Agonist binding and activation of the rat beta(1)-adrenergic receptor: role of Trp(134(3.28)), Ser(190(4.57)) and Tyr(356(7.43))

We investigated the role of Trp(134(3.28)), Ser(190(4.57)) and Tyr(356(7.43)) in agonist binding to, and activation of, the rat beta(1)-adrenergic receptor by comparing pK(i)s and functional responses of W134A, S190A and Y356F mutant receptors to wild type, all stably expressed in CHO cells. All three mutations significantly (P < 0.05) reduced adenylyl cyclase intrinsic activity (IA) compared to wild type in response to stimulation with both (-)-isoprenaline (53-88%) and (-)-RO363 (46-61%), and there was no significant correlation either between IA or pD(2) and pK(i) (P > 0.4), suggesting that changes in pK(i) were not sufficient to explain the fall in adenylyl cyclase activity. The most pronounced reduction in affinity (126-fold, P < 0.01) was displayed by xamoterol for the Y356F mutation, suggesting that xamoterol is able to directly interact with Tyr(356(7.43)). For the other agonists, the change in pK(i) values for the mutant receptors ranged from a 20-fold decrease to a 2-fold increase compared to the wild type. In a three-dimensional model of the rat beta(1)-adrenergic receptor, Trp(134(3.28)) and Tyr(356(7.43)) form part of a hydrophobic binding pocket involving residues in transmembrane helices 1, 2, 3 and 7. Our results suggest that Trp(134(3.28)) and Tyr(356(7.43)), together with Trp(353(7.40)), are able to interact via pi-pi interactions to stabilize the extracellular ends of transmembrane helices 3 and 7. Ser(190(4.57)) appears to be involved in a hydrogen bonding network, which maintains the spatial relationship between transmembrane helices 3 and 4. These interhelical interactions suggest that the three mutated residues stabilize the active receptor state by maintaining the proper packing of their respective transmembrane helix within the helix bundle, facilitating the appropriate movement and rotation of the transmembrane regions during the activation process.     

Intracellular generation of reactive oxygen species by mitochondria

Mitochondria have bioenergetic properties that strongly suggest their involvement in the cellular formation of reactive oxygen species (ROS). Apparent confirmation of this process has come from work with isolated mitochondria, which have been shown to produce H(2)O(2) from dismutating superoxide radicals. Two different sites were reported to shuttle single electrons to oxygen out of the normal respiratory sequence. However, the mechanisms for ROS formation at these two sites are controversial. Arguments against mitochondrial ROS formation in the living cell are based on the fact that bioenergetic alterations may result from the mechanical removal of mitochondria from their natural environment. Furthermore, the invasive detection methods that are generally used may be inappropriate because of the possible interaction of the detection system with mitochondrial constituents. The use of non-invasive detection methods has proved that ROS formation does not occur unless changes in the physical state of the membrane are established. The aim of this commentary is to discuss critically the arguments in favor of mitochondria as the main intracellular source of ROS. The pros and cons of working with isolated mitochondria, as well as the detection methodology are carefully analyzed to judge whether or not the above assumption is correct. The conclusion that mitochondria are the main ROS generators in the cell contradicts the fact that ROS release was not observed. However, if electron flow from ubiquinol to the bc(1) complex is hindered due to changes in lipid fluidity, single electrons may transfer to dioxygen and produce H(2)O(2) via superoxide radicals.     

L-arginine regulates neuronal nitric oxide synthase production of superoxide and hydrogen peroxide

Tetrahydrobiopterin (H(4)B) in the absence of L-arginine has been shown to be an important factor in promoting the direct formation of hydrogen peroxide (H(2)O(2)) at the expense of superoxide (O(2)(*-)) by neuronal nitric oxide synthase (NOS1) [Rosen GM, Tsai P, Weaver J, Porasuphatana S, Roman LJ, Starkov AA, et al. Role of tetrahydrobiopterin in the regulation of neuronal nitric-oxide synthase-generated superoxide. J Biol Chem 2002;277:40275-80]. Based on these findings, it is hypothesized that L-arginine also shifts the equilibrium between O(2)(*-) and H(2)O(2). Experiments were designed to test this theory. As the concentration of L-arginine and N(omega)-hydroxyl-L-arginine increases, the rate of NADPH consumption for H(4)B-bound NOS1 decreased resulting in lower rates of both O(2)(*-) and H(2)O(2) generation, while increasing the rate of nitric oxide (*NO) production. At saturating concentrations of L-arginine or N(omega)-hydroxyl-L-arginine (50microM), NOS1 still produced O(2)(*-) and H(2)O(2). Both L-arginine and N(omega)-hydroxyl-L-arginine have greater impact on the rate of generation of O(2)(*-) than on H(2)O(2).     

