Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER). ER localization of Nox4 is critical for the regulation of protein tyrosine phosphatase (PTP) 1B, also an ER resident, through redox-mediated signaling. Nox4-mediated oxidation and inactivation of PTP1B in the ER serves as a regulatory switch for epidermal growth factor (EGF) receptor trafficking and specifically acts to terminate EGF signaling. Consistent with this notion, PTP1B oxidation could also be modulated by ER targeting of antioxidant enzymes but not their untargeted counterparts. These data indicate that the specificity of intracellular ROS-mediated signal transduction may be modulated by the localization of Nox isoforms within specific subcellular compartments.

Obesity and type 2 diabetes are associated with chronic inflammation. Adiponectin is an adipocyte-derived hormone with antidiabetic and antiinflammatory actions. Here, we demonstrate what we believe to be a previously undocumented activity of adiponectin, facilitating the uptake of early apoptotic cells by macrophages, an essential feature of immune system function. Adiponectin-deficient (APN-KO) mice were impaired in their ability to clear apoptotic thymocytes in response to dexamethasone treatment, and these animals displayed a reduced ability to clear early apoptotic cells that were injected into their intraperitoneal cavities. Conversely, adiponectin administration promoted the clearance of apoptotic cells by macrophages in both APN-KO and wild-type mice. Adiponectin overexpression also promoted apoptotic cell clearance and reduced features of autoimmunity in lpr mice whereas adiponectin deficiency in lpr mice led to a further reduction in apoptotic cell clearance, which was accompanied by exacerbated systemic inflammation. Adiponectin was capable of opsonizing apoptotic cells, and phagocytosis of cell corpses was mediated by the binding of adiponectin to calreticulin on the macrophage cell surface. We propose that adiponectin protects the organism from systemic inflammation by promoting the clearance of early apoptotic cells by macrophages through a receptor-dependent pathway involving calreticulin.

As in many smooth muscle tissue preparations, single smooth muscle cells freshly dissociated from the stomach of the toad Bufo marinus contract when stretched. Stretch-activated channels have been identified in these cells using patch-clamp techniques. In both cell-attached and excised inside-out patches, the probability of the channel being open (Po) increases when the membrane is stretched by applying negative pressure to the extracellular surface through the patch pipette. The increase in Po is mainly due to a decrease in closed time durations, but an increase in open time duration is also seen. The open-channel current-voltage relationship shows inward rectification and is not appreciably altered when K+ is substituted for Na+ as the charge-carrying cation in Ca2+-free (2 mM EGTA) pipette solutions bathing the extracellular surface of the patch. The inclusion of physiological concentrations of Ca2+ (1.8 mM) in pipette solutions (containing high concentrations of Na+ and low K+) significantly decreases the slope conductance as well as the unitary amplitude. The channel also conducts Ca2+, since inward currents were observed using pipette solutions in which Ca2+ ions were the only inorganic cations. When simulating normal physiological conditions, we find that substantial ionic current is conducted into the cell when the channel is open. These characteristics coupled with the high density of the stretch-activated channels point to a key role for them in the initiation of stretch-induced contraction.

Large conductance Ca"'-activated K" channels in rabbit pulmonary artery smooth muscle cells are activated by membrane stretch and by arachidonic acid and other fatty acids, Activation by stretch appears to occur by a direct effect of stretch on the channel itself or a closely associated component. In excised inside-out patches stretch activation was seen under conditions which precluded possible mechanisms involving cytosolic factors, release of Ca"" from intracellular stores, or stretch induced transmombranc flux of Ca"" or other ions potentially capable of activatina the channel, Fatty acids al~o directly activate this channel, Like stretch activation, fatty acid activation occurs in excised inside.out patches in the absence of cytosolic constituents. Moreover, the channel is activated by fatty acids which, unlike arachidonic acid, are not subatrates for tile cyelo-oxygenase or lypoxygenasc pathways, indicating that oxygenated metabolltes do not mediate the response. Thus, four distinct types of stimuli (cytosolic Ca-". membrane potential, membrane stretch, and fatty acids) can directly affect the activity of this channel.

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