Original ContributionConstriction of pulmonary artery by peroxide: role of Ca2+ release and PKC
Introduction
Reactive oxygen species (ROS) play a key role in the signal transduction pathways of the normal cell, but in disease contribute to or cause the underlying pathophysiology. Several cellular mechanisms can generate ROS, but in the vasculature the major sources are NADPH oxidase, mitochondria, and uncoupled nitric oxide synthase [1], [2], [3]. The primogenitor of most ROS is the superoxide radical, formed by single-electron donation to molecular O2, but it is generally assumed that its highly reactive nature and short half-life obviate its direct involvement as an autocrine or paracrine agent. Instead, it is believed that the nonradical and less reactive hydrogen peroxide is the actual signaling moiety, formed by dismutation of superoxide by superoxide dismutase (SOD). ROS and other oxidants are tightly controlled within the cell by a series of mechanisms, including SOD, catalase (which breaks down peroxide), and glutathione peroxidase.
In the vasculature, ROS signaling has been implicated in a large number of pathways, including the response to growth factors, vasoactive agents such as angiotensin II, gene transcription, and smooth muscle proliferation [2]. Although exogenous peroxide has pronounced effects on vascular reactivity, the form that this takes is dependent on tissue bed, concentration, and vessel size [3]. In systemic arteries a common finding for concentrations of peroxide below 300 μM is a rapid transient constriction mediated by cyclooxygenase (COX), generally followed by a sustained relaxation most apparent in preconstricted arteries, though constriction is not always reported; the vasodilatation has been variously attributed to activation of K+ channels, COX, and/or cGMP-associated pathways and may or may not be endothelium-dependent [4], [5], [6], [7], [8], [9], [10], [11]. Concentrations of peroxide above 100 μM are commonly reported to cause irreversible changes in vasoreactivity [4].
Oxidant stress has been associated with acute lung injury, sepsis, COPD, and pulmonary hypertension [1], [12]. Modulation of intracellular ROS by acute hypoxia, particularly ROS generated by the mitochondria, is also believed by many to be the key signaling event for O2 sensing in pulmonary artery and hypoxic pulmonary vasoconstriction (HPV), an adaptive process that optimizes pulmonary ventilation–perfusion matching in the face of localized alveolar hypoxia [13], [14], [15]. The primary mechanisms of HPV reside within the pulmonary artery smooth muscle cell (PASMC) but are incompletely resolved, though there is strong evidence that they include Ca2+ release from ryanodine-sensitive stores, store-operated Ca2+ entry, and/or voltage-dependent Ca2+ channels and Rho kinase-mediated Ca2+ sensitization [16]. The redox hypothesis of HPV proposes that hypoxia causes decreased generation of mitochondrial ROS, a more reduced cytosol, and inhibition of K+ channels, with subsequent Ca2+ entry via voltage-dependent Ca2+ channels [15]. In contrast the ROS hypothesis suggests that mitochondrial ROS generation increases and that peroxide acts as the distal signaling moiety to cause Ca2+ release and entry [17]. There is increasing evidence for the latter, including the recent findings that overexpression of catalase or glutathione peroxidase in cultured PASMCs suppresses the hypoxia-mediated elevation of intracellular Ca2+ and that exogenous peroxide causes an elevation of intracellular Ca2+ [14], [18] due to Ca2+ release from ryanodine-sensitive stores and entry via a voltage-independent pathway [19].
There are fewer data concerning the effects of exogenous ROS on intact pulmonary arteries. Although a few studies have reported that peroxide elicits sustained constriction of pulmonary artery, the concentrations used were generally high. Moreover, unlike HPV the constriction developed very slowly over ∼1 h and was insensitive to ryanodine though suppressed by the myosin light-chain kinase (MLCK) inhibitor ML-9 [20], or was completely independent of Ca2+, insensitive to ML-9, and not accompanied by myosin light-chain (MLC20) phosphorylation [21]. However, the more rapid constriction elicited by flow in small porcine pulmonary arteries has been shown to be mediated by ROS [22]. In contrast, it has been reported that peroxide relaxes preconstricted bovine pulmonary artery via increased cGMP [23].
There have been no investigations as to whether peroxide (or any ROS) causes Rho kinase-mediated Ca2+ sensitization in pulmonary artery, a characteristic of sustained HPV. However, it has been reported that peroxide induces a Rho kinase-dependent contraction in tracheal smooth muscle [24], that superoxide causes activation of RhoA/Rho kinase and vasoconstriction in aorta [25], and that cold-induced vasoconstriction of skin arteries is initiated by increased generation of mitochondrial ROS and subsequent activation of Rho kinase [26].
We therefore investigated the vasoactive actions of low concentrations (≤100 μM) of peroxide on small intrapulmonary arteries (IPAs) of the rat, including its effects on Ca2+ mobilization and Ca2+ sensitization. Our key findings are that peroxide causes a biphasic but sustained vasoconstriction in IPA and that the sustained component is associated with Ca2+ release from ryanodine-sensitive stores and a PKC-dependent constriction that is independent of Rho kinase and changes in MLC20 phosphorylation.
