Deficiency of interleukin-19 exacerbates acute lung injury induced by intratracheal treatment of hydrochloric acid.

Interleukin (IL-19) belongs to the IL-10 family of cytokines and plays diverse roles in inflammation, cell development, viral responses, and lipid metabolism. Acute lung injury (ALI) is a severe respiratory condition associated with various diseases, including severe pneumonia, sepsis, and trauma, lacking established treatments. However, the role of IL-19 in acute inflammation of the lungs is unknown. We reported the impact of IL-19 functional deficiency in mice crossed with an ALI model using HCl. Lungs damages, neutrophil infiltration, and pulmonary edema induced by HCl were significantly worse in IL-19 knockout (KO) mice than in wild-type (WT) mice. mRNA expression levels of C-X-C motif chemokine ligand 1 (CXCL1) and IL-6 in the lungs were significantly higher in IL-19 KO mice than in WT mice. Little apoptosis was detected in lung injury in WT mice, whereas apoptosis was observed in exacerbated area of lung injury in IL-19 KO mice. These results are the first to show that IL-19 is involved in acute inflammation of the lungs, suggesting a novel molecular mechanism in acute respiratory failures. If it can be shown that neutrophils have IL-19 receptors and that IL-19 acts directly on them, it would be a novel drug target.

Inhibition of Drp1-Filamin Protein Complex Prevents Hepatic Lipid Droplet Accumulation by Increasing Mitochondria-Lipid Droplet Contact.

Lipid droplet (LD) accumulation in hepatocytes is one of the major symptoms associated with fatty liver disease. Mitochondria play a key role in catabolizing fatty acids for energy production through ß-oxidation. The interplay between mitochondria and LD assumes a crucial role in lipid metabolism, while it is obscure how mitochondrial morphology affects systemic lipid metabolism in the liver. We previously reported that cilnidipine, an already existing anti-hypertensive drug, can prevent pathological mitochondrial fission by inhibiting protein-protein interaction between dynamin-related protein 1 (Drp1) and filamin, an actin-binding protein. Here, we found that cilnidipine and its new dihydropyridine (DHP) derivative, 1,4-DHP, which lacks Ca2+ channel-blocking action of cilnidipine, prevent the palmitic acid-induced Drp1-filamin interaction, LD accumulation and cytotoxicity of human hepatic HepG2 cells. Cilnidipine and 1,4-DHP also suppressed the LD accumulation accompanied by reducing mitochondrial contact with LD in obese model and high-fat diet-fed mouse livers. These results propose that targeting the Drp1-filamin interaction become a new strategy for the prevention or treatment of fatty liver disease.

Pharmacological Activation of TRPC6 Channel Prevents Colitis Progression.

We recently reported that transient receptor potential canonical (TRPC) 6 channel activity contributes to intracellular Zn2+ homeostasis in the heart. Zn2+ has also been implicated in the regulation of intestinal redox and microbial homeostasis. This study aims to investigate the role of TRPC6-mediated Zn2+ influx in the stress resistance of the intestine. The expression profile of TRPC1-C7 mRNAs in the actively inflamed mucosa from inflammatory bowel disease (IBD) patients was analyzed using the GEO database. Systemic TRPC3 knockout (KO) and TRPC6 KO mice were treated with dextran sulfate sodium (DSS) to induce colitis. The Zn2+ concentration and the mRNA expression levels of oxidative/inflammatory markers in colon tissues were quantitatively analyzed, and gut microbiota profiles were compared. TRPC6 mRNA expression level was increased in IBD patients and DSS-treated mouse colon tissues. DSS-treated TRPC6 KO mice, but not TRPC3 KO mice, showed severe weight loss and increased disease activity index compared with DSS-treated WT mice. The mRNA abundances of antioxidant proteins were basically increased in the TRPC6 KO colon, with changes in gut microbiota profiles. Treatment with TRPC6 activator prevented the DSS-induced colitis progression accompanied by increasing Zn2+ concentration. We suggest that TRPC6-mediated Zn2+ influx activity plays a key role in stress resistance against IBD, providing a new strategy for treating colitis.

Fucoxanthin Ameliorates Atopic Dermatitis Symptoms by Regulating Keratinocytes and Regulatory Innate Lymphoid Cells.

