Vasoactivity of the gasotransmitters hydrogen sulfide and carbon monoxide in the chicken ductus arteriosus

Besides nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide (H(2)S) is a third gaseous messenger that may play a role in controlling vascular tone and has been proposed to serve as an O(2) sensor. However, whether H(2)S is vasoactive in the ductus arteriosus (DA) has not yet been studied. We investigated, using wire myography, the mechanical responses induced by Na(2)S (1 μM-1 mM), which forms H(2)S and HS(-) in solution, and by authentic CO (0.1 μM-0.1 mM) in DA rings from 19-day chicken embryos. Na(2)S elicited a 100% relaxation (pD(2) 4.02) of 21% O(2)-contracted and a 50.3% relaxation of 62.5 mM KCl-contracted DA rings. Na(2)S-induced relaxation was not affected by presence of the NO synthase inhibitor l-NAME, the soluble guanylate cyclase (sGC) inhibitor ODQ, or the K(+) channel inhibitors tetraethylammonium (TEA; nonselective), 4-aminopyridine (4-AP, K(V)), glibenclamide (K(ATP)), iberiotoxin (BK(Ca)), TRAM-34 (IK(Ca)), and apamin (SK(Ca)). CO also relaxed O(2)-contracted (60.8% relaxation) and KCl-contracted (18.6% relaxation) DA rings. CO-induced relaxation was impaired by ODQ, TEA, and 4-AP (but not by L-NAME, glibenclamide, iberiotoxin, TRAM-34 or apamin), suggesting the involvement of sGC and K(V) channel stimulation. The presence of inhibitors of H(2)S or CO synthesis as well as the H(2)S precursor L-cysteine or the CO precursor hemin did not significantly affect the response of the DA to changes in O(2) tension. Endothelium-dependent and -independent relaxations were also unaffected. In conclusion, our results indicate that the gasotransmitters H(2)S and CO are vasoactive in the chicken DA but they do not suggest an important role for endogenous H(2)S or CO in the control of chicken ductal reactivity.     

Mechanisms mediating the oxygen-induced vasoreactivity of the ductus arteriosus in the chicken embryo

The avian embryo provides a novel model for studying the ductus arteriosus (DA) during the transition from in ovo to ex ovo life. Here we examined the mechanisms regulating the vasoreactivity of the two morphologically distinct portions of the chicken DA (proximal and distal) in response to O(2). Oxygen-induced contraction is redox sensitive and reversed by the reducing agent dithiothreitol and the H(2)O(2) scavenger N-mercaptopropionylglycine. As in the mammalian DA, inhibiting mitochondrion-derived reactive oxygen species production with rotenone and antimycin A relaxed the O(2)-constricted DA. The contractile response to O(2) matures during hatching and is mimicked by the K(v) channel inhibitor 4-aminopyridine (4-AP) on day 19 and externally pipped (EP) embryos. Together, O(2) and 4-AP significantly increase DA tone above that observed with either alone. The O(2)-induced contraction is mediated by influx of extracellular Ca(2+) through l-type Ca(2+) and store-operated channels. Inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores play a minor role in the O(2)-induced contraction. The O(2)-induced contraction is mediated by the Rho kinase pathway, as fasudil and Y-27632 significantly relax the O(2) contracted DA. Prostaglandins E(2), F(2alpha), and D(2) produce significant contraction of the proximal DA. The O(2)-induced relaxation of the distal portion of the DA is mediated by an endothelial-derived nitric oxide/cGMP pathway. Both 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and endothelial cell removal inhibit O(2)-induced relaxation in the distal segment. Mechanisms regulating O(2)-induced contraction in chicken proximal DA are similar to those found in mammalian DA, making the chicken a useful model for studying development of this O(2)-sensitive vessel.     

