1 Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688; and 2 University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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Activation of store-operated Ca2+ entry inhibits type 6 adenylyl cyclase (EC 4.6.1.1; AC6; 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 AC6 and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca2+ entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca2+ inhibition of AC6 is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca2+ inhibition of AC6. Enzyme activity was assessed using PMVEC membranes, where Ca2+ and cAMP concentrations were independently controlled. Endogenous AC6 activity exhibited high- and low-affinity Ca2+ inhibition, similar to that observed in C6-2B cells, which predominantly express AC6. Ca2+ inhibition of AC6 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 Ca2+ inhibition of AC6 is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC6 to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition.
signal transduction; endothelial cells; permeability; lung
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INTRODUCTION |
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ENDOTHELIUM FORMS A METABOLICALLY active barrier that separates the circulation from underlying tissue. Emerging data intimate that lung macrovascular and microvascular endothelial cells arise from different embryological origins, giving rise to distinct phenotypic cell populations (9, 10, 33). Divergence in vascular origin contributes to unique functional attributes of the lung's macro- and microcirculations (23). To facilitate efficient gas exchange across the alveolar-capillary membrane, microvascular endothelium exhibits enhanced barrier function and restricts access of protein and water to interstitial sites (3, 8, 18, 27). Molecular determinants of microvascular endothelial barrier function are incompletely understood.
Activation of store-operated Ca2+ entry reduces macrovascular endothelial cell global cAMP, which is important for retraction of cell borders and increased protein and water permeability (32). This decrease in cAMP occurs after Ca2+ inhibition of type 6 adenylyl cyclase (AC6), directly implicating AC6 in control of cell-cell adhesion. Microvascular endothelial cells express AC6 and respond to inflammatory agonists with a rise in cytosolic Ca2+. In striking contrast to macrovascular endothelial cells, however, the activation of store-operated Ca2+ 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 AC6 in microvascular endothelial cells (5).
Microvascular endothelial cell phosphodiesterase activity is high and limits cAMP accumulation. We previously established that type 4 phosphodiesterase inhibition using rolipram produces a greater increase in cAMP than does adenylyl cyclase (AC) activation using forskolin or isoproterenol (28). The combination of forskolin and rolipram produces profound synergistic rises in cAMP, suggesting that microvascular endothelial cells exhibit high cAMP turnover. Significant cAMP gradients exist within single cells (25, 26). At least two cAMP compartments have been modeled, including its site of synthesis (e.g., plasmalemma) and the bulk cytosol. Such compartmentalization appears to be important for elevating cAMP to levels that regulate target activity, including the A-kinase, which is tethered to functional sites through A-kinase-anchoring proteins. In models developed by Rich and co-workers (25, 26), phosphodiesterase activity is an important determinant of the cAMP concentration that accumulates within the AC microdomain. Thus high phosphodiesterase activity in microvascular endothelial cells likely prevents cAMP from accumulating nearby AC6. The cAMP that escapes hydrolysis by phosphodiesterase(s) contributes to the global cAMP pool.
The purpose of our present studies was to determine the role of type 4 phosphodiesterase activity in AC6 Ca2+ inhibition. Rolipram reveals Ca2+ inhibition of global cAMP in microvascular endothelial cells. This effect of rolipram may occur because it increases cAMP within the AC microdomain to a level where Ca2+ inhibition of AC6 can be adequately resolved. Alternatively, the rise in cAMP within this microdomain may directly or indirectly impact AC6 activity. cAMP could activate A-kinase, which phosphorylates and inhibits AC6 (2), or rolipram may increase cAMP interaction with the AC6 catalytic core and unmask a high-affinity Ca2+-binding site (11, 17, 35, 36). Our results support the idea that rolipram increases cAMP to a level where AC6 Ca2+ inhibition can be resolved. These findings therefore support the emerging concept that phosphodiesterase activity controls functional microdomains of membrane-delimited cAMP.
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METHODS |
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Materials. Thapsigargin was obtained from Calbiochem (San Diego, CA) and rolipram from Biomol (Plymouth Meeting, PA). Unless otherwise noted, all other materials and reagents were obtained from Sigma (St. Louis, MO).
