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(Circulation Research. 1995;76:199-208.)
© 1995 American Heart Association, Inc.

Articles

cGMP and Nitric Oxide Modulate Thrombin-Induced Endothelial Permeability

Regulation via Different Pathways in Human Aortic and Umbilical Vein Endothelial Cells

Richard Draijer, Douwe E. Atsma, Arnoud van der Laarse, Victor W.M. van Hinsbergh

From the Gaubius Laboratory TNO-PG (R.D., V.W.M. van H.) and the Department of Cardiology, University Hospital (D.E.A., A. van der L.), Leiden, Netherlands.

Correspondence to Dr V.W.M. van Hinsbergh, Gaubius Laboratory TNO-PG, PO Box 430, 2300 AK Leiden, Netherlands.

	Abstract

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Abstract Previous studies have demonstrated that cGMP and cAMP reduce the endothelial permeability for fluids and macromolecules when the endothelial permeability is increased by thrombin. In this study, we have investigated the mechanism by which cGMP improves the endothelial barrier function and examined whether nitric oxide (NO) can serve as an endogenous modulator of endothelial barrier function. Thrombin increased the passage of macromolecules through human umbilical vein and human aortic endothelial cell monolayers and concomitantly increased [Ca]²⁺ in vitro. Inhibition of these increases by the intracellular Ca²⁺ chelator BAPTA indicated that cytoplasmic Ca²⁺ elevation contributes to the thrombin-induced increase in endothelial permeability. The cGMP-dependent protein kinase activators 8-bromo-cGMP (8-Br-cGMP) and 8-(4-chlorophenylthio)cGMP (8-PCPT-cGMP) decreased the thrombin-induced passage of macromolecules. Two pathways accounted for this observation. Activation of cGMP-dependent protein kinase by 8-PCPT-cGMP decreased the accumulation of cytoplasmic Ca²⁺ in aortic endothelial cells and hence reduced the thrombin-induced increase in permeability. On the other hand, in umbilical vein endothelial cells, cGMP-inhibited phosphodiesterase (PDE III) activity was mainly responsible for the cGMP-dependent reduction of endothelial permeability. The PDE III inhibitors Indolidan (LY195115) and SKF94120 decreased the thrombin-induced increase in permeability by 50% in these cells. Thrombin treatment increased cGMP formation in the majority of, but not all, cell cultures. Inhibition of NO production by N^G-nitro-L-arginine methyl ester (L-NAME) enhanced the thrombin-induced increase in permeability, which was restricted to those cell cultures that displayed an increased cGMP formation after addition of thrombin. Simultaneous elevation of the endothelial cGMP concentration by atrial natriuretic factor, sodium nitroprusside, or 8-Br-cGMP prevented the additional increase in permeability induced by L-NAME. These data indicate that cGMP reduces thrombin-induced endothelial permeability by inhibition of the thrombin-induced Ca²⁺ accumulation and/or by inhibition of cAMP degradation by PDE III. The relative contribution of these mechanisms differs in aortic and umbilical vein endothelial cells. NO can act in vitro as an endogenous permeability-counteracting agent by raising cGMP in endothelial cells of large vessels.

Key Words: permeability • human endothelial cells • cGMP-dependent protein kinase • cytoplasmic Ca²⁺ • nitric oxide

	Introduction

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The endothelium, the inner lining of blood vessels, regulates the extravasation of fluid and macromolecules. Impairment of the barrier function of the endothelium results in vascular leakage and edema. This can occur by exposure to toxic agents, after stimulation of the endothelium by vasoactive substances, or during inflammation, in particular in postcapillary venules. It is generally believed that the increase in endothelial permeability induced by vasoactive substances is caused by contraction of endothelial cells.^{1 2 3 4 5 6 7} Endothelial contraction involves interaction of actin and nonmuscle myosin, which is activated by a Ca²⁺/calmodulin- and ATP-dependent phosphorylation of the myosin light chain (MLC) by MLC kinase.^{5 8 9} The barrier function of endothelial cells is improved both in vivo and in vitro by agents that increase the intracellular cAMP concentration.^{10 11 12 13 14 15} An increase in cellular cAMP was found to be accompanied by a reduced degree of phosphorylation of MLC in cultured endothelial cells.⁹ Several in vitro studies have shown that elevation of the cGMP concentration also reduces endothelial permeability in large-vessel endothelial cells.^{16 17 18} The modulating effect of cGMP is most prominent when the endothelial permeability has been increased, for instance, by thrombin or oxidants, whereas it is minor or absent under basal conditions.^{16 18 19} In perfused rat lungs, stimulation of cGMP production by atrial natriuretic factor also reduced oxidant-induced vascular leakage.²⁰ However, the mechanism by which cGMP reduces oxidant- and thrombin-enhanced permeability is not known.

The process of endothelial cell contraction resembles the regulation of actin-myosin interaction in smooth muscle cells and platelets. The effects of cGMP on smooth muscle relaxation are thought to be mediated via cGMP-dependent protein kinase, which affects the intracellular Ca²⁺ metabolism.^{21 22 23} In smooth muscle and several other cell types, cGMP also contributes indirectly by inhibiting phosphodiesterase (PDE) type III, which results in a decreased breakdown of cAMP.^{24 25} In the present study, we have investigated, in human umbilical vein and aortic endothelial cells, whether cGMP regulates endothelial permeability by affecting the regulation of the cytoplasmic Ca²⁺ accumulation or by inhibiting PDE III activity.

