To clarify the mechanisms involved in relaxations mediated by endothelium-derived hyperpolarizing factor (EDHF), acetylcholine (ACh)-induced endothelium-dependent relaxations and hyperpolarizations were examined in the rat aorta, the main branch of the mesenteric artery (MBMA) and the first branch of the mesenteric aftery (FBMA). In the presence of 100 μM NG-nitro-L-arginine (L-NNA) and 10 μM indomethacin, ACh (1 nM to 100 μM) produced no relaxation in the phenylephrine-precontracted aorta. The L-NNA-resistant relaxations by ACh in MBMA precontracted with phenylephrine were eliminated in the presence of 1 μM nifedipine where contractions were independent of L-type Ca2+ channel activation. In FBMA precontracted with phenylephrine, the L-NNA-resistant relaxations were only partially inhibited by nifedipine. When vessels had been contracted with 300 nM phorbol-12,13-dibutyrate in the presence of nifedipine, ACh-induced L-NNA-resistant relaxations were observed in FBMA only. Pinacidil produced relaxations in all different-sized blood vessels, although sensitivity was inversely related to vessel size. The extent of the ACh hyperpolarizing responses was much smaller than that by pinacidil in the aorta. The membrane potential changes by ACh and pinacidil were almost the same in FBMA. These results indicate that the contribution of EDHF to endothelium-dependent relaxations increases as the vessel size decreases. This may be partly explained by precontractile processes dependent on Ca2+ entry through L-type Ca2+ channels, because Ca2+ channel deactivation seems to be involved as a major mechanism of EDHF-mediated vasorelaxations. However, EDHF may also generate vasorelaxations by an additional mechanism, probably a reduced Ca2+ sensitivity of contractile elements, as proposed for ATP-sensitive K+ channel openers.

Keywords:
Endothelium-dependent relaxation, Membrane hyperpolarization, Endothelium-derived hyperpolarizing factor, Pinacidil, Nifedipine, Phorbol ester, Rat vascular smooth muscle
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