RESUMO
The binding sites of antisera generated in the guinea pig against ovine luteinizing hormone (oLH) and its two subunits (oLHalpha and oLHbeta) have been localized in rat anterior pituitaries taken from normal or castrated males and from ovariectomized females with the peroxidase-labeled antibody method, using light and electron microscopy. With the light microscope, the cells positive with antiserum to ovine luteinizing hormone (A-oLH) were violet after the Alcian blue-periodic acid-Schiff (AB-PAS) staining; they were also positive for A-oLHalpha and for A-oLHbeta and, from castrated males, they displayed an increased affinity for A-oLHbeta. Another cell type which was blue after the AB-PAS method reacted with the A-oLHalpha only; these cells, presumably thyrotropic cells, were retracted after castration and, besides their affinity for A-oLHalpha, acquired an affinity for A-oLHbeta. As seen through the electron microscope, two cell types were positive for A-oLH, A-oLHbeta, and A-oLHalpha and may be identified as luteinizing hormone-secreting cells. Type A cells were characterized by two classes of rounded, secretory granules. Type B cells were smaller and contained only small secretory granules. 1 mo after the rats were castrated the type A cells were hypertrophied and vacuolized. In both cases the secretory granules were the main sites of the antigenicity with the three antisera. A positive reaction was also found in the cytoplasm, particularly in hypertrophied cells from ovariectomized females and with A-oLHbeta. The cisternae of the rough endoplasmic reticulum were usually negative, except in highly degranulated cells from ovariectomized females and with A-oLHbeta.
Assuntos
Sítios de Ligação de Anticorpos , Hormônio Luteinizante , Animais , Castração , Grânulos Citoplasmáticos , Retículo Endoplasmático , Feminino , Cobaias/imunologia , Histocitoquímica , Técnicas Imunológicas , Masculino , Microscopia , Microscopia Eletrônica , Ovário/fisiologia , Peroxidases , Hipófise/citologia , Hipófise/imunologia , Ratos , Ovinos/imunologiaRESUMO
The ability of 7,12-dimethylbenz(a)anthracene (DMBA), a potent inducer of mammary tumors, to mimic short term effects of estradiol (17 beta-E2) on the anterior pituitary, was tested in vitro. Incubation of anterior pituitaries from ovariectomized rats with DMBA resulted in a marked depletion of membrane dopamine receptors (labeled with [3H] spiperone) and a parallel stimulation of prolactin (PRL) release. Maximal receptor depletion and PRL release were obtained after 15-30 min of incubation with 10(-8) M DMBA. These effects were reversible and already significant after a 5-min incubation. Their magnitude, dose dependency, and time course were identical to those reported for 17 beta-E2. A structurally related noncarcinogenic polycyclic aromatic hydrocarbon, phenanthrene, had no effect on [3H]spiperone binding or PRL release. When DMBA and 17 beta-E2, at suboptimal concentrations, were simultaneously added to the culture medium, no synergistic effect could be observed. When 10(-8) M of both compounds were introduced simultaneously, the decrease in dopamine receptors and the increase in PRL release were not greater than those observed in the presence of 10(-8) M of only one compound, indicating that the same mechanism(s) can be involved. These data suggest that DMBA in desensitizing lactotrophs to dopamine and in releasing PRL, by direct estrogen-like actions on anterior pituitary, may provide a hormonal state conducive to tumor development.
