Influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus

The peptide hormone relaxin has been reported to depress the amplitude of contractile responses in the mouse gastric fundus by upregulating nitric oxide (NO) biosynthesis at the neural level. In the present study, we investigated whether relaxin also influenced nonadrenergic, noncholinergic (NANC) gastric relaxant responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. In carbachol precontracted strips from control mice and in the presence of guanethidine, electrical field stimulation (EFS) elicited fast relaxant responses that may be followed by a sustained relaxation. All relaxant responses were abolished by tetrodotoxin. Relaxin increased the amplitude of the EFS-induced fast relaxation without affecting either the sustained one or the direct smooth muscle response to papaverine. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), that abolished the EFS-induced fast relaxation without influencing the sustained one, relaxin was ineffective. In strips from relaxin-pretreated mice, EFS-induced fast relaxations were enhanced in amplitude with respect to the controls, while sustained ones as well as direct smooth muscle responses to papaverine were not changed. Further addition of relaxin to the bath medium did not influence neurally induced fast relaxant responses, whereas L-NNA did. In conclusion, in the mouse gastric fundus, relaxin enhances the neurally induced nitrergic relaxant responses acting at the neural level.     

Relaxin counteracts the altered gastric motility of dystrophic (mdx) mice: functional and immunohistochemical evidence for the involvement of nitric oxide

Impaired gastric motility ascribable to a defective nitric oxide (NO) production has been reported in dystrophic (mdx) mice. Since relaxin upregulates NO biosynthesis, its effects on the motor responses and NO synthase (NOS) expression in the gastric fundus of mdx mice were investigated. Mechanical responses of gastric strips were recorded via force displacement transducers. Evaluation of the three NOS isoforms was performed by immunohistochemistry and Western blot. Wild-type (WT) and mdx mice were distributed into three groups: untreated, relaxin pretreated, and vehicle pretreated. In strips from both untreated and vehicle-pretreated animals, electrical field stimulation (EFS) elicited contractile responses that were greater in mdx than in WT mice. In carbachol-precontracted strips, EFS induced fast relaxant responses that had a lower amplitude in mdx than in WT mice. Only in the mdx mice did relaxin depress the amplitude of the neurally induced excitatory responses and increase that of the inhibitory ones. In the presence of L-NNA, relaxin was ineffective. In relaxin-pretreated mdx mice, the amplitude of the EFS-induced contractile responses was decreased and that of the fast relaxant ones was increased compared with untreated mdx animals. Responses to methacholine or papaverine did not differ among preparations and were not influenced by relaxin. Immunohistochemistry and Western blotting showed a significant decrease in neuronal NOS expression and content in mdx compared with WT mice, which was recovered in the relaxin-pretreated mdx mice. The results suggest that relaxin is able to counteract the altered contractile and relaxant responses in the gastric fundus of mdx mice by upregulating nNOS expression.     

Reversal by relaxin of altered ileal spontaneous contractions in dystrophic (mdx) mice through a nitric oxide-mediated mechanism

Altered nitric oxide (NO) production/release is involved in gastrointestinal motor disorders occurring in dystrophic (mdx) mice. Since the hormone relaxin (RLX) can upregulate NO biosynthesis, its effects on spontaneous motility and NO synthase (NOS) expression in the ileum of dystrophic (mdx) mice were investigated. Mechanical responses of ileal preparations were recorded in vitro via force-displacement transducers. Evaluation of the expression of NOS isoforms was performed by immunohistochemistry and Western blot. Normal and mdx mice were distributed into three groups: untreated, RLX pretreated, and vehicle pretreated. Ileal preparations from the untreated animals showed spontaneous muscular contractions whose amplitude was significantly higher in mdx than in normal mice. Addition of RLX, alone or together with l-arginine, to the bath medium depressed the amplitude of the contractions in the mdx mice, thus reestablishing a motility pattern typical of the normal mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) or the guanylate cyclase inhibitor ODQ reversed the effects of RLX. In RLX-pretreated mdx mice, the amplitude of spontaneous motility was reduced, thus resembling that of the normal mice, and NOS II expression in the muscle coat was increased in respect to the vehicle-pretreated mdx animals. These results indicate that RLX can reverse the altered ileal motility of mdx mice to a normal pattern, likely by upregulating NOS II expression and NO biosynthesis in the ileal smooth muscle.     

