Enhanced Endothelium-dependent Relaxation Of Rat Pulmonary Artery Following β-adrenergic Overstimulation: Involvement Of The No/cgmp/vasp Pathway.

The aim of this study was to investigate whether β-adrenoceptor (β-AR) overstimulation induced by in vivo treatment with isoproterenol (ISO) alters vascular reactivity and nitric oxide (NO) production and signaling in pulmonary arteries. Vehicle or ISO (0.3mgkg(-1)day(-1)) was administered daily to male Wistar rats. After 7days, the jugular vein was cannulated to assess right ventricular (RV) systolic pressure (SP) and end diastolic pressure (EDP). The extralobar pulmonary arteries were isolated to evaluate the relaxation responses, protein expression (Western blot), NO production (diaminofluorescein-2 fluorescence), and cyclic guanosine 3',5'-monophosphate (cGMP) levels (enzyme immunoassay kit). ISO treatment induced RV hypertrophy; however, no differences in RV-SP and EDP were observed. The pulmonary arteries from the ISO-treated group showed enhanced relaxation to acetylcholine that was abolished by the NO synthase (NOS) inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME); whereas relaxation elicited by sodium nitroprusside, ISO, metaproterenol, mirabegron, or KCl was not affected by ISO treatment. ISO-treated rats displayed enhanced endothelial NOS (eNOS) and vasodilator-stimulated phosphoprotein (VASP) expression in the pulmonary arteries, while phosphodiesterase-5 protein expression decreased. ISO treatment increased NO and cGMP levels and did not induce eNOS uncoupling. The present data indicate that β-AR overactivation enhances the endothelium-dependent relaxation of pulmonary arteries. This effect was linked to an increase in eNOS-derived NO production, cGMP formation and VASP content and to a decrease in phosphodiesterase-5 expression. Therefore, elevated NO bioactivity through cGMP/VASP signaling could represent a protective mechanism of β-AR overactivation on pulmonary circulation.

Endothelial dysfunction in cardiovascular and endocrine-metabolic diseases: an update

The endothelium plays a vital role in maintaining circulatory homeostasis by the release of relaxing and contracting factors. Any change in this balance may result in a process known as endothelial dysfunction that leads to impaired control of vascular tone and contributes to the pathogenesis of some cardiovascular and endocrine/metabolic diseases. Reduced endothelium-derived nitric oxide (NO) bioavailability and increased production of thromboxane A2, prostaglandin H2 and superoxide anion in conductance and resistance arteries are commonly associated with endothelial dysfunction in hypertensive, diabetic and obese animals, resulting in reduced endothelium-dependent vasodilatation and in increased vasoconstrictor responses. In addition, recent studies have demonstrated the role of enhanced overactivation ofβ-adrenergic receptors inducing vascular cytokine production and endothelial NO synthase (eNOS) uncoupling that seem to be the mechanisms underlying endothelial dysfunction in hypertension, heart failure and in endocrine-metabolic disorders. However, some adaptive mechanisms can occur in the initial stages of hypertension, such as increased NO production by eNOS. The present review focuses on the role of NO bioavailability, eNOS uncoupling, cyclooxygenase-derived products and pro-inflammatory factors on the endothelial dysfunction that occurs in hypertension, sympathetic hyperactivity, diabetes mellitus, and obesity. These are cardiovascular and endocrine-metabolic diseases of high incidence and mortality around the world, especially in developing countries and endothelial dysfunction contributes to triggering, maintenance and worsening of these pathological situations.

Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling

Background: Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis. Results: Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability. Conclusion: A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.

Antioxidant activity of flavonoids in isolated mitochondria

Mitochondria are important intracellular sources and targets of reactive oxygen species (ROS), while flavonoids, a large group of secondary plant metabolites, are important antioxidants. Following our previous study on the energetics of mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, the present work addressed the antioxidant activity of these compounds (1-50 mu mol/L) on Fe2+/citrate-mediated membrane lipid peroxidation (LPO) in isolated rat liver mitochondria, running in parallel studies of their antioxidant activity in non-organelle systems. Only quercetin inhibited the respiratory chain of mitochondria and only galangin caused uncoupling. Quercetin and galangin were far more potent than taxifolin and catechin in affording protection against LPO (IC50 = 1.23 +/- 0.27 and 2.39 +/- 0.79 mu mol/L, respectively), although only quercetin was an effective scavenger of both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals. These results, together with the previous study, suggest that the 2,3-double bond in conjugation with the 4-oxo function in the flavonoid structure are major determinants of the antioxidant activity of flavonoids in mitochondria, the presence of an o-di-OH structure on the B-ring, as occurs in quercetin, favours this activity via superoxide scavenging, while the absence of this structural feature in galangin, favours it via a decrease in membrane fluidity and/or mitochondrial uncoupling. Copyright (c) 2008 John Wiley & Sons, Ltd.

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 mu M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca(2+) efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP(+) transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. (C) 2011 Elsevier Inc. All rights reserved.

