Search

Gestodene (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4,5-gonadien-3-one), the most potent progestin ever synthesized, stimulates breast cancer cell growth through an oestrogen receptor-mediated mechanism, and its use in hormonal contraception has been associated with side effects attributable to oestrogenic actions. These observations have remained controversial, since gestodene does not bind to the oestrogen receptor or exert oestrogen-like activities. Recently, we have demonstrated that non-phenolic gestodene derivatives interact with oestrogen receptors and induce oestrogenic effects in cell expression systems. To assess whether gestodene is biotransformed to metabolites with intrinsic oestrogenic potency, [3H]- and [14C]-labelled gestodene were incubated in vitro with rat anterior pituitary, hypothalamus and ventral prostate homogenates under different experimental conditions. The most remarkable finding was the isolation and identification of 3beta,5alpha-tetrahydrogestodene and 3alpha,5alpha-tetrahydrogestodene as metabolic conversion products of gestodene, presumably with 5alpha-dihydrogestodene as intermediate. The overall results seem to indicate that the weak oestrogenic effects attributable to gestodene could be mediated by its tetrahydro metabolites.

Background/objective: Injection of bacterial lipopolysaccharide (LPS) into male rats activates genes that in turn induce many enzymes that participate in the animals' response to LPS. There is induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in many tissues. This induction could result from combination with cell surface LPS receptors that directly induce both genes, or the nitric oxide (NO) released as a result of iNOS induction could induce COX-2., Methods: To distinguish between these two possibilities, specific inhibitors of iNOS and COX-2 activity, aminoguanidine (AG) and meloxicam (MLX), respectively, were injected either peripherally or intracerebroventricularly (i.c.v.), and their effect on NO and prostaglandin E (PGE) production induced by LPS in the medial basal hypothalamus (MBH) and anterior pituitary gland (AP) were determined., Results: Peripheral injection of AG blocked iNOS-derived NO production in the AP but not in the MBH. When AG was injected i.c.v., iNOS-derived NO production in the MBH was blocked. MLX injected peripherally blocked COX-2-derived PGE(2) production in the MBH and AP, whereas AG injected peripherally or i.c.v. was ineffective. Since AG was only effective in blocking iNOS-derived NO production in the MBH when injected i.c.v., AG apparently does not effectively cross the blood brain barrier, whereas MLX injected peripherally inhibited PGE production, probably by inhibiting COX-2 activity in both the MBH and AP. AG was ineffective in preventing the increase in PGE derived from COX-2 in either the MBH or AP., Conclusion: LPS directly induces both enzymes, iNOS and COX-2, in the hypothalamus and AP., (Copyright 2002 S. Karger AG, Basel)

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.

To identify the specific locations of type 2 deiodinase (D2) messenger RNA (mRNA) in the hypothalamus and pituitary gland and determine its regulation by thyroid hormone, we performed in situ hybridization histochemistry, Northern analysis, and quantitative RT-PCR in euthyroid, hypothyroid, and hyperthyroid rats. By in situ hybridization histochemistry, silver grains were concentrated over ependymal cells lining the floor and infralateral walls of the third ventricle extending from the rostral tip of the median eminence (ME) to the infundibular recess, surrounding blood vessels in the arcuate nucleus (ARC), and in the ME adjacent to the portal vessels and overlying the tuberoinfundibular sulci. Silver grains also accumulated over distinct cells in the midportion of the anterior pituitary. In hypothyroid animals, an increase in signal intensity was observed in the caudal hypothalamus, and a marked increase in the number of positive cells occurred in the anterior pituitary. Microdissection of the hypothalamus for Northern and PCR analysis established the authenticity of D2 mRNA in the caudal hypothalamus, and confirmed that the majority of D2 mRNA is concentrated in this region. The distribution of D2 mRNA suggests its expression in specialized ependymal cells, termed tanycytes, originating from the third ventricle. Thus, the tanycyte is the source of the high D2 activity previously found in the ARC-ME region of the hypothalamus. The results indicate that tanycytes may have a previously unrecognized integral role in feedback regulation of TSH secretion by T4.

