Suppression of histamine-induced relaxation of rat aorta and calcium signaling in endothelial cells by two-pore channel blocker trans-NED19 and hydrogen peroxide

The blocker of two-pore channels trans-NED19 and hydrogen peroxide were found to inhibit histamine-induced relaxation of rat aorta. The degree of inhibition depended on histamine concentration. The relaxation in response to 1 µM histamine of rat aorta preconstricted with 30 mM KCl, serotonin, or endothelin-1, was completely abolished by 30 µM trans-NED19. On the other hand, trans-NED19 decreased the relaxation of the aorta in the presence of 10 µM histamine only by 2.1to 2.4-fold, and there was almost no inhibition by trans-NED19 of the relaxation induced by 100 µM histamine. Relaxation of preconstricted with serotonin aorta in response to 10 and 100 µM histamine was reduced by hydrogen peroxide (200 µM) by 10and 2.5-fold, respectively. Suppression of aorta relaxation by trans-NED19 and H₂O₂ correlated with their inhibitory effect on the histamine-induced increase in the cytoplasmic free calcium concentration in human umbilical vein endothelial cells. With the use of a fluorescent probe LysoTracker, the cis-NED19 binding sites were demonstrated to be localized in endolysosomes of the endothelial cells. These data indicate that twopore calcium channels participate in the histamine-induced endothelium-dependent relaxation of rat aorta. Furthermore, their functional role is exhibited much more clearly at low histamine concentrations. We suggest that hydrogen peroxide evokes depletion of intracellular calcium depots thereby suppressing the response to histamine.