The Effect of Alkalosis on Intracellular Calcium Concentrations[Ca⁺⁺]_i in Pulmonary Microvascular Endothelial Cells • 1602

Background: The signaling mechanisms mediating alkalosis-induced pulmonary vasodilation are poorly understood. We have recently shown that an increase in pH from 7.4 to 7.7 stimulates cyclooxygenase (COX) and nitric oxide synthase (NOS) activity resulting in a 2-fold increase in cGMP and a 5-fold increase in prostacyclin production in pulmonary microvascular endothelial cells (EC) in culture. Objective: To test the hypothesis that alkalosis causes an increase in [Ca⁺⁺]_i by stimulating K⁺ channel activity resulting in membrane hyperpolarization. As EC do not have voltage-gated Ca⁺⁺ channels, Ca⁺⁺ entry in EC depends upon the electrochemical driving force. Methods: Bovine pulmonary microvascular EC were loaded for 1 hour at room temperature with 5 μM fura-2 AM and 0.01% Pluronic F127. Cultures were rinsed of free fura-2 and placed on the stage of an inverted microscope for measurement of[Ca⁺⁺]_i using a dual wavelength fluorescent imaging system(Intracellular Imaging, Inc.). Excitation wavelengths were altered between 340 and 380 nm and emission fluorescence was measured at 510 nm. Intracellular Ca⁺⁺ concentrations were computed from Ca⁺⁺ calibration curves generated from Ca⁺⁺ standard solutions and the 340/380 ratios using the InCyt IM2 image acquisition and analysis software.Results: Basal [Ca⁺⁺]_i was 52 ± 22 nM. There was a pH-dependent increase in [Ca⁺⁺]_i: pH 9.0 caused an abrupt 7-fold increase, while pH 7.7 caused a 2.2-fold increase over basal levels. The pH-dependent increase in [Ca⁺⁺]_i was abolished in Ca⁺⁺-free medium. TMB-8, an inhibitor of IP₃-mediated Ca⁺⁺ release, had no effect on the alkalosis-induced increase in[Ca⁺⁺]_i but abolished the Ca⁺⁺ transient induced by bradykinin. Depolarization of membrane potential with an isotonic KCl buffer(50 mM) abolished the increase in [Ca⁺⁺]_i mediated by alkalosis. Pretreatment of EC for 15 min with tetraethylammonium (TEA) partially inhibited the alkalosis-induced increase in [Ca⁺⁺]_i.Conclusion: Alkalosis induces a pH-dependent increase in[Ca⁺⁺]_i in pulmonary microvascular EC, which is dependent upon the presence of extracellular Ca⁺⁺. The alkalosis-induced increase in[Ca⁺⁺]_i is linked to K⁺ channel activation, as it is inhibited by membrane depolarization and partially blocked by TEA. These studies support the hypothesis that alkalosis mediates vasodilation by activating K⁺ channels, hyperpolarizing EC membrane potential, resulting in increased [Ca⁺⁺]_i, which ultimately activates Ca⁺⁺- dependent enzymes, such as COX and NOS.