Regulation of endothelial cell gap formation and barrier function by myosin-associated phosphatase activities

A. D. Verin, C. E. Patterson, M. A. Day, J. G.N. Garcia

Research output: Contribution to journalArticle

105 Scopus citations

Abstract

Thrombin-induced cultured bovine endothelial cell (EC) gap formation and albumin permeability is initiated by contraction, which is dependent upon myosin light chain kinase-mediated myosin light chain (MLC) phosphorylation. MLC are then rapidly dephosphorylated (J. G. N. Garcia, H. W. Davis, and C. E. Patterson. J. Cell. Physiol. 163: 510-522, 1995), suggesting a role for MLC dephosphorylation in regulation of EC barrier function. Therefore, we studied the effect of semiselective protein phosphatase (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 (three- to fourfold) and EC permeability (eightfold). EC homogenates were utilized to assess Ser/Thr PPase activities using either [32P]phosphorylase A or 32P-labeled 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 PPase 2A) 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 PPase 1 was present in both fractions, whereas PPase 2A 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.

Original languageEnglish (US)
Pages (from-to)L99-L108
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume269
Issue number1 13-1
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

Keywords

  • bovine pulmonary artery endothelium
  • endothelial cell permeability and myosin light chain phosphorylation
  • semiselective PPase inhibitors

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

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