REGENERATIVE CAFFEINE-INDUCED RESPONSES IN NATIVE RABBIT AORTIC ENDOTHELIAL-CELLS

1 Single native aortic endothelial cells obtained by enzymatic dispersion of the rabbit aortic endothelium were held under voltage clamp using patch pipette and whole-cell membrane currents were measured. In parallel experiments performed on cells from the same batches, the free internal calcium concentration, [Ca2+](i), in the cell was estimated by use of the Ca2+-sensitive fluorescent dye, fura-2. 2 Caffeine (20 mM) applied to the cell evoked an outward current and an initial peak in [Ca2+](i) followed by a lower sustained rise (plateau). Ca2+-free, EGTA-containing solution applied outside the cells did not reduce these responses. 3 Following caffeine stimulation there was a biphasic rising phase of outward current both in the presence and absence of extracellular Ca2+. 4 Application of graded doses of caffeine revealed all-or-none type responses of both the outward current and the rise in [Ca2+](i). 5 Preincubation with lower doses of caffeine reduced the magnitude of both the outward current and the [Ca2+](i) transient evoked by 20 mM caffeine. 6 Tetraethylammonium (3 mM) applied to the bathing solution blocked unitary and spontaneous transient outward currents (STOCs) stimulated by Ca2+-free solution, but only reduced the outward current evoked by caffeine (20 mM). 7 In conclusion, our results reveal the all-or-none nature of Ca2+ release from the endoplasmic reticulum (ER) in native aortic endothelial cells. Lower concentrations of caffeine (0.4-0.5 mM) may deplete intracellular Ca2+ stores. Extracellular Ca2+ is not necessary for maintaining the activity of spontaneous and caffeine-induced outward currents in native aortic endothelial cells. Spontaneous outward currents are believed to represent the sporadic release of calcium from store sites independent of both extracellular Ca2+ and the caffeine-sensitive Ca2+ stores which stimulate the outward current.