Fucoxanthin (FX) is a xanthophyll that is contained abundantly in marine plants. The biological action of FX includes its antioxidant and anti-lipogenic activities, while the precise action of its mechanisms on skin cells has not yet been clarified. The current study examined the effect of FX in comparison with tacrolimus (TAC) on NC/Nga mice, which are an atopic dermatitis (AD) model. FX topical treatment dramatically ameliorated itching behavior over the TAC treatment, which was insufficient for improvement of AD symptoms. In Nc/Nga mice, FX or TAC applied to the skin inhibited eosinophil infiltration with decreased expression of Il-33. FX also stimulated Il-2, Il-5, Il-13, Il-10, and TGF-ß expression levels, and Sca1+Il-10+TGF-ß+ regulatory innate lymphoid cells (ILCreg) were dominantly observed in FX treated skin epidermal keratinocytes and dermal layers. This combined evidence demonstrated that FX exerts anti-inflammatory effects on keratinocytes and ameliorates AD symptoms by regulating ILCreg to normalize immune responses in an atopic dermatitis model.

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-7.

Breakthroughs in Medicinal Chemistry [...].

Chronic kidney disease after 5/6 nephrectomy disturbs the intestinal microbiota and alters intestinal motility.

Organ-organ crosstalk is involved in homeostasis. Gastrointestinal symptoms are common in patients with renal failure. The aim of this study was to elucidate the relationship between gastrointestinal motility and gastrointestinal symptoms in chronic kidney disease. We performed studies in C57BL/6 mice with chronic kidney disease after 5/6 nephrectomy. Gastrointestinal motility was evaluated by assessing the ex vivo responses of ileum and distal colon strips to electrical field stimulation. Feces were collected from mice, and the composition of the gut microbiota was analyzed using 16S ribosomal RNA sequencing. Mice with chronic kidney disease after 5/6 nephrectomy showed a decreased amount of stool, and this constipation was correlated with a suppressed contraction response in ileum motility and decreased relaxation response in distal colon motility. Spermine, one of the uremic toxins, inhibited the contraction response in ileum motility, but four types of uremic toxins showed no effect on the relaxation response in distal colon motility. The 5/6 nephrectomy procedure disturbed the balance of the gut microbiota in the mice. The motility dysregulation and constipation were resolved by antibiotic treatments. The expression levels of interleukin 6, tumor necrosis factor-α, and iNOS in 5/6 nephrectomy mice were increased in the distal colon but not in the ileum. In addition, macrophage infiltration in 5/6 nephrectomy mice was increased in the distal colon but not in the ileum. We found that 5/6 nephrectomy altered gastrointestinal motility and caused constipation by changing the gut microbiota and causing colonic inflammation. These findings indicate that renal failure was remarkably associated with gastrointestinal dysregulation.

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes-6.

Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials that is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules [...].

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death.

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that also mediates cell death under oxidative stress. We reported previously that the active-site cysteine (Cys-152) of GAPDH plays an essential role in oxidative stress-induced aggregation of GAPDH associated with cell death, and a C152A-GAPDH mutant rescues nitric oxide (NO)-induced cell death by interfering with the aggregation of wild type (WT)-GAPDH. However, the detailed mechanism underlying GAPDH aggregate-induced cell death remains elusive. Here we report that NO-induced GAPDH aggregation specifically causes mitochondrial dysfunction. First, we observed a correlation between NO-induced GAPDH aggregation and mitochondrial dysfunction, when GAPDH aggregation occurred at mitochondria in SH-SY5Y cells. In isolated mitochondria, aggregates of WT-GAPDH directly induced mitochondrial swelling and depolarization, whereas mixtures containing aggregates of C152A-GAPDH reduced mitochondrial dysfunction. Additionally, treatment with cyclosporin A improved WT-GAPDH aggregate-induced swelling and depolarization. In doxycycline-inducible SH-SY5Y cells, overexpression of WT-GAPDH augmented NO-induced mitochondrial dysfunction and increased mitochondrial GAPDH aggregation, whereas induced overexpression of C152A-GAPDH significantly suppressed mitochondrial impairment. Further, NO-induced cytochrome c release into the cytosol and nuclear translocation of apoptosis-inducing factor from mitochondria were both augmented in cells overexpressing WT-GAPDH but ameliorated in C152A-GAPDH-overexpressing cells. Interestingly, GAPDH aggregates induced necrotic cell death via a permeability transition pore (PTP) opening. The expression of either WT- or C152A-GAPDH did not affect other cell death pathways associated with protein aggregation, such as proteasome inhibition, gene expression induced by endoplasmic reticulum stress, or autophagy. Collectively, these results suggest that NO-induced GAPDH aggregation specifically induces mitochondrial dysfunction via PTP opening, leading to cell death.

