[Expressions of pannexin proteins in I-10 Leydig tumor cells and TM3 Leydig cells and their regulatory effect on the channel function in mice].

ABSTRACT

OBJECTIVE: To investigate the expressions of the pannexin (Panx) proteins in I-10 Leydig tumor cells and TM3 Leydig cells and their regulatory effect on the Panx channel function in mice. METHODS: The expressions of the Panx-1 and Panx-2 proteins in the mouse Leydig tumor cells were determined by Western blot. The I-10 Leydig tumor cells were treated with carbenoxolone (CBX) at 100 µmol/L or probenecid (PBN) at 200 µmol/L, the fluorescence resonance energy transfer (FRET) detected by time-lapse fluorescence imaging, and the extracellular adenosine 5'-triphosphate (eATP) level measured with the commercial detection kit. Molecular biological methods were used to interfere with shRNA and overexpress mPanx-1 the Panx-1 gene and regulate the expression and function of the Panx-1 protein. RESULTS: The expressions of Panx-1 (ï¼»289.5 ± 55.8ï¼½%) and Panx-2 (ï¼»264.5 ± 24.6ï¼½%) were significantly increased in the I-10 Leydig tumor cells as compared with those in the normal TM3 Leydig cells (both P < 0.05). FRET was remarkably reduced after treated with CBX (ï¼»87.5 ± 17.7ï¼½%) and PBN (ï¼»89.3 ± 14.3ï¼½%) in comparison with that in the control group (both P < 0.01). At 8, 16 and 24 hours, the eATP level was decreased by (57.3 ± 7.2)%, (56.4 ± 9.6)% and (63.4 ± 6.4)% in the CBX group (P < 0.01) and (61.7 ± 2.5)%, (35.8 ± 1.6)% and (13.5 ± 8.3)% in the PBN group (P < 0.01). Molecular biological treatment down-regulated the expression of Panx-1 by (38.3 ± 5.2)% and (31.8 ± 5.1)% in the shRNA1 and shRNA2 groups, respectively (both P < 0.01), but up-regulated that of Panx-1 by (128.4 ± 7.5)% in the mPanx-1 group (P < 0.01) as compared with the negative control. FRET was reduced by (72.4 ± 39.4)% in the shRNA group (P < 0.01) and the eATP level by (14.7 ± 0.1)%, (13.7 ± 0.3)% and (13.1 ± 0.3)% at 8, 16 and 24 hours, respectively (P < 0.01) while FRET elevated by (122.5 ± 17.1)% in the mPanx-1 group (P < 0.01) and the eATP level by (886.1 ± 82.1)%, (885.8 ± 83.3)% and (841.5 ± 21.8)% at 8, 16 and 24 hours, respectively (P < 0.01). CONCLUSIONS: The expressions of Panx-1 and Panx-2 are increased in I-10 mouse Leydig tumor cells, and inhibiting the Panx channel with CBX, PBN and shRNA reduces FRET and the eATP level in the I-10 cells.