Mesangial cell-reduced Ca2+ signaling in high glucose is due to inactivation of phospholipase C-beta3 by protein kinase C.
In high glucose, glomerular mesangial cells (MCs) demonstrate impaired Ca(2+) signaling in response to seven-transmembrane receptor stimulation. To identify the mechanism, we first postulated decreased release from intracellular stores. Intracellular Ca(2+) 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 Ca(2+) response was reduced to 82% of that observed in normal glucose (NG). In NG 5.6 mM, Ca(2+) responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF) were unchanged in cells cultured in 50 nM Ca(2+) vs. 1.8 mM Ca(2+). Depletion of intracellular Ca(2+) stores with thapsigargin eliminated ET-1-stimulated Ca(2+) responses. Incubation in 30 mM glucose (HG) for 48 h or stimulation with phorbol myristate acetate (PMA) for 10 min eliminated the Ca(2+) 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 Ca(2+) response to ET-1. Because ET-1 and PDGF stimulate Ca(2+) signaling through different phospholipase C pathways, we hypothesized that, in HG, PKC selectively phosphorylates and inhibits PLC-beta(3). Using confocal immunofluorescence imaging, in NG, a 1.6- to 1.7-fold increase in PLC-beta(3) Ser(1105) phosphorylation was observed following PMA or ET-1 stimulation for 10 min. In HG, immunofluorescent imaging and immunoblotting showed increased PLC-beta(3) phosphorylation, without change in total PLC-beta(3), which was reversed with 24-h PMA or Gö6976. We conclude that reduced Ca(2+) signaling in HG cannot be explained by reduced Ca(2+) stores but is due to conventional PKC-dependent phosphorylation and inactivation of PLC-beta(3).
Institute of Medical Science, University Health Network, University of Toronto, ON, Canada M5S 1A8.
Helena
H
Frecker
Snezana
S
Munk
Hong
H
Wang
Catharine
C
Whiteside
D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D015536:Down-Regulation; D019332:Endothelin-1 / Q000502:physiology; D020869:Gene Expression Profiling; D005920:Glomerular Mesangium / Q000166:cytology / Q000502:physiology; D005947:Glucose / Q000502:physiology; D006943:Hyperglycemia / Q000150:complications*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D054799:Phospholipase C beta; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D019332:Endothelin-1; D007527:Isoenzymes; D011493:Protein Kinase C; D010738:Type C Phospholipases; D054799:Phospholipase C beta; C518041:Plcb3 protein, rat; D005947:Glucose