney and RCCT-28A cells express at least three distinct H-K-ATPases.

The effects of potassium depletion and supplementation on blood pressure: a clinical review.

Nonpharmacologic treatment currently is recognized as an important part in the treatment of hypertension, and the role of dietary potassium intake in blood pressure (BP) control is becoming quite evident. Clinical studies have examined the mechanism by which hypokalemia can increase BP and the benefit of a large potassium intake on BP control. Epidemiologic data suggest that potassium intake and BP are correlated inversely. In normotensive subjects, those who are salt sensitive or who have a family history of hypertension appear to benefit most from the hypotensive effects of potassium supplementation. The greatest hypotensive effect of potassium supplementation occurs in patients with severe hypertension. This effect is pronounced with prolonged potassium supplementation. The antihypertensive effect of increased potassium intake appears to be mediated by several factors, which include enhancing natriuresis, modulating baroreflex sensitivity, direct vasodilation, or lowering cardiovascular reactivity to norepinephrine or angiotensin II. Potassium repletion in patients with diuretic-induced hypokalemia improves BP control. An increase in potassium intake should be included in the nonpharmacologic management of patients with uncomplicated hypertension.

Renal expression of the gene encoding the gastric H(+)-K(+)-ATPase beta-subunit.

The gastric mucosal parietal cells and cells of the renal collecting duct both possess H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) activities. In the stomach, the H(+)-K(+)-ATPase (EC 3.6.1.3) is responsible for acidification of luminal contents. The kidney H(+)-K(+)-ATPase protein(s) contribute to potassium reabsorption and secretion of hydrogen ions to maintain potassium and acid-base homeostasis. The stomach H(+)-K(+)-ATPase is well defined and consists of an alpha-catalytic subunit of apparent molecular mass of 95 kDa and a highly glycosylated beta-subunit of 60-90 kDa. The molecular identity of the protein that mediates the H(+)-K(+)-ATPase activity in the kidney has been addressed in this paper. A combination of RNA hybridizations, polymerase chain reaction analysis of kidney RNA, and sequence analysis of cDNAs indicated that gastric H(+)-K(+)-ATPase beta-subunit mRNA is present in kidney. Immunoblotting with antibodies specific for the gastric H(+)-K(+)-ATPase beta-subunit detected proteins, which, after deglycosylation, had the same molecular mass as the gastric beta-subunit in membrane protein preparations from rabbit, pig, rat, and mouse kidneys. Furthermore, we have used transgenic mice to demonstrate that the gastric H(+)-K(+)-ATPase beta-subunit gene contains cis-acting regulatory sequences that are active in both gastric parietal cells and the renal collecting ducts. Overall, these data indicate that the gastric H(+)-K(+)-ATPase beta-subunit is found in the kidney and probably associates with the gastric H(+)-K(+)-ATPase alpha-subunit and/or other P-type ATPase alpha-subunits, thus contributing to acid-base and potassium homeostasis.

Effects of in vitro aldosterone on the rabbit cortical collecting tubule.

Considerable evidence indicates that the cortical collecting tubule is a target epithelium for aldosterone. Isolated perfused cortical collecting tubules from rabbits given large doses of deoxycorticosterone acetate (DOCA) for several days, or whose endogenous production of aldosterone is increased by dietary means, exhibit large lumen-negative transepithelial voltages, increased sodium (Na) absorption, and increased potassium (K) secretion compared with tubules from normal animals. However, controversy exists regarding the response of this nephron segment to acute in vitro administration of aldosterone. To address this issue we performed three groups of experiments: 1) clearance experiments on adrenalectomized rabbits to determine the minimum time required after in vivo aldosterone administration before significant changes in sodium excretion are observed; 2) microperfusion experiments on cortical collecting tubules from normal and adrenalectomized rabbits in which transepithelial voltage was measured before and after adding aldosterone to the bath; 3) microperfusion experiments on cortical collecting tubules from adrenalectomized rabbits in which transepithelial voltage, sodium and potassium flux were measured before and after in vitro exposure to aldosterone or dexamethasone. The clearance studies demonstrate that after a 2 hr latent period aldosterone produces significant antinatriuresis without change in K excretion. In vitro studies failed to reveal a steroid-induced change in the transepithelial voltage of cortical collecting tubules from either normal or adrenalectomized rabbits. However, aldosterone added in vitro to collecting tubules from adrenalectomized rabbits produced an increase in net Na absorption without a significant change in voltage or K secretion.(ABSTRACT TRUNCATED AT 250 WORDS)