Permeability of endothelial monolayers to albumin is increased by bradykinin and inhibited by prostaglandins

Pierre J. Farmer, Sylvie G. Bernier, Andrée Lepage, Gaétan Guillemette, Domenico Regoli, and Pierre Sirois

Institut de Pharmacologie de Sherbrooke, Medical School, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Using monolayers of bovine aortic endothelial cells (BAEC) in modified Boyden chambers, we examined the role of prostaglandins (PGs) in the bradykinin (BK)-induced increase of albumin permeability. BK induced a concentration-dependent increase of the permeability of BAEC, which reached 49.9 ± 1% at the concentration of 10-8 M. Two inhibitors of the prostaglandin G/H synthase, indomethacin (2.88 µM) and ibuprofen (10 µM), potentiated BK-induced permeability 1.8- and 3.9-fold, respectively. Exogenously administered PGE2 and iloprost, a stable analog of prostacyclin, attenuated the effect of BK in a concentration-dependent manner. Butaprost equally reduced the effect of BK, suggesting the participation of the EP2 receptor in this phenomenon. However, the EP4-selective antagonist AH-23848 did not significantly inhibit the protective effect of PGE2. The inhibitory effect of PGE2 was reversed by the adenylate cyclase inhibitor MDL-12330A (10 µM). These results suggest that BK-induced increase of permeability of BAEC monolayer to 125I-labeled albumin is negatively regulated by PGs. This postulated autocrine activity of PGs may involve an increase in the intracellular level of cAMP.

capillary permeability; iloprost; butaprost; prostaglandin E2; adenosine 3',5'-cyclic monophosphate; indomethacin; ibuprofen


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE INCREASE IN VASCULAR PERMEABILITY to solutes and macromolecules is one of the cardinal features of the inflammatory reaction. Permeabilization of the endothelium by inflammatory mediators such as thrombin (THR) and bradykinin (BK) is accomplished, in part, by the formation of intercellular gaps, permitting the passage of macromolecules (13). These changes in morphology are due to cytoskeleton rearrangements as a result of the phosphorylation of myosin light chains by associated myosin light chain kinase (MLCK) (14). This recently cloned enzyme is activated by Ca2+/calmodulin (CaM) kinase (15, 34). In addition to this paracellular transport, albumin is also conveyed transcellularly, bound to a specific membrane glycoprotein (gp60) (32). This route was shown to be responsible for 40% of the basal transport of albumin (32).

The mechanisms by which the increase in vascular permeability is counterregulated are, however, less well understood. Several studies have shown that beta -agonists could reduce the endothelial permeability. Indeed, Minnear et al. (24) demonstrated that isoproterenol attenuated the permeability of bovine pulmonary artery endothelial cell monolayers challenged with THR. Salmeterol and salbutamol were also shown to inhibit THR-induced permeability in experimental models using either bovine pulmonary artery endothelial monolayer (2) or bovine aortic endothelial cells (BAEC) (38). The protective effect of beta -agonist is associated with its capacity to activate protein kinase A (PKA) after de novo synthesis of cAMP (6). In support of these findings, Verin et al. (35) have presented data showing the inactivation of MLCK by a PKA-dependent phosphorylation.

Other reports suggest that the endothelium itself could regulate macromolecule transport. Nitric oxide (NO), synthesized by endothelial cells, was shown to decrease vascular permeability. Westendorp et al. (36) presented evidence that sodium nitroprusside and 8-bromo-cGMP could suppress the THR-induced increase in permeability of human umbilical and pulmonary artery endothelial cells. In addition, the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester enhanced the THR-induced increase in permeability. Similar results were obtained by Draijer et al. (10) using human vein umbilical cells and human aortic endothelial cells.

