Vitamin A Promotes the Fusion of Autophagolysosomes and Prevents Excessive Inflammasome Activation in Dextran Sulfate Sodium-Induced Colitis.

Vitamin A ensures intestinal homeostasis, impacting acquired immunity and epithelial barrier function; however, its role in innate immunity is mostly unknown. Here, we studied the impact of vitamin A in different dextran sulfate sodium (DSS)-induced colitis animal models. Interestingly, more severe DSS-induced colitis was observed in vitamin A-deficient (VAD) mice than in vitamin A-sufficient (VAS) mice; the same was observed in VAD severe combined immunodeficient mice lacking T/B cells. Remarkably, IL-1ß production, LC3B-II expression, and inflammasome activity in the lamina propria were significantly elevated in VAD mice. Electron microscopy revealed numerous swollen mitochondria with severely disrupted cristae. In vitro, non-canonical inflammasome signaling-induced pyroptosis, LC3B-II and p62 expression, and mitochondrial superoxide levels were increased in murine macrophages (RAW 264.7) pretreated with retinoic acid receptor antagonist (Ro41-5253). These findings suggest that vitamin A plays a crucial role in the efficient fusion of autophagosomes with lysosomes in colitis.

Tacrolimus Up-regulates Expression of TGFß Receptor Type II via ERK, Providing Protection Against Intestinal Epithelial Injury.

BACKGROUND/AIM: Transforming growth factor ß (TGFß) signaling plays a key role in modulating intestinal epithelial cell (IEC) homeostasis. The present study aimed to investigate the direct effect of tacrolimus on TGFß signaling in IECs. MATERIALS AND METHODS: The protective effects of tacrolimus, with or without anti-TGFß antibody, in dextran sulfate sodium (DSS)-induced colitis were evaluated. RESULTS: Tacrolimus ameliorated IEC apoptosis-mediated mucosal destruction despite anti-TGFß treatment. TGFß receptor type II (TGFß-RII), phosphor-SMAD family members 2/3, and phosphor-extracellular signal-regulated kinase (ERK) expression in IECs was enhanced in tacrolimus-treated mice, and these positive effects were maintained despite anti-TGFß treatment. Moreover, tacrolimus induced TGFß-RII up-regulation through ERK activation. CONCLUSION: Our data indicate that tacrolimus directly activated TGFß-SMAD signaling via the ERK pathway in IECs, thereby providing protection against apoptosis-mediated intestinal epithelial injury.

Polygonum tinctorium leaves suppress sodium dextran sulfate-induced colitis through interleukin-10-related pathway.

Indigo naturalis, a herbal medicine purified from indigo-containing plants, such as Strobilanthes cusia, Isatis tinctoria, and Polygonum tinctorium, has been reported to be useful in the treatment of ulcerative colitis by activating the aryl hydrocarbon receptor. However, the aryl hydrocarbon receptor pathway causes crucial side effects, such as pulmonary arterial hypertension. Although P. tinctorium is one of the plant derivatives of indigo naturalis, it is not identical to it. To date, the pure leaves of P. tinctorium have not been reported to ameliorate ulcerative colitis. Therefore, we investigated the effect of pure P. tinctorium leaves, which are consumed in some regions, on experimental colitis induced in mice using sodium dextran sulfate. We found that P. tinctorium leaves ameliorated weight loss (P < 0.01) and pathological inflammatory changes in the colon (P < 0.05), enhanced mRNA expression of interleukin-10 (P < 0.05), and decreased expression of tumor necrosis factor-in colonic tissues (P < 0.05), as determined using quantitative real-time reverse transcription polymerase chain reaction. The intraperitoneal administration of an aryl hydrocarbon receptor antagonist did not antagonize the inhibition of mucosal destruction, whereas an anti-interleukin-10 receptor antibody did. These results suggest that P. tinctorium ameliorate sodium dextran sulfate-induced intestinal inflammation via interleukin-10-related pathway, independent of the aryl hydrocarbon receptor pathway. P. tinctorium leaves have the potential to be a new, safe treatment for ulcerative colitis.

Long-term efficacy and tolerability of dose-adjusted thiopurine treatment in maintaining remission in inflammatory bowel disease patients with NUDT15 heterozygosity.

