Document Detail

H-K-ATPase in the RCCT-28A rabbit cortical collecting duct cell line.
MedLine Citation:
PMID:  9950954     Owner:  NLM     Status:  MEDLINE    
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.
W G Campbell; I D Weiner; C S Wingo; B D Cain
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The American journal of physiology     Volume:  276     ISSN:  0002-9513     ISO Abbreviation:  Am. J. Physiol.     Publication Date:  1999 Feb 
Date Detail:
Created Date:  1999-03-30     Completed Date:  1999-03-30     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370511     Medline TA:  Am J Physiol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  F237-45     Citation Subset:  IM    
Department of Biochemistry and Molecular Biology, and Division of Nephrology, Hypertension, and Transplantation, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Data Bank Information
Bank Name/Acc. No.:
GENBANK/AF023128;  AF023129
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MeSH Terms
Cell Line
Colon / metabolism
DNA, Complementary / genetics,  metabolism
H(+)-K(+)-Exchanging ATPase / genetics,  metabolism*
Hydrogen-Ion Concentration
Isoenzymes / genetics
Kidney Cortex / metabolism
Kidney Tubules, Collecting / cytology,  enzymology*
Potassium / pharmacology
RNA, Messenger / metabolism
Grant Support
Reg. No./Substance:
0/DNA, Complementary; 0/Isoenzymes; 0/RNA, Messenger; 7440-09-7/Potassium; EC ATPase

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