Increased free calcium in endothelial cells under stimulation with adenine nucleotides

✍ Scribed by Andreas Lückhoff; Rudi Busse

• Publisher: John Wiley and Sons
• Year: 1986
• Tongue: English
• Weight: 722 KB
• Volume: 126
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041260312

✦ Synopsis

7800 freiburg, federal Republic of Germany The release of vasodilating substances from the vascular endothelium has been postulated to depend on a rise in the level of intracellular free calcium (Cai+ +). We measured C a 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 C a i + + to a maximum with t h e calcium ionophore ionomycin (0.1 pM) 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 t h e cells was calculated from fluorescence at the isosbestic excitation wavelength (365 nm). Resting Cai+ + was 71 f 3 (SEM) nM. ATP (adenosine -5'-triphosphate) raised Cai+ + dose-dependently and reversibly to 458 60 nM at a concentration of 10 pM, and at 0.1 m M to values close to those that occurred under ionomycin. ADP (A-5'-PP) and AMP (A-5'-P) had smaller effects with a maximal C a i + + of 287 f 72 nM at 30 pM ADP and 176 & 17 nM at 0.1 mM AMP. At these concentrations, ADP and AMP attenuated significantly the increase of Cai+ + under ATP (10 pM). Adenosine (0.1 or 0.3 mM) and acetylcholine (0.1 to 30 pM) enhanced Cai+ + 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 Cai+ +, 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.