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

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-ζ did not alter Ca2+ signaling in HG. Furthermore, overexpression of conventional PKC-β or novel PKC-δ in NG diminished the [Ca2+] i response to ET-1, reflecting the condition observed in HG. Likewise, catalase or p47 phox 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 p47 phox antisense oligonucleotide. HG increased p47 phox mRNA by 1.7 ± 0.1-fold as measured by reverse transcriptase-PCR. In NG, H2O2 increased membrane-enriched PKC-β and -δ, suggesting activation of these isozymes. HG-enhanced immunoreactivity of PKC-δ 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. 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-ζ did not alter Ca2+ signaling in HG. Furthermore, overexpression of conventional PKC-β or novel PKC-δ in NG diminished the [Ca2+] i response to ET-1, reflecting the condition observed in HG. Likewise, catalase or p47 phox 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 p47 phox antisense oligonucleotide. HG increased p47 phox mRNA by 1.7 ± 0.1-fold as measured by reverse transcriptase-PCR. In NG, H2O2 increased membrane-enriched PKC-β and -δ, suggesting activation of these isozymes. HG-enhanced immunoreactivity of PKC-δ 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. High glucose (HG) 1The abbreviations used are: HG, high glucose; NG, normal glucose; ET-1, endothelin-1; ROS, reactive oxygen species; PKC, protein kinase C; DPI, diphenyleneiodium chloride; DAG, diacylglycerol; CM-H2DCFDA, fluo-3,5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester; PMA, phorbol 12-myristate 13-acetate; CCCP, carbonyl cyanide m-chlorophenylhydrazone; GFP, green fluorescent protein; DCF, carboxymethyl-H2-dichlorofluorescein diacetate.1The abbreviations used are: HG, high glucose; NG, normal glucose; ET-1, endothelin-1; ROS, reactive oxygen species; PKC, protein kinase C; DPI, diphenyleneiodium chloride; DAG, diacylglycerol; CM-H2DCFDA, fluo-3,5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester; PMA, phorbol 12-myristate 13-acetate; CCCP, carbonyl cyanide m-chlorophenylhydrazone; GFP, green fluorescent protein; DCF, carboxymethyl-H2-dichlorofluorescein diacetate. is the key factor contributing to long term complications of diabetes mellitus (1DCCT Research GroupN. Engl. J. Med. 2000; 342: 381-389Crossref PubMed Scopus (1380) Google Scholar). One of the phenotypic changes observed in mesangial cells exposed to HG is altered Ca2+ signaling. Several groups have shown that the Ca2+ signal induced by vasoactive compounds, including endothelin-1 (ET-1), is markedly reduced in the presence of HG. The mechanism(s) by which HG may depress Ca2+ signaling is unknown. One possible candidate is the activation of protein kinase C (PKC) in HG. Mené et al. (2Mené P. Pugliese G. Pricci F. Di Mario U. Cinotti G.A. Pugliese F. Diabetologia. 1997; 40: 521-527Crossref PubMed Scopus (49) Google Scholar) have shown that HG inhibits Ca2+ influx through store-operated channels via a PKC-dependent mechanism. An alternative postulate is the involvement of reactive oxygen species (ROS), which have been demonstrated to modify intracellular Ca2+ signaling responsiveness depending on the cell type, the species of ROS, and the magnitude and duration of ROS generation. HG induces dysfunction in mesangial cells and other target cells through enhanced synthesis of autocrine growth factors such as transforming growth factor-β1, ET-1, and altered signaling via pathways such as PKC (3Way K.J. Katai N. King G.L. Diabet. Med. 2001; 18: 945-959Crossref PubMed Scopus (268) Google Scholar, 4Chen S. Apostolova M.D. Cherian M.G. Chakrabarti S. Lab. Invest. 2000; 80: 1311-1321Crossref PubMed Scopus (77) Google Scholar). In the last few years, enhanced production of ROS in response to HG, identified in many target cells including mesangial cells (5Catherwood M.A. Powell L.A. Anderson P. McMaster D. Sharpe P.C. Trimble E.R. Kidney Int. 2002; 61: 599-608Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 6Ha H. Lee H.B. Kidney Int. Suppl. 2000; 77: S19-S25Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), has been postulated as a unifying mechanism causing diabetes complications (7Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (6861) Google Scholar, 8Nishikawa T. Edelstein D. Brownlee M. Kidney Int. Suppl. 2000; 77: 26-30Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 9Ha H. Kim K.H. Diabetes Res. Clin. Pract. 1999; 45: 147-151Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Although ROS have been implicated in causing cell damage and apoptosis, they also play a physiological role in intracellular signaling pathways (10Sauer H. Wartenberg M. Hescheler J. Cell Physiol. Biochem. 2001; 11: 173-186Crossref PubMed Scopus (892) Google Scholar, 11Hancock J.T. Desikan R. Neill S.J. Biochem. Soc. Trans. 2001; 29: 345-350Crossref PubMed Google Scholar). In particular, several growth factors including ET-1, angiotensin II, platelet-derived growth factor, and epidermal growth factor stimulate production of ROS as second messengers (12Fei J. Viedt C. Soto U. Elsing C. Jahn L. Kreuzer J. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1244-1249Crossref PubMed Scopus (67) Google Scholar, 13Seshiah P.N. Weber D.S. Rocic P. Valppu L. Taniyama Y. Griendling K.K. Circ. Res. 2002; 91: 406-413Crossref PubMed Scopus (632) Google Scholar). In several cell types, signaled ROS production is due to activation of NADPH oxidase, a multicomponent enzyme (14Griendling K.K. Ushio-Fukai M. Regul. Pept. 2000; 91: 21-27Crossref PubMed Scopus (358) Google Scholar). In phagocytic cells, the multiple subunits of NADPH oxidase are localized in subcellular compartments. gp91 phox , the catalytic moiety of the phagocyte oxidase, and p22 phox associate to form a flavocytochrome in the plasma membrane. The cytosol components p47 phox , p67 phox , p40 phox , and the small GTPase, Rac1 (or Rac2), are recruited to the membrane for assembly of a fully active oxidase (15Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar, 16Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Crossref PubMed Scopus (2577) Google Scholar, 17Forman H.J. Torres M. Fukuto J. Mol. Cell Biochem. 2002; 234: 49-62Crossref PubMed Scopus (204) Google Scholar). In nonphagocytic cells, most of the subunits of NADPH oxidase have been identified, although the precise mechanisms of regulation are not completely understood. A functional glomerular mesangial NADPH oxidase has been inferred through the use of diphenyleneiodium chloride (DPI), an inhibitor of flavoproteins, in response to cytokine (18Dorsam G. Taher M.M. Valerie K.C. Kuemmerle N.B. Chan J.C. Franson R.C. J. Pharmacol. Exp. Ther. 2000; 292: 271-279PubMed Google Scholar, 19Feng L. Xia Y. Garcia G.E. Hwang D. Wilson C.B. J. Clin. Invest. 1995; 95: 1669-1675Crossref PubMed Scopus (454) Google Scholar) and serotonin stimulation (20Grewal J.S. Mukhin Y.V. Garnovskaya M.N. Raymond J.R. Greene E.L. Am. J. Physiol. 