Melvyn S. Soloff

Uterine receptor for oxytocin: Effects of estrogen*

Summary The effect of estrogen treatment on the affinity and concentration of uterine binding sites for oxytocin was studied. The oxytocin receptor was present in the uterus of the ovariectomized rat but the affinity for oxytocin increased more than four-fold 24 hr after a single injection of diethylstilbestrol. An increase in affinity was apparent 6 hr after estrogen. An increase in the number of oxytocin binding sites per uterus was apparent only 12 hr after estrogen, reaching twice the initial value by 24 hr. These results suggest that the enhanced sensitivity of the rat uterus to oxytocin following estrogen treatment is the result of an increase in the affinity and number of oxytocin receptors in the uterus. The affinity of uterine binding sites for (lysine)vasopressin was less than for oxytocin, whether or not the rats were treated with estrogen.

Regulation of oxytocin action at the receptor level

Abstract Prior exposure of certain target cells to elevated concentrations of a hormone can cause a reduction in the concentration of receptors for that hormone. Following the initial findings (1–6), many examples of the regulation of receptor concentrations by hormones and drugs have been reported (for a partial list see 7). In most cases a hormone regulates its own receptor concentration. There are examples, however, of hormones that can regulate the concentration of receptors for other hormones. The oxytocin (OT) receptor is a prime example of a system that is regulated by estrogens and progestins. Although changes in receptor concentration can play an important role in modifying the response to a hormone, in most systems studied it has not been possible to relate the changes in receptor concentration to an altered biological response to the hormone. Regulation of the OT receptor system, however, has been shown to have a very important bearing in the initiation of labor and parturition and these aspects of OT action are part of the subject of this review.

The effect of radioactive contaminants on the estimation of binding parameters by Scatchard analysis.

Abstract 1. 1.|When protein-ligand interactions are measured with a radioactive ligand, the presence of a radioactive contaminant that is not bound can lead to errors in the determination of the concentration of bound and unbound ligand, and consequently to errors in estimation of the dissociation constant (Kd). The extent of errors caused by a non-binding contaminant was determined by computer simulation for five types of protein-ligand interaction: first order dissociation, two classes of binding sites with different dissociation constants, displacement, negative cooperativity and positive cooperativity. For each type of reaction several values of Kd and several concentrations of binding sites were assumed. 2. 2.|The presence of a contaminant results in Scatchard plots which are convex upward instead of linear for the first order dissociation reaction. Scatchard plots normally are concave upward with three of the reactions studied: two classes of binding sites, displacement reactions and negative cooperativity. The contaminant causes a reduction in the concavity and in certain instances the plots may appear linear; with larger amounts of a contaminant they may be convex upward. The shape of the Scatchard plots for positive cooperativity is convex upward in either the presence or absence of a contaminant. Except for the displacement reaction the distortion of the shape of the Scatchard plots increases as the concentration of binding sites increases. 3. 3.|A contaminant, therefore, can cause distortions of the Scatchard plots which can lead to: (1) misinterpretations of the type of protein-ligand interaction, (2) overestimation of the dissociation constants, and (3) errors in calculation of the concentration of binding sites. In some instances as little as 1% contaminant may have a profound effect on the apparent affinity and number of binding sites.

A comparison of the estrone-estradiol-binding proteins in the plasmas of prepubertal and pregnant rats

Abstract The specific binding substances for estrone and estradiol-17β in prepubertal and pregnancy plasma of the rat were compared. Property Prepubertal Plasma Pregnancy Plasma Sedimentation Coefficient (S) 4.1 4.1 Stokes Radius (A) 37 30 Molecular Weight 64,000 51,500 Ka, estradiol-17β (M−1) 108 109 Binding Capacity, estradiol-17β (μM) 23 0.36 Ligand Specificity estrone > estradiol-17β > > > any other estrogen or steroid The characteristics of the two binders are similar but not identical.

Binding of 17β-estradiol by variants of α-fetoprotein in rat amniotic fluid

Two variants of alpha-fetoprotein in rat amniotic fluid were separated by their different affinity for concanavalin A-Sepharose, which selectively binds alpha-D-manno-pyranosides and alpha-D-glucopyranosides. Both forms had the same mobility upon polyacrylamide gel electrophoresis. The binding of 17beta-estradiol per mg of alpha-fetoprotein, determined both immunologically and electrophoretically, was the same for both variants. These results indicate that a specific carbohydrate portion of the molecule is not necessary for steroid binding.

