Comparative pharmacology of endothelium-derived relaxing factor and nitric oxide

The present study was designed to characterize endothelium-derived relaxing factor (EDRF) and nitric oxide (NO) by employing both biological and chemical methods. EDRF was released by the calcium ionophore A23187 from cultured bovine pulmonary artery endothelium (BPAE) grown on microcarrier beads and then superfused in a cell column. The maximum relaxations induced by EDRF (83%) or NO (79%) on phenylephrine (PE)-contracted rabbit aorta were similar. In contrast, EDRF was only half as potent as NO in relaxing the KCl-contracted rabbit aorta. EDRF induced a concentration-dependent relaxation of both PE-contracted rabbit aorta and histamine-contracted guinea pig aorta that was accompanied by a marked elevation in cyclic GMP levels. However, EDRF was vascular selective and did not relax or increase cyclic GMP levels of the histamine-contracted taenia coli of either species. NO was not vascular selective and relaxed both aorta and taenia coli and also markedly increased cyclic GMP levels in each. NO also relaxed dog femoral artery and gastrointestinal smooth muscle preparation of the lower esophageal sphincter, whereas EDRF only relaxed the femoral artery. Experiments were also performed describing the actions of a series of different resins: anion exchange resins (NH2/NH, AG-1), cation exchange resin (-COOH), reversed phase resin (C18) and hemoglobin-agarose on EDRF- or NO-induced relaxation. NH2/NH, AG-1 and hemoglobin-agarose resins inhibited EDRF-induced relaxation, but -COOH and C18 did not. The inhibition was dependent on the amount of resin employed. NO-induced relaxation was blocked only by hemoglobin-agarose but by none of the other resins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of milrinone on contractile responses of guinea pig trachea, lung parenchyma and pulmonary artery

The effects of milrinone, a bipyridine with known vasodilator activity, on guinea pig tracheal-spirals, lung parenchymal strips and pulmonary artery rings in vitro were compared with the effects of isoproterenol and aminophylline on these tissues. The concentration of milrinone that produced 50% relaxation (IC50) of tracheal spirals constricted by carbachol was 3.6 X 10(-5) M. Isoproterenol (IC50, 9.5 X 10(-8) M) was significantly (P less than .001) more potent and aminophylline (IC50, 1.2 X 10(-4) M) was significantly (P less than .001) less potent than milrinone in this effect. The IC50 for milrinone for lung parenchymal strips contracted by histamine was 3.2 X 10(-5) M, whereas the IC50 for isoproterenol was significantly (P less than .001) less, 1.4 X 10(-7) M; aminophylline produced only limited relaxation of lung parenchymal strips. Milrinone relaxed pulmonary artery rings constricted by norepinephrine with an IC50 of 3.8 X 10(-6) M, whereas neither isoproterenol nor aminophylline produced a 50% relaxation. Pretreatment of tracheal spirals, lung parenchymal strips and pulmonary artery rings with 1.6 X 10(-4) M milrinone inhibited subsequent contraction by carbachol, histamine and norepinephrine, respectively. The relaxant effects of milrinone were not influenced by treatment with atropine, cimetidine, mepyramine, phentolamine or propranolol. However, indomethacin blocked milrinone's relaxant effects on tracheal spirals effectively, but not on pulmonary artery rings or lung parenchymal strips, suggesting distinct modes of action on various tissue types.     

Arachidonic acid metabolites and airway epithelium-dependent relaxant factor

Exogenous arachidonic acid (10(-8) to 10(-4) M) contracted epithelium-free guinea pig tracheal strips. Intact tracheal strips were contracted slightly by low concentrations of arachidonic acid (10(-8) to 10(-5) M), but higher concentrations relaxed them. In contrast, when tracheal strips were precontracted with histamine or carbachol, exogenous arachidonic acid had no effect on epithelium-free preparations but induced concentration-dependent (10(-8) to 10(-4) M) relaxation of intact tracheal strips. The effects of arachidonic acid both in epithelium-free and epithelium-containing trachea were blocked by either indomethacin (10(-6) M) or aspirin (10(-4) M). Studies on the effects of exogenous arachidonic acid, performed with a "sandwich protocol," demonstrated that the postulated airway epithelium-dependent relaxant factor released by an intact tracheal strip relaxes an adjacent epithelium-free strip in the same organ bath. This relaxation is antagonized by indomethacin suggesting the involvement of a cyclooxygenase product in this phenomenon. Comparison of concentration-response curves for contractile agonists in epithelium-free preparations and in one containing epithelium suggests the mobilization of airway epithelium-dependent relaxant factor by histamine but not by carbachol. The effects of cyclooxygenase and lipoxygenase inhibitors indicated that both relaxant and contractile arachidonic acid metabolites are generated by epithelial and nonepithelial cells alike in response to contractile agonists.     

Vasodilatory action of HA1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide], a novel calcium antagonist with no effect on cardiac function

A novel compound, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) was found to be a potent relaxant of blood vessels. Rabbit aortic strips contracted by 18 mM KCl relaxed in the presence of HA1004, with an ED50 value of 2.2 X 10(-6) M. The isolated guinea-pig atria on right ventricular papillary muscles did not respond to HA1004, even at a concentration of 3 X 10(-4) M. HA1004, like verapamil, produced a competitive inhibition of the Ca++-induced contraction of the depolarized rabbit aorta. The pA2 value of HA1004 for the Ca++-induced contraction was 6.60. Atropine, propranolol, theophylline or indomethacin had no effect on the HA1004-induced relaxation. HA1004 (3 X 10(-6) M) inhibited the contraction produced by Ca++ ionophore, A23187, whereas verapamil or diltiazem had no effect on this contraction, even at a concentration of 3 X 10(-5) M. Moreover, HA1004 produced a competitive inhibition of Ca++-induced contraction of the A23187-treated aorta and inhibited the phenylephrine-induced contraction elicited in ca++-free solution, thereby suggesting that this drug affects intracellular rather than extracellular Ca++. The present findings indicate that HA1004 is a novel calcium antagonistic vasodilator with no effect on cardiac function and is apparently of a different class of calcium antagonist from verapamil.     

Inhibition of release of vasoactive and inflammatory mediators in airway and vascular tissues and macrophages by a chinese herbal medicine formula for allergic rhinitis

Herbal therapies are being used increasingly for the treatment of allergic rhinitis. The aim of this study was to investigate the possible pharmacological actions and cellular targets of a Chinese herbal formula (RCM-101), which was previously shown to be effective in reducing seasonal allergic rhinitis symptoms in a randomized, placebo-controlled clinical trial. Rat and guinea pig isolated tissues (trachea and aorta) were used to study the effects of RCM-101 on responses to various mediators. Production of leukotriene B(4) in porcine neutrophils and of prostaglandin E(2) and nitric oxide (NO) in Raw 264.7 cells were also measured. In rat and guinea pig tracheal preparations, RCM-101 inhibited contractile responses to compound 48/80 but not those to histamine (guinea pig preparations) or serotonin (rat preparations). Contractile responses of guinea pig tracheal preparations to carbachol and leukotriene C(4,) and relaxant responses to substance P and prostaglandin E(2) were not affected by RCM-101. In rat aortic preparations, precontracted with phenylephrine, endothelium-dependent relaxant responses to acetylcholine and endothelium-independent relaxant responses to sodium nitroprusside were not affected by RCM-101. However, RCM-101 inhibited relaxations to l-arginine in endothelium-denuded rat aortic preparations, which had been pre-incubated with lipopolysaccharide. RCM-101 did not affect leukotriene B(4) formation in isolated porcine neutrophils, induced by the calcium ionophore A23187; however, it inhibited prostaglandin E(2) and NO production in lipopolysaccharide-stimulated murine macrophages (Raw 264.7 cells).The findings indicate that RCM-101 may have multiple inhibitory actions on the release and/or synthesis of inflammatory mediators involved in allergic rhinitis.     

Inhibitors of acyl-coenzyme A:lysolecithin acyltransferase activate the production of endothelium-derived vascular relaxing factor

Considerable acyl-CoA:lysolecithin acyltransferase (LAT) activity could be demonstrated in homogenates of cultured bovine endothelial cells. This LAT activity was inhibited by thimerosal and p-hydroxymercuribenzoate in a concentration-dependent manner. Preconstricted strips of rabbit aorta were relaxed by acetylcholine or the LAT inhibitors in a concentration-dependent fashion if the endothelium was intact (maximal effect of both LAT inhibitors at 10(-5) M). In rabbit aortic strips thimerosal also induced a concentration-dependent stimulation of the formation of 6-keto-prostaglandin F1 alpha, the major cyclooxygenase metabolite of this tissue. This effect of thimerosal was more pronounced in endothelium-intact than in endothelium-denuded preparations. Inhibition of prostaglandin synthesis with indomethacin (10(-5) M) did not impair the relaxation. Thimerosal and acetylcholine-induced relaxations were abolished when the endothelium was removed or when endothelium-intact preparations were pretreated with nordihydroguaiaretic acid (3 X 10(-5) M), gossypol (5 X 10(-6) M) or dithiothreitol (3 X 10(-4) M). In contrast, mepacrine (3 X 10(-5) M), that abolished the acetylcholine response, had no effect on the thimerosal relaxation. In other experiments bovine endothelial cells were grown to confluence on microcarrier beads and packed into columns. Adding thimerosal (5 X 10(-6) M) or bradykinin (10(-10) to 10(-8) M) to the medium superfusing the columns induced the release of an unstable nonprostanoid factor (or factors) that relaxed endothelium-denuded rabbit femoral artery segments. Bradykinin induced a transient effect whereas there was a strong and long-lasting release of the factor after administration of thimerosal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blockade of endothelium-dependent relaxation by the amiloride analog dichlorobenzamil: possible role of Na+/Ca++ exchange in the release of endothelium-derived relaxant factor

