Coordinate regulation of membrane cAMP by Ca2+-inhibited adenylyl cyclase and phosphodiesterase activities

Judy R. Creighton1, Nanako Masada2, Dermot M. F. Cooper2, and Troy Stevens1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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INTRODUCTION
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 alpha -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 [alpha -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).


    RESULTS
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INTRODUCTION
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DISCUSSION
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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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Fig. 1.   Activation of store-operated Ca2+ entry does not reduce basal ATP-to-cAMP conversion in pulmonary microvascular endothelial cells (PMVECs). A: ratiometric shifts of Ca2+-bound (340-nm) and Ca2+-unbound (380-nm) wavelengths of fura 2 indicate that 1 µM thapsigargin (TG) increased cytosolic Ca2+ concentration ([Ca2+]i) in PMVECs (n = 5 experiments). B: despite increasing [Ca2+]i through store-operated Ca2+ entry channels, thapsigargin had no discernible effect on ATP-to-cAMP conversion in otherwise unstimulated PMVECs (n = 6 experiments).

Because PMVECs possess high type 4 phosphodiesterase activity (28), we examined the effect of rolipram (type 4 phosphodiesterase inhibitor) on AC6 Ca2+ sensitivity. Rolipram evoked a time-dependent accumulation of cAMP and did not affect the Ca2+ response to thapsigargin (Fig. 2A). Activation of store-operated Ca2+ entry abolished the rolipram-induced rise in cAMP (Fig. 2B). Similar observations were made when rolipram and forskolin were added together. The combination of agonists did not alter the Ca2+ response to thapsigargin (Fig. 3A). Rolipram and forskolin converted >35% of the radiolabeled ATP pool to cAMP, indicating profound synergism (Fig. 3B). Nonetheless, thapsigargin abolished the subsequent rise in cAMP that was induced by rolipram and forskolin, suggesting that AC6 is the dominant AC isoform in PMVECs. Ca2+-insensitive AC activity is likely due to expression of other forskolin-stimulated AC isoforms (30). Because endothelial cells possess significant constitutive Ca2+ "leak" (20), we examined the role of such leak channels in control of cAMP. Rolipram unmasked Ca2+ sensitivity to constitutive leak channels, compatible with observations in conduit-derived endothelium (32). Whereas reducing extracellular Ca2+ from 2 mM to 100 nM increased the constitutive cAMP conversion by <10%, reducing extracellular Ca2+ in the presence of rolipram increased cAMP conversion by >75% (Fig. 4). These data suggest that type 4 phosphodiesterase activity is important for resolving AC6 Ca2+ sensitivity in intact PMVECs.


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Fig. 2.   Inhibition of type 4 phosphodiesterase activity using rolipram reveals Ca2+ inhibition of AC6. A: ratiometric shifts of Ca2+-bound (340-nm) and Ca2+-unbound (380-nm) wavelengths of fura 2 indicate that 10 µM rolipram (Rol) does not alter thapsigargin-induced rise in [Ca2+]i (n = 5 experiments). Inset: comparison of peak and plateau [Ca2+]i responses to thapsigargin in the absence (Con) and presence of rolipram. B: whereas rolipram increased ATP-to-cAMP conversion (e.g., cAMP accumulation), addition of 1 µM thapsigargin abolished stimulatory effect compatible with Ca2+ inhibition of AC6 (n = 6 experiments).



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Fig. 3.   Inhibition of type 4 phosphodiesterase activity and direct activation of adenylyl cyclase (AC) using forskolin reveals Ca2+ inhibition of type 6 AC (AC6). A: ratiometric shifts of Ca2+-bound (340-nm) and Ca2+-unbound (380-nm) wavelengths of fura 2 indicate that 10 µM rolipram and 100 µM forskolin (Fsk) do not alter thapsigargin-induced rise in [Ca2+]i (n = 5 experiments). Inset: comparison of peak and plateau [Ca2+]i responses to thapsigargin in the absence and presence of rolipram and forskolin. B: whereas rolipram and forskolin increased ATP-to-cAMP conversion, addition of 1 µM thapsigargin abolished stimulatory effect compatible with Ca2+ inhibition of AC6 (n = 6 experiments). Note difference in scale compared with Figs. 1 and 2. Combination of rolipram and forskolin converted >35% of radiolabeled ATP pool to cAMP.



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Fig. 4.   Decreasing Ca2+ leak into PMVECs reveals basal Ca2+ inhibition of AC6. Cells were treated with 10 µM rolipram for 10 min in the presence of physiological (2 mM) or nominal (100 nM) extracellular Ca2+ ([Ca2+]o). ATP-to-cAMP conversion was 10% greater under low-Ca2+ conditions, and this effect was significantly amplified in the presence of rolipram. * P < 0.05 (n = 6 experiments).

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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Fig. 5.   PMVEC membranes exhibit high- and low-affinity Ca2+ inhibition of AC6. PMVEC membranes were assayed for AC activity in the presence of the indicated free Ca2+ concentrations, established with 200 µM EGTA (see METHODS). Fitting of data to a nonlinear 2-site inhibition profile revealed high (0.06 µM; 68%)- and low (12 µM; 32%)-affinity inhibition by Ca2+, consistent with a predominance of AC6 in these membranes.

