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Cytosolic Ca2+ and adenylyl cyclase responses in phenotypically distinct pulmonary endothelial cells
Abstract
Pulmonary microvascular endothelium forms a tighter barrier than does pulmonary artery endothelium; the mechanism of this important phenotypic difference is uncertain. We examined two regulators of endothelial permeability, cytosolic Ca2+ concentration ([Ca2+]i) and adenosine 3',5'-cyclic monophosphate (cAMP), in microvascular (PMVEC) and pulmonary conduit artery (PAEC) endothelium. Both resting and stimulated [Ca2+]i were lower in PMVEC compared with PAEC (resting [Ca2+]i, 94 +/- 7 vs. 123 +/- 8 nM; ATP-stimulated peak, 1.04 +/- 0.14 vs. 1.98 +/- 0.13 microM). Sustained Ca2+ transients in response to either ATP or thapsigargin were reduced in PMVEC compared with PAEC (ATP, 199 +/- 22 vs. 411 +/- 43 nM; thapsigargin, 195 +/- 13 vs. 527 +/- 65 nM), suggesting reduced Ca2+ influx in PMVEC. Reduced Ca2+ influx in PMVEC was confirmed by Mn2+ quenching and patch-clamp experiments. mRNA for Ca(2+)-inhibitable and protein kinase C-stimulated adenylyl cyclases was detected in both cell types. Whereas ATP caused a [Ca2+]i-mediated decrease in cAMP in PAEC, ATP caused a protein kinase C-mediated increase in cAMP in PMVEC. We conclude that PMVEC express a unique phenotype that favors enhanced barrier function through attenuated Ca2+ influx and preservation of cAMP content.
... Mechanisms that regulate segmental barrier function are not known, although in vitro conduit ECs demonstrate a clear role for increased intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) as a catalyst for increased permeability. However, microvascular (e.g., Ͻ100 µm) ECs apparently do not respond to increased [Ca 2ϩ ] i with EC shape changes that promote permeability (18,29). This suggests that unique mechanisms account for pulmonary arterial EC versus pulmonary microvessel EC barrier disruption. ...
... Activation of SOC entry produces macrovascular but not microvascular EC gaps. Although an elevated [Ca 2ϩ ] i is generally thought to increase EC permeability, recent data (18,29) comparing the barrier properties of pulmonary macrovascular and microvascular ECs indicated that cultured microvascular cells form an intrinsically ''tighter'' barrier to macromolecular flux that is relatively unresponsive to inflammatory mediators. Our next studies therefore determined the influence of TG on EC gaps in situ as a means of investigating the 2). ...
... Thus, in contrast to conduit ECs, microvascular ECs express a unique phenotype that favors enhanced barrier func- ajplung.physiology.org tion associated with attenuated agonist-induced Ca 2ϩ influx (18,29). We determined whether similar differences in EC gaps occurred in macrovascular (Ͼ100 µm) compared with microvascular segments in the intact pulmonary circulation. ...
... Because increased [Ca 2ϩ ] i promotes endothelial disruption and increased cAMP opposes endothelial disruption, Ca 2ϩ inhibition of adenylyl cyclase may provide a mechanism by which [Ca 2ϩ ] i could decrease cAMP content and thus permissively increase permeability. RT-PCR cloning has revealed expression of multiple isoforms, including the type VI Ca 2ϩ -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 Ca 2ϩ , and studies using intact PAECs and PMVECs demonstrated that elevations in [Ca 2ϩ ] i also decreased cAMP content (29, 170,171). ...
... RT-PCR cloning has revealed expression of multiple isoforms, including the type VI Ca 2ϩ -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 Ca 2ϩ , and studies using intact PAECs and PMVECs demonstrated that elevations in [Ca 2ϩ ] i also decreased cAMP content (29, 170,171). These data substantiate the idea that Ca 2ϩ inhibition of adenylyl cyclase regulates endothelial cell cAMP content. ...
... These observations are significant because both arms of the phosphoinositide pathway, Ca 2ϩ 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 G q proteins and phosphoinositide turnover first cause Ca 2ϩ inhibition of cAMP content that is followed by PKC-dependent stimulation of cAMP content (Stevens, unpublished observations). ...
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.
... Mechanisms that regulate segmental barrier function are not known, although in vitro conduit ECs demonstrate a clear role for increased intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) as a catalyst for increased permeability. However, microvascular (e.g., Ͻ100 µm) ECs apparently do not respond to increased [Ca 2ϩ ] i with EC shape changes that promote permeability (18,29). This suggests that unique mechanisms account for pulmonary arterial EC versus pulmonary microvessel EC barrier disruption. ...
... Activation of SOC entry produces macrovascular but not microvascular EC gaps. Although an elevated [Ca 2ϩ ] i is generally thought to increase EC permeability, recent data (18,29) comparing the barrier properties of pulmonary macrovascular and microvascular ECs indicated that cultured microvascular cells form an intrinsically ''tighter'' barrier to macromolecular flux that is relatively unresponsive to inflammatory mediators. Our next studies therefore determined the influence of TG on EC gaps in situ as a means of investigating the L42 SEGMENTAL REGULATION OF PULMONARY VASCULAR PERMEABILITY site of vascular leak. ...
... Recently, Kelly et al. (18) demonstrated that activation of SOC entry is sufficient to produce gap formation in cultured pulmonary arterial ECs; however, activation of SOC entry was not sufficient to increase permeability in cultured pulmonary microvascular ECs (e.g., Ͻ100 µm). Thus, in contrast to conduit ECs, microvascular ECs express a unique phenotype that favors enhanced barrier func- (18,29). We determined whether similar differences in EC gaps occurred in macrovascular (Ͼ100 µm) compared with microvascular segments in the intact pulmonary circulation. ...
An intact endothelial cell barrier maintains normal gas exchange in the lung, and inflammatory conditions result in barrier disruption that produces life-threatening hypoxemia. Activation of store-operated Ca2+ (SOC) entry increases the capillary filtration coefficient (Kf,c) in the isolated rat lung; however, activation of SOC entry does not promote permeability in cultured rat pulmonary microvascular endothelial cells. Therefore, current studies tested whether activation of SOC entry increases macro- and/or microvascular permeability in the intact rat lung circulation. Activation of SOC entry by the administration of thapsigargin induced perivascular edema in pre- and postcapillary vessels, with apparent sparing of the microcirculation as evaluated by light microscopy. Scanning and transmission electron microscopy revealed that the leak was due to gaps in vessels >/= 100 micrometer, consistent with the idea that activation of SOC entry influences macrovascular but not microvascular endothelial cell shape. In contrast, ischemia and reperfusion induced microvascular endothelial cell disruption independent of Ca2+ entry, which similarly increased Kf,c. These data suggest that 1) activation of SOC entry is sufficient to promote macrovascular barrier disruption and 2) unique mechanisms regulate pulmonary micro- and macrovascular endothelial barrier functions.
... Calcium regulation of adenylyl cyclase and cAMP. Stevens and colleagues (40,41) have previously established that physiological rises in [Ca 2ϩ ] i , especially due to activation of store-operated calcium entry, inhibit PAEC adenylyl cyclase activity and reduce global cellular cAMP content. To assess the regulation of PMVEC adenylyl cyclase activity by calcium, thapsigargin was administered to both cell types to activate storeoperated calcium entry, and ATP-to-cAMP conversion was measured. ...
... The inability of calcium to regulate PMVEC cAMP could be due to the insensitivity of adenylyl cyclase to calcium, a reduced calcium response to thapsigargin, or an intrinsic feature of the activation state of adenylyl cyclase. A prior study by Stevens et al. (40) indicated that PMVECs express type VI calcium-inhibited adenylyl cyclase. We therefore next tested whether storeoperated calcium entry was suppressed in rat PMVECs compared with that in PAECs. ...
... Responsiveness to forskolin was decreased in PMVECs, indicating either reduced intrinsic activity of PMVEC adenylyl cyclase complexes or increased phosphodiesterase activity. Both cell types express similar isoforms of adenylyl cyclase, including types II, IV, VI, and VII (8,40). Expression of types VIII and IX have not been rigorously tested, and quantitative estimates of cyclase abundance have yet to be performed. ...
Pulmonary microvascular endothelial cells (PMVECs) form a more restrictive barrier to macromolecular flux than pulmonary arterial endothelial cells (PAECs); however, the mechanisms responsible for this intrinsic feature of PMVECs are unknown. Because cAMP improves endothelial barrier function, we hypothesized that differences in enzyme regulation of cAMP synthesis and/or degradation uniquely establish an elevated content in PMVECs. PMVECs possessed 20% higher basal cAMP concentrations than did PAECs; however, increased content was accompanied by 93% lower ATP-to-cAMP conversion rates. In PMVECs, responsiveness to beta-adrenergic agonist (isoproterenol) or direct adenylyl cyclase (forskolin) activation was attenuated and responsiveness to phosphodiesterase inhibition (rolipram) was increased compared with those in PAECs. Although both types of endothelial cells express calcium-inhibited adenylyl cyclase, constitutive PMVEC cAMP accumulation was not inhibited by physiological rises in cytosolic calcium, whereas PAEC cAMP accumulation was inhibited 30% by calcium. Increasing either PMVEC calcium entry by maximal activation of store-operated calcium entry or ATP-to-cAMP conversion with rolipram unmasked calcium inhibition of adenylyl cyclase. These data indicate that suppressed calcium entry and low ATP-to-cAMP conversion intrinsically influence calcium sensitivity. Adenylyl cyclase-to-cAMP phosphodiesterase ratios regulate cAMP at elevated levels compared with PAECs, which likely contribute to enhanced microvascular barrier function.
... BPAECs were seeded onto chambered glass coverslips (Nalge Nunc, Naperville, IL) and grown to confluence. [Ca 2ϩ ] i was estimated with the Ca 2ϩ -sensitive fluorophore fura 2-AM (Molecular Probes, Eugene, OR) according to methods previously described (19,25,40,41). Briefly, BPAECs were washed with 2 ml of a HEPES (Fisher Scientific, Atlanta, GA)-buffered physiological salt solution (PSS) containing (in g/l) 6.9 NaCl, 0.35 KCl, 0.16 KH 2 PO 4 , 0.141 MgSO 4 , and 2.0 D-glucose and 25 mM HEPES. ...
... After the spike, a sustained elevation in [Ca 2ϩ ] i above baseline was observed. In addition, the average sustained [Ca 2ϩ ] i level demonstrated some variability over time as indicated by the wide SE range, which was attributed to oscillatory changes in [Ca 2ϩ ] i at different rates of cycling for individual cells in the measurement field (40). Thapsigargin elicited a more gradual increase in [Ca 2ϩ ] i that likewise remained elevated above basal levels on activation of SOCs. ...
... Thrombin is one such mediator coupled to G q proteins that stimulates cultured pulmonary artery endothelial cell shape change to increase macromolecular permeability by activating multiple, parallel intracellular signaling pathways concurrent with increased [Ca 2ϩ ] i (21,22,27,33,36). As described here and consistent with previous observations ( 1, 14, 21, 22), the thrombin-induced increase in [Ca 2ϩ ] i occurs in response to Ins(1,4,5)P 3 -mediated Ca 2ϩ release and sustained Ca 2ϩ influx, which are distinguished with the Ca 2ϩ fluorophore fura 2 (14,21,22,40). With respect to Ca 2ϩ release, our data indicate that extracellular Ca 2ϩ levels do not influence peak responses to thrombin. ...
The present study evaluated the necessity of store-operated Ca(2+) entry in mediating thrombin-induced 20-kDa myosin light chain (MLC(20)) phosphorylation and increased permeability in bovine pulmonary artery endothelial cells (BPAECs). Thrombin (7 U/ml) and thapsigargin (1 microM) activated Ca(2+) entry through a common pathway in confluent BPAECs. Similar increases in MLC(20) phosphorylation were observed 5 min after thrombin and thapsigargin challenge, although thrombin produced a sustained increase in MLC(20) phosphorylation that was not observed in response to thapsigargin. Neither agonist increased MLC(20) phosphorylation when Ca(2+) influx was inhibited. Thrombin and thapsigargin induced inter-endothelial cell gap formation and increased FITC-dextran (molecular radii 23 A) transfer across confluent BPAEC monolayers. Activation of store-operated Ca(2+) entry was required for thapsigargin and thrombin receptor-activating peptide to increase permeability, demonstrating that activation of store-operated Ca(2+) entry is coupled with MLC(20) phosphorylation and is associated with intercellular gap formation and increased barrier transport of macromolecules. Unlike thrombin receptor-activating peptide, thrombin increased permeability without activation of store-operated Ca(2+) entry, suggesting that it partly disrupts the endothelial barrier through a proteolytic mechanism independent of Ca(2+) signaling.
... Cells were verified as endothelial by positive factor VIII staining and by the uptake of 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate-labeled, acetylated low density lipoprotein. Cells were cultured after standard procedures, as described elsewhere (14,15,36). ...
... Total RNA was extracted with RNA Stat-60 (Tel-Test 'B', Friendswood, TX) from A549 cells and human pulmonary artery endothelial cells were grown to 100% confluence (ϳ10 7 cells) on 75 cm 2 tissue culture flasks. First-strand synthesis was performed with SuperScript II reverse transcriptase (200 units) and oligo(dT) [12][13][14][15][16][17][18] primer (Life Technologies, Rockville, MD) on ϳ 1 g of DNase I-treated total RNA. PCR was conducted with the following sets of primers for Trp1/Trp: set A, 5Ј-TCG CCG AAC GAG GTG ATG G-3Ј (sense) and 5Ј-GTT ATG GTA ACA GCA TTT CTC C-3Ј (antisense); set B, 5Ј-GTG CTT GGG AGA AAT GCT G-3Ј (sense) and 5Ј-GGG GCT TGG GTA GAG ATA C-3Ј (antisense); PCR products were ligated into TA cloning vector pCR2.1 (Invitrogen, San Diego, CA) and transformed into chemically competent Escherichia coli. ...
... The thapsigargin response was associated with two clearly discernible increases in [Ca 2ϩ ] i , the first due to Ca 2ϩ release from intracellular stores and the second due exclusively to Ca 2ϩ entry across the cell membrane (Fig. 5A). In typical [Ca 2ϩ ] i responses to thapsigargin in other cell types (14,15,36), Ca 2ϩ release and entry phases are not clearly resolved when physiological concentrations of extracellular Ca 2ϩ are present. Individual 340 and 380 wave- Primer set A generated a product of expected size in both cell types. ...
Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, I(SOC), in A549 and endothelial cells. Twenty different 'chimeric' 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a I(SOC), which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 microM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca(2+) response to thapsigargin by 25% and similarly reduced I(SOC) by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion.
... These findings bring into question the independent roles of [Ca 2 ] i and cAMP in endothelial cell barrier disruption. Results from cloning and expression of a calcium-inhibited adenylyl cyclase (type 6 adenylyl cyclase [AC 6 ]*) in nonexcitable cells provided a plausible mechanism through which inflam- matory [Ca 2 ]i agonists could decrease cAMP (Yoshimura and Cooper, 1992 ). Endothelial cells express AC 6 as determined by in vivo (Chetham et al., 1997; Jourdan et al., 2001) and in vitro assays (Manolopoulos et al., 1995a,b; Stevens et al., 1995 Stevens et al., , 1997 Stevens et al., , 1999 ; unpublished data). Submicromolar calcium concentrations decrease cAMP accumulation by 30% in endothelial cell membranes (Stevens et al., 1995 ), and activation of store-operated calcium entry decreases cAMP and PKA activity 30–50% in intact cells (Stevens et al., 1995Stevens et al., , 1997Stevens et al., , 1999 unpublished data). ...
... Results from cloning and expression of a calcium-inhibited adenylyl cyclase (type 6 adenylyl cyclase [AC 6 ]*) in nonexcitable cells provided a plausible mechanism through which inflam- matory [Ca 2 ]i agonists could decrease cAMP (Yoshimura and Cooper, 1992 ). Endothelial cells express AC 6 as determined by in vivo (Chetham et al., 1997; Jourdan et al., 2001) and in vitro assays (Manolopoulos et al., 1995a,b; Stevens et al., 1995 Stevens et al., , 1997 Stevens et al., , 1999 ; unpublished data). Submicromolar calcium concentrations decrease cAMP accumulation by 30% in endothelial cell membranes (Stevens et al., 1995 ), and activation of store-operated calcium entry decreases cAMP and PKA activity 30–50% in intact cells (Stevens et al., 1995Stevens et al., , 1997Stevens et al., , 1999 unpublished data). Thus, calcium inhibition of AC 6 may importantly contribute to endothelial cell gap formation. ...
... Lung macro-and microvascular endothelial cells exhibit dis- tinct [Ca 2 ]i regulatory properties (Stevens et al., 1997 ), although the relative [Ca 2 ]i responses to G q -linked agonists are poorly understood. We therefore used pulmonary artery (PAECs) and microvascular endothelial cells (PMVECs) to examine thrombin-activated [Ca 2 ]i signaling. ...
Acute transitions in cytosolic calcium ([Ca2+]i) through store-operated calcium entry channels catalyze interendothelial cell gap formation that increases permeability. However, the rise in [Ca2+]i only disrupts barrier function in the absence of a rise in cAMP. Discovery that type 6 adenylyl cyclase (AC6; EC 4.6.6.1) is inhibited by calcium entry through store-operated calcium entry pathways provided a plausible explanation for how inflammatory [Ca2+]i mediators may decrease cAMP necessary for endothelial cell gap formation. [Ca2+]i mediators only modestly decrease global cAMP concentrations and thus, to date, the physiological role of AC6 is unresolved. Present studies used an adenoviral construct that expresses the calcium-stimulated AC8 to convert normal calcium inhibition into stimulation of cAMP, within physiologically relevant concentration ranges. Thrombin stimulated a dose-dependent [Ca2+]i rise in both pulmonary artery (PAECs) and microvascular (PMVEC) endothelial cells, and promoted intercellular gap formation in both cell types. In PAECs, gap formation was progressive over 2 h, whereas in PMVECs, gap formation was rapid (within 10 min) and gaps resealed within 2 h. Expression of AC8 resulted in a modest calcium stimulation of cAMP, which virtually abolished thrombin-induced gap formation in PMVECs. Findings provide the first direct evidence that calcium inhibition of AC6 is essential for endothelial gap formation.
... Microvascular endothelial cells express AC 6 and respond to inflammatory agonists with a rise in cytosolic Ca 2ϩ . In striking contrast to macrovascular endothelial cells, however, the activation of storeoperated Ca 2ϩ entry does not decrease microvascular endothelial cell global (e.g., whole cell) cAMP content (18,28,30), leading to questions regarding the physiological significance of AC 6 in microvascular endothelial cells (5). ...
