Pulmonary Vascular Responses to Chronic Intermittent Hypoxia in a Guinea Pig Model of Obstructive Sleep Apnea.

Experimental evidence suggests that chronic intermittent hypoxia (CIH), a major hallmark of obstructive sleep apnea (OSA), boosts carotid body (CB) responsiveness, thereby causing increased sympathetic activity, arterial and pulmonary hypertension, and cardiovascular disease. An enhanced circulatory chemoreflex, oxidative stress, and NO signaling appear to play important roles in these responses to CIH in rodents. Since the guinea pig has a hypofunctional CB (i.e., it is a natural CB knockout), in this study we used it as a model to investigate the CB dependence of the effects of CIH on pulmonary vascular responses, including those mediated by NO, by comparing them with those previously described in the rat. We have analyzed pulmonary artery pressure (PAP), the hypoxic pulmonary vasoconstriction (HPV) response, endothelial function both in vivo and in vitro, and vascular remodeling (intima-media thickness, collagen fiber content, and vessel lumen area). We demonstrate that 30 days of the exposure of guinea pigs to CIH (FiO2, 5% for 40 s, 30 cycles/h) induces pulmonary artery remodeling but does not alter endothelial function or the contractile response to phenylephrine (PE) in these arteries. In contrast, CIH exposure increased the systemic arterial pressure and enhanced the contractile response to PE while decreasing endothelium-dependent vasorelaxation to carbachol in the aorta without causing its remodeling. We conclude that since all of these effects are independent of CB sensitization, there must be other oxygen sensors, beyond the CB, with the capacity to alter the autonomic control of the heart and vascular function and structure in CIH.

Oxygen Sensing: Physiology and Pathophysiology.

Oxygen is such an essential element for life that multiple mechanisms have evolved to maintain oxygen homeostasis, including those which detect decreases in arterial O2 and generate adaptive responses to hypoxia [...].

Redox Regulation, Oxidative Stress, and Inflammation in Group 3 Pulmonary Hypertension.

Group 3 pulmonary hypertension (PH), which occurs secondary to hypoxia lung diseases, is one of the most common causes of PH worldwide and has a high unmet clinical need. A deeper understanding of the integrative pathological and adaptive molecular mechanisms within this group is required to inform the development of novel drug targets and effective treatments. The production of oxidants is increased in PH Group 3, and their pleiotropic roles include contributing to disease progression by promoting prolonged hypoxic pulmonary vasoconstriction and pathological pulmonary vascular remodeling, but also stimulating adaptation to pathological stress that limits the severity of this disease. Inflammation, which is increasingly being viewed as a key pathological feature of Group 3 PH, is subject to complex regulation by redox mechanisms and is exacerbated by, but also augments oxidative stress. In this review, we investigate aspects of this complex crosstalk between inflammation and oxidative stress in Group 3 PH, focusing on the redox-regulated transcription factor NF-κB and its upstream regulators toll-like receptor 4 and high mobility group box protein 1. Ultimately, we propose that the development of specific therapeutic interventions targeting redox-regulated signaling pathways related to inflammation could be explored as novel treatments for Group 3 PH.

Maladaptive Pulmonary Vascular Responses to Chronic Sustained and Chronic Intermittent Hypoxia in Rat.

Chronic sustained hypoxia (CSH), as found in individuals living at a high altitude or in patients suffering respiratory disorders, initiates physiological adaptations such as carotid body stimulation to maintain oxygen levels, but has deleterious effects such as pulmonary hypertension (PH). Obstructive sleep apnea (OSA), a respiratory disorder of increasing prevalence, is characterized by a situation of chronic intermittent hypoxia (CIH). OSA is associated with the development of systemic hypertension and cardiovascular pathologies, due to carotid body and sympathetic overactivation. There is growing evidence that CIH can also compromise the pulmonary circulation, causing pulmonary hypertension in OSA patients and animal models. The aim of this work was to compare hemodynamics, vascular contractility, and L-arginine-NO metabolism in two models of PH in rats, associated with CSH and CIH exposure. We demonstrate that whereas CSH and CIH cause several common effects such as an increased hematocrit, weight loss, and an increase in pulmonary artery pressure (PAP), compared to CIH, CSH seems to have more of an effect on the pulmonary circulation, whereas the effects of CIH are apparently more targeted on the systemic circulation. The results suggest that the endothelial dysfunction evident in pulmonary arteries with both hypoxia protocols are not due to an increase in methylated arginines in these arteries, although an increase in plasma SDMA could contribute to the apparent loss of basal NO-dependent vasodilation and, therefore, the increase in PAP that results from CIH.