Isolation and culture of rat pulmonary microvascular endothelial cells. Pulmonary microvascular endothelial cells (PMVECs) were isolated and cultured using a modification of the method described by Stevens et al. (28). Male Sprague-Dawley rats (350-400 g) were euthanized by intraperitoneal injection of 50 mg of pentobarbital sodium (Nembutal, Abbott Laboratories, Chicago, IL). After sternotomy, the heart and lungs were removed en bloc and placed in a DMEM (GIBCO BRL, Grand Island, NY) bath containing 100 U/ml penicillin and 100 µg/ml streptomycin (GIBCO BRL). Thin strips were removed from the lung parenchyma, finely minced, and transferred with 2-3 ml of DMEM to a 15-ml conical tube containing 3 ml of digestion solution [0.5 g of BSA, 10,000 units of type 2 collagenase (Worthington Biochemical, Lakewood, NJ), and Ca2+- and Mg2+-free PBS (GIBCO BRL) to make a total volume of 10 ml]. The digestion mixture was allowed to incubate at 37°C for 15 min before it was poured through an 80-mesh sieve into a sterile 200-ml beaker. An additional 5 ml of normal medium [10% fetal bovine serum (FBS; Hyclone, Logan, UT) with 100 U/ml penicillin and 100 µg/ml streptomycin (GIBCO BRL) in DMEM] was used to wash the sieve. The isolation mixture was transferred to a 15-ml conical tube and centrifuged at 300 g for 5 min, the medium was aspirated, and the cells were resuspended with 5 ml of complete medium consisting of a 1:3 mixture of microvascular conditioned medium-incomplete medium [80% RPMI 1640, 20% FBS, 12.3 U/ml heparin (Elkins-Sinn), and 6.7 µg/ml Endogro (Vec Technologies) with 100 U/ml penicillin and 100 µg/ml streptomycin (GIBCO BRL)]. Centrifugation-aspiration was repeated, and the cells were resuspended in 2-3 ml of complete medium and allowed to incubate at 37°C for 30 min before they were placed dropwise onto 35-mm culture dishes. After 1 h at 37°C in 5% CO2, 3 ml of complete medium were added. The dishes were checked daily for contaminating cells, which were removed by scraping and aspiration. Endothelial cell colonies were isolated with cloning rings, trypsinized, resuspended in 100 µl of complete medium, and placed as a drop in the center of a T-25 flask. The cells were allowed to attach (1 h at 37°C in 5% CO2) before addition of 5 ml of complete medium. Cultures were characterized using SEM, uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled low-density lipoprotein (DiI-acetylated low-density lipoprotein), and a lectin-binding panel, and were routinely passaged by scraping.
Isolation and culture of rat main pulmonary artery endothelial
cells.
Main pulmonary arteries were isolated as previously described
(28). Briefly, 300-g Sprague-Dawley rats were euthanized
using an overdose of pentobarbital sodium. The heart and lungs were excised en bloc after sternotomy, and the pulmonary arteries were isolated and removed. The arteries were opened, and the intimal lining
was carefully scraped using a scalpel. The harvested cells were then
placed into T-25 flasks (Corning, Corning, NY) containing F-12 nutrient
mixture-DMEM (1:1) supplemented with 10% FBS, 100 U/ml penicillin, and
100 µg/ml streptomycin (GIBCO BRL) and passaged up to 15 times. The
endothelial cell phenotype was confirmed by acetylated low-density
lipoprotein uptake, factor VIII-Rag immunocytochemical staining, a
lectin-binding panel, and the lack of immunostaining with smooth muscle
cell -actin antibodies (19).