... Initial studies, therefore, utilized thapsigargin to induce the passive depletion of endoplasmic reticulum Ca 2ϩ and activate store-operated Ca 2ϩ entry channels to assess whether activation of store-operated Ca 2ϩ entry reduces cAMP in PMVECs. Unlike our prior reports using pulmonary artery endothelial cells (PAECs), thapsigargin did not reduce cAMP conversion over a 40-min time course in PMVECs (28,30,32) (Fig. 1). ...
... Nonetheless, thapsigargin abolished the subsequent rise in cAMP that was induced by rolipram and forskolin, suggesting that AC 6 is the dominant AC isoform in PMVECs. Ca 2ϩ -insensitive AC activity is likely due to expression of other forskolin-stimulated AC isoforms (30). Because endothelial cells possess significant constitutive Ca 2ϩ "leak" (20), we examined the role of such leak channels in control of cAMP. ...
Activation of store-operated Ca(2+) entry inhibits type 6 adenylyl cyclase (EC; AC(6); Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712-6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC(6) and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca(2+) entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca(2+) inhibition of AC(6). Enzyme activity was assessed using PMVEC membranes, where Ca(2+) and cAMP concentrations were independently controlled. Endogenous AC(6) activity exhibited high- and low-affinity Ca(2+) inhibition, similar to that observed in C6-2B cells, which predominantly express AC(6). Ca(2+) inhibition of AC(6) in PMVEC membranes was observed after enzyme activation and inhibition of phosphodiesterase activity and was independent of the free cAMP concentration. Thus, under basal conditions, the constitutive type 4 phosphodiesterase activity rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC(6) to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition.
... Lung macro-and microvascular endothelial cells exhibit distinct Ca 2+ entry pathways that govern [Ca 2+ ] i transitions in response to G q -linked agonists (46,58). The typical global [Ca 2+ ] i response of endothelial cells to G q -linked agonists, e.g., thrombin, comprises the initial immediate [Ca 2+ ] i rise (first phase) that reflects release of Ca 2+ from inositol 1,4,5trisphosphate-sensitive intracellular Ca 2+ stores and the second phase of [Ca 2+ ] i elevation that reflects Ca 2+ entry across the cell membrane after Ca 2+ store depletion due to activation of store-and receptor-operated Ca 2+ channels. ...
... Prior studies reported heterogeneity of lung macro-and microvascular endothelial cells in both Ca 2+ and cAMP signaling pathways (45,46). Compared with PAECs, PMVECs exhibit a greater global [Ca 2+ ] i response to thrombin (9), but an attenuated intracellular cAMP response to forskolin or β-adrenergic receptor agonist isoproterenol (45,46), both activating transmembrane type 6 adenylyl cyclase. ...
... Prior studies reported heterogeneity of lung macro-and microvascular endothelial cells in both Ca 2+ and cAMP signaling pathways (45,46). Compared with PAECs, PMVECs exhibit a greater global [Ca 2+ ] i response to thrombin (9), but an attenuated intracellular cAMP response to forskolin or β-adrenergic receptor agonist isoproterenol (45,46), both activating transmembrane type 6 adenylyl cyclase. On the other hand, the intracellular cAMP response to the cAMP-specific type 4 phosphodiesterase inhibitor rolipram is more pronounced in PMVECs (45,46). ...
The T-type Ca2+ channel Cav3.1 subunit is present in pulmonary microvascular endothelial cells (PMVECs), but not in pulmonary artery endothelial cells (PAECs). The present study sought to assess the role of Cav3.1 in thrombin-induced Weibel-Palade body exocytosis and consequent von Willebrand factor (VWF) release. In PMVECs and PAECs transduced with a green fluorescent protein (GFP)-tagged VWF chimera, we examined the real-time dynamics and secretory process of VWF-GFP-containing vesicles in response to thrombin and the cAMP-elevating agent isoproterenol. Whereas thrombin stimulated a progressive decrease in the number of VWF-GFP-containing vesicles in both cell types, isoproterenol only decreased the number of VWF-GFP-containing vesicles in PAECs. In PMVECs, thrombin-induced decrease in the number of VWF-GFP-containing vesicles was nearly abolished by the T-type Ca2+ channel blocker mibefradil as well as by Cav3.1 gene silencing with small hairpin RNA. Expression of recombinant Cav3.1 subunit in PAECs resulted in pronounced increase in thrombin-stimulated Ca2+ entry, which is sensitive to mibefradil. Together, these data indicate that VWF secretion from lung endothelial cells is regulated by two distinct pathways involving Ca2+ or cAMP, and support the hypothesis that activation of Cav3.1 T-type Ca2+ channels in PMVECs provides a unique cytosolic Ca2+ source important for Gq-linked agonist-induced VWF release.
... Functional studies in in vitro models illustrate that lung microvascular endothelial cells possess a more restrictive barrier than their macrovascular counterparts (Chetham et al., 1999;Kelly et al., 1998;Moore et al., 1998b), and exhibit unique signaling responses to similar agonists (Chetham et al., 1999;Kelly et al., 1998;Moore et al., 1998a;Stevens et al., 1997Stevens et al., , 1999Stevens et al., , 2001. Distinct sitespecific vascular responses are observed in the intact lung (Chetham et al., 1999;Khimenko and Taylor, 1999;Qiao and Bhattacharya, 1991). ...
... We have previously observed that the thapsigargin-induced store operated calcium entry response is lower in PMVECs than it is in PAECs (Kelly et al., 1998;Moore et al., 1998b;Stevens et al., 1997Stevens et al., , 1999. Store-operated calcium entry pathways are activated by depletion of calcium in the endoplasmic reticulum (Putney, 1986). ...
... Our present finding that the plasmalemma and endoplasmic reticulum are immediately adjacent in PAECs lends further support for the necessity of direct coupling between the membrane and organelle in channel activation. Indeed, PAECs possess more prominent store operated calcium entry pathways than do PMVECs (Kelly et al., 1998;Stevens et al., 1997Stevens et al., , 1999. ...
Lung macro- and microvascular endothelial cells exhibit unique functional attributes, including signal transduction and barrier properties. We therefore sought to identify structural and functional features of endothelial cells that discriminate their phenotypes in the fully differentiated lung. Rat lung macro- (PAEC) and microvascular (PMVEC) endothelial cells each exhibited expression of typical markers. Screening for reactivity with nine different lectins revealed that Glycine max and Griffonia (Bandeiraea) simplicifolia preferentially bound microvascular endothelia whereas Helix pomatia preferentially bound macrovascular endothelia. Apposition between the apical plasmalemma and endoplasmic reticulum was closer in PAECs (8 nm) than in PMVECs (87 nm), implicating this coupling distance in the larger store operated calcium entry responses observed in macrovascular cells. PMVECs exhibited a faster growth rate than did PAECs and, once a growth program was initiated by serum, PMVECs sustained growth in the absence of serum. Thus, PAECs and PMVECs differ in their structure and function, even under similar environmental conditions.
... As described by Berkels et al. (2000) [24], NO production in vascular endothelial cell occurs simultaneously to the increase of intracellular Ca 2+ . Stevens et al. (1997) [25] had already shown that the increase of cAMP, via activation of isoform III enzyme adenylyl-cyclase, is related to increased Ca 2+ cytosol in some phenotypes of cells, such as vascular endothelium of rats [26], where six of these isoforms (types II-VI and VIII) can be found. ...
... As described by Berkels et al. (2000) [24], NO production in vascular endothelial cell occurs simultaneously to the increase of intracellular Ca 2+ . Stevens et al. (1997) [25] had already shown that the increase of cAMP, via activation of isoform III enzyme adenylyl-cyclase, is related to increased Ca 2+ cytosol in some phenotypes of cells, such as vascular endothelium of rats [26], where six of these isoforms (types II-VI and VIII) can be found. ...
Endothelium-derived relaxing factors contribute to smooth muscle relaxation. The aim of the present study was to investigate the contribution of nitric oxide (NO) produced in the endo-thelial cells to the vasodilatation stimulated with forskolin in rat aorta. Forskolin that directly activates adenylyl-cyclase, induced complete relaxation in phenylephrine-contracted aortas. En-dothelium removal reduced the potency (pEC 50) of forskolin without changes in the maximum effect (Emax). However, the inhibitor of endothe-lial NO-synthase (10 μM L-NG-Nitroarginine, L-NNA) reduced both Emax and pEC 50 in intact endothelium aortic rings. L-NNA or L-NNA plus cyclooxygenase inhibitor indomethacin (10 μM) reduced both Emax and pEC 50 of forskolin. For-skolin increased both the cytosolic Ca 2+ concentration and the cytosolic NO concentration ([NO]c) in the endothelial cells. The PKA inhibitor KT5720 reduced the NO production activated by forsko-lin in the endothelial cells. The enhanced [NO]c in the endothelial cells increased cyclic guano-sine-monophosphate (cGMP) in smooth muscle cells, which was abolished by L-NNA. Taken together , our results indicate that vasodilatation mediated by forskolin in rat aortic rings is po-tentiated by NO production in endothelial cells that increases the cGMP levels in the smooth muscle cells that along with cAMP contribute to the vasodilatation.
... One specific aim of this study is to expand the current knowledge of pH'" regulafion in microvascular endothelial cells. As stated previously, it is well accepted that differences exist between endothelial cells from macro-and microcirculation (Kumar et al., 1987;Garianda and Dejana, 1997;Stevens et al., 1997;Geiger et al., 1997). A substantial body of work exists on pH'" regulation in macrovascular endothelial cells; ...
