1 Department of Pharmacology and Lung Biology and Pathology Research Laboratory, University of South Alabama College of Medicine, Mobile, Alabama 36688; and 2 Department of Anesthesiology, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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Pulmonary endothelium forms a semiselective barrier that regulates fluid balance and leukocyte trafficking. During the course of lung inflammation, neurohumoral mediators and oxidants act on endothelial cells to induce intercellular gaps permissive for transudation of proteinaceous fluid from blood into the interstitium. Intracellular signals activated by neurohumoral mediators and oxidants that evoke intercellular gap formation are incompletely understood. Cytosolic Ca2+ concentration ([Ca2+]i) and cAMP are two signals that importantly dictate cell-cell apposition. Although increased [Ca2+]i promotes disruption of the macrovascular endothelial cell barrier, increased cAMP enhances endothelial barrier function. Furthermore, during the course of inflammation, elevated endothelial cell [Ca2+]i decreases cAMP to facilitate intercellular gap formation. Given the significance of both [Ca2+]i and cAMP in mediating cell-cell apposition, this review addresses potential sites of cross talk between these two intracellular signaling pathways. Emerging data also indicate that endothelial cells derived from different vascular sites within the pulmonary circulation exhibit distinct sensitivities to permeability-inducing stimuli; that is, elevated [Ca2+]i promotes macrovascular but not microvascular barrier disruption. Thus this review also considers the roles of [Ca2+]i and cAMP in mediating site-specific alterations in endothelial permeability.
adenosine 3',5'-cyclic monophosphate; pulmonary; adenylyl cyclase; phosphodiesterase; calcium entry; sarcoplasmic endoplasmic reticulum adenosinetriphosphatase
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INTRODUCTION |
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PULMONARY ENDOTHELIUM regulates the exchange of fluid, solutes, macromolecules, and cells between vascular and tissue spaces. With inflammation, the endothelial barrier becomes more permissive for exchange as the pathway for transport is increased. Studies using whole animal, isolated lung, and cultured endothelial cell models of inflammation suggest that neurohumoral mediators, oxidants, and leukocytes increase endothelial cell cytosolic Ca2+ concentration ([Ca2+]i). This change in [Ca2+]i is believed essential for the generation of endothelial cell paracellular gaps. Whereas elevated [Ca2+]i increases endothelial barrier permeability, increased cAMP has the opposite effect. Increased cAMP prevents or reverses permeability-induced pulmonary edema in nearly every animal species and model studied. Thus permissiveness of the endothelial barrier for exchange may depend on the relative levels of [Ca2+]i and cAMP at any given time.
Due to the significance of [Ca2+]i and cAMP levels on both pulmonary and systemic endothelial cell barrier integrity, this review focuses on putative sites of cross talk between these intracellular signals and their relationship to pulmonary endothelial permeability. Individual areas of focus are addressed in turn, and a summary of recent observations regarding [Ca2+]i and cAMP permeability regulation for pulmonary arterial endothelial cells (PAECs) vs. pulmonary microvascular endothelial cells (PMVECs) is given. To focus the review, however, recent emerging data linking these signal transduction pathways to the mechanisms of paracellular gap formation, such as activation of myosin light chain kinase, focal adhesion kinase phosphorylation, and decreased cell-cell and cell-matrix tethering, are not addressed. For recent reviews on general Ca2+ signaling, the cAMP pathway, or other signal transduction pathways having an influence on endothelial permeability, references are provided throughout the text.
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CYTOSOLIC FREE CALCIUM |
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Many endothelial cell first messengers activate specific receptors and increase [Ca2+]i. The [Ca2+]i response is characterized by two distinct phases, including a transient rise corresponding to the release of Ca2+ from intracellular stores and a more sustained increase due to entry of Ca2+ across the plasmalemma. Each phase, Ca2+ release and entry, can regulate discrete cellular functions. As an example, activation of endothelial cell phospholipase A2 depends on Ca2+ release, whereas activation of nitric oxide synthase and inhibition of adenylyl cyclase both require Ca2+ entry. In addition, [Ca2+]i is also determined by the activity of Ca2+ reuptake and extrusion enzymes. Altering the activities of these enzymes may result in prolonged or abridged Ca2+-mediated changes in cell function.
Intracellular Ca2+ Release
Inositol 1,4,5-trisphosphate, [Ca2+]i, and cAMP. Endothelial cell ligands, coupled through their receptors to the activation of phospholipase C, generate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol as breakdown products of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate. Ins(1,4,5)P3 is a highly hydrophilic molecule that faces little molecular or steric hinderance to diffusion in the cytosol (5). Limited hinderance to diffusion makes Ins(1,4,5)P3 an excellent second messenger because it has access to cytosolic compartments. Both nuclear and smooth endoplasmic reticulum (sER) membranes possess Ins(1,4,5)P3 receptors (60, 106-109, 113, 137, 166, 190, 194) that, when bound to Ins(1,4,5)P3, form an ion channel possessing high Ca2+ conductance (in order of divalent cations, Ba2+ > Sr2+ > Ca2+ > Mg2+). The Ins(1,4,5)P3 receptor likely exists as a tetramer that forms a Ca2+ channel on activation (28, 106, 107, 117, 118). The receptor protein possesses six-membrane-spanning domains so that both amino and carboxy termini reside in the cytosol. These physical properties, four subunits and six-membrane-spanning domains, support placement of the Ins(1,4,5)P3 receptor in the superfamily of voltage-gated and second messenger-gated channels. Recent evidence suggests that at least three distinct isoforms of the Ins(1,4,5)P3 receptor exist plus additional products that represent splice variants (18, 111, 151, 173, 201). It is presently unclear how the different isoforms and splice variants translate into different functions. Although highly conserved among species, expression of Ins(1,4,5)P3-receptor isoforms within a species is heterogeneous among organs. Expression of Ins(1,4,5)P3-receptor isoforms in endothelial cells is poorly characterized.
Given the large concentration gradient of Ca2+ from the sER (high µM to low mM Ca2+) to the cytosol (
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Ca2+ Entry
Perhaps most widely studied of the Ca2+ entry pathways are voltage-gated Ca2+ channels. In neurons and cardiac, skeletal, and smooth muscle cells, membrane depolarization occurring with action potential generation activates these channels and induces an inward Ca2+ flux. However, endothelial cells are nonexcitable and lack action potentials. Consequently, membrane depolarization decreases rather than increases Ca2+ entry because the driving force for Ca2+ flux is the electrochemical gradient existing across the plasmalemma. Although selected reports suggest the presence of voltage-gated Ca2+ channels in endothelial cells (17, 19-21), the abundance of data suggest that most endothelial cells do not possess voltage-gated Ca2+ channels (1, 134).Unstimulated endothelial cells normally maintain a low
[Ca2+]i,
around 60-110 nM. In the environment of the endothelial cell, higher extracellular Ca2+
concentration
([Ca2+]o)
levels are found, with values in the low millimolar range (1.8 mM),
thereby establishing an endothelial
[Ca2+]o-to-[Ca2+]i
gradient of
20,000:1. Clearly, the resting endothelial membrane is
restrictive to Ca2+ influx.
