Regenerative caffeine-induced responses in native rabbit aortic endothelial cells

Abstract

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 Ca(2+)-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). Ca(2+)-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 Ca(2+)-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.