The expression of the nitric oxide (NO)-synthase enzyme (NOS) was analyzed in several hypothalamic nuclei and in the pituitary gland using in situ hybridization and immunohistochemistry. The effects of physiological and experimental stimuli on the expression of NOS was also investigated. Moreover, the role of NO in the secretion of anterior pituitary hormone luteinizing hormone (LH) was studied using primary culture of pituitary cells. The findings indicate that the expression of neuronal NOS is hormonally regulated and suggest that NO plays a role in hormone secretion.

11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) is a microsomal enzyme that catalyzes the dehydrogenation of cortisol (F) to cortisone (E) in man and corticosterone (B) to 11-dehydrocorticosterone (A) in rats. 11 beta-OHSD has been identified in a wide variety of tissues. The differential distribution of 11 beta-OHSD suggests that this enzyme has locally defined functions that vary from region to region. The aim of this study was to investigate the effects of the glucocorticoids B and dexamethasone (DM), the mineralocorticoid deoxycorticosterone (DOC), and the inhibitors of 11 beta-OHSD glycyrrhizic acid (Gl) and glycyrrhetinic acid (GE) on 11 beta-OHSD bioactivity at the hypothalamus (HT) and anterior pituitary (AP). Male Wistar rats were treated with GI or were adrenalectomized (ADX) and treated with either B, DM, or DOC for 7 days. All treatments were in vivo except GE, which was used in vitro. At the end of treatment, homogenates of HT and AP were assayed for 11 beta-OHSD bioactivity, expressed as the percentage conversion of B to A in the presence of NADP, 11 beta-OHSD bioactivity is significantly higher (P < 0.0001) in the AP compared with the HT. Adrenalectomy significantly increased the enzyme activity in the AP (P < 0.05), an effect reversed by B or DM. ADX rats treated with DOC showed decreased enzyme activity in the AP (P < 0.001) but increased the activity in the HT (P < 0.0001). Gl increased activity in both HT and AP, whereas GE decreased activity significantly. We conclude that the modulation of 11 beta-OHSD is both steroid specific and tissue specific.

The activation of protein kinase A (PKA) by cAMP was estimated in supernatant fractions from the hypothalamus (Hyp) and anterior pituitary (AP) of the female rat during the oestrous cycle and in ovariectomized and ovariectomized, ovarian steroid hormone treated animals. In both structures, the largest activation of PKA was found in dioestrus-2, while the lowest one was in Hyp in dioestrus-1 and in AP in oestrus. Ovariectomy had no influence on cAMP-dependent activation of PKA from Hyp and AP. Treatment of ovariectomized rats with 17-beta-oestradiol (E2), progesterone (P) or both abolished the activation of PKA by cAMP from AP and had no effect on hypothalamic PKA. These results indicate that ovarian steroids act specifically on AP processes via cAMP dependent pathway and regulation of PKA activity.

Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) activities (NADH- and NADPH-linked) were measured in anterior pituitaries, hypothalami and brains from lactating rats (8 and 21 days postpartum) and non-lactating (60-day-old cycling) rats. Tissue levels of these three enzyme activities varied significantly among the three groups examined. In terms of pituitary, mean levels of both of its 3 alpha-HSOR activities were 40-140% higher in actively lactating rats (8 days postpartum) relative to mean levels in lactating rats at weaning (21 days postpartum) or in non-lactating rats. There were no differences in pituitary progesterone 5 alpha-reductase activity among the three experimental groups. In the hypothalamus, the NADPH-linked 3 alpha-HSOR was elevated (50%) at 8 days of lactation compared to the group at 21 days. Hypothalamic NADH-linked 3 alpha-HSOR levels did not vary among the 3 groups. Hypothalamic progesterone 5 alpha-reductase levels in the actively lactating and weaning groups were 30% lower than those of the non-lactating group. Brain levels of progesterone 5 alpha-reductase were also lower in these two lactating groups (35-55%) as compared to the non-lactating control group. In brain, NADPH 3 alpha-HSOR activity did not vary among the three groups, but levels of NADH 3 alpha-HSOR activity were lower (40-50%) in the weaning group as compared to the actively lactating and control groups. These findings suggest the possibility that tissue changes in these progesterone-metabolizing enzyme activities during lactation and at weaning are influencing the in situ supply of 3 alpha,5 alpha-tetrahydroprogesterone and 5 alpha-dihydroprogesterone and their derivative effects on GABAA receptor activity and prolactin and gonadotropin release. The decreased activity of progesterone 5 alpha-reductase in hypothalamus and brain would presumably reduce in situ 5 alpha-dihydroprogesterone formation while increases in 3 alpha-HSOR activity would suggest higher in situ 3 alpha,5 alpha-tetrahydroprogesterone formation, especially in the pituitary.

