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It is well-known that alpha-melanophore-stimulating hormone (alpha-MSH) release from the amphibian pars intermedia (PI) depends on the light condition of the animal's background, permitting the animal to adapt the colour of its skin to background light intensity. In the present study, we carried out nine experiments on the effect of low temperature on this skin adaptation process in the toad Xenopus laevis, using the skin melanophore index (MI) bioassay and a radioimmunoassay to measure skin colour adaptation and alpha-MSH secretion, respectively. We show that temperatures below 8 degrees C stimulate alpha-MSH secretion and skin darkening, with a maximum at 5 degrees C, independent of the illumination state of the background. No significant stimulatory effect of low temperature on the MI and alpha-MSH plasma contents was noted when the experiment was repeated with toads from which the neurointermediate lobe (NIL) had been surgically extirpated. This indicates that low temperature stimulates alpha-MSH release from melanotrope cells located in the PI. An in vitro superfusion study with the NIL demonstrated that low temperature does not act directly on the PI. A possible role of the central nervous system in cold-induced alpha-MSH release from the PI was tested by studying the hypothalamic expression of c-Fos (as an indicator for neuronal activity) and the coexistence of c-Fos with the regulators of melanotrope cell activity, neuropeptide Y (NPY) and thyrotrophin-releasing hormone (TRH), using double fluorescence immunocytochemistry. Upon lowering temperature from 22 degrees C to 5 degrees C, in white-adapted animals c-Fos expression decreased in NPY-producing suprachiasmatic-melanotrope-inhibiting neurones (SMIN) in the ventrolateral area of the suprachiasmatic nucleus (SC) but increased in TRH-containing neurones of the magnocellular nucleus. TRH is known to stimulate melanotrope alpha-MSH release. We conclude that temperatures around 5 degrees C inactivate the SMIN in the SC and activate TRH-neurones in the magnocellular nucleus, resulting in enhanced alpha-MSH secretion from the PI, darkening the skin of white-adapted X. laevis.

Brain-derived neurotrophic factor (BDNF) is involved as an autocrine factor in the regulation of the secretory activity of the neuroendocrine pituitary melanotrope cells of Xenopus laevis. We studied the subcellular distribution of BDNF in Xenopus melanotropes using a combination of high-pressure freezing, cryosubstitution and immunoelectron microscopy. Presence of BDNF, pro-opiomelanocortin (POMC) and alpha-melanophore-stimulating hormone (alphaMSH) within melanotrope secretory granules was studied by triple-labelling immunoelectron microscopy. In addition, intracellular processing of BDNF was investigated by quantifying the number of immunogold particles in different stages of secretory granule maturation, in animals adapted to black or white background light conditions. The high-pressure freezing technique provides excellent preservation of both cellular ultrastructure and antigenicity. BDNF coexists with POMC and alphaMSH within secretory granules. BDNF-immunoreactivity increases along the secretory granule maturation axis (i.e. from electron-dense, via moderately electron-dense, to electron-lucent secretory granules). Immature, low immunoreactive, electron-dense secretory granules are assumed to contain mainly or even exclusively proBDNF. Strongly immunoreactive electron-lucent secretory granules represent the mature granule stage in which proBDNF has been processed to mature BDNF. Furthermore, in moderately electron-dense secretory granules, immunoreactivity is markedly (+79%) higher in black-adapted than in white-adapted animals, indicating that stimulation of melanotrope cell activity by the black background condition speeds up processing of BDNF from its precursor in this granule stage. It is concluded that, in the Xenopus melanotrope, BDNF biosynthesis and processing occur along the secretory granule maturation axis, together with that of POMC-derived alphaMSH, and that the environmental light condition not only controls the biosynthesis and secretion of BDNF and of POMC end-products, but also regulates the rate of their intragranular processing.

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.

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., ((c) 2002 Elsevier Science (USA).)

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., (Copyright 2001 Wiley-Liss, Inc.)

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.

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.

