This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 99/9/988    most recent 01.RES.0000247065.11756.19v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Alvarez, D. F. Articles by Townsley, M. I. Search for Related Content PubMed PubMed Citation Articles by Alvarez, D. F. Articles by Townsley, M. I. Related Collections Other Vascular biology Genetically altered mice Ion channels/membrane transport Pulmonary biology and circulation Related Article

(Circulation Research. 2006;99:988.)
© 2006 American Heart Association, Inc.

Cellular Biology

Transient Receptor Potential Vanilloid 4–Mediated Disruption of the Alveolar Septal Barrier

A Novel Mechanism of Acute Lung Injury

Diego F. Alvarez, Judy A. King, David Weber, Emile Addison, Wolfgang Liedtke, Mary I. Townsley

From the Departments of Physiology (D.F.A., D.W., E.A., M.I.T.), Pharmacology (J.A.K.), and Pathology (J.A.K.) and Center for Lung Biology (D.F.A., J.A.K., M.I.T.), University of South Alabama, Mobile; and Departments of Medicine, Neurology, and Neurobiology (W.L.), Duke University, Durham, NC.

Correspondence to Mary I. Townsley, PhD, Department of Physiology, Center for Lung Biology, MSB 3074, 307 University Blvd, University of South Alabama, Mobile, AL 36688. E-mail mtownsley{at}usouthal.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Disruption of the alveolar septal barrier leads to acute lung injury, patchy alveolar flooding, and hypoxemia. Although calcium entry into endothelial cells is critical for loss of barrier integrity, the cation channels involved in this process have not been identified. We hypothesized that activation of the vanilloid transient receptor potential channel TRPV4 disrupts the alveolar septal barrier. Expression of TRPV4 was confirmed via immunohistochemistry in the alveolar septal wall in human, rat, and mouse lung. In isolated rat lung, the TRPV4 activators 4-phorbol-12,13-didecanoate and 5,6- or 14,15-epoxyeicosatrienoic acid, as well as thapsigargin, a known activator of calcium entry via store-operated channels, all increased lung endothelial permeability as assessed by measurement of the filtration coefficient, in a dose- and calcium-entry dependent manner. The TRPV antagonist ruthenium red blocked the permeability response to the TRPV4 agonists, but not to thapsigargin. Light and electron microscopy of rat and mouse lung revealed that TRPV4 agonists preferentially produced blebs or breaks in the endothelial and epithelial layers of the alveolar septal wall, whereas thapsigargin disrupted interendothelial junctions in extraalveolar vessels. The permeability response to 4-phorbol-12,13-didecanoate was absent in TRPV4^–/– mice, whereas the response to thapsigargin remained unchanged. Collectively, these findings implicate TRPV4 in disruption of the alveolar septal barrier and suggest its participation in the pathogenesis of acute lung injury.

Key Words: permeability • TRP channels • TRPV4 • acute lung injury

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Acute lung injury and its more severe form the adult respiratory distress syndrome are characterized by disruption of the alveolar septal barrier, leading to patchy alveolar flooding and hypoxemia.¹ Effective clinical treatments are limited and mortality remains high.^2,3 Such endothelial barrier disruption is often dependent on entry of Ca²⁺ from the extracellular space.^4,5 For example, in the intact rat lung, thapsigargin-induced store depletion and Ca²⁺ entry via store-operated channels increase endothelial permeability.^6,7 Store depletion targets extraalveolar vessel endothelium, although the septal microvasculature appears to be spared.^7,8 Although endothelial cells derived from extraalveolar pulmonary arteries and septal microvessels are phenotypically distinct^9,10 and both could potentially be targeted in acute lung injury, disruption of the septal barrier is more likely to promote alveolar flooding and impair gas exchange than disruption in extraalveolar vessels. Importantly, Ca²⁺ entry pathways involved in regulation of barrier integrity in the alveolar septal compartment have not been elucidated.

The consensus of data suggests that TRPC1 and TRPC4, members of the canonical subfamily of transient receptor potential (TRP) channels,^11–15 comprise subunits of store-operated Ca²⁺ channels in lung endothelium.^16–18 In the isolated rat lung, the permeability response to thrombin-induced store depletion was attenuated in TRPC4^–/– mice.¹⁸ Furthermore, loss of the permeability response to thapsigargin-induced store depletion in rat lung after heart failure was associated with downregulation of TRPC1 and TRPC4, channels which are normally expressed in extraalveolar endothelium.¹⁹ Retention of the permeability response to 14,15-epoxyeicosatrienoic acid (14,15-EET), a P450 epoxygenase-derived arachidonic acid metabolite, in this heart failure model was intriguing because the permeability responses to both thapsigargin-induced store depletion and to 14,15-EET are dependent on Ca²⁺ entry. This striking observation drives home the point that EETs must target Ca²⁺ entry pathways in lung endothelium distinct from store-operated channels.

We propose that Ca²⁺ entry via TRPV4, a member of the vanilloid subfamily of TRP channels,^13,20–24 contributes critically to regulation of endothelial permeability and barrier integrity in the lung. This hypothesis is based on several observations: 5,6-EET and 8,9-EET have been linked to activation of TRPV4 and subsequent Ca²⁺ entry in aortic endothelial cells,^25,26 5,6-EET and 14,15-EET increase endothelial permeability in rat and canine lung,^6,27 and the permeability response to 14,15-EET, at least, is dependent on entry of extracellular Ca²⁺.⁶ In the current study, we tested the hypothesis that Ca²⁺ entry via TRPV4 regulates lung endothelial permeability and barrier integrity. Furthermore, we tested whether Ca²⁺ entry via TRPV4 and that occurring through store-operated channels evoke barrier disruption in spatially distinct compartments of the pulmonary vasculature.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Protocols were approved by our Institutional Animal Care and Use Committee, conforming to the NIH Guide for the Care and Use of Laboratory Animals. Adult male CD rats (n=139, Charles River) or 8- to 10-week-old TRPV4 wild-type mice (TRPV4^+/+) and null littermates (TRPV4^–/–)²⁸ of either sex (n=39) were anesthetized with sodium pentobarbital (50 mg/kg body weight IP). Lungs were isolated for ex vivo perfusion as previously described.⁶

TRPV4 Expression
Human lung resection specimens (n=3), obtained under a protocol approved by the Institutional Review Board, were fixed by immersion in 10% formalin or 100% ethanol. Rat and mouse lungs (n=2 to 3 in each group) were perfusion fixed with 4% paraformaldehyde. Sections (5 µm) were processed for immunohistochemistry using a rabbit anti-TRPV4 polyclonal antibody (Alomone), stained with diaminobenzidine and counterstained with hematoxylin. Western blots were prepared from lysates (40 µg of total protein) of rat pulmonary artery and microvascular endothelium and TRPV4 detected using enhanced chemiluminescence. Total RNA from mouse lung was reverse transcribed, then PCR performed using primers designed to amplify the pore-loop region of TRPV4 (see the online data supplement, available at http://circres.ahajournals.org).

Microscopic Assessment of Acute Lung Injury
Light microscopy and transmission electron microscopy (EM) were used to evaluate structural changes in glutaraldehyde-fixed lung,¹⁹ after treatment with vehicle (910 µL of ethanol), 4PDD (3 or 10 µmol/L in rat or mouse lung, respectively), 14,15-EET (3 µmol/L, rat lung only), or thapsigargin (150 nmol/L) for 60 minutes (n=3 per group). Using 1-µm thick sections, extraalveolar vessel cuffing and alveolar flooding were evaluated. Cuff frequency and the cuff volume (V_c) fraction of total wall volume (V_c/V_w) were determined, the latter using a point-counting strategy.²⁹ Point-counting was used to determine alveolar fluid volume (V_af) fraction in the alveolar space (V_af/V_as). Thin sections (80 nm) from the same blocks were examined via transmission EM. Junctional discontinuities (gaps), blebs, or breaks in septal capillaries were enumerated. Means for cuff frequency, volume fractions and blebs or breaks per capillary were determined separately for each lung, then overall descriptive statistics derived for each group.

Isolated Lung and Assessment of Endothelial Permeability
Both rat and mouse lungs were perfused at constant flow with buffer (in mmol/L: 116.0 NaCl, 5.2 KCl, 0.9 MgSO₄, 1.0 Na₂HPO₄, 2.2 NaHCO₃, 8.3 D-glucose) containing 4% BSA and either physiological (2.2 mmol/L) or low (0.02 mmol/L) CaCl₂ at pH 7.4 (38°C). Hemodynamics and the filtration coefficient (K_f) were measured as previously described,^6,19,30,31 using zone 3 conditions. K_f was calculated as the rate of weight gain obtained 13 to 15 minutes after a 7 to 10 cmH₂O increase in pulmonary venous pressure, normalized per g lung dry weight. K_f, the product of specific endothelial permeability and surface area for exchange, is a sensitive measure of lung endothelial permeability when surface area is fully recruited.³²

Protocols
In rat lungs perfused with physiological [Ca²⁺], K_f and hemodynamics were measured at baseline and 60 minutes after treatment with the TRPV4 agonist 4-phorbol-12,13-didecanoate (4PDD) (1 to 10 µmol/L, n=3 per dose) or the TRPV1 agonist 4-phorbol-12,13-didecanoate-20 homovanillate (4PDDHV) (10 µmol/L, n=4). Using 0.02 mmol/L [Ca²⁺] perfusate, K_f and hemodynamics were measured at baseline and 45 minutes after treatment (n=5 per group) with 4PDD (3 µmol/L), 5,6-EET or 14,15-EET (3 µmol/L, Biomol), or thapsigargin (150 nmol/L), with or without the TRPV antagonist ruthenium red (1 µmol/L). Subsequently, CaCl₂ was added to achieve physiological [Ca²⁺] (Ca²⁺ add-back), and measurements repeated 15 minutes later. Lungs isolated from TRPV4^–/– mice or wild-type littermates (n=4 to 5 per group) were perfused with buffer containing 4% albumin and physiological [Ca²⁺]. K_f and hemodynamics were measured baseline and 60 minutes after treatment with vehicle (50 µL DMSO), 4PDD (10 µmol/L), or thapsigargin (150 nmol/L). Vehicle controls in rat lungs (n=5 each) included ethanol (910 µL or 2%) or DMSO (50 µL or 0.1%); DMSO was also evaluated in lungs from wild-type mice (n=4). All drugs were added to the perfusate; final circulating concentrations are noted.

Statistical Analysis
Quantitative data are presented as mean±SEM. Group means were compared using a paired t test (2 tailed) or ANOVA, as appropriate; the Newman–Keuls multiple comparison test was used to identify specific differences. Probability values of <0.05 were considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Baseline Parameters in Isolated Rat and Mouse Lung
Total pulmonary vascular resistance, the distribution of vascular resistance and baseline K_f were similar in rat and mouse lungs (Table 1) and were not impacted by the choice of perfusate [Ca²⁺]. There were no differences with respect to baseline measurements between TRPV4^+/+ and TRPV4^–/– mice.