Ixodid and argasid tick species and west nile virus

Control of West Nile virus (WNV) can only be effective if the vectors and reservoirs of the virus are identified and controlled. Although mosquitoes are the primary vectors, WNV has repeatedly been isolated from ticks. Therefore, tick-borne transmission studies were performed with an ixodid (Ixodes ricinus) and an argasid tick species (Ornithodoros moubata). Both species became infected after feeding upon viremic hosts, but I. ricinus ticks were unable to maintain the virus. In contrast, O. moubata ticks were infected for at least 132 days, and the infection was maintained through molting and a second bloodmeal. Infected O. moubata ticks transmitted the virus to rodent hosts, albeit at a low level. Moreover, the virus was nonsystemically transmitted between infected and uninfected O. moubata ticks co-fed upon uninfected hosts. Although ticks are unlikely to play a major role in WNV transmission, our findings suggest that some species have the potential to act as reservoirs for the virus.     

BCNU-induced gR2 DEFECT mediates S-glutathionylation of Complex I and respiratory uncoupling in myocardium

A deficiency of mitochondrial glutathione reductase (or GR2) is capable of adversely affecting the reduction of GSSG and increasing mitochondrial oxidative stress. BCNU [1,3-bis (2-chloroethyl)-1-nitrosourea] is an anticancer agent and known inhibitor of cytosolic GR ex vivo and in vivo. Here we tested the hypothesis that a BCNU-induced GR2 defect contributes to mitochondrial dysfunction and subsequent impairment of heart function. Intraperitoneal administration of BCNU (40 mg/kg) specifically inhibited GR2 activity by 79.8 ± 2.7% in the mitochondria of rat heart. However, BCNU treatment modestly enhanced the activities of mitochondrial Complex I and other ETC components. The cardiac function of BCNU-treated rats was analyzed by echocardiography, revealing a systolic dysfunction associated with decreased ejection fraction, decreased cardiac output, and an increase in left ventricular internal dimension and left ventricular volume in systole. The respiratory control index of isolated mitochondria from the myocardium was moderately decreased after BCNU treatment, whereas NADH-linked uncoupling of oxygen consumption was significantly enhanced. Extracellular flux analysis to measure the fatty acid oxidation of myocytes indicated a 20% enhancement after BCNU treatment. When the mitochondria were immunoblotted with antibodies against GSH and UCP3, both protein S-glutathionylation of Complex I and expression of UCP3 were significantly up-regulated. Overexpression of SOD2 in the myocardium significantly reversed BCNU-induced GR2 inhibition and mitochondrial impairment. In conclusion, BCNU-mediated cardiotoxicity is characterized by the GR2 deficiency that negatively regulates heart function by impairing mitochondrial integrity, increasing oxidative stress with Complex I S-glutathionylation, and enhancing uncoupling of mitochondrial respiration.     

Nitric oxide modulates the release of serotonin in the rat hypothalamus

To investigate the effect of nitric oxide (NO) on the release of serotonin and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), the posterior hypothalamus of the conscious rat was superfused through a push-pull cannula with drugs which either liberate NO, or inhibit NO synthase (NOS). The NO donors, linsidomine, diethylamine/nitric oxide (DEA/NO), S-nitroso-N-acetylpenicillamine (SNAP), S-nitroso-glutathione (SNOG) and sodium nitroprusside influenced the release of serotonin in a biphasic way. Low concentrations of drugs diminished, while higher concentrations of these compounds enhanced the outflow of serotonin. The NOS inhibitors N(G)-methyl-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NINA) enhanced the serotonin release. A high concentration of L-NAME slightly diminished the outflow of serotonin. Inhibition of the guanylyl cyclase by oxodiazolo[4, 3]quinoxaline-one (ODQ) abolished the changes in serotonin outflow induced by both low and high concentrations of linsidomine. The extracellular concentration of the 5-HIAA was not influenced by the compounds used. These data suggest that endogenous NO modulates the release of serotonin in a biphasic and cGMP-dependent way.     

