Developmental changes in the inhibition of cultured rat uterine cell proliferation by opioid peptides.

Opioid peptides are negative regulators of cell proliferation in several organs including the uterus. In the present study, the ontogeny of the direct inhibitory action of opioid peptides on the proliferation of cultured rat uterine cells was investigated. Uteri of 7, 14, 21, 28, 35 and 60-day-old rats were removed in a sterile way. Tissue blocks were dispersed by limited digestions with trypsin and collagenase. Cells were cultured in enriched Dulbecco's modified Eagle's medium (DMEM). Treatments were present during the entire culture period. Cell densities of the monolayers were determined by counting the cells following trypsinization and trypan blue exclusion. Rat uterine mixed cell cultures grew to confluence within 10 days. The average population doubling time gradually increased with the age of animals. Epidermal growth factor (EGF) increased cell densities of cultures from all age groups. The oestradiol (E2)-responsiveness appeared at 21 days of age. The effect of [D-Met2-Pro5]-enkephalinamide (ENK) was biphasic. ENK and [Met5]-enkephalin (OGF) decreased cell densities of both unstimulated and EGF-stimulated cultures from 7-day-old rats to the same extent. ENK failed to act in 14-day-old animals. From 21 days of age on, the E2- or EGF-stimulated proliferation was inhibited only by ENK and DAMGO, while 30 nm DPDPE, Dynorhin-A, OGF, [Leu5]-enkephalin, beta-endorphin, and morphiceptin were ineffective. The half-inhibitory concentration of ENK was 0.3 nm. The effects of ENK were prevented by concomitant treatment with naloxone. Our novel data demonstrate two different phases of the inhibitory action of opioid peptides on rat uterine cell proliferation during ontogeny with an insensitive interval in between.

Stimulation by oestradiol of soluble-protein synthesis in rat hypothalamus.

The effect of oestradiol treatment on protein synthesis in the cytosol fraction (10(5) g supernatant) from ovariectomized mature and developing female rat hypothalami were studied. Results obtained on adults by double-label technique and SDS polyacrylamide gel or cellogel electrophoresis show that, as in the uterus, oestradiol-induced protein synthesis (IP) occurred in the cytosol fraction of the hypothalamus. The molecular weight of IP was about 40 000 dalton. During postnatal development, IP was also observed in cytosol fractions from the hypothalami of female rats 14, 21 and 28 days old. No clear-cut effect of oestradiol was found in the 7-day-old animals.

Role of endogenous opioids in progesterone antagonism on oestradiol-induced DNA synthesis in the rat uterus.

To probe the possible involvement of endogenous opioid peptides (EOPs) in progesterone (PG) antagonism on oestradiol-17 beta-(OE) induced uterine cell proliferation, the opioid antagonist naltrexone hydrochloride (NTX) and anti-[Met5]-enkephalin antiserum (AME) were investigated for their effect on uterine DNA synthesis in ovariectomized rats pretreated with OE and PG 24 h before killing. As an index of DNA synthesis the rate of in vitro incorporation of [3H]thymidine ([3H]TdR) into DNA was measured. The inhibitory effect of PG on OE-induced DNA synthesis could be diminished by approximately 42 and approximately 20% by the NTX treatments given directly into the uterine horns 13 and 4h before killing, respectively. Intraluminal AME treatments were only effective when they were administered 13 h before decapitation. Systemic blockade of opioid receptors by intraperitoneal NTX injections given every 6 h to the OE + PG-treated rats did not result in the disinhibition of uterine [3H]TdR incorporation. Our results suggest the involvement of EOPs--including [Met5]-enkephalin--as autocrine/paracrine factors in the PG antagonism on OE-induced uterine DNA synthesis.

Changes of [3H]naloxone binding in oestrogen stimulated rat uterus.

