Nuclear FAK in endothelium: An intrinsic inhibitor of NF-κB activation in atherosclerosis.

BACKGROUND AND AIMS: Hyperlipidemia leads to the accumulation of oxidized low-density lipoprotein (oxLDL) within the vessel wall where it causes chronic inflammation in endothelial cells (ECs) and drives atherosclerotic lesions. Although focal adhesion kinase (FAK) is critical in proinflammatory NF-κB activation in ECs, it is unknown if hyperlipidemia alters FAK-mediated NF-κB activity in vivo to affect atherosclerosis progression. METHODS: We investigated changes in EC FAK and NF-κB activation using Apoe-/- mice fed a western diet (WD). Both pharmacological FAK inhibition and EC-specific FAK inhibited mouse models were utilized. FAK and NF-κB localization and activity were also analyzed in human atherosclerotic samples. RESULTS: ECs of hyperlipidemic mice clearly showed much higher levels of FAK activation in the cytoplasm, which was associated with increased NF-κB activation compared to normal diet (ND) group. On the contrary, FAK is mostly localized in the nucleus and inactive in ECs under healthy conditions with a low NF-κB activity. Both pharmacological and EC-specific genetic FAK inhibition in WD fed Apoe-/- mice exhibited a significant decrease in FAK activity and cytoplasmic localization, NF-κB activation, macrophage recruitment, and atherosclerotic lesions compared to the vehicle or FAK wild-type groups. Analyses of human atherosclerotic specimens revealed a positive correlation between increased active cytoplasmic FAK within ECs and NF-κB activation in the lesions. CONCLUSIONS: Hyperlipidemic conditions activate NF-κB pathway by increasing EC FAK activity and cytoplasmic localization in mice and human atherosclerotic samples. As FAK inhibition can efficiently reduce vascular inflammation and atherosclerotic lesions in mice by reversing EC FAK localization and NF-κB activation, these findings support a potential use for FAK inhibitors in treating atherosclerosis.

Focal Adhesion Kinase Activity and Localization is Critical for TNF-α-Induced Nuclear Factor-κB Activation.

While sustained nuclear factor-κB (NF-κB) activation is critical for proinflammatory molecule expression, regulators of NF-κB activity during chronic inflammation are not known. We investigated the role of focal adhesion kinase (FAK) on sustained NF-κB activation in tumor necrosis factor-α (TNF-α)-stimulated endothelial cells (ECs) both in vitro and in vivo. We found that FAK inhibition abolished TNF-α-mediated sustained NF-κB activity in ECs by disrupting formation of TNF-α receptor complex-I (TNFRC-I). Additionally, FAK inhibition diminished recruitment of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and the inhibitor of NF-κB (IκB) kinase (IKK) complex to TNFRC-I, resulting in elevated stability of IκBα protein. In mice given TNF-α, pharmacological and genetic FAK inhibition blocked TNF-α-induced IKK-NF-κB activation in aortic ECs. Mechanistically, TNF-α activated and redistributed FAK from the nucleus to the cytoplasm, causing elevated IKK-NF-κB activation. On the other hand, FAK inhibition trapped FAK in the nucleus of ECs even upon TNF-α stimulation, leading to reduced IKK-NF-κB activity. Together, these findings support a potential use for FAK inhibitors in treating chronic inflammatory diseases.

S100A6 is a positive regulator of PPP5C-FKBP51-dependent regulation of endothelial calcium signaling.

ISOC is a cation current permeating the ISOC channel. In pulmonary endothelial cells, ISOC activation leads to formation of inter-endothelial cell gaps and barrier disruption. The immunophilin FK506-binding protein 51 (FKBP51), in conjunction with the serine/threonine protein phosphatase 5C (PPP5C), inhibits ISOC . Free PPP5C assumes an autoinhibitory state, which has low "basal" catalytic activity. Several S100 protein family members bind PPP5C increasing PPP5C catalytic activity in vitro. One of these family members, S100A6, exhibits a calcium-dependent translocation to the plasma membrane. The goal of this study was to determine whether S100A6 activates PPP5C in pulmonary endothelial cells and contributes to ISOC inhibition by the PPP5C-FKBP51 axis. We observed that S100A6 activates PPP5C to dephosphorylate tau T231. Following ISOC activation, cytosolic S100A6 translocates to the plasma membrane and interacts with the TRPC4 subunit of the ISOC channel. Global calcium entry and ISOC are decreased by S100A6 in a PPP5C-dependent manner and by FKBP51 in a S100A6-dependent manner. Further, calcium entry-induced endothelial barrier disruption is decreased by S100A6 dependent upon PPP5C, and by FKBP51 dependent upon S100A6. Overall, these data reveal that S100A6 plays a key role in the PPP5C-FKBP51 axis to inhibit ISOC and protect the endothelial barrier against calcium entry-induced disruption.