The importance of extracellular calcium for the expression of endothelium-dependent relaxation was examined in isolated rat aortic rings contracted by methoxamine. The endothelium-dependent relaxation generated by acetylcholine or the calcium ionophore A23187 was eliminated when rings were placed in physiological buffer to which calcium had not been added. The endothelium-independent relaxation to sodium nitroprusside was still elicited in the presence of this "low calcium" buffer. Pretreatment of aortic rings with high concentrations of nifedipine (5 X 10(-7) M) or verapamil (10(-5) M) caused a comparable displacement to the right (2-3 times) in the relaxant dose-response curve for acetylcholine, A23187 and sodium nitroprusside with little or no changes in the maximal relaxation obtained with these vasodilators. Increasing concentrations of dichlorobenzamil, an analog of amiloride and a recently described inhibitor of calcium influx via sodium-calcium exchange, functionally antagonized and abolished the relaxations elicited by acetylcholine and A23187, but had no appreciable effect on the relaxations to sodium nitroprusside or atrial natriuretic factor (an endothelium-independent vasodilator). Similar results were obtained using isolated rabbit aortic rings. Thus, although the presence of extracellular calcium is critically required for the expression of endothelium-dependent relaxation, the associated calcium translocation is not blocked by the organic calcium entry blockers. The results with dichlorobenzamil suggest that sodium-calcium exchange may be an important mechanistic step in the release of endothelium-derived relaxant factor.     

Selective blockade of endothelium-dependent and glyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta

Hemoglobin at 1 microM reduced and at 10 microM abolished the endothelium-dependent relaxation induced by acetylcholine or by A23187 in rabbit aortic rings. Similarly, methylene blue at 10 microM reduced and at 50 microM abolished relaxation induced by acetylcholine and by A23187. Furthermore, hemoglobin (1-10 microM) and methylene blue (10-50 microM) each induced a dose-dependent inhibition of the endothelium-independent relaxation produced by glyceryl trinitrate, but neither had any effect on the relaxation produced by isoproterenol. The inhibitory effects of hemoglobin and methylene blue may be due to blockade of guanylate cyclase, as the rises in cyclic GMP content which accompany relaxation induced by acetylcholine, A23187 or glyceryl trinitrate were abolished. Isoproterenol-induced relaxation took place with no change in cyclic GMP content. Hemoglobin and methylene blue appear therefore to inhibit selectively vaso-relaxation induced by agents which increase cyclic GMP levels. Hemoglobin and methylene blue augment tone in aortic rings, particularly when endothelial cells are present, suggesting that the endothelium-derived relaxing factor (EDRF) might be released spontaneously in low concentrations. The possibility that hemoglobin inhibits endothelium-dependent and glyceryl trinitrate-induced relaxation by binding EDRF and nitric oxide, respectively, is discussed together with the proposal that methylene blue might produce its effects by oxidizing a component of guanylate cyclase, possibly a ferrous heme group linked to the enzyme molecule. Methylene blue might, in addition, interact directly with EDRF.     

Effects of selected bronchodilators on antigen- and A23187-induced contraction of guinea-pig trachea

