Hiroaki Kume

Actin Cytoskeleton Regulates Stretch-Activated Ca2+ Influx in Human Pulmonary Microvascular Endothelial Cells

During high tidal volume mechanical ventilation in patients with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), regions of the lung are exposed to excessive stretch, causing inflammatory responses and further lung damage. In this study, the effects of mechanical stretch on intracellular Ca(2+) concentration ([Ca(2+)](i)), which regulates a variety of endothelial properties, were investigated in human pulmonary microvascular endothelial cells (HPMVECs). HPMVECs grown on fibronectin-coated silicon chambers were exposed to uniaxial stretching, using a cell-stretching apparatus. After stretching and subsequent unloading, [Ca(2+)](i), as measured by fura-2 fluorescence, was transiently increased in a strain amplitude-dependent manner. The elevation of [Ca(2+)](i) induced by stretch was not evident in the Ca(2+)-free solution and was blocked by Gd(3+), a stretch-activated channel inhibitor, or ruthenium red, a transient receptor potential vanilloid inhibitor. The disruption of actin polymerization with cytochalasin D inhibited the stretch-induced elevation of [Ca(2+)](i). In contrast, increases in [Ca(2+)](i) induced by thapsigargin or thrombin were not affected by cytochalasin D. Increased actin polymerization with sphingosine-1-phosphate or jasplakinolide enhanced the stretch-induced elevation of [Ca(2+)](i). A simple network model of the cytoskeleton was also developed in support of the notion that actin stress fibers are required for efficient force transmission to open stretch-activated Ca(2+) channels. In conclusion, mechanical stretch activates Ca(2+) influx via stretch-activated channels which are tightly regulated by the actin cytoskeleton different from other Ca(2+) influx pathways such as receptor-operated and store-operated Ca(2+) entries in HPMVECs. These results suggest that abnormal Ca(2+) homeostasis because of excessive mechanical stretch during mechanical ventilation may play a role in the progression of ALI/ARDS.

Mechanical stretch enhances IL-8 production in pulmonary microvascular endothelial cells

In patients with acute respiratory distress syndrome, mechanical over-distension of the lung by a large tidal volume causes further damage and inflammation, called ventilator-induced lung injury (VILI), however, it is unclear how mechanical stretch affects the cellular functions or morphology in human pulmonary microvascular endothelial cells (HPMVECs). IL-8 has been proposed to play an important role in the progression of VILI by activating neutrophils. We demonstrated that HPMVECs exposed to cyclic uni-axial stretch produce IL-8 protein with p38 activation in strain- and time-dependent manners. The IL-8 synthesis was not regulated by other signal transduction pathways such as ERK1/2, JNK, or stretch-activated Ca(2+) channels. Moreover, cyclic stretch enhanced IL-6 and monocyte chemoattractant protein-1 production and reoriented cell perpendicularly to the stretch axis accompanied by actin polymerization. Taken together, IL-8 production by HPMVECs due to excessive mechanical stretch may activate neutrophilic inflammation, which leads to VILI.

Effect of ambulatory oxygen on exertional dyspnea in IPF patients without resting hypoxemia

BACKGROUND AND OBJECTIVEnThe effects of ambulatory oxygen for idiopathic pulmonary fibrosis (IPF) patients without resting hypoxemia have not been elucidated. The purpose of this study was to assess the effect of ambulatory oxygen on dyspnea in IPF patients without resting hypoxemia but with desaturation on exertion.nnnMETHODSnThis was a double-blind, placebo-controlled, randomized crossover trial of ambulatory oxygen versus ambulatory air. Patients with IPF who had a partial pressure of arterial oxygen (PaO2) between 60xa0mm Hg and 80xa0mm Hg at rest, and desaturation of 88% or less in a room-air 6-min walk test were eligible. Patients underwent a standardized 6-min walk test and a 6-min free walk test under each ambulatory gas. Oxygen and air were provided at 4xa0L/min intranasally. Dyspnea was evaluated immediately, 1, and 2xa0min after the tests.nnnRESULTSnTwenty patients (16 men), with a mean age of 73.5 (SD 4.1) years, % predicted forced vital capacity (FVC) of 71.0 (13.3) %, % predicted diffusion capacity for carbon monoxide (DLco) of 57.0 (13.3) %, and PaO2 of 72.5 (5.4) mm Hg were recruited. No significant differences in dyspnea were observed between ambulatory oxygen and air at each time point. However, some patients showed improvement in dyspnea with oxygen on an individual basis.nnnCONCLUSIONSnSince oxygen provides no additional benefit over air in terms of exertional dyspnea for IPF patients without resting hypoxemia, routine prescription of ambulatory oxygen is not recommended. However, assessment on an individual basis is necessary. Trial registration. UMIN Clinical Trial Registry; No.:UMIN000005098; URL:http://www.umin.ac.jp/ctr/.

