M. Zanisi

Differential effects of low- and high-dose estrogen treatments on vascular responses in female rats.

In an attempt to study the mechanisms by which estrogens affect vascular responses, we utilized aortic preparations from intact and ovariectomized female rats receiving low- and high-dose subcutaneous estrogen treatments. Oil-treated, as well as male rats, served as controls. In ovariectomized females, low-dose 17-beta-estradiol injections (5 microg/kg daily for two days) affected the basal release of nitric oxide, as evaluated by concentration-related curves to superoxide dismutase and N(G)-Methyl-L-arginine acetate, which was found to be greater in 17-beta-estradiol-treated females compared to oil-treated females or males. Conversely, the nitric oxide-related vascular relaxation evoked by acetylcholine and sodium nitroprusside was unchanged. Prostacyclin production was also evaluated. Aortic rings from ovariectomized 17-beta-estradiol-treated females released significantly more prostacyclin than those from oil-treated females. These results point out a possible role for nitric oxide and prostacyclin in the vascular protection brought about by physiological levels of estrogens. When intact females were treated with high doses of ethynilestradiol (100 microg/Kg daily for one month), a component of contraceptive pills, either the basal release of nitric oxide, or acetylcholine-induced relaxation underwent a significant decrease. Likewise, the relaxant responses to sodium nitroprusside were impaired in the aortic rings obtained from ethynilestradiol-treated animals when compared to controls. Similarly, the amount of prostacyclin released from aortic tissues obtained from ethynilestradiol-treated animals was significantly reduced. These results may provide a possible explanation for the higher incidence of cardiovascular disease in women who take contraceptive preparations containing high doses of estrogens.

5α-Reductase Type 2 and Androgen Receptor Expression in Gonadotropin Releasing Hormone GT1-1 Cells

Gonadal steroids are potent modulators of gonadotropin releasing hormone (GnRH) secretion, and androgen binding sites and 5α‐reductase activity have been found in the immortalized GnRH secreting cell line GT1‐1, suggesting the existence of a direct androgenic control of GnRH dynamics. Two isoforms of the 5α‐reductase have been cloned with very different biochemical/functional properties: 5α‐reductase type 1 (widely distributed in the body) and 5α‐reductase type 2 (confined in androgen target structures). We have analysed whether, in GT1‐1, androgen binding sites are linked to ‘classical’ androgen receptor, and which 5α‐reductase isoform is active. Reverse transcriptase‐polymerase chain reaction analysis showed that the mRNAs coding for androgen receptor and for the two 5α‐reductase isoforms are all expressed in GT1‐1 cells. However, the 5α‐reductase enzymatic reaction showed a peak of activity at a narrow pH around 5.5, the optimum for the 5α‐reductase type 2. The affinity for testosterone, of the enzyme present in GT1‐1 cells, was very similar to that observed for the recombinant type 2 isozyme expressed in yeasts. The data indicate that GT1‐1 cells (i) express a ‘classical’ androgen receptor and (ii) contain the 5α‐reductase type 2 isoform, a specific marker of androgen‐responsiveness.

Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression

BackgroundNeuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem.Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment.MethodsWe analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively.ResultsDoublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness.Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin.Conclusiona) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.

Effects of progesterone metabolites on gonadotrophin secretion.

The experiments described where planned to validate the hypothesis that dihydroprogesterone (DHP) and 5alpha-pregnan-3X-ol-20-one (3alpa-ol) might represent the mediators of progesterone (P) actions in the structures where the steroid exerts its feedback effects on luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion. 5alpha-Pregon-3beta-ol-20-one (3beta-ol) was also studied. This is the 3beta epimer of 3alpha-ol. These steroids were given to castrated estrogen-pretreated female rats and to castrated female rats without the estrogen pretreatment. Serum LH concentrations were determined by the double-antibody radioimmunoassay procedure. An antiserum against ovine LH and pure ovine LH iodinated with iodine-131 were used. Values were expressed in terms of NIH-LH-S17. FSH was measured using the NAIMD-RAT-FSH radioimmunoassy kit. Serum LH levels have been shown to be depressed by the injection of ethinyl estradiol (EE). The injection of P on Day 5 of estrogen priming more than doubled the serum levels of LH. Both 3alpha-ol and 3beat-ol increased serum levels of LH as much as P did. DHP was less effective. The 3beta-ol was most effective. Elevated levels of FSH found after ovariectomy were depressed by 5 daily injections of EE. DHP 3alpah-ol and 3beta-ol all exerted a stimulating effect on FSH release but less prnounced than that of P. P did not inhibit LH release but DHP 3alpha-ol and 3beta-ol did. P and 3beta-ol did not diminish serum levels of FSH in castrated female rats but DHP and 3alpha-ol did. Results suggest that P exerts its facilitatory effects on gonadotropin secretion after local conversion into DHP and particularly 3alpha-ol. That the central mechanisms that control LH and FSH secretion are not identical is indicated by the relative efficiency of the different metabolites of P.

Astrocyte-Neuron Interactions in Vitro: Role of Growth Factors and Steroids on LHRH Dynamics

The data here reviewed, obtained with in vitro models, indicate that growth factors and steroids play a significant role in astrocyte-neuron interactions. Different designs have been adopted: (1) GT1-1 cells (a cell line derived from a mouse hypothalamic LHRH-producing tumor) were cocultured with type 1 rat astrocytes; and (2) GT1-1 cells were exposed to the conditioned medium (CM) in which type 1 rat astrocytes had been grown for 24 h. LHRH release and mRNA LHRH levels were measured respectively in the medium and in cell homogenates, at different time intervals (LHRH release, by RIA; LHRH mRNA by Northern blot analysis). The data obtained show that type 1 astrocytes secrete in the medium TGFbeta, which is able to modulate the release and the gene expression of LHRH in GT1-1 cells; and that one or more LHRH-degrading enzymes is/are present in the conditioned medium of type 1 astrocytes. A second part of the experiments have indicated that type 1 astrocytes are also able to affect, in different directions, the metabolism of testosterone and progesterone into their 5alpha-reduced metabolites occurring in the GT1-1 cells. In particular, it has been observed that the conversion of testosterone into DHT is decreased by the coculture with type 1 astrocytes, while the conversion of progesterone into DHP is increased by the same coculture conditions. Moreover, type 1 astrocytes are sensitive to steroid hormones, and in particular to the 5alpha-reduced metabolites of progesterone; this has been shown by analyzing the effects exerted by different steroids on the gene expression of the typical astrocyte marker GFAP.

Sex steroids and the control of LHRH secretion

Gonadal steroids are important hormonal signals that regulate the activity of LHRH synthesizing and releasing neurons. Aside from a direct effect through the feedback mechanisms exerted at hypothalamic and/or anterior pituitary level, gonadal steroids may modify the rhythmic LHRH release by modulating other systems affecting LHRH neurons. 1. In ovariectomized E2-treated female rats, progesterone is able to evoke LHRH release from the perifused hypothalamus without affecting LH and FSH release. 2. Excitatory amino acids (EAA) and their related analogs (NMDA and kainate) are known to stimulate LH release in young rats. When tested in a perifusion system on hypothalamic and anterior pituitary tissues, they differentially stimulate the release of LHRH (NMDA) and of LH (KA); their effect on both structures is markedly reduced following orchidectomy. It appears that gonadal steroids might exert a facilitatory action on the neurosecretory activity of LHRH neurons as well as a modulatory influence on the effect of EAA.

The anterior pituitary gland as a possible site of action of kainic acid.

Abstract The purpose of the present study was to analyze the direct effect of kainic acid (KA), an agonist of L-Glutamate, on the secretion of LH and FSH from anterior pituitary (AP) of male rats perifused in vitro. At low concentrations (1 μM), KA was able to stimulate the release of both gonadotropins from AP of 50-day-old male rats, but the response to subsequent stimuli was markedly impaired. This, however, was not due to a neurotoxic action of KA, but seemed rather suggestive of a down-regulation or desensitization of KA receptors. The stimulatory action of KA on LH and FSH secretion was age-dependent, since the agonist was completely ineffective on the AP of 75-day- and 18-month-old male rats. DNQX (6,7-dinitroquinoxaline-2,3-dione), a specific antagonist of the KA receptor subtype, was able to block the KA-induced gonadotropin secretion; similarly, AP-5 (2-amino-5-phosphonovalerate), a competitive NMDA receptor antagonist, prevented the stimulatory effect of KA on LH and FSH release. An interaction between the opiatergic and the excitatory aminoacid (EAA) systems emerged from the observation that pulses of KA applied to AP of 50-day-old male rats during a continuous perifusion with a medium containing morphine (5 μM) failed to increase gonadotropin secretion. These results indicate that KA can, at low concentrations, directly stimulate LH and FSH secretion by acting at AP level; this effect disappears with progression of age, and might be exerted both through NMDA and non-NMDA receptor subtypes. Finally, the results provide evidence that opioids and excitatory aminoacids might influence gonadotropin secretion from AP by acting in opposite directions.

LHRF, LHRH, GnRH: what controls the secretion of this hormone?