Serine/threonine phosphatase 5 (PP5C/PPP5C) regulates the ISOC channel through a PP5C-FKBP51 axis.

Pulmonary endothelial cells express a store-operated calcium entry current ( Isoc), which contributes to inter-endothelial cell gap formation. Isoc is regulated by a heterocomplex of proteins that includes the immunophilin FKBP51. FKBP51 inhibits Isoc by mechanisms that are not fully understood. In pulmonary artery endothelial cells (PAECs) we have shown that FKBP51 increases microtubule polymerization, an event that is critical for Isoc inhibition by FKBP51. In neurons, FKBP51 promotes microtubule stability through facilitation of tau dephosphorylation. However, FKBP51 does not possess phosphatase activity. Protein phosphatase 5 (PP5C/PPP5C) can dephosphorylate tau, and similar to FKBP51, PP5C possesses tetratricopeptide repeats (TPR) that mediate interaction with heat shock protein-90 (HSP90) chaperone/scaffolding complexes. We therefore tested whether PP5C contributes to FKBP51-mediated inhibition of Isoc. Both siRNA-mediated suppression of PP5C expression in PAECs and genetic disruption of PP5C in HEK293 cells attenuate FKBP51-mediated inhibition of Isoc. Reintroduction of catalytically competent, but not catalytically inactive PP5C, restored FKBP51-mediated inhibition of Isoc. PAEC cell fractionation studies identified both PP5C and the ISOC heterocomplex in the same membrane fractions. Further, PP5C co-precipitates with TRPC4, an essential subunit of ISOC channel. Finally, to determine if PP5C is required for FKBP51-mediated inhibition of calcium entry-induced inter-endothelial cell gap formation, we measured gap area by wide-field microscopy and performed biotin gap quantification assay and electric cell-substrate impedance sensing (ECIS®). Collectively, the data presented indicate that suppression of PP5C expression negates the protective effect of FKBP51. These observations identify PP5C as a novel member of the ISOC heterocomplex that is required for FKBP51-mediated inhibition of Isoc.

Protective role of FKBP51 in calcium entry-induced endothelial barrier disruption.

Pulmonary artery endothelial cells (PAECs) express a cation current, ISOC (store-operated calcium entry current), which when activated permits calcium entry leading to inter-endothelial cell gap formation. The large molecular weight immunophilin FKBP51 inhibits ISOC but not other calcium entry pathways in PAECs. However, it is unknown whether FKBP51-mediated inhibition of ISOC is sufficient to protect the endothelial barrier from calcium entry-induced disruption. The major objective of this study was to determine whether FKBP51-mediated inhibition of ISOC leads to decreased calcium entry-induced inter-endothelial gap formation and thus preservation of the endothelial barrier. Here, we measured the effects of thapsigargin-induced ISOC on the endothelial barrier in control and FKBP51 overexpressing PAECs. FKBP51 overexpression decreased actin stress fiber and inter-endothelial cell gap formation in addition to attenuating the decrease in resistance observed with control cells using electric cell-substrate impedance sensing. Finally, the thapsigargin-induced increase in dextran flux was abolished in FKBP51 overexpressing PAECs. We then measured endothelial permeability in perfused lungs of FKBP51 knockout (FKBP51-/-) mice and observed increased calcium entry-induced permeability compared to wild-type mice. To begin to dissect the mechanism underlying the FKBP51-mediated inhibition of ISOC, a second goal of this study was to determine the role of the microtubule network. We observed that FKBP51 overexpressing PAECs exhibited increased microtubule polymerization that is critical for inhibition of ISOC by FKBP51. Overall, we have identified FKBP51 as a novel regulator of endothelial barrier integrity, and these findings are significant as they reveal a protective mechanism for endothelium against calcium entry-induced disruption.

Lectin-Based Characterization of Vascular Cell Microparticle Glycocalyx.

