Central role of intracellular calcium stores in acute flow- and agonist-evoked endothelial nitric oxide release

1 We have used a cascade bioassay system and isolated arterial ring preparations to investigate the contribution of Ca2+ release from endothelial intracellular stores to nitric oxide (NO) production evoked by increases in shear stress and by acetylcholine in rabbit aorta. 2 Experiments were performed before and following incubation with either the endoplasmic reticulum Ca2+-ATPase inhibitors cyclopiazonic acid (CPA, 10 mu M) and thapsigargin (TSG, 1 mu M) or ryanodine (30, 100 mu M) which binds to a specific endoplasmic reticulum Ca2+-release channel. 3 In cascade bioassay all three agents induced relaxations of the recipient ring (CPA, 24.4 +/- 3.8%; TSG, 51.5 +/- 10.6%; ryanodine, 17.4 +/- 1.6%) which were significantly attenuated by preincubation of the donor with 100 mu M N-G-nitro-L-arginine methyl ester (L-NAME). However, in isolated rings, only CPA and TSG induced L-NAME-sensitive relaxations (CPA 52.7 +/- 6.5%; TSG 61.3 +/- 7%). 4 Addition of superoxide dismutase (SOD) to the donor perfusate evoked relaxations of the recipient ring in cascade bioassay (13.3 +/- 1.4%, n=22). Prior administration of SOD attenuated relaxations to TSG 23.2 +/- 3.8%, n=4) and ryanodine (1.7 +/- 0.8%, n=4), and pre-incubation with TSG and ryanodine blunted SOD-induced responses (4 +/- 1.5%, n=4 and 8.9 +/- 1.1%, n=4, respectively). By contrast, no interaction was observed between the relaxations evoked by SOD and CPA. In isolated rings, SOD exerted no direct relaxant action and did not modulate relaxations to CPA, TSG or ryanodine. 5 In cascade bioassay studies time-averaged shear stress was manipulated with dextran (1-4% w/v, 80000 MW) to increase perfusate viscosity. No-dependent relaxation of the recipient ring induced by increased perfusate viscosity was significantly attenuated by CPA (P<0.01; n=6) and TSG (P<0.05; n=7), but not by ryanodine (n=6). 6 Endothelium-dependent relaxations to acetylcholine (0.1-30 mu M) in cascade bioassay and in isolated aortic ring preparations were markedly attenuated by pretreatment with CPA and TSG, but were unaffected by ryanodine. Ryanodine and CPA caused only a small attenuation of endothelium-independent relaxations to sodium nitroprusside (0.001-10 mu M), whereas TSG had no effect. 7 We conclude that release of Ca2+ from CPA-and TSG-sensitive endothelial stores is necessary for NO release evoked by acute flow changes and agonists in rabbit abdominal aorta. Ca2+-induced Ca2+ release via the ryanodine-sensitive release channel plays no direct role in these responses. Free radical interactions may complicate the interpretation of findings in cascade bioassay compared with isolated ring preparations.