Caffeine-evoked, calcium-sensitive membrane currents in rabbit aortic endothelial cells
1. Single cell photometry and whole-cell patch clamp recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+](i) in fura-2-loaded endothelial cells. Pretreatment of cells with 10 μM 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 Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current 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 currents (STOCs). 5. Inclusion of heparin (10 μg ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current. 6. Externally applied tetraethylammonium ions (TEA, 3-10 mM) reversibly inhibited unitary Ca2+-activated K+ currents and STOCs in endothelial 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 chloride channel blocker, did not affect unitary currents or STOCs but reduced the amplitude of the caffeine-evoked current by > 75% compared to control. Replacement of extracellular NaCl with Na gluconate also reduced the amplitude of the caffeine-induced outward current. Bath application of 0.1 mM Zn2+ and 10 mM TEA completely blocked the caffeine-evoked outward current in endothelial cells. 8. Caffeine-induced Ca2+ release from intracellular stores evokes a transient rise in [Ca2+](i) which is correlated with a large, transient outward current. The ionic dependence and inhibition of the caffeine-sensitive current by TEA and Zn2+ suggests that Ca2+-activated K+ and Cl- conductances contribute to the caffeine response in rabbit aortic endothelial cells.
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