Marietta Vértes

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.

Differential expression of Akt/protein kinase B, Bcl-2 and Bax proteins in human leiomyoma and myometrium

The expression and activation of serine/threonine protein kinase, Akt, in leiomyoma and in adjacent myometrium of human uteri was studied parallel with the changes of Bcl-2, Bax proteins, estrogen and progesterone receptors during menstrual cycle and early stage of the menopause. Abundant expression of Akt protein was detected in the studied tissues during menstrual cycle, the rate of increase was higher in leiomyoma than in corresponding myometrium. The expression of estrogen receptor alpha, progesterone receptor and of Bcl-2 protein changed parallel with that of Akt protein. The level of phosphorylated Akt (pAkt(473)) was seen only in leiomyoma samples from the growing period of tumors. At early stage of menopause levels of all studied proteins were lower than that in the menstrual cycle with the exception of Bax protein expression, which was high in leiomyoma. Our data suggest the involvement of phosphatidylinositol 3-kinase/Akt signaling in the pathomechanism of leiomyoma.

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.

Effect of estrogen and inhibition of phosphatidylinositol-3 kinase on Akt and FOXO1 in rat uterus.

The importance of FOXO transcription factors in regulating different aspects of cellular homeostasis and apoptosis has become apparent. Akt/protein kinase B has been shown to phosphorylate and inactivate members of FOXO family of transcription factors. Akt and its upstream regulator, phosphatidylinositol-3 kinase (PI3K) are involved in rapid action of estrogen (E2) in different cells and tissues. The aim of the present study was to analyze the E2/PI3K/Akt/FOXO pathway in rat uterus. In response to E2, phosphorylation of Akt/PKB on Ser473 and FOXO1 on Ser256 and Thr24 residues increased but with distinct kinetics, regulating the activation and inactivation of Akt and FOXO1 proteins, respectively. The antiestrogen ICI 182,780 prevented E2 induced Akt activation suggesting that estrogen receptors mediate this effect of E2. Intrauterine injection of Wortmannin caused a decrease in the phosphorylation of Ser473 of Akt, and attenuated phosphorylation of its downstream target FOXO1 at Ser256 and at Thr24. However, the effect of E2 on phosphorylation of Thr24 showed a kinetic pattern distinct from that of Ser256. Our results suggest that the E2/PI3K/Akt/FOXO1 pathway in rat uterus is functioning even at the lack of ovarian hormones and responses to E2 treatment. Estradiol increases Akt phosphorylation through a Wortmannin sensitive way, presumably involving PI3K. The present work shows that PI3K plays a crucial role in the phosphorylation and inactivation of FOXO1 in vivo, indicating that the regulation of this transcription factor is a more complex event in uterine cells requiring further investigations.

Phosphorylation of PTEN (phosphatase and tensin homologue deleted on chromosome ten) protein is enhanced in human fibromyomatous uteri

PTEN phosphatase, a product of PTEN tumor suppressor gene, exists in cells in phosphorylated and unphosphorylated form and has a central role in regulation of PI3K/Akt signalling which is involved in non-genomic action of estradiol. The purpose of this study was to analyze the level of total PTEN and phosphoPTEN parallel to phosphoAkt in leiomyoma and adjacent myometrium during menstrual cycle and at menopause. The expression of total PTEN in leiomyoma and myometrium did not change throughout the experiments. However, the level of phosphoPTEN was increased in leiomyoma during menstrual cycle. The phosphorylation of PTEN in myometrium was lower during secretory phase than that of proliferative phase. The phosphoAkt was abundant in leiomyoma, and its expression was higher during menstrual cycle than in myometrium. The phosphorylation of PTEN was directly related to phosphoAkt, suggesting a direct link between the inactivation of PTEN and activation of Akt. At the decline of sexual steroids, at menopause, no differences were observed in the expression of studied proteins between the two types of tissues. Our results suggest that the altered phosphorylation of PTEN protein and the consequent activation of survival signals may contribute to the pathomechanism of leiomyoma.

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.

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.

Expression and activation of Akt/protein kinase B in sexually immature and mature rat uterus

This study investigated the expression and activation of Akt/PKB in developing and adult rat uterus. Expression of Akt was observed in uteri from adult ovariectomized and 7-35-day-old rats and no changes were observed in response to in vivo estradiol treatment (1-100 microg/100g b.w.). To examine the mechanisms of PKB/Akt activation, phosphorylation at Thr(308) and Ser(473) regulatory sites were studied in uteri. Akt was constitutively phosphorylated on Ser(473) residue in the untreated, control uteri, while phosphorylation of Thr(308) was observed only after estradiol 17beta (E2) treatment. The effects of E2 treatment were age dependent, no response was induced in 11-day-old uteri, while in 28 days and older rats the activation of Akt at both regulatory sites, Ser(473) and Thr(308), increased, the first response was detected 2h after treatment, reaching the highest rate at 6h. The rate of phosphorylation was stronger at Ser(473) residue. The results suggest that the regulation of Akt activation at two regulatory sites in rat uteri are different, phosphorylation of Thr(308) seems to be entirely estrogen dependent, while the phosphorylation of Ser(473) is regulated by other factors as well as estrogen.

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.

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.