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Melanotrope cells of Xenopus laevis generate transitory increases in intracellular Ca2+, known as Ca2+ oscillations. These oscillations arise from the influx of Ca2+ through voltage-operated Ca2+ channels (VOCCs). Such oscillations are the driving force for secretion of α-melanophore-stimulating hormone (α-MSH) from the cell. The influx of Ca2+ through VOCCs initiates the mobilization of intracellular Ca2+ to generate a Ca2+ wave. The function of the Ca2+ 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 α-MSH. To determine the possible function of Ca2+ waves in Xenopus melanotropes, we addressed whether functional VOCCs, which are an established requirement for both secretion and Ca2+ wave initiation, are also required to maintain POMC gene expression. [ABSTRACT FROM AUTHOR]

It is well-known thatα-melanophore-stimulating hormone (α-MSH) release from the amphibian pars intermedia (PI) depends on the light condition of the animal's background, permitting the animal to adapt the colour of its skin to background light intensity. In the present study, we carried out nine experiments on the effect of low temperature on this skin adaptation process in the toadXenopus laevis, using the skin melanophore index (MI) bioassay and a radioimmunoassay to measure skin colour adaptation andα-MSH secretion, respectively. We show that temperatures below 8 °C stimulateα-MSH secretion and skin darkening, with a maximum at 5 °C, independent of the illumination state of the background. No significant stimulatory effect of low temperature on the MI andα-MSH plasma contents was noted when the experiment was repeated with toads from which the neurointermediate lobe (NIL) had been surgically extirpated. This indicates that low temperature stimulatesα-MSH release from melanotrope cells located in the PI. Anin vitrosuperfusion study with the NIL demonstrated that low temperature does not act directly on the PI. A possible role of the central nervous system in cold-inducedα-MSH release from the PI was tested by studying the hypothalamic expression of c-Fos (as an indicator for neuronal activity) and the coexistence of c-Fos with the regulators of melanotrope cell activity, neuropeptide Y (NPY) and thyrotrophin-releasing hormone (TRH), using double fluorescence immunocytochemistry. Upon lowering temperature from 22 °C to 5 °C, in white-adapted animals c-Fos expression decreased in NPY-producing suprachiasmatic-melanotrope-inhibiting neurones (SMIN) in the ventrolateral area of the suprachiasmatic nucleus (SC) but increased in TRH-containing neurones of the magnocellular nucleus. TRH is known to stimulate melanotropeα-MSH release. We conclude that temperatures around 5 °C inactivate the SMIN in the SC and activate TRH-neurones in the magnocellular nucleus, resulting in enhancedα-MSH secretion from the PI, darkening the skin of white-adaptedX. laevis. [ABSTRACT FROM AUTHOR]

Abstract Ca2+ oscillations regulate secretion of the hormone alpha-melanphore-stimulating hormone (α-MSH) by the neuroendocrine pituitary melanotrope cells of the amphibian Xenopus laevis . These Ca2+ oscillations are built up by discrete increments in the intracellular Ca2+ concentration, the Ca2+ steps, which are generated by electrical membrane bursting firing activity. It has been demonstrated that the patterns of Ca2+ oscillations and kinetics of the Ca2+ steps can be modulated by changing the degree of intracellular Ca2+ buffering. We hypothesized that neurotransmitters known to regulate α-MSH secretion also modulate the pattern of Ca2+ oscillations and related electrical membrane activity. In this study, we tested this hypothesis for the secretagogue sauvagine. Using high temporal-resolution Ca2+ imaging, we show that sauvagine modulated the pattern of Ca2+ signalling by increasing the frequency of Ca2+ oscillations and inducing a broadening of the oscillations through its effect on various Ca2+ step parameters. Second, we demonstrate that sauvagine caused a small but significant decrease in K+ currents measured in the whole-cell voltage-clamp, whereas Ca2+ currents remained unchanged. Third, in the cell-attached patch-clamp mode, a stimulatory effect of sauvagine on action current firing was observed. Moreover, sauvagine changed the shape of individual action currents. These results support the hypothesis that the secretagogue sauvagine stimulates the frequency of Ca2+ oscillations in Xenopus melanotropes by altering Ca2+ step parameters, an action that likely is evoked by an inhibition of K+ currents. [ABSTRACT FROM AUTHOR]

The neuroendocrine Caudo-Dorsal Cells (CDC) of the snail release an egg-laying hormone (CDCH) from their axon terminals by exocytosis. The CDC show 3 states of electrical activity, resting (inactive), active (electrical discharge) and inhibited (inexcitable). Exocytosis was first studied quantitatively with the tannic acid-glutaraldebyde-OO-method (TAGO method). Exocytosis activity appeared to be low in the resting state, very high in the active state, and moderate in the inhibited state. Active CDC particularly show multigranular exocytosis. Furthermore, with the freeze- fracture technique two stages of the exocytotic fusion process were identified: 1. the pre-exocytotic stage, characterized by particle-free areas in the P- and E-faces of the secretory granule and the opposing plasma membrane, and 2. the fusion step, visible as characteristic circular membrane discontinulties. Morphometry of freeze-fractured CDC confirmed that exocytoses are scarce during electrical rest, numerous during activity, and moderate during inhibition. The differences between the numbers of exocytoses In the 3 states were not that high as In the TAGO-studies, apparently because the freeze-fracture preparations did not allow quantitation of multigranular exocytosis. It is concluded that the TAGO-metbod appears very suitable for quantitation of exocytosis activity, whereas the freeze-fracture technique is particularly useful to study membrane events during exocytosis. The significance of exocytoals in the 3 CDC states for the control of egg-laying has been discussed. [ABSTRACT FROM PUBLISHER]

Juvenile specimens of Lymnaea stagnalis were exposed to 0 or 4 miracidia of Trichobilharzia ocellata. Dissection followed at day 22 post exposure. The effects of infection on the activity of the female gonadotropic hormone producing Dorsal Bodies (DB) were studied by phase-contrast and electron microscopy. Morphometry shows that the relative volume of DB cells of infected snails is 1.6 times as high as in controls. This is mainly due to a 45% higher cytoplasmic volume. The number of profiles of the Golgi apparatus increases by nearly 90% and the Golgi volume by 40% as a result of infection. Numerous omega-shaped indentations of the plasma membrane of the DB cell processes indicate the release of the contents of the DB hormone containing elementary granules. It is concluded that parasitic infection causes a clear increase in the synthetic activity of the DB. Most probably, the parasites exert the inhibiting effects on reproductive activity at the level of the targets of the reproductive hormones. [ABSTRACT FROM AUTHOR]

The cerebral caudodorsal cells of the pulmonate snail Lymnaea stagnalis control egg laying and egglaying behavior by releasing various peptides derived from two precursors. The biosynthesis, storage, intracellular breakdown and release of three caudodorsal cell peptides were studied by means of immuno-electron microscopy using antisera raised to fragments of these peptides: (1) Caudodorsal Cell Hormone-I (CDCH-I; derived from precursor I), (2) Caudodorsal Cell Hormone-II (CDCH-II; from precursor II), and (3) α-Caudodorsal Cell Peptide (α CDCP; from both precursors). After affinity purification of the antisera, the specificity of the sera was confirmed with dotting immunobinding assays. From the ultrastructural immunocytochemical data it has been concluded that the precursor molecules are cleaved at the level of the Golgi apparatus after which the C-terminal parts (containing α CDCP) and N-terminal parts (containing CDCH-I or CDCH-II) are sorted and preferentially packaged into large electron-dense granules (MD 150 nm), respectively. Very probably, the content of the large electron-dense granules is degraded within the cell body. The immunoreactivity of the secretory granules increases during discharge from the Golgi apparatus, indicating further processing. At least a portion of the secretory granules contains all three peptides, as shown by double and triple immunopositive stainings whereas other granules appear to contain only one or two of these peptides. The caudodorsal cells release multiple peptides via exocytosis from neurohemal axon terminals into the hemolymph and from blindly ending axon collaterals into the intercellular space of the cerebral commissure (nonsynaptic release). [ABSTRACT FROM AUTHOR]

To study the cellular defense mechanism against oxygen toxicity, an oxygen-tolerant cell line from Chinese hamster ovary (CHO) was obtained by multistep adaptation to increased O levels. The hyperoxia-adapted (HA) cells were able to proliferate under an atmosphere of 99% O/1% CO, an O tension lethal to the parental (control) cells. When grown under normoxic conditions (20% O/1% CO/79% N) the cells remained tolerant for at least 8 weeks, suggesting a genetic basis for the oxygen tolerance. Compared to the parental cells, the HA cells were irregularly shaped, had larger mitochondria, contained more lipid droplets and showed a reduced growth rate. Ultrastructural morphometry revealed a 1.8-fold ( p<0.001) increase of the mitochondrial volume fraction in the HA cells, resulting from an increase in both number and average volume of the mitochondria. The volume fraction of peroxisomes was increased over two-fold in the HA cells, as appeared from a ∼1.9-fold ( p< 0.001) increase in number and a 1.2-fold ( p<0.025) increase in size. There was no evidence for ultrastructural damage in the HA cells. Specific activities of antioxygenic enzymes were considerably higher in the HA cells compared to controls: CuZn-superoxide dismutase, x 2.5; Mn-superoxide dismutase, x 2.1; catalase, x 4.0; glutathione peroxidase, x 1.9. Oxygen tolerance in CHO cells is therefore associated with increased levels of antioxygenic enzymes, confirming the proposed important role of these enzymes in the defense against oxygen toxicity. [ABSTRACT FROM AUTHOR]

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. Postembedding 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. [ABSTRACT FROM AUTHOR]