Effects of a single dose of oestradiol (Oe) on [3H]naloxone (Nal) binding in ovariectomized rat uterus were studied. Specific [3H]Nal binding was assessed by saturation analysis in 800 g supernatants and pellets of uterine homogenates. Two binding sites with higher (Kd approximately 1 nM) and lower affinity (Kd approximately 15 nM) for Nal were observed, their binding capacities and affinities have changed after Oe treatment in a time-dependent manner. The high affinity binding sites, detected only in the cytoplasmic fraction, disappeared after 1 h and only became detectable again at 24 h after hormone treatment, the lower affinity binding sites, after an initial drop, slowly increased, peaking at the 9th hour of hormone injection. The competition experiments indicate the involvement of different opiate receptor subpopulations in Oe induced changes. In the nuclear fraction, the Bmax values started to increase at 15 h, reaching the highest level at 18 h. The Kd values of lower affinity sites, in both studied compartments, were increased, i.e. the affinity decreased in the second half of the examined period.

Opioids regulate cell proliferation in the developing rat uterus: effects during the period of sexual maturation.

The present studies demonstrate, for the first time, that the rate of DNA synthesis in rat uterus of 21-32 days of age is inhibited by opioid peptides [D-Met2, Pro5]enkephalinamide. At around the time of vaginal opening (approximately 33 days) the opioids failed to act. High-affinity nuclear [3H]naloxone binding sites with linear Scatchard plots were detected in the uteri during the opioid-sensitive periods of DNA synthesis. Characteristics of these binding sites and the opioid sensitivity of uterine DNA synthesis are dependent on the age of the animals, the level of circulatory oestradiol and/or the maturity of the nuclear oestrogen receptor system.

Antiestrogenic effect of opioid peptides in rat uterus.

The effects of a single injection or continuous infusion of opioid peptide, [D-Met(2),pro(5)]enkephalinamide (ENK) on the hormone binding and transcriptional properties of estrogen receptors were investigated in estradiol (E(2)) treated rat uterus. The level of estrogen- (ER) and progesterone receptor (PR) proteins, the hormone binding of E(2) receptors and the effects of single injection of ENK with or without naltrexone (NAL) on the E(2)-induced changes in the level of Fos and Jun proteins and the binding of AP-1 proteins to DNA were studied. The receptor proteins levels were determined by Western blots and the binding of AP-1 to DNA by electrophoretic mobility shift assay. Both the ER and PR protein concentrations and the [3H]Estradiol binding to the high affinity nuclear receptors decreased after ENK treatment during the first two days. At 72 h the PR concentration decreased further, while no significant changes were found in the level of ER, however, at this time the former competitive E(2) binding turned into positive cooperativity. The E(2)-induced increase in the level of Fos proteins and the binding of AP-1 proteins to DNA was inhibited by a single injection of ENK. We conclude that the endogenous opioid peptides may interact with E(2) in the gene regulation of rat uterus.

Nuclear [3H]naloxone binding in rat hypothalamus.

High affinity (Kd approximately 1 nM) and low capacity [3H]naloxone binding sites were detected in the nuclear fraction of rat hypothalamus. The profile of this binding changed with age. In immature rats from 11 days of age until vaginal opening (approximately 33 day) the Scatchard plots of saturation data were linear and the Hill coefficient was 1, while just after vaginal opening (< 6 h) Scatchard analysis of [3H]naloxone binding gave a curvilinear component, with the Hill coefficient approximately 2, followed by an increase in binding sites and a decrease in Kd. In adults, after ovariectomy, the pattern of [3H]naloxone binding was similar to that observed in immature rats before vaginal opening. Following oestradiol treatment, binding sites with linear Scatchard plots disappeared and returned at least 24 h after hormone administration.

The effect of prostaglandin F2 alpha treatment on the endometrial oestradiol receptor system.

