title: Endothelin-1-induced mesangial cell contraction involves activation of protein kinase C-alpha, -delta, and -epsilon.
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 microM 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.
authors.affiliation: Medical Research Council of Canada Group in Membrane Biology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors.forename: J A
authors.initials: JA
authors.lastname: Dlugosz
authors.forename: S
authors.initials: S
authors.lastname: Munk
authors.forename: X
authors.initials: X
authors.lastname: Zhou
authors.forename: C I
authors.initials: CI
authors.lastname: Whiteside
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D048429:Cell Size; D019332:Endothelin-1 / Q000494:pharmacology*; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D005920:Glomerular Mesangium / Q000166:cytology*; D015151:Immunoblotting; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D019332:Endothelin-1; D007527:Isoenzymes; D015759:Ionomycin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium