Pars Intermedia Of Xenopus Laevis Research Articles

Endogenous Production of Nitric Oxide and Effects of Nitric Oxide and Superoxide on Melanotrope Functioning in the Pituitary Pars Intermedia of Xenopus laevis

Previous studies have focused on the immunohistochemical detection of a nitric oxide (NO)-cyclic 3′,5′-monophosphate (cGMP) pathway in the brain and pituitary of the aquatic toad Xenopus laevis. We here investigate the endogenous production and possible involvement of NO signaling in the regulation of melanotrope cell activity in the pituitary pars intermedia of this amphibian. Using immunohistochemical staining of cultured cells with a polyclonal antiserum against inducible NO synthase (iNOS), immunoreactivity was observed both in melanotropes and in stellate-shaped cells. Part of these stellate-shaped cells is characterized as folliculo-stellate cells by their capacity of β-Ala-Lys-Nϵ-AMCA uptake. Using chemiluminescence detection we demonstrate the presence of NO and reaction products like nitrite (NO−2) or peroxynitrite (ONOO−) in the incubation medium of cultured melanotropes. Bacterial lipopolysaccharide (LPS) stimulates the generation of NO and reaction products, the effect of which was blocked by S-methyl-l-thiocitrulline hydrochloride, a potent general NOS inhibitor. With [3H]lysine incorporation and a superfusion technique, it is shown that peptide release from melanotropes is stimulated by administration of superoxide dismutase (SOD), which was added to the superfusion medium to prevent scavenging of NO by superoxide anions. Pretreating the cells with the general NOS inhibitor l-nitroarginine methyl ester for 48 h attenuated the SOD-induced stimulation, but did not affect the stimulation by sodium nitroprusside (SNP) or 3-morpholinylsydnoneimine chloride (SIN-1), whereas hemoglobin blocked the combined effect of SOD plus NO donors. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]-quinoxaline-1-one did not inhibit but even significantly potentiated the effect of NO donors on peptide release without affecting the SOD-induced stimulation of peptide release. In addition to the previously described neuronal NOS (nNOS) immunoreactivity in nerve fibers in the pars intermedia of Xenopus, the present data reveal iNOS and nNOS as potential sources of endogenous NO production in cultured cells of the pars intermedia. Our study shows that also in nonmammalian vertebrates endogenous NO production may be physiologically relevant under conditions where protection against oxidative damage is needed. The endocrine cells of the pars intermedia themselves, as well as the folliculo-stellate cells, under such conditions may dispose of a protective mechanism against oxidative stress. The sensitivity of the endogenous NO production to LPS suggests that NO may also play a role during systemic inflammation.

Serotonergic innervation of the pituitary pars intermedia of xenopus laevis.

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.

Acetylcholine autoexcites the release of proopiomelanocortin-derived peptides from melanotrope cells of Xenopus laevis via an M1 muscarinic receptor.

The release of POMC-derived peptides from the melanotrope cells in the pituitary pars intermedia of Xenopus laevis is regulated by multiple inhibitory and stimulatory neurochemical messengers. In this study we examined the role of acetylcholine (ACh) in regulating melanotrope cell activity. The origin of ACh was studied in in vitro biosynthesis experiments, where it was demonstrated that dissociated melanotropes can synthesize ACh. In addition, immunocytochemical studies showed the presence of the ACh-synthesizing enzyme choline acetyltransferase in the cytoplasm of melanotrope cells. Oscillations in the concentration of intracellular free calcium ions are thought to be the driving force for the secretion of POMC-derived peptides from Xenopus melanotrope cells. A technique was developed to study simultaneously calcium oscillations and peptide secretion from the same population of single melanotrope cells in vitro. ACh concentration-dependently raises the intracellular free calcium concentration by increasing both the frequency and amplitude of the calcium oscillations as well as the release of POMC-derived peptides. The actions of ACh on the melanotrope cell were mimicked by muscarine, but not by nicotine, indicating the exclusive presence of a muscarinic cholinergic receptor. Furthermore, selective muscarinic receptor antagonists showed that the actions of ACh are mediated through an M1-subtype muscarinic receptor. Immunofluorescence cytochemistry in combination with confocal laser scanning microscopy 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 way, acting via a muscarinic M1 receptor on the intracellular free calcium concentration.

