Effects of rikkunshi-to, a traditional Japanese medicine, on the delay of gastric emptying induced by N(G)-nitro-L-arginine.

We evaluated the effects of Rikkunshi-to and several of its ingredients on the delay of gastric emptying induced by a nitric oxide (NO) synthase inhibitor, N(G)-nitro-L-arginine (L-NNA). After oral administration of L-NNA to rats, the gastric emptying rate at 24 h was decreased from 82.8 +/- 2.4% to 53.3 +/- 5.7%. The decrease of the gastric emptying rate induced by L-NNA treatment was markedly ameliorated by administration of Rikkunshi-to (250 and 500 mg/kg, p.o.) in a dose-dependent manner. To identify the active ingredient of Rikkunshi-to, the components were separated according to polarity, and the effects of the respective fractions on gastric emptying were evaluated. Significant efficacy was found in the water and methanol fractions, but not in the 50% aqueous-methanol fraction. Furthermore, hesperidin (1 - 4.29 mg/kg, p.o.) contained in the methanol fraction and L-arginine (4.5 mg/kg, p.o.) contained in the water fraction ameliorated the decrease in the gastric emptying rate induced by L-NNA treatment. These results suggest that Rikkunshi-to ameliorated abnormalities of NO-mediated gastric functions such as delayed gastric emptying, and hesperidin and L-arginine were identified as two of the active ingredients contributing to the ability of Rikkunshi-to to facilitate gastric emptying.

Gastric cytoprotective activity of dehydroleucodine in rats. Role of nitric oxide.

Previously we reported that dehydroleucodine (DhL), a sesquiterpene lactone, shows gastric and duodenal cytoprotective activity. The mechanism is not mediated by antiacid secretory action; DhL stimulated mucus production and indomethacin pretreatment reduced cytoprotective action. In the present study we demonstrated that the gastric cytoprotective effect is antagonized by the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine. The inhibitory action of NG-nitro-L-arginine is reversed by L-arginine, but not D-arginine. The findings suggest that NO is involved in the gastroprotection induced by DhL.

Modulation of guinea-pig cardiac L-type calcium current by nitric oxide synthase inhibitors.

1. Electrophysiological (whole-cell clamp) techniques were used to study the effect of NO synthase (NOS) inhibitors on guinea-pig ventricular calcium current (ICa), and biochemical measurements (Western blot and citrulline synthesis) were made to investigate the possible mechanisms of action. 2. The two NOS inhibitors, NG-monomethyl-L-arginine (L-NMMA, 1 mM) and NG-nitro-L-arginine (L-NNA, 1 mM), induced a rapid increase in ICa when applied to the external solution. D-NMMA (1 mM), the stereoisomer of L-NMMA, which has no effect on NOS, did not enhance ICa. 3. Western blot experiments gave no indication of the presence of inducible NOS protein (iNOS) in our cell preparation, neither immediately after dissociation nor after more than 24 h. Statistically, there was no significant difference between electrophysiological experiments performed on freshly dissociated cells and experiments performed the next day. Moreover cells prepared and kept in the presence of dexamethasone (3 microM), to inhibit the expression of iNOS, gave the same response to L-NMMA as control cells. 4. The stimulatory effect of L-NMMA (1 mM) on basal ICa was reversed by competition with higher doses (5 mM) of externally applied L-arginine, the natural substrate of NOS. The effect of L-NMMA was also eliminated by L-arginine in the patch pipette solution. 5. Intracellular perfusion with GDP beta S (0.5 mM), which stabilizes the G-proteins in the inactive state, did not affect the L-NMMA-induced stimulation of ICa. 6. Carbachol (1 microM) reduced the ICa previously stimulated by L-NMMA, and intracellular cGMP (10 microM) prevented L-NMMA enhancement. 7. Simultaneous treatment with L-NMMA and isoprenaline (1 microM) induced a non-cumulative enhancement of ICa that could not be reversed by carbachol (1 microM). 8. NO synthesis, measured by the formation of [3H]citrulline from L-[3H]arginine during a 15 min incubation, showed a relatively high basal NO production, which was inhibited by L-NMMA but not affected by carbachol. 9. These results suggest that inhibitors of NOS are able to modulate the basal ventricular ICa in the absence of a receptor-mediated pathway, and that NO might be required for the muscarinic reduction of ICa under isoprenaline stimulation, even if NO production is not directly controlled by the muscarinic pathway.

Nitric oxide donors antagonize N-nitro-L-arginine and haloperidol catalepsy: potential implication for the treatment of Parkinsonism?

Nitric oxide (NO) synthase inhibitor, N-nitro-L-arginine (NNLA) produced dose-dependent, long-lasting catalepsy in rats, the effect being attenuated by NO donors L-arginine and molsidomine. Catalepsy induced by haloperidol (0.4 mg/kg i.p.), D2 receptor antagonist, was reduced dose-dependently by molsidomine (10.0-100.0 mg/kg) and by L-arginine at a dose of 100.0 mg/kg. Low, non-cataleptic doses of NNLA (0.1 mg/kg) and haloperidol (0.1 mg/kg) given in combination produced a marked and long-lasting catalepsy. The results suggest that NO plays a role in NNLA-induced catalepsy as well as in catalepsy elicited by haloperidol.

Possible involvement of nitric oxide in chlordiazepoxide-induced feeding in the mouse.

