RESUMEN
The Authors give an account of a case in which a newborn was affected by craniosynostosis of the sagittal suture. The ultrasonographic biometric measurements of cranium taken during the course of fetal development, when compared with those of the trunk and limbs, led the Authors to suspect the correct diagnosis even before birth; this permitted the immediate planning of therapeutic conduct.
Asunto(s)
Craneosinostosis/diagnóstico , Diagnóstico Prenatal , Femenino , Humanos , Recién NacidoRESUMEN
In estradiol-treated pituitary cells, progesterone enhances gonadotropin-releasing hormone (GnRH)-induced LH secretion from cultured rat pituitary cells during short-term treatment but attenuates this response during prolonged treatment. In the present study, the effects of gonadal steroids on GnRH-induced cytoplasmic calcium ([Ca2+]i) responses in gonadotrophs were analyzed in rat pituitary cells and immortalized (alpha T3-1) murine gonadotrophs. Ca2+ responses were measured in cell suspensions and single gonadotrophs, loaded with Fura-2 or Indo-1, respectively, and pretreated for 48 h with 1 nM estradiol with or without 100 nM progesterone, or for 48 h with 1 nM estradiol and then for 3 h with 100 nM progesterone. In cells of the alpha T3-1 gonadotroph lineage, GnRH elicited biphasic Ca2+ signals composed of an initial peak response followed by a prolonged plateau phase. The amplitudes of both the extracellular Ca(2+)-independent spike phase and the extracellular Ca(2+)-dependent plateau phase were enhanced or inhibited by short- or long-term progesterone treatment, respectively. In single pituitary gonadotrophs, GnRH (0.5 nM) elicited oscillatory responses due to intermittent release and uptake of Ca2+ from intracellular stores. Treatment with progesterone shifted the oscillatory signal toward biphasic (3 h) or subthreshold (48 h) response profiles, revealing a steroid-induced change in the pattern of Ca2+ mobilization. In addition to these agonist-induced responses, the transient [Ca2+]i responses of pituitary cells and individual gonadotrophs to high K+ were enhanced or inhibited after short- or long-term progesterone treatment, respectively. These actions were correlated with the effects of progesterone on K(+)-induced LH secretion. The [Ca2+]i and LH secretory responses to phorbol ester treatment were also enhanced by short-term exposure of the cells to progesterone. The results demonstrate that the stimulatory and inhibitory effects of progesterone on agonist-induced Ca2+ signaling result from changes in Ca2+ mobilization and entry, and contribute to the modulatory actions of the steroid on GnRH-induced LH secretion.
Asunto(s)
Calcio/metabolismo , Hormona Luteinizante/metabolismo , Hipófisis/metabolismo , Progesterona/fisiología , Transducción de Señal , Animales , Células Cultivadas , Citoplasma/metabolismo , Estradiol/fisiología , Exocitosis , Femenino , Potenciales de la Membrana , Hipófisis/citología , Hipófisis/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Gonadotropin-releasing hormone (GnRH) is released episodically into the pituitary portal vessels and from hypothalamic tissue of male and female rats in vitro. Perifused primary cultures of rat hypothalamic neurons, as well as the GT1-1 GnRH neuronal cell line, spontaneously exhibited episodic GnRH secretion of comparable frequency to that observed with perifused hypothalami. Such pulsatile GnRH release from GT1 cells indicates that GnRH neurons generate rhythmic secretory activity in the absence of input from other cell types. In primary hypothalamic cultures, the frequency of GnRH pulses increased with the duration of culture. The spontaneous pulsatility in GnRH release was abolished in Ca(2+)-deficient medium and was markedly attenuated in the presence of nifedipine, an antagonist of voltage-sensitive Ca2+ channels. The basal intracellular Ca2+ level of perifused GT1-1 cells cultured on coverslips was also dose-dependently reduced by nifedipine. Conversely, depolarization with high K+ increased intracellular Ca2+ and GnRH release in an extracellular Ca(2+)-dependent and nifedipine-sensitive manner. The dihydropyridine Ca2+ channel agonist Bay K 8644 increased basal and K(+)-induced elevations of intracellular Ca2+ concentration and GnRH secretion. These findings demonstrate that pulsatile neuropeptide secretion is an intrinsic property of GnRH neuronal networks and is dependent on voltage-sensitive Ca2+ influx for its maintenance.
Asunto(s)
Calcio/fisiología , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Animales , Canales de Calcio/fisiología , Células Cultivadas , Espacio Extracelular/fisiología , Femenino , Hipotálamo/embriología , Técnicas In Vitro , Masculino , Nifedipino/farmacología , Periodicidad , Potasio/fisiología , Ratas , Ratas Endogámicas , Tasa de Secreción/efectos de los fármacos , Transducción de SeñalRESUMEN
In agonist-stimulated clonal pituitary gonadotrophs (alpha T3-1 cells), cytoplasmic calcium ([Ca2+]i) exhibited rapid and prominent peak increases, followed by lower, but sustained, elevations for up to 15 min. The [Ca2+]i response to GnRH was rapidly inhibited by prior addition of a potent GnRH antagonist. In the absence of extracellular Ca2+ the initial peak [Ca2+]i response was only slightly decreased, but the prolonged increase in [Ca2+]i was abolished, indicating that the peak is derived largely from intracellular calcium mobilization and the sustained phase from Ca2+ influx. Application of the endoplasmic reticulum Ca(2+)-ATPase blocker thapsigargin caused progressive and dose-dependent elevation of [Ca2+]i and decreased the peak amplitude of the GnRH-induced Ca2+ response. On the other hand, addition of dihydropyridine calcium channel antagonists before or after GnRH treatment prevented or terminated the plateau phase, respectively, consistent with entry of Ca2+ through L-type voltage-sensitive Ca2+ channels (VSCC) as the major Ca2+ influx pathway during GnRH action. The presence of L-type VSCC in alpha T3-1 cells was further indicated by the ability of elevated extracellular K+ levels and the dihydropyridine calcium channel agonist Bay K 8644 to elevate [Ca2+]i in an extracellular calcium-dependent manner. These actions of depolarization and Bay K 8644 were inhibited by nifedipine, with an IC50 of 10 nM. High extracellular K(+)- and GnRH-induced Ca2+ entry was also attenuated by phorbol esters and permeant diacylglycerols, indicating that protein kinase-C exerts inhibitory modulation of VSCC activity. In contrast to normal pituitary gonadotrophs, in which GnRH induces a frequency-modulated oscillatory [Ca2+]i response, single alpha T3-1 cells exhibited a nonoscillatory amplitude-modulated signal during agonist stimulation. The [Ca2+]i responses observed in alpha T3-1 gonadotrophs indicate that the immortalized cells retain functional GnRH receptors and their coupling to the Ca2+ signaling pathway. Ca2+ influx through L-type channels maintains the plateau phase of the [Ca2+]i response during agonist stimulation and is inhibited by activation of protein kinase-C.
Asunto(s)
Calcio/metabolismo , Hormona Liberadora de Gonadotropina/farmacología , Hipófisis/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Línea Celular , Dihidropiridinas/farmacología , Retículo Endoplásmico/enzimología , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Ionomicina/farmacología , Nifedipino/farmacología , Hipófisis/efectos de los fármacos , Potasio/farmacología , Terpenos/farmacología , Acetato de Tetradecanoilforbol/farmacología , TapsigarginaRESUMEN
Specific receptors for endothelin (ET), localized by autoradiographic studies with [125I]ET in frozen sections of the rat pituitary gland, were abundant in the adenohypophysis, but not in the neurohypophysis. Specific binding of [125I]ET-1 and [125I]ET-3 was also demonstrable in 3-day-old primary cultures of anterior pituitary cells. The binding of [125I]ET-1 to its receptors was time and temperature dependent and was followed by rapid internalization of the receptor-ligand complex. Binding of [125I]ET-1 and [125I]ET-3 to pituitary tissues and cells was more effectively displaced by ET-1 and ET-2 than by ET-3. In cultured pituitary cells, ET-1 caused a rapid increase in polyphosphoinositide hydrolysis, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production, with a prompt rise in the cytoplasmic calcium concentration ([Ca2+]i) and LH secretion. The Ins(1,4,5)P3 response to 100 nM ET-1 was transient, with a spike at 10 sec followed by an exponential decrease toward the low steady state level. Ins(1,3,4)P3 and inositol bisphosphate (InsP2) increased more slowly, reaching peak values 30-40 sec after stimulation. The kinetics of the [Ca2+]i response to ET-1 were similar to those of the Ins(1,4,5)P3 response and more rapid than those of the Ins(1,3,4)P3 and InsP2 responses. In perifused cells, ET-stimulated increases in LH release showed the same biphasic patterns as the Ins(1,4,5)P3 and [Ca2+]i responses. ET-1 was more potent than ET-3 in stimulating [Ca2+]i and LH responses, consistent with its higher affinity for the pituitary ET receptors. The initial activation of Ca2+ signaling and LH exocytosis by ETs was followed by prolonged refractoriness to both ET-1 and ET-3. The development of desensitization occurred more rapidly in ET-1- than ET-3-stimulated cells and correlated temporally with endocytosis of the receptor-ligand complex. These findings indicate that stimulation of gonadotropin release by ETs occurs via activation of ETA-type receptors, which are coupled to polyphosphoinositide hydrolysis and [Ca2+]i mobilization, and undergo rapid internalization and profound desensitization.