Developmental changes in the effects of prostaglandin E<Subscript>2</Subscript> in the chicken ductus arteriosus

Prostaglandin E₂ (PGE₂) is the major vasodilator prostanoid of the mammalian ductus arteriosus (DA). In the present study we analyzed the response of isolated DA rings from 15-, 19- and 21-day-old chicken embryos to PGE₂ and other vascular smooth muscle relaxing agents acting through the cyclic AMP signaling pathway. PGE₂ exhibited a relaxant response in the 15-day DA, but not in the 19- and 21-day DA. Moreover, high concentrations of PGE₂ (≥3 μM in 15-day and ≥1 μM in 19-day and 21-day DA) induced contraction of the chicken DA. The presence of the TP receptor antagonist SQ29,548, unmasked a relaxant effect of PGE₂ in the 19- and 21-day DA and increased the relaxation induced by PGE₂ in the 15-day DA. The presence of the EP receptor antagonist AH6809 abolished PGE₂-mediated relaxation. The relaxant responses induced by PGE₂ and the β-adrenoceptor agonist isoproterenol, but not those elicited by the adenylate cyclase activator forskolin or the phosphodiesterase 3 inhibitor milrinone, decreased with maturation. High oxygen concentrations (95%) decreased the relaxation to PGE₂. The relaxing potency and efficacy of isoproterenol and milrinone were higher in the pulmonary than in the aortic side of the DA, whereas no regional differences were found in the response to PGE₂. We conclude that, in contrast to the mammalian situation, PGE₂ is a weak relaxant agent of the chicken DA and, with advancing incubation, it even stimulates TP vasoconstrictive receptors.     

Further investigation of a potential calcium channel modulator isolated from rat erythrocytes

The contractile effects of a peptide isolated from rat erythrocytes were further studied in rat aortic rings. Previous data showed that preincubation of aortic tissue with the peptide had no effect on resting tension, but significantly enhanced K+ and norepinephrine (NE) induced contraction. The calcium channel antagonist verapamil noncompetitively blocked the effect of the peptide, whereas nifedipine blockage appeared to be competitive. In the present study the peptide enhanced K+, NE, and phenylephrine (PE) induced contraction in a concentration-dependent manner, with a maximum enhancement at peptide concentrations of 10(-7)-10(-6) M. At a concentration as low as 10(-9) M, the peptide significantly enhanced K(+)-induced, but not NE- or PE-induced, contraction. The magnitude of maximal enhancement was greater for K(+)-induced contraction than that for NE- or PE-induced contraction. Preincubation of the tissues with the peptide caused a leftward shift of cumulative concentration-response curves to K+ and NE. The peptide enhancement of contraction increased with increasing K+ and NE concentration. The peptide potentiated the contractile response to Ca2+ in K(+)-depolarizing medium. It also enhanced the contractile response to NE in intracellular Ca2(+)-pool-depleted tissue following the replenishment of extracellular Ca2+, but had no apparent effect on the mobilization of intracellular calcium. Addition of nifedipine caused a rightward shift of both the peptide and Bay K 8644 concentration-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delayed rectifier potassium channels contribute to the depressed pulmonary artery contractility in pneumonia.

We investigated the role of K(+) channels in the attenuated pulmonary artery (PA) contractility characteristic of acute Pseudomonas pneumonia. Contractility of PA rings from the lungs of control or pneumonia rats was assessed in vitro by obtaining cumulative concentration-response curves to the contractile agonists KCl, phenylephrine, or PGF(2 alpha) on PA rings before and after treatment with K(+) channel blockers. In rings from pneumonia rats, paxilline (10 microM), tetraethylammonium (2 mM) (blockers of large-conductance Ca(2+)-activated K(+) channels), and glybenclamide (ATP-sensitive K(+) channel blocker, 80 microM) had no significant effect on the attenuated contractile responses to KCl, phenylephrine, and PGF(2 alpha). However, 4-aminopyridine (2 mM), a blocker of voltage-gated K(+) channels (delayed rectifier K(+) channel) reversed this depressed contractility. Therefore, large-conductance Ca(2+)-activated K(+) and ATP-sensitive K(+) channels do not contribute to the attenuated PA contractility observed in this model of acute pneumonia. In contrast, 4-aminopyridine enhances contraction in PA rings from pneumonia lungs, consistent with involvement of a voltage-gated K(+) channel in the depressed PA contractility in acute pneumonia. Unraveling the precise mechanism of attenuated contractility in pneumonia could lead to innovative therapies for the pulmonary vascular abnormalities associated with this disease.     

