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Papers by Bruce Jenks

Pflügers Archiv, 2001

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise ... more 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.

Pflügers Archiv European Journal of Physiology, 1994

The secretion of a-melanophore-stimulating hormone (a-MSH) from melanotrope cells in the pituitar... more The secretion of a-melanophore-stimulating hormone (a-MSH) from melanotrope cells in the pituitary gland of Xenopus laevis is regulated by various neural factors, both classical neurotransmitters and neuropeptides. The majority of these cells (80%) display spontaneous Ca 2+ oscillations. In order to gain a better understanding of the external regulation of intracellular Ca 2+ ([Ca2+]i) in the melanotrope cell, we have examined the action of well known a-MSH secretagogues on the Ca 2+ oscillations. It is shown that all secretagogues tested also control the oscillatory state of Xenopus melanotropes, that is, the secreto-inhibitors dopamine, isoguvacine ():-aminobutyric acid, GABAA agonist), baclofen (GABAB agonist) and neuropeptide Y evoked a rapid quenching of the spontaneous Ca 2+ oscillations, whereas the secreto-stimulant sauvagine, an amphibian peptide related to corticotropin releasing hormone, induced oscillatory activity in non-oscillating cells. Supporting argument is given for the idea that the regulation of Ca 2+ oscillations is a focal point in the regulation of secretory activity of melanotrope cells. There was considerable heterogeneity among melanotrope cells in the threshold of their Ca 2+ response to secretagogue treatment. This heterogeneity may be the basis for melanotrope cell recruitment observed during physiological adaptations of the animal to the light intensity of its background.

Peptides, 1995

The melanotrope cells in the pituitary gland of Xenopus laevis are innervated by neurons containi... more The melanotrope cells in the pituitary gland of Xenopus laevis are innervated by neurons containing neuropeptide Y (NPY). In the present study, the mechanism of action of NPY on the melanotropes has been investigated. NPY inhibited in vitro secretion from melanotropes in intact neurointermediate lobes as well as from isolated, single melanotropes. Inhibition of secretion from neurointermediate lobes was mimicked by the NPY analogues PYY and [Leu3l,Pro34]NPY, whereas NPY(13-36) was inactive. Secretion from isolated melanotropes was inhibited by [Leu31,Pro34]NPY and NPY(13-36), but NPY(13-36) was 10-fold less potent than [Leu3\Pro34]NPY. Studies on isolated cells revealed that NPY and its analogues inhibited the occurrence of intracellular Ca2+ oscillations with the same potency as they inhibited secretion from isolated cells. In addition to inhibiting basal secretion and spontaneous Ca2+ oscillations, NPY inhibited the basal production of cyclic AMP. On the basis of these results it is proposed that NPY inhibits secretion from Xenopus melanotropes by inhibiting cyclic AMP-dependent spontaneous Ca2+ oscillations through a Y r like receptor. Neuropeptide Y Melanotrope cells Xenopus laevis Ca2+ oscillations Cyclic AMP

Neuroscience Letters, 2005

Secretion of ␣-melanophore-stimulating hormone (␣-MSH) from the neuroendocrine melanotrope cells ... more Secretion of ␣-melanophore-stimulating hormone (␣-MSH) from the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland of the clawed frog Xenopus laevis is regulated by various inhibitory, stimulatory and autocrine factors. The neuropeptide sauvagine stimulates ␣-MSH secretion by changing the pattern of intracellular Ca 2+ oscillations and the electrical properties of the cell membrane. In the present study we investigated whether another secreto-stimulator, the extracellular Ca 2+-sensing receptor (CaR), also affects the Ca 2+ oscillatory pattern and electrical membrane properties. Using high-speed dynamic video-imaging we show that activation of the CaR with the specific agonist l-phenylalanine (l-Phe) changes the Ca 2+ oscillatory pattern by increasing the number of Ca 2+ steps, which are the "building blocks" of the oscillations. Moreover, using patch-clamp electrophysiology it is demonstrated that l-Phe affects membrane properties by increasing frequency and duration of action currents. Compared to sauvagine, the CaR has different effects on the action current parameters, suggesting that multiple mechanisms regulate the electrical properties of the melanotrope cell membrane and, thereby, the Ca 2+ oscillation-dependent level of ␣-MSH secretion.

