Michelle Connolly

Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca2+ sensitization

Reactive oxygen species play a key role in vascular disease, pulmonary hypertension, and hypoxic pulmonary vasoconstriction. We investigated contractile responses, intracellular Ca(2+) ([Ca(2+)](i)), Rho-kinase translocation, and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light chain (MLC(20)) in response to LY83583, a generator of superoxide anion, in small intrapulmonary arteries (IPA) of rat. LY83583 caused concentration-dependent constrictions in IPA and greatly enhanced submaximal PGF(2alpha)-mediated preconstriction. In small femoral or mesenteric arteries of rat, LY83583 alone was without effect, but it relaxed a PGF(2)alpha-mediated preconstriction. Constrictions in IPA were inhibited by superoxide dismutase and tempol, but not catalase, and were endothelium and guanylate cyclase independent. Constrictions were also inhibited by the Rho-kinase inhibitor Y27632 and the Src-family kinase inhibitor SU6656. LY83583 did not raise [Ca(2+)](i), but caused a Y27632-sensitive constriction in alpha-toxin-permeabilized IPA. LY83583 triggered translocation of Rho-kinase from the nucleus to the cytosol in pulmonary artery smooth muscle cells and enhanced phosphorylation of MYPT-1 at Thr-855 and of MLC(20) at Ser-19 in IPA. This enhancement was inhibited by superoxide dismutase and abolished by Y27632. Hydrogen peroxide did not activate Rho-kinase. We conclude that in rat small pulmonary artery, superoxide triggers Rho-kinase-mediated Ca(2+) sensitization and vasoconstriction independent of hydrogen peroxide.

Key role of the RhoA/Rho kinase system in pulmonary hypertension

Pulmonary hypertension (PH) is a general term comprising a spectrum of pulmonary hypertensive disorders which have in common an elevation of mean pulmonary arterial pressure (mPAP). The prototypical form of the disease, termed pulmonary arterial hypertension (PAH), is a rare but lethal syndrome with a complex aetiology characterised by increased pulmonary vascular resistance (PVR) and progressive elevation of mPAP; patients generally die from heart failure. Current therapies are inadequate and median survival is less than three years. PH due to chronic hypoxia (CH) is a condition separate from PAH and is strongly associated with chronic obstructive pulmonary disease (COPD). An early event in the pathogenesis of this form of PH is hypoxic pulmonary vasoconstriction (HPV), an acute homeostatic process that maintains the ventilation-perfusion ratio during alveolar hypoxia. The mechanisms underlying HPV remain controversial, but RhoA/Rho kinase (ROK)-mediated Ca²+-sensitisation is considered important. Increasing evidence also implicates RhoA/ROK in PASMC proliferation, inflammatory cell recruitment and the regulation of cell motility, all of which are involved in the pulmonary vascular remodelling occurring in all forms of PH. ROK is therefore a potential therapeutic target in treating PH of various aetiologies. Here, we examine current concepts regarding the aetiology of PAH and also PH due to CH, focusing on the contribution that RhoA/ROK-mediated processes may make to their development and on ROK inhibitors as potential therapies.

A role for voltage‐gated, but not Ca2+‐activated, K+ channels in regulating spontaneous contractile activity in myometrium from virgin and pregnant rats

The roles of voltage‐gated (KV) and large conductance Ca2+‐activated K+ (BKCa) channels in regulating basal contractility in myometrial smooth muscle are unresolved. The aim of this study was to determine the effects of inhibition of these channels on spontaneous rhythmic contraction in myometrial strips from four groups of rats: nonpregnant and during early (day 7), mid‐ (day 14), and late (day 21) pregnancy. BKCa channels were inhibited using iberiotoxin (1–100 nM), paxilline (1–10 μM) or penitrem A (1–500, or 3000 nM); KV channels were inhibited using tetraethylammonium (TEA; 1–10 mM) and/or 4‐aminopyridine (4‐AP; 1–5 mM). Contractility was assessed as mean integral tension (MIT). Time/vehicle controls were also performed. None of the selective BKCa channel inhibitors significantly affected contractility in myometrial strips from either nonpregnant or pregnant animals. 4‐AP caused concentration‐dependent increases in MIT in myometrium in all four groups. TEA (5 and 10 mM) significantly increased MIT in myometrium from nonpregnant, and mid‐ and late pregnant rats, but not in myometrium from early pregnant rats. TEA and 4‐AP still caused an increase in MIT following treatment with 3000 nM penitrem A or a combination of propranolol, phentolamine, atropine (all 1 μM) and capsaicin (10 μM) in myometrial strips from nonpregnant rats. These results indicate that whereas BKCa channels play little or no part in controlling basal rhythmicity in rat myometrium, KV channels appear to play a crucial role in this regard, especially during mid‐ and late pregnancy.

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.

Cell redox state and hypoxic pulmonary vasoconstriction: Recent evidence and possible mechanisms

During alveolar hypoxia, hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation near optimum via incompletely defined mechanisms. It is proposed that a hypoxia-induced rise in the intracellular concentration of reactive oxygen species (ROS) or an oxidising shift in the cytoplasmic redox state provides the signal which initiates the constriction of pulmonary arteries (PA), although this is controversial. Here, we review recent investigations demonstrating that hypoxia causes a rise in [ROS] in PA smooth muscle, and that ROS and antioxidants have effects on PA which would be predicted if cell oxidation causes contraction. We argue that intracellular Ca2+ release and Ca2+-sensitisation are the key effector mechanisms causing HPV, and discuss evidence that both processes are promoted by ROS or oxidative protein modifications. We conclude that while it is plausible that an increase in cytoplasmic [ROS] activates HPV effector mechanisms, proving this link will require the determination of whether hypoxia causes oxidative modifications of proteins involved in Ca2+ homeostasis and sensitisation.

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.