Measurement of ATP-to-cAMP conversion. ATP-to-cAMP conversion was performed as described previously (28). Briefly, PMVECs were seeded onto 35-mm six-well plates (Fisher Scientific, Atlanta, GA) at 40,000 cells/ml and grown to confluence over 3-4 days. Experiments were conducted in DMEM with physiological extracellular Ca2+ concentrations unless otherwise noted and pH balanced to 7.4 with osmolality of 285-300 mosM. The cells were incubated with 2 ml of DMEM containing 2.0 µCi/ml [3H]adenine (Moravek Biochemicals, Brea, CA) for 1 h at 37°C to radiolabel the ATP pool for assessment of ATP-to-cAMP conversion. [3H]adenine was aspirated, the cells were rinsed, and DMEM was reintroduced, with agents added at the concentrations and times indicated. Reactions were stopped using 1 ml of ice-cold 5% TCA with 1.5 nCi/ml [14C]cAMP as an internal standard. Wells were scraped, samples were collected in 1.5-ml centrifuge tubes, and wells were washed with 200 µl of distilled water, which was added to the sample for a total volume of 1.2 ml. Samples were vortexed, and 200 µl were aliquoted into 20-ml scintillation vials with 6 ml of scintillation fluid to evaluate total [3H]ATP and [14C]cAMP counts. The remaining sample volumes were centrifuged for 5 min, and the supernatants were poured over Dowex columns and washed with 3 ml of distilled H2O to remove ATP from the column. The Dowex columns were then placed over alumina columns, and the cAMP was transferred onto the alumina column using 4 ml of distilled H2O. cAMP was collected from the alumina column into 20-ml scintillation vials using 4 ml of 0.1 M imidazole (pH 7.1). Scintillation fluid (6 ml) was added to the vials before counting. The amount of [14C]cAMP recovered was expressed as a fraction of total [14C]cAMP (e.g., [14C]cAMP/total [14C]cAMP) and used to standardize [3H]cAMP to column efficiency. Results are expressed as the percent conversion of total [3H]ATP to [3H]cAMP and represent the cellular accumulation of cAMP that is synthesized over time. Because specified experiments were performed in the presence of cold cAMP at up to 1 mM, control studies were undertaken to ensure appropriate recovery from alumina columns. Indeed, addition of 1.5 mM cold cAMP to the sample buffer did not alter the cAMP elution profile (data not shown).
Preparation of plasma membranes.
Sucrose gradient-purified membranes were obtained using a modification
of a previously described method (32). Cells were grown to
confluence on 150-mm dishes rinsed with ice-cold buffer A
(137 mM NaCl, 2.7 mM KCl, 10.14 mM Na2HPO4, and
10.76 mM KH2PO4) and then incubated with
ice-cold buffer B (40 mM Tris · HCl, 1 mM EDTA, and 150 mM NaCl) for 10 min. Buffer B was aspirated
and replaced with buffer C (20 mM HEPES, pH 7.5, 5 mM EDTA,
1 mM EGTA, 2 mM dithiothreitol, 200 mM sucrose, and protease inhibitor
cocktail). Plates were scraped, and the contents were pooled into 50-ml
conical tubes and centrifuged at 4°C for 10 min at 250 g.
The remainder of the isolation process was carried out at 4°C. Cell
pellets were resuspended in 10-15 ml of reserved buffer
C and lysed using 10-20 strokes of a Dounce homogenizer. The
homogenate was further processed using four 10-s bursts from a Polytron
aggregate tissue grinder and then layered on top of a discontinuous
(5-45%) sucrose gradient and centrifuged at 25,000 rpm (2 h,
4°C; SW 28.1 rotor, Beckman Coulter, Fullerton, CA). The membrane
band at the 30-40% sucrose interface was removed, resuspended
(1:5) in buffer C, and recentrifuged for 30 min at 10,000 rpm. Buffer C was removed, and the final pellet was
resuspended in 2-3 ml of fresh buffer C and homogenized
with a Dounce tissue grinder. The membranes were then aliquoted and
frozen at 80°C. Protein content was determined using the method of
Lowry et al. (21).
Measurement of AC activity.
