Jesus Prieto-Lloret

Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role for intracellular Ca2+ release

•  Hypoxic pulmonary vasoconstriction (HPV) is a mechanism by which pulmonary arteries maintain blood oxygenation during alveolar hypoxia. •  HPV is generally studied using a vasoconstricting co‐stimulus that amplifies the HPV but may also distort its properties; we therefore characterised HPV in isolated rat intrapulmonary arteries during 40 min hypoxic challenges in the absence of any such stimulus. •  Immediate (phase 1) and sustained (phase 2) components of HPV were unaffected by blocking voltage‐gated Ca2+ channels but were abolished by depletion of sarcoplasmic reticulum Ca2+. Phase 2 was attenuated by blockade of store‐operated Ca2+ entry (SOCE), although it largely persisted in Ca2+‐free physiological saline solution. •  HPV was associated with an increase in the intrapulmonary artery ratio of oxidised to reduced glutathione and was inhibited by antioxidants. •  HPV resulted primarily from intracellular Ca2+ release, with SOCE making a contribution, particularly to phase 2. Sustained HPV involves oxidation of the pulmonary artery redox state.

Effects of Intermittent Hypoxia on Blood Gases Plasma Catecholamine and Blood Pressure

Obstructive sleep apnoea syndrome (OSAS) is a disorder characterized by repetitive episodes of complete (apnoea) or partial (hypopnoea) obstruction of airflow during sleep. The severity of OSAS is defined by the apnoea hypopnoea index (AHI) or number of obstructive episodes. An AHI greater than 30 is considered severe, but it can reach values higher than 100 in some patients. Associated to the OSA there is high incidence of cardiovascular and neuro-psychiatric pathologies including systemic hypertension, stroke, cardiac arrhythmias and atherosclerosis, diurnal somnolence, anxiety and depression. In the present study we have used a model of intermittent hypoxia (IH) of moderately high intensity (30 episodes/h) to evaluate arterial blood gases and plasma catecholamines as main effectors in determining arterial blood pressure. Male rats were exposed toIH with a regime of 80s, 20% O(2) // 40s, 10%O(2), 8 h/day, 8 or 15 days.Lowering the breathing atmosphere to 10% O(2) reduced arterial blood PO(2) to 56.9 mmHg (nadir HbO(2) 86, 3%). Plasma epinephrine (E) and norepinephrine (NE) levels at the end of 8 and 15 days of IH showed a tendency to increase, being significant the increase of norepinephrine (NE) levels in the group exposed to intermittent hypoxia during 15 days. We conclude that IH causes an increase in sympathetic activity and a concomitant increase in NE levels which in turn would generate an increase in vascular tone and arterial blood pressure.

Gap junctions support the sustained phase of hypoxic pulmonary vasoconstriction by facilitating calcium sensitization

Aims To determine the role of gap junctions (GJs) in hypoxic pulmonary vasoconstriction (HPV). Methods and results Studies were performed in rat isolated intrapulmonary arteries (IPAs) mounted on a myograph and in anaesthetized rats. Hypoxia induced a biphasic HPV response in IPAs preconstricted with prostaglandin F2α (PGF2α, 3 µM) or 20 mM K+. The GJ inhibitors 18β-glycyrrhetinic acid (18β-GA, 30 µM), heptanol (3.5 mM), or 2-aminoethoxydiphenyl borate (2-APB) (75 µM) had little effect on the transient Phase 1 of HPV, but abolished the sustained Phase 2 which is associated with Ca2+ sensitization. The voltage-dependent Ca2+ channel blocker diltiazem (10 µM) had no effect on HPV, and did not alter the inhibitory action of 18β-GA. Sustained HPV is enhanced by high glucose (15 mM) via potentiation of Ca2+ sensitization, in the presence of high glucose 18β-GA still abolished sustained HPV. Simultaneous measurement of tension and intracellular Ca2+ using Fura PE-3 demonstrated that whilst 18β-GA abolished tension development during sustained HPV, it did not affect the elevation of intracellular Ca2+. Consistent with this, 18β-GA abolished hypoxia-induced phosphorylation of the Rho kinase target MYPT-1. In anaesthetized rats hypoxia caused a biphasic increase in systolic right ventricular pressure. Treatment with oral 18β-GA (25 mg/kg) abolished the sustained component of the hypoxic pressor response. Conclusion These results imply that GJs are critically involved in the signalling pathways leading to Rho kinase-dependent Ca2+ sensitization during sustained HPV, but not elevation of intracellular Ca2+, and may explain the dependence of the former on an intact endothelium.

