Endothelin-1-induced mesangial cell contraction involves activation of protein kinase C-α, -δ, and -ε

Abstract

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-α, -δ, and -ε to 173 ± 30%, 162 ± 26%, and 166 ± 11% of basal ( P< 0.05 vs. basal), respectively, and decreased PKC-δ and PKC-ε in the cytosol to 56 ± 11% and 37 ± 6% of basal, respectively ( P < 0.05). ET-1 increased particulate PKC-δ and PKC-ε to 172 ± 15% and 187 ± 33% of basal ( P < 0.05), respectively. PKC-α 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-ζ 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-α, -δ, and -ε, but not PKC-ζ.