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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2068-2075.)
© 1995 American Heart Association, Inc.

Articles

Inhibition of Nitric Oxide Biosynthesis Promotes P-selectin Expression in Platelets

Role of Protein Kinase C

Toyoaki Murohara; Scott J. Parkinson; Scott A. Waldman; Allan M. Lefer

From the Departments of Physiology (T.M., A.M.L.) and Medicine, Division of Clinical Pharmacology (S.J.P., S.A.W.), Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa.

Correspondence to Dr Allan M. Lefer, Department of Physiology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust St, Philadelphia, PA 19107-6799.

	Abstract

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Abstract Inhibition of NO synthesis promotes P-selectin expression on endothelial cells; however, the precise mechanism is unclear. Because NO has been shown to inhibit protein kinase C (PKC) activity, we examined the hypothesis that the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) stimulates P-selectin expression on platelets via PKC activation. Ten-minute incubation with either phorbol 12-myristate 13-acetate (PMA), thrombin, or L-NAME significantly increased P-selectin expression on platelets (as assessed by flow-cytometric analysis) and PKC activity of platelet membranes. Increased P-selectin expression induced by either PMA, thrombin, or L-NAME was significantly attenuated by the selective PKC inhibitor UCN-01 (7-hydroxystaurosporine). Furthermore, L-NAME–induced P-selectin expression was significantly attenuated by either L-arginine, 8-bromo-cGMP, or sodium nitroprusside (SNP). Interestingly, L-NAME further potentiated P-selectin upregulation by thrombin. L-NAME, thrombin, and PMA also significantly increased polymorphonuclear leukocyte adherence to the coronary artery endothelium, an effect that was significantly attenuated by the anti–P-selectin monoclonal antibody PB1.3 or by UCN-01, L-arginine, 8-bromo-cGMP or SNP but not by D-arginine or the nonblocking anti–P-selectin monoclonal antibody NBP1.6. These results indicate that inhibition of NO synthesis induces rapid P-selectin expression, which appears to be at least partially mediated by PKC activation in platelets. Similar effects and mechanisms of L-NAME on P-selectin function were also observed in endothelial cells, another site of P-selectin expression. Thus, PKC activation may play an important role in cell-to-cell interaction when NO production is compromised.

Key Words: platelets • leukocytes • adherence • endothelium • nitric oxide synthase

	Introduction

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P-selectin plays an important pro-inflammatory role in mediating interactions among neutrophils, platelets, and the vascular endothelium.^{1 2 3} P-selectin is normally stored in the Weibel-Palade bodies of ECs and in -granules of platelets.^{1 3 4} However, once these cells are activated by thrombin, histamine, or H₂O₂, their intracellular granules fuse with the plasma membrane and P-selectin is rapidly translocated to the cell surface.^{4 5} Employing intravital microscopy, we have shown that the NO synthase inhibitor L-NAME induces endothelial P-selectin expression, which facilitates neutrophil "rolling" in the rat mesenteric microcirculation in vivo.⁶ Furthermore, either L-arginine, a precursor of NO, or 8-bromo-cGMP, a stable cGMP analogue, significantly attenuated the L-NAME–induced increase in P-selectin expression.⁶ NO may inhibit PMN-endothelium interactions by scavenging superoxide radicals, which promote PMN adherence to the endothelium.^{6 7} L-Arginine/NO can also attenuate monocyte adherence to the vascular endothelium in hypercholesterolemic rabbits.⁸ However, precise mechanism(s) by which inhibition of NO synthesis promotes P-selectin expression and PMN-endothelium interaction remain(s) poorly understood.

Thrombin, a well-known stimulator of P-selectin expression in platelets, activates a PI-specific phospholipase C.^{9 10} Thrombin thus promotes PI turnover and PKC activation, resulting in increases in intracellular Ca²⁺ and phosphorylation of substrate protein.^{9 10 11} Geng and coworkers¹² have shown that PMA, a PKC activator, stimulates rapid P-selectin expression and promotes neutrophil-endothelium interaction. Recently, N,N,N-trimethylsphingosine, an inhibitor of PKC, has been shown to attenuate thrombin-stimulated P-selectin expression in both platelets and ECs.^{13 14} Furthermore, Hannun and coworkers¹⁵ have suggested that PKC activation is a prerequisite for agonist-induced platelet activation. These studies collectively suggest that the PKC pathway may be significantly involved in the process of rapid P-selectin translocation to the cell surface after stimulation.