1999; 276: F922-F930PubMed Google Scholar). An earlier report identified the expression of components of human glomerular mesangial cell NADPH oxidase (21Radeke H.H. Cross A.R. Hancock J.T. Jones O.T. Nakamura M. Kaever V. Resch K. J. Biol. Chem. 1991; 266: 21025-21029Abstract Full Text PDF PubMed Google Scholar). To date, no report has described the role of NADPH oxidase in HG-induced altered mesangial cell phenotype. We reasoned that if HG causes enhanced and sustained ROS generation in mesangial cells, Ca2+ signaling responsiveness to ET-1 may be modified through a ROS-dependent mechanism. Since previous reports (22Meier M. King G.L. Vasc. Med. 2000; 5: 173-185Crossref PubMed Google Scholar, 23Koya D. Haneda M. Nakagawa H. Isshiki K. Sato H. Maeda S. Sugimoto T. Yasuda H. Kashiwagi A. Ways D.K. King G.L. Kikkawa R. FASEB J. 2000; 14: 439-447Crossref PubMed Scopus (408) Google Scholar, 24Koya D. King G.L. Diabetes. 1998; 47: 859-866Crossref PubMed Scopus (1141) Google Scholar, 25Sheetz M.J. King G.L. JAMA (J. Am. Med. Assoc.). 2002; 288: 2579-2588Crossref PubMed Scopus (803) Google Scholar, 26Ishii H. Koya D. King G.L. J. Mol. Med. 1998; 76: 21-31Crossref PubMed Scopus (255) Google Scholar), including work from our laboratory (27Whiteside C.I. Am. J. Physiol. 2002; PubMed Scopus Google Scholar, H. H.J. C.I. Diabetes. 2001; PubMed Scopus Google Scholar, J. T. T. C. J. Am. Soc. 1999; Google Scholar, T. J. C. Diabetes. 1998; 47: PubMed Scopus Google Scholar), have demonstrated enhanced PKC in response to HG, postulated a interaction between PKC and enhanced NADPH oxidase modified fluo-3,5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester DPI, catalase, phorbol 12-myristate carbonyl cyanide m-chlorophenylhydrazone and rotenone phox and p47 phox p47 phox antisense and a peptide that inhibits PKC-ζ S. H.J. C.I. Am. J. Physiol. 2002; PubMed Scopus Google Scholar) for for Cell rat mesangial cells from rat and as described S. J. M. C.I. Diabetes. 1995; PubMed Google Scholar). used for cells in modified to and growth-arrested in 5.6 mm glucose (NG) or 30 mm glucose (HG) for 48 of cells on to the and with in modified with for The was in a on the of a confocal and the cells to and the response to 15 for The confocal from condition and peak response of total cell [Ca2+] i cells from of Ca2+ was as for was nm C. S. T. J. Am. Soc. 1998; Google Scholar). The and the measured with nm Ca2+ with and Ca2+ mm the Ca2+ is the in the of measured by with was from the and S. J. M. C.I. Diabetes. 1995; PubMed Google Scholar, S. C.I. Am. J. Physiol. 1998; PubMed Google Scholar). of and PKC-ζ cells with or as by the To PKC-ζ mesangial cells with the PKC-ζ peptide inhibitor h) S. H.J. C.I. Am. J. Physiol. 2002; PubMed Scopus Google Scholar) in HG. with and for Ca2+ response to or of and was the as by the The was synthesis to the synthesis of of was by specific on the of L. Y. J. Biol. 2000; PubMed Scopus Google Scholar) including and The for the is as and on a and to reverse visualized by of and cells on to and growth-arrested in NG or HG. in and with To of cells with and with phox or PKC-δ The primary was with confocal of cells to on and with antisense or oligonucleotide for p47 phox or to the The to the p47 phox antisense oligonucleotide was from a report by and J. Res. 2000; Full Text Full Text PDF PubMed Google Scholar). The is as with a of the oligonucleotide for 48 and expression of p47 phox protein was by immunoreactivity of p47 phox confocal imaging. of of intracellular ROS was measured with the carboxymethyl-H2-dichlorofluorescein diacetate is a that is a by is to the fluorescent in the presence of intracellular mesangial cells for with in modified in NG or HG or 48 was measured by confocal nm, for of cells was and as a cells with membrane mesangial cells on in A mm and by through a and for 30 The was in A and for 30 The was as the plasma membrane are as ± was The of or groups by of of was in the of the multiple was described as are in NG and HG in to Ca2+ mesangial cells on and growth-arrested in 5.6 mm (NG) glucose or 30 mm (HG) glucose for 48 and with the Ca2+ was by the of the cell [Ca2+] i in nm the S. J. M. C.I. Diabetes. 