Epithelium-dependent regulation of airways smooth muscle function. A histamine-nitric oxide pathway.

The airway epithelium is responsible for the production of a number of arachidonic acid and non-prostanoid inhibitory factors. Epithelium synthesises nitric oxide (NO) which may be important in regulating the function of airways smooth muscles. We studied in vitro the effect of histamine (100 nM-100 microM) which increases the NO release on rabbit airway smooth muscles induced by 80 mM KC1 in the presence or not of 10(-5) Methylene blue (MB) (inactivator of guanylate cyclase) or N(G)-monomethyl L-arginine (L-NMMA), a NOS inhibitor. All experiments were done in tracheal muscle strips from 28 rabbits with epithelium and after epithelium removal. The additional use of histamine (1 microM) on KC1 contraction induced a relaxation of 10% of the initial contraction. The additional use of L-NMMA decreased the relaxation to 5% of initial contraction. MB rather than L-NMMA increased the contraction significantly (p<0.01). Epithelium removal increased the contraction induced by KC1 (80 mM) and histamine (1 microM) by about 30% (p<0.001). NO release especially from epithelium regulates the airways smooth muscle functions. Damage to the epithelium may contribute to an increase in airways sensitivity, observed in asthma.     

Influence of NG-monomethyl-L-arginine on endothelium-dependent relaxations in the perfused mesenteric vascular bed of the rat

Endothelium-dependent relaxation mediated by the formation of nitric oxide (NO) from L-arginine, is prevented by the arginine analog NG-monomethyl L-arginine (L-NMMA) (Palmer et al., Biochem. Biophys. Res. Comm. 153:1251-1256 (1988)). In the rat mesenteric arterial bed, incubation with L-NMMA did not prevent acetylcholine-induced relaxation, which, however, was reversed when L-NMMA was added during its maximum effect. A similar profile of action was observed with methylene blue, an inhibitor of guanylate cyclase. Methylene blue, but not L-NMMA, increased basal perfusion pressure. These data indicate that in the mesenteric arterial bed, NO formation via the L-NMMA-sensitive pathway occurs during stimulation with acetylcholine, but not under basal conditions.     

NG-nitro-L-arginine inhibits non-adrenergic, non-cholinergic relaxation in rabbit urethral smooth muscle.

Electrical field stimulation induced a relaxation response in female rabbit urethral smooth muscle strips precontracted with phenylephrine. The relaxation response was inhibited by tetrodotoxin, but not by atropine, propranolol, or hexamethonium. The relaxation response thus results from stimulation of inhibitory non-adrenergic, non-cholinergic nerves. The electrically induced relaxation response was inhibited by an inhibitor of nitric oxide biosynthesis, NG-nitro-L-arginine. This inhibition was overcome by addition of a precursor of nitric oxide, L-arginine. An inhibitor of soluble guanylate cyclase, methylene blue, reduced the relaxation response, and a selective cyclic GMP phosphodiesterase inhibitor, M & B 22948, potentiated the relaxation response. These data indicate that agents which affect the biosynthesis of nitric oxide are associated with the urethral relaxation response evoked by electrical field stimulation, and that cyclic GMP may mediate the relaxation response.     

红细胞抗高血压因子舒血管作用机制的研究

本实验研究了从Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠红细胞中提取的抗高血压因子（ａｎｔｉｈｙｐｅｒｔｅｎｓｉｖｅｆａｃｔｏｒ,ＡＨＦ）对苯肾上腺素引起的Ｗｉｓｔａｒ大鼠胸主动脉螺旋条预收缩的舒张作用。结果表明：ＡＨＦ对主动脉条的舒张呈内皮与剂量依赖性。左旋硝基精氨酸与美蓝均可阻断ＡＨＦ的舒血管作用,而铜锌超氧化物歧化酶对ＡＨＦ的舒血管效应有促进作用。提示ＡＨＦ是通过刺激内皮细胞产生一氧化氨或其类似物,从而激活血管平滑肌细胞内可溶性鸟苷酸环化酶这一途径引起血管舒张的。     

Relaxation of bovine coronary artery and activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside and a carcinogenic nitrosoamine.

