Magnesium Deficiency Causes Loss of Response to Intermittent Hypoxia in Paraganglion Cells

Magnesium deficiency is suggested to contribute to many age-related diseases. Hypoxia-inducible factor 1α (HIF-1α) is known to be a master regulator of hypoxic response. Here we show that hypomagnesemia suppresses reactive oxygen species (ROS)-induced HIF-1α activity in paraganglion cells of the adrenal medulla and carotid body. In PC12 cells cultured in the low magnesium medium and treated with cobalt chloride (CoCl₂) or exposed to intermittent hypoxia, ROS-mediated HIF-1α activity was suppressed. This suppression was due to up-regulation of inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS) that was caused by NF-κB activation, which resulted from ROS and calcium influx mainly through the T-type calcium channels. Induction of tyrosine hydroxylase, a target of HIF-1, by CoCl₂ injection was suppressed in the adrenal medulla of magnesium-deficient mice because of up-regulation of IPAS. Also in the carotid body of magnesium-deficient mice, CoCl₂ and chronic intermittent hypoxia failed to enhance the tyrosine hydroxylase expression. These results demonstrate that serum magnesium levels are a key determinant for ROS-induced hypoxic responses.Hypoxia-inducible factor 1α (HIF-1α)² and its family members are master regulators of hypoxic response (1–3). In hypoxia, the HIF-1, composed of HIF-1α and HIF-1β/Arnt, binds to hypoxia response element (HRE) to induce the gene expression of hypoxia-responsive proteins, such as erythropoietin and vascular endothelial growth factor. In addition to these proteins, tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, is induced in rat pheochromocytoma-derived PC12 cells and paraganglion cells in the adrenal medulla (AM) and carotid body (CB) in response to hypoxia (4). The CB acts as the primary peripheral chemoreceptor (5), and glomus cells of the CB are responsible for monitoring oxygen levels in arterial blood (5, 6). Through the release of neurotransmitters, including dopamine, the CB delivers information to the respiratory and cardiovascular networks in the brainstem, resulting in increases of ventilatory frequency and volume and also raising cardiac output.HIF-dependent hypoxic response is also caused by chronic intermittent hypoxia (CIH), which is a common feature of obstructive sleep apnea (OSA). There is accumulating evidence that CIH is associated with an increased oxidative stress (7, 8). Peng et al. (9) have shown that CIH induces reactive oxygen species (ROS) generation, thereby increasing HIF-1α expression, which is critical for eliciting CIH-induced cardiorespiratory responses by the CB. CIH also increases ROS generation and TH expression in the AM, although it is less sensitive than the CB (10).Recent studies have identified that IPAS, which is one of the alternatively spliced variants of HIF-3α, acts as a dominant negative inhibitor of HIF-1α by a direct interaction with HIF-1α and prevents its DNA binding (11). IPAS is predominantly expressed in the Purkinje cells of the cerebellum and corneal epithelium. In addition, because the IPAS gene has an HRE sequence in its promoter, IPAS can be induced by hypoxia in the heart and lung. Therefore, IPAS acts as a negative feedback inhibitor of HIF-1α (12).Magnesium deficiency is believed to be related to many diseases, such as hypertension, ischemic heart disease, and diabetes mellitus (13–16). However, the molecular mechanisms underlying the role of magnesium in the pathogenesis of these diseases have been largely undefined. Our analyses here demonstrate that magnesium deficiency causes a loss of ROS-induced HIF-1α activity by inducing IPAS gene expression.     

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a ��-Carotene 15,15��-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The K_m, k_cat, and k_cat/K_m values for β-carotene as substrate were 37 μm, 3.6 min⁻¹, and 97 mm⁻¹ min⁻¹, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₅ seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (3–5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23–28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.