In the present study, the possible direct effects of opioids on the release of LHRH have been studied utilizing the GT1-1 cell model. It has been shown that only opioid agonists which bind to δ receptors bring about a significant inhibition of the release of LHRH, when this is stimulated by forskolin- or PGE2. The effects of δ opioid agonists are dose-dependent, and are reversed by the δ specific antagonist naltrindole. If one assumes that the GT1-1 cells represent a good model for the study of LHRH-secreting neurons, the obvious conclusion from these data is that endogenous opioid peptides may influence LHRH secretion also by acting directly on LHRH-secreting neurons. In another study it has been shown that GT1-1 cells possess high affinity-low capacity binding sites for estradiol and androgens. Moreover, a two-fold induction of androgen-binding sites has been observed after treatment of GT1-1 cells with estradiol, indicating that estrogen receptors are functional. The present observations suggest that gonadal steroids might influence LHRH release acting directly on the cells which manufacture and release the hypothalamic hormone. In order to evaluate the possible existence of a humoral communication between glial cells and LHRH-secreting neurons, two different approaches have been used: (a) GT1-1 cells were co-incubated with purified cultures of type 1 astrocytes; (b) GT1-1 cells were exposed to the conditioned medium (CM, untreated or submitted to different experimental procedures) where type 1 astrocytes were grown for 24 h. In both sets of experiments LHRH was measured by RIA in the incubation media of GT1-1 cells. The data show that short periods of either co-culture or exposure to previously frozen or heated CM significantly increase the release of LHRH from the GT1-1 cells. The stimulatory effect on LHRH release appears to be specific for type 1 astrocytes. Surprisingly, fresh CM proved to be inactive; this suggested that the factor(s) involved needed to be ‘activated’. Since it is known that TGFβ may be liberated from its supporting proteins by freezing or heating, the effects of TGFβ and of a TGFβ-neutralizing antibody were analyzed. The stimulatory effect exerted by the frozen CM was completely abolished by the antibody, while TGFβ 1 proved able to significantly increase LHRH release from GT1-1 cells. The present data unequivocally show that type 1 astrocytes in culture release some principle(s), probably TGFβ, able to stimulate LHRH release from the LHRH-producing GT1-1 cell line. To the authors’ knowledge, the present data provide the first clear-cut demonstration that the glia may directly intervene in the control of LHRH release via the secretion of humoral factors.

Antihormonal activities of 5α-reductase and aromatase inhibitors

Abstract The problem of developing androgen antagonists has been tackled so far only by synthesizing steroids able to displace testosterone and other androgens from their specific receptor sites. The observation that testosterone has to be converted intracellularly either to 5α-reduced metabolites (DHT, 3α-diol, etc.) or to estrogens, in order to become fully active on androgen-dependent structures (both central and peripheral), has opened the possibility of creating molecules which prevent these conversions, and which could then block the actions of testosterone. The availability of these new compounds has allowed a better understanding of the selective physiological role of each of the metabolites of testosterone, and to provide the basis for the development of new hormone antagonists to be used in those clinical conditions for which an inhibition of the actions of testosterone is foreseen. The usefulness of these enzyme inhibitors is underlined by some examples described in this paper. The results obtained may permit the formulation of the following conclusions: (1) The conversion of testosterone to its 5α-reduced metabolites occurring in the neuroendocrine structures may represent an essential step for the appearance of the inhibitory feedback effect testosterone exerts on LH secretion; (2) Testosterone exhibits its negative feedback effect on FSH secretion as such and not following the local aromatization to estrogens; (3) Testosterone exerts its effect on the intrahypothalamic stores of LHRH acting as such and not following its local conversion either to 5α-reduced metabolites or to estrogenic molecules; (4) Some of the new enzyme inhibitors (e.g. 4-OH-A) may represent an interesting tool for the treatment and/or the prevention of BPH and possibly of other androgen-dependent diseases (prostate carcinoma, acne etc.), as shown by their ability to prevent the in vitro conversion of testosterone to its 5α-reduced metabolites both in the normal prostate of the rat and in the human BPH tissue.

Feedback Effects on Central Mechanisms Controlling Neuroendocrine Functions

The peptidergic neurons of the hypothalamus which synthesize the Releasing and Inhibiting Hormones receive three types of endocrine signals: a) from the peripheral target glands (long feedback mechanisms); b) from the anterior pituitary (short feedback mechanisms) (see for references 42 and 54); and c) from the Releasing and Inhibiting Hormones themselves (ultrashort feedback mechanisms) (20, 55; see also 42 and 54 for references). For brevity’s sake, the discussion in this paper will be limited to the presentation of some data obtained in the authors’ laboratory regarding the effects of sex steroids on the peptidergic neurons synthesizing LH-RH, and on the secretion of gonadotropins and of prolactin.