Role of Prefrontal Serotonergic and Dopaminergic Systems in Encounter-Induced Hyperactivity in Methamphetamine-Sensitized Mice.

Background: Isolation-reared mice show social encounter-induced hyperactivity with activation of prefrontal serotonergic and dopaminergic systems, but it is not known whether this stress response is observed in other pathological conditions. Here we examined whether the social encounter stimulation induces abnormal behavior during withdrawal in chronic methamphetamine-treated mice. Methods: To induce methamphetamine-induced behavioral sensitization, male mice were injected with methamphetamine (1 mg/kg) once daily for 7 days. Results: The encounter with an intruder elicited hyperactivity 24 h after the last injection of methamphetamine in methamphetamine-sensitized mice. This response was observed even as long as 2 weeks after withdrawal of methamphetamine. The encounter increased c-Fos expression in the prefrontal cortex, dorsal raphe nucleus and ventral tegmental area in methamphetamine-sensitized mice, while it did not in control mice. Furthermore, the encounter increased extracellular serotonin (5-HT) and dopamine, but not noradrenaline, levels in the prefrontal cortex in methamphetamine-sensitized mice. Local injection of 5,7-dihydroxytryptamine and 6-hydroxydopamine into the prefrontal cortex attenuated encounter-induced hyperactivity in methamphetamine-sensitized mice and it markedly decreased prefrontal 5-HT and dopamine levels, respectively. Pharmacological analysis showed that the encounter-induced hyperactivity is mediated by dopamine D1 receptors and 5-HT2A receptors and attenuated by anxiolytics and antidepressants such as diazepam, osemozotan and selective 5-HT reuptake inhibitors. The effect of paroxetine was blocked by the 5-HT3 receptor antagonist azasetron. Conclusions: The present study shows that psychological stress elicits hyperactivity with activation of prefrontal 5-HT and dopamine systems in methamphetamine-dependent mice and suggests that the abnormal behavior is associated with anxiety and depression.

Oxytocin attenuates deficits in social interaction but not recognition memory in a prenatal valproic acid-induced mouse model of autism.

Recent studies have reported that oxytocin ameliorates behavioral abnormalities in both animal models and individuals with autism spectrum disorders (ASD). However, the mechanisms underlying the ameliorating effects of oxytocin remain unclear. In this study, we examined the effects of intranasal oxytocin on impairments in social interaction and recognition memory in an ASD mouse model in which animals are prenatally exposed to valproic acid (VPA). We found that a single intranasal administration of oxytocin restored social interaction deficits for up to 2h in mice prenatally exposed to VPA, but there was no effect on recognition memory impairments. Additionally, administration of oxytocin across 2weeks improved prenatal VPA-induced social interaction deficits for at least 24h. In contrast, there were no effects on the time spent sniffing in control mice. Immunohistochemical analysis revealed that intranasal administration of oxytocin increased c-Fos expression in the paraventricular nuclei (PVN), prefrontal cortex, and somatosensory cortex, but not the hippocampal CA1 and CA3 regions of VPA-exposed mice, suggesting the former regions may underlie the effects of oxytocin. These findings suggest that oxytocin attenuates social interaction deficits through the activation of higher cortical areas and the PVN in an ASD mouse model.

Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger.

Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.

Prenatal Exposure to Histone Deacetylase Inhibitors Affects Gene Expression of Autism-Related Molecules and Delays Neuronal Maturation.

Valproic acid (VPA) is a multi-target drug and an inhibitor of histone deacetylase (HDAC). We have previously demonstrated that prenatal exposure to VPA at embryonic day 12.5 (E12.5), but not at E14.5, causes autism-like behavioral abnormalities in male mouse offspring. We have also found that prenatal VPA exposure causes transient histone hyperacetylation in the embryonic brain, followed by decreased neuronal cell numbers in the prefrontal and somatosensory cortices after birth. In the present study, we examined whether prenatal HDAC inhibition affects neuronal maturation in primary mouse cortical neurons. Pregnant mice were injected intraperitoneally with VPA (500 mg/kg) and the more selective HDAC inhibitor trichostatin A (TSA; 500 µg/kg) at E12.5 or E14.5, and primary neuronal cultures were prepared from the cerebral cortices of their embryos. Prenatal exposure to VPA at E12.5, but not at E14.5, decreased total number, total length, and complexity of neuronal dendrites at 14 days in vitro (DIV). The effects of VPA weakened at 21 DIV. Exposure to TSA at E12.5, but not at E14.5, also delayed maturation of cortical neurons. In addition, real-time quantitative PCR revealed that the prenatal exposure to TSA decreased neuroligin-1 (Nlgn1), Shank2, and Shank3 mRNA levels and increased contactin-associated protein-like 2 mRNA level. The delay in neuronal maturation was also observed in Nlgn1-knockdown cells, which were transfected with Nlgn1 siRNA. These findings suggest that prenatal HDAC inhibition causes changes in gene expression of autism-related molecules linked to a delay of neuronal maturation.

The role of Pak-interacting exchange factor-ß phosphorylation at serines 340 and 583 by PKCγ in dopamine release.

Protein kinase C (PKC) has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in PKCγ, shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is PKCγ-knock-out (KO) and that PKCγ-KO mice showed parkinsonian syndrome. However, the PKCγ substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the PKCγ substrates involved in dopamine release, we used PKCγ-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of PKCγ-KO mice. We focused on Pak-interacting exchange factor-ß (ßPIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKCγ directly phosphorylates ßPIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated ßPIX in vivo. Classical PKC inhibitors and ßPIX knock-down (KD) significantly suppressed Ca(2+)-evoked dopamine release in PC12 cells. Wild-type ßPIX, and not the ßPIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by ßPIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of ßPIX at Ser340 and Ser583 has pivotal roles in Ca(2+)-evoked dopamine release in the striatum. Therefore, we propose that PKCγ positively modulates dopamine release through ß2PIX phosphorylation. The PKCγ-ßPIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.

Identification of the role of bone morphogenetic protein (BMP) and transforming growth factor-ß (TGF-ß) signaling in the trajectory of serotonergic differentiation in a rapid assay in mouse embryonic stem cells in vitro.

The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock-in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet-1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate-based screening and identified BMP type I receptor kinase inhibitors LDN-193189 and DMH1 as activators of luciferase. LDN-193189 induced ES cells to express the genes encoding Pet-1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF-ß receptor inhibitor SB-431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN-193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF-ß target gene Lefty, whereas the opposite effect was observed with SB-431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF-ß receptor signaling are implicated in serotonergic differentiation. Candidate-based screening for serotonergic induction using a rapid assay in mouse embryonic stem cells revealed that the bone morphogenetic protein (BMP) type I receptor kinase inhibitors selectively induce serotonergic differentiation, whereas the TGF-ß receptor inhibitor SB-431542 inhibits the differentiation. These results suggest that inhibition of BMP type I receptors and concomitant activation of transforming growth factor-ß (TGF-ß) receptor signaling are involved in the early trajectory of serotonergic differentiation.

The Female Encounter Test: A Novel Method for Evaluating Reward-Seeking Behavior or Motivation in Mice.