Prostaglandins (PGs), also synthesized by the endothelium, are well known for their role in the inflammatory reaction. However, their effect on vascular permeability is complex and not well understood. PGs have been shown to produce little variation of plasma extravasation by themselves. Nevertheless, it has been reported that PGs potentiate the increase in vascular permeability induced by histamine and BK in guinea pig and rabbit skin (37). Such an effect was attributed mainly to the vasodilatory effect of prostanoids that leads to increases of capillary pressure and, consequently, to increases of plasma extravasation. However, Fantone et al. (11) reported that the systemic treatment of rats with PGE1 and the stable 15-(S)- 15-methylprostaglandin E1 markedly reduced the increases in vascular permeability induced by intradermal injection of histamine. Similarly, Gee et al. (16) observed that PGE1 inhibited sheep lung microvascular permeability induced by complement activation. More recently, Jiang et al. (19) have shown a protective effect of beraprost, a stable prostacyclin analog, on capillary permeability in a rabbit model of ischemic reperfusion. Their findings are in line with the observation of Moller and Grande (25), who described the protective effect of prostacyclin in cat skeletal muscle. Those findings are, however, in contrast with the work of Murohara et al. (27), who reported that prostacyclin appears to mediate, at least in part, the vascular permeability effect of vascular endothelial growth factor in a guinea pig model. These apparently contradictory results may be explained by the ubiquitous distribution of PG receptors (7). Activation of PG receptor subtypes is linked to either the Ca2+/CaM or the cAMP-PKA pathway. Surprisingly, although many reports have been published concerning the effects of endothelium-derived PGs on vascular smooth muscle activity, few studies have been aimed at understanding the autocrine effects of prostaglandins on the endothelium.

The aim of this study was to investigate the role of PG on BK-induced transendothelial transport of albumin. BK was selected as the increasing permeability agent because 1) it is an important proinflammatory mediator known to increase endothelial permeability in vivo (29) and in vitro (30), and 2) it is a powerful stimulant of PG secretion. Results presented in this paper show that PGs counteract the increase of endothelial permeability induced by BK, possibly via an E- prostanoid receptor (EP2) subtype.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation. Fresh bovine aortas were cut longitudinally and then fixed on a sterile base. Luminal cells were removed by gentle scraping with a sterile surgical scalpel. Cells were collected in a collagenase type V solution (1 mg/ml; Sigma, St. Louis, MO) and incubated at 37°C for a 15-min period. Cells were routinely cultured in medium 199 supplemented with L-glutamine (2 mM), fetal bovine serum (10%), thymidine (10-5 M), penicillin 100 U/ml, and streptomycin (100 µg/ml) at 37°C. Passages were done mechanically. Cultures with 6-19 passages were used in our experiments. Cells were characterized as endothelial by the specific incorporation of the 1,1'-dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate (Biomedical Technologies, Stoughton, MA) according to the procedure of Voyta et al. (35).

Preparation of bovine aortic endothelial monolayers. Polycarbonate filters (Fisher, Chicago, IL) of 0.80 µM porosity were incubated in a gelatin solution for 1 h. Filters were dried, glued onto the base of modified Boyden chambers, and sterilized overnight by ultraviolet radiation. The day of the seeding, filters were treated with a sterile fibronectin solution (Sigma; 30 µg/ml) in medium 199 for 1 h at room temperature. Cells were then seeded at a density of 6 × 105 cells/ml and grown to confluence for 2 days before the experiment.

Preparation of the 125I-labeled albumin. Bovine serum albumin was marked with Na125I by use of Iodogen (Sigma), as described by Fraker and Speck (12). Radioactive albumin was separated from free iodine by exclusion chromatography with a Sephadex G-25 column.

Permeability assay. The method used here is a slightly modified version of the technique described by Cooper et al. (8). The model consisted of a two-compartment system separated by a polycarbonate filter permeable to macromolecules on which a monolayer of cells was previously seeded. The lower chamber contained a solution (25 ml) of 0.5% albumin diluted in medium 199. The upper chamber held an identical (700-µl) solution with the addition of a trace amount of 125I-albumin. To measure the clearance rate of 125I-albumin, aliquots of 75 µl were taken from the lower chamber every 2 min. The radioactive content of these aliquots was evaluated with a gamma -counter (Wallac, Turku, Finland). Thirty minutes after the beginning of the sampling, the pharmacological agents were added in the upper chamber. Aliquots were then taken for an additional 30 min.

