Decrease of guanylyl cyclase ß1 subunit and nitric oxide (NO)-induced relaxation in mouse rectum with colitis and its reproduction on long-term NO treatment.

Nitric oxide (NO) influences motility in the colon in patients with ulcerative colitis, but the exact mechanism involved remains unknown. Colitis was induced in mice by the oral administration of 2.5% dextran sodium sulfate (DSS), and the motility in longitudinal preparations from rectum and distal colon and expression of ß1 subunit of soluble guanylyl cyclase (sGCß1) were analyzed. Electrical stimulation (ES) caused a transient relaxation via the NO pathway in both rectum and colon from control mice. Stimulation with sodium nitroprusside (SNP) caused relaxation in the two regions, and the half-time (T (1/2)) of the maximal relaxation induced by 100 µM SNP was 8.1 ± 1.0 s in rectum. DSS treatment (1) abolished the ES-induced relaxation, but not dibutyryl cyclic GMP-induced response, in both regions, (2) decreased the maximal response to SNP accompanied by a loss of immunoreactive sGCß1 protein in rectum, but did not affect the amplitude of the relaxant response or the protein in distal colon, and (3) caused an increase in the T (1/2) value in response to SNP in both regions. Pretreatment of both preparations from control mice with 600 µM SNP for 30 min decreased both ES- and SNP-induced relaxation, SNP-induced cyclic GMP formation, and immunoreactive sGCß1 levels. NO-mediated relaxation was impaired by a dysfunctional sGC with and without a loss of immunoreactivity to sGCß1 in rectum and colon from DSS-treated mice, respectively. Long-term exposure of the tissues with an excess amount of NO changes the sGC-mediated relaxation.

Effects of synthetic sphingosine-1-phosphate analogs on cytosolic phospholipase A2alpha-independent release of arachidonic acid and cell toxicity in L929 fibrosarcoma cells: the structure-activity relationship.

Sphingolipid metabolites including ceramide, sphingosine, and their phosphorylated products [sphingosine-1-phosphate (S1P) and ceramide-1-phosphate] regulate cell functions including arachidonic acid (AA) metabolism and cell death. The development of analogs of S1P may be useful for regulating these mediator-induced cellular responses. We synthesized new analogs of S1P and examined their effects on the release of AA and cell death in L929 mouse fibrosarcoma cells. Among the analogs tested, several compounds including DMB-mC11S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMB-mC9S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-nonyl)phenylpropyl phosphate] released AA within 1 h and caused cell death 6 h after treatment. The release of AA was observed in C12 cells [a L929 variant lacking a type alpha cytosolic phospholipase A(2) (cPLA(2)alpha)] and L929-cPLAalpha-siRNA cells (L929 cells treated with small interference RNA for cPLA(2)alpha). Treatment with pharmacological inhibitors of secretory and Ca(2+)-independent PLA(2)s decreased the DMB-mC11S-induced release of AA. The effect of the S1P analogs tested on the release of AA was comparable to that on cell death in L929 cells, and a high correlation coefficient was observed. Two analogs lacking a butoxycarbonyl moiety [DMAc-mC11S (dimethyl (2S,3R)-2-acetamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMAm-mC11S [dimethyl (2S,3R)-2-amino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate)] had inhibitory effects on the release of AA and cell toxicity induced by DMB-mC11S. Synthetic phosphorylated lipid analogs may be useful for studying PLA(2) activity and its toxicity in cells. [Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08284FP].

MGM-9 [(E)-methyl 2-(3-ethyl-7a,12a-(epoxyethanoxy)-9-fluoro-1,2,3,4,6,7,12,12b-octahydro-8-methoxyindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate], a derivative of the indole alkaloid mitragynine: a novel dual-acting mu- and kappa-opioid agonist with potent antinociceptive and weak rewarding effects in mice.

Mitragynine is a major indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that has opium-like properties, although its chemical structure is quite different from that of morphine. We attempted to develop novel analgesics derived from mitragynine, and thus synthesized the ethylene glycol-bridged and C10-fluorinated derivative of mitragynine, MGM-9 [(E)-methyl 2-(3-ethyl-7a,12a-(epoxyethanoxy)-9-fluoro-1,2,3,4,6,7,12,12b-octahydro-8-methoxyindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate]. We hypothesized that a dual-acting mu- and kappa-opioid agonist could produce potent antinociceptive effects with fewer rewarding effects compared with mu agonists. In this study, MGM-9 exhibited high affinity for mu- and kappa-opioid receptors with Ki values of 7.3 and 18 nM, respectively. MGM-9 showed a potent opioid agonistic effect, and its effects were meditated by mu- and kappa-opioid receptor mechanisms in in vitro assays. Subcutaneous and oral administration of MGM-9 produced potent antinociceptive effects in mouse tail-flick, hot-plate, and writhing tests. When administered orally, the antinociceptive effect of MGM-9 was seven to 22 times more potent than that of morphine. The antinociceptive effects of MGM-9 were mediated by both mu- and kappa-opioid receptors. Subcutaneous administration of MGM-9 twice daily for 5 days led to antinociceptive tolerance. In the gastrointestinal transit study, MGM-9 inhibited gastrointestinal transit, but its effect was weaker than that of morphine at equi-antinociceptive doses. Furthermore, MGM-9 induced less hyperlocomotion and fewer rewarding effects than morphine. The rewarding effect of MGM-9 was blocked by a mu antagonist and enhanced by a kappa antagonist. Taken together, the results suggest that MGM-9 is a promising novel analgesic that has a stronger antinociceptive effect and weaker adverse effects than morphine.

