Permeability and calcium signaling in lung endothelium: unpack the box .

This brief review assesses the role of Ca2+ signaling in lung endothelium in regulation of endothelial permeability. The disconnect between experimental and clinical outcomes to date may be due, in part, to the use of tools which yield information about aggregate permeability or Ca2+ responses in lung or in endothelial monolayers. The teaching point of this review is to "unpack the box," i.e. consider the many potential issues which could impact interpretation of outcomes. These include phenotypic heterogeneity and resultant segment-specific permeability responses, methodologic issues related to permeability measures, contributions from Ca2+ channels in cells other than endothelium-such as alveolar macrophages or blood leukocytes), Ca2+ dynamic patterns, rather than averaged Ca2+ responses to channel activation, and the background context, such as changes in endothelial bioenergetics with sepsis. Any or all of these issues might color interpretation of permeability and Ca2+ signaling in lung.

Endothelial hyperpermeability in severe pulmonary arterial hypertension: role of store-operated calcium entry.

Here, we tested the hypothesis that animals with severe pulmonary arterial hypertension (PAH) display increased sensitivity to vascular permeability induced by activation of store-operated calcium entry. To test this hypothesis, wild-type and transient receptor potential channel 4 (TRPC4) knockout Fischer 344 rats were given a single injection of Semaxanib (SU5416; 20 mg/kg) followed by 3 wk of exposure to hypoxia (10% oxygen) and a return to normoxia (21% oxygen) for an additional 2-3 wk. This Semaxanib/hypoxia/normoxia (i.e., SU5416/hypoxia/normoxia) treatment caused PAH, as evidenced by development of right ventricular hypertrophy, pulmonary artery medial hypertrophy, and occlusive lesions within precapillary arterioles. Pulmonary artery pressure was increased fivefold in Semaxanib/hypoxia/normoxia-treated animals compared with untreated, Semaxanib-treated, and hypoxia-treated controls, determined by isolated perfused lung studies. Thapsigargin induced a dose-dependent increase in permeability that was dependent on TRPC4 in the normotensive perfused lung. This increase in permeability was accentuated in PAH lungs but not in Semaxanib- or hypoxia-treated lungs. Fluid accumulated in large perivascular cuffs, and although alveolar fluid accumulation was not seen in histological sections, Evans blue dye conjugated to albumin was present in bronchoalveolar lavage fluid of hypertensive but not normotensive lungs. Thus PAH is accompanied by a TRPC4-dependent increase in the sensitivity to edemagenic agents that activate store-operated calcium entry.

Endothelial SK3 channel-associated Ca2+ microdomains modulate blood pressure.

Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductance Ca(2+)-activated potassium channels induces vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. Although the activation of SK3 and IK1 channels converges on EDH, their subcellular effects on signal transduction are different and not completely clear. In this study, a novel endothelium-specific SK3 knockout (SK3(-/-)) mouse model was utilized to specifically examine the contribution of SK3 channels to mesenteric artery vasorelaxation, endothelial Ca(2+) dynamics, and blood pressure. The absence of SK3 expression was confirmed using real-time quantitative PCR and Western blot analysis. Functional studies showed impaired EDH-mediated vasorelaxation in SK3(-/-) small mesenteric arteries. Immunostaining results from SK3(-/-) vessels confirmed the absence of SK3 and further showed altered distribution of transient receptor potential channels, type 4 (TRPV4). Electrophysiological recordings showed a lack of SK3 channel activity, while TRPV4-IK1 channel coupling remained intact in SK3(-/-) endothelial cells. Moreover, Ca(2+) imaging studies in SK3(-/-) endothelium showed increased Ca(2+) transients with reduced amplitude and duration under basal conditions. Importantly, SK3(-/-) endothelium lacked a distinct type of Ca(2+) dynamic that is sensitive to TRPV4 activation. Blood pressure measurements showed that the SK3(-/-) mice were hypertensive, and the blood pressure increase was further enhanced during the 12-h dark cycle when animals are most active. Taken together, our results reveal a previously unappreciated SK3 signaling microdomain that modulates endothelial Ca(2+) dynamics, vascular tone, and blood pressure.

Functional coupling of TRPV4, IK, and SK channels contributes to Ca(2+)-dependent endothelial injury in rodent lung.