The activities of monoamine oxidase and phenolsulfotransferase in the hypothalamus and anterior pituitary gland of spontaneously hypertensive rats and the normotensive control (Wistar Kyoto rat) rats were investigated. The monoamine oxidase activity (determined using dopamine as substrate) in both these tissues was not significantly different between the normo- and hypertensive animals. Hypothalamic phenolsulfotransferase does not sulfate-conjugate dopamine at pH of 6.5 and pituitary phenolsulfotransferase does not sulfate-conjugate dopamine or 3,4-dihydroxyphenylacetic acid at the same pH. Hypothalamic phenolsulfotransferase activity determined using 3,4-dihydroxyphenylacetic acid as substrate was significantly higher in the spontaneously hypertensive than the Wistar Kyoto rats, while pituitary enzyme (determined using phenol as substrate) was the same in both strains of animals. We proposed that in the spontaneously hypertensive rats the higher level of hypothalamic phenolsulfotransferase could (by removing 3,4-dihydroxyphenylacetic acid as sulfated acid) increase the deamination of dopamine by monoamine oxidase. This could in turn result in the presence of high amount of sulfated 3,4-dihydroxyphenylacetic acid in the anterior pituitary gland reported in our earlier study, and be partly responsible for the reduced central dopaminergic activity found in the hypertensive rats.

The effect of estradiol and testosterone on the activity of catechol-O- methyl transferase (COMT) of the superior cervical ganglion, pineal gland, anterior hypophysis and hypothalamus were examined in castrated rats. In female rats a single injection of 2 mug of estradiol caused a significant 25% increase of hypothalamic COMT, whereas after 3 daily injections no differences in enzyme activity were observed between hormone- and vehicle-injected controls. In male rats testosterone propionate (0.5 mg) increased hypothalamic COMT by 53%; this effect was not detectable in animals treated with the androgen for 3 days. COMT activity of the superior cervical ganglion, pineal gland and anterior hypophysis did not exhibit significant changes after any of the hormone schedules used.

The following experiments have been performed in order to verify whether the conversion of testosterone into its 5 alpha-reduced metabolites, 5 alpha-androstane-17 beta-ol-3-one (DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), in the hypothalamus and in the anterior pituitary is controlled by neural stimuli. Long-term castrated male rats have been submitted to anterior and total deafferentations of the hypothalamus and to the administration of the following centrally acting drugs: reserpine, p-chlorophenylalanine pCPA and atropine sulphate. The possible involvement of the central opioid system has also been investigated utilizing morphine and naloxone. Neither hypothalamic deafferentations, nor the treatment with reserpine, pCPA, atropine, morphine or naloxone produce any significant modification in the metabolism of testosterone in the hypothalamus. Hypothalamic deafferentations and treatments with reserpine, morphine and naloxone are also ineffective in changing the pattern of testosterone metabolism in the anterior pituitary. On the contrary, atropine and pCPA seem to affect the conversion of testosterone in the gland, both drugs producing an increased formation of DHT and 3 alpha-diol but not of 3 beta-diol. It is concluded that the 5 alpha-reductase-3-hydroxysteroid-dehydrogenase system of the hypothalamus does not appear to be controlled either neurally by inputs coming from other brain structures, or by variations of the neurotransmitter content in the hypothalamus itself. Serotonin and acetylcholine seem to participate in the control of testosterone metabolism at pituitary level, even if it is not clear whether their action takes place directly on the gland, or is mediated through some hypothalamic factor(s). Moreover, it does not appear that brain opioids are involved in the control of the enzymatic complex under consideration either in the hypothalamus or in the anterior pituitary.

