Intracellular Endothelin Type B Receptor-driven Ca2+ Signal Elicits Nitric Oxide Production in Endothelial Cells

Endothelin-1 exerts its actions via activation of ET_A and ET_B G_q/11 protein-coupled receptors, located in the plasmalemma, cytoplasm, and nucleus. Although the autocrine/paracrine nature of endothelin-1 signaling has been extensively studied, its intracrine role has been largely attributed to interaction with receptors located on nuclear membranes and the nucleoplasm. Because ET_B receptors have been shown to be targeted to endolysosomes, we used intracellular microinjection and concurrent imaging methods to test their involvement in Ca²⁺ signaling and subsequential NO production. We provide evidence that microinjected endothelin-1 produces a dose-dependent elevation in cytosolic calcium concentration in ET_B-transfected cells and endothelial cells; this response is sensitive to ET_B but not ET_A receptor blockade. In endothelial cells, the endothelin-1-induced Ca²⁺ response is abolished upon endolysosomal but not Golgi disruption. Moreover, the effect is prevented by inhibition of microautophagy and is sensitive to inhibitors of the phospholipase C and inositol 1,4,5-trisphosphate receptor. Furthermore, intracellular endothelin-1 increases nitric oxide via an ET_B-dependent mechanism. Our results indicate for the first time that intracellular endothelin-1 activates endolysosomal ET_B receptors and increase cytosolic Ca²⁺ and nitric oxide production. Endothelin-1 acts in an intracrine fashion on endolysosomal ET_B to induce nitric oxide formation, thus modulating endothelial function.