Physiological control of xunc18 expression in neuroendocrine melanotrope cells of Xenopus laevis

In mammals, the brain-specific protein munc18-1 regulates synaptic vesicle exocytosis at the synaptic junction, in a step before vesicle fusion. We hy pothesize that the rate of biosynthesis of munc18-1 messenger RNA (mRNA) an d the amount of munc18-1 present in neurons and neuroendocrine cells are re lated to the physiologically controlled state of activity. To test this hyp othesis, the homolog of munc18-1 in the clawed toad Xenopus laevis, xunc18, was studied in the brain and in the neuroendocrine melanotrope cells in th e intermediate lobe of the pituitary gland, at both the mRNA and the protei n level. In toads adapted to a black background, the melanotropes release t he peptide a-melanophore-stimulating hormone (alpha -MSH), which induces da rkening of the skin, whereas in animals adapted to a white background the c ells hardly release but store alpha -MSH, making the animal's skin look pal e. The intermediate pituitary lobe of black-adapted animals revealed a stro ng hybridization reaction with the xunc18 mRNA probe, whereas a much weaker hybridization was observed in the intermediate lobe of white-adapted anima ls (optical density black: 3.4 +/- 0.2 vs. white: 0.8 +/- 0.1; P < 0.02). I mmunocytochemically, Xenopus munc18-like protein has been detected througho ut the brain, in identified neuronal perikarya as well as in axon tracts. W estern blot analysis and immunocytochemistry further demonstrated the prese nce of xunc18 in the neural, intermediate and distal lobe of the pituitary gland. Xunc18 protein:was furthermore determined in immunoblots of homogena tes of melanotropes dissociated from the pituitary gland. In melanotropes o f toads adapted to a black background, the integrated optical density of th e xunc18 immunosignal was 2.7 +/- 0.5 times higher than in cells of white-a dapted toads (P < 0.0001). It is concluded that, in Xenopus melanotrope cel ls, the amounts of both xunc18 mRNA and xuncl8 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 ne uroendocrine messengers.