Pilar D’Ocon

Complex I Dysfunction and Tolerance to Nitroglycerin: An Approach Based on Mitochondrial-Targeted Antioxidants

Nitroglycerin (GTN) tolerance was induced in vivo (rats) and in vitro (rat and human vessels). Electrochemical detection revealed that the incubation dose of GTN (5×10−6 mol/L) did not release NO or modify O2 consumption when administered acutely. However, development of tolerance produced a decrease in both mitochondrial O2 consumption and the Km for O2 in animal and human vessels and endothelial cells in a noncompetitive action. GTN tolerance has been associated with impairment of GTN biotransformation through inhibition of aldehyde dehydrogenase (ALDH)-2, and with uncoupling of mitochondrial respiration. Feeding rats with mitochondrial-targeted antioxidants (mitoquinone [MQ]) and in vitro coincubation with MQ (10−6 mol/L) or glutathione (GSH) ester (10−4 mol/L) prevented tolerance and the effects of GTN on mitochondrial respiration and ALDH-2 activity. Biotransformation of GTN requires functionally active mitochondria and induces reactive oxygen species production and oxidative stress within this organelle, as it is inhibited by mitochondrial-targeted antioxidants and is absent in HUVEC&rgr;0 cells. Experiments analyzing complex I–dependent respiration demonstrate that its inhibition by GTN is prevented by mitochondrial-targeted antioxidants. Furthermore, in presence of succinate (10×10−3 mol/L), a complex II electron donor added to bypass complex I–dependent respiration, GTN-treated cells exhibited O2 consumption rates similar to those of controls, thus suggesting that complex I was affected by GTN. We propose that, following prolonged treatment with GTN in addition to ALDH-2, complex I is a target for mitochondrially generated reactive oxygen species. Our data also suggest a role for mitochondrial-targeted antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.

Endothelial NT-3 Delivered by Vasculature and CSF Promotes Quiescence of Subependymal Neural Stem Cells through Nitric Oxide Induction

Interactions of adult neural stem cells (NSCs) with supportive vasculature appear critical for their maintenance and function, although the molecular details are still under investigation. Neurotrophin (NT)-3 belongs to the NT family of trophic factors, best known for their effects in promoting neuronal survival. Here we show that NT-3 produced and secreted by endothelial cells of brain and choroid plexus capillaries is required for the quiescence and long-term maintenance of NSCs in the mouse subependymal niche. Uptake of NT-3 from irrigating vasculature and cerebrospinal fluid (CSF) induces the rapid phosphorylation of endothelial nitric oxide (NO) synthase present in the NSCs, leading to the production of NO, which subsequently acts as a cytostatic factor. Our results identify a novel interaction between stem cells and vasculature/CSF compartments that is mediated by an unprecedented role of a neurotrophin and indicate that stem cells can regulate their own quiescence in response to endothelium-secreted molecules.

