Oxytocin-stimulated phosphoinositide hydrolysis in human myometrial cells: involvement of pertussis toxin-sensitive and -insensitive G-proteins.
Phosphoinositide hydrolysis is important in mediating the actions of oxytocin and prostaglandin (PG) F2 alpha on uterine contractions during labour. We have measured the effect of oxytocin, PGF2 alpha and other agents on the formation of inositol phosphates (IPs) in cultured human myometrial cells labelled with [3H]inositol and on changes in intracellular free Ca2+ concentration ([Ca2+]i) in cells loaded with Fura-2. Oxytocin induced the formation of [3H]IPs in a concentration-dependent manner with an EC50 (concentration of agonist producing 50% of the maximal response) of 1.4 +/- 0.5 nmol/l (mean +/- S.E.M.). The maximal response was obtained with 1 mumol oxytocin/l and represented a stimulation of 670% over basal. PGF2 alpha also stimulated the formation of [3H]IPs and the response at 1 mumol/l was a 204% stimulation over basal. The effects of PGF2 alpha were independent of extracellular Ca2+ but the effect of oxytocin was reduced with low extracellular Ca2+. Cyclic AMP formation, induced by forskolin or PGE2, had no effect on the stimulated levels of [3H]IPs. Pertussis toxin (PT) reduced the oxytocin-stimulated formation of [3H]IPs in a concentration-dependent manner. The maximal effect of PT resulted in an 80% reduction in the formation of [3H]IPs. However, PGF2 alpha stimulation was not affected by PT treatment. To analyse the action of PT further, we studied its effect on oxytocin-induced changes in [Ca2+]i. The basal [Ca2+]i was 112 +/- 4 nmol/l (n = 225 cells) and was not affected by PT treatment (109 +/- 3 nmol/l; n = 200 cells). In the absence of PT, 1 mumol oxytocin/l increased [Ca2+]i to a peak of 522 +/- 26 nmol/l, and in PT-treated cells, the [Ca2+]i peak was reduced to 348 +/- 16 nmol/l. Similar inhibitory effects of PT were obtained at oxytocin concentrations ranging from 1 to 100 nmol/l. Our data suggest that in human myometrial cells, the oxytocin-induced production of [3H]IPs and increase in [Ca2+]i are mediated by a PT-sensitive G-protein. However, a significant fraction of the oxytocin response appears to be mediated by a PT-insensitive G-protein, possibly a member of the Gq family.
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, U.K.
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Phaneuf
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Europe-Finner
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Varney
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MacKenzie
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Watson
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López Bernal
D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D015237:Dinoprost / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D009215:Myometrium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010121:Oxytocin / Q000378:metabolism* / Q000494:pharmacology; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism*; D013268:Stimulation, Chemical; D010566:Virulence Factors, Bordetella / Q000378:metabolism* / Q000494:pharmacology
D010716:Phosphatidylinositols; D010566:Virulence Factors, Bordetella; D010121:Oxytocin; D015237:Dinoprost; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium