Bruce G. Jenks

Morphology of the pars intermedia and the melanophore-stimulating cells in Xenopus laevis in relation to background adaptation

The melanophore-stimulating hormone (MSH) cells of the amphibian pars intermedia secrete the peptide alpha-melanophore-stimulating hormone (alpha-MSH), which induces pigment dispersion in dermal melanophores. The purpose of the present study was to determine (1) whether prolonged activation of the secretory activity of the pars intermedia is associated with hypertrophy, hyperplasia, or both and (2) whether the MSH cells function as a homogeneous or heterogeneous population in meeting the physiological demand for MSH. The demand for MSH was manipulated by adapting animals for at least 3 weeks to white, two shades of grey, or black backgrounds. Morphometric analysis showed that the intermediate lobe volume was positively correlated with the degree of pigment dispersion in the melanophores. The number of MSH cells per lobe was not affected by the degree of pigment dispersion. Therefore, we conclude that enlargement of the tissue associated with MSH cell activation involves hypertrophy rather than hyperplasia. Ultrastructural examination indicated that the majority of MSH cells in black-adapted animals are biosynthetically active, whereas the cells of white-adapted animals are relatively inactive and show granule storage. The pars intermedia of grey-adapted toads contained both active and inactive cells, indicating that MSH cells respond as a heterogeneous cell population in meeting the endocrine demand imposed by background.

Dynamics of background adaptation in Xenopus laevis: Role of catecholamines and melanophore-stimulating hormone

The pars intermedia of the pituitary gland in Xenopus laevis secretes alpha-melanophore-stimulating hormone (alpha-MSH), which causes dispersion of pigment in dermal melanophores in animals on a black background. In the present study we have determined plasma levels of alpha-MSH in animals undergoing adaptation to white and black backgrounds. Plasma values of black-adapted animals were high and decreased rapidly after transfer to a white background, as did the degree of pigment dispersion in dermal melanophores. Plasma MSH values of white-adapted animals were below the detection limit of our radioimmunoassay. Transfer of white animals to a black background resulted in complete dispersion of melanophore pigment within a few hours, but plasma MSH levels remained low for at least 24 hr. This discrepancy between plasma MSH and degree of pigment dispersion suggested the involvement of an additional factor for stimulating dispersion. Results of in vitro and in vivo experiments with receptor agonists and antagonists indicated that a beta-adrenergic mechanism, functioning at the level of the melanophore, is involved in the stimulation of pigment dispersion during the early stages of background adaptation.

Regulation of proopiomelanocortin gene expression, an overview of the signaling cascades, transcription factors and responsive elements involved

Pituitary melanotroph and corticotroph cells produce and secrete peptides from the multifunctional precursor protein proopiomelanocortin (POMC). Stimulation of the secretory activity of both cell types involves production of cyclic 3′5′‐adenosine monophosphate (cAMP) and increases in the concentration of intracellular Ca2+. The increase in secretory activity is accompanied by enhanced expression of the POMC gene. Surprisingly, the POMC promoter lacks both cAMP‐responsive elements and Ca2+‐responsive elements through which the cAMP and Ca2+ signals could, in a relatively direct way, act on POMC gene expression. It is thus apparent that other, more indirect, mechanisms have evolved to utilize cAMP and Ca2+ signaling cascades to regulate POMC expression. This review gives an overview of the complex pathways and events that lead to the regulation of POMC gene expression in corticotrophs and melanotrophs. Another major site for POMC production is in hypothalamic neurons of the arcuate nucleus. In these neurons expression of the POMC gene relies on enhancer regions and responsive elements that differ from those utilized in the pituitary gland. In this review some attention will be given to progress made in unraveling the regulatory strategies acting on POMC expression in hypothalamic neurons. It is clear that the complexities of the promoter/enhancer structure of the POMC gene contribute to the versatility of this gene in participating in complex adaptation processes.

TRH acts as a multifunctional hypophysiotropic factor in vertebrates.