The toad Xenopus laevis is able to adapt its skin color to background light intensity. In this neuroendocrine reflex, the proopiomelanocortin (POMC)-derived peptide alpha-melanophore-stimulating hormone (alphaMSH) is a key regulatory factor. In animals adapting to a black background, release of alphaMSH from the pituitary pars intermedia causes dispersal of melanin in skin melanophores. To investigate the long-term in vivo dynamics of alphaMSH production during black background adaptation, the biosynthetic rate of POMC and the contents of POMC, alphaMSH and the POMC processing enzyme precursor convertase 2 (PC2) have been studied in the pars intermedia using pulse-labeling, Western blot and radioimmunoassay. In control animals, adapted to a white background, the rate of POMC biosynthesis and the POMC content were low, while high alphaMSH and PC2 contents were found. After 1 week of adaptation to a black background, the rate of POMC biosynthesis and the POMC protein content had increased 19- and 3.7-fold respectively. These parameters attained a maximum level (28- and 5. 8-fold higher than control) after 3 weeks and remained at these elevated levels for at least 12 weeks. After 1 week, the pars intermedia content of alphaMSH was only 30% of the control level, but after 6 and 12 weeks, the alphaMSH level had increased to the control level. The PC2 content decreased to 52% of control after 1 week and stabilized after 3 weeks at a level slightly lower than the control value. The results show that during long-term background adaptation a steady-state situation is reached, with a balance between the biosynthesis, enzymatic processing and release of alphaMSH. The in vivo dynamics of the processing enzyme PC2 suggest a parallel storage and release of alphaMSH and mature PC2 in the Xenopus pituitary pars intermedia.

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.

In the studies on Xenopus laevis presented here, the role of acetylcholine (ACh) in the regulation of pituitary activity was examined. The results are discussed in connection with other results on cholinergic regulation of pituitary functioning in vertebrates. It is demonstrated that dissociated melanotropes can synthesize ACh in vitro. In addition, immunocytochemical studies show the presence of the ACh-synthesizing enzyme choline acetyltransferase (ChAT) in the cytoplasm of melanotrope cells. ACh concentration-dependently raises the intracellular free calcium concentration as well as the release of proopiomelanocortin (POMC)-derived peptides. Selective muscarinic receptor antagonists showed that the actions of ACh are mediated through an M1-subtype muscarinic receptor. Immunofluorescence cytochemistry visualized muscarinic receptors on the surface of melanotrope cells. It is concluded that ACh stimulates the release of POMC-derived peptides from the Xenopus melanotrope cell in an autocrine, excitatory way. Finally, the possible mechanism through which ACh is released from melanotrope cells and the physiological significance of ACh in the pituitary gland are discussed.

The stimulatory effect of thyrotropin-releasing hormone (TRH) on alpha-melanocyte stimulating hormone (MSH) secretion from the frog pars intermedia is mediated through the phospholipase C (PLC) pathway but requires extracellular Ca2+. The aim of the present study was to investigate the respective contribution of extracellular and intracellular Ca2+ in the action of TRH on cytosolic calcium concentration ([Ca2+]i) and alpha-MSH release. In normal conditions, TRH (10(-7) M; 5 s) evoked two types of Ca2+ responses: in 63% of the cells, TRH caused a sustained and biphasic increase in [Ca2+]i while in 37% of the cells, TRH only induced a transient response. In the presence of EGTA or Ni2+, the stimulatory effect of TRH on [Ca2+]i and alpha-MSH secretion was totally suppressed. Nifedipine (10(-6) M) reduced by approximately 50% the amplitude of the two types of Ca2+ responses whereas omega-conotoxin GVIA (10(-7) M) suppressed the plateau-phase of the sustained response indicating that the activation of L-type Ca2+-channels (LCC) is required for initiation of the Ca2+ response while N-type Ca2+-channels (NCC) are involved in the second phase of the response. Paradoxically, neither nifedipine nor omega-conotoxin GVIA had any effect on TRH-induced alpha-MSH secretion. The PLC inhibitor U-73122 (10(-6) M) significantly reduced the transient increase in [Ca2+]i and totally suppressed the sustained phase of the Ca2+ response but had no effect on TRH-induced alpha-MSH secretion. The stimulatory effect of TRH on PLC activity was not effected by nifedipine and omega-conotoxin GVIA but was abolished in Ca2+-free medium. Ryanodine had no effect on the TRH-induced stimulation of [Ca2+]i and alpha-MSH secretion. Concomitant administration of nifedipine/omega-conotoxin GVIA or U-73122/omega-conotoxin GVIA markedly reduced the response to TRH but did not affect TRH-evoked alpha-MSH release. In contrast, concomitant administration of U-73122 and nifedipine significantly reduced the effect of TRH on both [Ca2+]i and alpha-MSH release. Taken together, these data indicate that, in melanotrope cells, activation of TRH receptors induces an initial Ca2+ influx through nifedipine- and omega-conotoxin-insensitive, Ni2+-sensitive Ca2+-channels which subsequently activates LCC and causes Ca2+ mobilization from intracellular pools by enhancing PLC activity. Activation of the PLC causes Ca2+ entry through NCC which is responsible for the plateau-phase of sustained Ca2+ response. Although nifedipine and U-73122, separately used, were devoid of effect on secretory response, Ca2+ entry through LCC and mobilization of intracellular Ca2+ are both involved in TRH-evoked alpha-MSH release because only one source of Ca2+ is sufficient for inducing maximal hormone release. In contrast, the Ca2+ influx through NCC does not contribute to TRH-induced alpha-MSH secretion.