PKC is a cytosolic enzyme that is abundant in platelets and is translocated to the plasma membrane after stimulation.^{16 17} Although PKC activity is physiologically regulated by a variety of factors (mainly lipid components in the plasma membrane), recent data suggest that NO inhibits PKC activity.¹⁸ The NO/soluble guanylate cyclase pathway exists in both ECs and platelets and regulates endothelial permeability and platelet aggregation.^{19 20} Previous studies have also shown that cGMP inhibits PKC activity.^{9 16 21} Thus, NO may be an important endogenous negative regulator of PKC in both platelets and ECs via S-nitrosylation of PKC, cGMP-dependent mechanisms, or other unknown mechanisms. On the basis of these considerations, we asked whether inhibition of NO synthesis could promote rapid P-selectin expression via a PKC-dependent mechanism.

Accordingly, the main purposes of the present study were to examine the effects of inhibition of NO synthesis by L-NAME on P-selectin expression in platelets and to determine whether this phenomenon could be related to platelet PKC activation. We also investigated the effects and mechanisms of L-NAME–induced adherence of PMNs to ECs as a reference system for comparison with P-selectin expression in platelets.²²

	Methods

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Flow-Cytometric Determination of P-selectin Expression on Feline Platelets
Flow-cytometric analysis of P-selectin expressed on feline platelets was performed by a previously described method.^{14 23} Ten adult male cats (2.8 to 3.6 kg) were anesthetized with sodium pentobarbital (30 mg/kg body weight IV). An intratracheal cannula was inserted, and the cats were placed on intermittent positive-pressure ventilation (Harvard Small Animal Respirator, Harvard Apparatus Co). A polyethylene catheter was inserted through the right femoral artery and placed in the abdominal aorta. Arterial blood (80 mL) was collected from the catheter and anticoagulated with sodium citrate (Sigma Chemical Co). Platelet-rich plasma was obtained by centrifuging the blood at 200g for 15 minutes. The platelet-rich plasma was recentrifuged at 2000g for 10 minutes to form a platelet-rich pellet, which was washed twice in calcium-free Tyrode's solution containing 0.2% (vol/vol) BSA. The final cell pellet was resuspended in DPBS containing 1 mmol/L CaCl₂.

Aliquots of platelet suspensions were incubated with a variety of pharmacological agents, including L-NAME, D-NAME, PMA, and thrombin, at 37°C for 10 minutes without stirring. In another set of experiments, platelet aliquots were first incubated with either L-arginine (1 mmol/L), D-arginine (1 mmol/L), the PKC inhibitor UCN-01 (100 nmol/L),²⁴ SNP (10 µmol/L), recombinant human superoxide dismutase (200 µg/mL, Grunenthal), 8-bromo-cGMP (1 mmol/L), or thrombin (2 U/mL) for 10 minutes. After this incubation, the platelets were incubated with L-NAME (1 mmol/L) for another 10 minutes. Subsequently, the platelets were fixed with an equal volume of 2% (vol/vol) paraformaldehyde in PBS at pH 7.2 and washed twice with PBS containing 0.2% (vol/vol) BSA. The platelet suspensions were treated with human block IgG (4.0 mg/mL, Sigma), and then the primary anti–P-selectin MAb PB1.3 (20 µg/mL, Cytel Corp) was added to the platelet suspensions. The platelets were maintained at 4°C for 60 minutes and then washed in DPBS with 0.2% (vol/vol) BSA to remove any excess primary antibody. F(ab')₂ fragments of a goat anti-mouse IgG-phycoerythrin conjugate (Tago) was used as the secondary antibody at a 1:100 dilution, and the cells were maintained at 4°C for 30 minutes. The stained platelets were washed twice, fixed in 1% (vol/vol) paraformaldehyde, and then analyzed by flow cytometry (FACScan, Becton-Dickinson).

Measurement of PKC Activity of the Platelet Membrane Fraction
The platelets were suspended in modified Tyrode's/HEPES buffer, pH 7.4, containing (in millimoles per liter) NaCl 134, KCl 2.9, NaHCO₃ 12, CaCl₂ 1, HEPES 5, and glucose 5. Aliquots of platelet suspension were then treated with thrombin, PMA, L-NAME, D-NAME, UCN-01, or vehicle at 37°C for 10 minutes without stirring. After incubation, the cells were lysed by sonication on ice eight times for 2 seconds each. The cell lysate was then centrifuged at 87 000g for 60 minutes to separate the membrane fraction, which was then resuspended in 300 µL modified Tyrode's/HEPES buffer by sonication.

