The possible roles of endothelial and smooth muscle cell hyperpolarization and nitric oxide (NO) in endothelium-dependent relaxation were examined in isolated rings of pig right coronary artery. 2. The effects of hyperpolarization were prevented with high K+ (30-125 mM), isotonic Krebs solutions. Functional antagonism due to high K(+)-induced smooth muscle contraction was prevented with 0.3 microM nifedipine (in all treatments, for consistency). All rings were contracted with the thromboxane-mimetic U46619, (1-100 nM) to bring them to an initial active force of within 30-50% of maximum contraction. 3. High K+ had no effects on the sensitivity (EC50) or time course of endothelium-dependent (substance P, SP; bradykinin, BK; calcimycin, A23187) and -independent (sodium nitroprusside, SNP) agents. Maximum relaxations (Rmax) to SP, BK and A23187 were reduced significantly by approximately 20% but only with 125 mM K+. 4. In normal K+ Krebs solution (5.9 mM), NG-nitro-L-arginine (L-NOARG; 100 microM) caused 40%, 20% and no reduction in Rmax for SP, BK and SNP respectively. EC50s for SP and BK were decreased significantly by approximately two fold whereas that for SNP was increased significantly by approximately ten fold. At all high K+ concentrations (30-125 mM), L-NOARG (100 microM) caused complete inhibition of relaxations to SP and BK but those to SNP were unaffected. 5. High K+ (30 mM) unmasked potent and concentration-dependent inhibition of relaxations of SP by L-NOARG. At 10 microM L-NOARG, all relaxation responses to SP were abolished and at the higher concentrations of SP (1-10 nM) small but significant contractions were observed. 6. N0-monomethyl-L-arginine (L-NMMA) had similar effects on relaxations to SP in the presence of 30 mM K+ except that maximum inhibition (40%) of Rmax was achieved at 10 MicroM L-NMMA and this was not increased with either 100 or 1000 MicroM L-NMMA. In normal K+, L-NMMA (1000 MicroM) only decreased the EC50 by approximately two fold, without affecting Rmax.7. High choline+ (25, 75 and 125 mM) isotonic Krebs also had no direct effect on the relaxations to SP,but like high K+, enabled L-NOARG (100 MicroM) to inhibit these responses completely. Neither charybdotoxin(30 nM) nor substitution of 25 mM NaCl with 50 mM sucrose had any direct effect on relaxations to SP or on the block of relaxations to SP by L-NOARG (100 MicroM).8. In conclusion, most if not all of the endothelium-dependent relaxation in the pig coronary artery in vitro is due to NO, but hyperpolarization can supplement 60% -80% of this response if NO synthesis is blocked. Multiple endothelium-derived factors could not only explain heterogeneity of the degree of block of endothelium-dependent relaxation responses by L-arginine analogues, but also constitute important 'back-up' mechanisms for control of arterial diameter.
摘要
在猪右冠状动脉离体血管环中研究了内皮细胞和平滑肌细胞超极化以及一氧化氮（NO）在内皮依赖性舒张中的可能作用。2. 用高钾（30 - 125 mM）等渗 Krebs 溶液可防止超极化的影响。为保持一致性，在所有处理中，用 0.3 μM 硝苯地平可防止高钾（K⁺）诱导的平滑肌收缩所导致的功能性拮抗作用。所有血管环均用血栓素类似物 U46619（1 - 100 nM）收缩，使其初始主动张力达到最大收缩力的 30 - 50%。3. 高钾对内皮依赖性（P 物质，SP；缓激肽，BK；离子霉素，A23187）和非内皮依赖性（硝普钠，SNP）药物的敏感性（EC50）或时间进程无影响。对 SP、BK 和 A23187 的最大舒张（Rmax）显著降低约 20%，但仅在 125 mM 钾时出现。4. 在正常钾（5.9 mM）的 Krebs 溶液中，NG - 硝基 - L - 精氨酸（L - NOARG；100 μM）分别使 SP、BK 和 SNP 的 Rmax 降低 40%、20%和无降低。SP 和 BK 的 EC50 显著降低约两倍，而 SNP 的 EC50 显著增加约十倍。在所有高钾浓度（30 - 125 mM）下，L - NOARG（100 μM）完全抑制对 SP 和 BK 的舒张，但对 SNP 的舒张无影响。5. 高钾（30 mM）揭示了 L - NOARG 对 SP 舒张的强效且浓度依赖性抑制作用。在 10 μM L - NOARG 时，对 SP 的所有舒张反应均被消除，在较高浓度的 SP（1 - 10 nM）时观察到小但显著的收缩。6. 在存在 30 mM 钾的情况下，N⁰ - 甲基 - L - 精氨酸（L - NMMA）对 SP 舒张的影响类似，不同的是在 10 μM L - NMMA 时达到最大抑制（40%）的 Rmax，且在 100 或 1000 μM L - NMMA 时未增加。在正常钾条件下，L - NMMA（1000 μM）仅使 EC50 降低约两倍，而不影响 Rmax。7. 高胆碱⁺（25、75 和 125 mM）等渗 Krebs 溶液对 SP 舒张也无直接影响，但与高钾一样，能使 L - NOARG（100 μM）完全抑制这些反应。大蝎毒素（30 nM）或用 50 mM 蔗糖替代 25 mM NaCl 对 SP 舒张或 L - NOARG（100 μM）对 SP 舒张的阻断均无直接影响。8. 总之，体外猪冠状动脉中大部分（如果不是全部）内皮依赖性舒张是由 NO 引起的，但如果 NO 合成被阻断，超极化可补充该反应的 60% - 80%。多种内皮衍生因子不仅可以解释 L - 精氨酸类似物对内皮依赖性舒张反应阻断程度的异质性，还构成控制动脉直径的重要“备用”机制。