... [Ca^^]'" regulafion (Stevens et al., 1997) and NO producfion (Geiger et al., 1997) depending on whether the cells are of macrovascular or microvascular origin. We reasoned that since angiogenesis is accepted to be a process that involves the microvasculature, the absence of proton pumps in macrovascular endothelial was consistent with the hypothesis. ...
... As described by Berkels et al. (2000) [24], NO production in vascular endothelial cell occurs simultaneously to the increase of intracellular Ca 2+ . Stevens et al. (1997) [25] had already shown that the increase of cAMP, via activation of isoform III enzyme adenylyl-cyclase, is related to increased Ca 2+ cytosol in some phenotypes of cells, such as vascular endothelium of rats [26], where six of these isoforms (types II-VI and VIII) can be found. ...
... As described by Berkels et al. (2000) [24], NO production in vascular endothelial cell occurs simultaneously to the increase of intracellular Ca 2+ . Stevens et al. (1997) [25] had already shown that the increase of cAMP, via activation of isoform III enzyme adenylyl-cyclase, is related to increased Ca 2+ cytosol in some phenotypes of cells, such as vascular endothelium of rats [26], where six of these isoforms (types II-VI and VIII) can be found. ...
Endothelium-derived relaxing factors contribute to smooth muscle relaxation. The aim of the present study was to investigate the contribu-tion of nitric oxide (NO) produced in the endo-thelial cells to the vasodilatation stimulated with forskolin in rat aorta. Forskolin that directly ac-tivates adenylyl-cyclase, induced complete rela-xation in phenylephrine-contracted aortas. En-dothelium removal reduced the potency (pEC 50) of forskolin without changes in the maximum effect (Emax). However, the inhibitor of endothe-lial NO-synthase (10 μM L-NG-Nitroarginine, L-NNA) reduced both Emax and pEC 50 in intact endothelium aortic rings. L-NNA or L-NNA plus cyclooxygenase inhibitor indomethacin (10 μM) reduced both Emax and pEC 50 of forskolin. For-skolin increased both the cytosolic Ca 2+ concen-tration and the cytosolic NO concentration ([NO]c) in the endothelial cells. The PKA inhibitor KT5720 reduced the NO production activated by forsko-lin in the endothelial cells. The enhanced [NO]c in the endothelial cells increased cyclic guano-sine-monophosphate (cGMP) in smooth muscle cells, which was abolished by L-NNA. Taken to-gether, our results indicate that vasodilatation mediated by forskolin in rat aortic rings is po-tentiated by NO production in endothelial cells that increases the cGMP levels in the smooth muscle cells that along with cAMP contribute to the vasodilatation.
... but also activated Ca 2+ -sensitive PKC isoforms (Lum et al. 1989(Lum et al. , 1992Lynch et al. 1990;Tiruppathi et al. 1992a;Stevens et al. 1997;Holda et al. 1998), thus enabling us to address the role of Ca 2+ signalling and activation of PKC in mediating the increase in endothelial permeability. ...
... The results are from four experiments carried out in duplicate; values are shown as means ± S.E.M. ATPase (Holda et al. 1998). Thapsigargin is known to promote intracellular store depletion and trigger the capacitative entry of Ca 2+ in cells (Stevens et al. 1997;Holda et al. 1998). We compared the results with thrombin, a mediator that increases endothelial permeability by a Ca 2+ -dependent mechanism (Lum et al. 1989). ...
The role of intracellular Ca ²⁺ mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca ²⁺ concentration ([Ca ²⁺ ] i ) . The rise in [Ca ²⁺ ] i in both cases was due to release of Ca ²⁺ from intracellular stores and influx of extracellular Ca ²⁺ .
Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial ¹²⁵ I‐albumin permeability. Thapsigargin induced activation of PKCα and discontinuities in VE‐cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCα activation and similar alterations in VE‐cadherin junctions, but in association with actin stress fibre formation.
Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC 20 ), whereas thrombin induced MLC 20 phosphorylation consistent with formation of actin stress fibres.
Calphostin C pretreatment prevented the disruption of VE‐cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca ²⁺ ] i elicited by thapsigargin and thrombin may activate a calphostin C‐sensitive PKC pathway that signals VE‐cadherin junctional disassembly and increased endothelial permeability.
Results suggest a critical role for Ca ²⁺ signalling and activation of PKCα in mediating the disruption of VE‐cadherin junctions, and thereby in the mechanism of increased endothelial permeability.
... Similar comparative observations were reported for bovine lung microvessel, vein, and artery ECs (34,35) and calf retina and aortic ECs (50). These data are consistent with our previous studies that showed our MVEC cultures formed barriers with superior integrity (10,44), as defined by a full spectrum of hydroxyl ethyl starch particle sizes. Our permeability comparisons contribute to an increasing body of data, suggesting that MVECs from various vascular origins form tighter, more restrictive barriers to macromolecules than do macrovascular ECs. ...
... The Ca 2ϩ transients obtained with the fura 2 ratio method differed in amplitude, as the MVEC response was less than the RPAEC response. Similar differences in cytosolic Ca 2ϩ responses to TG were found with the indo 1 Ca 2ϩ fluoroprobe L816 CALCIUM AND ENDOTHELIAL CELL PERMEABILITY using bovine pulmonary MVECs compared with bovine conduit artery ECs (44), but permeability was not determined in that study. Therefore, although numerous investigators have shown that a rise in [Ca 2ϩ ] i in response to various stimuli initiates vascular permeability in conduit ECs (12,23,32,37), the lack of TGinduced permeability in RPMVECs could be due to Ca 2ϩ regulation differences reflected as lower [Ca 2ϩ ] i . ...
Cytosolic Ca2+ concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+ induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 microM) produced higher [Ca2+]i levels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]i response in RPMVECs, we investigated whether reduced activation of store-operated Ca2+ entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+ followed by readdition media with 2 mM extracellular Ca2+ increased RPMVEC [Ca2+]i to a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]i in RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 microM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]i promotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]i signaling pathways or dominant Ca(2+)-independent mechanisms from controlling cellular gap formation and permeability.
... An elevation in [Ca 2ϩ ] i associated with activation of store-operated calcium entry is sufficient to increase endothelial cell permeability (4,13,18,28,29). Activation of store-operated calcium entry occurs after depletion of intracellular calcium stores either by stimulation of calcium release (e.g., histamine or thrombin) or by inhibition of calcium reuptake (e.g., thapsigargin) into storage sites. ...
... These cells were chosen for study because their morphology was consistent with RPAECs from a confluent monolayer. These single cells have previously been shown to possess electrophysiological recordings generally similar to those observed in confluent monolayers (28). The extracellular solution was composed of (in mM) 110 tetraethylammonium aspartate, 10 calcium aspartate, 10 HEPES, and 0.5 3,4diaminopyridine; and the pipette solution was composed of (in mM) 130 N-methyl-D-glucamine, 1.15 EGTA, 10 HEPES, and 1 Ca(OH) 2 with and without 2 Mg 2ϩ -ATP. ...
We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.
... Because the failure of thrombin to increase endothelial permeability in ECV304 cells may be due to an im-paired increase in [Ca 2ϩ ] i [see Thrombin-activated Ins (1,4,5)P 3 generation and Ca 2ϩ signaling], we studied the effects of thapsigargin [which is known to promote an increase in [Ca 2ϩ ] i by depleting intracellular store Ca 2ϩ and causing capacitative Ca 2ϩ entry in cells (11,19,25)] on the Ca 2ϩ transients and barrier function. Results indicated that thapsigargin increased [Ca 2ϩ ] i in both ECV304 cells and HUVECs (Fig. 6A). ...
... Because the absence of Ca 2ϩ signaling in ECV304 cells appears to be important in making these cells refractory to thrombin, we addressed the possibility of whether these cells could be "rescued" by treatment with thapsigargin, that is, the possibility that Ca 2ϩ influx activated by another means could activate the increase in endothelial permeability in these cells. Thapsigargin (an endoplasmic reticulum Ca 2ϩ -ATPase inhibitor) promotes intracellular store Ca 2ϩ depletion and triggers capacitative Ca 2ϩ entry in cells (11,19,25). We showed that thapsigargin resulted in decreased endothelial monolayer electrical resistance and increased 125 I-albumin permeability in ECV304 cells, a response similar to that observed in HUVECs. ...
We compared the thrombin-activated responses in human umbilical vein endothelial cells (HUVECs) and a HUVEC-derived cell line, ECV304. Thrombin induced a 40-50% decrease in transendothelial monolayer electrical resistance and a twofold increase in 125I-albumin permeability in HUVECs, whereas it failed to alter the endothelial barrier function in ECV304 cells. Thrombin produced a brisk intracellular Ca2+ concentration transient and phosphorylation of 20-kDa myosin light chain in HUVECs but not in ECV304 cells. Thrombin-induced phosphoinositide hydrolysis was comparable in ECV304 cells and HUVECs, indicating the activation of thrombin receptors in both cell types. La3+ reduced both the thrombin-induced decrease in endothelial monolayer electrical resistance and the increase in 125I-albumin permeability in HUVECs. Because the absence of Ca2+ signaling could explain the impairment in the permeability response in ECV304 cells, we studied the effect of increasing intracellular Ca2+ concentration in ECV304 cells with thapsigargin. Exposure of ECV304 cells to thapsigargin caused decreased endothelial monolayer electrical resistance and increased 125I-albumin permeability. These results indicate that Ca2+ influx and activation of Ca2+-dependent signaling pathways are important determinants of the thrombin-induced increase in endothelial permeability.