The effects of prostaglandin F2 alpha treatment on oestradiol receptor levels were studied in human decidual tissues from the first trimester of pregnancy. Two types of nuclear oestradiol binding sites in decidual tissue were detected. Type I receptors bind the oestradiol with high affinity and low capacity (Kd = 1.4 nM, Bmax = 0.3 pmol/mgDNA, Hillcoeff = 1), while type II binding sites have lower affinity and higher capacity for the hormone (Kd = 20.4 nM, Bmax = 2.0 pmol/mgDNA, Hillcoeff = 3). The nuclear oestrogen binding capacities of decidual tissue were affected by prostaglandin F2 alpha in a time dependent manner. Within the first 10 hrs after prostaglandin F2 alpha application the concentration of low affinity, high capacity binding sites (type II) was enhanced in comparison with the control values. Later, 20-24 hrs after the treatment, both types of nuclear oestrogen binding sites were increased significantly. When prostaglandin F2 alpha treatment failed to induce abortion no change in the concentration of nuclear oestrogen binding sites was observed. Oestradiol receptor concentration in the cytoplasm apparently was not affected by prostaglandin F2 alpha treatment.

Opioid peptides inhibit the estradiol-induced proliferation of cultured rat uterine cells.

The effect of opioid peptides on estradiol-induced cell proliferation in adult rat uterine primary cell cultures was studied. Estradiol increased cell density by 40%. This estradiol-induced stimulation of cell proliferation was decreased to control values by [D-Met2,Pro5]enkephalinamide. The opioid-induced inhibition of uterine cell proliferation was blocked completely by the specific opiate antagonist naloxone, while naloxone did not have any effect on its own. The inhibition of cell proliferation by enkephalinamide was apparent at each stimulatory estradiol concentration examined. This opioid effect was mediated mainly by the mu opiate receptor. The observed effects occurred within the physiological nanomolar concentration range. Enkephalinamide did not have any effect on the basal proliferation rate of adult rat uterine cells. However, enkephalinamide inhibited the basal rate of cell proliferation in cell cultures prepared from 7-day-old immature rats. In summary, here we present evidence of novel physiological direct cross-talk between the opioid and estrogenic signaling systems in the regulation of normal uterine growth.

Role of opioid peptides in the regulation of DNA synthesis in immature rat uterus.

The effects of a single dose of naloxone and of [D-Met2,Pro5]enkephalinamide on the DNA synthesis in the uterus of 7, 14 and 21-day-old rat were studied. After [D-Met2,Pro5]enkephalinamide treatment, an age-dependent decrease in in vitro [3H]thymidine incorporation into DNA was observed in all studied age groups. In the 21-day-old age group a reduced rate of DNA synthesis was detected for 12 h after [D-Met2,Pro5]enkephalinamide treatment followed by the return to control values at 24 h. The rate of inhibition was more marked in the younger age groups. The effect was also more pronounced in younger animals. Specific [3H]naloxone binding was detected both in membrane and nuclear fractions of uterine homogenates. While no age-related changes in binding affinities were found, the number of binding sites varied characteristically during development. Our data suggest the novel involvement of opioid peptides and their receptors in the regulation of uterine development.

Regulation of activator protein-1-DNA binding activity by opioid peptides in estrogen-sensitive cells of rat hypothalamus and uterus.

The present studies demonstrate, for the first time, that the binding of activator protein-1 (AP-1)-DNA in rat uterus and the estrogen-sensitive areas of the hypothalamus, as measured by electrophoretic mobility shift assay, is increased 2 h after intraperitoneal injection of [D-Met(2),Pro(5)]enkephalinamide. The effect was prevented by the opiate antagonist naltrexone given 30 min before the administration of [D-Met(2),Pro(5)]enkephalinamide, suggesting the involvement of opioid peptide receptors in the observed effects. The present findings support the role of opioid peptides in the regulation of transcription in estrogen-sensitive cells.

Anti-mitogenic action of opioid peptides on epidermal growth factor-stimulated uterine cells.

Endogenous opioid peptides are negative regulators of estradiol-induced uterine cell proliferation. To investigate the possible molecular target site(s) of their anti-mitogenic action, we examined the effect of opioid peptides on epidermal growth factor-induced cell proliferation both in uterine primary cell cultures prepared from adult rats and in human myometrial smooth muscle cell lines. Epidermal growth factor (EGF) significantly increased cell density in both types of cultured monolayers. This EGF-induced stimulation of cell proliferation was blocked by [D-Met(2)-Pro(5)]enkephalinamide in a time-dependent, receptor-mediated manner. The effective concentrations were within the physiological nanomolar range. Enkephalinamide did not have any effect on the basal rate of proliferation of the uterine cells. Our results on this novel physiological cross-talk suggest that shared step(s) of the mechanism of action of estradiol and EGF might be targeted by opioid peptides and not the general machinery of cell proliferation.