Relaxin depresses small bowel motility through a nitric oxide-mediated mechanism. Studies in mice

Gastrointestinal motility is reduced and the incidence of functional gastrointestinal disorders is increased in pregnancy, possibly due to hormonal influences. This study aims to clarify whether the hormone relaxin, which attains high circulating levels during pregnancy and has a nitric oxide-mediated relaxant action on vascular and uterine smooth muscle, also reduces bowel motility and, if it does, whether nitric oxide is involved. Female mice in proestrous or estrous were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Isolated ileal preparations from both groups were used to record contractile activity, either basal or after acute administration of relaxin (5 x 10(-8) M). Drugs inhibiting nitric oxide biosynthesis or neurotransmission were used in combination with relaxin. Expression of nitric oxide synthase isoforms by the ileum was assessed by immunocytochemistry and Western blot analysis. Relaxin caused a clear-cut decay of muscle tension and a reduction in amplitude of spontaneous contractions upon either chronic administration to mice or acute addition to isolated ileal preparations. These effects were significantly blunted by N(G)-nitro-L-arginine, but not by the neural blockers we used. Moreover, relaxin increased the expression of nitric oxide synthases II and III, but not synthase I. Relaxin markedly inhibits ileal motility in mice by exerting a direct action on smooth muscle through the activation of intrinsic nitric oxide biosynthesis.     

Relaxation of myometrium by calcitonin gene-related peptide is independent of nitric oxide synthase activity in mouse uterus

Calcitonin gene-related peptide (CGRP) inhibits myometrial contractile activity. However, the responsiveness of the mouse myometrium to CGRP is dependent on the hormonal and gestational stage. The inhibitory effect of CGRP in the myometrium is prominent during gestation and declines at parturition. The present study was undertaken to examine if nitric oxide (NO) production by nitric oxide synthase (NOS) isoforms mediates the inhibitory action of CGRP on uterine contractions as has been suggested earlier. Transgenic mice deficient in either of the three major NOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were used. Isometric force measurements on myometrial strips obtained from NOS-deficient mice were carried out and the inhibitory capacity of CGRP was monitored. CGRP inhibited KCl-induced contractions of the myometrial strips obtained from eNOS(-/-), iNOS(-/-), and nNOS(-/-) mice with equal efficiency as in wild-type animals. Additionally, NOS protein expression in the mouse uterus during gestation and during the estrous cycle was examined by means of Western immunoblot analysis. No correlation between NOS expression and inhibitory activity of CGRP was evident. The results suggest that the inhibitory action of CGRP in the mouse uterus is independent of the activity of these NOS isoforms.     

Interaction between nitric oxide and prostanoids in arterioles of rat cremaster muscle in vivo

We studied in vivo interactions of nitric oxide (NO), oxidative stress, and prostanoids derived from the cyclooxygenase pathway in the arterioles studied by intravital microscopy in peripheral muscle. Topical administration of NO synthase (NOS) inhibitor Nomega-nitro-l-arginine (l-NNA) or cyclooxygenase inhibitor mefenamic acid (MA) alone leads to vasoconstriction. We found that l-NNA after MA induced an additional constriction, whereas MA after l-NNA induced a relative dilation. Therefore, an additional constriction was found when MA was administered after l-NNA in the presence of the thromboxane A2 synthase-PGH2 (TP) receptor antagonist SQ-29548. We also found a relative dilation when the TP receptor antagonist was administered after NOS inhibition by l-NNA. In the presence of superoxide dismutase and catalase, l-NNA-induced vasoconstriction is reduced, and the dilation observed after addition of MA in presence of the reactive oxygen species is no longer present. Taken together, these results showed that NO inhibition induced a shift in the synthesis or in the effects of cyclooxygenase products, in favor of constrictor prostanoids. This effect of NO inhibition disappears when reactive oxygen species are scavenged by superoxide dismutase and catalase.     

Morinda lucida reduces contractility of isolated uterine smooth muscle of pregnant and non-pregnant mice.

The present work investigated the effect of Morinda lucida (M. lucida) extract on isolated uterine smooth muscle of pregnant and non-pregnant mice. Pregnant and non-pregnant mice were pretreated with oral stilboesterol (0.1 mg/kg body weight) and killed by cervical dislocation. Thin strips of the uterus were cut and mounted in a 20ml organ bath containing De Jalon solution bubbled with 95%O2-5% CO2 gas mixture. The strips were connected to a force transducer coupled to a Grass 7D Polygraph for the recording of isometric tension. Effects of graded concentrations of oxytocin (OXY; 10-5-10-2 mol/L), acetylcholine (ACh; 10-9-10-5 mol/L) and M. lucida extract (0.015-1.5 mg/ml) were recorded. Fresh uterine strips were then incubated with M. lucida extract for 5mins and cumulative response to OXY was repeated. Another set of fresh strips was incubated in L-NAME for 15mins and the cumulative responses to M.lucida extract were repeated. OXY resulted in increased contractile responses in both pregnant and non-pregnant uterine muscles. M. lucida resulted in relaxation of the uterine smooth muscle in both pregnant and non-pregnant mice at all doses. However, at 1.5mg/ml, M. lucida completely blocked spontaneous uterine contractions. Following incubation with L-NAME, M. lucida extract led to a slightly greater relaxation of the uterine strips. In conclusion, M. lucida reduced contractility of uterine smooth muscle in both pregnant and non-pregnant mice as well as blocking contractile responses to OXY and Ach in uterine smooth muscle of pregnant and non-pregnant mice. There was no significant alteration of M. lucida activity by L-NAME suggesting that the action of the compound on uterine muscle is not associated with impaired nitric oxide synthase.     

Loss of alpha2B-adrenoceptors increases magnitude of hypertension following nitric oxide synthase inhibition

Vascular alpha(2B)-adrenoceptors (alpha(2B)-AR) may mediate vasoconstriction and contribute to the development of hypertension. Therefore, we hypothesized that blood pressure would not increase as much in mice with mutated alpha(2B)-AR as in wild-type (WT) mice following nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-l-arginine (l-NNA, 250 mg/l in drinking water). Mean arterial pressure (MAP) was recorded in heterozygous (HET) alpha(2B)-AR knockout mice and WT littermates using telemetry devices for 7 control and 14 l-NNA treatment days. MAP in HET mice was increased significantly on treatment days 1 and 4 to 14, whereas MAP did not change in WT mice (days 0 and 14 = 113 +/- 3 and 114 +/- 4 mmHg in WT, 108 +/- 0.3 and 135 +/- 13 mmHg in HET, P < 0.05). MAP was significantly higher in HET than in WT mice days 10 through 14 (P < 0.05). Thus blood pressure increased more rather than less in mice with decreased alpha(2B)-AR expression. We therefore examined constrictor responses to phenylephrine (PE, 10(-9) to 10(-4) M) with and without NOS inhibition to determine basal NO contributions to arterial tone. In small pressurized mesenteric arteries (inner diameter = 177 +/- 5 microm), PE constriction was decreased in untreated HET arteries compared with WT (P < 0.05). l-NNA (100 microM) augmented PE constriction more in HET arteries than in WT arteries, and responses were not different between groups in the presence of l-NNA. Acetylcholine dilated preconstricted arteries from HET mice more than arteries from WT mice. Endothelial NOS expression was increased in HET compared with WT mesenteric arteries by Western analysis. Griess assay showed increased NO(x) concentrations in HET plasma compared with those in WT plasma. These data demonstrate that diminished alpha(2B)-AR expression increases the dependence of arterial pressure and vascular tone on NO production and that vascular alpha(2B)-AR either directly or indirectly regulates vascular endothelial NOS function.     

Modulation of nitrergic relaxant responses by peptides in the mouse gastric fundus