Microparticles (MPs) are released constitutively and from activated cells. MPs play significant roles in vascular homeostasis, injury, and as biomarkers. The unique glycocalyx on the membrane of cells has frequently been exploited to identify specific cell types, however the glycocalyx of the MPs has yet to be defined. Thus, we sought to determine whether MPs, released both constitutively and during injury, from vascular cells have a glycocalyx matching those of the parental cell type to provide information on MP origin. For these studies we used rat pulmonary microvascular and artery endothelium, pulmonary smooth muscle, and aortic endothelial cells. MPs were collected from healthy or cigarette smoke injured cells and analyzed with a panel of lectins for specific glycocalyx linkages. Intriguingly, we determined that the MPs released either constitutively or stimulated by CSE injury did not express the same glycocalyx of the parent cells. Further, the glycocalyx was not unique to any of the specific cell types studied. These data suggest that MPs from both normal and healthy vascular cells do not share the parental cell glycocalyx makeup.

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.

Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1.

Orai1 interacts with transient receptor potential protein of the canonical subfamily (TRPC4) and contributes to calcium selectivity of the endothelial cell store-operated calcium entry current (ISOC). Orai1 silencing increases sodium permeability and decreases membrane-associated calcium, although it is not known whether Orai1 is an important determinant of cytosolic sodium transitions. We test the hypothesis that, upon activation of store-operated calcium entry channels, Orai1 is a critical determinant of cytosolic sodium transitions. Activation of store-operated calcium entry channels transiently increased cytosolic calcium and sodium, characteristic of release from an intracellular store. The sodium response occurred more abruptly and returned to baseline more rapidly than did the transient calcium rise. Extracellular choline substitution for sodium did not inhibit the response, although 2-aminoethoxydiphenyl borate and YM-58483 reduced it by ∼50%. After this transient response, cytosolic sodium continued to increase due to influx through activated store-operated calcium entry channels. The magnitude of this sustained increase in cytosolic sodium was greater when experiments were conducted in low extracellular calcium and when Orai1 expression was silenced; these two interventions were not additive, suggesting a common mechanism. 2-Aminoethoxydiphenyl borate and YM-58483 inhibited the sustained increase in cytosolic sodium, only in the presence of Orai1. These studies demonstrate that sodium permeates activated store-operated calcium entry channels, resulting in an increase in cytosolic sodium; the magnitude of this response is determined by Orai1.

The serine-threonine phosphatase calcineurin is a regulator of endothelial store-operated calcium entry.

Disruption of the endothelium leads to increased permeability, allowing extravasation of macromolecules and other solutes from blood vessels. Calcium entry through a calcium-selective, store-operated calcium (SOC) channel, I soc, contributes to barrier disruption. An understanding of the mechanisms surrounding the regulation of I soc is far from complete. We show that the calcium/calmodulin-activated phosphatase calcineurin (CN) plays a role in regulation of SOC entry, possibly through the dephosphorylation of stromal interaction molecule 1 (STIM1). Phosphorylation has been implicated as a regulatory mechanism of activity for a number of canonical transient receptor potential (TRPC) and SOC channels, including I soc. Our results show that STIM1 phosphorylation increases in pulmonary artery endothelial cells (PAECs) upon activation of SOC entry. However, the phosphatases involved in STIM1 dephosphorylation are unknown. We found that a CN inhibitor (calcineurin inhibitory peptide [CIP]) increases the phosphorylation pattern of STIM1. Using a fura 2-acetoxymethyl ester approach to measure cytosolic calcium in PAECs, we found that CIP decreases SOC entry following thapsigargin treatment in PAECs. Luciferase assays indicate that thapsigargin induces activation of CN activity and confirm inhibition of CN activity by CIP in PAECs. Also, I soc is significantly attenuated in whole-cell patch-clamp studies of PAECs treated with CIP. Finally, PAECs pretreated with CIP exhibit decreased interendothelial cell gap formation in response to thapsigargin-induced SOC entry, as compared to control cells. Taken together, our data show that CN contributes to the phosphorylation status of STIM1, which is important in regulation of endothelial SOC entry and I soc activity.

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.

Regulation of store-operated calcium entry by FK506-binding immunophilins.

