The specific binding of [3H]oxytoxin to uterine membrane peparations derived from different species at late pregnancy was examined. The highest receptor density (b,,, value) was found in membranes derived from the myometria of guinea pigs between day 60 post-conception (b,,, = 3.6 f 0.1 pmol/mg) and day 65 (b,,, = 4.4 f 0.1 pmol/mg). The similarity of & values for oxytocin binding (Kd = 2.6 f 0.2 nM) and for vasopressin binding (& = 2.1 f 0.4 nM) to the same membranes derived from a guinea pig myometrium indicate a homogenous population of high-affinity binding sites which do not discriminate between these two hormones.Competitive binding experiments with specific oxytocin agonists containing either sarcosine or N-methylalanine in the place of Pro' demonstrated that these myometrial receptors have the pharmacological properties of oxytocin receptors.The analogue of 1 -deamino-[8-lysine]vasopressin containing a photoreactive azidophenylamidino group at the sidechain of Lys' retained roughly the same receptor affinity as oxytocin. In photoaffinity labelling experiments with the tritium-labelled analogue a membrane protein from guinea pig myometrium with an apparent relative molecular mass M , of 78000 f 5000 (n = 13) was preferentially labelled. The labelling of this protein was completely suppressed by a 100-fold molar excess of either oxytocin, or [Sar7]oxytocin or [Thr4, Sar7]oxytocin, but not by other peptide hormones. These results provide evidence that the labelled 78000-M, protein is a myometrial oxytocin-receptor protein.The neurohypophysial nonapeptide oxytocin stimulates the contraction of smooth muscle cells in the myometrial layer of the uterus. The existence of specific oxytocin receptors has been demonstrated in the animal and human myometrium [l, 21. The oxytocin sensitivity of the uterus increases during pregnancy and is accompanied by an estrogen-induced increase in the concentration of myometrial oxytocin receptors at late pregnancy and at early-term labor [3 -51.The molecular events which are triggered after binding of oxytocin to its myometrial receptor are not well understood. There is evidence for inositol phosphate production [6] and for Ca2+ influx in the myometrium [7]. Furthermore, oxytocin has been shown to inhibit a (CaZ+ + MgZ+) ATPase [8] and to increase the intercellular communication via gap junctions [9].Little is known about the oxytocin receptor's physical properties. Soloff and Fernstrom were able to solubilize with a relatively low yield (20 -25%) oxytocin binding sites from the pregnant rat myometrium and the rat mammary gland with a zwitterionic detergent [lo]. Gel filtration analysis indicated that the solubilized oxytocin binding components from rat mammary gland were present in multiple molecular mass forms: M, values ranged from 40000 to greater than 200000. The uterine oxytocin-receptor protein, however, has not yet been identified. The present study was performed to identify myometrial oxytocin-receptor proteins. For this purpose, a myometrial membrane from guinea pig c...
The affinity of 12 oxytocin analogues of similar structure but differences in conformational freedom and agonistic and antagonistic properties to receptors in guinea pig uterus and rat mammary gland membrane preparations was determined by competitive binding experiments using tritiated oxytocin. The values obtained for the apparent dissociation constant KD were compared to the values of biological activities from classical pharmacological tests ( uterotonic test in vitro and galactogogic test in vivo).
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