Thrombin stimulates PDGF production and monocyte adhesion through distinct intracellular pathways in human endothelial cells.

Thrombin stimulates PDGF production and monocyte adhesion through distinct intracellular pathways in human endothelial cells RAVI SHANKAR, CAROL A. DE LA MOTTE, AND PAUL Depwrtment of Vascular Cell Biology and Atherosclerosis Research, of The Cleveland Clinic Foundation, Cleveland, Ohio 44195 Shankar, Ravi, Carol A. de la Motte, and Paul E. DiCorleto. Thrombin stimulates PDGF production and monocyte adhesion through distinct intracellular pathways in human endothelial cells. Am. J. Physiol. 262 (CeZZ PhysioZ. 31): C199-C206, 1992.-Thrombin stimulates multiple functions in cultured endothelial cells (EC), including an increase in cell surface adhesion sites for monocytes and the production of platelet-derived growth factor (PDGF). We have initiated studies to define the intracellular signaling pathways involved in these two thrombin-induced EC functions by focusing on the possible roles of the Na’-H’ antiporter and guanine nucleotidebinding proteins (G proteins). Amiloride suppressed thrombinstimulated PDGF production by human aortic EC without affecting either basal PDGF production or overall protein synthesis. The steady-state mRNA levels of PDGF-A and PDGFB chain were not reduced by amiloride. In replicate EC cultures, amiloride had no effect on thrombin-stimulated monocyte adhesion. In addition, thrombin induction of PDGF production, but not monocyte adhesion, was abrogated in the absence of extracellular sodium. Thrombin stimulation of both monocyte adhesion and PDGF production appeared to involve a pertussis toxin-insensitive G protein. Thrombin induced an increase in [““Slguanosine Y-0-(3-thiotriphosphate) (GTPyS) binding to human EC membranes. GTPqS, in the presence of a suboptima1 concentration of thrombin, caused maximal stimulation of both monocyte adhesion and PDGF production. The effect of GTPyS on PDGF production was at the level of transcription. These results indicate that the EC is capable of responding to a pluripotent agonist such as thrombin through multiple signaling pathways, which converge and diverge to achieve differential cellular responses. second messengers; atherosclerosis;

leukocyte

adhesion

THE LAST DECADE, the vascular endothelium has emerged as a highly dynamic and interactive anatomical structure with several distinct endocrine properties. In response to its changing environment, the endothelium secretes a multitude of bioactive substances, which include vasodilator (27, 49, 54) and vasoconstrictor substances (39), pro- and anticoagulant molecules (3, 4, 7, 14, 35, 44, 45), cytokines (34), neutrophil and monocyte chemoattractants (2,31), and growth factors (11,17, 50). The coagulation protease thrombin is known to stimulate several endothelial cell (EC) functions. Thrombin stimulates EC in vitro to produce prostacyclin (21, 53), endothelin (39), platelet-activating factor (19,36), plasminogen activator, and plasminogen activator inhibitor (16, 18, 29). In addition, we have previously demonstrated that thrombin stimulates the adherence of the monocytic cell line U-937, as well as peripheral blood monocytes, to cultured arterial EC (13). Yet another EC function that is influenced by thrombin is the production of plateletderived growth factor (PDGF), a cationic polypeptide IN