The principal objective of this study was to test the hypothesis that nitroprusside relaxes vascular smooth muscle via the reactive intermediate, nitric oxide (NO), and that the biologic action of NO is associated with the activation of guanylate cyclase. Nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and NO elicit concentration-dependent relaxation of precontraced helical strips of bovine coronary artery. Nitroprusside, MNNG and NO also markedly activate soluble guanylate cyclase from bovine coronary arterial smooth muscle and, thereby, stimulate the formation of cyclic GMP. Three heme proteins, hemoglobin, methemoglobin and myoglobin, and the oxidant, methylene blue, abolish the coronary arterial relaxation elicited by NO. Similarly, these heme proteins, methylene blue and another oxidant, ferricyanide, markedly inhibit the activation of coronary arterial guanylate cyclase by NO, nitroprusside and MNNG. The following findings support the view that certain nitroso-containing compounds liberate NO in tissue:heme proteins, which cannot permeate cells, inhibit coronary arterial relaxation elicited by NO, but not by nitroprusside or MNNG; the vital stain, methylene blue, inhibits relaxation by NO, nitroprusside and MNNG; heme proteins and oxidants inhibit guanylate cyclase activation by NO, nitroprusside and MNNG in cell-free mixtures. The findings that inhibitors of NO-induced relaxation of coronary artery also inhibit coronary arterial guanylate cyclase activation suggest that cyclic GMP formation may be associated with coronary arterial smooth muscle relaxation.     

Absence of implication of L-arginine/nitric oxide pathway on neuronal cell injury induced by L-glutamate or hypoxia.

L-glutamate, N-methyl-D-aspartate (NMDA), kainate, quisqualate and sodium nitroprusside increased cyclic GMP (cGMP) level on rat whole brain cell culture. The accumulation of cGMP evoked by L-glutamate was inhibited by a NMDA antagonist MK-801, an inhibitor of guanylate cyclase methylene blue and two nitric oxide (NO) synthase inhibitors NG-monomethyl-L-arginine (L-NMMA) and L-NG-nitroarginine (NO2Arg). The inhibition of L-NMMA on cGMP level was reversed partially by addition of L-arginine. Although MK-801 was able to protect cells from neuronal injury induced by L-glutamate or by 5 h hypoxia, L-NMMA and NO2Arg were ineffective. The present study suggests that cGMP elevation mediated by NO following activation by L-glutamate is not involved in neuronal cell injury.     

Vasodilation by banhabackchulchunmatang, a Chinese medicine, is associated with negative modulation of PKCalpha activation and NO production

Banhabackchulchunmatang (BCT) is a widely used herbal medicine with vasodilatory actions. In the present study, we investigated the subcellular mechanisms of its vascular actions. Both in the presence and absence of endothelium, BCT relaxed vascular strips precontracted with phenylephrine, but the magnitude of relaxation was greater in the presence of endothelium. The relaxation was inhibited by either L-NAME, an NOS inhibitor, or methylene blue, a cGMP inhibitor, indicating the involvement of nitric oxide (NO). The involvement of NO was supported by the increased formation of nitrite from human umbilical vein endothelial cells in the presence of BCT. In vascular strips, BCT lowered the phosphorylation level of the 20 kDa myosin light chains. BCT also directly inhibited phenylephrine-induced protein kinase Calpha (PKCalpha) translocation in freshly isolated single ferret portal vein smooth muscle cells. Together, these effects are likely to contribute to the vasodilatory actions of BCT.     

L-arginine causes whereas L-argininosuccinic acid inhibits endothelium-dependent vascular smooth muscle relaxation

This study examined the actions of L-arginine, a putative precursor of endothelium-derived nitric oxide, and arginine analogs on endothelium-dependent relaxation of isolated rings of bovine pulmonary artery. L-Arginine did not consistently relax arterial rings unless rings were first rendered refractory to endothelium-dependent relaxation by pretreatment with 1 microM A23187 for 45 min. L-Arginine-elicited relaxation was endothelium-dependent, antagonized by oxyhemoglobin or methylene blue, and unaffected by indomethacin. L-Argininosuccinic acid caused endothelium-dependent contractions and irreversible inhibition of endothelium-dependent but not nitroglycerin-elicited relaxation, which was not overcome by addition of L-arginine. Inhibition of endothelium-dependent relaxation by L-NG-monomethyl arginine, however, was reversible and overcome by L-arginine. Therefore, endothelium-dependent relaxants may cause arginine depletion in endothelial cells and endogenous argininosuccinic acid may modulate the biosynthesis of endothelium-derived nitric oxide from arginine.     

