Influence of 4-aminopyridine on human ovarian luteinized granulosa cell proliferation, production, and apoptosis through inhibiting voltage-gated K+ channel

Abstract

To study the influence of 4-aminopyridine (4-AP) on proliferation, production, and apoptosis through inhibiting voltage-gated K(+) channel (Kv) in ovarian luteinized granulosa cells. Ovarian luteinized granulosa cells were recovered from 25 women with regular menses who underwent in vitro fertilization programme. The cultured granulosa cells were divided into 4 groups:blank group, 4-AP treated group, human chorionic gonadotropin (hCG)-induced group and hCG + 4-AP co-treated group. The final concentrations of hCG and 4-AP were 1250 U/L and 5 nmol/L respectively. The progesterone production was detected by the chemoluminescence method. The expression of Kv mRNA on human ovarian luteinized granulosa cell was detected by RT-PCR. The influence on the early apoptosis of granulosa cells by 4-AP was observed by flow cytometry. Cellular caspase-3 activities were observed with colorimetric method and the inhibition of the cell proliferation was studied using methyl thiazolyl tetrazolium (MTT) method. (1) Kv mRNA was expressed in granulosa cell. (2) The progesterone production of the blank group, 4-AP treated group, hCG-induced group and hCG + 4-AP co-treated group were (547 +/- 64), (206 +/- 32), (1991 +/- 172) and (763 +/- 79) nmol/L, respectively after 24 hours culture. Exposure of the granulosa cells to 4-AP reduced the production of progesterone in blank and hCG-induced granulosa cells. (3) The flow cytometry analysis and the cellular caspase-3 A(405) showed that 4-AP increased the percentage of early phase apoptosis (P < 0.01): 4-AP treated group vs blank group [(40 +/- 5)% and 0.049 +/- 0.009] vs [(17 +/- 4)% and 0.029 +/- 0.008], hCG + 4-AP co-treated group vs hCG-induced group [(25 +/- 4)% and 0.039 +/- 0.008] vs [(15 +/- 3)% and 0.022 +/- 0.007]. (4) 24 hours after treated with 4-AP and hCG, the inhibitory rate of cultured granulosa cells of 4-AP treated group was higher than the blank group (19.7% vs 0), and that of hCG + 4-AP co-treated group was obviously higher than hCG-induced group (34.6% vs 0, P < 0.01). The voltage-gated K(+) channels expressed by ovarian luteinized granulosa cell play an important role in cell proliferation, production, and apoptosis. 4-AP may inhibit differentiation of progesterone in granulosa cells through the inhibition of proliferation and induction of apoptosis.