Volume-sensitive myosin phosphorylation in vascular endothelial cells: correlation with Na-K-2Cl cotransport.
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-kDa 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 was 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.
Klein
J D
JD
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
O'Neill
W C
WC
D000818:Animals; D001381:Azepines / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D028021:Sodium-Potassium-Chloride Symporters
D001381:Azepines; D002352:Carrier Proteins; D006982:Hypertonic Solutions; D009281:Naphthalenes; D028021:Sodium-Potassium-Chloride Symporters; C070571:ML 7; D009219:Myosin-Light-Chain Kinase; D009218:Myosins