Effects of econazole on receptor-operated and depolarization-induced contractions in rat isolated aorta

In our previous study, econazole caused a decrease in serum nitrite levels in septic mice in vivo, but it enhanced the mortality rate. The aim of the study was to investigate the in vitro effects of econazole on receptor-operated and depolarization-induced contractions on endothelium-intact and -denuded rat isolated aorta. Econazole (0.1, 1 and 10 microM) significantly inhibited receptor-operated (phenylephrine, Phe) and depolarization (KCl)-induced contractions of endothelium-intact or -denuded rings in a noncompetitive and concentration-dependent manner. Removal of endothelium changed the pD'2 values only for KCl-induced responses. The pD'2 values of L-type calcium channel blocker nifedipine were significantly higher than the econazole on Phe concentration-response curves in endothelium-intact and -denuded rings. Econazole caused a biphasic response in precontracted by Phe or KCl in endothelium-intact and -denuded rings, first a transient contraction following sustained relaxation. Removal of endothelium did not affect the contractile responses induced by Phe. The contractile responses induced by 10 microM econazole in the KCl-precontracted rings were antagonized by the treatment of alpha-adrenergic receptor antagonist, phentolamine (10 microM). Deendothelization was significantly increased the IC50 values of econazole obtained from Phe- and KCl-precontractions. The relaxations induced by 10 microM econazole in endothelium-intact rings precontracted with Phe or KCl were not changed by NO synthase inhibitor, L-N(G)-nitroarginine (100 microM). The IC50 values of econazole were significantly higher than nifedipine in endothelium-intact and -denuded rings. These results suggest that econazole is a noncompetitive antagonist on alpha1-adrenoceptor-mediated and depolarization-induced contractions in rat isolated aorta by inhibiting Ca2+ entry through L-type calcium channels, and the endothelium seems to modulate vascular responses induced by this agent. The vascular effects of econazole may limit the usage of this agent in septic shock.     

Chronic in ovo hypoxia decreases pulmonary arterial contractile reactivity and induces biventricular cardiac enlargement in the chicken embryo

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic (15% O(2)) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 +/- 0.5 vs. 26.6 +/- 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane A(2) mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [pD(2) (= -log EC(50)): 6.51 +/- 0.1 vs. 7.05 +/- 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy.     

Maturation of the contractile response of the Emu ductus arteriosus

The avian embryo has a pair of ductus arteriosi that allow the blood to bypass the pulmonary circulation prior to the initiation of lung ventilation. Our objective was to characterize the factors regulating DA tone during the later stages of development in the emu embryo. We examined in vitro the reactivity of the emu ductus from day 39 through 49 of a 50-day incubation. Steady state tension was not altered by the COX inhibitor indomethacin or the nitric oxide synthase inhibitor l-NAME. However, prostaglandin E₂ (PGE₂) produced a significant relaxation. Norephinephrine and U-46619 produced strong significant contractions in the emu DA and the adrenergic response matured with development. The contractile response to oxygen matured as the embryo developed with significant oxygen-induced contraction on days 45 and 49, but not on day 39 of incubation. The Kv channel inhibitor 4-aminopyridine induced the contraction of the day 48–49 ductus of similar magnitude as the oxygen-induced contraction. The oxygen-induced contraction was reversed by the reducing agent DTT and the electron transport chain inhibitor rotenone. These results suggest that while the emu DA responds to PGE₂, locally produced PGE₂ are not the important regulators of vessel tone. Additionally, relaxation upon addition of the mitochondria electron transport chain inhibitor rotenone suggests that the mitochondria might be acting as vascular oxygen sensors in this system through the production of reactive oxygen species to stimulate the oxygen-induced contraction in a similar fashion to mammals.     