Photoaffinity labelling of the oxytocin receptor in plasma membranes from rat mammary gland

Plasma membranes from rat mammary gland containing a high concentration of [3H]oxytocin binding sites (2.8 pmol/mg protein) were used for photoaffinity labelling experiments. Competitive binding experiments show that these receptors bind with high affinity the specific oxytocin agonist [Thr4, Sar7]oxytocin and the analogue of 1‐deamino‐[8‐lysine]vasopressin containing a photoreactive azidobenzoyl group (Abz) at the side chain of lysine. The tritium‐labelled (50 Ci/mol) photoreactive analogue incorporated into a membrane protein with an apparent relative molecular mass of 65000 ± 3000 Da (n = 16). The labelling of this protein was completely suppressed by an excess of oxytocin.

Endocrine Control of Parturition

In their introduction to the previous edition, Thorburn et al. (1977) stated: “We now recognize that in late pregnancy a train of events is initiated that ultimately results in the delivery of the fetus. However, we still do not know exactly how and where the train starts, or exactly how it exerts its ultimate action on the myometrial cell.” Little has changed in the last decade to increase our understanding of these events. Although the initiation of parturition is generally understood, the precise trigger for labor is still unknown. In addition, labor is complicated by different mechanisms in different species. For example, the onset of labor in rats and rabbits is rapid: uterine contractions become intense immediately before delivery, and the newborn are expelled rapidly. In humans, monkeys, and guinea pigs, labor develops slowly and is protracted. Schofield (1968) suggested that in species with a large fetus relative to the mother, a more protracted delivery may be an advantage. In the human and monkey, uterine motility evolves gradually during the last trimester of pregnancy, and actual labor often precedes delivery by many hours. It is possible that different mechanisms are at play in rapid-onset and protracted-onset types of labor.

Binding of estrogens by α-fetoprotein in rat amniotic fluid

Amniotic fluid from 15–17-day rat fetuses bound estrone and 17β-estradiol specifically. Related steroids such as estriol, 6-ketoestradiol, 17α-estradiol and testosterone were not bound to any significant extent. The apparent Ka for 17β-estradiol was 2.6·108 M− at 4°C; 6 nmoles of 17β-estradiol were bound per ml of amniotic fluid. The binding component appears to be α-fetoprotein in that it migrates as an α1-globulin upon polyacrylamide gel electrophoresis and has an isoelectric pH of 4.7 as determined by isoelectric focusing. Furthermore, binding activity was precipitated by antiserum which was shown by immuno-electrophoresis to be specific for α-fetoprotein. Binding activity, partially purified by isoelectric focusing of amniotic fluid, was associated with one of two bands seen by polyacrylamide gel electrophoresis. This band migrated as an α1-globulin.

Oxytocin receptors in rat oviduct.

The binding of tritiated oxytocin was studied in rat oviduct homogen ates sedimenting between 1000 X g for 10 minutes and 48000 X g for 30 minutes. Results showed that oxytocin is specifically bound to oviducal particles with high affinity. In particles prepared from estrogen-treated rats apparent Kd for oxytocin binding was 1.8 X 10 M. About 215 fmoles of oxytocin were bound per mg of particulate protein. Preparations from untreated rats had about 1/4 the affinity for oxytocin. Oxytocin analogues were bound in the same rank order as their uterotonic potencies: (desamino) oxytocin greater than (4-threonine) oxytocin greater than oxytocin greater than (8-lysine) vasopressin greater than desaminotocinol. These regressions were parallel indicating a common set of binding sites. Desaminotocinol the alcohol of the (desamino) oxytocin ring did not compete for binding sites in the dose range studied. Less than 20% of the oxytocin-binding activity of the oviducal 1000-48000 X g particles was lost during the 1-hour incubation period. The present studies have not demonstrated oxytocin receptors in ovarian particles under conditions which demonstrated binding with oviducal particles. This illustrates tissue specificity of oxytocin binding sites.

Oxytocin Receptors in the Uterus

Oxytocin is a nonapeptide that is synthesized by magnocellular neurons of the hypothalamus and, as shown within the past 10 years, by ovarian luteal cells of certain species.1–3 The peptide is similar in structure to arginine vasopressin, or antidiuretic hormone (Fig. 1), accounting for each having overlapping activities on the other’s target cells. Thus, vasopressin will cause uterine contractions in experimental animals, but considerably higher doses are required to obtain the same response as with oxytocin. Labor at term can be induced either by giving exogenous oxytocin or by stimulating endogenous oxytocin release.4 Despite these findings, many investigators have felt that oxytocin is not a physiological initiator of labor because fluctuations in its concentration in the blood immediately before labor do not correspond to changes in uterine activity. It now seems clear, from data collected in several species, that oxytocin’s activity during pregnancy and labor depends more on the sensitivity of myometrial cells than on the concentration of the hormone in the circulation.