However, many neurohumoral first messengers alter endothelial membrane
Ca2+ permeability to allow for
increased
[Ca2+]i
that is critical for intraendothelial processes. The change in
Ca2+ permeability is accomplished
by the opening of Ca2+-permeable
cationic channels. Several different
Ca2+-permeable channels have been
suggested to exist in endothelial cells, although no gene product for
an endothelial Ca2+ channel has
been identified.
Leak channels. Conditions dictating
the
[Ca2+]o-to-[Ca2+]i
gradient are not static. Rather, a dynamic equilibrium exists as
Ca2+ cycles from the extracellular
millieu through the cell, through the intracellular storage organelles,
and back into the vascular and interstitial spaces (13). Therefore,
Ca2+ leaks across the endothelial
cell membrane, and a leak rate has been estimated at 16 pmol · 106
cells1 · s
1
for bovine (B) PAECs (82). The rate of
Ca2+ leak for endothelial cells
depends on membrane potential and pH because depolarization decreases
(71, 82, 169) and alkalosis increases (41), respectively,
Ca2+ leak.
The precise role for passive Ca2+ leak in regulating endothelial function in situ is unclear, although it is known that constitutive Ca2+ leak in cultured BPAECs regulates both basal and stimulated endothelial cAMP production (171) (Fig. 2, A and B). In addition, BPAEC monolayer permeability to [3H]sorbitol is decreased when the Ca2+ leak pathway is blocked with La3+ (171) (Fig. 2C). The effects of Ca2+ leak on cAMP levels and macromolecular permeability are believed to be mediated, in part, by Ca2+ inhibition of type VI adenylyl cyclase (171). Thus normal Ca2+ leak may help establish in situ pulmonary endothelial barrier permeability by controlling some proportion of endothelial cAMP production. This tonic effect of Ca2+ leak on cAMP levels would provide the endothelial barrier with the ability to undergo bidirectional changes in vascular permeability because inhibiting or increasing Ca2+ leak would decrease or increase, respectively, permeability. Changes in endothelial barrier fluid and solute permeability could then be accomplished in response to changes in the electrochemical driving force for Ca2+ entry.
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To date, the molecular structure of the endothelial leak channel(s) is not known. One possibility is that mammalian counterparts of the transient receptor potential-like (trp-L) gene product, a constitutively active cation (including Ca2+) conductance pathway in the Drosophila melanogaster retina (142), mediate Ca2+ leak in the endothelium. Overexpression of Drosophila trp-L in nonexcitable cell lines increases unstimulated cation conductance (76, 95, 97). The mammalian Trp3 channel shares a high sequence homology (147) and ion selectivity profile (16, 76, 206) with trp-L, i.e., nonselective with respect to Na+ and Ca2+. Overexpression of human Trp3 (hTrp3 or TRPC3) in HEK 293 (16) or Chinese hamster ovary cells (206) increases unstimulated cation transmembrane currents. Thus Trp3 would seem to be an attractive candidate for mediating endothelial Ca2+ leak. Recent data indicate that endothelial cells of systemic vascular origin express Trp3 and that the rat lung also possesses the Trp3 message (58). However, Trp3 may not be expressed in rat (R) or human (H) PAECs (123). Future studies are certainly necessary to determine the gene product(s) responsible for forming pulmonary endothelial leak channels.
Mechanosensitive
Ca2+-permeable
channels.
Increased vascular shear stress is known to mediate changes in vascular
tone via increased nitric oxide production (37, 50, 93, 143, 195). One
mechanism that partly explains shear stress regulation of nitric oxide
production involves activation of mechanosensitive
Ca2+-permeable cation channels in
endothelial cells. Indeed, shear stress induces cationic currents in
human umbilical venous endothelial cells with a
Ca2+-to-Na+
permeability ratio of 12 (156). Interestingly, the
shear stress-activated Ca2+ influx
is lost when sialic acid is removed from the endothelial glycocalyx,
suggesting that channel activation is dependent on a physical coupling
of the channel to some stress-sensing glycocalyx structure (74).
Store-operated Ca2+ entry. Whereas endothelial first messengers promote increased [Ca2+]i via receptor-coupled Ins(1,4,5)P3 generation and/or receptor-gated channel activation, the depletion of intracellular Ca2+ stores appears to stimulate Ca2+ entry by activation of capacitative or SOCs. Specifically, Ins(1,4,5)P3-mediated liberation of stored Ca2+ promotes intracellular store depletion. In turn, Ca2+-permeable membrane channels are activated to allow for refilling of the Ca2+ storage pools. This Ca2+ entry pathway may be distinct from the previously discussed receptor-operated pathway because store depletion, independent of receptor activation, can be accomplished with Ca2+-ATPase inhibitors and membrane cationic currents can be measured (134).
