Increased free calcium in endothelial cells under stimulation with adenine nucleotides

Abstract

The release of vasodilating substances from the vascular endothelium has been postulated to depend on a rise in the level of intracellular free calcium (Ca_i⁺⁺). We measured Ca_i⁺⁺ in intact monolayers of calf endothelial cells, grown in culture, that were loaded with the fluorescent calcium indicator quin 2. Fluorescence (excitation wavelength 340 nm, emission wavelength 492 nm) was calibrated by raising Ca_i⁺⁺ to a maximum with the calcium ionophore ionomycin (0.1 μM) and by lowering it to a minimum with ionomycin plus manganese (0.4 mM), which quenches quin 2 fluorescence completely. Loss of fluorescent dye from the cells was calculated from fluorescence at the isosbestic excitation wavelength (365 nm). Resting Ca_i⁺⁺ was 71 ± 3 (SEM) nM. ATP (adenosine ‐5′‐triphosphate) raised Ca_i⁺⁺ dose‐dependently and reversibly to 458 ± 60 nM at a concentration of 10 μM, and at 0.1 mM to values close to those that occurred under ionomycin. ADP (A‐5′‐PP) and AMP (A‐5′‐P) had smaller effects with a maximal Ca_i⁺⁺ of 287 ± 72 nM at 30 μM ADP and 176 ± 17 nM at 0.1 mM AMP. At these concentrations, ADP and AMP attenuated significantly the increase of Ca_i⁺⁺ under ATP (10 μM). Adenosine (0.1 or 0.3 mM) and acetylcholine (0.1 to 30 μM) enhanced Ca_i⁺⁺ inconsistently, by a maximum of 50 nM. These effects were abolished by theophylline and atropine, respectively. In the absence of extracellular calcium, ATP still raised Ca_i⁺⁺, although endothelial responsiveness declined after repetitive stimulations. We conclude that activation of purinergic receptors increases intracellular free calcium in endothelial cells, and that this increase is probably an essential trigger for synthesis of prostacyclin and the labile endothelium‐derived relaxant factor.