Endocrine and behavioral effects of neuromedin S

The present experiments focused on the effects of neuromedin S on hypothalamic–pituitary–adrenal (HPA) activation and behavior. The peptide (0.25–1 nmol) was administered intracerebroventricularly to rats, the behavior of which was monitored by means of telemetry, open field observations and an elevated plus-maze (EPM) test. Autonomic functions such as the temperature and the heart rate were recorded by telemetry. The action on the HPA axis was assessed via measurements of the plasma corticosterone and ACTH levels. To reveal the transmission of the endocrine responses, animals were pretreated with corticotrophin releasing hormone receptor (CRHR) antagonists (1 nmol). In the open field test, the animals were pretreated with either a CRHR₁ antagonist (antalarmin) or haloperidol (10 μg/kg), while in the EPM test they were pretreated with antalarmin or diazepam (1 mg/kg). The dopamine release from striatal and amygdala slices after peptide treatment was measured with a superfusion apparatus. Neuromedin S exerted dose-dependent effects on the HPA system, which were inhibited by antalarmin. It also activated grooming and decreased the entries to and time spent in the open arms during the EPM test. The grooming response was abolished by haloperidol and antalarmin pretreatment, while diazepam and antalarmin showed a tendency to attenuate the response evoked in the EPM test. In the superfusion studies, neuromedin S enhanced the dopamine release from the amygdala slices. These results demonstrate that neuromedin S stimulates the HPA axis through the CRHR₁ pathway and evokes stereotyped behavior and anxiety through mesolimbic dopamine and corticotrophin releasing hormone release.     

Endocrine, behavioral and autonomic effects of neuropeptide AF

The actions of neuropeptide AF (NPAF), on the hypothalamic-pituitary-adrenal (HPA) axis, behavior and autonomic functions were investigated. NPAF (0.25, 0.5, 1, 2 nmol) was administered intracerebroventricularly to rats, the behavior of which was monitored by means of telemetry, open-field (OF) observations and elevated plus-maze (EPM) tests. The temperature and heart rate were recorded by telemetry, and the plasma ACTH and corticosterone levels were used as indices of the HPA activation. The dopamine release from striatal and amygdala slices after peptide treatment (100 nM and 1 μM) was measured with a superfusion apparatus. To establish the transmission of the HPA response, animals were pretreated with the corticotrophin-releasing hormone (CRH) receptor antagonist antalarmin or astressin 2B (0.5 nmol). In the OF test, the animals were pretreated with antalarmin or haloperidol (10 μg/kg), while in the EPM test they were pretreated with antalarmin or diazepam (1 mg/kg). NPAF stimulated ACTH and corticosterone release, which was inhibited by antalarmin. It activated exploratory locomotion (square crossings and rearings) and grooming in OF observations, and decreased the entries to and the time spent in the open arms during the EPM tests. The antagonists inhibited the locomotor responses, and also attenuated grooming and the EPM responses. NPAF also increased spontaneous locomotion, and tended to decrease the core temperature and the heart rate in telemetry, while it augmented the dopamine release from striatal and amygdala slices. These results demonstrate, that acute administration of exogenous NPAF stimulates the HPA axis and behavioral paradigms through CRH and dopamine release.     

