Biomedical subjects
K Stuart-Smith
Publications and source records attributed to K Stuart-Smith.
Role of the endothelium and nitric oxide synthases in modulating superoxide formation induced by endotoxin and cytokines in porcine pulmonary arteries.
BACKGROUND: The interactive roles of cytokines, endotoxins, superoxide (O(2)(*-) ) and nitric oxide (NO) in the pathogenesis of adult respiratory distress syndrome (ARDS) have not been fully elucidated. The effects of tumour necrosis factor-alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), and lipopolysaccharide (LPS) and the role of NO and the endothelium in mediating O(2)(*-) formation were therefore investigated in intact porcine pulmonary arteries in vitro. METHODS: Intrapulmonary artery (PA) segments were obtained from White Landrace pigs (25-35 kg) and incubated with LPS, IL-1alpha, and TNF-alpha and O(2)(*-) release was measured by the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c. The source of O(2)(*-) formation was determined using a number of enzyme inhibitors. The role of NO was explored using NO synthase (NOS) inhibitors and the distribution of NOS isoforms and peroxynitrite (ONOO(-), an index of NO-O(2)(*-) interactions) assessed by immunocytochemistry. RESULTS: LPS, IL-1alpha, and TNF-alpha promoted the formation of O(2)(*-) from PA compared with untreated controls in a time and dose dependent manner, an effect markedly enhanced by removal of the endothelium but completely inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI). L-NAME and the eNOS inhibitor N(5)-(1-iminoethyl)-ornithine (L-NIO) enhanced O(2)(*-) formation from PA (with endothelium) in response to IL-1alpha and TNF-alpha but had no effect on LPS mediated O(2)(*-) formation, whereas L-NAME and the iNOS inhibitor L-N(6)-(1-iminoethyl)-lysine-HCl (L-NIL) enhanced O(2)(*-) formation only in response to LPS. CONCLUSIONS: LPS, IL-1alpha, and TNF-alpha promote O(2)(*-) formation through an upregulation of NADPH oxidase activity which is augmented by removal of the endothelium, as well as the inhibition of eNOS (in the case of cytokines) and iNOS (in the case of LPS). The concomitant expression of NOS isoforms (and NO formation) with that of NADPH oxidase may therefore constitute a protective system designed to remove O(2)(*-) through the formation of ONOO(-). If this is so, the integrity of the endothelium may be axiomatic in the progression and severity of ARDS.
Demystified. Nitric oxide.
The discovery of nitric oxide (NO) demonstrated that cells could communicate via the manufacture and local diffusion of an unstable lipid soluble molecule. Since the original demonstration of the vascular relaxant properties of endothelium derived NO, this fascinating molecule has been shown to have multiple, complex roles within many biological systems. This review cannot hope to cover all of the recent advances in NO biology, but seeks to place the discovery of NO in its historical context, and show how far our understanding has come in the past 20 years. The role of NO in mitochondrial respiration, and consequently in oxidative stress, is described in detail because these processes probably underline the importance of NO in the development of disease.
Microvessel damage in acute respiratory distress syndrome: the answer may not be NO.
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The role of cGMP in the relaxation to nitric oxide donors in airway smooth muscle.
The aim of this study was to determine the effect of the soluble guanylyl cyclase inhibitors methylene blue and LY83583 (6-anilino-5,8-quinolinedione) on relaxation and increases in intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration ([cGMP]i) induced by sodium nitroprusside, 3-morpholinosydnonimine (SIN-1) and diethylamine-nitric oxide (NO) in porcine tracheal smooth muscle in vitro. We measured (1) the effect of NO donors on isometric force and [cGMP]i and (2) the ability of methylene blue and LY83583 to antagonize these effects. In muscle strips contracted with carbachol (0.1-0.3 microM), both sodium nitroprusside and diethylamine-NO caused relaxation and an increase in [cGMP]i. By contrast, SIN-1 caused a relaxation which was not associated with a concomitant increase in [cGMP]i. Methylene blue (10 microM) and LY83583 (10 microM) completely blocked the increase in [cGMP]i induced by sodium nitroprusside and diethylamine-NO; however substantial relaxation remained. It is concluded that in porcine airway smooth muscle, (1) relaxation induced by some NO donors may occur without a concomitant increase in [cGMP]i; and (2) whereas relaxation induced by some NO donors may be associated with increases in [cGMP]i, the relaxation is not completely dependent upon it.
Oxygen tension may be crucial in relaxation of the human umbilical artery.
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Maturation of the contractile response and its endothelial modulation in newborn porcine intrapulmonary arteries.
Pulmonary vascular reactivity is thought to be greater in the newborn than adult lung. To determine the influence of the endothelium on smooth muscle cell contractility, responses of rings of isolated intrapulmonary arteries were studied from pigs at birth aged < 2 h, 2 d, 3 d, and 10 d (n = 4 per age group) and from eight adult animals. At birth, the response to KCl (25 mM) and prostaglandin F2 alpha (PGF2 alpha) (3 microM) but not histamine (0.1 mM) was greater in rings with endothelium (E+) than without (E-). The response to PGF2 alpha decreased between birth and 3 d by which time the contraction was less in E+ rings than E-, but L-nitro monomethyl arginine augmented contraction at all ages. In the immature piglets, the response to phenylephrine was less in E+ rings than E-, an effect which was reversed by L-N omega-nitro-L-arginine methyl ester. The response to all contractile agonists increased between 10 d and adulthood. The concentration of plasma endothelin-1 was determined in all animals by RIA and was higher at birth than at 3 d or later. In summary, 1) at birth, endothelium enhanced contractility, when plasma endothelin was greatest, but released NO in the presence of phenylephrine and PGF2 alpha; 2) contractile response to all agonists was small at birth; and 3) a complex interaction existed between the contractile agonist and the effect of endothelial maturation.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenoceptor-stimulated endothelium-dependent relaxation in porcine intrapulmonary arteries.
The responses of rings of isolated adult porcine intrapulmonary arteries to noradrenaline were observed. The effects of prazosin (alpha-1 adrenoceptor antagonist), yohimbine (alpha-2 adrenoceptor agonist) and N-omega-nitro-L-arginine methyl ester (L-NAME) on the noradrenaline responses were studied. In addition, following contraction with prostaglandin PGF2 alpha, the responses to noradrenaline or UK 14304 (alpha-2 adrenoceptor agonist) were observed alone and in the presence of either prazosin or yohimbine. The effects of UK14304 were also observed following precontraction with phenylephrine. Noradrenaline produced an initial increase in vascular tone followed by a decrease and then a second increase in tone at high concentrations. The initial contractile response was inhibited by prazosin or yohimbine. L-NAME or endothelium removal enhanced the contractile responses and abolished the mid range reduction in tone. Following PGF2 alpha pre-contraction, UK14304 further increased the tone, with a reduction in tone at higher concentrations. the contractile effect was augmented by prazosin. Following phenylephrine pre-contraction, UK14304 only produced reduction in tone. In conclusion, in porcine intrapulmonary arteries, L-NAME inhibited noradrenaline induced endothelium dependent reduction in tone, which was also inhibited by alpha-1 and alpha-2 antagonists. UK14304 demonstrated partial alpha-1 agonism.
Differential effects of nitrovasodilators and nitric oxide on porcine tracheal and bronchial muscle in vitro.
Nitrovasodilators and nitric oxide relax airway smooth muscle. The mechanism by which nitrovasodilators are thought to act is by release of nitric oxide, but the importance of nitric oxide in nitrovasodilator-induced airway smooth muscle relaxation is unclear. The aim of this study was to compare the relaxing effects of nitric oxide itself with those of nitrovasodilators in porcine tracheal muscle and intrapulmonary airways and to investigate the mechanisms involved. Strips of porcine tracheal smooth muscle, rings of bronchi, and strips of bronchi from the same animal were suspended in organ chambers in modified Krebs Ringer solution (95% O2-5% CO2, 37 degrees C). Tissues were contracted with carbachol, and concentration-response curves to nitric oxide, sodium nitroprusside, and SIN-1 (an active metabolite of molsidomine) were obtained. All tissues relaxed to sodium nitroprusside, SIN-1, and nitric oxide. The relaxation to nitric oxide but not to SIN-1 or sodium nitroprusside was inhibited by methylene blue. Tissues pretreated with methylene blue that failed to relax to nitric oxide were, however, relaxed by sodium nitroprusside. These results demonstrate that nitrovasodilators relax airways by a mechanism other than by or in addition to the release of nitric oxide.
Role of Ca2+ entry in the modulation of airway tone by hypoxia.
To evaluate the cellular mechanisms involved in hypoxic relaxation of airway smooth muscle, we investigated the effects of hypoxia on the behavior of third- and fourth-order porcine bronchial rings contracted with either carbachol or KCl. In one series of experiments, hypoxia (95% N2-5% CO2) was imposed and rings were then exposed to increasing concentrations of carbachol or KCl. In separate experiments, rings were first contracted with carbachol (10(-6) M) or KCl (40 mM) and were then exposed to solutions bubbled with decreasing concentrations of O2. The CO2 concentration was maintained constant at 5% in all experiments. The initial magnitude of KCl-induced but not carbachol-induced contractions was profoundly reduced by 95% N2-5% CO2. The sensitivity of the airway to carbachol was unchanged. In rings precontracted with either carbachol or KCl, hypoxia caused similar losses of airway smooth muscle tone in a reversible and concentration-dependent manner. The effects of hypoxia were independent of the presence of an intact epithelium and were not inhibited by the cyclooxygenase inhibitor indomethacin (5 microM), the soluble guanylate cyclase inhibitor methylene blue (50 microM), or the beta-adrenoceptor antagonist propranolol (1 microM). The impairment by hypoxia of the initiation phase of KCl-induced contractions and of the maintenance phase of both KCl- and carbachol-induced contractions, but not the initiation phase of carbachol-induced contractions, suggests that changes in O2 tension modulate airway tone by altering the entry of extracellular calcium into the airway smooth muscle.