Inhibition of oestradiol-induced DNA synthesis by opioid peptides in the rat uterus.

Opioid drugs and peptides were investigated for their effect on uterine DNA synthesis induced by a single injection of 17 beta-oestradiol given to ovariectomized rats 24 h prior to decapitation. [D-Met2,Pro5]-enkephalinamide administered 12, 2 or 1 h before killing resulted in a significant (approximately 50%) inhibition of in vitro [3H]-thymidine incorporation into DNA, while injections given 24 or 6 h before decapitation were ineffective. Non-linear regression of the dose-effect curves resulted in an ED50 of approximately 0.26 and approximately 0.45 microgram/100 g b.wt. for the opioid treatments given 12 or 2 h before killing, respectively. These effects could be completely reversed by the opioid antagonist naloxone injected 30 min prior to the agonist treatment, while naloxone itself had no effect. Morphine and [D-Ala2,D-Leu5]-enkephalin administered 12 h, as well as dynorphin A fragment 1-13 given 2 h before decapitation also inhibited oestradiol-induced uterine DNA synthesis. In ovariectomized animals without 17 beta-oestradiol priming no significant effect of [D-Met2,Pro5]-enkephalinamide or naloxone on [3H]TdR incorporation was found.

Effect of naloxone and D-met2-pro5-enkephalinamide treatment on the DNA synthesis in the developing rat brain.

Effects of a single dose of naloxone and of D-Met2-Pro5-enkephalinamide on the DNA synthesis in the forebrain, hypothalamus and cerebellum of 11 day old female rats were studied. As an index of DNA synthesis the rate of incorporation of 3H-thymidine into DNA was measured 30 min after a sc. injection of 40 muCi/100 g b.w.. A time dependent effect of naloxone administration on cerebral DNA synthesis was observed. In the forebrain at 1 and 3 hrs after naloxone injection an increased rate of 3H-thymidine incorporation into DNA was found followed by a marked decrease at 9 and 12 hrs. The effect in the hypothalamus was similar but the initial increase at 1 hr was absent. On cerebellar DNA synthesis naloxone had no effect. The administration of D-Met2-Pro5-enkephalinamide resulted in a marked reduction in the labelling of cerebral and hypothalamic DNA between 1 to 12 hrs. Except a decrease at 1 hr no effect was found in the cerebellum.

Biochemical effects of neonatal testosterone treatment on the female rat hypothalmus during postnatal development. I. DNA synthesis.

The effect of 1 mg testosterone propionate (TP) administered on the 2nd day of life was studied on DNA synthesis in the anterior and posterior pats of the hypothalamus, in the forebrain, anterior pituitary and liver in 5, 7, 14 and 21 days old female rats. As an index of DNA synthesis the rate of 2-14C-thymidine (40 muCi/100 g body weight) incorporation into DNA was measured 1 hr after a subcutaneous injection . As an effect of TP treatment in both hypothalamic regions the rate of thymidine incorporation into DNA, markedly decreased during the first two weeks of life, while at 21 days no difference from the control value was found. In the anterior pituitary too therate of DNA synthesis decreased at 5 and 7 days but at 14 and 21 days the values were similar to the controls. TP treatment had no effect on the rate of DNA synthesis in the forebrain or in the liver.

In vivo nuclear 3H-estradiol binding in brain areas of the rat: reduction by endogenous and exogenous androgens.