The melanotrope cell of Xenopus laevis is innervated by nerve terminals that contain, among other transmitter substances, the neurotransmitter gamma-aminobutyric acid (GABA). Postsynaptically the melanotrope cell possess both GABAA and GABAB receptors. Activation of either receptor type leads to an inhibition of alpha MSH release from the cell. The present study concerns the functional significance of the existence of two types of GABA receptors on the melanotrope regarding two questions: 1) do the different receptor types have different effects on the melanotrope? and 2) can the endogenous ligand GABA differentially activate these receptors? Concerning the first question, we have tested the hypothesis that the GABAA receptor (a chloride ion channel) and the GABAB receptor (a G protein-coupled receptor negatively linked to adenylyl cyclase) may have differential effects on the sensitivity of the cell to stimulation by cAMP-dependent mechanisms. We show that treatments with either isoguvacine (GABAA agonist) or baclofen (GABAB agonist) inhibit intracellular Ca2+ oscillations and peptide secretion from melanotrope cells. Treatments known to increase intracellular cAMP in the melanotrope (e.g. use of the peptide sauvagine or the cAMP analog 8-bromo-cAMP) completely overcame the inhibition induced by baclofen, but not that caused by isoguvacine. We conclude that the GABAA and GABAB receptors have different effects on the Xenopus melanotrope cell by differentially affecting the sensitivity the cell shows to stimulation by cAMP-dependent mechanisms. Concerning possible differential activation of the receptor types, we found that we could use a membrane potential probe (from the bis-oxonol family) to differentiate between GABAA and GABAB receptor activation. Using this probe we showed that low GABA concentrations (< 10(-7) M) give a response indicative of the GABAB receptor, whereas at high GABA concentrations (> 10(-7) M), the GABAA receptor response predominates. We, therefore, conclude that GABA can differentially activate the two types of GABA receptors on the Xenopus melanotrope cell.

In the South African clawed toad Xenopus laevis, background adaptation is regulated by alpha MSH, a POMC-derived peptide. After transfer of the animal from a black to a white background, secretion of alpha MSH from the intermediate pituitary lobe is inhibited by the hypothalamic neurotransmitter neuropeptide Y (NPY). The neurointermediate lobe in vitro is also subject to inhibitory regulation by dopamine and gamma-aminobutyric acid (GABA). In the nerve terminals contacting the intermediate lobe of the pituitary, GABA is contained in electron-lucent vesicles, whereas dopamine and NPY coexist in electron-dense vesicles. To study the role of these secreto-inhibitors in the regulation of POMC biosynthesis, the rate of incorporation of radioactive amino acids into POMC protein was determined after in vitro treatment of the neurointermediate pituitary with NPY, apomorphine (dopamine D2 receptor agonist), isoguvacine (GABAA receptor agonist) and baclofen (GABAB receptor agonist). After 24 h of treatment, inhibition of POMC biosynthesis by NPY and apomorphine was 77% and 74%, respectively. Isoguvacine treatment resulted in an inhibition of 59%, whereas no significant effect of baclofen was observed. When neurointermediate lobes were treated for 3 days, inhibition of POMC biosynthesis by NPY was maintained, and inhibition by apomorphine was even stronger, whereas isoguvacine gave an inhibition of 52%, and baclofen produced 34% inhibition. Superfusion experiments on alpha MSH secretion showed that prolonged treatment with the GABA receptor agonists results in a desensitization of GABA receptor-mediated signal transduction mechanisms, whereas the NPY receptor does not show desensitization. The observations indicate differential actions of the secreto-inhibitors NPY, apomorphine, and GABA agonists on POMC biosynthesis in the Xenopus intermediate pituitary, suggesting a major role for dopamine and NPY, whereas GABA, acting via two receptor types, does not seem to have a major function in long term control of POMC biosynthesis.

