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ispartof: FRONTIERS IN NEUROSCIENCE vol:14 ispartof: location:Switzerland status: published

The toad Xenopus laevis is able to adapt its skin color to background light intensity. In this neuroendocrine reflex, the proopiomelanocortin (POMC)-derived peptide alpha-melanophore-stimulating hormone (alphaMSH) is a key regulatory factor. In animals adapting to a black background, release of alphaMSH from the pituitary pars intermedia causes dispersal of melanin in skin melanophores. To investigate the long-term in vivo dynamics of alphaMSH production during black background adaptation, the biosynthetic rate of POMC and the contents of POMC, alphaMSH and the POMC processing enzyme precursor convertase 2 (PC2) have been studied in the pars intermedia using pulse-labeling, Western blot and radioimmunoassay. In control animals, adapted to a white background, the rate of POMC biosynthesis and the POMC content were low, while high alphaMSH and PC2 contents were found. After 1 week of adaptation to a black background, the rate of POMC biosynthesis and the POMC protein content had increased 19- and 3.7-fold respectively. These parameters attained a maximum level (28- and 5. 8-fold higher than control) after 3 weeks and remained at these elevated levels for at least 12 weeks. After 1 week, the pars intermedia content of alphaMSH was only 30% of the control level, but after 6 and 12 weeks, the alphaMSH level had increased to the control level. The PC2 content decreased to 52% of control after 1 week and stabilized after 3 weeks at a level slightly lower than the control value. The results show that during long-term background adaptation a steady-state situation is reached, with a balance between the biosynthesis, enzymatic processing and release of alphaMSH. The in vivo dynamics of the processing enzyme PC2 suggest a parallel storage and release of alphaMSH and mature PC2 in the Xenopus pituitary pars intermedia.

Summary Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.

1. 1. Ppeitde biosynthesis in neurointermediate lobes of black adapted Xenopus laevis was studied using pulse-chase incubation and reversed-phase, high-performance liquid chromatographic analysis. 2. 2. During the pulse period one major product was synthesized, which was subsequently converted to 12 chase peptides, suggesting a precursor-product mode of biosynthesis for this tissue. 3. 3. Chase peptides I, II and IV possessed high melanotropic activity. Alpha-MSH did not appear to be among the chase peptides. Peptide II had also high corticotropic activity. 4. 4. Peptides I and II are probably small, since they were TCA-soluble and ran faster on acid-urea gels than α-MSH. They may, however, well be structurally related to this latter hormone.

To study in vivo biosynthesis of pars intermedia peptides in Xenopus laevis, [3H]lysine was administered by an osmotic minipump via a cannula inserted near the pituitary gland. Following extraction of the neurointermediate lobe, high-performance liquid chromatography was used to separate the newly synthesized peptides. In black-background adapted animals, [3H]lysine was incorporated into a number of peptides. The elution characteristics of these peptides corresponded exactly with those of peptides synthesized during in vitro incubation of neurointermediate lobes, and which were identified as des-N alpha-acetyl-alpha-MSH, a gamma-MSH-like peptide, two corticotropin-like intermediate lobe peptides, and two forms of endorphin. In white-background adapted Xenopus, practically no synthesis of pars intermedia peptides occurred. Transfer of black-adapted toads to a white background at the beginning of infusion led to storage of newly synthesized peptides. When such animals were maintained on a white background for 10 days, des-N alpha-acetyl-alpha-MSH, but not alpha-MSH, was present in the pars intermedia; this supports the notion that des-N alpha-acetyl-alpha-MSH constitutes the "storage form" of alpha-MSH.

1. Release of [3H]lysine-labelled peptides from the neurointermediate lobe of black adapted Xenopus laevis was studied using a microsuperfusion system and high-performance liquid chromatographic analysis. 2. It appeared that all newly synthesized peptides, inclusive the precursor for these peptides, were concomitantly released. 3. On the basis of the total release of labelled peptides as a function of superfusion incubation time, 3 time-periods were selected to study the effects of secretagogues on the release-process. 4. Dopamine inhibited the release of all newly synthesized peptides, except the precursor, leading to an enhanced level of these peptides remaining in the lobe. Cyclic AMP stimulated the release of all peptides, except the precursor, leading to reduced levels of lobe-peptides.

Most amphibians can adapt their colour to that of their background by a mechanism which is controlled by melanophore stimulating hormone (MSH), a product of the pars intermedia of the pituitary gland1. Although the melanotropic peptides of amphibians have never been sequenced, immunological evidence indicates that at least some of them are structurally related to α-MSH2–4. In our studies of the biosynthesis and structure of melanotropic peptides in the pars intermedia of Xenopus laevis, we have found evidence, reported here, for des-Nα-acetyl-α-MSH as the immediate precursor of α-MSH, with the acetylation taking place just before or during release from the pituitary.