Volume-sensitive myosin phosphorylation in vascular endothelial
cells: correlation with na-k-2cl cotransport.
Klein, Janet D., and W. Charles O'neill.
Renal Division, Department of Medicine, and Department of
Physiology, Emory University School of Medicine, Atlanta, GA
30322
APStracts 2:0304C, 1995.
To identify protein kinases that are regulated by cell volume, we
examined protein phosphorylation in hypertonically shrunken aortic
endothelial cells. Shrinkage reversibly increased, and swelling
decreased, phosphorylation of a 19 kD cytoskeletal protein identified
as myosin light chain (MLC) by immune precipitation and
immunoblotting. Shrinkage also increased MLC phosphorylation in human
umbilical vein endothelial cells, rat aortic smooth muscle cells, and
human dermal fibroblasts. Phosphorylation was blocked by ML-7, an
inhibitor of MLC kinase (MLCK). Neither inhibition of protein kinase
C nor inhibition of myosin phosphatase (with calyculin) altered MLC
phosphorylation. Peptide mapping of MLC indicated phosphorylation by
MLCK. Na-K-2Cl cotransport activation paralleled MLC phosphorylation
in hypertonic medium. Na-K-2Cl was stimulated by low concentrations
of ML-7 with no further stimulation by hypertonic shrinkage, and
inhibited by higher concentrations, paralleling inhibition of MLC
phosphorylation. Shrinkage-induced phosphorylation of the
cotransporter was not blocked by ML-7. We conclude that cell volume
regulates MLC phosphorylation by MLCK. MLCK influences Na-K-2Cl
cotransport but independently of cotransporter phosphorylation. These
data suggest an important link between cell volume, volume-regulatory
transporters, and the contractile state of the cytoskeleton.
Received 9 March 1995; accepted in final form 28 July 1995.
APS Manuscript Number C132-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 24 August 1995.