Biomedical subjects

E W Roubos

Publications and source records attributed to E W Roubos.

At least 37 records · Page 2Linked to original sources

PubMed2002

TRH signal transduction in melanotrope cells of Xenopus laevis.

TRH is a neuropeptide that activates phospholipase C and, when acting on secretory cells, usually induces a biphasic response consisting of a transitory increase in secretion (due to IP(3) mobilization of Ca(2+) from intracellular stores), followed by a sustained plateau phase of stimulated secretion (by protein kinase C-dependent influx of extracellular Ca(2+) through voltage-operated Ca(2+) channels). The melanotrope cell of the amphibian Xenopus laevis displays a unique secretory response to TRH, namely a broad transient but no sustained second phase, consistent with the observation that TRH induces a single Ca(2+) transient rather than the classic biphasic increase in [Ca(2+)](i). The purpose of the present study was to determine the signal transduction mechanism utilized by TRH in generating this Ca(2+) signaling response. Our hypothesis was that the transient reflects the operation of only one of the two signaling arms of the lipase (i.e., either IP(3)-induced mobilization of internal Ca(2+) or PKC-dependent influx of external Ca(2+)). Using video-imaging microscopy it is shown that the TRH-induced Ca(2+) transient is dramatically attenuated under Ca(2+)-free conditions and that thapsigargin has no noticeable effect on the TRH-induced transient. These observations indicate that an IP(3)-dependent mechanism plays no important role in the action of TRH. PKC also does not seem to be involved because an activator of PKC did not induce a Ca(2+) transient and an inhibitor of PKC did not affect the TRH response. Experiments with a bis-oxonol membrane potential probe showed that the TRH response also does not underlie a PKC-independent mechanism that would induce membrane depolarization. We conclude that the action of TRH on the Xenopus melanotrope does not rely on the classical phospholipase C-dependent mechanism.

Animals↗

PubMed2002

New aspects of signal transduction in the Xenopus laevis melanotrope cell.

Light and temperature stimuli act via various brain centers and neurochemical messengers on the pituitary melanotrope cells of Xenopus laevis to control distinct subcellular activities such as the biosynthesis, processing, and release of alpha-melanophore-stimulating hormone (alphaMSH). The melanotrope signal transduction involves the action of a large repertoire of neurotransmitter and neuropeptide receptors and the second messengers cAMP and Ca(2+). Here we briefly review this signaling mechanism and then present new data on two aspects of this process, viz. the presence of a stimulatory beta-adrenergic receptor acting via cAMP and the egress of cAMP from the melanotrope upon a change of alphaMSH release activity.

Animals↗

PubMed2002

Multiple control and dynamic response of the Xenopus melanotrope cell.

Some amphibian brain-melanotrope cell systems are used to study how neuronal and (neuro)endocrine mechanisms convert environmental signals into physiological responses. Pituitary melanotropes release alpha-melanophore-stimulating hormone (alpha-MSH), which controls skin color in response to background light stimuli. Xenopus laevis suprachiasmatic neurons receive optic input and inhibit melanotrope activity by releasing neuropeptide Y (NPY), dopamine (DA) and gamma-aminobutyric acid (GABA) when animals are placed on a light background. Under this condition, they strengthen their synaptic contacts with the melanotropes and enhance their secretory machinery by upregulating exocytosis-related proteins (e.g. SNAP-25). The inhibitory transmitters converge on the adenylyl cyclase system, regulating Ca(2+) channel activity. Other messengers like thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH, from the magnocellular nucleus), noradrenalin (from the locus coeruleus), serotonin (from the raphe nucleus) and acetylcholine (from the melanotropes themselves) stimulate melanotrope activity. Ca(2+) enters the cell and the resulting Ca(2+) oscillations trigger alpha-MSH secretion. These intracellular Ca(2+) dynamics can be described by a mathematical model. The oscillations travel as a wave through the cytoplasm and enter the nucleus where they may induce the expression of genes involved in biosynthesis and processing (7B2, PC2) of pro-opiomelanocortin (POMC) and release (SNAP-25, munc18) of its end-products. We propose that various environmental factors (e.g. light and temperature) act via distinct brain centers in order to release various neuronal messengers that act on the melanotrope to control distinct subcellular events (e.g. hormone biosynthesis, processing and release) by specifically shaping the pattern of melanotrope Ca(2+) oscillations.

