Topical therapy with negative allosteric modulators of the calcium-sensing receptor (calcilytics) for the management of asthma: the beginning of a new era?

In this review article we present the evidence to date supporting the role of the calcium-sensing receptor (CaSR) as a key, pluripotential molecular trigger for asthma and speculate on the likely benefits of topical therapy of asthma with negative allosteric modulators of the CaSR: calcilytics.

Characterization of Negative Allosteric Modulators of the Calcium-Sensing Receptor for Repurposing as a Treatment of Asthma.

Asthma is still an incurable disease, and there is a recognized need for novel small-molecule therapies for people with asthma, especially those poorly controlled by current treatments. We previously demonstrated that calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs), calcilytics, uniquely suppress both airway hyperresponsiveness (AHR) and inflammation in human cells and murine asthma surrogates. Here we assess the feasibility of repurposing four CaSR NAMs, which were originally developed for oral therapy for osteoporosis and previously tested in the clinic as a novel, single, and comprehensive topical antiasthma therapy. We address the hypotheses, using murine asthma surrogates, that topically delivered CaSR NAMs 1) abolish AHR; 2) are unlikely to cause unwanted systemic effects; 3) are suitable for topical application; and 4) inhibit airway inflammation to the same degree as the current standard of care, inhaled corticosteroids, and, furthermore, inhibit airway remodeling. All four CaSR NAMs inhibited poly-L-arginine-induced AHR in naïve mice and suppressed both AHR and airway inflammation in a murine surrogate of acute asthma, confirming class specificity. Repeated exposure to inhaled CaSR NAMs did not alter blood pressure, heart rate, or serum calcium concentrations. Optimal candidates for repurposing were identified based on anti-AHR/inflammatory activities, pharmacokinetics/pharmacodynamics, formulation, and micronization studies. Whereas both inhaled CaSR NAMs and inhaled corticosteroids reduced airways inflammation, only the former prevented goblet cell hyperplasia in a chronic asthma model. We conclude that inhaled CaSR NAMs are likely a single, safe, and effective topical therapy for human asthma, abolishing AHR, suppressing airways inflammation, and abrogating some features of airway remodeling. SIGNIFICANCE STATEMENT: Calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs) reduce airway smooth muscle hyperresponsiveness, reverse airway inflammation as efficiently as topical corticosteroids, and suppress airway remodeling in asthma surrogates. CaSR NAMs, which were initially developed for oral therapy of osteoporosis proved inefficacious for this indication despite being safe and well tolerated. Here we show that structurally unrelated CaSR NAMs are suitable for inhaled delivery and represent a one-stop, steroid-free approach to asthma control and prophylaxis.

Hypoxic pulmonary vasoconstriction.

It has been known for more than 60 years, and suspected for over 100, that alveolar hypoxia causes pulmonary vasoconstriction by means of mechanisms local to the lung. For the last 20 years, it has been clear that the essential sensor, transduction, and effector mechanisms responsible for hypoxic pulmonary vasoconstriction (HPV) reside in the pulmonary arterial smooth muscle cell. The main focus of this review is the cellular and molecular work performed to clarify these intrinsic mechanisms and to determine how they are facilitated and inhibited by the extrinsic influences of other cells. Because the interaction of intrinsic and extrinsic mechanisms is likely to shape expression of HPV in vivo, we relate results obtained in cells to HPV in more intact preparations, such as intact and isolated lungs and isolated pulmonary vessels. Finally, we evaluate evidence regarding the contribution of HPV to the physiological and pathophysiological processes involved in the transition from fetal to neonatal life, pulmonary gas exchange, high-altitude pulmonary edema, and pulmonary hypertension. Although understanding of HPV has advanced significantly, major areas of ignorance and uncertainty await resolution.

Transforming growth factor-ß enhances Rho-kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1.