1995; PubMed Google Scholar). was no between the Ca2+ response in NG and HG to nm not a of the peak total cell [Ca2+] i response in NG was ± nm, and in HG, the response was reduced to ± nm PKC Inhibition in to the Ca2+ response of mesangial cells to the vasoactive peptide ET-1, cells on and growth-arrested in NG or HG glucose for 48 and with ET-1 (50 In from total cell [Ca2+] i from and peak response are In NG, the total cell peak [Ca2+] i response to ET-1 (50 nm) was 630 ± 102 nm, which was attenuated to 159 ± 15 nm in HG NG To PKC was in Ca2+ signaling in HG, PKC by of nm) in HG for 48 Inhibition of PKC the in HG. The peak total cell [Ca2+] i response was to 541 ± 74 nm HG Since PKC the role of PKC-ζ in Ca2+ signaling was a peptide demonstrated to be specific for PKC S. H.J. C.I. Am. J. Physiol. 2002; PubMed Scopus Google Scholar). cells growth-arrested in HG with or the PKC-ζ inhibitor Pretreatment with the PKC-ζ inhibitor in HG did not alter Ca2+ signaling in response to ET-1 ± nm, NG To a role of PKC in Ca2+ in response to ET-1, mesangial cells with green fluorescent protein or to the conventional and novel of PKC isozymes. cells functional and in these as they to stimulation by to the the of PKC activation In cells, expression of the was from the cells with ET-1, cells with or a Ca2+ response was in the Conversely, cells with cells including the with a of Ca2+ Thus, the did not the of of or attenuated the Ca2+ response in cells with or in NG, with and with ET-1 or cells ET-1 stimulation are by an cells ET-1 stimulation are by an cells cells ROS in in HG by or the of HG on ROS mesangial cells on growth-arrested in 30 mm glucose for with carboxymethyl-H2-dichlorofluorescein diacetate and with confocal HG increased ROS generation as measured by as as The was sustained in long term with HG to 48 cells with catalase h) or HG-induced ROS generation was reduced ± ± cells, HG ± cells, and HG ± cells, of ROS is inhibited by catalase and cells growth-arrested on in HG, HG with catalase or HG with with and HG HG of condition measured as HG. of and by a role of NADPH oxidase, the p47 phox in HG-induced production of ROS, the expression of p47 phox in mesangial imaging that mesangial cells p47 phox , which is localized in a HG increased of the that the cell in NG was ± in HG, the was ± cells To the precise of p47 phox , mesangial cells with p47 phox antisense that was to expression of p47 phox in a Since catalase and HG-induced ROS the of NADPH oxidase was that the HG-enhanced ± was inhibited mesangial cells with p47 phox antisense oligonucleotide ± HG, of the of p47 phox antisense oligonucleotide did not ± NG, Inhibition of ROS the of HG on in to the role of ROS on the observed reduced Ca2+ mesangial cells growth-arrested in HG and with the of catalase In these the peak total cell [Ca2+] i response in NG to ET-1 (50 nm) was ± nm. In HG, the was reduced to ± nm. the Ca2+ response in HG to 521 ± 58 nm HG peak To the role of ROS in Ca2+ to the p47 phox of NADPH oxidase is for the of HG on Ca2+ mesangial cells with p47 phox antisense in HG and with In NG, the peak total cell [Ca2+] i response was ± nm. In HG, the peak was reduced to ± nm. Inhibition of p47 phox by down-regulation with antisense the of HG to a that was not the response in NG ± 48 nm, HG of the of p47 phox antisense oligonucleotide did not reverse the [Ca2+] i observed in HG ± nm, peak NG Thus, ROS from NADPH oxidase a role in