0363-6143/92

$2.00 Copyright

E. DICORLETO Research Institute

that has both mitogenic and chemotactic properties toward cells of mesenchymal origin (10, 22, 38). The only transmembrane signal that has been shown to influence thrombin-stimulated PDGF production is the activation of adenyl cyclase (9, 10, 28, 43). However, this pathway is inhibitory for thrombin-induced PDGF production, and no evidence exists for the activation of adenyl cyclase by thrombin. Most mammalian cells possess an inwardly sodiumdriven transport system referred to as the Na+-H+ exchanger (20). This exchanger, which is activated in response to external stimuli and intracellular acidification, results in an increase in intracellular pH and activation of many cell functions. This exchanger is blocked by amiloride and its analogues (41). In EC, fibroblasts, and platelets, thrombin stimulates the Na+-H+ exchange process in addition to Ca2+ influx and phospholipase Cmediated protein kinase C activation (5, 24, 42). An amiloride-sensitive Na+-H+ antiporter has recently been shown to play an integral role in thrombin-stimulated platelet-activating factor production in EC (19) and mitogenesis in fibroblasts (47). Although we have demonstrated that thrombin stimulates monocyte adhesion to EC through a protein kinase C-mediated pathway, the possible role of other thrombin-induced transmembrane signals, especially the activation of the Na+-H+ antiporter, in monocyte adhesion and in PDGF production remains to be determined. Many macromolecular agonists achieve their cellular effects through coupling cell surface receptors to second messenger systems via a guanine nucleotide-binding protein (G protein). The activation of adenyl cyclase, phospholipase C, and ion channels are some examples of such G protein-coupled effector systems. Evidence for receptor-coupled G protein activation is emerging for many agonist-induced EC functions. Further, the susceptibility of these functions to specific toxins indicates that distinct G proteins are associated with different agonists. While bradykinin-induced phosphoinositide turnover in EC is insensitive to pertussis toxin (52) both ATPstimulated prostacyclin production (33) and histaminestimulated phosphatidylinositol turnover (51) are pertussis toxin sensitive. Brock and Capasso (5) have recently demonstrated that thrombin stimulates phosphatidylinositol turnover through a pertussis toxin-insensitive G protein in permeabilized human EC. In the present study, we have examined the possibility that both a G protein and the amiloride-sensitive Na+-H+ antiporter play a role in two thrombin-stimulated EC functions, namely, PDGF production and monocyte adhesion.

0 1992 the American

Physiological

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Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on November 21, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.

c200 MATERIALS

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Materials. Bovine cu-thrombin was purchased from United States Biochemical (Cleveland, OH). Sodium-free Dulbecco’s modified Eagle’s medium/F-12 was specially prepared by GIBCO (Grand Island, NY). In this medium, sodium chloride was isosmotically replaced with choline chloride. Other sodium salts were substituted with respective potassium salts without altering the overall K+ molarity. MCDB 105 and amiloride were procured from Sigma Chemical (St. Louis, MO). Na1251 and L [ 4,5-3H] leucine were purchased from ICN Biochemical (Irvine, CA). [35S]guanosine 5’-O-(3-thiotriphosphate) (GTPyS), [32P]deoxycytidine 5’-triphosphate (dCTP), [a-32P]NAD, and [“Crlsodium chromate were the products of New England Nuclear Research Products (Boston, MA). All tissue culture plastic was purchased from Costar (Cambridge, MA). Culture of human aortic EC. Human aortic EC were isolated by collagenase digestion from infrarenal arteries, obtained from the surgical transplant unit of The Cleveland Clinic Foundation, by a modification of a published procedure (25). Briefly, aortic segments were opened longitudinally and rinsed in serum-free medium, and the exposed intimal surface was digested with collagenase (2 mg/ml in serum-free medium) for 15 min at 37°C. The detached EC patches were gently collected with a rounded spatula or cotton swab and grown in primary culture with MCDB 107 containing 15% fetal bovine serum (FBS), 90 pg/ml heparin, and 150 pg/ml bovine hypothalamic endothelial cell growth supplement in 5% CO, at 37°C. MCDB 107 was prepared by supplementing MCDB 105 with glycine and KC1 as described (30). At confluence, the cells were subcultured at a I:3 ratio and used between passages 3-5. U-937 ceZZs.U-937 cells (48), originally derived from a human histiocytic lymphoma, were procured from the American Type Culture Collection and grown in suspension culture in RPM1 1640 media containing 5% FBS and routinely subcultured at a 1:5 ratio three times per week. Assay for monocytic ceZZadhesion to EC. U-937 cell adhesion to cultured human aortic EC was measured as previously described (12). Briefly, human aortic EC were plated into 24-well plates in MCDB 107 medium 48-96 h prior to the assay and grown to confluence (-2 x IO5 cells/well). On the day of the assay, U-937 cells (50 x 106/ml) were labeled for 90 min at 37°C with 100 &i of 51Cr as sodium chromate in culture medium (1 ml). The labeled cells were washed by centrifugation and lo6 viable U-937 cells were added to each well of EC after removal of the incubation medium. The binding was performed at 4°C for 1 h, the wells were washed, and the cells were lysed with 1% Triton X-100 prior to removing an aliquot for yradiation counting. The number of U-937 cells bound per well was calculated from the initial specific radioactivity (cpm/lO” cells). All data points represent triplicate determinations, with SE ~10%. Spontaneous release of 51Cr from the monocytic cells during the assay incubation was less than 5% of the total count. Radioreceptor assay for PDGI;‘. Confluent human aortic EC in 12-well plates were incubated in MCDB 107 with 5% FBS for 8 h at 37°C in 5% CO,. The conditioned media were collected, and the PDGF concentration was measured by the ability of the unlabeled PDGF in the conditioned medium to compete with 1251-PDGF for receptor sites on cultured human foreskin fibroblasts (11). Homogeneous PDGF was purified from human platelets by a modification of the method of Raines and Ross (37) and used to standardize the assay system. PDGF was radiolabeled with Na1251 as described (23). Protein synthesis. Human aortic EC protein synthesis was determined by the incorporation of [3H]leucine into trichloroacetic acid-precipitable material (15). Briefly, human aortic EC were incubated for 8 h under the same conditions for the PDGF assay in the presence of 2 &i/ml [“Hlleucine, and the cell-