The effects of pituitary adenylate cyclase-activating peptide (PACAP-38) and vasoactive intestinal polypeptide (VIP) were investigated in the gastric fundus strips of the mouse. In carbachol (CCh) precontracted strips, in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor L-N(G)-nitro arginine (L-NNA) or by the guanylate cyclase inhibitor (ODQ), the sustained one by alpha-chymotrypsin. alpha-Chymotrypsin also increased the amplitude of the EFS-induced fast relaxation. PACAP-38 and VIP caused tetrodotoxin-insensitive sustained relaxant responses that were both abolished by alpha-chymotrypsin. Apamin did not influence relaxant responses to EFS nor relaxation to both peptides. PACAP 6-38 abolished EFS-induced sustained relaxations, increased the amplitude of the fast ones and antagonized the smooth muscle relaxation to both PACAP-38 and VIP. VIP 10-28 and [D-p-Cl-Phe6,Leu17]-VIP did not influence the amplitude of both the fast or the sustained response to EFS nor influenced the relaxation to VIP and PACAP-38. The results indicate that in strips from mouse gastric fundus peptides, other than being responsible for EFS-induced sustained relaxation, also exerts a modulatory action on the release of the neurotransmitter responsible for the fast relaxant response, that appears to be NO.     

Knock out of neuronal nitric oxide synthase exacerbates intestinal ischemia/reperfusion injury in mice

Recent investigation of the intestine following ischemia and reperfusion (I/R) has revealed that nitric oxide synthase (NOS) neurons are more strongly affected than other neuron types. This implies that NO originating from NOS neurons contributes to neuronal damage. However, there is also evidence of the neuroprotective effects of NO. In this study, we compared the effects of I/R on the intestines of neuronal NOS knockout (nNOS(-/-)) mice and wild-type mice. I/R caused histological damage to the mucosa and muscle and infiltration of neutrophils into the external muscle layers. Damage to the mucosa and muscle was more severe and greater infiltration by neutrophils occurred in the first 24?h in nNOS(-/-) mice. Immunohistochemistry for the contractile protein, α-smooth muscle actin, was used to evaluate muscle damage. Smooth muscle actin occurred in the majority of smooth muscle cells in the external musculature of normal mice but was absent from most cells and was reduced in the cytoplasm of other cells following I/R. The loss was greater in nNOS(-/-) mice. Basal contractile activity of the longitudinal muscle and contractile responses to nerve stimulation or a muscarinic agonist were reduced in regions subjected to I/R and the effects were greater in nNOS(-/-) mice. Reductions in responsiveness also occurred in regions of operated mice not subjected to I/R. This is attributed to post-operative ileus that is not significantly affected by knockout of nNOS. The results indicate that deleterious effects are greater in regions subjected to I/R in mice lacking nNOS compared with normal mice, implying that NO produced by nNOS has protective effects that outweigh any damaging effect of this free radical produced by enteric neurons.     

Role of nitric oxide in beta3-adrenoceptor activation on basal tone of internal anal sphincter

Effects of activation of beta3-adrenoceptor (beta3-AR) have not been determined in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The effects of disodium (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), a selective beta3-AR agonist, on the basal smooth muscle tone and direct release of nitric oxide (NO) by circular smooth muscle strips of the opossum IAS were determined. We also examined the presence of endothelial nitric oxide synthase (eNOS) protein by Western blot studies. CL 316243 produced a concentration-dependent relaxation of the smooth muscle that remained unmodified by different neurohumoral antagonists. The smooth muscle relaxation by CL 316243 was selectively antagonized by L 748337, a beta3-AR antagonist. Such relaxation was several times longer than by isoproterenol. The effect of CL 316243 was significantly attenuated by a nonselective NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) and by putative inhibitor of eNOS l-N5-(1-iminoethyl)-ornithine dihydrochloride (l-NIO). Inhibitors of iNOS [N-(3-aminomethyl)benzyl acetamide 2HCl] and nNOS [1-[2-(trifluoromethylphenyl)imidazole]] had no effect on this relaxation. Relaxation of the IAS smooth muscle induced by CL 316243 was accompanied by an increased release of NO; this was attenuated by l-NNA and l-NIO. In addition, Western blot studies revealed the presence of eNOS in the circular smooth muscle of the IAS. These data demonstrate potent and protracted IAS smooth muscle relaxation by beta3-AR activation, which is partly transduced via NOS, possibly smooth muscle eNOS. Multiple signal-transduction pathways including NOS activation may explain the characteristic IAS relaxation by beta3-AR activation. The studies may have therapeutic implications in anorectal motility disorders.     