... 19 Finally, while PMVECs have been observed to have a resting membrane potential centered at Ϫ22 mV, PAECs have a bimodal distribution of resting potentials peaking at Ϫ63 and Ϫ24 mV. 20 This piece of information makes the data of Wu et al, 21 in this issue of Circulation Research, especially intriguing: they show that the ion channel responsible for thrombin-mediated increases in the cytosolic calcium of lung microcirculation endothelial cells is the T-type voltage-dependent calcium channel. T-type calcium channels are normally associated with excitable cells that have resting potentials close to the reversal potential for potassium ions (typically Ϫ85 mV). ...
... Consequently, T-type channels are usually activated by depolarizing stimuli. In the case of PMVECs, with a resting potential close to Ϫ20 mV, 20 activation of T-type channels occurs when thrombin causes an initial hyperpolarization by opening calcium-sensitive potassium channels, followed by depolarization due to cyclic nucleotide-gated ion channels into the range of voltages in which T-type currents are active. ...
In 1910, James Herrick made the first report of a case of sickle cell anemia.1 He described thin, elongated, sickle-shaped, and crescent-shaped red blood corpuscles. Soon afterward, the observation of sickle cells in the asymptomatic father of a sickle cell anemia patient raised the possibility of an inherited disorder.2 The distinction between symptomatic sickle cell anemia and the asymptomatic sickle cell trait was established in 1933.3 The difference between normal hemoglobin (HbAA) and sickle hemoglobin (HbSS) was recognized in 1959 to be the substitution of a valine residue for a glutamic acid in the β-chain amino terminus.4 Patients who have the sickle cell trait are heterozygotes (HbAS), having an abnormal, as well as a normal, β-globin gene. In sickle cell anemia, sickling may start at an oxygen saturation as high as 85%, while in the trait the desaturation has to be more severe before sickling is induced.
Sickling leading to vaso-occlusion and infarction occurs in many organs but was first described in the lungs.5 Initially it was thought that deformation and increased rigidity of the erythrocytes, related to polymerization of HbSS, was sufficient to cause mechanical obstruction. However, the work of Hebbel et al6,7 focused attention on erythrocyte adherence to the endothelium as a mechanism promoting microvascular occlusion. A key observation in this regard is that sickle erythrocytes adhere more readily to microvascular endothelium than to endothelium from conduit vessels.8 If endothelial cells circulating in the blood can be taken as representative of the sedentary population, the endothelium is activated in sickle cell patients, whether in steady state or in an acute crisis.9 This is shown by expression of the adhesion molecules, ICAM-1, VCAM-1, E-selectin, and P-selectin.
More recent work has implicated inflammation as a factor in the pathogenesis of vaso-occlusive crises. …
... In the lung, the population of endothelial cells exhibits significant diversity (Ofori-Acquah, King, Voelkel, Schaphorst, & Stevens, 2008;Potente & M€ akinen, 2017;Stevens, 2011), including physiologic differences evident in each type of vascular bed (macrovasculature, microvasculature, or lymphatics) and differences at the level of individual cells, which is not yet well explored. Heterogeneity between endothelial cells includes, but is not limited to, differences in gene expression patterns, membrane potential, calcium and oxidant signaling, intercellular junctional proteins, and caveolar density (Chi et al., 2003;Grishko, Solomon, Wilson, LeDoux, & Gillespie, 2001;Mehta & Malik, 2006;Ofori-Acquah et al., 2008;Stevens et al., 1997). Thus, although eNOS and Cav-1 are expressed throughout the lung vascular network, its expression may vary from vessel-to-vessel and even between endothelial cell subpopulations, and may represent an important factor capable of regulating the switch from resting to activated endothelial cell phenotype. ...
Pulmonary vascular diseases are associated with several factors including infection, cigarette smoking, abuse of dietary suppressants and drugs, prolonged exposure to high altitude, and other causes which in part induce significant oxidative stress resulting in endothelial cell injury, apoptosis, hyperproliferation, and vaso-occlusive disease. Maintenance of normal endothelial cell function is a critical role of endothelial nitric oxide synthase (eNOS) activity and physiologic nitric oxide (NO) signaling in the vascular wall. eNOS expression and activity is regulated by the membrane-associated scaffolding protein caveolin-1 (Cav-1), the main protein constituent of caveolae. This chapter summarizes the literature and highlights unanswered questions related to how inflammation-associated oxidative stress affects Cav-1 expression and regulatory functions, and how dysregulated eNOS enzymatic activity promotes endothelial dysfunction. Focus is given to how the conversion of eNOS from a NO-producing enzyme to a transient oxidant-generating system is associated twith Cav-1 depletion, endothelial cell injury, and pulmonary vascular diseases. Importantly, the vascular defects observed in absence of Cav-1 that give rise to injured or hyperproliferative endothelial cells and promote remodeled vasculature can be rescued by “re-coupling,” inhibiting, or genetically deleting eNOS, supporting the notion that strict control of Cav-1 expression and eNOS activity and signaling is critical for maintaining pulmonary vascular homeostasis.
... The integrity of the endothelial layer is essential for its normal function; once this is compromised, the permeability of the endothelial cell layer may be altered, which may in turn lead to problems such as edema and exudation [54][55][56]. In this study, PMVECs were cultured in DMEM (low-glucose) using the tissue block method [57][58][59]. We also used DMEM (low-glucose) to establish the OGD model in astrocytes, and this was followed by CM collection. ...
Brain damage can cause lung injury. To explore the mechanism underlying the lung injury induced by acute cerebral ischemia (ACI), we established a middle cerebral artery occlusion (MCAO) model in male Sprague-Dawley rats. We focused on glia maturation factor β (GMFB) based on quantitative analysis of the global rat serum proteome. Polymerase chain reaction, western blotting, and immunofluorescence revealed that GMFB was over-expressed in astrocytes in the brains of rats subjected to MCAO. We cultured rat primary astrocytes and confirmed that GMFB was also up-regulated in primary astrocytes after oxygen-glucose deprivation (OGD). We subjected the primary astrocytes to Gmfb RNA interference before OGD and collected the conditioned medium (CM) after OGD. We then used the CM to culture pulmonary microvascular endothelial cells (PMVECs) acquired in advance and assessed their status. The viability of the PMVECs improved significantly when Gmfb was blocked. Moreover, ELISA assays revealed an elevation in GMFB concentration in the medium after OGD. Cell cultures containing recombinant GMFB showed increased levels of reactive oxygen species and a deterioration in the state of the cells. In conclusion, GMFB is up-regulated in astrocytes after ACI, and brain-derived GMFB damages PMVECs by increasing reactive oxygen species. GMFB might thus be an initiator of the lung injury induced by ACI.
Electronic supplementary material
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... The activation of β-ARs and consequent activation of PKA induce Ltype Ca 2+ channel phosphorylation, which results in a Ca 2+ influx into endothelial cells [41]. The nitric oxide production in vascular endothelial cells occurs simultaneously to the increase in intracellular Ca 2+ [42]. ...
... In the pulmonary circulation, for example, endothelial cells lining the blood vessels with a diameter Ͼ25 m are morphologically and functionally different than those endothelial cells lining the capillaries. They have different gene expression patterns (7), lectin binding capacity (15), resting membrane potentials (25), intracellular calcium (8) and cAMP dynamics (24), hydraulic conductance (21), caveolar density (19), and even vasculogenic capacity (1). The fact that all pulmonary endothelia have a pool of cold-stable microtubules brings into question how these microtubule pools are differentially regulated. ...
Microtubules are composed of α-tubulin and β-tubulin dimers. Microtubules yield tubulin dimers when exposed to cold, which reassemble spontaneously to form microtubule fibers at 37°C. However, mammalian neurons, glial cells, and fibroblasts have cold-stable microtubules. While studying the microtubule toxicity mechanisms of the exotoxin Y from Pseudomonas aeruginosa in pulmonary microvascular endothelial cells, we observed that some endothelial microtubules were very difficult to disassemble in the cold. As a consequence, we designed studies to test the hypothesis that microvascular endothelium has a population of cold-stable microtubules. Pulmonary microvascular endothelial cells and HeLa cells (control) were grown under regular cell culture conditions, followed by exposure to an ice-cold water bath and a microtubule extraction protocol. Polymerized microtubules were detected by immunofluorescence confocal microscopy and Western blot analyses. After cold exposure, immunofluorescence revealed that the majority of HeLa cell microtubules disassembled, whereas a smaller population of endothelial cell microtubules disassembled. Immunoblot analyses showed that microvascular endothelial cells express the microtubule cold-stabilizing protein N-STOP (neuronal stable tubule-only polypeptides), and that N-STOP binds to endothelial microtubules after cold exposure, but not if microtubules are disassembled with nocodazole before cold exposure. Hence, pulmonary endothelia have a population of cold-stable microtubules.
... Since dipyridamole can elevate intracellular adenosine levels by inhibiting adenosine metabolism (50), we used a more specific adenosine transporter antagonist, NBTI, and found that NBTI partially attenuated the barrier-enhancing effect of adenosine, suggesting that adenosine uptake via transporters is also involved in mediating adenosine barrier enhancement. cAMP has been shown to serve as a barrier-enhancing agent in the lung in vivo (1) and in pulmonary endothelium in vitro (11,48,51,57). cAMP-induced endothelial barrier enhancement is thought to act through Epac/Rap-mediated Rac1 activation (4 -6, 14, 24). ...