Assuntos
9,10-Dimetil-1,2-benzantraceno/farmacologia , Estradiol/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Receptores Dopaminérgicos/efeitos dos fármacos , Animais , Feminino , Técnicas In Vitro , Fenantrenos/farmacologia , Prolactina/metabolismo , Ratos , Ratos Endogâmicos , Espiperona/metabolismoRESUMO
The effect of 17 beta-estradiol (17 beta E2) on anterior pituitary dopaminergic receptor (D2) content was studied in vitro in relation to PRL secretion. Anterior pituitaries from ovariectomized rats were incubated for short periods in medium 199, with or without the steroid. Dopamine (DA) receptors in partially purified pituitary membranes were quantified by equilibrium binding using [3H]spiperone; the PRL released into the incubation medium was analyzed by RIA. Addition of 10(-10) to 10(-6) M 17 beta E2 to the incubation medium of anterior pituitaries rapidly and reversibly decreased the number of DA receptors (P less than 0.01 to 0.001), while increasing PRL release, in a dose-related fashion. The maximal effect on both receptor numbers and PRL secretion was obtained with 10(-8) M 17 beta E2. This effect involved no change in receptor affinity (Kd = 0.11 +/- 0.01 nM in presence or in absence of 17 beta E2). This estrogen-induced decrease in DA-binding capacity was apparently not the result of the occupation of spiperone binding sites by the steroid, since after a 30-min incubation with 10(-8) M [3H]17 beta E2, no radioactivity was detectable on the partially purified membranes. Moreover, the presence of 17 beta E2 at the same time as the labeled D2 ligand did not modify the kinetics of association or dissociation of spiperone with pituitary membranes. This decrease in anterior pituitary DA receptor content and the increase in PRL release were already significant after a 7-min incubation in the presence of 10(-8) M 17 beta E2. Finally, these effects of 17 beta E2 were not mimicked by its 17 alpha-stereoisomer, nor by progesterone, or testosterone. These results suggest that the stimulatory effect of 17 beta E2 on PRL secretion may be due, at least in part, to the desensitization of anterior pituitary cells to DA. The steroid may produce this desensitization directly by decreasing the number of D2. The short latency of this effect likely discards the possibility of a genomic action of 17 beta E2.
Assuntos
Estradiol/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Receptores Dopaminérgicos/efeitos dos fármacos , Animais , Dopamina/fisiologia , Feminino , Técnicas In Vitro , Cinética , Ovariectomia , Adeno-Hipófise/fisiologia , Prolactina/metabolismo , Ratos , Receptores Dopaminérgicos/metabolismo , Taxa Secretória/efeitos dos fármacos , Espiperona/metabolismo , Esteroides/farmacologiaRESUMO
A single ip injection (0.2 ml) of a rabbit antiserum to LHRH was given at noon on proestrus (day 0) to 4-day cycling rats. A blockade of the preovulatory surge of LH and FSH was observed, as well as inhibition of ovulation; the prolactin (PRL) surge was unaffected. Four days later, palsma LH and FSH were still low and hypoprolactinemia appeared, while the estrous cycle was unaffected. Then, after a transient diestrous period, vaginal smears indicated a pattern of persistent estrus for at least 240 days starting from day 8 after treatment. Hypothalamic LHRH remained low throughout this period, with the exception of a transient rise around day 36; hypothalamic serotonin and dopamine were unchanged. A peak of FSH in serum appeared on day 28 and an elevation of serum LH on day 36; this latter rise was concomitant with the increase of hypothalmic LHRH and with ovarian luteinization. Except for this short period, the ovaries showed a picture of persistent estrus, with large, cystic follicles. During the whole experimental period, basal serum FSH levels were higher than those of LH. Pituitary FSH and LH contents remained in the range usually found during the normal estrous cycle. Starting from the 8th day after treatment, a marked hyperprolactinemia appeared. Serum estradiol and progesterone were assayed on days 10 and 40 after injection; no significant increase in either steroid was observed on day 10, but on day 40 the levels of estradiol increased to values similar to those of progestrus, while progesterone showed only a small, but significant, increase. Thus, a single injection of LHRH antiserum (AS) in a normal cycling rat provoked a long-term alteration in hypothalamic function. The results so far obtained suggest that the long-lasting hyperprolactinemia induced by this treatment, might be due to an abnormal ovarian secretion.