ADHESION

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associated and secreted protein syntheses were determined. Northern analysis. Human aortic EC were grown to confluence in 15-cm diameter plates with MCDB 107 containing 15% FBS, 90 pg/ml heparin, and 150 pg/ml bovine hypothalamic endothelial cell growth supplement at 37°C in 5% CO,. On the day of the experiment, the monolayers were once washed with serum-free medium and incubated with appropriate doses of the test reagents in medium in the presence or absence of thrombin for 4 h at 37°C. The conditioned medium was then aspirated, the cells were washed once with phosphate-buffered saline, and the total RNA was extracted with guanidinium isothiocyanate. RNA was obtained by isopycnic centrifugation over CsCl (8) and separated by electrophoresis on a formaldehyde denaturing gel. RNA was then transferred from the gel to a Nytran (Schleicher & Schuell, New Haven, CT) membrane by capillary transfer and hybridized with [32P]dCTP random primer-labeled cDNA probes for human PDGF-A chain [ 1.3 kb cDNA in PUC-13(amp’)], PDGF-B chain [c-sis-2.9 kb cDNA in pcDV1 (amp’)], and chicken cu-tubulin [ 1.4 kb cDNA in pBR322 (tet’)]. PDGF-A chain probe was a generous gift from C. Betsholtz of the Univ. of Uppsala, Sweden. ADP ribosylation. Confluent human aortic EC in 15-cm diameter dishes were incubated in MCDB 107 with 5% FBS in the absence or presence of pertussis toxin (1 mg/ml) for 1 h at 37°C. The medium was then aspirated and the monolayers were washed twice with serum-free medium. The cells were collected by scraping and homogenized in a Wheaton glass homogenizer with 2 ml of IO mM tris(hydroxymethyl)aminomethane (Tris) . HCl buffer (pH 7.5), containing 5 mM MgC1,, 1 mM EDTA, and 1 mM dithiothreitol (DTT). The homogenate was centrifuged at 12,000 g for 5 min at 4°C and the pellet was washed twice with the homogenizing buffer (52). The crude membrane suspension (20 rug protein) was incubated either alone or in the presence of preactivated pertussis toxin with ADP ribosylation buffer (final vol, 50 ~1) containing 50 mM Tris HCl, 2 mM MgC12, I mM EDTA, 0.1% polyoxyethylene ether Wl, 10 mM DTT, 1 mM ATP, 10 mM thymidine, 1 mM NADP, 10 PM NAD, 1 &i [a- ““PINAD, and 10 PM GTP+ for 1 h at 37°C. Pertussis toxin was activated by incubation with 50 mM DTT for 30 min at 37°C. The ribosylation reaction was terminated by the addition of 15-~1 stop buffer containing 5% sodium dodecyl sulfate (SDS), 0.1 M sodium phosphate, 5% mercaptoethanol, 10% glycerol, and 0.1% bromphenol blue. The samples were boiled for 5 min, and the proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (11% running gel and 4% stacking gel). After fixation, staining, and drying, the radioactive bands were visualized by autoradiography at -70°C for 16-18 h. [““S]GTPyS binding. GTPyS binding was performed essentially as described by Sternweis and Robishaw (46). Briefly, crude EC membranes (20 pug protein) were incubated with 50 ~1 buffer containing 24 mM Tris HCl, pH 7.4, 5.7 mM MgCl,, 0.6 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’tetraacetic acid (EGTA), 0.1 mM EDTA, 0.6 mM DTT, and 400 nM GTP+ (-1.0 x lo6 cpm) at 25°C. At the end of the incubation period, the reaction mixture was rapidly filtered through a 0.45-pm Millipore membrane filter and washed three times with ice-cold 24 mM Tris HCl buffer, pH 7.4, containing 5.0 mM MgC1,. The membranes were dissolved in Liquid Stint and counted for radioactivity. Nonspecific binding was determined in the presence of excess unlabeled GTPyS (0.1 mM). l

