The structure-activity relationship between oxycoumarin derivatives showing inhibitory effects on iNOS in mouse macrophage RAW264.7 cells.

We have investigated the structure-activity relationship between 63 natural oxycoumarin derivatives and their effects on the expression of inducible-nitric oxide synthase (iNOS) induced by lipopolysaccharide. The protein expression of iNOS was screened by Western blot analysis, and four 5,7-dimethoxycoumarins were selected as potent inhibitors of iNOS expression. In terms of structural specificity, the methoxyl group on C-5 and C-7 and the short alkyl chain (1-5 carbons) on C-6 may be essential for the potent activities. These compounds also showed inhibitory effects on nitric oxide generation and mRNA expression of inflammatory mediators, namely, iNOS and COX-2. Interestingly, the inhibitory effect on mRNA expression was specific for iNOS and was not detected for neuronal NOS. It is expected that these compounds will show anti-inflammatory activities via inhibition of the expressions of iNOS and COX-2.

Morphine-6-glucuronide induces contraction of the ileal circular muscle more potently than morphine in mice.

It has been reported that morphine-6-glucuronide inhibits small intestinal transit in mice more potently than morphine. In this study, we investigated the effects of morphine, morphine-6-glucuronide and morphine-3-glucuronide on the contractile response of the circular muscle isolated from mouse ileum. Morphine and morphine-6-glucuronide induced tonic contraction dose-dependently, and the contractile force of morphine-6-glucuronide was greater than that of morphine. Morphine-3-glucuronide induced slight contraction at high dose. These results suggest that the strong contraction induced by morphine-6-glucuronide contributed to the inhibition of small intestinal transit in mice.

Contractile effect of TRPA1 receptor agonists in the isolated mouse intestine.

TRPA1 is a member of the transient receptor potential (TRP) channel family expressed in sensory neurons. The present study focused on the effects of TRPA1 activation on contractile responses in isolated mouse intestine preparations. The jejunum, ileum, and proximal and distal colon were surgically isolated from male ddY mice. Intestinal motility was recorded as changes in isotonic tension. TRPA1, TRPM8, and TRPV1 expressions were examined by reverse transcription-polymerase chain reaction (RT-PCR). A TRPA1 agonist allyl isothiocyanate (AITC) dose-dependently induced contractions in the proximal and distal colon, whereas in the jejunum and ileum, even 100 muM AITC caused very little contraction. Likewise, a TRPA1 and TRPM8 agonist icilin, a TRPA1 agonist allicin, and a TRPV1 agonist capsaicin induced contractions in the colon. However, a TRPM8 agonist menthol induced long-lasting relaxation in the colon. Repeated exposure to AITC produced desensitization of its own contraction in the colon. Moreover, contractions induced by AITC generate cross-desensitization with icilin and capsaicin. Tetrodotoxin completely abolished AITC-induced contractions in the colon, whereas atropine significantly attenuated AITC-induced contractions in the distal colon, but not in the proximal colon. Menthol-induced relaxation in the colon was not inhibited by tetrodotoxin and atropine. RT-PCR analysis revealed the expression of TRPA1 and TRPV1, but not TRPM8, throughout the mouse intestine. These results suggest that TRPA1, but not TRPM8, are functionally expressed in the enteric nervous system throughout the mouse intestine on neurons that may also co-express TRPV1, yet the contractile responses to TRPA1 activation differ depending on their location along the intestine.

Morphine leads to contraction of the ileal circular muscle via inhibition of the nitrergic pathway in mice.

Morphine inhibits small intestinal transit in mice, although few mu-opioid receptors are present in the ileum. The present study focused on the action of morphine in the isolated mouse ileum to reveal the mechanism by which morphine inhibits mouse small intestinal transit. In the isolated circular muscle, morphine caused tonic contraction. This contraction was potently inhibited by naloxone and the mu-opioid receptor antagonist cyprodime. Moreover, the response was almost completely inhibited by tetrodotoxin and N(G)-nitro-L-arginine, but only moderately inhibited by atropine and indomethacin. In the isolated longitudinal muscle, morphine caused no or only slight contractions. Furthermore, electrically induced contraction was dose-dependently depressed by morphine, an effect that was not reversed by naloxone. These findings indicate that 1) morphine-induced circular muscle contraction occurs in the mouse ileum, 2) the contraction occurs through mu-opioid receptors mainly by inhibiting the release of nitric oxide from nitrergic nerves, although cholinergic nerves are at least partly involved in this contractile mechanism, and 3) inhibition of descending relaxation of peristalsis by morphine may slow small intestinal transit.

Regionally differential effects of sennoside A on spontaneous contractions of colon in mice.

