Current Issue

Volume 19, Number 3, Oct-Dec (Autumn) 2017, Serial Number: 75 Pages: 443-451

Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia


Tamar Barbakadze, Ph.D, 1, 2, Galina Goloshvili, M.Sc, 1, Nana Narmania, Ph.D, 1, Elene Zhuravliova, Ph.D, 1, 2, David Mikeladze, Ph.D., D.Sc, 1, 2, *,
Institute of Chemical Biology, School of Natural Sciences and Engineering, Ilia State University, Tbilisi, Georgia
Department of Biochemistry, I. Beritashvili Center of Experimental Biomedicine, Tbilisi, Georgia
*Corresponding Address: 3/5 K. Cholokashvili Ave. 0162 Institute of Chemical Biology School of Natural Sciences and Engineering Ilia State University Tbilisi Georgia Email:davit_mikeladze@iliauni.edu.ge

Abstract

Objective

Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated S-nitrosylated-H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-H-Ras in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions.

Materials and Methods

In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATP-production as estimated by the luminometric method.

Results

Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn’t change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras.

Conclusion

According to the results, it may be proposed that hypoxia can decrease S-nitrosylation of soluble-H-Ras in D PC12 cells and abolish the inhibitory effect of NO-H-Ras in mitochondrial oxidative metabolism.