RESUMO
The cytosolic free calcium concentration, [Ca2+]i, was monitored in single rat lactotrophs in primary culture with the fluorescent probe Fura 2. It was found that lactotrophs are very heterogeneous in their [Ca2+]i response to TRH and dopamine, the major physiological regulators of PRL secretion. While in most lactotrophs TRH raises [Ca2+]i, the kinetics of this rise and the magnitude of its first and second phases vary considerably. For dopamine two clearly divergent response types can be observed. In part of the lactotrophs dopamine causes a lowering of [Ca2+]i from elevated levels, whereas in about 40% of the lactotrophs dopamine leads to a transient rise of [Ca2+]i. The present study reveals subclasses of lactotrophs with distinct [Ca2+]i response characteristics. It is suggested that such response type heterogeneity is a means of optimizing the secretory response to the complex regulatory influences on the pituitary.
Assuntos
Cálcio/metabolismo , Dopamina/farmacologia , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Hormônio Liberador de Tireotropina/farmacologia , Animais , Células Cultivadas , Citosol/efeitos dos fármacos , Citosol/metabolismo , Cinética , Masculino , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
Although it is known that LH secretion starts at 17 days of gestation in the fetal rat and that this first LH release is most likely driven by hypothalamic GnRH, an earlier role for GnRH during fetal life has been postulated with the observation that presence of GnRH is important before day 13 of gestation for the differentiation of the pituitary anlage. In order to clarify the different roles of GnRH during fetal life, we have studied the first appearance of GnRH in the fetal brain, the expression of GnRH receptors in the fetal pituitary gland, and the presence of GnRH immunoreactivity within the fetal gonadotrophs. GnRH was present in the earliest brain tissue examined (12 days of gestation). From 12-17 days, GnRH content of fetal brain remained low and then increased markedly by the end of gestation. No immunoreactive GnRH-like material could be detected in rat placental tissue throughout gestation. Binding sites for GnRH were detected as early as 12 days of gestation in fetal pituitary glands. However, binding was very low until 16 days. At 17 days, Scatchard analysis indicated the presence of high affinity, low capacity binding sites [affinity constant (Ka) = 10(10) M-1]. Intracellular presence of GnRH as seen by immunocytochemistry using ultrathin sections prepared by cryoultramicrotomy was first visible at 14 days and started to increase at 16 days. LH was first detectable in the fetal pituitary by RIA at 17 days; FSH was first detectable at 21 days, and PRL at 1 day of postnatal life. Thereafter, neonatal pituitary contents of LH, FSH, and PRL increased linearly with-time, as did the number of pituitary GnRH receptors. At 10 days of postnatal life, pituitary contents of LH and FSH were significantly higher in females than in males. In summary, hypothalamic GnRH appears early in fetal life and potentially can induce differentiation of the pituitary anlage. Conversely, the presence at 15 days of gestation of specific binding sites for GnRH and of intracellular GnRH immunoreactivity in gonadotrophs indicates that the hypophysiotropic action of GnRH clearly precedes the start of LH biosynthesis.
Assuntos
Animais Recém-Nascidos/metabolismo , Feto/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Hipófise/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Feminino , Hormônio Foliculoestimulante/metabolismo , Histocitoquímica , Imunoquímica , Hormônio Luteinizante/metabolismo , Masculino , Hipófise/citologia , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Placenta/metabolismo , Gravidez , Radioimunoensaio , Ratos/embriologiaRESUMO
Solubilization in an active form of the pituitary plasma membrane protein carrying binding sites for gonadotropin-releasing hormone (GnRH) was possible by using as detergent CHAPS (3[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate), which is a zwitterionic derivative of cholic acid. In contrast, the use of the non-ionic detergent Triton X-100 resulted in the loss of GnRH binding sites. CHAPS at a concentration of 5-10 mM allowed the solubilization of up to 80% of available sites, and remaining non-solubilized sites retained unaltered binding properties. Characterization of solubilized binding sites of GnRH was achieved by using as radioactive ligand and standard a stable GnRH analog. [D-Trp6,(NEt)Pro9,desGly10]-GnRH. Maximum binding of GnRH to solubilized binding sites was possible in the presence of a very low amount of CHAPS in the incubation medium. Scatchard analysis of classical saturation experiments indicated the presence of a single class of high affinity, low capacity binding sites. The mean value for the affinity constant KA was 0.36 +/- 0.07 X 10(10) M -1 (n = 5) for solubilized GnRH binding sites, when analysed in the presence of 2 mM CHAPS; when solubilized GnRH binding sites were analysed in the absence of CHAPS, significantly higher KA values were observed, ranging from 1.2 to 5.9 X 10(10) M -1. Parallel analysis of the plasma membranes prior to solubilization indicated a mean KA value of 0.53 +/- 0.13 X 10(10) M -1 (n = 5). The binding specificity of the pituitary GnRH binding sites as seen by evaluating cross-reactions with a panel of agonists and antagonists of GnRH was not altered by solubilization. Gel filtration on Sephadex G-25 of solubilized binding sites preincubated with radioiodinated GnRH analog demonstrated the presence of a large molecular weight complex. In conclusion, CHAPS appears to be quite suitable for the solubilization of the binding site moiety of the pituitary GnRH receptor with good retention of the binding characteristics, thus offering a new possibility for the purification and characterization of the GnRH receptor protein.
Assuntos
Ácidos Cólicos , Detergentes , Proteínas de Membrana/isolamento & purificação , Hipófise/metabolismo , Receptores de Superfície Celular/isolamento & purificação , Tensoativos , Animais , Ligação Competitiva , Bovinos , Cromatografia em Gel , Receptores LHRH , SolubilidadeRESUMO
In normal pituitary cells somatostatin (SRIF) blocks the spontaneous oscillations in [Ca++]i by inhibiting the generation of action potentials. This is sufficient to explain the inhibitory effect on basal, but not entirely that on stimulated pituitary hormone secretion. In insulin secreting cells, which, in contrast to pituitary cells, only fire action potentials on stimulus-evoked depolarization, SRIF hyperpolarization and lowering of [Ca++]i is only transient. The marked inhibition of insulin secretion is suggested to be due to a coordinated action of SRIF on membrane potential and [Ca++]i as well as a direct interference with late secretory events.
Assuntos
Cálcio/metabolismo , Hipófise/fisiologia , Taxa Secretória/fisiologia , Somatostatina/fisiologia , Animais , Células Cultivadas , Citosol/metabolismo , Pâncreas/fisiologia , Ratos , Somatostatina/farmacologiaRESUMO
Transient activation of the outward K+ current caused by a rise in the cytosolic free Ca2+ concentration, [Ca2+]i was the predominant change in plasma membrane ion flux during the first phase of thyrotropin-releasing hormone (TRH) action on pituitary cells. Following the intracellular application of inhibitors of 1,2-diacylglycerol (DG) kinase, R59022 and 1-oleyl-2-acetyl glycerol (OAG) the outward K+ current response to TRH in cells of the pituitary line GH3B6 was potentiated. This potentiation was analyzed further with the combination of microfluorimetric and electrophysiological recording techniques. Receptor-induced changes in [Ca2+]i and ion channel activation were monitored simultaneously in the same cell. It was found that R59022 and OAG altered in parallel the TRH-induced transient rise in [Ca2+]i and outward K+ current. This resulted in a significant correlation between the kinetic parameters (speed of onset, duration) of the [Ca2+]i and the K+ current responses to TRH. Intracellular application of vanadate abolished the rapid start of the TRH response presumably by its block of Ca2+ uptake into the endoplasmic reticulum, leading to depletion of a Ca2+ pool mobilizable by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). The use of vanadate unmasked a slowly developing response to TRH, which was still potentiated by OAG and R59022. Together, these observations suggest that Ca2+ mobilization during the first phase of TRH action is mediated by two distinct processes, one of which is linked to receptor stimulation of DG production.
Assuntos
Cálcio/farmacologia , Fosfotransferases/antagonistas & inibidores , Canais de Potássio/efeitos dos fármacos , Hormônio Liberador de Tireotropina/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Diacilglicerol Quinase , Fator de Ativação de Plaquetas/antagonistas & inibidores , Pirimidinonas/farmacologia , Tiazóis/farmacologia , Vanadatos/farmacologiaRESUMO
The kinetic features of the changes in the cytosolic free Ca2+ concentration, [Ca2+]i, following stimulation by thyrotropin releasing hormone (TRH) were analysed in single cells of a pituitary line (GH3B6) by dual excitation microfluorimetry [Tsien, Rink & Poenie (1985) Cell Calcium 6, 145-157], using fura 2 as intracellular Ca2+ probe. Two phases were observed: initially, [Ca2+]i is raised in a single rapid transient to a maximum averaging 8.0 microM, and in a second phase TRH causes a series of rapid [Ca2+]i oscillations with maxima around 1.0 microM, which are probably due to the enhanced firing of action potentials. TRH triggers both phases independently, i.e. it can elicit either the first or the second phase exclusively. This is also the case in those cells in which [Ca2+]i undergoes rhythmic oscillations due to the firing of spontaneous action potentials [Schlegel, Winiger, Mollard, Vacher, Wuarin, Zahnd, Wolheim & Dufy (1987) Nature (London) 329, 719-721]. The sudden onset of the first phase of TRH action on [Ca2+]i shows that Ca2+ mobilization due to enhanced production of inositol phosphate may occur as rapidly as the firing of action potentials, i.e. in the ms time range. Due to a marked response type heterogeneity and to the randomness of the rapid events, previous monitoring of [Ca2+]i in cell populations had misleadingly suggested small and maintained changes due to TRH. It is concluded that stimulatory regulation of secretion is provided by the generation of rapid [Ca2+]i transients, the frequency of which determines secretory rate. Furthermore, it is demonstrated that the regulation of [Ca2+]i by hormones and neurotransmitters in pituitary and many other cell types will have to be studied at the single cell level in order to appreciate its role in cell activation.
