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Mesangial cell-reduced Ca²⁺ signaling in high glucose is due to inactivation of phospholipase C-₃ by protein kinase C

Institute of Medical Science, University Health Network, Department of Medicine, University of Toronto, Toronto, Canada

Submitted 7 December 2004 ; accepted in final form 1 July 2005

In high glucose, glomerular mesangial cells (MCs) demonstrate impaired Ca²⁺ signaling in response to seven-transmembrane receptor stimulation. To identify the mechanism, we first postulated decreased release from intracellular stores. Intracellular Ca²⁺ was measured in fluo-3-loaded primary cultured rat MCs using confocal fluorescence microscopy. In high glucose (HG) 30 mM for 48 h, the 25 nM ionomycin-stimulated intracellular Ca²⁺ response was reduced to 82% of that observed in normal glucose (NG). In NG 5.6 mM, Ca²⁺ responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF) were unchanged in cells cultured in 50 nM Ca²⁺ vs. 1.8 mM Ca²⁺. Depletion of intracellular Ca²⁺ stores with thapsigargin eliminated ET-1-stimulated Ca²⁺ responses. Incubation in 30 mM glucose (HG) for 48 h or stimulation with phorbol myristate acetate (PMA) for 10 min eliminated the Ca²⁺ response to ET-1 but had no effect on the PDGF response. Downregulation of protein kinase C (PKC) with 24-h PMA or inhibition with Gö6976 in HG normalized the Ca²⁺ response to ET-1. Because ET-1 and PDGF stimulate Ca²⁺ signaling through different phospholipase C pathways, we hypothesized that, in HG, PKC selectively phosphorylates and inhibits PLC-₃. Using confocal immunofluorescence imaging, in NG, a 1.6- to 1.7-fold increase in PLC-₃ Ser¹¹⁰⁵ phosphorylation was observed following PMA or ET-1 stimulation for 10 min. In HG, immunofluorescent imaging and immunoblotting showed increased PLC-₃ phosphorylation, without change in total PLC-₃, which was reversed with 24-h PMA or Gö6976. We conclude that reduced Ca²⁺ signaling in HG cannot be explained by reduced Ca²⁺ stores but is due to conventional PKC-dependent phosphorylation and inactivation of PLC-₃.

endothelin-1; platelet-derived growth factor; thapsigargin; ionomycin; fluo-3

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