The role of endothelial nitric oxide synthase (eNOS) uncoupling on leukocyte‐endothelial interactions in rat mesenteric postcapillary venules

Endothelial derived nitric oxide (NO) is essential in the regulation of blood pressure and attenuates leukocyte‐endothelial interactions associated with vascular injury. Endothelial NO synthase (eNOS) is coupled to L‐arginine in the presence of tetrahydrobiopetrin (BH 4 ) to produce NO. However, when BH 4 is oxidized to dihydrobiopetrin (BH 2 ) under conditions of oxidative stress, the ratio of BH 2 to BH 4 is increased causing the uncoupling of eNOS to use molecular oxygen as a substrate, instead of L‐arginine, to produce superoxide. This study examined the role of eNOS uncoupling by superfusing BH 2 (100 or 200 μM) by itself and BH 2 (100 μM) combined with BH 4 (100 μM) in rat mesenteric venules on leukocyte rolling, adherence, and transmigration by using intravital microscopy. The effects of BH 2 were compared to Krebs' buffer, to NOS inhibitor N G ‐nitro‐L‐arginine methyl ester (L‐NAME, 50 μM), and to the combination of BH 2 /BH 4 . We found that superfusion of BH 2 (100 μM n=6, 200 μM n=5, both P<0.05) significantly increased leukocyte rolling, adherence, and transmigration, similar to L‐NAME (n=5, P<0.05), within a 2 hr period compared to Krebs' buffer control rats (n=5, P<0.05). The BH 2 induced response was significantly attenuated by BH 4 (n=, P<0.05). The data suggest that eNOS uncoupling may be an important mechanism mediating inflammation induced vascular injury. This study was supported by the Center for Chronic Disorders of Aging and the Department of Pathology/Microbiology/Immunology & Forensic Medicine at PCOM.