BACKGROUND/AIMS: Thiopurines are key drugs for inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD). Recently, NUDT15 polymorphism (R139C, c.415C > T) has been shown to be associated with thiopurineinduced adverse events in Asian populations. In patients with the C/T genotype, low-dose thiopurine treatment is recommended, but its long-term efficacy and tolerability remain unclear. This study aimed to uncover the long-term efficacy and appropriate dosage of thiopurine for IBD patients with the C/T genotype. METHODS: A total of 210 patients with IBD (103 UC and 107 CD) determined to have NUDT15 R139C variants were enrolled. Clinical data were retrospectively reviewed from medical records. RESULTS: Of 46 patients (21.9%) with the C/T genotype, 30 patients (65.2%) were treated with thiopurines. Three of whom (10.0%) discontinued thiopurine treatment due to adverse events and 27 of whom continued. The median maintenance dosage of 6-mercaptopurine was 0.25 mg/kg/day (range, 0.19-0.36 mg/kg/day), and 6-thioguanine nucleotides level was 230 (104-298) pmol/8 × 108 red blood cells. Cumulative thiopurine continuation rates for 120 months for patients with the C/C and C/T genotypes were not significantly different (P= 0.895). Cumulative non-relapse rates in the patients with UC treated with thiopurine monotherapy and surgery-free rates in CD patients treated with combination therapy (thiopurines and anti-tumor necrosis factor-α agents) for maintenance remission were not significantly different at 60 months (C/C vs. C/T, P= 0.339 and P= 0.422, respectively). CONCLUSIONS: Low-dose thiopurine treatment is an effective and acceptable treatment for patients with C/T genotype.

Hepatic Macrophages Express Melanoma Differentiation-Associated Gene 5 in Nonalcoholic Steatohepatitis.

The activation of innate immune system is essential for the pathogenesis of nonalcoholic steatohepatitis (NASH). Among pattern recognition receptors, it is well-characterized that toll-like receptors (TLRs) are deeply involved in the development of NASH to reflect exposure of the liver to gut-driven endotoxins. In contrast, it has not been elucidated whether retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are similarly implicated in the disease progression. In the present study, we examined the expression of melanoma differentiation-associated antigen 5 (MDA5), known to be a member of RLRs, in a diet-induced murine model of NASH. The liver tissues were collected from C57BL/6 J mice at 1, 3, and 6 weeks after choline-deficient L-amino acid-defined high-fat diet (CDAHFD), and the expression of MDA5 was analyzed by western blotting, immunofluorescence (IF), and real-time quantitative PCR (qPCR). The results of western blotting showed that hepatic expression of MDA5 was increased at 3 and 6 weeks. In IF, MDA5-positive cells co-expressed F4/80 and CD11b, indicating they were activated macrophages, and these cells began to appear at 1 week after CDAHFD. The mRNA expression of MDA5 was significantly upregulated at 1 week. Additionally, we performed IF using liver biopsy specimens collected from 11 patients with nonalcoholic fatty liver diseases (NAFLD), and found that MDA5-positive macrophages were detected in eight out of eleven patients. In an in vitro study, MDA5 was induced upon stimulation with lipopolysaccharide in murine bone marrow-derived macrophages and THP-1 cells. Our findings suggest that MDA5 may be involved in the inflammation of NASH.

Cyclosporine protects from intestinal epithelial injury by modulating butyrate uptake via upregulation of membrane monocarboxylate transporter 1 levels.

