Regulation of endothelial cell gap formation and barrier function
by myosin-associated phosphatase activities.
Verin, Alexander D., Carolyn E. Patterson, Melissa A. Day, and Joe G.
N. Garcia.
Department of Medicine, Indiana University School of Medicine,
Richard L. Roudebush Veterans Administration Medical Center,
Indianapolis, Indiana 46202
APStracts 2:0037L, 1995.
Thrombin-induced cultured bovine endothelial cells (EC) gap formation
and albumin permeability is initiated by contraction, which is depend
upon myosin light chain kinase-mediated myosin light chain (MLC)
phosphorylation. MLC are then rapidly dephosphorylated (16),
suggesting a role for MLC dephosphorylation in regulation of EC
barrier function. Therefore, we studied the effect of semi-selective
PPase inhibitors, calyculin A and okadaic acid, on MLC
phosphorylation status, myosin-associated PPase activity and EC
monolayer permeability. Calyculin A (0.1-10 nM), but not okadaic acid
(1-100 nM), produced significant dose-dependent enhancement of both
MLC phosphorylation (3-4 fold) and EC permeability (8 fold). EC
homogenates were utilized to assess Ser/Thr PPase activities using
either 32P-phosphorylase A or 32P-skeletal MLC as substrates.
Calyculin A at 5 nM (sufficient to inhibit type 1 and type 2A PPase)
produced 95% inhibition of all EC PPase activity against both
substrates, whereas 2 nM okadaic acid (selective for type 2A PPase)
only partially inhibited EC PPase activity (40-60%). Fractionation of
EC homogenates produced a supernatant fraction containing < 10% of
total myosin and a pellet fraction with > 90% of total myosin.
PPase activity in the myosin-enriched pellet was insensitive to 2 nM
okadaic acid (0% inhibition) but sensitive to 5 nM calyculin (> 95%
inhibition). Immunoreactive type 1 PPase was present in both
fractions whereas type 2A PPase was present only in the myosin
-depleted fraction. We conclude that a type 1 myosin-associated PPase
is involved in regulation of EC contractility and barrier function.
Received 23 January 1995; accepted in final form 9 March 1995.
APS Manuscript Number L20-5.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 21 March 1995.