Background adaptation and synapse plasticity in the pars intermedia of xenopus laevis

The amphibian Xenopus laevis adapts the colour of its skin to the colour of its background, by the release of the pro-opiomelanocortin-derived peptide α-melanophore-stimulating hormone from the pars intermedia of the pituitary gland. Suprachiasmatic neurons play an important role in adaptation to a light background as they produce the neurotransmitters GABA, dopamine and neuropeptide Y, which inhibit the release of α-melanophore-stimulating hormone. These factors are transported to axon varicosities contacting the melanotrope cells. In these varicosities GABA resides in electron-lucent vesicles and neuropeptide Y and dopamine coexist in electron-dense vesicles. In this study the effects of background adaptation on the morphology of the varicosities in the pars intermedia were studied, using immunoelectron microscopy and morphometry with freeze-substitution-fixed material. Varicosities were found singly and in clusters throughout the pars intermedia. Varicosities were identified by the presence of electron-dense and electron-lucent secretory vesicles, the latter being immunopositive for anti-GABA. Both varicosity types revealed active zones with exclusively GABA-containing vesicles in contact with the presynaptic membrane. When white- and black-adapted animals were compared, significant background effects were found with respect to the organization of the varicosities: the density of varicosity profiles was twice as high in white-adapted animals as in black-adapted ones, due to an increase in density of the clustered varicosities. Furthermore, in white-adapted animals varicosities were about twice as large as in black-adapted animals. With respect to vesicle types, single and clustered varicosities showed a differential effect. For both the population of electron-lucent and electron-dense vesicles, single varicosities showed equal numbers in white- and black-adapted animals, but clustered varicosities showed higher numbers of electron-lucent and electron-dense vesicles in black-adapted animals, indicating storage of neurotransmitters. Finally, in varicosities of white-adapted animals the number and size of the active zones and the number of electron-lucent vesicles attached to the active zones, were about twice as high as in black-adapted animals, indicating a stronger GABA release. It is concluded that the profound effects of environmental light conditions on synaptic structure and substructure in the Xenopus pars intermedia are related to a changed release activity of neurotransmitters inhibiting the activity of the melanotrope cells.

α,N-Acetyl β-Endorphin [1-8] Is the Terminal Product of Processing of Endorphins in the Melanotrope Cells of Xenopus laevis, as Demonstrated by FAB Tandem Mass Spectrometry

The major N-terminal acetylated endorphin of the pars intermedia of Xenopus laevis was purified and submitted to fast-atom bombardment tandem mass spectroscopy. The collisionally induced dissociation MS/MS spectrum of the [M+H]+ ion revealed sufficient fragment ions to determine unambiguously the identity of the peptide as α,N-acetyl β-endorphin [1-8], the sequence of which was predicted on the basis of the nucleotide sequence of Xenopus POMC cDNA. The determination was confirmed by showing that the synthetic peptide of this structure had identical FAB tandem mass spectrometric characteristics as the endogenous endorphin. We conclude that α,N-acetyl β-endorphin [1-8] is the terminal product of processing of endorphins in the melanotrope cell of Xenopus laevis.

The processing of beta-endorphin and alpha-melanotrophin in the pars intermedia of Xenopus laevis is influenced by background adaptation.

beta-Endorphin- and alpha-melanotrophin (alpha-MSH)-related peptides were extracted from the pars intermedia of Xenopus laevis maintained for 2, 4 or 6 weeks on a white background and for the same periods on a black background. The peptides were resolved under dissociating conditions by gel exclusion chromatography on Sephadex G-50 and they were detected by radioimmunoassay with antibodies to beta-endorphin, alpha,N-acetyl beta-endorphin and alpha-MSH. The beta-endorphin-related peptides separated into two fractions of different molecular size. Further purification of the peptides in each fraction was by ion exchange chromatography on SP-Sephadex C-25 and by high-pressure liquid chromatography. The alpha-MSH-related peptides were resolved by gel exclusion and ion exchange chromatography. The purified beta-endorphin- and alpha-MSH-immunoreactive peptides were identified by comparison of their chromatographic properties with the corresponding peptides from porcine pituitary or by comparison with synthetic peptides. The major form of beta-endorphin in the pars intermedia of the frog adapted to a white background was identified as alpha,N-acetyl beta-endorphin (1-8); it was accompanied by a small quantity of acetylated peptides with molecular size similar to beta-endorphin. In contrast, the pars intermedia of the frogs adapted to a black background contained approximately equal amounts of alpha,N-acetyl beta-endorphin (1-8) and the larger forms of beta-endorphin. The higher molecular weight forms were identified as the alpha,N-acetyl derivatives of beta-endorphin (1-26), (1-27) and (1-31); however after 6 weeks of white adaptation the sole remaining peptide in this group was the 26-residue peptide. An additional beta-endorphin immunoreactive peptide, provisionally identified as beta-endorphin (10-26), was present in both black- and white-adapted animals; the amounts of this peptide increased during white adaptation. Major differences in the processing of alpha-MSH were also observed. In the frogs adapted to a black background des-acetyl alpha-MSH greatly predominated over the acetyl form whereas after 6- weeks adaptation to a white background the acetylated peptide proved to be the principal component. The results demonstrate that the proteolytic processing of beta-endorphin and the acetylation of alpha-MSH in Xenopus laevis are influenced by background adaptation. The formation of beta-endorphin (1-8) appears to reflect the action of an endopeptidase that acts at the single arginine residue present at position 9.(ABSTRACT TRUNCATED AT 400 WORDS)

Demonstration of coexisting catecholamine (dopamine), amino acid (GABA), and peptide (NPY) involved in inhibition of melanotrope cell activity in Xenopus laevis: a quantitative ultrastructural, freeze- substitution immunocytochemical study

This quantitative ultrastructural immunocytochemical study demonstrates the coexistence of a catecholamine [dopamine (DA)], an amino acid (GABA), and a neuropeptide [neuropeptide Y (NPY)] in axon varicosities innervating the pars intermedia of Xenopus laevis. The varicosities are assumed to control the pars intermedia melanotrope cells, which regulate skin color during the physiological process of background adaptation. Varicosity profiles appear to abut melanotrope cells and folliculostellate cells, star-shaped cells that intimately contact the melanotropes. All varicosity profiles contain two morphological types of vesicle. Monolabeling studies on routinely fixed and freeze-substituted tissues showed that the small, electron-lucent vesicles store GABA, whereas DA and NPY occur in larger, electron-dense ones. Double and triple labeling experiments, in which the degree of immunoreactivity was quantified per varicosity profile and per vesicle, led to the conclusion that (1) DA, GABA, and NPY coexist within almost all varicosity profiles and (2) DA and NPY are costored within electron-dense vesicles. Varicosity profiles that about melanotrope cells show a much higher ratio between the numbers of electron-lucent and electron-dense vesicles than varicosities contacting folliculostellate cells (15.8 and 3.3, respectively). This differential distribution is in line with the previous demonstration that, in contrast to GABA, NPY does not act directly on the melanotrope cells but indirectly, by controlling the activity of the folliculostellate cells.

