December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Mitochondrial Activity Supplies Glutamate for Glutathione Synthesis in Rat Müller Cells
Author Affiliations & Notes
  • J Li
    Eye Research Institute Oakland University Rochester MI
  • CA Starnes
    Eye Research Institute Oakland University Rochester MI
  • BS Winkler
    Eye Research Institute Oakland University Rochester MI
  • Footnotes
    Commercial Relationships   J. Li, None; C.A. Starnes, None; B.S. Winkler, None. Grant Identification: NIH Grant EY10015
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 3622. doi:
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      J Li, CA Starnes, BS Winkler; Mitochondrial Activity Supplies Glutamate for Glutathione Synthesis in Rat Müller Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3622.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: To determine the extent to which the mitochondrial dependent production of L-glutamate (L-glu) contributes to the synthesis of reduced glutathione (GSH), an important tripeptide (L-Glu-L-Gly-L-Cys) with antioxidant and free radical scavenger activities, in cultured rat Müller cells. Methods: Passaged rat Müller cells (rMC-1) were grown to confluency in DMEM with 15% serum. The cells were then incubated in serum-free media for up to 4 hrs under the following conditions: 1) control DMEM; 2) with 1 mM buthiomine sulfoximine (BSO), an inhibitor of the synthesis of GSH; 3) in the absence of L-glutamine (L-Gln) and L-Glu or in the absence of cystine; and 4) with an inhibitor of mitochondrial activity (1 x10-5 M Antimycin A). Time-course measurements were made of the content of GSH, oxidized glutathione (GSSG) and ATP, together with measurements of the appearance of lactate in the media and enzyme activities. Results: In the control DMEM condition, the content of GSH in the Müller cells remained high over the 4 hrs incubation period (12.2 nmoles/million cells), and GSSG was virtually undetectable. In BSO-treated cells the GSH content declined by 60% after 4 hrs, but GSSG content remained extremely low. A quantitatively similar loss in GSH (without an increase in GSSG) was observed when cells were incubated for 4 hrs in cystine-free DMEM, indicating that extracellular cystine appears to be a good source of intracellular cysteine (L-Cys). In contrast, omitting L-Gln and/or L-Glu from DMEM did not lead to a statistically significant decrease in GSH content over 4 hrs. However, when mitochondrial activity was inhibited with Antimycin A, the content of GSH declined by 60% after 4 hrs, a decrease similar to that observed with BSO, without any increase in GSSG. In the presence of Antimycin A, the content of ATP in the Müller cells remained as high as in the control (uninhibited) condition, and lactate production increased by 1.6-fold. Thus, the decrease in GSH in mitochondrially-inhibited cells was not accompanied by a depletion of ATP. Inhibition of aspartate aminotransferase (AAT) with aminooxyacetic acid did not alter the level of GSH, suggesting that glutamate dehydrogenase (GDH) is the major route by which mitochondrially derived alpha-ketoglutarate is converted to L-Glu, This result is consistent with the specific activity of GDH being 2.5-fold higher than AAT in the mitochondria of Müller cells. Conclusion: These results suggest that mitochondrial production of L-Glu is an important source of the L-Glu used for the synthesis of GSH in rat Müller cells.

Keywords: 479 Muller cells • 475 mitochondria • 321 antioxidants 

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