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 (Zn(2+)) 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 Zn(2+) 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 (ZnCl(2)) to PAEC. We observed that Zn(2+)-exposed PAECs exhibited a dose-dependent increase in superoxide (O(2)(-).) generation that was localized to the mitochondria. Furthermore, we found Zn(2+)-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 Zn(2+)-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a Zn(2+)-selective chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and Zn(2+)-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of Zn(2+) homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of Zn(2+) levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress.

Oxidative stress has been associated with multiple pathologies and disease states, including those involving the cardiovascular system. Previously, we showed that pulmonary artery endothelial cells (PAECs) undergo apoptosis after acute exposure to H(2)O(2). However, the underlying mechanisms regulating this process remain unclear. Because of the prevalence of H(2)O(2) in normal physiological processes and apparent loss of regulation in disease states, the purpose of this study was to develop a more complete understanding of H(2)O(2)-mediated adverse effects on endothelial cell survival. Acute exposure of PAECs to H(2)O(2) caused a dose-dependent increase in cellular release of lactate dehydrogenase and a significant increase in production of superoxide ions, which appear to be generated within the mitochondria, as well as a significant loss of mitochondrial membrane potential and activity. Subsequent to the loss of mitochondrial membrane potential, PAECs exhibited significant caspase activation and apoptotic nuclei. We also observed a significant increase in intracellular free Zn(2+) after bolus exposure to H(2)O(2). To determine whether this increase in Zn(2+) was involved in the apoptotic pathway induced by acute H(2)O(2) exposure, we developed an adenoviral construct for overexpression of the Zn(2+)-binding protein metallothionein-1. Our data indicate that chelating Zn(2+), either pharmacologically with N,N,N',N-tetrakis(2-pyridylmethyl)ethylene diamine or by overexpression of the Zn(2+)-binding protein metallothionein-1, in PAECs conferred significant protection from induction of apoptosis and cell death associated with the effects of acute H(2)O(2) exposure. Our results show that the acute toxicity profile of H(2)O(2) can be attributed, at least in part, to liberation of Zn(2+) within PAECs. We speculate that regulation of Zn(2+) levels may represent a potential therapeutic target for cardiovascular disease associated with acute oxidative stress.

contributed equally to this work.Receptor endocytosis is regulated by ligand binding, and receptors may signal after endocytosis in signaling endosomes. We hypothesized that signaling endosomes containing different types of receptors may be distinct from one another and have different physical characteristics. To test this hypothesis, we developed a high-resolution organelle fractionation method based on mass and density, optimized to resolve endosomes from other organelles. Three different types of receptors undergoing ligand-induced endocytosis were localized predominately in endosomes that were resolved from one another using this method. Endosomes containing activated receptor tyrosine kinases (RTKs), TrkA and EGFR, were similar to one another. Endosomes containing p75 NTR (in the tumor necrosis receptor superfamily) and PAC1 (a G-protein-coupled receptor) were distinct from each other and from RTK endosomes. Receptorspecific endosomes may direct the intracellular location and duration of signal transduction pathways to dictate response to signals and determine cell fate.

A large fraction of anthropogenic CO 2 emissions comes from large point sources such as power plants, petroleum refineries, and large industrial facilities. The existence and locations of these facilities depend on a variety of factors that include the distribution of natural resources and the economy of scale of operating large facilities. These large facilities provide goods and/or services well beyond the political jurisdiction in which they reside and their emissions to the global atmosphere are not a simple reflection of the consumption of goods and services within the geographic region in which they reside. And yet many accounting schemes do not distinguish between emissions for local consumption and emissions for export. Looking at the geographic distribution of large point sources of CO 2 emissions in the U.S. suggests that per capita emissions from a geographic area are not necessarily a good indication of the mitigation responsibility of the residents. The design of effective and fair mitigation strategies needs to consider that emissions embodied in the products of large facilities, such as electric power and refined petroleum products, are often transferred across accounting boundaries; e.g. the CO 2 emissions occur in one jurisdiction even though the electricity is used in another. We close with a short discussion of how two sub-national emissions trading schemes in the U.S. have confronted the issue of embodied emissions crossing their jurisdictional boundaries.