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Address correspondence to Troy Stevens, Department of Pharmacology, MSB 3364, University of South Alabama College of Medicine, Mobile, AL 36688. Tel.: (251) 460-6010. Fax: (251) 460-6798. E-mail: tstevens{at}jaguar1.usouthal.edu
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Abstract |
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Key Words: adenosine 3',5'-cyclic monophosphate; cAMP; store-operated calcium entry; thrombin; permeability
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Introduction |
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Inflammatory [Ca2+]i agonists only increase permeability in the absence of a rise in cAMP (Carson et al., 1989; Casnocha et al., 1989; Minnear et al., 1989; Morel et al., 1989; Stelzner et al., 1989; Allsup and Boarder, 1990; Oliver, 1990). cAMP-elevating agents improve constitutive barrier function and prevent the endothelial cell disruption that occurs in response to a rise in [Ca2+]i. Moreover, either inhibition of cAMP synthesis or its protein kinase (e.g., PKA) is sufficient to decrease adhesion and increase permeability (Stelzner et al., 1989; Stevens et al., 1995), suggesting that cAMP primarily regulates sites of cellcell tethering. These findings bring into question the independent roles of [Ca2+]i and cAMP in endothelial cell barrier disruption. Results from cloning and expression of a calcium-inhibited adenylyl cyclase (type 6 adenylyl cyclase [AC6]*) in nonexcitable cells provided a plausible mechanism through which inflammatory [Ca2+]i agonists could decrease cAMP (Yoshimura and Cooper, 1992). Endothelial cells express AC6 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al., 1997; Jourdan et al., 2001) and in vitro assays (Manolopoulos et al., 1995a,b; Stevens et al., 1995, 1997, 1999; unpublished data).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store-operated calcium entry decreases cAMP and PKA activity 3050% in intact cells (Stevens et al., 1995, 1997, 1999; unpublished data). Thus, calcium inhibition of AC6 may importantly contribute to endothelial cell gap formation.
Evidence for regulation of endothelial cell barrier function by AC6 is indirect and has been hampered by the inability to specifically control cAMP concentrations within a physiologically relevant range. A mechanism(s) accounting for calcium inhibition of AC6 is unresolved (Guillou et al., 1999; Gu and Cooper, 2000), although calcium inhibition does not require calmodulin or other calcium binding proteins (Caldwell et al., 1992). Recent studies suggest the presence of two distinct calcium binding sites on AC6, with apparent high and low affinities (Guillou et al., 1999; Gu and Cooper, 2000). Presumably the high affinity binding site accounts for enzyme calcium inhibition, because even calcium-insensitive isoforms of AC possess a low affinity binding site that inhibits enzyme activity at millimolar calcium concentrations. Structural assignment of the high affinity binding site is incomplete, making site-directed mutagenesis an untenable approach to evaluate the physiological role of AC6.
Fagan et al. (2000a) recently developed an adenovirus that expresses a calcium calmodulinstimulated (AC8) enzyme. In their studies, adenovirus infection resulted in significant AC8 expression in the excitable GH4C1 cell type. Enzyme activity was intimately regulated by calcium entry through store-operated calcium entry pathways, relatively insensitive to regulation by calcium entry through voltage-gated calcium channels, and not stimulated by calcium release from intracellular stores. Their findings indicated that calcium-sensitive ACs functionally colocalize with store-operated calcium entry pathways (Fagan et al., 1996, 1998, 2000b), and also demonstrated that AC8 expression may represent a tenable approach to reversing the endogenous calcium inhibition of AC6. Presently, we used the adenoviral construct that expresses AC8 to examine the physiological role of endogenously expressed AC6 in nonexcitable endothelial cells. Our results indicate that thrombin-induced gap formation only proceeds when a rise in [Ca2+]i inhibits AC6 and is not observed when a rise in [Ca2+]i stimulates AC8. These results provide the first direct evidence that calcium inhibition of AC6 plays a central, dominant role in regulation of intercellular gap formation.
