High Glucose-suppressed Endothelin-1 Ca2+ Signaling via NADPH Oxidase and Diacylglycerol-sensitive Protein Kinase C Isozymes in Mesangial Cells

Abstract

High glucose (HG) is the underlying factor contributing to long term complications of diabetes mellitus. The molecular mechanisms transforming the glomerular mesangial cell phenotype to cause nephropathy including diacylglycerol-sensitive protein kinase C (PKC) are still being defined. Reactive oxygen species (ROS) have been postulated as a unifying mechanism for HG-induced complications. We hypothesized that in HG an interaction between ROS generation, from NADPH oxidase, and PKC suppresses mesangial Ca2+ signaling in response to endothelin-1 (ET-1). In primary rat mesangial cells, growth-arrested (48 h) in 5.6 mM (NG) or 30 mm (HG) glucose, the total cell peak [Ca2+]i response to ET-1 (50 nM) was 630 +/- 102 nM in NG and was reduced to 159 +/- 15 nM in HG, measured by confocal imaging. Inhibition of PKC with phorbol ester down-regulation in HG normalized the ET-1-stimulated [Ca2+]i response to 541 +/- 74 nM. Conversely, an inhibitory peptide specific for PKC-zeta did not alter Ca2+ signaling in HG. Furthermore, overexpression of conventional PKC-beta or novel PKC-delta in NG diminished the [Ca2+]i response to ET-1, reflecting the condition observed in HG. Likewise, catalase or p47phox antisense oligonucleotide normalized the [Ca2+]i response to ET-1 in HG to 521 +/- 58 nM and 514 +/- 48 nM, respectively. Pretreatment with carbonyl cyanide m-chlorophenylhydrazone or rotenone did not restore Ca2+ signaling in HG. Detection of increased intracellular ROS in HG by dichlorofluorescein was inhibited by catalase, diphenyleneiodonium, or p47phox antisense oligonucleotide. HG increased p47phox mRNA by 1.7 +/- 0.1-fold as measured by reverse transcriptase-PCR. In NG, H2O2 increased membrane-enriched PKC-beta and -delta, suggesting activation of these isozymes. HG-enhanced immunoreactivity of PKC-delta visualized by confocal imaging was attenuated by diphenyleneiodium chloride. Thus, mesangial cell [Ca2+]i signaling in response to ET-1 in HG is attenuated through an interaction mechanism between NADPH oxidase ROS production and diacylglycerol-sensitive PKC.