Assuntos
Cálcio/metabolismo , Hipófise/metabolismo , Hormônio Liberador de Tireotropina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Linhagem Celular , Citofotometria , Citosol/metabolismo , Cinética , Hipófise/citologia , Hipófise/efeitos dos fármacos , Ratos , Estimulação QuímicaRESUMO
Thyrotropin-releasing hormone (TRH) acts on pituitary cells to raise the cytosolic free Ca2+ concentration ([Ca2+]i) and causes simultaneously a transient hyperpolarization of the plasma membrane. The combination of the microfluorimetric monitoring of [Ca2+]i with electrophysiological recordings obtained using the patch clamp technique in its whole cell configuration, allows the analysis of the correlation between changes in [Ca2+]i and the alterations in ionic currents at the plasma membrane. It was shown that in the absence of hormone stimulation, a depolarization-induced change in steady state [Ca2+]i, as well as the internal perfusion with Ca2+ at microM levels at constant membrane potential led to the activation of outward K+ current. TRH stimulation resulted in a marked but transient rise in [Ca2+]i; concomitantly, there was an increase in membrane conductance and an enhancement of outward current. During the time course of an individual response, an excellent correlation between the changes in [Ca2+]i and those in conductance or current was observed. The relative changes of current and conductance during the TRH response were consistent with the activation of a single type of ionic current, the apparent reversal potential of which coincided with the equilibrium potential for K+. A strong correlation between the TRH-induced changes in [Ca2+]i and K+, conductance was demonstrated in a large number of cells with varied kinetic features: significant correlation coefficients were found both for the transition time from basal to maximal values (r = 0.85, p less than 0.001) as well as for the total duration of the responses (r = 0.68, p less than 0.002). It is concluded that during the early phase of TRH action, the hormone-induced rise in [Ca2+]i is the principal cause of enhanced K+ channel activation.
Assuntos
Cálcio/metabolismo , Canais de Potássio/fisiologia , Tireotropina/farmacologia , Aciltransferases/genética , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Condutividade Elétrica , Potenciais da Membrana/efeitos dos fármacos , Neoplasias Hipofisárias , Canais de Potássio/efeitos dos fármacos , Sulfurtransferases/genética , Transaminases/genéticaRESUMO
Daily administration of melatonin (MT) markedly delays sexual maturation in the male Wistar rat. In this study, we have evaluated pituitary responsiveness to GnRH and the level of tonic inhibition by endogenous opioids in normal juvenile male rats and in rats with delayed sexual development induced by daily afternoon MT injection (100 micrograms, s.c.) starting at 20 days of life. Plasma LH responses to repetitive intravenous GnRH administration (100 ng/100 g body weight), or to different doses of GnRH administered subcutaneously (5-100 ng/100 g body weight) were normal in MT-treated rats both at 30 and 40 days of life despite significantly lower number of pituitary GnRH receptors and decreased pituitary gonadotropin content. One naloxone (NAL) injection (2.5-5.0 mg/kg, s.c.) produced a significant increase of plasma LH in normal 40- and 55-day-old rats, which was not seen in MT-treated rats of the same age. In contrast, no increase of plasma LH was seen in 30-day-old control rats nor in MT-treated rats at this age. Pretreatment with morphine sulfate (10 mg/kg, s.c.), or with the potent Met-enkephalin analog FK 33-824 (1.0 mg/kg, s.c.) prevented the NAL-induced rise of plasma LH in control rats at day 40 of life. In all instances, plasma PRL levels were decreased after NAL both in untreated and in MT-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Envelhecimento/fisiologia , Hormônio Luteinizante/sangue , Melatonina/farmacologia , Naloxona/farmacologia , Hormônios Liberadores de Hormônios Hipofisários/farmacologia , Maturidade Sexual/efeitos dos fármacos , Animais , Masculino , Melatonina/administração & dosagem , Prolactina/sangue , Ratos , Ratos Endogâmicos , Receptores do Hormônio Hipofisário/fisiologiaRESUMO