Animals↗

PubMed2002

Sauvagine regulates Ca2+ oscillations and electrical membrane activity of melanotrope cells of Xenopus laevis.

Ca2+ oscillations regulate secretion of the hormone alpha-melanphore-stimulating hormone (alpha-MSH) by the neuroendocrine pituitary melanotrope cells of the amphibian Xenopus laevis. These Ca2+ oscillations are built up by discrete increments in the intracellular Ca2+ concentration, the Ca2+ steps, which are generated by electrical membrane bursting firing activity. It has been demonstrated that the patterns of Ca2+ oscillations and kinetics of the Ca2+ steps can be modulated by changing the degree of intracellular Ca2+ buffering. We hypothesized that neurotransmitters known to regulate alpha-MSH secretion also modulate the pattern of Ca2+ oscillations and related electrical membrane activity. In this study, we tested this hypothesis for the secretagogue sauvagine. Using high temporal-resolution Ca2+ imaging, we show that sauvagine modulated the pattern of Ca2+ signalling by increasing the frequency of Ca2+ oscillations and inducing a broadening of the oscillations through its effect on various Ca2+ step parameters. Second, we demonstrate that sauvagine caused a small but significant decrease in K+ currents measured in the whole-cell voltage-clamp, whereas Ca2+ currents remained unchanged. Third, in the cell-attached patch-clamp mode, a stimulatory effect of sauvagine on action current firing was observed. Moreover, sauvagine changed the shape of individual action currents. These results support the hypothesis that the secretagogue sauvagine stimulates the frequency of Ca2+ oscillations in Xenopus melanotropes by altering Ca2+ step parameters, an action that likely is evoked by an inhibition of K+ currents.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2002

Demonstration of postsynaptic receptor plasticity in an amphibian neuroendocrine interface.

Pituitary pars intermedia melanotrope cells are often used as a model to study mechanisms of neuroendocrine integration. In the amphibian Xenopus laevis, the synthesis and release of alpha-melanophore-stimulating hormone (alpha-MSH) from these cells is a dynamic process dependent upon the colour of background. In animals on a black background, there is a higher level of synthesis and secretion of alpha-MSH than in animals on a white background, and, consequently, there is skin darkening in animals on a black background. The melanotropes are innervated by hypothalamic neurones that produce neuropeptide Y (NPY), a peptide that inhibits alpha-MSH secretion via the NPY Y1 receptor. The inhibitory neurones have a higher expression of NPY in animals adapted to a white background and both the size and the number of inhibitory synapses on the melanotrope cells are enhanced. The purpose of the present study was to determine if this presynaptic plasticity displayed by the inhibitory neurones is reciprocated by postsynaptic plasticity (i.e. if there is an enhanced expression of the Y1 receptor in melanotropes of animals adapted to a white background). For this purpose quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the level of Y1 receptor mRNA in melanotropes of animals undergoing the process of background adaptation. The results showed that there is a higher Y1 receptor mRNA expression in melanotropes of white-adapted animals. We conclude that the inhibitory neuroendocrine interface in the Xenopus pars intermedia displays postsynaptic plasticity in response to changes of background colour. To our knowledge, this is the first demonstration of a physiological environmental change leading to changes in postsynaptic receptor expression in a fully identified vertebrate neuroendocrine reflex.

Animals↗

PubMed2001

Dynamics and plasticity of peptidergic control centres in the retino-brain-pituitary system of Xenopus laevis.

This review deals particularly with the recent literature on the structural and functional aspects of the retino-brain-pituitary system that controls the physiological process of background adaptation in the aquatic toad Xenopus laevis. Taking together the large amount of multidisciplinary data, a consistent picture emerges of a highly plastic system that efficiently responds to changes in the environmental light condition by releasing POMC-derived peptides, such as the peptide alpha-melanophore-stimulating hormone (alpha-MSH), into the circulation. This plasticity is exhibited by both the central nervous system and the pituitary pars intermedia, at the level of molecules, subcellular structures, synapses, and cells. Signal transduction in the pars intermedia of the pituitary gland of Xenopus laevis appears to be a complex event, involving various environmental factors (e.g., light and temperature) that act via distinct brain centres and neuronal messengers converging on the melanotrope cells. In the melanotropes, these messages are translated by specific receptors and second messenger systems, in particular via Ca(2+) oscillations, controlling main secretory events such as gene transcription, POMC-precursor translation and processing, posttranslational peptide modifications, and release of a bouquet of POMC-derived peptides. In conclusion, the Xenopus hypothalamo-hypophyseal system involved in background adaptation reveals how neuronal plasticity at the molecular, cellular and organismal levels, enable an organism to respond adequately to the continuously changing environmental factors demanding physiological adaptation.

