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 Ca2+-imaging and perforated patch-clamp. We observed a resting intracellular Ca2+-concentration ([Ca2+]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 [Ca2+]i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP3-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y2 and P2Y4 receptors and that the [Ca2+]i increase was a prerequisite for hyperpolarization. Inhibitors of Ca2+-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 P2Y2 and P2Y4 receptors in granulosa cells modulate Cl− permeability by regulating Ca2+-release.
KeywordsGranulosa cells Purinergic receptors Cl− channels Follicle maturation Perforated patch-clamp Ca2+-imaging
- Bintig W, Buchholz V, Schlie S, Baumgart J, Heisterkamp A, Ngezahayo A (2007) Acta Physiologica 188(Suppl. 653):P10Google Scholar
- Burnstock G, Williams MJ (2002) Pharmacol Exp Ther 295:862–869Google Scholar
- Grynkiewicz G, Poenie M, Tsien RY (1985) J Biol Chem 260:3440–3450Google Scholar
- King BF, Townsend-Nicholson A (2003) Tocris Reviews 23:1–12Google Scholar