Identification of a myometrial oxytocin‐receptor protein

Abstract

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...

Oxytocin Receptors in Guinea Pig Myometrium Near Term and During Labor1

“…The absolute values for B max in the present study are similar to those reported by Chwalisz et al [29] and Fuchs et al [17], fourfold lower than those published by Fahrenholz et al [18], and 25 times higher than those obtained by one-point determinations [16]. Values of Hill coefficients close to unity, as assessed in our experiments, are similar to published values of 1.1-1.8 at 60-65 days of gestation [18,29].…”

“…As the dissociation constant (K d ) of myometrial oxytocin receptors was not determined in that study [16], the biological significance of these findings remains uncertain. Dissociation constants of 1.9 ϫ 10 Ϫ9 M [17] and of 6.9 Ϯ 0.3 ϫ 10 Ϫ9 M [18] at 60 days of gestation and of 2.6 Ϯ 0.2 x 10 Ϫ9 M at 65 days [18] were reported in later studies in which binding of oxytocin was studied over a limited range of oxytocin concentrations (10 Ϫ10 to 10 Ϫ7 M). This range did not permit the investigators to determine whether binding sites with K d s Ͼ 10 Ϫ8 M exist.…”

Reconstitution of the myometrial oxytocin receptor into proteoliposomes

“…Myometrial plasma membranes were prepared from myometrial tissue from pregnant guinea pigs on day 50 of gestation by modifications of the method used for preparation of rat myometrial membranes (Alexandrova and Soloff, 1980), as described (Fahrenholz et al, 1988), and stored frozen at -70°C.…”

“…Protein chemical studies, especially photoaffinity labeling experiments and enzymic deglycosylation of the myometrial oxytocin receptor from guinea pig, showed that the oxytocin receptor is highly glycosylated, with an apparent molecular mass between 68-80kDa (Kojro et al, 1991). At present, the functional domains of the receptor protein responsible for hormone binding and coupling with G-proteins are unknown, mainly because the purification of the receptor protein has not been achieved.Guinea pig uterus is the tissue of choice for purification of the myometrial oxytocin receptor because of its high receptor density at late pregnancy (Fahrenholz et al, 1988). In contrast to the solubilization of the oxytocin receptor from rat mammary gland (Soloff and Fernstrom, 1987), it has so far not been possible to achieve solubilization of the oxytocin receptor from guinea pig uterus without losing its binding activity.…”

“…Guinea pig uterus is the tissue of choice for purification of the myometrial oxytocin receptor because of its high receptor density at late pregnancy (Fahrenholz et al, 1988). In contrast to the solubilization of the oxytocin receptor from rat mammary gland (Soloff and Fernstrom, 1987), it has so far not been possible to achieve solubilization of the oxytocin receptor from guinea pig uterus without losing its binding activity.…”