Antigen and the calcium ionophore A23187 contract isolated tracheal spirals from sensitized guinea pigs and A23187 also contracts normal airways. The contractions appear to be the result of released bronchoconstrictor mediators, likely products of the lipoxygenase pathway of arachidonic acid metabolism. Pretreatment (10-30 min) with a series of bronchodilators resulted in inhibition of ovalbumin-induced airways contractions. The order of potency on the trachea was isoprenaline greater than forskolin greater than prostaglandin E2 much greater than aminophylline greater than dibutyryl cyclic AMP congruent to prostacyclin. The IC50 values and order of potency were similar to their capacity to relax tracheal smooth muscle. Only isoprenaline (10(-6) M), forskolin (10(-6) M) and aminophylline (10(-3) M) inhibited A23187-induced contraction of trachea. These agents lost their inhibitory capacity 20 to 30 min after A23187 challenge. This was not observed with ovalbumin challenge because the membrane stimulation with ovalbumin occurs immediately whereas A23187 induction of Ca++ influx is continual. The recovery from inhibition is probably not receptor desensitization as recovery also occurs in the presence of forskolin, a direct activator of adenylate cyclase. Sensitized trachea responded to A23187 in the presence of bronchodilators differently than normal tissue. Low concentrations of isoprenaline (10(-8) to 10(-7) M), forskolin (10(-8) to 10(-7) M), prostaglandin E2 (10(-7) to 10(-5) M), aminophylline (10(-4) M) and dibutyryl cyclic AMP (10(-3) M) all enhanced A23187-induced contraction. Pools of cyclic AMP are known to be involved both in secretion and in modulation of mediator release involved in airways smooth muscle contraction and sensitization may affect the differential availability of these pools. That this enhancement occurred only in sensitized trachea emphasized an intrinsic difference between sensitized and normal tissues.     

Co-regulation of tracheal tone by cyclic AMP- and cyclic GMP-dependent mechanisms

The regulation of guinea pig tracheal muscle tone by cyclic AMP-dependent and cyclic GMP-dependent relaxant mechanisms was investigated by studying the tracheal relaxant activities of forskolin, nitroprusside, N6-2'-O-dibutyryl-cyclic AMP and 8-bromoguanosine-cyclic GMP. In carbachol (3 X 10(-6) M)-contracted isolated tracheal rings, N6-2'-O-dibutyryl-cyclic AMP and 8-bromoguanosine-cyclic GMP each caused biphasic relaxation responses, which consisted of an acute relaxation followed by a sustained but lesser degree of relaxation. The biphasic nature of this response is suggested to result from a functional counter-balancing of cyclic nucleotide-dependent relaxant mechanisms and the contractile mechanisms stimulated by carbachol. The sensitivity of carbachol-contracted tracheal rings to forskolin and nitroprusside (activators of adenylate and guanylate cyclase, respectively) was generally not influenced by N6-2'-O-dibutyryl-cyclic AMP or 8-bromoguanosine-cyclic GMP in concentrations that induced up to 50% relaxation of the trachea. Furthermore, the partial relaxation of tracheal tension with one cyclic nucleotide analog did not alter the sensitivity of the tracheal rings to the other. These results demonstrate that cyclic AMP- and cyclic GMP-dependent mechanisms induce relaxations of the trachea that are functionally additive, each neither potentiating nor depressing the effects of the other. In the presence of 3 X 10(-6) M carbachol, the effectiveness of cyclic AMP- and cyclic GMP-dependent relaxant mechanisms appears to be fixed, and independent of the amount of active tension being maintained by the tracheal muscle itself.     

The methylenedioxyindenes, a novel class of "intracellular calcium antagonists": effects on contractility and on processes involved in regulating intracellular calcium homeostasis

The 2-substituted methylenedioxyindenes are a novel class of "calcium antagonists" which have been characterized as acting intracellularly. In the present study, the effect of 2-propyl-methylenedioxyindene (p-MDI) on the contractility of isolated rabbit papillary muscles and aortic rings as well as on several metabolic processes believed to be important in regulating calcium movement within the cell was examined. p-MDI was found to exert a dose-dependent negative inotropic effect in the range of 1.0 X 10(-5) to 1.0 X 10(-3) M (1.0 X 10(-3) M p-MDI produced a total cessation of contractility). At a concentration of 1.0 X 10(-4) M p-MDI, this depressant effect could be partially reversed by increasing the level of calcium in the tissue bath. p-MDI also relaxed aortic rings previously contracted with 50 mM KCl or 10 microM norepinephrine, although the concentration of p-MDI required to relax norepinephrine-contracted rings was 3 times greater than that required to relax KCl-contracted rings. Such selectivity was also observed when the effects of verapamil on norepinephrine- and KCl-contracted rings were examined, but was not observed with papaverine. To determine whether the effects of p-MDI on cardiac and vascular smooth muscle were due to direct interference with any of the metabolic processes which regulate intracellular calcium homeostasis, the effects of p-MDI on isolated cardiac sarcoplasmic reticulum and mitochondria and on the intracellular calcium-binding protein calmodulin were examined. At concentrations which depressed cardiac contractility, p-MDI had no effect on 1) calcium transport, uptake or ethyleneglycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid-induced calcium release by sarcoplasmic reticulum, 2) calcium uptake by mitochondria or 3) calmodulin. p-MDI did, however, exert a biphasic effect of glutamate-supported mitochondrial respiration; stimulating oxygen consumption at concentrations of 1.0 X 10(-5) and 1.0 X 10(-4) M and inhibiting respiration at 1.0 X 10(-3) M. It is therefore concluded that p-MDI has no apparent direct effect on intracellular calcium movement per se and that the calcium antagonist effect of p-MDI may be due in part to calcium channel blockage. Inhibition of mitochondrial respiration may also contribute to the negative inotropic effect of high concentrations of p-MDI.     

