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To investigate the possibility that smoke also induces endothelial dysfunction, a finding common to other forms of pulmonary hypertension, we exposed guinea pigs to smoke or air (control) daily for 2 wk and then prepared precision-cut lung slices. After exposure to endothelin-1, a vasoconstrictor, intra-acinar arteries in lung slices derived from smoke-exposed animals constricted more rapidly (greater constriction at a given concentration of endothelin) than did vessels from air-exposed animals. To examine relaxation responses, arteries were constricted with the vasconstrictor U-46619 and then relaxed with progressively increasing doses of acetylcholine. Vessels from smokers had a delayed response to acetylcholine compared with vessels from controls. The NO synthase inhibitor N<jats:sup>G<\/jats:sup>-nitro-l-arginine methyl ester reduced relaxation in both control and smoke-exposed arteries, whereas the NO donor sodium nitroprusside increased relaxation of the smoke-exposed arteries, confirming that endothelial dysfunction with decreased effective NO production is present. These findings show that precision cut lung slices can be used to examine the physiological effects of cigarette smoke on intra-acinar pulmonary arteries and indicate that even relatively short-term exposure to smoke produces endothelial dysfunction with a resulting tendency to earlier constriction and later relaxation in cigarette smokers. 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