RESULTS

Role of the Na+-H+ antiporter in EC production of PDGF and monocyte adhesion. PDGF production by human aortic EC was found to be increased two- to threefold by bovine cu-thrombin (half-maximal response


ENDOTHELIAL

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at 5 U/ml), which is consistent with the observations of others using human umbilical vein EC and kidney microvascular EC (10, 22). Thrombin-stimulated PDGF production by human aortic EC was inhibited in a dosedependent manner by the presence of amiloride (P < 0.002). Similar inhibitory response to thrombin stimulation was also observed in the presence of an amiloride analogue 5-(N-methyl-N-isobutyl)amiloride (MIA) (data not shown). Furthermore, amiloride had no significant effect on basal PDGF production (P > 0.18; Fig. 1). The viability of these cells under the imposed test conditions was unaltered as assessed by de novo protein synthesis. Amiloride under identical conditions failed to inhibit thrombin-stimulated U-937 cell adhesion to human aortic EC (Fig. 2). A role for the Na’-H+ exchanger was further examined by testing the ability of thrombin to stimulate both monocyte adhesion and PDGF production by human aortic EC in the absence of extracellular Na+. At the r^

0.5

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AND

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c201

highest effective concentration used (30 U/ml), the ability of thrombin to stimulate monocyte adherence to EC was unaltered in sodium-depleted medium (P > 0.116). However, at 12 U/ml, thrombin appears to cause a slight increase in monocyte adhesion (P > 0.025). Whereas, thrombin-augmented PDGF production was inhibited by -65% under the same conditions without adverse effects on cell-associated protein synthesis (P < 0.007; Fig. 3). The percentage reduction in stimulated PDGF production in the presence of 100 PM amiloride was comparable with that obtained in the absence of extracellular Na+. r^ 1.4 5 1.2 5 3 1.0 F

- 0.8 k t; 0.6 z

,o 0.4 n

b 0.2 n n 0.0

0

20

40

60 Amiloride

80

100

120

(PM)

Fig. 1. Influence of amiloride on basal and thrombin-stimulated platelet-derived growth factor (PDGF) production. Confluent human aortic endothelial cells (EC) were preincubated either with MCDB 107 containing 5% FBS alone or with indicated concentrations of amiloride at 37°C for 30 min. Bovine-a thrombin (12 U/ml) (A) or medium (e) was added to appropriate wells, and incubation continued for an additional 8 h. PDGF was quantitated as described under MATERIALS AND METHODS. Extent of [“Hlleucine incorporation into a trichloroacetic acid precipitate was also measured under both basal (0) and thrombinstimulated (0) conditions. Each data point represents mean t SD (n

= 3).