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Results |
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The thrombin-induced [Ca2+]i responses were different in PMVECs than in PAECs. Thrombin induced a sigmoidal [Ca2+]i dose response curve in PMVECs, both in calcium release and entry (Fig. 2). There was good association between calcium release and entry over the entire dose range (Fig. 2 D), generally compatible with the mechanism of store-operated calcium entry. PMVECs were more sensitive to thrombin than were PAECs. Even though thrombin did not initiate a [Ca2+]i rise in PMVECs until 0.1 U/ml (versus 0.01 U/ml in PAECs), the microvascular cell dose response curve was left shifted, indicating greater thrombin sensitivity; in PMVECs the EC50 = 0.05 U/ml and in PAECs the EC50 = 0.1 U/ml.
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Fig. 5 illustrates thrombin-induced gap formation in PAECs and PMVECs. Acute application of 1 U/ml (EC100) thrombin to PAEC monolayers produced small gaps between cells that resealed over a 1-h time course (Fig. 5 A). Gaps were difficult to clearly resolve by light microscopy, though they were evident by the appearance of translucent cellcell borders. These findings are compatible with prior studies indicating that increased macromolecular permeability occurs through only minor ultrastructural disturbances in adherens junctions (McDonald et al., 1999; Moy et al., 2000). Higher thrombin concentrations (100 U/ml) produced larger, more visible intercellular gaps that persisted and, indeed, increased over a 2-h time course (Fig. 5, B and D). Because [Ca2+]i responses are lower in response to 100 U/ml than to 1 U/ml thrombin (Fig. 1 D), these data indicate that the large gaps generated in response to higher thrombin concentrations occur after activation of calcium-independent signaling pathways.
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Thrombin induced PAEC and PMVEC gaps that varied in size, though clearly the largest gaps were observed in PMVECs. A direct comparison between cell types is shown in Fig. 5 D, where equivalent gap sizes are plotted. Whereas gap size progressively increased over 2 h in PAECs, gap size progressively decreased after 10 min in PMVECs. As shown in Fig. 5 E, even the largest PMVEC gaps were nearly completely resealed by 2 h. These findings indicate that PMVECs possess a unique capacity to repair or reseal intercellular gaps, even in the continued presence of thrombin.
Calcium-sensitive ACs and endothelial cell gap formation
Because thrombin activates multiple intracellular signaling cascades, we sought to examine the specific role of [Ca2+]i in gap formation. In PAECs, low thrombin concentrations (0.01 U/ml; EC20) desensitized the subsequent [Ca2+]i response to maximal doses (1.0 U/ml). In PMVECs, higher thrombin doses (0.10 U/ml) were required to desensitize subsequent [Ca2+]i responses. However, the use of higher concentrations elicited a large [Ca2+]i response and therefore the desensitization approach could not resolve between [Ca2+]i-dependent and -independent signal transduction pathways (Fig. 6, A and B), indicating that more direct approaches were required to specifically divulge the requirement of [Ca2+]i in thrombin-induced gap formation.
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Expression of the AC8/adenovirus construct did not change basal PMVEC cAMP concentrations (control = 124 ± 9 fmol cAMP vs. AC8 = 120 ± 11 fmol cAMP). Forskolin and rolipram synergistically increase cAMP in PMVECs, and allow easy resolution of AC6 Ca2+ inhibition (Stevens et al., 1999; unpublished data). Similar to our previous reports (Stevens et al., 1995, 1997, 1999; unpublished data), thrombin inhibited cAMP in PMVECS that endogenously expressed AC6 (Fig. 7 A). However, in cells expressing the AC8/adenovirus construct, the thrombin-induced Ca2+ inhibition was converted into Ca2+ stimulation of cAMP (Fig. 7 B). Thus, cells infected with the AC8/adenoviral construct provided the first available model to specifically examine the physiological role of AC6 in regulation of endothelial cell barrier apposition.
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It is not clear in AC8-expressing cells how small rises in global cAMP could, with such strength, oppose thrombin-induced gap formation. Recent studies (Rich et al., 2000, 2001) implicate discrete cAMP pools, including its site of synthesis (e.g., plasmalemma) and the bulk cytosol, in location-specific function. Thus, AC localization is an important determinant of enzyme (and cAMP) function independent of global cAMP concentrations. We examined localization of AC8 by expressing it as a fusion with yellow fluorescent protein (YFP). Fig. 8 illustrates nearly uniform expression of every cell infected with the YFPAC8/adenoviral construct. Confocal imaging from abluminal-to-luminal cell aspects reveals punctate staining at abluminal and luminal cell aspects, with staining enrichment at cellcell borders, suggesting that the enzyme localizes to sites of cell contact. Indeed, the video (Video 6) accompanying Fig. 8 E demonstrates dynamic movement of YFPAC8 at sites of cellcell contact. As seen in this video, YFPAC8 localization to cellcell borders is retained in accordance with cell movement in response to thrombin, including membrane association during small, transient gap formation. Taken together, these data support the idea that AC localization to sites of cellcell contact is an important determinant of how localized changes in calcium-dependent cAMP concentrations regulate intercellular gap formation.
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Discussion |
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Emerging evidence suggests that PAECs and PMVECs arise from distinct embryological origins and retain phenotypic differences even when their environments are the same (deMello et al., 1997; deMello and Reid, 2000; Stevens et al., 2001; Maeda et al., 2002). Previous studies using these cells have demonstrated that their calcium and cyclic nucleotide regulatory properties significantly differ (Stevens et al., 1997, 1999), although they both possess store-operated calcium entry pathways and they both express AC6. PMVECs form a more restrictive barrier in vivo and in vitro to protect the alveolar-capillary network from fluid accumulation and to optimize gas exchange (Kelly et al., 1998; Chetham et al., 1999). Direct activation of store-operated calcium entry using thapsigargin increases PAEC, but does not increase PMVEC, macromolecular permeability (Kelly et al., 1998; Chetham et al., 1999). Our present studies therefore used the inflammatory [Ca2+]i agonist thrombin, which, like thapsigargin, activates store-operated calcium entry. In addition, however, thrombin activates Gi and G12/13 signaling pathways that inhibit AC (Brass et al., 1991) and activate Rho signaling cascades (Vouret-Craviari et al., 1998; Carbajal and Schaeffer, 1999; Carbajal et al., 2000; Sah et al., 2000; van Nieuw Amerongen et al., 2000a, b; Wojciak-Stothard et al., 2001), respectively. Activation of Rho and its kinase promotes the cytoskeletal reorganization that is important for induction of intercellular gap formation.
We found that thrombin induces gap formation in both PAECs and PMVECs. High thrombin concentrations were required to break cellcell adhesions in microvascular cells. Indeed, our dose response curves were conducted over thrombin concentrations that exceeded its normal range. High thrombin concentrations revealed unique responses in PAECs and PMVECs. Calcium regulatory mechanisms are different between these cell types at high thrombin concentrations; PAECs posses a feedback inhibitory mechanism not present in PMVECs. In addition, gap formation in PAECs was slow and progressive, whereas gap formation in PMVECs was rapid and quickly resolved, suggesting that microvascular cells possess a motility/repair mechanism not present in their macrovascular counterparts. The gaps that formed in PMVECs were substantially larger than those present in PAECs. While speculative, PMVECs may possess a higher resting tension so that the disruption of cellcell adhesion produces large gaps that form rapidly.
