Reoxygenation stimulates EDRF(s) release from endothelial cells of rabbit aorta

We have reported that hypoxia stimulates EDRF(s) release from endothelial cells and the release may be augmented by previous hypoxia. As a mechanism, it was hypothesized that reoxygenation can stimulate EDRF(s) release from endothelial cells and we tested the hypothesis via bioassay experiment. In the bioassay experiment, rabbit aorta with endothelium was used as EDRF donor vessel and rabbit carotid artery without endothelium as a bioassay test ring. The test ring was contracted by prostaglandin F2a (3 X 10-6 M) which was added to the solution perfusing through the aorta. Hypoxia was evoked by switching the solution aerated with 95% O2/5% CO2 mixed gas to one aerated with 95% N2/5% CO2 mixed gas Hypoxia/reoxygenation were interexchanged at intervals of 2 minutes (intermittent hypoxia). In some experiments, endothelial cells were exposed to 10-minute hypoxia (continuous hypoxia) and then exposed to reoxygenation and intermittent hypoxia. In other experiments, the duration of re oxygenation was extended from 2 minutes to 5 minutes. When the donor aorta was exposed to intermittent hypoxia, hypoxia stimulated EDRF(s) release from endothelial cells and the hypoxia-induced EDRF(s) release was augmented by previous hypoxia/reoxygenation. When the donor aorta was exposed to continuous hypoxia, there was no increase of hypoxia-induced EDRF(s) release during hypoxia. But, after the donor aorta was exposed to reoxygenation, hypoxia-induced EDRF(s) release was markedly increased. When the donor aorta was pretreated with nitro-L-arginine (10-5 M for 30 minutes), the initial hypoxia-induced EDRF(s) release was almost completely abolished, but the mechanism for EDRF(s) release by the reoxygenation and subsequent hypoxia still remained to be clarified. TEA also blocked incompletely hypoxia-induced and hypoxia/reoxygenation-induced EDRF(s) release EDRF(s) release by repetitive hypoxia and reoxygenation was completely blocked by the combined treatment with nitro-L-arginine and TEA. Cytochrome P450 blocker, SKF-525A, inhibited the EDRF(s) release reversibly and endothelin antgonists, BQ 123 and BQ 788, had no effect on the release of endothelium-derived vasoactive factors. Superoxide dismutase (SOD) and catalase inhibited the EDRF(s) release from endothelial cells. From these data, it could be concluded that reoxygenation stimulates EDRF(s) release and hypoxia/reoxygenation can release not only NO but also another EDRF from endothelial cells by the production of oxygen free radicals.

Different mechanisms for K+-induced relaxation in various arteries

(K+)o can be increased under a variety of conditions including subarachnoid hemorrhage. The increase of (K+)o in the range of 5 ~ 15 mM may affect tensions of blood vessels and cause relaxation of agonist-induced precontracted vascular smooth muscle (K+-induced relaxation). In this study, effect of the increase in extracellular K+ concentration on the agonist-induced contractions of various arteries including resistant arteries of rabbit was examined, using home-made Mulvany-type myograph. Extracellular K+ was increased in three different ways, from initial 1 to 3 mM, from initial 3 to 6 mM, or from initial 6 to 12 mM. In superior mesenteric arteries, the relaxation induced by extracellular K+ elevation from initial 6 to 12 mM was the most prominent among the relaxations induced by the elevations in three different ways. In cerebral arteries, the most prominent relaxation was produced by the elevation of extracellular K+ from initial 1 to 3 mM and a slight relaxation wasp rovoked by the elevation from initial 6 to 12 mM. In superior mesenteric arteries, K+-induced relaxation by the elevation from initial 6 to 12 mM was blocked by Ba2+ (30 muM) and the relaxation by the elevation from 1 to 3 mM or from 3 to 6 mM was not blocked by Ba2+. In cerebral arteries, however, K+-induced relaxation by the elevation from initial 3 to 6 mM was blocked by Ba2+, whereas the relaxation by the elevation from 1 to 3 mM was not blocked by Ba2+. Ouabain inhibited all of the relaxations induced by the extracellular K+ elevations in three different ways. In cerebral arteries, when extracellular K+ was increased to 14 mM with 2 or 3 mM increments, almost complete relaxation was induced at 1 or 3 mM of initial K+ concentration and slight relaxation occurred at 6 mM. TEA did not inhibit Ba2+/-sensitive relaxation at all and NMMA or endothelial removal did not inhibit K+-induced relaxation. Most conduit arteries such as aorta, carotid artery, and renal artery were not relaxed by the elevation of extracellular K+. Among conduit arteries, trunk of superior mesenteric artery and basilar artery were relaxed by the elevations of (K+)o. These data suggest that K+-induced relaxation has two independent components, Ba2+-sensitive and Ba2+-insensitive one and there are different mechanisms for K+-induced relaxation in various arteries.

Sensitivity of rabbit cerebral artery to serotonin is increased with the moderate increase of extracellular K+

(K+)O can be increased under a variety of conditions including subarachnoid hemorrhage. The increase of (K+)O in the range of 5~15 mM may affect tensions of blood vessels and can change their sensitivity to various vasoactive substances. Therefore, it was examined in the present study whether the sensitivity of cerebral arteries to vasoactive substances can be changed with the moderate increase of (K+)O, using Mulvany-type myograph and (Ca2+)c measurement. The contractions of basilar artery and branch of middle cerebral artery induced by histamine were not increased with the elevation of (K+)O from 6 mM to 9 mM or 12 mM. On the contrary, the contractions induced by serotonin were significantly increased with the elevation of (K+)O. The contractions were also significantly increased by the treatment with nitro-L-arginine (10-4 M for 20 minutes). In the nitro-L-arginine treated arteries, the contractions induced by serotonin were significantly increased with the elevation of (K+)O from 6 mM to 12 mM. K+-induced relaxation was evoked with the stepwise increment of extracellular K+ from 0 or 2 mM to 12 mM by 2 mM in basilar arterial rings, which were contracted by histamine. But (K+)O elevation from 4 or 6 mM to 12 mM by the stepwise increment evoked no significant relaxation. Basal tension of basilar artery was increased with (K+)O elevation from 6 mM to 12 mM by 2 mM steps or by the treatment with ouabain and the increase of basal tension was blocked by verapamil. The cytosolic free Ca2+ level was not increased by the single treatment with serotonin or with the elevation of (K+)O from 4 mM to 8 or 12 mM. In contrast to the single treatment, the Ca2+ level was increased by the combined treatment with serotonin and the elevation of (K+)O. The increase of free Ca2+ concentration was blocked by the treatment with verapamil. These data suggest that the sensitivity of cerebral artery to serotonin is increased with the moderate increase of (K+)O and the increased sensitivity to serotonin is due to the increased (Ca2+)i induced by extracellular Ca2+ influx.