Assuntos
Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Gonadotropina/imunologia , Soros Imunes/farmacologia , Hormônio Luteinizante/metabolismo , Prolactina/metabolismo , Animais , Anticorpos/análise , Castração , Estradiol/sangue , Estro , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Ovário/fisiologia , Hipófise/metabolismo , Gravidez , Proestro , Progesterona/sangue , Coelhos , Ratos , Fatores de TempoRESUMO
The high speed supernatant fraction from homogenates of the steer anterior pituitary was shown to contain a high affinity (Kd = 0.10-0.20 nM), saturable (Bmax - 1.2-3.5 fmol/mg protein), stereoselective binding site for 3H-spiperone (3H-SPIP). The Kd (0.10 nM) calculated from the experimentally determined rate constants k1 and k2 was in excellent agreement with that derived from equilibrium measurements. The rank order of potencies of dopaminergic agents to compete for binding was consistent with known dopamine (DA) receptors. Soluble binding sites represented 3% of the total specific binding found in the anterior pituitary, and several lines of evidence suggested they were not due to enzymatic, mechanical or osmotic displacement of membrane receptors. Further studies are necessary to elucidate the possible significance of these soluble binding sites; however, this observation is intriguing since both DA and membrane bound DA receptors enter the cell.
Assuntos
Adeno-Hipófise/metabolismo , Receptores Dopaminérgicos/metabolismo , Animais , Ligação Competitiva , Bovinos , Núcleo Caudado/metabolismo , Citosol/metabolismo , Cinética , Masculino , Especificidade de Órgãos , Espiperona/metabolismoRESUMO
Changes in serum and pituitary LH and FSH concentrations have been measured in the newborn male rat before, during, and up to 24 h after birth. A sudden and transient increase of serum and pituitary gonadotropins is observed at birth, which is followed by a rapid increase of absolute and relative testicular weights between 2--12 h (P less than 0.0001) and by a transient increase of serum testosterone between 0 h in utero (810 +/- 26 pg/ml) and 2 h (2820 +/- 318 pg/ml; P less than 0.0001). Similarly, premature newborn rats obtained by cesarian delivery on day 20 of gestation also exhibited an increase in testicular weight between 0--6 h and an increase in serum testosterone levels between 0 h (730 +/- 170 pg/ml) and 2 h (3400 +/- 300 pg/ml; P less than 0.001) with only a slight increase in serum LH. These results show that the hypophyseo-testicular axis of the rats is stimulated at the moment of birth. The factors responsible for this stimulation are discussed. This transient testicular crisis occurring at birth could affect the process of masculinization of the central nervous system of the rat.
Assuntos
Hormônio Foliculoestimulante/sangue , Hormônio Luteinizante/sangue , Testículo/anatomia & histologia , Animais , Animais Recém-Nascidos , Parto Obstétrico , Feminino , Feto , Masculino , Tamanho do Órgão , Gravidez , Ratos , Fatores de TempoRESUMO
Complete deafferentation of the medial basal hypothalamus (MBH) was performed in male rats to ascertain the location of the cell bodies of nerve terminals containing luteinizing hormone-releasing hormone (LH-RH) in the external layer of the median eminence and the organum vasculosum of the lamina terminalis (OVLT). Radioimmunoassayable LH-RH in the MBH decreased to one-fourth of the control value 10 days after complete deafferentation, whereas, no change was observed in an area coextensive with the OVLT. Immunohistochemical staining of LH-RH appeared less intense within the median eminence of deafferented animals but was normal in the OVLT. These results suggest that the cell bodies of neurons producing LH-RH found in terminals in the OVLT lie outside the MBH. The possible explanations for the decreased LH-RH content in the median eminence are discussed.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Terminações Nervosas/metabolismo , Neurônios Aferentes/fisiologia , Neurônios/fisiologia , Animais , Denervação , Hipotálamo/fisiologia , Masculino , Eminência Mediana/metabolismo , RatosRESUMO
The precursor molecule to the GnRH contains a peptide named GnRH-associated peptide (GAP) with PRL-inhibiting properties. In this work, we have studied the electrophysiological properties and responses to GAP of three different types of PRL-secreting cells: 1) the rat tumor cell line GH3, 2) normal rat pituitary cells in primary culture, and 3) human PRL-secreting adenoma cells. Using different but complementary techniques we show that GAP reduces intracellular Ca++ levels, [Ca++]i, and inhibits Ca++ transients in these cells. This reduction of [Ca++]i results from coordinate actions of GAP on K+ and Ca++ conductances and may explain the inhibitory effect of GAP on hormonal secretion by PRL-secreting cells.
Assuntos
Adenoma/metabolismo , Cálcio/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Adeno-Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Prolactina/metabolismo , Precursores de Proteínas/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Potenciais da Membrana/efeitos dos fármacos , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/fisiologia , Ratos , Ratos Endogâmicos , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/fisiologiaRESUMO
The distribution of tachykinin-like immunoreactivity (LI) was studied in the adrenal gland of the frog Rana ridibunda using the immunofluorescence technique. A dense network of varicose fibers immunoreactive to both substance-P (SP) and neurokinin-A (NKA) was found in the adrenal tissue. In contrast, no positive fibers could be detected using antineurokinin-B (NKB) antibodies. At the electron microscope level, the immunogold technique revealed that tachykinin-LI was sequestered in dense core vesicles of 50-70 nm. Bilateral transection of either splanchnic or vagus nerves or total lesion of celiac sympathetic ganglion did not suppress tachykinin-LI. A combination of HPLC analysis and RIA detection was used to characterize tachykinin-LI in frog adrenal extracts. Two major peaks were resolved, which coeluted, respectively, with synthetic ranakinin, a novel tachykinin previously isolated from the frog brain, and [Leu3,Ile7]NKA previously isolated from the frog gut. No NKB could be detected in the extracts. The effects of various synthetic tachykinins on corticosteroid secretion were studied using perifused frog adrenal slices. For concentrations ranging from 10(-8)-10(-4) M, SP induced a dose-dependent stimulation of corticosterone and aldosterone release. A desensitization phenomenon was observed when iterative or prolonged infusions of SP were administered to the tissue. All mammalian or amphibian tachykinin-related peptides tested in our model also enhanced corticosteroid production. The effectiveness of the tachykinins tested was: [Pro7] NKB > NKA > ranakinin > [Pro9]SP > SP > kassinin > physalaemin > NKB > [Leu3,Ile7]NKA. SP also enhanced prostaglandin E2 and prostacyclin release in the effluent perifusate and the response preceded by 10-15 min the increase in corticosteroid output. Indomethacin (5 x 10(-6) M), a specific blocker of cyclooxygenase activity, totally suppressed SP-evoked steroid secretion. These data indicate that tachykinin-induced stimulation of steroidogenesis was mediated through activation of the arachidonic acid cascade. Taken together, our results show that the frog adrenal gland is innervated by a dense network of peptidergic fibers containing both ranakinin and [Leu3,Ile7]NKA, which, in vitro, stimulates corticosteroid secretion by adrenocortical cells through a prostaglandin-dependent mechanism. The present results support the view that tachykinins released by nerve fibers exert a neuroendocrine control on corticosteroid release in amphibians.
