Contraction Of Pulmonary Arteries Research Articles

Effect of chronic perinatal hypoxia on the role of rho-kinase in pulmonary artery contraction in newborn lambs

Exposure to chronic hypoxia during gestation predisposes infants to neonatal pulmonary hypertension, but the underlying mechanisms remain unclear. Here, we test the hypothesis that moderate continuous hypoxia during gestation causes changes in the rho-kinase pathway that persist in the newborn period, altering vessel tone and responsiveness. Lambs kept at 3,801 m above sea level during gestation and the first 2 wk of life were compared with those with gestation at low altitude. In vitro studies of isolated pulmonary arterial rings found a more forceful contraction in response to KCl and 5-HT in high-altitude compared with low-altitude lambs. There was no difference between the effects of blockers of various pathways of extracellular Ca(2+) entry in low- and high-altitude arteries. In contrast, inhibition of rho-kinase resulted in significantly greater attenuation of 5-HT constriction in high-altitude compared with low-altitude arteries. High-altitude lambs had higher baseline pulmonary artery pressures and greater elevations in pulmonary artery pressure during 15 min of acute hypoxia compared with low-altitude lambs. Despite evidence for an increased role for rho-kinase in high-altitude arteries, in vivo studies found no significant difference between the effects of rho-kinase inhibition on hypoxic pulmonary vasoconstriction in intact high-altitude and low-altitude lambs. We conclude that chronic hypoxia in utero results in increased vasopressor response to both acute hypoxia and serotonin, but that rho-kinase is involved only in the increased response to serotonin.

EP3 receptor-mediated contraction of human pulmonary arteries and inhibition of neurogenic tachycardia in pithed rats

BackgroundThe aim of our study was (1) the pharmacological characterization of EP3 receptors in human pulmonary arteries and (2) the examination of the potential involvement of these receptors in the regulation of neurogenic tachycardia in pithed rats. L-826266 served as the EP3 receptor antagonist. MethodsExperiments were performed on isolated human pulmonary arteries and pithed rats. ResultsThe prostanoid EP1/EP3 receptor agonist sulprostone (1nM – 100μM) concentration-dependently contracted isolated human pulmonary arteries (pEC50, 6.88±0.10). The EP1 receptor antagonist SC 19920 (100μM) did not affect the vasoconstriction induced by sulprostone, the TP receptor antagonist sulotroban (10μM) only slightly attenuated the effects elicited by sulprostone >3μM, whereas L-826266 (10μM) shifted its concentration-response curve to the right (apparent pA2 value 6.18; incubation time 0.5h). In rings exposed to L-826266 (0.1,1 or 10μM) for 3h, a concentration-dependent inhibitory effect against the sulprostone-induced vasoconstriction was obtained, yielding a Schild plot-based pA2 value of 7.39. In pithed rats, sulprostone (10 – 1,000nmol/kg), but not the IP/EP1 receptor agonist iloprost (1-100nmol/kg), inhibited the electrically evoked increase in heart rate (HR) dosedependently, maximally by at least 80%. L-826266 (3μmol/kg) did not affect basal HR and diastolic blood pressure, but reduced the inhibitory effect of sulprostone 1,000nmol/kg by about 20%. ConclusionEP3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibers supplying the heart of pithed rats strongly inhibit the neurogenic tachycardia.

Peroxisome Proliferator-Activated Receptor-γ Ameliorates Pulmonary Arterial Hypertension by Inhibiting 5-Hydroxytryptamine 2B Receptor

An elevated plasma level of 5-hydroxytryptamine (5-HT) or upregulation of 5-HT receptor signaling or both is implicated in vascular contraction and remodeling in pulmonary arterial hypertension (PAH). Recently, peroxisome proliferator-activated receptor-γ (PPARγ) agonists have been shown to ameliorate PAH. However, their effects on the 5-HT-induced contraction of pulmonary arteries remain unknown. Here, we examined the role of PPARγ in inhibiting 5-HT2B receptor (5-HT2BR) to ameliorate PAH. Pulmonary arteries from PAH rats induced by monocrotaline or chronic hypoxia showed an enhanced vasoconstriction in response to BW723C86, a specific agonist for 5-HT2BR. Expression of 5-HT2BR was also increased in pulmonary arteries from the PAH rats, accompanied by vascular remodeling and right ventricular hypertrophy. Treatment with the PPARγ agonist rosiglitazone in vivo reversed the expression and the vasocontractive effect of 5-HT2BR as well as the thickening of pulmonary arteries. In pulmonary artery smooth muscle cells, 5-HT induced the gene expression of 5-HT2BR, which was inhibited by rosiglitazone, pioglitazone, or adenovirus-mediated overexpression of constitutively activated PPARγ. The pharmacological effect of PPARγ was through the suppression of the 5-HT-induced activator protein-1 activity. These results demonstrated the beneficial effect of PPARγ on 5-HT2BR-mediated vasocontraction, providing a new mechanism for the potential use of PPARγ agonists in PAH.

