Acute hypoxia-reoxygenation and vascular oxygen sensing in the chicken embryo.

Fetal/perinatal hypoxia is one of the most common causes of perinatal morbidity and mortality and is frequently accompannied by vascular dysfunction. However, the mechanisms involved have not been fully delineated. We hypothesized that exposure to acute hypoxia-reoxygenation induces alterations in vascular O2 sensing/signaling as well as in endothelial function in the chicken embryo pulmonary artery (PA), mesenteric artery (MA), femoral artery (FA), and ductus arteriosus (DA). Noninternally pipped 19-day embryos were exposed to 10% O2 for 30 min followed by reoxygenation with 21% O2 or 80% O2 Another group was constantly maintained at 21% O2 or at 21% O2 for 30 min and then exposed to 80% O2 Following treatment, responses of isolated blood vessels to hypoxia as well as endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside and forskolin) relaxation were investigated in a wire myograph. Hypoxia increased venous blood lactate from 2.03 ± 0.18 to 15.98 ± 0.73 mmol/L (P < 0.001) and reduced hatchability to 0%. However, ex vivo hypoxic contraction of PA and MA, hypoxic relaxation of FA, and normoxic contraction of DA were not significantly different in any of the experimental groups. Relaxations induced by acetylcholine, sodium nitroprusside, and forskolin in PA, MA, FA, and DA rings were also similar in the four groups. In conclusion, exposure to acute hypoxia-reoxygenation did not affect vascular oxygen sensing or reactivity in the chicken embryo. This suggests that direct effects of acute hypoxia-reoxygenation on vascular function does not play a role in the pathophysiology of hypoxic cardiovascular injury in the perinatal period.

Neutral sphingomyelinase, NADPH oxidase and reactive oxygen species. Role in acute hypoxic pulmonary vasoconstriction.

The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium-denuded PA segments and their inhibition prevented hypoxia-induced voltage-gated potassium channel (K(V) ) inhibition and pulmonary vasoconstriction. Consistently, H(2) O(2) , or its analogue t-BHP, decreased K(V) currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production, K(V) channel inhibition and vasoconstriction. Hypoxia induced p47(phox) phosphorylation and its interaction with caveolin-1. Inhibition of nSMase (GW4869) or PKCζ prevented p47(phox) phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase-PKCζ-NADPH oxidase as a necessary step required for ROS production and vasoconstriction.

Hypoxia sensing in the fetal chicken femoral artery is mediated by the mitochondrial electron transport chain.

Vascular hypoxia sensing is transduced into vasoconstriction in the pulmonary circulation, whereas systemic arteries dilate. Mitochondrial electron transport chain (mETC), reactive O(2) species (ROS), and K(+) channels have been implicated in the sensing/signaling mechanisms of hypoxic relaxation in mammalian systemic arteries. We aimed to investigate their putative roles in hypoxia-induced relaxation in fetal chicken (19 days of incubation) femoral arteries mounted in a wire myograph. Acute hypoxia (Po(2) approximately 2.5 kPa) relaxed the contraction induced by norepinephrine (1 microM). Hypoxia-induced relaxation was abolished or significantly reduced by the mETC inhibitors rotenone (complex I), myxothiazol and antimycin A (complex III), and NaN(3) (complex IV). The complex II inhibitor 3-nitroproprionic acid enhanced the hypoxic relaxation. In contrast, the relaxations mediated by acetylcholine, sodium nitroprusside, or forskolin were not affected by the mETC blockers. Hypoxia induced a slight increase in ROS production (as measured by 2,7-dichlorofluorescein-fluorescence), but hypoxia-induced relaxation was not affected by scavenging of superoxide (polyethylene glycol-superoxide dismutase) or H(2)O(2) (polyethylene glycol-catalase) or by NADPH-oxidase inhibition (apocynin). Also, the K(+) channel inhibitors tetraethylammonium (nonselective), diphenyl phosphine oxide-1 (voltage-gated K(+) channel 1.5), glibenclamide (ATP-sensitive K(+) channel), iberiotoxin (large-conductance Ca(2+)-activated K(+) channel), and BaCl(2) (inward-rectifying K(+) channel), as well as ouabain (Na(+)-K(+)-ATPase inhibitor) did not affect hypoxia-induced relaxation. The relaxation was enhanced in the presence of the voltage-gated K(+) channel blocker 4-aminopyridine. In conclusion, our experiments suggest that the mETC plays a critical role in O(2) sensing in fetal chicken femoral arteries. In contrast, hypoxia-induced relaxation appears not to be mediated by ROS or K(+) channels.