8T 8.0 X

; 5 6.0 ti 2 4.0 3

8.0 r

G 2.0 s

6

m 4.0 c

Amiloride (PM) Fig. 2. Effect of amiloride on basal and thrombin-stimulated U-937 cell adhesion. Confluent human aortic EC were preincubated with MCDB 107 containing 5% FBS alone or with indicated concentrations of amiloride for 30 min at 37°C. Incubation was continued in presence (0) or absence (0) of bovine cu-thrombin (30 U/ml) for 6 h at 37°C. U937 cell adhesion was measured as described under MATERIALS AND METHODS. Each data point represents mean t SD (n = 3).

Fig. 3. Effect of Na+-depleted medium on thrombin-stimulated PDGF production and U-937 cell adhesion. Confluent human aortic EC were incubated with either Dulbecco’s modified Eagle’s medium (DMEM)Ham’s F-12 medium containing 5 mg/ml bovine albumin alone or with Na’-depleted DMEM-Ham’s F-12 with 5 mg/ml bovine albumin in presence or absence of indicated concentrations of bovine cu-thrombin for 6 h and 24 h, respectively, for U-937 cell adhesion and PDGF production at 37°C. At end of incubation period, U-937 cell adhesion and PDGF production in conditioned medium were assayed as described. Results are means zt SD (n = 3).


c202

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PRODUCTION

These results suggest that, whereas thrombin utilizes the Na+-H+ exchange process to enhance PDGF production by human aortic EC, the thrombin-induced signals that lead to monocyte adhesion are independent of this antiporter. Thrombin is known to act at the level of transcription in stimulating PDGF production by EC (28, 43). We therefore examined the effect of amiloride on the steadystate levels of PDGF-A and -B chains (Fig. 4). Northern analysis confirmed that thrombin at 12 U/ml caused a

A PDGF-

ru A

0.01 0

2

4 Time

PDGF-

B

TUBULIN

PDGF-

B

TUBULIPI

Fig. 4. Influence of amiloride and Na+-depleted medium on basal and thrombin-stimulated steady-state levels of PDGF-A chain and PDGFB chain mRNA. A: confluent human aortic EC were pretreated with medium alone or indicated concentrations of amiloride for 30 min at 37°C. Bovine a-thrombin was then added to appropriate dishes and incubated for an additional 4 h. B: confluent human aortic EC were incubated with either DMEM-Ham’s F-12 or Na+-depleted DMEMHam’s F-12 in absence or presence of bovine ol-thrombin for 4 h at 37°C. Total cellular RNA was isolated and Northern analysis performed as describedin MATERIALS AND METHODS.

a

6

I 10

12

(min)

Fig. 5. [“S]guanosine 5’-O(3thiotriphosphate) (GTPrS) binding to human aortic EC membranes. Crude human aortic EC membranes (20 mg protein) were incubated alone (0) or with bovine Lu-thrombin (5 U) (w) in Tris buffer, pH 7.4, for 10 min at 25°C. [%]GTP$!I binding was quantitated as described in MATERIALS AND METHODS. Results represent means of triplicate measurements. Data are means -+ SD (n = 3).

significant increase in both A- and B-chain mRNA levels compared with the basal level. Neither amiloride nor amiloride in combination with thrombin caused any appreciable change in the steady-state levels of PDGF-A and -B chain mRNA as determined by quantitative densitometry of the presented autoradiograph (densitometric results not shown). Similar results were observed when incubations were performed in the absence of extracellular Na+ (Fig. 4). These results indicate that thrombin utilizes the Na+-H+ exchanger to regulate PDGF production posttranscriptionally. The role of G proteins in thrombin-induced EC functions. In many cell lines, the cellular effects of thrombin are mediated through a G protein(s) (1). We have therefore examined the possibility that thrombin induces increased PDGF production and monocyte adhesion through activation of a G protein. Initial studies revealed that [35S]GTPyS binding to human aortic EC membranes reached saturation at 400 nM (data not shown). In the absence of thrombin, a minimal exchange of [35S]GTPyS for bound guanine nucleotides associated with the human aortic EC membranes was observed over a lo-min period (Fig. 5). However, when preincubated with thrombin (5 U/ml) to allow receptor-G protein coupling, EC membranes rapidly exchanged [35S]GTPyS. At the end of the lo-min period, thrombin appeared to have stimulated exchange of -80% of the total [35S]GTPyS binding capacity. These results suggest that, in this in vitro system using crude membrane preparations, thrombin is able to accelerate guanine nucleotide exchange. Having established an association between a G protein and thrombin signaling in EC, we tested whether a G protein activator, GTP+, would stimulate either monocyte adhesion or PDGF production. GTPyS (25 PM) alone was without effect in either assay system; however, in the presence of a suboptimal concentration of thrombin, GTPrS stimulated both EC functions (Fig. 6). These results suggest a need for receptor occupancy to allow the interaction of GTP$S with the thrombin-G protein complex. The stimulatory effect of GTPyS on PDGF


