Role of calcium in endothelium-dependent relaxation of arterial smooth muscle

Endothelium-dependent relaxation was studied in rings of rabbit thoracic aorta. Relaxation responses were induced with methacholine, the calcium ionophore A23187 and maitotoxin before and after removal of Ca ++ from the external medium; in the presence of calcium-channel entry blockers (verapamil and nifedipine); or with trifluoperazine. Deletion of Ca ++ greatly impaired responses to all 3 agonists while trifluoperazine only blocked cholinergic-induced relaxation. The calcium-channel blockers had effects that were concentration- and time-dependent, but their action included blockade of A23187. Cytosolic-free Ca ++ concentrations were measured in cultured endothelial cells after incubation of the cells with 10 μM Fura-2/AM or 50 μM Quin 2/AM. Bradykinin (1 × 10 −10 to 1 × 10 −7 M) and melittin (0.5 to 5 μg/ml) caused dose-dependent increases in intracellular Ca ++ with maximal responses at 3 × 10 −8 M and 3 μg/ml, respectively. Both agents were able to induce an increase in cytosolic-free Ca ++ in the presence of EGTA (1.5 × 10 −3 M) or verapamil (1 × 10 −5 M). The plateau phase of the Ca ++ transient appeared to be modified slightly by verapamil, while the peak responses and plateau were attenuated by ‘0’ Ca ++/EGTA. To assess a function of the endothelium, production of endothelium-derived relaxing factor (EDRF) was studied in cells grown on microcarrier beads supervised in a column, and the column effluent was bioassayed on aortic rings. The cells used in studies of receptor-mediated changes in intracellular Ca ++ produced and released EDRF in response to bradykinin and A23187. The EDRF release required extracellular Ca ++. An increase in cytosolic-free Ca ++ correlated with the production or release of EDRF by bovine and rabbit aortic endothelium.