Adaptation, Physiological↗

PubMed2001

Functional organization of the suprachiasmatic nucleus of Xenopus laevis in relation to background adaptation.

The process of background adaptation in the toad Xenopus laevis is controlled by neurons in the suprachiasmatic nucleus (SC) that inhibit the release of alpha-melanophore-stimulating hormone from the neuroendocrine melanotrope cells in the pituitary gland. We have identified the structural and functional organization of different neuropeptide Y (NPY)-containing cell groups in the Xenopus SC in relation to background adaptation. A ventrolateral, a dorsomedial, and a caudal group were distinguished, differing in location as well as in number, size, and shape of their cells. They also show different degrees of NPY immunoreactivity in response to different background adaptation conditions. In situ hybridization using a Xenopus mRNA probe for the exocytosis protein DOC2 revealed that melanotrope cells of black-adapted animals have a much higher expression of DOC2-mRNA than white-adapted ones. This establishes that the degree of DOC2-mRNA expression is a good parameter to measure cellular secretory activity in Xenopus. We show that in the ventrolateral SC group, more NPY-positive neurons express DOC2-mRNA in white- than in black-adapted animals. In contrast, NPY-positive neurons in the dorsomedial group have a high secretory activity under the black-adaptation condition. We propose that in black-adapted animals, NPY-positive neurons in the ventrolateral group, known to inhibit the melanotrope cells in white-adapted animals synaptically, are inhibited by NPY-containing interneurons in the dorsmedial group. NPY-positive neurons in the caudal group have similar secretory dynamics as the dorsomedial NPY neurons, indicating that they also play a role in background adaptation, distinct from that exerted by the ventrolateral and dorsomedial group.

Adaptation, Physiological↗

PubMed2001

Intracellular calcium buffering shapes calcium oscillations in Xenopus melanotropes.

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.

Animals↗

PubMed2001

Membrane-initiated Ca(2+) signals are reshaped during propagation to subcellular regions.

An important aspect of Ca(2+) signaling is the ability of cells to generate intracellular Ca(2+) waves. In this study we have analyzed the cellular and subcellular kinetics of Ca(2+) waves in a neuroendocrine transducer cell, the melanotrope of Xenopus laevis, using the ratiometric Ca(2+) probe indo-1 and video-rate UV confocal laser-scanning microscopy. The purpose of the present study was to investigate how local Ca(2+) changes contribute to a global Ca(2+) signal; subsequently we quantified how a Ca(2+) wave is kinetically reshaped as it is propagated through the cell. The combined kinetics of all subcellular Ca(2+) signals determined the shape of the total cellular Ca(2+) signal, but each subcellular contribution to the cellular signal was not constant in time. Near the plasma membrane, [Ca(2+)](i) increased and decreased rapidly, processes that can be described by a linear and exponential function, respectively. In more central parts of the cell slower kinetics were observed that were best described by a Hill equation. This reshaping of the Ca(2+) wave was modeled with an equation derived from a low-pass RC filter. We propose that the differences in spatial kinetics of the Ca(2+) signal serves as a mechanism by which the same cellular Ca(2+) signal carries different regulatory information to different subcellular regions of the cell, thus evoking differential cellular responses.

Animals↗

PubMed2001

Minimal model for intracellular calcium oscillations and electrical bursting in melanotrope cells of Xenopus laevis.