Neuroendocrinology, 1991

A number of neurochemical messengers have been shown to act directly on the melanotrope cells of ... more A number of neurochemical messengers have been shown to act directly on the melanotrope cells of the pars intermedia of Xenopus laevis to regulate alpha MSH secretion. In the present study the possibility that the melanotropes are also indirectly controlled has been examined. For this purpose, the characteristics of alpha MSH release from superfused intact lobes, cultured lobe and isolated melanotropes were compared after treatment with elevated potassium. Isolated melanotropes responded with an increased secretion of alpha MSH, whereas intact lobes showed a profound inhibitory response, probably caused by potassium-induced release of inhibitory factors from nerve terminals. Cultured lobes displayed a biphasic response characterized by an initial activation followed by a strong inhibition; the stimulatory phase likely reflects a direct action of potassium on the melanotropes, before being overridden by an inhibitory mechanism. The inhibitory phase must originate from the action of nonneuroendocrine cells because the cultured lobes lack functionally active nerve terminals, as verified by immunocytochemistry and electron microscopy. The most likely candidates for this action are folliculo-stellate cells which are in intimate contact with the melanotropes and are innervated by neuropeptide Y-containing nerve terminals. Like elevated potassium, neuropeptide Y inhibited alpha MSH secretion from fresh and cultured lobes but not from isolated melanotropes. This indicates that NPY acts indirectly, in a nonpresynaptic way, to inhibit alpha MSH secretion.

Neuroendocrinology, 2007

Melanotrope cells of the amphibian pituitary pars intermedia produce α-melanophore-stimulating ho... more Melanotrope cells of the amphibian pituitary pars intermedia produce α-melanophore-stimulating hormone (α-MSH), a peptide which causes skin darkening during adaptation to a dark background. The secretory activity of the melanotrope of the South African clawed toad Xenopus laevis is regulated by multiple factors, both classical neurotransmitters and neuropeptides from the brain. This review concerns the plasticity displayed in this intermediate lobe neuroendocrine interface during physiological adaptation to the environment. The plasticity includes dramatic morphological plasticity in both pre- and post-synaptic elements of the interface. Inhibitory neurons in the suprachiasmatic nucleus, designated suprachiasmatic melanotrope-inhibiting neurons (SMINs), possess more and larger synapses on the melanotrope cells in white than in black-background adapted animals; in the latter animals the melanotropes are larger and produce more proopiomelanocortin (POMC), the precursor of α-MSH. On a ...

Neuroendocrinology, 2004

Dopamine inhibits pituitary melanotrope cells of the amphibian Xenopus laevis through activation ... more Dopamine inhibits pituitary melanotrope cells of the amphibian Xenopus laevis through activation of a dopamine (D2) receptor that couples to a Gi protein. Activated Gi protein subunits are known to affect voltage-operated Ca2+ currents (ICa). In the present study we investigated which Ca2+ currents are regulated by D2-receptor activation and which Gi protein subunits are involved. Whole-cell voltage-clamp patch-clamp experiments from holding potentials (HPs) of –80 and –30 mV show that 28.6 and 36.9%, respectively, of the total ICa was inhibited by apomorphin, a D2-receptor agonist. The inhibited current had fast activation and inactivation kinetics. From an HP of –80 mV, inhibition of N-type Ca2+ currents with ω-conotoxin GVIA and R-type current by SNX-482 reduced the efficacy of the apomorphin-induced inhibition. From an HP of –30 mV this reduction for ω-conotoxin GVIA was still observed. Blocking L-type current by nifedipine or P/Q-type current by ω-agatoxin IVA did not affect ap...

Journal of Neuroendocrinology, 1994

The secretory activity of endocrine cells largely depends on the concentration of free cytosolic ... more The secretory activity of endocrine cells largely depends on the concentration of free cytosolic calcium. We have studied the mechanisms that are involved in supplying the calcium necessary for the secretion of α‐melanophore‐stimulating hormone (α‐MSH) from melanotrope cells in the pituitary intermediate lobe of the amphibian Xenopus laevis. Using whole‐cell voltage clamp, high‐voltage activated calcium currents were observed, with a peak current between 0 and +20 mV. Two types of Ca2 +‐currents appeared, depending on the experimental setup. An inactivating current, which was observed after a 10 msec depolarizing prepulse, resembled currents through N‐type channels as it was clearly inhibited by 1 μM ω‐conotoxin. The second type was a non‐inactivating current, which was blocked up to 50% by 1 μM nifedipine, indicating its L‐type nature. Only a small component of this inactivating current could be blocked by ω‐conotoxin. No evidence was found for the presence of transient, low‐voltag...

Journal of Cellular Physiology, 2011

The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the productio... more The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was >100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.

Journal of Biological Chemistry, 1998

It is believed that specific patterns of changes in the cytosolic-free calcium concentration ([Ca... more It is believed that specific patterns of changes in the cytosolic-free calcium concentration ([Ca 2؉ ] i) are used to control cellular processes such as gene transcription, cell proliferation, differentiation, and secretion. We recently showed that the Ca 2؉ oscillations in the neuroendocrine melanotrope cells of Xenopus laevis are built up by a number of discrete Ca 2؉ rises, the Ca 2؉ steps. The origin of the Ca 2؉ steps and their role in the generation of long-lasting Ca 2؉ patterns were unclear. By simultaneous, noninvasive measuring of melanotrope plasma membrane electrical activity and the [Ca 2؉ ] i , we show that numbers, amplitude, and frequency of Ca 2؉ 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 [Ca 2؉ ] i. Under Na ؉-free conditions, a depolarizing pulse of 20 mM K ؉ induced repetitive action currents and stepwise increases in the [Ca 2؉ ] i. The Ca 2؉ channel blocker CoCl 2 eliminated action currents and stepwise increases in the [Ca 2؉ ] i in both the absence and presence of high K ؉. We furthermore demonstrate that the speed of Ca 2؉ removal from the cytoplasm depends on the [Ca 2؉ ] i , also between Ca 2؉ steps during the rising phase of an oscillation. It is concluded that Ca 2؉ channels, and not Na ؉ channels, are essential for the generation of specific step patterns and, furthermore, that the frequency and shape of Ca 2؉ action currents in combination with the Ca 2؉ removal rate determine the oscillatory pattern.

