EFFECT OF ACID BASE BALANCE ON H-ATPASE 31 KD SUBUNIT MESSENGER-RNA LEVELS IN COLLECTING DUCT CELLS/

The cortical collecting duct (CCD) adapts to disturbances of acid/base balance by adjusting the direction and magnitude of its HCO3 transpor t. The molecular events involved in this adaptation are incompletely u nderstood, but it seems that adaptation is accompanied by changes in t he activity and intracellular distribution of the vacuolar H-ATPase. T he goal of this study was to examine the effects of metabolic acidosis and alkali load on the expression of the mRNA encoding the 31 kD subu nit of the vacuolar H-ATPase in rabbit CCD cells, Pairs of rabbits rec eived either a NH4Cl load or a NaHCO3 load for 16 hours, resulting in a urinary pH of 5.53 +/- 0.38 and 8.42 +/- 0.10, respectively. CCD cel ls were isolated by immunodissection and mRNA levels of the H-ATPase 3 1 kD subunit and of beta-actin were determined from the same cDNA samp les by quantitative RT-PCR. H-ATPase mRNA levels were significantly hi gher in CCD cells from acidotic than alkali-loaded rabbits (2.51 +/- 1 .3 vs. 0.65 +/- 0.2; P < 0.05). Similar differences in the H-ATPase 31 kD subunit mRNA levels were observed by Northern blotting. beta-actin mRNA levels were comparable in CCD cells of the two groups. The distr ibution of the H-ATPase 31 kD subunit mRNA was determined among the th ree cell types of the CCD, that is in alpha- and beta-intercalated cel ls (alpha-ICC and beta-ICC) and principal cells (PC) isolated by fluor escence-activated cell sorting. The level of expression was comparable in alpha-ICCs and beta-ICCs, whereas PCs contained very low levels of H-ATPase mRNA. In both alpha-ICC and beta-ICC the levels of the 31 kD H-ATPase mRNA were significantly higher in acidotic than in alkali lo aded rabbits. These results indicate that in the rabbit CCD changes in acid/base balance not only regulate the subcellular distribution of t he vacuolar H-ATPase but also alter its expression, at least at the mR NA level.