Resting Cytosolic and Nuclear Reactive Oxygen Species (ROS) Are Regulated by the Basal Activity of ET-1 Receptors in...

Endothelin-1 (ET-1) is a potent vasoconstrictor that exerts its numerous biological actions through two receptors, ET_A and ET_B. However, the implication and role of each receptor in ROS generation remain ambiguous. Previously, our group reported that blocking the basal activity of ET_A and ET_B receptors with their respective peptidic antagonists increased basal intracellular calcium (Ca²⁺⁾ levels, an effect inhibited by chelating extracellular Ca²⁺. Since a crosstalk between Ca²⁺ and reactive oxygen species (ROS) exists, the purpose of the present work was to investigate whether this increase in basal resting Ca²⁺ level induced by the blockade of ET_A and ET_B receptors is associated with an increase in resting ROS level. Our results showed that the basal activity of ET_A and ET_B receptors contributes negatively to the resting level of cytosolic and nuclear ROS, and that each receptor appears to act as the other's physiological antagonist. Furthermore, our results showed that ET-1 receptor blockade increases ROS via a receptor insensitive to ET_A and ET_B receptor antagonists. This type of receptor could be the one reported by our group, ET_C, or simply a heterodimeric ET_A/ET_B receptor. Moreover, blocking the heterodimerized ET_A/ET_B binding site is sufficient to unblock the physiological antagonism that each receptor exerts on the other. Furthermore, our results showed that blocking both ETA and ETB receptors, thereby preventing heterodimerization, prevented the increase in resting ROS, supporting the existence of a heterodimerized ET-1 receptor. Since human vascular smooth muscle cells (VSMCs) express only ETB receptors at the nuclear membrane, it is possible to suggest that nuclear ETB receptors are homodimers that regulate the resting nuclear ROS level. In conclusion, our results showed that the regulation of resting ROS levels by ET-1 and its receptors can be mediated by homodimerized and/or heterodimerized receptor activation; hence, the importance of developing drugs targeting this receptor type.