General and Comparative Endocrinology, 2012

Brain-derived neurotrophic factor (BDNF) is, despite its name, also found outside the central ner... more Brain-derived neurotrophic factor (BDNF) is, despite its name, also found outside the central nervous system (CNS), but the functional significance of this observation is largely unknown. This review concerns the expression of BDNF in the pituitary gland. While the presence of the neurotrophin in the mammalian pituitary gland is well documented its functional significance remains obscure. Studies on the pars intermedia of the pituitary of the amphibian Xenopus laevis have shown that BDNF is produced by the neuroendocrine melanotrope cells, its expression is physiologically regulated, and the melanotrope cells themselves express receptors for the neurotrophin. The neurotrophin has been shown to act as an autocrine factor on the melanotrope to promote cell growth and regulate gene expression. In doing so BDNF supports the physiological function of the cell to produce and release a-melanophore-stimulating hormone for the purpose of adjusting the animal's skin color to that of its background.

General and Comparative Endocrinology, 2008

We have tested the hypothesis that the type and kinetics of voltage-activated Ca 2+ channels in a... more We have tested the hypothesis that the type and kinetics of voltage-activated Ca 2+ channels in a neuroendocrine cell depend on the cell's long-term external input. For this purpose, the presence and kinetics of both low (LVA) and high-voltage-activated (HVA) L-type Ca 2+ channels have been assessed in melanotrope pituitary cells of the amphibian Xenopus laevis. The secretory activity of this cell type can readily be manipulated in vivo by changing the animal's environmental light condition, from a black to a white background. We here show that, compared to white background-adapted Xenopus, melanotropes from black background-adapted frogs have (1) a much larger size, as revealed by their 2.5 times larger membrane capacitance (P < 0.001), (2) a 2 times higher HVA current density (P < 0.05), (3) a clearly smaller Ca 2+-dependent inactivation (10%; P < 0.05), (4) L-type channels with 5 times slower activation and inactivation kinetics (P < 0.05), and (5) slower kinetics of L-type channels that become faster and more similar to those in white-background adapted cells when the intracellular Ca 2+-buffering capacity is reduced. Furthermore, white-adapted melanotropes possess LVA-type Ca 2+ channels, which are lacking from cells from black-adapted animals. The melanotrope calmodulin mRNA level does not differ between the two adaptation states. These results indicate that HVA L-type channel kinetics differ in relation to environmentally induced changes in cellular secretory state, probably mediated via intracellular Ca 2+-buffering, whereas the occurrence of LVA Ca 2+ channels may depend on environmentally controlled channel gene expression.

General and Comparative Endocrinology, 2011

This review gives an overview of the functioning of the hypothalamo-hypophyseal neuroendocrine in... more This review gives an overview of the functioning of the hypothalamo-hypophyseal neuroendocrine interface in the pituitary neurointermediate lobe, as it relates to melanotrope cell function in two amphibian species, Rana ridibunda and Xenopus laevis. It primarily but not exclusively concerns the work of two collaborating laboratories, the Laboratory for Molecular and Cellular Neuroendocrinology (University of Rouen, France) and the Department of Cellular Animal Physiology (Radboud University Nijmegen, The Netherlands). In the course of this review it will become apparent that Rana and Xenopus have, for the most part, developed the same or similar strategies to regulate the release of α-melanophore-stimulating hormone (α-MSH). The review concludes by highlighting the molecular and cellular mechanisms utilized by thyrotropin-releasing hormone (TRH) to activate Rana melanotrope cells and the function of autocrine brain-derived neurotrophic factor (BDNF) in the regulation of Xenopus melanotrope cell function.