Sphingosylphosphorylcholine potentiates vasoreactivity and voltage-gated Ca2+ entry via NOX1 and reactive oxygen species

Aims Sphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (≤1 µmol/L), however, it does not constrict intrapulmonary arteries (IPAs), but strongly potentiates vasoreactivity. Our aim was to determine whether this also occurs in a systemic artery and to delineate the signalling pathway. Methods and results Rat mesenteric arteries and IPAs mounted on a myograph were challenged with ∼25 mmol/L [K+] to induce a small vasoconstriction. SPC (1 µmol/L) dramatically potentiated this constriction in all arteries by ∼400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C (PLC; U73122), PKCε (inhibitory peptide), Src (PP2), and NADPH oxidase (VAS2870), and also by Tempol (superoxide scavenger), but not by inhibition of Rho kinase (Y27632). Potentiation was lost in mesenteric arteries from p47phox–/–, but not NOX2−/–, mice. The intracellular superoxide generator LY83583 mimicked the effect of SPC. SPC elevated reactive oxygen species (ROS) in vascular smooth muscle cells, and this was blocked by PP2, VAS2870, and siRNA knockdown of PKCε. SPC (1 µmol/L) significantly reduced the EC50 for U46619-induced vasoconstriction, an action ablated by Tempol. In patch-clamped mesenteric artery cells, SPC (200 nmol/L) enhanced Ba2+ current through L-type Ca2+ channels, an action abolished by Tempol but mimicked by LY83583. Conclusion Our results suggest that low concentrations of SPC activate a PLC-coupled and NOX1-mediated increase in ROS, with consequent enhancement of voltage-gated Ca2+ entry and thus vasoreactivity. We speculate that this pathway is not specific for SPC, but may also contribute to vasoconstriction elicited by other G-protein coupled receptor and PLC-coupled agonists.

Hypoxic pulmonary vasoconstriction in isolated rat pulmonary arteries is not inhibited by antagonists of H2S‐synthesizing pathways

We evaluated the hypothesis that an increase in the hydrogen sulphide concentration in pulmonary artery smooth muscle cells (PASMCs) causes hypoxic pulmonary vasoconstriction (HPV) by examining the effects of the sulphide donor cysteine and sulphide‐synthesis blockers on HPV in isolated rat intrapulmonary arteries (IPAs). Cysteine (1 mm) enhanced HPV and also the contraction to prostaglandin F2α (PGF2α) and both effects were abolished by the cystathionine γ‐lyase (CSE) blocker propargylglycine (PAG, 1 mm), which had little or no non‐selective effect on contraction at this concentration. Neither PAG nor the cysteine aminotransferase (CAT) antagonist aspartate affected HPV in normal physiological saline solution (PSS), or in PSS containing physiological concentrations of cysteine, cystine and glutamate, whereas dithiothreitol (DTT), proposed to enhance HPV by converting mitochondrial thiosulphate to sulphide, instead abolished HPV. PAG markedly diminished whereas DTT did not affect cysteine‐induced sulphide release from liver pieces. The results do not support the proposal that hydrogen sulphide plays a role in HPV.

Divergent modulation of Rho-kinase and Ca2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle

The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src‐family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR‐mediated ASM contraction and associated signalling events.

Potentiation of Hypoxic Pulmonary Vasoconstriction by Hydrogen Sulfide Precursors 3-Mercaptopyruvate and D-Cysteine Is Blocked by the Cystathionine γ Lyase Inhibitor Propargylglycine