The effects of a number of steroids on the conversion of progesterone to 5alpha-dihydroprogesterone by hypothalamic and pituitary progesterone 5alpha-reductase have been investigated. Using enzyme preparations from female rats and 3H-progesterone as substrate, 5alpha-reduced products (5alpha-dihydroprogesterone and 3alpha-hydroxy-5alpha-pregnan-20-one) were analyzed by reverse isotopic dilution analysis. The amount of total 5alpha-reduced products formed was compared in the presence and absence of the test steroid. Derivatives lacking the delta4 and/or the 3-keto moiety were without effect. Corticosterone had no effect. 16beta-Methylprogesterone inhibited progesterone 5alpha-reduction in both tissues by at least 65%, while the 2alpha-, 6alpha-, and 7alpha-methylated derivatives had lesser effects. 3-Oxo-4-pregnene-20beta-carboxaldehyde and 21-fluoroprogesterone were potent inhibitors. 17-Hydroxyprogesterone was a competitive inhibitor (substrate) with Ki's of 0.27 micrometer (pituitary) and 0.29 micrometer (hypothalamus). Medroxyprogesterone exerted little inhibitory effect. Of the 19-nor-steroids examined, only norethindrone appreciably inhibited the 5alpha-reduction. These results suggest that some natural delta4-3-ketosteroids can modify enzymatic activity. Also, inhibitory analogues may be useful for studies on the role of this 5alpha-reduction of progesterone.

Pituitary 5 alpha-reductase activity in intact male rats increases after the pituitary is incubated with the hypothalamus. Incubating the pituitary of castrate rats with the hypothalamus of intact rats relatively inhibits pituitary 5alpha-reductase activity. Coincubation of the pituitary and hypothalamus of castrate rats, or the pituitary of intact with the hypothalamus of castrate males, does not elicit changes in pituitary 5alpha-reductase activity. Different amounts of LH-RH in the incubation medium can modify 5alpha-reductase activity, i.e., activate it in the intact pituitary and inhibit it in the castrate pituitary. Hypothalamus from intact rats, which according to SHIN et al. [1974] is 'rich' in LH-RH, induces changes in pituitary 5alpha-reductase activity. The LH-RH-'poor' hypothalamus of castrate rats does not cause changes in this enzyme activity. The results suggest that there is a very close relationship between LH-RH and 5alpha-reductase activity in the pituitary.

The possibility that the activity of GABA-related enzymes in the hypothalamus, glutamic acid decarboxylase (GAD) and GABA-transaminase (GABA-T), may be modified by circulating prolactin levels was investigated in castrated female and male rats. Estradiol benzoate was chosen to induce an increase, while bromergocriptine was used to obtain a decrease of serum prolactin levels. Acute treatment with estradiol benzoate brought about 3 h later, a significant increase of serum prolactin, and a slight, non-significant increase of GAD activity in the hypothalamus. Chronic treatment with estradiol benzoate for 12 days induced significant increases in serum prolactin and GAD activity in the hypothalamus. Bromergocriptine significantly blocked this increase of GAD activity. Bromergocriptine-treated rats had significant increases of GABA-T activity in the hypothalamus. The results of this investigation support the possibility of the existence of a relationship between GABA-related enzymes in the hypothalamus and circulating prolactin levels.

In the present study we investigated the possible participation of endo-oligopeptidase B (poline-endopeptidase) in the control of gonadotrophin secretion through the control of LH-RH inactivation. This enzyme selectively hydrolyzes the Pro9-Gly10-NH2 peptide bond of LH-RH, thereby inactivating this substance. The enzyme activity was evaluated using a specific colorimetric substrate, i.e., Z-Gly-Pro-SM. Female adult Wistar rats were submitted to castration, experimental situations that are known to produce changes in gonadotrophin secretion. Hypothalamic and pituitary endo-oligopeptidase B activity was shown to be present predominantly in the soluble fraction of the enzyme preparations. The results also indicated that endo-oligopeptidase B activity adult female rat pituitary decreased after castration and increased after administration of estradiol and progesterone to castrated animals. The present results lead us to suggest that anterior pituitary endo-oligopeptidase B may be related to the control gonadotrophin secretion in female rats.

