Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

Background Isoflurane pretreatment can induce protection against lipopolysaccharide and interferon gamma (IFNgamma)-induced injury and activation of mouse microglial cells. This study's goal was to determine whether delayed isoflurane treatment is protective. Methods Mouse microglial cells were exposed to various concentrations of isoflurane for 1 h immediately after the initiation of lipopolysaccharide (10 or 1000 ng/ml) and IFNgamma (10 U/ml) stimulation or to 2% isoflurane for 1 h at various times after initiation of the stimulation. Nitrite production, lactate dehydrogenase release, and cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were assessed after stimulation with lipopolysaccharide and IFNgamma for 24 h. Inducible nitric oxide synthase (iNOS) protein expression was quantified by Western blotting. The iNOS expression in mouse brain was also studied. Results Isoflurane applied 0 and 2 h after the initiation of lipopolysaccharide and IFNgamma stimulation improved cell viability. Isoflurane at 2%, but not at 1 or 3%, reduced the lipopolysaccharide and IFNgamma-induced nitrite production and decreased cell viability. Aminoguanidine, an iNOS inhibitor, also attenuated this decreased cell viability. Chelerythrine and bisindolylmalemide IX, protein kinase C inhibitors, abolished isoflurane effects on cell viability and iNOS expression after lipopolysaccharide and IFNgamma application. Isoflurane also decreased lipopolysaccharide-induced iNOS expression in mouse brain. Late isoflurane application to microglial cells reduced lipopolysaccharide and IFNgamma-induced lactate dehydrogenase release that was not inhibited by aminoguanidine. Conclusions These results suggest that delayed isoflurane treatment can reduce lipopolysaccharide and IFNgamma-induced activation and injury of microglial cells. These effects may be mediated by protein kinase C.

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Protein kinase C-α inhibits the repair of oxidative phosphorylation after S-(1,2-dichlorovinyl)-l-cysteine injury in renal cells

AJP Renal Physiology ◽

10.1152/ajprenal.00216.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. F64-F73 ◽

Cited By ~ 18

Author(s):

Xiuli Liu ◽

Malinda L. Godwin ◽

Grażyna Nowak

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Oxidative Phosphorylation ◽

Atpase Activity ◽

Mitochondrial Function ◽

Recovery Period ◽

Β Subunit ◽

Atp Production ◽

Kinase C ◽

Mitochondrial Functions

Previously, we showed that physiological functions of renal proximal tubular cells (RPTC) do not recover following S-(1,2-dichlorovinyl)-l-cysteine (DCVC)-induced injury. This study investigated the role of protein kinase C-α (PKC-α) in the lack of repair of mitochondrial function in DCVC-injured RPTC. After DCVC exposure, basal oxygen consumption (Qo2), uncoupled Qo2, oligomycin-sensitive Qo2, F1F0-ATPase activity, and ATP production decreased, respectively, to 59, 27, 27, 57, and 68% of controls. None of these functions recovered. Mitochondrial transmembrane potential decreased 53% after DCVC injury but recovered on day 4. PKC-α was activated 4.3- and 2.5-fold on days 2 and 4, respectively, of the recovery period. Inhibition of PKC-α activation (10 nM Go6976) did not block DCVC-induced decreases in mitochondrial functions but promoted the recovery of uncoupled Qo2, oligomycin-sensitive Qo2, F1F0-ATPase activity, and ATP production. Protein levels of the catalytic β-subunit of F1F0-ATPase were not changed by DCVC or during the recovery period. Amino acid sequence analysis revealed that α-, β-, and ε-subunits of F1F0-ATPase have PKC consensus motifs. Recombinant PKC-α phosphorylated the β-subunit and decreased F1F0-ATPase activity in vitro. Serine but not threonine phosphorylation of the β-subunit was increased during late recovery following DCVC injury, and inhibition of PKC-α activation decreased this phosphorylation. We conclude that during RPTC recovery following DCVC injury, 1) PKC-α activation decreases F0F1-ATPase activity, oxidative phosphorylation, and ATP production; 2) PKC-α phosphorylates the β-subunit of F1F0-ATPase on serine residue; and 3) PKC-α does not mediate depolarization of RPTC mitochondria. This is the first report showing that PKC-α phosphorylates the catalytic subunit of F1F0-ATPase and that PKC-α plays an important role in regulating repair of mitochondrial function.

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Targeting protein kinase C by Enzastaurin restrains proliferation and secretion in human pancreatic endocrine tumors

Endocrine Related Cancer ◽

10.1530/erc-11-0055 ◽

2011 ◽

Vol 18 (4) ◽

pp. 439-450 ◽

Cited By ~ 14

Author(s):

Daniela Molè ◽

Teresa Gagliano ◽

Erica Gentilin ◽

Federico Tagliati ◽

Claudio Pasquali ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Kinase C ◽

Insulin Secretion ◽

Protein Kinase ◽

Cell Viability ◽

Cell Line ◽

Primary Cultures ◽

Endocrine Tumors ◽

Kinase C ◽

Pancreatic Endocrine Tumors

Dysregulation of the protein kinase C (PKC) signaling pathway has been implicated in tumor progression. In this study, we investigate the effects of a PKC inhibitor, Enzastaurin, in human pancreatic neuroendocrine neoplasms (PNN) primary cultures and in the human pancreatic endocrine cancer cell line, BON1. To this aim six human PNN dispersed in primary cultures and BON1 cells were treated without or with 1–10 μM Enzastaurin and/or 100 nM IGF1 in the presence or absence of serum. Cell viability and apoptosis were evaluated after 48–72 h; Chromogranin A (CgA) and/or insulin secretion was assessed after 6 h of incubation. PKC expression was investigated by immunofluorescence and western blot. We found that Enzastaurin significantly reduced human PNN primary culture cell viability, as well as CgA and insulin secretion. Moreover, in the BON1 cell line Enzastaurin inhibited cell proliferation at 5 and 10 μM by inducing caspase-mediated apoptosis, and reduced phosphorylation of glycogen synthetase kinase 3β (GSK3β) and of Akt, both downstream targets of PKC pathway and pharmacodynamic markers for Enzastaurin. In addition, Enzastaurin blocked the stimulatory effect of IGF1 on cell proliferation, and reduced CgA expression and secretion in BON1 cells. Two different PKC isoforms are expressed at different levels and have partially different subcellular localization in BON1 cells. In conclusion, Enzastaurin reduces cell proliferation by inducing apoptosis, with a mechanism likely involving GSK3β signaling, and inhibits secretory activity in PNN in vitro models, suggesting that Enzastaurin might represent a possible medical treatment of human PNN.

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Isoflurane Preconditioning Reduces the Rat NR8383 Macrophage Injury Induced by Lipopolysaccharide and Interferon γ

Anesthesiology ◽

10.1097/aln.0b013e318167aeb4 ◽

2008 ◽

Vol 108 (4) ◽

pp. 643-650 ◽

Cited By ~ 26

Author(s):

Xuebing Xu ◽

Jifeng Feng ◽

Zhiyi Zuo

Keyword(s):

Nitric Oxide ◽

Protein Kinase C ◽

Protein Kinase ◽

Nitric Oxide Synthase ◽

Cell Viability ◽

Interferon Gamma ◽

Inducible Nitric Oxide Synthase ◽

Kinase C ◽

Oxide Synthase ◽

Inducible Nitric Oxide

Background Isoflurane exposure before an insult can reduce the insult-induced injury in various organs. This phenomenon is called isoflurane preconditioning. The authors hypothesize that isoflurane can precondition macrophages, cells that travel to all tissues and are important in the host defense and inflammation responses. Methods Rat NR8383 macrophages were pretreated with or without 1-3% isoflurane for 1 h at 30 min before they were incubated with or without 100 ng/ml lipopolysaccharide plus 50 U/ml interferon gamma for 24 h. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was performed after cells were stained with annexin V and propidium iodide. Inducible nitric oxide synthase protein expression in macrophages was quantified by Western blotting. Results Lipopolysaccharide plus interferon gamma decreased cell viability by approximately 50%. This decrease was dose-dependently inhibited by aminoguanidine, an inducible nitric oxide synthase inhibitor. Lipopolysaccharide plus interferon gamma caused inducible nitric oxide synthase expression. This expression was inhibited by pretreatment with 2% but not 1% or 3% isoflurane. Isoflurane at 2% inhibited lipopolysaccharide plus interferon gamma-induced accumulation of nitrite, an oxidation product of nitric oxide. Pretreatment with 2% but not 1% or 3% isoflurane improved cell viability. Lipopolysaccharide plus interferon gamma increased the number of propidium iodide-positive staining cells. This increase was attenuated by 2% isoflurane pretreatment. The protective effect of 2% isoflurane was abolished by chelerythrine, calphostin C, or bisindolylmaleimide IX, protein kinase C inhibitors. Conclusions Lipopolysaccharide plus interferon gamma causes an inducible nitric oxide synthase-dependent macrophage injury. Isoflurane induces preconditioning effects that may be mediated by protein kinase C in macrophages.

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