Multiple studies (13, 16, 149) have attempted to define both the signal responsible for regulating channel function in response to store depletion as well as the molecular identity of the SOC(s). However, fewer studies have addressed the functional consequences of Ca2+ influx accomplished by SOC activation, even though Ca2+ entry through this pathway is part of the normal signaling response to Ca2+-elevating agonists. One effect of SOC activation in endothelial cells may be to alter intracellular cAMP content and stimulate endothelial cell shape change, similar to the function of Ca2+ entry through the other discussed pathways. There is now evidence to support the notion that SOC regulation of the cAMP second messenger pathway in nonexcitable cells occurs by Ca2+ effects on adenylyl cyclase activity. Studies by Cooper et al. (35), Chiono et al. (31), and Fagan et al. (46) have revealed that SOC activation can either increase or decrease intracellular cAMP when Ca2+ influx through these channels stimulates or inhibits, respectively, different isoforms of adenylyl cyclase. In fact, the type VI Ca2+-inhibitable cyclase is selectively inhibited by store-operated Ca2+ entry and unaffected by Ca2+ release in C6-2B cells (31). This observation carries strong implications for SOC regulation of pulmonary endothelial cell cAMP content and cell shape because the type VI cyclase is expressed in pulmonary endothelium (171), Ca2+ entry regulates both basal and stimulated cAMP content (171), and Ca2+ entry increases transit of fluid and macromolecules across pulmonary endothelial barriers both in situ and in vitro (29, 88, 122, 123, 171). Because an important site of cross talk between the Ca2+ and cAMP signaling pathways may therefore exist as an SOC-adenylyl cyclase interaction, it is possible that inflammatory mediator-induced changes in pulmonary endothelial cell shape and permeability are accomplished by specific activation of endothelial SOCs. In the intact isolated rat lung, thapsigargin, an agonist of store-operated Ca2+ entry, produces increased solvent permeability, provided [Ca2+]o values are sufficient (29). Furthermore, cultured RPAECs exhibit store-operated Ca2+ entry pathways that, when activated, configure the F-actin cytoskeleton and myosin light chain to promote changes in endothelial cell shape and increase monolayer permeability to macromolecules (123; T. M. Moore, N. R. Norwood, J. R. Creighton, P. Babal, G. H. Brough, D. M. Shasby, and T. Stevens, unpublished observations). Thus activation of endothelial SOCs independent of membrane-receptor activation is sufficient to increase endothelial barrier permeability. As with the other Ca2+ entry pathways, no membrane protein or cellular gene product has been identified as the endothelial SOC. Again, the leading candidates to fulfill this identity belong to the Trp family of proteins. Cloning and expression of the transient receptor potential (trp) gene product from the D. melanogaster retina reveals that this product forms a Ca2+-permeant cation channel that mediates Ca2+ entry when intracellular Ins(1,4,5)P3 is generated and Ca2+ is liberated from the intracellular stores (51, 70, 121). This channel is different functionally than the previously discussed Trp-L because Trp is activated by Ca2+ store depletion, whereas Trp-L is not (164, 186). Six mammalian trp gene products, some with splice variants, have been identified (16). Of these, Trp1, Trp3, Trp4, and Trp6 have been reported in lung tissue (22, 55, 205). Neither Trp3 nor Trp6 forms SOCs (22, 206), and neither appears to be present in pulmonary endothelial cells (123), although it is currently unclear whether Trp4 is expressed in pulmonary endothelial cells. Recent data indicate that Trp1 and possibly a splice variant may form pulmonary endothelial SOCs. Trp1 and TRPC1A are expressed in HPAECs (123). Likewise, Trp1 is expressed in cultured RPAECs (Moore et al., unpublished observations). Unequivical proof is still needed, however, to determine whether Trp1 or any other Trp protein constitutes pulmonary endothelial cell SOCs and, more importantly, whether these proteins are involved in pulmonary inflammation and the development of increased pulmonary vascular permeability.Ca2+ Reuptake
Sarco(endo)plasmic reticulum Ca2+-ATPase. Termination of a Ca2+ signal, whether due to Ca2+ release or entry, involves [Ca2+]i resequestration into intracellular stores. Biochemical data demonstrate that intracellular Ca2+ stores in excitable and nonexcitable cells are maintained by a class of ion-motive ATPases known as sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). To date, five SERCA isoforms have been characterized, and these are derived through alternative splicing of three gene products: SERCA1, -2 and -3. SERCA1a is the major adult fast-twitch skeletal isoform expressed in high levels in striated muscle sarcoplasmic reticulum (33). SERCA1b is developmentally regulated and only expressed in neonatal fast-twitch skeletal muscle (7). SERCA2a is the major isoform found in slow-twitch and cardiac muscles, whereas SERCA2b is ubiquitously expressed and is considered to be the "housekeeping"-type Ca2+ pump of nonexcitable cells (104, 182). SERCA3 is also expressed in nonexcitable cells (104, 182); however, SERCA2b appears to be the major candidate to act as the Ca2+ pump for Ins(1,4,5)P3-sensitive Ca2+ stores (48). SERCA3 expression is most abundant in the intestine, thymus, and cerebellum, with lower levels of expression in the spleen, lymph node, and lung (196). Quantification of SERCA isoform transcripts from rat lung reveals that SERCA2b (59%) is the predominant isoform followed by SERCA3 (28%) and SERCA2a (13%) (196). Although there is no direct evidence, SERCA3 expression may be limited to a subpopulation of airway secretory epithelial cells (6, 196). However, SERCA3 expression has been demonstrated in endothelial cells from the rat aorta and cardiac microvascular circulation (6, 196). Therefore, the SERCA isoform(s) relevant to lung endothelial cells has yet to be determined.
Biochemical, sequence, and three-dimensional crystal analyses of the SERCAs have yielded a reasonable model of the protein (105, 185). Each of the SERCAs possesses three structural domains including 1) the large cytosolic head containing the ATP-binding site, 2) the transmembrane domain containing a high-affinity Ca2+-binding site that forms the Ca2+ transport pathway, and 3) the stalk region connecting the transmembrane domain to the cytosolic head. MacLennan et al. (105) proposed a "marionette" model that describes cytosolic nucleotide binding and the transmission of conformational changes to transmembrane Ca2+ translocation domains. In E1 conformations, Ca2+ has access to a high-affinity binding site in the transmembrane domain but does not have access to the lumen. Binding of Ca2+ at these sites permits ATP hydrolysis in the cytosolic domain that phosphorylates the enzyme in a high-energy form. Phosphorylation triggers a series of conformational changes, providing Ca2+ access to the lumen and disruption of cytosolic Ca2+-binding sites (E2 conformation). Dephosphorylation and subsequent ATP binding then "resets" the pump. Binding of regulatory proteins [e.g., phospholamban (PLN)] to the SERCA protein may impinge on the cytosolic phosphorylation domain (105). SERCAs maintain sER Ca2+ in high micromolar to low millimolar concentrations, thereby establishing a Ca2+ concentration gradient favorable for stimulus-response coupling as occurs after generation of Ins(1,4,5)P3 (see Inositol 1,4,5-trisphosphate, [Ca2+]i, and