View this table: [in this window] [in a new window]   Table 1. Baseline Hemodynamics and Permeability in Isolated Rat and Mouse Lung

Expression of TRPV4 in Lung: Functional Consequences
TRPV4 was expressed in the septal compartment of lungs from humans, rats and mice (Figure 1A through 1C, respectively), as well as in bronchiolar epithelium (not shown). TRPV4 was also expressed in smooth muscle in human and rat extraalveolar vessels. Although TRPV4 expression in cultured rat pulmonary artery endothelium was similar to that in microvascular endothelium (Figure 1D), TRPV4 was not consistently expressed in extraalveolar vessel endothelium in intact lung (Figure 1A through 1C). In rat lung, 1, 5, and 10 µmol/L 4PDD increased K_f by 1.7-, 4.2-, and 5.6-fold, respectively (Figure 1E), supporting the notion that TRPV4 may play a role in regulating endothelial permeability. In contrast, the TRPV1 agonist 4PDDHV had no impact on endothelial permeability despite use of a >EC₁₀₀ dose.³³ To determine whether activation of TRPV4 increased permeability in a Ca²⁺ entry-dependent fashion, K_f was reevaluated using the low Ca²⁺/Ca²⁺ add-back strategy. The Ca²⁺ concentration chosen for the low Ca²⁺ segment of these experiments was based on the lowest concentration that allowed stable K_f for at least 1 hour (see the online data supplement). Ca²⁺ add-back provides a normal inward Ca²⁺ gradient, and if Ca²⁺ permeant channels have been activated by the treatment, Ca²⁺ entry results and endothelial permeability increases. The 3-fold increase in K_f induced by 4PDD was clearly dependent on Ca²⁺ entry (Figure 2A) and was blocked by ruthenium red (Figure 2B), which potently blocks TRPV4 by binding to an extracellular domain on the channel.^24,33 We confirmed that the permeability response to 14,15-EET and thapsigargin in rat lung is dose dependent (see the online data supplement) and Ca²⁺ entry dependent (Figure 2A) and documented a similar pattern for 5,6-EET (online data supplement and Figure 2A). At 0.02 mmol/L [Ca²⁺], both 5,6-EET and 14,15-EET evoked a small increase in permeability, although Ca²⁺ add-back was required for the development of the normal permeability response to the EETs.⁶ In the absence of Ca²⁺ add-back, the increase in K_f induced by 14,15-EET in 0.02 mmol/L [Ca²⁺] (0.010±0.001 to 0.024±0.003 mL/min per cm H₂O per gram dry weight, P=0.0007, n=5) was transient and K_f returned to baseline within 15 minutes (P=0.007). Ruthenium red (Figure 2B) blocked the Ca²⁺ entry-dependent component of the permeability response to 5,6- and 14,15-EET but had no impact on the Ca²⁺ entry-dependent permeability response to thapsigargin. In the absence of treatment, 0.02 mmol/L [Ca²⁺] had no impact on K_f with or without Ca²⁺ add-back (see the online data supplement). EETs activate large conductance Ca²⁺-activated potassium channels (BK_Ca), providing an increased driving gradient for Ca²⁺ entry.^34,35 However, blockade of BK_Ca channels did not alter the EET-induced permeability response (see the online data supplement).

View larger version (74K): [in this window] [in a new window]   Figure 1. Activation of TRPV4 expressed in lung increases endothelial permeability. In human (A), rat (B), and mouse (C) lung, TRPV4 was expressed in the alveolar septal compartment (left panels) and bronchial epithelium (not shown). TRPV4 expression in vascular smooth muscle in extraalveolar vessels (right panels) was observed in human and rat lung, whereas little was seen in mouse lung. Western blotting (D) showed similar TRPV4 expression in rat microvascular (MV) and pulmonary artery (PA) endothelium (TRPV4/ß-actin band density was 1.1 in both groups). However, TRPV4 was not consistently expressed in extraalveolar vessel endothelium in intact lung (A through C). The TRPV4 agonist 4PDD (E) increased the filtration coefficient Kf in isolated rat lung (P=0.021) in a dose-dependent fashion (P=0.002); *P<0.05 vs 1 or 5 µmol/L. The TRPV1 agonist 4PDDHV had no effect (P=0.201, paired t test).

View larger version (30K): [in this window] [in a new window]   Figure 2. Activation of TRPV4 or store-operated channels increases permeability in a Ca2+ entry-dependent fashion. In low (0.02 mmol/L) extracellular [Ca2+] (hatched bars), Kf was measured at baseline (BL) and 45 minutes after treatment with the TRPV4 agonists 4PDD or EETs, or thapsigargin, which activates store-operated channels. A final Kf was measured 15 minutes after Ca2+ add-back (2.2 mmol/L, filled bars). Vehicle (A) or the TRPV antagonist ruthenium red (1 µmol/L) (B) was added 15 minutes before treatment. Ca2+ entry was required for the permeability response to all agonists (A), yet only the response to 4PDD or EETs was blocked by ruthenium red (B). Probability values (ANOVA) are shown above each group; post hoc tests identified specific differences: *P<0.05 vs BL, #P<0.05 vs agonist in low Ca2+.

Compartmentalization of Barrier Disruption
Results of the morphometric analysis from light microscopy (Figure 3) and transmission EM (Figure 4) are shown in Table 2. 4PDD and thapsigargin resulted in frequent extraalveolar cuffs in rat (P=0.006), but not in mouse lung, compared with control. Nonetheless, V_c/V_w was not different between groups in either rat or mouse lung. Activation of TRPV4 resulted in blebs or breaks in septal endothelium in both rat and mouse lung (P=0.009 and P=0.019, respectively, versus control or thapsigargin). Furthermore, the TRPV4 agonists disrupted type I alveolar epithelial cells, manifested as separation of the epithelium from the basal lamina. 4PDD increased V_af/V_as, compared with control and thapsigargin, in both rat and mouse lung (P=0.003 and 0.011, respectively). Whereas 14,15-EET caused significant septal injury, the increase in V_af/V_as was more variable, and as a result the 4-group ANOVA was not significant. In contrast, thapsigargin primarily targeted interendothelial junctional complexes in extraalveolar vessels, leading to formation of gaps; the septal capillary network was spared, and V_af/V_as did not increase. Together, these data support the notion that activation of TRPV4 (via 4PDD or EETs) and activation of store-operated channels (via thapsigargin) promote barrier disruption in discrete vascular compartments of the lung even though these agents cause similar increases in total lung K_f in a Ca²⁺ entry-dependent fashion.

