Agonist-induced changes of [Ca2+]i and membrane currents in single bovine aortic endothelial cells

Cultured bovine aortic endothelial cells (BAECs) possess an inward rectifier K+ current (IK1), a Ca(2+)-activated K+ current, a nonselective cation current (INS), and a Ca(2+)-activated Cl- current; however, their relative roles remain to be established. In single BAECs, cytosolic free Ca2+ concentration ([Ca2+]i) [K5-fura 2 (50 microM), ratio 340/380 nm] was measured simultaneously with whole cell currents at 22 degrees C. Bradykinin (BK, 2 microM), ATP (10 microM), ionomycin (100 nM), or 2,5-di-(tert-butyl)-1,4-benzohydroquinone (10 microM) were used as agonists. In physiological salt solution (PSS), agonist exposure caused a rapid [Ca2+]i increase, followed by an increase in outward current (greater than -50 mV) and a smaller increase in inward current (greater than -80 mV). Chelation of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated agonist-induced [Ca2+]i and current responses. Inactivation of the cyclooxygenase pathway by acetylsalicyclic acid and ibuprofen (50 microM each) did not affect the BK-induced [Ca2+]i transient but abolished the current response. In K(+)-free solution, agonist-stimulated outward currents (at +50 mV) were 10 times smaller than in PSS and were consistent with the activation of both INS and a Cl- current. In Cl(-)-free solution, the outward current response following agonist exposure was virtually abolished; at the same time, a linear inward current component with a reversal potential near the equilibrium potential for Na+ was activated. The maximal amplitude of the agonist-induced outward current decreased with decreasing symmetrical Cl- concentrations. Our results suggest that 1) IKI is the dominant current in resting BAECs; 2) K+, Cl-, and nonselective cation conductances contribute to the agonist-induced current response; and 3) most of the agonist-induced activation of currents depends on increased [Ca2+]i and is sensitive to cyclooxygenase inhibitors.