Assuntos
Glândulas Suprarrenais/química , Taquicininas/análise , 6-Cetoprostaglandina F1 alfa/metabolismo , Corticosteroides/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Dinoprostona/metabolismo , Imunofluorescência , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Neurocinina A/análise , Neurocinina A/farmacologia , Neurocinina B/análise , Neurocinina B/farmacologia , Rana ridibunda , Substância P/análise , Substância P/farmacologia , Taquicininas/farmacologia , Distribuição TecidualRESUMO
The present study examined the effects of 17 beta-estradiol benzoate (E2B) on the hypothalamic (HT) and anterior pituitary (AP) content of GnRH precursor (pro-GnRH-GAP), GnRH, GnRH-associated peptide (GAP), and substance-P (SP) during the various phases of the E2B-induced LH surge in cynomolgus monkeys. Changes in GnRH and GnRH-associated peptide (GAP) at both hypothalamic and AP levels were closely related at all times after E2B treatment. However, the pattern of change in the AP was very different from that in the HT. In the HT, pro-GnRH-GAP levels did not change significantly throughout the experimental period. In the AP, the pro-GnRH-GAP increased 48 h post-E2B treatment, the time of initiation of the LH surge. An 8-fold increase in AP GnRH occurred 30 h post-E2B treatment. There were no significant changes in the HT content of SP at any time after E2B treatment. However, there was a depletion of AP content by 48 h post-E2B, the time of the LH surge. These results demonstrate that E2 activates and deactivates in a coordinated manner the GnRH and SP systems of the HT-AP complex during initiation of the E2-induced LH surge. The observation that more significant changes occur in the AP than in the HT suggests that an important component of the E2 effect in inducing the LH surge may be directly at the AP level. This action involves changes in the contents of GnRH and SP in the AP.
Assuntos
Estradiol/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Hormônio Luteinizante/metabolismo , Ovariectomia , Adeno-Hipófise/metabolismo , Substância P/metabolismo , Animais , Estradiol/análogos & derivados , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Foliculoestimulante/metabolismo , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Cinética , Hormônio Luteinizante/sangue , Macaca fascicularis , Adeno-Hipófise/efeitos dos fármacos , Prolactina/sangue , Prolactina/metabolismo , Precursores de Proteínas/metabolismo , Fatores de TempoRESUMO
The effect of chronic estrogen treatment on the anterior pituitary domperidone-binding sites was studied in female rats. The rats were implanted from 1-6 months with a Silastic capsule containing 17 beta-estradiol. The Feldman analysis of [3H]domperidone binding to anterior pituitary membranes of control or estrogenized rats revealed the presence of two sites. The binding characteristics of the higher affinity site were identical for both groups (Kd of the high affinity site, 0.30-0.45 nM; maximum number of binding sites of the high affinity site, 74-95 fmol/mg protein); however, those of the lower affinity site were affected by the estrogen treatment: the Kd of the low affinity site increased from 17.4 +/- 3.2 to 41.5 +/- 9 (+/- SE) nM, and the maximum number of binding sites of the low affinity site increased from 214 +/- 22 to 343 +/- 35 fmol/mg protein. Thus, in chronic estrogenized rats, the total number of binding sites was increased by 54%. These changes, induced by chronic estrogenization, were reversible, since 2 weeks after removal of the 17 beta-estradiol pellet, the binding characteristics were no longer different from those observed in control rats. In contrast to chronic estrogen treatment, ovariectomy reduced markedly the total number of [3H]domperidone-binding sites in anterior pituitary membranes (-70%). Feldman analysis revealed that this reduction resulted from the complete disappearance of the low affinity sites in those membranes. No significant change in the binding characteristics of the high affinity site was detected in ovariectomized rats. Since estradiol induces a decrease in the anterior pituitary content of dopamine, a denervation supersensitivity-like mechanism might be responsible for the increase in pituitary domperidone-binding sites in estrogenized rats. Conversely, a hyposensitivity mechanism could be implicated in the decrease in the total number of the pituitary domperidone-binding sites in ovariectomized rats, since pituitary dopamine levels are increased in those animals. Whether the antidopaminergic properties of estrogen are also involved in this modulation after chronic estradiol treatment requires further investigation.
