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3 BY PROTEIN KINASE C
1 Institute of Medical Science, University Health Network, Department of Medicine, University of Toronto, Toronto, Ontario, Canada
* To whom correspondence should be addressed. E-mail: catharine.whiteside{at}utoronto.ca.
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 NG. In normal glucose (NG) 5.6 mM, Ca2+ responses to endothelin
(ET)-1 and platelet-derived growth factor (PDGF) were unchanged in cells cultured in 50nM
Ca2+ versus 1.8mM 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 24h PMA or
inhibition with Go6976 in HG normalized the Ca2+ response to ET-1. Since 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 1.6-1.7 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 24h PMA or Go6976. 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.
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