If cAMP accumulation is important to resolve AC6 sensitivity to Ca2+ inhibition, then such inhibition should be greatest at high cAMP concentrations. Basal cytosolic cAMP concentrations are estimated to be in the low nanomolar range (1), although the presence of an AC-to-bulk cytosolic cAMP gradient indicates that AC6-associated cAMP may be higher than previously anticipated on the basis of whole cell or cytosolic estimates (25). Endothelial cell intracellular free Ca2+ concentration is ~100 nM under basal conditions (29). PMVEC membranes were utilized to directly examine the effect of cAMP on AC6 activity. In initial studies, buffer solutions contained 1 and 100 nM cAMP and Ca2+, respectively, to mimic the unstimulated physiological estimates of these intracellular messengers. Compatible with whole cell studies, PMVEC membranes exhibited a substantially higher rate of cAMP synthesis than did PAEC membranes (data not shown). PMVEC membranes also possessed AC activity that was stimulated by forskolin (Fig. 6A). Rolipram slightly amplified forskolin stimulation of cAMP, indicating that the membranes possess modest type 4 phosphodiesterase activity (not significant). Prior studies established that PMVECs predominantly possess type 4 phosphodiesterase activity (37); rolipram and IBMX elicit similar rises in cAMP (data not shown). Ca2+ inhibition of cAMP could not be resolved under basal conditions, inasmuch as 1 nM-1 mM Ca2+ had no effect on the rate of cAMP synthesis (Fig. 6B). Rolipram and forskolin stimulated AC activity at 100 nM Ca2+, but AC activity in the presence of rolipram and forskolin was decreased at higher Ca2+ concentrations (Fig. 6C). Thus, similar to studies in whole cells, stimulation of AC6 with forskolin and rolipram reveals AC6 Ca2+ inhibition.


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Fig. 6.   Ca2+ does not inhibit constitutive AC6 activity in PMVEC membranes. PMVEC membranes isolated from a 30-40% sucrose gradient were incubated with buffer containing 1 nM cAMP and 100 nM Ca2+ (see METHODS). A: 10 min of treatment with 100 µM forskolin and 10 µM rolipram increased cAMP, indicating that membranes retained AC activity. B: under unstimulated conditions, accumulation of cAMP was not affected by Ca2+, suggesting that AC6 was not inhibited by Ca2+ (n = 6 experiments). C: rolipram and forskolin treatments amplified AC production of cAMP and revealed Ca2+ inhibition of AC6 (n = 6 experiments).

We next assessed whether cAMP directly impacts Ca2+ inhibition of AC activity. The Ca2+ concentration was buffered at 100 nM, while cAMP was adjusted from 1 nM to 1 mM. cAMP dose dependently increased AC activity at buffer concentrations up to 1 µM (Fig. 7A). Higher concentrations of buffer cAMP inhibited cAMP synthesis. Because these studies were conducted without phosphodiesterase inhibitors, the decrease in cAMP synthesis could be due to inhibition of AC or activation of phosphodiesterase activities by the buffer cAMP. Type 4 phosphodiesterases possess a Michaelis-Menten constant of 1-3 µM (14), within the range in which cAMP accumulation dropped significantly. However, a similar response to increasing buffer cAMP concentrations was also observed when PMVEC membranes were incubated with rolipram (data not shown). Thus the decrease in cAMP accumulation observed in Fig. 7 in the presence of >10 µM buffer cAMP is likely due to inhibition of AC activity, similar to previous kinetic studies (11).


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Fig. 7.   cAMP regulates AC activity but does not influence AC6 Ca2+ sensitivity. A: AC activity in PMVEC membranes was assessed in the presence of 100 nM Ca2+ at various cAMP concentrations: <1 µM cAMP promoted AC activity, and >1 µM cAMP inhibited AC activity. B: increasing cAMP concentrations altered AC activity but did not change high-affinity Ca2+ inhibition of AC6. AC activity was determined in the presence of 10 and 1,000 nM cAMP. Fitting of data as in Fig. 5 revealed the presence of high- and low-affinity inhibition by Ca2+ in both cases, which were not significantly affected by cAMP concentration [67% at 0.03 µM and 33% at 17 µM (low cAMP concentration) vs. 70% at 0.0 µM and 30% at 66 µM (high cAMP concentration), not significant]. C: data from B, shown as percentage of maximal response, reveals that high-affinity Ca2+ inhibition of AC6 is not altered over a range of cAMP concentrations. open circle , 10 nM cAMP; , 100 nM cAMP.

Under basal physiological Ca2+ concentrations, maximal AC activity was observed at 1 µM cAMP. To examine whether cAMP concentration is an independent determinant of AC6 Ca2+ sensitivity, studies were performed over a range of cAMP concentrations and AC activity was measured in the presence of increasing Ca2+ concentrations (Fig. 7B). Irrespective of cAMP concentrations, similar high-affinity Ca2+ inhibition of AC6 was observed (Fig. 7C). Protein kinase A phosphorylates and inhibits AC6 and could independently decrease cAMP synthesis (2). However, H89 did not independently reduce cAMP synthesis (data not shown). Thus cAMP does not appear to directly alter Ca2+ inhibition of AC6, indicating in whole cell and membrane studies that rolipram improves resolution of AC6 Ca2+ inhibition by regulating the amount of cAMP within the AC microdomain.


    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 Gialpha 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.


    ACKNOWLEDGEMENTS

We thank Dr. Michael Chinkers for careful review and constructive comments.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
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REFERENCES

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