The flavonoid quercetin is metabolized into isorhamnetin, tamarixetin, and kaempferol, the vascular effects of which are unknown. In the present study, the effects of quercetin and its metabolites were analyzed on isometric tension in isolated rat thoracic and abdominal aorta, in isolated intact and ␤-escinpermeabilized iliac arteries, and on perfusion pressure in the isolated mesenteric resistance vascular bed. In noradrenalineprecontracted vessels, the four flavonoids produced a vasodilator effect, which was inversely correlated with the diameter of the vessel studied; i.e., quercetin, isorhamnetin, tamarixetin, and kaempferol were 5-, 25-, 4-, and 6-fold, respectively, more potent in the resistance mesenteric bed (Ϫlog IC 50 ϭ 5.35 Ϯ 0.15, 5.89 Ϯ 0.11, 5.34 Ϯ 0.10, and 5.66 Ϯ 0.06, respectively) than in the thoracic aorta (Ϫlog IC 50 ϭ 4.68 Ϯ 0.08, 4.61 Ϯ 0.08, 4.73 Ϯ 0.11, and 4.81 Ϯ 0.13, respectively; n ϭ 4 -6). The vasodilator responses of quercetin and isorhamnetin were not significantly modified after removal of the endothelium in the thoracic aorta or in the mesenteric bed. Furthermore, the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 10 Ϫ6 M), the adenylate cyclase inhibitor SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine; 10Ϫ6 M], KCl (40 mM), or ouabain (10 Ϫ3 M) had no effect on isorhamnetin-induced vasodilation in the mesenteric bed. In permeabilized iliac arteries stimulated with Ca 2ϩ (pCa of 5.9), isorhamnetin was also significantly more potent (Ϫlog IC 50 ϭ 5.27 Ϯ 0.15) than quercetin (Ϫlog IC 50 ϭ 4.56 Ϯ 0.15). In conclusion, quercetin and its metabolites showed vasodilator effects with selectivity toward the resistance vessels. These effects are not due to or modulated by endothelial factors and are unrelated to changes in cytosolic Ca 2ϩ .Flavonoids comprise a large group of secondary metabolites widely distributed throughout the plant kingdom, including food plants. The daily flavonoid intake in the human diet (mainly from onions, apples, grapes, wine, tea, berries, herbs, and spices) is highly variable, with estimations ranging from 23 mg (flavonols plus flavones; Hertog et al., 1993b) to more than 500 mg (total flavonoids;

AimsThe mechanisms involved in hypoxic pulmonary vasoconstriction (HPV) are not yet fully defined. The aim of the study was to determine the role of protein kinase C ζ (PKCζ) and neutral sphingomyelinase (nSMase) in HPV.Methods and resultsCeramide content was measured by immunocytochemistry and voltage-gated potassium channel (KV) currents were recorded by the patch clamp technique in isolated rat pulmonary artery smooth muscle cells (PASMC). Contractile responses were analysed in rat pulmonary arteries mounted in a wire myograph. Pulmonary pressure was recorded in anesthetized open-chest rats. Protein and mRNA expression were measured by western blot and RT–PCR, respectively. We found that hypoxia increased ceramide content in PASMC which was abrogated by inhibition of nSMase, but not acid sphingomyelinase (aSMase). The hypoxia-induced vasoconstrictor response in isolated pulmonary arteries and the inhibition of KV currents were strongly reduced by inhibition of PKCζ or nSMase but not aSMase. The nSMase inhibitor GW4869 prevented HPV in vivo. The vasoconstrictor response to hypoxia was mimicked by exogenous addition of bacterial Smase and ceramide. nSMase2 mRNA expression was ∼10-fold higher in pulmonary compared with mesenteric arteries. In mesenteric arteries, hypoxia failed to increase ceramide but exogenous SMase induced a contractile response.ConclusionnSMase-derived ceramide production and the activation of PKCζ are early and necessary events in the signalling cascade of acute HPV.

The present study aimed to characterize the contractile reactivity of the chicken ductus arteriosus (DA) from the last stage of prenatal development and throughout the perinatal period. Isolated DA rings from 15-day, noninternally-pipped 19-day, and externally-pipped 21-day embryos were studied using myograph techniques. On embryonic day 15, the chicken DA did not respond to O 2 (0 to 21%), norepinephrine (NE), or phenylephrine (Phe) but contracted in response to high-K ϩ solution, the inhibitor of voltage-gated channels 4-aminopyridine, U-46619, and endothelin (ET)-1. These responses increased with advancing incubation age. Contractile responses to O 2, NE, and Phe were present in the 19-and 21-day embryo. Oxygen-induced contraction was restricted to the pulmonary side of the DA and was augmented by the nitric oxide synthase inhibitor N -nitro-L-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and reduced by the peptidic ET A and ETB-receptor antagonist PD-142,893. Transmural electrical stimulation of nerves, the nonselective cyclooxygenase (COX) inhibitor indomethacin, the COX-1 inhibitor valeryl salicylate, the COX-2 inhibitor nimesulide, the inhibitor of ATP-sensitive K ϩ channels glibenclamide, and the inhibitor of Ca 2ϩ -activated K ϩ channels tetraethylammonium did not cause contraction of the DA rings at any age. We conclude that transition to ex ovo life is accompanied by dramatic changes in chicken DA reactivity. At 0.7 incubation, excitation-contraction and pharmacomechanical coupling for several contractile agonists are already present, whereas the constrictor effects of O 2 and cathecolamines appear later in development and are located in the pulmonary side of the DA. ductus arteriosus; chicken embryo; potassium channels; oxygen sensing; cathecolamines.THE DUCTUS ARTERIOSUS (DA) is a vessel that connects the pulmonary artery to the aorta and provides, during the fetal life, a pulmonary-to-systemic diversion that shunts more than half of the right ventricle output away from the nonventilated lungs into the systemic circulation (43, 47). The main factors maintaining patency of the DA in utero are low O 2 tension, high levels of circulating PGE 2 , and locally produced PGE 2 and PGI 2 (24, 43). Failure of DA closure after birth is a common complication of premature delivery that is still presenting challenges in terms of diagnosis, assessment, and treatment options (43).Although the isolated DA is sensitive to a wide range of contractile agonists, the major factor actively stimulating contraction at birth is increasing O 2 tension, which has a profound effect on the DA, both directly and by modulating its response to vasodilators and vasoconstrictors (43). To constrict properly after birth, the DA prepares itself for this specific task from a quite early onset during development (3). This preparation is reflected by changes in responsiveness with advancing gestational age. These have been extensively characterized in numerous mammalian specie...