View larger version (66K): [in this window] [in a new window]   Figure 3. Microscopic assessment of perivascular cuffing and fluid. Perivascular cuffs were infrequently observed in control rat lungs (A). Perivascular cuffing induced by 4PDD (B), 14,15-EET (C), or thapsigargin (D) was heterogeneous (arrows), and cuff volume fraction was no different among groups (see Table 2). Alveolar fluid volume fraction was increased by 4PDD and 14,15-EET, (Table 2). Scale bars=100 µm. PA indicates pulmonary arteriole; Br, bronchiole; L, lymphatic.

View larger version (47K): [in this window] [in a new window]   Figure 4. Assessment of endothelial ultrastructure in rat lung. Endothelial cell integrity was assessed using transmission electron microscopy. Random blocks were selected from glutaraldehyde-fixed lung 1 hour after treatment with vehicle or drug. Kf was measured to document permeability prior to fixation. Representative micrographs from extraalveolar vessels (A through D) and septal capillaries (E through H) are shown. Endothelial ultrastructure was retained in control lung (A and E), although occasional blebs or breaks in septal capillaries were observed. 4PDD (B and F) and 14,15-EET (C and G) rarely altered junctional morphology. However, both agonists resulted in endothelial breaks and blebs (asterisk) in the septal capillary endothelium (F and G), as well as blebs in the alveolar epithelium (inset in G). Thapsigargin resulted in development of gaps at junctions between endothelial cells in extraalveolar vessels (arrowhead) (D) but had no impact in the septal compartment (H). Scale bars: 2 µm (A through H); 500 nm (inset in G).

View this table: [in this window] [in a new window]   Table 2. Extraalveolar Cuffs and Alveolar Septal Barrier Disruption

The 4PDD and EET-mediated permeability responses were inhibited with ruthenium red, a pleiotropic antagonist that blocks TRPV4. Although activation of TRPV1 had no impact on endothelial permeability in rat lung, it was possible that ruthenium red blocked other TRPV isoforms, contributing to its impact on the permeability response. Thus, we used mice genetically engineered for a targeted deletion of exon 12 in the trpv4 gene,²⁸ encoding the transmembrane domains 5 and 6, which includes the predicted pore loop region of the channel (TRPV4^–/–). Genotyping (see the online data supplement) and PCR (Figure 5A) were used to document expression of TRPV4 in wild-type mice. Whereas 3 µmol/L 4PDD had no impact on permeability in TRPV4^+/+ mice, 10 µmol/L 4PDD increased K_f 2.8-fold (Figure 5B); this dose did not alter K_f in lungs from TRPV4^–/– littermates. Thapsigargin increased K_f 3-fold in both TRPV4^+/+ and TRPV4^–/– mice. These results confirm that activation of TRPV4 increases endothelial permeability in the mouse lung.

View larger version (42K): [in this window] [in a new window]   Figure 5. Activation of TRPV4 increases endothelial permeability in mouse lung. A, Using primers designed to amplify across the pore-loop region of TRPV4 mRNA, the predicted 612-bp product was observed in wild-type and heterozygote mice but not in null animals. B, In isolated mouse lung, we showed that the TRPV4 agonist 4PDD (10 µmol/L) increased endothelial permeability (Kf) in only TRPV4+/+ mice (P=0.036, paired t test). In contrast, activation of store-operated channels via thapsigargin (150 nmol/L) increased permeability in both wild-type and null mice (P=0.032 and 0.008, respectively, paired t test). These results confirm the role of TRPV4 in mediating acute lung injury. *P<0.05 vs baseline (paired t test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study provides the first evidence of a functional role for TRPV4 in lung endothelium, implicating TRPV4 in Ca²⁺ entry-dependent regulation of endothelial permeability and barrier integrity in the alveolar septal network. Despite similar impact on K_f, activation of TRPV4 and store-operated channels led to injury in distinct vascular compartments. Activation of TRPV4 by 4PDD and 14,15-EET preferentially targeted the alveolar septal microvessels, whereas thapsigargin-induced store depletion targeted extraalveolar vessels. The TRPV4 agonists typically caused endothelial blebs and/or breaks, whereas thapsigargin induced formation of gaps at endothelial cell junctions. These data suggest that distinct endothelial compartments, and possibly distinct subcellular compartments of lung endothelial cells, must be targeted subsequent to Ca²⁺ entry via these TRP channels. The observation that the Ca²⁺ entry-dependent component of the permeability response to 5,6- and 14,15-EET, as well as the TRPV4 agonist 4PDD, was ablated by pretreatment of rat lung with ruthenium red suggests an important role for TRPV4 in regulation of lung endothelial permeability. This notion is corroborated by the loss of the permeability response to the TRPV4 agonist 4PDD in lungs from TRPV4^–/– mice. In light of the critical vulnerability of the alveolar septal barrier in acute lung injury,^2,3 our findings in the rodent models of lung injury and the finding of TRPV4 expression in human alveolar septum lead us to hypothesize that TRPV4 is likely to play a role in the development of acute lung injury and the acute respiratory distress syndrome in humans.