Our previous work has shown that the increased lung endothelial permeability response to 14,15-epoxyeicosatrienoic acid (14,15-EET) in rat lung requires Ca(2+) entry via vanilloid type-4 transient receptor potential (TRPV4) channels. Recent studies suggest that activation of TRPV4 channels in systemic vascular endothelium prolongs agonist-induced hyperpolarization and amplifies Ca(2+) entry by activating Ca(2+)-activated K(+) (KCa) channels, resulting in vessel relaxation. Activation of endothelial KCa channels thus has potential to increase the electrochemical driving force for Ca(2+) influx via TRPV4 channels and to amplify permeability responses to TRPV4 activation in lung. To examine this hypothesis, we used Western blot analysis, electrophysiological recordings, and isolated-lung permeability measurements to document expression of TRPV4 and KCa channels and the potential for functional coupling. The results show that rat pulmonary microvascular endothelial cells express TRPV4 and 3 KCa channels of different conductances: large (BK), intermediate (IK), and small (SK3). However, TRPV4 channel activity modulates the IK and SK3, but not the BK, channel current density. Furthermore, the TRPV4-mediated permeability response to 14,15-EET in mouse lung is significantly attenuated by pharmacologic blockade of IK and SK3, but not BK, channels. Collectively, this functional coupling suggests that endothelial TRPV4 channels in rodent lung likely form signaling microdomains with IK and SK3 channels and that the integrated response dictates the extent of lung endothelial injury caused by 14,15-EET.

The effects of mitochondrial complex I blockade on ATP and permeability in rat pulmonary microvascular endothelial cells in culture (PMVEC) are overcome by coenzyme Q1 (CoQ1).

In isolated rat lung perfused with a physiological saline solution (5.5mM glucose), complex I inhibitors decrease lung tissue ATP and increase endothelial permeability (Kf), effects that are overcome using an amphipathic quinone (CoQ1) [Free Radic. Biol. Med.65:1455-1463; 2013]. To address the microvascular endothelial contribution to these intact lung responses, rat pulmonary microvascular endothelial cells in culture (PMVEC) were treated with the complex I inhibitor rotenone and ATP levels and cell monolayer permeability (PS) were measured. There were no detectable effects on ATP or permeability in experimental medium that, like the lung perfusate, contained 5.5mM glucose. To unmask a potential mitochondrial contribution, the glucose concentration was lowered to 0.2mM. Under these conditions, rotenone decreased ATP from 18.4±1.6 (mean±SEM) to 4.6±0.8nmol/mg protein, depolarized the mitochondrial membrane potential (Δψm) from -129.0±3.7 (mean±SEM) to -92.8±5.5mV, and decreased O2 consumption from 2.0±0.1 (mean±SEM) to 0.3±0.1nmol/min/mg protein. Rotenone also increased PMVEC monolayer permeability (reported as PS in nl/min) to FITC-dextran (~40kDa) continually over a 6 h time course. When CoQ1 was present with rotenone, normal ATP (17.4±1.4nmol/mg protein), O2 consumption (1.5±0.1nmol/min/mg protein), Δψm (-125.2±3.3mV), and permeability (PS) were maintained. Protective effects of CoQ1 on rotenone-induced changes in ATP, O2 consumption rate, Δψm, and permeability were blocked by dicumarol or antimycin A, inhibitors of the quinone-mediated cytosol-mitochondria electron shuttle [Free Radic. Biol. Med.65:1455-1463; 2013]. Key rotenone effects without and with CoQ1 were qualitatively reproduced using the alternative complex I inhibitor, piericidin A. We conclude that, as in the intact lung, PMVEC ATP supply is linked to the permeability response to complex I inhibitors. In contrast to the intact lung, the association in PMVEC was revealed only after decreasing the glucose concentration in the experimental medium from 5.5 to 0.2mM.

Role of MMP2 and MMP9 in TRPV4-induced lung injury.

Ca(2+) entry through transient receptor potential vanilloid 4 (TRPV4) results in swelling, blebbing, and detachment of the epithelium and capillary endothelium in the intact lung. Subsequently, increased permeability of the septal barrier and alveolar flooding ensue. In this study, we tested the hypothesis that TRPV4 activation provides a Ca(2+) source necessary for proteolytic disruption of cell-cell or cell-matrix adhesion by matrix metalloproteinases (MMPs) 2 and 9, thus increasing septal barrier permeability. In our study, C57BL/6 or TRPV4(-/-) mouse lungs were perfused with varying doses of the TRPV4 agonist GSK-1016790A (Sigma) and then prepared for Western blot. Lung injury, assessed by increases in lung wet-to-dry weight ratios and total protein levels in the bronchoalveolar lavage fluid, was increased in a dose-dependent fashion in TRPV4(+/+) but not TRPV4(-/-) lungs. In concert with lung injury, we detected increased active MMP2 and MMP9 isoforms, suggesting that TRPV4 can provide the Ca(2+) source necessary for increased MMP2/9 activation. Furthermore, tissue inhibitor of metalloproteinases (TIMP) 2 levels in the TRPV4-injured lungs were decreased, suggesting that TRPV4 activation increases the availability of these active MMPs. We then determined whether MMP2 and MMP9 mediate TRPV4-induced lung injury. Pharmacological blockade (SB-3CT, 1 µM; Sigma) of MMP2 and MMP9 resulted in protection against TRPV4-induced lung injury. We conclude that TRPV4 activation and the subsequent Ca(2+) transient initiates a rapid cascade of events leading to release and activation of the gelatinase MMPs, which then contribute to lung injury.

TRPV4 calcium entry and surface expression attenuated by inhibition of myosin light chain kinase in rat pulmonary microvascular endothelial cells.

In previous studies, blockade or gene deletion of either myosin light chain kinase (MLCK) or the mechanogated transient receptor potential vanilloid 4 (TRPV4) channel attenuated mechanical lung injury. To determine their effects on calcium entry, rat pulmonary microvascular endothelial cells (RPMVEC) were labeled with fluo-4 and calcium entry initiated with the TRPV4 agonist, 4α-phorbol 12, 13-didecanoate (4αPDD). Mean calcium transients peaked at ∼25 sec and persisted ∼500 sec. The 4αPDD response was essentially abolished in calcium-free media, or after pretreatment with the MLCK inhibitor, ML-7. ML-7 also attenuated the 4αPDD-induced inward calcium current measured directly using whole-cell patch clamp. Pretreatment with dynasore, an inhibitor of dynamin produced an initial calcium transient followed by a 4αPDD transient of unchanged peak intensity. Automated averaging of areas under the curve (AUC) of calcium transients in individual cells indicated total calcium activity with a relationship between treatment groups of ML-7 + 4αPDD < 4αPDD only < dynasore + 4αPDD. Measurement of biotinylated surface TRPV4 protein indicated a significant reduction after ML-7 pretreatment, but no significant change with dynasore treatment. RPMVEC monolayer electrical resistances were decreased by only 3% with 10 µmol/L 4αPDD and the response was dose-related. Dynasore alone produced a 29% decrease in resistance, but neither ML-7 nor dynasore affected the subsequent 4αPDD resistance response. These studies suggest that MLCK may inhibit mechanogated calcium responses through reduced surface expression of stretch activated TRPV4 channels in the plasma membrane.

Depleted energy charge and increased pulmonary endothelial permeability induced by mitochondrial complex I inhibition are mitigated by coenzyme Q1 in the isolated perfused rat lung.

Mitochondrial dysfunction is associated with various forms of lung injury and disease that also involve alterations in pulmonary endothelial permeability, but the relationship, if any, between the two is not well understood. This question was addressed by perfusing isolated intact rat lung with a buffered physiological saline solution in the absence or presence of the mitochondrial complex I inhibitor rotenone (20 µM). Compared to control, rotenone depressed whole lung tissue ATP from 5.66 ± 0.46 (SEM) to 2.34 ± 0.15 µmol · g(-1) dry lung, with concomitant increases in the ADP:ATP and AMP:ATP ratios. Rotenone also increased lung perfusate lactate (from 12.36 ± 1.64 to 38.62 ± 3.14 µmol · 15 min(-1) perfusion · g(-1) dry lung) and the lactate:pyruvate ratio, but had no detectable impact on lung tissue GSH:GSSG redox status. The amphipathic quinone coenzyme Q1 (CoQ1; 50 µM) mitigated the impact of rotenone on the adenine nucleotide balance, wherein mitigation was blocked by NAD(P)H-quinone oxidoreductase 1 or mitochondrial complex III inhibitors. In separate studies, rotenone increased the pulmonary vascular endothelial filtration coefficient (Kf) from 0.043 ± 0.010 to 0.156 ± 0.037 ml · min(-1) · cm H2O(-1) · g(-1) dry lung, and CoQ1 protected against the effect of rotenone on Kf. A second complex I inhibitor, piericidin A, qualitatively reproduced the impact of rotenone on Kf and the lactate:pyruvate ratio. Taken together, the observations imply that pulmonary endothelial barrier integrity depends on mitochondrial bioenergetics as reflected in lung tissue ATP levels and that compensatory activation of whole lung glycolysis cannot protect against pulmonary endothelial hyperpermeability in response to mitochondrial blockade. The study further suggests that low-molecular-weight amphipathic quinones may have therapeutic utility in protecting lung barrier function in mitochondrial insufficiency.

Transient receptor potential channels and regulation of lung endothelial permeability.

This review highlights our current knowledge regarding expression of transient receptor potential (TRP) cation channels in lung endothelium and evidence for their involvement in regulation of lung endothelial permeability. Six mammalian TRP families have been identified and organized on the basis of sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPA (ankyrin). To date, only TRPC1/4, TRPC6, TRPV4, and TRPM2 have been extensively studied in lung endothelium. Calcium influx through each of these channels has been documented to increase lung endothelial permeability, although their channel-gating mechanisms, downstream signaling mechanisms, and impact on endothelial structure and barrier integrity differ. While other members of the TRPC, TRPV, and TRPM families may be expressed in lung endothelium, we have little or no evidence linking these to regulation of lung endothelial permeability. Further, neither the expression nor functional role(s) of any TRPML, TRPP, and TRPA family members has been studied in lung endothelium. In addition to this assessment organized by TRP channel family, we also discuss TRP channels and lung endothelial permeability from the perspective of lung endothelial heterogeneity, using outcomes of studies focused on TRPC1/4 and TRPV4 channels. The diversity within the TRP channel family and the relative paucity of information regarding roles of a number of these channels in lung endothelium make this field ripe for continued investigation.

An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.

Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

Structure and composition of pulmonary arteries, capillaries, and veins.

The pulmonary vasculature comprises three anatomic compartments connected in series: the arterial tree, an extensive capillary bed, and the venular tree. Although, in general, this vasculature is thin-walled, structure is nonetheless complex. Contributions to structure (and thus potentially to function) from cells other than endothelial and smooth muscle cells as well as those from the extracellular matrix should be considered. This review is multifaceted, bringing together information regarding (i) classification of pulmonary vessels, (ii) branching geometry in the pulmonary vascular tree, (iii) a quantitative view of structure based on morphometry of the vascular wall, (iv) the relationship of nerves, a variety of interstitial cells, matrix proteins, and striated myocytes to smooth muscle and endothelium in the vascular wall, (v) heterogeneity within cell populations and between vascular compartments, (vi) homo- and heterotypic cell-cell junctional complexes, and (vii) the relation of the pulmonary vasculature to that of airways. These issues for pulmonary vascular structure are compared, when data is available, across species from human to mouse and shrew. Data from studies utilizing vascular casting, light and electron microscopy, as well as models developed from those data, are discussed. Finally, the need for rigorous quantitative approaches to study of vascular structure in lung is highlighted.

TRPV4 channels augment macrophage activation and ventilator-induced lung injury.

We have previously implicated transient receptor potential vanilloid 4 (TRPV4) channels and alveolar macrophages in initiating the permeability increase in response to high peak inflation pressure (PIP) ventilation. Alveolar macrophages were harvested from TRPV4(-/-) and TRPV4(+/+) mice and instilled in the lungs of mice of the opposite genotype. Filtration coefficients (K(f)) measured in isolated perfused lungs after ventilation with successive 30-min periods of 9, 25, and 35 cmH(2)O PIP did not significantly increase in lungs from TRPV4(-/-) mice but increased >2.2-fold in TRPV4(+/+) lungs, TRPV4(+/+) lungs instilled with TRPV4(-/-) macrophages, and TRPV4(-/-) lungs instilled with TRPV4(+/+) macrophages after ventilation with 35 cmH(2)O PIP. Activation of TRPV4 with 4-alpha-phorbol didecanoate (4alphaPDD) significantly increased intracellular calcium, superoxide, and nitric oxide production in TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. Cross-sectional areas increased nearly 3-fold in TRPV4(+/+) macrophages compared with TRPV4(-/-) macrophages after 4alphaPDD. Immunohistochemistry staining of lung tissue for nitrotyrosine revealed increased amounts in high PIP ventilated TRPV4(+/+) lungs compared with low PIP ventilated TRPV4(+/+) or high PIP ventilated TRPV4(-/-) lungs. Thus TRPV4(+/+) macrophages restored susceptibility of TRPV4(-/-) lungs to mechanical injury. A TRPV4 agonist increased intracellular calcium and reactive oxygen and nitrogen species in harvested TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. K(f) increases correlated with tissue nitrotyrosine, a marker of peroxynitrite production.

Alpha1G T-type calcium channel selectively regulates P-selectin surface expression in pulmonary capillary endothelium.