Demonstration of dopamine in electron-dense synaptic vesicles in the pars intermedia of Xenopus laevis, by freeze substitution and postembedding immunogold electron microscopy

The presence of dopamine in the pituitary of the clawed toad Xenopus laevis was studied by light and electron microscope immunocytochemistry, using pre- and postembedding techniques. Light microscopy showed the presence of an intricate, anti-dopamine-positive fibre network throughout the pars intermedia. In preembedded stained material, dopamine appeared to occur in varicosities which make synaptic contacts with both folliculo-stellate cells and melanotrope cells. Post-embedding immunogold staining of freeze-substituted material permitted the localization of anti-dopamine reactivity in electron-dense vesicles in these varicosities. This finding supports the hypothesis that dopamine is involved in the (inhibitory) control of melanotrope cell activity in X. laevis.

Indirect action of elevated potassium and neuropeptide Y on alpha MSH secretion from the pars intermedia of Xenopus laevis: a biochemical and morphological study.

A number of neurochemical messengers have been shown to act directly on the melanotrope cells of the pars intermedia of Xenopus laevis to regulate alpha MSH secretion. In the present study the possibility that the melanotropes are also indirectly controlled has been examined. For this purpose, the characteristics of alpha MSH release from superfused intact lobes, cultured lobe and isolated melanotropes were compared after treatment with elevated potassium. Isolated melanotropes responded with an increased secretion of alpha MSH, whereas intact lobes showed a profound inhibitory response, probably caused by potassium-induced release of inhibitory factors from nerve terminals. Cultured lobes displayed a biphasic response characterized by an initial activation followed by a strong inhibition; the stimulatory phase likely reflects a direct action of potassium on the melanotropes, before being overridden by an inhibitory mechanism. The inhibitory phase must originate from the action of nonneuroendocrine cells because the cultured lobes lack functionally active nerve terminals, as verified by immunocytochemistry and electron microscopy. The most likely candidates for this action are folliculo-stellate cells which are in intimate contact with the melanotropes and are innervated by neuropeptide Y-containing nerve terminals. Like elevated potassium, neuropeptide Y inhibited alpha MSH secretion from fresh and cultured lobes but not from isolated melanotropes. This indicates that NPY acts indirectly, in a nonpresynaptic way, to inhibit alpha MSH secretion.

GABAergic regulation of melanocyte-stimulating hormone secretion from the pars intermedia of Xenopus laevis: immunocytochemical and physiological evidence.

alpha-MSH secretion from the amphibian pars intermedia is under inhibitory hypothalamic control, and the catecholamine dopamine is thought to be the physiological MSH release-inhibiting factor. In the present study we evaluated the possible role of the neurotransmitter gamma-aminobutyric acid (GABA) in the regulation of the pars intermedia of Xenopus laevis. Immunocytochemical staining with antibodies to glutamic acid decarboxylase showed the presence of a rich GABAergic network in the intermediate lobe of the pituitary gland. Administration of GABA to superfused neurointermediate lobes caused a rapid and dose-dependent inhibition of basal release of MSH and immunoreactive endorphin. Pulse-chase experiments revealed that GABA gave a coordinate inhibition of the release of all peptides derived from proopiomelanocortin. In vivo administration of GABA resulted in almost complete pigment aggregation in dermal melanophores of both adults and larvae. Altogether, our results indicate that GABA is a physiologically important factor for regulation of the pars intermedia in Xenopus laevis.

N alpha-acetylation is linked to alpha-MSH release from pars intermedia of the amphibian pituitary gland.

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.

Early Changes in the Ultrastructure of the Pars Intermedia of the Pituitary of <i>Xenopus laevi</i><i>s</i> after Change of Background Color

Stereological analysis of the secretory cells of the pars intermedia of Xenopus laevis over a period of 3 days following the transfer of animals from a white to a black background has revealed that significant alterations in the ultrastructural appearance of these cells can be detected 8 h after the transfer. In particular, changes in the secretory granules and the rough endoplasmic reticulum were found to correlate well with previous reports concerning the melanocyte-stimulating hormone (MSH) content and the capacity for protein synthesis of the pars intermedia.

