Expression of the colonic h-k-atpase mrna in the cortical collecting duct: regulation by acid/base balance. Fejes-T[grave]uth, G[umlaut]aza, Erzs[umlaut]abet Rusvai, Kenneth A. Longo, and Anik[grave]u N[cedilla]cray-Fejes-T[grave]uth. Department of Physiology, Dartmouth Medical School, Lebanon, NH 03756
APStracts 2:0085F, 1995.
In addition to the gastric isoform of H-K-ATPase, the colonic isoform is also expressed in the kidney, but its intrarenal localization and exact function is not known. The goal of this study was to determine whether the colonic H-K-ATPase is expressed in the rabbit cortical collecting duct (CCD), and whether it is regulated by changes in acid/base balance. Using quantitative reverse transcriptase (RT) PCR with RNA isolated from immunodissected rabbit CCD cells and degenerate oligonucleotide primers, a PCR product of the predicted size (430 bp) was amplified. The amplified DNA was further characterized by nested PCR and sequencing. Direct sequencing of the 434 bp PCR product revealed 83% identity at the nucleotide level and a 80.4% identity at the deduced amino acid level to the rat colonic H-K-ATPase. Using the same primers and cDNA originating from rabbit distal colon, a DNA fragment with a size and nucleotide sequence identical to that originating from CCD cells was amplified. Furthermore, using PCR screening we isolated and sequenced a 1.5 kb cDNA clone from a rabbit CCD library. The predicted amino acid sequence of the protein encoded by this cDNA is 85% and 82% identical to the corresponding regions of the guinea pig and rat colonic H-K -ATPase, respectively, 70% identical to the H-K-ATPase recently cloned from Bufo marinus, whereas it shows only 45% and 42% homology to the rat Na-K-ATPase a1 subunit and the rat gastric H-K-ATPase, respectively. Northern analysis revealed that an 4.4 kb mRNA hybridizing to a H-K-ATPase cRNA probe is present in rabbit CCD cells as well as in the distal colon. Within the kidney, the colonic H-K -ATPase mRNA was not detectable in proximal tubule, distal convoluted tubule and TALH cells, or the papilla. To determine if changes in acid/base homeostasis regulate mRNA levels of the colonic H-K-ATPase in the CCD, metabolic acidosis or alkalosis was induced in rabbits and H-K-ATPase and -actin mRNA levels were determined in cDNA samples derived from immunodissected CCD cells. The relative abundance of the H-K-ATPase mRNA was calculated from the ratio of [32]dCTP incorporated into the H-K-ATPase PCR product vs. the -actin PCR product. The levels of H-K-ATPase mRNA in CCD cells were significantly higher in alkali-loded than in acid-loaded rabbits (0.01 0.001 vs. 0.0041 0.001; p < 0.001) whereas no significant change was observed in the levels of -actin product. The average differences in H-K-ATPase mRNA levels between alkali-loded and acid -loaded rabbits was 4.5-fold. These results indicate that the colonic form of H-K-ATPase is expressed in rabbit CCD cells, and its levels are higher in animals with metabolic alkalosis vs. acidosis. The cellular localization and the physiological role of the colonic H-K -ATPase in the CCD remain to be elucidated. 

Received 25 April 1994; accepted in final form 28 April 195.
APS Manuscript Number F133-4.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 26 May 1995.