Cyclic-AMP is known to be released from cells and tissues and the amounts released have been reported to reflect intracellular cAMP levels. To measure cAMP release the phosphodiesterase inhibitor IBMX is often used to increase the amount of cAMP to a detectable level. Using this method to follow cAMP dynamics of melanotrope cells in the neurointermediate pituitary lobe of the amphibian Xenopus laevis we show that the alpha-MSH secreto-inhibitors baclofen (GABAB receptor agonist) and dopamine inhibit cAMP release, confirming the idea that these factors inhibit alpha-MSH secretion by reducing adenylyl cyclase activity. Using a sensitive cAMP radioimmunoassay we were able to measure cAMP release from Xenopus neurointermediate lobes in the absence of IBMX. Both baclofen and dopamine appeared to inhibit alpha-MSH secretion but strongly stimulated the release of cAMP. This indicates that the extracellular cAMP level is not a reliable parameter to measure the intracellular cAMP level in the absence of IBMX. The data furthermore suggest that cAMP release is a physiologically regulated process, which might be involved in lowering intracellular cAMP levels associated with a cellular secretory compartment. No apparent differences could be found in the lobe content of cAMP at the termination of secreto-inhibitor treatment, leading to the idea that the cAMP compartment associated with secretion in small relative to the total amount of cAMP present in the lobe.

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 [Leu31,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 [Leu31,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 Y1-like receptor.

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 alpha-melanophore-stimulating hormone (alpha-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 Ca(2+)-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 microM omega-conotoxin. The second type was a non-inactivating current, which was blocked up to 50% by 1 microM nifedipine, indicating its L-type nature. Only a small component of this inactivating current could be blocked by omega-conotoxin. No evidence was found for the presence of transient, low-voltage activated currents. The spontaneous secretion of alpha-MSH from superfused neurointermediate lobes was dependent on extracellular calcium, as low calcium conditions (10(-4)-10(-8) M) rapidly inhibited this process. Under these conditions, secretion was not affected by depolarizing concentrations of potassium chloride. The calcium ionophore A23187 increased secretion under low calcium conditions, but had no effect on spontaneous alpha-MSH release. These results suggest that spontaneous alpha-MSH release depends on influx of calcium through voltage-operated calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

The amphibian Xenopus laevis is able to adapt the colour of its skin to the light intensity of the background, by releasing alpha-melanophore-stimulating hormone from the pars intermedia of the hypophysis. In this control various inhibitory (dopamine, gamma-aminobutyric acid, neuropeptide Y, noradrenaline) and stimulatory (thyrotropin-releasing hormone and corticotropin-releasing hormone) neural factors are involved. Dopamine, gamma-aminobutyric acid and neuropeptide Y are present in suprachiasmatic neurons and co-exist in synaptic contacts on the melanotrope cells in the pars intermedia, whereas noradrenaline occurs in the locus coeruleus and noradrenaline-containing fibres innervate the pars intermedia. Thyrotropin-releasing hormone and corticotropin-releasing hormone occur in axon terminals in the pars nervosa. In the present study, the neuronal origins of these factors have been identified using axonal tract tracing. Application of the tracers 1,1'dioctadecyl-3,3,3',3' tetramethyl indocarbocyanine and horseradish peroxidase into the pars intermedia resulted in labelled neurons in two brain areas, which were immunocytochemically identified as the suprachiasmatic nucleus and the locus coeruleus, indicating that these areas are involved in neural inhibition of the melanotrope cells. Thyrotropin-releasing hormone and corticotropin-releasing hormone were demonstrated immunocytochemically in the magnocellular nucleus. This area appeared to be labelled upon tracer application into the pars nervosa. This finding is in line with the idea that corticotropin-releasing hormone and thyrotropin-releasing hormone stimulate melanotrope cell activity after diffusion from the neural lobe to the pars intermedia. After anterograde filling of the optic nerve with horseradish peroxidase, labelled axons were traced up to the suprachiasmatic area where they showed to be in contact with suprachiasmatic neurons. These neurons showed a positive reaction with anti-neuropeptide Y and the same held for staining with anti-tyrosine hydroxylase. It is suggested that a retino-suprachiasmatic pathway is involved in the control of the melanotrope cells during the process of background adaptation.

