title: 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.
authors.affiliation: Institute of Medical Science, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Hong
authors.initials: H
authors.lastname: Hua
authors.forename: Snezana
authors.initials: S
authors.lastname: Munk
authors.forename: Howard
authors.initials: H
authors.lastname: Goldberg
authors.forename: I George
authors.initials: IG
authors.lastname: Fantus
authors.forename: Catharine I
authors.initials: CI
authors.lastname: Whiteside
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004075:Diglycerides / Q000378:metabolism / Q000494:pharmacology*; D015536:Down-Regulation; D004579:Electron Transport; D019332:Endothelin-1 / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007678:Kidney Glomerulus / Q000166:cytology; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009861:Onium Compounds / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D064546:Protein Kinase C beta; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D000099221:Protein Kinase C zeta
chemical_list: D000199:Actins; D002118:Calcium; D002374:Catalase; D004075:Diglycerides; D019332:Endothelin-1; D005947:Glucose; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D015759:Ionomycin; D007476:Ionophores; D008164:Luminescent Proteins; D019255:NADPH Oxidases; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D009861:Onium Compounds; D010455:Peptides; D010750:Phosphoproteins; D020033:Protein Isoforms; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D000099221:Protein Kinase C zeta; C007517:diphenyleneiodonium; C060505:neutrophil cytosolic factor 1