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Orexin is a neuropeptide that has been implicated in several processes, such as induction of appetite, arousal and alertness and sleep/wake regulation. Multiple lines of evidence also suggest that orexin is involved in the stress response. When orexin is administered intracerebroventricular it activates the hypothalamic pituitary adrenal (HPA)-axis, which is the main regulator of the stress response. The HPA-axis is not the only player in the stress response evidence suggests that urocortin 1 (Ucn1), a member of the corticotropin releasing factor (CRF) neuropeptide family, also plays an important role in the stress response adaptation. Ucn1 is primarily synthetized in the centrally projecting Edinger-Westphal nucleus (EWcp), which also receives dense innervation by orexin terminals. In this study we tested the hypothesis that orexin would directly shape the response of EWcp-Ucn1 neurons to acute cold stress. To test this hypothesis, we first assessed whether orexinergic axon terminals would innervate EWcp-Ucn1/CART neurons, and next we exposed orexin deficient (orexin-KO) male mice and their male wild-type (WT) littermates to acute cold stress for 2h. We also assessed stress-associated changes in plasma corticosterone (CORT), as well as the activation of Ucn1/CART neurons in the EWcp nucleus. We found that orexin immunoreactive axon terminals were juxtaposed to EWcp-Ucn1/CART neurons, which also expressed orexin receptor 1 mRNA. Furthermore, acute stress strongly activated the EWcp-Ucn1/CART neurons and increased plasma CORT in both WT littermates and orexin-KO mice, however no genotype effect was found on these indices. Taken together our data show that orexin in general is not involved in the animal's acute stress response (plasma CORT) and it does not play a direct role in shaping the response of EWcp-Ucn1 neurons to acute stress either., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An important aspect of Ca(2+) signaling is the ability of cells to generate intracellular Ca(2+) waves. In this study we have analyzed the cellular and subcellular kinetics of Ca(2+) waves in a neuroendocrine transducer cell, the melanotrope of Xenopus laevis, using the ratiometric Ca(2+) probe indo-1 and video-rate UV confocal laser-scanning microscopy. The purpose of the present study was to investigate how local Ca(2+) changes contribute to a global Ca(2+) signal; subsequently we quantified how a Ca(2+) wave is kinetically reshaped as it is propagated through the cell. The combined kinetics of all subcellular Ca(2+) signals determined the shape of the total cellular Ca(2+) signal, but each subcellular contribution to the cellular signal was not constant in time. Near the plasma membrane, [Ca(2+)](i) increased and decreased rapidly, processes that can be described by a linear and exponential function, respectively. In more central parts of the cell slower kinetics were observed that were best described by a Hill equation. This reshaping of the Ca(2+) wave was modeled with an equation derived from a low-pass RC filter. We propose that the differences in spatial kinetics of the Ca(2+) signal serves as a mechanism by which the same cellular Ca(2+) signal carries different regulatory information to different subcellular regions of the cell, thus evoking differential cellular responses.

We have previously shown that the stimulatory effect of TRH on alpha-MSH secretion from the frog pars intermedia is associated with Ca2+ influx through voltage-dependent Ca2+ channels, activation of a phospholipase C and mobilization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (AC), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A2, and protein tyrosine kinase (PTK) in TRH-induced alpha-MSH release. Incubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13-acetate (24 h), which causes desensitization of PKC, or with the PKC inhibitor NPC-15437, reduced by approximately 50% of the effect of TRH on alpha-MSH release. In most melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase of the Ca2+ response. Incubation of NILs with TRH (10(-6) M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is involved in TRH-induced alpha-MSH release. The calmodulin inhibitor W-7 and the CAM KII inhibitor KN-93 did not significantly reduce the response to TRH. Similarly, the phospholipase A2 inhibitors quinacrine and 7-7'-DEA did not impair the effect of TRH on alpha-MSH secretion. The PTK inhibitors ST638 and Tyr-A23 had no effect on TRH-induced [Ca2+]i increase but inhibited in a dose-dependent manner TRH-evoked alpha-MSH release (ED50 = 1.22x10(-5) M and ED50 = 1.47x10(-5) M, respectively). Taken together, these data indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH-induced alpha-MSH secretion. Activation of PKC is responsible for the sustained phase of the increase in [Ca2+]i, whereas activation of PTK does not affect Ca2+ mobilization.