< 0.001. Two-tailed Student's t-test (last normoxia time versus hypoxia times in KO + pNCLX; d, e): & P < 0.05, && P < 0.01. In f, h, statistical comparisons are shown only for normoxia versus 0-10 groups. AU, arbitrary units; NS, not significant.

Abstract-Multiple lines of evidence indicate that serotonin (5-hydroxytryptamine ) and voltage-gated K ϩ (K V ) channels play a central role in the pathogenesis of pulmonary hypertension (PH). We hypothesized that 5-HT might modulate the activity of K V channels, therefore establishing a link between these pathogenetic factors in PH. Here, we studied the effects of 5-HT on K V channels present in rat pulmonary artery smooth muscle cells (PASMC) and on hK V 1.5 channels stably expressed in Ltk Ϫ cells. 5-HT reduced native K V and hK V 1.5 currents, depolarized cell membrane, and caused a contraction of isolated pulmonary arteries. The effects of 5-HT on K V currents and contraction were markedly prevented by the 5-HT 2A receptor antagonist ketanserin. Incubation with inhibitors of phospholipase C (U73122), classic protein kinase Cs (Gö6976), or tyrosine kinases (genistein and tyrphostin 23), the cholesterol depletion agent ␤-cyclodextrin or concanavalin A, an inhibitor of endocytotic processes, also prevented the effects of 5-HT. In homogenates from pulmonary arteries, 5-HT 2A receptors and caveolin-1 coimmunoprecipitated with K V 1.5 channels, and this was increased on stimulation with 5-HT. Moreover, K V 1.5 channels were internalized when cells were stimulated with 5-HT, and this was prevented by concanavalin A. These findings indicate that activation of 5-HT 2A receptors inhibits native K V and hK V 1.5 currents via phospholipase C, protein kinase C, tyrosine kinase, and a caveolae pathway. K V channel inhibition accounts, at least partly, for 5-HT-induced pulmonary vasoconstriction and might play a role in PH. Key Words: potassium ion channels hypertension Ⅲ pulmonary arteries Ⅲ receptors P ulmonary hypertension (PH) is a heterogeneous group of disorders characterized by a sustained increase in pulmonary artery (PA) pressure leading to progressive right ventricular failure and death. Several lines of evidence indicate that serotonin (5-hydroxytryptamine ) plays a central role in the pathogenesis of PH. 1,2 First, 5-HT is an effective pulmonary vasoconstrictor and induces vascular smooth muscle hyperplasia. [2][3][4][5] Moreover, plasma levels of 5-HT are increased in patients with primary PH. 6 Conversely, mild pulmonary hypertension has been reported in some series of patients with carcinoid syndrome, a tumor of enterochromaffin cells releasing large amounts of 5-HT. 7 Patients treated with fenfluramine or dexfenfluramine, anorectic drugs that induce platelet 5-HT release, inhibit the 5-HT transporter (5-HTT) and stimulate 5-HT receptors, also have a 23-fold increased risk of PH. 8 In addition, 5-HTT overexpression or polymorphisms in the gene encoding the 5-HTT are associated with PH. 9,10 Furthermore, mice lacking 5-HTT or 5-HT receptors (eg, 5-HT 1B or 5-HT 2B ) show attenuated PH induced by hypoxia. 3,11,12 (K V ) channels present in pulmonary artery smooth muscle cells (PASMC) are inhibited by hypoxia, endothelin-1, thromboxane A 2 , and anorectic drugs. 16,19 -21 Moreover, decreased expression or fun...

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Recent data suggest that diabetes is a risk factor for pulmonary hypertension. The aim of the present study was to analyze whether diabetes induces endothelial dysfunction in pulmonary arteries and the mechanisms involved. Male Sprague-Dawley rats were randomly divided into a control (saline) and a diabetic group (70 mg/kg(-1) streptozotocin). After 6 wk, intrapulmonary arteries were mounted for isometric tension recording, and endothelial function was tested by the relaxant response to acetylcholine. Protein expression and localization were measured by Western blot and immunohistochemistry and superoxide production by dihydroethidium staining. Pulmonary arteries from diabetic rats showed impaired relaxant response to acetylcholine and reduced vasoconstrictor response to the nitric oxide (NO) synthase inhibitor L-NAME, whereas the response to nitroprusside and the expression of endothelial NO synthase remained unchanged. Endothelial dysfunction was reversed by addition of superoxide dismutase or the NADPH oxidase inhibitor apocynin. An increase in superoxide production and increased expression of the NADPH oxidase regulatory subunit p47(phox) were also found in pulmonary arteries from diabetic rats. In conclusion, the pulmonary circulation is a target for diabetes-induced endothelial dysfunction via enhanced NADPH oxidase-derived superoxide production.