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.
Bintig
Willem
W
Institute of Biophysics, Leibniz University Hannover, Herrenhäuser-Str. 2, D-30419 Hannover, Germany.
Baumgart
Judith
J
Walter
Wilhelm J
WJ
Heisterkamp
Alexander
A
Lubatschowski
Holger
H
Ngezahayo
Anaclet
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D011983:Receptors, Purinergic / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D002712:Chlorides; D004963:Estrenes; D011760:Pyrrolidinones; D011983:Receptors, Purinergic; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D002118:Calcium