Effects of Halothane and Isoflurane on Bradykinin-evoked Calcium2+Influx in Bovine Aortic Endothelial Cells

Background : Volatile anesthetics, such as halothane and isoflurane, have been reported to affect the endothelium-mediated relaxation of vascular smooth muscle cells. Because the activity of the constitutive nitric oxide synthase in endothelial cells depends on the availability of intracellular Ca 2+ , there is a definite possibility that the observed inhibitory effect of volatile anesthetics involves an action on the agonist-evoked internal Ca 2+ mobilization and/or Ca 2+ influx in these cells. Therefore, a study was undertaken to determine how halothane and isoflurane affect the Ca 2+ signalling process in vascular endothelial cells. Methods : The effect of halothane and isoflurane on the Ca 2+ response to bradykinin of bovine aortic endothelial (BAE) cells was investigated using the fluorescent Ca 2+ indicator fura-2. Halothane or isoflurane was applied either to resting cells or after bradykinin stimulation. The agonist-evoked Ca 2+ influx in BAE cells was estimated by measuring either the rate of fura-2 quenching induced by Mn 2+ or the increase in cytosolic Ca 2+ concentration initiated after readmission of external Ca 2+ after a brief exposure of the cells to a Ca 2+ -free external medium. The effects of halothane on cell potential and intracellular Ca 2+ concentration were measured in cell-attached patch-clamp experiments in which a calcium-activated K + channel and an inward rectifying Ca 2+ -independent K + channel were used as probes to simultaneously monitor the intracellular Ca 2+ concentration and the cell transmembrane potential. In addition, combined fura-2 and patch-clamp cell-attached recordings were carried out, to correlate the variations in internal Ca 2+ caused by halothane and the activity of the Ca 2+ -dependent K + channels, which are known in BAE cells to regulate intracellular potential. Finally, a direct action of halothane and isoflurane on the gating properties of the Ca 2+ -activated K + channel present in these cells was investigated in patch-excised inside-out experiments. Results : The results of the current study indicate that the initial Ca 2+ increase in response to bradykinin stimulation is not affected by halothane, but that pulse applications of halothane (0.4-2 mM) or isoflurane (0.5-1 mM) reversibly reduce the sustained cytosolic Ca 2+ increase initiated either by bradykinin or by the Ca 2+ pump inhibitor thapsigargin. In addition, halothane appeared to dose-dependently inhibit the Ca 2+ influx evoked by bradykinin, and to cause, concomitant to a decrease in cytosolic Ca 2+ concentration, a depolarization of the cell potential. Halothane failed, however, to affect internal Ca 2+ concentration in thapsigargin-treated endothelial cells, which were depolarized using a high K + external solution. Finally, halothane and isoflurane decreased the open probability of the Ca 2+ -dependent K + channel present in these cells. Conclusions : These observations suggest that the effects of halothane and isoflurane on Ca 2+ homeostasis in BAE cells reflect, for the most part, a reduction of the thapsigargin- or bradykinin-evoked Ca 2+ influx, which would be consequent to a cellular depolarization caused by an inhibition of the Ca 2+ -dependent K + channel activity initiated after cell stimulation.