In this immunocytochemical study, we used light and electron microscopic observations in combination with morphometry to analyze the processing of pro-opiomelanocortin (POMC) in melanotrope cells of the intermediate pituitary of Xenopus laevis adapted to either a white or a black background. An antiserum was raised against a synthetic peptide including the cleavage site between ACTH and beta-lipotropic hormone in Xenopus. Western blotting revealed that this antiserum recognizes only a 38-kD protein, the POMC prohormone, from extracts of Xenopus neurointermediate pituitary. Light immunocytochemistry showed differential immunostaining for anti-POMC compared to anti-alpha-MSH. Anti-POMC was predominantly found in the perinuclear region, whereas anti-alpha-MSH yielded staining throughout the cytoplasm. Immunogold double labeling revealed that electron-dense secretory granules (DGs) show high immunoreactivity for anti-POMC and low immunoreactivity for anti-alpha-MSH. Electron-lucent granules (LGs) are immunoreactive to anti-alpha-MSH only. Moderately electron-dense granules (MGs) revealed intermediate reactivity compared to DGs and LGs. Background light intensity has significant effects on the morphology and the immunoreactivity of the secretory granules. Black-adapted animals have 4.5 times as many DGs and MGs as white-adapted animals. In addition, the MGs in black animals show 42% more anti-alpha-MSH immunogold than the MGs in white animals. Together, these findings indicate that the three granule types represent subsequent stages in granule maturation. Adaptation to a black background stimulates the formation of young immature granules, while at the same time the processing rate during granule maturation increases.

The melanotrope cell of Xenopus laevis is innervated by nerve terminals that contain, among other transmitter substances, the neurotransmitter gamma-aminobutyric acid (GABA). Postsynaptically the melanotrope cell possess both GABAA and GABAB receptors. Activation of either receptor type leads to an inhibition of alpha MSH release from the cell. The present study concerns the functional significance of the existence of two types of GABA receptors on the melanotrope regarding two questions: 1) do the different receptor types have different effects on the melanotrope? and 2) can the endogenous ligand GABA differentially activate these receptors? Concerning the first question, we have tested the hypothesis that the GABAA receptor (a chloride ion channel) and the GABAB receptor (a G protein-coupled receptor negatively linked to adenylyl cyclase) may have differential effects on the sensitivity of the cell to stimulation by cAMP-dependent mechanisms. We show that treatments with either isoguvacine (GABAA agonist) or baclofen (GABAB agonist) inhibit intracellular Ca2+ oscillations and peptide secretion from melanotrope cells. Treatments known to increase intracellular cAMP in the melanotrope (e.g. use of the peptide sauvagine or the cAMP analog 8-bromo-cAMP) completely overcame the inhibition induced by baclofen, but not that caused by isoguvacine. We conclude that the GABAA and GABAB receptors have different effects on the Xenopus melanotrope cell by differentially affecting the sensitivity the cell shows to stimulation by cAMP-dependent mechanisms. Concerning possible differential activation of the receptor types, we found that we could use a membrane potential probe (from the bis-oxonol family) to differentiate between GABAA and GABAB receptor activation. Using this probe we showed that low GABA concentrations (< 10(-7) M) give a response indicative of the GABAB receptor, whereas at high GABA concentrations (> 10(-7) M), the GABAA receptor response predominates. We, therefore, conclude that GABA can differentially activate the two types of GABA receptors on the Xenopus melanotrope cell.

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, the 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.

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.

Qualitative and quantitative ultrastructural effects of the platinum compounds cisplatin (Platinol), carboplatin (JM8), and iproplatin (JM9) were studied on two types of identified peptidergic neuron (caudodorsal cells, light green cells) in the pond snail Lymnaea stagnalis. Depending on the parameter under investigation, either one or both cell types were studied. Central nervous systems of the snail were incubated for 5 and 20 h in various identical and equitoxic drug concentrations. Cisplatin had the most severe effects. Platinol, i.e., cisplatin dissolved in NaCl solution with the addition of HCl (pH 2.0-3.0), as well as cisplatin dissolved in snail Ringer's solution (pH 7.8), caused swelling of axons and distensions of the intercellular spaces. This drug induced an increase in chromatin clump size in the caudodorsal cells (20-h incubation), while carboplatin and iproplatin induced the formation of many small chromatin clumps. Incubation in snail Ringer's solution (controls) and cisplatin affect the morphology of the nucleoli. At high dosages of cisplatin, the nucleoli of light green cells were transformed into homogeneous dense structures. The data indicate that platinum compounds react with nuclear and nucleolar DNA. All three drugs affected the activity and organization of the rough endoplasmic reticulum and the Golgi apparatus of the peptidergic neurons studied (qualitative observations). These effects, which point to a reduced neuropeptide synthesis, may be secondary, i.e., exerted via inhibition of RNA synthesis and ribosome formation (nucleoli). The fact that the number of neuropeptide granules in the cytoplasm of the cells remained constant (both cell types) may indicate that granule transport was also inhibited. Cisplatin and iproplatin induced an increase in the number of lysosomes in the light green cells. The number of lipid droplets in these cells was not affected by drug treatment. The results corroborate clinical data indicating that cisplatin is highly neurotoxic. Despite conflicting clinical data, observations on the snail neurons suggest that iproplatin is also neurotoxic, although less than cisplatin. Carboplatin is minimally neurotoxic, which is in accordance with clinical data. The central nervous system of Lymnaea is a suitable model for studying possible neurotoxic effects of platinum compounds.

In the pars intermedia of the pituitary the prohormone proopiomelanocortin (POMC) is tissue-specifically processed to, among other peptides, alpha-melanotropin (alpha MSH). In the South African clawed toad Xenopus laevis this hormone mediates the process of background adaptation: release of alpha-MSH causes darkening of the animal, while inhibition of alpha-MSH release results in a pale toad. Elevated release of alpha-MSH coincides with a higher rate of POMC gene transcription. The present study aims to find possible transcriptional regulatory elements in the Xenopus POMC gene. For that purpose the complete nucleotide sequence of the POMC gene and its 5'- and 3'- flanking regions were determined and analyzed. The Xenopus POMC gene promoter contains several regions which may be regulatory DNA elements in view of their similarity with corresponding regions of mammalian POMC gene promoters. In the rat POMC gene promoter, many of these regions represent protein-binding sequences. Besides the promoter sequence and the protein-coding sequences, no other segments with significant identity between the Xenopus and human POMC genes were found. Intron A of the Xenopus POMC gene contains a simple sequence, (TATC)76, and a JH12 repetitive element, while the 3'-flanking region contains a repetitive-EcoRI-monomer-2 element. Comparison of the JH12 sequence of the POMC gene with JH12 sequences from other Xenopus genes revealed a 335-bp consensus sequence which is flanked by a 30-bp inverted repeat. This JH12 consensus sequence is significantly larger than the previously reported JH12 core region. Alignment of intron B of the Xenopus POMC gene with database sequences revealed a consensus sequence of a novel Xenopus repetitive element of 330 bp flanked by a nearly perfect inverted repeat, indicating that this element may be a transposon-like element.

A D2 dopamine receptor pharmacologically different from the mammalian D2 receptor has previously been characterized in the amphibian Xenopus laevis. Here we report the cloning of a Xenopus D2 receptor which revealed about 75% amino acid sequence identity with its mammalian counterpart and the presence of an additional 33 amino acid sequence in the 3rd cytoplasmic loop instead of the additional 29 residues in the large form of the mammalian D2 receptor. All 7 predicted transmembrane domains are highly conserved between the Xenopus and mammalian D2 receptors, as are the 1st and 2nd intracellular loop, the 1st and 3rd extracellular loop and the carboxy-terminal portion of the receptors. The amino-terminal portion, the 2nd extracellular loop and the middle portion of the 3rd intracellular loop of these receptors, however, differ considerably. Knowledge of the locations of these regions of conservation and divergence within the D2 receptors of Xenopus and mammals will help to delineate portions of the receptor molecule that are functionally important. Interestingly, the 5'-untranslated region of the Xenopus D2 receptor mRNA contains 4 small open reading frames which may affect translational efficiency.

The neuroendocrine protein 7B2 is highly conserved and widely present in neurons and endocrine cells. It is coexpressed with the prohormone proopiomelanocortin (POMC) in the intermediate lobe of the pituitary gland of Xenopus laevis. To study the biosynthesis of 7B2 in this amphibian, an anti-7B2 monoclonal antibody was used in immunoprecipitation analysis of newly synthesized radiolabeled proteins, produced by pulse and pulse-chase-incubated neurointermediate lobes. Following a 15-min pulse incubation, a single immunoprecipitable protein of 25 kDa was synthesized. During subsequent chase incubation, this newly synthesized 7B2 protein was processed to an 18-kDa immunoprecipitable form. Analysis of the chase incubation medium revealed that only the 18-kDa processed product of 7B2, and not 7B2 itself, had been secreted. This secretion is a regulated process because it was blocked completely by the dopamine receptor agonist apomorphine. A study of protein biosynthesis in lobes treated with tunicamycin to prevent N-linked glycosylation showed that in contrast to POMC and an 18-kDa derivative of POMC, neither 7B2 nor its 18-kDa derivative was glycosylated. Chemical and enzymatic peptide mapping showed that processing of 7B2 occurs in the carboxyl-terminal region. The function of the 7B2 protein is unknown; the present results show that 7B2 itself is a precursor molecule and can only have an intracellular function whereas an extracellular function can only be attributed to 7B2-derived peptides.

Eleven- to 12-wk-old rats were treated twice a week with cisplatin/saline or with cisplatin plus ORG.2766 during 12.5 wk. Cisplatin and ORG.2766 were administered at a final concentration of 0.04 mg/ml (i.p.) and 10 micrograms/ml (s.c.), respectively. Control animals were treated with saline. In this period the cisplatin-treated animals developed a peripheral neuropathy resulting in impairment of sensory functions. Estimates of the motor (MNCV) and sensory (SNCV) nerve conduction velocity were made after 0, 7.5, 10, and 12.5 wk. It appeared that the MNCV of the control, cisplatin-, and cisplatin plus ORG.2766-treated rats increased from 50 to 59 m/s. In contrast, the SNCV of the cisplatin-treated rats decreased significantly (P less than 0.001) from 63 to 56 m/s, whereas that of the control animals increased from 62 to 84 m/s. Rats which received cisplatin plus ORG.2766 showed an increase in SNCV up to control levels. After 12.5 wk the animals were perfused with a mixture of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.05 M phosphate buffer. At the level of L5 and L6, 5 mm of spinal cord tissue and three dorsal root ganglia were removed and processed for electron microscopy. With the point-counting method the volume fraction (v/v) of somata and myelin in spinal ganglia was estimated. No significant change in the volume fraction of somata of the control (0.42), cisplatin (0.33)-, and cisplatin plus ORG.2766 (0.39)-treated rats was found. The same held true for the volume fraction of myelin of the control (0.53), cisplatin (0.59)-, and cisplatin plus ORG.2766 (0.58)-treated rats. In addition, the number of lysosomes per 100 microns 2 was estimated in spinal ganglion neurons and in spinal cord motor neurons of a total of 120 randomly chosen neurons. It was found that the number of lysosomes in the spinal ganglion neurons of the control animals was lower (10 per 100 microns 2) than in cisplatin-treated (30 per 100 microns 2) and in cisplatin plus ORG.2766-treated rats (28 per 100 microns 2) (P less than 0.05). No difference was observed in the number of lysosomes between cisplatin- and cisplatin plus ORG.2766-treated rats. The number of lysosomes in spinal cord tissue of cisplatin-treated rats (2.4 per 100 microns 2) did not differ from controls (0.1 per 100 microns 2) and from cisplatin plus ORG.2766-treated rats (0.8 per 100 microns 2).(ABSTRACT TRUNCATED AT 400 WORDS)

The central nervous system of the snail Lymnaea stagnalis was studied in order to develop a test system to predict the neurotoxic side effects of the three cytostatic Vinca alkaloids, vincristine (VCR), vindesine (VDS), and vinblastine (VLB). Vinca alkaloids appear to interfere with microtubule formation by the induction of paracrystalline inclusions. After in vitro incubation the numbers of these inclusions were counted in cross-sections of the cerebral commissure using electron microscopy. For each compound the number of paracrystalline profiles increases with increasing concentrations and incubation times. At equimolar concentrations (0.15 mM), VCR induces more paracrystals than VDS, and VDS induces more than VLB. These effects are clear after short periods of incubation (e.g., after 2 h, VCR:VDS:VLB = 5:2:1). Equitoxic concentrations of VCR, VDS, and VLB induce similar numbers of paracrystals. Furthermore, morphological changes in the cell bodies of identified neurons (light green cells) in the cerebral ganglia were observed. Quantitative analysis shows that at equimolar concentrations the surface area of nuclear chromatin of all Vinca alkaloid-treated cells is approximately 30% lower than that of the controls. The lamellae of the rough endoplasmic reticulum are swollen and have lost their regular arrangement. For VDS and VLB this swelling is accompanied by a strong increase (about 3-fold) in the total surface area of the rough endoplasmic reticulum. No increase was observed for VCR. The compounds do not affect the number of secretory granules. In contrast to the controls, all Vinca-treated cells show lipid droplets. After VCR treatment they are about 5-fold as numerous as after treatment with VDS or VLB. The total surface area of lysosomes increases about 1.3-fold by VDS and VLB treatment and about 3-fold by VCR treatment. From these quantitative data it is concluded that VCR is more neurotoxic than VDS and VLB. VDS appears to be more neurotoxic than VLB as judged from the data on paracrystal induction. On the basis of a comparison of these data with clinical data on Vinca-induced neurotoxicities, it is proposed that neurons of the snail L. stagnalis may be suitable for the development of a test system to predict the degree of clinical neurotoxicity induced by Vinca antitumor drugs.

The suitability of neurons of the freshwater snail Lymnaea stagnalis as a test system for the neurotoxic side effects of antitumour Vinca alkaloids has been investigated, by studying the process of paracrystal induction by Vinca antitumour agents. Three Vinca alkaloids have been compared: the natural vinblastine and vincristine and the semisynthetic vindesine. They appear to induce two types of inclusion. The first type is paracrystalline and has a rod-like shape with a width of 0.3-2.5 micron and a length of 1-10 micron. It consists of hexagonally arranged tubules with a lattice constant of approximately 28 nm. The second type appears as ladder-like profiles with a periodicity of approximately 30 nm. It is proposed that the ladder-like profiles are in fact helical structures and are precursors of the paracrystals. Both types of inclusion may fill up large parts of the axons; they are rare in axon terminals and almost absent from the neuronal somata. It has been concluded that the process of paracrystal induction by Vinca alkaloids in Lymnaea neurons is very much the same as in mammalian neurons and may be largely responsible for the neurotoxic effects of the Vinca drugs, because it impairs axonal transport of neuronal secretory granules. Apparently, in this respect vindesine behaves in a similar way as the conventional vincristine and vinblastine drugs. Vincristine induces clearly more paracrystals than vindesine, whereas the least paracrystals occur in vinblastine-treated material. These differences correlate with the different clinical neurotoxicities of these drugs. Therefore, because Lymnaea neurons can be considered as excellent model systems for studies of the functioning of neurons in general, it is expected that counting the number of paracrystals in Lymnaea nervous tissue will prove to be a good method to predict the degree of clinical neurotoxicity of newly developed antitumor Vinca alkaloids.