Thyrotropin-releasing hormone (TRH) is the first hypothalamic hypophysiotropic neuropeptide whose sequence has been chemically characterized. The primary structure of TRH (pGlu-His-Pro-NH(2)) has been fully conserved across the vertebrate phylum. TRH is generated from a large precursor protein that contains multiple repeats of the TRH progenitor tetrapeptide Gln-His-Pro-Gly. In all tetrapods, TRH-expressing neurons located in the hypothalamus project towards the external zone of the median eminence while in teleosts they directly innervate the pars distalis of the pituitary. In addition, in frogs and teleosts, a bundle of TRH-containing fibers terminate in the neurointermediate lobe of the pituitary gland. Although TRH was originally named for its ability to trigger the release of thyroid-stimulating hormone (TSH) in mammals, it later became apparent that it exerts multiple, species-dependent hypophysiotropic activities. Thus, in fish TRH stimulates growth hormone (GH) and prolactin (PRL) release but does not affect TSH secretion. In amphibians, TRH is a marginal stimulator of TSH release in adult frogs, not in tadpoles, and a major releasing factor for GH and PRL. In birds, TRH triggers TSH and GH secretion. In mammals, TRH stimulates TSH, GH and PRL release. In fish and amphibians, TRH is also a very potent stimulator of alpha-melanocyte-stimulating hormone release. Because the intermediate lobe of the pituitary of amphibians is composed by a single type of hormone-producing cells, the melanotrope cells, it is a suitable model in which to investigate the mechanism of action of TRH at the cellular and molecular level. The occurrence of large amounts of TRH in the frog skin and high concentrations of TRH in frog plasma suggests that, in amphibians, skin-derived TRH may exert hypophysiotropic functions.

Physiologically-induced changes in proopiomelanocortin mRNA levels in the pituitary gland of the amphibian Xenopuslaevis

In the pars intermedia of the pituitary gland of the amphibian Xenopus laevis the level of mRNA encoding proopiomelanocortin (POMC), the precursor protein for alpha-melanophore-stimulating hormone (alpha-MSH), is shown to be dependent on physiological parameters. POMC mRNA levels in the pars intermedia of black-background-adapted Xenopus are much higher than those of white-adapted animals. These physiological changes in POMC mRNA levels are tissue-specific because they were not found in the pars distalis of the pituitary gland. Background transfer experiments revealed that modulation of POMC gene activity is much slower than changes in the secretion of alpha-MSH.

Gaba and neuropeptide Y co-exist in axons innervating the neurointermediate lobe of the pituitary of Xenopus laevis : an immunoelectron microscopic study

The neural innervation of the neurointermediate lobe of the pituitary of the amphibian Xenopus laevis has been studied at the light and electron microscopic level. In the pars intermedia melanotropes and stellate cells are abutted by varicosities originating from GABA- and neuropeptide Y-producing neurons. The varicosities contain two types of vesicle: electron-lucent vesicles (mean diameter 50 nm) which are immunopositive for GABA and larger (80 nm) electron-dense vesicles which are immunopositive for neuropeptide Y. Double immunogold labeling established that GABA and neuropeptide Y co-exist within the varicosities. In the pars nervosa similar varicosities, though low in number, occur. They are associated with neurosecretory nerve terminals, pituicytes and blood vessels. The possible significance of GABA and neuropeptide Y for the neural regulation of melanophore stimulating hormone-release from the pars intermedia is discussed.

Spatial and temporal aspects of Ca2+ oscillations in Xenopus laevis melanotrope cells.

Spatio-temporal aspects of Ca2+ signaling in melanotrope cells of Xenopus laevis have been studied with confocal laser-scanning microscopy. In the whole-frame scanning mode, two major intracellular Ca2+ compartments, the cytoplasm and the nucleus, were visualized. The basal [Ca2+] in the nucleus appeared to be lower than that in the cytoplasm and Ca2+ oscillations seemed to arise synchronously in both compartments. The N-type channel blocker omega-conotoxin eliminated oscillations in both regions, indicating a strong coupling between the two compartments with respect to Ca2+ dynamics. Line-scanning mode, which gives higher time resolution, revealed that the rise phase of a Ca2+ oscillation is not a continuous process but consists of 3 or 4 discrete steps. Each step can be seen as a Ca(2+)-wave starting at the cell membrane and going through the cytoplasm at a speed of 33.3 +/- 4.3 microns/s. Before the Ca(2+)-wave enters the nucleus, a delay of 120.0 +/- 24.1 ms occurred. In the nucleus, the speed of a wave was 80.0 +/- 3.0 microns/s. Treatment with the Ca(2+)-ATPase inhibitor thapsigargin (1 MicroM) almost completely eliminated the apparent difference in the basal [Ca2+] in the cytoplasm and the nucleus, reduced the delay of a Ca(2+)-wave before entering the nucleus to 79.8 +/- 8.7 ms, and diminished the nuclear wave speed to 35.0 +/- 4.9 microns/s. These results indicate that a cytoplasmic thapsigargin-sensitive ATPase near the nuclear envelope is involved in buffering Ca2+ before the Ca2+ wave enters the nucleus. After sensitizing IP3 receptors by thimerosal (10 microM) the speed of the cytoplasmic Ca(2+)-wave was increased to 70.3 +/- 3.6 microns/s, suggesting that IP3 receptors may be involved in the propagation of the cytoplasmic Ca2+ wave. Our results indicate that in melanotropes the generation and propagation of Ca2+ oscillations is a complex event involving influx of Ca2+ through N-type Ca2+ channels, propagation of the cytoplasmic Ca2+ wave through mobilization of intracellular stores and a regulated Ca2+ entry into the nucleus. We propose that Ca(2+)-binding proteins may act as a Ca2+ store for propagation of the wave in the nucleus.