CHARACTERIZATION OF TWO DIFFERENT CA2+ ENTRY PATHWAYS DEPENDENT ON DEPLETION OF INTERNAL CA2+ POOLS IN RAT AORTA

Abstract Ryanodine (10 μM), thapsigargin (1 μM) and cyclopiazonic acid (10 μM) produced a slow, sustained contractile response in rat aorta that only can be observed in Ca2+-containing solution. In Ca2+-free medium, no response to the drugs was obtained, which suggests that the contraction elicited in presence of Ca2+ is mainly due to the contribution of extracellular influx. This Ca2+ entry does not depend on the opening of dihydropyridine-dependent Ca2+-channels for nimodipine does not affect this.Noradrenaline (1 μM) induced a biphasic response in Ca2+-free medium that was mediated by two different Ca2+ compartments, one of which is common to caffeine (10 mM), and is also depleted by ryanodine (10 μM), thapsigargin (1 μM) and cyclopiazonic acid (10 μM). This compartment loses its Ca2+ content after long exposure (65 min) to Ca2+-free EDTA-containing solution and its refilling was also affected by the three agents tested. The other compartment depleted by noradrenaline, but not by caffeine, was also insensitive to ryanodine, thapsigargin and cyclopiazonic acid, and did not lose its Ca2+ after 65 min in Ca2+-free medium.Contractions induced by noradrenaline (1 μM) or caffeine (10 mM) in Ca2+-free medium were not affected by ryanodine, thapsigargin and cyclopiazonic acid when these agents were added 1 min before or during the response to each agonist. After depletion of internal Ca2+ stores sensitive to noradrenaline, an increase in the resting tone (IRT) of rat aorta was observed when Ca2+ was added again in absence of the agonist. This IRT was not affected by treatment with ryanodine, thapsigargin and cyclopiazonic acid, and represents a Ca2+ entry pathway dependent on the depletion of the noradrenaline-sensitive Ca2+ compartment. In conclusion, we can differentiate two Ca2+ entry pathways in rat aorta that depend on the previous depletion of two internal Ca2+ compartments: One corresponds to the classic capacitative Ca2+ entry model and is promoted by depletion of the internal pool sensitive to noradreanline, caffeine, ryanodine, thapsigargin and cyclopiazonic acid, the other is dependent only on depletion of an α1-adrenoceptor-sensitive Ca2+ pool.

Capacitative Ca2+ entry associated with α1-adrenoceptors in rat aorta

Abstract In rat aorta, depletion of internal Ca2+ stores by addition of noradrenaline (1μM) induces a biphasic response (an initial phasic response and a tonic one) mediated by two different intracellular Ca2+ pools. This response cannot be repeated, suggesting a depletion of internal Ca2+ stores sensitive to noradrenaline. In absence of the agonist, this depletion is the signal for the entry of extracellular Ca2+, not only to refill the stores but also, under our experimental conditions, to activate the contractile proteins thus inducing an increase in the resting tone (IRT) that constitutes functional evidence of this Ca2+ entry. The ionic channels involved in the mechanism of the IRT have been studied in the present work.The fact that the addition of nimodipine (10–15–10–11M) selectively inhibits the IRT suggests that this mechanical response is mediated by Ca2+ influx through dihydropyridine-sensitive Ca2+ channels. Moreover, the inhibitory action of nimodipine is attenuated by glibenclamide (10μM). Cromakalim (10–10–10–6M) also inhibits the IRT concentration dependently, and this inhibition is antagonized by glibenclamide (10μM). These results relate the ATP-dependent K+ channels to the mechanism of the IRT.The refilling of the two internal Ca2+ compartments sensitive to noradrenaline was, like the IRT, altered in presence of the compounds tested, since the subsequent contractile response to noradrenaline was decreased. The present results suggest that nimodipine treatment inhibits the refilling of the Ca2+ compartment responsible for the tonic contraction induced by noradrenaline in Ca2+-free medium, whereas the refilling of the Ca2+ pool responsible for the phasic response to noradrenaline remained unaltered. Both the phasic and tonic responses to noradrenaline in Ca2+-free medium decreased after treatment with cromakalim. We can therefore assume that the refilling of both Ca2+ compartments sensitive to noradrenaline was inhibited.In conclusion, these results are consistent with the contraction of the rat aorta in response to noradrenaline in Ca2+-free medium consisting of an initial phasic response and a tonic one. The former is due to the release of internal Ca2+ from a compartment refilled through a special channel that is cromakalim but not dihydropyridine sensitive. The tonic response is due to Ca2+ release from another compartment refilled through a cromakalim- and dihydropyridine-sensitive Ca2+ channel. The Ca2+ entry through this latter channel intervenes in the IRT observed during the refilling of these stores previously depleted by noradrenaline, and the opening state of this channel is also modulated by ATP-dependent K2+ channels.

Myocardial G protein receptor-coupled kinase expression correlates with functional parameters and clinical severity in advanced heart failure.