Endothelial nitric oxide synthase is protective in the initiation of caerulein-induced acute pancreatitis in mice

The effect of inhibiting nitric oxide (NO) synthase (NOS) or enhancing NO on the course of acute pancreatitis (AP) is controversial, in part because three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). We investigated whether inhibition or selective gene deletion of NOS isoforms modified the initiation phase of caerulein-induced AP in mice and explored whether this affected pancreatic microvascular blood flow (PMBF). We investigated the effects of nonspecific NOS inhibition with N(omega)-nitro-l-arginine (l-NNA; 10 mg/kg ip) or targeted deletion of eNOS, nNOS, or iNOS genes on the initiation phase of caerulein-induced AP in mice using in vivo and in vitro models. Western blot analysis was performed to assess eNOS phosphorylation status, an indicator of enzyme activity, and microsphere studies were used to measure PMBF. l-NNA and eNOS deletion, but not nNOS or iNOS deletion, increased pancreatic trypsin activity and serum lipase during the initiation phase of in vivo caerulein-induced AP. l-NNA and eNOS did not affect trypsin activity in caerulein-hyperstimulated isolated acini, suggesting that nonacinar events mediate the effect of NOS blockade in vivo. The initiation phase of AP in wild-type mice was associated with eNOS Thr(495) residue dephosphorylation, which accompanies eNOS activation, and a 178% increase in PMBF; these effects were absent in eNOS-deleted mice. Thus eNOS is the main isoform influencing the initiation of caerulein-induced AP. eNOS-derived NO exerts a protective effect through actions on nonacinar cell types, most likely endothelial cells, to produce greater PMBF.     

姜黄素对小鼠离体十二指肠平滑肌舒缩活动的影响

目的:本研究采用小鼠离体十二指肠平滑肌观察姜黄素对胃肠道蠕动的影响,探讨其作用机制。方法:取小鼠离体十二指肠平滑肌条,放入37℃Krebs液浴槽中,通入95%氧气和5%二氧化碳混合气体,分组进行下列实验:对照组和分别加入10-40 M姜黄素组,测量记录十二指肠平滑肌的自主收缩变化;另取一组平滑肌条,分对照组、乙酰胆碱组、乙酰胆碱+姜黄素组、阿托品组、阿托品+姜黄素组,采用张力换能器连接多通道生理信号采集处理系统,测量比较十二指肠平滑肌舒缩的变化。结果:小鼠十二指肠平滑肌加入姜黄素孵育后,其自主收缩幅度有明显下降(P0.01),而且降低的幅度与姜黄素剂量相关;给与乙酰胆碱引起十二指肠收缩后,再加姜黄素孵育,十二指肠平滑肌的收缩幅度明显的下降(P0.01);给予阿托品引起小鼠平滑肌舒张后,再给予姜黄素孵育,平滑肌收缩幅度进一步降低。结论:姜黄素对小鼠离体十二指肠平滑肌具有直接舒张作用。     

Chronic inhibition of nitric oxide synthase activity by N(G)-nitro-L-arginine induces nitric oxide synthase expression in the developing rat cerebellum

Studies on chronic inhibition of nitric oxide synthase (NOS) in the CNS suggest a plastic change in nitric oxide (NO) synthesis in areas related to motor control, which might protect the animal from the functional and behavioral consequences of NO deficiency. In the present study, the acute and chronic effect of the substrate analogue inhibitor N(G)-nitro-l-arginine (l-NNA) was examined on NO production, NO-sensitive cyclic guanosine monophosphate (cGMP) levels and the expression of NOS isoforms in the developing rat cerebellum. Acute intraperitoneal administration of the inhibitor (5-200mg/kg) to 21-day-old rats reduced NOS activity and NO concentration dose dependently by 70-90% and the tissue cGMP level by 60-80%. By contrast, chronic application of l-NNA between postnatal days 4-21 diminished the total NOS activity and NO concentration only by 30%, and the tissue cGMP level by 10-50%. Chronic treatment of 10mg/kg l-NNA induced neuronal (n)NOS expression in granule cells, as revealed by in situ hybridization, NADPH-diaphorase histochemistry and Western-blot, but it had no significant influence on tissue cGMP level or on layer formation of the cerebellum. However, a higher concentration (50mg/kg) of l-NNA decreased the intensity of the NADPH-diaphorase reaction in granule cells, significantly reduced cGMP production, and retarded layer formation and induced inducible (i)NOS expression & activity in glial cells. Treatments did not affect endothelial (e)NOS expression. The administration of the biologically inactive isomer D-NNA (50mg/kg) or saline was ineffective. The present findings suggest the existence of a concentration-dependent compensatory mechanism against experimentally-induced cronich inhibition of NOS, including nNOS or iNOS up-regulation, which might maintain a steady-state NO level in the developing cerebellum.     