Sennosides, the most popular irritant laxatives, cause purgative actions in the intestine through biotransformation to rhein anthrone; however, the underlying mechanisms remain unclear. The purpose of this study was to define colonic motor actions of sennoside A with special reference to purgative action. Mice received a single oral dose of 30 mg/kg sennoside A, and the colon was removed about 6 hr later. Contractions of longitudinal and circular muscles were recorded using an isometric force transducer and a pressure transducer, respectively. In longitudinal muscle preparations, spontaneous contractions were augmented in distal colon compared to control. In circular muscle preparations, contractions were reduced in the proximal colon, but increased in the distal colon. Particularly in the proximal colon, the frequency of high-amplitude contraction was reduced. In the control group, non-adrenergic, non-cholinergic treatment decreased the amplitude of contractions in the proximal colon, but not in the distal colon. In the sennoside A group, non-adrenergic, non-cholinergic treatment only slightly depressed the amplitude of contractions in the proximal and distal colon. To confirm a causal relationship between luminal prostaglandin level and purgative action of sennoside A, the mice were treated with indomethacin. Significant changes induced by sennoside A were attenuated by indomethacin treatment. The present study indicates that spontaneous motility is inhibited by sennoside A in the proximal colon, but accelerated in the distal colon, and that effects are associated with luminal prostanoid level and only partially with cholinergic nerve mediation.

Quercetin-induced PC12 cell death accompanied by caspase-mediated DNA fragmentation.

Flavonoids have been reported to be potent antioxidants and beneficial in oxidative stress related diseases. Quercetin, a major flavonoid in food, deserves much attention because of its antioxidative activity. However, the actions of flavonoids including quercetin are complex and paradoxical. Quercetin caused apoptosis and/or cell death in various cells including cancer cells and normal cells. In this study, we investigated the effects of quercetin with or without hydrogen peroxide (H2O2) on cell death of PC12 cells, a neuronal cell line. We showed that quercetin at 10-30 microM alone caused cell death accompanied by caspase-mediated DNA fragmentation in undifferentiated PC12 cells. Quercetin did not inhibit and rather enhanced 0.1 mM H2O2-induced cell death. The toxic effect of quercetin was not inhibited by antioxidants such as N-acetylcysteine and GSH, although H2O2-induced cell death was inhibited by the antioxidants. Quercetin-induced cell death was reduced by 2 h treatment with nerve growth factor and serum. In addition, quercetin caused cell death in differentiated PC12 cells that were cultured with nerve growth factor for 6 d. Genistein, a soy isoflavone that has the pro-apoptotic activity, also caused cell death with DNA fragmentation. Further evaluation of the potential of dietary flavonoids as neuroprotective reagents is needed.

Capsaicin- and anandamide-induced gastric acid secretion via vanilloid receptor type 1 (TRPV1) in rat brain.

The activation of transient receptor potential vanilloid receptor 1 (TRPV1) by capsaicin in rat brain stimulates gastric acid secretion via tachykinin NK2 receptors and the vagus cholinergic nerve, but the involvement of other receptor systems has not been elucidated. We investigated the role of the glutamate and gamma-amino-butyric acid (GABA) receptor systems on the capsaicin response. Gastric acid secretion stimulated by the injection of capsaicin (30 nmol) into the lateral cerebroventricle (i.c.v.) was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an antagonist of non-N-methyl-D-aspartate (non-NMDA) receptors, 10.9 nmol, i.c.v.) and bicuculline (a GABA(A) receptor antagonist, 222 microg kg(-1) 10 min(-1), i.v. infusion). Secretion stimulated by the injection of capsaicin (50 nmol) into the fourth cerebroventricle was inhibited by CNQX and bicuculline. I.c.v. injection of anandamide (an endogenous ligand of TRPV1 and cannabinoid receptors, 30 and 100 nmol) stimulated gastric acid secretion, and the response was inhibited by an antagonist of TRPV1 and in the capsaicin-treated rats, but not by an antagonist of cannabinoid receptors. In conclusion, the TRPV1 system, which is activated by capsaicin and anandamide, is preferentially coupled with non-NMDA and GABA(A) receptor systems in the brain and stimulates gastric acid secretion in rats.

Role of neuropeptide receptor systems in vanilloid VR1 receptor-mediated gastric acid secretion in rat brain.