The disappearance rate of [D-Ser(t-bu)6,des-Gly10]GnRH ethylamide (Buserelin, HOE 766) was studied in plasma and urine after intranasal (300 micrograms) or sc (10 micrograms/kg) administration. A radioimmunoassay for HOE 766 was developed using 125I[D-Trp6,Des-Gly10]GnRH ethylamide as tracer and an antiserum raised against HOE 766. Cross-reaction with native GnRH was only 1.7%. Sensitivity was 1 pg/tube. In 6 male adolescents, the mean plasma HOE 766 concentration (+/- SEM) was 0.46 +/- 0.08, 0.50 +/- 0.10, 0.28 +/- 0.04, 0.24 +/- 0.04, 0.13 +/- 0.03, and 0.08 +/- 0.02 micrograms/l 30, 60, 90, 120 and 180 min after the intranasal administration, respectively. Concomitant urinary excretion of HOE 766-like material was 9.43 +/- 1.96 micrograms/4 h. There was a good correlation between integrated plasma levels and urinary excretion (r = 0.92). In the same 6 volunteers, the plasma HOE 766 levels were 21.2 +/- 3.0, 25.9 +/- 0.8, 21.2 +/- 0.9, 17.1 +/- 0.7, 12.8 +/- 1.1, 8.9 +/- 0.4, and 5.9 +/- 0.8 micrograms/l 20, 40, 60, 90, 120, 180 and 240 min after sc injection, respectively. The mean urinary excretion was 543 +/- 61 micrograms/4 h. In two girls with precocious puberty treated during 12 to 15 months with intranasal administration of HOE 766, urinary excretion of HOE 766-like material was shown to correlate well with the degree of inhibition of plasma 17 beta-E2 and of plasma LH and FSH responses to a GnRH challenge. Thus, monitoring of HOE 766 in urine appears to be helpful for evaluating of intranasal therapy with a GnRH analog in precocious puberty.
Assuntos
Busserrelina/metabolismo , Puberdade Precoce/tratamento farmacológico , Administração Intranasal , Adolescente , Adulto , Busserrelina/administração & dosagem , Busserrelina/uso terapêutico , Criança , Pré-Escolar , Feminino , Humanos , Injeções Subcutâneas , Masculino , Puberdade Precoce/metabolismo , RadioimunoensaioRESUMO
Receptor-mediated alterations in the cytosolic free calcium concentration, [Ca2+]i are monitored with the intracellular fluorescent calcium probe fura 2 by dual excitation microfluorimetry. The calcium dependence on the excitation spectrum of fura 2 allows us to choose two wavelengths, lambda 1 and lambda 2, at which an increase in [Ca2+]i causes either a rise or a fall in fluorescence; the ratio of fluorescence at lambda 1 and lambda 2 (R = F lambda 1/F lambda 2) is a measure of [Ca2+]i. It appears essential for such measurements that the alteration between the two excitation wavelengths is done rapidly, to allow us to distinguish between effects on [Ca2+]i and other effects on fluorescence. In addition, specific problems relating to the calibration of fura 2 measurements, such as its relative insensitivity at basal [Ca2+]i, the role of intracellular viscosity on fura 2 fluorescence, and the difficulties encountered in establishing calibration constants have to be appreciated. In spite of these potential drawbacks, it appears that monitoring receptor-mediated [Ca2+]i regulation in single cells will prove essential for the further comprehension of stimulus-secretion coupling in pituitary and probably many other cell types.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Fluorometria , Hipófise/metabolismo , Animais , Benzofuranos/metabolismo , Fluorometria/instrumentação , Fura-2 , Masculino , Microquímica/instrumentação , Hipófise/citologia , RatosRESUMO
Electrical activity in non-neuronal cells can be induced by altering the membrane potential and eliciting action potentials. For example, hormones, nutrients and neurotransmitters act on excitable endocrine cells. In an attempt to correlate such electrical activity with regulation of cell activation, we report here direct measurements of cytosolic free Ca2+ changes coincident with action potentials. This was achieved by the powerful and novel combination of two complex techniques, the patch clamp and microfluorimetry using fura 2 methodology. Changes in intracellular calcium concentration were monitored in single cells of the pituitary line GH3B6. We show that a single action potential leads to a marked transient increase in cytosolic free calcium. The size of these short-lived maxima is sufficient to evoke secretory activity. The striking kinetic features of these transients enabled us to identify oscillations in intracellular calcium concentration in unperturbed cells resulting from spontaneous action potentials, and hence provide an explanation for basal secretory activity. Somatostatin, an inhibitor of pituitary function, abolishes the spontaneous spiking of free cytosolic Ca2+ which may explain its inhibitory effect on basal prolactin secretion. Our data therefore demonstrate that electrical activity can stimulate Ca2+-dependent functions in excitable non-neuronal cells.