Maternal high-altitude hypoxia and suppression of ryanodine receptor-mediated Ca2+ sparks in fetal sheep pulmonary arterial myocytes

Ca(2+) sparks are fundamental Ca(2+) signaling events arising from ryanodine receptor (RyR) activation, events that relate to contractile and dilatory events in the pulmonary vasculature. Recent studies demonstrate that long-term hypoxia (LTH) can affect pulmonary arterial reactivity in fetal, newborn, and adult animals. Because RyRs are important to pulmonary vascular reactivity and reactivity changes with ontogeny and LTH we tested the hypothesis that RyR-generated Ca(2+) signals are more active before birth and that LTH suppresses these responses. We examined these hypotheses by performing confocal imaging of myocytes in living arteries and by performing wire myography studies. Pulmonary arteries (PA) were isolated from fetal, newborn, or adult sheep that lived at low altitude or from those that were acclimatized to 3,801 m for > 100 days. Confocal imaging demonstrated preservation of the distance between the sarcoplasmic reticulum, nucleus, and plasma membrane in PA myocytes. Maturation increased global Ca(2+) waves and Ca(2+) spark activity, with sparks becoming larger, wider, and slower. LTH preferentially depressed Ca(2+) spark activity in immature pulmonary arterial myocytes, and these sparks were smaller, wider, and slower. LTH also suppressed caffeine-elicited contraction in fetal PA but augmented contraction in the newborn and adult. The influence of both ontogeny and LTH on RyR-dependent cell excitability shed new light on the therapeutic potential of these channels for the treatment of pulmonary vascular disease in newborns as well as adults.

Differential Effects of Selexipag and Prostacyclin Analogs in Rat Pulmonary Artery

{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid (ACT-333679) is the main metabolite of the selective prostacyclin (PGI(2)) receptor (IP receptor) agonist selexipag. The goal of this study was to determine the influence of IP receptor selectivity on the vasorelaxant efficacy of ACT-333679 and the PGI(2) analog treprostinil in pulmonary artery under conditions associated with pulmonary arterial hypertension (PAH). Selexipag and ACT-333679 evoked full relaxation of pulmonary artery from control and monocrotaline (MCT)-PAH rats, and ACT-333679 relaxed normal pulmonary artery contracted with either endothelin-1 (ET-1) or phenylephrine. In contrast, treprostinil evoked weaker relaxation than ACT-333679 of control pulmonary artery and failed to induce relaxation of pulmonary artery from MCT-PAH rats. Treprostinil did not evoke relaxation of normal pulmonary artery contracted with either ET-1 or phenylephrine. Expression of prostaglandin E(3) (EP(3)) receptor mRNA was increased in pulmonary artery from MCT-PAH rats. In contraction experiments, the selective EP(3) receptor agonist sulprostone evoked significantly greater contraction of pulmonary artery from MCT-PAH rats compared with control rats. The presence of a threshold concentration of ET-1 significantly augmented the contractile response to sulprostone in normal pulmonary artery. ACT-333679 did not evoke direct contraction of rat pulmonary artery, whereas treprostinil evoked concentration-dependent contraction that was inhibited by the EP(3) receptor antagonist (2E)-3-(3',4'-dichlorobiphenyl-2-yl)-N-(2-thienylsulfonyl)acrylamide. Antagonism of EP(3) receptors also revealed a relaxant response to treprostinil in normal pulmonary artery contracted with ET-1. These data demonstrate that the relaxant efficacy of the selective IP receptor agonist selexipag and its metabolite ACT-333679 is not modified under conditions associated with PAH, whereas relaxation to treprostinil may be limited in the presence of mediators of disease.

