SAT-174 - Role of Large-Conductance Ca2+-Activated K+ (Bkca) Channel in Steroidogenesis of Murine Non-Senescent and Senescent Leydig Cells

We have previously validated the involvement of the large-conductance Ca2+-activated K+ (BKCa) channel activity in testicular steroidogenesis. Here, we show that BKCa-α (pore-forming subunit) expression was positively correlated with senescence markers (p16/p21/p53) in Ames dwarf and GHRH-KO mice testes and isolated Leydig cells (LC), while serum testosterone levels were decreased. Moreover, mRNA expression of key steroidogenic proteins/enzymes (Star/Sf1/Dax1/Cyp11a1/Cyp17a1/Hsd17b) was significantly reduced, and testosterone in vitro production was positively correlated with BKCa-α expression in LC.
We performed functional studies to assess the role of BKCa in steroidogenesis in non-senescent and senescent LC. Thus, we generated a physiological senescent-LC model (LC isolated from 18-month-old C57BL/6J mice Vs. LC isolated from 3-month-old C57BL/6J mice) and a pharmacological senescent-LC model (TM3 LC line: 100 µM H2O2 for 48h Vs. Control conditions). In both LC models, senescent cells were characterized by increased expression levels of p21/p53 and decreased in vitro testosterone production.
Non-senescent LC (TM3 or 3-month-old mice) were incubated in the absence or presence of Iberiotoxin (IbTx 100 nM; 3h), a highly specific BKCa blocker. IbTx decreased in vitro testosterone production [TM3: Co=2.0±0.5, IbTx=0.8±0.1*; 3-month/old mice LC: Co=1.3±0.4, IbTx=0.6±0.1*; pmol/106 cells ± SEM; *p < 0.05]. IbTx also reduced key steroidogenic proteins/enzymes expression (StAR/Cyp11a1/Hsd3b/Hsd17b3) and increased senescence markers expression (p16/p21/p53) without altering BKCa-α expression. Senescent LC (TM3 or 18-month-old-mice) were incubated in the absence or presence of NS11021 (10 µM; 3h), a specific BKCa activator. NS11021 increased in vitro testosterone production [TM3: Co=1.3±0.4, NS=3.5±0.9*; 18-month-old-mice LC: Co=1.44±0.01, NS=1.99±0.1*; pmol/106 cells ± SEM; *p < 0.05]. NS11021 also increased key steroidogenic proteins/enzymes expression (StAR/Cyp11a1/Cyp17a1/Hsd17b3) and overall decreased some senescence markers expression (p16/p21/p53). BKCa-α expression, on the other hand, was decreased by NS11021 incubation in senescent-TM3 LC, while induced in 18-month-old-mice senescent LC.
Overall, our results demonstrate: 1) the existence of a positive correlation between testosterone in vitro production and BKCa-α expression in murine LC; 2) the negative impact of BKCa inactivation in non-senescent LC steroidogenesis; 3) the positive impact of BKCa activation in senescent-LC steroidogenesis.

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