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 mu M) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells. (C) 2010 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Mitochondrial energy metabolism and redox responses to hypertriglyceridemia

In this work we review recent findings that explain how mitochondrial bioenergetic functions and redox state respond to a hyperlipidemic in vivo environment and may contribute to the maintenance of a normal metabolic phenotype. The experimental model utilized to evidence these adaptive mechanisms is especially useful for these studies since it exhibits genetic hypertriglyceridemia and avoids complications introduced by high fat diets. Liver from hypertrigliceridemic (HTG) mice have a greater content of glycerolipids together with increased mitochondrial free fatty acid oxidation. HTG liver mitochondria have a higher resting respiration rate but normal oxidative phosphorylation efficiency. This is achieved by higher activity of the mitochondrial potassium channel sensitive to ATP (mitoK(ATP)). The mild uncoupling mediated by mitoK(ATP) accelerates respiration rates and reduces reactive oxygen species generation. Although this response is not sufficient to inhibit lipid induced extra-mitochondrial oxidative stress in whole liver cells it avoids amplification of this redox imbalance. Furthermore, higher mitoK(ATP) activity increases liver, brain and whole body metabolic rates. These mitochondrial adaptations may explain why these HTG mice do not develop insulin resistance and obesity even under a severe hyperlipidemic state. On the contrary, when long term high fat diets are employed, insulin resistance, fatty liver and obesity develop and mitochondrial adaptations are inefficient to counteract energy and redox imbalances.

Classical and alternative components of the mitochondrial respiratory chain in pathogenic fungi as potential therapeutic targets

The frequency of opportunistic fungal infection has increased drastically, mainly in patients who are immunocompromised due to organ transplant, leukemia or HIV infection. In spite of this, only a few classes of drugs with a limited array of targets, are available for antifungal therapy. Therefore, more specific and less toxic drugs with new molecular targets is desirable for the treatment of fungal infections. In this context, searching for differences between mitochondrial mammalian hosts and fungi in the classical and alternative components of the mitochondrial respiratory chain may provide new potential therapeutic targets for this purpose.

Impaired calcium influx despite hyper-reactivity in contralateral carotid following balloon injury: eNOS involvement

Balloon catheter injury results in hyper-reactivity to phenylephrine in contralateral carotids. Decreased nitric oxide (NO) modulation and/or increased intracellular calcium concentration triggers vascular smooth muscle contraction. Therefore, this study explores the participation of NO signaling pathway and calcium mobilization on hyper-reactivity to phenylephrine in contralateral carotids. Concentration-response curves for calcium (CaCl(2)) and phenylephrine were obtained in control and contralateral carotids four days after balloon injury, in the presence and absence of the inhibitors (L-NAME, L-NNA, 1400W, 7-NI, Oxyhemoglobin, ODQ or Tiron). Confocal microscopy using Fluo-3AM or DHE was performed to detect the intracellular levels of calcium and reactive oxygen species, respectively. The modulation of NO on phenylephrine-induced contraction was absent in the contralateral carotid. Phenylephrine-induced intracellular calcium mobilization was not altered in contralateral carotids. However, extracellular calcium mobilization by phenylephrine was reduced in the contralateral carotid compared to control arteries, and this result was confirmed by confocal microscopy. L-NAME increased phenylephrine-induced extracellular calcium mobilization in the contralateral carotid to the control levels. Results obtained with L-NNA, 1400W, 7-NI, OxyHb, ODQ or Tiron showed that this response was mediated by products from endothelial NOS (eNOS) different from NO and without soluble guanylate cyclase activation, but it involved superoxide anions. Furthermore. Tiron or L-NNA reduced the levels of reactive oxygen species in contralateral carotids. Data suggest that balloon catheter injury promoted eNOS uncoupling in contralateral carotids, which generates superoxide rather than NO, and reduces phenylephrine-induced extracellular calcium mobilization, despite the hyper-reactivity to phenylephrine in contralateral carotids. (C) 2010 Elsevier B.V. All rights reserved.

Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel

Fast synaptic neurotransmission is mediated by transmitter-activated conformational changes in ligand-gated ion channel receptors, culminating in opening of the integral ion channel pore. Human hereditary hyperekplexia, or startle disease, is caused by mutations in both the intracellular or extracellular loops flanking the pore-lining M2 domain of the glycine receptor alpha 1 subunit. These flanking domains are designated the M1-M2 loop and the M2-M3 loop respectively. We show that four startle disease mutations and six additional alanine substitution mutations distributed throughout both loops result in uncoupling of the ligand binding sites from the channel activation gate. We therefore conclude that the M1-M2 and M2-M3 loops act in parallel to activate the channel. Their locations strongly suggest that they act as hinges governing allosteric control of the M2 domain. As the members of the ligand-gated ion channel superfamily share a common structure, this signal transduction model may apply to all members of this superfamily.