Effects of chronic endothelin-1 stimulation on cardiac myocyte contractile function

Endothelin-1 (ET-1) has acute positive inotropic effects, but consequences of chronically increased ET-1 on contractile function of cardiac myocytes are largely unknown. In the present study, effects of long-term treatment with ET-1 (10 nM) for 5 days on both force development [force of contraction (FOC)] and kinetics of contraction were determined in heart tissue reconstituted from rat cardiac cells. Isometric force was measured in response to cumulative concentrations of Ca(2+) and isoprenaline. ET-1 augmented basal FOC by 64 +/- 11% (P < 0.05), which was associated with a significantly blunted contractile response to Ca(2+) and isoprenaline. Moreover, ET-1 significantly prolonged relaxation (62 +/- 3 vs. 53 +/- 2 ms). Selective ET(A) (BQ-123) and ET(B) receptor blockade (BQ-788) demonstrated that effects of ET-1 on contractile function were mediated through the ET(A) receptor subtype. Effects of ET-1 were prevented by cotreatment with either Ro31-8425, a PKC inhibitor, or dimethylamiloride, an inhibitor of the Na(+)/H(+) exchanger. In contrast to long-term ET-1 treatment, no changes in contractile parameters were observed after ET-1 treatment for 3 h before force measurement. These data suggest that chronic ET-1 stimulation has dual effects on contractility: improvement of basal force but impairment of twitch kinetics and inotropic responsiveness to beta-adrenoceptor stimulation. The signaling pathways involved include ET(A) receptors, PKC, and the Na(+)/H(+) exchanger. The present in vitro findings raise the possibility that ET-1 may exert both adaptive and maladaptive effects in the failing myocardium in which local accumulation of ET-1 is present.     

Direct effects of acute hypoxia on the reactivity of peripheral arteries of the chicken embryo

In the chicken embryo, acute hypoxemia results in cardiovascular responses, including an increased peripheral resistance. We investigated whether local direct effects of decreased oxygen tension might participate in the arterial response to hypoxemia in the chicken embryo. Femoral arteries of chicken embryos were isolated at 0.9 of incubation time, and the effects of acute hypoxia on contraction and relaxation were determined in vitro. While hypoxia reduced contraction induced by high K(+) to a small extent (-21.8 +/- 5.7%), contractile responses to exogenous norepinephrine (NE) were markedly reduced (-51.1 +/- 3.2%) in 80% of the arterial segments. This effect of hypoxia was not altered by removal of the endothelium, inhibition of NO synthase or cyclooxygenase, or by depolarization plus Ca(2+) channel blockade. When arteries were simultaneously exposed to NE and ACh, hypoxia resulted in contraction (+49.8 +/- 9.3%). Also, relaxing responses to ACh were abolished during acute hypoxia, while the vessels became more sensitive to the relaxing effect of the NO donor sodium nitroprusside (pD(2): 5.81 +/- 0.21 vs. 5.31 +/- 0.27). Thus, in chicken embryo femoral arteries, acute hypoxia blunts agonist-induced contraction of the smooth muscle and inhibits stimulated endothelium-derived relaxation factor release. The consequences of this for in vivo fetal hemodynamics during acute hypoxemia depend on the balance between vasomotor influences of circulating catecholamines and those of the endothelium.     

Interaction between endothelial heme oxygenase-2 and endothelin-1 in altered aortic reactivity after hypoxia in rats

