EXPRESSION OF COLONIC H-K-ATPASE MESSENGER-RNA IN CORTICAL COLLECTING DUCT - REGULATION BY ACID/BASE BALANCE

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 are 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. With quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from immunodissected rabbit CCD cells and degenerate oligonucleotide primers, a PCR product of the predicted size (similar to 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 an 80.4% identity at the deduced amino acid level to the rat colonic H-K-ATPase. With 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, and 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 alpha(1)-subunit and the rat gastric H-K-ATPase, respectively. Northern analysis revealed that an similar to 4.4-kb mRNA hybridizing to a H-K-ATPase cRNA probe is present in rabbit CCD cells, as well as in distal colon. Within the kidney, the colonic H-K-ATPase mRNA was not detectable in proximal tubule, distal convoluted tubule, thick ascending limb of Henle's loop cells, or papilla. To determine whether 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 beta-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 [P-32]dCTP incorporated into the H-K-ATPase PCR product vs. the beta-actin PCR product. The levels of H-K-ATPase mRNA in CCD cells were significantly higher in alkali-loaded 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 beta-actin product. The average difference in H-K-ATPase mRNA levels between alkali-loaded and acid-loaded rabbits was similar to 4.5-fold. These results indicate that the colonic form of H-K-ATPase is expressed in rabbit CCD cells and that its levels are higher in animals with metabolic alkalosis vs. acidosis. The cellular localization and physiological role of the colonic H-K-ATPase in the CCD remain to be elucidated.