The secretion of alpha-melanophore-stimulating hormone (alpha-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 Ca2+ oscillations. In order to gain a better understanding of the external regulation of intracellular Ca2+ ([Ca2+]i) in the melanotrope cell, we have examined the action of well known alpha-MSH secretagogues on the Ca2+ oscillations. It is shown that all secretagogues tested also control the oscillatory state of Xenopus melanotropes, that is, the secreto-inhibitors dopamine, isoguvacine (gamma-aminobutyric acid, GABAA agonist), baclofen (GABAB agonist) and neuropeptide Y evoked a rapid quenching of the spontaneous Ca2+ 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 Ca2+ 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 Ca2+ 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.

Placing the clawed toad Xenopus laevis on a black background stimulates the melanotrope cells in the pars intermedia of the pituitary gland to release proopiomelanocortin (POMC)-derived peptides, including alpha-MSH and N-acetyl-beta-endorphin. In this study three types of secretory granules, electron-dense (approximately 130 nm phi), moderately electron-dense (approximately 160 nm phi) and electron-lucent (approximately 180 nm phi), have been identified in these cells. Apparently, only dark granules are formed by the Golgi apparatus and lucent granules release their contents via exocytosis. Immuno-electron microscopy (immunogold double labelling) of glutaraldehyde-fixed and freeze-substituted material shows that desacetyl-alpha-MSH and N-acetyl-beta-endorphin coexist in all three granule types. Quantification of immunostaining revealed that immunoreactivities to these peptides are lowest in the dark granules and highest in the light ones. It is proposed that intragranular processing of POMC to immunoreactive desacetyl-alpha-MSH and N-acetyl-beta-endorphin involves an increase in granule size and a decrease in granule electron density. Black background-induced activation of the melanotrope cell is reflected by an increase in immunoreactivity of the secretory granules to each of the antisera. This suggests that cell activation stimulates the formation of peptides by intragranular processing of POMC and/or of intermediate POMC-processing products. In addition, cell activation evoked an increase in the percentage of the granule population that reacts with anti-N-acetyl-beta-endorphin, probably by stimulating intragranular acetylation of beta-endorphin. Apparently, this acetylation is a regulated event that occurs in the cytoplasm, independently from the acetylation of desacetyl-alpha-MSH which takes place near the plasmalemma at the time of granule exocytosis.

The effects of GABAa receptor activation on the secretion of alpha-melanocyte-stimulating hormone (alpha-MSH) from the superfused pars intermedia of the amphibian Xenopus laevis were examined. The GABAa receptor agonist isoguvacine inhibited secretion of alpha-MSH from the pars intermedia, an action completely antagonized by the chloride channel blocker picrotoxin. Isoguvacine stimulated secretion from picrotoxin-treated tissue. Both effects were blocked by the GABAA receptor antagonist bicuculline, indicating that it is a specific action via the GABAA receptor. We conclude that, besides the inhibitory chloride channel, there is a stimulatory signaling property associated with the GABAA receptor. Isoguvacine stimulated the production of c-AMP, an action that was not blocked by picrotoxin. This suggests that the stimulatory mechanism of the GABAA receptor involves, directly or indirectly, the generation of c-AMP.