The common-866G > A variant in the promoter of UCP2 is associated with decreased risk of coronary artery disease in type 2 diabetic men

OBJECTIVE-Uncoupling protein 2 (UCP2) is a physiological downregulator of reactive oxygen species generation and plays an antiatherogenic role in the vascular wall. A common variant in the UCP2 promoter (-866G>A) modulates mRNA expression, with increased expression associated with the A allele. We investigated association of this variant with coronary artery disease (CAD) in two cohorts of type 2 diabetic subjects. RESEARCH DESIGN AND METHODS-We studied 3,122 subjects from the 6-year prospective Non-Insulin-Dependent Diabetes, Hypertension, Microalbuminuria, Cardiovascular Events, and Ramipril (DIABHYCAR) Study (14.9% of CAD incidence at follow-up). An independent, hospital-based cohort of 335 men, 52% of whom had CAD, was also studied. RESULTS-We observed an inverse association of the A allele with incident cases of CAD in a dominant model (hazard risk 0.88 [95% CI 0.80-0.96]; P = 0.006). Similar results were observed for baseline cases of CAD. Stratification by sex confirmed an allelic association with CAD in men, whereas no association was observed in women. All CAD phenotypes considered-myocardial infarction, angina pectoris, coronary artery bypass graft (CABG), and sudden death-contributed significantly to the association. Results were replicated in a cross-sectional study of an independent cohort (odds ratio 0.47 [95% CI 0.25-0.89]; P = 0.02 for a recessive model). CONCLUSIONS-The A allele of the -866G>A variant of UCP2 was associated with reduced risk of CAD in men with type 2 diabetes in a 6-year prospective study. Decreased risk of myocardial infarction, angina pectoris, CABG, and sudden death contributed individually and significantly to the reduction of CAD risk. This association was independent of other common CAD risk factors.

Aspirin prevents diabetic oxidative changes in rat lacrimal gland structure and function

The aim of this study is to evaluate whether aspirin reduces Diabetis Mellitus (DM) oxidative damage in the lacrimal gland (LG), and ocular surface (OS). Ten weeks after streptozotocin induced DM and aspirin treatment, LG and OS of rats were compared for tear secretion, hidtology, peroxidase activity, and expression of uncoupling proteins (UCPs). DM reduction of tear secretion was prevented by aspirin (P < 0.01). Alterations of LG morphology and increased numbers of lipofucsin-like inclusions were observed in diabetic but not in aspirin-treated diabetic rats. Peroxidase activity levels were higher and UCP-2 was reduced in DM LG but not in aspirin treated (P = 0.0025 and P < 0.05, respectively). The findings prevented by aspirin indicate a direct inhibitory effect on oxidative pathways in LG and their inflammatory consequences, preserving the LG structure and function against hyperglycemia and/or insulin deficiency damage.

Effect of weight reduction on liver histology and biochemistry in patients with chronic hepatitis C

Background: Steatosis occurs in more than 50% of patients with chronic hepatitis C and is associated with increased hepatic fibrosis. In many of these patients the pathogenesis of steatosis appears to be the some as for patients with non-alcoholic fatty liver disease-that is, related to visceral adiposity and obesity. Methods: The effect of a three month weight reduction programme on liver biochemistry and metabolic parameters was examined in 19 subjects with steatosis and chronic hepatitis C. Paired liver biopsies were performed in 10 subjects, prior to and 3-6 months following the intervention, to determine the effect of weight loss on liver histology. Results: There was a mean weight loss of 5.9 (3.2) kg and a mean reduction in waist circumference of 9.0 (5.0) cm. In 16 of the 19 patients, serum alanine aminotransferase levels fell progressively with weight loss. Mean fasting insulin fell from 16 (7) to 11 (4) mmol/l (p

Bioactivation of tamoxifen by recombinant human cytochrome P450 enzymes

Tamoxifen is a major drug used for adjuvant chemotherapy of breast cancer; however, its use has been associated with a small but significant increase in risk of endometrial cancer. In rats, tamoxifen is a hepatocarcinogen, and DNA adducts have been observed in both rat and human tissues. Tamoxifen has been shown previously to be metabolized to reactive products that have the potential to form protein and DNA adducts. Previous studies have suggested a role for P450 3A4 in protein adduct formation in human liver microsomes, via a catechol intermediate; however, no clear correlation was seen between P450 3A4 content of human liver microsomes and adduct formation. In the present study, we investigated the P450 forms responsible for covalent drug-protein adduct formation and the possibility that covalent adduct formation might occur via alternative pathways to catechol formation. Recombinant P450 3A4 catalyzed adduct formation, and this correlated with the level of uncoupling in the P450 incubation, consistent with a role of reactive oxygen species in potentiating adduct formation after enzymatic formation of the catechol metabolite. Whereas P450s 1AI, 2D6, and 3A5 generated catechol metabolite, no covalent adduct formation was observed with these forms. By contrast, P450 2136, 2C19, and rat liver microsomes catalyzed drug-protein adduct formation but not catechol formation. Drug protein adducts formed specifically with P450 3A4 in incubations using membranes isolated from bacteria expressing P450 3A4 and reductase, as well as in reconstitutions of purified 3A4, suggesting that the electrophilic species reacted preferentially with the P450 enzymes concerned.

Mechanism of mitosis-specific activation of MEK1

Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G(2)/M progression. The MAPK cascade has well established roles in entry and exit from mitosis in Xenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G(2)/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr(286). These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis.