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Am J Physiol Cell Physiol. 2004 Jul;287(1):C97-105.

Glucose activates H(+)-ATPase in kidney epithelial cells.

Author information

1
Department of Medicine, University of Florida, College of Medicine, Gainesille, Florida 32610, USA. suguru.nakamura@murraystate.edu

Abstract

The vacuolar H(+)-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H(+)-ATPase remain largely unknown. The present study examines the effect of glucose on H(+)-ATPase activity in the pig kidney epithelial cell line LLC-PK(1). Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH(3)/NH(4)(+) prepulse, and rates of intracellular pH (pH(i)) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 microM ethylisopropylamiloride and were K(+) free to eliminate Na(+)/H(+) exchange and H(+)-K(+)-ATPase activity. After NH(3)/NH(4)(+)-induced acidification, LLC-PK(1) cells had a significant pH(i) recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pH(i) recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pH(i) recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H(+)-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-d-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 microM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification.

PMID:
15189820
DOI:
10.1152/ajpcell.00469.2003
[Indexed for MEDLINE]
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