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.
Highlights
Ca2ϩ Signaling in NG and HG in Response to Ionomycin—To study Ca2ϩ signaling, mesangial cells were grown on glass coverslips and growth-arrested in 5.6 mM (NG) glucose or 30 mM (HG) glucose for 48 h and stimulated with the calcium ionophore, ionomycin
We have demonstrated that Ca2ϩ signaling in response to vasoactive peptides such as ET-1 is suppressed in HG through a mechanism requiring both NADPH oxidase Reactive oxygen species (ROS) and DAG-sensitive protein kinase C (PKC) isozymes
DAGsensitive PKC isozymes, known to be activated in HG, are necessary for suppression of Ca2ϩ signaling to ET-1 in HG, since down-regulation of DAG-sensitive PKC isozymes by chronic exposure to phorbol 12-myristate 13-acetate (PMA) in HG restored the Ca2ϩ response, and a PKC- peptide inhibitor failed to normalize the Ca2ϩ signaling in response to ET-1 in HG
Summary
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. Protein Expression of p47phox and Modified Expression by Antisense Oligonucleotide—To investigate a role of NADPH oxidase, the p47phox subunit, in HG-induced production of ROS, we first determined the expression of p47phox in mesangial cells. Inhibition of ROS Formation Reverses the Effect of HG on Ca2ϩ Signaling in Response to ET-1—To determine the role of ROS on the observed reduced Ca2ϩ signaling, mesangial cells were growth-arrested in HG and treated with the addition of catalase (100 units, 1 h).
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