Modulation of Expression of Rat Mitochondrial 2-Oxoglutarate Carrier in NRK-52E Cells Alters Mitochondrial Transport and Accumulation of Glutathione and Susceptibility to Chemically Induced Apoptosis
- Department of Pharmacology (F.X., D.A.P., L.H.M., L.H.L.) and Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute (L.H.M.), Wayne State University School of Medicine, Detroit, Michigan
- Address correspondence to:
Dr. Lawrence H. Lash, Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Ave., Detroit, MI 48201. E-mail: l.h.lash{at}wayne.edu
Abstract
We previously showed that two anion carriers of the mitochondrial inner membrane, the dicarboxylate carrier (DIC; Slc25a10) and oxoglutarate carrier (OGC; Slc25a11), transport glutathione (GSH) from cytoplasm into mitochondrial matrix. In the previous study, NRK-52E cells, derived from normal rat kidney proximal tubules, were transfected with the wild-type cDNA for the DIC expressed in rat kidney; DIC transfectants exhibited increased mitochondrial uptake and accumulation of GSH and were markedly protected from chemically induced apoptosis. In the present study, cDNAs for both wild-type (WT) and a double-cysteine mutant of rat OGC (rOGC and rOGC-C221,224S, respectively) were expressed in Escherichia coli, purified, and reconstituted into proteoliposomes to assess their function. Although both WT rOGC and rOGC-C221,224S exhibited transport properties for GSH and 2-oxoglutarate that were similar to those found in mitochondria of rat kidney proximal tubules, rates of transport and mitochondrial accumulation of substrates were reduced by >75% in rOGC-C221,224S compared with the WT carrier. NRK-52E cells were stably transfected with the cDNA for WT-rOGC and exhibited 10- to 20-fold higher GSH transport activity than nontransfected cells and were markedly protected from apoptosis induced by tert-butyl hydroperoxide (tBH) or S-(1,2-dichlorovinyl)-l-cysteine (DCVC). In contrast, cells stably transfected with the cDNA for rOGC-C221,224S were not protected from tBH- or DCVC-induced apoptosis. These results provide further evidence that genetic manipulation of mitochondrial GSH transporter expression alters mitochondrial and cellular GSH status, resulting in markedly altered susceptibility to chemically induced apoptosis.
Footnotes
-
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Grant R01-DK40725 (to L.H.L. and L.H.M.). Core facilities funded by the National Institute of Environmental Health Sciences, National Institutes of Health Center for Molecular Toxicology with Human Applications (Grant P30-ES06639) at Wayne State University were used for some of these experiments.
-
doi:10.1124/jpet.105.094599.
-
ABBREVIATIONS: GSH, glutathione; DIC, dicarboxylate carrier; 2-OG, 2-oxoglutarate; OGC, oxoglutarate carrier; rDIC, rat dicarboxylate carrier; tBH, tert-butyl hydroperoxide; WT, wild-type; rOGC, rat 2-oxoglutarate carrier; DCVC, S-(1,2-dichlorovinyl)-l-cysteine; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); Mes, 4-morpholineethanesulfonic acid; TMD, transmembrane domain; PLP, pyridoxal 5′-phosphate; PhSucc, phenylsuccinate.
-
- Received August 24, 2005.
- Accepted November 11, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