The PKC activity of the platelet membrane fraction was measured colorimetrically (Colorimetric PKC Assay Kit, Pierce), modified from the assay methods of Toomik et al.²⁵ Ten microliters of platelet membrane suspension was added to the PKC reaction solution containing 2 mmol/L ATP, 10 mmol/L MgCl₂, 0.1 mmol/L CaCl₂, 0.002% (vol/vol) Triton X-100, 20 mmol/L Tris, 0.2 mg/mL phosphatidyl-L-serine (PKC activator), and dye-labeled glycogen synthase peptide (PKC substrate). The mixture was then incubated at 30°C for 30 minutes. After incubation, 20 µL of each sample was applied directly to a specific affinity membrane in an individual separation unit (Pierce). This unique membrane is a ferric adsorbent paper and binds only phosphorylated substrate and all excess ATP, effectively stopping the PKC-mediated phosphorylation reaction of the substrate peptide.²⁵ The remaining (unbound) nonphosphorylated substrate was washed and extracted from the affinity membrane by centrifugation into 500 µL phosphopeptide binding buffer containing 0.1 mol/L sodium acetate, 0.5 mol/L NaCl, and 0.02% (vol/vol) NaN₃, pH 5.0,. Finally, the (bound) phosphorylated substrate was separated from the affinity membrane by centrifugation into a phosphopeptide elution buffer containing 0.1 mol/L NH₄HCO₃ and 0.02% (vol/vol) NaN₃ at pH 8.0. The phosphorylated substrate in the phosphopeptide elution buffer was then measured spectrophotometrically at 570 nm. The value of the absorbance was divided by the total protein concentration in the membrane suspension and standardized. Relative PKC activity was expressed as a percentage of phosphorylation of the PKC substrate by platelet membranes that had been stimulated with PMA (100 nmol/L).

Determinations of Platelet cGMP
For measurement of platelet cGMP, 0.5 mL of platelet suspension was incubated with L-NAME (1 mmol/L) for 10 minutes at 37°C. Effects of co-incubation of either L-arginine or D-arginine (1 mmol/L) with L-NAME were also examined. As a positive control, platelets were treated with SNP (100 µmol/L) for 10 minutes. Following these incubations, platelet suspensions were treated with 100 µmol/L 3-isobutyl 1-methylxanthine (Sigma), a phosphodiesterase inhibitor, at 37°C for an additional 10 minutes. The reaction was terminated by addition of 0.5 mL ice-cold trichloroacetic acid (6%, wt/vol). The samples were stored at -80°C until cGMP levels (picomoles per milligram protein) were measured by radioimmunoassay as described previously.²⁶

Preparation of Feline Coronary Artery Segments
Immediately after arterial blood was drawn for PMN and platelet isolation as described above, a rapid midsternal thoracotomy was performed and the heart was rapidly excised and immersed into warm (37°C), oxygenated K-H solution. The coronary arteries were removed and placed into warm K-H solution consisting of (in millimoles per liter) NaCl 118, KCl 4.75, CaCl₂·2H₂O 2.54, KH₂PO₄ 1.19, MgSO₄·7H₂O 1.19, NaHCO₃ 12.5, and glucose 10.0. Isolated coronary arteries were cleaned of fat and connective tissue and cut into rings 2 to 3 mm long for subsequent studies of PMN-endothelium adherence.

Autologous Feline Neutrophil Isolation
Autologous feline neutrophils were isolated by a previously described Percoll density-gradient method^{22 27} from peripheral blood (80 mL) collected in citrate-phosphate-dextrose solution. PMN isolation was performed at 4°C to minimize neutrophil activation and L-selectin shedding.²² PMNs were collected from the 62% to 80% interface of a Percoll–platelet-poor plasma gradient and washed with DPBS before being assayed for viability by trypan blue exclusion. PMN preparations obtained by this method were >95% pure and >95% viable. The PMN pellet was finally suspended in 2 mL DPBS, and the number of cells was counted.

Autologous Feline PMN Adherence to Coronary Artery Endothelium
PMNs isolated by the aforementioned procedure were labeled with fluorescent dye PKH2-GL (Sigma Immunochemical Co) according to the method of Yuan and Fleming.²⁸ One milliliter of the dye diluent was added to a loose cell pellet containing 10⁷ PMNs. One milliliter of PKH2-GL dye (4 µmol/L) was added to the cell suspension, mixed, and incubated for 7 minutes. Two milliliters of DPBS containing 10% (wt/vol) platelet-poor plasma was added to stop the labeling reaction, and another 5 mL DPBS was added to the suspension. The cells were then centrifuged at 400g for 10 minutes at room temperature. The supernatant was removed, and the cells were resuspended in DPBS and recounted. This labeling procedure does not affect the normal morphology or function of feline PMNs.^{23 28}

