Novel Imaging Approaches Provide New Windows to View Subcellular Changes in the Redox State of Smooth Muscle Cells
See related articles, pages 526–535
In this issue of Circulation Research, Schumacker and colleagues1 describe the development of novel genetic targeting approaches that record unique information on subcellular changes in the thiol redox state. Ratiometric rhoGFP fluorescence detectors were used to examine hypoxia-induced redox changes in cultured rat pulmonary and systemic (renal) arterial smooth muscle cells. The role and the origins of peroxide contributing to the redox changes were dissected by genetically overexpressing catalase in subcellular compartments. Although the study was designed to provide further insight into the controversial hypothesis that hypoxia promotes contraction of pulmonary arterial smooth muscle by increasing the release of mitochondrial-derived hydrogen peroxide, the methodological approaches used may be viewed as a more important aspect of the work. In particular, the molecular transfection approaches used provide new windows of opportunity to examine the nature of subcellular redox signaling. Overall, the study contained impressive data showing that hypoxia increases the concentration of cytosolic hydrogen peroxide, based on the inhibition of thiol oxidation-dependent fluorescence responses by catalase overexpressed in the cytosol. Hypoxia also increased oxidation in the mitochondrial intermembrane space but, decreased it in the mitochondrial matrix in a manner that was not affected by the overexpression of catalase in the mitochondrial matrix. These results underscore the need to consider carefully the complexity of subcellular redox processes, the processes that control redox changes, and the methods used in evaluating the data that is obtained.
Most recent studies on hypoxic responses in pulmonary and systemic vascular smooth muscle are in agreement. Collectively, they support roles for sensing changes in oxygen tension through metabolic-redox processes that are regulated by signaling mechanisms controlling contractile function. However, convincing evidence has emerged which supports several theories that contain conflicting proposals for mechanisms involved in sensing hypoxia. For example, previous work by Schumacker and colleagues strongly supported the view that hypoxia increases the release of mitochondrial-derived peroxide, which promotes rat pulmonary arterial contraction by activating mechanisms that are regulated by intracellular calcium.2 In contrast, studies by the groups of Archer, Weir, and colleagues provided substantial evidence for hypoxia lowering the release of mitochondrial-derived peroxide in rat pulmonary arterial smooth muscle. In this mechanism, contraction in response to hypoxia is thought to be elicited by a decrease in peroxide levels that in turn close the voltage-gated potassium channels that are regulated by the redox state of their thiol residues.3,4 Archer and colleagues also provided evidence that hypoxia relaxes systemic (rat renal) arteries by increasing mitochondrial-derived reactive oxygen species, which appear to promote hyperpolarization by opening the voltage-gated potassium channels. In contrast, work in systemic (bovine coronary) arteries suggests that the primary mechanism of hypoxic sensing involves a metabolic stress eliciting the oxidation of cytosolic NADPH.5 In this mechanism, hypoxia could promote relaxation as a result of cytosolic NADPH oxidation, which coordinates multiple mechanisms that lower intracellular calcium. Although our work in bovine pulmonary arteries supports the model of Archer and Weir for hypoxia lowering hydrogen peroxide, we have hypothesized that Nox4 oxidase may be the key source of peroxide removal by hypoxia.6 The decrease in the generation of peroxide under hypoxia is hypothesized to promote contraction by potentially removing the relaxing mechanisms controlled by cGMP-protein kinase and perhaps increasing the ratios of cytosolic NADPH/NADP.6 New observations reported in this issue of Circulation Research by Schumacker and colleagues1 provide additional support for their previous hypotheses that hypoxia promotes pulmonary arterial contraction through cytosolic oxidation processes originating from increased mitochondrial peroxide release. In addition, their study also provides new evidence that subcellular redox changes in systemic arterial smooth muscle cells are similar to the smooth muscle cells of the pulmonary system. However, these redox changes in systemic arterial smooth muscle cells appear to be associated with decreases in intracellular calcium under the conditions examined.
One needs to consider the complexity of cellular redox systems and the experimental conditions under which hypoxic responses are examined to understand the origins of the controversial theories that exist in the vascular hypoxia sensing field and the origins of what might be detected by rhoGFP protein targeting. Many of the issues that could potentially influence thiol redox processes controlling both rhoGFP fluorescence and hypoxia-associated redox signaling are listed in the Table. The production of reactive oxygen species by oxidases thought to be involved in vascular oxygen sensing can be directly modulated by the balance between availability of electron donors (eg, NAD[P]H) and oxygen concentration. We have observed that the availability of NADH seems to be a key factor in controlling mitochondrial superoxide production in bovine coronary arteries,7 and both cytosolic NADH and NADPH redox appear to control superoxide production by Nox oxidases.8 Because the ratiometric fluorescence detectors function through conversion of adjacent thiols to disulfides, it is likely that the behavior of cellular thiol redox control mechanisms is likely to be a major factor in what is actually detected. Recent studies show that rhoGFP fluorescence can be efficiently activated in the proximity of thiol based peroxidases of the glutathione peroxidase and peroxiredoxin families that are actively metabolize peroxides.9 However, it is also logical to assume that fundamental systems regulating the redox state of protein thiols could also control rhoGFP fluorescence. For example, the redox status of glutathione and thioredoxin is likely to influence protein thiol redox state through glutaredoxin and protein disulfide isomerases.9 In addition, NADPH redox markedly influences the status of thiol redox systems through enzymes such as glutathione and thioredoxin reductases.10 Because cellular signaling mechanisms controlled by thiol redox state may be regulated by processes similar to those controlling rhoGFP fluorescence, the probe used by Schumacker and colleagues is likely to be a detector for many of the thiol redox controlled processes that influence vascular function. Combinations of molecular knockout and genetic targeting approaches for imaging of subcellular changes in redox detection should open new windows for sorting out how redox systems controlling signaling mechanisms contribute to hypoxia-elicited vascular responses and other aspects of cellular regulation.
Sources of Funding
Recent studies from the laboratory of M.S.W. have been funded by US Public Health Service grants L31069, HL43023, and HL66331.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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