Smooth muscle NOS, colocalized with caveolin-1, modulates contraction in mouse small intestine

Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca(2+)] through L-type Ca(2+) channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 muM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-beta cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-omega-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 muM) blocked the responses to KCl and LNNA confirming the role of L-type Ca(2+) channels. ODQ (1 muM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP-dependent mechanism.     

Dopamine enhances mtNOS activity: Implications in mitochondrial function

Dopamine and nitric oxide systems can interact in different processes in the central nervous system. Dopamine and oxidation products have been related to mitochondrial dysfunction. In the present study, intact mitochondria and submitochondrial membranes were incubated with different DA concentrations for 5 min. Dopamine (1 mM) increased nitric oxide production in submitochondrial membranes and this effect was partially prevented in the presence of both DA and NOS inhibitor N_ω-nitro-l-arginine (l-NNA). A 46% decrease in state 3 oxygen uptake (active respiration state) was found after 15 mM dopamine incubation. When mitochondria were incubated with 15 mM dopamine in the presence of l-NNA, state 3 respiratory rate was decreased by only 17% showing the involvement of NO. As shown for O₂ consumption, the inhibition of cytochrome oxidase by 1 mM DA was mediated by NO. Hydrogen peroxide production significantly increased after 15 mM DA incubation, being mainly due to its metabolism by MAO. Also, DA-induced depolarization was prevented by the addition of l-NNA showing the involvement of nitric oxide in this process too. This work provides evidence that in the studied conditions, dopamine modifies mitochondrial function by a nitric oxide-dependent pathway.     

Different responsiveness of excitatory and inhibitory enteric motor neurons in the human esophagus to electrical field stimulation and to nicotine

To compare electrical field stimulation (EFS) with nicotine in the stimulation of excitatory and inhibitory enteric motoneurons (EMN) in the human esophagus, circular lower esophageal sphincter (LES), and circular and longitudinal esophageal body (EB) strips from 20 humans were studied in organ baths. Responses to EFS or nicotine (100 microM) were compared in basal conditions, after N(G)-nitro-l-arginine (l-NNA; 100 microM), and after l-NNA and apamin (1 microM). LES strips developed myogenic tone enhanced by TTX (5 microM) or l-NNA. EFS-LES relaxation was abolished by TTX, unaffected by hexamethonium (100 microM), and enhanced by atropine (3 microM). Nicotine-LES relaxation was higher than EFS relaxation, reduced by TTX or atropine, and blocked by hexamethonium. After l-NNA, EFS elicited a strong cholinergic contraction in circular LES and EB, and nicotine elicited a small relaxation in LES and no contractile effect in EB. After l-NNA and apamin, EFS elicited a strong cholinergic contraction in LES and EB, and nicotine elicited a weak contraction amounting to 6.64 +/- 3.19 and 9.20 +/- 5.51% of that induced by EFS. EFS elicited a contraction in longitudinal strips; after l-NNA and apamin, nicotine did not induce any response. Inhibitory EMN tonically inhibit myogenic LES tone and are efficiently stimulated both by EFS and nicotinic acetylcholine receptors (nAChRs) located in somatodendritic regions and nerve terminals, releasing nitric oxide and an apamin-sensitive neurotransmitter. In contrast, although esophageal excitatory EMN are efficiently stimulated by EFS, their stimulation through nAChRs is difficult and causes weak responses, suggesting the participation of nonnicotinic mechanisms in neurotransmission to excitatory EMN in human esophagus.     