Postnatal adrenoreceptor maturation in porcine intrapulmonary arteries.
The effect of postnatal age on norepinephrine-induced alpha 2-adrenoreceptor-mediated release of endothelium-derived relaxing factor in porcine intramuscular pulmonary arteries was studied. Rings of pulmonary artery from fetal, newborn, 3-d-, 10-d-, 9-wk-, and 15-wk-old pigs with and without endothelium were suspended for isometric force measurement in Krebs-Ringer bicarbonate solution (37 degrees C, 95% O2-5% CO2). In 15- and 10-wk-old pigs, norepinephrine increased tone in arteries with and without endothelium but produced relaxations at high concentrations only in arteries with endothelium. These relaxations were not inhibited by the alpha 1-antagonist prazosin but were completely abolished by the alpha 2-antagonist yohimbine and the inhibitor of nitric oxide release N-omega-nitro-L-arginine methyl ester. This confirms that the norepinephrine-induced relaxations were due to alpha 2-mediated release of endothelium-derived relaxing factor. Arteries from only 50% of the 10-d-old animals showed endothelium-dependent relaxations, and at 3 d of age and younger no relaxations were seen. In animals less than 10 d old, only some of the vessels contracted to norepinephrine and the contractile response was diminished compared with 15-wk-old animals, whereas the response to prostaglandin F2 alpha and histamine was similar in the neonatal group (newborn, 3 d old, and 10 d old). This group showed a dose-dependent relaxation to nitric oxide, with 10-d-old animals more sensitive to nitric oxide than newborn animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Heterogeneity in epithelium-dependent responses.
The airway epithelium exerts a profound influence on the responsiveness of bronchial smooth muscle to both contracting and relaxing agents. This may be due to the release of an epithelium-derived factor or factors. There is a considerable heterogeneity in the effects of the epithelium between orders of bronchi, between species, and between pharmacologic agents. Such heterogeneity may reflect variations in the release and/or effect of the epithelium-derived relaxing factor(s). This report demonstrates that: (1) there is a basal and a stimulated release of the factor, (2) the prominence of different types of release varies between species, (3) the effect of the epithelium on relaxation of bronchial smooth muscle is greatest in the presence of high degrees of cholinergic tone, (4) the effects of the epithelium are not mediated via cyclic GMP, and (5) the epithelium-derived relaxing factor is not nitric oxide.
Epithelium, contractile tone, and responses to relaxing agonists in canine bronchi.
Experiments were conducted in canine bronchi to determine whether the effect of epithelium removal on relaxations was affected by the contractile agent used to induce active force and the degree of contraction. Pairs of fourth-order bronchi with and without epithelium were suspended in organ chambers in physiological salt solution (95% O2-5% CO2, 37 degrees C). The bronchi were contracted to the concentration of acetylcholine or 5-hydroxytryptamine that resulted in a contraction that was 40 or 80% of the response to 10(-4) M of the agonist (ED40 or ED80). Epithelium removal reduced relaxations to isoproterenol and sodium nitroprusside during contraction to the ED80 but not the ED40 of acetylcholine. Responses to forskolin were not affected. Bronchi were significantly more sensitive to relaxing agonists in the presence of 5-hydroxytryptamine; there was no effect on epithelium removal or level of contraction. Thus 1) the influence of epithelium on bronchial relaxation is greatest during high degrees of cholinergic tone and 2) experimental conditions influence the effect of epithelium removal on relaxation.
Airway epithelium modulates the responsiveness of porcine bronchial smooth muscle.
The effect of epithelium removal on the responses of porcine airways to exogenously applied agonists and nerve stimulation was examined. Paired rings of third- (segmental), fourth- and fifth-order (subsegmental) bronchi, with and without epithelium, were placed in organ chambers in physiological salt solution (95% O2-5% CO2, 37 degrees C). Removal of the epithelium caused a leftward shift in the concentration-effect curve for acetylcholine (3rd and 4th order). A similar shift occurred for histamine (3rd and 5th order). The relaxation to isoproterenol was reduced by epithelium removal in a similar fashion in the three orders. Removal of the epithelium reduced the maximal response to KCl (3rd and 4th order) and acetylcholine (5th order). The peak response to nerve stimulation showed a significant rightward shift in the absence of epithelium. In fifth-order bronchi, tissues with epithelium showed a significantly greater degree of fade of the response to sustained electrical stimulation. Thus both epithelium-derived relaxing and contracting factors may be released in porcine airways.