LY 83583 interferes with the release of endothelium-derived relaxing factor and inhibits soluble guanylate cyclase

LY 83583 (6-anilino-5,8-quinolinedione) has been reported to lower intracellular cyclic GMP by an unknown mechanism. The objective of the present study was to investigate the effect of LY 83583 on different types of vasorelaxation and to study its mechanism of action. Low concentrations of LY 83583 (less than or equal to 0.1 microM) inhibited endothelium-dependent relaxations of rabbit aortic strips induced by acetylcholine or by the calcium ionophore A23187. Higher concentrations (greater than or equal to 0.3 microM) were required to produce partial inhibition of relaxation to sodium nitroprusside and glyceryl trinitrate. Cyclic AMP-mediated relaxations, induced by isoprenaline or forskolin, were not affected by LY 83583 (10 microM). The site of interference of LY 83583 with endothelium-dependent relaxation was examined with endothelium-derived relaxing factor (EDRF) released from cultured endothelial cells that were grown on microcarrier beads and stimulated by superfusion with ATP or thimerosal. EDRF in the superfusate was detected by endothelium-denuded segments of rabbit femoral artery, which responded with dilation and, simultaneously, by purified soluble guanylate cyclase (GC) in test tubes, which was activated by EDRF. When LY 83583 was added to the glutathione-containing GC-assay or to the superfusate from cultured endothelial cells, it did not affect stimulation of soluble GC by EDRF but it slowly reversed the dilator response of the arterial detector segment. Superfusion of cultured endothelial cells with LY 83583 (1 microM), rapidly and reversibly inhibited EDRF release.(ABSTRACT TRUNCATED AT 250 WORDS)     

家兔离体主动脉血管对天麻水煎剂干预的反应

背景:现代医学研究发现天麻(Gastrodiaelata B1)具有明显地心肌保护和血管扩张作用,可用于冠心病、高血压的治疗,但其作用机制仍需明确.目的:观察天麻水煎剂对去甲肾上腺素、KCl预收缩血管反应的舒张作用,并分析其作用途径.设计:分组设计、离体动物组织对照实验.单位:川北医学院生理学教研室.材料:选用健康家兔12只,兔龄七、八个月,雌雄不拘,由川北医学院实验动物中心提供.实验所用药品天麻水煎剂由慧仁堂药店鉴定提取,将其配制成10%,20%.40%和80%浓度备用.β肾上腺素能受体阻断剂普萘洛尔为北京第二制药厂产品:一氧化氮合酶抑制剂左旋硝基精氨酸为Sigma公司产品:甲烯蓝、环氧酶抑制剂吲哚美辛为江苏太仓制药厂产品.方法:实验于2006-01/2007-01在川北医学院药物研究所完成.采用家兔离体主动脉平滑肌标本,以去甲肾上腺素(1×10-6mol/L)预收缩主动脉后给予1.0,2.0,4.0,8.0和16.0g/L天麻水煎剂观察其张力变化,并观察去除血管内皮、给予1×10-4mol/L左旋硝基精氨酸、1×10-5mol/L甲烯蓝、1×10-5mol/L吲哚美辛和1×10-5mol/L普萘洛尔对血管张力的影响:血管张力经张力换能器输入BL-410智能型生物信号处理系统进行处理.另外观察8g/L天麻水煎剂对无内皮细胞血管环NA和KCl的量效收缩反应.主要观察指标:离体血管张力.结果:天麻对血管环静息张力无明显影响.但不同剂量的天麻可使1×10-6mol/L去甲肾上腺素预收缩血管产生明显舒张(r=0.85,t=18.45,P＜0.01).去除血管内皮、1×10-4mol/L左旋硝基精氨酸或1×10-5mol/L甲烯蓝可减弱天麻的舒张作用,但吲哚美辛和普萘洛尔对其无影响.另外,8g/L天麻水煎剂对无内皮细胞血管环去甲肾上腺素和KCI的量效收缩反应明显降低,且去甲肾上腺素和KCl的PD2(-log游离药物半数有效浓度)分别由温育前(6.90±0.93)mol/L和(1.53±0.55)mmol/L,降低为(5.61±0.70)mol/L和(1.10±0.25)mmol/L,(t=2.41,3.82,P＜0.05).结论:天麻水煎剂对主动脉的舒张是内皮依赖性的,并与内皮一氧化氮的释放有关;也可通过拮抗ROC和PDC通道,抑制Ca2 的内流和释放等机制有关.     

Characterization of endothelium-derived relaxing factor involvement in the potentiating effect of parathyroidectomy on norepinephrine-induced rat aortic contraction

Summary— Previous results have shown that the contractile response to norepinephrine (NE) was enhanced in isolated aortae from SHR and normotensive Wistar parathyroidectomized rats. In this work we sought to characterize the contribution of endothelium-derived relaxing factor (EDRF) release to this effect which is not linked to hypertension. Parathyroidectomy (PTX) was performed by surgery on 5 week-old male Wistar rats. Five weeks later intact (E+) and rubbed (E–) aortic rings were mounted in an organ chamber for isometric tension recording. KCl-induced contractions were potentiated in PTX E+ aortae compared to sham operated (SO), (P < 0.05), but not in denuded E- aortae. Similarly NE (1.nM- 10 μM) induced a potentiated contractile response in PTX E+ (P < 0.01), but not in PTX E- rings; nevertheless the sensitivity did not change. After removal of endothelium, the expected enhanced contraction and sensitivity observed in SO rats was not present in PTX. The NO synthase inhibitor L-NAME (20 μM), enhanced sensitivity to NE in SO but not in PTX E+ aortic rings. In addition, hemoglobin (Hb, 10 μM) enhanced NE contraction in SO (P < 0.01) aortic rings, but to a lesser extent in PTX rat aortae. Moreover, in the presence of L-NAME or Hb, SO and PTX aortae displayed a similar contraction. Superoxide dismutase (SOD, 150 U/ml) diminished the NE contraction since NO was protected from degradation but the difference was still present between SO and PTX rat aortae, ruling out the possible implication of superoxide anions in the hyperreactivity of PTX aortae. On the other hand, A23187, which induces EDRF release, reduced the level of NE contraction as expected, but suppressed the PTX enhancing effect and in calcium-free solution the enhancement of contraction after PTX was not observed. These experiments extend to the rat the observations previously obtained in rabbit aorta: extracellular calcium is a major determining factor in NO production. Acetylcholine and A23187 (cumulative doses) produced an endothelium-dependent relaxation which was not significantly modified in NE-pre-contracted PTX aortae compared to SO aortae. L-arginine (100 μM), reversed the L-NAME inhibitory effect and induced an attenuated endothelium-dependent relaxation in PTX vessels (P < 0.01). In conclusion, in rat isolated aortae the enhancing effect of parathyroidectomy on norepinephrine and KCl contractions is due to a diminished endothelial nitric oxide production. This might arise via a decrease of the constitutive NO synthase activity in an extracellular calcium-dependent manner.     

Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor

Removal of endothelial cells on rings of rat aorta increased the sensitivity to the selective alpha-1 adrenoceptor agonist phenylephrine, to the nonselective alpha adrenoceptor agonist norepinephrine and to the selective alpha-2 adrenoceptor agonist clonidine. In the case of the first two, which are strong agonists for the alpha-1 adrenoceptor-mediating contraction, removal of endothelium increased sensitivity 4- and 6-fold at the EC30 level, but produced little or no increase in maximum. In the case of clonidine, a partial agonist for the alpha-1 adrenoceptor, which gave only about 15% of the maximum given by phenylephrine on endothelium-containing rings, removal of the endothelium not only shifted the curve to the left but also increased the maximum to about 50% of that given by phenylephrine. The depression of sensitivity to these agonists in rings with endothelium appeared to be due to the vasodepressor action of endothelium-derived relaxing factor (EDRF), as hemoglobin, a specific blocking agent of EDRF, abolished this depression. It is unlikely that the endothelium-dependent depression was due to stimulation of release of EDRF, because clonidine did not produce endothelium-dependent relaxation in precontracted rings even when its contractile action was blocked by the alpha-1 adrenoceptor antagonist prazosin. Further evidence against alpha adrenoceptor agents stimulating release of EDRF was that neither phenylephrine nor clonidine induced a rise in cyclic GMP in aortic rings, whereas acetylcholine, which does release EDRF, caused a large rise in cyclic GMP content. The possibility that the muscle cells of intact rat aortic rings were under the tonic influence of released EDRF was supported by the finding that, in the absence of any contractile agent, hemoglobin induced a fall in the basal level of cyclic GMP in endothelium-containing rings. Also consistent with EDRF being released spontaneously was the finding that contraction induced by 5-hydroxytryptamine, like that by alpha-adrenergic agonists, was also depressed in endothelium-containing rings of aorta. When the efficacy of phenylephrine as an alpha-1 agonist was reduced to about the initial efficacy of clonidine by irreversible inactivation of a very large fraction of alpha-1 adrenoceptors of the smooth muscle cells by pretreatment with dibenamine, the concentration-contraction curves for phenylephrine for both endothelium-containing rings and for endothelium-denuded rings now became very similar to the corresponding curves obtained for clonidine before receptor inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mast cell tryptase and chymase reverse airway smooth muscle relaxation induced by vasoactive intestinal peptide in the ferret