Calcium entry from the extracellular space into cells is an important signaling mechanism in both physiological and pathophysiological functions. In non-excitable cells, store-operated calcium (SOC) entry represents a principal mode of calcium entry. Activation of SOC entry in pulmonary artery endothelial cells leads to the formation of inter-endothelial cell gaps and subsequent endothelial barrier disruption. Regulation of endothelial SOC entry is poorly understood. In this work, we identify two large molecular weight immunophilins, FKBP51 and FKBP52, as novel regulators of SOC entry in endothelial cells. Using cell fractionation studies and immunocytochemistry we determined that a fraction of these largely cytosolic proteins localize to the plasma membrane where SOC entry channels are found. That FKBP51 and FKBP52 associate with SOC entry channel protein complexes was supported by co-precipitation of the immunophilins with TRPC4, a subunit of the calcium-selective, SOC entry channel ISOC. Dexamethasone-induced upregulation of FKBP51 expression in pulmonary artery endothelial cells reduced global SOC entry as well as ISOC. Similar results were observed when FKBP51 was over-expressed in an inducible HEK293 cell line. On the other hand, when FKBP52 was over-expressed SOC entry was enhanced. When expression of FKBP52 was inhibited, SOC entry was decreased. Collectively, our observations support regulatory roles for these large molecular weight immunophilins in which FKBP51 inhibits, whereas FKBP52 enhances, SOC entry in endothelial cells.

Orai1 determines calcium selectivity of an endogenous TRPC heterotetramer channel.

RATIONALE: Canonical transient receptor potential 4 (TRPC4) contributes to the molecular composition of a channel encoding for a calcium selective store-operated current, I(SOC), whereas Orai1 critically comprises a channel encoding for the highly selective calcium release activated calcium current, I(CRAC). However, Orai1 may interact with TRPC proteins and influence their activation and permeation characteristics. Endothelium expresses both TRPC4 and Orai1, and it remains unclear as to whether Orai1 interacts with TRPC4 and contributes to calcium permeation through the TPRC4 channel. OBJECTIVE: We tested the hypothesis that Orai1 interacts with TRPC4 and contributes to the channel's selective calcium permeation important for endothelial barrier function. METHODS AND RESULTS: A novel method to purify the endogenous TRPC4 channel and probe for functional interactions was developed, using TRPC4 binding to protein 4.1 as bait. Isolated channel complexes were conjugated to anti-TRPC protein antibodies labeled with cy3-cy5 pairs. Förster Resonance Energy Transfer among labeled subunits revealed the endogenous protein alignment. One TRPC1 and at least 2 TRPC4 subunits constituted the endogenous channel (TRPC1/4). Orai1 interacted with TRPC4. Conditional Orai1 knockdown reduced the probability for TRPC1/4 channel activation and converted it from a calcium-selective to a nonselective channel, an effect that was rescued on Orai1 reexpression. Loss of Orai1 improved endothelial cell barrier function. CONCLUSION: Orai1 interacts with TRPC4 in the endogenous channel complex, where it controls TRPC1/4 activation and channel permeation characteristics, including calcium selectivity, important for control of endothelial cell barrier function.

Terminal sialic acids are an important determinant of pulmonary endothelial barrier integrity.

The surface of vascular endothelium bears a glycocalyx comprised, in part, of a complex mixture of oligosaccharide chains attached to cell-surface proteins and membrane lipids. Importantly, understanding of the structure and function of the endothelial glycocalyx is poorly understood. Preliminary studies have demonstrated structural differences in the glycocalyx of pulmonary artery endothelial cells compared with pulmonary microvascular endothelial cells. Herein we begin to probe in more detail structural and functional attributes of endothelial cell-surface carbohydrates. In this study we focus on the expression and function of sialic acids in pulmonary endothelium. We observed that, although pulmonary microvascular endothelial cells express similar amounts of total sialic acids as pulmonary artery endothelial cells, the nature of the sialic acid linkages differs between the two cell types such that pulmonary artery endothelial cells express both α(2,3)- and α(2,6)-linked sialic acids on the surface (i.e., surficially), whereas microvascular endothelial cells principally express α(2,3)-linked sialic acids. To determine whether sialic acids play a role in endothelial barrier function, cells were treated with neuraminidases to hydrolyze sialic acid moieties. Disruption of cell-cell and cell-matrix adhesions was observed following neuraminidase treatment, suggesting that terminal sialic acids promote endothelial barrier integrity. When we measured transendothelial resistance, differential responses of pulmonary artery and microvascular endothelial cells to neuraminidase from Clostridium perfringens suggest that the molecular architecture of the sialic acid glycomes differs between these two cell types. Collectively our observations reveal critical structural and functional differences of terminally linked sialic acids on the pulmonary endothelium.

Organization and function of the FKBP52 and FKBP51 genes.