Stimulation of the influx of Ca²⁺ in endothelial cells not only causes endothelial cell contraction but also results in the release of several endothelial products, including prostacyclin and nitric oxide (NO). Production of NO is due to the Ca²⁺/calmodulin-dependent activation of the constitutive NO synthase, which is predominantly present in muscular vessel endothelial cells.^{26 27} The production of NO not only reduces the contraction of smooth muscle cells and counteracts platelet activation, but it also stimulates guanylate cyclase in the endothelial cell itself. Because the cGMP thereby generated may counteract the stimulus-induced increase in permeability, we wondered whether the production of NO attenuates the contraction of endothelial cells. Our data point to a possible counterregulatory role of NO on the regulation of endothelial permeability.

	Materials and Methods

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Medium 199 supplemented with 20 mmol/L HEPES was obtained from Flow Laboratories; tissue culture plastics, from Corning or Costar; and Transwells (diameter, 0.65 cm; pore size, 3 µm), from Costar. A crude preparation of endothelial cell growth factor was prepared from bovine brain as described by Maciag et al.²⁸ Human serum was obtained from the local blood bank and was prepared from fresh blood taken from healthy donors; the sera were pooled and stored at 4°C. Newborn calf serum was obtained from GIBCO and heat-inactivated before use (30 minutes, 56°C). Pyrogen-free human serum albumin was purchased from the Central Laboratory of Blood Transfusion Service. Horseradish peroxidase EC 1.11.1.7 type I (HRP), sodium nitroprusside (SNP), 8-bromo-cGMP (8-Br-cGMP), N^G-nitro-L-arginine methyl ester (L-NAME), and fluorescein isothiocyanate (FITC)-dextrans with molecular masses of 35 600, 38 900, and 487 000 D were obtained from Sigma Chemical Co; bovine -thrombin, from LEO Pharmaceutical Products; forskolin, from Hoechst; isobutyl methylxanthine (IBMX), from Janssen Chimica; SKF96365, from Biomol Research Laboratories; BAPTA-AM and fura 2-AM, from Molecular Probes; 8-(4-chlorophenylthio)cGMP (8-PCPT-cGMP), from Biolog Life Science Institute; ionomycin, from Calbiochem Corp; [¹⁴C]sucrose, from Dupont NEN; and human atrial natriuretic factor-(99-128), from Bissendorf Peptide GmbH. SKF94120 was a gift from Smith Kline & French Laboratories Ltd; Rolipram (ZK62711), a gift from Schering Aktiengesellschaft; and Indolidan (LY195115), a gift from Lilly Research Laboratories.

Isolation and Culture of Endothelial Cells
Human umbilical vein endothelial cells were isolated by the method of Jaffe et al²⁹ and characterized as described previously.³⁰ Isolation and characterization of human endothelial cells from the pulmonary artery and aorta were performed as described previously.³¹ The blood vessels of human origin were obtained according to the guidelines of the institutional review board of University Hospital Leiden. Cells were cultured on fibronectin-coated dishes in medium 199 supplemented with 10% human serum, 10% newborn calf serum, 150 µg/mL crude endothelial cell growth factor, 5 U/mL heparin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Cells were kept at 37°C under 5% CO₂/95% air. For the evaluation of the barrier function, confluent monolayers of endothelial cells from umbilical vein (primary), pulmonary artery (first, second, or third passage), or aorta (fourth or fifth passage) were released with trypsin-EDTA and seeded in high density on fibronectin-coated polycarbonate filters of the Transwell system and cultured as described by Langeler and colleagues.^{32 33} Medium was renewed every other day.

Evaluation of the Barrier Function
Endothelial cells cultured on filters were used between 4 and 6 days after seeding. Exchange of macromolecules through the endothelial monolayers was investigated by assay of the transfer of HRP and FITC-dextran. Passage of HRP through human endothelial cell monolayers was performed as described previously.³² Briefly, endothelial cell monolayers were cultured on porous membranes (0.33 cm²; pore size, 3 µm) to form a tight monolayer. Before the start of the experiment, cells were incubated for 1 hour in medium 199 with 1% albumin. In pretreatment, the cells were incubated for 15 minutes with 8-Br-cGMP (1 to 1000 µmol/L), 8-PCPT-cGMP (1 to 1000 µmol/L), SNP (0.1 mmol/L), atrial natriuretic factor (10^-7 mol/L), SKF94120 (100 µmol/L), Indolidan (100 µmol/L), or Rolipram (100 µmol/L) in the upper and lower compartment. BAPTA-AM (10 µmol/L) and L-NAME (100 µmol/L) were preincubated for 1 hour to achieve sufficient loading. At the start of the experiment, 5 µg/mL HRP in medium 199 with 1% albumin was added to the upper compartment of the Transwell system in the presence or absence of thrombin (1 U/mL). Samples were taken from the lower compartment (at the other side of the endothelial monolayer) at various time intervals, and an equal volume of medium 199 containing 1% albumin was readded to this lower compartment. Cells were kept at 37°C under 5% CO₂/95% air. All passage experiments were performed in triplicate. The concentration of HRP was derived from the HRP activity in each sample with peroxide and tetramethylbenzidine as substrate and expressed as nanograms passed per square centimeter in a certain time interval. The permeability coefficient (PC) was derived from Fick's law of diffusion and was determined by the following:

