BACKGROUND AND PURPOSEHypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca 2+ concentration ([Ca 2+ ]i) by opening of K channels and release of H2S. EXPERIMENTAL APPROACHPorcine coronary arteries without endothelium were mounted for measurement of isometric tension and [Ca 2+ ]i, and the expression of voltage-gated K channels KV7 channels (encoded by KCNQ genes) and large-conductance calcium-activated K channels (KCa1.1) was examined. Voltage clamp assessed the role of KV7 channels in hypoxia. KEY RESULTSGradual reduction of oxygen concentration from 95 to 1% dilated the precontracted coronary arteries and this was associated with reduced [Ca 2+

Endogenous hydrogen sulfide (H 2 S) is involved in the regulation of vascular tone. We hypothesized that the lowering of calcium and opening of potassium (K) channels as well as calciumindependent mechanisms are involved in H 2 S-induced relaxation in rat mesenteric small arteries. Amperometric recordings revealed that free [H 2 S] after addition to closed tubes of sodium hydrosulfide (NaHS), Na 2 S, and GYY4137 [P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid] were, respectively, 14%, 17%, and 1% of added amount. The compounds caused equipotent relaxations in isometric myographs, but based on the measured free [H 2 S], GYY4137 caused more relaxation in relation to released free H 2 S than NaHS and Na 2 S in rat mesenteric small arteries. Simultaneous measurements of [H 2 S] and tension showed that 15 mM of free H 2 S caused 61% relaxation in superior mesenteric arteries. Simultaneous measurements of smooth muscle calcium and tension revealed that NaHS lowered calcium and caused relaxation of NE-contracted arteries, while high extracellular potassium reduced NaHS relaxation without corresponding calcium changes. In NE-contracted arteries, NaHS (1 mM) lowered the phosphorylation of myosin light chain, while phosphorylation of myosin phosphatase target subunit 1 remained unchanged. Protein kinase A and G, inhibitors of guanylate cyclase, failed to reduce NaHS relaxation, whereas blockers of voltage-gated K V 7 channels inhibited NaHS relaxation, and blockers of mitochondrial complex I and III abolished NaHS relaxation. Our findings suggest that low micromolar concentrations of free H 2 S open K channels followed by lowering of smooth muscle calcium, and by another mechanism involving mitochondrial complex I and III leads to uncoupling of force, and hence vasodilation.

These novel PDE1 inhibitors induce vasodilation and lower BP, suggesting a potential use of these vasodilators in the treatment of hypertension and vasospasm.

The voltage-gated K V 7 (KCNQ) potassium channels are activated by ischemia and involved in hypoxic vasodilatation. We investigated the effect of K V 7 channel modulation on cardiac ischemia and reperfusion injury and its interaction with cardioprotection by ischemic preconditioning (IPC). Reverse-transcription polymerase chain reaction revealed expression of K V 7.1, K V 7.4, and K V 7.5 in the left anterior descending rat coronary artery and all K V 7 subtypes (K V 7.1-K V 7.5) in the left and right ventricles of the heart. Isolated hearts were subjected to no-flow global ischemia and reperfusion with and without IPC. Infarct size was quantified by 2,3,5-triphenyltetrazolium chloride staining. Two blockers of K V 7 channels, XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone] (10 mM) and linopirdine (10 mM), reduced infarct size and exerted additive infarct reduction to IPC. An opener of K V 7 channels, flupirtine (10 mM) abolished infarct size reduction by IPC. Hemodynamics were measured using a catheter inserted in the left ventricle and postischemic left ventricular recovery improved in accordance with reduction of infarct size and deteriorated with increased infarct size. XE991 (10 mM) reduced coronary flow in the reperfusion phase and inhibited vasodilatation in isolated small branches of the left anterior descending coronary artery during both simulated ischemia and reoxygenation. K V 7 channels are expressed in rat coronary arteries and myocardium. Inhibition of K V 7 channels exerts cardioprotection and opening of K V 7 channels abrogates cardioprotection by IPC. Although safety issues should be further addressed, our findings suggest a potential role for K V 7 blockers in the treatment of ischemiareperfusion injury.

Persistent pulmonary hypertension in newborn (PPHN) is a serious and possibly fatal syndrome characterized by sustained foetal elevation of pulmonary vascular resistance at birth. PPHN may manifest secondary to other conditions as meconium aspiration syndrome, infection and congenital diaphragmatic hernia. This MiniReview provides the reader with an overview of current and future treatment options for patients with PPHN without congenital diaphragmatic hernia. The study is based on systematic searches in the databases PubMed and Cochrane Library and registered studies on Clinicaltrials.gov investigating PPHN. Inhaled nitric oxide (iNO) is well documented for treatment of PPHN, but 30% fail to respond to iNO. Other current treatment options could be sildenafil, milrinone, prostaglandin analogues and bosentan. There are several ongoing trials with sildenafil, but evidence is lacking for the other treatments and/or for the combination with iNO. Currently, there is no evidence for effect in PPHN of other treatments, for example tadalafil, macitentan, ambrisentan, riociguat and selexipag used for pulmonary arterial hypertension in adults. Experimental studies in animal models for PPHN suggest effect of a series of approaches including recombinant human superoxide dismutase, L-citrulline, Rho-kinase inhibitors and peroxisome proliferator-activated receptor-γ agonists. We conclude that iNO is the most investigated and the only approved pulmonary vasodilator for infants with PPHN. In the iNO non-responders, sildenafil currently seems to be the best alternative either alone or in combination with iNO. Systematic and larger clinical studies are required for testing the other potential treatments of PPHN.

