Functional characterisation of an ovine endometrial oxytocin receptor cDNA transiently expressed in Cos-7 cells.
The entire coding region of an ovine endometrial oxytocin receptor (OTR) cDNA was generated by PCR, subcloned into the SV40 major late promoter expression vector pSVLJ and transiently expressed in Cos-7 cells. A specific OTR antagonist, 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin (OTA), was used to describe the binding kinetics of the expressed receptor which had a Kd of 4.5 nM and Bmax of 2.4 nM/mg protein (6.8 x 10(5) receptor molecules/transfected cell). The functional properties of the expressed OTR were determined by measuring oxytocin-induced phosphoinositide (PI) hydrolysis. Oxytocin increased PI turnover in OTR transfected cells fourfold in excess of residual endogenous activity, and stimulated phospholipase C (PLC) activity in a dose- and time-dependent manner, confirming that the expressed OTR cDNA was functional. Arginine vasopressin also stimulated PI turnover in a dose-dependent manner; thresholds of responses to oxytocin and arginine vasopressin were 10(-9) M and 10(-7) M respectively. OTA did not increase PI turnover and competitively inhibited the oxytocin-induced response. Direct activation of the pathway by aluminium fluoride and guanosine (3'-O-thio)-triphosphate (GTP gamma S) confirmed that the OTR was G-protein linked. Co-incubation of GTP gamma S with oxytocin shifted the PI-response threshold from 10(-7) M to 10(-9) M and significantly increased the level of response, suggesting that maximum PI turnover was agonist-dependent. The G-protein involved in mediating the signal transduction pathway was pertussis toxin-insensitive and, therefore, probably a member of the Gq subfamily. The PLC inhibitor, U73122, had no effect on oxytocin-induced PI turnover, consistent with the response in endometrial tissue. These data suggest that the signalling pathway mediated by expressed OTR is similar to that attributed to OTR occupancy in ovine endometrium.
Riley
P R
PR
Department of Physiology, Royal Free Hospital School of Medicine, London, UK.
Abayasekara
D R
DR
Stewart
H J
HJ
Flint
A P
AP
D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004717:Endometrium / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D007295:Inositol Phosphates / Q000378:metabolism; D010121:Oxytocin / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D018045:Receptors, Oxytocin / Q000235:genetics / Q000378:metabolism*; D012756:Sheep / Q000378:metabolism*; D015398:Signal Transduction
D004270:DNA, Circular; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D018045:Receptors, Oxytocin; D001127:Arginine Vasopressin; D010121:Oxytocin; D019204:GTP-Binding Proteins