General and Comparative Endocrinology, 2008

The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pitui... more The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pituitary cell, but the intracellular mechanism this G protein-coupled receptor uses in these cells is not known. In the present study we investigated possible intracellular signal transduction pathway(s) utilized by the CaR of the endocrine melanotrope cells in the intermediate pituitary lobe of the South African-clawed toad Xenopus laevis. For this purpose, the effects of various pharmacological agents on CaR-evoked secretion of radiolabeled secretory peptides from cultured melanotrope cells were assessed. CaR-evoked secretion, induced by the potent CaR agonist L-phenylalanine (L-Phe), could not be inhibited by cholera toxin, nor by NPC-15437 and PMA, indicating that neither G s /PKA nor G q /PKC pathways are involved. However, pertussis toxin (G i/o protein inhibitor), genistein (inhibitor of PTKs), wortmannin/LY-294002 (PI3-K inhibitor) and U-0126 (inhibitor of extracellular signal-regulated kinase, ERK) all substantially inhibited CaRevoked secretion, indicating that the Xenopus melanotrope cell possesses a PI3-K/MAPK system that plays some role in CaR-signaling. Since no direct effect of L-Phe on ERK phosphorylation could be shown it is concluded that CaR must act primarily through another, still unknown, signaling pathway in Xenopus melanotropes. Our results indicate that the PI3-K/MAPK system has a facilitating effect on CaR-induced secretion, possibly by sensitizing the CaR.

General and Comparative Endocrinology, 2002

TRH is a neuropeptide that activates phospholipase C and, when acting on secretory cells, usually... more 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.

General and Comparative Endocrinology, 2010

Pituitary melanotrope cells of the amphibian Xenopus laevis are neuroendocrine cells regulating t... more Pituitary melanotrope cells of the amphibian Xenopus laevis are neuroendocrine cells regulating the animal&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s skin color adaptation through secretion of α-melanophore-stimulating hormone (α-MSH). To fulfill this function optimally, the melanotrope cell undergoes plastic changes in structure and secretory activity in response to changed background light conditions. Xenopus melanotrope cells display Ca(2+) oscillations that are thought to drive α-MSH secretion and gene expression. They also produce brain-derived neurotrophic factor (BDNF), which stimulates in an autocrine way the biosynthesis of the α-MSH precursor, pro-opiomelanocortin (POMC). We have used this physiological adaptation mechanism as a model to investigate the role of BDNF in the regulation of Ca(2+) kinetics and Ca(2+)-dependent gene expression. By dynamic video imaging of isolated cultured melanotropes we demonstrated that BDNF caused a dose-dependent increase in Ca(2+) oscillation frequency up to 64.7±2.3% of control level. BDNF also induced a transient Ca(2+) peak in Ca(2+)-free medium, which was absent when calcium stores were blocked by thapsigargin and 2-aminoethoxydiphenyl borate, indicating that BDNF stimulates acute release of Ca(2+) from IP(3)-sensitive intracellular Ca(2+) stores. Moreover, we show that thapsigargin inhibits the expression of BDNF transcript IV (by 61.1±28.8%) but does not affect POMC transcript. We conclude that BDNF mobilizes Ca(2+) from IP(3)-sensitive intracellular Ca(2+) stores and propose the possibility that the resulting Ca(2+) oscillations selectively stimulate expression of the BDNF gene.

General and Comparative Endocrinology, 1992

The secretion of most pituitary hormones is under the control of feedback mechanisms. The feedbac... more The secretion of most pituitary hormones is under the control of feedback mechanisms. The feedback control of a-melanophore-stimulating hormone (cr-MSH) from melanotrope cells is controversial. The possible existence of an autofeedback exerted by o-MSH or other POMC-derived peptides on melanotrope cells of the amphibian Xenopus luevis has been investigated. o(-MSH or its potent agonist 4-norleucine,7-D-phenylalanine-o-MSH has no effect on the release of radiolabeled POMC-derived peptides or immunoreactive @endorphin from superfused neurointermediate pituitary lobes. Melanin concentrating hormone, previously reported to have an u-MSH-like effect on melanophores, did not affect o.-MSH secretion. Neurointermediate lobe superfusate, which contains a mixture of POMC-derived peptides, failed to affect the secretory activity of melanotropes. It is concluded that in X. laevis the secretory activity of melanotropes is not under the control of short-term autofeedback mechanisms involving (Y-MSH or other POMC-derived peptides.

General and Comparative Endocrinology, 2002

Light and temperature stimuli act via various brain centers and neurochemical messengers on the p... more 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.

Endocrinology, 2011

We tested whether double cortin-like kinase-short (DCLK-short), a microtubule-associated Ser/Thr ... more We tested whether double cortin-like kinase-short (DCLK-short), a microtubule-associated Ser/Thr kinase predominantly expressed in the brain, is downstream of the ERK signaling pathway and is involved in proopiomelanocortin gene (POMC) expression in endocrine pituitary melanotrope cells of Xenopus laevis. Melanotropes form a well-established model to study physiological aspects of neuroendocrine plasticity. The amphibian X. laevis adapts its skin color to the background light intensity by the release of α-MSH from the melanotrope cell. In frogs on a white background, melanotropes are inactive but they are activated during adaptation to a black background. Our results show that melanotrope activation is associated with an increase in DCLK-short mRNA and with phosphorylation of DCLK-short at serine at position 30 (Ser-30). Upon cell activation phosphorylated Ser-30-DCLK-short was translocated from the cytoplasm into the nucleus, and the ERK blocker U0126 inhibited this process. The mu...

