REGULATION OF LUNG ENDOTHELIAL CONTRACTION/PERMEABILITY

DESCRIPTION: (Adapted from the applicant's abstract and Specific Aims.)
A key vascular endothelial cell (EC) function is the regulation of
exchanges across the capillary wall between circulating blood and the
interstitial fluid. EC barrier dysfunction is an important hallmark of
inflammation and results in tissue edema and organ dysfunction and is
central development of life-threatening pulmonary edema, vascular
ischemia and atherosclerosis. Barrier dysfunction induced by the
multifunctional serine protease, thrombin, involves contraction-induced
intercellular gap formation linked to modulation of actin polymerization
and myosin phosphorylation. Thrombin-induced EC contractile events were
dependent upon G protein-coupled phosphoinositide metabolism resulting
in increased [Ca2+i] and protein kinase C (PKC) activation. Direct PKC
activation by phorbol esters produced gap formation and EC barrier
dysfunction, whereas agents which increased cAMP were found to protect
EC against thrombin-induced gap formation and barrier dysfunction. The
mechanisms by which both PKC and cAMP-dependent protein kinase A (PKA)
regulate actomyosin interactions are unknown. Preliminary data indicate
both PKC and PKA modulate myosin light chain kinase activity and that
EC MLCK has specific biochemical and regulatory features which indicate
it is distinct from MLCKs derived from other tissues. The application
proposes to examine early, intermediate and terminal activation
sequences involved in the phosphorylation of myosin light chains (MLC20)
and the actin binding protein, caldesmon77, resulting in productive
actomyosin interaction, cellular contraction, gap formation, and loss of
EC barrier function. The Specific Aims are: 1) to investigate
potential mechanisms of cAMP-dependent protein kinase A (PKA)-mediated
barrier protection; 2) to characterize protein kinase C-mediated
regulation of actomyosin interactions; 3) to examine the role of
Ca2+/calmodulin/phosphocalmodulin in regulation of MLCK activity; and 4)
to clone, sequence, and purify myosin light chain kinase (MLCK) from
vascular EC. Because preservation of a cellular barrier to plasma
proteins is vital, these studies which define the biochemical basis of
EC actomyosin activation may further enhance understanding of the role
of contractile proteins in control of EC barrier function.