Assuntos
Domperidona/metabolismo , Estradiol/farmacologia , Adeno-Hipófise/metabolismo , Animais , Apomorfina/farmacologia , Sítios de Ligação , Castração , Estradiol/sangue , Feminino , Prolactina/sangue , Ratos , Ratos EndogâmicosRESUMO
Specific saturable high affinity receptors for substance-P (SP) are present in human pituitaries obtained post-mortem. The human pituitary SP receptor bound SP and two of its analogs, [D-Tyr0]SP and [D-Tyr0,Nor,Leu11]SP, with similar affinities (Kd, 1.76 X 10(-8), 3.53 X 10(-8), and 1.93 X 10(-8) mol/L, respectively). The fragments SP-(3-11) and SP-(7-10) had lower affinities for the receptor than SP (Kd, 9.24 X 10(-8) and 2.86 X 10(-7) mol/L, respectively). SP-(5-8) and SP-(1-4) were poorly bound by the receptor (Kd, greater than 10(-6) mol/L). The progressive decrease in binding with the removal of amino acid residues from the N- or C-termini of the molecule is similar to that observed in the rat. GnRH and TRH did not compete for the SP-binding sites. SP was able to inhibit GnRH-stimulated LH secretion from cultured human pituitary cells in a dose-dependent manner, while having no effect on GnRH-stimulated FSH secretion. Basal release of TSH, PRL, GH, and ACTH was unaffected by SP. These findings suggest that SP may have a physiological role in the regulation of LH secretion in man.
Assuntos
Hormônio Luteinizante/metabolismo , Adeno-Hipófise/metabolismo , Receptores de Neurotransmissores/metabolismo , Substância P/metabolismo , Adulto , Ligação Competitiva , Membrana Celular/metabolismo , Humanos , Cinética , Hormônio Luteinizante/antagonistas & inibidores , Masculino , Receptores da Neurocinina-1RESUMO
The effects of Substance P (SP) and of a specific nonpeptide antagonist of the NK1 receptor (RP 67580) on preovulatory gonadotropin surges and on the in vitro GnRH induced LH surge were investigated in cycling female rats. A subcutaneous injection of SP (0.5 mg/kg body weight) at 12.00 h on the proestrous day significantly decreased the LH preovulatory surge. RP 67580 (1.5 mg/kg) significantly increased this LH surge. However, when SP and its antagonist were administered together, LH preovulatory surge was normal. The FSH preovulatory surge at 18.00 h and also at 19.00 h was significantly inhibited by SP administration. RP 67580 alone had no effect on the FSH preovulatory surge. When SP and RP 67580 were both administered, there was no diminution of FSH plasma levels at 18.00 h and 19.00 h. In vitro perifusions of anterior pituitaries showed that SP inhibits GnRH-induced LH release via a NK1 receptor. Thus, SP inhibits the LH preovulatory surge via NK1 receptors and SP modulation of gonadotropin surges is at least partly exerted at the pituitary.
Assuntos
Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Indóis/farmacologia , Hormônio Luteinizante/metabolismo , Substância P/farmacologia , Análise de Variância , Animais , Feminino , Hormônio Foliculoestimulante/sangue , Isoindóis , Cinética , Hormônio Luteinizante/sangue , Antagonistas dos Receptores de Neurocinina-1 , Proestro , Radioimunoensaio , Ratos , Ratos Wistar , Substância P/antagonistas & inibidoresRESUMO
Human IM-9 B-lymphoblasts have been shown to express the receptor for the neuropeptide substance P (SP). The present study was undertaken to evaluate the effect of sex hormones on this receptor. Testosterone inhibited [125I]SP binding in a dose-dependent manner with an IC50 of approximately 100 nM, while both estradiol and progesterone failed to inhibit SP binding even at concentrations as high as 1 microM. Furthermore, Scatchard analysis indicated that the dissociation constant (Kd) of the SP receptor was markedly increased from 0.25 nM to 2.2 nM when cells were incubated with testosterone. These data indicate a unique neuro-endocrine interaction on lymphocytes involving the substance P receptor.
