Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the α-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1α is unstable in cells maintained at 21% O2, but is progressively stabilized as the O2 concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 µM) stabilize HIF-1α at all O2 concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (ρ0-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1α stabilized by exposure of the cells to 3% O2. This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1α by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance.

Background—: Hypoxia is an important microenvironmental factor influencing atherosclerosis progression by inducing foam-cell formation, metabolic adaptation of infiltrated macrophages, and plaque neovascularization. Therefore, imaging plaque hypoxia could serve as a marker of lesions at risk. Methods and Results—: Advanced aortic atherosclerosis was induced in 18 rabbits by atherogenic diet and double balloon endothelial denudation. Animals underwent ¹⁸ F-fluoromisonidazole positron emission tomographic and ¹⁸ F-fluorodeoxyglucose positron emission tomographic imaging after 6 to 8 months (atherosclerosis induction) and 12 to 16 months (progression) of diet initiation. Four rabbits fed standard chow served as controls. Radiotracer uptake of the abdominal aorta was measured using standardized uptake values. After imaging, plaque hypoxia (pimonidazole), macrophages (RAM-11), neovessels (CD31), and hypoxia-inducible factor-1α were assessed by immunohistochemistry. ¹⁸ F-fluoromisonidazole uptake increased with time on diet (standardized uptake value mean, 0.10±0.01 in nonatherosclerotic animals versus 0.20±0.03 [ P =0.002] at induction and 0.25±0.03 [ P <0.001] at progression). Ex vivo positron emission tomographic imaging corroborated the ¹⁸ F-fluoromisonidazole uptake by the aorta of atherosclerotic rabbits. ¹⁸ F-fluorodeoxyglucose uptake also augmented in atherosclerotic animals, with an standardized uptake value mean of 0.43±0.02 at induction versus 0.35±0.02 in nonatherosclerotic animals ( P =0.031) and no further increase at progression. By immunohistochemistry, hypoxia was mainly located in the macrophage-rich areas within the atheromatous core, whereas the macrophages close to the lumen were hypoxia-negative. Intraplaque neovessels were found predominantly in macrophage-rich hypoxic regions (pimonidazole ⁺ /hypoxia-inducible factor-1α ⁺ /RAM-11 ⁺ ). Conclusions—: Plaque hypoxia increases with disease progression and is present in macrophage-rich areas associated with neovascularization. ¹⁸ F-fluoromisonidazole positron emission tomographic imaging emerges as a new tool for the detection of atherosclerotic lesions.

We have assessed the relative contribution of Ca²⁺ entry and Ca²⁺ release from internal stores to the [Ca²⁺]_i transients evoked by purinergic receptor activation in bovine adrenal chromaffin cells. The [Ca²⁺]_i was recorded from single cells using ratiometric fura-2 microfluorometry. Two discrete groups of ATP-sensitive cells could be distinguished on the basis of their relative capacity to respond to ATP in the virtual absence of extracellular Ca²⁺. One group of cells (group I) failed to respond to ATP in the absence of Ca²⁺, was completely insensitive to UTP, and displayed suramin-blockable [Ca²⁺]_i transients when challenged with ATP in the presence of external Ca²⁺. ATP activated a prominent and rapidly inactivating Mn²⁺ influx pathway in group I cells, as assessed by monitoring Mn²⁺ quenching of fura-2 fluorescence. In contrast, a second group of ATP-sensitive cells (group II) exhibited pronounced [Ca²⁺]_i rises when challenged with ATP and UTP in the absence of Ca²⁺ and was completely insensitive to suramin. ATP and UTP activated a delayed and less prominent Mn²⁺ influx pathway in group II cells. Contrary to the nicotinic receptor agonist DMPP, which evoked a preferential release of epinephrine, ATP evoked a preferential release of norepinephrine, and UTP had no effect on secretion. Suramin nearly suppressed ATP-evoked norepinephrine release. We conclude that chromaffin cells contain two distinct and cell-specific purinoceptor subtypes. Although some cells express a P_2U-type purinoceptor coupled to Ca²⁺ release from internal stores and to the associated slow Ca²⁺ refilling mechanism, other cells express a suramin-sensitive and UTP-insensitive purinoceptor exclusively coupled to Ca²⁺ influx, probably an ATP-gated channel. It is suggested that the ATP-gated channel is preferentially localized to norepinephrine-secreting chromaffin cells and supports specifically hormone output from these cells. Thus, the biochemical pathways involved in the exocytotic release of the two major stress-related hormones appear to be regulated by distinct signaling systems.

Nitric oxide (NO), generated endogenously in NO-synthase-transfected cells, increases the reduction of mitochondrial cytochrome c oxidase (CcO) at O2 concentrations ([O2]) above those at which it inhibits cell respiration. Thus, in cells respiring to anoxia, the addition of 2.5 μM L-arginine at 70 μM O2 resulted in reduction of CcO and inhibition of respiration at [O2] of 64.0±0.8 and 24.8±0.8 μM, respectively. This separation of the two effects of NO is related to electron turnover of the enzyme, because the addition of electron donors resulted in inhibition of respiration at progressively higher [O2], and to their eventual convergence. Our results indicate that partial inhibition of CcO by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO. Thus, respiration is maintained without compromising the bioenergetic status of the cell. We suggest that this is a physiological mechanism regulated by the flux of electrons in the mitochondria and by the changing ratio of O2:NO, either during hypoxia or, as a consequence of increases in NO, as a result of cell stress.