The multifunctional prohormonc, proopiomclanocortin (POMC), is processsd in the melanotropc cells of the pituitary pars intprmedia at pairs of basic amino acid residues to give a number of pcptides, including a-melanophorc-stimulating hormone (o-MSH). This hormone causes skin darkening in amphibians during background adaptation. Here WC report the complete structure of XLWU~ILS IueGs prohonnonc convcrtasc PC?, the enzyme thought to be responsible for processing of POMC to a-MSH. A comparative structural analysis revealed an overall aminoacid sequence identity of85-87% between Xctt~pus PC2 and its mammalian counterparts, with the lowest degree ofidcntity in the signal peptidrsequcmx (28-368) and the region amino+zrmintil to the catalytic domain (59-608). The occurrence of a second, structurally different PC2 protein reflects the expression of two ,%'crro(l~rs PC2 penes. The expression pattern of PC2 in the XLWII~US pituitary gland of black-and white-adapted anhnals was found to be similar to that of POMC, namely high expression in active mclanotrope cells of black animals. This observation is in lint with a physiological role for PC2 in processing POMC to a-MSH.

M elanin-concentrating horm one (M CH) is a neuro peptide involved in background adaptation in teleost fish, and in m ultiple regulatory functions in mammals and fish. To study the expression of the M C H preprohorm one (ppM C H ) in teleosts, we first cloned a hypothalamic cD N A encoding the com plete ppM C H of tilapia (Oreochromis mossambicus), and a cRNA probe derived from a 270 bp ppM C H cD N A fragment was used for the expression studies. T he level of ppM C H m R N A expression in tilapia hypothalamus, m easured by dot blot analy sis, was significantly higher in fish adapted to a white background than in black-adapted animals, which is in accordance with the reported M CH plasma and tissue concentrations in fish. N orthern blot analysis not only revealed a strong ppM C H mRNA signal in the hypothalam us, but also the presence of ppM C H m R N A in the neurointerm edi ate lobe (N IL) of the pituitary. In situ hybridization and immunocytochemistry showed that ppM C H mRNA as well as M C H immunoreactivity are located in perikarya of two hypothalamic regions, namely in the nucleus lateralis tuberis (N L T ) and the nucleus recessus lateralis (NRL). Quantitative analysis by dot blot hybridization revealed about eight times more ppM C H m R N A in the N L T than in the N R L and N IL of mature tilapias. ppM C H m RNA in the N IL couLd be localized to cell bodies of the neurohypophysis, which were also M CH immunoreactive.

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.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of neuronal cell survival and differentiation factors but is thought to be involved in neuronal cell proliferation and myelination as well. To explore the role of BDNF in vivo, we employed the intermediate pituitary melanotrope cells of the amphibian Xenopus laevis as a model system. These cells mediate background adaptation of the animal by producing high levels of the prohormone proopiomelanocortin (POMC) when the animal is black adapted. We used stable X. transgenesis in combination with the POMC gene promoter to generate transgenic frogs overexpressing BDNF specifically and physiologically inducible in the melanotrope cells. Intriguingly, an approximately 25-fold overexpression of BDNF resulted in hyperplastic glial cells and myelinated axons infiltrating the pituitary, whereby the transgenic melanotrope cells became located dispersed among the induced tissue. The infiltrating glial cells and axons originated from both peripheral and central nervous system sources. The formation of the phenotype started around tadpole stage 50 and was induced by placing white-adapted transgenics on a black background, i.e. after activation of transgene expression. The severity of the phenotype depended on the level of transgene expression, because the intermediate pituitaries from transgenic animals raised on a white background or from transgenics with only an approximately 5-fold BDNF overexpression were essentially not affected. In conclusion, we show in a physiological context that, besides its classical role as neuronal cell survival and differentiation factor, in vivo BDNF can also induce glial cell proliferation as well as axonal outgrowth and myelination.

To study in vivo the dynamics of the biosynthetic and secretory processes in a neuroendocrine cell, we use the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis. The activity of these cells can be simply manipulated by adapting the animal to a white or a black background, resulting in inactive and hyperactive cells respectively. Here, we applied differential display proteomics and field emission scanning electron microscopy (FESEM) to examine the changes in architecture accompanying the gradual transition of the inactive to the hyperactive melanotrope cells.The proteomic analysis showed differential expression of neuroendocrine secretory proteins, endoplasmic reticulum (ER)-resident chaperones, and housekeeping and metabolic proteins. The FESEM study revealed changes in the ultrastructure of the ER and Golgi and the number of secretory granules. We conclude that activation of neuroendocrine cells tunes their molecular machineries and organelles to become professional secretors.