Hydrogen saline is protective for acute lung ischaemia/reperfusion injuries in rats

Protective effects of saturated hydrogen (H(2)) saline on cardiac ischaemia-reperfusion (I/R) injury have been demonstrated previously. This study was designed to show that hydrogen-rich saline is protective in preventing lung I/R injury in rats. Adult male Sprague-Dawley rats underwent 45 min occlusion of the right lung roots and 120 min reperfusion. Rats were divided randomly into three groups: sham-operated control group, I/R plus saline treatment, and I/R plus hydrogen-rich saline treatment (0.6 mmol/L, 0.5 ml/kg/d). Three days of intraperitoneal injection of hydrogen-rich saline before the reperfusion combined with immediate administration of hydrogen-rich saline after the reperfusion were performed. Following reperfusion, the lung tissue and the pulmonary artery was immediately obtained and the W/D ratio, pulmonary artery contraction and relaxation ability, H-E staining, TUNEL staining, caspase-3, MDA, 8-OHdG content and measurement of such biomarkers as WBC, CRP were measured or carried out. Hydrogen saline significantly protected vasoactivity of the pulmonary artery, reduced pulmonary oedema, decreased lung malondialdehyde (MDA), 8-OHdG concentration, alleviated lung epithelial cell apoptosis and lowered the level of such biomarkers as WBC, CRP, ALT and TBiL. It is concluded that hydrogen-rich saline is a novel, simple, safe and effective method to attenuate pulmonary I/R injury.

MAChR Dependent Contraction of Pulmonary Arteries with Functional Endothelium from Chronically Hypoxic Fetal and Adult Sheep

Muscarinic acetylcholine receptor (mAChR)‐dependent arterial relaxation is often used to test for a functional endothelium, where inability to relax is indicative of endothelial damage. The present studies test the hypothesis that mAChR activation can contract pulmonary arteries (PA) with intact endothelium from chronically hypoxic sheep. The hypothesis was tested in PA isolated from term‐fetal sheep, ~10 days old newborns, and in adult ewes that lived at 3,200 meters for <100 days, which provides a chronic hypoxic insult. Dose‐dependent vasoreactivity to carbachol, a mAChR agonist and bradykinin (BK) were examined in PA pre‐constricted with 10 μM phenylephrine (PE). Carbachol caused robust dose‐dependent contraction of PA from adult sheep, fetal PA contracted somewhat, and PA from newborns relaxed. Bradykinin (1 μM) relaxed carbachol treated PA, indicating the endothelium was functional. Bradykinin caused dose‐dependent relaxation of PE‐constricted newborn and adult arteries to a similar extent, while fetal arteries relaxed somewhat. Carbachol (100 μM) contracted BK‐relaxed arteries from fetal and adult, but not newborn sheep. mAChR‐dependent contraction may therefore provide a novel mechanism to match ventilation to perfusion in the lung. Targeted therapies could increase lung perfusion and improve oxygenation in patients with pulmonary vascular disease. NIH P01HD031226, R01HD003807 (LDL)

Maternal Hypoxemia Suppresses Muscarinic Acetylcholine Receptor Dependent Contraction of Pulmonary Arteries from Fetal Sheep

Muscarinic acetylcholine (ACh) receptor (mAChR) activation relaxes arteries through endothelium‐dependent signaling pathways, yet ACh can contract pulmonary arteries (PA). The impact of development and chronic hypoxia (CH) on PA contraction and intimacy between endothelium and myocyte function led to the hypothesis that chronic hypoxia (CH) reduces mAChR‐dependent PA contractility. This was tested by performing wire‐myography on endothelium‐denuded PA rings from term‐fetal or adult sheep that lived at low altitude or 3,200 meters for <100 days. The roles for the three mAChR isoforms were assessed using the mAChR agonist carbachol and various antagonists, including non‐selective (atropine), or M1 (Pirenzepine), M2 (AFDX‐116), and M3 (Dau5884, 4‐DAMP) selective blockers. Carbachol responses as a function of arterial diameter were also examined. Carbachol contracted very few vessels from fetal CH sheep. However, in fetal normoxic and adult CH sheep, smaller arteries contracted more than larger ones, and in adult normoxic medium arteries contracted the most. The M2 receptor predominated in fetal normoxic while M3 was most important in adult normoxic and CH arteries. These results suggest M2 mAChR reactivity contributes to high pulmonary vascular resistance in the fetus and loss of contractility in the CH fetus may influence pulmonary vascular disease of the newborn. NIH P01HD031226, R01HD003807 (LDL).

