Purinergic signalling in rat GFSHR-17 granulosa cells: an in vitro model of granulosa cells in maturing follicles.

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca(2+)-imaging and perforated patch-clamp. We observed a resting intracellular Ca(2+)-concentration ([Ca(2+)](i)) of 100 nM and a membrane potential of -40 mV. This was consistent with high K(+)- and Cl(-) permeability and a high intracellular Cl(-) concentration of 40 mM. Application of ATP for 5-15 s every 3 min induced repeated [Ca(2+)](i) increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP(3)-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y(2) and P2Y(4) receptors and that the [Ca(2+)](i) increase was a prerequisite for hyperpolarization. Inhibitors of Ca(2+)-activated channels or K(+) channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl(-) channels hyperpolarized cells to -70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y(2) and P2Y(4) receptors in granulosa cells modulate Cl(-) permeability by regulating Ca(2+)-release.