cAMP). The sER of nonexcitable cells also possesses a high passive Ca2+ conductance, suggesting that SERCA function, by regulating the amount of sER Ca2+, may also represent a physiologically relevant and efficient mode of Ca2+ signaling. Indeed, exogenous Ca2+-ATPase inhibitors such as thapsigargin, cyclopiazonic acid, and dibutylhydroquinone promote sER Ca2+ release. PLN regulates SERCA2a activity in cardiac myocytes and thus modulates cardiac contractility. A plausible structural model for PLN regulation has recently emerged (172). PLN is a pentameric structure juxtaposed to SERCA and may represent a storage form of the protein. According to this model, PLN is in dynamic equilibrium with a monomeric pool. These monomers interact with SERCA via transmembrane helices and at a specific site in the cytoplasmic head. Phosphorylation of the PLN monomer results in structural and/or electrostatic changes that disrupt the SERCA interaction and push the monomers back toward pentamers. Therefore, PLN in the unphosphorylated state inhibits SERCA function, and after phosporylation by PKA, PKC, or Ca2+/calmodulin-dependent kinase, SERCA2a activity is increased. Thus, in the heart, elevated cAMP activates SERCA2a and lowers [Ca2+]i. Coexpression of SERCA2b with PLN in COS 1 cells has demonstrated that this isoform is susceptible to inhibition by PLN (188). A study (96) utilizing PLN knockout mice demonstrated decreased agonist-induced aortic contractile responses, suggesting a PLN regulatory role in smooth muscle. The relevance of these observations to endothelial cell function is unclear, however, because neither PLN expression nor function has been demonstrated in these cells; in fact, SERCA3 activity is entirely resistant to PLN (184, 197). Direct phosphorylation of SERCA2b by Ca2+/calmodulin-dependent kinase has been shown to increase enzyme activity and therefore represents an alternative form of endogenous regulation (67, 200). The role of direct phosphorylation of endothelial cell-associated SERCA(s) has yet to be determined. Intracellular oxidant concentrations represent an additional mechanism by which endothelial cell SERCA function may be regulated (65). Studies (116, 138) utilizing lung- and neuronal-derived microsomes demonstrated that SERCA activity is inhibited by superoxide and hydrogen peroxide in pulmonary endothelial cells, although differences exist as to the susceptibility to oxidant-mediated SERCA inhibition, with SERCA3 being more resistant to peroxide than SERCA2b (64, 66). Furthermore, H2O2-induced Ca2+ release is correlated to increased macromolecular permeability of cultured bovine PMVECs (163), suggesting that in microvascular endothelial cells the intracellular oxidant load may regulate barrier properties via inhibition of Ca2+-ATPase activity. ![]() |
ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE |
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The adenine nucleotide cAMP is a conserved, ubiquitous intracellular second messenger. Intracellular cAMP content at any given time is a reflection of the balance between production and degradation (35, 179, 180). Production of cAMP is regulated by ligand-receptor-G protein activation of adenylyl cyclases, both stimulatory via Gs and inhibitory via Gi. The production of cAMP is therefore susceptible to regulation by ligand concentrations, ligand-receptor occupancies, the degree of Gs versus Gi activation, processes that regulate the intrinsic cycling of G proteins (e.g., GTP vs. GDP bound state), and the quantity and activity of adenylyl cyclase isoform(s). cAMP degradation is due to phosphodiesterase (PDE) activity, although membrane-associated cAMP transporters that extrude cAMP have been identified (68). Emerging evidence indicates that both cAMP production and degradation may be influenced by [Ca2+]i.
The effect of cAMP on endothelial cell barrier properties, at this time, seems unambiguous. Whereas [Ca2+]i and the degree of endothelial barrier "leakiness" often are not related, cAMP levels and pulmonary endothelial barrier integrity are usually highly coupled, i.e., lower cAMP concentration = relatively leaky and higher cAMP concentration = tight barrier. Elevation of cAMP due to receptor-coupled or direct activation of adenylyl cyclase, activation of Gs proteins, inhibition of PDEs, or application of cAMP mimetics all appear to increase cell-cell and cell-matrix tethering, decrease isometric tension development, decrease intercellular gap formation, and decrease permeability in multiple experimental preparations (2, 3, 10, 26, 27, 29, 47, 56, 72, 86, 87, 90, 98, 119, 120, 131, 139, 141, 160-162, 167, 171). Often, the cAMP target that confers the barrier-enhancing effect is poorly understood, but the message that elevated cAMP decreases permeability is clear.
cAMP Production
Adenylyl cyclase(s). Adenylyl cyclase is widely recognized as the enzyme responsible for cAMP synthesis and has been utilized as a pharmacological target for treatment of multiple and diverse clinical problems including heart failure, circulatory collapse, urticaria, and asthma. However, only recently has the complexity of adenylyl cyclase-mediated cell signaling been realized. Since the early 1990s, nine isoforms of the enzyme have been described, each exhibiting limited tissue distribution and distinct regulatory properties. These regulatory properties can be generally characterized by their sensitivity to Ca2+ and PKC. Types I (178) and VIII (46) are stimulated by submicromolar Ca2+, whereas types V and VI are inhibited by submicromolar Ca2+ (78, 203, 204). Type III is Ca2+ stimulated when Gs is activated in cell membrane preparations (32) and Ca2+ is inhibited in intact cells (191, 193), although in some preparations the type III enzyme exhibits no Ca2+ sensitivity (46). Types II and VII are stimulated by PKC (203, 204), and type IV possesses neither Ca2+ nor PKC sensitivity (53). The most recently cloned species, type IX, is not directly sensitive to Ca2+ but is inhibited by the Ca2+-sensitive phosphatase calcineurin (146). Little work in regard to cell-specific expression and function of adenylyl cyclase species has been completed, although it is clear that cells generally possess multiple isoforms of the enzyme. Such diversity of adenylyl cyclase species may engender a greater appreciation of how cell-specific cAMP responses exist and provoke a reexamination of cell- or organ-specific cAMP signaling mechanisms.Recent studies (29, 170, 171) using lung endothelial cells have been conducted to determine whether elevated [Ca2+]i, as occurs during inflammation, decreases cellular cAMP content by inhibiting adenylyl cyclase activity. Because increased [Ca2+]i promotes endothelial disruption and increased cAMP opposes endothelial disruption, Ca2+ inhibition of adenylyl cyclase may provide a mechanism by which [Ca2+]i could decrease cAMP content and thus permissively increase permeability. RT-PCR cloning has revealed expression of multiple isoforms, including the type VI Ca2+-inhibited adenylyl cyclase in cultured PAECs (170, 171) and PMVECs (29, 170), whereas immunostains similarly revealed expression of the type VI enzyme in endothelial cells throughout the intact pulmonary circulation (29). Studies using PAEC membrane fractions demonstrated that adenylyl cyclase activity is in fact inhibited by Ca2+, and studies using intact PAECs and PMVECs demonstrated that elevations in [Ca2+]i also decreased cAMP content (29, 170, 171). These data substantiate the idea that Ca2+ inhibition of adenylyl cyclase regulates endothelial cell cAMP content.
One important regulatory feature of type VI adenylyl cyclase is its
inhibition by compartmentalized
Ca2+ responses. Nonspecific
elevations in
[Ca2+]i
with ionophores, for example, are capable of inhibiting enzyme activity
(56, 171). However, Ca2+ release
from intracellular stores is insufficient but activation of
Ca2+ entry across the cell
membrane is sufficient to inhibit adenylyl cyclase activity (35). The
reason for the disparate effect was not initially apparent, although
recent elegant work by Cooper et al. (35) demonstrated that activation
of Ca2+ entry results in sustained
increases in membrane-associated
Ca2+ levels ranging from 1 to 40 µM, whereas activation of Ca2+
release resulted in transient increases in membrane-associated Ca2+ levels in the range of
0.5-1 µM. Therefore,
Ca2+ entry rather than
Ca2+ release most likely regulates
type VI adenylyl cyclase activity because
1) high concentrations of
membrane-associated Ca2+ levels
are achieved with stimulation of
Ca2+ entry and
2) diffusion limitations of
Ca2+ released from storage
organelles are not in close proximity to the type VI adenylyl cyclase.
Certain receptor-coupled agonists may also activate Gi proteins to decrease cAMP. Work by Manolopoulos et al. (110) suggested that thrombin is negatively coupled to adenylyl cyclase through a Gi protein that decreases cAMP independent of Ca2+ entry and inhibits isoproterenol stimulation of adenylyl cyclase. Experiments conducted by other investigators (187) using thrombin-challenged macro- and microvascular pulmonary endothelial cells have shown thrombin-induced inhibition of cAMP accumulation requires the presence of extracellular Ca2+. Thus agonists receptor coupled to phospholipase C may decrease adenylyl cyclase activity by activation of a Gi protein and/or stimulation of Ca2+ entry. Future studies will be required to determine whether extracellular Ca2+ influences Gi protein coupling to adenylyl cyclase.
As indicated, cAMP responses in endothelial cells are complicated
because of the expression of multiple adenylyl cyclase isoforms, and
thus the specific contribution of type VI adenylyl cyclase to cAMP
content is difficult to assess. Recently, pertussis toxin was shown to
possess differential sensitivities for adenylyl cyclase isoforms (179,
180). Gi greatly suppresses the
activity of Ca2+-inhibited
adenylyl cyclases compared with other isoforms, indicating that types V
and VI adenylyl cyclase are most likely the targets for pertussis
toxin-sensitive Gi proteins.
Interestingly, pertussis toxin 1) is
generally considered a potent cAMP-elevating agent in endothelial cells
and 2) prevents thrombin-induced
inhibition of cAMP accumulation in cells expressing type VI adenylyl
cyclase (110). These data suggest that a pertussis toxin-sensitive
adenylyl cyclase, likely type VI, regulates endothelial cell cAMP
content. These data may also explain how both
Ca2+ entry and
Gi-coupled receptors
synergistically or redundantly regulate cAMP content, i.e., by
converging on the same isoform of adenylyl cyclase.
Ogawa et al. (135) have provided a link between pertussis toxin-sensitive adenylyl cyclase activity and endothelial cell permeability. These investigators have shown that exposure of endothelial cells to hypoxia results in decreased cAMP content and increased macromolecular permeability. Pertussis toxin apparently uncouples hypoxia from decreasing cAMP and thus preserves endothelial barrier function. A theoretical enigma exists because other investigators (169, 171) have shown that hypoxia reduces Ca2+ leak into endothelial cells and thus might be expected to increase cAMP levels. However, the conflicting data may be resolved by examining the time dependence of both [Ca2+]i and cAMP changes. Still, these data provide additional support for the idea that type VI adenylyl cyclase plays a privotal role in regulating cAMP content, important for control of endothelial cell barrier function.
Some diversity in responsiveness to pertussis toxin has been seen between endothelial cells from different vascular beds, different sites within a vascular bed, between species, and between experimental conditions. However, if pertussis toxin regulates type VI adenylyl cyclase and represents an important regulatory mechanism of endothelial barrier properties, then differential sensitivities to pertussis toxin should predict the endothelial permeability response. In support of this idea, pertussis toxin produces barrier disruption in endothelial cells, which respond with little to no increase in cAMP, and barrier enhancement in endothelial cells, which respond with a large increase in cAMP (139, 140). Future studies will be required to carefully dissect the nature of interaction among pertussis toxin, type VI adenylyl cyclase activity, and endothelial barrier function.
In addition to a Ca2+-inhibited isoform of adenylyl cyclase, endothelial cells express two adenylyl cyclase isoforms (types II and VII) that are stimulated by PKC. Indeed, RT-PCR studies using RNA from cultured cells indicated expression of types II and VII adenylyl cyclase, whereas immunostains of lung sections detected type II adenylyl cyclase in macro- and microvascular pulmonary endothelial cells. These observations are significant because both arms of the phosphoinositide pathway, Ca2+ and PKC, appear to influence cAMP responses. The contribution of types II and VII adenylyl cyclases to endothelial cell cAMP homeostasis is generally poorly understood, although recent work indicates that inhibition of PKC reduces cAMP content and direct stimulation of PKC increases cAMP content, consistent with the idea that PKC regulates adenylyl cyclase activity (170). Furthermore, preliminary data indicate that neurohumoral inflammatory mediators coupled to Gq proteins and phosphoinositide turnover first cause Ca2+ inhibition of cAMP content that is followed by PKC-dependent stimulation of cAMP content (Stevens, unpublished observations). Future studies will be required to more carefully address the independent and combined effects of Ca2+-inhibited and PKC-stimulated isoforms of adenylyl cyclase on endothelial cell function because the relative expression of these isoforms may have profound effects on the endothelial cell permeability responses to neurohumoral inflammatory mediators.
cAMP Degradation
PDEs. Research involving the cyclic nucleotide PDE component of pulmonary endothelial cell cyclic nucleotide metabolism has progressed during the 1990s in two main areas: the biochemistry of endothelial isoforms and the pharmacology of PDE inhibitors. It is important to appreciate that cyclic nucleotide PDEs consist of complex families of isozymes designated 1-7 (12, 34, 59, 183). The families have been defined over the last three decades using several criteria including kinetics (e.g., high affinity for cAMP or cGMP with or without both positive and negative cooperative behaviors), substrate preference (cAMP by PDE3, -4, and -7 or cGMP by PDE1, -2, -5, and -6), regulatory properties (Ca2+/calmodulin activation of PDE1 or cGMP stimulation versus cGMP inhibition by PDE2 and -3, respectively), hormonal sensitivity [insulin or tumor necrosis factor (TNF)-
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Seven gene families express >50 cyclic nucleotide PDEs in various mammalian tissues and species (34), thereby making biochemical studies on endothelial PDEs difficult. Endothelial cells have an added complexity in that different complements of PDE isoforms are expressed in different vascular beds or even areas of the same tissue. Although the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine has been widely used to inhibit cAMP degradation in many studies, the development of selective inhibitors to characterize different isoforms has progressed rapidly in recent years. Agents such as rolipram and indolidan may be extremely useful for identifying specific roles for various isoforms in intact cells and tissues but, as yet, selective inhibitors have not been widely employed for endothelial functional analysis. The present data on the regulation of cAMP (and cGMP) metabolism in endothelial cells, however, do support a pivotal role for PDEs.
