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

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 (MLC20) phosphorylation, or Ca2+ 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) mum(2) in NG, P<0.01, n=31] with no contractile response to 0.1 <mu>M 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 Ca2+ signaling was not altered in HG vs. NG. Immunoblotting of phosphorylated MLC20 showed that HG increased mono- and decreased unphosphorylated MLC20 (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 P-32 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 Ca2+ signaling and normal MLC20 phosphorylation. In HG, altered F-actin and contractile functions in MCs are mediated by PKC-<zeta>.