Isolated feline coronary artery rings were cut, carefully opened, and placed endothelial surface up in culture dishes filled with 3 mL oxygenated K-H buffer (37°C). First, coronary artery segments were incubated with either 2 U/mL thrombin, 1 mmol/L L-NAME, 1 mmol/L D-NAME, thrombin (2 U/mL) plus L-NAME (1 mmol/L), the PKC activator PMA (100 nmol/L), or vehicle for 10 minutes. After incubation, coronary artery segments were rapidly removed; placed into another cell-culture dish filled with fresh, oxygenated K-H solution; and incubated with labeled, autologous PMNs for 20 minutes. During this period, the culture dishes were agitated in a metabolic shaker bath at 37°C. After incubation each coronary artery ring was removed, placed onto a glass slide, and covered with a coverslip. Labeled PMNs that had adhered to the endothelial surface were counted by epifluorescence microscopy (Nikon Diaphot, Nikon Inc). Adherent neutrophils on five regions of each vessel segment were randomly counted and expressed as the mean number of PMNs per square millimeter of endothelial surface area.²²

In the second series of experiments, coronary artery segments were first incubated with either L-arginine (1 mmol/L), D-arginine (1 mmol/L), 8-bromo-cGMP (1 mmol/L), SNP (10 µmol/L), the PKC inhibitor UCN-01 (100 nmol/L, Kyowa Hakko Kogyo Co),²⁴ or vehicle for 10 minutes. After incubation, L-NAME (1 mmol/L) was added to each bath and co-incubated with the coronary artery segments for 10 minutes. Subsequently, the segments were transferred to culture dishes containing fresh K-H solution, and labeled PMNs were incubated for 20 minutes. Finally, adherent PMNs were counted and expressed as described above. We also examined the effects of pre-incubation with the selective PKC activator UCN-01 (100 nmol/L) on PMN adherence to thrombin-stimulated coronary artery endothelium. To examine the role of P-selectin–mediated PMN-endothelium interactions in the L-NAME– or thrombin-stimulated coronary artery segments, we also tested the effects of a MAb directed against P-selectin (MAb PB1.3, 20 µg/mL) on PMN adherence to L-NAME– or thrombin-treated coronary artery endothelium.

Antibodies and Reagents
All of the following reagents were purchased from Sigma: thrombin, L-NAME, D-NAME, L-arginine, D-arginine, 8-bromo-cGMP, SNP, and PMA. The selective PKC inhibitor UCN-01 (7-hydroxystaurosporine) was a gift from the Kyowa Hakko Kogyo Co, Tokyo, Japan. MAb PB1.3 binds to P-selectin and blocks the interaction between P-selectin and carbohydrate ligands, whereas control MAb NBP1.6 binds to P-selectin but does not inhibit P-selectin–mediated adhesive interactions. PB1.3 and NBP1.6 are murine IgG1 MAbs raised against human P-selectin and were gifts from Dr J.C. Paulson at Cytel Corp, San Diego, Calif. Our previous flow-cytometric analysis demonstrated that MAb PB1.3 binds avidly to feline platelets.^{14 22 23}

Statistical Analysis
All values in the text, figures, and tables are presented as mean±SEM based on n independent experiments. All data were subjected to ANOVA followed by Fisher's t test for evaluation of differences between groups. Values of P.05. were considered statistically significant for differences between groups. The procedures in the present study are in accordance with the guidelines of the Thomas Jefferson University, Committee on the Use and Care of Experimental Animals.

	Results

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Flow-Cytometric Analysis of P-selectin Expressed on Thrombin- or PMA-Stimulated Platelets
Inflammatory mediators such as thrombin or histamine are well-known stimulators of P-selectin expression. Thus, we examined P-selectin translocation on the surface of thrombin-stimulated platelets. Thrombin-stimulated feline platelets incubated with phycoethrythrin-conjugated anti-mouse IgG alone (ie, omission of the primary MAb PB1.3) revealed only 3% positive staining for platelets and a mean channel fluorescence of 4.0, indicating a small extent of nonspecific background fluorescent antibody binding. Table 1 shows the mean channel fluorescence and percent positive staining for platelets after incubation with thrombin (2 U/mL) or the PKC activator PMA (100 nmol/L) in the absence or presence of the PKC inhibitor UCN-01 (100 nmol/L). The percent positive staining and mean channel fluorescence of background fluorescence (ie, omission of the primary MAb) were subtracted from each value. P-selectin expression was markedly enhanced after stimulation with either thrombin (2 U/mL) or PMA (100 nmol/L) for 10 minutes compared with nonstimulated platelets. After incubation with thrombin (2 U/mL), the binding of PB1.3 to feline platelets markedly increased, to a mean channel fluorescence of 42±8 compared with 7±3 in nonstimulated platelets. Pretreatment with the PKC inhibitor UCN-01 (100 nmol/L) significantly attenuated both percent positive cells and mean channel fluorescence of P-selectin on thrombin-stimulated platelets (Table 1).

View this table: [in this window] [in a new window]   Table 1. Flow-Cytometric Analysis of P-selectin Expression on Feline Platelets After Stimulation With Thrombin or PMA

After treatment with the PKC activator PMA (100 nmol/L), binding of PB1.3 to platelets was markedly increased, yielding a mean channel fluorescence of 46±7 compared with 6±4 in nonstimulated platelets that had been given vehicle only (ie, the same concentration of DMSO). Pretreatment with the PKC inhibitor UCN-01 (100 nmol/L) significantly attenuated both the percent positive cells and the mean channel fluorescence of P-selectin on PMA-stimulated platelets. Thus, the PKC activator PMA appears to mimic the effects of thrombin on P-selectin expression.

Flow-Cytometric Analysis of P-selectin Expression on L-NAME–Treated Platelets
Next, we examined the effects of NO synthase inhibition by L-NAME on platelet P-selectin expression. Table 2 shows the mean channel fluorescence and percent positive staining for P-selectin on platelets after incubation with L-NAME or D-NAME for 10 minutes in the absence or presence of various agents. After incubation with L-NAME but not with D-NAME, binding of MAb PB1.3 to feline platelets was significantly increased to a mean channel fluorescence of 36±13 compared with 7±3 for the nonstimulated control platelets. The percent positive staining of cells was also significantly increased to 39±5% compared with 6±3% for nonstimulated platelets. Increases in P-selectin expression induced by L-NAME (as indicated by increases in both percent positive cells and mean channel fluorescence) was significantly attenuated by co-incubation of platelets with either L-arginine (1 mmol/L), the PKC inhibitor UCN-01 (100 nmol/L), 8-bromo-cGMP (1 mmol/L), or the NO donor SNP (10 µmol/L). In contrast, increases in P-selectin expression induced by L-NAME were not attenuated by co-incubation with D-arginine or recombinant human superoxide dismutase (200 µg/mL) (Table 2). When L-NAME was added to thrombin-stimulated platelets, the percent positive cells and mean channel fluorescence to P-selectin were significantly increased when compared with corresponding values for either L-NAME alone or thrombin alone (Table 2). Representative fluorescence histograms are shown in Fig 1. Stimulation with thrombin or L-NAME significantly increased the fluorescence intensity of P-selectin on feline platelets, as indicated by a rightward shift in the pattern of Fig 1a and 1b. This increased expression of P-selectin induced by L-NAME was significantly attenuated by co-incubation with L-arginine but not with D-arginine (Fig 1c). P-selectin expression was also significantly attenuated by the selective PKC inhibitor UCN-01 (Fig 1d).

View this table: [in this window] [in a new window]   Table 2. Flow-Cytometric Analysis of P-selectin Expression on Feline Platelets After Stimulation With L-NAME

View larger version (14K): [in this window] [in a new window]   Figure 1. Representative fluorescence histograms of P-selectin on feline platelets as assessed by flow cytometry. Both thrombin (2 U/mL) and L-NAME (1 mmol/L) significantly increased P-selectin expression (a and b), which was significantly attenuated by co-incubation with either L-arginine (L-Arg, 1 mmol/L) or the selective PKC inhibitor UCN-01 (100 nmol/L) (c and d) but not by co-incubation with D-arginine (D-Arg, 1 mmol/L; c).

PKC Activity of Platelet Membrane Fraction After Stimulation
Because NO can inhibit PKC activity by S-nitrosylation and phorbol ester can significantly increase P-selectin expression, we examined the relationship between inhibition of NO by L-NAME and platelet PKC activity. The PKC activity of each platelet membrane fraction was measured after a 10-minute incubation with either thrombin (2 U/mL), L-NAME (1 mmol/L), D-NAME (1 mmol/L), L-NAME (1 mmol/L) plus thrombin (2 U/mL), or PMA (100 nmol/L), as this time frame correlates well with the rapid expression of P-selectin. Fig 2 illustrates platelet membrane PKC activity expressed as a percent of maximum PKC activation after exposure to PMA (100 nmol/L). Thrombin significantly increased membrane PKC activity to 52±5% compared with nontreated control platelets (8±2%, P<.05). Moreover, L-NAME incubation also increased PKC activity to 25±4% (P<.05 versus control). However, D-NAME did not significantly increase PKC activity. Co-incubation with both L-NAME and thrombin further increased PKC activity to 70±8%. Thus, endogenous NO appears to act as a "brake" on PKC activation in platelet membranes after thrombin stimulation.

