Reactive oxygen species (ROS) have been proposed to participate in the induction of cardiac preconditioning. However, their source and mechanism of induction are unclear. We tested whether brief hypoxia induces preconditioning by augmenting mitochondrial generation of ROS in chick cardiomyocytes. Cells were preconditioned with 10 min of hypoxia, followed by 1 h of simulated ischemia and 3 h of reperfusion. Preconditioning decreased cell death from 47 ؎ 3% to 14 ؎ 2%. Return of contraction was observed in 3/3 preconditioned versus 0/6 non-preconditioned experiments. During induction, ROS oxidation of the probe dichlorofluorescin (sensitive to H 2 O 2 ) increased ϳ2.5-fold. As a substitute for hypoxia, the addition of H 2 O 2 (15 mol/liter) during normoxia also induced preconditioning-like protection. Conversely, the ROS signal during hypoxia was attenuated with the thiol reductant 2-mercaptopropionyl glycine, the cytosolic Cu,Zn-superoxide dismutase inhibitor diethyldithiocarbamic acid, and the anion channel inhibitor 4,4-diisothiocyanato-stilbene-2,2-disulfonate, all of which also abrogated protection. ROS generation during hypoxia was attenuated by myxothiazol, but not by diphenyleneiodonium or the nitric-oxide synthase inhibitor L-nitroarginine. We conclude that hypoxia increases mitochondrial superoxide generation which initiates preconditioning protection. Furthermore, mitochondrial anion channels and cytosolic dismutation to H 2 O 2 may be important steps for oxidant induction of hypoxic preconditioning.Myocardial preconditioning was initially described as an adaptive response of the heart to brief episodes of ischemia that decreased necrosis during subsequent prolonged ischemia (1). Reactive oxygen species (ROS 1 ; e.g. superoxide, H 2 O 2 , hydroxyl radicals) generated from brief ischemia/reperfusion have been recognized as possible "triggers" in the initiation of preconditioning (2). Evidence for this role includes intact heart studies where exposure to superoxide or H 2 O 2 caused preconditioninglike protection (2, 3), and other studies demonstrating that antioxidants abolished the induction of preconditioning (4, 5). Few studies have directly measured ROS generation during brief hypoxia or ischemia induction (6). Such direct measures are needed to clarify important questions that remain regarding the role of ROS as inducing agents, including their source, where they are metabolized, and the relative contributions of different oxidant species to the induction of preconditioning protection.Within the intact heart, possible sources of ROS include the cardiomyocytes, endothelial cells, neutrophils, or the auto-oxidation of catecholamines (7,8). Within cardiomyocytes, sources of ROS could include superoxide generation from NAD(P)H or other oxidases such as cytochrome P450 (9 -11), the mitochondrial electron transport chain (12), or even nitric-oxide synthase under conditions where arginine is depleted (13-15). Although it is likely that superoxide is the initial oxidant generated from these systems, the rel...

Cardiomyocytes suppress contraction and O 2 consumption during hypoxia. Cytochrome oxidase undergoes a decrease in V max during hypoxia, which could alter mitochondrial redox and increase generation of reactive oxygen species (ROS). We therefore tested whether ROS generated by mitochondria act as second messengers in the signaling pathway linking the detection of O 2 with the functional response. Contracting cardiomyocytes were superfused under controlled O 2 conditions while fluorescence imaging of 2,7-dichlorofluorescein (DCF) was used to assess ROS generation. Compared with normoxia (PO 2 ϳ 107 torr, 15% O 2 ), graded increases in DCF fluorescence were seen during hypoxia, with responses at PO 2 ‫؍‬ 7 torr > 20 torr > 35 torr. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline attenuated these increases and abolished the inhibition of contraction. Superfusion of normoxic cells with H 2 O 2 (25 M) for >60 min mimicked the effects of hypoxia by eliciting decreases in contraction that were reversible after washout of H 2 O 2 . To test the role of cytochrome oxidase, sodium azide (0.75-2 M) was added during normoxia to reduce the V max of the enzyme. Azide produced graded increases in ROS signaling, accompanied by graded decreases in contraction that were reversible. These results demonstrate that mitochondria respond to graded hypoxia by increasing the generation of ROS and suggest that cytochrome oxidase may contribute to this O 2 sensing.Alterations in oxygen tension (PO 2 ) elicit a variety of functional responses in different cell types, including gene expression, altered metabolic function, altered ion channel activation, and release of neurotransmitters (1). In spontaneously contracting embryonic cardiomyocytes, we previously found significant decreases in contractile activity during prolonged moderate hypoxia (PO 2 ϭ 20 torr for Ͼ2 h) (2). This inhibition was not associated with a depletion of ATP or phosphocreatine stores, and was reversible when normoxic conditions were restored. Similar findings of decreased contractile function during hypoxia (48 h at 1% O 2 ) have also been seen in rat cardiac myocytes (3), which suggests that this response is not unique to embryonic cells. An ability to respond to changes in oxygen tension within the physiological range implies the existence of a cellular O 2 sensor linked to a signal transduction pathway. When activated by hypoxia, the sensor presumably would initiate a signaling cascade which ultimately leads to the functional response (e.g. diminished contractile activity). However, the O 2 sensing mechanism and the subsequent signal transduction pathways involved in the cardiomyocyte responses to hypoxia are not known.A number of different potential mechanisms of cellular O 2 sensing have been identified (1). Mitochondria are responsible for most of the O 2 consumption by the cell and would seem to be well suited because their local PO 2 responds to changes in the ratio of O 2 supply to demand. However, the low apparent K m of cytochrome oxidas...