BACKGROUND: Reduced motivation is an important marker of psychiatric disorders, including depression. We describe the female encounter test, a novel method of evaluating reward-seeking behavior in mice. METHODS: The test apparatus consists of three open chambers, formed with partitions that allow the animal to move freely from one chamber to another. A test male mouse is habituated in the apparatus, and subsequently a female and male mouse are introduced into a wire-mesh box in the left and right chamber, respectively. The time the test male mouse spends in the female or male area is measured for 10 min. RESULTS: All six strains of mice tested showed a significant preference for female encounters. The preference was observed in 7-30-week-old mice. The preference was blocked by castration of the resident male test mouse, and was not affected by the phase of the menstrual cycle of the female intruder. The preference was impaired in mouse models of depression, including social isolation-reared, corticosterone-treated, and lipopolysaccharide-treated mice. The impairment was alleviated by fluvoxamine in isolation-reared and lipopolysaccharide-treated mice, and it was improved by the metabotropic glutamate 2/3 receptor antagonist LY341495 in corticosterone-treated mice. Encounter with a female, but not male, mouse increased c-Fos expression in the nucleus accumbens shell of test male mice. Furthermore, both the preference and encounter-induced increases in c-Fos expression were blocked by dopamine D1 and D2 receptor antagonists. CONCLUSIONS: These findings indicate that motivation in adult male mice can be easily evaluated by quantitating female encounters.

Pharmacological profile of encounter-induced hyperactivity in isolation-reared mice.

We have recently found that isolation-reared mice show hyperactivity during an encounter with an intruder. However, it is not known whether encounter-induced hyperactivity may model some aspects of psychiatric disorders. The present study examined the pharmacological profile of encounter-induced hyperactivity in isolation-reared mice. Encounter-induced hyperactivity was reduced by acute administration of various antidepressants including the tricyclic antidepressant desipramine (10 mg/kg), the selective serotonin (5-HT) reuptake inhibitors fluvoxamine (10 mg/kg) and paroxetine (10 mg/kg), the 5-HT/noradrenaline reuptake inhibitors venlafaxine (10 mg/kg) and duloxetine (10 mg/kg), the antipsychotic drug risperidone (0.01 mg/kg), the 5-HT2 antagonist ritanserin (1 mg/kg), and the glucocorticoid receptor antagonist RU-43044 (30 mg/kg). The α2 adrenoceptor agonist clonidine (0.03 mg/kg) and the 5-HT4 receptor agonist BIMU8 (30 mg/kg) also reduced encounter-induced hyperactivity. The effect of desipramine was blocked by the α2 adrenoceptor antagonist idazoxan (0.3 mg/kg). The effect of fluvoxamine was blocked by the 5-HT4 receptor antagonist GR125487 (3 mg/kg), but not the 5-HT1A receptor antagonist WAY100635 (1 mg/kg), the 5-HT3 receptor antagonist azasetron (3 mg/kg), or the 5-HT6 receptor antagonist SB399885 (3 mg/kg). The effect of venlafaxine was blocked by the simultaneous administration of idazoxan (0.3 mg/kg) and GR125487 (3 mg/kg), but not by either compound alone. These findings suggest that encounter-induced hyperactivity in isolation-reared mice is a robust model for testing the pharmacological profile of antidepressants, although the range of antidepressants tested is limited and some non-antidepressants are also effective. The present study also shows a key role of α2 and 5-HT4 receptors in the antidepressant effect in this model.

Role of the 5-HT1A autoreceptor in the enhancement of fluvoxamine-induced increases in prefrontal dopamine release by adrenalectomy/castration in mice.

We have found that fluvoxamine-induced increases in prefrontal dopamine release are enhanced by adrenalectomy/castration and 5-HT1A receptors are involved in the enhancement. This study examined which 5-HT1A autoreceptors or postsynaptic receptor play a key role in the enhancement in mice. Adrenalectomy/castration-induced enhancement of fluvoxamine-induced increase in the dopamine release was not blocked by local perfusion with the 5-HT1A receptor antagonist WAY100635 (10 µM), while it was blocked by systemic administration of WAY100635 at low dose (0.1 mg/kg) which blocked preferentially autoreceptor-mediated responses. These finding suggests that 5-HT1A autoreceptors play a key role in the enhancement of prefrontal dopamine release.

Galantamine promotes adult hippocampal neurogenesis via M1 muscarinic and α7 nicotinic receptors in mice.