The role of lysolecithin in the relaxation of vascular smooth muscle

The effect of lysolecithin (lysophosphatidylcholine) on the relaxation of rabbit aortic strip closely resembled that produced by acetylcholine (ACh) which releases the endothelium-derived relaxing factor (EDRF). Relaxation induced by lysolecithin depended on the presence of endothelium and was inhibited by hemoglobin and methylene blue. It appeared to be mediated by the second messenger, c-GMP. Lysolecithin induced relaxation was slower but more persistent than that resulting from the endothelium-derived relaxing factor (EDRF) produced by acetylcholine (ACh). Like lysolecithin, Triton X-100, a non-ionic detergent, also preferentially relaxed aortic strips with intact endothelium. The results demonstrate the importance of phospholipids derived from cell membranes in vascular smooth muscle relaxation. Endothelium-derived relaxing factors appear as a group of heterogeneous substances.     

A relaxing factor released by phospholipase A2 in the absence of endothelium

Summary Phospholipase A₂ (PLA₂) produced slow dose dependent relaxation in intact and endothelium-deprived precontracted rabbit aortic strips. In endothelium-deprived preparations, relaxation induced by PLA₂ is inhibited by hemoglobin, methylene blue and parabromophenacylbromide (PBPB), and is potentiated by superoxide dismutase (SOD). Indomethacin has no effect. Relaxation is accompanied by a rise in c-GMP. Phospholipase C causes a significant increase in tension, while Phospholipase D has no effects. In intact aortic strips PLA₂ causes a biphasic response with no elevation in c-GMP. The results indicate several common features of the PLA₂ released factor with endothelium-derived relaxing factor (EDRF). However PLA₂ induced relaxation is not dependent on endothelial cells. Apparently in addition to nitric oxide which may be the endothelium-derived relaxing factor, a second smooth muscle relaxing factor exists which is initiated by PLA₂ and is independent of endothelium. The production of the PLA₂ produced relaxation is dependent on its specific hydrolytic activity. We call this relaxing factor the phospholipid-derived relaxing factor (PDRF).     

Cyanide and sulfide interact with nitrogenous compounds to influence the relaxation of various smooth muscles

Sodium nitroprusside relaxed guinea pig ileum after the segment had been submaximally contracted by either histamine or acetylcholine, intact isolated rabbit gall bladder after submaximal contraction by either acetylcholine or cholecystokinin octapeptide, and rat pulmonary artery helical strips after submaximal contraction with norepinephrine. In each of these cases the relaxation produced by nitroprusside was at least partially reversed by the subsequent addition of excess sodium cyanide. Cyanide, however, in nontoxic concentrations did not reverse the spasmolytic effects of hydroxylamine hydrochloride, sodium azide, nitroglycerin, sodium nitrite, or nitric oxide hemoglobin on guinea pig ileum, nor did cyanide alone in the same concentrations have any effect. The similar interaction between nitroprusside and cyanide on rabbit aortic strips is not dependent on the presence of an intact endothelial cell layer. Also, on rabbit aortic strips and like cyanide, sodium sulfide reversed the spasmolytic effects of azide and hydroxylamine, but it had little or no effect on the relaxation induced by papaverine. Unlike cyanide, however, sulfide augmented the relaxation induced by nitroprusside, and it reversed the effects of nitric oxide hemoglobin, nitroglycerin, and nitrite. A direct chemical reaction between sulfide and nitroprusside may account for the difference between it and cyanide. Although evidence was obtained also for a direct chemical reaction between sulfide and norepinephrine, that reaction does not seem to have played a role in these results. These observations suggest the existence of at least three distinct subclasses of so-called nitric oxide vasodilators. At least in some cases cyanide and sulfide cannot be acting by the same mechanism in their modifications of the responses to the agonists.     

Vascular smooth muscle-derived relaxing factor (MDRF) and its close similarity to nitric oxide

The principal finding in this study is that vascular smooth muscle generates a labile relaxing factor that possesses pharmacological and chemical properties that are similar to those of authentic nitric oxide. MDRF was generated by perfusion of endothelium-denuded bovine pulmonary artery as assessed by bioassay. In addition, endothelium-denuded arterial rings that were incubated at 37 degrees C for 24 hr to lower endogenous L-arginine levels relaxed in response to L-arginine but not D-arginine. Freshly mounted, endothelium-denuded arterial rings were not relaxed by L-arginine but did relax in response to the dipeptide L-arginyl-L-alanine. Relaxant responses were accompanied by increases in smooth muscle levels of cyclic GMP and nitrite, and were inhibited by oxyhemoglobin, methylene blue, and NG-nitro-L-arginine. NG-Nitro-L-arginine also caused endothelium-independent contractile responses. Thus, a relaxing factor with the properties of nitric oxide can be generated from vascular smooth muscle.     

Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation

This study examined the influence of lowered arterial levels of L-arginine on endothelium-dependent relaxation of isolated rings of bovine pulmonary artery. Incubation of arterial rings under tension for 24 hr in oxygenated Krebs bicarbonate solution at 37 degrees C resulted in the development of marked or complete tolerance to A23187 (calcium ionophore)- and acetylcholine-elicited relaxation. Relaxant responses to nitric oxide were unaffected. Addition of L-arginine did not relax control rings but did elicit marked endothelium-dependent relaxation of tolerant rings that was inhibited by oxyhemoglobin or methylene blue. L-Arginine also restored acetylcholine-elicited relaxation. Inclusion of L-canavanine in the 24 hr incubations protected against the development of tolerance. The tissue concentration of arginine was 3-fold lower in tolerant than control arterial rings and L-canavanine restored arterial arginine levels to control values. Therefore, depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation.     

白细胞介素-2引起离体大鼠主动脉环舒张及其作用机制

本文旨在研究白细胞介素－2（interleukin-2,IL-2）以离体大鼠胸主动脉环收缩张力的作用及其可能机制。采用累积加药法,检测IL－2对去氧肾上腺素（PE）和KCl预收缩的胸主动脉环收缩张力的影响。结果表明,IL－2（1、10、100、1000U／ml）对PE（10μmol/L）预收缩的内皮完整血管环产生浓度依赖性的舒张作用,而对KCl (120mmol/L）预收缩的血管无作用,去除内皮后,IL－2的舒张作用被取消。用一氧化氮合酶抑制剂L－NAME（0.1mmol/L）和鸟苷酸环化酶抑制剂亚甲蓝（10μmol/L）预处理,均可阻断IL－2的舒张血管作用。用环氧合酶抑制剂吲哚美辛（Indo,10μmol/L）预处理可阻断IL－2的血管舒张作用。从上述观察结果推论,IL－2通过NO－鸟苷酸环化酶和环氧合酶途径产生内皮依赖的血管舒张作用。     

Endothelin-1- and endothelin-3-induced vasorelaxation via common generation of endothelium-derived nitric oxide.

The vasorelaxant effects by endothelin-1 (ET-1) and endothelin-3 (ET-3), and their mechanisms of action were studied in isolated porcine pulmonary arterial strips. ET-1 and ET-3 dose-dependently (10(-9) - 10(-8) M) relaxed vascular strips precontracted with norepinephrine only in the presence of endothelium. The maximal vasorelaxant effect by ET-1 was about 70% of that by ET-3. The ET-1- and ET-3- induced vasorelaxation was blocked by NG-nitro-L-arginine, an inhibitor of nitric oxide synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The present data suggest that vascular smooth muscle relaxation induced by ET-1 and ET-3 is mainly ascribed to synthesis and release of nitric oxide from L-arginine in endothelium.     

Nitric oxide from endothelium and smooth muscle modulates responses to sympathetic nerve stimulation: implications for endotoxin shock.

The influence of nitric oxide (NO) on vascular responses to transmural stimulation (TNS) of noradrenergic nerves was studied in isolated rings of rat iliac arteries. TNS produced frequency-dependent contractions in all vessels. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) significantly enhanced TNS responses in intact vessels, but not in those in which the endothelium had been removed. However, in endothelium-denuded rings incubated for 8 hours, L-NMMA increased the contractions induced by nerve stimulation, an effect which was prevented by treatment with dexamethasone or cycloheximide, and enhanced by incubation with lipopolysaccharide and gamma-interferon. Addition of L-arginine reversed the effect of L-NMMA in intact rings; however, it significantly decreased below control values TNS-induced contractions in vessels without endothelium. The results indicate that a) the arterial response to noradrenergic nerve stimulation is modulated by NO originating either in endothelial cells or in smooth muscle cells after induction of NO synthase activity, and b) once NO synthase is induced, the limiting step in NO production is the availability of the substrate L-arginine. An overproduction of vascular NO in the presence of endotoxin or other inflammatory stimuli may prevent the vascular response to sympathetic stimuli and contribute to the vasodilation observed in inflammation or endotoxic shock.     