Recent evidence suggests that nonadrenergic airway relaxation may be controlled by vasoactive intestinal peptide (VIP). The magnitude and duration of smooth muscle relaxation in response to VIP may be influenced by rates of peptide degradation after release from efferent peptidergic neurons. To explore the potential role of mast cell mediators in modulating neural control of airway tone, we studied the effect of the mast cell proteases tryptase and chymase on airway smooth muscle relaxation induced by VIP in ferret airway. Tracheal rings precontracted by serotonin (10(-6) M) in a muscle bath were relaxed by VIP (10(-7) M). We found that protease-rich supernatant obtained by degranulation of dog mastocytoma cells reversed VIP-induced relaxation, as did highly purified tryptase and chymase incubated with the tracheal rings. Either enzyme completely reversed the effect of VIP, but tryptase was more potent than chymase, paralleling previous test tube observations on the relative rates of VIP cleavage by the two enzymes. Inhibitors of mast cell tryptase and chymase preincubated with the supernatant or with the purified proteases prevented reversal of VIP-induced relaxation. Mast cell proteases did not reverse the tracheal relaxation caused by the nonpeptide adrenergic agonist isoproterenol. These findings show that mast cell proteases tryptase and chymase counteract the smooth muscle relaxant effects of VIP in ferret trachea and suggest a potential role for the mast cell proteases in the modulation of nonadrenergic neural control of airway tone by VIP.     

Platelet-activating factor-acether-induced relaxation of guinea pig airway muscle: role of prostaglandin E2 and the epithelium

A fixed concentration of paf-acether (platelet-activating factor; 4 microM) relaxed isolated guinea pig tracheal preparations which had been contracted with histamine (50 microM), serotonin (1 microM) or leukotriene D4 (0.1 microM). The relaxations were approximately 43, 100 and 57%, respectively. We did not observe any relaxant effect of paf-acether (4 microM) in tissues contracted with acetylcholine (50 microM). Both lyso paf-acether (10 microM) and bovine serum albumin (25 micrograms/ml) were without effect on histamine-contracted preparations. In the presence of indomethacin (1.7 microM; 30 min) or aspirin (0.1 mM; 30 min) the relaxant effect of paf-acether (4 microM) in tissues contracted with histamine was significantly reduced to approximately 10 and 12%, respectively. When paf-acether (4 microM) was added to histamine-contracted tracheal preparations in the presence of noradrenaline (0.1 microM) or prostaglandin E2 (PGE2, 10 nM) the relaxations were 62 and 82%, respectively. Noradrenaline and PGE2 alone had only a slight relaxant effect in these tissues (7 and 14%, respectively). In the presence of indomethacin (1.7 microM) the synergistic effect of paf-acether and PGE2 was still observed. The basal production of PGE2 in isolated guinea pig tracheal preparations was 4.6 +/- 1.4 pg/mg of tissue. In the presence of paf-acether (4 microM) increased levels of this prostanoid were detected (11.2 +/- 2.4 pg/mg of tissue). Isolated guinea pig tracheal preparations when contracted with histamine released PGE2 (17.6 +/- 4.1 pg/mg of tissue). In the presence of histamine and paf-acether there was a significant increase in detectable levels of PGE2 (48.6 +/- 13.2 pg/mg of tissue).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of electrical field stimulation on antigen-induced contraction and mediator release in the guinea pig isolated superfused trachea and bronchus