Research and development of bronchodilators for asthma and COPD with a focus on G protein/KCa channel linkage and β2-adrenergic intrinsic efficacy.

Bronchodilators are used to improve symptoms and lung function in asthma and COPD. Airway smooth muscle tone is regulated by both muscarinic and β2-adrenergic receptor activity. Large-conductance Ca(2+)-activated K(+) (KCa) channels are activated by β2-adrenergic receptor agonists, via Gs, and suppressed by muscarinic receptor antagonists via Gi. This functional antagonism converges on the G protein/KCa channel linkages. Membrane potential regulated by KCa channels contributes to airway smooth muscle tension via Ca(2+) influx passing through voltage-dependent Ca(2+) (VDC) channels. The Gs/KCa/VDC channel linkage is a key process in not only physiological effects, but also in dysfunction of β2-adrenergic receptors and airway remodeling. Moreover, this pathway is involved in the synergistic effects between β2-adrenergic receptor agonists and muscarinic receptor antagonists. Intrinsic efficacy is also an important characteristic for both maintenance and loss of β2-adrenergic action. Allosteric modulators of G protein-coupled receptors contribute not only to this synergistic effect between β2-adrenergic and muscarinic M2 receptors, but also to intrinsic efficacy. The effects of weak partial agonists are suppressed by lowering receptor number, disordering receptor function, and enhancing functional antagonism; in contrast, those of full or strong partial agonists are not suppressed. Excessive exposure to full agonists causes β2-adrenergic desensitization; in contrast, exposure to partial agonists does not cause desensitization. Intrinsic efficacy may provide the rationale for the clinical use of β2-adrenergic receptor agonists in asthma and COPD. In conclusion, the G protein/KCa linkage and intrinsic efficacy (allosteric effects) may be therapeutic targets for research and development of novel agents against both airway obstruction and airway remodeling.

Activation of the Prefrontal Cortex Is Associated with Exertional Dyspnea in Chronic Obstructive Pulmonary Disease

Background: Exertional dyspnea is the primary symptom that limits exercise in patients with chronic obstructive pulmonary disease (COPD). It is unknown which activated brain area is associated with this symptom in COPD patients. Objectives: To investigate the activation of cortical areas associated with dyspnea during exercise in COPD patients. Methods: COPD patients (n = 10) and age-matched controls (n = 10) performed mild-intensity constant work rate cycle exercise (40% of their symptom-limited peak work rates) for 10 min, while cerebral hemodynamics and oxygenation were measured by near-infrared spectroscopy (NIRS). Ventilatory responses (breathing pattern and pulmonary gas exchange) and Borg scale ratings of dyspnea and leg fatigue were measured during exercise. Three NIRS probes were placed over the prefrontal and temporoparietal cortical regions of the subjects’ heads. Changes in cortical oxyhemoglobin (oxy-Hb), deoxyhemoglobin (deoxy-Hb), and total hemoglobin (total Hb) concentrations from baseline recordings were measured. Increased oxy-Hb (oxygenation) was assumed to reflect cortical activation. Results: Oxy-Hb concentration was significantly increased in the prefrontal region during exercise in both groups but not in the temporoparietal regions. The change in prefrontal oxy-Hb concentration of COPD patients was not different from that of controls. Dyspnea scores were positively correlated with changes in oxy-Hb concentrations of the prefrontal regions in both groups. Multivariate analysis showed that oxy-Hb concentration in the prefrontal region was the best predictor of dyspnea in both groups. Conclusions: Exertional dyspnea was related to activation (oxygenation) of the prefrontal cortex in COPD patients and control subjects.

Influence of leukotriene pathway polymorphisms on clinical responses to montelukast in Japanese patients with asthma

What is known and Objective:u2002 Montelukast, a cysteinyl leukotriene receptor 1 antagonist, is safe and efficacious in patients with asthma. The mechanisms underlying the significant interpatient variability in response to montelukast are not clear but are believed to be, in part, because of genetic variability.

Exertional dyspnoea and cortical oxygenation in patients with COPD

This study was designed to investigate the association of perceived dyspnoea intensity with cortical oxygenation and cortical activation during exercise in patients with chronic obstructive pulmonary disease (COPD) and exertional hypoxaemia. Low-intensity exercise was performed at a constant work rate by patients with COPD and exertional hypoxaemia (n=11) or no hypoxaemia (n=16), and in control participants (n=11). Cortical oxyhaemoglobin (oxy-Hb) and deoxyhaemoglobin (deoxy-Hb) concentrations were measured by multichannel near-infrared spectroscopy. Increased deoxy-Hb is assumed to reflect impaired oxygenation, whereas decreased deoxy-Hb signifies cortical activation. Exercise decreased cortical deoxy-Hb in control and nonhypoxaemic patients. Deoxy-Hb was increased in hypoxaemic patients and oxygen supplementation improved cortical oxygenation. Decreased deoxy-Hb in the pre-motor cortex (PMA) was significantly correlated with exertional dyspnoea in control participants and patients with COPD without hypoxaemia. In contrast, increased cortical deoxy-Hb concentration was correlated with dyspnoea in patients with COPD and hypoxaemia. With the administration of oxygen supplementation, exertional dyspnoea was correlated with decreased deoxy-Hb in the PMA of COPD patients with hypoxaemia. During exercise, cortical oxygenation was impaired in patients with COPD and hypoxaemia compared with control and nonhypoxaemic patients; this difference was ameliorated with oxygen supplementation. Exertional dyspnoea was related to activation of the pre-motor cortex in COPD patients. Exertional dyspnoea was related to activation of the pre-motor cortex in COPD patients http://ow.ly/QUHfC

Fat-free mass index predicts survival in patients with idiopathic pulmonary fibrosis.

Detailed body composition, such as fat‐free mass, has not been examined in idiopathic pulmonary fibrosis (IPF). We investigated whether the fat‐free mass index (FFMI), an index of lean body mass, predicted survival.

Role of Sphingosine-1-Phosphate in β-adrenoceptor Desensitization via Ca2+ Sensitization in Airway Smooth Muscle

BACKGROUNDnThe correlation between inflammatory cells and airway smooth muscle plays fundamental roles in the pathophysiology of asthma. This study was designed to determine whether pre-exposure of airway smooth muscle to sphingosine-1-phosphate (S1P), which is released from mast cells by allergic reactions, causes a deterioration of β-adrenoceptor function.nnnMETHODSnIsometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F(340)/F(380)), an indicator of intracellular Ca2+ levels, were simultaneously measured using fura-2 loaded guinea-pig tracheal tissues. Intracellular cAMP levels were also measured.nnnRESULTSnPre-exposure to S1P caused a reduction in the inhibitory effects of 0.3μM isoprenaline, a β-adrenoceptor agonist, and 10μM forskolin, a direct activator of adenylyl cyclase, against 1μM methacholine-induced contraction in concentration- and time- dependent manners. In contrast, the values of F(340)/F(380) were not augmented under this experimental condition. After incubation with S1P in the presence of 0.001-1μM Y-27632, a Rho-kinase inhibitor, the reduced responsiveness to forskolin induced by S1P was reversed in a concentration-dependent manner. Moreover, pre-treatment with pertussis toxin (PTX), an inhibitor of G(i), suppressed the loss of forskolin-induced relaxation induced by S1P. Pre-exposure to S1P markedly inhibited the augmentation of cAMP accumulation induced by forskolin. However, addition of Y-27632 and pre-exposure to PTX returned forsokin-induced cAMP accumulation to the control level.nnnCONCLUSIONSnPre-exposure to S1P causes heterologus desensitization of β-adrenoceptors by increasing the sensitivity of airway smooth muscle to intracellular Ca2+. Ca2+ sensitization regulated by G(i) and Rho-kinase is involved in this phenomenon.

Large-Conductance Calcium-Activated Potassium Channels

Large-conductanceCa2+-activated K+ (BKCa, MaxiK) channels are abundantly distributed on the cell membrane and ubiquitously expressed in a variety of tissues except cardiac muscle. These channels have a conductance of approximately 250 pS and are intracellular Ca2+- and membrane potential-sensitive. Activation of these channels causes membrane hyperpolarization mediated by an increase in outward K+ currents, leading to a reduction in tension in smooth muscle such as vessels and airways. The BKCa channels are a tetramer of a pore-forming α-subunit encoded by a single gene (Slo, KCNMA1) associated with modulatory β-subunits (KCNMB1-4). The α-subunit consists of a transmembrane (TM) domain-sensing membrane potential and conducting ion, and a cytoplasmic domain-sensing Ca2+. The β-subunits comprise two TMs, and their expression is tissue specific. In smooth muscle cells, the BKCa channels are markedly activated due to increased local Ca2+ concentrations via Ca2+ release from the sarcoplasmic reticulum (Ca2+ sparks). The coupling of Ca2+ sparks to hundreds of BKCa channels causes spontaneous outward currents (STOCs), leading to relaxation via membrane hyperpolarization. The activity and expression of the BKCa channels are modulated by a variety of factors such as phosphorylation (e.g., protein kinase A, protein kinase G) and other metabolites (e.g., reactive oxygen species, estrogen, nitric oxide). Therefore, altered BKCa channels play a key role in vital body functions (e.g., development, pregnancy) and diseases (e.g., hypertension, diabetes). These channels may be a therapeutic target for a variety of diseases.