View larger version (20K): [in this window] [in a new window]   Figure 2. PKC activity of feline platelet membrane fraction expressed as a percentage of PMA (100 nmol/L)-induced PKC activity, which was set at 100%. Heights of bars are means, brackets indicate SEM, and numbers of experiments are within bars. *P<.05 vs nonstimulated control platelets.

cGMP Content in the Platelet Suspension After Stimulation
Table 3 summarizes the cGMP concentration in platelet suspensions as measured by radioimmunoassay. After stimulation with SNP (100 µmol/L), cGMP content was significantly elevated when compared with that in nonstimulated platelets. After incubation with L-NAME (1 mmol/L), the cGMP concentration had decreased slightly but not significantly from baseline levels (Table 3). This lower cGMP content was somewhat restored by co-incubation with L-arginine (1 mmol/L) but not D-arginine (1 mmol/L), although these values were not significantly different compared with those in the L-NAME group (Table 3).

View this table: [in this window] [in a new window]   Table 3. Platelet cGMP Content

Adherence of Nonstimulated PMNs to Stimulated Feline Coronary Artery Endothelium
Another important site of P-selectin expression is vascular ECs, in which P-selectin is rapidly translocated to the cell surface after stimulation with inflammatory mediators, thereby facilitating leukocyte adherence to the endothelial surface. We examined the effects of NO synthase inhibition on PMN adherence to feline coronary artery endothelium that had been stimulated with either thrombin, L-NAME, D-NAME, thrombin plus L-NAME, or the PKC activator PMA. Fig 3 summarizes these results. PMN adherence was markedly enhanced by endothelial stimulation with either thrombin (2 U/mL), L-NAME (1 mmol/L), thrombin plus L-NAME, or PMA (100 nmol/L) but not with D-NAME. The PMA-induced increase in PMN adherence to the endothelium was significantly attenuated by the PKC inhibitor UCN-01 (1 µmol/L). Interestingly, thrombin plus L-NAME significantly enhanced PMN adherence to the coronary artery endothelium to a greater extent than did either thrombin or L-NAME alone.

View larger version (24K): [in this window] [in a new window]   Figure 3. Labeled, autologous PMN adherence to feline coronary artery endothelium after stimulation by NO or P-selectin–modulating agents. Heights of bars are means, brackets indicate SEM, and numbers of coronary segments are within bars. **P<.01 vs nonstimulated control coronary endothelium; P<.05 vs thrombin- or L-NAME–treated group.

Adherence of Nonstimulated PMNs to L-NAME–Stimulated Feline Coronary Artery Endothelium: Role of NO Inhibition
We then examined the mechanism of PMN adherence to feline coronary artery endothelium that had been stimulated with L-NAME (1 mmol/L) for 10 minutes. Fig 4 summarizes the results of nonstimulated-PMN adherence to L-NAME–stimulated coronary artery endothelium with or without the relevant modulating agents. PMN adherence was markedly enhanced by treatment with L-NAME compared with nonstimulated control endothelium (P<.01). This increase in PMN adherence to the endothelium was significantly attenuated by treatment with the anti–P-selectin MAb PB1.3 (P<.01 versus MAb vehicle). In contrast, treatment with MAb NBP1.6 (20 µg/mL), a nonblocking control antibody directed against P-selectin, did not attenuate PMN adhesion (Fig 4). These results indicate that P-selectin expression on the endothelial surface plays an important role in the L-NAME–stimulated increase in the PMN-endothelium interaction after 10 minutes. The increase in PMN adherence to L-NAME–stimulated coronary artery endothelium was also significantly attenuated by the selective PKC inhibitor UCN-01. Thus, L-NAME–mediated P-selectin expression may be at least partially mediated by PKC-dependent mechanism(s). Further examination revealed that the greater PMN adherence to L-NAME–stimulated endothelium was significantly attenuated by co-incubation with either L-arginine (1 mmol/L), 8-bromo-cGMP (1 mmol/L), or the NO donor SNP (10 µmol/L). In contrast, greater PMN adherence to L-NAME–treated endothelium was not significantly attenuated by D-arginine. These results suggest that lower NO content and the consequent decrease in cGMP may contribute to the L-NAME–induced increase in PMN adherence to coronary artery endothelium.

View larger version (25K): [in this window] [in a new window]   Figure 4. Labeled, autologous PMN adherence to feline coronary artery endothelium after stimulation by L-NAME (1 mmol/L) with or without various pharmacological agents. Heights of bars are means, brackets indicate SEM, and numbers of coronary segments are within bars. *P<.05, **P<.01 vs L-NAME–stimulated coronary endothelium.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
P-selectin is expressed on platelets and ECs and plays a key role in platelet adherence and neutrophil rolling on the vascular endothelium during inflammation.^{1 2 3 4 5 29 30 31} P-selectin on platelet surfaces may also be involved in tumor cell metastasis in vivo, presumably by tumor cell–platelet aggregation.^{32 33} P-selectin is stored in the Weibel-Palade bodies of ECs and in the -granules of platelets.^{1 2 3 4 5} When these cells are stimulated with inflammatory mediators, such as thrombin, histamine, H₂O₂, or components of the complement cascade, intracellular granules fuse with the plasma membrane and P-selectin is rapidly mobilized to the cell surface.^{1 2 3 4 5 34 35} Recently, in vivo neutralization of P-selectin was shown to attenuate acute inflammatory tissue injury in myocardial ischemia/reperfusion²³ and immune complex–induced lung injury.³⁶