Previously, we reported that the injection of capsaicin into the lateral cerebroventricle (i.c.v.) stimulated gastric acid secretion via vanilloid VR1 receptors and the vagal cholinergic pathways in anesthetized rats. In the present study, we investigated the involvement of receptor systems for neurokinin A, calcitonin gene-related peptide (CGRP) and glutamate in the vanilloid VR1 receptor-mediated response. The i.c.v. injection of neurokinin A (30 nmol) stimulated gastric acid secretion in the presence of cis-2-(diphenylmethyl)-N-[(2-iodophenyl)methyl]-1-azabicyclo[2.2.2]octan-3-amine oxalate (L-703606, a tachykinin NK1 receptor antagonist, 30 nmol) and the effect was inhibited by cyclo[Gln-Trp-Phe-Gly-Leu-Met] (L-659877, a tachykinin NK2 receptor antagonist, 30 nmol); the values were 145.9 +/- 32.3 and 21.1 +/- 16.6 microEq HCl per 120 min, respectively. The value in the control group was 14.3 +/- 3.8 microEq HCl. The tachykinin NK2 receptor-mediated secretion was inhibited by i.c.v. injections of antagonists of the CGRP1 receptor (human CGRP fragment 8-37, 15 nmol) and non-N-methyl-D-aspartate (non-NMDA)-type glutamate receptor (6-cyano-7-nitroquinoxaline-2,3-dione, 10.9 nmol); the values were 30.8+/-29.8 and 5.7+/-16.9 microEq HCl, respectively. Gastric acid secretion induced by the i.c.v. injection of 30 nmol capsaicin (178.4 +/- 34.0 microEq HCl) was inhibited by antagonists of tachykinin NK2 (23.7 +/- 6.2) and CGRP1 (21.2 +/- 8.5), but not tachykinin NK1 (181.4 +/- 37.0), receptors. The gastric acid secretion induced by capsaicin was decreased by the i.c.v. pre-injection of low doses of neurokinin A or CGRP, which alone had no effect on the secretion. These findings suggest the involvement of tachykinin NK2, CGRP and non-NMDA receptor systems in the vanilloid VR1 receptor-mediated regulation of gastric acid secretion in the rat brain regions close to the lateral cerebroventricle.

Involvement of glutamate and gamma-amino-butyric acid receptor systems on gastric acid secretion induced by activation of kappa-opioid receptors in the central nervous system in rats.

1. Various neurotransmitters in the brain regulate gastric acid secretion. Previously, we reported that the central injection of kappa-opioid receptor agonists stimulated this secretion in rats. Although the existence of kappa(1)-kappa(3)-opioid receptor subtypes has been proposed, the character is not defined. We investigated the interactions between kappa-opioid receptor subtypes and glutamate, gamma-amino-butyric acid (GABA) or 5-hydroxy tryptamine (5-HT) receptors in the rat brain. 2. Gastric acid secretion induced by the injection of U69593 (8.41 nmol, a putative kappa(1)-opioid receptor agonist) into the lateral cerebroventricle was completely inhibited by the central injection of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10.9 nmol, an antagonist for non-N-methyl-D-aspartate (non-NMDA) receptors) and by bicuculline infusion (222 micro g kg(-1) per 10 min, i.v., GABA(A) receptor antagonist). The secretion induced by bremazocine (8.52 nmol, a putative kappa(2)-opioid receptor agonist) was inhibited by bicuculline infusion, but not by CNQX. The secretion induced by naloxone benzoylhydrazone (224 nmol, a putative kappa(3)-opioid receptor agonist) was slightly and partially inhibited by CNQX and bicuculline. 3. Treatment with CNQX and bicuculline inhibited gastric acid secretion induced by the injection of dynorphin A-(1-17) into the lateral, but not the fourth, cerebroventricle. Antagonists for NMDA, GABA(B) and 5-HT(2/1C) receptors did not inhibit the secretions by kappa-opioid receptor agonists. 4. In rat brain regions close to the lateral cerebroventricle, kappa-opioid receptor systems (kappa(1)>kappa(3)>>kappa(2)) are regulated by the non-NMDA type of glutamate receptor system, and kappa(1)- and kappa(2)-opioid receptor systems are regulated by the GABA(A) receptor system. The present findings show pharmacological evidence for kappa-opioid receptor subtypes that regulate gastric acid secretion in the rat brain.

Stimulatory effects of centrally injected nitric oxide donors on gastric acid secretion in anesthetized rats.

The effects of centrally injected nitric oxide (NO) donors on gastric acid secretion were investigated in continuously perfused stomach of anesthetized rats. The lateral cerebroventricular (LV) injection of NOC5 (30 - 100 microg) and NOC12 (10 - 100 microg) dose-dependently stimulated gastric acid secretion. The LV injection of NOC18 (30 microg) also stimulated gastric acid secretion. The other type of NO donor, sodium nitroprusside (3 - 30 microg, LV), also dose-dependently stimulated gastric acid secretion. The effect of NOC5 at 100 microg was blocked by carboxy-PTIO, an NO scavenger, and by cervical vagotomy. Furthermore, NOC12 (30, 100 microg) dose-dependently stimulated gastric acid secretion in pylorus-ligated conscious rats. These results suggest that centrally injected NO donors stimulate gastric acid secretion in both conscious and anesthetized rats through vagus activation.

Effect of mitragynine, derived from Thai folk medicine, on gastric acid secretion through opioid receptor in anesthetized rats.