Regulated P-selectin surface expression provides a rapid measure for endothelial transition to a proinflammatory phenotype. In general, P-selectin surface expression results from Weibel-Palade body (WPb) exocytosis. Yet, it is unclear whether pulmonary capillary endothelium possesses WPbs or regulated P-selectin surface expression and, if so, how inflammatory stimuli initiate exocytosis. We used immunohistochemistry, immunofluorescence labeling, ultrastructural assessment, and an isolated perfused lung model to demonstrate that capillary endothelium lacks WPbs but possesses P-selectin. Thrombin stimulated P-selectin surface expression in both extra-alveolar vessel and alveolar capillary endothelium. Only in capillaries was the thrombin-stimulated P-selectin surface expression considerably mitigated by pharmacologic blockade of the T-type channel or genetic knockout of the T-type channel alpha(1G)-subunit. Depolarization of endothelial plasma membrane via high K(+) perfusion capable of eliciting cytosolic Ca(2+) transients also provoked P-selectin surface expression in alveolar capillaries that was abolished by T-type channel blockade or alpha(1G) knockout. Our findings reveal an intracellular WPb-independent P-selectin pool in pulmonary capillary endothelium, where the regulated P-selectin surface expression is triggered by Ca(2+) transients evoked through activation of the alpha(1G) T-type channel.

Impact of epoxyeicosatrienoic acids in lung ischemia-reperfusion injury.

OBJECTIVE: Epoxyeicosatrienoic acids (EETs) are protective in both myocardial and brain ischemia, variously attributed to activation of K(ATP) channels or blockade of adhesion molecule upregulation. In this study, we tested whether EETs would be protective in lung ischemia-reperfusion injury. METHODS: The filtration coefficient (K(f)), a measure of endothelial permeability, and expression of the adhesion molecules vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) were measured after 45 minutes ischemia and 30 minutes reperfusion in isolated rat lungs. RESULTS: K(f) increased significantly after ischemia-reperfusion alone vs time controls, an effect dependent upon extracellular Ca(2+) although not on the EET-regulated channel TRPV4. Inhibition of endogenous EET degradation or administration of exogenous 11,12- or 14,-15-EET at reperfusion significantly limited the permeability response to ischemia-reperfusion. The beneficial effect of 11,12-EET was not prevented by blockade of K(ATP) channels nor by blockade of TRPV4. Finally, 11,12-EET-dependent alteration in adhesion molecules expression is unlikely to explain its beneficial effect, since the expression of the adhesion molecules VCAM and ICAM in lung after ischemia-reperfusion was similar to that in controls. CONCLUSION: EETs are beneficial in the setting of lung ischemia-reperfusion, when administered at reperfusion. However, further study will be needed to elucidate the mechanism of action.

Ca2+ entry via alpha1G and TRPV4 channels differentially regulates surface expression of P-selectin and barrier integrity in pulmonary capillary endothelium.

Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca(2+) influx in response to diverse environmental stimuli. However, it is not clear whether Ca(2+) influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the alpha(1G) T-type Ca(2+) channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca(2+), immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K(+) perfusate induces Ca(2+) entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca(2+) entry in septal endothelium evoked by the selective TRPV4 activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of alpha(1G) abolishes depolarization-induced Ca(2+) entry and surface expression of P-selectin but does not prevent 4alpha-PDD-activated Ca(2+) entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4alpha-PDD-activated Ca(2+) entry and the increase in permeability, while not impacting depolarization-induced Ca(2+) entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca(2+) entry through alpha(1G) and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.

Physiological determinants of the pulmonary filtration coefficient.

Current emphasis on translational application of genetic models of lung disease has renewed interest in the measurement of the gravimetric filtration coefficient (K(f)) as a means to assess vascular permeability changes in isolated perfused lungs. The K(f) is the product of the hydraulic conductivity and the filtration surface area, and is a sensitive measure of vascular fluid permeability when the pulmonary vessels are fully recruited and perfused. We have observed a remarkable consistency of the normalized baseline K(f) values between species with widely varying body weights from mice to sheep. Uniformity of K(f) values can be attributed to the thin alveolar capillary barrier required for gas exchange and the conserved matching of lung vascular surface area to the oxygen requirements of the body mass. An allometric correlation between the total lung filtration coefficient (K(f,t)) and body weight in several species (r(2)=1.00) had a slope that was similar to those reported for alveolar and pulmonary capillary surface areas and pulmonary diffusion coefficients determined by morphometric methods in these species. A consistent K(f) is dependent on accurately separating the filtration and vascular volume components of lung weight gain, then K(f) is a consistent and repeatable index of lung vascular permeability.

High vascular pressure-induced lung injury requires P450 epoxygenase-dependent activation of TRPV4.

High vascular pressure targets the lung septal network, causing acute lung injury. While calcium entry in septal endothelium has been implicated, the channel involved is not known. This study tested the hypothesis that the vanilloid transient receptor potential channel, TRPV4, is a critical participant in the permeability response to high vascular pressure. Isolated lungs from TRPV4(+/+) or TRPV4(-/-) mice were studied at baseline or during high pressure challenge. Permeability was assessed via the filtration coefficient. Endothelial calcium transients were assessed using epifluorescence microscopy of the lung subpleural network. Light microscopy and point counting were used to determine the alveolar fluid volume fraction, a measure of alveolar flooding. Baseline permeability, calcium intensity, and alveolar flooding were no different in TRPV4(+/+) versus TRPV4(-/-) lungs. In TRPV4(+/+) lungs, the high pressure-induced permeability response was significantly attenuated by low calcium perfusate, the TRPV antagonist ruthenium red, the phospholipase A(2) inhibitor methyl arachidonyl fluorophosphonate, or the P450 epoxygenase inhibitor propargyloxyphenyl hexanoic acid. Similarly, the high pressure-induced calcium transient in TRPV4(+/+) lungs was attenuated with ruthenium red or the epoxygenase inhibitor. High vascular pressure increased the alveolar fluid volume fraction compared with control. In lungs from TRPV4(-/-) mice, permeability, calcium intensity, and alveolar fluid volume fraction were not increased. These data support a role for P450-derived epoxyeicosatrienoic acid-dependent regulation of calcium entry via TRPV4 in the permeability response to high vascular pressure.

TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs.

We have previously implicated calcium entry through stretch-activated cation channels in initiating the acute pulmonary vascular permeability increase in response to high peak inflation pressure (PIP) ventilation. However, the molecular identity of the channel is not known. We hypothesized that the transient receptor potential vanilloid-4 (TRPV4) channel may initiate this acute permeability increase because endothelial calcium entry through TRPV4 channels occurs in response to hypotonic mechanical stress, heat, and P-450 epoxygenase metabolites of arachidonic acid. Therefore, permeability was assessed by measuring the filtration coefficient (K(f)) in isolated perfused lungs of C57BL/6 mice after 30-min ventilation periods of 9, 25, and 35 cmH(2)O PIP at both 35 degrees C and 40 degrees C. Ventilation with 35 cmH(2)O PIP increased K(f) by 2.2-fold at 35 degrees C and 3.3-fold at 40 degrees C compared with baseline, but K(f) increased significantly with time at 40 degrees C with 9 cmH(2)O PIP. Pretreatment with inhibitors of TRPV4 (ruthenium red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole) prevented the increases in K(f). In TRPV4(-/-) knockout mice, the high PIP ventilation protocol did not increase K(f) at either temperature. We have also found that lung distention caused Ca(2+) entry in isolated mouse lungs, as measured by ratiometric fluorescence microscopy, which was absent in TRPV4(-/-) and ruthenium red-treated lungs. Alveolar and perivascular edema was significantly reduced in TRPV4(-/-) lungs. We conclude that rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation.

Special topics issue of microcirculation: ion channels and pulmonary vascular function.

Unique features of the pulmonary circulation impact its function in health and disease, not the least of which is the existence of developmentally distinct, functionally heterogeneous extra-alveolar and septal capillary networks. The impact of ion channel expression and regulation in lung vascular smooth muscle or endothelium in these vascular compartments provides a focus for this special topics issue. Reviews and original contributions from experts in the field discuss two broad groups of ion channels, drawing on studies utilizing biophysical and molecular approaches in heterologous expression systems, in vitro approaches in pulmonary vascular smooth muscle and endothelial cells, and physiologic studies in animal models of chronic pulmonary hypertension. First, channels involved in membrane depolarization and related alterations in vascular tone, and shear sensing or exocytosis by endothelium are discussed: voltage-gated potassium channels, ATP-regulated potassium channels and L- and T- type voltage-gated calcium channels. The second series of reviews discusses the role of calcium influx pathways provided by transient receptor potential channels in regulation of pulmonary vascular tone or vascular remodeling, and endothelial barrier function. Understanding the role of ion channels in pulmonary vascular pathophysiology may be critical to development of new therapeutic strategies.