Asunto(s)
Endotelinas/farmacología , Hormona Luteinizante/metabolismo , Adenohipófisis/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Animales , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Endocitosis , Endotelinas/metabolismo , Femenino , Fosfatos de Inositol/biosíntesis , Adenohipófisis/metabolismo , Ratas , Ratas Endogámicas , Receptores de EndotelinaRESUMEN
Single pituitary gonadotrophs exhibit episodes of spontaneous fluctuations in cytoplasmic calcium concentration [( Ca2+]i) due to entry through voltage-sensitive calcium channels (VSCC) and show prominent agonist-induced oscillations in [Ca2+]i that are generated by periodic release of intracellular Ca2+. Gonadotropin releasing hormone (GnRH) elicited three types of Ca2+ responses: at low doses, subthreshold, with an increase in basal [Ca2+]i; at intermediate doses, oscillatory, with dose-dependent modulation of spiking frequency; and at high doses, biphasic, without oscillations. Elevation of [Ca2+]i or activation of protein kinase C (PKC) did not influence the frequency of agonist-induced [Ca2+]i spikes but caused dose-dependent reductions in amplitude for all types of Ca2+ response. Stimulation of transient Ca2+ spikes by GnRH was followed by inhibition of the spontaneous fluctuations. GnRH also reduced the ability of high extracellular K+ to promote Ca2+ influx through VSCC. Activation of PKC by phorbol esters stimulated Ca2+ influx in quiescent cells but inhibited influx when VSCC were already activated, either spontaneously or by high K+. In contrast to their biphasic actions on [Ca2+]i, phorbol esters exerted only stimulatory actions on gonadotropin release, even when Ca2+ influx was concomitantly reduced. However, pituitary cells had to be primed with an appropriate [Ca2+]i level before exocytosis could be amplified by PKC. In PKC-depleted cells, all actions of phorbol esters on Ca2+ entry and amplitude modulation, and on LH release, were abolished. GnRH-induced LH secretion was also significantly reduced, especially the plateau phase of the response. These data indicate that Ca2+ and PKC serve as interacting signals during the cascade of cellular events triggered by agonist stimulation, in which Ca2+ turns cell responses on or off, and PKC amplifies the positive and negative effects of Ca2+.
Asunto(s)
Calcio/metabolismo , Adenohipófisis/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Alcaloides/farmacología , Animales , Transporte Biológico , Western Blotting , Canales de Calcio/metabolismo , Células Cultivadas , Activación Enzimática , Femenino , Hormona Liberadora de Gonadotropina/metabolismo , Hormona Luteinizante/antagonistas & inhibidores , Hormona Luteinizante/metabolismo , Potenciales de la Membrana , Adenohipófisis/citología , Adenohipófisis/fisiología , Potasio/farmacología , Ratas , Ratas Endogámicas , Estaurosporina , Temperatura , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Endothelin (ET) receptors are present in pituitary cells and stimulate hormone release through the phosphoinositide/Ca2+ signaling system. In pituitary cell suspensions, ET caused [Ca2+]i elevations of much higher amplitudes than those induced by other vasoactive hormones, including angiotensin II, vasopressin, and noradrenalin. The action of ET was coupled to rapid and transient activation of exocytosis in gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. In contrast, angiotensin II did not stimulate luteinizing hormone release, and luteinizing hormone responses to vasopressin and noradrenalin were very small. Single gonadotrophs exhibited three types of [Ca2+]i responses to increasing doses of ET, (a) subthreshold responses, with amplitude modulation; (b) threshold-oscillatory responses, with frequency modulation; and (c) threshold-biphasic responses, as the summation of single Ca2+ spikes. The same [Ca2+]i patterns were also seen in gonadotropin-releasing hormone (GnRH)-stimulated cells. In the presence of [Ca2+]e, the amplitudes of the Ca2+ spikes progressively decreased during continuous stimulation with ET or GnRH, reaching the nonoscillatory plateau level after 200-400 sec of stimulation. In cells stimulated with GnRH, subsequent exposure to ET, GnRH, or ionomycin during the plateau phase did not elicit further increases in [Ca2+]i, whereas cells stimulated with ET responded partially to all three agents. In addition, cells exposed to ET or GnRH for 30 min, followed by a 30-min recovery period, were able to mount a full [Ca2+]i response to GnRH, but not to ET-1. Similarly, both peptides elicited rapid increases in LH release, with comparable potencies, but the response to ET decreased much more rapidly during sustained stimulation and gonadotrophs became refractory to further ET stimulation. This is in part attributable to rapid endocytosis of ET receptors during continuous agonist stimulation. These data indicate that ET exerts potent but transient secretory actions in several pituitary cell types and is a potential regulator of gonadotropin release. The initial receptor-coupling events in both ET- and GnRH-stimulated cells are similar, but the differences observed during continuous or repetitive stimulation indicate that the ET receptor pathway undergoes rapid desensitization that is critical in determining the distinct cellular responses to the two peptides.