where UC is the upper compartment and LC is the lower compartment. The mass flux of HRP is expressed in nanograms per square centimeter per hour. Because the initial passage of molecules proceeds linearly in time, the mass flux of peroxidase was calculated from the initial hour of passage, and the mean concentrations of the upper and lower compartments during this period were used to calculate the concentration difference. PC was corrected for the contribution of the filter membrane (<0.5%):

where PC_EC represents the PC of the endothelial cell monolayer; PC_F, the PC of the empty filter; and PC_EC-F, the PC determined for the filter and the endothelial cells together. The PC_F was determined at 37°C under identical conditions with separate fibronectin-coated filters that had been preincubated in culture medium for 24 hours. The passage of FITC-dextran (input upper compartment, 1 mg/mL) was determined similarly with the use of an inverted fluorescence microscope equipped with a photometer and a scanning stage and operated by a microprocessor.³⁴

Extraction and Assays of Cyclic Nucleotides
Cultured human endothelial cells were grown to confluence in 5-cm² wells. Medium of the monolayers was renewed with medium 199 supplemented with 1% albumin, with or without 100 µmol/L L-NAME, 1 hour before the incubation period. Cells were preincubated for 15 minutes with IBMX (1 mmol/L) to prevent degradation of cyclic nucleotides by PDEs. At the start of the experiment, thrombin was added to the medium and incubated for 15 minutes. Immediately on removal of the medium, 3.5% perchloric acid (0.5 mL) and a small known amount of [³H]cGMP or [³H]cAMP were added to each well for the determination of the intracellular cyclic nucleotide concentration. The cell lysates were transferred to Eppendorf reaction tubes and neutralized by using potassium hydrogen carbonate (50% saturated). After centrifugation, the supernatants were collected and dried under a stream of nitrogen gas. The concentration of the intracellular cyclic nucleotides was determined by radioimmunoassays (Amersham), according to Steiner et al,³⁵ and corrected for the recoveries in the various samples.

Measurement of [Ca²⁺]_i
Endothelial cells were cultured on 5-cm² glass coverslips and loaded with fura 2 by incubation with 2 µmol/L fura 2-AM for 45 to 60 minutes at 37°C in medium 199 supplemented with 1% human serum albumin. Then, the cells were washed three times with Tyrode's buffer. The coverslips were mounted in a Teflon two-compartment incubation dish, incubated in 1 mL Tyrode's buffer, and placed in a temperature-controlled microincubator.^{36 37} The two-compartment dish allows the exposure of the two halves of the same culture to different treatment. In this way, the effect of thrombin on [Ca²⁺]_i in one half can be compared with the effect of thrombin, in the presence of 8-PCPT-cGMP, 8-Br-cGMP, BAPTA, or SKF96365 in the other half of the same culture. Fura 2 fluorescence was measured with an imaging dual-wavelength fluorescence microscope, which consisted of an inverted microscope body (Leitz Diavert) equipped with a x20 fluorite objective (Nikon) and a mercury light source (HBO-100, Osram). A filterwheel (Sutter) allowed the selection of excitation filters of 340 and 380 nm. Emission fluorescence was led through a 490-nm high-pass filter and imaged by a high-sensitivity SIT camera (Hamamatsu C2400-08). The resulting video signal was digitized by a frame-grabber board (PCVISIONplus, Imaging Technologies) in a PC-AT 486 computer. Spatial resolution of the images was 256x256 pixels, with an eight-bit intensity resolution. Every 3.6 seconds, a pair of images at 340- and 380-nm excitation wavelength was made. Off-line, background fluorescence was subtracted, and the 340-nm image was divided by the 380-nm image on a pixel-by-pixel basis, yielding a ratio image. Statistical analysis was performed by using dedicated image processing software (TIM, Difa). The mean [Ca²⁺]_i was determined from a field of 50 cells and was calculated by the following equation (in nanomoles per liter):

where R represents the ratio of the fluorescence values at 340 and 380 nm; R_max and R_min are the maximal and minimal ratio values, respectively, being determined after each experiment by addition of 1 µmol/L ionomycin and 10 mmol/L EGTA, respectively; and ß represents the ratio of the fluorescence at 380 nm of free fura 2 and fura 2 completely saturated with Ca²⁺ (3.6). K_d, the dissociation constant of the fura 2–Ca²⁺ complex, was assumed to be 224 nmol/L at 37°C, according to Grynkiewicz et al.³⁸

Statistical Analysis
Data are presented as mean±SEM. Statistical analysis as indicated in the text was performed with the Mann-Whitney and Wilcoxon rank sum tests. Statistical significance was assumed at P<.05.