The activity of many endocrine cells is regulated by gamma-aminobutyric acid (GABA). The effects of GABA are mediated by GABAA and/or GABAB receptors. While GABAB receptors in the central nervous system have now been extensively characterized, little is known of the function and pharmacology of GABAB receptors on endocrine cells. In the amphibian Xenopus laevis, GABA inhibits the release of alpha MSH from the endocrine melanotrope cells through both GABAA and GABAB receptors. We have investigated the following aspects of the GABAB receptor of the melanotrope cells of X. laevis: 1) the pharmacology of this receptor, using antagonists previously established to demonstrate GABAB receptors in the mammalian central nervous system; 2) the relative contribution to the regulation of hormone secretion by the GABAA and GABAB receptors on melanotrope cells in vitro; and 3) the role of the GABAB receptor with respect to the physiological function of the melanotrope cell in vivo, i.e. regulation of pigment dispersion in skin melanophores in relation to background color. Our results demonstrate that phaclofen, 2-hydroxysaclofen, and 4-aminobutylphosphonic acid dose-dependently blocked the inhibition of alpha MSH release by GABAB receptor activation, but not by GABAA receptor activation. The GABAB receptor antagonist delta-aminovaleric acid appeared to be a selective agonist on the GABAB receptor of melanotrope cells. The inhibitory secretory response to a low dose of GABA (10(-5) M) was not affected by bicuculline, but was significantly reduced by phaclofen, indicating that at a low GABA concentration, the GABAB receptor mechanism would dominate in inhibiting the melanotrope cells. Different thresholds of activation may form the basis for differential action of GABA through both GABA receptor types. The tonic inhibition of alpha MSH release in animals adapted to a white background was not affected by 4-aminobutylphosphonic acid, indicating that the GABAB receptor is not (solely) involved in the in vivo inhibition of alpha MSH release in animals on a white background.

The secretion of alpha-MSH from the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis is under complex neural control. Three neurotransmitters, dopamine, GABA and NPY, coexist in nerve terminals that contact the melanotrope cells. All three neurotransmitters inhibit alpha-MSH release. We have investigated the significance of this neurotransmitter coexistence for the regulation of alpha-MSH release, using an in vitro superfusion system. From experiments where lobes were treated with various combinations of receptor agonists we conclude that the transmitters act in an additive way but have clear, differential actions. Inhibition of secretion by either dopamine, isoguvacine (GABAA receptor agonist) or baclofen (GABAB receptor agonist) occurs rapidly and alpha-MSH secretion rapidly returns when treatment is terminated (recovery from baclofen being relatively fast, that from dopamine relatively slow); in contrast, inhibition by NPY and recovery from NPY-induced inhibition occurs only very slowly. Differential effects of the transmitters were also seen in experiments with 8-bromo-cyclic AMP, which strongly stimulates alpha-MSH secretion from isoguvacine- or baclofen-treated lobes, but is relatively ineffective in stimulating secretion from lobes treated with dopamine or NPY. NPY, furthermore, enables a short phasic stimulation of secretion by isoguvacine and attenuates the inhibitory action of dopamine and baclofen. Altogether it is concluded that the coexisting factors differentially affect the secretory process of the melanotrope cells of Xenopus laevis. NPY has a slow, sustained action whereas dopamine and GABA act fast.

This quantitative ultrastructural immunocytochemical study demonstrates the coexistence of a catecholamine [dopamine (DA)], an amino acid (GABA), and a neuropeptide [neuropeptide Y (NPY)] in axon varicosities innervating the pars intermedia of Xenopus laevis. The varicosities are assumed to control the pars intermedia melanotrope cells, which regulate skin color during the physiological process of background adaptation. Varicosity profiles appear to abut melanotrope cells and folliculostellate cells, star-shaped cells that intimately contact the melanotropes. All varicosity profiles contain two morphological types of vesicle. Monolabeling studies on routinely fixed and freeze-substituted tissues showed that the small, electron-lucent vesicles store GABA, whereas DA and NPY occur in larger, electron-dense ones. Double and triple labeling experiments, in which the degree of immunoreactivity was quantified per varicosity profile and per vesicle, led to the conclusion that (1) DA, GABA, and NPY coexist within almost all varicosity profiles and (2) DA and NPY are costored within electron-dense vesicles. Varicosity profiles that about melanotrope cells show a much higher ratio between the numbers of electron-lucent and electron-dense vesicles than varicosities contacting folliculostellate cells (15.8 and 3.3, respectively). This differential distribution is in line with the previous demonstration that, in contrast to GABA, NPY does not act directly on the melanotrope cells but indirectly, by controlling the activity of the folliculostellate cells.