A minimal model is presented to explain changes in frequency, shape, and amplitude of Ca2+ oscillations in the neuroendocrine melanotrope cell of Xenopus Laevis. It describes the cell as a plasma membrane oscillator with influx of extracellular Ca2+ via voltage-gated Ca2+ channels in the plasma membrane. The Ca2+ oscillations in the Xenopus melanotrope show specific features that cannot be explained by previous models for electrically bursting cells using one set of parameters. The model assumes a KCa-channel with slow Ca2+-dependent gating kinetics that initiates and terminates the bursts. The slow kinetics of this channel cause an activation of the Kca-channel with a phase shift relative to the intracellular Ca2+ concentration. The phase shift, together with the presence of a Na+ channel that has a lower threshold than the Ca2+ channel, generate the characteristic features of the Ca2+ oscillations in the Xenopus melanotrope cell.

Animals↗

PubMed2001

Physiological control of Xunc18 expression in neuroendocrine melanotrope cells of Xenopus laevis.

In mammals, the brain-specific protein munc18-1 regulates synaptic vesicle exocytosis at the synaptic junction, in a step before vesicle fusion. We hypothesize that the rate of biosynthesis of munc18-1 messenger RNA (mRNA) and the amount of munc18-1 present in neurons and neuroendocrine cells are related to the physiologically controlled state of activity. To test this hypothesis, the homolog of munc18-1 in the clawed toad Xenopus laevis, xunc18, was studied in the brain and in the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland, at both the mRNA and the protein level. In toads adapted to a black background, the melanotropes release the peptide alpha-melanophore-stimulating hormone (alpha-MSH), which induces darkening of the skin, whereas in animals adapted to a white background the cells hardly release but store alpha-MSH, making the animal's skin look pale. The intermediate pituitary lobe of black-adapted animals revealed a strong hybridization reaction with the xunc18 mRNA probe, whereas a much weaker hybridization was observed in the intermediate lobe of white-adapted animals (optical density black: 3.4 +/- 0.2 vs. white: 0.8 +/- 0.1; P < 0.02). Immunocytochemically, Xenopus munc18-like protein has been detected throughout the brain, in identified neuronal perikarya as well as in axon tracts. Western blot analysis and immunocytochemistry further demonstrated the presence of xunc18 in the neural, intermediate and distal lobe of the pituitary gland. Xunc18 protein was furthermore determined in immunoblots of homogenates of melanotropes dissociated from the pituitary gland. In melanotropes of toads adapted to a black background, the integrated optical density of the xunc18 immunosignal was 2.7 +/- 0.5 times higher than in cells of white-adapted toads (P < 0.0001). It is concluded that, in Xenopus melanotrope cells, the amounts of both xunc18 mRNA and xunc18 protein are up-regulated in conjunction with the induction of exocytosis of alpha-MSH as a result of a physiological stimulation (environmental light condition). We propose that xunc18 is involved in physiologically controlled exocytotic secretion of neuroendocrine messengers.

Animals↗

PubMed2000

Localization and physiological regulation of the exocytosis protein SNAP-25 in the brain and pituitary gland of Xenopus laevis.

In mammals, the synaptosomal-associated protein of 25 kDa, SNAP-25, is generally thought to play a role in synaptic exocytosis of neuronal messengers. Using a polyclonal antiserum against rat SNAP-25, we have shown the presence of a SNAP-25-like protein in the brain of the South-African clawed toad Xenopus laevis by Western blotting and immunocytochemistry. Xenopus SNAP-25 is ubiquitously present throughout the brain, where its distribution in various identified neuronal perikarya and axon tracts is described. Western blot analysis and immunocytochemistry also demonstrated the presence of SNAP-25 in the neural, intermediate and distal lobes of the pituitary gland. Intensity line plots of confocal laser scanning microscope images of isolated melanotropes indicated that SNAP-25 is produced and processed in the rough endoplasmatic reticulum and Golgi apparatus, and is associated with the plasma membrane. Immunoelectron microscopy substantiated the idea that SNAP-25 is present in the plasma membrane but also showed a close association of SNAP-25 with the bounding membrane of peptide-containing secretory granules in both the neurohemal axon terminals in the neural lobe and the endocrine melanotropes in the intermediate lobe. Quantitative Western blotting revealed that adapting Xenopus to a dark background has a clear stimulatory effect on the expression of SNAP-25 in the neural lobe and in the melanotrope cells. This background light intensity-dependent stimulation of SNAP-25 expression was confirmed by the demonstration of increased immunofluorescence recorded by confocal laser scanning microscopy of individual melanotropes of black background-adapted toads. On the basis of this study on Xenopus laevis, we conclude that SNAP-25 (i) plays a substantial role in the secretion of a wide variety of neuronal messengers; (ii) functions in the central nervous system but also in neurohormonal and endocrine systems; (iii) acts at the plasma membrane but possibly also at the membrane of synaptic vesicles and peptide-containing secretory granules; (iv) acts not only locally (as in synapses), but at various sites of the plasma membrane (as in the endocrine melanotrope cell); and (v) can be upregulated in its expression by physiological stimuli that increase the extent of the molecular machinery involved in exocytosis.