In vitro relaxation of rabbit and human internal anal sphincter by rutaecarpine, an alkaloid isolated from Evodia rutaecarpa

Rutaecarpine, a compound extracted from the Chinese medicinal herb Evodia rutaecarpa, has been shown to possess relaxing action on vascular smooth muscle from rat thoracic aorta. The internal anal sphincter is a specialized smooth muscle regulating important anorectal physiology. To investigate the effect and underlying mechanisms of rutaecarpine on internal anal sphincter, muscle strips from rabbit internal anal sphincter were used. The results showed that rutaecarpine (1 x 10(-10) M to 1 x 10(-4) M) produced a concentration-dependent muscular relaxation effect in our preparations, which were precontracted with acetylcholine. This muscular relaxation effect was not affected by treatment with L-N(G)-nitro-arginine methyl ester (a nitric oxide synthase inhibitor), methylene blue (a guanylate cyclase inhibitor), N-ethylmaleimide (an adenylate cyclase inhibitor), or by removal of the mucosa and submucosa tissue. Pretreatment with nifedipine (a calcium channel blocker) or extracellular Ca+2 removal by ethylenediaminetetraacetic acid (EDTA) greatly attenuated the relaxation effect, suggesting that calcium ion might be involved. In experiments using strips from human internal anal sphincter, an even more prominent relaxation effect was shown. It is thus concluded that rutaecarpine caused relaxation on internal anal sphincter from rabbits and human subjects. The relaxation action was not related to NO-cGMP pathway, instead calcium ion might play an important role and shed insight into clinical implications for those anorectal disorders with hyperactive anal tone.     

Vascular smooth muscle relaxation by endothelium-dependent beta 1-adrenergic action

1. Norepinephrine (NE) (10(-5) M) in rabbit aorta relaxed ring segments with endothelium precontracted with 10(-6) M NE, but not segments without endothelium. 2. The relaxation was inhibited with metoprolol and methylene blue, but not inhibited with yohimbine and indomethacin. 3. NE (10(-5) M) significantly elevated tissue c-GMP levels in segments with endothelium. 4. These studies suggest that the vascular relaxation by high doses of NE is mediated by the release of endothelium-derived relaxing factor (EDRF) induced by the stimulation of beta 1-adrenoceptor.     

Indomethacin alters the effects of substance-P and VIP on isolated airway smooth muscle

Both substance-P and vasoactive intestinal peptide (VIP) have previously been demonstrated to contract and relax, respectively, the isolated guinea pig trachea. In addition, substance-P and VIP have been localized within the pulmonary innervation of various species. In the present studies, substance-P was found to cause a concentration-related contraction of isolated lung parenchymal strips of the guinea pig, as well as isolated tracheal strips. VIP caused a significant concentration-related relaxation of the isolated tracheal strip, but not the lung parenchymal strip. Indomethacin, a prostaglandin synthetase inhibitor, potentiated the contractile response of the trachea to substance-P and inhibited the VIP- and isoproterenol-induced relaxation. These studies are potentially important in understanding the pathogenesis of bronchospastic disorders, since alterations in prostaglandin biosynthesis may result in hyperreactivity of airways to contractile agonists such as neurotransmitters, as well as an inhibition of relaxation induced by endogenous substances such as VIP or β agonists.     

Endothelium-derived relaxing factor (nitric oxide) has protective actions in the stomach

The role that nitric oxide, an endothelium-derived relaxing factor, may play in the regulation of gastric mucosal defence was investigated by assessing the potential protective actions of this factor against the damage caused by ethanol in an ex vivo chamber preparation of the rat stomach. Topical application of glyceryl trinitrate and sodium nitroprusside, which have been shown to release nitric oxide, markedly reduced the area of 70% ethanol-induced hemorrhagic damage. Topical application of a 0.01% solution of authentic nitric oxide also significantly reduced the severity of mucosal damage. Pretreatment with indomethacin precluded the involvement of endogenous prostaglandins in the protective effects of these agents. The protective effects of NO were transient, since a delay of 5 minutes between NO administration and ethanol administration resulted in a complete loss of the protective activity. The protection against ethanol afforded by 10 micrograms/ml nitroprusside could be completely reversed by intravenous infusion of either 1% methylene blue or 1 mM hemoglobin, both of which inhibit vasodilation induced by nitric oxide. Intravenous infusion of 1% methylene blue significantly increased the susceptibility of the mucosa to damage induced by topical 20% ethanol. These results indicate that ethanol-induced gastric damage can be significantly reduced by nitric oxide. The mechanisms underlying the protective actions of nitric oxide are unclear, but may be related to its vasodilator or anti-aggregatory properties.