BACKGROUND AND AIMS: A relationship between treatment outcomes and intestinal microbiota in patients with inflammatory bowel diseases has been demonstrated. Cyclosporine treatment leads to rapid improvement in severe ulcerative colitis. We hypothesized that the potent effects of cyclosporine would be exerted through relationships between intestinal epithelial cells (IECs) and the host microbiota. The present study was designed to elucidate the effects of cyclosporine on monocarboxylate transporter 1 (MCT1) regulation and butyrate uptake by IECs. METHODS: Colitis was induced in C57BL6 mice via the administration of 4% dextran sulfate sodium in drinking water, following which body weights, colon lengths, and histological scores were evaluated. To examine the role of butyrate in the protective effects of cyclosporine, MCT1 inhibitor and an antibiotic cocktail was administered and tributyrin (TB; a prodrug of butyrate) was supplemented; MCT1 protein expression and acetylated histone 3 (AcH3) signals in IECs, as well as the MCT1-membrane fraction of Caco-2 cells, were evaluated. To explore butyrate uptake, as s butyrate derivatives, 3-bromopyruvic acid (3-BrPA) and 1-pyrenebutyric acid were used. RESULTS: Treatment with cyclosporine inhibited body weight loss and colon length shortening. However, treatment with MCT1 inhibitor and the antibiotic cocktail negated the efficacy of cyclosporine, whereas TB supplementation restored its protective effect. Furthermore, cyclosporine upregulated MCT1 expression in the membrane and the AcH3 signal in IECs, while also inducing higher anti-inflammatory cytokine production compared to that in the vehicle-treated mice. The transcription level of MCT1 mRNA in IECs and Caco-2 cells did not increase with cyclosporine treatment; however, cyclosporine treatment increased membrane MCT1 expression in these cells and uptake of butyrate derivative. CONCLUSION: Cyclosporine treatment modulates butyrate uptake via the post-transcriptional upregulation of membrane MCT1 levels in IECs.

Diagnostic approach for patients with unidentified fever according to the classical criteria of fever of unknown origin in the field of autoimmune disorders.

Fever of unknown origin (FUO) is caused by various diseases, making differential diagnosis difficult. This study aimed to determine the clinical features of patients with FUO for use in daily medical practice. Medical records of patients who first visited our department for FUO between January 2008 and December 2017 were reviewed. We classified the diagnostic categories as infection, non-infectious inflammation, neoplasm, others, and unidentified through definitive diagnosis and compared the clinical characteristics of patients who fulfilled the criteria of classical FUO and those who did not. The most prevalent diseases in patients who fulfilled the criteria were adult-onset Still's disease, Behçet's disease (BD), and polymyalgia rheumatica, which do not have any specific image inspection or specific serological markers. BD and familial Mediterranean fever were most prevalent in patients who did not fulfill the criteria. All neoplasms fulfilled the criteria of classical FUO. The most useful diagnostic procedure was determined according to the criteria of each disease. The key factor that did not fulfill the criteria was periodic fever continuing for less than 3 weeks. When examining patients with FUO, we should strictly diagnose in accordance with the criteria of each disease and consider diseases that cause periodic fever.

A novel prophylactic effect of furosemide treatment on autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).

The transgenic rat strain S284L-TG harbors the S284L mutant of the neuronal nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4), which is responsible for human autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). S284L-TG rats have epileptic seizure phenotypes during slow-wave sleep, similar to those in NFLE. We previously demonstrated that γ-aminobutyric acid (GABA)ergic action of these rats was suppressed before the onset of ADNFLE seizures, and that glutamate release in the epileptic focus lesion was increased at the onset of epilepsy. Here, mRNA analysis revealed that Cl(-)-accumulating Na-K-2Cl cotransporter 1 (NKCC1) levels were increased and Cl(-)-extruding K-Cl cotransporter 1 and 2 (KCC1 and KCC2) levels were decreased at the onset of ADNFLE seizures in S284L-TG rat frontal cortexes, which perturbed the GABAergic inhibitory system. The reversal potentials (EGABA) of GABAA receptor-mediated currents in cortical layer V pyramidal neurons of S284L-TG rats also changed their polarity from hyperpolarization to depolarization, and S284L-TG miniature excitatory postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), significantly increased in both amplitude and frequency. Administration of 25mg/kg/day furosemide before, but not after, the onset of interictal discharges prevented idiopathic epileptic activity, reversed the depolarizing shift of EGABA and increased mEPSC amplitude to normal levels. These data indicate that early treatment with an agent that normalizes pathogenesis has a prophylactic effect on epilepsy. We propose a strategy for prophylactic medication against idiopathic epilepsy through the suppression of epileptogenesis and/or ictogenesis.

Dysfunction of extrasynaptic GABAergic transmission in phospholipase C-related, but catalytically inactive protein 1 knockout mice is associated with an epilepsy phenotype.

Phospholipase C-related, but catalytically inactive protein (PRIP) was first identified as a novel inositol 1,4,5-triphosphate binding protein. The PRIP-1 subtype is expressed predominantly in the central nervous system and binds directly to the GABA type A receptor (GABA(A)-R) ß-subunit and several other proteins involved in the trafficking of GABA(A)-Rs to the plasma membrane. We found that the PRIP-1 knockout mouse showed an epileptic phenotype, confirmed by electroencephalogram. These ictal seizures were completely suppressed by diazepam (DZP), but the interictal discharges could not be abolished. We studied the electrophysiological properties of GABAergic transmission in hippocampal CA1 pyramidal neurons, using a slice patch-clamp technique. There was no difference in the effect of up to 1 µM DZP on the amplitude and frequency of miniature inhibitory postsynaptic currents between PRIP-1 knockout neurons versus wild-type neurons. In contrast, the amplitude of the tonic GABA current in PRIP-1 knockout neurons was markedly reduced compared with that in wild-type neurons. Consequently, the effect of DZP on PRIP-1 knockout mice was reduced. Dysfunction of extrasynaptic GABAergic transmission probably is involved in the epileptic phenotype of PRIP-1 knockout mice.

Effects of valproate on neurotransmission associated with ryanodine receptors.

To clarify the antiepileptic mechanisms of valproate (VPA), we determined the effects of acute and sub-acute administrations of VPA on ryanodine receptor (RyR)-associated hippocampal releases of GABA and glutamate using microdialysis, as well expression of mRNA and protein of RyR subtypes in the rat hippocampus. Acute administration of therapeutic-relevant VPA did not affect the hippocampal extracellular levels of GABA or glutamate, whereas sub-acute administration increased GABA level without affecting that of glutamate. Perfusion with ryanodine increased the hippocampal extracellular level of glutamate (ryanodine concentration range: 1-1000µM) concentration-dependently; however, that of GABA was increased by 1-100µM ryanodine concentration-dependently but the stimulatory effects of 1000µM ryanodine on GABA release was not observed. Both acute and sub-acute administrations of therapeutic-relevant VPA inhibited ryanodine-induced responses of hippocampal extracellular glutamate level without affecting that of GABA. Especially, both acute and sub-acute administrations of VPA prevented the breakdown of GABA release induced by 1000µM ryanodine. Sub-acute administration of therapeutically-relevant dose VPA weakly increased RyR mRNA expression but we could not detect the changes of RyR protein expression in rat hippocampus. These results suggest that VPA inhibited the neurotransmitter release associated with RyR without affecting the expression of RyR protein. Therefore, the antiepileptic action of VPA seems to be mediated, at least in part, by an increase in basal GABA release and inhibition of RyR-associated glutamate release.

Rats harboring S284L Chrna4 mutation show attenuation of synaptic and extrasynaptic GABAergic transmission and exhibit the nocturnal frontal lobe epilepsy phenotype.

Mutations of genes encoding alpha4, beta2, or alpha2 subunits (CHRNA4, CHRNB2, or CHRNA2, respectively) of nAChR [neuronal nicotinic ACh (acetylcholine) receptor] cause nocturnal frontal lobe epilepsy (NFLE) in human. NFLE-related seizures are seen exclusively during sleep and are characterized by three distinct seizure phenotypes: "paroxysmal arousals," "paroxysmal dystonia," and "episodic wandering." We generated transgenic rat strains that harbor a missense mutation S284L, which had been identified in CHRNA4 in NFLE. The transgenic rats were free of biological abnormalities, such as dysmorphology in the CNS, and behavioral abnormalities. The mRNA level of the transgene (mutant Chrna4) was similar to the wild type, and no distorted expression was detected in the brain. However, the transgenic rats showed epileptic seizure phenotypes during slow-wave sleep (SWS) similar to those in NFLE exhibiting three characteristic seizure phenotypes and thus fulfilled the diagnostic criteria of human NFLE. The therapeutic response of these rats to conventional antiepileptic drugs also resembled that of NFLE patients with the S284L mutation. The rats exhibited two major abnormalities in neurotransmission: (1) attenuation of synaptic and extrasynaptic GABAergic transmission and (2) abnormal glutamate release during SWS. The currently available genetically engineered animal models of epilepsy are limited to mice; thus, our transgenic rats offer another dimension to the epilepsy research field.

Carbamazepine prevents breakdown of neurotransmitter release induced by hyperactivation of ryanodine receptor.

To clarify the mechanisms of the pharmacological action of carbamazepine (CBZ), we determined the effect of CBZ on GABA and glutamate release associated with the ryanodine receptor (Ryr)-sensitive Ca(2+)-induced Ca(2+)-releasing system (CICR) in the rat hippocampus using microdialysis. The therapeutically relevant concentration of CBZ increased basal GABA release without affecting basal glutamate release; however, K(+)-evoked releases were concentration-dependently reduced by CBZ. Lower-concentration ryanodine increased basal and K(+)-evoked releases of GABA and glutamate in a concentration dependent manner, whereas higher-concentration ryanodine reduced them. These inflection points in the concentration-response curves of ryanodine for neurotransmitter release (critical concentrations) were shifted to the left by K(+)-evoked stimulation. The critical concentration of ryanodine in GABA release was lower than that in glutamate release. During the resting stage, the critical concentrations of ryanodine were unaffected by inhibition of L-type, N-type and P-type voltage-sensitive Ca(2+) channels (VSCCs) but were prevented by CBZ; however, during the neuronal hyperexcitable stage, the critical concentration was increased by CBZ, L-type and P-type VSCC inhibitors but not the N-type VSCC inhibitor. Therefore, a therapeutically relevant concentration of CBZ protects against the breakdown of the neurotransmitter release mechanism induced by hyperactivation of Ryr via inhibition of L-type and P-type VSCCs as well as inhibition of Ryr-sensitive CICR. These actions of CBZ appear to be involved, at least partially, in its anti-seizure mechanisms.

Effects of interleukin-1beta on hippocampal glutamate and GABA releases associated with Ca2+-induced Ca2+ releasing systems.

Recent clinical and basic studies have demonstrated that hyperactivation of interleukin-1beta (IL-1beta) plays important roles in generation of febrile and epileptic seizures. To clarify this mechanism, the present study determined the effects of IL-1beta on Ca2+-associated releases of glutamate and GABA in mouse hippocampus. Both basal and K+-evoked GABA releases were regulated by Ca2+ influx and Ca2+-induced Ca2+ releasing system (CICR). The K+-evoked glutamate release was also regulated by Ca2+ influx and CICR, whereas basal glutamate release was not affected by them. IL-1beta increased basal releases of glutamate and GABA depending on the activation of Ca2+ influx and ryanodine receptor (RyR)-sensitive CICR, but reduced K+-evoked releases depending on Ca2+ influx, RyR-sensitive and inositol 1,4,5-trisphosphate receptor (IP3R)-sensitive CICRs. During neuronal hyperexcitability, the effect of IL-1beta on GABA release was more predominantly modulated by Ca2+ influx and RyR-sensitive CICR than that on glutamate. These results indicate that hyperactivation of IL-1beta leads to imbalance between glutamatergic and GABAergic transmission via toxic overload response of Ca2+ influx and CICR.

Involvement of Ca(2+)-induced Ca2+ releasing system in interleukin-1beta-associated adenosine release.

Interleukin-1beta (IL-1beta) plays an important role in neuroprotective and neurodegenerative events in the central nervous system. To clarify the mechanism of controversial actions of IL-1beta, we determined the effect of IL-1beta, as well as the interaction between IL-1beta and Ca(2+)-induced Ca2+ releasing system (CICR), on adenosine releases in mice hippocampus using mini-slices method. Basal and K(+)-stimulated adenosine releases were regulated by two types of CICRs, including inositol-1,4,5-trisphosphate (IP3) receptor and ryanodine receptor. Lower concentration of IL-1beta increased both adenosine releases, whereas higher concentration did not affect their releases. The stimulatory effect of IL-1beta on basal adenosine release was reduced by removal of extracellular Ca2+ and IP3 receptor inhibitor, while the stimulatory effect of IL-1beta on K(+)-stimulated adenosine release was reduced by ryanodine receptor inhibitor. These results suggest that the potent effect of IL-1beta upon adenosine release might contribute to the neuroprotective action of IL-1beta, whereas IL-1beta-induced neurodegeneration might be due to the overload response of Ca2+ mobilization and the inactivation of adenosine exocytosis.

Effects of zonisamide on neurotransmitter exocytosis associated with ryanodine receptors.

To clarify the antiepileptic and neuroprotective actions of zonisamide (ZNS), we determined acute effects of ZNS on exocytosis of GABA and glutamate associated with ryanodine-receptor (Ryr) in rat hippocampus using microdialysis. ZNS increased basal GABA release concentration-dependently without affecting basal glutamate release; however, K(+)-evoked glutamate and GABA releases were reduced by ZNS concentration-dependently. Inhibition of Ryr reduced K(+)-evoked GABA and glutamate releases without affecting their basal releases. Ryanodine affected GABA and glutamate releases biphasic concentration-dependently: lower concentration of ryanodine increased both basal and K(+)-evoked releases of GABA and glutamate, whereas higher concentration reduced them. The therapeutically relevant concentration of ZNS inhibited ryanodine-induced GABA and glutamate releases, and abolished the inflection point in concentration-response curve for ryanodine on neurotransmitter exocytosis. These data suggest that ZNS elevates seizure threshold via enhancement of GABAergic transmission during resting stage. ZNS inhibits propagation of epileptic hyperexcitability and Ryr-associated neuronal damage during neuronal hyperexcitable stage. These demonstrations indicate that the indirect inhibition of Ryr activities by ZNS during neuronal hyperexcitability appear to be involved in the mechanisms of action of antiepileptic and neuroprotective actions of ZNS.

[Methodological consideration in studying the exocytosis mechanisms using microdialysis].

Microdialysis has become an effective and frequently used technique to study the extracellular levels of monoamine, i.e. dopamine, serotonin and norepinephrine in the central nervous system. However, the detailed exocytosis mechanisms of monoamine using microdialysis has remained to be clarified. The present report introduces methods for administration of voltage-sensitive calcium channel (VSCC) inhibitors and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) inhibitors to clarify the mechanisms of monoamine exocytosis using in vivo microdialysis. The N-type and P-type VSCCs are inhibited by perfusion with omega-conotoxin GVIA and omega-agatoxin IVA, respectively; however, their diffusion rate from internal to external spaces of the microdialysis probe is lower than 1%. Unlike VSCC inhibitors, SNAP-25, synaptobrevin and syntaxin can be cleavaed with botulinum toxin type A, B and C, respectively. These toxins (with molecular weights over 500,000) bind specifically to the presynaptic membrane via the heavy chain, while the light chain enters the cytosol, where it displays zinc-endopeptidase activity directed to proteins of the neuroexocytosis apparatus. Therefore, to prevent SNARE activity, botulinum toxins are microinjected. These two methods, perfusion with VSCC inhibitors and microinjection with botulinum toxins, can contribute to the clarification of the mechanisms of monoaminergic exocytosis.

Protein kinase associated with gating and closing transmission mechanisms in temporoammonic pathway.

The entorhinal cortex (EC) is a major source of afferent input to the hippocampus via the perforant and temporoammonic pathways; however, the detailed transmission mechanism in the temporoammonic pathway remains to be clarified. Thus, we determined interaction among GABA(A), AMPA/glutamate receptors and protein kinases (PKA and PKC) in the exocytosis of GABA and glutamate using multiprobe microdialysis, as well as propagation of neuronal excitability using optical recording in the EC-Hippocampal formation. Multiprobe microdialysis demonstrated that EC-evoked GABA release in ventral CA1 was predominantly regulated by the PKC-related rather than PKA-related exocytosis mechanism and was augmented by the activation of glutamatergic transmission. Contrary to GABA release, EC-evoked glutamate release was predominantly regulated by PKA-related rather than PKC-related mechanisms and was suppressed by activation of GABAergic transmission. Optical recording demonstrated that there are two sub-pathways in the temporoammonic pathway; direct projects from EC layers (II-IV) to dendrites on pyramidal cells and GABAergic interneurons in ventral hippocampal CA1. PKC activation enhanced trisynaptic transmission, whether the GABA(A) receptor was functional or blocked, whereas PKC activation enhanced and inhibited temporoammonic transmission when the GABA(A) receptor was functional and blocked, respectively. Thus, GABAergic inhibition, which is regulated by PKC activity, in the temporoammonic pathway is more significant than that in the trisynaptic pathway.

Determination of exocytosis mechanisms of DOPA in rat striatum using in vivo microdialysis.