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C203

PDGF- A

Aa

PDGF- B

Fig. 7. Effect of GTP-yS on steady-state mRNA levels of PDGF-A and PDGF-B chains. Confluent human aortic EC were preincubated with either MCDB 107 containing 5% fetal bovine serum (FBS) alone or indicated concentration of GTP-yS at 37°C for 90 min. Thrombin was then added to appropriate dishes and incubation continued for an additional 4 h. Total cellular RNA was then isolated, electrophoretically separated, and probed with respective [32P]dCTP-labeled cDNA as describedin MATERIALS AND METHODS.

s

s

Fig. 6. Influence of GTPrS on PDGF production and U-937 cell adhesion. Top: confluent human aortic EC were preincubated with medium alone or with indicated concentration of GTPrS for 90 min at 37°C. Bovine ol-thrombin was then added and incubation continued for an additional 8 h. PDGF concentration in conditioned medium was measured as described. Results are expressed as means + SD (n = 3). Bottom: treatment protocol was same as for top panel but EC were incubated for only 6 h. U-937 cell adhesion to EC was determined as described under MATERIALS AND METHODS. Results are means + SD (n = 3).

production was mirrored in an increase in the steadystate mRNA levels for the two chains of PDGF (Fig. 7). In the presence of a suboptimal concentration of thrombin and GTPyS (25 PM), PDGF-A and-B chain mRNAs were increased to a level similar to that seen with a maximal dose of thrombin. To examine further the nature of the G protein(s) involved in thrombin stimulation of EC, we tested for the sensitivity of the responses to pertussis toxin. Thrombin is thought to mediate its effect in several cell lines through activation of a phospholipase C pathway via a pertussis toxin-sensitive G protein. Pertussis toxin (1 pg/ml) did not inhibit either thrombin-induced PDGF production or monocyte adhesion (Fig. 8). Thrombinstimulated and basal steady-state levels of PDGF-A and

-B chain mRNA were also unaffected by pertussis toxin (data not shown). These results indicate that thrombin stimulates these two responses through a pertussis toxininsensitive G protein. We then addressed whether pertussis toxin was in fact ADP ribosylating a G protein in intact EC. Pertussis toxin treatment of crude human aortic EC membranes catalyzed ADP ribosylation of a 40-kDa membrane protein. More importantly, preincubation of intact human aortic EC with pertussis toxin (1 pg/ml) prevented the subsequent in vitro ADP ribosylation (Fig. 9), suggesting that the G protein had been completely ADP ribosylated in intact EC. DISCUSSION

Activation of the Na+-H+ antiporter and phospholipase C through receptor-G protein coupling are two important transcellular signals elicited by thrombin in several cell lines including EC. The results of the current study demonstrate that in human aortic EC, thrombininduced PDGF production is regulated by the Na+-H+ antiporter while monocyte adhesion is independent of this pathway. This antiporter is a nonelectrogenic transport system that is widespread in the plasmalemma of eukaryotic cells and plays a pivotal role in cellular regulation through modulation of intracellular pH (pHi) and cell volume (20). Thrombin is believed to cause an increase in pHi in EC via the activation of this antiporter, which is considered a mediator of some of thrombin’s cellular effects such as platelet-activating factor produc-


C204

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Mw @Da)

200

-

Fig. 9. ADP rihosylation of human aortic EC membrane proteins by pertussis toxin. Crude membranes were prepared from human aortic EC that were untreated or pretreated with pertussis toxin (1 pg/ml) for 1 h at 37°C. EC membranes (20 rg protein) were then incubated with 32P-NAD in presence or absence of preactivated pertussis toxin for 1 h at 37°C. Reaction was stopped and membrane proteins were separated by SDS-PAGE and analyzed by autoradiography. I: Untreated EC membranes alone; 2: membranes from untreated EC that were incubated in vitro with preactivated pertussis toxin; 3: membranes from pertussis toxin-pretreated EC that were incubated in vitro with preactivated pertussis toxin.

