REGULATION OF MYOSIN PHOSPHORYLATION IN ENDOTHELIUM

Project: Research project

Description

Increased vascular permeability is a prominent feature of the adult
respiratory distress syndrome, ischemic coronary artery disease, and
atherosclerosis. The applicant has shown that alpha-thrombin, an important
coagulation protease, induces a reversible reorganization of endothelial
cell microfilaments and conformational changes intimately reorganization of
endothelial cell microfilaments and conformational changes intimately
associated with an increased albumin clearance across endothelial
monolayers. The biochemical events by which thrombin induces cellular
contraction, however, are unclear. The proposed research will test the
hypothesis that thrombin-induced endothelial contractile events are
mediated by G protein-coupled phosphoinositide hydrolysis resulting in
increased [Ca++]i and Ca++- activated protein kinase-induced caldesmon and
myosin light chain phosphorylation. Thrombin-mediated phosphorylation of
endothelial myosin (light and heavy chain) and the actin binding protein
caldesmon, will be measured in human umbilical vein and bovine pulmonary
endothelial cells by radioimmunoblotting and 2-dimensional SDS PAGE
techniques. The intracellular distribution of actin, myosin, and caldesmon
will be analyzed by immunofluorescent microscopy and functional assessment
of in vitro endothelial cell permeability will utilize a novel system
developed by the applicant. Specific Aim #1 will investigate the role of
guanine nucleotide regulatory proteins (G proteins) in the reorganization
of the endothelial cytoskeleton using G protein activators (fluoride, GTP
gamma S) as well as G protein inhibitors (GDP beta S, pertussis and cholera
toxins). Specific Aim #2 will examine the role of the phosphoinositol
metabolites, inositol triphosphate and diacylglycerol, in the transduction
of signals which alter the endothelial cytoskeleton. The regulation of
[Ca++]i, a critical second messenger regulating important cellular
processes including myosin phosphorylation, will be examined in Specific
Aim #3. In Specific Aim #4 the Ca++-dependent and -independent protein
phosphorylation of caldesmon and myosin by protein kinase C, cAMP-protein
kinase, Ca++-calmodulin protein kinase II, and myosin light chain kinase
will be examined. The identity of the relevant kinase-phosphorylation
profiles, and phosphopeptide mapping techniques. These studies which
investigate the regulation of endothelial actomyosin activation may further
enhance understanding of the role of contractile proteins in control of
endothelial cell barrier function.
StatusFinished
Effective start/end date4/1/903/31/96

Funding

  • National Institutes of Health
  • National Institutes of Health: $106,260.00
  • National Institutes of Health
  • National Institutes of Health: $93,870.00
  • National Institutes of Health

Fingerprint

Myosins
GTP-Binding Proteins
Thrombin
Endothelium
Calmodulin-Binding Proteins
Phosphorylation
Myosin Light Chains
Protein Kinases
Cytoskeleton
Actin Cytoskeleton
Actins
Myosin-Light-Chain Kinase
Endothelial Cells
Contractile Proteins
Phosphopeptides
Actomyosin
Calcium-Calmodulin-Dependent Protein Kinases
Umbilical Veins
Myosin Heavy Chains
Diglycerides

ASJC

  • Medicine(all)