Arachidonic acid evokes epithelium-dependent relaxations in canine airways.
Responses to arachidonate were examined in rings with and without epithelium of lobar, segmental, and subsegmental canine bronchi. Arachidonate evoked epithelium-dependent relaxations, which were less pronounced in subsegmental bronchi and abolished by indomethacin and meclofenamate. Nordihydroguairetic acid (NDGA) and nafazatrom reduced epithelium-dependent relaxations only in lobar but unmasked epithelium-independent relaxations to arachidonate in all bronchi. Prostaglandin E2 and prostacyclin relaxed all tissues similarly. In lobar bronchi without epithelium, basal release of prostaglandin E2 was reduced by indomethacin but unaffected by NDGA. Arachidonate augmented prostaglandin E2 release more in subsegmental than in lobar bronchi with epithelium; in bronchi without epithelium the rise was absent (lobar) or attenuated (subsegmental). Arachidonate augmented the release of 6-ketoprostaglandin F1 alpha more in lobar bronchi with than without epithelium; this was inhibited by indomethacin, but not NDGA. Thus arachidonate releases prostaglandin E2 (possibly produced by cyclooxygenase inaccessible to inhibitors and activated by lipoxygenase products) but not prostacyclin from the epithelium. Heterogeneity in response to arachidonate is not due to different sensitivity to, or production of, prostaglandins.
Heterogeneity in the effects of epithelium removal in the canine bronchial tree.
The effect of removing the airway epithelium on the responses of canine airways of decreasing diameter to contractile and relaxing agonists was explored. Three orders of canine bronchus were studied: second order (lobar bronchus), third order (segmental bronchus), and fourth order (subsegmental bronchus). Paired rings of tissue, with and without epithelium, were placed in organ chambers in physiological salt solution gassed with 95% O2-5% CO2 and maintained at 37 degrees C. For second- and third-order bronchi, epithelium removal caused significant left-ward shifts of the concentration-effect curves for 5-hydroxytryptamine, histamine, and acetylcholine. In fourth-order bronchi, there was no significant shift for any of the contractile agonists. Isoproterenol (during contractions evoked by acetylcholine) induced concentration-dependent relaxations that were significantly greater in bronchi with than in those without epithelium. This effect was most prominent in fourth-order bronchi. These results suggest that 1) the canine airway epithelium releases a relaxing factor, 2) in larger airways the major effect is reduction of contractile responses, and 3) in smaller airways the major effect is enhancement of relaxing responses.
Contraction of human umbilical artery, but not vein, by oxygen.
The O2 tension of umbilical arterial blood in utero is 15 mmHg. The contractile effect of increasing the O2 tension above this value was studied quantitatively in vitro in preparations of human umbilical artery and vein. The umbilical arterial smooth muscle was contracted in a concentration-related manner by stepped increments in O2 tension. The threshold for O2-induced contraction was estimated to be 36 mmHg at pH 7.28 and the maximum contraction occurred at 297 mmHg. The sensitivity of the preparations to low O2 tensions (less than 100 mmHg) was reduced by increasing the pH of the bathing medium from 7.28 to 7.36, at which the threshold was 69 mmHg and the maximum contraction occurred at 282 mmHg. Reducing the pH to 7.18 did not significantly change the sensitivity from that found at pH 7.28. Indomethacin (0.1 microM) virtually abolished responses to O2. The results indicate that the increase in the physiological O2 tension at birth, from 15 to 100 mmHg, may be an adequate stimulus to effect the closure of the umbilical artery. In the physiological range O2 did not contract the umbilical venous smooth muscle. This may allow the transfusion of blood from the placenta to the fetus at birth when the O2 tension of umbilical cord blood increases after the onset of breathing.
Characterization of the receptor mediating contraction of human umbilical artery by 5-hydroxytryptamine.
The 5-hydroxytryptamine (5-HT) receptor in human umbilical artery was found to be similar to that in rabbit aorta. The pD2 was 7.45, pA2 for methysergide 8.63 and pA2 for phentolamine 6.21. Noradrenaline gave only very weak contractions at non-physiological concentrations. Amidephrine and xylazine did not contract human umbilical artery. It is concluded that there is no significant population of functional alpha-adrenoceptors in this vessel. The implications of these findings are discussed in relation to the control of the umbilical circulation.