These studies examined the ability of electrical field stimulation (EFS) to influence antigen-induced responses in the guinea pig isolated trachea and main-stem bronchi. Airways isolated from guinea pigs actively sensitized to ovalbumin were superfused and stimulated transmurally with square pulses of 1 msec duration at a frequency of 16 pulses per sec. In the trachea, EFS caused an atropine-sensitive contraction followed by a maintained relaxation. The relaxation consisted of adrenergic and nonadrenergic components. In the bronchus, EFS caused a maintained contraction. This contraction was due to a combination of cholinergic (atropine-sensitive) and noncholinergic (capsaicin-sensitive) mechanisms. Histamine could not be detected in superfusate samples during electrical stimulation alone of either the trachea or bronchus. EFS significantly inhibited ovalbumin-induced tracheal contractions by about 30% without altering ovalbumin-induced histamine or immunoreactive peptido-leukotriene release from the tissues. EFS had a similar inhibitory effect on the contraction induced by application of exogenous histamine (10(-5) M). The electrical stimulus-induced inhibition of the antigen-induced contraction was abolished by tetrodotoxin and propranolol and reduced by a combination of atropine, propranolol and phentolamine. Norepinephrine (5 x 10(-6) M) inhibited ovalbumin-induced histamine release by about 30% without altering the contraction. Carbamylcholine had no effect on ovalbumin-induced histamine release. In the guinea pig bronchus, EFS stimulation had no effect on either histamine release or contraction induced by ovalbumin. These results demonstrate that in the guinea pig trachea nerve stimulation can significantly antagonize antigen-induced contractions and suggest that this is due to a functional antagonism by adrenergic and nonadrenergic relaxant neurotransmitters at the level of the airway smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence against serotonin-induced endothelium-dependent relaxation in bovine coronary artery.

Bovine coronary artery rings were mounted in tissue baths for the measurement of isometric contraction in order to test for serotonin-induced endothelium-dependent relaxation. A23187 caused a concentration-dependent relaxation in precontracted artery rings when the endothelium was intact, but not when it was removed. Endothelium removal had no effect on serotonin concentration contractile response curves (CRC) in normal Krebs' solution, but enhanced the response to serotonin in artery rings precontracted with 25 mM K+ Krebs' solution. In endothelium-intact, precontracted artery rings, ketanserin shifted the serotonin CRC to the right, but did not convert the contraction to relaxation. 5-Carboxamidotryptamine caused a weak contraction that was blocked by ketanserin, but not converted to relaxation. Inactivation of monoamine oxidase enhanced the contractile response to serotonin in precontracted artery rings in a manner identical to that caused by endothelium removal. Inactivation of monoamine oxidase had no effect in endothelium-denuded artery rings. De-endothelialized rabbit aorta strips pretreated with phenoxybenzamine were placed in close apposition to endothelium-intact coronary artery strips. Superoxide dismutase, acetylcholine and A23187 caused the precontracted rabbit aorta strip to relax, whereas serotonin had no effect. These results support the conclusion that the endothelium of the bovine coronary artery releases a relaxing factor both spontaneously and in response to acetylcholine and A23187. Serotonin appears to be incapable of releasing endothelium-derived relaxing factor or causing an endothelium-dependent relaxation. However, bovine coronary artery endothelium appears to be able to take up and metabolize serotonin.     

An examination of the ability of d-tubocurarine to evoke contraction and mediator release from superfused trachea and parenchymal strips isolated from the guinea pig

The relationship between curare-induced mediator release and contraction in superfused guinea pig trachea and parenchymal strips was examined. In trachea, curare produced histamine release and contraction with peak release occurring in the first 90 sec (collection period 1) after challenge. Peak contraction developed later (collection periods 4-6). Curare-induced contraction of parenchymal strips was inconsistent and smaller than that found in trachea. No histamine could be detected in parenchymal strip superfusate samples. Curare also was selective in releasing histamine from monodispersed airway cells vs. peripheral lung cells. No leukotriene bioactivity or immunoreactivity could be detected after curare challenge of tissues or cell suspensions. Tracheal contractions, but not histamine release, occurring early (first 5 or 6 collection periods) after challenge were antagonized by mepyramine, 10(-6) M, and phenoxybenzamine, 3 X 10(-5) M. Combination of FPL55712, 10(-5) M, with mepyramine did not further alter tracheal contraction. Contractions occurring later after challenge and total histamine release were enhanced by indomethacin, 5 X 10(-6) M. Indomethacin also increased contractions in the presence of mepyramine. With mepyramine and indomethacin, LY171883, 1 and 3 X 10(-6) M, and nordihydroguaiaretic acid, 3 X 10(-5) M, antagonized tracheal contractions to curare, 3 X 10(-3) M, but not to 1 X 10(-3) M, without altering histamine release. Indomethacin prolonged return to base line of tracheal tension after challenge with exogenous histamine. After addition of LY171883 or nordihydroguaiaretic acid, the return of tracheal tension after histamine was not different from that seen without drug pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)