In vitro determination of AC activity was performed on isolated
membranes as previously described (32), with the following modifications (unless otherwise noted). AC activity was measured in 1 ml of buffer containing 0.15 mg protein/ml membrane, 1 × 106 counts per min (cpm)/ml [-32P]ATP (NEN
Life Sciences Products, Boston, MA), 0.1 mM ATP, 0.1 mM cAMP (unless
otherwise noted), 2.5 U/ml creatine phosphokinase, 12 mM
phosphocreatine, 1 U/ml adenosine deaminase, 2 mM MgCl2, 25 mM HEPES, and 0.04 mM GTP. Time controls were performed to ensure ATP
regeneration. Indeed, basal and forskolin-stimulated cAMP conversion
was linear over a 40-min time course. Free Ca2+
concentrations were determined using the Fabiato free ion calculator (12). The buffer was adjusted for intracellular ionic
composition and strength (0.150 M), and pH was adjusted to 7.3 to
approximate intracellular physiological conditions. Reactions were
carried out at 30°C for 20 min and stopped with 200 µl of ice-cold
30% TCA. [3H]cAMP (15,000 cpm; NEN Life Sciences
Products) was added as the internal recovery standard.
[32P]cAMP was purified as described in Measurement
of ATP-to-cAMP conversion and quantified (32).
Accumulation of cAMP is expressed as picomoles of cAMP formed per
milligram of protein per minute and represents the rate of cAMP
synthesis. Basal cAMP production was constant over a 40-min time course.
Assessment of cytosolic Ca2+ concentration. Cells were seeded onto 25-mm coverslips and grown to confluence as described in Measurement of ATP-to-cAMP conversion. The cells were rinsed with Krebs buffer before being loaded (20 min at 37°C in 5% CO2) with the Ca2+-sensitive fluorophore fura 2-AM (3 µM; Molecular Probes, Eugene, OR) and 10% pluronic acid. Deesterification of the fluorophore was achieved by rinsing and then incubating the cells with Krebs buffer (37°C in 5% CO2) for an additional 20 min. After an additional rinse, the cells were utilized to evaluate cytosolic Ca2+ concentration as previously described (34). Experiments were routinely performed in Krebs buffer (pH 7.4) with 25 mM HEPES and 2 mM Ca2+ unless otherwise noted.
Graphing and statistical analysis. One-way ANOVA with Student's t-test was performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA).
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RESULTS |
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Ca2+ inhibition of cAMP in intact
PMVECs.
Store-operated Ca2+ entry channels provide the
Ca2+ source that inhibits AC6 (4, 6, 13,
24). Whereas activation of store-operated Ca2+ entry
induces a rise in global cytosolic Ca2+ to submicromolar
concentrations, AC6-associated Ca2+
concentration reaches intermediate micromolar levels (24). Endothelial cells express AC6, and store-operated
Ca2+ entry pathways represent the predominant mode of
Ca2+ entry (23). Initial studies, therefore,
utilized thapsigargin to induce the passive depletion of endoplasmic
reticulum Ca2+ and activate store-operated Ca2+
entry channels to assess whether activation of store-operated Ca2+ 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).
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AC activity in PMVEC plasma membranes.
We initially sought to characterize the effect of Ca2+ on
AC6 in PMVEC membranes isolated from a 30-40% sucrose
gradient. As seen in previous studies using C6-2B cell membranes, which
almost exclusively express AC6 (7),
Ca2+ strongly inhibits AC6 in PMVEC membranes
(Fig. 5). Fitting the data to a kinetic
model reveals a curve that is consistent with high- and low-affinity
Ca2+-binding sites, discerned using a two-site competition
model (15). The high-affinity Ca2+-binding
site exhibited strong inhibition, with an initial plateau at ~750 nM
Ca2+. Such inhibition is greater than that observed in
C6-2B cell membranes (15). Thus AC6 expressed
in PMVECs can be readily inhibited by high-affinity binding to
Ca2+ at submicromolar Ca2+ concentrations.
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DISCUSSION |
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Endothelial cells form a semipermeable barrier that separates blood from interstitium. Such barrier function is partly maintained by cell-cell adhesion, regulated directly by intracellular cAMP concentrations (16, 23). Inflammatory Ca2+ mediators that disrupt the endothelial barrier decrease cAMP through inhibition of AC6 (28, 30, 32). Indeed, submicromolar Ca2+ concentrations reduce PAEC cAMP content by 30-50%, sufficient to initiate intercellular gap formation (5, 32). Endothelial phenotypes differ vastly, however, partly on the basis of the environment in which they reside and their embryological origin (33). Whereas the lung macrocirculation arises from angiogenesis, the lung microcirculation arises from vasculogenesis (9, 10). Microvascular endothelium possesses a more restrictive barrier than does macrovascular endothelium, which is necessary to optimize gas exchange within the lung's microcirculation. cAMP conversion is ~10-fold higher in PMVECs than in PAECs, and although PMVECs express AC6, we have observed that the activation of store-operated Ca2+ entry using thapsigargin does not decrease global cAMP content.