Galantamine, an inhibitor of acetylcholinesterase, promotes hippocampal neurogenesis, but the exact mechanism for this is not known. In the present study, we examined the mechanisms underlying the effects of acute galantamine on neurogenesis in the mouse hippocampus. Galantamine (3 mg/kg) increased the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells in the subgranular zone of the dentate gyrus. This effect was blocked by the muscarinic receptor antagonist scopolamine and the preferential M1 muscarinic receptor antagonist telenzepine, but not by the nicotinic receptor antagonists mecamylamine and methyllycaconitine. Galantamine did not alter the ratio of neuronal nuclei (NeuN)- or glial fibrillary acidic protein (GFAP)-positive cells to BrdU-labeled cells in the subgranular zone and granule cell layer. Galantamine (1, 3 mg/kg) promoted the survival of 2-wk-old newly divided cells in mice in the granule cell layer of the dentate gyrus, whereas it did not affect the survival of newly divided cells at 1 and 4 wk. Galantamine-induced increases in cell survival were blocked by the α7 nicotinic receptor antagonist methyllycaconitine, but not by scopolamine. Bilateral injection of recombinant IGF2 into the dentate gyrus of the hippocampus mimicked the effects of galantamine. The effects of galantamine were blocked by direct injection of the IGF1 receptor antagonist JB1. These findings suggest that galantamine promotes neurogenesis via activation of the M1 muscarinic and α7 nicotinic acetylcholine receptors. The present study also suggests that IGF2 is involved in the effects of galantamine on the survival of 2-wk-old immature cells in the granule cell layer.

Involvement of prefrontal AMPA receptors in encounter stimulation-induced hyperactivity in isolation-reared mice.

We recently showed that social encounter stimulation induces hyperactivity in mice reared in social isolation from early life and this is associated with the transient activation of prefrontal dopaminergic and serotonergic systems. In the present study, we examined the effect of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 2, 3-dioxo-6-nitro-1, 2, 3, 4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) on encounter-induced behavioural and neurochemical changes to study the role of the receptor in abnormal behaviours in isolation-reared mice. The encounter to an intruder mouse induced hyperactivity with transient increases in prefrontal dopamine and serotonin levels in isolation-reared mice. NBQX attenuated the encounter-induced hyperactivity and the associated neurochemical changes in isolation-reared mice. In addition, NBQX reduced aggressive behaviour and cognitive impairment in isolation-reared mice, but did not affect depressive-like behaviour or spontaneous hyper-locomotion in these animals. The AMPA receptor agonist (S)-AMPA increased prefrontal dopamine and serotonin release, and this effect was higher in isolation-reared mice than in the group-reared mice, suggesting higher prefrontal AMPA receptor activity in isolation-reared mice. Furthermore, isolation rearing increased the expression of AMPA receptor subunits (GluR1, GluR2 and GluR3) and GluR1 Ser845 phosphorylation in the prefrontal cortex, but not in the hippocampus or nucleus accumbens. Taken together, these results suggest that an increase in AMPA receptor activity in the prefrontal cortex contributes to some, but not all, abnormal behaviours in isolation-reared mice.

Atomoxetine-induced increases in monoamine release in the prefrontal cortex are similar in spontaneously hypertensive rats and Wistar-Kyoto rats.

Spontaneously hypertensive rats (SHRs) are used as a model for attention-deficit/hyperactivity disorder (ADHD), since SHRs are hyperactive and show defective sustained attention in behavioral tasks. The psychostimulants amphetamine and methylphenidate and the selective norepinephrine reuptake inhibitor atomoxetine are used as ADHD medications. The effects of high K(+) stimulation or psychostimulants on brain norepinephrine or dopamine release in SHRs have been previously studied both in vitro and in vivo, but the effects of atomoxetine on these neurotransmitters have not. The present study examined the effects of administration of atomoxetine on extracellular norepinephrine, dopamine, and serotonin levels in the prefrontal cortex of juvenile SHRs and Wistar-Kyoto (WKY) rats. Baseline levels of prefrontal norepinephrine, dopamine, and serotonin were similar in SHRs and WKY rats. Systemic administration of atomoxetine (3 mg/kg) induced similar increases in prefrontal norepinephrine and dopamine, but not serotonin, levels in both strains. Furthermore, there was no difference in high K(+)-induced increases in extracellular norepinephrine, dopamine, and serotonin levels in the prefrontal cortex between SHRs and WKY rats. These findings indicate that monoamine systems in the prefrontal cortex are similar between SHRs and WKY rats.