We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A(1) or A(3) antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A(2A) and A(2B), respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A(2A) and A(2B) receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A(2A)/A(2B) receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A(2A) and A(2B) agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A(2A) and A(2B) receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by alpha-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the development of edema before ALI but also partially reversed edema after ALI. The data suggest that adenosine deaminase inhibition may be useful in treatment of pulmonary edema in settings of ALI.
... There are multiple levels of complexity in the activation of the signaling pathways. These may include multiple combinations of the G protein subunits (22) and the expression of multiple isoforms of the key enzymes such as PLC and adenylate cyclase (167,289). In addition, a signaling molecule (e.g., diacylglycerol) may arise from more than one pathway [e.g., from PLC (short acting) or phospholipase D (long term)] with each source of the signaling molecule itself activating a different pathway (166). These mechanisms may account in part for different patterns of permeability increase initiated by different inflammatory mediators. ...
This review addresses classical questions concerning microvascular permeabiltiy in the light of recent experimental work on intact microvascular beds, single perfused microvessels, and endothelial cell cultures. Analyses, based on ultrastructural data from serial sections of the clefts between the endothelial cells of microvessels with continuous walls, conform to the hypothesis that different permeabilities to water and small hydrophilic solutes in microvessels of different tissues can be accounted for by tortuous three-dimensional pathways that pass through breaks in the junctional strands. A fiber matrix ultrafilter at the luminal entrance to the clefts is essential if microvascular walls are to retain their low permeability to macromolecules. Quantitative estimates of exchange through the channels in the endothelial cell membranes suggest that these contribute little to the permeability of most but not all microvessels. The arguments against the convective transport of macromolecules through porous pathways and for the passage of macromolecules by transcytosis via mechanisms linked to the integrity of endothelial vesicles are evaluated. Finally, intracellular signaling mechanisms implicated in transient increases in venular microvessel permeability such as occur in acute inflammation are reviewed in relation to studies of the molecular mechanisms involved in signal transduction in cultured endothelial cells.
... Indeed, we previously established that the [Ca 2+ ] i response to thapsigargin is significantly lower in pulmonary microvascular endothelial cells than it is in pulmonary artery endothelial cells, even under similar environmental conditions [85,86] (Fig. 8A). We also demonstrated that microvascular endothelia possess a relatively depolarized resting membrane potential [87], suggesting the decreased electrochemical gradient for Ca 2+ entry could contribute to the lower thapsigargin [Ca 2+ ] i response. To address this concern we adapted a recalcification procedure that clamped membrane potential in both cell types. ...
Ca2+ store depletion activates both Ca2+ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca2+ selective current, I(SOC). Indeed, I(SOC) is a relatively small inward Ca2+ current that exhibits an approximate +40mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I(SOC), although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I(SOC) requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca2+ store depletion and channel activation. Thus, at present findings indicate a TRPC4-protein 4.1 physical linkage regulates I(SOC) activation following Ca2+ store depletion.
... However, it is now clear that endothelial cells along the vascular tree are phenotypically distinct based on environmental and epigenetic control of their behavior, 19 including dynamic regulation of Ca 2ϩ transitions. 1,18,21,35,36 Thapsigargin activates a larger SOC entry response in PAECs than in PMVECs, although the mechanism for this observation is not clear. To discern the SOC entry pathways in PAECs and PMVECs, divalent cation entry was examined in intact cells. ...
Store-operated calcium (SOC) entry is sufficient to disrupt the extra-alveolar, but not the alveolar, endothelial cell barrier. Mechanism(s) underlying such insensitivity to transitions in cytosolic calcium ([Ca2+]i) in microvascular endothelial cells are unknown. Depletion of stored Ca2+ activates a larger SOC entry response in extra-alveolar (pulmonary artery; PAECs) than alveolar (pulmonary microvascular; PMVECs) endothelial cells. In vivo permeation studies revealed that Ca2+ store depletion activates similar nonselective cationic conductances in PAECs and PMVECs, while only PAECs possess the calcium-selective, store-operated Ca2+ entry current, I(SOC). Pretreatment with the type 4 phosphodiesterase inhibitor, rolipram, abolished thapsigargin-activated I(SOC) in PAECs, and revealed I(SOC) in PMVECs. Rolipram pretreatment shifted the thapsigargin-induced fluid leak site from extra-alveolar to alveolar vessels in the intact pulmonary circulation. Thus, our results indicate I(SOC) provides a [Ca2+]i source that is needed to disrupt the endothelial cell barrier, and demonstrate that intracellular events controlling I(SOC) activation coordinate the site-specific vascular response to inflammation.
... Transient intercellular gap formation and apoptosis both increase permeability that contributes to pulmonary edema, decreasing the efficiency of gas exchange. The mutually opposing actions of intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and intracellular cAMP concentrations ([cAMP] i ) control barrier function and are the focus of active investigations (2,5,14,20,(35)(36)(37). However, signaling events that trigger endothelial cell apoptosis are still poorly understood. ...
Sustained increases in intracellular cGMP concentrations ([cGMP]i) inhibit cell growth and induce apoptosis. We now report that a cGMP-specific phosphodiesterase, PDE5, plays a dominant role in regulating [cGMP]i transitions that inhibit cell growth and control susceptibility to apoptosis in pulmonary endothelium. Atrial natriuretic peptide (ANP) activates guanylyl cyclase A/B and induces a rapid [cGMP]i rise 2-5 min after its application, in both pulmonary arterial endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs). However, increased [cGMP]i in PAECs is transient and decays within 10 min due to cytosolic PDE5 hydrolytic activity. Increased [cGMP]i in PMVECs is sustained for >3 h due to the absence of PDE5. Indeed, at any ANP concentration, the sustained (30 min) [cGMP]i rise is greater in PMVECs than in PAECs, unless PAECs are also treated with the PDE5 inhibitor zaprinast. Using RT-PCR, Western blot analysis, immunoprecipitation, and DEAE chromatography, we resolved the expression and activity of PDE 5A1/A2 only in PAECs. Similarly, PDE5 expression was restricted to extra-alveolar endothelium in vivo. ANP induced growth inhibition and apoptosis in PMVECs, but similar effects were not seen in PAECs unless ANP treatment was combined with zaprinast. ANP blocked the VEGF-induced proliferation and migration in PMVECs. Collectively, these data suggest that PDE5-regulated [cGMP]i controls endothelial cell growth and apoptosis, representing a mechanism of heterogeneity between two endothelial phenotypes.
... This increases the fluid pump function of the endothelium (Fischbarg et al., 1977;Riley et al., 1996), which could be part of a response to counter deleterious effects of stress on endothelial function. In pulmonary artery endothelial cells, A 2b R activation (Stevens et al., 1997) also decreases paracellular permeability via cAMP dependent and cAMP independent pathways. Similarly, a recent study has indicated that adenosine decreases endothelial barrier permeability through a cAMP dependent pathway that induces myosin light chain dephosphorylation in cultured BCE (Srinivas et al., 2004). ...
Previous studies indicated that adenosine can increase [cAMP](i) and stimulate fluid transport by corneal endothelium. The purpose of this study was to determine which adenosine receptor subtype(s) are expressed and to examine their functional roles in modulating [cAMP](i), [Ca(2+)](i) and effects on Cl(-) permeability in corneal endothelium. We screened bovine corneal endothelium (BCE) for adenosine receptor subtypes by RT-PCR and immunoblotting, and examined the effects of pharmacological agents on adenosine stimulated Cl(-) transport, [cAMP](i) and [Ca(2+)](i). RT-PCR indicated the presence of A(1) and A(2b) adenosine receptors, while A(2a) and A(3) were negative. Western blot (WB) confirmed the presence of A(2b) ( approximately 50 kDa) and A(1) ( approximately 40 kDa) in fresh and cultured BCE. Ten micromolar adenosine increased [cAMP](i) by 2.7-fold over control and this was inhibited 66% by 10 microm alloxazine, a specific A(2b) blocker. A(1) activation with 1 micromN(6)-CPA (a specific A(1) agonist) or 100 nm adenosine decreased [cAMP](i) by 23 and 6%, respectively. Adenosine had no effect on [Ca(2+)](i) mobilization. Indirect immunofluorescence localized A(2b) receptors to the lateral membrane and A(1) to the apical surface in cultured BCE. Adenosine significantly increased apical Cl(-) permeability by 2.2 times and this effect was nearly abolished by DMPX (10 microm), a general A(2) blocker. Adenosine-induced membrane depolarization was also inhibited by 33% (n=6) in the presence of alloxazine. Bovine corneal endothelium expresses functional A(1) and A(2b) adenosine receptors. A(1), preferentially activated at <1 microm adenosine, acts to decrease [cAMP](i) and A(2b), activated at >1 microm adenosine, increase [cAMP](i).
... This site-specific functional heterogeneity can be partly attributed to phenotypically distinct signal transduction cas-cades. As compared to lung macrovascular endothelial cells, the magnitude of store-operated calcium entry is substantially reduced in endothelial cells of the pulmonary microcirculation (Stevens et al. 1997;Kelly et al. 1998). As a result, lung microvascular endothelial cells possess an intrinsic capacity to preserve intracellular cyclic adenosine monophosphate (cAMP) concentrations ) which enhances their barrier function ). ...
In recent decades, it has become evident that the endothelium is by no means a passive inner lining of blood vessels. This 'organ' with a large surface (approximately 350 m2) and a comparatively small total mass (approximately 110 g) is actively involved in vital functions of the cardiovascular system, including regulation of perfusion, fluid and solute exchange, haemostasis and coagulation, inflammatory responses, vasculogenesis and angiogenesis. The present chapter focusses on two central aspects of endothelial structure and function: (1) the heterogeneity in endothelial properties between species, organs, vessel classes and even within individual vessels and (2) the composition and role of the molecular layer on the luminal surface of endothelial cells. The endothelial lining of blood vessels in different organs differs with respect to morphology and permeability and is classified as 'continuous', 'fenestrated' or 'discontinuous'. Furthermore, the mediator release, antigen presentation or stress responses of endothelial cells vary between species, different organs and vessel classes. Finally there are relevant differences even between adjacent endothelial cells, with some cells exhibiting specific functional properties, e.g. as pacemaker cells for intercellular calcium signals. Organ-specific structural and functional properties of the endothelium are marked in the vascular beds of the lung and the brain. Pulmonary endothelium exhibits a high constitutive expression of adhesion molecules which may contribute to the margination of the large intravascular pool of leucocytes in the lung. Furthermore, the pulmonary microcirculation is less permeable to protein and water flux as compared to large pulmonary vessels. Endothelial cells of the blood-brain barrier exhibit a specialised phenotype with no fenestrations, extensive tight junctions and sparse pinocytotic vesicular transport. This barrier allows a strict control of exchange of solutes and circulating cells between the plasma and the interstitial space. It was observed that average haematocrit levels in muscle capillaries are much lower as compared to systemic haematocrit, and that flow resistance of microvascular beds is higher than expected from in vitro studies of blood rheology. This evidence stimulated the concept of a substantial layer on the luminal endothelial surface (endothelial surface layer, ESL) with a thickness in the range of 0.5-1 microm. In comparison, the typical thickness of the glycocalyx directly anchored in the endothelial plasma membrane, as seen in electron micrographs, amounts to only about 50-100 microm. Therefore it is assumed that additional components, e.g. adsorbed plasma proteins or hyaluronan, are essential in constituting the ESL. Functional consequences of the ESL presence are not yet sufficiently understood and acknowledged. However, it is evident that the thick endothelial surface layer significantly impacts haemodynamic conditions, mechanical stresses acting on red cells in microvessels, oxygen transport, vascular control, coagulation, inflammation and atherosclerosis.
... Such heterogeneity has been illustrated in Ca 2ϩdependent signal transduction pathways. 20,22 In the present study, we demonstrate the selective expression of Ca v 3.1 T-type voltage-gated Ca 2ϩ channels in lung microvascular endothelial cells. Functional expression of Ca v 3.1 is based on the following observations: (1) T-type Ca 2ϩ currents are present in PMVECs and not PAECs; (2) PMVEC currents are poorly sensitive to Ni 2ϩ blockade (IC 50 380.25 mol/L), which discriminates Ca v 3.1 among three T-type Ca 2ϩ channel isoforms; and (3) only Ca v 3.1 is expressed in PMVECs. ...
In the present study, we demonstrate that lung microvascular endothelial cells express a Cav3.1 (alpha1G) T-type voltage-gated Ca2+ channel, whereas lung macrovascular endothelial cells do not express voltage-gated Ca2+ channels. Voltage-dependent activation indicates that the Cav3.1 T-type Ca2+ current is shifted to a positive potential, at which maximum current activation is -10 mV; voltage-dependent conductance and inactivation properties suggest a "window current" in the range of -60 to -30 mV. Thrombin-induced transitions in membrane potential activate the Cav3.1 channel, resulting in a physiologically relevant rise in cytosolic Ca2+. Furthermore, activation of the Cav3.1 channel induces a procoagulant endothelial phenotype; eg, channel inhibition attenuates increased retention of sickled erythrocytes in the inflamed pulmonary circulation. We conclude that activation of the Cav3.1 channels selectively induces phenotypic changes in microvascular endothelial cells that mediate vaso-occlusion by sickled erythrocytes in the inflamed lung microcirculation.
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This chapter focuses on the cellular and biochemical mechanisms that normally usher in and maintain the intact, quiescent, barrier competent state, as well as those factors that directly interfere with or reverse endothelial activation and injury. Beneficial effects of the endothelial protective agents on lung injury and acute respiratory distress syndrome (ARDS) are discussed in the chapter. Most cultured endothelial cells require serum with its incompletely defined and variable growth factors and hormones. Serum-free media, with defined partial replications of these factors, have limited, variable success in promoting growth for some cell lines, but more generally result in growth arrest. Even serum-supplemented cells usually require additional factors such as vascular endothelial growth factor (VEGF), which is absolutely essential for fetal vessel development, angiopoietin-1, acidic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). The chapter also discusses interactions between the endothelium and the matrix.
cAMP was the first second messenger discovered. Since its original description,elements of the cAMP signal transduction cascade have grown in complexity. The idea that cAMP quickly and evenly disperses within the cytosolic compartment to achieve a uniform cell signal has slowly faded. The challenge before us is to understand how CAMP signaling is achieved with spatio-temporal fidelity. This fidelity is cell-type specific and in accordance with any cell's unique function. To fully accomplish such a high level of understanding, identifying the molecular anatomy of the cAMP signaling scaffold must be put in context with a cell's specialized phenotype. Pulmonary microvacular endothelial cells must maintain a tight barrier function to limit fluid, solute and macromolecular flux, and preserve alveolar gas exchange. Therefore, membrane cAMP concentrations are maintained at a high level, and CAMP spillover into the bulk cytosol is limited by high, membrane-associated phosphodiesterase activity. This arrangement allows CAMP to achieve concentrations high enough to activate localized effectors that promote barrier function, while maintaining bulk cytosolic cAMP concentrations below a threshold required to activate effectors that disrupt barrier function. Calcium inhibition of membrane cAMP allows transient gap formation. Generation of cAMP outside the membrane compartment produces a sustained increase in microvascular endothelial cell permeability. In this context, pulmonary microvascular endothelium has developed a highly ordered and specialized mechanism for regulating cAMP production to dynamically control alveolar-capillary stability on a moment-to-moment basis.
This chapter introduces some of the primary players and pathways for the molecular integration and coordination of the membrane and cellular signaling of integrity and acute activation. The very complex and dynamic existence of endothelium—based on two states, quiescence and activation—makes a simplistic division of signaling into acute and long-term mechanisms. By “acute,” it means processes that can be altered in a matter of seconds or perhaps minutes, involving the release of intracellular second messengers and rapid responses, such as altered phosphorylation, redox, and conformational states of proteins. Such signals are often triggered by the binding of external mediators to membrane receptors and the consequent changes in cellular proteins often result in their translocation to membranes or to complexes with other proteins. Signaling is also divided based on the two states of the endothelium. Some pathways are primarily associated with the establishment of the architecture of quiescent endothelial cells, formation of cell–cell junctions, and maintenance of barrier integrity. The chapter describes the signaling of quiescence and barrier integrity.
Acute lung injury is a general term that describes injurious conditions that can range from mild interstitial edema to massive inflammatory tissue destruction. This review will cover theoretical considerations and quantitative and semi-quantitative methods for assessing edema formation and increased vascular permeability during lung injury. Pulmonary edema can be quantitated directly using gravimetric methods, or indirectly by descriptive microscopy, quantitative morphometric microscopy, altered lung mechanics, high-resolution computed tomography, magnetic resonance imaging, positron emission tomography, or x-ray films. Lung vascular permeability to fluid can be evaluated by measuring the filtration coefficient (Kf) and permeability to solutes evaluated from their blood to lung clearances. Albumin clearances can then be used to calculate specific permeability-surface area products (PS) and reflection coefficients (σ). These methods as applied to a wide variety of transgenic mice subjected to acute lung injury by hyperoxic exposure, sepsis, ischemia-reperfusion, acid aspiration, oleic acid infusion, repeated lung lavage, and bleomycin are reviewed. These commonly used animal models simulate features of the acute respiratory distress syndrome, and the preparation of genetically modified mice and their use for defining specific pathways in these disease models are outlined. Although the initiating events differ widely, many of the subsequent inflammatory processes causing lung injury and increased vascular permeability are surprisingly similar for many etiologies. © 2011 American Physiological Society. Compr Physiol 1:835-882, 2011.
In recent years there has been an increasing appreciation of the functional heterogeneity that exists between extraalveolar and alveolar endothelial cells. One of the most striking features of pulmonary microvascular endothelial cells is that they possess a highly impermeable barrier with respect to pulmonary artery or vein endothelial cells. This cellular feature is observed in culture and in the intact microcirculation, prompting a reevaluation of the key physiological principles that control permeability and the fate of fluid (or exudate) once it leaves the circulation. Pulmonary microvascular endothelial cells express calcium channels not found in extraalveolar endothelial cells, including the vanilloid family transient receptor potential 4 channel and the α1G T-type calcium channel. Whereas activation of the TRPV4 channel causes alveolar flooding, activation of the α1G T-type calcium channel promotes P-selectin surface translocation, events specific to the microcirculation. Although endothelium is an attractive therapeutic target in acute lung injury and other vascular disorders, the growing awareness of pulmonary endothelial cell heterogeneity increasingly suggests that a panendothelial cell approach is suboptimal. Rather, development of novel therapeutics based upon anatomically restricted expression of molecular signatures may be developed to better combat vascular disease.
