Chloride influx mediates the effect of 4-AP on granulosa cell membrane potential and progesterone production. A) Effect of 4-AP (2 mM) on membrane potential of granulosa cells (GC) bathed in Tyrode solution modified to contain a low concentration (8 mM) of chloride (Low [Cl-]out). Data represent DiBAC4(3) fluorescence of untreated GC (n = 8) and GC exposed to 4-AP for 0.5 h (n = 7). B) Effect of DIDS (100 μM) and 4-AP (2 mM) on membrane potential (DiBAC4(3) fluorescence) of GC (n = 6) cultured for 24 h in the presence or absence of drugs. Decreased DiBAC4(3) fluorescence indicates hyperpolarization of resting membrane potential, while increased DiBAC4(3) fluorescence indicates depolarization of resting membrane potential. C) Progesterone (P4) accumulation by 24 h GC cultures maintained in the basic culture media (CON) alone or with 4-AP (2 mM), DIDS (100 μM) or DIDS+4AP. Data were obtained from 4 (CON, 4-AP) or 6 (DIDS, DIDS+4-AP) culture wells from a single GC isolation. Different superscripts indicate significant difference (P < 0.05).