Mesangial cell filamentous actin disassembly and hypocontractility in high glucose are mediated by PKC-ζ

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In high glucose (HG), mesangial cells (MCs) lose their contractile response to endothelin-1 (ET-1) coincidently with filamentous (F)-actin disassembly. We postulated that these MC phenotypic changes are mediated by altered protein kinase C (PKC) isozyme activity, myosin light chain (MLC(20)) phosphorylation, or Ca(2+) signaling. MCs were growth arrested for 24 h in 0.5% fetal bovine serum (FBS)-DMEM in 5.6 (normal glucose; NG) or 30 mM glucose (high glucose; HG). In HG, the planar area was reduced [2,608 +/- 135 vs. 3,952 +/- 225 (SE) microm(2) in NG, P < 0.01, n = 31] with no contractile response to 0.1 microM ET-1. Mannitol did not affect cell size or ET-1 response. Confocal imaging of fluo 3- loaded cells revealed that the peak intensity of ET-1-induced Ca(2+) signaling was not altered in HG vs. NG. Immunoblotting of phosphorylated MLC(20) showed that HG increased mono- and decreased unphosphorylated MLC(20) (42 +/- 16 and 49 +/- 15 vs. 13 +/- 3 and 80 +/- 4% of total in NG, P < 0.05, n = 3), but the peak phosphorylation responses to ET-1 were identical in NG and HG. ET-1 stimulated translocation of PKC-delta and -epsilon from cytosolic to membrane and particulate fractions identically in NG and HG but did not cause PKC-zeta translocation. In HG, membrane accumulation of PKC-zeta was observed. Membrane PKC-zeta activity measured by immunoprecipitation and (32)P phosphorylation of PKC-epsilon pseudosubstrate peptide was 190 +/- 18% of NG (P < 0.01, n = 4), which was completely inhibited by pretreatment with a myristoylated peptide inhibitor (ZI). In HG, pretreatment with ZI for 24 h restored normal MC size and contractile and F-actin disassembly responses to ET-1. In conclusion, in HG, decreased MC size is due to decreased F-actin assembly, and loss of contractile response to ET-1 occurs in the presence of normal Ca(2+) signaling and normal MLC(20) phosphorylation. In HG, altered F-actin and contractile functions in MCs are mediated by PKC-zeta.