Assuntos
Linfócitos/fisiologia , Sistemas Neurossecretores/fisiologia , Receptores de Neurotransmissores/metabolismo , Testosterona/farmacologia , Relação Dose-Resposta a Droga , Humanos , Linfócitos/metabolismo , Concentração Osmolar , Receptores da Neurocinina-1 , Substância P/metabolismoRESUMO
Using a lipid-adapted ELISA, antiphosphatidylinositol (PtdIns) antibodies (Ab) have been found in sera of cancer patients. With the same procedure, we have checked their possible raising in plasma of female rats during the appearance of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors. Twenty days after DMBA administration, the mean anti-PtdIns Ab level was already higher than that of control rats (oil). Immunochemical analysis of the anti-PtdIns Ab site showed a rather high Ab avidity (8 x 10(-9) M, at half-displacement) and high specificity for glyceryl phosphate inositol residues. Other phospholipids (PL) were not recognized by the anti-PtdIns Ab induced by malignant cell transformation.
Assuntos
Autoanticorpos/análise , Neoplasias Mamárias Experimentais/imunologia , Fosfatidilinositóis/imunologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Feminino , Neoplasias Mamárias Experimentais/induzido quimicamente , Ratos , Ratos Endogâmicos , Valores de Referência , Fatores de TempoRESUMO
The effects of substance P (SP) on the preovulatory surge of LH and on the inhibitory and stimulatory effects of oestradiol-17 beta and progesterone on gonadotrophin-releasing hormone (GnRH)-induced LH release were investigated in vivo and in vitro in the rat. A single s.c. injection of 100 micrograms SP at 12.00 h on the day of pro-oestrus significantly decreased the preovulatory surge of LH. In vitro, the inhibitory effect of oestradiol-17 beta on GnRH-induced LH release was not modified by treatment with SP. The stimulatory effect of progesterone on GnRH-induced LH release was reduced by treatment with SP. It is concluded that SP may play a modulatory role in the neuroendocrine control of the preovulatory LH surge.
Assuntos
Estradiol/farmacologia , Hormônio Liberador de Gonadotropina/fisiologia , Hormônio Luteinizante/metabolismo , Progesterona/farmacologia , Substância P/farmacologia , Animais , Feminino , Proestro/fisiologia , Ratos , Ratos Endogâmicos , Taxa Secretória/efeitos dos fármacosRESUMO
A kinetic study of the influence of thyreotrope-releasing hormone (TRH) on prolactin turnover and synthesis by a new rat pituitary prolactin cell line (SD1) has been performed by means of pulse-chase experiments. After a 10-min [3H]leucine pulse, the chase was carried out in the presence or absence of TRH (54 nM), cycloheximide (3.6 X 10(-5)M) and/or [14C]-proline. The prolactin content of the cells in the medium was estimated using a radioimmunoassay technique. The specific radioactivity of prolactin in the medium was estimated after its isolation by disc gel electrophoresis. This kinetic study demonstrated, firstly, a rapid intracellular transit of newly synthesized prolactin (15 + 10 min or less); secondly, the existence of at least two intracellular prolactin pools; thirdly, a rapid effect of TRH on release of stored prolactin, which is independent of de novo protein synthesis, and fourthly, a delayed stimulating effect of TRH on prolactin synthesis.
Assuntos
Prolactina/metabolismo , Hormônio Liberador de Tireotropina/farmacologia , Linhagem Celular , Cicloeximida/farmacologia , Cinética , Leucina/metabolismo , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
Normal female and ovariectomized rats were infused into the 3rd ventricle with [3H]glycine or [3H]alanine. Some rats were pretreated with cycloheximide. Hypothalamus, anterior pituitary and fragments of cortex were excised, homogenized, extracted and treated with specific antiserum to GnRH, bound to Sepharose. The radioactivity of immuno-absorbed products was counted either immediately of after extraction and thin-layer chromatography by using two different solvent systems. With the two systems, the location of the immuno-absorbed radioactivity always coincided with the spot of synthetic GnRH. Our results show that [3H]glycine was incorporated, as a function of time, into GnRH isolated from rat hypothalami. The amount was incorporated, as a function of time, into GnRH isolated from rat hypothalami. The amount of radioactivity incorporated into hypothalami from diestrous-I rats was similar to that of ovariectomized rats and twice as high as in late proestrous rats. Only minute amounts of radioactivity were incorporated into the immuno-absorbed product. Cycloheximide inhibited incorporation of [3H]glycine into the immuno-absorbed product to the same extent as its incorporation into the total protein from the hypothalamus. Our experimental results support the hypothesis of ribosomal mechanisms being involved in the biosynthesis of GnRH. They also suggest that the rate of accumulation of newly synthesized labeled GnRH is of the same order in the hypothalamus of ovariectomized rats as in diestrous-I rats.