Pflügers Archiv, 2001

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise ... more 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.

Pflügers Archiv European Journal of Physiology, 1994

The secretion of a-melanophore-stimulating hormone (a-MSH) from melanotrope cells in the pituitar... more The secretion of a-melanophore-stimulating hormone (a-MSH) from melanotrope cells in the pituitary gland of Xenopus laevis is regulated by various neural factors, both classical neurotransmitters and neuropeptides. The majority of these cells (80%) display spontaneous Ca 2+ oscillations. In order to gain a better understanding of the external regulation of intracellular Ca 2+ ([Ca2+]i) in the melanotrope cell, we have examined the action of well known a-MSH secretagogues on the Ca 2+ oscillations. It is shown that all secretagogues tested also control the oscillatory state of Xenopus melanotropes, that is, the secreto-inhibitors dopamine, isoguvacine ():-aminobutyric acid, GABAA agonist), baclofen (GABAB agonist) and neuropeptide Y evoked a rapid quenching of the spontaneous Ca 2+ oscillations, whereas the secreto-stimulant sauvagine, an amphibian peptide related to corticotropin releasing hormone, induced oscillatory activity in non-oscillating cells. Supporting argument is given for the idea that the regulation of Ca 2+ oscillations is a focal point in the regulation of secretory activity of melanotrope cells. There was considerable heterogeneity among melanotrope cells in the threshold of their Ca 2+ response to secretagogue treatment. This heterogeneity may be the basis for melanotrope cell recruitment observed during physiological adaptations of the animal to the light intensity of its background.

Peptides, 1995

The melanotrope cells in the pituitary gland of Xenopus laevis are innervated by neurons containi... more The melanotrope cells in the pituitary gland of Xenopus laevis are innervated by neurons containing neuropeptide Y (NPY). In the present study, the mechanism of action of NPY on the melanotropes has been investigated. NPY inhibited in vitro secretion from melanotropes in intact neurointermediate lobes as well as from isolated, single melanotropes. Inhibition of secretion from neurointermediate lobes was mimicked by the NPY analogues PYY and [Leu3l,Pro34]NPY, whereas NPY(13-36) was inactive. Secretion from isolated melanotropes was inhibited by [Leu31,Pro34]NPY and NPY(13-36), but NPY(13-36) was 10-fold less potent than [Leu3\Pro34]NPY. Studies on isolated cells revealed that NPY and its analogues inhibited the occurrence of intracellular Ca2+ oscillations with the same potency as they inhibited secretion from isolated cells. In addition to inhibiting basal secretion and spontaneous Ca2+ oscillations, NPY inhibited the basal production of cyclic AMP. On the basis of these results it is proposed that NPY inhibits secretion from Xenopus melanotropes by inhibiting cyclic AMP-dependent spontaneous Ca2+ oscillations through a Y r like receptor. Neuropeptide Y Melanotrope cells Xenopus laevis Ca2+ oscillations Cyclic AMP

Neuroscience Letters, 2005

Secretion of ␣-melanophore-stimulating hormone (␣-MSH) from the neuroendocrine melanotrope cells ... more Secretion of ␣-melanophore-stimulating hormone (␣-MSH) from the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland of the clawed frog Xenopus laevis is regulated by various inhibitory, stimulatory and autocrine factors. The neuropeptide sauvagine stimulates ␣-MSH secretion by changing the pattern of intracellular Ca 2+ oscillations and the electrical properties of the cell membrane. In the present study we investigated whether another secreto-stimulator, the extracellular Ca 2+-sensing receptor (CaR), also affects the Ca 2+ oscillatory pattern and electrical membrane properties. Using high-speed dynamic video-imaging we show that activation of the CaR with the specific agonist l-phenylalanine (l-Phe) changes the Ca 2+ oscillatory pattern by increasing the number of Ca 2+ steps, which are the "building blocks" of the oscillations. Moreover, using patch-clamp electrophysiology it is demonstrated that l-Phe affects membrane properties by increasing frequency and duration of action currents. Compared to sauvagine, the CaR has different effects on the action current parameters, suggesting that multiple mechanisms regulate the electrical properties of the melanotrope cell membrane and, thereby, the Ca 2+ oscillation-dependent level of ␣-MSH secretion.

