Background-Recent studies in vitro have demonstrated that endothelium-derived hydrogen peroxide (H 2 O 2 ) is an endothelium-derived hyperpolarizing factor (EDHF) in animals and humans. The aim of this study was to evaluate our hypothesis that endothelium-derived H 2 O 2 is an EDHF in vivo and plays an important role in coronary autoregulation. Methods and Results-To test this hypothesis, we evaluated vasodilator responses of canine (nϭ41) subepicardial small coronary arteries (Ն100 m) and arterioles (Ͻ100 m) with an intravital microscope in response to acetylcholine and to a stepwise reduction in coronary perfusion pressure (from 100 to 30 mm Hg) before and after inhibition of NO synthesis with N G -monomethyl-L-arginine (L-NMMA). After L-NMMA, the coronary vasodilator responses were attenuated primarily in small arteries, whereas combined infusion of L-NMMA plus catalase (an enzyme that selectively dismutates H 2 O 2 into water and oxygen) or tetraethylammonium (TEA, an inhibitor of large-conductance K Ca channels) attenuated the vasodilator responses of coronary arteries of both sizes. Residual arteriolar dilation after L-NMMA plus catalase or TEA was largely attenuated by 8-sulfophenyltheophylline, an adenosine receptor inhibitor. Conclusions-These results suggest that H 2 O 2 is an endogenous EDHF in vivo and plays an important role in coronary autoregulation in cooperation with NO and adenosine.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several relaxing factors, such as prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have previously demonstrated in animals and humans that endothelium-derived hydrogen peroxide (H 2 O 2 ) is an EDHF that is produced in part by endothelial NO synthase ( eNOS). In this study, we show that genetic disruption of all three NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]) abolishes EDHF responses in mice.The contribution of the NOS system to EDHFmediated responses was examined in eNOS ؊ / ؊ , n/eNOS ؊ / ؊ , and n/i/eNOS ؊ / ؊ mice. EDHFmediated relaxation and hyperpolarization in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased, whereas vascular smooth muscle function was preserved. Loss of eNOS expression alone was compensated for by other NOS genes, and endothelial cell production of H 2 O 2 and EDHF-mediated responses were completely absent in n/i/eNOS ؊ / ؊ mice, even after antihypertensive treatment with hydralazine. NOS uncoupling was not involved, as modulation of tetrahydrobiopterin (BH 4 ) synthesis had no effect on EDHF-mediated relaxation, and the BH 4 /dihydrobiopterin (BH 2 ) ratio was comparable in mesenteric arteries and the aorta. These results provide the fi rst evidence that EDHF-mediated responses are dependent on the NOSs system in mouse mesenteric arteries.

We developed a portable needle-probe videomicroscope with a charge-coupled device (CCD) camera to visualize the subendocardial microcirculation. In 12 open-chest anesthetized pigs, the sheathed needle probe with a doughnut-shaped balloon and a microtube for flushing away the intervening blood was introduced into the left ventricle through an incision in the left atrial appendage via the mitral valve. Images of the subendocardial microcirculation of the beating heart magnified by 200 or 400 on a 15-in. monitor were obtained. The phasic diameter change in subendocardial arterioles during cardiac cycle was from 114±46 ,um (mean+SD) in end diastole to 84±26 ,um in end systole (p<0.001, n=13, ratio of change=24%) and that in venules from 134±60 ,m to 109±45 ,um (p<0.001, n=15, ratio of change=17%). In contrast, the diameter of subepicardial arterioles was almost unchanged (2% decrease, n=5, p<0.01), and the venular diameter increased by 19%1 (n=8, p<0.001) from end diastole to end systole. Partial kinking and/or pinching of vessels was observed in some segments of subendocardial arterioles and venules. The percentage of systolic decrease in the diameter from diastole in the larger (>100 ,um) subendocardial arterioles and venules was greater than smaller (50-100 ,m) vessels (both p<0.05). In conclusion, using a newly developed microscope system, we were able to observe the subendocardial vessels in diastole and systole. The vascular compression by cardiac contraction decreased the diameters of subendocardial arterioles and venules by about 20o, whereas subepicardial arterial diameter changed very little during the cardiac cycle and subepicardial venules increased in diameter during systole. (Circulation Research 1993;72:939-946) KEY WORDS * subendocardial microcirculation heart * needle-probe videomicroscope he phasic flows in the left coronary artery and T vein are unlike those of other organs; the arterial inflow is greatest during diastole, whereas the venous outflow is greatest during systole.1-6 This unique pattern of coronary arterial and venous flow was inferred in 1695 by Scaramucci,7 who is considered the founder of coronary physiology. He hypothesized that the myocardial vessels are squeezed by the contraction of the muscle fibers around them, which