Enhanced 5‐HT‐induced contraction of the rat pulmonary artery in hyperthyroidism involved activation of calcium‐activated chloride channels

Experimentally‐induced hyperthyroidism in rodents is associated with pulmonary hypertension. The main objective of this study was to investigate reactivity of the pulmonary artery to 5‐HT and the possible underlying signaling pathway in artery segments from thyroxine‐treated rats. 5‐HT concentration‐dependently contracted artery segments from control and thyroxine‐treated rats. The maximum response was significantly greater in artery segments from thyroxine‐treated rats but there was no change in pD2 values (5.04±0.19 and 5.34±0.14, in control and thyroxine treated rats, respectively). BW 723C86, a 5‐HT2B‐receptor agonist and CP 93129, a selective 5‐HT1B‐receptor agonist had no effect on pulmonary artery segments from both groups. There was no significant difference in the level of expression of 5‐HT2A‐receptor protein between the two groups. Ketanserin antagonized 5‐HT‐induced contractions in both groups with equal potency. Nifedipine (10−6M) inhibited 5‐HT‐induced contractions in artery segments from control and thyroxine‐treated rats and was more effective against 5‐HT‐induced contraction in artery segments for thyroxine‐treated rats. The calcium‐activated chloride channel blocker, niflumic acid (10−4M) also inhibited 5‐HT‐induced contractions in artery segments from control and thyroxine‐treated rats and was more effective against 5‐HT‐induced contraction in artery segments for thyroxine‐treated rats. It was concluded that hyperthyroidism enhanced 5‐HT‐induced contractions of the rat pulmonary artery via activation of calcium‐activated chloride channels.

Mechanism of hydrogen sulfide mediated contraction in rat small pulmonary arteries

The gas H2S (sulfide) causes a biphasic contraction of unknown mechanism in pulmonary arteries (PA). We assessed this mechanism in rings of rat 2nd order PA using myography to measure tension and NaHS as a source of sulfide. Reactive oxygen species (ROS) levels and the mitochondrial membrane potential (Δψ) were recorded using the indicators LO12 and TMRE, respectively. NaHS (500μM) evoked a small contraction and then a much larger 2nd contraction which gradually relaxed. The 2nd contraction was strongly reduced by 10μg/ml antimycin (AM) and 50μM dantrolene but was unaffected by 1μM rotenone (ROT). 10 or 30μM sulfide caused an immediate transient rise in [ROS]; at 100–1000μM this was followed by a 2nd larger rise in ROS which fell to baseline within ~10 min. The biphasic increases in tension and ROS had similar timecourses. The rise in [ROS] was slightly blocked by 1μM ROT but was almost abolished by 10μg/ml AM. 500 μM sulphide caused a triphasic effect on Δψ: a rapid very transient hyperpolarization was followed by a depolarization, a 2nd slower hyperpolarization, and then a return to baseline. Sulfide oxidation by the mitochondrial flavoprotein sulfide‐quinone oxoreductase (SQR), which is expressed in PA, has been shown in some cells to generate electrons that are passed to complex 3. We propose that metabolism of sulfide by SQR gives rise to a complex 3‐mediated increase in ROS that causes a contraction by activating the RyR.

Cellular mechanisms and intracellular signaling pathways for the modulation of eNOS in pulmonary arteries by 15-HETE

The 15-hydroxyeicosatetraenoic acid (15-HETE), a lipid metabolite and vasoconstrictor, plays an important role in hypoxic contraction of pulmonary arteries (PAs) through working on smooth muscle cells (SMCs). Previous studies have shown that vascular endothelium is also involved in PAs tone regulation. However, little is known as to how the pulmonary artery endothelial cells (PAECs) are related to the 15-HETE-induced vasoconstriction and that which intracellular signaling systems are critical. To test this hypothesis, we examined PAs constriction in isolated rat PAs rings, the expression and activity of endothelial nitric oxide synthase (eNOS) with western blot, and nitric oxide (NO) production using the DAF-FM DA fluorescent indicator. The results showed that the 15-HETE-induced PAs constriction was diminished in endothelium-intact rings. In the presence of the eNOS inhibitor L-NAME, vasoconstrictor responses to KCl were greater than the control. The activation of eNOS was activated by Ca2+ released from intracellular stores and the PI3K/Akt pathway. Phosphorylations of the eNOS at Ser-1177 and Akt at Ser-473 were necessary for their activity. A prolonged 15-HETE treatment (30 min) led to a decrease in NO production by phosphorylation of eNOS at Thr-495, leading to augmentation of PAs constriction. Therefore, 15-HETE initially inhibited the PAs constriction through the endothelial NO system, and both Ca2+ and the PI3K/Akt signaling systems are required for the effects of 15-HETE on PAs tone regulation.

Alpha2C-adrenoceptors play a prominent role in sympathetic constriction of porcine pulmonary arteries

Enhanced pulmonary vasoconstriction in response to injuries of the central nervous system and hypoxia result in pulmonary edema due to increased sympathetic activation. This study aimed to characterize α(2)-adrenoceptor (AR)-mediated responses in porcine pulmonary arteries. α(2)-AR-mediated vasoconstriction was studied using a tissue bath protocol. α(2)-AR protein was determined by Western blotting. UK14304 (α(2)-AR agonist) elicited only a slight contraction in pulmonary arteries compared to veins. Verapamil (voltage-operated Ca(2+) channel blocker), 2-APB (store-operated Ca(2+) channel inhibitor), and P1075 (K(ATP) channel opener) induced a marked decrease of the basal tone in veins, but not in arteries. The UK14304-induced contraction in arteries was enhanced by (S)-(-)-Bay K 8644 (L-type Ca(2+) channel activator), N (ω)-nitro-L: -arginine methyl ester hydrochloride (L-NAME, eNOS inhibitor), and (S)-(-)-Bay K 8644 plus L-NAME to the same extent. Endothelium denudation failed to affect the UK14304 response. (S)-(-)-Bay K 8644 did not increase the maximal noradrenaline (non-selective α-AR agonist) or phenylephrine (α(1)-AR agonist) response. The rightward shift of the concentration-response curve to noradrenaline by prazosin (α(1)-AR antagonist) plus (S)-(-)-Bay K 8644 was smaller and non-parallel compared to that in the presence of prazosin alone. UK14304 responses were inhibited by MK912 (α(2C)-AR antagonist). Affinity of MK912 (pA(2) 9.76) and Western blotting analysis argue for an involvement of α(2C)-ARs in noradrenaline-induced contraction of pulmonary arteries. It is concluded that postjunctional α(2C)-ARs predominantly mediate contraction in porcine pulmonary arteries when the cytosolic Ca(2+) concentration is elevated. α(2C)-AR antagonists may be beneficial in the treatment of pulmonary edema.

Ambrisentan and Tadalafil Synergistically Relax Endothelin-Induced Contraction of Rat Pulmonary Arteries

Endothelin receptor antagonists and phosphodiesterase type 5 inhibitors are used to treat pulmonary arterial hypertension. We tested the hypothesis that a selective endothelin type A receptor antagonist (ambrisentan) and a phosphodiesterase type 5 inhibitor (tadalafil) may act synergistically to relax endothelin-constricted pulmonary arteries. Rat isolated intrapulmonary arterial rings contracted with 8 nmol/L endothelin-1 were relaxed by 10 nmol/L ambrisentan and 30 nmol/L tadalafil alone by 26±3% and 21±1%, respectively, whereas both drugs in combination acted synergistically to relax arterial rings by 83±6%. The nonselective endothelin type A and B receptor antagonists bosentan (100 nmol/L) and macitentan (30 nmol/L) alone relaxed endothelin-contracted rings by 30±5% and 24±3%, respectively. Combinations of 30 nmol/L tadalafil with 100 nmol/L bosentan or 30 nmol/L macitentan relaxed endothelin-contracted rings by 53±5% or 46±7%, respectively; these values are similar to the calculated sums of the individual effects of these compounds. Denudation of endothelium from pulmonary arterial rings abolished the vasodilator response to 30 nmol/L tadalafil and the synergistic vasorelaxant effect of tadalafil with ambrisentan. In the presence of 1 μmol/L BQ-788, a selective endothelin type B receptor antagonist, the vasorelaxant effects of 10 nmol/L ambrisentan and 30 nmol/L tadalafil were additive but not synergistic. These data can be interpreted to suggest that ambrisentan and tadalafil synergistically inhibit endothelin-1-induced constriction of rat intrapulmonary arteries and that endothelin type B receptors in endothelium are necessary to enable a synergistic vasorelaxant effect of the drug combination.

Improved pulmonary vascular reactivity and decreased hypertrophic remodeling during nonhypercapnic acidosis in experimental pulmonary hypertension

Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in decreased responsiveness of pulmonary circulation to vasodilator therapies. We have shown that extracellular acidosis inhibits VSMC proliferation and migration in vitro. Here we tested whether induction of nonhypercapnic acidosis in vivo ameliorates PH and the underlying pulmonary vascular remodeling and dysfunction. Adult male Sprague-Dawley rats were exposed to hypoxia (8.5% O(2)) for 2 wk, or injected subcutaneously with monocrotaline (MCT, 60 mg/kg) to develop PH. Acidosis was induced with NH(4)Cl (1.5%) in the drinking water 5 days prior to and during the 2 wk of hypoxic exposure (prevention protocol), or after MCT injection from day 21 to 28 (reversal protocol). Right ventricular systolic pressure (RVSP) and Fulton's index were measured, and pulmonary arteriolar remodeling was analyzed. Pulmonary and mesenteric artery contraction to phenylephrine (Phe) and high KCl, and relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) were examined ex vivo. Hypoxic and MCT-treated rats demonstrated increased RVSP, Fulton's index, and pulmonary arteriolar thickening. In pulmonary arteries of hypoxic and MCT rats there was reduced contraction to Phe and KCl and reduced vasodilation to ACh and SNP. Acidosis prevented hypoxia-induced PH, reversed MCT-induced PH, and resulted in reduction in all indexes of PH including RVSP, Fulton's index, and pulmonary arteriolar remodeling. Pulmonary artery contraction to Phe and KCl was preserved or improved, and relaxation to ACh and SNP was enhanced in NH(4)Cl-treated PH animals. Acidosis alone did not affect the hemodynamics or pulmonary vascular function. Phe and KCl contraction and ACh and SNP relaxation were not different in mesenteric arteries of all groups. Thus nonhypercapnic acidosis ameliorates experimental PH, attenuates pulmonary arteriolar thickening, and enhances pulmonary vascular responsiveness to vasoconstrictor and vasodilator stimuli. Together with our finding that acidosis decreases VSMC proliferation, the results are consistent with the possibility that nonhypercapnic acidosis promotes differentiation of pulmonary VSMCs to a more contractile phenotype, which may enhance the effectiveness of vasodilator therapies in PH.

Mitochondrial complex II is essential for hypoxia-induced pulmonary vasoconstriction of intra- but not of pre-acinar arteries

Alveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecular mechanisms underlying this hypoxic pulmonary vasoconstriction (HPV) are still incompletely understood. Here, we investigated the role of succinate dehydrogenase (SDH; synonymous to mitochondrial complex II) in HPV, with particular emphasis on regional differences along the vascular bed and consequences for PAP and perfusion-to-ventilation matching, using mutant mice heterozygous for the SDHD subunit of complex II (SDHD(+/-)). Western blots revealed reduced protein content of complex II subunits SDHA, SDHB, and SDHC in lungs of SDHD(+/-) mice, despite unaffected mRNA content as determined by real-time PCR. Hypoxic pulmonary vasoconstriction of small (20-50 µm) intra-acinar and larger (51-100 µm) pre-acinar arteries was evaluated by videomorphometric analysis of precision-cut lung slices. The hypoxic response was detectable in pre-acinar arteries but absent from intra-acinar arteries of SDHD(+/-) mice. In isolated perfused lungs, basal PAP and its hypoxia-induced increase were indistinguishable between both mouse strains. Arterial oxygenation was measured after provocation of regional ventilatory failure by tracheal fluid instillation in anaesthetized mice, and it declined more in SDHD(+/-) than in wild-type mice. SDHD is required for the formation of a stable mitochondrial complex II and it is selectively important for HPV of intra-acinar vessels. This specialized vascular segment participates in perfusion-to-ventilation matching but does not significantly contribute to the acute hypoxic rise in PAP that results from more proximal vasoconstriction.