TRP Channels and Permeability
Ca²⁺ entry into endothelial cells occurs via activation of selective Ca²⁺ channels or nonselective cation channels, such as those encoded by the large superfamily of TRP channel proteins. Much of the focus in lung endothelium has been on the TRPC subfamily. Endothelial cells express mRNA and protein for TRP channels, including TRPC1, TRPC3, TRPC4, TRPC6, and TRPC7.^14,36,37 In rat lung, TRPC1, TRPC3, TRPC4, and TRPC6/7 protein is expressed in endothelium of extraalveolar vessels. Although mRNA for several members of the TRPV subfamily, including TRPV4, are expressed in human pulmonary artery endothelium,³⁶ only a subset of TRP proteins may actually play a physiological role in regulation of lung endothelial permeability. Two studies have addressed a role for TRPC channels in regulation of permeability in the intact lung. Tiruppathi et al reported a partial loss of the permeability response to thrombin in TRPC4^–/– mice,¹⁸ and we recently found that in an aortocaval fistula model of heart failure, endothelial TRPC1 and TRPC4 expression in extraalveolar vessels is downregulated and the permeability response to thapsigargin is lost.¹⁹ Although Ca²⁺ entry can occur via receptor-operated TRP channels (TRPC3, TRPC6, and TRPC7),³⁸ and these channels are indeed expressed in rat lung endothelium, activation of these channels with a diacylglycerol analog had no impact on endothelial permeability in rat lung.¹⁹ A role in regulation of lung endothelial permeability has not been explored for TRP proteins outside the canonical TRP family. Aside from thrombin and thapsigargin, we do not know whether the Ca²⁺ entry-dependent increases in endothelial permeability are attributable to gating of TRP channels, nor which TRP channels are involved. Our previous work evaluating the impact of EETs, P450 epoxygenase derivatives of arachidonic acid, on lung endothelial permeability in normal rats and rats with chronic heart failure^6,19 supports the notion of heterogeneity in Ca²⁺-dependent regulation of endothelial permeability in lung. P450 epoxygenases metabolize arachidonic acid to form four regioisomers: 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET.³⁹ EETs are released from human lung after inflammatory challenge,⁴⁰ suggesting their potential involvement in acute lung injury. In canine lung, blockade of EET synthesis attenuated the pulmonary edema and hypoxemia resulting from ethchlorvynol.⁴¹ Furthermore, exogenous 5,6-EET and 14,15-EET increased endothelial permeability in canine and rat lung,^6,27 in a Ca²⁺ entry-dependent manner. The notion that EETs must target Ca²⁺ permeant channels distinct from TRPC1/TRPC4 is based on the following evidence: (1) the permeability response to both EETs and thapsigargin in rat lung requires Ca²⁺ entry; (2) the response to 14,15-EET was retained after experimentally induced heart failure, although that to store depletion was lost; and (3) TRPC1 and TRPC4 expression were downregulated in extraalveolar vessels from rats with heart failure.^6,19 Our current results support the notion that TRPV4 expressed in lung septal microvascular endothelium plays a critical role in the permeability response to EETs and 4PDD.

Functional Role for TRPV4 Channels in the Alveolar Septal Network
TRPV4, a channel originally described as activated by hypotonicity,^20,23,42 appears to participate in detection of changes in extracellular fluid osmolality.²⁸ Although TRPV channels are appreciated for their role in sensory transduction,^43–46 nonsensory functions are now recognized.^21,47–50 Work from Nilius and colleagues^25,26,51 suggests that TRPV4 likely plays a significant role in endothelial Ca²⁺ signaling. Both 4PDD and 5,6-EET activate TRPV4 heterologously expressed in HEK-293 cells as well as endogenous TRPV4 in aortic endothelial cells, promoting Ca²⁺ entry which was inhibited by ruthenium red.²⁶ Furthermore, EET-induced Ca²⁺ entry in aortic endothelial cells was diminished in endothelium derived from TRPV4^–/– mice.²⁵ Collectively, these data suggested that TRPV4 could play a role in Ca²⁺ entry-dependent regulation of endothelial permeability. Our results provide a functional role for TRPV4 expressed in the lung alveolar septal network. The TRPV4 agonist 4PDD increased endothelial permeability in rat lung in a Ca²⁺ entry-dependent fashion. This response, and the Ca²⁺ entry-dependent permeability response to 5,6- and 14,15-EET, could be blocked with ruthenium red. Furthermore, the permeability response to 4PDD observed in TRPV4^+/+ mice was lacking in TRPV4^–/– littermates.

Transmission EM documented that both 4PDD and 14,15-EET caused disruption of the alveolar epithelium, in addition to an impact on the septal endothelial barrier, which very likely contributed to the increase in K_f and alveolar flooding induced by these TRPV4 agonists. TRPV4 has previously been demonstrated to be expressed in respiratory epithelial cells,^52–54 although its functional role in bronchiolar or alveolar epithelium in the intact lung has not been clarified. Our results suggest that an exploration of the signaling cascade linking Ca²⁺ entry via TRPV4 in alveolar type I cells to detachment of those cells from the basement membrane would be informative.

In summary, this work provides critical evidence of a functional role for TRPV4 in regulation of barrier integrity in the lung alveolar septal network. Although Ca²⁺ entry via store-operated TRP channels causes gap formation in extraalveolar vessels, the functional consequences of barrier disruption in this compartment are likely distinct from those resulting from TRPV4-dependent barrier disruption in the septal compartment. Activation of store-operated channels in extraalveolar endothelium leads to fluid accumulation in perivascular cuffs, although this likely has little impact on alveolar gas exchange. In contrast, disruption of the vulnerable alveolar septal barrier, such as that resulting from Ca²⁺ influx via TRPV4, leads to alveolar flooding and thus would be predicted to impair gas exchange. Indeed this is a hallmark of acute lung injury. The implication of this work for translational biomedical research is that TRPV4 is likely a novel molecular target for therapeutic intervention in acute lung injury.