Modulation of endothelial calcium-activated potassium (KCa) channels has been proposed as an approach to restore endothelial function. The present study investigated whether novel openers of KCa channels with small (KCa2.x) and intermediate (KCa3.1) conductance, NS309 and NS4591, improve endothelium-dependent relaxation and erectile function. Rat corpus cavernosum (CC) strips were mounted for isometric tension recording and processed for immunoblotting. Mean arterial pressure (MAP), intracavernosal pressure (ICP), and electrocardiographic (ECG) measurements were conducted in anesthetized rats. Immunoblotting revealed the presence of KCa2.3 and large KCa conductance (KCa1.1) channels in the corpus cavernosum. NS309 and NS4591 increased current in CC endothelial cells in whole cell patch clamp experiments. Relaxation induced by NS309 (<1 μM) was inhibited by endothelial cell removal and high extracellular potassium. An inhibitor of nitric oxide (NO) synthase, and blockers of KCa2.x and KCa1.1 channels, apamin and iberiotoxin also inhibited NS309 relaxation. Incubation with NS309 (0.5 μM) markedly enhanced acetylcholine relaxation. Basal erectile function (ICP/MAP) increased during administration of NS309. Increases in ICP/MAP after cavernous nerve stimulation with NS309 were unchanged, whereas NS4591 significantly improved erectile function. Administration of NS309 and NS4591 caused small changes in the electrocardiogram, but neither arrhythmic events nor prolongation of the QTc interval were observed. The present study suggests that openers of KCa2.x and KCa3.1 channels improve endothelial and erectile function. The effects of NS309 and NS4591 on heart rate and ECG are small, but will require additional safety studies before evaluating whether activation of KCa2.3 channels has a potential for treatment of erectile dysfunction.

BackgroundThe systemic vascular response to hypoxia is vasodilation. However, reports suggest that the potent vasoconstrictor endothelin-1 (ET-1) is released from the vasculature during hypoxia. ET-1 is reported to augment superoxide anion generation and may counteract nitric oxide (NO) vasodilation. Moreover, ET-1 was proposed to contribute to increased vascular resistance in heart failure by increasing the production of asymmetric dimethylarginine (ADMA). We investigated the role of ET-1, the NO pathway, the potassium channels and radical oxygen species in hypoxia-induced vasodilation of large coronary arteries.ResultsIn prostaglandin F2α (PGF2α, 10 μM)-contracted segments with endothelium, gradual lowering of oxygen tension from 95 to 1% O2 resulted in vasodilation. The vasodilation to O2 lowering was rightward shifted in segments without endothelium at all O2 concentrations except at 1% O2. The endothelin receptor antagonist SB217242 (10 μM) markedly increased hypoxic dilation despite the free tissue ET-1 concentration in the arterial wall was unchanged in 1% O2 versus 95% O2. Exogenous ET-1 reversed hypoxic dilation in segments with and without endothelium, and the hypoxic arteries showed an increased sensitivity towards ET-1 compared to the normoxic controls. Without affecting basal NO, hypoxia increased NO concentration in PGF2α-contracted arteries, and an NO synthase inhibitor, L-NOARG,(300 μM, NG-nitro-L-Arginine) reduced hypoxic vasodilation. NO-induced vasodilation was reduced in endothelin-contracted preparations. Arterial wall ADMA concentrations were unchanged by hypoxia. Blocking of potassium channels with TEA (tetraethylammounium chloride)(10 μM) inhibited vasodilation to O2 lowering as well as to NO. The superoxide scavenger tiron (10 μM) and the putative NADPH oxidase inhibitor apocynin (10 μM) leftward shifted concentration-response curves for O2 lowering without changing vasodilation to 1% O2. PEG (polyethylene glycol) catalase (300 u/ml) inhibited H2O2 vasodilation, but failed to affect vasodilation to O2 lowering. Neither did PEG-SOD (polyethylene glycol superoxide dismutase)(70 u/ml) affect vasodilation to O2 lowering. The mitochondrial inhibitors rotenone (1 μM) and antimycin A (1 μM) both inhibited hypoxic vasodilatation.ConclusionThe present results in porcine coronary arteries suggest NO contributes to hypoxic vasodilation, probably through K channel opening, which is reversed by addition of ET-1 and enhanced by endothelin receptor antagonism. These latter findings suggest that endothelin receptor activation counteracts hypoxic vasodilation.

ObjectiveIn vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension.Approach and ResultMale wild type and KCa3.1−/−/KCa2.3T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1−/−/KCa2.3T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype.ConclusionDespite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of KCa2.3/KCa3.1 activators for the treatment of pulmonary hypertension.