Hydroxycobalamin Reveals the Involvement of Hydrogen Sulfide in the Hypoxic Responses of Rat Carotid Body Chemoreceptor Cells.

Carotid body (CB) chemoreceptor cells sense arterial blood PO2, generating a neurosecretory response proportional to the intensity of hypoxia. Hydrogen sulfide (H2S) is a physiological gaseous messenger that is proposed to act as an oxygen sensor in CBs, although this concept remains controversial. In the present study we have used the H2S scavenger and vitamin B12 analog hydroxycobalamin (Cbl) as a new tool to investigate the involvement of endogenous H2S in CB oxygen sensing. We observed that the slow-release sulfide donor GYY4137 elicited catecholamine release from isolated whole carotid bodies, and that Cbl prevented this response. Cbl also abolished the rise in [Ca2+]i evoked by 50 µM NaHS in enzymatically dispersed CB glomus cells. Moreover, Cbl markedly inhibited the catecholamine release and [Ca2+]i rise caused by hypoxia in isolated CBs and dispersed glomus cells, respectively, whereas it did not alter these responses when they were evoked by high [K⁺]e. The L-type Ca2+ channel blocker nifedipine slightly inhibited the rise in CB chemoreceptor cells [Ca2+]i elicited by sulfide, whilst causing a somewhat larger attenuation of the hypoxia-induced Ca2+ signal. We conclude that Cbl is a useful and specific tool for studying the function of H2S in cells. Based on its effects on the CB chemoreceptor cells we propose that endogenous H2S is an amplifier of the hypoxic transduction cascade which acts mainly by stimulating non-L-type Ca2+ channels.

Role of reactive oxygen species and sulfide-quinone oxoreductase in hydrogen sulfide-induced contraction of rat pulmonary arteries.

Application of H2S ("sulfide") elicits a complex contraction in rat pulmonary arteries (PAs) comprising a small transient contraction (phase 1; Ph1) followed by relaxation and then a second, larger, and more sustained contraction (phase 2; Ph2). We investigated the mechanisms causing this response using isometric myography in rat second-order PAs, with Na2S as a sulfide donor. Both phases of contraction to 1,000 µM Na2S were attenuated by the pan-PKC inhibitor Gö6983 (3 µM) and by 50 µM ryanodine; the Ca2+ channel blocker nifedipine (1 µM) was without effect. Ph2 was attenuated by the mitochondrial complex III blocker myxothiazol (1 µM), the NADPH oxidase (NOX) blocker VAS2870 (10 µM), and the antioxidant TEMPOL (3 mM) but was unaffected by the complex I blocker rotenone (1 µM). The bath sulfide concentration, measured using an amperometric sensor, decreased rapidly following Na2S application, and the peak of Ph2 occurred when this had fallen to ~50 µM. Sulfide caused a transient increase in NAD(P)H autofluorescence, the offset of which coincided with development of the Ph2 contraction. Sulfide also caused a brief mitochondrial hyperpolarization (assessed using tetramethylrhodamine ethyl ester), followed immediately by depolarization and then a second more prolonged hyperpolarization, the onset of which was temporally correlated with the Ph2 contraction. Sulfide application to cultured PA smooth muscle cells increased reactive oxygen species (ROS) production (recorded using L012); this was absent when the mitochondrial flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using small interfering RNA. We propose that the Ph2 contraction is largely caused by SQR-mediated sulfide metabolism, which, by donating electrons to ubiquinone, increases electron production by complex III and thereby ROS production.

Hydrogen Sulfide as an O2 Sensor: A Critical Analysis.

There is increasing interest in the physiological actions and therapeutic potential of the gasotransmitter hydrogen sulfide (H2S). In addition to exerting antihypertensive, anti-inflammatory, antioxidant, and pro-angiogenic effects, H2S has been suggested to play a central and ubiquitous role in O2 sensing. According to this concept, because H2S is metabolized by oxidation, its cellular concentration varies inversely with the ambient pO2 such that hypoxia causes a rise in intracellular [H2S]; this then acts to induce appropriate cellular responses. In particular, it has been proposed that H2S underpins O2 sensing in the carotid body, which triggers increases in ventilation in response to hypoxemia, and also in pulmonary arteries, which constrict in response to local alveolar hypoxia. This process, termed hypoxic pulmonary vasoconstriction (HPV), acts to divert blood to better-oxygenated regions of the lung, thereby maintaining the ventilation-perfusion ratio and minimizing hypoxia-induced falls in blood O2 saturation. In this chapter, we present a critical review of the evidence supporting and questioning this model in both HPV and the carotid body.