Potential role of serotonin1A receptors in post-weaning social isolation-induced abnormal behaviors in rodents.

Post-weaning social isolation in mice induces behavioral abnormalities such as hyperactivity, aggression, depression- and anxiety-like behaviors, deficits of prepulse inhibition, and reduced pain sensitivity to the noxious stimuli. Then, this mouse is considered to be a model of psychiatric disorders including schizophrenia. We have found that serotonin (5-HT)(1A)-receptor ligands attenuate these abnormalities, suggesting the pharmacological role of the receptor in treatment of psychiatric disorders. Furthermore, we have recently found that isolation-reared mice show social encounter-induced hyperactivity, a novel phenotype of the abnormal behaviors, and the hyperactivity is triggered by activation of the serotonergic system from the dorsal raphe to the frontal cortex. This review summarizes the effects of 5-HT(1A) receptor ligands on aggressive behavior, deficits of prepulse inhibition, reduced pain sensitivity to the noxious stimuli, and encounter-induced hyperactivity in social isolation-reared mice. These findings suggest that the 5-HT(1A) receptor is a potential target molecule for treatment of psychiatric disorders and pain.

Autism-like behaviours with transient histone hyperacetylation in mice treated prenatally with valproic acid.

Maternal use of valproic acid (VPA) during pregnancy has been implicated in the aetiology of autism spectrum disorders in children, and rodents prenatally exposed to VPA showed behavioural alterations similar to those observed in humans with autism. However, the exact mechanism for VPA-induced behavioural alterations is not known. To study this point, we examined the effects of prenatal exposure to VPA and valpromide, a VPA analog lacking histone deacetylase inhibition activity, on behaviours, cortical pathology and histone acetylation levels in mice. Mice exposed to VPA at embryonic day 12.5 (E12.5), but not at E9 and E14.5, displayed social interaction deficits, anxiety-like behaviour and memory deficits at age 4-8 wk. In contrast to male mice, the social interaction deficits (a decrease in sniffing behaviour) were not observed in female mice at age 8 wk. The exposure to VPA at E12.5 decreased the number of Nissl-positive cells in the middle and lower layers of the prefrontal cortex and in the lower layers of the somatosensory cortex at age 8 wk. Furthermore, VPA exposure caused a transient increase in acetylated histone levels in the embryonic brain, followed by an increase in apoptotic cell death in the neocortex and a decrease in cell proliferation in the ganglionic eminence. In contrast, prenatal exposure to valpromide at E12.5 did not affect the behavioural, biochemical and histological parameters. Furthermore, these findings suggest that VPA-induced histone hyperacetylation plays a key role in cortical pathology and abnormal autism-like behaviours in mice.

Methylphenidate and venlafaxine attenuate locomotion in spontaneously hypertensive rats, an animal model of attention-deficit/hyperactivity disorder, through α2-adrenoceptor activation.

Recent clinical studies have shown that serotonin-norepinephrine reuptake inhibitors such as venlafaxine and duloxetine are effective against symptoms of attention-deficit/hyperactivity disorder such as inattention, oppositionality, and hyperactivity. We have recently found that these serotonin-norepinephrine reuptake inhibitors, like methylphenidate, reduced the hyperactivity in spontaneously hypertensive rats (SHR), an animal model of attention-deficit/hyperactivity disorder. The present study investigated whether the α2-adrenoceptor and the dopamine-D1 receptor are involved in the behavioral effects of methylphenidate and venlafaxine in SHR. Adolescent male SHR showed greater horizontal locomotion in a familiar open field than male Wistar Kyoto and Wistar rats, and methylphenidate (0.3 mg/kg) and venlafaxine (30 mg/kg) reduced horizontal locomotion in SHR, but not Wistar Kyoto or Wistar rats. The effects of methylphenidate and venlafaxine were blocked by idazoxan (an α2-adrenoceptor antagonist), but not by SCH23390 (a dopamine-D1 receptor antagonist). These findings suggest that the α2-adrenoceptor plays a key role in the effects of methylphenidate and venlafaxine on enhanced locomotion in SHR.

The selective metabotropic glutamate 2/3 receptor agonist MGS0028 reverses psychomotor abnormalities and recognition memory deficits in mice lacking the pituitary adenylate cyclase-activating polypeptide.

Previous studies suggest that metabotropic glutamate 2/3 receptors are involved in psychiatric disorders. In this study, we examined the effects of the selective metabotropic glutamate 2/3 (mGlu2/3) receptor agonist MGS0028 on behavioral abnormalities in mice lacking the pituitary adenylate cyclase-activating polypeptide (PACAP), an experimental model of psychiatric disorders such as schizophrenia and attention-deficit/hyperactivity disorder. We found that PACAP-deficient mice showed impairments in the novel object recognition test and these impairments were improved by MGS0028 (0.1 mg/kg). Similarly, MGS0028 improved hyperactivity and jumping behaviors, but did not reverse increased immobility times in the forced swim test in PACAP-deficient mice. These results suggest that MGS0028 may be a potential, novel treatment for psychiatric disorders.

Effect of overexpression of the brain-specific Na(+)/Ca(2+) exchanger splice variant NCX1.5 on NO cytotoxicity in HEK293 cells.

Nitric oxide (NO) induces cytotoxicity in neuronal and glial cells via activation of the Na(+)/Ca(2+) exchanger (NCX). This study examined the role of the predominant brain-specific NCX splice variant NCX1.5 in NO-induced cytotoxicity in the HEK293 cell expression system. Cells were transfected with the plasmid construct pcDNA3.1/V5-His containing full-length rat NCX1.5 cDNA. There was no difference in the cytotoxic effects of the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine between control and transfected cells. These results suggest that NO cytotoxicity is not dependent on NCX1.5.

Effects of SEA0400 on arrhythmogenicity in a Langendorff-perfused 1-month myocardial infarction rabbit model.

BACKGROUND: The effects of SEA0400, a Na(+) /Ca(2+) exchanger (NCX) blocker, on dynamic factors and arrhythmogenic alternans in 1-month myocardial infarction (MI) hearts remain unknown. METHODS: Simultaneous voltage and intracellular Ca(2+) (Cai ) optical mapping was performed in 12 rabbit hearts with MI for 1 month and six normal rabbit hearts as control. Western-blot studies were performed in both groups in an additional six hearts for each. Action potential duration (APD) restitution was constructed and arrhythmogenic alternans was induced by dynamic pacing. SEA0400 (0.03, 3 µM) was administered after baseline studies. RESULTS: SEA0400 suppressed pacing-induced ventricular premature beats in a concentration-dependent manner. SEA0400 at 0.03 µM steepened APD restitution slopes and enhanced spatially discordant alternans (SDA), which became insignificant at 3 µM. The VF inducibility was seven of nine at baseline, nine of nine at 0.03 µM SEA0400, and five of nine at 3 µM SEA0400 (P = NS). Significant upregulation of NCX in the remote but not periinfarct zone and less degree downregulation of DHP1α in the remote versus periinfarct zone may play a role in enhancing SDA induction by SEA0400 in 1-month MI hearts. CONCLUSIONS: In 1-month MI hearts, SEA0400 suppresses pacing-induced ventricular premature beats, but also is proarrhythmic by steepening APD restitution and enhancing SDA via NCX inhibition. Heterogeneous upregulation of NCX and downregulation of DHP1α may contribute to SDA augmentation by SEA0400 in this model. The insignificant effect of SEA0400 on VF inducibility suggests that suppression of both reentry and triggered activity is required to suppress VF induction in this model.

