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Am J Physiol Renal Physiol 275: F423-F432, 1998;
0363-6127/98 $5.00
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Vol. 275, Issue 3, F423-F432, September 1998

Endothelin-1-induced mesangial cell contraction involves activation of protein kinase C-alpha , -delta , and -epsilon

John A. Dlugosz, Snezana Munk, Xiaopeng Zhou, and Catharine I. Whiteside

Medical Research Council of Canada Group in Membrane Biology, University of Toronto, Toronto, Ontario, Canada M5S 1A8

In endothelin-1 (ET-1)-stimulated mesangial cells, to identify the independent roles of calcium and protein kinase C (PKC) causing contraction, the changes in planar surface area in response to ET-1, ionomycin, or phorbol 12-myristate 13-acetate (PMA) were compared. ET-1, PMA, and ionomycin reduced planar area to 49 ± 3%, 56 ± 3%, and 78 ± 2% of basal (means ± SE, n = 40-50 cells), respectively. ET-1 or ionomycin increased cytosolic calcium from 80 ± 7 to 220 ± 30 nM or 97 ± 10 to 192 ± 10 nM, respectively. The myosin light chain kinase inhibitor, ML-7, blunted ET-1- but not PMA-stimulated contraction (82 ± 3% and 48 ± 6% of time 0, respectively). Cells pretreated with 10 µM chelerythrine for 1 h or PMA for 24 h failed to contract to either ET-1 or PMA. To identify the specific PKC isoform response to ET-1, cytosolic, membrane, and particulate fractions of mesangial cell lysates were immunoblotted with PKC isoform-specific polyclonal antibodies. ET-1 increased membrane PKC-alpha , -delta , and -epsilon to 173 ± 30%, 162 ± 26%, and 166 ± 11% of basal (P < 0.05 vs. basal), respectively, and decreased PKC-delta and PKC-epsilon in the cytosol to 56 ± 11% and 37 ± 6% of basal, respectively (P < 0.05). ET-1 increased particulate PKC-delta and PKC-epsilon to 172 ± 15% and 187 ± 33% of basal (P < 0.05), respectively. PKC-alpha in the cytosol and particulate fractions was not altered by ET-1, but translocation to the nucleus and cell periphery was observed by confocal immunofluorescence imaging. Ionomycin did not change PKC isoform distribution. PKC-zeta was expressed but unaltered by ET-1. Therefore, mesangial cell ET-1-stimulated contraction not only involves a calcium-dependent pathway but also includes the activation of one or more PKC-alpha , -delta , and -epsilon , but not PKC-zeta .

calcium; ionomycin; phorbol 12-myristate 13-acetate; protein kinase C-alpha ; protein kinase C-delta ; protein kinase C-epsilon ; protein kinase C-zeta


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