Data are limited on the expression of PDE enzymes in human endothelial cells. To the authors' knowledge, there are no detailed PDE characterizations in human endothelial cells. However, PDE3, characterized by its high affinity for cAMP as a substrate and the unique regulatory property of inhibition by cGMP, has been implicated as mediating cGMP-dependent reduction in thrombin-induced increased permeability of human umbilical venous endothelial cell monolayers (44, 175). A similar role for PDE3 in human aortic endothelial cells has not been observed (44). These observations emphasize the differences in PDE expression throughout the vasculature, although differences in umbilical venous and aortic endothelial cell isolation and culture may be considered.
Cyclic nucleotide PDE2 and -4 appear to be the major enzymes found in bovine and porcine endothelial cells, whereas PDE1, -3, -5, and -7 show relatively minor activity (8, 89, 91, 101, 165). Significant PDE3 activity has been detected in the particulate fraction of porcine endothelial cells, implicating a potential role for this isozyme in endothelial cAMP level regulation (177). PDE2 represents a major cGMP-receptor site and a potential site for cross talk between the cGMP and cAMP pathways. PDE2 hydrolyzes both cAMP and cGMP equally at saturating substrate concentrations. However, the enzyme contains two noncatalytic, high-affinity cGMP-binding sites (12), the occupation of which causes a conformational change in the enzyme and increases cAMP hydrolysis at low substrate concentrations. The effect of cGMP binding is to alleviate positive cooperativity of cAMP hydrolysis in the absence of cGMP. Because of this mechanism, activation occurs only at low cAMP substrate concentrations. PDE2 has been identified as the major endothelial cGMP-degrading enzyme in porcine PAECs (176) based on the observation of a synergistic reduction in monolayer permeability with atrial natriuretic peptide and the PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine. PDE2 cloning from brain tissues has shown two splice variants with hydrophobic amino-terminal substitutions that are thought to allow membrane association (202). As yet, an endothelial PDE2 has not been cloned.
The PDE4 gene family is far more complex than the PDE2 family. Four subfamilies designated PDE4A-D are known, with 15-20 splice variants occurring in these subfamilies. Subfamilies A-D are not necessarily expressed in all human or rat tissues (45). All isoforms show high-affinity cAMP-specific catalysis, and endothelial PDE4 exhibits Michaelis-Menten kinetic behavior and lacks the negative cooperativity or multiple affinity kinetics characteristic of some PDE4 enzymes. It has been proposed that one variant of PDE4 shows a membrane locus (99), therefore supporting the possibility of subcellular compartmentalization, presumably related to regional cAMP-controlled phosphorylation cascades.
Studies on the regulation of PDE2 and -4 in endothelial cells and their
role in regulating endothelial barrier function are even more limited
than biochemical characterizations. However, some studies
(11, 91, 177) have shown that both of these isoforms may participate in
agonist-induced changes in endothelial function. TNF- treatment of
bovine aortic endothelial cell monolayers increases permeability and
decreases thrombomodulin activity in a time- and dose-dependent manner.
Both of these effects are associated with a decrease in cAMP and
correlate with increased PDE2 and -4 activities, although variation in
the PDE2 and -4 contributions to the TNF-
effect has been reported
(91). PDE3 and -4 inhibitors have been shown to attenuate
H2O2-induced
permeability alterations in porcine PAECs (177). Furthermore, a
specific PDE4 inhibitor, rolipram, both prevents and reverses
ischemia-reperfusion (I/R)-induced increased vascular
permeability in isolated rat lungs (11). Thus at least PDE4 activation
may be important for allowing increased lung vascular permeability.
The importance of differential PDE expression and activity within endothelial cells in conjunction with fluctuations in [Ca2+]i raises an interesting question. Because Ca2+ influx regulates cAMP production to affect endothelial barrier properties (171), can increased [Ca2+]i influence cAMP degradation to regulate permeability? The effects of [Ca2+]i on PDE activity potentially involve two opposing cellular events: 1) increased [Ca2+]i may synergistically promote permeability via PDE activation or 2) it may inhibit PDE activity and thus limit a Ca2+-induced permeability response. Significant Ca2+/calmodulin-stimulated cAMP PDE activity (PDE1) exists in smooth muscle cells (89, 144), and PDE1 has been detected in lysates of cultured porcine aortic endothelial cells (157). However, ionomycin has been reported to inhibit PDE4 in permeabilized endothelial cells (85). Therefore, future studies are needed to determine the precise effects of [Ca2+]i on PDE activities in endothelial cells and how Ca2+ regulation of PDE function relates to regulation of pulmonary vascular permeability.
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CONDUIT-VESSEL AND MICROVASCULAR PULMONARY ENDOTHELIAL CELLS |
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Even though substantial data indicate that Ca2+ promotes endothelial permeability and cAMP reduces permeability, it remains unclear how the intact pulmonary vascular endothelial barrier specifically responds to different inflammatory events. Considerable effort has been made to better understand the normal physiology of pulmonary endothelium and to elucidate mechanisms responsible for producing endothelial dysfunction with the following experimental tools: 1) whole animal and isolated lung models that can be studied both in the physiological and pathological settings with respect to describing the biophysical characteristics of the entire vascular bed and 2) cultured endothelial cells that can be used to describe specific mechanisms of various inflammatory mediator effects.
Until recently, only conduit vessel-derived (i.e., pulmonary artery) endothelial cells have been readily available for mechanistic studies related to endothelial cell permeability regulation. A recurrent concern, however, has been that the data obtained from these cells do not adequately represent cells in situ existing at the putative edemagenic sites, i.e., the microvasculature. Indeed, microscopic evaluation of intact pulmonary circulations has shown microvascular endothelial cells are phenotypically distinct from conduit-vessel endothelial cells (40). Several laboratories have now isolated and cultured endothelial cells from the pulmonary microcirculation (168) and are providing evidence to show that microvascular cells are, in fact, distinct, even in culture, from pulmonary artery-derived endothelial cells.
