We examined the effect of Cl- depletion metabolic
alkalosis (CDA) on
H(+)-ATPase and
band 3 protein localization in intercalated cells (IC) of the rat cortical collecting duct (CCD) and the outer medullary collecting duct (OMCD). After 30 min of
peritoneal dialysis against 0.15 M NaHCO3 to produce CDA, or Ringer
bicarbonate to serve as controls (CON), both groups were infused intravenously with an 80 mM Cl-
solution for 90 min. For CDA vs. CON, physiological parameters were as follows: plasma total CO2, 38.0 +/- 1.1 vs. 27.8 +/- 0.6 meq/l (P less than 0.001); urinary total CO2 excretion, 141 +/- 89 vs. 20 +/- 3 neq.min-1.100 g body wt-1; and urinary Cl- excretion, 20 +/- 10 vs. 486 +/- 144 neq.min-1.100 g body wt-1 (P less than 0.001).
H(+)-ATPase was localized in thin sections using a rabbit polyclonal antibody against the 70-kDa subunit of bovine brain
H(+)-ATPase.
Band 3 protein was localized using a polyclonal antibody against the 43-kDa subunit of the cytoplasmic domain of human erythrocyte
band 3 protein. In CON rats,
H(+)-ATPase localized along the apical plasma membrane and over the apical cytoplasmic vesicles of type A ICs in the CCD and ICs of the OMCD.
H(+)-ATPase was observed along the basolateral plasma membrane and over cytoplasmic vesicles throughout type B ICs. In CDA rats,
H(+)-ATPase was only observed over apical cytoplasmic vesicles in type A ICs and in the majority of OMCD ICs. In type B ICs,
H(+)-ATPase staining was intensified along the basal plasma membrane in CDA.
Band 3 protein was consistently localized in the basolateral plasma membrane of all type A cells in the CCD and ICs of the OMCD in both CON and CDA. In summary, stimulation of HCO3- secretion in rats caused withdrawal of
H(+)-ATPase from the apical plasma membrane and storage in apical cytoplasmic vesicles of ICs of the OMCD and type A ICs of the CCD.
H(+)-ATPase appeared to be inserted into the basal plasma membrane of type B ICs. These findings suggest that, during correction of CDA,
proton secretion by type A and OMCD ICs is suppressed and
proton transport across the basolateral plasma membrane of type B ICs is stimulated.