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Transgenic animals are commonly employed to explore the function of individual proteins. Transgenic animal models include the mouse, the zebrafish, and the South African clawed toad Xenopus laevis. In contrast to mice and zebrafish, with Xenopus transgenesis DNA integration is mostly achieved in the one-cell stage. Moreover, Xenopus (as well as zebrafish) eggs are relatively large, the embryos are transparent, a large offspring is generated, and maintenance of the offspring is easy. In our transgenic studies in Xenopus, we focus on the well-characterized neuroendocrine melanotrope cells of the pituitary pars intermedia that are regulated during the process of adaptation of Xenopus to a changing environment. When the animal is placed on a black background, the melanotrope cells produce and process large amounts of the prohormone proopiomelanocortin (POMC). We apply stable melanotrope-specific transgenesis that is achieved by mixing a Xenopus POMC-promoter/transgene construct with sperm nuclei and injecting this mixture into unfertilized eggs. Since in the melanotrope cells the POMC promoter is much more active in black-adapted animals, the level of transgene expression can be manipulated by placing the animal on either a black or a white background. In this paper we review the possibilities of the Xenopus melanotrope-specific transgenic approach. Following a brief overview of the functioning of Xenopus melanotrope cells, stable melanotrope-specific transgenesis is discussed and our transgenic studies on brain-derived neurotrophic factor and secretory pathway components are described as examples of the transgenic approach in a physiological context and close to the in vivo situation.

We examined the distribution of the mRNAs encoding proTRH and the three TRH receptor subtypes (xTRHR1, xTRHR2, and xTRHR3) in the Xenopus laevis CNS and pituitary. A positive correlation was generally observed between the expression patterns of proTRH and xTRHR mRNAs. xTRHRs were widely expressed in the telencephalon and diencephalon, where two or even three xTRHR mRNAs were often simultaneously observed within the same brain structures. In the pituitary, xTRHR2 was selectively expressed in the distal lobe, and xTRHR3 was found exclusively in the intermediate lobe of white background-adapted animals, indicating that, in amphibians, the effect of TRH on alpha-melanotropin (alpha-MSH) secretion from melanotrope cells is mediated through the novel receptor subtype xTRHR3.

We previously characterized the primary structure of neurotensin (NT) from an extract of the intestine of the frog Rana esculenta. In this study, we provide evidence for the involvement of NT in the neurocrine regulation of the secretory activity of frog adrenocortical cells. Immunohistochemical studies revealed that the adrenal gland of R. esculenta is innervated by a dense network of NT-immunoreactive fibers. Graded concentrations of frog NT induced a dose-dependent stimulation of corticosterone and aldosterone secretion by frog adrenocortical explants through activation of two receptors with pEC(50) of 9.8 and 6.9. These data support the view that NT, released by nerve fibers within the frog adrenal gland, acts locally to control corticosteroid secretion.

Flow cytometry was used to separate Xenopus melanotrope cells according to their size. The cells were then submitted to real-time RT-PCR to determine the level of POMC-gene expression. The results show a positive correlation between cell size and gene expression for cells from black-background (but not white-background) adapted animals.

Melanotrope cells of Xenopus laevis generate transitory increases in intracellular Ca(2+), known as Ca(2+) oscillations. These oscillations arise from the influx of Ca(2+) through voltage-operated Ca(2+) channels (VOCCs). Such oscillations are the driving force for secretion of a-melanophore-stimulating hormone (alpha-MSH) from the cell. The influx of Ca(2+) through VOCCs initiates the mobilization of intracellular Ca(2+) to generate a Ca(2+) wave. The function of the Ca(2+) wave in the melanotrope is unknown, but its presence in the nucleus suggests a role in the regulation of gene expression, perhaps that of proopiomelanocortin (POMC), the precursor protein for alpha-MSH. To determine the possible function of Ca(2+) waves in Xenopus melanotropes, we addressed whether functional VOCCs, which are an established requirement for both secretion and Ca(2+) wave initiation, are also required to maintain POMC gene expression.

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 present study examined inositol phosphate metabolism in melanotrope cells of Xenopus laevis to determine if inositol phosphates are involved in regulating the biosynthetic or secretory activity of these cells. No correlation could be found between inositol phosphate metabolism and the secretory activity of the melanotrope cells. Therefore, we conclude that inositol phosphate production is not directly involved in the regulation of release of alpha-MSH from these cells. However, there were dramatic differences in the capacity of the melanotrope cells to produce inositol phosphates dependent on the state of background adaptation of the animals from which the melanotropes were derived; cells from white-adapted animals had a low capacity to produce inositol phosphates, whereas melanotropes from black-adapted animals had a high capacity in this regard. During adaptation of animals from a white to a black background, the capacity of the melanotrope cells to produce inositol phosphates was only very slowly acquired, reminiscent of the slow acquisition displayed by these cells to produce POMC during background adaptations. Likewise, during black to white background adaptation, the melanotrope cells very slowly lost the capacity to phosphorylate inositol, which correlates with the slow loss of the biosynthetic capacity of melanotrope cells during such adaptations. Altogether we conclude that inositol phospholipid metabolism is likely involved in the regulation of the biosynthetic processes of melanotrope cells of Xenopus laevis.