Influence of orexin A on the mechanical activity of mouse gastric strips

The presence of orexins and orexin receptors has been revealed not only in the central nervous system but also in the gastrointestinal tract. The present study was aimed to investigate the influence of orexin A (OXA) on the mechanical activity of fundal and antral strips of the mouse stomach. In the fundus, electrical field stimulation (EFS) elicited tetrodotoxin (TTX)-sensitive, frequency-dependent contractile responses whose amplitude was markedly reduced by OXA and enhanced by the orexin-1 type receptor antagonist SB-334867. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), OXA was no longer effective. Methacholine caused a sustained contracture whose amplitude was not influenced by OXA, TTX or L-NNA. In carbachol-precontracted strips, the neurally-induced relaxant responses elicited during EFS were increased in amplitude by OXA. Antral strips showed a spontaneous contractile activity that was unaffected by TTX or L-NNA and transiently depressed by EFS. OXA did not influence either the spontaneous motility or the EFS-induced effects. The results indicate that OXA exerts region-specific effects and that, in the fundus, depresses EFS-induced contractile responses by acting at the nervous level. It is likely that NO is involved in the effects of the peptide.     

Inducible nitric oxide synthase is involved in acid-induced gastric hyperemia in rats and mice

The role of different isoforms of nitric oxide synthase (NOS) in the gastric mucosal hyperemia, induced by 155 mM luminal hydrochloric acid (pH approximately 0.8) without a barrier breaker, was investigated. Rats were anesthetized with Inactin (120 mg/kg ip), and mice were anesthetized with Forene (2.2% in 40% oxygen gas at 150 ml/min); the gastric mucosa was exteriorized. Gastric mucosal blood flow was measured with laser-Doppler flowmetry (LDF) in rats treated with Nomega-nitro-l-arginine (l-NNA; unspecific NOS inhibitor), l-N6-(1-iminoethyl)lysine [l-NIL; inducible (i) NOS inhibitor], or S-methyl-l-thiocitrulline [SMTC; neuronal (n) NOS inhibitor], 10 mg/kg, followed by 3 mg. kg-1. h-1 iv, in iNOS-deficient (-/-) and nNOS(-/-) mice. mRNA was isolated from the gastric mucosa in iNOS(-/-) and wild-type (wt) mice, and real-time RT-PCR was performed. The effect of 155 mM acid on gastric mucosal permeability was determined by measuring the clearance of 51Cr-EDTA from blood to lumen. LDF increased by 48 +/- 13% during 155 mM HCl luminally, an increase that was abolished by l-NNA, SMTC, or l-NIL. In iNOS wt mice, LDF increased by 33 +/- 8% during luminal acid. The blood flow increase was attenuated substantially in iNOS(-/-) mice. RT-PCR revealed iNOS mRNA expression in the gastric mucosa in the iNOS wt groups. The blood flow increase in response to acid was not abolished in nNOS(-/-) mice (nNOS-sufficient mice, 39 +/- 18%; heterozygous mice, 25 +/- 19%; -/- mice, 19 +/- 7%). Mucosal permeability was transiently increased during 155 mM HCl. The results suggest that iNOS is constitutively expressed in the gastric mucosa and is involved in acid-induced hyperemia, suggesting a novel role for iNOS in gastric mucosal protection.     

Enhanced vasoconstrictor responses in eNOS deficient mice.

Previous studies suggest that vasoconstriction is modulated by nitric oxide (NO). Contractions to ET-1 and/or thromboxane may be enhanced during chronic deficiency in expression or activity of NO synthase (NOS). Multiple isoforms of NOS are expressed within the vessel wall and purely pharmacological approaches cannot define the role of each. We tested the hypothesis that vasoconstriction to endothelin-1 (ET-1) and/or the thromboxane mimetic, U46619, is enhanced under conditions of chronic, selective deficiency in endothelial NOS (eNOS-/-) by examining responses in aorta from eNOS-/- mice compared to wild type (eNOS+/+). ET-1 produced dose-dependent contraction of aorta from eNOS+/+ mice that was increased twofold following acute inhibition of all NOS isoforms with N(G)-nitro-L-arginine (L-NNA). In eNOS-/- mice, contractions to ET-1 were increased twofold compared to eNOS+/+. L-NNA had no effect. Although contraction of the aorta to thromboxane mimetic U46619 was increased at lower concentrations, maximal contractions to U46619 were not increased following acute inhibition of NOS or in eNOS-/- mice. These studies provide direct evidence that vasoconstriction to ET-1 and thromboxane is augmented in the face of eNOS deficiency, demonstrating that eNOS normally inhibits vascular contractile responses.