Effects of cortisol on uterine eosinophilia and other oestrogenic responses

The effects of cortisol on several oestrogenic responses of the rat uterus were measured. Whether injected i.v., simultaneously with oestradiol-17β, or i.p., 12 h before the oestradiol, cortisol had no effects on the oestrogen-induced increases in uterine glycogen, protein and DNA contents. In contrast, cortisol inhibited both uterine eosinophilia and the increase of wet weight. Both responses show the same higher sensitivity to i.v. injection than to i.p. injection of cortisol. Inhibition of both responses by cortisol follows identical dose-response curves. These data support our hypothesis that the water-imbibition effect of oestrogen is mediated by uterine eosinophilia and is thus related to the eosinophil receptor system.     

Nafoxidine-responsive uterine protein: further characterization and distinction from the "estrogen-induced" protein (IP)

In a previous paper, we reported that nafoxidine (UA) stimulated the synthesis of a uterine protein showing the same electrophoretic mobility as the "estrogen-induced protein" (E2-IP) first described by Notides and Gorski. In the present work, we analyzed the IP-containing electrophoretic zone by SDS polyacrylamide-gel electrophoresis, and found that estradiol-17 beta (E2) and nafoxidine stimulated the synthesis of different proteins. As expected, estradiol-17 beta stimulated the synthesis of the E2-IP of 46 000 Mr. On the other hand, UA stimulated the synthesis of 27 000 and 30 000 Mr proteins (UA-IP). These UA-IP were not precipitated by an antiserum raised against E2-IP. Therefore, UA-IP appear to be independent entities and not degradation or precursor products of E2-IP. Both UA-IP are constitutively present in the uterus and even in higher relative amounts in rat brain. The present finding, that an "anti-estrogen", such as nafoxidine, stimulates the synthesis of different proteins than estrogen, provides a new approach to the study of the molecular mechanism of estrogen action.     

Multiple forms of nuclear estrogen receptor in the hypothalamus and pituitary after in vitro exchange with [3H]estradiol or [3H] hydroxytamoxifen

We have examined the sedimentation properties of hypothalamic and pituitary estrogen receptors (ER) translocated to the nucleus by in vivo estradiol (E₂) or nafoxidine treatments in the immature female rat. Nuclear ER were extracted with 0.4 M KC1 and incubated in vitro with saturating concentrations of [³H]E₂under conditions which allowed exchange with endogenous unlabeled ligands (23°C for 1–16 h). Sucrose gradient centrifugation of pituitary extracts from rats treated for 1, 24 or 48 h with E₂ yielded two components, sedimenting at 3 and 4–5S; in hypothalamic extracts the 3S form and variable amounts of a heavier shoulder were observed. After nafoxidine treatment the 4–5S species was present in greater proportions than after E₂. Both species interacted with monoclonal antibodies prepared against cytoplasmic ER from MCF-7 cells to yield more rapidly sedimenting complexes (7–8S). The 4–5S receptor was preferentially labeled in nuclear extracts subjected to exchange with [³H]E₂ for l h in vitro. In addition, nuclear ER from rats treated with [³H]E₂for l h in vivo sedimented at about 5S. We also investigated the direct binding of [³H]4-hydroxytamoxifen to nuclear ER. A single 4.2–4.3S component was present in both the hypothalamus and pituitary; this peak was displaced to about 7.5S after incubation with antibodies to MCF-7 ER. In the same nuclear preparations the 3S receptor predominated after exchange with [³H]E₂;however, a 1000-fold excess of unlabeled nafoxidine or hydroxytamoxifen abolished the 3S peak. These results suggest that 4–5S nuclear ER may give rise to the 3S form through degradation by endogenous proteases and that binding of antiestrogens to the larger species may interfere with this conversion.     

Oestrogen-induced increase in uterine cGMP content: A true hormonal action?