Characterization of hydroxyl radical formation by microsomal enzymes using a water-soluble trap, terephthalate

Using terephthalic acid as a water-soluble trap, we characterized hydroxyl radicals (HO?) formation by liver microsomal enzymes from isoniazid-treated rats. We found that HO? formation was entirely dependent on intact microsomal enzymes, the presence of NADPH, and iron complexed with EDTA. In contrast to the other radical traps, we found no evidence that terephthalate is a substrate for cytochrome P450. Cumene hydroperoxide, an artificial supporter of cytochrome P450-catalyzed oxidation, failed to maintain HO(.-) formation. HO(.-) formation in liver microsomes was inhibited by the HO(.-) radical scavengers: dimethyl sulfoxide (DMSO), mannitol, and citrulline. It was abolished by catalase, but not superoxide dismutase (SOD), indicating that hydrogen peroxide was the sole precursor of the HO(.-). Therefore, the generation of hydroxyl radicals by microsomal enzymes appears to be dependent on two processes: (1) the rate of hydrogen peroxide production; and (2) the availability of iron ions or other transition metals for Fenton type reactions.     

No 4 gene dose effect on hippocampal atrophy in a large MRI database of healthy elderly subjects

The effect of ApoE genotype on grey matter (GM) atrophy was studied on a cohort of 750 healthy elderly volunteers (age range 63–75 years). High-resolution T1-weighted MR images were processed using both voxel-based morphometry and region of interest analysis for hippocampal volume estimation. Significant decrease of grey matter in http://www.sciencedirect.com/scidirimg/entities/25b.gif" alt="var epsilon" title="var epsilon" border="0">₄ homozygous subjects (n = 12), as compared both to http://www.sciencedirect.com/scidirimg/entities/25b.gif" alt="var epsilon" title="var epsilon" border="0">₄ heterozygous subjects (n = 175) and to noncarrier (n = 563) subjects, was found bilaterally in the medial temporal lobe, including the hippocampus, and extending over the superior temporal gyrus. By contrast, no significant difference was observed between http://www.sciencedirect.com/scidirimg/entities/25b.gif" alt="var epsilon" title="var epsilon" border="0">₄ heterozygous subjects and noncarriers at the level of the medial temporal lobe. Follow-up of the cohort cognitive performances over 4 years after their MRI exam revealed that, as compared to noncarrier subjects, the relative risk of cognitive impairment was 5.9 for http://www.sciencedirect.com/scidirimg/entities/25b.gif" alt="var epsilon" title="var epsilon" border="0">₄ homozygous subjects (P = 0.03), while it was not different from 1 for http://www.sciencedirect.com/scidirimg/entities/25b.gif" alt="var epsilon" title="var epsilon" border="0">₄ heterozygous subjects (P = 0.92). These findings indicate that, in the age range of this cohort, ApoE-4 effects on cortical atrophy and cognitive performances of healthy elderly are limited to http://www.sciencedirect.com/scidirimg/entities/25b.gif" alt="var epsilon" title="var epsilon" border="0">₄ homozygous subjects.     

NMR and pattern recognition studies on liver extracts and intact livers from rats treated with alpha-naphthylisothiocyanate

The metabolite profiles from livers of toxin-treated rats were investigated using high resolution 1H NMR spectroscopy of aqueous (acetonitrile/water), lipidic (chloroform/methanol) extracts and magic angle spinning (MAS)-NMR spectroscopy of intact tissue. Rats were treated with the model cholestatic hepatotoxin, alpha-naphthylisothiocyanate (ANIT, 150 mg/kg) and NMR spectra of liver were analysed using principal components analysis (PCA) to extract novel toxicity biomarker information. 1H NMR spectra of control aqueous extracts showed signals from a range of organic acids and bases, amino acids, sugars, and glycogen. Chloroform/methanol extracts showed signals from a range of saturated and unsaturated triglycerides, phospholipids and cholesterol. The MAS 1H NMR spectra of livers showed a composite of signals found in both aqueous and lipophilic extracts. Following ANIT treatment, 1H NMR-PCA of aqueous extracts indicated a progressive reduction in glucose and glycogen, together with increases in bile acid, choline, and phosphocholine signals. 1H NMR-PCA of chloroform/methanol extracts showed elevated triglyceride levels. The 1H MAS-NMR-PCA analysis allowed direct detection of all of the ANIT-induced tissue perturbations revealed by 1H NMR of extracts, enabling metabolic characterisation of the lesion, which included steatosis, bile duct obstruction and altered glucose/glycogen metabolism. MAS-NMR spectroscopy requires minimal sample preparation and, unlike 1H NMR spectroscopy of tissue extracts, does not discriminate metabolites based on their solubility in a particular solvent and so this is a particularly useful exploratory tool in biochemical toxicology.