Data analysis. The permeability coefficient (PC) was determined according to Casnocha et al. (6) by use of the formula derived from Fick's law of diffusion, where A represents the area of membrane, Vl is the volume of luminal chamber, t is sample time, Vs is albumin concentration in the subluminal chamber, Cs(t) is the albumin concentration at each sample time and TP(t) is the total mass of protein (albumin) at each sample time
ln<FENCE><FR><NU><IT>1</IT></NU><DE><IT>1−</IT>(V<SUB>s</SUB><IT>+</IT>V<SUB>l</SUB>)Cs(<IT>t</IT>)/TP(<IT>t</IT>)</DE></FR></FENCE><IT>=</IT>PC<FENCE><IT>A </IT><FR><NU>(V<SUB>s</SUB><IT>+</IT>V<SUB>l</SUB>)</NU><DE>V<SUB>l</SUB>/V<SUB>s</SUB></DE></FR></FENCE>
Results are expressed as the percent difference between the clearance rates before and after pharmacological treatments. Significance of clearance changes between groups was calculated by the nonparametric Wilcoxon's ranked sum test.

Experimental protocol: prostaglandin G/H inhibitors. Cells were preincubated with either indomethacin (2.88 µM) or ibuprofen (10 µM) 60 min before the first sampling of lower-chamber medium. The cells were washed 30 min before the beginning of the experiment, and the medium was replaced with fresh medium containing the inhibitors and the radioactive tracer. At the same moment, captopril (10 µM) was added into the Boyden chambers, whereas THR and BK were added 30 min after the beginning of the sampling period (Fig. 1).


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Fig. 1.   Schematic representation of the protocol used for the experiments with prostaglandin G/H inhibitors. COX, cyclooxygenase; capto., captopril; 125I-albu., 125I-labeled albumin; BK, bradykinin; start, starting point of the sampling period.

Chemicals and drugs. Captopril, purchased from Sigma, was dissolved in water to a concentration of 1 mg/ml as a stock solution. Indomethacin (Sigma) was initially reconstituted in a Tris base solution (0.2 M; Boehringer Mannheim, Laval, Canada) and then further diluted with medium 199 to obtain a final concentration of 1 mg/ml (2.88 µM). Ibuprofen (Sigma) was solubilized with ethanol to a concentration of 1 mg/ml and then further diluted with medium 199 to obtain a final concentration of 10 µM. Iodogen (Sigma) was dissolved in dichloroethylene to a concentration of 40 µg/ml. The following compounds were purchased from Sigma and solubilized in water: BK, THR, collagenase type V, thymidine (diluted in medium 199) and salbutamol. L-Glutamine, fetal bovine serum, penicillin, streptomycin, and medium 199 were purchased from GIBCO BRL, (Grand Island, NY) and stored at -20°C. Sephadex G-25 was purchased from Pharmacia (Uppsala, Sweden). Iloprost, received as a generous gift from Schering (Berlin, Germany), was obtained as an aqueous solution and diluted in medium 199 containing 0.5% albumin solution on the day of the experiment. Prostaglandin E2, generously supplied by Upjohn (Kalamazoo, MI), was initially diluted as a 1 M stock solution in absolute ethanol and was further diluted to appropriate concentrations. The EP4 antagonist AH-23848 (Calbiochem, La Jolla, CA) was diluted in a 1% bicarbonate solution to a final concentration of 30 µM. A stock solution of adenylate cyclase inhibitor, MDL-12330A hydrochloride (Calbiochem), was reconstituted in DMSO (1 mg/ml); further dilutions were done in the culture medium.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of BK and inhibitors of prostaglandin G/H synthase (cyclooxygenase). The effect of BK on the permeability of a monolayer of BAEC to albumin is shown in Fig. 2. Results are expressed as the percent difference between the monolayer permeability coefficient after the pharmacological treatment relative to its baseline permeability coefficient. The basal permeability coefficient varied from 7.0 × 10-6 to 1.3 × 10-5cm/s. Medium 199 produced a nonsignificant decrease in the permeability (3.0 ± 5%), and THR (0.1 U/ml) increased the permeability by 8.7 × 10-6 ± 4 × 10-7 cm/s to 1.5 × 10-5 ± 3.2 × 10-6 cm/s (an increase of 84%). BK alone elevated the endothelial permeability by 23 ± 7 and 49 ± 9% (P < 0.05) at the concentrations of 10-9 and 10-8 M, respectively, but there was no further increase at 10-7 M (35 ± 13%). Pretreatment of the BAEC monolayer with the (COX) inhibitor indomethacin (2.88 µM) potentiated the effect of BK (10 nM) from 49 ± 9 to 86 ± 12% and that of BK (100 nM) from 34 ± 13 to 62 ± 4%. To further confirm the implication of PGs in this process, we also studied the effect of ibuprofen, another COX inhibitor with a chemical structure unrelated to indomethacin. Ibuprofen (10 µM) also potentiated the effect of BK (10 nM) on endothelial permeability from 43 ± 9 to 101 ± 35% and that of BK (100 nM) from 33 ± 9 to 133 ± 31%.