Role of Secretory Phospholipase A2 in Rhythmic Contraction of Pulmonary Arteries of Rats With Monocrotaline-Induced Pulmonary Arterial Hypertension

Excessive stretching of the vascular wall in accordance with pulmonary arterial hypertension (PAH) induces a variety of pathogenic cellular events in the pulmonary arteries. We previously reported that indoxam, a selective inhibitor for secretory phospholipase A2 (sPLA2), blocked the stretch-induced contraction of rabbit pulmonary arteries by inhibition of untransformed prostaglandin H2 (PGH2) production. The present study was undertaken to investigate involvement of sPLA2 and untransformed PGH2 in the enhanced contractility of pulmonary arteries of experimental PAH in rats. Among all the known isoforms of sPLA2, sPLA2-X transcript was most significantly augmented in the pulmonary arteries of rats with monocrotaline-induced pulmonary hypertension (MCT-PHR). The pulmonary arteries of MCT-PHR frequently showed two types of spontaneous contraction in response to stretch; 27% showed rhythmic contraction, which was sensitive to indoxam and SC-560 (selective COX-1 inhibitor), but less sensitive to NS-398 (selective COX-2 inhibitor); and 47% showed sustained incremental tension (tonic contraction), which was insensitive to indoxam and SC-560, but sensitive to NS-398 and was attenuated to 45% of the control. Only the rhythmically contracting pulmonary arteries of MCT-PHR produced a substantial amount of untransformed PGH2, which was abolished by indoxam. These results suggest that sPLA2-mediated PGH2 synthesis plays an important role in the rhythmic contraction of pulmonary arteries of MCT-PHR.

Depolarization‐Dependent Contraction Increase after Birth and Preservation following Long‐Term Hypoxia in Sheep Pulmonary Arteries

Membrane depolarization is critical to pulmonary arterial (PA) contraction. Both L-type Ca2+ channels (CaL) and Rho-kinase are important signaling components of this process and mitochondrial and non-mitochondrial generated superoxides can be part of the signaling process. Maturation and long-term hypoxia (LTH) each can modify depolarization-dependent contraction and the role of superoxides. By the use of wire myography, we tested the hypothesis that maturation and LTH increase pulmonary arterial reactivity to high-K+-induced membrane depolarization through enhancements in the importance of CaLand Rho-kinase-dependent pathways. The data show that maturation, but not LTH, increases contraction to 125 mM KCl (high-K+) without altering the EC50. High-K+-dependent contraction was inhibited to a similar extent in fetal and adult PA by multiple CaL blockers, including 10 μM diltiazem, 10 μM verapamil, and 10 μM nifedipine. Postnatal maturation increased the role for 10 μM nifedipine-sensitive CaL, and decreased that for 10 μM Y-27632-sensitive Rho-kinase. In all groups, the combination of nifedipine and Y-27632 effectively inhibited high-K+ contraction. Tempol (3 mM) but not 100 μM apocynin slightly reduced contraction in arteries from fetal hypoxic and adult normoxic and hypoxic sheep, indicating a limited role for non-mitochondrial derived superoxide to high-K+-induced contraction. Western immunoblot for alpha smooth muscle actin indicated small increases in relative abundance in the adult. The data suggest that while CaL therapies more effectively vasodilate PA in adults and rho-kinase therapies are more effective in newborns, combination therapies would provide greater efficacy in both young and mature patients regardless of normoxic or hypoxic conditions.

Role of thromboxane A2-activated nonselective cation channels in hypoxic pulmonary vasoconstriction of rat

Hypoxia-induced pulmonary vasoconstriction (HPV) is critical for matching of ventilation/perfusion in lungs. Although hypoxic inhibition of K(+) channels has been a leading hypothesis for depolarization of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, pharmacological inhibition of K(+) channels does not induce significant contraction in rat pulmonary arteries. Because a partial contraction by thromboxane A(2) (TXA(2)) is required for induction of HPV, we hypothesize that TXA(2) receptor (TP) stimulation might activate depolarizing nonselective cation channels (NSCs). Consistently, we found that 5-10 nM U46619, a stable agonist for TP, was indispensible for contraction of rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K(+) channel (K(v)). Whole cell voltage clamp with rat PASMC revealed that U46619 induced a NSC current (I(NSC,TXA2)) with weakly outward rectifying current-voltage relation. I(NSC,TXA2) was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid-related channel (TRPV) subfamily. 2-Aminoethoxydiphenyl borate, an agonist for TRPV1-3, consistently activated NSC channels in PASMCs. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (α-PDD) rarely induced an increase in the membrane conductance of PASMCs. RT-PCR analysis showed the expression of transcripts for TRPV2 and -4 in rat PASMCs. Finally, it was confirmed that pretreatment with RR largely inhibited HPV in the presence of U46619. The pretreatment with agonists for TRPV1 (capsaicin) and TRPV4 (α-PDD) was ineffective as pretone agents for HPV. Taken together, it is suggested that the concerted effects of I(NSC,TXA2) activation and K(v) inhibition under hypoxia induce membrane depolarization sufficient for HPV. TRPV2 is carefully suggested as the TXA(2)-activated NSC in rat PASMC.

