Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca 2؉ currents and the recombinant ␣ 1C subunit of the human cardiac L-type Ca 2؉ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca 2؉ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.CO is an established and important signaling molecule in both the heart and vasculature as well as other tissues (1, 2). Cardiac atrial and ventricular myocytes express heme oxygenases HO-1 4 and HO-2, which generate CO along with biliverdin and free Fe 2ϩ by heme catabolism, and HO-1 levels can be increased by various stress factors (3), including myocardial infarction (4). CO limits the cellular damage of ischemia/reperfusion injury in the heart (5). Indeed, greater cardiac damage is seen following ischemia/reperfusion injury in HO-1 knock-out mice (6). Conversely, HO-1 overexpression in the heart reduces infarct size and other markers of damage following ischemia/ reperfusion injury (7). CO also improves cardiac blood supply through coronary vessel dilation (8, 9) and reduces cardiac contractility (9). However, the mechanisms underlying this cardioprotective effect of CO are not understood.In the vasculature, CO also exerts numerous beneficial effects. Its ability to dilate blood vessels is long established (9 -11) and endothelium-independent (12) and not due to development of hypoxia through displacement of O 2 (see Ref. 13). CO has clear, protective effects in various vascular diseases, such as systemic and pulmonary hypertension, development of atherosclerosis, and neointimal hyperplasia due to proliferation of vascular smooth muscle cells following vascular injury (all reviewed in Refs. 2, 13, and 14). Import...

Transporter-mediated glutamate uptake is a principal function of astrocytes. Our previous studies have shown that this process is compromised under hypoxic conditions through the NF-kappaB mediated inhibition of expression of the glutamate transporters EAAT-1 and EAAT-2. Here, we demonstrate that identical conditions of hypoxia (1% O(2), 24 h) lead to a dramatic increase in TNFalpha production from astrocytes without altering their viability. This hypoxia-evoked production of TNFalpha was prevented in the presence of any of three mechanistically distinct NF-kappaB inhibitors. Exogenous application of TNFalpha was without effect on EAAT-1 expression as determined by Western blotting, but mimicked the effects of hypoxia to suppress expression of EAAT-2. Furthermore thalidomide, which prevents TNFalpha production, was without effect on hypoxic suppression of EAAT-1 but prevented hypoxic suppression of EAAT-2. These data indicate that regulation of glutamate transporter expression in astrocytes by hypoxia is subtype specific. Regulation of both EAAT-1 and EAAT-2 is mediated by NF-kappaB, and this transcriptional regulator is also required for increased production of TNFalpha. However, while TNFalpha is essential for hypoxic suppression of EAAT-2, hypoxic modulation of EAAT-1 expression is unaffected by this cytokine.

Activation of transient receptor potential melastatin 2 (TRPM2), a non-selective, Ca(2+)-permeable cation channel, is implicated in cell death. Channel opening is stimulated by oxidative stress, a feature of numerous disease states. The wide expression profile of TRPM2 renders it a potentially significant therapeutic target in a variety of pathological settings including cardiovascular and neurodegenerative diseases. HEK293 cells transfected with human TRPM2 (HEK293/hTRPM2) were more vulnerable to H(2)O(2)-mediated cell death than untransfected controls in which H(2)O(2)-stimulated Ca(2+) influx was absent. Flufenamic acid partially reduced Ca(2+) influx in response to H(2)O(2) but had no effect on viability. N-(p-Amylcinnamoyl) anthranilic acid substantially attenuated Ca(2+) influx but did not alter viability. Poly(adenosine diphosphate ribose) polymerase inhibitors (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone and nicotinamide) reduced Ca(2+) influx and provided a degree of protection but also had some protective effects in untransfected controls. These data suggest H(2)O(2) triggers cell death in HEK293/hTRPM2 cells by a mechanism that is in part Ca(2+) independent, as blockade of channel opening (evidenced by suppression of Ca(2+) influx) did not correlate well with protection from cell death. Determining the underlying mechanisms of TRPM2 activation is pertinent in elucidating the relevance of this channel as a therapeutic target in neurodegenerative diseases and other pathologies associated with Ca(2+) dysregulation and oxidative stress.

No abstract

[Ca(2+)](i) (cytosolic [Ca(2+)]) and OS (oxidative stress) were measured simultaneously in calf pulmonary artery endothelial cells using fura-2 and carboxy-2',7'-dichlorodihydrofluorescein. ATP stimulated a [Ca(2+)](i) increase that was followed a few seconds later by an increase in OS. Pre-exposure to 5 microM H(2)O(2) potentiated these responses to ATP. Elevating or removing extracellular Ca(2+) increased or reduced the [Ca(2+)](i) response to ATP and caused parallel changes in the OS response, suggesting that this response was a consequence of the [Ca(2+)](i) response. Inhibition of mitochondria with rotenone or antimycin A affected the responses but not in a manner that allowed a simple interpretation of the role of mitochondria. These data show an intimate connection between [Ca(2+)](i) and OS that can be modulated by low levels of exogenously applied OS, allowing the possibility of positive feedback.