Evidence for differential roles of nitric oxide (NO) and hyperpolarization in endothelium-dependent relaxation of pig isolated coronary artery

Abstract

1. 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.