Behavioral and neuroendocrine actions of the Met-enkephalin-related peptide MERF

The effects and the mediation of the action of the proenkephalin derivative Met(5)-enkephalin-Arg(6)-Phe(7) (MERF) on the hypothalamo-pituitary-adrenal (HPA) system and open-field behavior were investigated in mice. Intracerebroventricular injection of the heptapeptide increased square crossing, rearing, and plasma corticosterone level. To characterize the receptors involved in these neuroendocrine processes, animals were pretreated either with the nonselective opioid antagonist naloxone or the kappa-antagonist nor-binaltorphimine (nor-BNI). Both antagonists dose-dependently attenuated the HPA activation elicited by MERF. Naloxone also blocked the behavioral responses, but nor-binaltorphimine did not elicit a significant inhibition. The dopamine antagonist haloperidol and a corticotropin-releasing hormone (CRH) antagonist were also preadministered to shed light on the transmission of the actions of MERF. Both the motor responses and the HPA activation were diminished by the preadministration of the CRH antagonist, while haloperidol attenuated only square crossing and rearing. To investigate the direct effect of MERF on the dopaminergic system, dopamine release of striatal slices was measured in a superfusion system. Neither the basal nor the electric impulse-evoked dopamine release was modified by MERF. The results suggest that opioid-mediation predominate in the neuroendocrine actions of MERF, and the effect of the heptapeptide on the HPA system seems to be mediated by kappa-receptors. In the behavioral responses evoked by MERF, both CRH release and the action of the dopaminergic neurons of the subcortical motor system might be involved. MERF also appears to activate the paraventricular CRH neurons, but dopaminergic transmission does not seem to play a significant role in its hypothalamic action.     

Asparagus racemosus Attenuates Anxiety-Like Behavior in Experimental Animal Models

Asparagus racemosus Linn. (AR) is used worldwide as a medicinal plant. In the present study, the anxiolytic activity of standardized methanolic extract of root of AR (MAR) was evaluated in open-field test (OFT), hole-board, and elevated plus maze (EPM) tests. Rats received oral pretreatment of MAR in the doses of 50, 100, and 200 mg/kg daily for 7 days and then were evaluated for the anxiolytic activity in different animal models. Both MAR (100 and 200 mg/kg) and diazepam (1 mg/kg, p.o.) increased the grooming behavior, number of central squares crossed, and time spent in the central area during OFT. Further, MAR (100 and 200 mg/kg) increased the head-dip and head-dip/sniffing behavior, and decreased sniffing activity in hole-board test. Furthermore, MAR (100 and 200 mg/kg) increased the percentage entries and time spent to open arm in EPM test paradigm. The anxiolytic activity in the experimental models was similar to that of diazepam. MAR (100 and 200 mg/kg) enhanced the level of amygdalar serotonin and norepinephrine. It also increased the expression of 5-HT_2A receptors in the amygdala. In another set of experiment, flumazenil attenuated the anxiolytic effect of minimum effective dose of MAR (100 mg/kg) in OFT, hole-board, and EPM tests, indicating GABA_A-mediated mechanism. Moreover, the anxiolytic dose of MAR did not show sedative-like effect in OFT and EPM tests compared to diazepam (6 mg/kg, p.o.). Thus, the anxiolytic response of MAR may involve GABA and serotonergic mechanisms. These preclinical data show that AR can be a potential agent for treatment of anxiety disorders.     

Orexin A-induced anxiety-like behavior is mediated through GABA-ergic, α- and β-adrenergic neurotransmissions in mice

Orexins are hypothalamic neuropeptides, which are involved in several physiological functions of the central nervous system, including anxiety and stress. Several studies provide biochemical and behavioral evidence about the anxiogenic action of orexin A. However, we have little evidence about the underlying neuromodulation. Therefore, the aim of the present study was to investigate the involvement of neurotransmitters in the orexin A-induced anxiety-like behavior in elevated plus maze (EPM) test in mice. Accordingly, mice were pretreated with a non-selective muscarinic cholinergic antagonist, atropine; a γ-aminobutyric acid subunit A (GABA-A) receptor antagonist, bicuculline; a D2, D3, D4 dopamine receptor antagonist, haloperidol; a non-specific nitric oxide synthase (NOS) inhibitor, nitro-l-arginine; a nonselective α-adrenergic receptor antagonist, phenoxybenzamine and a β-adrenergic receptor antagonist, propranolol 30 min prior to the intracerebroventricular administration of orexin A. The EPM test started 30 min after the i.c.v. injection of the neuropeptide. Our results show that orexin A decreases significantly the time spent in the arms (open/open + closed) and this action is reversed by bicuculline, phenoxybenzamine and propranolol, but not by atropine, haloperidol or nitro-l-arginine. Our results provide evidence for the first time that the orexin A-induced anxiety-like behavior is mediated through GABA-A-ergic, α- and β-adrenergic neurotransmissions, whereas muscarinic cholinergic, dopaminergic and nitrergic neurotransmissions may not be implicated.     

CRF1-R Activation of the Dynorphin/Kappa Opioid System in the Mouse Basolateral Amygdala Mediates Anxiety-Like Behavior

Stress is a complex human experience and having both rewarding and aversive motivational properties. The adverse effects of stress are well documented, yet many of underlying mechanisms remain unclear and controversial. Here we report that the anxiogenic properties of stress are encoded by the endogenous opioid peptide dynorphin acting in the basolateral amygdala. Using pharmacological and genetic approaches, we found that the anxiogenic-like effects of Corticotropin Releasing Factor (CRF) were triggered by CRF₁-R activation of the dynorphin/kappa opioid receptor (KOR) system. Central CRF administration significantly reduced the percent open-arm time in the elevated plus maze (EPM). The reduction in open-arm time was blocked by pretreatment with the KOR antagonist norbinaltorphimine (norBNI), and was not evident in mice lacking the endogenous KOR ligand dynorphin. The CRF₁-R agonist stressin 1 also significantly reduced open-arm time in the EPM, and this decrease was blocked by norBNI. In contrast, the selective CRF₂-R agonist urocortin III did not affect open arm time, and mice lacking CRF₂-R still showed an increase in anxiety-like behavior in response to CRF injection. However, CRF₂-R knockout animals did not develop CRF conditioned place aversion, suggesting that CRF₁-R activation may mediate anxiety and CRF₂-R may encode aversion. Using a phosphoselective antibody (KORp) to identify sites of dynorphin action, we found that CRF increased KORp-immunoreactivity in the basolateral amygdala (BLA) of wildtype, but not in mice pretreated with the selective CRF₁-R antagonist, antalarmin. Consistent with the concept that acute stress or CRF injection-induced anxiety was mediated by dynorphin release in the BLA, local injection of norBNI blocked the stress or CRF-induced increase in anxiety-like behavior; whereas norBNI injection in a nearby thalamic nucleus did not. The intersection of stress-induced CRF and the dynorphin/KOR system in the BLA was surprising, and these results suggest that CRF and dynorphin/KOR systems may coordinate stress-induced anxiety behaviors and aversive behaviors via different mechanisms.     

Dopamine depletion in nucleus accumbens reduces ACTH1–24-induced excessive grooming

Animals were pretreated with 6-OHDA or ascorbate vehicle injected into the nucleus accumbens and tested 10 days later for excessive grooming induced by intracerebroventricular injection of ACTH_1–24. The animals pretreated with 6-OHDA showed a significant decrease in excessive grooming produced by the neuropeptide and this reduction was seen only in the last 30 minutes of a 60-min test session. The results suggest an interaction of ACTH with dopamine systems on the onset and maintenance of excessive grooming.     

Mediation of the behavioral, endocrine and thermoregulatory actions of ghrelin

The action of ghrelin on telemetrically recorded motor activity and the transmission of the effects of this neuropeptide on spontaneous and exploratory motor activity and some related endocrine and homeostatic parameters were investigated. Different doses (0.5-5 microg) of ghrelin administered intracerebroventricularly caused significant increases in both square crossing and rearing activity in the "open-field" apparatus, while only the dose of 5 microg evoked a significant increase in the spontaneous locomotor activity recorded by telemetry. Ghrelin also induced significant increases in corticosterone release and core temperature. To determine the transmission of these neuroendocrine actions, the rats were pretreated with different antagonists, such as a corticotropin-releasing hormone (CRH) antagonist (alpha-helical CRH(9-41)), the nitric oxide synthase inhibitor Nomega-nitro-L-arginine-methyl ester (L-NAME), haloperidol, cyproheptadine or the cyclooxygenase inhibitor noraminophenazone (NAP). The open-field and biotelemetric observations revealed that the motor responses were diminished by pretreatment with the CRH antagonist and haloperidol. In the case of HPA (hypothalamic pituitary adrenal) activation, only cyproheptadine pretreatment proved effective; haloperidol and L-NAME did not modify the corticosterone response. NAP had only a transient, while cyproheptadine elicited a more permanent impact on the hyperthermic response evoked by ghrelin; the other antagonists proved to be ineffective. The present data suggest that both CRH release and dopaminergic transmission may be involved in the ghrelin-evoked behavioral responses. On the other hand, ghrelin appears to have an impact on the HPA response via a serotonergic pathway and on the hyperthermic response via a cyclooxygenase and a serotonergic pathway.     

Haloperidol-induced suppression of carotid chemoreception in vitro

Effects of antagonism of endogenous dopamine with haloperidol on single-unit frequency, interspike interval distribution, and interval serial dependency of the cat sinus nerve were tested using an in vitro carotid body-sinus nerve superfusion technique. A dose dependency of inhibition by haloperidol (0.05-2.0 microgram/ml) was observed. Superfusion with 1-2 microgram/ml haloperidol significantly reduced frequency within 5 min (P less than 0.05) and caused a complete cessation of firing within 25 min in 5 of 10 chemoreceptor units. Frequency recovered to control during drug washout. Acetylcholine (10-micrograms/ml superfusion or 500-micrograms bolus) increased sinus nerve activity under control conditions but not during superfusion with haloperidol. No effect of haloperidol on impulse serial dependency was detected. However, interval distribution was significantly altered by haloperidol in five of six chemoreceptor units. Our results suggest an excitatory role for dopamine in carotid chemoreception.     

Catecholaminergic component of the action of a heptapeptide analog of ACTH4-10 on the open-field behavior of rats

Heptapeptide Met-Glu-His-Phe-Pro-Gly-Pro (ACTH4-10 analog) at a dose of 0.015 mg/kg failed to alter open field behaviour of rats in the first test series. The peptide abolished amphetamine-induced stimulation of the exploratory and grooming behaviour. Extinction of the rats' exploratory behaviour during second test series in the open field (7 days later) was disturbed when haloperidol or apomorphine were injected before the first test series. When the peptide was administered with haloperidol or apomorphine, the extinction tended to become normal. Heptapeptide failed to change noradrenaline, dopamine or 5-hydroxytryptamine content in the rat forebrain. However, this peptide at a concentration of 10(-4) M moderately diminished tyrosine hydroxylation velocity in the rat striatal or hypothalamic synaptosomes, the effect depending on tyrosine concentration. These data suggest the involvement of catecholaminergic component into the heptapeptide action on the behaviour of rats.     

A single exposure to immobilization causes long-lasting pituitary-adrenal and behavioral sensitization to mild stressors

We have previously reported that a single exposure to immobilization (IMO) in rats causes a long-term desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the same (homotypic) stressor. Since there are reports showing that a single exposure to other stressors causes sensitization of the HPA response to heterotypic stressors and increases anxiety-like behavior, we studied in the present work the long-term effects of IMO on behavioral and HPA response to mild superimposed stressors. In Experiments 1 and 2, adult male Sprague–Dawley rats were subjected to 2 h of IMO and then exposed for 5 min to the elevated plus-maze (EPM) at 1, 3 or 7 days after IMO. Blood samples were taken at 15 min after initial exposure to the EPM. Increases in anxiety-like behavior and HPA responsiveness to the EPM were found at all times post-IMO. Changes in the resting levels of HPA hormones did not explain the enhanced HPA responsiveness to the EPM (Experiment 3). In Experiments 4 and 5, we studied the effects of a single exposure to a shorter session of IMO (1 h) on behavioral and HPA responses to a brief and mild session of foot-shocks done 10 days after IMO. Neither previous IMO nor exposure to shocks in control rats modified behavior in the EPM. However, a brief session of shocks in previously IMO-exposed rats dramatically increased anxiety in the EPM. HPA and freezing responses to shocks were similar in control and previous IMO groups. Therefore, a single exposure to IMO appears to induce long-lasting HPA and behavioral sensitization to mild superimposed stressors, although the two responses are likely to be at least partially independent. Long-term effects of IMO on the susceptibility to stress-induced endocrine and emotional disturbances may be relevant to the characterization of animal models of post-traumatic stress.     

A single exposure to immobilization causes long-lasting pituitary-adrenal and behavioral sensitization to mild stressors

We have previously reported that a single exposure to immobilization (IMO) in rats causes a long-term desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the same (homotypic) stressor. Since there are reports showing that a single exposure to other stressors causes sensitization of the HPA response to heterotypic stressors and increases anxiety-like behavior, we studied in the present work the long-term effects of IMO on behavioral and HPA response to mild superimposed stressors. In Experiments 1 and 2, adult male Sprague–Dawley rats were subjected to 2 h of IMO and then exposed for 5 min to the elevated plus-maze (EPM) at 1, 3 or 7 days after IMO. Blood samples were taken at 15 min after initial exposure to the EPM. Increases in anxiety-like behavior and HPA responsiveness to the EPM were found at all times post-IMO. Changes in the resting levels of HPA hormones did not explain the enhanced HPA responsiveness to the EPM (Experiment 3). In Experiments 4 and 5, we studied the effects of a single exposure to a shorter session of IMO (1 h) on behavioral and HPA responses to a brief and mild session of foot-shocks done 10 days after IMO. Neither previous IMO nor exposure to shocks in control rats modified behavior in the EPM. However, a brief session of shocks in previously IMO-exposed rats dramatically increased anxiety in the EPM. HPA and freezing responses to shocks were similar in control and previous IMO groups. Therefore, a single exposure to IMO appears to induce long-lasting HPA and behavioral sensitization to mild superimposed stressors, although the two responses are likely to be at least partially independent. Long-term effects of IMO on the susceptibility to stress-induced endocrine and emotional disturbances may be relevant to the characterization of animal models of post-traumatic stress.     

Ghrelin and Nicotine Stimulate Equally the Dopamine Release in the Rat Amygdala

The orexigenic peptide ghrelin plays a prominent role in the regulation of energy balance and in the mediation of reward processes and reinforcement for addictive drugs, such as nicotine. Nicotine is the principal psychoactive component in tobacco, which is responsible for addiction and relapse of smokers. Ghrelin and nicotine activates the mesolimbicocortical dopaminergic pathways via growth hormone secretagogue receptors (GHS-R1A) and nicotinic acetylcholine receptors (nAchR), respectively, resulting in the release of dopamine in the nucleus accumbens, the amygdala and the prefrontal cortex. In the present study an in vitro superfusion of rat amygdalar slices was performed in order to investigate the direct action of ghrelin and nicotine on the amygdalar dopamine release. Ghrelin increased significantly the dopamine release from the rat amygdala following electrical stimulation. This effect was inhibited by both the selective GHS-R1A antagonist GHRP-6 and the selective nAchR antagonist mecamylamine. Under the same conditions, nicotine also increased significantly the dopamine release from the rat amygdala. This effect was antagonized by mecamylamine, but not by GHRP-6. Co-administration of ghrelin and nicotine induced a similar increase of amygdalar dopamine release. This stimulatory effect was partially reversed by both GHRP-6 and mecamylamine. The present results demonstrate that both ghrelin and nicotine stimulates directly the dopamine release in the amygdala, an important dopaminergic target area of the mesolimbicocortical pathway.     

GABAA receptors in nucleus accumbens shell mediate the hyperphagia and weight gain following haloperidol treatment in rats

AimsWeight gain is a common outcome of antipsychotics therapy in schizophrenic patients. However, the underlying neuronal mechanisms are unclear. The present study was undertaken to investigate the role of GABA_A receptors within the framework of nucleus accumbens shell (AcbSh) in haloperidol-induced hyperphagia and body weight gain in sated rats.Main methodsIn acute studies, GABA_A receptor agonists muscimol, diazepam or antagonist bicuculline were administered by AcbSh route, alone or in combination with haloperidol (intraperitoneal/ip). Immediately after these treatments, preweighed food was offered to the animals at commencement of dark phase. Cumulative food intake was measured at 2 and 6 h post-injection time-points. Furthermore, effects of subacute haloperidol treatment, alone or in combination with muscimol, diazepam or bicuculline, on food intake and body weight were investigated.Key findingsWhile acute treatment with haloperidol, muscimol or diazepam dose dependently stimulated the food intake, bicuculline suppressed the same. Prior administration of muscimol (20 ng/rat, intra-AcbSh) and diazepam (5 µg/rat, intra-AcbSh) significantly potentiated, whereas bicuculline (40 ng/rat, intra-AcbSh) negated the hyperphagic effect of acute haloperidol (0.005 or 0.01 mg/kg/rat, ip). Subacute administration of haloperidol (0.01 mg/kg/rat/day, ip) for 15 days produced increase in food intake and body weight. Although, concomitant administration of muscimol (20 ng/rat/day, intra-AcbSh) or diazepam (5 μg/rat/day, intra-AcbSh) markedly enhanced, bicuculline (40 ng/rat/day, intra-AcbSh) prevented the subacute haloperidol-induced hyperphagia and weight gain.SignificanceThe results of present study suggest that increased food intake and body weight following haloperidol treatment in rats, may be mediated via AcbSh GABA_A receptors.     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

Involvement of dopaminergic receptors in action of corticotropin releasing factor (CRF) under in vitro conditions.

ACTH release under the effect of median eminence extract (ME) was studied in both incubation and superfusion experiments. ACTH content of the incubation medium was measured by radioimmunoassay or by the corticosterone production of trypsinisolated adrenocortical cells. Dopamine at low concentration led to a slight increase of basal ACTH secretion, while at higher concentration failed to influence ACTH release. The dopamine agonist CB-154 produced a significant rise of ACTH secretion and augmented the ME extract-induced increase of pituitary ACTH release. Chlorpromazine and haloperidol suppressed basal ACTH secretion and inhibited the ME extract-induced release. The simultaneous administration of CB-154 and haloperidol into the incubation medium prevented the haloperidol-induced inhibition of ACTH release. The observations indicate that dopaminergic receptors play a role in the activation of CRF-induced ACTH secretion under in vitro experimental conditions.     

The Effects of Corticoptropin-Releasing Factor and the Urocortins on Striatal Dopamine Release Induced by Electrical Stimulation—An in vitro Superfusion Study

The members of the CRF peptide family, corticotropin-releasing factor (CRF), urocortin I (Ucn I), urocortin II (Ucn II) and urocortin III (Ucn III) coordinate endocrine and behavioral responses to stress. CRF has also been demonstrated to stimulate dopamine (DA) synthesis.In our study, a superfusion system was used to investigate the effects of this peptide family on striatal DA release following electrical stimulation. The involvement of the CRF receptors was studied by pretreatment of rat striatal slices with selective CRF antagonists. CRF and Ucn I increased the release of [³H]DA while Ucn II and Ucn III were ineffective. The CRFR1 antagonist antalarmin inhibited the [³H]DA release induced by electrical stimulation and enhanced by CRF and Ucn I. The CRFR2 antagonist astressin-2B was ineffective.These results suggest that CRF and Ucn I mediate DA release through the activation of CRFR1. Ucn II and Ucn III are not involved in this process.Special Issue Dedicated to Miklós Palkovits.     

Urocortin increased LPS‐induced endothelial permeability by regulating the cadherin–catenin complex via corticotrophin‐releasing hormone receptor 2

Urocortin (Ucn1), a member of corticotrophin‐releasing hormone (CRH) family, has been reported to be upregulated in inflammatory diseases and function as an autocrine or paracrine inflammatory mediator. Growing evidence shows that Ucn1 increases the endothelial permeability in inflammatory conditions; however, the detailed mechanisms are not clear. In the present study, we investigated the mechanisms of increased endothelial permeability by Ucn1 in human umbilical vein endothelial cells (HUVECs) exposed to lipopolysaccharide (LPS). Pretreatment of HUVECs with Ucn1 increased the endothelial cell permeability, which was augmented by LPS synergistically. Significant downregulation of VE‐cadherin expression was also observed. Moreover, Ucn1 increased phosphorylation of protein kinase D (PKD) and heat shock protein 27 (HSP27) in a time‐ and CRHR₂‐dependent manner. Inhibition of PKD and HSP27 drastically attenuated Ucn1‐induced downregulation of VE‐cadherin expression. Further investigations demonstrated that Ucn1 phosphorylated β‐catenin at Ser552 to disrupt the cadherin–catenin complex and hence promote the disassociation of β‐catenin and VE‐cadherin. Disassociation of β‐catenin and VE‐cadherin resulted in decreased VE‐cadherin expression while on the contrary β‐catenin was increased, which may due to the inactivation of GSK‐3β. Increased β‐catenin translocated into the nucleus and subsequently bound to TCF/LEF site, contributing to the elevated expression of vascular endothelial growth factor (VEGF). The above effects of Ucn1 were completely reversed by CRHR₂ receptor blocker, antisauvagine‐30. Taken together, our data suggest that Ucn1 increase LPS‐induced endothelial permeability by disrupting the VE‐cadherin–β‐catenin complex via activation of CRHR₂ and PKD‐HSP27 signaling pathway. J. Cell. Physiol. 228: 1295–1303, 2013. © 2012 Wiley Periodicals, Inc.     

Inhibition by Diazepam and γ-Aminobutyric Acid of Depolarization-Induced Release of [14C]Cysteine Sulfinate and [3H]Glutamate in Rat Hippocampal Slices

Effects of diazepam and gamma-aminobutyric acid-related compounds on the release of [14C]cysteine sulfinate and [3H]glutamate from preloaded hippocampal slices of rat brain were examined by a superfusion method. Diazepam markedly inhibited the release of cysteine sulfinate and glutamate evoked either by high K+ or veratridine without affecting that of other neurotransmitter candidates, e.g., gamma-aminobutyric acid, acetylcholine, noradrenaline, and dopamine; IC50 values for the release of cysteine sulfinate and glutamate were about 20 and 7 microM, respectively. gamma-Aminobutyric acid (1 to 10 microM) and muscimol (100 microM) significantly reduced high K+-stimulated release of glutamate. Bicuculline, which had no effect on the release at a concentration of 50 microM by itself, antagonized the inhibitor effects of diazepam and gamma-aminobutyric acid on glutamate release. Similar results were obtained with the release of cysteine sulfinate except that a high concentration (100 microM) of gamma-aminobutyric acid was required for the inhibition. These results indicate the modulation by gamma-aminobutyric acid innervation of the release of excitatory amino acids in rat hippocampal formation, and also suggest that some of the pharmacological effects of diazepam may be a consequence of inhibition of excitatory amino acid transmission.     

Corticosterone does not change open elevated plus maze-induced antinociception in mice

It has been demonstrated that the exposure of rodents to the standard elevated plus-maze (sEPM: 2 open and 2 enclosed arms) elicits defensive behavioral reactions and antinociception and also activates the hypothalamo-pituitary-adrenal (HPA) axis. We have recently reported that EPM-induced antinociception is particularly observed when rats and mice are exposed to a totally open EPM (oEPM: 4 open arms). Given that the oEPM seems to be a more aversive situation than the sEPM, we hypothesized that oEPM exposure would induce higher plasma levels of corticosterone than sEPM exposure in mice. In this study, we investigated the influence of exposure to eEPM (enclosed EPM: 4 enclosed arms), sEPM or oEPM on plasma corticosterone levels in mice, with or without prior nociceptive stimulation (2.5% formalin injection into the right hind paw). We also tested whether the nociceptive response in the formalin test and oEPM-induced antinociception are altered by adrenalectomy. Results showed that oEPM-exposed mice spent less time licking the injected paw than sEPM- and eEPM-exposed animals. All three types of EPM exposure increased plasma corticosterone when compared to the basal group, but sEPM- and oEPM-exposed mice showed higher corticosterone levels than eEPM-exposed mice. Prior nociceptive stimulation (formalin injection) did not enhance the plasma corticosterone response induced by the three types of EPM exposure. Indeed, formalin injection appeared to provoke a ceiling effect on plasma corticosterone concentration. Furthermore, neither the nociceptive response in the formalin test nor oEPM-induced antinociception was changed by adrenalectomy. Present results suggest that oEPM antinociception does not depend on corticosterone release in mice.