Asunto(s)
Calcio/metabolismo , Endotelinas/farmacología , Adenohipófisis/efectos de los fármacos , Angiotensina II/farmacología , Animales , Arginina Vasopresina/farmacología , Canales de Calcio/fisiología , Endocitosis , Exocitosis , Femenino , Ionomicina/farmacología , Hormona Luteinizante/metabolismo , Adenohipófisis/metabolismo , Adenohipófisis/fisiología , Hormonas Liberadoras de Hormona Hipofisaria/farmacología , Ratas , Receptores de Superficie Celular/metabolismo , Receptores de EndotelinaRESUMEN
The relationships between receptor-mediated endocytosis and the generation of intracellular signals were analyzed in angiotensin II (AII)-stimulated adrenal glomerulosa cells. In cells equilibrated with 125I-AII analogs at 4 degrees C, specifically bound agonist but not antagonist AII derivatives were rapidly internalized at 37 degrees C. AII-induced internalization was not influenced by the presence or absence of extracellular Ca2+ but was inhibited by treatment with phenylarsine oxide (PAO) or by arresting coated pit formation with hypotonic shock and potassium depletion. Inhibition of internalization by PAO was prevented by the bifunctional sulfhydryl reagent dithiothreitol but only partially reversed by mercaptoethanol, and readdition of K+ restored internalization in K(+)-depleted cells. Treatment with PAO did not impair the initial AII-induced elevations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and cytoplasmic calcium [( Ca2+]i) but reduced the sustained phase of the Ins(1,4,5)P3 response by 85% and abolished the second phase of the cytoplasmic Ca2+ response; these responses were restored by concomitant treatment with dithiothreitol. Inhibition of AII-receptor internalization by K+ depletion also caused selective loss of the sustained phase of the AII-induced Ca2+ response. Thus, blockade of AII-receptor internalization has similar effects as extracellular Ca2+ deficiency, which abolishes the sustained but not the early AII-induced increases in Ins(1,4,5)P3 production and [Ca2+]i. The close correlations between AII-induced internalization and the generation of Ins(1,4,5)P3 and [Ca2+]i responses suggest that endocytosis of the agonist-receptor complex is necessary to maintain the production of these intracellular signals. It is also possible that receptor-operated vesicular uptake of extracellular Ca2+ makes a significant contribution to the sustained [Ca2+]i responses of certain agonist-stimulated target cells.
Asunto(s)
Angiotensina II/farmacología , Calcio/metabolismo , Endocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Zona Glomerular/metabolismo , Animales , Arsenicales/farmacología , Transporte Biológico , Bovinos , Células Cultivadas , Ditiotreitol/farmacología , Mercaptoetanol/farmacología , Potasio/metabolismo , Zona Glomerular/citología , Zona Glomerular/efectos de los fármacosRESUMEN
The presence of endothelin, a vasoconstrictor peptide, in the hypothalamus and posterior pituitary suggests that it also regulates neural and other nonvascular target cells. In pituitary gonadotrophs, low doses of endothelin evoked oscillations in the intracellular calcium concentration, and high doses induced a biphasic calcium response. Mobilization of intracellular calcium predominated during the spike phase of the calcium response to endothelin, whereas calcium entry through dihydropyridine-sensitive channels contributed to both the spike and plateau phases of the calcium response. Endothelin was a potent as hypothalamic gonadotropin-releasing hormone (GnRH) in stimulation of gonadotropin release in perifused pituitary cells. Endothelin bound specifically to pituitary cells with a dissociation constant of 70 picomolar, and induced rapid formation of inositol trisphosphate and diacyglycerol. Although intracellular calcium concentration and gonadotropin secretory responses to endothelin were independent to the GnRH receptor, endothelin and GnRH appeared to have a common signal transduction mechanism. These observations suggest that endothelin can act as a neuropeptide to regulate anterior pituitary function.