Anesthetic drugs modulate feeding behavior and hypothalamic expression of the POMC polypeptide precursor and the NPY neuropeptide.

BACKGROUND: Several hypnotic drugs have been previously identified as modulators of food intake, but exact mechanisms remain unknown. Feeding behavior implicates several neuronal populations in the hypothalamic arcuate nucleus including orexigenic neuropeptide Y and anorexigenic pro-opiomelanocortin producing neurons. The aim of this study was to investigate in mice the impact of different hypnotic drugs on food consumption and neuropeptide Y or pro-opiomelanocortine mRNA expression level in the hypothalamic arcuate nucleus. METHODS: Saline control, isoflurane, thiopental, midazolam or propofol were administered to C57Bl/6 mice. Feeding behavior was evaluated during 6 h. In situ hybridization of neuropeptide Y and pro-opiomelanocortine mRNAs in the hypothalamus brain region was also performed. Data were analyzed by Kruskal Wallis test and analysis of variance (p < 0.05). RESULTS: Midazolam, thiopental and propofol induced feeding behavior. Midazolam and thiopental increased neuropeptide Y mRNA level (respectively by 106 and 125%, p < 0.001) compared with control. Propofol and midazolam decreased pro-opiomelanocortine mRNA level by 31% (p < 0,01) compared with control. Isoflurane increased pro-opiomelanocortine mRNA level by 40% compared with control. CONCLUSION: In our murine model, most hypnotics induced food consumption. The hypnotic-induced regulation of neuropeptide Y and pro-opiomelanocortine hypothalamic peptides is associated with this finding. Our data suggest that administration of some hypnotic drugs may affect hypothalamic peptide precursor and neuropeptide expression and concomittantly modulate food intake. Thus, this questions the choice of anesthetics for better care management of patients undergoing major surgery or at risk of undernutrition.

[Interaction between hypnotic agents and the hypothalamic-pituitary-adrenocorticotropic axis during surgery]. / Modulation de l'axe hypothalamo-hypophyso-surrénalien par l'utilisation des agents hypnotiques dans le contexte chirurgical.

During stress, the relationship between the central nervous system and the immune system is essential to maintain homeostasis. The main neuroendocrine system involved in this interaction is the hypothalamic-pituitary-adrenal axis (HPA), which via the synthesis of glucocorticoids will modulate the intensity of the inflammatory response. Anaesthetic agents could be interacting with the HPA axis during surgery. Although etomidate currently remains in the center of the discussions, it seems, at least experimentally, that most hypnotics have the capacity to modulate the synthesis of adrenal steroids. Nevertheless, with the large literature on this subject, etomidate seems to be the most deleterious hypnotic agent on the HPA axis function. Its use should be limited when HPA axis is already altered.

Chemotherapy-induced long-term alteration of executive functions and hippocampal cell proliferation: role of glucose as adjuvant.

In patients, cancer and treatments provoke cognitive impairments referred to "chemofog". Here a validated neurobehavioral animal model, the unique way to explore causal direct links between chemotherapy used in clinical practices and brain disorders, allowed investigation of the direct long-term impact of colo-rectal cancer chemotherapy on cognition and cerebral plasticity. Young and aged mice received three injections every 7 days during 2 weeks of 5-fluorouracil either alone (5-FU, 37.5 mg/kg) or in combination with oxaliplatin (3 mg/kg) or with glucose (5%). The long-term effects (from day 24 to day 60) of chemotherapy were tested on emotional reactivity, learning and memory, behavioral flexibility and hippocampal cell plasticity. 5-FU (in saline)-treated aged and also young mice exhibited specific altered cognitive flexibility and behavioral hyper-reactivity to novelty, whereas the combination 5-FU (in saline)/oxaliplatin (in glucose) did not provoke any cognitive dysfunction. We thus observed that glucose counteracted 5-FU-induced altered executive functions and hippocampal cell proliferation in vivo, and protected neural stem cells in vitro from toxicity of 5-FU or oxaliplatin. In conclusion, these data suggest that the lasting chemotherapy-induced selective impairment of executive functions, whatever the age, and associated with a reduced number of hippocampal proliferating cells, can be counteracted by co-administration with glucose.

The octadecaneuropeptide stimulates somatolactin release from cultured goldfish pituitary cells.

The present study aimed to investigate the distribution of the octadecaneuropeptide (ODN) in the goldfish brain and to look for a possible effect of ODN on somatolactin (SL) release from pituitary cells. A discrete population of ODN-immunoreactive neurones was localised in the lateral part of the nucleus lateralis tuberis. These neurones sent projections through the neurohypophyseal tract towards the neurohypophysis, and nerve fibres were seen in the close vicinity of SL-producing cells in the pars intermedia. Incubation of cultured goldfish pituitary cells with graded concentrations of ODN (10(-9) -10(-5 ) m) induced a dose-dependent stimulation of SL-ß, but not SL-α, release. ODN-evoked SL release was blocked by the metabotrophic endozepine receptor antagonist cyclo(1-8) [DLeu(5) ]OP but was not affected by the central-type benzodiazepine receptor antagonist flumazenil. ODN-induced SL release was suppressed by treatment with the phospholipase C (PLC) inhibitor U-73122 but not with the protein kinase A (PKA) inhibitor H-89. These results indicate that, in fish, ODN produced by hypothalamic neurones acts as a hypophysiotrophic neuropeptide stimulating SL release. The effect of ODN is mediated through a metabotrophic endozepine receptor positively coupled to the PLC/inositol 1,4,5-trisphosphate/protein kinase C-signalling pathway.

The octadecaneuropeptide exerts an anxiogenic-like action in goldfish.

I.c.v. administration of the octadecaneuropeptide (ODN), a peptide derived from diazepam-binding inhibitor (DBI), induces anorexigenic and anxiogenic-like actions in rodents. We have recently shown that, in goldfish, i.c.v. injection of ODN also reduces food consumption via the metabotropic endozepine receptor. However, there is little information regarding the structure of DBI and the psychophysiological roles of endozepines in fish. Therefore, in the present study, we isolated and cloned a cDNA encoding goldfish DBI. The deduced sequence exhibits high similarity with non-mammalian DBIs, and we investigated the effect of homologous ODN on psychomotor activity in goldfish. i.c.v. injection of synthetic goldfish ODN at 10 pmol/g body weight (BW) stimulated locomotor activity. Since intact goldfish placed in a tank with both black and white background areas prefers the black compartment, we developed a method for measuring the time taken for fish to move from the black to the white area. I.c.v. administration of diazepam (35 and 350 pmol/g BW) decreased, whereas i.c.v. administration of ODN (10 pmol/g BW) or the central-type benzodiazepine receptor inverse agonist FG-7142 (9 pmol/g BW) increased the time taken to move from the black to the white background area. The anxiogenic-like effect of ODN was blocked by the central-type benzodiazepine receptor antagonist flumazenil (100 pmol/g BW), but was not affected by the metabotropic endozepine receptor antagonist cyclo1-8[d-Leu(5)]octapeptide (100 pmol/g BW). These data indicate that ODN can potently affect locomotor and psychomotor activities in goldfish and that this action is mediated via the central-type benzodiazepine receptor-signaling pathway.

Infections as triggers and complications of systemic lupus erythematosus.

A growing body of experimental and clinical evidence supports the pivotal role of infections in the induction or exacerbation of systemic lupus erythematosus (SLE). Infections can be responsible for aberrant immune response leading to a loss of tolerance towards native proteins. Molecular mimicry, especially between Sm or Ro autoantigens and EBV Nuclear Antigen-1 response, as well as the over-expression of type 1 INF genes are among the major contributors to SLE development. On the other hand infections are very common in SLE patients, where they are responsible for 30-50% of morbidity and mortality. Several factors, either genetic, including complement deficiencies or mannose-binding lectin deficiency or acquired such as severe disease manifestations or immunosuppressant use, predispose SLE patients to infections. All types of infections, including bacterial, viral and opportunistic infections, have been reported and the most frequently involved sites of infections are the same as those observed in the general population, including respiratory, skin, and urinary tract infections. Some preventive measures could be adopted in order to reduce the rate of infections in SLE patients: i.e. screening for Mycobacterium tuberculosis and for some chronic viral infections before immunosuppressive treatment; adequate prophylaxes or drug adjustments when indicated, and pneumococcal and influenza vaccinations in patients with stable disease.

Identification and characterization of two novel (neuro)endocrine long coiled-coil proteins.

We have identified a novel vertebrate-specific gene by applying a Differential Display method on two distinct subtypes of pituitary melanotropes showing divergent secretory phenotypes of hypo- and hypersecretion. A paralogue of this gene was also identified. The existence of a long coiled-coil domain and a C-terminal transmembrane domain in the sequences, together with the Golgi distribution of the proteins in transfected cells, suggest that they can be considered as new members of the golgin family of proteins. Both genes were primarily expressed in (neuro)endocrine tissues in vertebrates thus supporting a role for these proteins in the regulated secretory pathway.

Differential expression and processing of chromogranin A and secretogranin II in relation to the secretory status of endocrine cells.

Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.

In vivo action of a new octadecaneuropeptide antagonist on neuropeptide Y and corticotropin-releasing hormone mRNA levels in rat.

It has been reported that several of the effects induced by an octadecaneuropeptide (ODN), derived from an 86-amino-acid polypeptide termed diazepam-binding inhibitor, could be mediated by activation of a metabotropic receptor. In order to investigate the role and mechanism of action of ODN in the regulation of corticotropin-releasing factor (CRH) and neuropeptide Y (NPY) expression in the paraventricular nucleus and arcuate nucleus, respectively, we studied the effects of the acute intracerebroventricular administration of ODN (2 microg/rat) and the ODN antagonist to metabotropic receptor, cyclo(1-8)[Dleu5]OP (20 microg/rat), on the gene expression of the two neuropeptides in castrated male rat. ODN administration resulted in a 45% increase in CRH mRNA expression, an effect which was reversed by cyclo(1-8)[Dleu5]OP. When cyclo(1-8)[Dleu5]OP was administered alone, it induced a 19% decrease in CRH mRNA levels. ODN administration induced a 17% decrease in NPY mRNA expression while cyclo(1-8)[Dleu5]OP increased by 21% the hybridization signal. The administration of both ODN and ODN antagonist completely abolished the depressing effect of ODN on NPY mRNA. These data suggest that the effects of ODN on CRH and NPY mRNA might be mediated by interaction with metabotropic receptors. Moreover, since cyclo(1-8)[Dleu5]OP can by itself influence the expression of two peptide mRNAs, it might be suggested that ODN is exerting a tonic influence on NPY and CRH neurons.

Analysis of Rab18 and a new golgin in the secretory pathway.

Two new amphibian genes have been isolated and characterized from frog melanotropes, and the level of expression of these genes is related to the secretory status of the cells. Both genes, Rab18 and a novel member of the golgin family of proteins, are ubiquitously expressed in endocrine and nonendocrine tissues, and their corresponding proteins appear to show intracellular distributions associated with discrete vesicular and tubular structures, respectively, suggesting that they may play relevant roles in the regulation of the secretory pathway.

In vivo action of a new octadecaneuropeptide (ODN) antagonist on gonadotropin-releasing hormone gene expression in the male rat brain.

It has been reported that several of the effects induced by octadecaneuropeptide (ODN) could be mediated by an activation of a metabotropic receptor. In order to investigate the role and mechanism of action of ODN in gonadotropin-releasing hormone (GnRH) neuron regulation, we studied the effects of the acute i.c.v. administration of ODN and of a new ODN antagonist to metabotropic receptor, cyclo(1-8)[Dleu(5)]OP, on GnRH mRNA expression as evaluated by in situ hybridization in castrated male rats. The administration of ODN produced a decrease in the hybridization signal while the administration of cyclo(1-8)[Dleu(5)]OP alone produced an 18% increase. When administrated concomitantly with ODN, the antagonist both inhibited the depressing effect of ODN and induced a 22% increase over the values detected in ODN-treated rats. The data suggest that the effect of ODN on GnRH mRNA expression might be mediated by interaction with metabotropic receptors.

Two different transduction pathways are activated by C3a and C5a anaphylatoxins on astrocytes.

C3a and C5a anaphylatoxins are two proinflammatory peptides generated during complement system activation. C3a and C5a exert several biological activities through binding to their specific receptors, named C3aR and C5aR, respectively. We have previously shown that C3aR and C5aR are constitutively expressed by astrocytes, a cell type that actively participates in inflammatory events in the central nervous system. In this article, we focus on the transduction signal pathways activated by these two receptors on astrocytes. We show that the stimulation of C3aR or C5aR results in the activation of the mitogen activated protein kinase pathway by phosphorylation of the p44 and p42 kinases. On the contrary, the binding of C3a or C5a to their receptors on astrocytes decreases the production of cAMP, revealing an inhibition of the adenylyl cyclase pathway. Stimulation of C3aR and C5aR induces an increase in intracellular calcium concentration, arising from the opening of intracellular calcium channels. The observed calcium wave results from the activation of the phospholipase C pathway. Taken together, our results suggest that the binding of C3a or C5a to their receptors on astrocytes would be of functional importance since it induces the activation of two important transduction pathways leading to several cellular events such as neurotrophin and cytokine production.

Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN. Design of a potent endozepine antagonist.

The octadecaneuropeptide (ODN; QATVGDVNTDRPGLLDLK) and its C-terminal octapeptide (OP; RPGLLDLK), which exert anxiogenic activity, have been previously shown to increase intracellular calcium concentration ([Ca2+]i) in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C. It has also been found that the [d-Leu5]OP analog possesses a weak antagonistic activity. The aim of the present study was to synthesize and characterize cyclic analogs of OP and [d-Leu5]OP. On-resin homodetic backbone cyclization of OP yielded an analog, cyclo1-8 OP, which was three times more potent and 1.4-times more efficacious than OP to increase [Ca2+]i in cultured rat astrocytes. Cyclo1-8 OP also mimicked the effect of both OP and ODN on polyphosphoinositide turnover. Conversely, the cyclo1-8 [d-Leu5]OP analog was totally devoid of agonistic activity but suppressed the effect of OP and ODN on [Ca2+]i and phosphoinositide metabolism in astrocytes. The structure of these cyclic analogs has been determined by two-dimensional 1H-NMR and molecular dynamics. Cyclo1-8 OP exhibited a single conformation characterized by a gamma turn comprising residues Pro2-Leu4 and a type III beta turn encompassing residues Leu5-Lys8. Cyclo1-8 [d-Leu5]OP was present as two equimolar conformers resulting from cis/trans isomerization of the Arg-Pro peptide bond. These pharmacological and structural data should prove useful for the rational design of non peptidic ODN analogs.

Melanotrope cell plasticity: a key mechanism for the physiological adaptation to background color changes.

The intermediate lobe of the pituitary secretes the melanotropic hormone alpha-MSH, which in amphibians plays a crucial role in skin color adaptation. It has been previously demonstrated that, in the frog Rana ridibunda, the intermediate lobe is composed of two distinct subpopulations of melanotrope cells that can be separated in vitro by using Percoll density gradients. These two melanotrope cell subsets, referred to as high-density (HD) and low-density (LD) cells, differ in their ultrastructural characteristics as well as in their biosynthetic and secretory activity. However, the specific, physiological role of the heterogeneity displayed by melanotrope cells remains elusive. In the present study, we investigated the effects of background color adaptation on melanotrope cell subpopulations. We found that adaptation of frogs to dark or white environment did not modify either the overall number of cells per intermediate lobe or the apoptotic and proliferation rates of melanotrope cells. On the other hand, adaptation of the animals to a white background significantly increased the proportion of hormone-storage HD cells and caused a concomitant decrease in that of LD cells (which exhibit higher levels of alpha-MSH release and POMC messenger RNA than HD cells). Conversely, after black-background adaptation the proportion of LD cells was markedly increased, suggesting that interconversion of HD cells to LD cells occurs during physiological activation of the intermediate lobe. In addition, black-background adaptation also enhanced alpha-MSH release by both cell subpopulations and increased inositol phosphate production in LD cells. These data indicate that, in frog, the proportions of the two melanotrope cell subsets undergo marked modifications during skin color adaptation, likely reflecting the occurrence of a secretory cell cycle whose dynamics are highly correlated to the hormonal demand imposed by the environment.

The octadecaneuropeptide ODN stimulates neurosteroid biosynthesis through activation of central-type benzodiazepine receptors.

Neurosteroids may play a major role in the regulation of various neurophysiological and behavioural processes. However, while the biochemical pathways involved in the synthesis of neuroactive steroids in the central nervous system are now elucidated, the mechanisms controlling the activity of neurosteroid-producing cells remain almost completely unknown. In the present study, we have investigated the effect of the octadecaneuropeptide (ODN), an endogenous ligand of benzodiazepine receptors, in the control of steroid biosynthesis in the frog hypothalamus. Glial cells containing ODN-like immunoreactivity were found to send their thick processes in the close vicinity of neurones expressing the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase. Exposure of frog hypothalamic explants to graded concentrations of ODN (10(-10)-10(-5) M) produced a dose-dependent increase in the conversion of tritiated pregnenolone into various radioactive steroids, including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone and dihydrotestosterone. The ODN-induced stimulation of neurosteroid biosynthesis was mimicked by the central-type benzodiazepine receptor (CBR) inverse agonists methyl beta-carboline-3-carboxylate (beta-CCM) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The stimulatory effects of ODN, beta-CCM and DMCM on steroid formation was markedly reduced by the CBR antagonist flumazenil. The ODN-evoked stimulation of neurosteroid production was also significantly attenuated by GABA. Collectively, these data indicate that the endozepine ODN, released by glial cell processes in the vicinity of 3 beta-hydroxysteroid dehydrogenase-containing neurones, stimulates the biosynthesis of neurosteroids through activation of central-type benzodiazepines receptors.

The triakontatetraneuropeptide (TTN) stimulates thymidine incorporation in rat astrocytes through peripheral-type benzodiazepine receptors.

Astrocytes and astrocytoma cells actively express the diazepam-binding inhibitor (DBI) gene, suggesting that DBI-processing products may regulate glial cell activity. In the present study, we have investigated the possible effect of one of the DBI-derived peptides, the triakontatetraneuropeptide (TTN), on [(3)H]thymidine incorporation in cultured rat astrocytes. Reversed-phase HPLC analysis of incubation media indicated that TTN is the major form of DBI-derived peptides released by cultured astrocytes. At very low concentrations (10(-14)-10(-11) M), TTN induced a dose-dependent increase in [(3)H]thymidine incorporation, whereas at higher concentrations (10(-10)-10(-5) M) the effect of TTN gradually declined. In the same range of concentrations, the specific peripheral-type benzodiazepine receptor (PBR) agonist Ro 5-4864 mimicked the bell-shaped stimulatory effect of TTN on [(3)H]thymidine incorporation. The PBR antagonist PK11195 (10(-6) M) suppressed the stimulatory action of both TTN and Ro 5-4864 on [(3)H]thymidine incorporation, whereas the central-type benzodiazepine receptor antagonist flumazenil (10(-6) M) had no effect. The present study demonstrates that the endozepine TTN stimulates DNA synthesis in rat glial cells through activation of PBRs. These data strongly suggest that TTN exerts an autocrine/paracrine stimulatory effect on glial cell proliferation.

Neurosteroid progesterone is up-regulated in the brain of jimpy and shiverer mice.

Concentrations of neurosteroids have been measured in the brains of postnatal myelin mutants jimpy (jp) and shiverer (shi) mice and of their normal controls. Progesterone (PROG) concentrations were increased more than threefold in the brains of mutant mice. Marked astroglial reaction occurs in the brains of jp mice and to a much smaller extent in shi ones. Whereas the mitochondrial benzodiazepine/diazepam binding inhibitor (DBI) receptor (MBR) was below the immunohistochemical detection limit in normal mice (except in the choroid plexus and ependyma cells), it was significantly expressed in many reactive astrocytes of jp and shi mice brains. DBI-like peptides, investigated either by immunohistochemistry or by radioimmunoassay, were expressed to similar extents in mutant and control mice. Reversed-phase HPLC indicated that DBI-like peptides were almost exclusively of the triakontatetraneuropeptide size. It was concluded that the increased expression of MBR (involved in the intramitochondrial delivery of cholesterol to P450scc) likely accounts for the large PROG content in mutant mice brain. The role of PROG in myelin repair is discussed.

Involvement of protein kinase C and protein tyrosine kinase in thyrotropin-releasing hormone-induced stimulation of alpha-melanocyte-stimulating hormone secretion in frog melanotrope cells.

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.

The endozepine ODN stimulates [3H]thymidine incorporation in cultured rat astrocytes.

High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10(-14) to 10(-11) M), ODN caused a dose-dependent increase of [3H]thymidine incorporation. At higher doses (10(-10) to 10(-5) M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10(-6) M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10(-6) M) had no effect. The ODN-induced stimulation of [3H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The GABAA receptor antagonist bicuculline (10(-4) M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABAA agonist 3APS (10(-10) to 10(-4) M) also induced a dose-related increase of [3H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABAA receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation.

Immunohistochemical localization and biochemical characterization of two novel decapeptides derived from POMC-A in the trout hypothalamus.

Several vertebrate species which underwent duplication of their genome, such as trout, salmon and Xenopus, possess two proopiomelanocortin (POMC) genes. In the trout, one of the POMC molecules, called POMC-A, exhibits a unique C-terminal extension of 25 amino acids which has no equivalent in other POMCs characterized so far. This C-terminal peptide contains three pairs of basic residues, suggesting that it may be the source of novel regulatory peptides. The aim of the present study was to investigate the occurrence of these peptides in the brain of the trout Oncorhynchus mykiss by using specific antibodies raised against two epitopes derived from the C-terminal extension of POMC-A, i.e., EQWGREEGEE and YHFQ-NH2. Immunohistochemical labeling of brain sections revealed the presence of EQWGREEGEE- and YHFQ-NH2-immunoreactive cell bodies in the anterior part of the nucleus lateralis tuberis of the hypothalamus. Immunoreactive fibers were observed in the dorsal hypothalamus, the thalamus, the telencephalon, the optic tectum and the medulla oblongata. In contrast, no labeling was detected using antibodies against the non-amidated peptide YHFQG. Biochemical characterization was performed by combining high-performance liquid chromatography (HPLC) analysis with radioimmunoassay (RIA) quantification. Two peptides exhibiting the same retention time as synthetic EQWGREEGEE and ALGERKYHFQ-NH2 were resolved. However, no peptide co-eluting with YHFQ-NH2 or YHFQG could be detected. These results demonstrate that, in the trout brain, post-translational processing of POMC-A generates the two decapeptides EQWGREEGEE and ALGERKYHFQ-NH2. The wide distribution of immunoreactive fibers in the diencephalon, telencephalon, optic tectum and medulla oblongata suggests that these peptides may exert neurotransmitter and/or neuromodulator activities.