Granulosa cells have calcium-dependent action potentials and a calcium-dependent chloride conductance

Abstract

We have found chicken granulosa cells to be excitable. Experiments using the whole-cell patch-clamp technique showed that they had membrane resting potentials of -62 +/- 3 mV (n = 8) and generated action potentials, either in response to 10-ms depolarizing current pulses or, on occasion, spontaneously. The action potentials persisted in a Na(+)-free bath and were reversibly blocked by 4 mM Co2+. They lasted 0.9-3.0s with 64 mM Cl- in the pipette, were shortened 67 +/- 8% by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 20 microM), and lengthened to 8.7 +/- 2.2 when the Cl- equilibrium potential (Vcl) was changed from -20 mV to -2 mV by using 134 mM Cl- in the pipette. With conventional whole-cell voltage-clamp, slowly activating and inactivating currents, which reached maximum amplitude after 0.35-1.40 s, were evoked by depolarizing voltage steps. These slow currents activated between voltage steps of -60 mV and -50 mV and reached a maximum inward amplitude at about -40 mV. Changing the Cl- concentration in the pipette (VCl of -2MV or -20 mV) or bath (VCl of -2 mV or + 18 mV) shifted their reversal potential in a direction consistent with a Cl- electrode. They were inhibited by the Cl- channel antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.5 mM), NPPB (20 microM), and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 0.5 mM). The slow currents were blocked by Ca2+ deprivation, or by CO2+ (4 mM), or by replacing external Ca2+ with Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)