title: Caffeine-evoked, calcium-sensitive membrane currents in rabbit aortic endothelial cells.
abstract: 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 microM 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+. 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 micrograms 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, 3-10mM) 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+1, 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.
authors.affiliation: Department of Molecular & Cellular Pharmacology, University of Miami School of Medicine, FL 33101, USA.
authors.forename: J
authors.initials: J
authors.lastname: Rusko
authors.forename: G
authors.initials: G
authors.lastname: Van Slooten
authors.forename: D J
authors.initials: DJ
authors.lastname: Adams
mesh_terms: D000818:Animals; D001011:Aorta; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005470:Fluorometry; D006493:Heparin / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D011817:Rabbits; D012433:Ryanodine / Q000494:pharmacology
chemical_list: D012433:Ryanodine; D002110:Caffeine; D006493:Heparin; D002118:Calcium