the reduced Ca2+ response in HG. Since several reports demonstrated the of ROS from a in the the of CCCP, an of and rotenone on Ca2+ signaling in response to cells in In these the peak total cell [Ca2+] i response was ± nm, which was attenuated to ± nm peak NG in HG nm, h) did not alter the peak total cell [Ca2+] i in NG ± nm, peak NG or in HG ± nm, peak HG Pretreatment with rotenone h) the and peak total cell [Ca2+] i in NG and was not used in HG. of PKC on mRNA and ROS on PKC the of HG on p47 phox mesangial cells growth-arrested in NG or HG, and p47 phox mRNA was measured by reverse transcriptase-PCR. HG increased p47 phox mRNA by 1.7 ± 0.1-fold To PKC a role in p47 phox mRNA mesangial cells with nm, 48 that down-regulation of mesangial cell PKC did not alter the of HG regulation on p47 phox mRNA ± Since PKC did not p47 phox mRNA postulated that ROS of PKC. shown in our the immunoreactivity of PKC-δ is enhanced in HG J. T. T. C. J. Am. Soc. 1999; Google Scholar), in the confocal Pretreatment of mesangial cells with the in cells also with and membrane-enriched cell for PKC isozymes. a of H2O2 increased PKC-δ in the cell a or H2O2 was for PKC-β to be in the membrane The of in the cell membrane is in with our previous report activation of PKC in HG T. J. C. Diabetes. 1998; 47: PubMed Scopus Google Scholar). In have demonstrated that Ca2+ signaling in response to vasoactive such as ET-1 is in HG through a mechanism NADPH oxidase ROS and PKC isozymes. We have that mesangial cell generation of ROS in HG is inhibited by catalase or Furthermore, of p47 phox antisense a that reduced expression of p47 phox protein the increased generation of ROS in HG. Likewise, of p47 phox antisense oligonucleotide was to reverse the of HG-induced Ca2+ signaling in response to with catalase also normalized the Ca2+ response in HG. PKC to be in HG, are for of Ca2+ signaling to ET-1 in HG, down-regulation of PKC by to in HG the Ca2+ and a PKC-ζ peptide inhibitor to the Ca2+ signaling in response to ET-1 in HG. overexpression of a conventional or novel PKC increased observed in HG, attenuated Ca2+ signaling in response to the functional between enhanced ROS generation in HG through NADPH oxidase and PKC-dependent of Ca2+ signaling. Several reports that HG Ca2+ signaling in response to vasoactive in cell types, although the hypothesized mechanism(s) for the attenuated Ca2+ signaling to be In mesangial cells, and J. Am. Soc. 2000; 11: Google Scholar) that and to HG ET-1-stimulated Ca2+ signaling that is by In mesangial cells, transforming growth was shown to the expression of to a reduced Ca2+ response K. L. M. V. G. G. Am. J. Physiol. 2000; Google Scholar). We have observed that ET-1-stimulated Ca2+ signaling measured by confocal imaging from with the Ca2+ S. H. G. and C. Mené et al. (2Mené P. Pugliese G. Pricci F. Di Mario U. Cinotti G.A. Pugliese F. Diabetologia. 1997; 40: 521-527Crossref PubMed Scopus (49) Google Scholar) that HG inhibited store-operated Ca2+ influx in mesangial cells by a PKC-dependent mechanism. are also observed that reduced Ca2+ signaling in HG is is the to that PKC are of PKC-ζ did not Ca2+ in response to We are the to that overexpression of is to Ca2+ signaling. A of that ROS play a role in several of signaling R.C. Biol. Med. 2000; PubMed Scopus Google Scholar, G. K.J. Mol. 2002; PubMed Scopus Google Scholar). In cells, to HG was shown to Ca2+ in response to C. M. Y. Circ. Res. 1998; PubMed Scopus Google Scholar). The that diminished Ca2+ by Ca2+ from intracellular and Ca2+ Ca2+ In human cells, was shown that NADPH ROS are to the generation of Ca2+ by the of the to K. K. R.C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). have that in HG, ROS diminished Ca2+ by ET-1 in mesangial cells as catalase, and antisense oligonucleotide to p47 phox Ca2+ signaling in HG, to that in In to the role of ROS, used that the as by Edelstein D. Brownlee M. Diabetes. 