Neuroendocrinology, 1991

A number of neurochemical messengers have been shown to act directly on the melanotrope cells of ... more A number of neurochemical messengers have been shown to act directly on the melanotrope cells of the pars intermedia of Xenopus laevis to regulate alpha MSH secretion. In the present study the possibility that the melanotropes are also indirectly controlled has been examined. For this purpose, the characteristics of alpha MSH release from superfused intact lobes, cultured lobe and isolated melanotropes were compared after treatment with elevated potassium. Isolated melanotropes responded with an increased secretion of alpha MSH, whereas intact lobes showed a profound inhibitory response, probably caused by potassium-induced release of inhibitory factors from nerve terminals. Cultured lobes displayed a biphasic response characterized by an initial activation followed by a strong inhibition; the stimulatory phase likely reflects a direct action of potassium on the melanotropes, before being overridden by an inhibitory mechanism. The inhibitory phase must originate from the action of nonneuroendocrine cells because the cultured lobes lack functionally active nerve terminals, as verified by immunocytochemistry and electron microscopy. The most likely candidates for this action are folliculo-stellate cells which are in intimate contact with the melanotropes and are innervated by neuropeptide Y-containing nerve terminals. Like elevated potassium, neuropeptide Y inhibited alpha MSH secretion from fresh and cultured lobes but not from isolated melanotropes. This indicates that NPY acts indirectly, in a nonpresynaptic way, to inhibit alpha MSH secretion.

Neuroendocrinology, 2007

Melanotrope cells of the amphibian pituitary pars intermedia produce α-melanophore-stimulating ho... more Melanotrope cells of the amphibian pituitary pars intermedia produce α-melanophore-stimulating hormone (α-MSH), a peptide which causes skin darkening during adaptation to a dark background. The secretory activity of the melanotrope of the South African clawed toad Xenopus laevis is regulated by multiple factors, both classical neurotransmitters and neuropeptides from the brain. This review concerns the plasticity displayed in this intermediate lobe neuroendocrine interface during physiological adaptation to the environment. The plasticity includes dramatic morphological plasticity in both pre- and post-synaptic elements of the interface. Inhibitory neurons in the suprachiasmatic nucleus, designated suprachiasmatic melanotrope-inhibiting neurons (SMINs), possess more and larger synapses on the melanotrope cells in white than in black-background adapted animals; in the latter animals the melanotropes are larger and produce more proopiomelanocortin (POMC), the precursor of α-MSH. On a ...

Neuroendocrinology, 2004

Dopamine inhibits pituitary melanotrope cells of the amphibian Xenopus laevis through activation ... more Dopamine inhibits pituitary melanotrope cells of the amphibian Xenopus laevis through activation of a dopamine (D2) receptor that couples to a Gi protein. Activated Gi protein subunits are known to affect voltage-operated Ca2+ currents (ICa). In the present study we investigated which Ca2+ currents are regulated by D2-receptor activation and which Gi protein subunits are involved. Whole-cell voltage-clamp patch-clamp experiments from holding potentials (HPs) of –80 and –30 mV show that 28.6 and 36.9%, respectively, of the total ICa was inhibited by apomorphin, a D2-receptor agonist. The inhibited current had fast activation and inactivation kinetics. From an HP of –80 mV, inhibition of N-type Ca2+ currents with ω-conotoxin GVIA and R-type current by SNX-482 reduced the efficacy of the apomorphin-induced inhibition. From an HP of –30 mV this reduction for ω-conotoxin GVIA was still observed. Blocking L-type current by nifedipine or P/Q-type current by ω-agatoxin IVA did not affect ap...

Journal of Neuroendocrinology, 1994

The secretory activity of endocrine cells largely depends on the concentration of free cytosolic ... more The secretory activity of endocrine cells largely depends on the concentration of free cytosolic calcium. We have studied the mechanisms that are involved in supplying the calcium necessary for the secretion of α‐melanophore‐stimulating hormone (α‐MSH) from melanotrope cells in the pituitary intermediate lobe of the amphibian Xenopus laevis. Using whole‐cell voltage clamp, high‐voltage activated calcium currents were observed, with a peak current between 0 and +20 mV. Two types of Ca2 +‐currents appeared, depending on the experimental setup. An inactivating current, which was observed after a 10 msec depolarizing prepulse, resembled currents through N‐type channels as it was clearly inhibited by 1 μM ω‐conotoxin. The second type was a non‐inactivating current, which was blocked up to 50% by 1 μM nifedipine, indicating its L‐type nature. Only a small component of this inactivating current could be blocked by ω‐conotoxin. No evidence was found for the presence of transient, low‐voltag...

Journal of Cellular Physiology, 2011

The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the productio... more The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.

Journal of Biological Chemistry, 1998

It is believed that specific patterns of changes in the cytosolic-free calcium concentration ([Ca... more It is believed that specific patterns of changes in the cytosolic-free calcium concentration ([Ca 2؉ ] i) are used to control cellular processes such as gene transcription, cell proliferation, differentiation, and secretion. We recently showed that the Ca 2؉ oscillations in the neuroendocrine melanotrope cells of Xenopus laevis are built up by a number of discrete Ca 2؉ rises, the Ca 2؉ steps. The origin of the Ca 2؉ steps and their role in the generation of long-lasting Ca 2؉ patterns were unclear. By simultaneous, noninvasive measuring of melanotrope plasma membrane electrical activity and the [Ca 2؉ ] i , we show that numbers, amplitude, and frequency of Ca 2؉ 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 [Ca 2؉ ] i. Under Na ؉-free conditions, a depolarizing pulse of 20 mM K ؉ induced repetitive action currents and stepwise increases in the [Ca 2؉ ] i. The Ca 2؉ channel blocker CoCl 2 eliminated action currents and stepwise increases in the [Ca 2؉ ] i in both the absence and presence of high K ؉. We furthermore demonstrate that the speed of Ca 2؉ removal from the cytoplasm depends on the [Ca 2؉ ] i , also between Ca 2؉ steps during the rising phase of an oscillation. It is concluded that Ca 2؉ channels, and not Na ؉ channels, are essential for the generation of specific step patterns and, furthermore, that the frequency and shape of Ca 2؉ action currents in combination with the Ca 2؉ removal rate determine the oscillatory pattern.