Within glomeruli, the initial sites of synaptic integration in the olfactory pathway, olfactory sensory axons terminate on dendrites of projection and juxtaglomerular (JG) neurons. JG cells form at least two major circuits: the classic intraglomerular circuit consisting of external tufted (ET) and periglomerular (PG) cells and an interglomerular circuit comprised of the long-range connections of short axon (SA) cells. We examined the projections and the synaptic inputs of identified JG cell chemotypes using mice expressing green fluorescent protein (GFP) driven by the promoter for glutamic acid decarboxylase (GAD) 65 kDa, 67 kDa, or tyrosine hydroxylase (TH). Virtually all (97%) TH+ cells are also GAD67+ and are thus DAergic–GABAergic neurons. Using a combination of retrograde tracing, whole-cell patch-clamp recording, and single-cell three-dimensional reconstruction, we show that different JG cell chemotypes contribute to distinct microcircuits within or between glomeruli. GAD65 + GABAergic PG cells ramify principally within one glomerulus and participate in uniglomerular circuits. DAergic–GABAergic cells have extensive interglomerular projections. DAergic–GABAergic SA cells comprise two subgroups. One subpopulation contacts 5–12 glomeruli and is referred to as “oligoglomerular.” Approximately one-third of these oligoglomerular DAergic SA cells receive direct olfactory nerve (ON) synaptic input, and the remaining two-thirds receive input via a disynaptic ON→ET→SA circuit. The second population of DAergic–GABAergic SA cells also disynaptic ON input and connect tens to hundreds of glomeruli in an extensive “polyglomerular” network. Although DAergic JG cells have traditionally been considered PG cells, their interglomerular connections argue that they are more appropriately classified as SA cells.

Although a burst of oxidants has been well described with reperfusion, less is known about the oxidants generated by the highly reduced redox state and low O(2) of ischemia. This study aimed to further identify the species and source of these oxidants. Cardiomyocytes were exposed to 1 h of simulated ischemia while oxidant generation was assessed by intracellular dihydroethidine (DHE) oxidation. Ischemia increased DHE oxidation significantly (0.7 +/- 0.1 to 2.3 +/- 0.3) after 1 h. Myxothiazol (mitochondrial site III inhibitor) attenuated oxidation to 1.3 +/- 0.1, as did the site I inhibitors rotenone (1.0 +/- 0.1), amytal (1.1 +/- 0.1), and the flavoprotein oxidase inhibitor diphenyleneiodonium (0.9 +/- 0.1). By contrast, the site IV inhibitor cyanide, as well as inhibitors of xanthine oxidase (allopurinol), nitric oxide synthase (nitro-L-arginine methyl ester), and NADPH oxidase (apocynin), had no effect. Finally, DHE oxidation increased with Cu- and Zn-containing superoxide dismutase (SOD) inhibition using diethyldithiocarbamate (2.7 +/- 0.1) and decreased with exogenous SOD (1.1 +/- 0.1). We conclude that significant superoxide generation occurs during ischemia before reperfusion from the ubisemiquinone site of the mitochondrial electron transport chain.

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