Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells

Abstract

Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632, suggesting protease-activated receptor coupling to Gq and G12/13, respectively. These data suggest a vascular bed specific differential coupling of protease-activated receptors to the signaling pathways that regulate endothelial nitric oxide synthase and nitric oxide production that may be responsible for endothelial dysfunction associated with cardiovascular disease.

Keywords: Endothelial nitric-oxide synthase; endothelial dysfunction; nitric oxide; protease-activated receptors; thrombin.

Figure 1

Effects of thrombin on eNOS phosphorylation and NO production in HCAECs (a) HCAECs were stimulated with thrombin (10 U/ml) for the indicated time points and phosphorylated eNOS-ser1177 and eNOS-Thr495 were determined using Western blot analysis with phosphospecific antibodies. Representative immunoblots are shown with densitometric analyses. Mean +/− SEM of measurements in cultures of five different passages; ****P < 0.0001 Thrombin 0.5 min versus Basal. (b) NO production was determined indirectly by measuring total nitrate/nitrite using media from HCAECs treated with thrombin (10 U/ml) for the indicated time points. n = 5, ***P < 0.05 Thrombin versus Basal. (c) Cells were pretreated with SCH-79797 (10 µmol/L) or vehicle (0.01% DMSO) for 1 h prior to stimulation with thrombin (10 U/ml) for 0.5 min. Phosphorylated eNOS-Ser1177 and eNOS-Thr495 were determined using Western blot analysis with phosphospecific antibodies. Representative immunoblots are shown with densitometric analyses. Mean +/− SEM of measurements in cultures of four different passages; *P < 0.001 Thrombin versus Vehicle. (d) Total nitrate/nitrite production was measured using media from HCAECs pretreated with SCH-79797 (10 µmol/L) or vehicle (0.01% DMSO) for 1 h prior to stimulation with thrombin (10 U/ml) for 10 min n = 3, **P < 0.05 Thrombin versus Basal and SCH + Thrombin 10 min

Figure 2

Effects of PAR-1 activating peptide on eNOS phosphorylation and NO production in HCAECs (a) HCAECs were stimulated with TFLLR (10 µmol/L) for the indicated time points and phosphorylated eNOS-Ser1177 and eNOS-Thr495 were determined using Western blot analysis with phosphospecific antibodies ****P < 0.0001 TFLLR 0.5 min versus Basal, ***P < 0.001 TFLLR Thr495p 0.5 min, 2 min and 10 min versus Basal. (b) NO production was determined indirectly by measuring total nitrate/nitrite using media from HCAECs treated with TFLLR (10 µmol/L) for the indicated time points n = 3, ****P < 0.0001 TFLLR 30 min versus Basal. (c) Cells were pretreated with SCH-79797 (10 µmol/L) or vehicle (0.01% DMSO) for 1 hour prior to stimulation with TFLLR (10 µmol/L) for 0.5 min. Phosphorylated eNOS-Ser1177 was determined using Western blot analysis with phosphospecific antibody. Representative immunoblots are shown with densitometric analyses. Mean +/− SEM of measurements in cultures of six different passages; **P < 0.001 TFLLR 0.5 min versus Vehicle, *P < 0.005 TFLLR versus SCH + TFLLR 0.5 min. (d) Total nitrate/nitrite production was measured using media from HCAECs pretreated with SCH-79797 (10 µmol/L) or vehicle (0.01% DMSO) for 1 hour prior to stimulation with TFLLR (10 µmol/ml) for 30 min n = 3, *P < 0.01 TFLLR 30 min versus Vehicle; TFLLR 30 min versus SCH + TFLLR 30 min

Figure 3

Effects of PAR-2 activating peptide on eNOS phosphorylation and NO production in HCAECs (a) HCAECs were stimulated with SLIGRL (50 µmol/L) for the indicated time points and phosphorylated eNOS-ser1177 and eNOS-Thr495 was determined using Western blot analysis with phosphospecific antibodies. Representative immunoblots are shown with densitometric analyses. Mean +/− SEM of measurements in cultures of six different passages; ***P < 0 .001 SLIGRL Thr495p 10 min versus Basal, *P < 0.05 SLIGRL 15 min and 30 min versus Basal, *P < 0.05 SLIGRL Ser1177p 0.5 min and 2 min versus Basal. (b) NO production was determined indirectly by measuring total nitrate/nitrite using media from HCAECs treated with SLIGRL (50 µmol/L) for the indicated time points, n = 6, NS