Preservation of Serotonin-Mediated Contractility in Adult Sheep Pulmonary Arteries Following Long-Term High-Altitude Hypoxia

Long-term hypoxia (LTH) can increase serotonin (5-HT) signaling as well as extracellular calcium entry in adult rodent pulmonary arteries (PA), and 5-HT is associated with pulmonary hypertension. Because LTH, 5-HT, and calcium entry are related, we tested the hypothesis that LTH increases 5-HT-mediated PA contractility and associated calcium influx through L-type Ca2+ channels, nonselective cation channels (NSCC), and reverse-mode sodium-Ca2+ exchange. We performed wire myography and confocal calcium imaging on pulmonary arteries from adult ewes that lived near sea level or were maintained at high-altitude (3801 m) for ∼110 days. LTH did not increase the arterial medial wall thickness, nor did it affect the potency or efficacy for 5-HT-induced PA contraction. Ketanserin (100 nM), a 5-HT2A antagonist, shifted the 5-HT potency to a far greater extent than 1 μM GR-55562, a 5-HT1B/D inhibitor. These influences were unaffected by LTH. The rank order for reducing 5-HT-induced PA contraction in normoxic animals was extracellular calcium removal≈10 mM Ni2+≈10 μM verapamil≈10 μM nifedipine with 50 μM SKF 96365>30 μM KB-R7943≈100 μM flufenamic acid≈10 μM nifedipine≈100 μM Gd3+> 100 μM La3+>500 μM Ni2+≈10 μM diltiazem≈50 μM 2-APB≈100 μM LOE 908. Contraction was not reduced by 100 μM spermine or 30 μM SN-6. LTH increased the effects of KB-R7943 and mitigated those of nifedipine but did not affect calcium responses in imaging studies. Overall, in adult sheep, arterial structure and 5-HT2A and 5HT1B/D functions are preserved following LTH while the role of NSCC-related calcium-dependent contraction is increased. These elements indicate preservation of PA contractility in LTH with minimal functional changes.

Long-term maternal hypoxia: the role of extracellular Ca2+ entry during serotonin-mediated contractility in fetal ovine pulmonary arteries.

Antenatal maternal long-term hypoxia (LTH) can alter serotonin (5-HT) and calcium (Ca(2+)) signaling in fetal pulmonary arteries (PAs) and is associated with persistent pulmonary hypertension of the newborn (PPHN). In humans, the antenatal maternal hypoxia can be secondary to smoking, anemia, and chronic obstructive pulmonary disorders. However, the mechanisms of antenatal maternal hypoxia-related PPHN are unresolved. Because both LTH and 5-HT are associated with PPHN, we tested the hypothesis that antenatal maternal LTH can increase 5-HT-mediated PA contraction and associated extracellular Ca(2+) influx through L-type Ca(2+) channels (Ca(L)), nonselective cation channels (NSCCs), and reverse-mode sodium-calcium exchanger (NCX) in the near-term fetus. We performed wire myography and confocal-Ca(2+) imaging approaches on fetal lamb PA (∼ 140 days of gestation) from normoxic ewes or those acclimatized to high-altitude LTH (3801 m) for ∼110 days. Long-term hypoxia reduced the potency but not the efficacy of 5-HT-induced PA contraction. Ketanserin (100 nmol/L), a 5-HT(2A) antagonist, shifted 5-HT potency irrespective of LTH, while GR-55562 (1 µmol/L), a 5-HT(1B/D) inhibitor, antagonized 5-HT-induced contraction in normoxic fetuses only. Various inhibitors for Ca(L), NSCC, and reverse-mode NCX were used in contraction studies. Contraction was reliant on extracellular Ca(2+) regardless of maternal hypoxia, NSCC was more important to contraction than Ca(L), and reverse-mode NCX had little or no role in contraction. Long-term hypoxia also attenuated the effects of 2-APB and flufenamic acid and reduced Ca(2+) responses observed by imaging studies. Overall, LTH reduced 5HT(1B/D) function and increased NSCC-related Ca(2+)-dependent contraction in ovine fetuses, which may compromise pulmonary vascular function in the newborn.