	Results

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Elevation of [Ca²⁺]_i During Thrombin-Induced Increase in Endothelial Permeability
FITC-dextrans and HRP, which has a Stokes radius similar to that of albumin, were used as marker molecules to assay the permeability of human endothelial cell monolayers for macromolecules. On addition of 1 U/mL thrombin, the permeability of human umbilical vein endothelial cell monolayers for [¹⁴C]sucrose (360 D), FITC-dextran (38 900 D), HRP, and FITC-dextran (487 000 D) increased 2-, 5-, 7-, and 15-fold, respectively (average values of 10 different cultures, not shown). The increase in permeability was detectable rapidly after the addition of thrombin (Fig 1A) and lasted for at least 1 hour. It was accompanied by an immediate decrease in the transendothelial electrical resistance (40% to 60% reduction, not shown). Thrombin also rapidly enhanced the passage rate of HRP 5-fold through monolayers of human aortic endothelial cells (Fig 1D).

View larger version (21K): [in this window] [in a new window]   Figure 1. Graphs showing effects of thrombin on the [Ca2+]i and permeability of human umbilical vein (A through C) and human aortic (D through F) endothelial cell monolayers. A and D, Early time courses show the passage of horseradish peroxidase (HRP) under basal conditions () and after stimulation with 1 U/mL thrombin (), which is added at t=0 (mean±SEM of six determinations). The thrombin-induced permeability is significantly different from the basal permeability after 3 minutes (P<.05). Passage of HRP was determined as described in "Materials and Methods." B and E, The increase in cytoplasmic calcium ion concentration after addition, indicated by an arrow, of 1 U/mL thrombin (), was prevented by addition of the intracellular Ca2+ chelator BAPTA-AM (10 µmol/L, ). In the presence of the Ca2+ entry blocker SKF96365 (100 µmol/L, ) [Ca2+]i was markedly reduced. Each graph represents the mean of three representative recordings with different batches of umbilical vein endothelial cells and one representative recording with aortic endothelial cells. Similar results were obtained with 25 µmol/L EGTA in Ca2+-free buffer instead of SKF96365 (not shown). C and F, Time courses show the passage of HRP in hours under basal conditions () and after stimulation with 1 U/mL thrombin (). The thrombin-induced passage of HRP through endothelial cell monolayers was partly prevented in monolayers that were preincubated for 1 hour with BAPTA-AM (1 µmol/L, ; 10 µmol/L, ) (mean±SEM of triplicate cultures).

Thrombin induced an immediate rise in [Ca²⁺]_i in both endothelial cell types (Fig 1B and 1E). This increase was abolished by the intracellular Ca²⁺ chelator BAPTA. The elevation of the [Ca²⁺]_i was caused by a rapid release of Ca²⁺ from intracellular stores and an influx of extracellular Ca²⁺ (Fig 1B), since [Ca²⁺]_i accumulation was reduced by the Ca²⁺ entry blocker SKF96365³⁹ and by incubation in Ca²⁺-free medium supplemented with EGTA (not shown). Further evidence that elevation of the [Ca²⁺]_i is also important for the prolonged thrombin-mediated increase in endothelial permeability was obtained by using the intracellular Ca²⁺ chelator BAPTA. BAPTA reduced the thrombin-mediated increase in permeability in a concentration-dependent manner (Fig 1C and 1F). In the presence of 10 µmol/L BAPTA, the thrombin-induced increase in permeability was reduced to 50±9% in umbilical vein endothelial cells (five independent cultures, P<.05) and to 53±20% in aortic endothelial cells (three independent experiments with cells from two different donors).

cGMP Induces a Simultaneous Reduction of Thrombin-Enhanced Permeability and Rise of [Ca²⁺]_i in Aortic Endothelial Cells
The thrombin-enhanced permeability was reduced in human umbilical vein and aortic endothelial cell monolayers by the cell membrane–permeant cGMP analogue 8-Br-cGMP (Table 1). Under basal conditions, 8-Br-cGMP was less or not effective on endothelial permeability. When another cGMP-analogue, 8-PCPT-cGMP, was used, the thrombin-increased permeability was reduced in aortic endothelial cell monolayers to 50±3% and 33±8% in the presence of 0.1 and 1 mmol/L 8-PCPT-cGMP, respectively (four experiments) but was not affected in umbilical endothelial cell monolayers (with 1 mmol/L 8-PCPT-cGMP, 101±14% of thrombin-stimulated counterparts; seven experiments). The cGMP analogues activate cGMP-dependent protein kinase with a similar potency but have relatively little effect on cAMP-dependent protein kinase.^{40 41} In addition, 8-PCPT-cGMP acts selectively on cGMP-dependent protein kinase compared with cGMP-regulated PDEs, whereas 8-Br-cGMP is less specific in this respect.⁴⁰ Both 8-PCPT-cGMP and 8-Br-cGMP decreased the thrombin-enhanced permeability for macromolecules at low concentrations (1 to 30 µmol/L) in aortic endothelial cells (Fig 2). This suggests that activation of cGMP-dependent protein kinase is indeed involved.

View this table: [in this window] [in a new window]   Table 1. Effect of 8-Bromo-cGMP (1 mmol/L) on the Passage of Horseradish Peroxidase Through Human Umbilical Vein and Human Aortic Endothelial Cell Monolayers Under Basal Conditions and After Stimulation With 1 U/mL Thrombin

View larger version (19K): [in this window] [in a new window]   Figure 2. Graph showing the passage of a fluorescein isothiocyanate (FITC)-labeled dextran (38 900 D) through human aortic endothelial cell monolayers after stimulation with 1 U/mL thrombin, measured in the presence of different concentrations of 8-bromo-cGMP () or 8-PCPT-cGMP (). The basal passage (bar) increased fourfold after thrombin stimulation. The elevated passage was reduced in a concentration-dependent manner by the cGMP analogues. The passage of HRP was reduced similarly (not shown). Data are means of two different cultures performed in duplicate.

