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Inhibition of serine‐threonine protein phosphatases decreases barrier function of rat pulmonary microvascular endothelial cells
Author(s) -
Diwan Abdul H.,
Honkanen Richard E.,
Schaeffer Richard C.,
Strada Samuel J.,
Thompson William J.
Publication year - 1997
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(199706)171:3<259::aid-jcp4>3.0.co;2-n
Subject(s) - threonine , serine , phosphatase , function (biology) , phosphorylation , microbiology and biotechnology , chemistry , barrier function , protein phosphatase 1 , biochemistry , protein phosphatase 2 , biology
The flux of multisized fluorescein‐isothiocyanate‐labeled hydroxy ethyl starch (FITC‐HES) macromolecules was used to assess changes in barrier function of rat pulmonary microvascular endothelial cell (RPMVEC) monolayers exposed to protein phosphatase (PP) inhibitors or cGMP analogs and atriopeptin (ANF). Two potent PP inhibitors, calyculin A (CalA) and okadaic acid (OA), increased RPMVEC permeability in a dose‐ and time‐dependent manner, and CalA had a higher intrinsic activity than OA. In contrast, ANF and potent cGMP analogs had no effect on basal RPMVEC permeability. The phosphohistone PP activity contained in RPMVEC sonicates was inhibited by OA with an inhibition profile that suggested at least two components were present, with PP2A accounting for ∼70% of the OA‐inhibitable phosphohistone phosphatase activity. Following separation with heparin‐Sepharose chromatography, PP activity exhibited equipotent inhibition by CalA and differential inhibition by OA. Differential inhibition of PP1 and PP2A by OA suggested that PP1 is involved in regulating RPMVEC barrier function. Permeabilized RPMVEC showed increased phosphorylation of several proteins in the presence of phosphatase inhibitors. Treatment with KT 5926, a myosin light chain (MLC) kinase (MLCK) inhibitor, or rolipram, a phosphodiesterase inhibitor, decreased 32 P incorporation into immunoprecipitated MLC by CalA and OA. However, this effect did not abolish either the CalA‐ or OA‐induced decrease in the RPMVEC barrier function. Localization of filamentous (F) actin was at the periphery as well as in the cytoplasm and perinuclear region, whereas nonmuscle myosin was seen in the perinuclear region. Neither of these patterns was changed in the presence of CalA. Thus, cGMP does not alter RPMVEC permeability, but inhibition of PP activity results in loss of barrier function by a mechanism independent from MLC phosphorylation. J. Cell. Physiol. 171:259–270, 1997. © 1997 Wiley‐Liss, Inc.