Activation of endothelial nitric oxide synthase by the pro-apoptotic drug embelin: Striking discrepancy between nitric oxide-mediated cyclic GMP accumulation and l-citrulline formation

The benzoquinone derivative embelin is a multifunctional drug that not only induces apoptosis by inhibiting XIAP, the X chromosome-linked inhibitor of apoptosis protein, but also blocks nuclear factor-κB signaling pathways, thereby leading to down-regulation of a variety of gene products involved in tumor cell survival, proliferation, invasion, angiogenesis, and inflammation. Here, we report that embelin activates and modulates l-arginine/nitric oxide/cyclic GMP signaling in cultured endothelial cells. Embelin elicited a rapid increase of intracellular free Ca2+, leading to activation of endothelial nitric oxide synthase (eNOS) and NO-induced cGMP accumulation. While the cGMP response was comparable to that caused by other Ca2+-mobilizing agents, the stimulatory effect of embelin on l-citrulline formation (∼4-fold) was substantially lower than that observed upon activation of eNOS with the Ca2+ ionophore A23187 (∼18-fold), the receptor agonist ATP (∼16-fold) or the sarco-endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (∼14-fold). The apparent discrepancy between NO/cGMP and l-citrulline formation in embelin-treated cells was not due to enhanced metabolism and/or efflux of l-citrulline, increased NO bioavailability, inhibition of cGMP hydrolysis, sensitization of soluble guanylate cyclase (sGC) to NO, or enhanced formation of a sGC/eNOS complex. Our puzzling observations suggest that embelin improves coupling of endothelial NO synthesis to sGC activation through mobilization of an as yet unrecognized signaling pathway.