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 (I(K1)), a Ca²⁺-activated K⁺ current, a nonselective cation current (I(NS)), and a Ca²⁺-activated Cl^- current; however, their relative roles remain to be established. In single BAECs, cytosolic free Ca²⁺ concentration ([Ca²⁺](i)) [K₅-fura 2 (50 μM), ratio 340/380 nm] was measured simultaneously with whole cell currents at 22°C. Bradykinin (BK, 2 μM), ATP (10 μM), ionomycin (100 nM), or 2,5-di-(tert-butyl)-1,4- benzohydroquinone (10 μM) 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 μM 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 I(NS) 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) I(K1) 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.