Endothelial na-k-cl cotransport regulation by hypertonicity and
vasopressin: phosphorylation of cotransport protein.
O'donnell, Martha E., Anthony Martinez, and Dandan Sun.
Department of Human Physiology, School of Medicine, University of
California, Davis, CA 95616-8644
APStracts 2:0295C, 1995.
The Na-K-Cl cotransport system of vascular endothelial cells plays a
central role in maintenance and regulation of intracellular volume.
Activity of the cotransporter is modulated both by hormones and by
extracellular tonicity. Vasopressin and other hormones which
stimulate the endothelial cotransporter act via a Ca- and calmodulin
-dependent pathway. Little is known, however, about the mechanisms
which mediate cell shrinkage-induced stimulation of cotransport
activity. In the present study, we evaluated the Ca-dependence of
cell shrinkage-stimulated Na-K-Cl cotransport activity and cell
volume recovery of cultured bovine aortic endothelial cells, and also
the effects of protein kinase and phosphatase inhibitors on these
processes. In addition, to investigate the possibility that hormones
and/or hypertonicity regulate endothelial Na-K-Cl cotransport via
direct phosphorylation of the cotransporter protein, we employed a
monoclonal antibody to the human colonic T84 epithelial cell Na-K-Cl
cotransport protein (T4 antibody) for Western blot analysis and
immunoprecipitation of phosphoprotein. Our studies revealed that both
cell shrinkage-stimulated net K uptake and recovery of intracellular
volume were Ca-dependent. We also found that hypertonicity-induced
stimulation of cotransport activity was blocked by several inhibitors
of Ca- and calmodulin-dependent protein kinases. Further, inhibitors
of myosin light chain kinase blocked cell shrinkage-stimulated
cotransport and recovery of intracellular volume, while having no
effect on vasopressin-stimulated cotransport. Western blot analysis
of bovine aortic and cerebral microvascular endothelial cell membrane
preparations revealed a 170 kDa protein recognized by the T4
antibody. In addition, we found that hypertonicity induced a marked
increase in phosphorylation of the endothelial cotransport protein,
as did vasopressin, bradykinin, okadaic acid and calyculin A. Our
findings indicate that modulation of endothelial cell Na-K-Cl
cotransport activity by hypertonicity and by stimulatory hormones
occurs via pathways involving Ca- and calmodulin-dependent protein
kinases and direct phosphorylation of the cotransport protein.
Received 19 June 1995; accepted in final form 2 August 1995.
APS Manuscript Number C353-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 14 August 1995.