Mitragynine, an indole alkaloid from Thai folk medicine Mitragyna speciosa, exerts agonistic effects on opioid receptors. Gastric acid secretion is proposed to be regulated by opioid receptors in the central nervous system (CNS). Previously, we reported the dual roles (inhibition via micro-opioid receptors and stimulation via kappa-opioid receptors) of the opioid system in the central control of gastric acid secretion. We investigated whether mitragynine affects gastric acid secretion via opioid receptors in the CNS. Injection of mitragynine (30 microg) alone into the lateral cerebroventricle did not have a significant effect on basal gastric acid secretion in the perfused stomach of anesthetized rats. Injection of mitragynine (3-30 microg) into the fourth cerebroventricle, like morphine, inhibited 2-deoxy-D-glucose-stimulated gastric acid secretion. The inhibitory effect of mitragynine (30 microg) was reversed by naloxone (100 microg). These results suggest that mitragynine has a morphine-like action on gastric acid secretion in the CNS.

Gastric acid secretion stimulated by centrally injected nociceptin in urethane-anesthetized rats.

Nociceptin is a preferred endogenous ligand for the orphan opioid receptor-like 1 (ORL1) receptor. Central administration of nociceptin showed various pharmacological effects on analgesia, cardiovascular and renal responses, food intake, and so on. In the present study, we investigated the effect of nociceptin injected into the central nervous system (CNS) on gastric acid secretion in the perfused stomach of urethane-anesthetized rats. Injection of nociceptin (0.55-5.52 nmol per rat) into the fourth cerebroventricle stimulated gastric acid secretion and the secretion was inhibited in atropine-treated (1 mg/kg, i.v.) and vagotomized rats. The secretion induced by nociceptin (1.65 nmol) was not inhibited by the central injection of naloxone (275 nmol, a non-selective antagonist of opioid receptors). The secretion was significantly inhibited by the central injection of [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)-NH(2) ([F/G]nociceptin-(1-13), 0.21 nmol, an antagonist of ORL1 receptor), although [F/G]nociceptin-(1-13) alone at higher doses (2.10 and 7.31 nmol) markedly stimulated gastric acid secretion. In the 0-40 min period, the secretion induced by nociceptin was inhibited at least partially by CompB (68.8 nmol, a nonpeptidic antagonist of ORL1 receptor). Injection of nociceptin (5.52 nmol) into the lateral cerebroventricle also stimulated the secretion. Injection of nociceptin did not modify gastric acid secretion stimulated by 2-deoxy-D-glucose (200 mg/kg, i.v.). In conclusion, nociceptin injected into the CNS stimulated gastric acid secretion in rats via the ORL1 receptors and through mechanisms involving the vagus nerve.

Phenolic compounds from Gastrodia rhizome and relaxant effects of related compounds on isolated smooth muscle preparation.

Gastrol (1), together with 10 known phenolic compounds, has been isolated from the MeOH extract of the rhizomes of Gastrodia elata Blume (Orchidaceae), and their structures were elucidated by detailed spectral analyses including by 2D NMR spectroscopic analyses. The relaxant effects of these constituents on smooth muscle preparations isolated from guinea-pig ileum were also studied in order to reveal their characteristic pharmacological activities.

Mesaconitine-induced relaxation in rat aorta: involvement of Ca2+ influx and nitric-oxide synthase in the endothelium.

Aconiti tuber, roots of aconite (Aconitum japonicum), is an oriental herbal medicine used for centuries in Japan and China to improve the health of persons with a weak constitution and poor metabolism. We investigated the effects of mesaconitine, one of the aconite alkaloids in Aconiti tuber, on the contraction and free intracellular Ca2+ concentration ([Ca2+]i) level in isolated rat thoracic aorta. Mesaconitine at 30 microM inhibited 3 microM phenylephrine-induced contraction in the endothelium-intact, but not endothelium-denuded, aortic rings. The effect of mesaconitine was dependent on external Ca2+ concentrations. The relaxation induced by mesaconitine was abolished by N(omega)-nitro-L-arginine methyl ester (0.1 mM, an inhibitor of nitric-oxide synthase), as well as the relaxation induced by acetylcholine. Acetylcholine induced relaxation in two phases in our conditions; the initial phase was transient and external Ca2+ -independent, and the second phase was sustained and external Ca2+ -dependent. Treatment with 100 nM thapsigargin, which depleted intracellular Ca2+ stores, inhibited acetylcholine-induced, but not mesaconitine-induced, relaxation. Mesaconitine increased the [Ca2+]i level in endothelial cells by influx of Ca2+ from extracellular spaces. These findings suggest that mesaconitine-induced Ca2+ influx and activation of nitric-oxide synthase in endothelial cells and, thus, induced vasorelaxation in rat aorta.