Best established as components of steroid hormone receptor complexes, it is now clear that the large molecular weight immunophilins, FKBP52 and FKBP51, play important regulatory roles elsewhere in the cell. This review outlines what is known about the organization of the genes, FKBP4 and FKBP5, respectively, encoding these proteins and describes their diverse actions in the nervous system, reproduction, and cancer. The organization of FKBP4 and FKBP5 is very similar among the chordates, and gene expression is influenced by both genetic and epigenetic mechanisms. Recent studies identifying roles of FKBP52 and FKBP51 in the regulation of the microtubule-associated protein tau and microtubule assembly are discussed, as is their interaction with and influence on the transient receptor potential canonical (TRPC) subfamily of ion channel proteins.

Redox regulation of endothelial canonical transient receptor potential channels.

The endothelium is a highly dynamic structure lining the inside of blood vessels that exhibits physical and chemical properties that are critical determinants of overall vascular function. Physically, the endothelium constitutes a semipermeable barrier. Chemically, the endothelium synthesizes numerous factors such as reactive oxygen species (ROS) that can act as autocrine and paracrine signaling molecules. Oxidative stress results when ROS levels increase to levels that cause cellular injury, and, in the endothelium oxidative stress leads to barrier disruption. Endothelial barrier disruption also results from increased cytosolic calcium through store-operated calcium (SOC) entry channels. Although it is known that ROS can interact with and regulate some ion channels, relatively little is known about the interaction of these species with components of endothelial SOC entry channels, the canonical transient receptor potential (TRPC) proteins. Here we review our current understanding of ROS-mediated TRPC channel function and how it affects SOC entry and endothelial barrier disruption.

Activation of glucose-6-phosphate dehydrogenase promotes acute hypoxic pulmonary artery contraction.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O(2) tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose-6-phosphate dehydrogenase (Glc-6-PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95% N(2), 5% CO(2)) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH, and HPV, substantiating the idea that glucose metabolism and Glc-6-PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD(+) and decrease in the ATP-to-ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated Glc-6-PD. Inhibition of Glc-6-PD decreased Ca(2+) sensitivity to the myofilaments and diminished Ca(2+)-independent and -dependent myosin light chain phosphorylation otherwise increased by hypoxia. Silencing Glc-6-PD expression in PA using a targeted small interfering RNA abolished HPV and decreased extracellular Ca(2+)-dependent PA contraction increased by hypoxia. Similarly, Glc-6-PD expression and activity were significantly reduced in lungs from Glc-6-PD(mut(-/-)) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating Glc-6-PD activity and the NADPH-to-NADP(+) ratio to the HPV response clearly indicated a positive linear relationship between Glc-6-PD activity and HPV. Based on these findings, we propose that Glc-6-PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension.

Store-operated calcium entry channels in pulmonary endothelium: the emerging story of TRPCS and Orai1.

Cells of diverse origin utilize shifts in cytosolic calcium concentrations as intracellular signals to elicit physiological responses. In endothelium, inflammatory first messengers increase cytosolic calcium as a signal to disrupt cell-cell borders and produce inter-cellular gaps. Calcium influx across the plasma membrane is required to initiate barrier disruption, although the calcium entry mechanism responsible for this effect remains poorly understood. This chapter highlights recent efforts to define the molecular anatomy of the ion channel responsible for triggering endothelial cell gap formation. Resolving the identity and function of this calcium channel will pave the way for new anti-inflammatory therapeutic targets.

TRPing on the lung endothelium: calcium channels that regulate barrier function.

Rises in cytosolic calcium are sufficient to initiate the retraction of endothelial cell borders and to increase macromolecular permeability. Although endothelial cell biologists have recognized the importance of shifts in cytosolic calcium for several decades, only recently have we gained a rudimentary understanding of the membrane calcium channels that change cell shape. Members of the transient receptor potential family (TRP) are chief among the molecular candidates for permeability-coupled calcium channels. Activation of calcium entry through store-operated calcium entry channels, most notably TRPC1 and TRPC4, increases lung endothelial cell permeability, as does activation of calcium entry through the TRPV4 channel. However, TRPC1 and TRPC4 channels appear to influence the lung extraalveolar endothelial barrier most prominently, whereas TRPV4 channels appear to influence the lung capillary endothelial barrier most prominently. Thus, phenotypic heterogeneity in ion channel expression and function exists within the lung endothelium, along the arterial-capillary-venous axis, and is coupled to discrete control of endothelial barrier function.