In PAECs, gaps formed in response to the high thrombin concentrations that also inhibited calcium entry. Thus, intercellular gap formation can be induced by [Ca2+]i-independent as well as [Ca2+]i-dependent signaling cascades. AC6 is a calcium entrysensitive target that coordinates [Ca2+]i and cAMP signaling pathways. AC6 functionally colocalizes with store-operated calcium entry channels (Fagan et al., 1996, 1998, 2000b), indicating that thrombin may inhibit AC6 activity by stimulating calcium entry and activating Gi.
Our present studies sought to specifically determine the relevance of AC6 in thrombin-induced gap formation. Despite the activation of multiple signaling pathways, we found that converting calcium inhibition to modest stimulation of cAMP nearly abolished thrombin-induced gap formation in PMVECs. In AC8-expressing cells, AC8 localized to sites of cellcell contact. Digitally time-compressed videos revealed that although AC8-expressing cells did not form gaps, they still exhibited significant movement that was generally consistent with activation of motor function. It therefore appears that AC6 decreases the cAMP necessary to break cellcell adhesions without significantly altering actomyosin function per se. cAMP activation of its protein kinase reduces endothelial cell myosin light chain kinase activity (Garcia et al., 1997), indicating that cAMP is capable of reducing endothelial cell motor function. However, our findings suggest that the principal role of calcium-sensitive cAMP synthesis is not to regulate actomyosin interaction, but rather to control cellcell adhesion. In future studies, it will be important to determine relevant A-kinase targets that may control development of intercellular gap formation.
In summary, our studies have used two distinct endothelial cell phenotypes, PAECs and PMVECs, to evaluate the role of AC6 in thrombin-induced gap formation. Thrombin activates store-operated calcium entry in both cell types, along with other reported signaling cascades. Nonetheless, calcium entry across the cell membrane is an important determinant of gap formation. We have established for the first time that calcium entry is required to inhibit AC6 activity, which decreases cAMP necessary for generation of intercellular gaps. These findings firmly establish a physiological role for AC6 in nonexcitable endothelial cells: AC6 critically regulates gap formation.
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Materials and methods |
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Isolation and culture of pulmonary endothelial cells
PAECs and PMVECs were isolated and cultured using a method described in detail by Stevens et al. (1999). Cells were routinely passaged by scraping. Cultures were characterized using SEM, uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyaninelabeled low-density lipoprotein (DiI-acetylated LDL), and a lectin binding panel.
Construction of the virus expressing AC8
A full-length description of the adenovirus expressing AC8 has been reported in detail elsewhere (Fagan et al., 2000a). However, for construction of an adenovirus-transducing vector encoding the YFPAC8 fusion protein, the YFPAC8 fusion protein gene was excised from the plasmid pEYFP-C1 by digesting with NheI, filling in with T4 DNA polymerase in the presence of dNTPs, and digesting with XbaI. The plasmid pShuttleCMV (He et al., 1998) was digested with XhoI, blunt ended with T4 DNA polymerase in the presence of dNTPs, and digested with XbaI. The YFPAC8 gene and pShuttleCMV fragments were resolved by low melting point agarose gel electrophoresis, excised, and the DNA fragments were recovered by melting the agarose followed by phenol extraction and ethanol precipitation. The DNA fragments were ligated to generate the plasmid pShuttleCMV-YFPAC8. The YFPAC8 gene was removed from pShuttleCMV-YFPAC8 by digestion with NotI and XbaI and ligated with pShuttleE1A DNA (Schaack et al., 2001) that had been digested with the same restriction enzymes. The shuttle vectors encoding YFPAC8 were then linearized by digestion with PmeI, and 20 ng of each was used to electroporate Escherichia coli strain BJ5183 carrying the plasmid pAdEasy-1(He et al., 1998), a modification that we previously found helpful for plasmid construction (Orlicky and Schaack, 2001), to introduce