KEY POINTS: Transforming growth-factor-ß (TGF-ß) and RhoA/Rho-kinase are independently implicated in the airway hyper-responsiveness associated with asthma, but how these proteins interact is not fully understood. We examined the effects of pre-treatment with TGF-ß on expression and activity of RhoA, Rho-kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin-induced contraction, in airway smooth muscle. TGF-ß enhanced bradykinin-induced RhoA translocation, Rho-kinase-dependent phosphorylation and contraction, but partially suppressed bradykinin-induced RhoA activity (RhoA-GTP content). TGF-ß enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF-ß on RhoA and Rho-kinase activity and contraction, respectively. ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin-sensitized mice. ARHGEF1 is a key TGF-ß target gene, an important regulator of Rho-kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper-responsiveness. ABSTRACT: Transforming growth factor-ß (TGF-ß), RhoA/Rho-kinase and Src-family kinases (SrcFK) have independently been implicated in airway hyper-responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF-ß pre-treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho-kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF-ß pre-treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20 , MYPT-1 and the actin-severing protein cofilin, but not of RhoA, ROCK2 or c-Src. In hASMCs, acute treatment with BK triggered subcellular translocation of ARHGEF1 and RhoA and enhanced auto-phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20 , but induced de-phosphorylation of cofilin. TGF-ß pre-treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA-GTP content, SrcFK auto-phosphorylation and cofilin de-phosphorylation. In hASMCs, an ARHGEF1 small interfering RNA suppressed the effects of BK and TGF-ß on RhoA-GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF-ß on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMCs of asthmatic patients and in lungs of ovalbumin-sensitized mice. Our data indicate that TGF-ß enhances BK-induced contraction, RhoA translocation and Rho-kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA-GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper-responsiveness is worthy of further investigation.

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.

Profiling of healthy and asthmatic airway smooth muscle cells following interleukin-1ß treatment: a novel role for CCL20 in chronic mucus hypersecretion.

Chronic mucus hypersecretion (CMH) contributes to the morbidity and mortality of asthma, and remains uncontrolled by current therapies in the subset of patients with severe, steroid-resistant disease. Altered cross-talk between airway epithelium and airway smooth muscle cells (ASMCs), driven by pro-inflammatory cytokines such as interleukin (IL)-1ß, provides a potential mechanism that influences CMH. This study investigated mechanisms underlying CMH by comparing IL-1ß-induced gene expression profiles between asthma and control-derived ASMCs and the subsequent paracrine influence on airway epithelial mucus production in vitroIL-1ß-treated ASMCs from asthmatic patients and healthy donors were profiled using microarray analysis and ELISA. Air-liquid interface (ALI)-cultured CALU-3 and primary airway epithelial cells were treated with identified candidates and mucus production assessed.The IL-1ß-induced CCL20 expression and protein release was increased in ASMCs from moderate compared with mild asthmatic patients and healthy controls. IL-1ß induced lower MIR146A expression in asthma-derived ASMCs compared with controls. Decreased MIR146A expression was validated in vivo in bronchial biopsies from 16 asthmatic patients versus 39 healthy donors. miR-146a-5p overexpression abrogated CCL20 release in ASMCs. CCL20 treatment of ALI-cultured CALU-3 and primary airway epithelial cells induced mucus production, while CCL20 levels in sputum were associated with increased levels of CMH in asthmatic patients.Elevated CCL20 production by ASMCs, possibly resulting from dysregulated expression of the anti-inflammatory miR-146a-5p, may contribute to enhanced mucus production in asthma.

Physiological redox signalling and regulation of ion channels: implications for pulmonary hypertension.

NEW FINDINGS: What is the topic of this review? The review concerns the role of reactive oxygen species as physiological second messengers in potentiating G-protein-coupled receptor-mediated vasoconstriction and its potential dysregulation by oxidant stress in pulmonary hypertension. What advances does it highlight? The review highlights the concept that physiological signalling by reactive oxygen species must normally be highly compartmentalized to prevent self-regenerating oxidant stress and promiscuous and uncontrolled signalling, which contribute to the aetiology. Pulmonary hypertension is associated with oxidant stress and increased generation of reactive oxygen species (ROS) by NADPH oxidases (NOX), mitochondria and other sources. There is considerable evidence that these contribute to the aetiology via promotion of pulmonary vascular remodelling, endothelial dysfunction and enhanced vasoreactivity. However, it is now recognized that ROS act as important signalling mediators and second messengers in normal physiological conditions. Many ion channels and protein kinases crucial to pulmonary vascular function are directly or indirectly affected by redox/ROS, including K+ , Ca2+ and non-selective cation channels and Rho kinase. However, the inherent difficulties in quantifying ROS, particularly in subcellular compartments, make it uncertain whether these reported effects are of relevance in physiological rather than pathological conditions. In an attempt to address such issues, we have focused on the role of physiologically generated ROS in the regulation of G-protein-coupled receptor (GPCR)-activated vasoconstrictor pathways. We have recently reported a novel mechanism whereby low concentrations of GPCR-linked vasoconstrictors greatly potentiate Ca2+ entry via a NOX1- and ROS-mediated pathway parallel to the classical vasoconstrictor pathways of Ca2+ mobilization and activation of Rho kinase. Our findings imply that ROS signalling is highly compartmentalized in physiological conditions, but that this may be compromised by pathological increases in oxidant production, for example in pulmonary hypertension, leading to promiscuous actions that contribute to the aetiology. This model is consistent with the proposal that targeted antioxidants could prove to be an effective therapy for pulmonary hypertension.

From Physiological Redox Signalling to Oxidant Stress.

Oxidant stress is strongly associated with cardiovascular disease, including pulmonary hypertension, but antioxidant therapies have so far proven ineffective. This is partly due to a lack of understanding of the key role played by reactive oxygen species (ROS) in physiological cell signalling, and partly to the complex interrelationships between generators of ROS (e.g. mitochondria and NADPH oxidases, NOX), cellular antioxidant systems and indeed Ca2+ signalling. At physiological levels ROS reversibly affect the function of numerous enzymes and transcription factors, most often via oxidation of specific protein thiols. Importantly, they also affect pathways that promote ROS generation by NOX or mitochondria (ROS-induced ROS release), which has an inherent propensity for positive feedback and uncontrolled oxidant production. The reason this does not occur under normal conditions reflects in part a high level of compartmentalisation of ROS signalling within the cell, akin to that for Ca2+. This article considers the physiological processes which regulate NOX and mitochondrial ROS production and degradation and their interactions with each other and Ca2+ signalling pathways, and discusses how loss of spatiotemporal constraints and activation of positive feedback pathways may impact on their dysregulation in pulmonary hypertension.

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.

Sensory neuron downregulation of the Kv9.1 potassium channel subunit mediates neuropathic pain following nerve injury.

Chronic neuropathic pain affects millions of individuals worldwide, is typically long-lasting, and remains poorly treated with existing therapies. Neuropathic pain arising from peripheral nerve lesions is known to be dependent on the emergence of spontaneous and evoked hyperexcitability in damaged nerves. Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.

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

Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist-induced preconstriction ('pretone'). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPAs) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L-type Ca2+ channel antagonists diltiazem (10 and 30 µm) or nifedipine (3 µm). Application of the store-operated Ca2+ entry (SOCE) blockers BTP2 (10 µm) or SKF96365 (50 µm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca2+-free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (30 µm) was also applied during the 30 min incubation in Ca2+-free physiological saline solution. Pretreatment with 10 µm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 µm ryanodine attenuated both phases. The two-pore channel blocker NED-19 (1 µm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 µm) had no effect on either phase of HPV. The lysosomal Ca2+-depleting agent concanamycin (1 µm) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8-bromo-cyclic ADP ribose (30 µm) had no effect on either phase of HPV. Neither the Ca2+-sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 µm) nor FK506 (10 µm), a drug which displaces FKBP12.6 from ryanodine receptor 2 (RyR2), had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y-27632 (1 µm) and attenuated by the protein kinase C inhibitor Gö6983 (3 µm). Hypoxia for 45 min caused a significant increase in the ratio of oxidised to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase inhibitor VAS2870 (10 µm), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 µm) and TEMPOL (3 mm). We conclude that both phases of HPV in this model are mainly dependent on [Ca2+]i release from the sarcoplasmic reticulum. Neither phase of HPV requires voltage-gated Ca2+ entry, but SOCE contributes to phase 2. We can detect no requirement for cyclic ADP ribose, NAADP-dependent lysosomal Ca2+ release, activation of the CaSR, or displacement of FKBP12.6 from RyR2 for either phase of HPV. Sustained HPV is associated with an oxidising shift in the GSSG/GSH redox potential and is inhibited by the antioxidants ebselen and TEMPOL, consistent with the concept that it requires an oxidising shift in the cell redox state or the generation of reactive oxygen species.

Diminished sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma.

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)). A rise in [Ca(2+)](i) is normalized by rapid reuptake of Ca(2+) into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca(2+) (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca(2+)](i), cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca(2+)](i) following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca(2+) re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca](i) after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.

Ca(2+) homeostasis and structural and functional remodelling of airway smooth muscle in asthma.

Asthma is characterised by airway hyper-responsiveness and remodelling, and there is mounting evidence that alterations in the phenotype of airway smooth muscle (ASM) play a central role in these processes. Although the concept that dysregulation of ASM Ca(2+) homeostasis may underlie at least part of these alterations has been around for many years, it is only relatively recently that the availability of ASM biopsies from subjects with mild and moderate asthma has allowed it to be properly investigated. In this article, critical components of the pathobiology of asthmatic ASM, including contractile function, proliferation, cell migration and secretion of proinflammatory cytokines and chemokines, are reviewed and related to associated changes in ASM Ca(2+) homeostasis. Based on this evidence, it is proposed that a unifying mechanism for the abnormal asthmatic phenotype is dysregulation of Ca(2+) homeostasis caused at least in part by a downregulation in expression and function of sarcoendoplasmic Ca(2+) ATPases (SERCAs).

Mechanisms of hypoxic pulmonary vasoconstriction and their roles in pulmonary hypertension: new findings for an old problem.

Hypoxic pulmonary vasoconstriction (HPV) normally optimises ventilation-perfusion matching in the lung, but leads to pulmonary hypertension (PH) under conditions of global hypoxia. The past few years have provided some major advances in our understanding of this complex phenomenon, but significant controversy remains concerning many of the key underlying mechanisms. On balance, recent evidence is most consistent with an elevation in mitochondria-derived reactive oxygen species as a key event for initiation of HPV, with consequent Ca2+ release from intracellular ryanodine-sensitive stores, although the activation pathways and molecular identity of the associated Ca2+ entry pathways remain unclear. Recent studies have also raised our perception of the critical role played by Rho kinase (ROCK) in both sustained HPV and the development of PH, further promoting ROCK and the pathways regulating its activity and expression as important therapeutic targets.

Oxygen sensors in context.

The ability to adapt to changes in the availability of O2 provides a critical advantage to all O2-dependent lifeforms. In mammals it allows optimal matching of the O2 requirements of the cells to ventilation and O2 delivery, underpins vital changes to the circulation during the transition from fetal to independent, air-breathing life, and provides a means by which dysfunction can be limited or prevented in disease. Certain tissues such as the carotid body, pulmonary circulation, neuroepithelial bodies and fetal adrenomedullary chromaffin cells are specialised for O2 sensing, though most others show for example alterations in transcription of specific genes during hypoxia. A number of mechanisms are known to respond to variations in PO2 over the physiological range, and have been proposed to fulfil the function as O2 sensors; these include modulation of mitochondrial oxidative phosphorylation and a number of O2-dependent synthetic and degradation pathways. There is however much debate as to their relative importance within and between specific tissues, whether their O2 sensitivity is actually appropriate to account for their proposed actions, and in particular their modus operandi. This review discusses our current understanding of how these mechanisms may operate, and attempts to put them into the context of the actual PO2 to which they are likely to be exposed. An important point raised is that the overall O2 sensitivity (P50) of any O2-dependent mechanism does not necessarily correlate with that of its O2 sensor, as the coupling function between the two may be complex and non-linear. In addition, although the bulk of the evidence suggests that mitochondria act as the key O2 sensor in carotid body, pulmonary artery and chromaffin cells, the signalling mechanisms by which alterations in their function are translated into a response appear to differ fundamentally, making a global unified theory of O2 sensing unlikely.

Hypoxic pulmonary vasoconstriction--invited article.

Hypoxic pulmonary vasoconstriction (HPV) is an adaptive mechanism that in the face of localised alveolar hypoxia diverts blood away from poorly ventilated regions of the lung, thereby preserving ventilation/perfusion matching. HPV has been recognised for many years, but although the underlying mechanisms are known to reside within the arteries themselves, their precise nature remains unclear. There is a growing consensus that mitochondria act as the oxygen sensor, and that Ca(2+) release from ryanodine-sensitive stores and Rho kinase-mediated Ca(2+) sensitisation are critical for sustained vasoconstriction, though Ca(2+) entry via both voltage-dependent and/or -independent pathways has also been implicated. There is, however, controversy regarding the signalling pathways that link the oxygen sensor to its effectors, with three main hypotheses. The AMP-activated protein kinase (AMPK) hypothesis proposes that hypoxic inhibition of mitochondrial function increases the AMP/ATP ratio and thus activates AMPK, which in turn mediates cADPR-dependent mobilisation of ryanodine-sensitive sarcoplasmic reticulum Ca(2+) stores. In contrast the two other hypotheses invoke redox signalling, albeit in mutually incompatible ways. The Redox hypothesis proposes that hypoxia suppresses mitochondrial generation of reactive oxygen species (ROS) and causes the cytosol to become more reduced, with subsequent inhibition of K(V) channels, depolarisation and voltage-dependent Ca(2+) entry. In direct contrast the ROS hypothesis proposes that hypoxia causes an apparently paradoxical increase in mitochondrial ROS generation, and it is this increase in ROS that acts as the signalling moiety. In this article we describe our current understanding of HPV, and evidence in support of these models of oxygen-sensing.

Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery.

AIMS: We investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway. METHODS AND RESULTS: Intra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F(2 alpha) (PGF(2 alpha)) in alpha-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF(2 alpha) were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF(2 alpha) enhanced phosphorylation of three srcFK proteins at tyr-416. In alpha-toxin-permeabilized IPAs, PGF(2 alpha) enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF(2 alpha) enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF(2 alpha)-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF(2 alpha) triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. CONCLUSIONS: srcFK are activated by PGF(2 alpha) in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1.

Constriction of pulmonary artery by peroxide: role of Ca2+ release and PKC.

Reactive oxygen species are implicated in pulmonary hypertension and hypoxic pulmonary vasoconstriction. We examined the effects of low concentrations of peroxide on intrapulmonary arteries (IPA). IPAs from Wistar rats were mounted on a myograph for recording tension and estimating intracellular Ca2+ using Fura-PE3. Ca2+ sensitization was examined in alpha-toxin-permeabilized IPAs, and phosphorylation of MYPT-1 and MLC(20) was assayed by Western blot. Peroxide (30 microM) induced a vasoconstriction with transient and sustained components and equivalent elevations of intracellular Ca2+. The transient constriction was strongly suppressed by indomethacin, the TP-receptor antagonist SQ-29584, and the Rho kinase inhibitor Y-27632, whereas sustained constriction was unaffected. Neither vasoconstriction nor elevation of intracellular Ca2+ was affected by removal of extracellular Ca2+, whereas dantrolene suppressed the former and ryanodine abolished the latter. Peroxide-induced constriction of permeabilized IPAs was unaffected by Y-27632 but abolished by PKC inhibitors; these also suppressed constriction in intact IPAs. Peroxide caused translocation of PKCalpha, but had no significant effect on MYPT-1 or MLC(20) phosphorylation. We conclude that in IPAs peroxide causes transient release of vasoconstrictor prostanoids, but sustained constriction is associated with release of Ca2+ from ryanodine-sensitive stores and a PKC-dependent but Rho kinase- and MLC(20)-independent constrictor mechanism.

ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery: Role of Src-family kinases and ARHGEF1.

The role of reactive oxygen species (ROS) in smooth muscle contraction is poorly understood. We hypothesised that G-protein coupled receptor (GPCR) activation and hypoxia induce Rho-kinase activity and contraction in rat intra-pulmonary artery (IPA) via stimulation of ROS production and subsequent Src-family kinase (SrcFK) activation. The T-type prostanoid receptor agonist U46619 induced ROS production in pulmonary artery smooth muscle cells (PASMC). U46619 also induced c-Src cysteine oxidation, SrcFK auto-phosphorylation, MYPT-1 and MLC20 phosphorylation and contraction in IPA, and all these responses were inhibited by antioxidants (ebselen, Tempol). Contraction and SrcFK/MYPT-1/MLC20 phosphorylations were also inhibited by combined superoxide dismutase and catalase, or by the SrcFK antagonist PP2, while contraction and MYPT-1/MLC20 phosphorylations were inhibited by the Rho guanine nucleotide exchange factor (RhoGEF) inhibitor Y16. H2O2 and the superoxide-generating quinoledione LY83583 both induced c-Src oxidation, SrcFK auto-phosphorylation and contraction in IPA. LY83583 and H2O2-induced contractions were inhibited by PP2, while LY83583-induced contraction was also inhibited by antioxidants and Y16. SrcFK auto-phosphorylation and MYPT-1/MLC20 phosphorylation was also induced by hypoxia in IPA and this was blocked by mitochondrial inhibitors rotenone and myxothiazol. In live PASMC, sub-cellular translocation of RhoA and the RhoGEF ARHGEF1 was triggered by both U46619 and LY83583 and this translocation was blocked by antioxidants and PP2. RhoA translocation was also inhibited by an ARHGEF1 siRNA. U46619 enhanced ROS-dependent co-immunoprecipitation of ARHGEF1 with c-Src. Our results demonstrate a link between GPCR-induced cytosolic ROS or hypoxia-induced mitochondrial ROS and SrcFK activity, Rho-kinase activity and contraction. ROS and SrcFK activate RhoA via ARHGEF1.

Dietary soy modulates endothelium-dependent relaxation in aged male rats: Increased agonist-induced endothelium-derived hyperpolarising factor and basal nitric oxide activity.

We examined the effects of dietary soy on the contributions of endothelium-derived hyperpolarising factor (EDHF), nitric oxide (NO), and oxidative stress to vascular tone in isolated aortic rings and small mesenteric and pulmonary arteries in vitro. Male Wistar rats were either continuously fed a soy-deficient diet (SD) or switched from a soy-deficient diet to a soy-rich one for 6 months (SW). Contractile responses were generally smaller in arteries from SW rats. In mesenteric arteries, this difference was blunted by L-NAME, but not by charybdotoxin and apamin. Preconstricted SW mesenteric arteries were more sensitive to acetylcholine (ACh) than SD ones. This difference was unaffected by L-NAME but was abolished by charybdotoxin and apamin. Exogenous superoxide dismutase (SOD) and catalase induced powerful relaxations in aortic rings, which were smaller in those from SW rats. In mesenteric and pulmonary arteries, however, they partially inhibited ACh-mediated relaxation, and enhanced PGF(2alpha)-mediated contraction, respectively. Our results suggest that feeding aging male rats a soy-rich diet results in improved agonist-mediated EDHF production and a generalized reduction in contractile force, which is partly due to elevated basal NO. Our data also suggest a prorelaxant role for endogenous H(2)O(2) in small arteries, which is modulated by a soy diet.