In vivo specific nuclear binding of [2, 4, 6, 7 (n)-3H]estradiol (3H-E2) as indicated by diethylstilbestrol (DES)-blockable radioactivity, was measured 1 h after injection in adult intact male, untreated and testosterone propionate (TP)-pretreated or 5alpha-dihydrotestosterone (DHT)-pretreated gonadectomized (GDX) male and female rats. There was no sex difference in specific 3H-E2 binding in brain areas and anterior pituitary (PIT) of untreated GDX animals. DHT pretreatment had no effect on binding in any tissue. Specific nuclear binding in the preoptic area-anterior hypothalamus (POA-AH), median eminence-basal hypothalamus (ME-BH), amygdala (AMYG), septum (SEP), and dorsal hypothalamus (DH) was lower in intact males and TP-pretreated GDX males and females than in untreated GDX animals. Uptake in the PIT was not affected by TP pretreatment or by the presence of the testes, suggesting that the reduction in nuclear 3H-E2 binding observed in other tissues was caused by competition by estradiol formed in situ from TP or endogenous androgens for nuclear binding sites. Thus, the reduction in 3H-E2 uptake caused by the presence of TP or the testes could be used to estimate the degree of aromatization that occurred in these brain areas. In males, specific nuclear binding of 3H-E2 was reduced by the presence of the testes by 60-70% in the POA-AH and AMYG, by 30-40% in the ME-BH and SEP and by 0-20% in the DH. After s.c. administration of 1 mg/kg TP in GDX males, the amount of estrogen formed after 1,5 h in these same tissues approximated that present in the steady state in intact males. This suggests that, in the intact adult male rat, a high percent of estradiol nuclear binding sites in some brain areas is normally occupied by the products of aromatization.

Low dose RU486 prevents progesterone antagonism on uterine growth and concomitant type II oestradiol binding induction by oestradiol in rats.

The effect of RU486, a synthetic antisteroid, on the antagonism of progesterone (Pg) and dexamethasone (Dex) against oestradiol (Oe) induced uterine growth, and on uterine oestradiol binding (type I and type II sites) was studied in ovariectomized CFY rats. Changes of hypothalamic low affinity [3H]Oe binding have also been evaluated. Inhibitory effects of Pg but not of Dex on uterine growth and type II Oe binding site induction were prevented by RU486. Antiprogestin effect of RU486 could also be demonstrated on low affinity [3H]Oe binding in hypothalami. The inhibitory effect of dexamethasone on type II Oe binding was not opposed by antisteroid, on the contrary, RU486 seemed to potentiate this effect of Dex. Evaluation of type I binding was complicated by the distorting effect of type II binding at the saturation curve. Changes of type I binding seemed to parallel those of type II binding except after Oe+Dex treatment where an increased level of uterine cytoplasmic type I sites and a simultaneous decrease of type II sites were found. Blockage of [3H]Oe binding at high RU486 concentrations was found in vitro in the uterine cytoplasmic fraction. A less pronounced effect was observed in the nuclear fraction. Possible mechanisms of the RU486 effect on type II Oe binding are discussed.

On the mechanism of opioid-oestradiol interactions.

Characteristics of opioid binding and possible relationships between oestradiol and opioid binding sites were studied in rat oestrogen sensitive tissues(uterus, preoptic area-anterior hypothalamus, median eminence-basal hypothalamus). Naloxone (Nal) and oestradiol (Oe) bindings were assessed by in vitro saturation analyses. In 800 g supernatants of both uterine and hypothalamic tissues homogenates high affinity (Kd: 2-4 X 10(-9) M) and low capacity [3H]Nal binding sites were found. These binding sites were sedimented from 800 g supernatant by further centrifugation at 10(5) g for 1 h. In competition studies [3H]Nal binding was completely prevented by morphine, while met-enkephalin and leu-enkephalin caused only a partial inhibition. [3H]Nal binding was increased by ovariectomy and decreased by Oe treatment (10 micrograms/100 g b.wt) in both tissues. The cytoplasmic [3H]Oe binding in the studied tissues seems to be affected by the naloxone binding system. After in vitro saturation of naloxone binding sites by naloxone the [3H]Oe binding to low affinity sites (type II) in hypothalamus as well as in uterus has been increased by 8- and 2-fold, respectively. These results indicate the presence of specific [3H]Nal binding in rat uterus with similar properties to those found in the hypothalamus. Furthermore an interaction between opioid and oestradiol receptor systems could be also suggested.