General and Comparative Endocrinology, 2012

Brain-derived neurotrophic factor (BDNF) is, despite its name, also found outside the central ner... more Brain-derived neurotrophic factor (BDNF) is, despite its name, also found outside the central nervous system (CNS), but the functional significance of this observation is largely unknown. This review concerns the expression of BDNF in the pituitary gland. While the presence of the neurotrophin in the mammalian pituitary gland is well documented its functional significance remains obscure. Studies on the pars intermedia of the pituitary of the amphibian Xenopus laevis have shown that BDNF is produced by the neuroendocrine melanotrope cells, its expression is physiologically regulated, and the melanotrope cells themselves express receptors for the neurotrophin. The neurotrophin has been shown to act as an autocrine factor on the melanotrope to promote cell growth and regulate gene expression. In doing so BDNF supports the physiological function of the cell to produce and release a-melanophore-stimulating hormone for the purpose of adjusting the animal's skin color to that of its background.

General and Comparative Endocrinology, 2008

We have tested the hypothesis that the type and kinetics of voltage-activated Ca 2+ channels in a... more We have tested the hypothesis that the type and kinetics of voltage-activated Ca 2+ channels in a neuroendocrine cell depend on the cell's long-term external input. For this purpose, the presence and kinetics of both low (LVA) and high-voltage-activated (HVA) L-type Ca 2+ channels have been assessed in melanotrope pituitary cells of the amphibian Xenopus laevis. The secretory activity of this cell type can readily be manipulated in vivo by changing the animal's environmental light condition, from a black to a white background. We here show that, compared to white background-adapted Xenopus, melanotropes from black background-adapted frogs have (1) a much larger size, as revealed by their 2.5 times larger membrane capacitance (P < 0.001), (2) a 2 times higher HVA current density (P < 0.05), (3) a clearly smaller Ca 2+-dependent inactivation (10%; P < 0.05), (4) L-type channels with 5 times slower activation and inactivation kinetics (P < 0.05), and (5) slower kinetics of L-type channels that become faster and more similar to those in white-background adapted cells when the intracellular Ca 2+-buffering capacity is reduced. Furthermore, white-adapted melanotropes possess LVA-type Ca 2+ channels, which are lacking from cells from black-adapted animals. The melanotrope calmodulin mRNA level does not differ between the two adaptation states. These results indicate that HVA L-type channel kinetics differ in relation to environmentally induced changes in cellular secretory state, probably mediated via intracellular Ca 2+-buffering, whereas the occurrence of LVA Ca 2+ channels may depend on environmentally controlled channel gene expression.

General and Comparative Endocrinology, 2011

This review gives an overview of the functioning of the hypothalamo-hypophyseal neuroendocrine in... more This review gives an overview of the functioning of the hypothalamo-hypophyseal neuroendocrine interface in the pituitary neurointermediate lobe, as it relates to melanotrope cell function in two amphibian species, Rana ridibunda and Xenopus laevis. It primarily but not exclusively concerns the work of two collaborating laboratories, the Laboratory for Molecular and Cellular Neuroendocrinology (University of Rouen, France) and the Department of Cellular Animal Physiology (Radboud University Nijmegen, The Netherlands). In the course of this review it will become apparent that Rana and Xenopus have, for the most part, developed the same or similar strategies to regulate the release of α-melanophore-stimulating hormone (α-MSH). The review concludes by highlighting the molecular and cellular mechanisms utilized by thyrotropin-releasing hormone (TRH) to activate Rana melanotrope cells and the function of autocrine brain-derived neurotrophic factor (BDNF) in the regulation of Xenopus melanotrope cell function.