Determination of the [Ca²⁺]_i in fura 2–loaded endothelial cells revealed that the thrombin-induced elevation of [Ca²⁺]_i was markedly reduced by a preincubation with 8-PCPT-cGMP in aortic endothelial cells. On the other hand, the [Ca²⁺]_i rise was only marginally attenuated by 8-PCPT-cGMP in umbilical vein endothelial cells (Table 2, Fig 3). The cAMP analogue 8-Br-cAMP (0.1 to 1 mmol/L) did not change the thrombin-induced elevation of the [Ca²⁺]_i in either cell type (not shown).

View this table: [in this window] [in a new window]   Table 2. Reduction of Thrombin-Induced Rise in [Ca2+]i in Endothelial Cells by 8-(4-Chlorophenylthio) cGNP

View larger version (14K): [in this window] [in a new window]   Figure 3. Effect of 8-(4-chlorophenylthio)cGMP (8-PCPT-cGMP) on [Ca2+]i in thrombin-stimulated human aortic endothelial cells. Top, Graph showing that preincubation of endothelial cells with 100 µmol/L 8-PCPT-cGMP () reduced the increase in [Ca2+]i induced by 1 U/mL thrombin (). The arrow indicates the time point of thrombin addition. Bottom, Video microscope image of a part of the culture before (A and C) and 10 seconds after stimulation with thrombin (B and D). The intensity of the fluorescence ratio 340/380 nm, which is represented in pixels, is reduced by 15-minute preincubation of the cells with 8-PCPT-cGMP (C and D). The fluorescence intensity bar represents, from left to right, an increase in [Ca2+].

cGMP-Inhibited cAMP PDE Activity in Umbilical Vein Endothelial Cells
The discrepancy between the effects of 8-PCPT-cGMP and 8-Br-cGMP on thrombin-enhanced permeability of human umbilical vein endothelial cells suggests the existence of an additional regulatory target by which cGMP may affect permeability. Therefore, we investigated whether a cGMP-inhibited cAMP PDE activity (PDE III) contributed, additionally, to the reducing effect of cGMP on the passage of macromolecules through thrombin-stimulated endothelial cell monolayers. SKF94120 and Indolidan, two specific inhibitors of PDE III, were used. When cAMP levels were measured after thrombin stimulation in umbilical vein endothelial cells, cAMP increased from 1.2±0.2 pmol in the absence of PDE III inhibitors to 1.9±0.4 pmol/3.5x10⁵ cells in the presence of thrombin and SKF94120 (P<.05) and tended to increase to 1.9±0.5 pmol in the presence of thrombin and Indolidan (eight different cell cultures). In the absence of thrombin, both inhibitors slightly increased cAMP 30% compared with the control value. SKF94120 and Indolidan, as well as Rolipram, an inhibitor of the PDE type IV (cAMP-specific PDE), inhibited the thrombin-induced increase of the passage of macromolecules through umbilical vein endothelial cell monolayers (Fig 4). SKF94120 slightly reduced the thrombin-induced HRP passage in aortic endothelial cells to 89±7% (four cultures). The basal permeability was not changed by SKF94120 in either cell type.

View larger version (30K): [in this window] [in a new window]   Figure 4. Bar graph showing the effect of phosphodiesterase inhibitors on the passage of horseradish peroxidase and fluorescein isothiocyanate (FITC)-dextran (35 600 D) through monolayers of umbilical vein endothelial cells. The endothelial cells were stimulated with 1 U/mL thrombin in the presence of Rolipram (100 µmol/L, filled bars), SKF94120 (100 µmol/L, widely hatched bars), or Indolidan (100 µmol/L, narrowly hatched bar) or without the addition of phosphodiesterase inhibitors (open bars). Permeability values after stimulation with thrombin (=100%) were 76±19 ng · cm-2 · h-1 for horseradish peroxidase and 7.6±0.7 µg · cm-2 · h-1 for FITC-dextran. All conditions reduced the passage of both tracer molecules (P<.05 for Rolipram and Indolidan, P<.01 for SKF94120). Data are mean±SEM of the indicated number of different cultures.

Inhibition of NO Synthesis by L-NAME Intensifies the Thrombin-Induced Elevation of Endothelial Permeability
The rise in [Ca²⁺]_i after addition of thrombin stimulates the constitutive Ca²⁺/calmodulin-dependent NO synthase. NO activates guanylate cyclase, which leads to cGMP generation. In agreement with observations by other authors,⁴² we found that thrombin augmented the intracellular cGMP concentration in tight endothelial cell monolayers of umbilical vein from 1.3±0.2 to 2.4±0.4 pmol/3.5x10⁵ cells (P<.01; 11 different cultures, assayed after 15 minutes in the presence of IBMX). This increase in cGMP concentration is apparently due to NO generation, because (pre)incubation of the cells with the competitive NO synthase inhibitor L-NAME (100 µmol/L) prevented the thrombin-induced increase in these cells (1.4±0.2 pmol cGMP/3.5x10⁵ cells; P<.05 compared with thrombin-stimulated cells). cGMP was not significantly altered when these cells were (pre)incubated with L-NAME alone (1.6±0.2 pmol cGMP/3.5x10⁵ cells). Therefore, we wondered whether the thrombin-induced increase in permeability was partly attenuated/counteracted by the generation of NO. If so, the addition of L-NAME would be expected to increase thrombin-induced permeability. In 37 different cultures of human endothelial cell monolayers, the thrombin-enhanced permeability increased by 51±13% after preincubation of the cells for 1 hour with 100 µmol/L L-NAME (P<.005). Although this effect is highly significant, considerable variation was observed between cultures. In Fig 5, the effect of L-NAME on the thrombin-enhanced permeability is plotted as a function of the thrombin-enhanced permeability. The effect of L-NAME was significant in 23 cultures that had a thrombin-enhanced permeability for HRP that was <100 ng · cm^-2 · h^-1 (42±4 versus 69±9 ng · cm^-2 · h^-1). This effect could not be demonstrated in the cultures that displayed a relatively high permeability after thrombin stimulation (14 cultures with a mean permeability of 217±21 ng · cm^-2 · h^-1). In the latter cultures, the thrombin-induced increase in permeability could still be reduced by an elevation of the intracellular cGMP content by 8-Br-cGMP or atrial natriuretic factor (not shown). In the responsive cultures, L-NAME enhanced the thrombin-induced increase in permeability in a concentration-dependent manner (Fig 6). Furthermore, the additional increase caused by L-NAME was completely prevented by agents that raise cGMP: atrial natriuretic factor, SNP, and 8-Br-cGMP (Fig 7). This was also observed in human pulmonary arterial endothelial cells (Fig 7B). Additionally, a significant increase of the passage of HRP through umbilical vein endothelial cell monolayers after thrombin stimulation by L-NAME from 100% (thrombin) to 130±18% (thrombin with L-NAME) was decreased by SNP to 105±12% (thrombin with L-NAME and SNP, seven cultures of different donors). L-NAME was ineffective on the basal permeability, regardless of the basal passage rate. To evaluate whether the observed lack of response to L-NAME was associated with an impaired formation of NO and/or cGMP, cGMP and thrombin-enhanced permeability were determined in 10 independent cultures of umbilical vein endothelial cells. The cGMP concentration was increased after thrombin stimulation from 0.8±0.1 to 2.5±0.4 pmol/3.5x10⁵ cells in cultures with a low thrombin-induced permeability (P<.05, five different cultures) but remained unchanged in cultures with a high permeability (0.6±0.1 versus 0.9±0.4 pmol/3.5x10⁵ cells, respectively; five different cultures).

View larger version (17K): [in this window] [in a new window]   Figure 5. Graph showing the effect of NG-nitro-L-arginine methyl ester (L-NAME) on thrombin-stimulated endothelial permeability in cultures of three endothelial cell types. The ratio of the passage of horseradish peroxidase (HRP) after 1 U/mL thrombin stimulation with and without the nitric oxide synthase inhibitor L-NAME (100 µmol/L) is plotted against the HRP passage in nanograms per square centimeter per hour after thrombin stimulation without L-NAME. No additional effect of L-NAME on the thrombin-induced passage is marked with a horizontal line at a ratio of 1. The passage is shown 1 hour after thrombin-stimulation for umbilical vein (), aortic (), and pulmonary artery () endothelial cell monolayers. The response to L-NAME is inversely correlated with the permeability of HRP in the presence of thrombin. Each point represents the mean of a triplicate determination.

View larger version (15K): [in this window] [in a new window]   Figure 6. Graph showing the effect of NG-nitro-L-arginine methyl ester (L-NAME) on the thrombin-induced increase in endothelial permeability. The time courses of the horseradish peroxidase (HRP) passage through human umbilical vein endothelial cell monolayers are presented, under basal conditions (medium 199 supplemented with 1% human serum albumin) or after stimulation with 1 U/mL thrombin, in the presence or absence of L-NAME. The basal HRP passage () is increased on addition of thrombin () and is further elevated in combination with L-NAME (1 µmol/L, ; 10 µmol/L, ; and 100 µmol/L, ). Data are mean±SEM of triplicate filters.

View larger version (28K): [in this window] [in a new window]   Figure 7. Bar graphs showing that cGMP-elevating agents reduce the NG-nitro-L-arginine methyl ester (L-NAME)–dependent enhancement of thrombin-stimulated permeability of human aortic and pulmonary arterial endothelial cell monolayers. L-NAME (100 µmol/L, hatched bars) enhances the increase in permeability induced by 1 U/mL thrombin (open bars); the permeability under control conditions is indicated by filled bars. Simultaneous addition of the cGMP-elevating agents atrial natriuretic factor (ANP, 10-7 mol/L), sodium nitroprusside (SNP, 10-4 mol/L), and 8-bromo-cGMP (8-Br-cGMP, 1 mmol/L) reduced the increased permeability induced by thrombin and L-NAME. A, Human aortic endothelial cell monolayers (two different cultures). B, Human pulmonary arterial endothelial cell monolayers (three different cultures). Data are mean±SEM.