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 (30µM) relaxed arteries pre-constricted with the thromboxane A2 analog U46619 (0.2µM) by ~60% in an endothelium-independent manner. Relaxation to 30µM glabridin was abolished by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (1µM) and by the BKCa channel blocker tetraethyammonium (1mM) but was unaffected by the estrogen receptor antagonist ICI182780. The concentration-response curve to glabridin (0.1 to 30µM) was downshifted by the KATP channel blocker glibenclamide (10µM), the KV channel blocker 4-aminopyridine (300µM), and the KIR blocker BaCl2 (30µM). In U46619-contracted arteries partially relaxed by 0.1µM sodium nitroprusside, application of 10 and 30nM glabridin caused additional vasorelaxation. Glabridin (30µM) 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.

Reactive oxygen species facilitate the EDH response in arterioles by potentiating intracellular endothelial Ca(2+) release.

There is abundant evidence that H2O2 can act as an endothelium-derived hyperpolarizing factor in the resistance vasculature. However, whilst scavenging H2O2 can abolish endothelial dependent hyperpolarization (EDH) and the associated vascular relaxation in some arteries, EDH-dependent vasorelaxation can often be mimicked only by using relatively high concentrations of H2O2. We have examined the role of H2O2 in EDH-dependent vasodilatation by simultaneously measuring vascular diameter and changes in endothelial cell (EC) [Ca(2+)]i during the application of H2O2 or carbachol, which triggers EDH. Carbachol (10µM) induced dilatation of phenylephrine-preconstricted rat cremaster arterioles was largely (73%) preserved in the presence of indomethacin (3µM) and l-NAME (300µM). This residual NO- and prostacyclin-independent dilatation was reduced by 89% upon addition of apamin (0.5µM) and TRAM-34 (10µM), and by 74% when an extracellular ROS scavenging mixture of SOD and catalase (S&C; 100Uml(-1) each) was present. S&C also reduced the carbachol-induced EC [Ca(2+)]i increase by 74%. When applied in Ca(2+)-free external medium, carbachol caused a transient increase in EC [Ca(2+)]i. This was reduced by catalase, and was enhanced when 1µM H2O2 was present in the bath. H2O2 -induced dilatation, which occurred only at concentrations ≥100µM, was reduced by a blocking antibody to TRPM2, which had no effect on carbachol-induced responses. Similarly, iberotoxin and Rp-8bromo cGMP reduced the vasodilatation induced by H2O2, but not by carbachol. Inhibiting PLC, PLA2 or CYP450 2C9 each greatly reduced the carbachol-induced increase in EC [Ca(2+)]i and vasodilatation, but adding 10µM H2O2 during PLA2 or CYP450 2C9 inhibition completely restored both responses. The nature of the effective ROS species was investigated by using Fe(2+) chelators to block the formation of ∙OH. A cell permeant chelator was able to inhibit EC Ca(2+) store release, but cell impermeant chelators reduced both the vasodilatation and EC Ca(2+) influx, implying that ∙OH is required for these responses. The results indicate that rather than mediating EDH by acting directly on smooth muscle, H2O2 promotes EDH by acting within EC to enhance Ca(2+) release.

Role of Hydrogen Sulfide in Systemic and Pulmonary Hypertension: Cellular Mechanisms and Therapeutic Implications.

There is increasing interest in the possible therapeutic benefits of the gasotransmitter hydrogen sulfide (H2S sulfide), and drugs which release sulfide are currently being evaluated in both preclinical and clinical studies as treatments for conditions ranging from cancer to cardiovascular disease to inflammation. Sulfide is generally considered to act as a vasodilator, and it is therefore not surprising that the effects of sulfide donors on both systemic and pulmonary hypertension are being examined. In this review, we provide a critical evaluation of both the evidence that H2S may be of benefit in treating systemic and pulmonary hypertension, and of current hypotheses regarding the cellular mechanisms underlying sulfide's effects on the systemic and pulmonary vascular systems.

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.

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 p47(phox-/-), 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 H2 S-synthesizing pathways.

An increase in the H2 S (hydrogen sulphide, hereafter sulphide) concentration in pulmonary artery smooth muscle cells (PASMCs) has been proposed to mediate hypoxic pulmonary vasoconstriction (HPV). We evaluated this hypothesis in isolated rat intrapulmonary arteries (IPAs) by examining the effects of the sulphide precursor cysteine and sulphide-synthesis blockers on HPV and also on normoxic pulmonary vasoconstriction (NPV) stimulated by prostaglandin F2α (PGF2α ) and by the drug LY83583, which causes contraction in IPAs by increasing cellular reactive oxygen species levels. Experiments with several blockers of cystathionine γ-lyase (CSE), the enzyme responsible for sulphide synthesis in the vasculature, demonstrated that propargylglycine (PAG, 1 mm) had little or no effect on the NPV caused by PGF2α or LY83583. Conversely, other CSE antagonists tested, aminooxyacetic acid (AOAA, 100 µm), ß-cyanoalanine (BCA, 500 µm) and hydroxylamine (HA, 100 µm), altered the NPV to PGF2α (BCA increased, HA inhibited) and/or LY83583 (BCA increased, AOAA and HA inhibited). Preincubating IPAs in physiological saline solution (PSS) containing 1 mm cysteine increased the amplitude of the NPV to PGF2(α) by ∼50%, and had a similar effect on HPV elicited by hypoxic challenge with 0% O2 . The enhancement of both responses by cysteine was abolished by pretreatment with 1 mm PAG. Measurements carried out with an amperometric electrode demonstrated that incubation with 1 mm cysteine under anoxic conditions (to minimize sulphide oxidation) greatly potentiated the release of sulphide from pieces of rat liver and that this release was strongly antagonized by PAG, indicating that at this concentration PAG could enter cells intact and antagonize CSE. PAG at 1 mm had no effect on HPV recorded in control PSS, or in PSS supplemented with physiological concentrations of cysteine (10 µm), cystine (50 µm) and glutamate (100 µm) in order to prevent the possible depletion of intracellular cysteine during experiments. Application of a combination of 1 mm cysteine and 1 mm α-ketoglutarate to promote sulphide synthesis via the cysteine aminotransferase/mercaptopyruvate sulphurtransferase (CAT/MST) pathway caused an increase in HPV similar to that observed for cysteine. This was partially blocked by the CAT antagonist aspartate (1 mm) and also by PAG. However, HPV was not increased by 1 mm α-ketoglutarate alone, and HPV in the absence of α-ketoglutarate and cysteine was not attenuated by aspartate. Pretreatment of IPAs with dithiothreitol (DTT, 1 mm), proposed to promote the conversion of mitochondrial thiosulphate to sulphide, did not increase the release of sulphide from pieces of rat liver in either the presence or the absence of 1 mm cysteine, and virtually abolished HPV. The results provide evidence that the sulphide precursor cysteine can promote both NPV and HPV in rat IPA by generating sulphide via a PAG-sensitive pathway, presumably CSE. However, HPV evoked under control conditions was unaffected by the blockade of CSE. Moreover, HPV was not affected by the CAT antagonist aspartate and was blocked rather than enhanced by DTT. The data therefore indicate that sulphide generated by CSE or CAT/MST or from thiosulphate is unlikely to contribute to O2 sensing during HPV in these arteries.

Investigating the potential role of TRPA1 in locomotion and cardiovascular control during hypertension.

Radiotelemetry was used to investigate the in vivo cardiovascular and activity phenotype of both TRPA1 (transient receptor potential ankyrin 1) wild-type (WT) and TRPA1 knockout (KO) mice. After baseline recording, experimental hypertension was induced using angiotensin II infusion (1.1 mg(-1) kg(-1) a day, for 14 days). TRPA1 WT and KO mice showed similar morphological and functional cardiovascular parameters, including similar basal blood pressure (BP), heart rate, size, and function. Similar hypertension was also displayed in response to angiotensin II (156 ± 7 and 165 ± 11 mmHg, systolic BP ± SEM, n = 5-6). TRPA1 KO mice showed increased hypertensive hypertrophy (heart weight:tibia length: 7.3 ± 1.6 mg mm(-1) vs. 8.8 ± 1.7 mg mm(-1)) and presented with blunted interleukin 6 (IL-6) production compared with hypertensive WT mice (151 ± 24 vs. 89 ± 16 pg mL(-1)). TRPA1 expression in dorsal root ganglion (DRG) neurones was upregulated during hypertension (163% of baseline expression). Investigations utilizing the TRPA1 agonist cinnamaldehyde (CA) on mesenteric arterioles isolated from näive mice suggested a lack of TRPA1-dependent vasoreactivity in this vascular bed; a site with notable ability to alter total peripheral resistance. However, mesenteric arterioles isolated from TRPA1 KO hypertensive mice displayed significantly reduced ability to relax in response to nitric oxide (NO) (P < 0.05). Unexpectedly, naïve TRPA1 KO mice also displayed physical hyperactivity traits at baseline, which was exacerbated during hypertension. In conclusion, our study provides a novel cardiovascular characterization of TRPA1 KO mice in a model of hypertension. Results suggest that TRPA1 has a limited role in global cardiovascular control, but we demonstrate an unexpected capacity for TRPA1 to regulate physical activity.