A number of diverse biological compounds involved in the regulation of the hypothalamo-hypophyseal-ovarian axis have been examined for effects on the conversion of 3H-progesterone to 3H-5 alpha-dihydro-progesterone and 3H-3 alpha-hydroxy-5 alpha-pregnan-20-one by female rat hypothalamus and/or anterior pituitary. Broken cell preparations were incubated with 3H-progesterone and NADPH, and product 5 alpha-reduced progestins were quantitated by reverse isotopic dilution analysis. Progesterone 5 alpha-reductase activity was reduced up to 50% in the presence of 10(-2) to 10(-3) M serotonin in both preparations. At 10(-3) M, various indoles including n-acetylserotonin, melatonin, 5-methoxytryptamine, 5-methoxytryptophol, and 5-hydroxyindole acetic acid decreased by 10 to 30% 5 alpha-reduced product formation. At 10(-2) M, carbamylcholine and norepinephrine were without effect, while 10(-2) M dopamine reduced by 20% the 5 alpha-reduction of progesterone only in pituitary homogenates. The LHRH protease inhibitor bacitracin (2 X 10(-3) M) decreased by 10 to 40% progesterone 5 alpha-reductase activity in both tissues. By itself, LHRH did not affect the 5 alpha-reduction of progesterone nor did it potentiate the bacitracin effect. In the presence of 1 mM ATP, 100 micronM cAMP and 100 micronM cGMP increased 5 alpha-reduced product formation in the hypothalamus by 19 and 14%. The gonadotropins LH and FSH and the prostaglandins E1, E2, F1 alpha, and F2 alpha were without effect. Thus, these results and others indicate that a number of cellular components and other factors can affect the in vitro 5 alpha-reduction of progesterone in broken cell preparations.

The conversion of T4 to T3 by the anterior pituitary gland appears to be of considerable physiological importance in the control of pituitary function. To determine a possible role of hypothalamic factors in controlling this enzymatic process, iodothyronine 5'-deiodinase (I5'D) activity was studied in rats 6 weeks after homologous transplantation of pituitary (implanted animals) or muscle tissue (sham animals) under the renal capsule. Intrasellar pituitaries remained intact, and serum T3, T4, and TSH levels were similar in both groups. I5'D activity was determined by quantifying T3 production rates in tissue homogenates at T4 concentrations of 0.002-4 microM, and with 20 mM dithiothreitol. Sellar pituitaries from sham and implanted animals displayed similar nonlinear reaction kinetics, suggesting the presence of two enzymatic processes having approximate Michaelis-Menten constant (Km) values of 2 nM and 0.3 microM. Maximum velocity (Vmax) was 51.3 +/- (SE) 4.0 fmol T3/min X mg protein (units) and 40 +/- 6 U for the low and high Km components, respectively. In transplanted pituitary tissue, I5'D activity was markedly altered; the low Km activity was significantly decreased (Km, 6 nM; Vmax, 13.0 +/- 1.1 U; P less than 0.001 compared to sellar pituitaries), whereas the high Km activity was increased 15-fold (Km, 5 microM; Vmax, 620 U). The in vitro addition of 6-n-propyl-2-thiouracil (0.1 mM) inhibited high Km I5'D activity in homogenates of both transplanted pituitary and renal tissue by approximately 50% (P less than 0.001), but had no effect on low Km I5'D activity in either sellar or transplanted pituitaries. In sham and implanted animals rendered hypothyroid by the inclusion of 1 g/dl NaClO4 in their drinking water for 6 weeks, low Km I5'D activity was increased approximately 3-fold in sellar and transplanted pituitary tissue. The levels of activity reached in transplanted tissue, however, were only 20-30% of those noted in sellar pituitary homogenates (P less than 0.001). High Km I5'D activity was estimated to be decreased 55% in transplanted tissue from hypothyroid animals. These studies demonstrate that transplantation of the anterior pituitary gland under the renal capsule in the rat results in marked alterations in two distinct components of pituitary I5'D activity. This suggests that neuroendocrine factors are important in the control of pituitary T4 to T3 conversion. Furthermore, it provides evidence for a unique mechanism whereby the hypothalamus, by modulating local thyroid hormone metabolism, may influence pituitary function.