Although the gasotransmitter hydrogen sulfide (H(2)S) generally dilates systemic arteries in mammals, it causes constriction of pulmonary arteries. In isolated rat pulmonary arteries, we have shown that the H(2)S precursor cysteine enhances both hypoxic pulmonary vasoconstriction and tension development caused by the agonist prostaglandin F(2α) under normoxic conditions. These effects were blocked by propargylglycine (PAG), a blocker of the enzyme cystathionine γ lyase which metabolises cysteine to sulfide. In the present study, we evaluated whether 3-mercaptopyruvate (3-MP), a sulfide precursor which is thought to give rise to sulfide when it is metabolised by the enzyme mercaptopyruvate sulfurtransferase, also enhanced contraction. Application of 3-MP prior to hypoxic challenge caused a marked enhancement of HPV which was completely blocked by both L- and D,L-PAG (both 1 mM). Cumulative application of 3-1,000 μM 3-MP during an ongoing contraction to PGF(2α) under normoxic conditions also caused a marked increase in tension. Application of D-cysteine (1 mM) also enhanced HPV, and this effect was prevented by both the D-amino acid oxidase inhibitor sodium benzoate (500 μM) and 1 mM L-PAG.

Glabridin-induced vasorelaxation: Evidence for a role of BKCa channels and cyclic GMP

Background and purpose Glabridin is a major flavonoid in Glycyrrhiza glabra (licorice) root, a traditional Asian medicine. Glabridin is reported to have anti-atherogenic, anti-inflammatory and anti-nephritic properties; however its effects on vascular tone remain unexplored. Experimental approach We examined the effect of glabridin on rat main mesenteric artery using isometric myography and also ELISA to measure cGMP levels. Key results Glabridin (30 μM) relaxed arteries pre-constricted with the thromboxane A2 analog U46619 (0.2 μM) by ~ 60% in an endothelium-independent manner. Relaxation to 30 μM glabridin was abolished by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (1 μM) and by the BKCa channel blocker tetraethyammonium (1 mM) but was unaffected by the estrogen receptor antagonist ICI182780. The concentration-response curve to glabridin (0.1 to 30 μM) was downshifted by the KATP channel blocker glibenclamide (10 μM), the KV channel blocker 4-aminopyridine (300 μM), and the KIR blocker BaCl2 (30 μM). In U46619-contracted arteries partially relaxed by 0.1 μM sodium nitroprusside, application of 10 and 30 nM glabridin caused additional vasorelaxation. Glabridin (30 μM) approximately doubled tissue [cyclic GMP]. Application of the phosphodiesterase inhibitor isobutylmethylxanthine caused a much larger rise in [cyclic GMP], and glabridin failed to cause vasorelaxation or a further rise in [cGMP] when co-applied with IBMX. Conclusions and implications Vasorelaxation to glabridin is dependent on the opening of K+ channels, particularly BKCa, probably caused by a rise in cellular [cyclic GMP] owing to phosphodiesterase inhibition. In the presence of sodium nitroprusside an effect of glabridin is observed at nM concentrations, similar those measured in plasma following human ingestion of licorice flavonoid oil.

Intracellular remodelling of Ca2+ stores in pulmonary hypertension

This editorial refers to ‘Stretch-induced Ca2+ signalling in vascular smooth muscle cells depends on Ca2+ store segregation’ by G. Gilbert et al. , pp. 313–323, this issue. The low resistance of the pulmonary circulation, a function of its unique structure and regulation, normally allows it to accept the entire cardiac output in the pulmonary vascular bed while maintaining an internal pressure that is low enough to prevent pulmonary oedema. In pulmonary hypertension (PH), however, pulmonary vascular resistance can increase enormously due to structural remodelling and abnormalities in the regulation of pulmonary artery (PA) diameter. Both changes are driven, at least in part, by increases in the intracellular Ca2+ concentration ([Ca2+]i) in PA smooth muscle cells (PASMCs). This is associated with the up-regulation of multiple pathways mediating the influx of Ca2+ into these cells.1 [Ca2+]i, however, is not determined by Ca2+ influx alone; there is recent evidence2 that the expression of SERCA2a, which mediates Ca2+ uptake into the sarcoplasmic reticulum (SR), is decreased in PASMCs from humans with PH. An elegant study by Gilbert et al. 3 in the current issue of Cardiovascular Research now demonstrates that the abnormalities in intracellular Ca2+ handling in PH go well beyond SERCA; they find that two animal models of PH exhibit a re-organization of intracellular Ca2+ stores of …

L-phenylalanine restores vascular function in spontaneously hypertensive rats through activation of the GCH1-GFRP complex

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