Action of stimulatory and inhibitory α-MSH secretagogues on spontaneous calcium oscillations in melanotrope cells of Xenopus laevis

The secretion of α-melanophore-stimulating hormone (α-MSH) from melanotrope cells in the pituitary gland of Xenopus laevis is regulated by various neural factors, both classical neurotransmitters and neuropeptides. The majority of these cells (80%) display spontaneous Ca2+ oscillations. In order to gain a better understanding of the external regulation of intracellular Ca2+ ([Ca2+]i) in the melanotrope cell, we have examined the action of well known α-MSH secretagogues on the Ca2+ oscillations. It is shown that all secretagogues tested also control the oscillatory state of Xenopus melanotropes, that is, the secreto-inhibitors dopamine, isoguvacine (γ-aminobutyric acid, GABAA agonist), baclofen (GABAB agonist) and neuropeptide Y evoked a rapid quenching of the spontaneous Ca2+ oscillations, whereas the secreto-stimulant sauvagine, an amphibian peptide related to corticotropin releasing hormone, induced oscillatory activity in non-oscillating cells. Supporting argument is given for the idea that the regulation of Ca2+ oscillations is a focal point in the regulation of secretory activity of melanotrope cells. There was considerable heterogeneity among melanotrope cells in the threshold of their Ca2+ response to secretagogue treatment. This heterogeneity may be the basis for melanotrope cell recruitment observed during physiological adaptations of the animal to the light intensity of its background.

Identification of POMC processing products in single melanotrope cells by matrix-assisted laser desorption/ionization mass spectrometry

The use of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) in identifying proopiomelanocortin (POMC) processing products in melanotrope cells of the pituitary intermediate lobe of Xenopus laevis was explored. Mass spectra were obtained with such a high sensitivity of detection that the peptides could be identified in a single melanotrope cell. In addition to known POMC processing products of the Xenopus melanotrope cell, t he presence of previously unidentified POMC‐derived peptides was demonstrated. Together these POMC processing products accounted for the entire length of the POMC precursor. Furthermore, Xenopus possesses two genes for POMC and the sensitivity and accuracy of the MALDI‐MS technique allowed identification of processing products of both the POMCA and POMCB gene. In addition, differences were obtained between the mass spectra of melanotrope cells from Xenopus laevis adapted to different conditions of background illumination. These results show that MALDI‐MS is a valuable tool in the study of the expression of peptides in single (neuroendocrine) cells.

Spontaneous calcium oscillations in Xenopus laevis melanotrope cells are mediated by ω-conotoxin sensitive calcium channels

The dynamics of intracellular Ca2+ signalling in single melanotrope cells of the pituitary gland of the amphibian Xenopus laevis have been studied by means of a digital imaging technique using the fluorescent dye Fura-2. When placed in vitro, the majority of the cells (77%) displayed spontaneous oscillatory changes in the free cytosolic Ca2+ concentration with a frequency of 1 +/- 0.25 (SD) min-1. The oscillations rapidly stopped when extracellular Ca2+ was reduced to nanomolar concentrations, revealing their complete dependence on Ca2+ influx. The fact that the Ca2+ oscillations were blocked by 1 microM omega-conotoxin, but not by nifedipine, at concentrations up to 50 microM, indicated that Ca2+ entered the cell via N-type rather than L-type voltage operated Ca2+ channels. Thapsigargin, a putative inhibitor of intracellular Ca(2+)-ATPase activity, elevated the baseline Ca2+ concentration but had no effect on the occurrence of the spontaneous oscillations. This suggests that intracellular Ca2+ pools are not involved in the mechanism underlying spontaneous Ca2+ oscillations. This is the first report showing spontaneous Ca2+ oscillations mediated by N-type Ca2+ channels in melanotrope cells.