Fig. 8. Effect of pertussis toxin on basal and thrombin-stimulated PDGF production and U-937 cell adhesion. Confluent human aortic EC were preincubated with or without pertussis toxin (1 mg/ml) for 1 h at 37°C prior to addition of bovine oc-thrombin to appropriate wells. EC were incubated for a further 8 h for PDGF production and 6 h for U-937 cell adhesion. PDGF concentration in conditioned medium and U-937 cell adhesion was determined as described in MATERIALS AND METHODS. Results are means + SD (n = 3).

tion (19). Intracellular pH measurements with the fluorescent dye 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein not only substantiate this claim but also show that thrombin-induced increases in pHi can be blocked either by amiloride and its analogues or by excluding extracellular Na+ (19, 20). Amiloride and its analogues are believed to be competitive inhibitors with respect to extracellular Na+ (20). Studies involving the Na+-H+ antiporter are generally conducted in bicarbonate-free media (40). We observed no qualitative differences in the presence or absence of bicarbonate in terms of the ability of amiloride or Na+free medium to influence either monocyte adhesion to, or PDGF production by, EC (data not shown). When human aortic EC were exposed to thrombin in Na+-free

medium, the observed inhibition in PDGF production was similar to that seen with either amiloride or MIA. However, thrombin-stimulated PDGF production by human aortic EC was not significantly inhibited below the basal level of production by either amiloride or Na+-free medium, suggesting that the Na+-H+ antiporter plays a role only in thrombin-stimulated PDGF production and that constitutive PDGF production is refractory to the Na+-H+ exchange process (12). Several agonists, including thrombin, have been shown to induce phospholipase C activation through a receptorG protein-coupled process. Although there has been a recent surge of interest in signal transduction in EC, the relationship between agonist-mediated G protein activation and specific functional end points in these cells is just beginning to be established. Our [35S]GTP$3 exchange studies suggest that a G protein is positively associated with a thrombin receptor in EC. Further, we have provided evidence for a role of thrombin-G protein coupling in PDGF production and monocyte adhesion by demonstrating that, in the presence of a suboptimal concentration of thrombin, GTPyS can substantially induce both these EC responses. The need for receptor occupancy in thrombin-GTPyS activation was also observed by Brock and Capasso (5) who investigated the effect of GTPyS on thrombin-stimulated inositol phosphate accumulation in permeabilized human EC. The observed effect of GTP+ cannot be attributed to its possible interaction with purinergic receptors on EC