The precise intracellular signals responsible for P-selectin translocation are unclear; however, Geng and coworkers¹² have shown that PMA, an activator of PKC, significantly induces P-selectin expression and facilitates PMN adhesion to the endothelium. Thrombin, a known stimulator of P-selectin expression, also activates PKC and increases cytosolic Ca²⁺ via stimulation of phospholipase C in platelets.^{9 10 11} Recent studies have also shown that the PKC inhibitor N,N,N-trimethylsphingosine significantly attenuates P-selectin expression in both stimulated platelets and ECs.^{13 14 22} These studies implicate PKC as a major contributor to the process of rapid P-selectin translocation to the cell surface.

We have recently shown that inhibition of NO synthesis by L-NAME induces P-selectin expression and facilitates PMN rolling in the rat mesenteric microcirculation and that these effects are significantly reversed by either L-arginine or 8-bromo-cGMP.⁶ However, the precise mechanism(s) by which inhibition of NO synthesis promotes endothelial P-selectin expression is unclear. Moreover, it is not known whether inhibition of NO synthesis also upregulates P-selectin on platelets. This issue is important, because platelets have also been shown to generate NO in a manner that regulates platelet function,¹⁹ and platelet P-selectin plays an important role in inflammatory reactions, such as fibrin deposition, platelet-leukocyte aggregation, thrombus formation, and atherogenesis.^{29 30}

In the present study, not only thrombin or PMA but also L-NAME significantly increased the P-selectin expression on platelets, which was significantly reduced by the selective PKC inhibitor UCN-01. Furthermore, L-NAME significantly stimulated PKC activity in platelets, although this effect was less than that produced by either thrombin or PMA. Because D-NAME failed to stimulate either PKC or P-selectin expression and L-arginine but not D-arginine reversed these effects, the impact of L-NAME is very likely related to the inhibition of NO production in platelets. Furthermore, the NO donor SNP, which increases cGMP content in platelets, completely reverses the L-NAME–induced increase in P-selectin expression. The PKC inhibitor UCN-01 significantly attenuated P-selectin expression by either thrombin or PMA and also attenuated PKC levels in platelets that had been stimulated by these compounds. These results suggest that activation of platelets and increases in P-selectin expression by thrombin, PMA, or L-NAME involve a common signal-transduction pathway that is related to PKC activation. These results are in accord with a previous report by Hannun and coworkers,¹⁵ who have suggested that PKC activation is a necessary and common event in agonist-induced activation of human platelets.

There are several possible mechanisms whereby L-NAME could stimulate PKC in platelets. First, NO can directly inhibit PKC activity by S-nitrosylation of protein thiols.¹⁸ It is then conceivable that if physiological concentrations of NO in platelets reduce PKC activity, inhibition of NO by L-NAME may then result in relative PKC activation. Second, cGMP derived from NO via the action of soluble guanylate cyclase directly inhibits PKC activity.^{9 16 21} Takai and coworkers⁹ have shown that either SNP or 8-bromo-cGMP significantly inhibit PI hydrolysis, diacylglycerol formation, PKC activation, and serotonin release from thrombin-stimulated platelets. Rösen and coworkers³⁷ further suggest that NO and a cGMP analogue can attenuate P-selectin expression in stimulated platelets. In the present study, SNP and 8-bromo-cGMP significantly attenuated P-selectin expression in L-NAME–treated platelets; however, cGMP concentrations in platelet suspensions after treatment with L-NAME were not significantly decreased. Therefore, L-NAME–induced PKC activation and P-selectin expression in platelets may be primarily mediated by direct inhibition by NO and only secondarily or partially by decreased cGMP.

We also considered that when platelets are activated by agonists, L-NAME may interact synergistically with these agonists to further increase P-selectin expression. To test this hypothesis, we examined the effects of co-incubation of platelets with L-NAME and thrombin on PKC activation as well as P-selectin expression. Interestingly, L-NAME significantly enhanced thrombin-induced PKC activation and P-selectin expression compared with thrombin alone. These results indicate that NO inhibition can further enhance PKC activity and P-selectin expression when platelets are stimulated by other agonists. Increases in P-selectin expression induced by thrombin plus L-NAME were also significantly attenuated by PKC inhibition. We thus propose that when platelets are activated, NO may play a more significant regulatory role in preventing cells from further activation. In other words, if biosynthesis of NO is compromised, agonist-induced platelet activation may be enhanced.