Involvement of mu-opioid receptors in antinociception and inhibition of gastrointestinal transit induced by 7-hydroxymitragynine, isolated from Thai herbal medicine Mitragyna speciosa.

7-hydroxymitragynine, a constituent of the Thai herbal medicine Mitragyna speciosa, has been found to have a potent opioid antinociceptive effect. In the present study, we investigated the mechanism of antinociception and the inhibitory effect on gastrointestinal transit of 7-hydroxymitragynine, and compared its effects with those of morphine. When administered subcutaneously to mice, 7-hydroxymitragynine produced antinociceptive effects about 5.7 and 4.4 times more potent than those of morphine in the tail-flick (ED50=0.80 mg/kg) and hot-plate (ED50=0.93 mg/kg) tests, respectively. The antinociceptive effect of 7-hydroxymitragynine was significantly blocked by the mu1/mu2-opioid receptor antagonist beta-funaltrexamine hydrochloride (beta-FNA) and the mu1-opioid receptor-selective antagonist naloxonazine in both tests. Thus, 7-hydroxymitragynine acts predominantly on mu-opioid receptors, especially on mu1-opioid receptors. Isolated tissue studies further supported its specificity for the mu-opioid receptors. Further, 7-hydroxymintragynine dose-dependently (ED50=1.19 mg/kg, s.c.) and significantly inhibited gastrointestinal transit in mice, as morphine does. The inhibitory effect was significantly antagonized by beta-FNA pretreatment, but slightly antagonized by naloxonazine. The ED50 value of 7-hydroxymitragynine on gastrointestinal transit was larger than its antinociceptive ED50 value. On the other hand, morphine significantly inhibits gastrointestinal transit at a much smaller dose than its antinociceptive dose. These results suggest that mu-opioid receptor mechanisms mediate the antinociceptive effect and inhibition of gastrointestinal transit. This compound induced more potent antinociceptive effects and was less constipating than morphine.

Antinociception, tolerance and withdrawal symptoms induced by 7-hydroxymitragynine, an alkaloid from the Thai medicinal herb Mitragyna speciosa.

7-Hydroxymitragynine is a potent opioid analgesic alkaloid isolated from the Thai medicinal herb Mitragyna speciosa. In the present study, we investigated the opioid receptor subtype responsible for the analgesic effect of this compound. In addition, we tested whether development of tolerance, cross-tolerance to morphine and naloxone-induced withdrawal signs were observed in chronically 7-hydroxymitragynine-treated mice. Subcutaneous (s.c.) administration of 7-hydroxymitragynine produced a potent antinociceptive effect mainly through activation of mu-opioid receptors. Tolerance to the antinociceptive effect of 7-hydroxymitragynine developed as occurs to morphine. Cross-tolerance to morphine was evident in mice rendered tolerant to 7-hydroxymitragynine and vice versa. Naloxone-induced withdrawal signs were elicited equally in mice chronically treated with 7-hydroxymitragynine or morphine. 7-Hydroxymitragynine exhibited a potent antinociceptive effect based on activation of mu-opioid receptors and its morphine-like pharmacological character, but 7-hydroxymitragynine is structurally different from morphine. These interesting characters of 7-hydroxymitragynine promote further investigation of it as a novel lead compound for opioid studies.

Indole alkaloids of a Thai medicinal herb, Mitragyna speciosa, that has opioid agonistic effect in guinea-pig ileum.

Recently, we found that mitragynine, a major constituent of Mitragyna speciosa, has an opioid agonistic activity, but its weak potency could not explain the opium-like effect of this plant. In the present study, bioassay-guided fractionation of the crude extract of the leaves of M. speciosa was carried out to search for potent opioid agonists other than mitragynine. Opioid agonistic activities were evaluated using twitch contraction induced by electrical stimulation in guinea-pig ileum. The crude extract of M. speciosa inhibited the twitch contraction in a concentration-dependent manner. The inhibition was reversed by naloxone. The opioid effect was detected only in the crude base fraction, which was followed by the isolation of five indole alkaloids. Among these alkaloids, 7-hydroxymitragynine showed the most potent opioid effect on the electrically-stimulated contraction (pD (2) = 8.38 +/- 0.12). The potency, calculated using pD (2) values, was 30- and 17-fold higher than that of mitragynine and morphine, respectively. Antagonism of naloxone on concentration-response curves for 7-hydroxymitragynine confirmed its opioid effect. These results suggest that the opioid effect of M. speciosa is mostly based on the activity of 7-hydroxymitragynine.

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.

Studies on the synthesis and opioid agonistic activities of mitragynine-related indole alkaloids: discovery of opioid agonists structurally different from other opioid ligands.

Mitragynine (1) is a major alkaloidal component in the Thai traditional medicinal herb, Mitragyna speciosa, and has been proven to exhibit analgesic activity mediated by opioid receptors. By utilizing this natural product as a lead compound, synthesis of some derivatives, evaluations of the structure-activity relationship, and surveys of the intrinsic activities and potencies on opioid receptors were performed with guinea pig ileum. The affinities of some compounds for mu-, delta-, and kappa-receptors were determined in a receptor binding assay. The essential structural moieties in the Corynanthe type indole alkaloids for inducing the opioid agonistic activity were also clarified. The oxidative derivatives of mitragynine, i.e., mitragynine pseudoindoxyl (2) and 7-hydroxymitragynine (12), were found as opioid agonists with higher potency than morphine in the experiment with guinea pig ileum. In addition, 2 induced an analgesic activity in the tail flick test in mice.

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.