Figure 4

Effects of PAR-3 activating peptide on eNOS phosphorylation and NO production in HCAECs (a) HCAECs were stimulated with TFRGAP (500 µmol/L) for the indicated time points and phosphorylated eNOS-Ser1177 and eNOS-Thr495 was determined using Western blot analysis with phosphospecific antibodies. Representative immunoblots are shown with densitometric analyses. Mean +/− SEM of measurements in cultures of four different passages; *P < 0.05 TFRGAP Thr495p 10 min versus Basal. (b) NO production was determined indirectly by measuring total nitrate/nitrite using media from HCAECs treated with TFRGAP (500 µmol/L) for the indicated time points, n = 4, NS

Figure 5

Effects of NOS inhibitor, L-NAME, on PAR-1 mediated NO production in HCAECs. (a) HCAECs were pretreated with L-NAME (100 µmol/L) for 1 h prior to stimulation with thrombin (10 U/ml) for 0.5 min and 10 min. Phosphorylation of eNOS-Ser1177 was determined using Western blot analysis with phosphospecific antibody. Representative immunoblots are shown with densitometric analyses. Mean +/− SEM of measurements in cultures three of different passages; *P < 0.05 Thrombin 0.5 min versus Basal, **P < 0.01 L-NAME + Thrombin 0.5 min versus Basal. (b) NO production was determined indirectly by measuring total nitrate/nitrite using media from HCAECs pretreated with L-NAME (100 µmol/L) for 1 h prior to stimulation with thrombin (10 U/ml) for 0.5 min and 10 min ****P < 0.001 Thrombin 0.5 min versus Basal, ****P < 0.001 L-NAME + Thrombin 0.5 min versus Basal. (c) HCAECs were pretreated with L-NAME (100 µmol/L) for 1 h prior to stimulation with TFLLR (10 µmol/ml) for 0.5 min and 10 min. Phosphorylation of eNOS-Ser1177 was determined using Western blot analysis with phosphospecific antibody. Representative immunoblots are shown with densitometric analyses, n = 3, ****P < 0.0001 TFLLR 0.5 min and L-NAME + TFLLR 0.5 min versus Basal. (d) Total nitrate/nitrite production was measured using media from HCAECs pretreated with L-NAME (100 µmol/L) for 1 h prior to stimulation with TFLLR (10 µmol/L) for 0.5 min and 30 min, n = 3, TFLLR 0.5 min, no detection (nd) ****P < 0.0001 TFLLR 30 min and L-NAME + TFLLR 30 min versus basal

Figure 6

Effects of calcium inhibitor, BAPTA, on PAR-mediated eNOS-Ser-1177 phosphorylation in HCAECs. HCAECs were pretreated with BAPTA-AM (10 µmol/L) for 30 min and then stimulated with (a) thrombin (10 U/ml) and (b) TFLLR (10 µmol/L) for 0.5 min, and (c) SLIGRL (50 µmol/L) for 2 min. Phosphorylation of eNOS-Ser1177 was determined using Western blot analysis with phosphospecific antibody. Representative immunoblots are shown with densitometric analyses. Mean +/− SEM of measurements in cultures of three different passages; *P < 0.05 Thrombin versus BAPTA + Thrombin and TFLLR versus BAPTA + TFLLR, **P < 0.001 SLIGRL versus BAPTA + SLIGRL, n = 3

Figure 7

Effects of ROCK inhibitor, Y-27632, on PAR-mediated eNOS-Thr-495 phosphorylation in HCAECs. HCAECs were pretreated with Y-27632 (10 µmol/L) for 90 min and then stimulated with (a) thrombin (10 U/ml) for 10 min, (b) TFLLR (10 µmol/L) for 2 min, (c) SLIGRL (50 µmol/L) for 10 min and (d) TRAGAP (500 µmol/L) for 10 min. Phosphorylation of eNOS-Thr-495 was determined using Western blot analysis with phosphospecific antibody. Representative immunoblots are shown with densitometric analyses. Mean +/− SEM of measurements in cultures of three different passages; *P < 0.05 TFLLR 2 min, SLIGRL 10 min, TFRGAP 10 min versus Basal, or Y-27 versus Y-27 + TFLLR 2 min, SLIGRL 10 min, or TFRGAP 10 min, n = 3

Figure 8

Proposed signal transduction cascade of PAR-1, PAR-2 and PAR-3 in Adult HCAECs. In adult HCAECs, PAR-1 and PAR-2 couple to both G_q and G_12/13 to regulate the signaling pathways that phosphorylate eNOS and cause NO production. A Ca²⁺-dependent-mediated mechanism, which is coupled to G_q, leads to the phosphorylation of eNOS-Ser-1177 and causes an increase in NO production. A ROCK-dependent pathway, which is coupled to G_12/13, leads to eNOS-Thr-495 phosphorylation and a decrease in NO production. PAR-3 activation is coupled to G_12/13 and the ROCK signaling pathway, which leads to a suppression of NO production via phosphorylation of eNOS-Thr-495. (A color version of this figure is available in the online journal.)