In regard to normal barrier function, cultured PMVECs are
more restrictive to basal macromolecular diffusion than similarly cultured PAECs (84). Although macromolecular convective flux and fluid
flux measurements have yet to be performed, it is not unexpected that
cultured PMVECs form a highly restrictive monolayer on reaching
confluence. In situ estimates of the microvascular filtration
coefficient
(Kf,c) from
intact pulmonary circulations from numerous species (181) indicate that
pulmonary hydraulic conductances average 0.21 ± 0.05 ml · min1 · mmHg
1 · 100 g lung wet weight
1. Because
this average Kf,c
value is much greater than the values reported for the intact skeletal
muscle and cerebral vascular beds but is comparable to
Kf,c estimates
from the liver, heart, and intestinal circulations, the lung at first
appears to be a relatively "leaky" organ. However, this is not at
all the case because the lungs contain a much higher surface area for
fluid exchange relative to other vascular beds. When quantitative
estimates of the total capillary exchange area for the lung are
compared with those of the skeletal muscle and the hydraulic
conductivity (Lp,c; in
cm3 · dyn
1 · s
1)
values are calculated, the microcirculation of the lung
(Lp,c = 0.51 ± 0.09) is actually much "tighter" than that of skeletal muscle (Lp,c = 2.2). It is not surprising that PMVECs form tight barriers because
normal gas exchange would be impaired without a microcirculation highly
restrictive to fluid and solute leakage.
Two important questions now are: 1) how do inflammatory mediators affect microvascular endothelial function? and 2) are PMVEC responses to classic inflammatory mediators different from conduit vessel-derived endothelial cell responses, especially when phenotype differences exist? These questions are just beginning to be answered, and some initial data specifically related to [Ca2+]i regulation and permeability responses are emerging.
Responses to both the receptor-coupled agonist ATP and the receptor-independent, SOC-activating agonist thapsigargin have been compared with the use of cultured PMVECs and PAECs (84, 170). [Ca2+]i responses to both agonists are decreased in microvascular endothelial cells compared with the conduit vessel-derived endothelial cells. In particular, store-operated Ca2+ entry is greatly suppressed in the microvascular cells under identical physiological conditions (84). Investigation into the mechanisms responsible for the suppressed Ca2+ entry reveals that microvascular cells exist at a relatively depolarized resting membrane potential and exhibit an attenuated hyperpolarization in response to agonist stimulation (170). Thus this decreased electrochemical driving gradient for Ca2+ entry may underlie the suppressed Ca2+ responses in the pulmonary microvascular endothelium, consistent with the previously reported effects of membrane potential on agonist-induced [Ca2+]i responses in endothelium of isolated microvessels (38, 71, 73).
Elevated [Ca2+]i has been clearly linked to increased permeability in conduit vessel-derived cells (62, 84, 102, 103, 122, 152, 153, 155, 158) as well as in isolated perfused lungs (29, 80, 88). However, the findings that pulmonary microvascular cells exhibit attenuated agonist-induced [Ca2+]i responses suggest that these cells may display attenuated permeability responses. Store-operated Ca2+ entry mediates cell shape change and increases macromolecular flux across cultured PAEC monolayers (84, 122, 123). However, activation of store-operated Ca2+ entry does not promote these effects in identically treated PMVEC monolayers (84). Interestingly, when experimental measures are taken to produce equivalent [Ca2+]i levels in both cell types by SOC activation, the pulmonary microvascular cell monolayers are still resistant to cell shape change and increased macromolecular flux. Therefore, one could speculate that inflammatory agonists activating SOCs may have preferential actions for different pulmonary vascular segments in situ, and the functional consequences of SOC activation may show heterogeneity between microvascular and conduit-vessel endothelial cells. Important future studies will be necessary to determine whether ex vivo culture conditions influence the differential pulmonary endothelial [Ca2+]i and permeability responses.
The observation that equivalent [Ca2+]i levels are achieved by SOC activation in pulmonary conduit-vessel endothelial cells and PMVECs without the production of identical effects on cell shape or macromolecular permeability suggests that microvascular endothelial cell shape and barrier regulation may be relatively Ca2+ insensitive. This concept deviates from studies that show that PAEC shape change and barrier disruption occurs in response to Ca2+-driven, myosin light chain-dependent active "contraction" (54, 61, 130, 131, 160, 198, 199). However, nonspecific elevations in [Ca2+]i with ionophore treatment at doses sufficient to produce cultured PAEC shape change fail to produce cell shape change and increase macromolecular flux in cultured PMVECs (57, 84). These data suggest that microvascular endothelial cell shape may not be influenced directly by changes in [Ca2+]i and are consistent with a previous report (43) that showed that inhibition of phosphatase activity in microvascular endothelial cells increases macromolecular permeability via a myosin light chain-independent mechanism.
In addition to differing [Ca2+]i responses, cAMP responses vary between conduit-vessel and microvascular endothelial cells, although both PAECs and PMVECs express a Ca2+-inhibitable adenylyl cyclase (29, 170). In PAECs, endogenous cAMP levels appear to decrease in response to activation of store-operated Ca2+ entry (29, 170). However, neurohumoral inflammatory mediators that activate store-operated Ca2+ entry do not decrease cAMP content in microvascular endothelial cells (170), although stimulation of cAMP production or inhibition of cAMP degradation decreases permeability in both cell types (128). We predict that preserved cAMP responses represent an important determinant of the more restrictive barrier formed by PMVECs. This speculation is supported by previous key studies indicating the importance of cAMP in regulating vascular permeability because strategies aimed at elevating cAMP prevent or reverse agonist-evoked and I/R-induced leak (2, 3, 10, 26, 27, 29, 47, 56, 72, 86, 87, 90, 98, 119, 120, 131, 139, 141, 160-162, 167, 171).
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IMPLICATIONS |
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How can we begin to interpret the data from cultured pulmonary endothelial cells and integrate these findings with those from whole organ studies to enhance our knowledge of the mechanisms responsible for permeability-induced pulmonary edema? This integration process is critical because the diagnosis of clinical acute lung injury and adult respiratory distress syndrome is based, in part, on a patient's level of refractory hypoxemia, which reflects alveolar edema and microvascular dysfunction. Postmortem assessment of lung structure indicates that patients whose death is associated with various forms of noncardiogenic pulmonary edema certainly have severely disrupted microvascular architecture. The precipitating factors causing this condition are many, and overwhelming inflammation is usually associated with severe permeability-induced pulmonary edema (42, 92). However, the precise mechanisms causing the pulmonary microvascular dysfunction are still not clear, and thus the therapeutic regimen for treating the adult respiratory distress syndrome patient is largely supportive care with ventilatory management only.