An important factor in regulating secretion from endocrine cells is the cytoplasmic concentration of cyclic-AMP. Many regulatory substances are known to either stimulate or inhibit the production of this second messenger through activation of their receptors. In the present study, we have monitored changes in cyclic-AMP efflux from melanotrope cells of Xenopus laevis in response to established neurochemical regulators of alpha-MSH secretion. In vitro superfusion of neurointermediate lobes allows for a dynamic recording of cyclic-AMP production in relation to hormone secretion. Unlike alpha-MSH secretion, the efflux of cyclic-AMP was not dependent on the concentration of extracellular calcium, indicating that hormone release and cyclic-AMP efflux are mediated by different mechanisms. The phosphodiesterase inhibitor IBMX and the adenylate cyclase activator forskolin stimulated cyclic-AMP efflux, but had no stimulatory effect on alpha-MSH release. This indicates that an increase in cyclic-AMP production in melanotrope cells is not necessarily accompanied by an increase in the rate of alpha-MSH release. Corticotropin-releasing factor stimulated cyclic-AMP efflux with dynamics similar to that induced by the amphibian peptide sauvagine. Dopamine and the GABAB receptor agonist baclofen both inhibited cyclic-AMP efflux and alpha-MSH release, with similar dynamics of inhibition and similar dose-response relationships. It is proposed that an inhibition of cyclic-AMP efflux is coupled to an inhibition of alpha-MSH secretion.

The melanotrope cells in the pars intermedia in the pituitary of Xenopus laevis synthesize and release the melanophore-stimulating hormone (alpha MSH), a small peptide that causes skin darkening during the process of background adaptation. Evidence has been found for a heterogeneity in biosynthetic activity of the melanotrope cells. In the present study two questions were addressed: (1) does the melanotrope cell population also show heterogeneous alpha MSH-release, and (2) can this heterogeneity be changed by extracellular messengers? Since dopamine is known to inhibit alpha MSH-release, this messenger is used to study the regulation of the heterogeneity. To quantify alpha MSH-release from individual cells, a cell blotting procedure has been developed for the binding and relative quantification of the small alpha MSH peptide. The immunoblotting procedure involves binding of the cells to a carrier slide and binding of released alpha MSH to a nitrocellulose filter. After immunostaining, the amount of alpha MSH per cell was quantitated by image analysis. Untreated melanotrope cells reveal a distinct variability in alpha MSH-release, some cells showing low secretory activity, whereas others are strongly secreting, indicating heterogeneity of alpha MSH-release. Dopamine treatment strongly inhibits alpha MSH-release from individual cells, resulting in a clearly less pronounced melanotrope cell heterogeneity. The effect of dopamine appears to be dose-dependent as a low dopamine concentration has only a moderate effect on the alpha MSH-release. It is proposed that dopamine is a physiological regulator of the degree of melanotrope cell heterogeneity in alpha MSH-release.

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.

7B2 is a highly conserved protein present in many secretory cells. Using in situ hybridization techniques and immunocytochemistry, parameters concerning the biosynthesis and storage of the 7B2 protein were studied in the pituitary gland and median eminence of the clawed toad Xenopus laevis, in relation to the physiological process of background adaptation. 7B2-like immunoreactivity was present in the median eminence, in the neural and anterior pituitary lobes and, particularly, in the melanotrope cells of the intermediate pituitary lobe. In these cells, it coexisted with immunoreactivity to proopiomelanocortin (POMC)-derived alpha-melanocyte stimulating hormone (alpha MSH). The melanotropes of black-adapted animals had abundant 7B2-mRNA and POMC-mRNA; melanotropes of white-adapted toads had only low levels of these mRNAs. The presence of 7B2 in nerve terminals and endocrine cells supports the idea that the protein has a general function in the cellular secretory process. In X. laevis, 7B2 appears to be particularly associated with POMC and alpha MSH and, therefore, may play a role in the regulation of background adaptation.