Adaptation of the outer medullary collecting duct to metabolic
acidosis in vitro.
Tsuruoka, Shuichi, and George J. Schwartz.
Departments of Pediatrics and Medicine, University of Rochester
School of Medicine, Rochester, NY 14642
APStracts 5:0162F, 1998.
Metabolic acidosis in vivo, as well as in vitro (1 h at pH 6.8
followed by 2 h at pH 7.4) stimulates H+- ATPase-dependent H+
secretion in outer medullary collecting ducts from the inner stripe
(OMCDi) (J. Clin. Invest. 99:1420, 1997). Bastani et al have shown
that the adaptation to metabolic acidosis in vivo is mediated by an
apical polarization of H+ pumps without an increase in total H+ pump
mRNA or protein (J. Clin. Invest. 88:126, 1991). To further address
the mechanism of adaptation, we measured net HCO3- absorption before
and after applying protein/RNA synthesis and signal transduction
inhibitors during the 1 h of low pH and a cytoskeletal inhibitor
duiring the entire 3 h incubation. Net HCO3- transport, measured by
microcalorimetry, increased 33% after in vitro acidosis. This
increase was prevented by application during the first h of
anisomycin (10 M) or actinomycin D (4 M), but not by anisomycin
applied during the 2 h incubation at pH 7.4. Similar results were
obtained with the cell calcium chelator, BAPTA-AM (20 M), the
calmodulin antagonist, calmidazolium (30 nM), the endoplasmic
reticulum Ca-ATPase inhibitor, thapsigargin (100 nM), and the protein
kinase C inhibitor, staurosporine (100 nM), applied during the 1 h at
pH 6.8, but not with BAPTA-AM or thapsigargin used during the 2 h
incubation at pH 7.4. Colchicine (10 M) applied during the entire 3 h
incubation also prevented this adaptive increase in H+ secretion
while lumicolchicine (10 M, the inactive congener) did not.
Colchicine also reversibly prevented any adaptive increases in
transepithelial positive voltage. Thus, the adaptation to acidosis in
vitro required RNA and protein synthesis, changes in intracellular
calcium and protein kinase C activity, and intact microtubules. Time
was required for the adaptation to occur as the increase in HCO3-
transport was small after < 3 h incubation. Protein synthesis and
changes in cell calcium were critical during the initial period of
low pH but not once the acid stimulus had been removed. Exocytosis of
H+ pumps appears to occur continually during the entire 3 h
incubation. These data would suggest that the synthesis and
regulation of proteins involved in shuttling H+ pumps in cytoplasmic
vesicles to the apical membrane via exocytosis are important for the
OMCDi to adapt to low pH in vitro and probably to metabolic acidosis
in vivo.
Received 28 April 1998; accepted in final form 10 September 1998.
APS Manuscript Number F96-8.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 20 October 1998