	Acknowledgments

 
We thank Sue Barnes, Freda McDonald, and Doug Drake for technical support.

Sources of Funding

This work was supported by grants from the NIH (HL081851 and MH064702) and a fellowship from the American Heart Association (0315049B).

Disclosures

None.

	Footnotes

 
Original received May 17, 2006; revision received August 16, 2006; accepted September 14, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, Lamy M, LeGall JR, Morris A, Spragg R. The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med. 1994; 149: 818–824.[Abstract]
2. Matthay MA, Zimmerman GA, Esmon C, Bhattacharya J, Coller B, Doerschuk CM, Floros J, Gimbrone MA Jr, Hoffman E, Hubmayr RD, Leppert M, Matalon S, Munford R, Parsons P, Slutsky AS, Tracey KJ, Ward P, Gail DB, Harabin AL. Future research directions in acute lung injury: summary of a National Heart, Lung, and Blood Institute working group. Am J Respir Crit Care Med. 2003; 167: 1027–1035.[Abstract/Free Full Text]
3. Ware LB, Matthay MA. Clinical practice. Acute pulmonary edema. N Engl J Med. 2005; 353: 2788–2796.[Free Full Text]
4. Moore TM, Chetham PM, Kelly JJ, Stevens T. Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP. Am J Physiol. 1998; 275: L203–L222.[Medline] [Order article via Infotrieve]
5. Tiruppathi C, Minshall RD, Paria BC, Vogel SM, Malik AB. Role of Ca²⁺ signaling in the regulation of endothelial permeability. Vascul Pharmacol. 2002; 39: 173–185.[CrossRef][Medline] [Order article via Infotrieve]
6. Alvarez DF, Gjerde EA, Townsley MI. Role of EETs in regulation of endothelial permeability in rat lung. Am J Physiol Lung Cell Mol Physiol. 2004; 286: L445–L451.[Abstract/Free Full Text]
7. Chetham PM, Babal P, Bridges JP, Moore TM, Stevens T. Segmental regulation of pulmonary vascular permeability by store-operated Ca²⁺ entry. Am J Physiol. 1999; 276: L41–L50.[Medline] [Order article via Infotrieve]
8. Alvarez DF, King JA, Townsley MI. Evaluation of endothelial permeability by a corrosion casting technique. Microsc Microanal. 2004; 10 (suppl 2): 200–201.
9. Gebb S, Stevens T. On lung endothelial cell heterogeneity. Microvasc Res. 2004; 68: 1–12.[CrossRef][Medline] [Order article via Infotrieve]
10. King J, Hamil T, Creighton J, Wu S, Bhat P, McDonald F, Stevens T. Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes. Microvasc Res. 2004; 67: 139–151.[CrossRef][Medline] [Order article via Infotrieve]
11. Clapham DE, Runnels LW, Strübing C. The TRP ion channel family. Nat Rev Neurosci. 2001; 2: 387–396.[Medline] [Order article via Infotrieve]
12. Freichel M, Philipp S, Cavalie A, Flockerzi V. TRPC4 and TRPC4-deficient mice. Novartis Found Symp. 2004; 258: 189–199.[Medline] [Order article via Infotrieve]
13. Harteneck C, Plant TD, Schultz G. From worm to man: three subfamilies of TRP channels. Trends Neurosci. 2000; 23: 159–166.[CrossRef][Medline] [Order article via Infotrieve]
14. Nilius B, Droogmans G, Wondergem R. Transient receptor potential channels in endothelium: solving the calcium entry puzzle? Endothelium. 2003; 10: 5–15.[CrossRef][Medline] [Order article via Infotrieve]
15. Vazquez G, Wedel BJ, Aziz O, Trebak M, Putney JW Jr. The mammalian TRPC cation channels. Biochim Biophys Acta. 2004; 1742: 21–36.[Medline] [Order article via Infotrieve]
16. Brough GH, Wu S, Cioffi D, Moore TM, Li M, Dean N, Stevens T. Contribution of endogenously expressed Trp1 to a Ca²⁺-selective, store-operated Ca²⁺ entry pathway. FASEB J. 2001; 15: 1727–1738.[Abstract/Free Full Text]
17. Cioffi DL, Wu S, Stevens T. On the endothelial cell I_SOC. Cell Calcium. 2003; 33: 323–336.[CrossRef][Medline] [Order article via Infotrieve]
18. Tiruppathi C, Freichel M, Vogel SM, Paria BC, Mehta D, Flockerzi V, Malik AB. Impairment of store-operated Ca²⁺ entry in TRPC4^–/- mice interferes with increase in lung microvascular permeability. Circ Res. 2002; 91: 70–76.[Abstract/Free Full Text]
19. Alvarez DF, King JA, Townsley MI. Resistance to store depletion-induced endothelial injury in rat lung after chronic heart failure. Am J Respir Crit Care Med. 2005; 172: 1153–1160.[Abstract/Free Full Text]
20. Liedtke W, Choe Y, Marti-Renom MA, Bell AM, Denis CS, Sali A, Hudspeth AJ, Friedman JM, Heller S. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell. 2000; 103: 525–535.[CrossRef][Medline] [Order article via Infotrieve]
21. Liedtke W. TRPV4 plays an evolutionary conserved role in the transduction of osmotic and mechanical stimuli in live animals. J Physiol. 2005; 567: 53–58.[Abstract/Free Full Text]
22. Nilius B, Vriens J, Prenen J, Droogmans G, Voets T. TRPV4 calcium entry channel: a paradigm for gating diversity. Am J Physiol Cell Physiol. 2004; 286: C195–C205.[Abstract/Free Full Text]
23. Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD. OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol. 2000; 2: 695–702.[CrossRef][Medline] [Order article via Infotrieve]
24. Voets T, Prenen J, Vriens J, Watanabe H, Janssens A, Wissenbach U, Bödding M, Droogmans G, Nilius B. Molecular determinants of permeation through the cation channel TRPV4. J Biol Chem. 2002; 277: 33704–33710.[Abstract/Free Full Text]
25. Vriens J, Owsianik G, Fisslthaler B, Suzuki M, Janssens A, Voets T, Morisseau C, Hammock BD, Fleming I, Busse R, Nilius B. Modulation of the Ca²⁺ permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium. Circ Res. 2005; 97: 908–915.[Abstract/Free Full Text]
26. Watanabe H, Vriens J, Prenen J, Droogmans G, Voets T, Nilius B. Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature. 2003; 424: 434–438.[CrossRef][Medline] [Order article via Infotrieve]
27. Ivey CL, Stephenson AH, Townsley MI. Involvement of cytochrome P-450 enzyme activity in the control of microvascular permeability in canine lung. Am J Physiol. 1998; 275: L756–L763.[Medline] [Order article via Infotrieve]
28. Liedtke W, Friedman JM. Abnormal osmotic regulation in trpv4^–/- mice. Proc Natl Acad Sci U S A. 2003; 100: 13698–13703.[Abstract/Free Full Text]
29. Weibel ER. Morphometry: sterological theory and practical methods. In: Gil J, ed. Models of Lung Disease. Microscopy and Structural Methods. New York: Marcel Dekker Inc; 1990: 199–252.
30. Townsley MI, Korthuis RJ, Rippe B, Parker JC, Taylor AE. Validation of double vascular occlusion method for P_c,i in lung and skeletal muscle. J Appl Physiol. 1986; 61: 127–132.[Abstract/Free Full Text]
31. Townsley MI, Fu Z, Mathieu-Costello O, West JB. Pulmonary microvascular permeability. Responses to high vascular pressure after induction of pacing-induced heart failure in dogs. Circ Res. 1995; 77: 317–325.[Abstract/Free Full Text]
32. Parker JC, Townsley MI. Evaluation of lung injury in rats and mice. Am J Physiol Lung Cell Mol Physiol. 2004; 286: L231–L246.[Abstract/Free Full Text]
33. Watanabe H, Davis JB, Smart D, Jerman JC, Smith GD, Hayes P, Vriens J, Cairns W, Wissenbach U, Prenen J, Flockerzi V, Droogmans G, Benham CD, Nilius B. Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives. J Biol Chem. 2002; 277: 13569–13577.[Abstract/Free Full Text]
34. Harder DR, Campbell WB, Roman RJ. Role of cytochrome P-450 enzymes and metabolites of arachidonic acid in the control of vascular tone. J Vasc Res. 1995; 32: 79–92.[Medline] [Order article via Infotrieve]
35. Roman RJ. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev. 2002; 82: 131–185.[Abstract/Free Full Text]
36. Fantozzi I, Zhang S, Platoshyn O, Remillard CV, Cowling RT, Yuan JX. Hypoxia increases AP-1 binding activity by enhancing capacitative Ca²⁺ entry in human pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol. 2003; 285: L1233–L1245.[Abstract/Free Full Text]
37. Yao X, Garland CJ. Recent developments in vascular endothelial cell transient receptor potential channels. Circ Res. 2005; 97: 853–863.[Abstract/Free Full Text]
38. Trebak M, Bird GS, McKay RR, Birnbaumer L, Putney JW Jr. Signaling mechanism for receptor-activated canonical transient receptor potential 3 (TRPC3) channels. J Biol Chem. 2003; 278: 16244–16252.[Abstract/Free Full Text]
39. Spector AA, Fang X, Snyder GD, Weintraub NL. Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog Lipid Res. 2004; 43: 55–90.[CrossRef][Medline] [Order article via Infotrieve]
40. Kiss L, Schütte H, Mayer K, Grimm H, Padberg W, Seeger W, Grimminger F. Synthesis of arachidonic acid-derived lipoxygenase and cytochrome P450 products in the intact human lung vasculature. Am J Respir Crit Care Med. 2000; 161: 1917–1923.[Abstract/Free Full Text]
41. Stephenson AH, Sprague RS, Weintraub NL, McMurdo L, Lonigro AJ. Inhibition of cytochrome P-450 attenuates hypoxemia of acute lung injury in dogs. Am J Physiol. 1996; 270: H1355–H1362.[Medline] [Order article via Infotrieve]
42. Wissenbach U, Bödding M, Freichel M, Flockerzi V. Trp12, a novel Trp related protein from kidney. FEBS Lett. 2000; 485: 127–134.[CrossRef][Medline] [Order article via Infotrieve]
43. Alessandri-Haber N, Dina OA, Yeh JJ, Parada CA, Reichling DB, Levine JD. Transient receptor potential vanilloid 4 is essential in chemotherapy-induced neuropathic pain in the rat. J Neurosci. 2004; 24: 4444–4452.[Abstract/Free Full Text]
44. Lee H, Iida T, Mizuno A, Suzuki M, Caterina MJ. Altered thermal selection behavior in mice lacking transient receptor potential vanilloid 4. J Neurosci. 2005; 25: 1304–1310.[Abstract/Free Full Text]
45. Liedtke W, Tobin DM, Bargmann CI, Friedman JM. Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2003; 100: 14531–14536.[Abstract/Free Full Text]
46. Suzuki M, Mizuno A, Kodaira K, Imai M. Impaired pressure sensation in mice lacking TRPV4. J Biol Chem. 2003; 278: 22664–22668.[Abstract/Free Full Text]
47. Caterina MJ. Vanilloid receptors take a TRP beyond the sensory afferent. Pain. 2003; 105: 5–9.[CrossRef][Medline] [Order article via Infotrieve]
48. Liu X, Bandyopadhyay B, Nakamoto T, Singh B, Liedtke W, Melvin JE, Ambudkar I. A role for AQP5 in activation of TRPV4 by hypotonicity: concerted involvement of AQP5 and TRPV4 in regulation of cell volume recovery. J Biol Chem. 2006; 281: 15485–15495.[Abstract/Free Full Text]
49. Nilius B, Watanabe H, Vriens J. The TRPV4 channel: structure-function relationship and promiscuous gating behaviour. Pflügers Arch. 2003; 446: 298–303.[CrossRef][Medline] [Order article via Infotrieve]
50. O’Neil RG, Heller S. The mechanosensitive nature of TRPV channels. Pflügers Arch. 2005; 451: 193–203.[CrossRef][Medline] [Order article via Infotrieve]
51. Vriens J, Watanabe H, Janssens A, Droogmans G, Voets T, Nilius B. Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4. Proc Natl Acad Sci U S A. 2004; 101: 396–401.[Abstract/Free Full Text]
52. Delany NS, Hurle M, Facer P, Alnadaf T, Plumpton C, Kinghorn I, See CG, Costigan M, Anand P, Woolf CJ, Crowther D, Sanseau P, Tate SN. Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2. Physiol Genomics. 2001; 4: 165–174.[Abstract/Free Full Text]
53. Fernandez-Fernandez JM, Nobles M, Currid A, Vazquez E, Valverde MA. Maxi K⁺ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line. Am J Physiol Cell Physiol. 2002; 283: C1705–C1714.[Abstract/Free Full Text]
54. Sidhaye VK, Güler AD, Schweitzer KS, D’Alessio F, Caterina MJ, King LS. Transient receptor potential vanilloid 4 regulates aquaporin-5 abundance under hypotonic conditions. Proc Natl Acad Sci U S A. 2006; 103: 4747–4752.[Abstract/Free Full Text]
55. Parker JC, Ivey CL, Tucker JA. Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lungs. J Appl Physiol. 1998; 84: 1113–1118.[Abstract/Free Full Text]
56. Parker JC, Gillespie MN, Taylor AE, Martin SL. Capillary filtration coefficient, vascular resistance, and compliance in isolated mouse lungs. J Appl Physiol. 1999; 87: 1421–1427.[Abstract/Free Full Text]

Related Article:

TRP Channels and the Regulation of Vascular Permeability: New Insights From the Lung Microvasculature

Fitz-Roy E. Curry and Catherine A. Glass
Circ. Res. 2006 99: 915-917. [Full Text] [PDF]

This article has been cited by other articles:

				J. Yin, J. Hoffmann, S. M. Kaestle, N. Neye, L. Wang, J. Baeurle, W. Liedtke, S. Wu, H. Kuppe, A. R. Pries, et al. Negative-Feedback Loop Attenuates Hydrostatic Lung Edema via a cGMP-Dependent Regulation of Transient Receptor Potential Vanilloid 4 Circ. Res., April 25, 2008; 102(8): 966 - 974. [Abstract] [Full Text] [PDF]

				M.-Y. Jian, J. A. King, A.-B. Al-Mehdi, W. Liedtke, and M. I. Townsley High Vascular Pressure-Induced Lung Injury Requires P450 Epoxygenase-Dependent Activation of TRPV4 Am. J. Respir. Cell Mol. Biol., April 1, 2008; 38(4): 386 - 392. [Abstract] [Full Text] [PDF]

				V. K. Sidhaye, K. S. Schweitzer, M. J. Caterina, L. Shimoda, and L. S. King Shear stress regulates aquaporin-5 and airway epithelial barrier function PNAS, March 4, 2008; 105(9): 3345 - 3350. [Abstract] [Full Text] [PDF]

				D. F. Alvarez, L. Huang, J. A. King, M. K. ElZarrad, M. C. Yoder, and T. Stevens Lung microvascular endothelium is enriched with progenitor cells that exhibit vasculogenic capacity Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L419 - L430. [Abstract] [Full Text] [PDF]

				T. Miyahara, K. Hamanaka, D. S. Weber, M. Anghelescu, J. R. Frost, J. A. King, and J. C. Parker Cytosolic phospholipase A2 and arachidonic acid metabolites modulate ventilator-induced permeability increases in isolated mouse lungs J Appl Physiol, February 1, 2008; 104(2): 354 - 362. [Abstract] [Full Text] [PDF]

				B. Troyanovsky, D. F. Alvarez, J. A. King, and K. L. Schaphorst Thrombin enhances the barrier function of rat microvascular endothelium in a PAR-1-dependent manner Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L266 - L275. [Abstract] [Full Text] [PDF]

				J. R. Klinger, J. D. Murray, B. Casserly, D. F. Alvarez, J. A. King, S. S. An, G. Choudhary, A. N. Owusu-Sarfo, R. Warburton, and E. O. Harrington Rottlerin causes pulmonary edema in vivo: a possible role for PKC{delta} J Appl Physiol, December 1, 2007; 103(6): 2084 - 2094. [Abstract] [Full Text] [PDF]

				K. Hamanaka, M.-Y. Jian, D. S. Weber, D. F. Alvarez, M. I. Townsley, A. B. Al-Mehdi, J. A. King, W. Liedtke, and J. C. Parker TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L923 - L932. [Abstract] [Full Text] [PDF]

				S. Chatterjee, S. Baeter, and J. Bhattacharya Endothelial and epithelial signaling in the lung Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L517 - L519. [Abstract] [Full Text] [PDF]

				W. Liedtke Hydromineral Neuroendocrinology: Role of TRPV ion channels in sensory transduction of osmotic stimuli in mammals Exp Physiol, May 1, 2007; 92(3): 507 - 512. [Abstract] [Full Text] [PDF]