Brain-derived neurotrophic factor stimulates growth of pituitary melanotrope cells in an autocrine way

Brain-derived neurotrophic factor (BDNF) is expressed in the mammalian pituitary gland, in both the anterior and intermediate lobes, where its functional significance is unknown. Melanotrope cells in the intermediate pituitary lobe of the amphibian Xenopus laevis also produce BDNF, which co-exists in secretory granules with α-melanophore-stimulating hormone (α-MSH), a peptide that causes pigment dispersion in dermal melanophores during adaptation of the toad to a dark background. Xenopus melanotropes are highly plastic, undergoing very strong growth to support the high biosynthesis and release of α-MSH in black-adapted animals. In this study we have tested our hypothesis that this enhanced growth of the melanotrope is maintained by autocrine release of BDNF. Furthermore, since the extracellular-regulated kinase (ERK) pathway is a major component of BDNF signaling in neuronal plasticity, we investigated its involvement in melanotrope cell growth. For these purposes melanotropes were treated for 3 days in vitro, with either an anti-BDNF serum or a recombinant tropomyosin-receptor kinase B (TrkB) receptor fragment to eliminate released BDNF, or with the ERK inhibitor U0126. We also applied a novel inhibitor of the TrkB receptor, cyclotraxin-B, to test this receptor’s involvement in melanotrope cell growth regulation. All treatments markedly reduced melanotrope cell growth. Therefore, we conclude that autocrine release of BDNF and subsequent TrkB-dependent ERK-mediated signaling is important for melanotrope cell growth during its physiologically induced activation.     

Light modulates the melanophore response to α-MSH in Xenopus laevis: An analysis of the signal transduction crosstalk mechanisms involved

Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or α-MSH. We have previously demonstrated that the initial biochemical steps of light and α-MSH signaling are distinct, since the increase in cAMP observed in response to α-MSH was not seen after light exposure. cAMP concentrations in response to α-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence of an adenylyl cyclase (AC) in the Xenopus melanophore, similar to the mammalian type IX which is inhibited by Ca²⁺-calmodulin-activated phosphatase. This finding supports the hypothesis that the cyclase could be negatively modulated by a light-promoted Ca²⁺ increase. In fact, the activity of calcineurin PP2B phosphatase was increased by light, which could result in AC IX inhibition, thus decreasing the response to α-MSH. St-Ht31, a disrupting agent of protein kinase A (PKA)-anchoring kinase A protein (AKAP) complex totally blocked the melanosome dispersing response to α-MSH, but did not impair the photo-response in Xenopus melanophores. Sequence comparison of a melanophore AKAP partial clone with GenBank sequences showed that the anchoring protein was a gravin-like adaptor previously sequenced from Xenopus non-pigmentary tissues. Co-immunoprecipitation of Xenopus AKAP and the catalytic subunit of PKA demonstrated that PKA is associated with AKAP and it is released in the presence of α-MSH. We conclude that in X. laevis melanophores, AKAP12 (gravin-like) contains a site for binding the inactive PKA thus compartmentalizing PKA signaling and also possesses binding sites for PKC. Light diminishes α-MSH-induced increase of cAMP by increasing calcineurin (PP2B) activity, which in turn inhibits adenylyl cyclase type IX, and/or by activating PKC, which phosphorylates the gravin-like molecule, thus destabilizing its binding to the cell membrane.     

Pituitary and melanophore responses to background in poecilia latipinna (Teleostei): Role of the pars intermedia PAS cell

Fish were adapted to black (B) or white (W) backgrounds for 28 days, or moved between B and W for shorter periods. Compared to W, B induced a darker dorsal surface, the recruitment of new ventral melanophores, and the dispersion of melanin in denervated caudal fin melanophores. These responses were identical in fish adapted to seawater (SW), one-third seawater ($\text{1}\text{3}$ SW), or fresh water (FW). The effects of B were abolished (melanophore recruitment, melanin dispersion) or attenuated (dorsal darkening) by hypophysectomy. B did not affect pituitary MSH content or plasma cortisol levels. Examination of the pituitary showed that while the prolactin cells were activated by reduced salinity, only the pars intermedia PAS-positive cell (PIPAS cell) was activated by B, the PI melanotrope (PIPbH cell) and all the pars distalis cells being similar in structure on B and W. Neither the PIPAS nor the PIPbH cell was affected by the different salinities. The neurohypophysis showed no response to background. It is concluded that the melanophore responses to B are mediated by an unknown hormone secreted by the PIPAS cell, and reasons are adduced for believing that prolactin, ACTH, and MSH are not involved in these responses.     

Social behavior, chemical communication, and adult neurogenesis: studies of scent mark function in Podarcis wall lizards

Our previous studies showed that in barfin flounder, α-melanocyte-stimulating hormone (α-MSH) stimulates pigment dispersion in xanthophores, while it shows negligible effects in melanophores. The present study was undertaken to evaluate whether these results are limited to barfin flounder by using Japanese flounder. Three subtypes of proopiomelanocortin gene encoding melanocortins (MCs) were expressed in the Japanese flounder pituitary, one of which was also expressed in the skin. Expression of melanocortin 5 receptor gene (Mc5r) was observed in isolated xanthophores, while that of Mc1r and Mc5r was found in melanophores. In the xanthophores of Japanese flounder skin, α-MSH as well as desacetyl (Des-Ac)-α-MSH and diacetyl (Di-Ac)-α-MSH exhibited dose-dependent pigment-dispersing activities, indicating that the signals of α-MSH-related peptides were mediated by MC5R. On the other hand, α-MSH did not stimulate pigment dispersion in melanophores, while Des-Ac-α-MSH and Di-Ac-α-MSH did, thus indicating that the expression of two different types of Mcr is related to the decrease in α-MSH activity. Thus, the molecular repertoire in MC system observed in Japanese flounder is similar to that in barfin flounder. Moreover, the relationship between the pigment-dispersing activities of α-MSH-related peptides and the expression of Mcr subtypes in xanthophores and melanophores were also similar between Japanese flounder and barfin flounder. Consequently, we hypothesize that inhibition of α-MSH activity could be due to the formation of heterodimers comprising MC1R and MC5R, often observed in G-protein-coupled receptors.     

Dynamics of background adaptation in Xenopus laevis: role of catecholamines and melanophore-stimulating hormone

The pars intermedia of the pituitary gland in Xenopus laevis secretes alpha-melanophore-stimulating hormone (alpha-MSH), which causes dispersion of pigment in dermal melanophores in animals on a black background. In the present study we have determined plasma levels of alpha-MSH in animals undergoing adaptation to white and black backgrounds. Plasma values of black-adapted animals were high and decreased rapidly after transfer to a white background, as did the degree of pigment dispersion in dermal melanophores. Plasma MSH values of white-adapted animals were below the detection limit of our radioimmunoassay. Transfer of white animals to a black background resulted in complete dispersion of melanophore pigment within a few hours, but plasma MSH levels remained low for at least 24 hr. This discrepancy between plasma MSH and degree of pigment dispersion suggested the involvement of an additional factor for stimulating dispersion. Results of in vitro and in vivo experiments with receptor agonists and antagonists indicated that a beta-adrenergic mechanism, functioning at the level of the melanophore, is involved in the stimulation of pigment dispersion during the early stages of background adaptation.     

Analysis of the melanotrope cell neuroendocrine interface in two amphibian species, Rana ridibunda and Xenopus laevis: a celebration of 35 years of collaborative research

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.     

Endorphins supersensitize frog skin melanophores to isoproterenol, but subsensitize them to alpha-melanocyte-stimulating hormone

In view of known antagonisms between opiates and melanocyte-stimulating hormone (MSH) on nervous tissue, the effect of opiates on other MSH target cells, the melanophores of frog skin, has been studied. Reflectometry of isolated frog skin accompanied by microscopy was used to study melanophore responses. Although opiates had no darkening effect by themselves, endorphins greatly supersensitized dermal spot and epidermal melanophores to the melanosome-dispersing action of isoproterenol. The response to epinephrine was unaffected. The order of potency for supersensitization was [d-Ala²-d-Leu⁵]-enkephalin > [d-Ala²-MePhe⁴-Met-(O)⁵-ol]-enkephalin > β-endorphin > [Leu⁵]-enkephalin. Morphine, α-endorphin, and [d-Ala²-Met⁵]-enkephalinamide lacked this effect. The effect of [d-Ala²-d-Leu⁵]-enkephalin was blocked by propranolol or naloxone, indicating that both β-adrenoceptors and opiate receptors are required. The effect was obtained with two species of frog, Rana berlandieri and Xenopus laevis. If these results can be extended to other β-adrenergic responses, they may provide more information about the physiological and pathological roles of the endorphins. In contrast, endorphins, but not morphine, subsensitized R. berlandieri melanophores to α-MSH, as expected from the established antagonisms between morphine and MSH. Naloxone failed to eliminate this subsensitization, although it reduced it somewhat. Naloxone alone also had a small subsensitization effect. These results indicate that melanophores may possess opiate receptors, although there is as yet no direct evidence for this.     

Expression and physiological regulation of BDNF receptors in the neuroendocrine melanotrope cell of Xenopus laevis

Brain-derived neurotrophic factor (BDNF) and alpha-melanophore-stimulating hormone (alpha-MSH) are co-sequestered in secretory granules in melanotrope cells of the pituitary pars intermedia of the amphibian Xenopus laevis. alpha-MSH is responsible for pigment dispersion in dermal melanophores during the process of black-background adaptation. BDNF-production in melanotrope cells is increased by placing animals on a black background, and BDNF acts as an autocrine stimulatory factor on the melanotrope cells. However, the repertoire of possible neurotrophin receptors of the melanotrope is unknown. In this study we have established the expression of full length TrkB (TrkB.FL), truncated TrkB (TrkB.T) and p75(NTR) receptors in the Xenopus neurointermediate lobe by RT-PCR. In situ hybridization reveals the presence of TrkB.FL mRNA and p75(NTR) mRNA in melanotrope cells. Quantitative RT-PCR shows that in animals on a black background the amounts of TrkB.T and p75(NTR) mRNA are about three times higher than in white background-adapted animals. We suggest that the amount of p75(NTR) sets the sensitivity of the melanotrope cells for the stimulatory action of BDNF during physiological adaptation to background light intensity.     

Methionine enkephalin-induced changes in pigmentation of zebrafish (Cyprinidae, Brachydanio rerio) and related species and varieties, measured videodensitometrically. I. Zebrafish

The ability of MSH and of methionine enkephalin (met-E) to induce dispersion of pigment granules was examined in melanophores and in xanthophores of the zebrafish Brachydanio rerio using the melanophore index (MI) and videodensitometry. Both methods gave similar results. In B. rerio both MSH and met-E induced pigment dispersion in dermal melanophores, in fin and peritoneal melanophores, and in xanthophores. Darkening lasted a few hours. However, met-E-induced darkening developed 40-50 min later and faded more slowly than the effect of MSH. Both effects were dose related. Naloxone prevented met-E-induced darkening while it did not interfere with the MSH-induced effect. Epidermal melanophores did not react to either MSH or met-E. Thus met-E proved to induce changes of coloration when injected into a fish. Our data suggest a central mechanism involved in met-E-mediated change of coloration in zebrafish under the conditions examined. A new approach was suggested for objective measurement of the mean body darkness of the fish with the help of computational videodensitometry. Our fist results indicate a proportionality between the MI evaluation and videodensitometry.     

Influence of the epithalamic brain region of amphibian chromatophore behavior.

The possible influence of the epithalamic area on melanophore-stimulating hormone (MSH) release was investigated in Rana berlandieri forreri (Rana pipiens, sensu lato). Electrical stimulation of a specific region on the diencephalic roof resulted in a reversible darkening of skin melanophores. The latency between stimulation and onset of melanophore dispersion averaged from 1 to 3 min. Hypophysectomy, however, prevented the electrical stimulation-induced melanophore response from occurring. Thus, a neuronal, humoral, or integrated neuroendocrine pathway apparently develops from the diencephalon, in proximity to the pineal-subcommissural complex, to regulate pars intermedia MSH secretion.     

The effects of melanophore-stimulating hormone and cyclic nucleotides on teleost fish chromatophores

The effects of melanophore-stimulating hormone (MSH) and cyclic nucleotides on pigment translocation within the isolated scale chromatophores of the medaka, Oryzias latipes, were examined in relation to the second-messenger hypothesis. In leucophores, MSH, cyclic AMP, dibutyryl cyclic AMP (DBcAMP), and methylxanthines brought about a rapid pigment dispersal. β-Adrenergic blockers completely abolished methylxanthine-mediated dispersion without inhibiting the response induced by MSH or DBcAMP. In melanophores and xanthophores, MSH did not accelerate pigment dispersion. Dibutyryl cyclic GMP (DBcGMP) was more effective than cyclic AMP or DBcAMP in producing pigment dispersal. In the presence of DBcGMP, epinephrine was incapable of stimulating pigment aggregation in melanophores whereas the presence of DBcAMP did not interfere with the epinephrine-induced aggregation. The results implicate the involvement of the adenylate cyclase-cyclic AMP system in the regulation of leucophore response while in melanophores and xanthophores, cyclic GMP appears to mediate pigment displacement.     

Effect of adenosine 3',5'-monophosphate on melanin dispersion in the shore crab Carcinus maenas (L)

The mode of action of the melanin-dispersing hormone (MDH) in the shore crab Carcinus maenas was studied. The dose-response relationship for this hormone obtained from the sinus gland was compared to the response of the melanophores to injected adenosine 3′,5′-monophosphate (cyclic AMP). In agreement with data reported on the effect of cyclic AMP on amphibian melanophores, millimolar concentrations of this nucleotide were required to cause melanin dispersion in the crab. Although the activity of adenyl cyclase in epidermal preparations of the shore crab was increased about 8-fold when fresh sinus gland extracts were added to the incubation medium, this stimulation does not appear to be related to the MDH activity of these extracts. These findings are discussed with reference to the concept that the action of MDH on the melanophore may be mediated by cyclic AMP.     

