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Physiological Control of Xunc18 Expression in Neuroendocrine Melanotrope Cells of Xenopus laevis¹

Department of Cellular Animal Physiology (S.M.K., C.A.F.M.B., E.W.R.), Nijmegen Institute for Neurosciences, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands; European Institute for Peptide Research (IFRMP23) (H.V.), INSERM U413, University of Rouen, 76821 Mont-Saint-Aignan, France; and Department of Medical Pharmacology (M.V.), Rudolf Magnus Institute for Neurosciences, University of Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands

Address all correspondence and requests for reprints to: Sharon M. Kolk, Department of Cellular Animal Physiology, Nijmegen Institute for Neurosciences, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. E-mail: smkolk{at}sci.kun.nl

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 -melanophore-stimulating hormone (-MSH), which induces darkening of the skin, whereas in animals adapted to a white background the cells hardly release but store -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 -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.

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