Figure 2.
Figure 2. NSAIDs enhance the inhibitory effect of L-NAME on aortic ring relaxation to acetylcholine, and platelet P-selectin expression is significantly elevated in mice given both L-NAME and celecoxib but decreased following L-NAME and indomethacin. (A) Effect of celecoxib on L-NAME inhibition of relaxation. Aortic ring relaxation responses to acetylcholine were determined. Control (▪), L-NAME (300 μM; ○), celecoxib (10 μM; ▵), or L-NAME (300 μM) and celecoxib (10 μM; ▴). (B) Effect of indomethacin on L-NAME inhibition of relaxation. Aortic ring relaxation responses to acetylcholine were determined. Control (▪), L-NAME (300 μM; ○), indomethacin (10 μM; ▵), or L-NAME (300 μM) and indomethacin (10 μM; ▴). (C) Effect of aspirin on L-NAME inhibition of relaxation. Aortic ring relaxation responses to acetylcholine were determined. Control (▪), L-NAME (300 μM; ○), aspirin (10 μM;▵), or L-NAME (300 μM) and aspirin (10 μM;▴). *P < .05 compared with wild-type group, using 2-wayANOVAto isolate differences between groups. For all experiments, n = 5-9 per group, mean ± SEM. (D) Effect of celecoxib on constriction of endothelium-denuded rings. Aortic ring constriction responses to phenylephrine were determined (n = 5-9 per group). WT denuded (▪), WT denuded + celecoxib (10 μM; ▴). (E) Effect of celecoxib on relaxation of endothelium-denuded rings. Aortic ring relaxation responses to acetylcholine were determined (n = 5-9 per group). WT denuded (▴), WT denuded + celecoxib (10 μM; ▾). (F) Immunohistochemistry of COX-2 in aortic rings. Rings were stained using anti–COX-2 (i) or isotype control IgG (ii). Inset shows corresponding transmission images. (G) Identification of platelets in murine whole blood. Mice were administered L-NAME, indomethacin, or both or celecoxib for 3 days. Platelets were gated in mouse whole blood by FACS analysis using anti–mouse αIIβ-FITC (shown as R1). (H) Representative data showing P-selectin expression following 3 days' administration with celecoxib and L-NAME. Platelets identified by αIIβ expression as in panel G were analyzed for P-selectin expression using anti–mouse P-selectin–FITC, or isotype rat anti–IgG-FITC. (I) Platelet P-selectin expression is significantly increased on day 3 following coadministration of celecoxib and L-NAME. Percentage of P-selectin–expressing cells were defined as those in M1, shown on Panel B with subtraction of percentage of isotype values from all samples. (J) Platelet P-selectin expression is significantly decreased on day 3 following coadministration of indomethacin and L-NAME. Percentage of P-selectin–expressing cells were defined as those in M1, shown on Panel B with subtraction of percentage of isotype values from all samples (for both panels, n = 5, mean ± SEM, *P < .05 compared with control using Student 2-tailed t test).

NSAIDs enhance the inhibitory effect of L-NAME on aortic ring relaxation to acetylcholine, and platelet P-selectin expression is significantly elevated in mice given both L-NAME and celecoxib but decreased following L-NAME and indomethacin. (A) Effect of celecoxib on L-NAME inhibition of relaxation. Aortic ring relaxation responses to acetylcholine were determined. Control (▪), L-NAME (300 μM; ○), celecoxib (10 μM; ▵), or L-NAME (300 μM) and celecoxib (10 μM; ▴). (B) Effect of indomethacin on L-NAME inhibition of relaxation. Aortic ring relaxation responses to acetylcholine were determined. Control (▪), L-NAME (300 μM; ○), indomethacin (10 μM; ▵), or L-NAME (300 μM) and indomethacin (10 μM; ▴). (C) Effect of aspirin on L-NAME inhibition of relaxation. Aortic ring relaxation responses to acetylcholine were determined. Control (▪), L-NAME (300 μM; ○), aspirin (10 μM;▵), or L-NAME (300 μM) and aspirin (10 μM;▴). *P < .05 compared with wild-type group, using 2-wayANOVAto isolate differences between groups. For all experiments, n = 5-9 per group, mean ± SEM. (D) Effect of celecoxib on constriction of endothelium-denuded rings. Aortic ring constriction responses to phenylephrine were determined (n = 5-9 per group). WT denuded (▪), WT denuded + celecoxib (10 μM; ▴). (E) Effect of celecoxib on relaxation of endothelium-denuded rings. Aortic ring relaxation responses to acetylcholine were determined (n = 5-9 per group). WT denuded (▴), WT denuded + celecoxib (10 μM; ▾). (F) Immunohistochemistry of COX-2 in aortic rings. Rings were stained using anti–COX-2 (i) or isotype control IgG (ii). Inset shows corresponding transmission images. (G) Identification of platelets in murine whole blood. Mice were administered L-NAME, indomethacin, or both or celecoxib for 3 days. Platelets were gated in mouse whole blood by FACS analysis using anti–mouse αIIβ-FITC (shown as R1). (H) Representative data showing P-selectin expression following 3 days' administration with celecoxib and L-NAME. Platelets identified by αIIβ expression as in panel G were analyzed for P-selectin expression using anti–mouse P-selectin–FITC, or isotype rat anti–IgG-FITC. (I) Platelet P-selectin expression is significantly increased on day 3 following coadministration of celecoxib and L-NAME. Percentage of P-selectin–expressing cells were defined as those in M1, shown on Panel B with subtraction of percentage of isotype values from all samples. (J) Platelet P-selectin expression is significantly decreased on day 3 following coadministration of indomethacin and L-NAME. Percentage of P-selectin–expressing cells were defined as those in M1, shown on Panel B with subtraction of percentage of isotype values from all samples (for both panels, n = 5, mean ± SEM, *P < .05 compared with control using Student 2-tailed t test).

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