2001; PubMed Scopus Google Scholar). In our to reverse Ca2+ signaling. that production of ROS is not in the Ca2+ response to ET-1 in HG. reduced Ca2+ response to ET-1 in NG and not be used in HG. Although ROS from a was shown to be in cells in HG (7Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (6861) Google Scholar, 8Nishikawa T. Edelstein D. Brownlee M. Kidney Int. Suppl. 2000; 77: 26-30Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar), other play In a of the increased production with the and the was inhibited by Lee Kim Lee Kim Diabetes. 2002; PubMed Scopus Google Scholar). Furthermore, expression of p22 phox mRNA Lee Kim Lee Kim Diabetes. 2002; PubMed Scopus Google Scholar) as as gp91 phox U. H. H. M. M. M. F. A. T. Griendling K. U. T. Circ. Res. 2001; PubMed Google Scholar) was Likewise, production in HG was inhibited by T. P. F. M. M. T. T. T. M. H. H. H. Diabetes. 2000; PubMed Scopus Google Scholar). In immunoreactivity of p47 phox was increased in the A. A. T. Kidney Int. 2002; 61: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, increased generation of ROS in from was by p47 phox antisense oligonucleotide S. T. Diabetes. 2002; PubMed Scopus Google Scholar). We that HG-induced ROS generation in rat mesangial cells was most through enhanced NADPH oxidase catalase and reduced in HG. In of p47 phox antisense oligonucleotide also ROS stimulation by HG in rat mesangial Although the NADPH oxidase is a multicomponent several reports that down-regulation of a is to Inhibition of p22 phox by of antisense p22 phox cells in reduced angiotensin production M. T. N. Griendling K.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells from p47 phox ROS in response to the was p47 phox was the cells S. F. Circ. Res. 2002; PubMed Scopus Google Scholar). cells from p47 phox also diminished production 2001; PubMed Scopus Google Scholar) and the response to growth suggesting that from NADPH oxidase a role in C. M. J. Clin. Invest. 2001; PubMed Scopus Google Scholar). In the have shown that of NADPH oxidase with p47 phox antisense oligonucleotide was to HG-induced ROS to an altered Ca2+ response to In may NADPH oxidase, p47 phox , by A. M.A. J. 2002; PubMed Scopus Google Scholar). In nonphagocytic cells, the role of PKC in NADPH oxidase activation is et al. T. P. F. M. M. T. T. T. M. H. H. H. Diabetes. 2000; PubMed Scopus Google Scholar) that in cells and cells, HG and stimulate ROS through PKC-dependent activation of NADPH oxidase by the use of In our HG activation of PKC p47 We that down-regulation of PKC with was to HG-induced of p47 phox Conversely, with the visualized by confocal imaging of PKC-δ in HG. Likewise, H2O2 increased of PKC-δ and in the membrane-enriched that ROS stimulate these mesangial cell PKC isozymes. of PKC is an mechanism for HG-induced a in the of complications (3Way K.J. Katai N. King G.L. Diabet. Med. 2001; 18: 945-959Crossref PubMed Scopus (268) Google Scholar, 24Koya D. King G.L. Diabetes. 1998; 47: 859-866Crossref PubMed Scopus (1141) Google Scholar, 25Sheetz M.J. King G.L. JAMA (J. Am. Med. Assoc.). 2002; 288: 2579-2588Crossref PubMed Scopus (803) Google Scholar, G.L. M. Y. T. T. Xia P. Diabetes. 45: PubMed Google Scholar). In the of PKC is in the response of mesangial cells to HG. a of HG Ca2+ signaling in response to ET-1 is on PKC and NADPH oxidase We to T. and M.

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