Potassium channel antagonists influence porcine granulosa cell proliferation, differentiation, and apoptosis

This investigation determined the effects of K(+) channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells. The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol I(Ks) antagonist 293B, the benzodiazepine I(Ks) antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX). Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively. Granulosa cell apoptosis was evaluated using flow cytometry. Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27(kip1) and the caspase-3 substrate poly(ADP-ribose) polymerase. The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells. However, the broad spectrum K(+) channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis. All three I(Ks) antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide. In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell. These data demonstrate that selective antagonism of granulosa cell K(+) channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis. Thus, K(+) channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.