To obtain further mechanistic information, cyclic nucleotides and [Ca²⁺]_i were assayed after addition of thrombin and L-NAME in aortic and umbilical vein endothelial cells, in the absence of IBMX. A transient (50% to 100%) increase in cGMP was observed, which peaked at 5 to 6 minutes after the addition of thrombin. In aortic endothelial cells, the cGMP concentration increased from 0.39±0.09 to 0.62±0.06 pmol cGMP/3.5x10⁵ cells 5 minutes after thrombin addition. Preincubation of the cells with L-NAME reduced the cellular cGMP concentration to 0.24±0.13 pmol in those cells. In the same aortic endothelial cell culture, the peak value of [Ca²⁺]_i after stimulation by thrombin (606±170 nmol/L) was additionally increased to 1015±184 nmol/L by preincubation of the cells with L-NAME (P<.05, 24 determinations). These observations are consistent with the suggestion that NO-mediated cGMP generation partially reduces the accumulation of [Ca²⁺]_i after stimulation of aortic endothelial cells by thrombin.

In umbilical vein endothelial cells, no change in [Ca²⁺]_i was observed. On the other hand, in the absence of IBMX, cAMP increased after stimulation with thrombin from 1.9±0.5 to 2.6±0.2 pmol/3.5x10⁵ cells (three different cultures). The thrombin-induced increase in cAMP was reduced by L-NAME to 2.1±0.1 pmol/3.5x10⁵ cells.

	Discussion

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In the present study, we have presented evidence that endogenous production of NO attenuates the thrombin-induced increase in permeability by a cGMP-dependent mechanism. Elevation of the cGMP concentration acts on the regulation of permeability by suppressing the elevation of [Ca²⁺]_i via cGMP-dependent kinase and by elevation of the cellular cAMP concentration via the cGMP-dependent inhibition of PDE III. The relative contribution of these mechanisms is different in human aortic and umbilical vein endothelial cells.

Mechanisms Involved in cGMP-Dependent Reduction of Thrombin-Stimulated Endothelial Permeability
Previous reports have shown that elevation of the cellular cGMP concentration reduces the increase in endothelial permeability induced by thrombin.^{16 19} Our data confirm these observations and identify two mechanisms by which cGMP acts on endothelial permeability: (1) reduction of the increase in [Ca²⁺]_i induced by thrombin and (2) elevation of the cellular cAMP concentration by inhibition of PDE III. Ca²⁺ is involved in the induction of endothelial contraction.^{4 8 17} Our data with the intracellular Ca²⁺ chelator BAPTA demonstrate a direct relation between the rise in [Ca²⁺]_i and a rapid and prolonged increase in endothelial permeability after exposure to thrombin. The sustained elevation of the permeability, after [Ca²⁺]_i has returned to basal level, suggests the onset of other intracellular events.⁶ Two lines of evidence indicate that cGMP interferes with the Ca²⁺-dependent increase in permeability, in particular in human aortic endothelial cells. First, the increase in permeability induced by thrombin was reduced by 8-PCPT-cGMP and 8-Br-cGMP at concentrations at which they selectively activate cGMP-dependent protein kinase compared with the activation of the cAMP-dependent protein kinase.^{40 41} We recently found that endothelial cells from human aorta, but not from the umbilical vein, contain a considerable amount of cGMP-dependent protein kinase (authors' unpublished data). Second, direct assay of [Ca²⁺]_i in fura 2–loaded endothelial cells demonstrated a reduced accumulation of Ca²⁺ in the presence of 8-PCPT-cGMP. Reduction of [Ca²⁺]_i by activation of cGMP-dependent protein kinase is expected to reduce the Ca²⁺/calmodulin-dependent phosphorylation of MLC kinase and the subsequent actin–nonmuscle myosin interaction.^{5 8 9} It is unlikely that cGMP reduces the Ca²⁺ response via interaction with the regulatory subunit of the cAMP-dependent protein kinase,⁴³ because 1 mmol/L 8-Br-cAMP did not influence the thrombin-stimulated Ca²⁺ response. This observation is in accordance with the inability of cAMP-elevating agents to reduce accumulation of cytoplasmic Ca²⁺ induced by histamine in endothelial cells.⁴⁴ The mechanism by which cGMP affects the accumulation of Ca²⁺ in aortic endothelial cells is not known. Analogous with findings in smooth muscle cells, it may be expected that in endothelial cells the cyclic nucleotide can induce a decrease in Ca²⁺ influx or an increase in Ca²⁺ efflux.^{45 46} Ca²⁺ efflux from vascular smooth muscle cells was found to be stimulated by cGMP via Na⁺-Ca²⁺-exchange.⁴⁷ Alternatively, it has been suggested that cGMP-dependent protein kinase activity causes reduction of cytoplasmic Ca²⁺ via suppression of inositol 1,4,5-trisphosphate formation⁴⁸ or via the stimulation of Ca²⁺-ATPase pumps.^{22 23} Further studies are needed to elucidate whether one or several of these mechanisms are involved in the cGMP-dependent reduction of [Ca²⁺]_i in endothelial cells.

In addition to reducing cytoplasmic Ca²⁺ accumulation, cGMP also affects endothelial cell permeability by inhibiting cGMP-inhibited cAMP phosphodiesterase (PDE III). PDE III has been demonstrated previously in endothelial cells^{49 50} and has been implicated in the control of endothelial permeability.⁴⁹ Inhibition of PDE III lowers the cellular breakdown of cAMP and enhances the steady state level of cAMP. Many studies have demonstrated that elevation of the cAMP concentration in endothelial cells can reduce endothelial permeability in vivo^{10 11 51} and in vitro.^{14 15} cAMP activates the cAMP-dependent protein kinase, which interferes with endothelial contraction by several mechanisms, including reduction of the phosphorylation of MLC.⁹ Involvement of PDE III in cGMP-dependent reduction of the increased permeability mediated by thrombin was demonstrated in our study by using two specific PDE III inhibitors, Indolidan and SKF94120.^{52 53} PDE III inhibition was found in human umbilical vein endothelial cells in particular, whereas only a small effect of the PDE III inhibitors was observed in human aortic endothelial cells. Thus, the PDE III activity may be different in various endothelial cell types. Alternatively, the PDE III activity of endothelial cells from aorta and umbilical vein may have been altered to a different degree during subculturing of the cells. In umbilical vein endothelial cells, 8-PCPT-cGMP did not decrease the thrombin-induced permeability and in parallel reduced the thrombin-stimulated [Ca²⁺]_i rise only slightly. The fact that 8-Br-cGMP reduced the permeability of these cells can be explained by an inhibitory action of 8-Br-cGMP on PDE III, a property that is less prominent for 8-PCPT-cGMP.^{40 41}

NO Acts as an Endogenous Modulator of Endothelial Cell Function
The notion that cGMP can modulate endothelial [Ca²⁺]_i puts forward the following question: Does NO, which induces cGMP generation by activation of soluble guanylate cyclase not only in smooth muscle cells and platelets^{54 55} but also in endothelial cells,^{56 57} act as an endogenous counterregulatory molecule? Under normal noninflammatory conditions, NO is generated in endothelial cells by the constitutive NO synthase, the activity of which depends, among others, on Ca²⁺/calmodulin.^{58 59} Thrombin evokes a rapid increase in [Ca²⁺]_i in endothelial cells. In accordance with the aforementioned feature of the constitutive NO synthase, thrombin causes a rapid and sustained elevation of NO generation⁶⁰ and an increase of the cGMP level (Reference 42⁴² and the present study) in human endothelial cells. Inhibition of NO synthase by L-NAME^{61 62} prevented cGMP accumulation. The enhancement of the thrombin-induced increase of endothelial permeability caused by preincubation of the cells with L-NAME suggests that the NO/cGMP generation indeed modulates endothelial contraction, at least partly by attenuating the cytoplasmic Ca²⁺ accumulation. This suggestion is further strengthened by the observation that the L-NAME–induced increase in permeability was abolished by adding agents that increase the cellular cGMP production independent of NO synthase. Furthermore, a preincubation with L-NAME caused an additional increase of the thrombin-induced [Ca²⁺]_i accumulation. Shin et al⁵⁶ obtained comparable results with bovine aortic endothelial cells, in which ATP-induced [Ca²⁺]_i accumulation was enhanced by the NO-synthesis inhibitor N^G-monomethyl-L-arginine. Thrombin-induced NO/cGMP formation may, in umbilical vein endothelial cells, increase intracellular cAMP via inhibition of cAMP degradation. This was suggested by cAMP accumulation after thrombin stimulation in the presence of the PDE III inhibitors SKF94120 and Indolidan. Additionally, thrombin-induced cAMP accumulation was blocked by L-NAME. A counterregulatory role of NO/cGMP is probably to be found not only in the regulation of endothelial permeability but also in other Ca²⁺-dependent processes in the endothelial cells, such as the generation of prostacyclin,⁶³ platelet-activating factor,⁶⁴ and NO itself⁵⁹ and the release of von Willebrand factor and tissue-type plasminogen activator.⁶⁵ Indeed, Buga et al⁶⁶ reported recently that NO is able to modulate its own generation.

Our observation that those endothelial cell monolayers that displayed a rather high permeability after exposure to thrombin (permeability coefficient, >5.5x10^-6 cm/s) were not affected by L-NAME was surprising but not contrary to our previous findings. These cells, for unknown reasons, are probably defective in the generation of NO and/or cGMP. This suggestion is favored by the observations that thrombin did not enhance the cellular cGMP concentration in such cells and that the thrombin-induced increase in permeability is excessively high. It further strengthens the hypothesis that Ca²⁺-regulated NO production prevents excessive contraction of endothelial cells and impairment of their barrier function.

In conclusion, cGMP elevation attenuates the thrombin-induced increase in permeability of endothelial monolayers in vitro. cGMP can act via two pathways: cGMP reduces elevation of thrombin-stimulated [Ca²⁺]_i and reduces cAMP degradation by inhibition of the PDE III activity. We postulate that autocrine NO can act as a permeability-counterregulatory agent in endothelial cells.

	Acknowledgments

 
This study was supported by Netherlands Heart Foundation grants 90.085 (Dr Draijer) and 90.089 (Dr Atsma). We would like to thank Bea van der Vecht and Annette Seffelaar for excellent technical assistance.

Received March 2, 1994; accepted October 3, 1994.

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