Assuntos
Hipotálamo/metabolismo , Hormônios Liberadores de Hormônios Hipofisários/biossíntese , Aminoácidos/metabolismo , Animais , Castração , Cromatografia em Camada Fina , Cicloeximida/farmacologia , Feminino , Cinética , Adeno-Hipófise/metabolismo , Hormônios Liberadores de Hormônios Hipofisários/metabolismo , RatosRESUMO
In humans, transcription of the gonadotropin-releasing hormone (GnRH) gene can be initiated at two transcription start sites to produce different GnRH mRNAs. The upstream transcription start site is used only in reproductive tissues and tumors. To determine if a similar pattern of GnRH gene expression exists in non-human primates, we cloned GnRH cDNA from rhesus monkey hypothalamic RNA using reverse transcriptase-polymerase chain reaction (RT-PCR) and the 5' flanking region of the monkey GnRH gene by PCR. A 96% similarity between monkey and human GnRH cDNA was found with 94% similarity in the upstream promoter region. An upstream transcriptional start site, was identified in cynomolgus monkey testicular mRNA, 504 base pairs upstream from the hypothalamic site, which was different from that identified in the human GnRH gene. Various cynomolgus monkey reproductive tissues were found to utilize this upstream transcriptional start site. In contrast, no evidence was found for the use of upstream transcriptional start sites in rat testis or placenta, suggesting that the reproductive tissue specificity of the upstream transcription start site may be a primate specific feature.
Assuntos
Regulação da Expressão Gênica , Hormônio Liberador de Gonadotropina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Humanos , Hipotálamo/metabolismo , Macaca fascicularis , Macaca mulatta , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro , Ratos , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Using a sensitive RNase protection assay, the simultaneous quantification of hypothalamic beta-, gamma-preprotachykinin (PPT) and SP receptor NK-1 transcripts was performed throughout the estrous cycle. The amounts of these 3 transcripts were up-regulated during diestrus II-proestrus night (2-, 1.5- and 1.3-fold, respectively). These levels returned to low values during the proestrous day in the case of gamma-PPT mRNA and during the estrus-diestrus I night in the cases of beta-PPT and NK-1 mRNAs. These results implicate a differential regulation in amounts of the two alternatively spliced PPT transcripts. The 160 hypothalami of this study had been previously assayed for amounts of substance P (SP) and neurokinin A (NKA) peptides [P. Duval, V. Lenoir, S. Moussaoui, C. Garret and B. Kerdelhué, Substance P and neurokinin A variations throughout the rat estrous cycle; comparison with ovariectomized and male rats: I. Plasma, hypothalamus, anterior and posterior pituitary, J. Neurosci. Res., 45 (1996) 598-609]. Variations in mRNA and peptide levels were compared by statistical analysis. Surprisingly, variations in SP level paralleled those in beta-PPT mRNA level and variations in NKA level paralleled those of gamma-PPT mRNA level, although beta- and gamma-PPT transcripts encode both SP and NKA. Furthermore, the level of NK-1 mRNA was positively correlated with the level of beta-PPT mRNA (r = 0.90, P < 10(-58)) and with the level of SP peptide (r = 0.30, P < 10(-3)) but not with the level of NKA peptide. This analysis suggests that SP receptor NK-1 mRNA could be physiologically regulated by SP peptide in the rat hypothalamus.