Absence of a readily observable AC6 Ca2+
inhibition in PMVECs leads to important questions regarding the
enzyme's physiological function. We recently addressed this issue
using PMVECs treated with thrombin. Thrombin differs from thapsigargin
(e.g., direct activation of store-operated Ca2+ entry)
because it promotes store-operated Ca2+ entry and activates
Gi proteins, which inhibit AC6
(22); these thrombin effects induce intercellular gap
formation in PMVECs (5). To address the physiological role
of AC6 in PMVEC barrier function, the
Ca2+-calmodulin-stimulated AC (AC8) was
expressed using adenovirus. Expression of AC8 was enriched
at the lateral membrane of cell-cell contacts, suggesting that ACs
target to sites of cell-cell adhesion. AC8 converted the
endogenous thrombin-induced decrease in cAMP to a modest stimulation of
cAMP. AC8 expression nearly abolished thrombin-induced gap
formation, providing direct evidence that Ca2+-sensitive
ACs regulate cell-cell apposition in endothelial cells. Interestingly,
expression of AC8 did not prevent cell "contraction" (31), functionally separating the role of contraction from
the role of adhesion in intercellular gap formation. Thus
Ca2+ inhibition of endogenously expressed AC6
reduces cAMP within the enzyme's microdomain, and this decrease in
cAMP allows gaps to form between cells.
The evidence for the requisite role of AC6 in endothelial cell gap formation appears to contradict the evidence that thapsigargin does not alone reduce global cAMP content. However, present studies established for the first time that AC6 is the dominant AC isoform in PMVECs. In intact cells and in cell membranes, rolipram revealed AC6 Ca2+ inhibition. This effect of rolipram was to increase cAMP to level where Ca2+ inhibition of AC6 could be resolved, because increasing cAMP concentrations independent of phosphodiesterase activity did not alter high-affinity Ca2+ binding to AC6. Similarly, membrane incubation with H89 did not alter AC6 Ca2+ inhibition. These findings, therefore, strongly suggest that rolipram reveals AC6 Ca2+ inhibition in PMVECs by allowing cAMP to accumulate within the AC microdomain to levels sufficient to resolve enzyme inhibition.
Our findings are compatible with the idea that Ca2+ inhibition of AC6 works cooperatively with phosphodiesterase(s) to regulate cAMP concentrations within microdomain compartments. PMVECs possess highly robust phosphodiesterase type 4 activity (28). However, this constitutive activity is not sufficient to lower membrane-delimited cAMP pools to a level that reduces cell-cell adhesion and promotes intercellular gap formation. Indeed, gap formation requires Ca2+ inhibition of AC6 (5).
In summary, the present studies were undertaken to examine regulation of Ca2+-inhibited AC6 by phosphodiesterase activity in PMVECs. Although Ca2+ inhibition of cAMP was not resolved under basal conditions, activation of AC with forskolin and rolipram revealed Ca2+ inhibition of AC6. Independently increasing cAMP concentrations altered AC activity but did not impact high-affinity Ca2+ binding or Ca2+ inhibition of AC6. These findings suggest that the high constitutive phosphodiesterase activity in PMVECs contributes, with Ca2+ inhibition of AC6, to control membrane-delimited cAMP pools critical for control of cell-cell apposition.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Chinkers for careful review and constructive comments.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-66299 and HL-60024.
Address for reprint requests and other correspondence: T. Stevens, Center for Lung Biology, Dept. of Pharmacology, MSB 3364, University of South Alabama College of Medicine, Mobile, AL 36688 (E-mail: tstevens{at}jaguar1.usouthal.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00083.2002
Received 19 March 2002; accepted in final form 18 July 2002.
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