Two experiments investigated a possible role of nitric oxide (NO) in chlordiazepoxide (CP)-induced feeding in nondeprived male ICR mice in independent groups designs. Experiment 1 demonstrated a dose-related decrease in CP-induced solid food intake over a 60-min test period with increasing dose (10, 25, and 50 mg/ kg SC) of the NO-synthase (NOS) inhibitor, L-NG-nitro arginine (L-NOARG), reaching statistical significance at 10 mg/kg L-NOARG when compared to vehicle control. Identical doses of L-NOARG failed to significantly affect normal feeding in vehicle treated mice. In Experiment 2, initial pretreatment with L-arginine (500 and 1000 mg/kg IP) partially or completely restored the feeding inhibitory action of a challenge dose (25 mg/kg SC) of L-NOARG; D-arginine (500 mg/kg IP) was ineffective, thus supporting a stereospecific action of arginine. Arginine isomers did not differentially affect intake in normal feeding animals. These results implicate involvement of NO in CP-induced hyperphagia; they are consistent with and extend research linking NO and ingestive behaviors.

Characterization of the apamin- and L-nitroarginine-resistant NANC inhibitory transmission to the circular muscle of guinea-pig colon.

1. The aim of this study was a pharmacological characterization of the multiple NANC inhibitory transmission systems producing relaxation of the circular muscle of guinea-pig proximal colon. In the presence of atropine (1 microM), guanethidine (3 microM) and of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (0.3 microM) and MEN 10627 (1 microM), respectively, electrical field stimulation (EFS) produced a frequency-dependent (0.1-3 Hz) relaxation. During a cumulative frequency-response curve, the maximal relaxant effect was produced at 3 Hz and approached the maximal relaxation to 1 microM isoprenaline. In the presence of both apamin (0.3 microM) and L-nitroarginine (L-NOARG, 100 microM), EFS failed to evoke relaxation up to 1 Hz; at 1-10 Hz, a slowly developing relaxation ensured which approached 50% of the Emax to isoprenaline. The EFS-evoked NANC relaxation, either in the presence or absence of apamin and L-NOARG, was unaffected by in vitro capsaicin pretreatment (10 microM for 15 min). 2. Three protocols of EFS were developed for further pharmacological analysis: (a) EFS at 1 Hz for 5 s in the presence of L-NOARG, producing a transient fast apamin-sensitive relaxation; (b) EFS at 1 Hz for 5 s in the presence of apamin, producing a transient fast L-NOARG-sensitive relaxation; and (c) EFS at 10 Hz for 5 s in the presence of both apamin and L-NOARG, producing a transient but slowly developing and more sustained relaxation. 3. The neutral endopeptidase inhibitor, thiorphan (1-10 microM), enhanced and prolonged the apamin- and L-NOARG-resistant NANC relaxation produced by EFS at 10 Hz, without affecting that evoked at 1 Hz in the presence of apamin or L-NOARG. The angiotensin converting enzyme inhibitor, captopril (1-10 microM) was without effect. 4. The cAMP analogue inhibitor of protein kinase A, Rp-cAMPs (100-300 microM) significantly reduced and shortened the NANC relaxation produced by 10 Hz EFS in the presence of L-NOARG without affecting that produced by 1 Hz EFS in the presence of apamin or L-NOARG. 5. The inhibitor of sarcoplasmic reticulum Ca-ATPase, cyclopiazonic acid (CPA, 3-10 microM for 60 min) abolished the 1 Hz EFS-induced relaxation in the presence of L-NOARG, and greatly inhibited that produced by 10 Hz EFS in the presence of both apamin and L-NOARG. The relaxation produced by 1 Hz EFS in the presence of apamin was inhibited by about 32% at 10 microM only. 6. Nifedipine (1 microM) did not affect the EFS-induced NANC relaxations. In the presence of nifedipine, tetraethylammonium (TEA, 1 mM) enhanced the 1 Hz EFS-induced relaxation in the presence of L-NOARG (158% of control) and that produced by 10 Hz EFS in the presence of apamin and L-NOARG (215% of control) while that evoked by 1 Hz EFS in the presence of apamin was slightly affected (109% of control). 7. In the presence of atropine, guanethidine, SR 140333 and MEN 10627, bath application of human vasoactive intestinal polypeptide (VIP, 0.1 nM-10 nM) produced a concentration-dependent, slowly developing relaxation of colonic strips. The relaxation to VIP was unaffected by apamin (0.3 microM), L-NOARG (100 microM), nifedipine (1 microM) or nifedipine plus TEA (1 mM); it was inhibited by CPA (10 microM) and Rp-cAMPs (100 microM) and was potentiated by thiorphan (10 microM). 8. The putative VIP receptor antagonist, VIP(10-28) (10 microM) did not affect the VIP-induced relaxation nor the NANC relaxation to 10 Hz EFS in the presence of apamin and L-NOARG. 9. The present findings provide evidence that three distinct NANC inhibitory mechanisms mediate relaxation of the circular muscle of the guinea-pig proximal colon. The first system provides a fast relaxation in response to low frequency of stimulation and may involve the action of a transmitter(s) (possibly ATP) which mobilizes intracellular Ca2+ from sarcoplasmic reticulum leading to the activation of apamin-sensitive K+ channels. The second system likewise provides a fast relaxation of the colon in