To explore the exocytosis mechanism of striatal 3,4-dihydroxyphenylalanine (DOPA), this study determined the interaction between voltage-sensitive Ca2+-channel (VSCC) and SNARE on releases of DOPA and glutamate in rat striatum using microdialysis. Inhibitors of VSCCs and SNAREs did not affect basal glutamate release but decreased basal DOPA release, however, blocking effects of P-type-VSCC and synaptobrevin inhibitors were weaker than those of N-type-VSCC and syntaxin. The K+-evoked releases of DOPA and glutamate were reduced by inhibitors of P-type-VSCC and synaptobrevin predominantly and by inhibitors of N-type-VSCC and syntaxin weakly. However, interaction study between VSCC and SNARE on K+-evoked DOPA release indicates that DOPA release is regulated by different exocytosis mechanism from glutamate and monoamine during the depolarization stage (N-type-VSCC/P-type-VSCC/synaptobrevin and/or combination with N-type-VSCC/synaptobrevin and P-type-VSCC/synaptobrevin). Therefore we conclude that striatal DOPA release might be regulated by its specific exocytosis mechanism via different from dopaminergic presynaptic vesicle.

Pharmacological discrimination of protein kinase associated exocytosis mechanisms between dopamine and 3,4-dihydroxyphenylalanine in rat striatum using in vivo microdialysis.

To explore the exocytosis mechanism of dopamine and its precursor, 3,4-dihydroxyphenylalanine (DOPA), we determined the effects of protein-kinase, cyclic-AMP-dependent protein-kinase (PKA), Ca(2+)-phospholipid-dependent protein-kinase (PKC) and Ca(2+)-calmodulin-dependent protein-kinase II (CaMK-II) on dopamine and DOPA releases in rat striatum using microdialysis. Basal DOPA and dopamine releases were reduced by PKC and CaMK-II inhibitors predominantly, and PKA inhibitor weakly. Ca(2+)-evoked releases were reduced by PKC and CaMK-II inhibitors, but not by PKA inhibitor. K(+)-evoked (20 min) releases were reduced by PKA and CaMK-II inhibitors predominantly, and PKC inhibitor weakly. Sustained K(+)-evoked (120 min) releases of DOPA and dopamine were reduced by CaMK-II inhibitor, but not by PKC or PKA. DOPA accumulation was reduced by PKA and CaMK-II inhibitors strongly, and PKC inhibitor weakly. Therefore, the present study demonstrates that striatal DOPA exocytosis is regulated by a similar protein kinase-associated exocytosis mechanism as that of dopamine.

Hyperactivity of endoplasmic reticulum associated exocytosis mechanism contributes to acute phencyclidine intoxication.

Phencyclidine (PCP) produces schizophrenia-like psychosis and acute PCP-intoxications; however, whether glutamate/NMDA-receptor blockade by PCP modulates or not these mechanisms has remained to be clarified. To clarify this mechanism, we determined interaction among voltage-gated Na(+)-channel inhibitor, tetrodotoxin (TTX), Golgi-disturbing-agent, brefeldin-A (BFA), and PCP on releases of glutamate, GABA, and monoamine in prefrontal-cortex (pFC), using microdialysis. PCP increased basal monoamine release, whereas it decreased basal GABA release, without affecting glutamate release. PCP increased K(+)-evoked monoamine release, whereas it decreased K(+)-evoked glutamate and GABA releases. TTX reduced basal monoamine and GABA releases without affecting glutamate release, whereas BFA did not affect them. Interestingly, BFA and TTX inhibited PCP-associated basal monoamine release and abolished PCP-induced reduction of basal GABA release without affecting glutamate release. BFA and TTX reduced K(+)-evoked releases of all neurotransmitters. BFA inhibited PCP-associated K(+)-evoked monoamine release, but TTX did not affect them. PCP-induced reduction of K(+)-evoked GABA and glutamate releases was abolished by TTX and BFA. These results indicate that PCP reduces GABAergic transmission via NMDA-receptor blockade and activates intracellular endoplasmic-reticulum-associated signal-transduction, resulting in enhancement of monoaminergic transmission in pFC. Thus, these PCP properties support the hypothesis that mechanisms of the neurological symptoms of acute PCP-intoxication, convulsion, and rhabdomyolysis may be involved in both reduction of GABAergic-transmission and activation of endoplasmic-reticulum-associated signal-transduction induced by PCP.