Animals↗

PubMed1999

Analysis by mass spectrometry of POMC-derived peptides in amphibian melanotrope subpopulations.

We have previously shown that the melanotrope population of the pituitary intermediate lobe of Rana ridibunda is composed of two subpopulations, of low (LD) and high density (HD), that show distinct ultrastructural features and display different synthetic and secretory rates. To investigate whether LD and HD melanotrope cells also differ in proopiomelanocortin (POMC) processing, we have analyzed the POMC-end products in single cells from both subpopulations by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mass spectra revealed the presence of 8 POMC-derived peptides in HD and LD melanotrope cells, indicating a similar processing of the precursor in both subpopulations. However, the relative abundance of three POMC-end products (i.e. lys-gamma1-MSH, acetyl-alpha-MSH, and CLIP fragment) was higher in the HD subset. Moreover, two peptides with molecular weights of 1030 and 1818 Da, respectively, were detected that could not be assigned to any product deduced from the frog POMC sequence. The relative amount of the 1030 Da peptide was higher in LD melanotrope cells. Taken together, our results suggest that POMC processing is differentially regulated in the two melanotrope cell subsets.

Animals↗

PubMed1999

Co-expression in Xenopus neurons and neuroendocrine cells of messenger RNA homologues of exocytosis proteins DOC2 and munc18-1.

The proteins munc18-1 and DOC2 are assumed to play a role in docking of synaptic vesicles in neurotransmitter exocytosis at the presynaptic junction. As the proteins are known to interact, they should co-exist within neurons. We have tested this hypothesis for exocytosis of both classical and peptidergic messengers, by investigating the distribution of the messenger RNAs of munc 18-1 and DOC2 homologues in the brain and pituitary gland of the clawed toad Xenopus laevis, using in situ hybridization. For this purpose we cloned a partial complementary DNA encoding Xenopus unc18 (xunc18) and used a corresponding RNA probe, together with an RNA probe for Xenopus DOC2. At the messenger RNA level DOC2 and xunc18 were found to be expressed throughout the Xenopus brain. All brain nuclei expressing DOC2-messenger RNA showed xunc18-messenger RNA expression as well. Co-expression was shown at the individual cell level in consecutive sections of large-sized neurons. A strong expression was demonstrated in the suprachiasmatic and magnocellular nuclei and in peptidergic endocrine cells in the intermediate and anterior lobes of the pituitary gland, suggesting roles of DOC2 and xunc18 in messenger release from peptidergic secretory systems. Combined in situ hybridization and immunocytochemical analyses show that neuropeptide Y-containing cells in the suprachiasmatic nucleus also express DOC2 and xunc18 messenger RNAs. Since these cells have a high secretory activity, controlling the activity of the pituitary pars intermedia, the levels of expression of DOC2 and xunc18 may be indicators for neuronal secretory activity. The present data represent the first evidence for the co-existence of DOC2 and munc18-1 and suggest co-ordinate action of these proteins at the level of brain nuclei, individual neurons and endocrine cells.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Serotonergic innervation of the pituitary pars intermedia of xenopus laevis.

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.

Animals↗

PubMed1999

Involvement of protein kinase C and protein tyrosine kinase in thyrotropin-releasing hormone-induced stimulation of alpha-melanocyte-stimulating hormone secretion in frog melanotrope cells.

