CAFFEINE-EVOKED, CALCIUM-SENSITIVE MEMBRANE CURRENTS IN RABBIT AORTICENDOTHELIAL-CELLS

1 Single cell photometry and whole-cell patch clamp recording were use d to study caffeine-induced intracellular Ca2+ signals and membrane cu rrents, respectively, in endothelial cells freshly dissociated from ra bbit aorta. 2 Caffeine (5 mM) evoked a transient increase in [Ca2+](i) in fura-2-loaded endothelial cells. Pretreatment of cells with 10 mu M ryanodine did not alter resting [Ca2+](i) but irreversibly inhibited the caffeine-induced rise in [Ca2+](i). The caffeine-induced increase in [Ca2+](i) was not attenuated by the removal of extracellular Ca2and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3 Bath application of caffeine evoked a dose- and voltage-dependent ou tward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and memb rane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the c hannels underlying the current are voltage-sensitive. 4 In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward c urrent was reduced by approximately 50% but the duration of the curren t was prolonged compared to that observed in the presence of external Ca2+. Ca2+-free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward curr ents (STOCs). 5 Inclusion of heparin (10 mu g ml(-1)) in the patch pip ette abolished the acetylcholine (ACh)-induced outward current but fai led to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, hepari n did not affect either STOCs or the caffeine-induced outward current. 6 Externally applied tetraethylammonium ions (TEA, 3-10 mM) reversibl y inhibited unitary Ca2+-activated K+ currents and STOCs in endothelia l cells but failed to inhibit completely the outward current evoked by 20 mM caffeine. 7 Bath application of 0.1 mM zinc ion (Zn2+), a chlor ide channel blocker, did not affect unitary currents or STOCs but redu ced the amplitude of the caffeine-evoked current by >75% compared to c ontrol. Replacement of extracellular NaCl with Na gluconate also reduc ed the amplitude of the caffeine-induced outward current. Bath applica tion of 0.1 mM Zn2+ and 10 mM TEA completely blocked the caffeine-evok ed outward current in endothelial cells. 8 Caffeine-induced Ca2+ relea se from intracellular stores evokes a transient rise in [Ca2+](i) whic h is correlated with a large, transient outward current. The ionic dep endence and inhibition of the caffeine-sensitive current by TEA and Zn 2+ suggests that Ca2+-activated K+ and Cl- conductances contribute to the caffeine response in rabbit aortic endothelial cells.