the YFPAC8 genes into the adenovirus chromosome by homologous recombination (Chartier et al., 1996; Crouzet et al., 1997; He et al., 1998). Resulting colonies were screened by agarose gel electrophoresis of undigested plasmid DNAs. Supercoiled plasmids that migrated slower than linear 12-kb marker DNA were then used to transform E. coli DH5, and the plasmids tested by restriction digestion, and grown in large scale. The resulting plasmids were named pAdEasyCMV-YFPAC8 and pAdEasyE1A-YFPAC8. The plasmid DNAs were digested with PacI to liberate the adenovirus chromosomes and used to transfect (Jordan et al., 1996) 293 cells (Graham et al., 1977) to make viruses. Despite repeated attempts, no plaques were isolated from cells transfected with pAdEasyCMV-YFPAC8. However, fluorescent plaques were readily obtained from cells transfected with pAdEasyE1A-YFPAC8, suggesting that the level of overexpression of the YFPAC8 gene that results from the use of the CMV major immediate early promoter inhibited virus production (Schaack et al., 2001). AdEasyE1A-YFPAC8 virus was plaque purified and grown in large scale using 293 cells, purified by successive banding on CsCl step and isopycnic gradients, and dialyzed using three changes of 10 mM Tris-HCl, pH 8.0, 135 mM NaCl, 1 mM MgCl2, 50% vol/vol glycerol at 4°C. The particle concentration of the virus was quantitated by determination of the absorbance at 260 nm, where one A260 unit was considered equal to 1012 virus particles. The particle/plaque forming unit ratio for viruses prepared in this manner is reproducibly close to 100:1. The virus was stored at -20°C.
Infection with adenovirus/AC8 construct
PMVECs were seeded onto 25-mm glass coverslips and grown to 60% confluence, at which point they were infected with the adenoviral vector constructed to express Ca2+-stimulated AC8 at a titer of 100 plaque forming units/cell. Initial studies using PAECs (unpublished data) and GH4C1 cells established a dose-dependent increase in calcium-dependent cAMP synthesis over a multiplicity of infection range from 0 to 200. A multiplicity of infection of 100 provided reproducible calcium-stimulated cAMP responses and was therefore selected as the dose used in our present studies. Cells were used 48 h after infection for cAMP measurement and videomicroscopy studies.
Calcium measurement
Calcium measurements and endothelial cell calibrations have been described elsewhere (Stevens et al., 1994).
cAMP measurements
cAMP content was determined using a Biotrack cAMP competitive enzyme-immunoassay system (Amersham Pharmacia Biotech).
Confocal fluorescence microscopy
Fluorescent images were acquired on a Leica TCS SP2 confocal laser scanning microscope at a 514-nm excitation. Slices were taken through cells at 0.5-µm sections.
Time-lapsed fluorescence microscopy
4048 h after infection with the YFPAC8 construct, media was removed from cells and replaced with Krebs/2 mM Ca2+ buffer. Fluorescent images were acquired on an Olympus IX70 inverted microscope at 488-nm excitation and processed using Spot Software (Diagnostic Instruments). Time-lapse experiments were performed using the MetaMorph® (Universal Imaging Corp.) system. Images were collected at 40-s intervals over 30 min.
Online supplemental material
Supplemental videos are available at http://www.jcb.org/cgi/content/full/jcb.200204022/DC1. Videos 13 are time-compressed movies of PMVECs, demonstrating that thrombin-induced gap formation occurs only when arise in [Ca2+] reduces cAMP. Videos 46 illustrate localization of AC (YFPAC8) primarily at sites of cellcell adhesion.
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Footnotes |
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* Abbreviations used in this paper: AC, adenylyl cyclase; InsP3, inositol 1,4,5-triphosphate; PAEC, pulmonary artery endothelial cell; PMVEC, pulmonary microvascular endothelial cell; YFP, yellow fluorescent protein.
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Acknowledgments |
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This work was supported by PO1 HL66299 and RO1 HL60024 (to T. Stevens) and RO1 GM32483 (to D.M.F. Cooper).
Submitted: 4 April 2002
Revised: 2 May 2002
Accepted: 7 May 2002
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References |
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