We have previously shown that the stimulatory effect of TRH on alpha-MSH secretion from the frog pars intermedia is associated with Ca2+ influx through voltage-dependent Ca2+ channels, activation of a phospholipase C and mobilization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (AC), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A2, and protein tyrosine kinase (PTK) in TRH-induced alpha-MSH release. Incubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13-acetate (24 h), which causes desensitization of PKC, or with the PKC inhibitor NPC-15437, reduced by approximately 50% of the effect of TRH on alpha-MSH release. In most melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase of the Ca2+ response. Incubation of NILs with TRH (10(-6) M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is involved in TRH-induced alpha-MSH release. The calmodulin inhibitor W-7 and the CAM KII inhibitor KN-93 did not significantly reduce the response to TRH. Similarly, the phospholipase A2 inhibitors quinacrine and 7-7'-DEA did not impair the effect of TRH on alpha-MSH secretion. The PTK inhibitors ST638 and Tyr-A23 had no effect on TRH-induced [Ca2+]i increase but inhibited in a dose-dependent manner TRH-evoked alpha-MSH release (ED50 = 1.22x10(-5) M and ED50 = 1.47x10(-5) M, respectively). Taken together, these data indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH-induced alpha-MSH secretion. Activation of PKC is responsible for the sustained phase of the increase in [Ca2+]i, whereas activation of PTK does not affect Ca2+ mobilization.

Animals↗

PubMed1998

Action currents generate stepwise intracellular Ca2+ patterns in a neuroendocrine cell.

It is believed that specific patterns of changes in the cytosolic-free calcium concentration ([Ca2+]i) are used to control cellular processes such as gene transcription, cell proliferation, differentiation, and secretion. We recently showed that the Ca2+ oscillations in the neuroendocrine melanotrope cells of Xenopus laevis are built up by a number of discrete Ca2+ rises, the Ca2+ steps. The origin of the Ca2+ steps and their role in the generation of long-lasting Ca2+ patterns were unclear. By simultaneous, noninvasive measuring of melanotrope plasma membrane electrical activity and the [Ca2+]i, we show that numbers, amplitude, and frequency of Ca2+ steps are variable among individual oscillations and are determined by the firing pattern and shape of the action currents. The general Na+ channel blocker tetrodotoxin had no effect on either action currents or the [Ca2+]i. Under Na+-free conditions, a depolarizing pulse of 20 mM K+ induced repetitive action currents and stepwise increases in the [Ca2+]i. The Ca2+ channel blocker CoCl2 eliminated action currents and stepwise increases in the [Ca2+]i in both the absence and presence of high K+. We furthermore demonstrate that the speed of Ca2+ removal from the cytoplasm depends on the [Ca2+]i, also between Ca2+ steps during the rising phase of an oscillation. It is concluded that Ca2+ channels, and not Na+ channels, are essential for the generation of specific step patterns and, furthermore, that the frequency and shape of Ca2+ action currents in combination with the Ca2+ removal rate determine the oscillatory pattern.

Action Potentials↗

PubMed1998

Identification of suprachiasmatic melanotrope-inhibiting neurons in Xenopus laevis: a confocal laser-scanning microscopy study.

The amphibian Xenopus laevis is able to adjust its skin color to the light intensity of the environment. Paling of the skin is achieved by inhibiting the release of alpha-melanophore-stimulating hormone (alpha-MSH) from the melanotrope cells in the pars intermedia of the pituitary gland. The release of alpha-MSH is inhibited by gamma-aminobutyric acid (GABA), neuropeptide Y (NPY), and dopamine (DA). To locate and identify neurons that might be responsible for the inhibitory input, double and triple immunocytochemistry, retrograde tracing from the pars intermedia with the carbocyanine membrane probe 1,1'dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzene-sulfonate (Fast DiI), and confocal laser-scanning microscopy were combined. Glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and NPY were found to coexist in an axonal network innervating the pars intermedia. The suprachiasmatic nucleus (SC) contained different populations of neurons that were single, double, or triple labelled for GAD, NPY, and TH. In the lateral SC, NPY+ neurons were observed. TH-immunoreactive (TH-IR) neurons occurred in the medial, dorsolateral, lateral, and ventrolateral SC. Neurons that were double labelled for NPY and TH and triple labelled for Fast DiI, NPY, and TH were present in the ventrolateral SC. This same area contained neurons that were triple labelled for GAD, NPY, and TH. It is concluded that the triple-labelled and probably the double-labelled ventrolateral SC neurons (suprachiasmatic melanotrope-inhibiting neurons) innervate the pituitary pars intermedia and are responsible for the NPY-, DA-, and GABA-mediated inhibition of melanotrope cell activity in Xenopus laevis.

Animals↗