General and Comparative Endocrinology, 2008

The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pitui... more The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pituitary cell, but the intracellular mechanism this G protein-coupled receptor uses in these cells is not known. In the present study we investigated possible intracellular signal transduction pathway(s) utilized by the CaR of the endocrine melanotrope cells in the intermediate pituitary lobe of the South African-clawed toad Xenopus laevis. For this purpose, the effects of various pharmacological agents on CaR-evoked secretion of radiolabeled secretory peptides from cultured melanotrope cells were assessed. CaR-evoked secretion, induced by the potent CaR agonist L-phenylalanine (L-Phe), could not be inhibited by cholera toxin, nor by NPC-15437 and PMA, indicating that neither G s /PKA nor G q /PKC pathways are involved. However, pertussis toxin (G i/o protein inhibitor), genistein (inhibitor of PTKs), wortmannin/LY-294002 (PI3-K inhibitor) and U-0126 (inhibitor of extracellular signal-regulated kinase, ERK) all substantially inhibited CaRevoked secretion, indicating that the Xenopus melanotrope cell possesses a PI3-K/MAPK system that plays some role in CaR-signaling. Since no direct effect of L-Phe on ERK phosphorylation could be shown it is concluded that CaR must act primarily through another, still unknown, signaling pathway in Xenopus melanotropes. Our results indicate that the PI3-K/MAPK system has a facilitating effect on CaR-induced secretion, possibly by sensitizing the CaR.

General and Comparative Endocrinology, 2002

TRH is a neuropeptide that activates phospholipase C and, when acting on secretory cells, usually... more 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.

General and Comparative Endocrinology, 2010

Pituitary melanotrope cells of the amphibian Xenopus laevis are neuroendocrine cells regulating t... more Pituitary melanotrope cells of the amphibian Xenopus laevis are neuroendocrine cells regulating the animal&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s skin color adaptation through secretion of α-melanophore-stimulating hormone (α-MSH). To fulfill this function optimally, the melanotrope cell undergoes plastic changes in structure and secretory activity in response to changed background light conditions. Xenopus melanotrope cells display Ca(2+) oscillations that are thought to drive α-MSH secretion and gene expression. They also produce brain-derived neurotrophic factor (BDNF), which stimulates in an autocrine way the biosynthesis of the α-MSH precursor, pro-opiomelanocortin (POMC). We have used this physiological adaptation mechanism as a model to investigate the role of BDNF in the regulation of Ca(2+) kinetics and Ca(2+)-dependent gene expression. By dynamic video imaging of isolated cultured melanotropes we demonstrated that BDNF caused a dose-dependent increase in Ca(2+) oscillation frequency up to 64.7±2.3% of control level. BDNF also induced a transient Ca(2+) peak in Ca(2+)-free medium, which was absent when calcium stores were blocked by thapsigargin and 2-aminoethoxydiphenyl borate, indicating that BDNF stimulates acute release of Ca(2+) from IP(3)-sensitive intracellular Ca(2+) stores. Moreover, we show that thapsigargin inhibits the expression of BDNF transcript IV (by 61.1±28.8%) but does not affect POMC transcript. We conclude that BDNF mobilizes Ca(2+) from IP(3)-sensitive intracellular Ca(2+) stores and propose the possibility that the resulting Ca(2+) oscillations selectively stimulate expression of the BDNF gene.

General and Comparative Endocrinology, 1992

The secretion of most pituitary hormones is under the control of feedback mechanisms. The feedbac... more The secretion of most pituitary hormones is under the control of feedback mechanisms. The feedback control of a-melanophore-stimulating hormone (cr-MSH) from melanotrope cells is controversial. The possible existence of an autofeedback exerted by o-MSH or other POMC-derived peptides on melanotrope cells of the amphibian Xenopus luevis has been investigated. o(-MSH or its potent agonist 4-norleucine,7-D-phenylalanine-o-MSH has no effect on the release of radiolabeled POMC-derived peptides or immunoreactive @endorphin from superfused neurointermediate pituitary lobes. Melanin concentrating hormone, previously reported to have an u-MSH-like effect on melanophores, did not affect o.-MSH secretion. Neurointermediate lobe superfusate, which contains a mixture of POMC-derived peptides, failed to affect the secretory activity of melanotropes. It is concluded that in X. laevis the secretory activity of melanotropes is not under the control of short-term autofeedback mechanisms involving (Y-MSH or other POMC-derived peptides.

General and Comparative Endocrinology, 2002

Light and temperature stimuli act via various brain centers and neurochemical messengers on the p... more 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.

Endocrinology, 2011

We tested whether double cortin-like kinase-short (DCLK-short), a microtubule-associated Ser/Thr ... more We tested whether double cortin-like kinase-short (DCLK-short), a microtubule-associated Ser/Thr kinase predominantly expressed in the brain, is downstream of the ERK signaling pathway and is involved in proopiomelanocortin gene (POMC) expression in endocrine pituitary melanotrope cells of Xenopus laevis. Melanotropes form a well-established model to study physiological aspects of neuroendocrine plasticity. The amphibian X. laevis adapts its skin color to the background light intensity by the release of α-MSH from the melanotrope cell. In frogs on a white background, melanotropes are inactive but they are activated during adaptation to a black background. Our results show that melanotrope activation is associated with an increase in DCLK-short mRNA and with phosphorylation of DCLK-short at serine at position 30 (Ser-30). Upon cell activation phosphorylated Ser-30-DCLK-short was translocated from the cytoplasm into the nucleus, and the ERK blocker U0126 inhibited this process. The mu...