PROTEIN-KINASE-C-BETA MODULATES THROMBIN-INDUCED CA2+ SIGNALING AND ENDOTHELIAL PERMEABILITY INCREASE

We investigated the function of the Ca2+-dependent protein kinase C (P KC) beta(1) in the regulation of endothelial barrier property. Human d ermal microvascular endothelial cells (HMEC-1) were transduced with fu ll-length PKC beta(1) antisense (AS) cDNA or control pLNCX vector to g enerate stable cell lines (HMEC-AS and HMEC-pLNCX, respectively). Anal yses indicated that HMEC-AS expressed the antisense PKC beta(1) transc ript with decreased PKC beta protein level (without a change in PKC al pha or PKC epsilon). The baseline transendothelial I-125-albumin clear ance rates of HMEC-1, HMEC-pLNCX, and HMEC-AS were 5.0 +/- 0.5 x 10(-2 ), 6.8 +/- 0.4 x 10(-2), and 6.9 +/- 0.6 x 10(-2) mu l/min, respective ly. Activation of HMEC-1 and HMEC-pLNCX with phorbol 12-myristate 13-a cetate (PMA) increased the rates to the respective 14.5 +/- 1.7 x 10(- 2) mu l/min and 16.9 +/- 2.8 x 10-(2) mu l/min (corresponding to 191% and 149% increases over baseline). However, in HMEC-AS, PMA increased the rate to 9.8 +/- 1.0 x 10(-2) mu l/min (420io). When HMEC-1 and HME C-pLNCX were activated with thrombin, the rates increased to 10.8 +/- 1.4 x 10(-2) and 14.0 +/- 1.9 x 10-2 mu l/min, respectively (116% and 106%). In contrast, thrombin stimulation of HMEC-AS more than doubled the increase to 27.2 +/- 3.5 x 10(-2) mu l/min (294%). Furthermore, th e thrombin-induced peak increase in the [Ca2+](i) in HMEC-AS was great er than in control cells. Fluorescence-activated cell sorter analysis of thrombin receptor expression indicated that the augmented thrombin- induced responses were not attributable to altered receptor density in HMEC-AS. These results indicate that PKC beta functions in a negative feedback manner to inactivate thrombin-generated signals and thereby modulates the endothelial permeability increase. Because decreased PKC beta expression significantly reduced the PMA-induced permeability in crease, PKC beta may downregulate thrombin receptor function upstream of PKC activation (i.e., Ca2+). (C) 1998 Wiely-Liss, Inc.