Oxytocin receptors on cultured astroglial cells. Regulation by a guanine-nucleotide-binding protein and effect of Mg2+.
Specific binding sites for the radio-iodinated oxytocin (OT) antagonist d(CH2)5-[Tyr(Me)2,Thr4, Tyr-NH2(9)]OVT ([125I]OTA) have been characterized on cultured hypothalamic astroglial cell membranes. The rate of association of the ligand to OT-binding sites was identical in the presence and the absence of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p, 0.1 mM), whereas the monophasic dissociation reaction became biphasic in the presence of Gpp[NH]p. Scatchard analysis of equilibrium binding of [125I]OTA resulted in a linear plot with a single class of binding sites (Kd 0.06 nM) which were insensitive to the addition of Gpp[NH]p. Unlabelled OT and [Arg8]vasopressin (AVP) bound to high- (H) and low- (L) affinity states with a dissociation constant ratio (KL/KH) of 100 for both hormones. Binding with both high and low affinity required the presence of Mg2+ in the incubation buffer, and the addition of Gpp[NH]p decreased the KL/KH ratio to 10 and increased the percentage of low-affinity binding sites. On the other hand, neither omission of Mg2+ from the buffer nor the addition of Gpp[NH]p altered the binding of either OT or V1 AVP antagonists to OT receptors. In the presence of a G-protein inactivator (N-ethylmaleimide; 3 mM) during OT competition studies the affinities of the two OT-binding sites were unchanged, but 90% of the high-affinity binding sites were converted into the low-affinity state. These results obtained with cultured hypothalamic astroglial cells provide further evidence for a coupling of OT receptors with a guanine-nucleotide-binding protein, with a requirement for Mg2+.
Di Scala-Guenot
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Laboratoire de Physiologie, URA 1446 C.N.R.S., Strasbourg, France.
Strosser
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D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D002413:Cations, Divalent; D002478:Cells, Cultured; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007031:Hypothalamus / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D010121:Oxytocin / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000378:metabolism*; D018045:Receptors, Oxytocin
D002413:Cations, Divalent; D011945:Receptors, Angiotensin; D018045:Receptors, Oxytocin; D001127:Arginine Vasopressin; D010121:Oxytocin; D019204:GTP-Binding Proteins; D008274:Magnesium