Expression of colonic H-K-ATPase mRNA 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 (approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000136:Acid-Base Equilibrium; D000471:Alkalosis / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003106:Colon / Q000201:enzymology*; D018076:DNA, Complementary / Q000302:isolation & purification; D006168:Guinea Pigs; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics*; D007672:Kidney Cortex; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D014018:Tissue Distribution
D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D017506:H(+)-K(+)-Exchanging ATPase