Endothelial response to stress from exogenous Zn2+resembles that of NO-mediated nitrosative stress, and is protected by MT-1 overexpression

Abstract

While nitric oxide (NO)-mediated biological interactions have been intensively studied, the underlying mechanisms of nitrosative stress with resulting pathology remain unclear. Previous studies have demonstrated that NO exposure increases free zinc ions (Zn2+) within cells. However, the resulting effects on endothelial cell survival have not been adequately resolved. Thus the purpose of this study was to investigate the role of altered zinc homeostasis on endothelial cell survival. Initially, we confirmed the previously observed significant increase in free Zn2+with a subsequent induction of apoptosis in our pulmonary artery endothelial cells (PAECs) exposed to the NO donor N-[2-aminoethyl]- N-[2-hydroxy-2-nitrosohydrazino]-1,2-ethylenediamine. However, NO has many effects upon cell function and we wanted to specifically evaluate the effects mediated by zinc. To accomplish this we utilized the direct addition of zinc chloride (ZnCl2) to PAEC. We observed that Zn2+-exposed PAECs exhibited a dose-dependent increase in superoxide (O2−·) generation that was localized to the mitochondria. Furthermore, we found Zn2+-exposed PAECs exhibited a significant reduction in mitochondrial membrane potential, loss of cardiolipin from the inner leaflet, caspase activation, and significant increases in TdT-mediated dUTP nick end labeling-positive cells. Furthermore, using an adenoviral construct for the overexpression of the Zn2+-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a Zn2+-selective chelator, N, N,N′, N′-tetrakis(2-pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and Zn2+-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of Zn2+homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of Zn2+levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress.