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Caffeine‐evoked, calcium‐sensitive membrane currents in rabbit aortic endothelial cells
Author(s) -
Rusko J.,
Slooten G.,
Adams D.J.
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb16330.x
Subject(s) - caffeine , ryanodine receptor , extracellular , biophysics , chemistry , patch clamp , depolarization , membrane potential , acetylcholine , intracellular , calcium , medicine , endocrinology , biochemistry , biology , receptor , organic chemistry
1 Single cell photometry and whole‐cell patch clamp recording were used to study caffeine‐induced intracellular Ca 2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2 Caffeine (5 mM) evoked a transient increase in [Ca 2+ ] i in fura‐2‐loaded endothelial cells. Pretreatment of cells with lO μ M ryanodine did not alter resting [Ca 2+ ] i but irreversibly inhibited the caffeine‐induced rise in [Ca 2+ ] i . The caffeine‐induced increase in [Ca 2+ ] i was not attenuated by the removal of extracellular Ca 2+ and did not stimulate the rate of Mn 2+ 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 Ca 2+ , 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 Ca 2+ . Ca 2+ ‐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 Ca 2+ , heparin did not affect either STOCs or the caffeine‐induced outward current. 6 Externally applied tetraethylammonium ions (TEA, 3–10 mM) reversibly inhibited unitary Ca 2+ ‐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 (Zn 2+ ), 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 Zn 2+ and 10 mM TEA completely blocked the caffeine‐evoked outward current in endothelial cells. 8 Caffeine‐induced Ca 2+ release from intracellular stores evokes a transient rise in [Ca 2+ ] i which is correlated with a large, transient outward current. The ionic dependence and inhibition of the caffeine‐sensitive current by TEA and Zn 2+ suggests that Ca 2+ ‐activated K + and Cl − conductances contribute to the caffeine response in rabbit aortic endothelial cells.