On the basis of preliminary data from cultured pulmonary microvascular cells, receptor-linked Ca2+-promoting inflammatory mediators may not induce significant PMVEC tension development, although Ca2+-driven tension development is associated with increased macromolecular and fluid flux across PAEC monolayers (54, 61, 130, 131, 160, 198, 199) and in isolated lungs (88), respectively. Experiments dedicated to resolving these puzzling observations may help to accomplish two goals: 1) identify specific in situ leak sites occurring with noncardiogenic pulmonary edema of different etiologies and 2) describe novel functions for pulmonary conduit-vessel endothelial cells with respect to cell shape change in response to physiological agonists.
Recent data support the notion that different inflammatory events can
produce distinct patterns of pulmonary vascular leak. In one scenario,
pulmonary conduit vessels appear to become leaky without a discernible
contribution of the alveolar microcirculation to the leak. In another
scenario, leak occurs in all segments of the pulmonary vasculature.
Individual examples of each scenario can be represented by the
thapsigargin-induced (store-operated Ca2+ entry-induced) (29) and the
I/R-induced lung edema models (2, 3, 11, 87, 88, 124-129, 161),
respectively. In isolated lungs, both thapsigargin and I/R produce
significant increases in the
Kf,c, a
biophysical index of fluid and small-solute permeability of the
vascular wall. In fact, when an
EC50 dose of thapsigargin is
compared with a 30-min period of postischemic reperfusion, changes in
total vascular permeability are nearly identical. However, morphological changes in the two lung edema models are not identical (Fig. 3).
Histological inspection of thapsigargin-challenged lungs reveals
perivascular cuffing of larger vessels, with no apparent disruption of
microvessels. Electron micrographs appear to support this view,
demonstrating that endothelial cells of larger arteries and veins
possess gaps, whereas smaller (150 µm and less) arteriolar, venular, and capillary cells remain intact (P. M. Chetham, P. Babal, J. P. Bridges, I. F. McMurtry, and T. Stevens, unpublished observations).
In contrast, a different histological profile is revealed on inspection
of I/R-challenged lungs. Perivascular cuffing of larger vessels is
still present, but this is accompanied by capillary disruption and
alveolar infiltrate (127). Detailed transmission electron microscopy
reveals that the capillary disruption is characterized by dissociation
of endothelial cells from the underlying basement membrane but that
intercellular gap formation is not discernable (Chetham et al.,
unpublished observations).
|
In addition to the histological differences between the two models, mechanistic differences with respect to the induction of vascular leak are also apparent. As expected, the thapsigargin-induced increased permeability requires extracellular Ca2+, and interventions designed to decrease Ca2+ entry diminish the increase in vascular permeability (29). Furthermore, extracellular antioxidants do not prevent the thapsigargin-induced permeability increase (29). In contrast, I/R-induced increased permeability is oxygen radical dependent (4, 94, 127), and initial data indicate that I/R injury occurs even when extracellular Ca2+ levels are diminished (Chetham et al., unpublished observations). However, the Ca2+ regulatory component to pulmonary microvascular endothelial barrier integrity during I/R may be highly complex. Superoxide is known to inhibit some microsomal SERCA isozyme activity to promote Ca2+ release (116, 138). In addition, calmodulin antagonists block I/R-induced permeability changes in whole lungs (88), and an H2O2-induced pulmonary microvascular permeability increase in cultured monolayers is correlated to Ca2+ release. These observations suggest that a critical component to I/R-induced pulmonary microvascular damage may be endothelial microsomal Ca2+ release. In a preliminary report, it has been shown that depletion of intracellular Ca2+ stores before exposure of isolated lungs to I/R significantly reduces the I/R-induced permeability edema (Chetham et al., unpublished observations). Thus oxidant production during I/R and endothelial [Ca2+]i may, in fact, be linked in promoting microvascular barrier dysfunction, but the mechanism may not involve Ca2+ entry.
These two models of noncardiogenic pulmonary edema, store-operated Ca2+ entry activation and I/R, have brought into focus critical issues related to lung endothelial cell function: 1) activation of Ca2+ entry may produce intercellular gap formation in selective vascular segments located away from gas-exchange areas, and 2) the pulmonary oxidant burden during a pulmonary inflammatory event may determine the degree of microvascular involvement in the overall permeability response. These observations are significant because multiple inflammatory stimuli, including thrombin, complement, platelet-activating factor, and substance P, may activate store-operated Ca2+ entry, promote oxidant generation, or both.
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SUMMARY |
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This review identifies the sites of potential cross talk between [Ca2+]i and cAMP in lung endothelial cells. It is clear that although such sites of cross talk exist, their relevance to endothelial cell function are, for the most part, poorly understood. In particular, although both [Ca2+]i and cAMP clearly influence endothelial cell barrier function, little is known about how [Ca2+]i and cAMP responses are balanced during development of intercellular gap formation and in the reformation of an intact barrier. Future studies will be required to address these important issues.
Perhaps most strikingly, emerging data support the idea that PMVECs do not respond to increases in [Ca2+]i in a manner similar to PAECs. This concept strays from the traditional view that elevated [Ca2+]i activates contraction-dependent alterations in cell shape, studies conducted solely to this point with PAECs. Models of oxidant-induced lung injury clearly demonstrate microvascular endothelial cell disruption in the setting of lung injury, but it is now unclear whether these cells respond to receptor-coupled inflammatory agonists in a similar way. Important future studies will determine whether inflammatory agonists are capable of increasing microvascular endothelial cell permeability by decreasing cell-cell and cell-matrix tethering, to determine whether different responses to inflammatory mediators in PAECs versus PMVECs are due to cAMP regulation, and to understand why pulmonary conduit-vessel endothelial cells readily respond to Ca2+ entry-driven changes in cell shape.
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ACKNOWLEDGEMENTS |
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We thank Drs. W. J. Thompson and M. N. Gillespie for constructive advice.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-56050, HL-60024 (both to T. Stevens), and HL-46494; a Parker B. Francis Pulmonary Fellowship (to T. Stevens); and American Heart Association-Alabama Affiliate Fellowships (to T. M. Moore and J. J. Kelly).
Address for reprint requests: T. Stevens, Dept. of Pharmacology, College of Medicine, MSB 3130, Univ. of South Alabama, Mobile, AL 36688.
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