Localization of bradykinin B2 receptors in the endometrium and myometrium of rat uterus and the effects of estrogen and progesterone.

In the uterus, bradykinin is a potent inducer of smooth muscle contraction, which is mediated by the bradykinin B2 receptor subtype. However, little is known about the distribution or regulation of this receptor in this tissue. The aim of this study was to localize the B2 receptor in the uterus and determine whether the levels of this receptor were altered during the estrous cycle and modulated by estrogen and/or progesterone in ovariectomized rats. At diestrus, uterine B2 receptors were localized to both the circular and longitudinal smooth muscle layers of the myometrium, the endometrial stroma, the glandular epithelium, and the layer subjacent to the luminal epithelium. B2 receptor levels in both myometrium and endometrium were lowest during early proestrus, when estrogen levels are low, whereas myometrial B2 receptor protein and messenger RNA levels were highest during late proestrous, when estrogen levels peak. Similar findings were observed for the estrogen-supplemented group after ovariectomy, with progesterone appearing to inhibit the estrogen-induced rise in bradykinin B2 receptor density in estrogen/progesterone-treated animals. Using in vitro receptor autoradiography employing the specific B2 receptor antagonist analog, HPP-HOE140, immunostaining with specific antipeptide antibodies generated against the B2 receptor, and in situ hybridization using a specific bradykinin B2 receptor riboprobe, our findings show a discrete distribution of the bradykinin B2 receptor throughout the different layers of the uterus and suggest that bradykinin B2 receptor levels in the rat uterus are regulated by estrogen, and possibly progesterone, in both myometrium and endometrium.     

Multiple mechanisms of regulation of estrogen action in the rat uterus: effects of insulin

Eosinophils appear in the rat uterus in the presence of estrogen. The level of these cells in the uterus depends on the number of blood eosinophils. Insulin is an eosinopenic hormone in the blood and, therefore, could regulate estrogenic responses mediated by these cells in the uterus. Estrogen-induced uterine edema and eosinophilia at doses of 0.01,. 0.1, 1, 10, and 30 micrograms 17 beta-estradiol (E2)/100 g BW are inhibited by insulin. Estrogen binding by uterine eosinophils in vitro decreases in the presence of insulin, suggesting another explanation for the observations in the uterus in vivo. Injection of insulin alone or in combination with 0.01, 0.01, 0.1, or 1 microgram E2/100 g BW increases uterine RNA and protein contents by 6 h. Inactive insulin does not modify any of these stimulatory effects of estrogen. The results support the idea of two separate receptor systems for estrogens in the rat uterus: the eosinophil receptor system, which mediates estrogen-induced uterine edema, and the cytosol-nuclear receptor system, which mediates estrogen-induced uterine RNA and protein syntheses.     

Effects of in vivo estrogen treatment on adipose tissue metabolism and nuclear estrogen receptor binding in isolated rat adipocytes.

We have previously demonstrated the existence of nuclear estrogen receptors in isolated adipocytes (Pedersen et al. (1991) Biochim. Biophys. Acta 1093, 80-86). In the present study we have investigated the regulatory properties of these nuclear estrogen receptors, in addition to the metabolic effects of estrogen on adipose tissue metabolism. Estrogen treatment (20 micrograms 17 beta-estradiol in NaCl for 7 days) decreased lipoprotein lipase activity (LPL) in the adipose tissue by 62% (p less than 0.05), decreased adipocyte size by 27% (p less than 0.01) and diminished the normal postovariectomy weight gain. Furthermore, estrogen treatment increased the nuclear estrogen receptor binding in adipocytes; in addition, there was a tendency for increased cytosolic estrogen receptor content as well. Time course studies revealed that already 6 h after a single estrogen injection the Bmax increased from 3.82 +/- 0.3 fmol/10(6) cells to 9.8 +/- 3.6 fmol/10(6) cells (p less than 0.1) and 24 h after a single injection the Bmax was maximally increased to 12.7 +/- 5.5 fmol/10(6) cells (p less than 0.05). The Kd was similar at all time points (about 3-5 nM). Furthermore, the specific insulin receptor binding was increased in adipocytes from estrogen treated rats. The specific insulin binding was maximally increased by 149 +/- 6% (p less than 0.001) after 4 days of daily estrogen injections. The increased binding seemed to be due to an increased number of insulin receptors on adipocytes from estrogen treated rats with no alteration of the ED50 value. In conclusion it was found that estrogen treatment has a positive feedback effect on its own nuclear receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin D-dependent calcium binding protein in rat uterus: differential effects of estrogen, tamoxifen, progesterone, and pregnancy on accumulation and cellular localization

The present studies were undertaken to characterize the expression of calcium binding protein (CaBP or calbindin-D9k) in uterine tissues. Using immunohistochemical techniques, calbindin-D9k was localized to the uterine (luminal) epithelium of pregnant rats, but not present in the uterine epithelium of nonpregnant rats. Calbindin was found also in the uterine smooth muscle and endometrial stromal cells of pregnant animals. These latter localizations were reproduced in uteri of 21-day-old nonpregnant rats by administration of tamoxifen or physiological doses of estrogens. Estrogen and tamoxifen produced half-maximal increases of uterine calbindin at daily doses of 0.1 and 10 micrograms, respectively, and maximal responses at 0.3 and 40 micrograms/day. Testosterone and progesterone, at doses which increased the growth of the uterus, did not induce calbindin-D, and both hormones blocked estradiol's effect on uterine calbindin-D appearance. The epithelial localization of calbindin in pregnant uteri was not reproduced in nonpregnant animals by either estradiol (3 micrograms/day) or progesterone (1 mg/day). The localization of calbindin in uterine epithelium during pregnancy appears to be dependent upon an as yet unknown factor. In view of the large surface area of the luminal epithelium in pregnant animals, and the pregnancy-related expression of calbindin in these cells, we propose that uterine epithelium plays an important role in transport of calcium during pregnancy.     