Aggressive Crosstalk Between Fatty Acids and Inflammation in Macrophages and Their Influence on Metabolic Homeostasis.

From the immunological point of view, macrophages are required to maintain metabolic homeostasis. Recently, there has been an increased focus on the influence of macrophage phenotypes in adipose tissue on the maintenance of metabolic homeostasis in healthy conditions because dysregulated metabolic homeostasis causes metabolic syndrome. This review notes several types of inflammatory and anti-inflammatory mediators in metabolic homeostasis. M1 macrophage polarization mediates inflammation, whereas M2 macrophage polarization mediates anti-inflammation. Fatty acids and their related factors mediate both inflammatory and anti-inflammatory responses. Saturated fatty acids and polyunsaturated fatty acids mediate inflammation, whereas marine-derived n-3 fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid, mediate anti-inflammation. In this review, we discuss the current understanding of the crosstalk between fatty acids and inflammation in macrophages and their influence on metabolic homeostasis.

Lauric Acid Alleviates Neuroinflammatory Responses by Activated Microglia: Involvement of the GPR40-Dependent Pathway.

In several neurodegenerative diseases such as Alzheimer's disease (AD), microglia are hyperactivated and release nitric oxide (NO) and proinflammatory cytokines, resulting its neuropathology. Mounting evidence indicates that dietary supplementation with coconut oil (CNO) reduces the cognitive deficits associated with AD; however, the precise mechanism(s) underlying the beneficial effect of CNO are unknown. In the present study, we examined the effects of lauric acid (LA), a major constituent of CNO, on microglia activated experimentally by lipopolysaccharide (LPS), using primary cultured rat microglia and the mouse microglial cell line, BV-2. LA attenuated LPS-stimulated NO production and the expression of inducible NO synthase protein without affecting cell viability. In addition, LA suppressed LPS-induced reactive oxygen species and proinflammatory cytokine production, as well as phosphorylation of p38-mitogen activated protein kinase and c-Jun N-terminal kinase. LA-induced suppression of NO production was partially but significantly reversed in the presence of GW1100, an antagonist of G protein-coupled receptor (GPR) 40, which is an LA receptor on the plasma membrane. LA also decreased LPS-induced phagocytosis, which was completely reversed by co-treatment with GW1100. Moreover, LA alleviated amyloid-ß-induced enhancement of phagocytosis. These results suggest that attenuation of microglial activation by LA may occur via the GPR40-dependent pathway. Such effects of LA may reduce glial activation and the subsequent neuronal damage in AD patients who consume CNO.

The Role of Interleukin-19 in Contact Hypersensitivity.

Interleukin (IL)-19 is a member of the IL-10 family of interleukins and is an immuno-modulatory cytokine produced by the main macrophages. The gastrointestinal tissues of IL-19 knockout mice show exacerbated experimental colitis mediated by the innate immune system and T cells. There is an increasing focus on the interaction and relationship of IL-19 with the function of T cells. Contact hypersensitivity (CHS) is T cell-mediated cutaneous inflammation. Therefore, we asked whether IL-19 causes CHS. We investigated the immunological role of IL-19 in CHS induced by 1-fluoro-2,4-dinitrofluorobenzene as a hapten. IL-19 was highly expressed in skin exposed to the hapten, and ear swelling was increased in IL-19 knockout mice. The exacerbation of the CHS response in IL-19 knockout mice correlated with increased levels of IL-17 and IL-6, but no alterations were noted in the production of interferon (IFN)γ and IL-4 in the T cells of the lymph nodes. In addition to the effect on T cell response, IL-19 knockout mice increased production of inflammatory cytokines. These results show that IL-19 suppressed hapten-dependent skin inflammation in the elicitation phase of CHS.

Extracellular poly(ADP-ribose) is a neurotrophic signal that upregulates glial cell line-derived neurotrophic factor (GDNF) levels in vitro and in vivo.

Synthesis of poly(ADP-ribose) (PAR) is catalyzed by PAR polymerase-1 (PARP-1) in neurons. PARP1 plays a role in various types of brain damage in neurodegenerative disorders. In neurons, overactivation of PARP-1 during oxidative stress induces robust PAR formation, which depletes nicotinamide adenine dinucleotide levels and leads to cell death. However, the role of the newly-formed PAR in neurodegenerative disorders remains elusive. We hypothesized that the effects of PAR could occur in the extracellular space after it is leaked from damaged neurons. Here we report that extracellular PAR (EC-PAR) functions as a neuroprotective molecule by inducing the synthesis of glial cell line-derived neurotrophic factor (GDNF) in astrocytes during neuronal cell death, both in vitro and in vivo. In primary rat astrocytes, exogenous treatment with EC-PAR produced GDNF but not other neurotrophic factors. The effect was concentration-dependent and did not affect cell viability in rat C6 astrocytoma cells. Topical injection of EC-PAR into rat striatum upregulated GDNF levels in activated astrocytes and improved pathogenic rotation behavior in a unilateral 6-hydroxydopamine model of Parkinson disease in rats. These findings indicate that EC-PAR acts as a neurotrophic enhancer by upregulating GDNF levels. This effect protects the remaining neurons following oxidative stress-induced brain damage, such as that seen with Parkinson disease.

Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-ß Amyloidogenesis in Alzheimer Disease.

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-ß peptide (Aß). Numerous studies have established Aß amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aß amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aß40 with small amounts of GAPDH aggregates significantly enhanced Aß40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aß40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aß40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aß40 co-incubated with GAPDH aggregates exhibited Aß40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome c from mitochondria. Finally, in the 3×Tg-AD mouse model of AD, GAPDH/Aß co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aß aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aß amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD.

Nuclear-translocated Glyceraldehyde-3-phosphate Dehydrogenase Promotes Poly(ADP-ribose) Polymerase-1 Activation during Oxidative/Nitrosative Stress in Stroke.

In addition to its role in DNA repair, nuclear poly(ADP-ribose) polymerase-1 (PARP-1) mediates brain damage when it is over-activated by oxidative/nitrosative stress. Nonetheless, it remains unclear how PARP-1 is activated in neuropathological contexts. Here we report that PARP-1 interacts with a pool of glyceradehyde-3-phosphate dehydrogenase (GAPDH) that translocates into the nucleus under oxidative/nitrosative stress both in vitro and in vivo. A well conserved amino acid at the N terminus of GAPDH determines its protein binding with PARP-1. Wild-type (WT) but not mutant GAPDH, that lacks the ability to bind PARP-1, can promote PARP-1 activation. Importantly, disrupting this interaction significantly diminishes PARP-1 overactivation and protects against both brain damage and neurological deficits induced by middle cerebral artery occlusion/reperfusion in a rat stroke model. Together, these findings suggest that nuclear GAPDH is a key regulator of PARP-1 activity, and its signaling underlies the pathology of oxidative/nitrosative stress-induced brain damage including stroke.

Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.

Overexpression of Na+/Ca2+ exchanger 1 display enhanced relaxation in the gastric fundus.

In gastric smooth muscles, the released Ca2+ activates the contractile proteins and Ca2+ taken up from the cytosol cause relaxation. The Na+/Ca2+ exchanger (NCX) is an antiporter membrane protein that controls Ca2+ influx and efflux across the membrane. However, the possible relation of NCX in gastric fundus motility is largely unknown. Here, we investigated electric field stimulation (EFS)-induced relaxations in the circular muscles of the gastric fundus in smooth muscle-specific NCX1 transgenic mice (Tg). EFS caused a bi-phasic response, transient and sustained relaxation. The sustained relaxation prolonged for an extended period after the end of the stimulus. EFS-induced transient relaxation and sustained relaxation were greater in Tg than in wild-type mice (WT). Disruption of nitric oxide component by N-nitro-l-arginine, EFS-induced transient and sustained relaxations caused still marked in Tg compared to WT. Inhibition of PACAP by antagonist, EFS-induced sustained relaxation in Tg was not seen, similar to WT. Nevertheless, transient relaxation remained more pronounced in Tg than in WT. Next, we examined responses to NO and PACAP in smooth muscles. The magnitudes of NOR-1, which generates NO, and PACAP-induced relaxations were greater in Tg than in WT. In this study, we demonstrate that NCX1 regulates gastric fundus motility.

Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death.

IL-19 reduces ligation-mediated neointimal hyperplasia by reducing vascular smooth muscle cell activation.

We tested the hypothesis that IL-19, a putative member of the type 2 helper T-cell family of anti-inflammatory interleukins, can attenuate intimal hyperplasia and modulate the vascular smooth muscle cell (VSMC) response to injury. Ligated carotid artery of IL-19 knockout (KO) mice demonstrated a significantly higher neointima/intima ratio compared with wild-type (WT) mice (P = 0.04). More important, the increased neointima/intima ratio in the KO could be reversed by injection of 10 ng/g per day recombinant IL-19 into the KO mouse (P = 0.04). VSMCs explanted from IL-19 KO mice proliferated significantly more rapidly than WT. This could be inhibited by addition of IL-19 to KO VSMCs (P = 0.04 and P < 0.01). IL-19 KO VSMCs migrated more rapidly compared with WT (P < 0.01). Interestingly, there was no type 1 helper T-cell polarization in the KO mouse, but there was significantly greater leukocyte infiltrate in the ligated artery in these mice compared with WT. IL-19 KO VSMCs expressed significantly greater levels of inflammatory mRNA, including IL-1ß, tumor necrosis factor α, and monocyte chemoattractant protein-1 in response to tumor necrosis factor α stimulation (P < 0.01 for all). KO VSMCs expressed greater adhesion molecule expression and adherence to monocytes. Together, these data indicate that IL-19 is a previously unrecognized counterregulatory factor for VSMCs, and its expression is an important protective mechanism in regulation of vascular restenosis.

Botulinum neurotoxin A subtype 2 reduces pathological behaviors more effectively than subtype 1 in a rat Parkinson's disease model.

Recent reports indicate that interruption of acetylcholine release by intrastriatal injection of botulinum neurotoxin type A (BoNT/A) in a rat Parkinson's disease model reduces pathogenic behavior without adverse side effects such as memory dysfunction. Current knowledge suggests that BoNT/A subtype 1 (BoNT/A1) and BoNT/A subtype 2 (BoNT/A2) exert different effects. In the present study, we compared the effects of BoNT/A1 and BoNT/A2 on rotation behavior and in vivo cleavage of presynaptic protein SNAP-25 in a rat unilateral 6-hydroxydopamine-induced Parkinson's disease model. BoNT/A2 more effectively reduced pathogenic behavior by efficiently cleaving SNAP-25 in the striatum compared with that of BoNT/A1. Our results suggest that BoNT/A2 has greater clinical therapeutic value for treating subjects with Parkinson's disease compared to that of BoNT/A1.

Evidence that nitric oxide is a non-adrenergic non-cholinergic inhibitory neurotransmitter in the circular muscle of the mouse distal colon: a study on the mechanism of nitric oxide-induced relaxation.

The gastrointestinal tract is composed of outer longitudinal muscle layers and inner circular muscle layers. Nitric oxide (NO), carbon monoxide (CO), and ATP play major roles as non-adrenergic non-cholinergic (NANC) inhibitory neurotransmitters in the longitudinal muscle of the mouse distal colon, whereas it is unclear which NANC inhibitory neurotransmitters are in its circular muscle. We investigated the electric field stimulation (EFS)-induced relaxations in the circular smooth muscle of the distal colon under NANC conditions. In the experiments in which N(ω)-nitro-L-arginine, an inhibitor of NO synthase, was added, the EFS-induced relaxation decreased in a concentration-dependent manner and finally vanished. In contrast, CO, purinergic receptor ligands, and peptidergic substances do not play major roles as NANC neurotransmitters in the circular muscle of the mouse distal colon. ODQ, an inhibitor of soluble guanylate cyclase, strongly attenuated EFS-induced relaxation. Ryanodine, a Ca(2+) release modulator at the sarcoplasmic reticulum, strongly attenuated EFS-induced relaxation as well. Relaxation induced by NOR-1, which generates NO, was inhibited by ODQ and ryanodine. Next, we performed experiments that simultaneously measured tension and the cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt). NOR-1 decreased the tension and [Ca(2+)]cyt levels in the circular muscle. ODQ and ryanodine strongly attenuated the NOR-1-induced change in both tension and [Ca(2+)]cyt levels. In this study, we demonstrate that NO functions as a NANC inhibitory neurotransmitter in the circular muscle obtained from the mouse distal colon.