The modulatory effects of heat shock protein 70 on membrane calcium homeostasis of PC12 cells induced by ischemic/hypoxia

Abstract

Objective To investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on voltage-gated calcium channels on the cell membrane of pheochromocytoma cell 12 (PC12 cells) induced by ischemic/hypoxia and its mechanisms. Methods PC12 cells at logarithmic phase were collected, which were challenged with hypoxia for 6 hours and reoxygenation for 12 hours to stimulate the nerve cells suffered ischemia/reperfusion pathological process in vitro. PC12 cells were divided into non-infection group, infected by lentivirus containing green fluorescent protein (GFP) without HSP70 gene lentivirus control group (GFP lentivirus control group), and infected by lentivirus containing HSP70 and GFP gene recombined lentiviral infection group (HSP70 recombined lentiviral infection group). Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to determine mRNA and protein expressions of L-type voltage-gated Ca2+ channel subunits cav1.2 and cav1.3, receptor gated channel subunits NR1 and NR2, and Na+/Ca2+ exchange (NCX) in PC12 cells. Results After being challenged with hypoxia/reoxygenation, the mRNA and protein expressions of cav1.2, NR1 and NR2 in the PC12 cells were significantly lower in HSP70 recombined lentiviral infection group than those of GFP lentivirus control group and non-infection group [cav1.2 mRNA (2-ΔΔCt): 3.13±0.46 vs. 5.12±0.52, 5.13±0.66; NR1 mRNA (2-ΔΔCt): 1.61±0.44 vs. 3.23±0.82, 3.31±0.78; NR2 mRNA (2-ΔΔCt): 2.09±0.41 vs. 3.91±0.64, 3.88±0.62; cav1.2 protein (gray value): 2.82±0.39 vs. 3.98±0.23, 3.96±0.24; NR1 protein (gray value): 1.84±0.35 vs. 2.79±0.21, 2.86±0.23; NR2 protein (gray value): 0.87±0.24 vs. 1.57±0.31, 1.33±0.44; all P 0.01). There were no statistical differences in the mRNA and protein expressions of cav1.3 and NCX among non-infection group, GFP lentivirus control group and HSP70 recombined lentiviral infection group [cav1.3 mRNA (2-ΔΔCt): 4.82±0.32, 4.72±0.36, 4.82±0.29; NCX mRNA (2-ΔΔCt): 3.49±0.78, 3.47±0.71, 3.56±0.65; cav1.3 protein (gray value): 2.63±0.40, 2.64±0.39, 2.68±0.39; NCX protein (gray value): 3.27±0.48, 3.34±0.48, 3.31±0.42; all P > 0.01]. Conclusion Exogenous HSP70 affects the expression of L-type voltage-gated Ca2+ channel subunit cav1.2 and receptor gated channel subunits NR1 and NR2, which may protect PC12 cells from the injury caused by ischemic/hypoxia. Key words: Heat shock protein 70; Lentivirus; Ischemic/hypoxia; L-type voltage-gated Ca2+ channel; Receptor gated channel; Na+/Ca2+ exchange; Pheochromocytoma cell 12