The dependency of the oestrogen-induced increase in uterine cGMP content towards the cytosol-nuclear receptor system was investigated. The following observations were made: (1) With oestradiol-17 beta (E2-17 beta), U11-100A (UA) or CI-628 (CI) the cGMP response elicited in the uterus of immature rats followed a course that was parallel to (yet delayed by about 1 h from it) the known time-course evolution of nuclear occupancy by the complex formed by each compound with the oestrogen-receptor. (2) While a marked (about 2-fold) increase in uterine cGMP content was obtained with 0.1 microgram E2-17 beta, oestradiol-17 alpha (E2-17 alpha) given at the same dose had no effect on uterine cGMP. (3) The 2--3 h response to E2-17 beta (or to UA) could not be obtained in animals that had received a first injection of E2-17 beta, 2 h, or of one of the anti-oestrogens UA or tamoxifen, 20--22 h prior to the test injection of E2-17 beta. Those 3 treatments have in common that, at the time indicated, they create a state of depletion in the uterine cytosolic receptor population. The cGMP response to E2-17 beta was restored 20--22 h following a first injection of E2-17 beta. This time is known, in this case, to correspond to full replenishment of the cytosol-receptor population. In all those tests, the wet weight increase, measured in the same organs, behaves exactly as did the cGMP response. These results support the conclusion that the increase in uterine cGMP after oestrogen administration to the immature rat, represents a true hormonal action which, like other uterotrophic actions of oestrogens, involves binding of the hormone by the cytosol receptor.     

Estrogen-induced proteins in luminal epithelium,endometrial stroma and myometrium of the rat uterus

The early effect of estrogen on the synthesis of cytosolic proteins was investigated in the luminal epithelium, endometrial stroma and myometrium of the uterus in adult ovariectomized rats. The procedure of Reiss and Kaye (1981) was followed (involving two-step fractionation of ³⁵S-labelled proteins and fluorographic analysis) except that the uteri were fractionated into their three main tissue components before homogenization. The results show that E₂ stimulates the synthesis of BB-CK (brain-type creatine kinase), the major component of IP (estrogen-induced protein), in the three tissues. This suggests that BB-CK is related to a function that is common to the estrogen responses (such as hypertrophy) of all three uterine tissues in ovariectomized adult animals. The synthesis of two unidentified proteins of 37000 and 27000 M_r was markedly stimulated in the epithelium. These proteins are probably rate-limiting in responses to estrogen treatment that are specific to the epithelium. The 27000 M_r protein has the same charge as that of the 27000 M_r nafoxidine-induced protein described previously (Mairesse et al., 1981) and is probably therefore the same protein.     

Interaction between the hepatic growth hormone receptor and concanavalin A.

The interaction between the plant lectin concanavalin A (Con A) and hepatic receptors for human growth hormone (GH) has been studied in particulate and soluble microsomal membrane preparations from rabbit and rat liver. Con A shows a dose-dependent, partial (30%) inhibition of 125I-human GH binding which is reversed by the Con A competitor, alpha-methyl mannoside. The Con A effect is dependent on the receptor concentration. The inhibition by Con A in rabbit liver is a reflection of a decreased number of available binding sites--there is no effect on binding affinity. It would appear that Con A binds directly to the GH-binding protein and not to an adjacent membrane glycoprotein. The GH receptor may consist of more than one molecular species, differing only in the carbohydrate type or content.     

Differential involvement of signaling pathways in the regulation of growth hormone release by somatostatin and growth hormone-releasing hormone in orange-spotted grouper (Epinephelus coioides)

Somatostatin is the most effective inhibitor of GH release, and GHRH was recently identified as one of the primary GH-releasing factors in teleosts. In this study, we analyzed the possible intracellular transduction pathways that are involved in the mechanisms induced by SRIF and GHRH to regulate GH release. Using a pharmacological approach, the blockade of the PLC/IP/PKC pathway reversed the SRIF-induced inhibition of GH release but did not affect the GHRH-induced stimulation of GH release. Furthermore, SRIF reduced the GH release induced by two PKC activators. Inhibitors of the AC/cAMP/PKA pathway reversed both the SRIF- and GHRH-induced effects on GH release. Moreover, the GH release evoked by forskolin and 8-Br-cAMP were completely abolished by SRIF. The blockade of the NOS/NO pathway attenuated the GHRH-induced GH release but had minimal effects on the inhibitory actions of SRIF. In addition, inhibitors of the sGC/cGMP pathway did not modify the SRIF- or GHRH-induced regulation of GH release. Taken together, these findings indicate that the SRIF-induced inhibition of GH release is mediated by both the PLC/IP/PKC and the AC/cAMP/PKA pathways and not by the NOS/NO/sGC/cGMP pathway. In contrast, the GHRH-induced stimulation of GH secretion is mediated by both the AC/cAMP/PKA and the NOS/NO pathways and is independent of the sGC/cGMP pathway and the PLC/IP/PKC system.