Kinesin is responsible for centrifugal movement of pigment granules in melanophores.

Kinesin is a mechanochemical ATPase that induces translocation of latex beads along microtubules and microtubule gliding on a glass surface. This protein is thought to be a motor for the movement of membranous organelles in cells. Recently Hollenbeck and Swanson [Hollenbeck, P. J. & Swanson, J. A. (1990) Nature (London) 346, 864-866] showed that kinesin is involved in the positioning of tubular lysosomes in macrophages. However, the role of this protein in the movement of organelles was not yet clear. We used a polyclonal antibody against the kinesin heavy chain that inhibited kinesin-dependent microtubule gliding in vitro to study the role of kinesin in the movement of pigment granules in melanophores of the teleost black tetra (Gymnocorymbus ternetzi). Microinjection of the antibody into cultured melanophores did not produce any specific effect on the aggregation of pigment granules in melanophores, but it did result in a strong dose-dependent inhibition of the dispersion. Immunoblotting of melanophore extracts showed that the kinesin antibody reacted in these cells with a single protein component with a molecular mass of 135 kDa. Thus, kinesin is responsible for the movement of pigment granules from the center to the periphery of the melanophore.     

Calcium requirement for α-MSH action on tail-fin melanophores of xenopus tadpoles

The role of Ca²⁺ in α-MSH action on melanophores was studied, in vitro, with a bioassay on ventral tail-fin pieces from tadpoles of Xenopus laevis. Melanosome dispersion induced by α-MSH required 1–2 mM extracellular Ca²⁺. Gradual lowering of the extracellular Ca²⁺ levels produced a concentration-dependent inhibition of the α-MSH response; complete inhibition was obtained in a Ca²⁺-free medium containing 10^?4 M EGTA. In Mg²⁺-free medium, normal dispersion was observed. The Ca²⁺ antagonists verapamil (10^?4 M), methoxy-verapamil (10^?4 M) and La³⁺ (10^?3 M) inhibited the dispersion induced by 3 × 10^?9 M α-MSH, whereas ruthenium red (10^?3 M) was without effect. The ionophore A23187 mimicked the effect of the hormone. Melanosome movement per se was evidently independent of Ca²⁺, because cAMP and dibutyryl-cAMP induced a full dispersion in the absence of Ca²⁺. These results show that extracellular Ca²⁺ is specifically required for α-MSH action on tail-fin melanophores in vitro and suggests a Ca²⁺ influx concomitant with the action of the hormone. Possible intra- and extra-cellular Ca²⁺ sites are discussed.     

Continuous illumination through larval development suppresses dopamine synthesis in the suprachiasmatic nucleus, causing activation of α-MSH synthesis in the pituitary and abnormal metamorphic skin pigmentation in flounder

In order to better understand the endocrine aberrations related to abnormal metamorphic pigmentation that appear in flounder larvae reared in tanks, this study examined the effects of continuous 24-h illumination (LL) through larval development on the expression of tyrosine hydroxylase-1 (th1), proopiomelanocortin (pomc), α-melanophore-stimulating hormone (α-MSH) and melanin concentrating hormone (MCH), which are known to participate in the control of background adaptation of body color. We observed two conspicuous deviations in the endocrine system under LL when compared with natural light conditions (LD). First, LL severely suppressed th1 expression in the dopaminergic neurons in the anterior diencephalon, including the suprachiasmatic nucleus (SCN). Second, pomc and α-MSH expression in the pars intermedia melanotrophs was enhanced by LL. Skin color was paler under LL than LD before metamorphic pigmentation, and abnormal metamorphic pigmentation occurred at a higher ratio in LL. We therefore hypothesize that continuous LL inhibited dopamine synthesis in the SCN, which resulted in up-regulation of pomc mRNA expression in the melanotrophs. In spite of the up-regulation of pomc in the melanotrophs, larval skin was adjusted to be pale by MCH which was not affected by LL. Accumulation of α-MSH in the melanotrophs is caused by uncoupling of α-MSH synthesis and secretion due to inhibitory role of MCH on α-MSH secretion, which results in abnormal metamorphic pigmentation by affecting differentiation of adult-type melanophores. Our data demonstrate that continuous illumination at the post-embryonic stage has negative effects on the neuroendocrine system and pituitary in flounder.     

The role of brain-derived neurotrophic factor in the regulation of cell growth and gene expression in melanotrope cells of Xenopus laevis

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 α-melanophore-stimulating hormone for the purpose of adjusting the animal's skin color to that of its background.