Section snippets
Animals and tissue isolation
This study conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Wistar rats (200–250 g) were killed by lethal overdose of pentobarbital (ip). Small IPAs (third to fourth branch; 150–450 μm i.d.) were mounted in a myograph (Danish MyoTechnology, Denmark) containing physiological salt solution (PSS) gassed with 95% air/5% CO2 (pH 7.4) at 37°C, as previously described [27]. When
Results
Peroxide (30 μM) elicited a sustained vasoconstriction in IPA, consisting of an initial short transient that reached a peak of 16.3 ± 1.7 %KPSS at around 2 min and a sustained component of 16.9 ± 1.6 %KPSS (25 min, n = 46; Figs. 1A and 1D). The vasoconstriction was relatively variable (coefficient of variation ∼30%). A cumulative concentration–response relationship revealed that half-maximal tension (sustained) was obtained at ∼12 μM peroxide (Fig. 1B; n = 11); concentrations above 100 μM caused
Discussion
Reports concerning the vasoactive properties of exogenous hydrogen peroxide are variable and often compromised by use of high concentrations, especially when examining signaling pathways. In many cases the use of conduit arteries (aorta, main pulmonary artery) may limit translation to resistance arteries, which are known to differ in their response to a variety of stimuli. In this study we therefore utilized a relatively low concentration of peroxide (30 μM) and small IPAs (150–450 μm i.d.),
Acknowledgments
This work was funded by the British Heart Foundation and the Wellcome Trust (078075).
References (46)
- et al.
Superoxide in the pulmonary circulation
Pharmacol. Ther.
(1999) - et al.
Characterization of four different effects elicited by H2O2 in rat aorta
Vasc. Pharmacol.
(2005) - et al.
Endothelium-dependent relaxation to hydrogen peroxide in canine basilar artery: a potential new cerebral dilator mechanism
Brain Res. Bull.
(1998) - et al.
Mechanisms of hydrogen peroxide-induced relaxation in rabbit mesenteric small artery
Eur. J. Pharmacol.
(2001) Oxygen sensors in context
Biochim. Biophys. Acta
(2008)- et al.
Role of mitochondrial reactive oxygen species in hypoxia-dependent increase in intracellular calcium in pulmonary artery myocytes
Free Radic. Biol. Med.
(2007) - et al.
Direct effects of hydrogen peroxide on airway smooth muscle tone: roles of Ca2+ influx and Rho-kinase
Eur. J. Pharmacol.
(2007) - et al.
Protein kinases in vascular smooth muscle tone—role in the pulmonary vasculature and hypoxic pulmonary vasoconstriction
Pharmacol. Ther.
(2004) Investigation of the mechanisms underlying H2O2-evoked contraction in the isolated rat aorta
Gen. Pharmacol.
(1998)- et al.
Mechanisms of hydrogen peroxide-induced contraction of rat aorta
Eur. J. Pharmacol.
(1998)
Isoprostanes constrict human radial artery by stimulation of thromboxane receptors, Ca2+ release, and RhoA activation
J. Thorac. Cardiovasc. Surg.
ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor: a primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction.
J. Biol. Chem.
Hydrogen peroxide stimulates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum
J. Biol. Chem.
Mitochondrial ROS–PKCɛ signaling axis is uniquely involved in hypoxic increase in [Ca 2+]i in pulmonary artery smooth muscle cells
Biochem. Biophys. Res. Commun.
Modulation of vascular smooth muscle signaling by reactive oxygen species
Physiology (Bethesda)
Hydrogen peroxide as a paracrine vascular mediator: regulation and signaling leading to dysfunction
Exp. Biol. Med. (Maywood)
Mechanisms of hydrogen-peroxide-induced biphasic response in rat mesenteric artery
Br. J. Pharmacol.
The effect of hydrogen peroxide in human internal thoracic arteries: role of potassium channels, nitric oxide and cyclooxygenase products
Cardiovasc. Drugs Ther.
Contractile responses elicited by hydrogen peroxide in aorta from normotensive and hypertensive rats: endothelial modulation and mechanism involved
Br. J. Pharmacol.
Hydrogen peroxide induces contraction and raises [Ca2+]i in canine cerebral arterial smooth muscle: participation of cellular signaling pathways
Naunyn-Schmiedebergs Arch. Pharmacol.
Hydrogen peroxide-dependent arteriolar dilation in contracting muscle of rats fed normal and high salt diets
Microcirculation
Role of oxidants in lung injury during sepsis
Antioxid. Redox Signal.
Increases in mitochondrial reactive oxygen species trigger hypoxia-induced calcium responses in pulmonary artery smooth muscle cells
Circ. Res.
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