We examined whether these relationships also occurred in ECs, another site of P-selectin upregulation. We used a biologic assay system in which autologous PMN adherence to stimulated coronary artery endothelium was examined.²² Previously, we showed that PMN adherence to thrombin- or H₂O₂-stimulated ECs is mediated mainly by a P-selectin–dependent mechanism.²² In the present study, PMN adherence to the coronary artery endothelium was significantly increased by thrombin, PMA, or L-NAME and significantly attenuated by an anti–P-selectin MAb but not by a nonblocking control MAb. These results suggest that P-selectin plays a role in PMN adherence to the endothelium that has been stimulated by these substances. This increase in PMN adherence was significantly attenuated by UCN-01. Furthermore, the L-NAME–induced increase in PMN adherence was significantly attenuated by L-arginine, 8-bromo-cGMP, or SNP but not by D-arginine. Moreover, D-NAME failed to increase PMN adherence to the coronary artery endothelium. Collectively, these results suggest that (1) NO inhibition may significantly promote P-selectin expression and enhance adhesiveness in ECs, presumably by PKC activation, and (2) physiological concentrations of NO in ECs may also reduce PKC activity and act as endogenous regulators of cell function. Because L-NAME significantly enhanced thrombin-induced PMN adherence to the coronary artery endothelium in our study, the NO/cGMP system may also be an important physiological inhibitor of PKC and adhesive interactions in stimulated ECs. In this regard, Draijer and coworkers³⁸ have recently demonstrated that L-NAME significantly enhances thrombin-induced increases in endothelial permeability, suggesting that NO generation indeed modulates EC activity after stimulation by agonists. These recent findings are consistent with the data obtained with platelets in the present study. Our current data extend these findings and provide additional insight into the mechanism of this effect.

Our results do not indicate that PKC activation is the sole factor in determining the extent of P-selectin expression. The precise intracellular mechanism of rapid P-selectin expression remains to be further determined. For example, thrombin-induced signal transduction consists of a variety of signaling pathways in addition to PKC activation.¹¹ Thus, thrombin can activate PI hydrolysis and thereby greatly increase intracellular Ca²⁺ levels. Therefore, although PMA is a more potent stimulator of PKC than is thrombin, the degree of P-selectin expression is similar to that of thrombin in our study.

The biologic properties of NO in this study are particularly important if they occur in vivo. Several reports indicate that this is indeed the case. For example, endothelial dysfunction occurs after coronary artery occlusion/reperfusion, during which both basal and agonist-induced releases of NO are reduced.^{39 40} We recently observed a significant increase in coronary endothelium P-selectin expression 10 to 20 minutes after myocardial reperfusion.²³ Although reperfusion-induced generation of oxygen free radicals, thrombin, and other inflammatory mediators (eg, complement factors) also participate in P-selectin expression,^{6 8} it is likely that diminished NO further stimulates endothelial PKC in concert with these inflammatory stimuli, thus increasing P-selectin expression. In this regard, Numaguchi and coworkers⁴¹ recently reported that PKC activation may be involved in the coronary endothelial dysfunction that occurs after myocardial ischema/reperfusion in rat hearts. Interestingly, PKC downregulates endothelial NO synthase activity by direct phosphorylation, resulting in further decreased production of NO.^{42 43 44} In this connection, we have recently demonstrated that the PKC inhibitor N,N,N-trimethylsphingosine given intravenously protects coronary endothelial function, endothelial P-selectin expression, neutrophil accumulation, and myocardial necrosis during ischemia and reperfusion in vivo.¹⁴

In conclusion, inhibition of NO synthesis by L-NAME promotes P-selectin translocation to the cell surface in platelets and appears to be at least partially mediated by PKC activation. Therefore, NO may participate in regulating the PKC-mediated signal-transduction pathway, thus playing an important physiological role in the regulation of cellular function.

	Selected Abbreviations and Acronyms

 

DPBS = Dulbecco's PBS EC(s) = endothelial cell(s) K-H = Krebs-Henseleit L-NAME = NG-nitro-L-arginine methyl ester MAb(s) = monoclonal antibod(y/ies) PI = phosphatidylinositol PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate PMN(s) = polymorphonuclear leukocyte(s) SNP = sodium nitroprusside

	Acknowledgments

 
This work was supported by research grant No. GM-45434 from the National Institutes of Health, Bethesda, Md. T. Murohara was a recipient of a Japan Heart Foundation Postdoctoral Fellowship. We thank Dr J.C. Paulson, Cytel Corp, San Diego, Calif, for the generous supply of anti–P-selectin MAb PB1.3. We thank Robert Craig and John Margiotta for their expert technical assistance during the course of these investigations.

Received April 6, 1995; accepted September 19, 1995.

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