Neuropharmacologic studies on the brain serotonin1A receptor using the selective agonist osemozotan.

Alterations in serotonin (5-HT) neurochemistry have been implicated in the etiology of major neuropsychiatric disorders such as anxiety-spectrum disorders, depression, and schizophrenia. The neuromodulatory effects of 5-HT are mediated through 14 receptor subtypes, and those receptors, including the 5-HT1A receptor, are considered to be potential targets for the treatment of psychiatric disorders. We developed the novel 5-HT1A receptor agonist MKC-242 (called osemozotan) and characterized its neurochemical and pharmacological profiles. 5-HT1A receptor agonists modulate the release of amine neurotransmitters through the activation of presynaptic or postsynaptic 5-HT1A receptors in the brain. The agonist has antianxiety and antidepressant effects and improves abnormal behaviors such as aggressive behavior and deficits of prepulse inhibition in isolation-reared mice. We also demonstrated that spinal 5-HT1A receptor activation is involved in isolation rearing-induced hypoalgesia. Concerning the mechanism for induction of isolation-induced abnormal behaviors, we have recently found that the raphe-prefrontal 5-HT system plays a key role in encounter stimulation-induced hyperactivity in isolation-reared mice. Furthermore, we showed that osemozotan attenuates psychostimulant-induced behavioral sensitization and that prefrontal dopamine release is enhanced by functional interaction between the 5-HT1A receptor and other receptors. This review summarizes the neuropharmacology of the 5-HT1A receptor, focusing on our studies using osemozotan, and suggests that the 5-HT1A receptor may be a target molecule for the treatment of psychiatric disorders, pain, and drug dependence.

Ca2+ entry mode of Na+/Ca2+ exchanger as a new therapeutic target for heart failure with preserved ejection fraction.

AIMS: Left ventricular (LV) fibrosis and stiffening play crucial roles in the development of heart failure with preserved ejection fraction (HFPEF). Plasma level of digitalis-like factors (DLFs) is increased in patients with hypertension, a principal underlying cardiovascular disease of HFPEF. Digitalis-like factors inhibit ion-pumping function of Na(+)/K(+)-ATPase and activate the Ca(2+) entry mode of Na(+)/Ca(2+) exchanger (NCX). Digitalis-like factors are known to promote collagen production in fibroblasts. The aim of this study was to explore whether the pharmacological inhibition of the NCX entry mode is effective in the prevention of LV fibrosis and in the development of HFPEF. METHODS AND RESULTS: (i) Dahl salt-sensitive rats fed 8% NaCl diet from age 6 weeks served as hypertensive HFPEF model. In this model, 24 h urine excretion of DLFs was greater than that in the age-matched control at compensatory hypertrophic and heart failure stages. (ii) Continuous administration of ouabain for 14 weeks developed LV fibrosis without affecting blood pressure in Sprague-Dawley rats. (iii) Ouabain elevated intracellular Ca(2+) concentration through the entry of extracellular Ca(2+), increased the phosphorylation level of p42/44 mitogen-activated protein kinases, and enhanced (3)H-proline incorporation in cardiac fibroblast; and SEA0400, the inhibitor of the NCX entry mode, suppressed these effects. (iv) In the HFPEF model, administration of SEA0400 at subdepressor dose improved the survival rate in association with the attenuation of LV fibrosis and stiffening. CONCLUSION: Digitalis-like factors and the subsequently activated NCX entry mode may play an important role in the development of hypertensive HFPEF, and the blockade of the NCX entry mode may be a new therapeutic strategy for this phenotype of heart failure.