QUANTITATIVE RT-PCR ANALYSIS OF MESSENGER-RNAS ENCODING A COLONIC PUTATIVE H,K-ATPASE ALPHA-SUBUNIT ALONG THE RAT NEPHRON - EFFECT OF K+ DEPLETION

The rat nephron displays two ouabain-sensitive K-ATPases: one, which i s present in proximal tubules and thick ascending limbs of normal rats , is specifically activated by K+ and is down-regulated by K+ depletio n. whereas the other one appears in collecting ducts of K+-depleted ra ts and is activated by either Na+ or K+ To determine which of these tw o ATPases is similar to colonic-type H,K-ATPase, we quantitated by rev erse transcriptase-polymerase chain reaction (RT-PCR) the mRNAs encodi ng the colonic H,K-ATPase alpha-subunit in microdissected nephron segm ents, In normal rats, statistically significant amounts of colonic H,K -ATPase mRNAs were detected exclusively in cortical thick ascending li mbs and cortical collecting ducts (200-500 copies/mm). Because these l evels of expression were low (1-1.2 copies/target cell), they probably have no physiological relevance. In rats fed a K+-depleted diet for 2 weeks, expression of colonic H,K-ATPase was markedly enhanced in cort ical and medullary collecting ducts (5000-12,000 copies/mm or 30-40 co pies per cell), whereas ii remained low in all other nephron segments. Thus, colonic H,K-ATPase a subunit is specifically expressed in corti cal and outer medullary collecting ducts of K+-depleted rats where it likely accounts fur the ouabain-sensitive K-ATPase activity.