The aim of this study was to determine whether increased expression of heme oxygenase (HO) contributes to impairment of aortic contractile responses after hypoxia through effects on reactivity to endothelin-1 (ET-1). Thoracic aortas from normoxic rats and rats exposed to hypoxia (10% O2) for 16 or 48 h were mounted in organ bath myographs for contractile studies, fixed in paraformaldehyde, or frozen in liquid nitrogen for protein extraction. In rings from normoxic rats, the HO inhibitor tin protoporphyrin IX (SnPP IX, 10 microM) did not alter the response to phenylephrine or ET-1. In rings from rats exposed to 16-h hypoxia, maximum tension generated in response to these agonists was higher in endothelium-intact but not -denuded rings in the presence of SnPP IX. In rings from rats exposed to 48-h hypoxia SnPP IX increased contraction in endothelium-intact but not -denuded rings. In endothelium-intact aortic rings from rats exposed to 16-h hypoxia incubated with endothelin A receptor-specific antagonist BQ-123 (10(-7) M), SnPP IX did not alter phenylephrine-induced contraction. Aortic ET-1 protein levels, measured by radioimmunoassay, were increased in rats exposed to hypoxia for 16 and 48 h. Western blotting showed that HO-1 and HO-2 protein were increased after 16 h of hypoxia and returned to near-control levels after 48 h. Increase in HO-1 protein was detected in endothelium-intact and -denuded rings. Removal of endothelium abolished the increase in HO-2 immunoreactivity. Immunohistochemistry localized expression of HO-1 protein to vascular smooth muscle, whereas HO-2 was only detected in endothelium. HO-2 is expressed by aortic endothelial cells early during hypoxic exposure and impairs ET-1-mediated potentiation of contraction to alpha-adrenoceptor stimulation.     

Effect of interleukin-6 (IL-6) on the vascular smooth muscle contraction in abdominal aorta of rats with streptozotocin-induced diabetes

Patients with type 1 diabetes are at a risk of hypertension. However, the mechanisms behind the findings are not completely known. The aim of the present study was to investigate involvement of interleukin-6 (IL-6) on the contraction of abdominal aorta in rats with type 1 diabetes. IL-6 levels in the plasma of rats with streptozotocin (STZ)-induced diabetes were determined by ELISA. The abdominal aorta was dissected free of fat and connective tissues and then cut into spiral rings. The endothelium-denuded strip was vertically suspended in tissue chambers containing 5 ml Krebs solution at 37 degrees C and bubbled continuously with 95% O2-5% CO2. The effects of phenylephrine (Phe) on the contractile responses of abdominal aorta were recorded. The effects of IL-6 and anti-rat IL-6 antibody on the Phe-induced response were also examined. Plasma levels of IL-6 increased time-dependently in rats with STZ-induced diabetes. Phe caused concentration-dependent contraction in aortic rings. Phe-induced contractions were higher in vascular strips of STZ-induced diabetic rats than that of control rats. Pretreatment of vascular strips with IL-6 for 1 h did not cause contraction but enhanced the contraction in response to Phe. Treatment of the vascular strips with an anti-IL-6 antibody for 1 h decreased the Phe-induced contractions. These results suggest that IL-6 causes vascular smooth muscle contraction in abdominal aorta of rats with type 1 diabetes.     

Vascular hyporesponsiveness in simulated microgravity: role of nitric oxide-dependent mechanisms.

Simulated microgravity depresses the ability of arteries to constrict to norepinephrine (NE). In the present study the role of nitric oxide-dependent mechanisms on the vascular hyporesponsiveness to NE was investigated in peripheral arteries of the rat after 20 days of hindlimb unweighting (HU). Blood vessels from control rats and rats subjected to HU (HU rats) were cut into 3-mm rings and mounted in tissue baths for the measurement of isometric contraction. Mechanical removal of the endothelium from carotid artery rings, but not from aorta or femoral artery rings, of HU rats restored the contractile response to NE toward control. A 10-fold increase in sensitivity to ACh was observed in phenylephrine-precontracted carotid artery rings from HU rats. In the presence of the nitric oxide synthase (NOS) substrate L-arginine, the inducible NOS inhibitor aminoguanidine (AG) restored the contractile responses to NE to control levels in the femoral, but not carotid, artery rings from HU rats. In vivo blood pressure measurements revealed that the peak blood pressure increase to NE was significantly greater in the control than in the HU rats, but that to AG was less than one-half in control compared with HU rats. These results indicate that the endothelial vasodilator mechanisms may be upregulated in the carotid artery, whereas the inducible NOS expression/activity may be increased in the femoral artery from HU rats. These HU-mediated changes could produce a sustained elevation of vascular nitric oxide levels that, in turn, could contribute to the vascular hyporesponsiveness to NE.     

Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels

This study examines the roles of voltage-dependent Ca(2+) channels (VDCC), ryanodine receptors (RyRs), large-conductance Ca(2+)-activated K(+) (BK) channels, and small-conductance Ca(2+)-activated K(+) (SK) channels in the regulation of phasic contractions of guinea pig urinary bladder smooth muscle (UBSM). Nisoldipine (100 nM), a dihydropyridine inhibitor of VDCC, abolished spontaneous UBSM contractions. Ryanodine (10 microM) increased contraction frequency and thereby integrated force and, in the presence of the SK blocker apamin, had a greater effect on integrated force than ryanodine alone. Blocking BK (iberiotoxin, 100 nM) or SK (apamin, 100 nM) channels increased contraction amplitude and duration but decreased frequency. The contractile response to iberiotoxin was more pronounced than to apamin. The increases in contraction amplitude and duration to apamin were substantially augmented with ryanodine pretreatment. These results indicate that BK and SK channels have prominent roles as negative feedback elements to limit UBSM contraction amplitude and duration. RyRs also appear to play a significant role as a negative feedback regulator of contraction frequency and duration, and this role is influenced by the activity of SK channels.     

库容性Ca2+内流参与ACh诱导的大鼠远端结肠平滑肌收缩

应用生物换能技术和Ca^2＋通道特异性阻断剂观察并记录大鼠离体远端结肠平滑肌收缩张力的变化,分析库容性Ca^2＋内流（capacitative Ca^2＋ entry,CCE）是否与ACh诱导的离体远端结肠平滑肌收缩反应有关。结果表明,以无钙的Krebs液灌流或应用EGTA螯合细胞外Ca^2＋后,高K^＋及ACh引起的远端结肠平滑肌收缩几乎完全消失。电压操纵性Ca^2＋通道阻断剂verapamil也能减弱高K^＋及ACh引起的远端结肠平滑肌收缩,其减弱的程度分别为74％和41％。在无钙的Krebs液中,5μmol／LACh可引起离体肠管瞬时性收缩,这是由肌质网（sarcoplasmic reticulum,SR）释放钙所致：然后加入10μmol／L阿托品（atropine）,并在此基础上恢复细胞外Ca^2＋（2．5mmol／L）,结肠平滑肌则出现持续性收缩,待收缩反应达峰值时,加入5μmol／L verapamil,收缩无明显变化,且该收缩反应对钙库操纵性通道（store-operated Ca^2＋ channel,socc）阻断剂La^3＋敏感,20,50和100μmol／L的La^3＋使上述收缩张力分别降低15％,23％和36％,且呈浓度依赖性,但对Cd^2＋不敏感。研究结果提示,细胞外Ca^2＋内流对高K^＋及ACh介导的离体远端结肠平滑肌持续性收缩是必需的,由ACh诱导的远端结肠平滑肌收缩至少包括SR释放钙引起的短暂性收缩及受体操纵性Ca^2＋通道（receptor-operated Ca^2＋ channel,ROCC）、电压操纵性Ca^2＋通道（voltage-operated Ca^2＋ channel,VOCC）和CCE介导的胞外Ca^2＋ 内流等途径。这将从通道水平进一步分析消化管平滑肌收缩的机制和特征,亦将为预防和控制因胃肠动力紊乱所致的消化管疾病寻求有针对性的药物干预和治疗提供理论依据。     

