Faculty Articles

Title

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

ISBN or ISSN

0002-9513

Publication Title

The American journal of physiology

Volume

276

Issue

2 Pt 2

Publication Date / Copyright Date

2-1-1999

First Page

237

Last Page

245

Publisher

American Physiological Society

Abstract

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.

Disciplines

Medical Specialties | Medicine and Health Sciences | Osteopathic Medicine and Osteopathy

Keywords

Animals, Cell Line, Colon, DNA, Complementary, H(+)-K(+)-Exchanging ATPase, Hydrogen-Ion Concentration, Isoenzymes, Kidney Cortex, Kidney Tubules, Collecting, Potassium, RNA, Messenger, Rabbits

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