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Fig. 2.   Effect of prostaglandin G/H synthase inhibitors on bradykinin (BK)-induced permeability of bovine aortic endothelial cells (BAEC) to 125I-albumin. Open bar, effect of vehicle (V); filled bar, effect of thrombin [THR (T); 0.1 U/ml]; gray bars, effect of BK alone; hatched bars, BK in the presence of indomethacin (2.88 µM/ml); cross-hatched bars, effect of BK in the presence of ibuprofen (10 µM). Results are means ± SE expressed in percent increase of monolayer permeability coefficient in relation to its basal value; n = 5-10. Experiments were done in the presence of 10 µM captopril. Significant difference from vehicle: * P < 0.05; ** P < 0.01. Significant difference from BK: dagger  P < 0.05; dagger dagger  P < 0.01.

Effects of iloprost, butaprost, and PGE2. Prostacyclin (PGI2) and, to a lesser extent, PGE2 are two prostanoids synthesized by the BAEC. Therefore, a series of experiments was undertaken to further explore the effect of exogenous PGs in our experimental system. Iloprost, a PGI2 mimetic, or PGE2 had no significant effect on basal endothelial permeability (data not shown), although a tendency to decrease the permeability was observed. However, when the monolayer was stimulated with BK, either PG reduced the endothelial permeability in a concentration-dependent manner (Figs. 3 and 4). Whereas concentrations of 10-10 and 10-9 M iloprost did not significantly modulate the activity of BK (Fig. 3), higher concentrations of iloprost attenuated the increase in permeability (25 ± 9, 5 ± 9, and -14 ± 14% at the respective concentrations of 10-8, 10-7, and 10-6 M vs. 47 ± 12% for BK alone). The EC50 of iloprost was approximated to 11 nM. THR (0.1 U/ml) increased BAEC permeability by 42 ± 10%, thus confirming the biological responsiveness of the BAEC monolayer. PGE2 was more potent than iloprost in inhibiting BK-induced permeability, with an EC50 value estimated at 0.6 nM (Fig. 4). A concentration of 10-9 M was sufficient to abolish the pharmacological effect of BK (-5 ± 1% vs. 53 ± 13% for BK only). Interestingly, higher concentrations of PGE2 had a slightly reduced inhibitory property (4 ± 7, 7 ± 8, and 11 ± 9% at the respective concentrations of 10-8, 10-7, and 10-6 M). The EC50 of PGE2 on the BK-induced permeability was estimated at 0.6 nM. Permeability-reducing agents are often associated with their ability to generate cAMP. A least two receptor subtypes are known to be coupled with the Gs protein: the EP2 and the EP4 receptor types. Therefore, we tested the biological effect of an EP2 agonist and an EP4-selective antagonist. Butaprost, an EP2-selective agonist, also inhibited in a concentration-dependent manner the effect of BK on albumin permeability (Fig. 5). At the concentrations of 10-10, 10-9, and 10-8 M, butaprost did not significantly modify the effect of BK, but at a higher concentration (10-7 M), this agonist significantly reduced the effect of BK (21 ± 6, P < 0.05). BK alone increased the permeability by 46 ± 6%. The EC50 of butaprost was approximated to 100 nM.


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Fig. 3.   Effect of iloprost on BK-induced permeability of BAEC cells to [125I]albumin. Open bar, effects of vehicle (V); black bar, THR (T; 0.1 U/ml); gray bar, BK (10-8 M); hatched bars, BK (10-8 M) in the presence of iloprost. Results are means ± SE expressed in percent increase of the monolayer permeability coefficient in relation to its basal value; n = 7-16. Experiments were done with 10 µM captopril. Significant difference from vehicle: * P < 0.05; ** P <=  0.01. Significance difference from BK: dagger  P < 0.05; dagger dagger  P < 0.01.



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Fig. 4.   Effect of PGE2 on BK-induced permeability of BAEC cells to 125I-albumin. Open bar, effects of vehicle; black bar, THR (T; 0.1 U/ml); gray bar, BK (10-8 M); hatched bars, BK (10-8 M) in the presence of iloprost. Results are means ± SE expressed in percent increase of the monolayer permeability coefficient in relation to its basal value; n = 7-16. Experiements were done with 10 µM captopril. Significant difference from vehicle: * P < 0.05; ** P < 0.01. Significant difference from BK: dagger  P < 0.05; dagger dagger  P < 0.01.



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Fig. 5.   Effect of butaprost on BK-induced permeability of BAEC cells to 125I-albumin. Open bar, effects of vehicle; black bar, THR (T; 0.1 U/ml); gray bar, BK (10-8 M); hatched bar, BK (10-8 M) in the presence of iloprost. Results are means ± SE expressed in percent increase of the monolayer permeability coefficient in relation to its basal value; n = 7-16. Experiments were done with 10 µM captopril. Significant difference from vehicle: * P < 0.05; ** P <=  0.01. Significant difference from BK, dagger  P < 0.05.

As shown in Fig. 6, the EP4 antagonist AH-23848 (30 µM) had a minimal effect on the inhibitory activity of PGE2. After preincubation of the cells with 30 µM AH-23848, the BAEC permeability to albumin increased from 8 ± 6 to 18 ± 4% when PGE2 (10-9 M) and BK (10-8 M) were coadministered and from 3 ± 6 to 7 ± 4% when PGE2 (10-8 M) was administered with BK (10-8 M). No statistical difference was observed between the two sets of experiments (n = 6-7).


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Fig. 6.   Effect of the E-prostanoid (EP4) receptor-selective antagonist AH-23848 on the protective effect of PGE2 on BK (10-8 M)-induced BAEC permeability to 125I-albumin. open circle , Effect of PGE2 on BK-induced permeability in absence of the antagonist; , effect of PGE2 in the presence of the EP4 antagonist. Results are means ± SE expressed in percent increase of the monolayer permeability coefficient in relation to its basal value; n = 7-10. Experiments were done in the presence of 10 µM captopril.

Effects of cAMP modulatory agents on endothelial cell permeability. To better understand the inhibitory mechanism of PGs on BK-induced BAEC permeability, the effect of salbutamol, an agonist known to increase intracellular cAMP, was studied for comparative purposes. This beta 2-adrenergic agonist inhibited (data not shown) the rise in endothelial permeability induced by BK (10-8 M) in a concentration-dependent manner. In the presence of 10-10 M salbutamol, BK (10-8 M) increased endothelial permeability by 42.3 ± 7%. When the concentrations of salbutamol were augmented to 10-9, 10-8, 10-7, and 10-6 M, changes in permeability of 18 ± 8, -22 ± 11, -15 ± 12, and -50 ± 10% were observed.

In the last set of experiments, we verified whether or not the inhibitory effect of PGE2 on BK-induced increases of endothelial permeability was related to its capacity to activate the adenylate cyclase enzyme (Fig. 7). Results showed that the adenylate cyclase inhibitor MDL-12330A (10 µM) reduced the inhibitory activity of PGE2 on BK-induced permeability to albumin. At the concentration of 10-8 M, BK increased the cell permeability by 56 ± 12%, but this was reduced to 13 ± 85% by the addition of PGE2 (10 nM). However, when the cells were preincubated with the adenylate cyclase inhibitor, the combined effect of BK and PGE2 was restored to 63 ± 7%. Furthermore, compound MDL-12330A also potentiated the effect of BK from 56 ± 12 to 109 ± 23% but did not modify the activity of either control or THR. Interestingly, the increase in permeability induced by MDL-12330A was very similar to the effect produced by the two COX inhibitors (see Fig. 2). The adenylate cyclase inhibitor was less efficient in potentiating the response of the cells to THR. Finally, compound MDL-12330A did not significantly change the effect of the vehicle on albumin permeability (63 ± 13 vs. 78 ± 19% in the presence of the compound MDL-12330A).


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Fig. 7.   Effect of adenylate cyclase inhibitor MDL-12330A (10 µM) on endothelial permeability to 125I-albumin. Effect of different agonist in the absence (open bars) and in the presence (filled bars) of the inhibitor. Results are means ± SE expressed in percent increase of the monolayer permeability coefficient in relation to its basal value; n = 6-13. Experiments were done in the presence of 10 µM captopril.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The autoregulatory role of PGs on vascular permeability was studied. Our results show that BK significantly increases the permeability of a BAEC monolayer to albumin and confirm previous reports by Schaeffer et al. (30), who described that BK permeabilized a monolayer of bovine pulmonary artery endothelial cells. These in vitro results are in accord with many in vivo observations on the effects of kinins on plasma extravasation (11, 28). Furthermore, Northover (27a) measured the morphology variations of cultured endothelial cells of the guinea pig inferior vena cava after exposure to BK. It was observed that the cell contractions were dependent on the activation of the kinin B2-receptor subtype.

The contribution of PGs to the BK effect was investigated using inhibitors of prostaglandin G/H synthase. These compounds potentiated the effect of BK, thus suggesting that PGs behave as negative-feedback autoregulatory agents. This conclusion is based on the observations that indomethacin or ibuprofen potentiated the BK effect. Moreover, exogenous PGs inhibited the BK-induced increase in endothelial permeability. It appeared that it would be helpful to characterize the type of PG receptor that is involved. It must be remembered that PGs of the E series exert their effects through four receptor subtypes called EP1, EP2, EP3, and EP4. Considering that PGE2 increased intracellular levels of cAMP in BAEC, we therefore verified the implication of both EP2 and EP4 receptor subtypes because other subtypes do not use this signaling pathway. To discriminate between the two subtypes, an EP2-selective agent, butaprost, was used and was found to inhibit the BK-induced increase of permeability; the EP4-selective antagonist (AH-23848) was inactive. The EP2-selective agonist butaprost inhibited BK-induced permeability but was 166 times less potent than PGE2. This finding of lower activity of butaprost is in agreement with many reports (4, 7, 9). These preliminary results suggested the implication of the EP2 receptor in the control of permeability of BAEC, but the participation of the EP4 receptor could not be completely excluded. Endogenous PGs or rapid receptor desensitization may impair the detection of this receptor in our experimental model. Our results could also suggest the presence of a mixed PG receptor population. Indeed, high concentrations of PGE2 were less efficient than lower concentrations in reducing the BK-induced permeability. Furthermore, the high sensitivity of the cells to iloprost may suggest the presence of a prostacyclin (IP) receptor. Further studies are needed to characterize the various PG receptors present on BAEC. This protective effect of PGs does not seem to be specific to BK. Casnocha et al. (6) reported that pretreatment of a human umbilical vein endothelial cell monolayer with iloprost reduced the permeabilizing activity of THR. Using a similar experimental model, we have observed that PGE2 inhibited platelet-activating factor-induced increases in permeability of BAEC to Evans blue-labeled albumin (unpublished results). Very few studies have characterized PG receptors located on the endothelium. Jumblatt (20) has shown that the inhibition of rabbit corneal endothelial cell mitosis by PGE2 was mediated by an EP2 receptor subtype. Binding sites for iloprost were also reported on human vascular umbilical cord endothelial cells (17). This study is in agreement with that by Komhoff et al. (21), who reported the presence of IP receptor in human kidney endothelial cells. Bhattacharya et al. (5) presented data showing the presence of the EP3 and EP4 receptor subtypes on the nuclear envelope and plasma membrane of porcine cerebral microvascular endothelial cells. In their study, the stimulation of nuclear EP4 receptors increased the transcription of the inducible NO synthase gene. However, the role of nuclear prostaglandin receptors regarding microvascular permeability is unknown.

The present findings suggest that cAMP is probably the second messenger of the inhibitory activity of PGE2. This conclusion is based on the observation that the beta 2-adrenoceptor agonist salbutamol, an agent known to increase intracellular cAMP, reproduced the inhibitory effect of the prostanoids on BK-induced permeability. Furthermore, the adenylate cyclase inhibitor MDL-12330A inhibited the activity of PGE2. It is noteworthy that the adenylate cyclase inhibitor potentiated the BK-induced permeability to a level similar to that obtained with indomethacin. However, the MDL-12330A compound did not potentiate THR-induced permeability. This may be explained by the relatively poor capability of THR to induce PG synthesis in BAEC (3). This observation may be explained, in part, by the fact that THR more potently inhibits adenylate cyclase than does BK. Indeed, if the THR receptor coupling efficiency for protein Gi is much higher than the coupling efficiency for protein Gq, as the work of Mattera et al. (23) suggests, the administration of the MDL-12330A compound would be unnoticed because the adenylate cyclase is already inhibited. But the net effect on the permeability would be the same because the increase in Ca2+ (Gq) or decrease in cAMP (Gi) are both linked to an increase in endothelial permeability (23).

The protective effect of cAMP on the increase in endothelial permeability has been well described in the literature. Isoproterenol was shown to reduce THR-induced pulmonary endothelial permeability in vivo (24), a finding that is in line with results obtained with salbutamol. Langeler et al. (22) reported that forskolin reduced the transendothelial transport of low-density lipoproteins in an in vitro model that makes use of endothelial cells isolated from human carotid artery. In vivo modulation of cAMP was also associated with a reduction of plasma extravasation. Isoproterenol, salbutamol, forskolin, and theophylline inhibited the increased vascular permeability caused by allergic mediators such as histamine, serotonin, and leukotriene C4 in rats (18). The inhibition of vascular permeability may be explained through a PKA-dependent phosphorylation that inactivates the regulatory MLCK (26, 31). Inhibition of the MLCK enzyme prevents intercellular gap formation and thus may reduce albumin leakage. Furthermore, Adamson et al. (1) observed that the elevation of cAMP leads in the increase in number and stability of tight junctions between endothelial cells of the frog mesentery capillary systems. All of the aforementioned findings point to an inhibitory role of cAMP in the endothelial increase in permeability.

In summary, we have found that the inhibition of PG synthesis in endothelial cells resulted in the potentiation of the permeabilizing activity of BK. Our results suggest that PGs released by the endothelium possibly interact with EP2 and IP receptors, resulting in the increase of intracellular cAMP and, consequently, a reduction of transendothelium albumin transport. Our findings may provide an explanation for the protective effect of prostaglandins of the E series and PGI2 observed in some in vivo studies (11, 16, 19). The proinflammatory activities of PGs, including their effects on vascular permeability, could also be attributed to activation of receptors on other sites of action, such as vascular smooth muscle cells or sensory nerve terminals, that could modulate either directly or indirectly the vascular events (33, 37). This interpretation may help us understand the different results obtained in various in vivo studies.


    ACKNOWLEDGEMENTS

We acknowledge the Medical Research Council of Canada, the Fonds pour la Formation de Chercheur et l'Aide à la Recherche/Fonds de la Recherche en Santé du Québec, and the Canadian Heart and Stroke foundation for financial support. We equally acknowledge Upjohn for the generous gift of PGE2 and Schering for the kind gift of iloprost, Drs. Takashi Kawaguchi and A. B. Malik for their advice, and, finally, Charlène Bélanger for technical assistance.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Sirois, Institut de Pharmacologie de Sherbrooke, Medical School, Univ. of Sherbrooke, Sherbrooke, PQ, Canada J1H 5N4 (E-mail: p.sirois{at}courrier.usherb.ca).

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.

Received 22 June 2000; accepted in final form 14 November 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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