Endothelin-1-induced contraction in veins is independent of hydrogen peroxide

Reactive oxygen species (ROS), such as superoxide and H(2)O(2), are capable of modifying vascular tone, although the response to ROS can vary qualitatively among vascular beds, experimental procedures, and species. Endothelin-1 (ET-1) induces superoxide production, which can be dismutated to H(2)O(2). The RhoA/Rho kinase pathway partially mediates ET-1-induced contraction and recently was implicated in superoxide-induced contraction. We hypothesized that H(2)O(2), not superoxide, mediates venous ET-1-induced contraction. Rat thoracic aorta and vena cava contracted to exogenously added H(2)O(2) (1 microM-1 mM) [maximum aortic contraction = 10 +/- 3% of phenylephrine (10 microM) contraction; maximum venous contraction = 85 +/- 13% of norepinephrine (10 microM) contraction]. (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632, 10 microM), a Rho kinase inhibitor, significantly reduced venous H(2)O(2)-induced contraction (15 +/- 1% of control maximum) and reduced maximum ET-1-induced contraction by 59 +/- 1%. However, neither the H(2)O(2) scavenger catalase (100 and 2,000 U/ml) nor cell permeable polyethylene glycol-catalase (163 and 326 U/ml) reduced ET-1-induced contraction in the vena cava. The catalase inhibitor 3-aminotriazole (3-AT) also had no effect on maximal venous ET-1-induced contraction. Basal H(2)O(2) levels were three times higher in the vena cava than in the aorta (vena cava, 0.74 +/- 0.09 nmol H(2)O(2)/mg protein; aorta, 0.24 +/- 0.05 nmol H(2)O(2)/mg protein). ET-1 (100 nM) increased H(2)O(2) in the vena cava but not in the aorta (vena cava, 154.10 +/- 17.29% of control H(2)O(2); aorta, 83.72 +/- 20.20%). Antagonism of either ET(A) or ET(B) receptors with the use of atrasentan (30 nM) or BQ-788 (100 nM), respectively, reduced ET-1 (100 nM)-induced increases in venous H(2)O(2). In summary, ET-1 increased H(2)O(2) in veins but not arteries, and venous ET-1-induced H(2)O(2) production was independent of the contractile properties of ET-1.     

H2O2-induced redox-sensitive coronary vasodilation is mediated by 4-aminopyridine-sensitive K+ channels

Hydrogen peroxide (H(2)O(2)) is a proposed endothelium-derived hyperpolarizing factor and metabolic vasodilator of the coronary circulation, but its mechanisms of action on vascular smooth muscle remain unclear. Voltage-dependent K(+) (K(V)) channels sensitive to 4-aminopyridine (4-AP) contain redox-sensitive thiol groups and may mediate coronary vasodilation to H(2)O(2). This hypothesis was tested by studying the effect of H(2)O(2) on coronary blood flow, isometric tension of arteries, and arteriolar diameter in the presence of K(+) channel antagonists. Infusing H(2)O(2) into the left anterior descending artery of anesthetized dogs increased coronary blood flow in a dose-dependent manner. H(2)O(2) relaxed left circumflex rings contracted with 1 muM U46619, a thromboxane A(2) mimetic, and dilated coronary arterioles pressurized to 60 cmH(2)O. Denuding the endothelium of coronary arteries and arterioles did not affect the ability of H(2)O(2) to cause vasodilation, suggesting a direct smooth muscle mechanism. Arterial and arteriolar relaxation by H(2)O(2) was reversed by 1 mM dithiothreitol, a thiol reductant. H(2)O(2)-induced relaxation was abolished in rings contracted with 60 mM K(+) and by 10 mM tetraethylammonium, a nonselective inhibitor of K(+) channels, and 3 mM 4-AP. Dilation of arterioles by H(2)O(2) was antagonized by 0.3 mM 4-AP but not 100 nM iberiotoxin, an inhibitor of Ca(2+)-activated K(+) channels. H(2)O(2)-induced increases in coronary blood flow were abolished by 3 mM 4-AP. Our data indicate H(2)O(2) increases coronary blood flow by acting directly on vascular smooth muscle. Furthermore, we suggest 4-AP-sensitive K(+) channels, or regulating proteins, serve as redox-sensitive elements controlling coronary blood flow.     