Response of chicken ductus arteriosus to hypercarbic and normocarbic acidosis.

BACKGROUND: Changes in pH can have profound effects on vascular tone and reactivity, but their influence on the ductus arteriosus (DA) remains unknown. OBJECTIVE: To analyzethe effects of hypercarbic and normocarbic acidosis in the reactivity of the chicken DA. METHODS: DA rings from 19-day chicken fetuses (total incubation time, 21 days) were mounted in a wire myograph for isometric tension recording. RESULTS: In DA rings (pulmonary side) stimulated with O(2), norepinephrine (NE), KCl, or U46619, changes from control conditions (5% CO(2), 24 mM NaHCO(3), pH 7.4) to 7.5% CO(2) (pH 7.25) or 10% CO(2) (pH 7.14) induced a concentration-dependent relaxation that reached 43.0% (SD 21.3) of the O(2)-, 28.6% (SD 23.1) of the NE-, 10.4% (SD 18.7) of the KCl-, and 6.8% (SD 12.6) of the U46619-induced contraction. Hypercarbic-acidosis-induced relaxation was impaired by the non-selective K(+) channel blocker tetraethylammonium or the BK(Ca) channel inhibitor iberiotoxin. Normocarbic acidosis (5% CO(2), 12 mM NaHCO(3), pH 7.13) induced transient relaxation of the DA, which was not affected by the presence of tetraethylammonium or iberiotoxin. Euhydric hypercarbia (10% CO(2), 48 mM NaHCO(3), pH 7.46) induced a transient contraction of the DA. CONCLUSIONS: Our results indicate that the chicken DA is very sensitive to changes in extracellular pH, and that stimulation of BK(Ca) channels may account for the ductal-relaxing effects of hypercarbic acidosis.

Maturation of O2 sensing and signaling in the chicken ductus arteriosus.

The increase in O(2) tension after birth is a major factor stimulating ductus arteriosus (DA) constriction and closure. Here we studied the role of the mitochondrial electron transport chain (ETC) as sensor, H(2)O(2) as mediator, and voltage-gated potassium (K(V)) channels and Rho kinase as effectors of O(2)-induced contraction in the chicken DA during fetal development. Switching from 0% to 21% O(2) contracted the pulmonary side of the mature DA (mature pDA) but had no effect in immature pDA and relaxed the aortic side of the mature DA (mature aDA). This contraction of the pDA was attenuated by inhibitors of the mitochondrial ETC and by the H(2)O(2) scavenger polyethylene glycol (PEG)-catalase. Moreover, O(2) increased reactive oxygen species (ROS) production, measured with the fluorescent probes dihydroethidium and 2',7'-dichlorofluorescein, only in mature pDA. The H(2)O(2) analog t-butyl-hydroperoxide mimicked the responses to O(2) in the three vessels. In contrast to immature pDA cells, mature pDA cells exhibited high-amplitude O(2)-sensitive potassium currents. The K(V) channel blocker 4-aminopyridine prevented the current inhibition elicited by O(2). The L-type Ca(2+) (Ca(L)) channel blocker nifedipine and the Rho kinase inhibitors Y-27632 and hydroxyfasudil induced a similar relaxation when mature pDA were stimulated with O(2) or H(2)O(2). Moreover, the sensitivity to these drugs increased with maturation. Our results indicate the presence of a common mechanism for O(2) sensing/signaling in mammalian and nonmammalian DA and favor the idea that, rather than a single mechanism, a parallel maturation of the sensor and effectors is critical for O(2) sensitivity appearance during development.

Activation of BKCa channels by nitric oxide prevents coronary artery endothelial dysfunction in ouabain-induced hypertensive rats.

OBJECTIVE: Chronic-ouabain administration to rats induces hypertension and increases the endothelial modulation of vasoconstrictor responses. The aim of this study was to analyze whether ouabain-treatment affects the mechanisms involved in endothelium-dependent relaxation of coronary arteries. METHODS: Coronary arteries from control and ouabain-treated rats (approximately 8.0 microg/day, 5 weeks) were used. Vascular reactivity was analyzed by isometric tension recording and membrane currents were measured using the whole-cell configuration of the patch-clamp technique. RESULTS: In 5-hydroxytryptamine (5-HT) precontracted arteries, acetylcholine (ACh, 1 nmol/l-10 micromol/l) induced a similar relaxant response in coronary arteries from both groups that was abolished by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (100 micromol/l). However, when arteries were contracted with high KCl (60 mmol/l) or preincubated with the large-conductance Ca2+-activated K+ (BKCa) channels-blocker iberiotoxin (0.1 micromol/l), the relaxation elicited by ACh was more reduced in ouabain-treated than control rats. After iberiotoxin preincubation, the relaxant response of the nitric-oxide donor, DEA-NO (10 nmol/l-100 micromol/l) was significantly inhibited in ouabain-treated coronary arteries but not in control vessels. The soluble guanylyl cyclase activator BAY 41-2272 (10 nmol/l-30 micromol/l) induced relaxant responses that were inhibited by iberiotoxin. In coronary-artery myocytes isolated from ouabain-treated rats DEA-NO (1 micromol/l) markedly increased the amplitude of the iberiotoxin-sensitive current in the whole range of test potentials, compared with nontreated rats. CONCLUSION: Our results indicate that chronic ouabain treatment increases activation of BKCa currents by nitric oxide and this effect might contribute to preserve the endothelial function in coronary arteries in this hypertension model.

Developmental changes in the effects of prostaglandin E2 in the chicken ductus arteriosus.

Prostaglandin E(2) (PGE(2)) 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 PGE(2) and other vascular smooth muscle relaxing agents acting through the cyclic AMP signaling pathway. PGE(2) exhibited a relaxant response in the 15-day DA, but not in the 19- and 21-day DA. Moreover, high concentrations of PGE(2) (>or= 3 microM in 15-day and >or= 1 microM 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 PGE(2) in the 19- and 21-day DA and increased the relaxation induced by PGE(2) in the 15-day DA. The presence of the EP receptor antagonist AH6809 abolished PGE(2)-mediated relaxation. The relaxant responses induced by PGE(2) and the beta-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 PGE(2). 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 PGE(2). We conclude that, in contrast to the mammalian situation, PGE(2) is a weak relaxant agent of the chicken DA and, with advancing incubation, it even stimulates TP vasoconstrictive receptors.

Ontogeny of chicken ductus arteriosus response to oxygen and vasoconstrictors.

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(+) 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(omega)-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 ET(B)-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(+) channels glibenclamide, and the inhibitor of Ca(2+)-activated K(+) 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.

Postnatal maturation of phosphodiesterase 5 (PDE5) in piglet pulmonary arteries: activity, expression, effects of PDE5 inhibitors, and role of the nitric oxide/cyclic GMP pathway.

After birth and during the first days of extrauterine life, pulmonary arterial pressure is progressively reduced to reach the adult values. We hypothesized that changes in PDE5 activity might be involved in the pulmonary postnatal maturation of the nitric oxide (NO)/cGMP pathway. The PDE5 inhibitor sildenafil produced vasorelaxant responses in isolated pulmonary arteries. These effects were similar in newborn (3-18 h) and 2-wk-old piglets, unchanged by endothelium removal, and markedly inhibited by the soluble guanylyl cyclase inhibitor ODQ. The peak of the transient vasorelaxant response to NO gas increased with postnatal age but was unaffected by PDE inhibition. However, the duration of the response to NO was significantly increased. The vasorelaxant response to sodium nitroprusside was potentiated by sildenafil in both age groups. The PDE5 inhibitors dipyridamole and zaprinast, produced qualitatively similar effects but with lower potency. Both total and PDE5-dependent cGMP hydrolytic activity and PDE5 protein expression increased with postnatal age. All these results suggest that PDE5 is a key regulator of NO-induced vasodilation in the postnatal pulmonary arteries. PDE5 inhibition is able to produce pulmonary vasodilation even in the absence of a functional endothelium and potentiates the vasorelaxant response to exogenous NO and nitroprusside. However, PDE5 is not responsible for the maturational increase of NO bioactivity during the first days of extrauterine life.

Cytosolic Ca2+ and phosphoinositide hydrolysis linked to constitutively active alpha 1D-adrenoceptors in vascular smooth muscle.

In the present study, we analyzed changes in intracellular Ca2+ levels and inositol phosphate accumulation related to a population of alpha 1d-adrenoceptors in rat aorta resembling constitutively active receptors. Following intracellular Ca2+ store depletion by noradrenaline in Ca2+-free medium and removal of the agonist, restoration of extracellular Ca2+ induced four signals: a biphasic (transient and sustained) increase in [Ca2+]i, inositol phosphate accumulation, and a contractile response in the aorta. The transient increase in Ca2+, the inositol phosphate accumulation, and the contractile response were not observed in aortae incubated with prazosin or BMY 7378 [8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione] (a selective alpha 1d-adrenoceptor ligand), relating the three signals to alpha 1d-adrenoceptor activity. In the presence of nimodipine, only the sustained increase in Ca2+ and the inositol phosphate accumulation were observed, relating both signals to calcium entry through L-channels. The four signals were abolished by Ni2+. In the rat tail artery, where alpha 1d-adrenoceptors are not functionally active, restoration of extracellular Ca2+ after store depletion induced only a sustained increase in [Ca2+]i without inositol phosphate accumulation nor contractile response. Taken together these results suggest that in the aorta, Ca2+ entry is required for the recovery of cytosolic calcium levels and the display of the membrane signals related to the constitutive activity of alpha 1d-adrenoceptors, i.e., inositol phosphate formation and Ca2+ entry through L-type channels, which maintains a contractile response once the agonist has been removed.

Endothelium-independent vasodilator effects of the flavonoid quercetin and its methylated metabolites in rat conductance and resistance arteries.

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 beta-escin-permeabilized iliac arteries, and on perfusion pressure in the isolated mesenteric resistance vascular bed. In noradrenaline-precontracted 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 (-log IC(50) = 5.35 +/- 0.15, 5.89 +/- 0.11, 5.34 +/- 0.10, and 5.66 +/- 0.06, respectively) than in the thoracic aorta (-log IC(50) = 4.68 +/- 0.08, 4.61 +/- 0.08, 4.73 +/- 0.11, and 4.81 +/- 0.13, respectively; n = 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(-6) M), the adenylate cyclase inhibitor SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine; 10(-6) M], KCl (40 mM), or ouabain (10(-3) M) had no effect on isorhamnetin-induced vasodilation in the mesenteric bed. In permeabilized iliac arteries stimulated with Ca(2+) (pCa of 5.9), isorhamnetin was also significantly more potent (-log IC(50) = 5.27 +/- 0.15) than quercetin (-log IC(50) = 4.56 +/- 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(2+).