Effect of cytochalasin B on hexose transport and glucose metabolism in pancreatic islets

Summary The effect of cytochalasin B on hexose transport into rat pancreatic islets isolated by collagenase digestion has been investigated. A double radioactive label procedure has permitted correction for label present in the extracellular space and determined that the permeability of the B-cell membrane of collagenase treated islets to L-glucose and sucrose is insignificant under the conditions employed. Cytochalasin B (15 μg/ml) significantly inhibits the initial rate of uptake of D-glucose, 3-0-methyl-D-glucose, and 2-deoxy-D-glucose at temperature of 20°C and 37°C when islets are pre-incubated (20 min) in the presence of cytochalasin B. No significant inhibition is observed on the initial rate of uptake of D-glucose at a temperature of 37°C if cytochalasin B is present only in the incubation medium. The production of¹⁴CO₂ from uniformly¹⁴C labeled D-glucose is unaltered in the presence of cytochalasin B over a 2 h incubation period. This paper was supported by U.S.P.H.S. Grants AM 03373 and AM 06181     

Characterization of membrane estrogen binding proteins from rabbit uterus

Estrogens exert fast non-genomic actions in their target tissues which may involve the participation of receptors located at the cell membrane. Studies were performed to identify and characterize membrane-associated 17beta-estradiol binding proteins in rabbit uterus. Specific and saturable [3H]17beta-estradiol binding sites of high affinity (Kd = 0.36 nM) were detected in uterine microsomes at higher concentration than in cytosol (370 +/- 98 vs. 270 +/- 87 fmol/mg protein, respectively). Various other steroid hormones, the stereoisomer 17alpha-estradiol and the antiestrogen tamoxifen were significantly less effective than 17beta-estradiol to compete with the radioactive ligand for binding to the membranes. The microsome binding sites were trypsin-sensitive and could be extracted to a great extent (80-90%) with 0.4/0.6 M KCl. Assays of the marker enzyme glucose-6-P dehydrogenase excluded membrane contamination with cytosolic soluble components. Immunoblot analysis of particulate and soluble fractions using monoclonal antibodies against the transactivation, heat shock protein recognition, and steroid binding domains of the nuclear estrogen receptor (ER; 67 kDa), revealed lower concentrations of the ER in membranes and the presence of five additional immunoreactive proteins of 57, 50, 32, 28, and 11 kDa which were absent in cytosol. Moreover, the antibody against the steroid binding domain was as effective as an inhibitor for cytosolic and membrane specific radioligand binding. Extraction of microsomes with the nondenaturing detergent CHAPS allowed a 2-fold enrichment of ER-like binding proteins as shown by antibody labeling and [3H]17beta-estradiol binding analysis. The results of this work are consistent with the existence of novel 17beta-estradiol membrane binding proteins structurally related to the intracellular ER. Future studies should investigate whether any of these proteins are involved in the primary events (e.g. receptor function) mediating nongenomic estrogen effects.     

Specificity of the effects of cytochalasin B on transport and motile processes.

The effects of cytochalasin B (CB) and dihydrocytochalasin B (H2CB) on a variety of transport and motile processes have been compared. CB inhibited transport of D-glucose and L-glucose but not transport of thymidine in human erythrocytes. In contrast, H2CB, which differs from CB by the absence of a single double bond, had little or no effect on any of these processes. Both cytochalasins, however, affected the morphology of cultured fibroblasts and inhibited motile processes such as membrane ruffling, axon growth cone activity, blood clot retraction, cytoplasmic streaming, photodinesis, and cytokinesis. Determination of the partition coefficient of the two cytochalasins in several organic solvent/phosphate-buffered saline systems showed that H2CB has a higher affinity for the hydrophobic phase than CB. These results indicate that the inhibitory effects of CB on sugar transport and on cell motility and morphology are separable and independent events, mediated by the binding of the drug to specific cellular receptors.     

Dose-response effects of 2-methoxyestradiol on estrogen target tissues in the ovariectomized rat

In three experiments, we evaluated the pharmacological effects of 2-methoxyestradiol (2ME(2)) on several estrogen target tissues. Experiment 1: we gavaged recently ovariectomized (OVX) 9.5-wk-old rats with 2ME(2) at doses of 0, 0.1, 1, 4, 20, and 75 mg/kg in a 21-d dose-response study. 2ME(2) reduced body weight and serum cholesterol, increased uterine weight and epithelial cell height, and inhibited longitudinal and radial bone growth compared with values in the untreated OVX rat. All doses of 2ME(2) maintained cancellous bone mass at the baseline level, the lowest effective dose being 20-fold less than a uterotrophic dose. Experiment 2: in an 8-wk experiment in adult OVX rats, a nonuterotrophic dose of 2ME(2) (4 mg/kg x d) suppressed body weight gain, inhibited bone formation in cancellous bone and partially prevented bone loss in the tibial metaphysis. Experiment 3: in weanling rats, ICI 182,780 did not antagonize the effect of 2ME(2). We conclude that 2ME(2) antagonizes the skeletal changes that follow OVX at doses that have minimal or no effects in the uterus in both young and adult rats; 2ME(2) does not appear to act via estrogen receptors and is active on bone at doses well below those required for tumor suppression in mice. 2ME(2), through a novel pathway, may be a useful alternative to conventional hormone replacement therapy for prevention of postmenopausal bone loss.