Quiescent pulmonary endothelium establishes an antithrombotic, anti-inflammatory surface that promotes blood flow. However, the endothelium rapidly responds to injury and inflammation by promoting thrombosis and enabling the directed transmigration of inflammatory cells, such as neutrophils, into the alveolar airspace. Although the endothelial cell signals responsible for establishing a prothrombotic surface are distinct from those responsible for recognizing circulating neutrophils, these processes are highly interrelated. Von Willebrand factor (VWF)-stimulated secretion plays an important role in thrombus formation, and P-selectin surface expression plays a key role in neutrophil binding necessary for transmigration. Both VWF and P-selectin are located within Weibel-Palade bodies in pulmonary arteries and arterioles, yet Weibel-Palade bodies are absent in capillaries. Despite the absence of the Weibel-Palade bodies, pulmonary capillaries express both VWF and P-selectin. The physiological and pathophysiological significance of these observations is unclear. In this review, we address some anatomical and physiological features that distinguish pulmonary artery, capillary, and vein endothelium. In addition, we review our current understanding regarding the stimulated secretion of VWF and P-selectin in pulmonary artery and capillary endothelium. This information is considered in the context of vasculitis and pneumonia, two pathophysiological processes to which the stimulated secretion of VWF and P-selectin contribute.
Thesis (M.S.)--Texas Tech University Health Sciences Center, 2000. "December, 2000." Includes bibliographical references (leaves 50-57).
The purpose of this review is to examine whether our current knowledge of the higher order control of gene expression and nuclear organization can help us understand the mechanisms of genomic imprinting. Imprinting involves the inheritance of a silenced allele of a gene through either a paternal or maternal germline. We have approached the problem of imprinting using a model based on the dynamic attachment of chromatin loops to immobilized RNA polymerases and control elements. We have combined the information from different experimental approaches, examining primarily the IGF2-H19 locus, in an attempt to simplify the complexity of the imprinting data that has accumulated. It is hoped that a unified model may generate predictions amenable to experimental testing and contribute to the interpretation of future experiments.
Discovery of the pathophysiologic mechanisms leading to pulmonary edema and identification of effective strategies for prevention remain significant clinical concerns. Endothelial barrier function is a key component for maintenance of the integrity of the vascular boundary in the lung, particularly since the gas exchange surface area of the alveolar-capillary membrane is large. This review is focused on new insights in the pulmonary endothelial response to injury and recovery, reversible activation by edemagenic agents, and the biochemical/structural basis for regulation of endothelial barrier function. This information is discussed in the context of fundamental concepts of lung fluid balance and pulmonary function.
The expression of low-voltage-gated T-type calcium channel alpha1G-subunit (Cav3.1) has recently been revealed in pulmonary microvascular endothelial cells (PMVECs). In endothelial cells, changes in membrane potential may be caused by a variety of physiological or pathophysiological vascular stimuli. Thus, the characteristic low-voltage range of activation, transient kinetics of inactivation, and conducting continuous Ca2+ entry in a range of low membrane potentials of this channel may confer its temporal and spatial control of cytosolic Ca2 + important for a variety of cellular processes in vascular endothelial cells. The goal of this review is to provide a succinct description of the endothelial cell T-type calcium channel, including its molecular characteristics, biophysical and pharmacological profiles, and involvement in certain endothelial cell functions.
Evidence was provided, in the preceding paper (Thuringer & Sauvé, 1992), that the external Ca(2+)-dependent phase of the Ca2+ signals evoked by bradykinin (BK) or caffeine in bovine aortic endothelial cells (BAE), differ in their respective sensitivity to procaine. To examine whether the emptying of the InsP3-sensitive Ca2+ store is the signal for activating the agonist-evoked Ca2+ entry, we have investigated the effects of thapsigargin (TSG), a known inhibitor of the microsomal Ca(2+)-ATPase activity in a variety of cell types, via the activity of calcium-activated potassium channels [K(Ca2+) channels]. In cell-attached experiments, the external application of TSG caused a sustained or oscillatory activation of K(Ca2+) channels depending on both the cells and doses tested. The TSG-evoked channel activity could be reversibly blocked by removing extracellular Ca2+, and strongly decreased by adding 10 mM procaine to the bath medium. In Ca(2+)-free external conditions, TSG did not promote an apparent Ca2+ discharge from internal stores but prevented in a dose- and time-dependent manner the subsequent agonist-evoked channel activity related to the release of internally sequestered Ca2+. These results confirm that TSG and BK release Ca2+ from the same internal stores but with different kinetics. Because the channel response to caffeine was found to be poorly sensitive to procaine, in contrast to that evoked by BK and TSG, it may be concluded that both BK and TSG activate the same Ca2+ entry pathway. Therefore, the emptying of the InsP3-sensitive Ca2+ store is likely to be the main signal for activating the agonist-evoked Ca2+ entry in BAE cells.
The regulation of cytoplasmic free calcium concentration [( Ca2+]i) in endothelial cells (EC) derived from human umbilical vein, aorta, and pulmonary artery, or from bovine pulmonary artery, was studied by means of the fluorescent Ca2+ indicator indo-1. Histamine and thrombin caused a rapid transient elevation in [Ca2+]i in the EC of all the human blood vessels tested. In aortic EC, [Ca2+]i also rose in response to ATP and bradykinin. It was shown that in bovine pulmonary artery EC [Ca2+]i rises in response to platelet-activating factor (PAF) and thrombin. For a more detailed investigation of the receptor-mediated mechanism of [Ca2+]i increase in EC we used histamine as a stimulating agent. Histamine effects were seen at concentrations ranging from 5 X 10(-7) to 10(-4) M [50% effective dose (ED50) approximately 2-4 microM)] and were mediated by H1-receptors. The histamine-induced increase in [Ca2+]i was not markedly diminished when the extracellular calcium was bound by excess ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The data obtained indicate that the histamine effect is best explained by Ca2+ mobilization from intracellular stores. The histamine-induced increase in [Ca2+]i was not influenced by elevating the intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) or cyclic guanylic acid (cGMP) by use of isobutylmethylxanthine and forskolin or by nitroprusside preincubation, respectively. However, the protein kinase C stimulator, phorbol myristate acetate (PMA), strongly inhibits [Ca2+]i elevation. It is assumed that a negative feedback mechanism that blocks receptor-mediated [Ca2+]i increase is triggered as a result of the activation of protein kinase C.
Experiments were performed on isolated maximally vasodilated perfused rat hindquarters to evaluate the role of calcium and magnesium for the capillary permeability increase(s) elicited by histamine. Changes in capillary permeability were quantified by determinations of capillary filtration coefficient (CFC) with gravimetric technique, and capillary diffusion capacity (PS) for vitamin B12 (MW = 1,355) with a single injection indicator dilution technique. During control, vascular resistance was 2.2 PRU100 at a flow of 9.4 ml min-1 per 100 g, and PS for B12 was 3.7 +/- 0.1 ml min-1 per 100 g, while CFC was 0.0377 +/- 0.0004 ml min-1 mmHg-1 per 100 g. Perfusion with 'Mg-free' solution for 1 h caused a 24% increase in CFC, while neither 'Ca-free' perfusion nor perfusion with verapamil (5 X 10(-5) M) nor felodipine (1 X 10(-6) M) induced any changes in CFC. Histamine (100-200 microM) caused in all preparations a 150-200% increase in CFC with only small changes in PS for B12. This histamine effect was absent after 1 h of 'Ca-free' perfusion and was partially blocked after 1 h of perfusion with 0.1 mM calcium, while the calcium antagonists verapamil and felodipine had no effects on the histamine-induced changes. The results imply that histamine exerts its action on the endothelial cells through a calcium-dependent process, probably involving low affinity calcium sites but this process could not be inhibited by the calcium antagonists used. Thus, endothelial cell contractility, which probably is responsible for the histamine-induced increase in capillary permeability, exhibits unique characteristics, differing from those of vascular smooth muscle.
Calcium-sensitive fluorophores are used to estimate cytosolic free Ca2+ in many cell types under various conditions. We tested the effect of reduced PO2 on the behavior of Fura-2 and Indo-1 in cultured bovine pulmonary artery endothelial cells. Reduced PO2 (PO2 25-35 mmHg) caused a significant upward shift of in vivo calibration curves for both fluorophores. The in vivo emission spectrum of Fura-2 indicated that the effect was principally due to attenuated emission at the Ca(2+)-unbound 380 nm wavelength, with no shift in position of the emission maxima for either Ca(2+)-bound or unbound forms of the fluorophore. Reduced PO2 did not directly alter the behavior of the dyes, as no shift of in vitro calibration curves was seen. Neither decreased photobleaching nor altered autofluorescence accounted for the shift. We investigated several potential indirect effects, including cellular acidification, reduced viscosity, inhibition of oxidative energy production and reductive stress. In contrast to lowered PO2, acidification in vitro produced a leftward but not an upward shift. Estimation of intracellular pH with SNAFL-calcein under reduced PO2 showed no apparent acidification in these cells, further strengthening the argument that altered intracellular pH was not causing the shift. Others have shown that decreases in viscosity in vitro may shift the calibration curve for Fura-2 upward, similar to our finding with reduced PO2. However, for Indo-1 we found that decreased viscosity in vitro attenuated fluorescence emission at the Ca(2+)-bound 405 nm wavelength, thus producing the opposite effect on fluorescence ratio and indicating that reduced PO2 was not acting through changes in cellular microviscosity.(ABSTRACT TRUNCATED AT 250 WORDS)