SYNTHESIS AND SECRETION OF A LARGE GLYCOPROTEIN IN THE PARS INTERMEDIA

The biosynthesis, intracellular transport and release of [3-H]-leucine-containing secretory product has been followed in the pars intermedia of Xenopus laevis and, in particular, the synthesis and secretion of a large molecular weight glycoprotein secretory product was demonstrated. However, if Xenopus adrenocorticotrophin does contain a leucine residue the results obtained provided no support for the view that it serves as a precursor for melanocytestimulating hormone in this species.

Localization of adrenergic fibers in the amphibian pars intermedia by electron microscope autoradiography and their selective removal by 6-hydroxydopamine

Electron microscope autoradiography has been used to demonstrate directly the adrenergic nature of the type B fibers in the pars intermedia of Xenopus laevis. Using 6-hydroxydopamine, it has been shown that these fibers can be selectively removed leaving the A type fibers morphologically unchanged without affecting the adrenergic elements in that part of the central nervous system protected by the blood-brain barrier.

Studies on secretory activity in the pars intermedia of Xenopus laevis: 2: A biochemical and electron cytochemical investigation of acid hydrolase activity following the stimulation of secretory activity in vivo

In the MSH cell at the onset of secretory activity, acid hydrolase activity increases. This increased activity, shown quantitatively by assaying beta-glycerophosphatase and R-glucuronidase within the stimulated gland, has been shown by electron cytochemical methods for beta-glycerophosphatase (acid phosphatase) and aryl sulphatase to be related to the production of large numbers of dense bodies. Cytochemical evidence also supports the view that these lytic bodies arise from GERL-like cisternal elements since it is shown that in addition to the flattened, parallel Golgi cisternae these elements are also R-glycerophosphatase-positive. The similarities between the dense bodies and those of other cell types are described and discussed.

Studies on secretory activity in the pars intermedia of Xenopus laevis: 3. The synthesis and release of melanocyte stimulating hormone (MSH) in vitro

Methods of bioassay and electron microscopy have been employed in the study of secretion of MSH from single pars intermedia/nervosa lobes maintained in vitro. It is shown that in a modified Krebs-Ringer medium the tissue displays an endogenous capacity for the synthesis and release of MSH. The amount of MSH produced is directly related to the development and functioning of the endoplasmic reticulum within the MSH cell, and although a net increase in the amount of MSH is adversely affected by anaerobic conditions and low temperature it is independent of an exogenous amino acid supply. The release of MSH, which is studied both independently and in association with concomitant synthesis, is also significantly reduced by anaerobic conditions and low temperature. Of the inorganic ions present in the medium only Ca(++) is essential for the synthesis and release of MSH. The evidence suggests that more than one step in the secretory process is dependent upon an exogenous supply of Ca(++) since in its absence the inhibition of MSH release is immediate but reversible, whereas although the inhibition of synthesis is delayed it is eventually complete and irreversible.

Studies on secretory activity in the pars intermedia of Xenopus laevis: 1: Fine structural changes related to the onset of secretory activity in vivo

Changes in the fine structural organization of the pars intermedia related to the onset of secretory activity within the gland have been studied. It is shown that during the first seven days, following the onset of secretory activity, there is an extensive membraneogenesis within the cytoplasm of the parenchymatous cells of the pars intermedia which results in the formation of a well-organized array of endoplasmic reticulum and an increased development of the Golgi complex. Simultaneously the large population of secretory granules present in the cells in the inactive condition is reduced, the granules fusing with the plasma membrane and releasing their contents into the extracellular space. During the process of intracellular reorganization, in addition to the elaboration of those components of the Golgi complex which are believed to be concerned with the formation of the secretory granules, a second distinctive cisternal element develops within the Golgi area. This component which remains confined to the Golgi area for only a short time (days 2–4) appears to be responsible for the production of membrane-bound dense bodies with a finely granular content. The dense bodies, in turn, become transformed into the larger heterogeneous structures which are a prominent feature of actively secreting pars intermedia cells.