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because neither PDGF nor monocyte adhesion was influenced by ATP (unpublished observation). It is generally believed that cells need to be permeabilized for GTPyS to gain entry into the cell to bind to intracellular sites. Our experiments suggest that a sufficient intracellular concentration of GTPyS can be attained by preincubating cells with GTPyS for 60-90 min without prior permeabilization. Similar results were reported by Voyno-Yasenetskaya et al. (52) using pulmonary artery EC. We chose not to permeabilize the cells because our initial studies revealed that the tested permeabilization procedures caused a significant reduction in viability of the EC in the time scale required to study either PDGF production or monocyte adhesion (4-8 h). Despite mounting evidence for G protein involvement in thrombin-mediated effects, little is known about the exact nature of the G protein that couples the thrombin receptor to phospholipase C. Thrombin stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis through a pertussis toxin-sensitive G protein in fibroblasts and vascular smooth muscle cells (26, 32); whereas, in osteosarcoma cells (1) and in EC (6), pertussis toxin fails to inhibit stimulation by thrombin. Further, Voyno-Yasenetskaya et al. (51) have shown that histamine-induced, but not bradykinin-induced, phosphoinositide turnover is pertussis toxin-sensitive in human umbilical vein EC. In human aortic EC, neither thrombin-stimulated PDGF production nor monocyte adhesion is pertussis toxin sensitive. However, ADP ribosylation studies have demonstrated the presence of a pertussis toxin substrate in intact human aortic EC. In summary, our results suggest the involvement of a pertussis toxin-insensitive G protein in transducing signals that are important for thrombin-stimulated PDGF production and monocyte adhesion. This G protein mediated step appears to transcriptionally regulate thrombin-stimulated PDGF production. The activation of a thrombin-stimulated amiloride-sensitive Na+-H+ antiporter, on the other hand, plays a role PDGF production at a posttranscriptional level. The differential regulation of multiple signals generated in EC by a single agonist may govern the specific response of the cell to its environment. For example, EC may preferentially respond to thrombin in vivo by increasing monocyte adhesion sites under circumstances when the pHi may not be conducive for the activation of the Na+-H+ antiporter. Under different environmental conditions, PDGF gene expression may accompany the induction of monocyte adhesion sites. Thus specific paracrine functions of the EC may be activated temporally to catalyze such processes as wound healing, inflammation, and atherosclerosis. We thank Drs. Robert Graham, Mi Jae Im, and Alan Wolfman for helpful discussions about the G protein studies. These studies were supported by National Heart, Lung, and Blood Institute Grants HL-29582 and HL-34727 and by a postdoctoral fellowship grant to R. Shankar from the American Heart Association, Northeast Ohio Affiliate. P. E. DiCorleto was the recipient of a Research Career Development Award (HL-1561) from the National Institutes of Health during the course of these studies. Address for reprint requests: P. E. DiCorleto, Cleveland Clinic

ADHESION

AND

Foundation, OH 44195. Received

PDGF

Research 22 April

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PRODUCTION Institute

1991; accepted

(NCl), in final

9500 form

Euclid 8 August

Ave.,

Cleveland,

1991.

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Thrombin and parathyroid hormone mobilize intracellular calcium in rat osteosarcoma cells by distinct pathways.

Thrombin induces platelet adhesion to endothelial cells.

A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates intracellular calcium and stimulates prostacyclin production in human endothelial cells.

Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

Thrombin stimulates inositol phosphate production and intracellular free calcium by a pertussis toxin-insensitive mechanism in osteosarcoma cells.

CGRP stimulates the adhesion of leukocytes to vascular endothelial cells.

Neuropeptide Y stimulates prostacyclin production in porcine vascular endothelial cells.

Thrombin stimulates inositol phosphate formation, intracellular calcium fluxes and DNA synthesis in cultured fetal human non-pigmented ciliary epithelial cells.

Human milk stimulates prostacyclin production by cultured human vascular endothelial cells.

Artemisinin inhibits monocyte adhesion to HUVECs through the NF-κB and MAPK pathways in vitro.

Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).

Human β-Defensin 3 Reduces TNF-α-Induced Inflammation and Monocyte Adhesion in Human Umbilical Vein Endothelial Cells.

MnTBAP stimulates angiogenic functions in endothelial cells through mitofusin-1.

Differential induction of the c-fos promoter through distinct PDGF receptor-mediated signaling pathways.

Thrombin receptor 14-amino acid peptide binds to endothelial cells and stimulates calcium transients.

Pingyangmycin stimulates apoptosis in human hemangioma-derived endothelial cells through activation of the p53 pathway.

Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion.

Intracellular signal transduction for interleukin-1 beta-induced endothelin production in human umbilical vein endothelial cells.

Minimally modified low density lipoprotein stimulates monocyte endothelial interactions.

Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar-endothelial co-cultures.

Endothelin-1 stimulates its own synthesis in human endothelial cells.

Glucose reduces PDGF production and cell proliferation of cultured vascular endothelial cells.

Single- and double-walled carbon nanotubes enhance atherosclerogenesis by promoting monocyte adhesion to endothelial cells and endothelial progenitor cell dysfunction.

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.