Endogenous prostaglandins regulate spontaneous contractile activity of uterine strips isolated from non-pregnant pigs

Myometrial strips isolated from non-pregnant pigs show spontaneous contractile activity. In the present study, the involvement of endogenous prostaglandins in regulation of uterine spontaneous contraction was investigated using mechanical, immunohistochemical and biochemical approaches. Immunohistochemical study and Western blot analysis for immunoreactive cyclooxygenase (COX) indicated that COX-1 but not COX-2 was expressed predominantly in the myometrium of non-pregnant pigs in a muscle layer-dependent manner (longitudinal muscle>circular muscle). Pretreatment of uterine strips with indomethacin and selective COX-1 inhibitors (SC-560 and FR122047) significantly reduced both the amplitude and frequency of spontaneous contraction in the longitudinal muscle, but inhibition by COX inhibitors was negligible in the circular muscle. On the other hand, CAY10404, a COX-2 inhibitor, did not change the spontaneous contraction in either of the muscle layers. Pretreatment with SC-560 reduced myometrial PGF(2alpha) and PGE(2) levels. Contractile FP and EP(3) receptors were expressed in a muscle layer-dependent manner (longitudinal muscle>circular muscle), similar to the expression pattern of COX-1. In conclusion, endogenous prostaglandins produced by COX-1 regulate spontaneous contractile activity of non-pregnant porcine uterine longitudinal muscle selectively due to the heterogeneous expression of contractile prostanoid receptors and COX-1.     

Homocyst(e)ine impairs endocardial endothelial function

Homocyst(e)ine injured vascular endothelium and modulated endothelial-dependent vascular function. Endothelium plays an analogous role in both the vessel and the endocardium. Therefore, we hypothesized that homocyst(e)ine modulated endocardial endothelium (EE) dependent cardiac function. The ex vivo cardiac rings from normal male Wistar-Kyoto rats were prepared. The contractile responses of left and right ventricular rings were measured in an isometric myobath, using different concentrations of CaCl2. The response was higher in the left ventricle than right ventricle and was elevated in endocardium without endothelium. The half effective concentration (EC50) and maximum tension generated by homocyst(e)ine were 10(6) and 5-fold lower than endothelin (ET) and angiotensin II (AII), respectively. However, in endothelial-denuded endocardium, homocyst(e)ine response was significantly increased (p<0.005, compared with intact endothelium) and equal to the response to ET and AII. To determine the physiological significance of ET, AII, homocyst(e)ine, and endothelial nitric oxide in EE function, cardiac rings were pretreated with AII (10(-10) M) or ET (10(-13) M) and then treated with homocyst(e)ine (10(-8) M). Results suggested that at these concentrations AII, ET, or homocyst(e)ine alone had no effect on cardiac contraction. However, in the presence of 10(-10) M AII or 10(-13) M ET, the cardiac contraction to homocyst(e)ine (10(-8) M) was significantly enhanced (p<0.01, compared with without pretreatment) and further increased in the endocardium without endothelium. The pretreatment of cardiac ring with the inhibitor of nitric oxide, Nomega-nitro-L-arginine methyl ester (L-NAME), increased contractile response to homocyst(e)ine. These results suggested that homocyst(e)ine impaired EE-dependent cardiac function and acted synergistically with AII and ET in enhancing the cardiac contraction.     

Interleukin-1 impairs both vascular contraction and relaxation in rabbit isolated aorta.

Incubation of rabbit aortic rings with interleukin-1 (100 U/ml) in vitro led to a depressed contractile response to norepinephrine, whether the endothelium was present or not. In both cases norepinephrine-induced contraction was restored in the presence of NG-methyl-L-arginine (300 microM), an inhibitor of nitric oxide synthesis. In interleukin-1-treated rings precontracted with norepinephrine (1 microM), the relaxing response to acetylcholine was totally suppressed independently on the presence of endothelium. High concentrations of acetylcholine (greater than 1 microM) induced a slight contraction which was of lower amplitude than that obtained in control endothelium-denuded rings and was increased in the presence of NG-methyl-L-arginine. These results show that interleukin-1 (i) affects not only vascular contraction but also relaxation and (ii) involves both endothelial and non-endothelial factors. These observations suggest an impairment of the whole vascular reactivity during septic shock.