BACKGROUND In heart failure (HF), sympathetic hyperactivation induces deleterious effects in myocardial β-adrenergic signaling, with receptor down-regulation and desensitization mediated by G protein receptor-coupled kinases (GRKs). We hypothesised that changes in GRK isoforms may be associated with clinical status in advanced HF, using the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) scale. METHODS We included 31 patients with advanced HF undergoing transplantation. According to INTERMACS profiles, mRNA and protein levels of GRK isoforms in left ventricular (LV) myocardium were analyzed and compared with nonfailing LV samples. RESULTS In failing LV myocardium, GRK2 and GRK5 (but not GRK3) protein was up-regulated compared with control samples. Among HF patients, an increase in GRK2 and GRK5 mRNA and protein abundance was observed in β-agonist-treated patients (vs β-blockers: P < .05) and in higher-risk INTERMACS status (profiles 2 and 3 vs 4 and 5: P < .05). A significant negative correlation of GRK2 expression with LV stroke volume supported these findings. CONCLUSIONS Increased GRK2 correlates with clinical severity using the INTERMACS scale and LV stroke volume, supporting it as a potential target in advanced HF. These changes are paralleled by GRK5 expression in the failing myocardium, suggesting a relevant role in human HF.

Role of endothelial nitric oxide in pulmonary and systemic arteries during hypoxia

UNLABELLED Our aim was to investigate the role played by endothelial nitric oxide (NO) during acute vascular response to hypoxia, as a modulator of both vascular tone (through guanylate cyclase (sGC) activation) and mitochondrial O2 consumption (through competitive inhibition of cytochrome-c-oxydase (CcO)). Organ bath experiments were performed and O2 consumption (Clark electrode) was determined in isolated aorta, mesenteric and pulmonary arteries of rats and eNOS-knockout mice. All pre-contracted vessels exhibited a triphasic hypoxic response consisting of an initial transient contraction (not observed in vessels from eNOS-knockout mice) followed by relaxation and subsequent sustained contraction. Removal of the endothelium, inhibition of eNOS (by L-NNA) and inhibition of sGC (by ODQ) abolished the initial contraction without altering the other two phases. The initial hypoxic contraction was observed in the presence of L-NNA+NO-donors. L-NNA and ODQ increases O2 consumption in hypoxic vessels and increases the arterial tone in normoxia but not in hypoxia. When L-NNA+mitochondrial inhibitors (cyanide, rotenone or myxothiazol) were added, the increase in tone was similar in normoxic and hypoxic vessels, which suggests that inhibition of the binding of NO to reduced CcO restored the action of NO on sGC. CONCLUSION A complex equilibrium is established between NO, sGC and CcO in vessels in function of the concentration of O2: as O2 falls, NO inhibition of mitochondrial O2 consumption increases and activation of sGC decreases, thus promoting a rapid increase in tone in both pulmonary and systemic vessels, which is followed by the triggering of NO-independent vasodilator/vasoconstrictor mechanisms.

Differences in the Signaling Pathways of α1A- and α1B-Adrenoceptors Are Related to Different Endosomal Targeting

Aims To compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. Methods Using CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). Results and Conclusions Constitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin

Antithrombin activity and outcomes in patients undergoing cardiac surgery with cardiopulmonary bypass.

We recently reported prospective results from a cohort of patients scheduled for elective cardiac surgery with cardiopulmonary bypass (CPB) in which most baseline clinical parameters of patients and surgery outcomes failed to demonstrate relationships with post-CPB antithrombin (AT) activity. In this extension study, a larger sample size (250 patients) was analyzed following general linear models. Patients’ sociodemographic and pre-CPB clinical data as well as pre/post-CPB AT activity and outcomes were collected. There was a significant decrease of post-CPB AT activity (95.6 ± 13.7–64.6 ± 12.1%; P < 0.001). Univariate and multivariate analyses revealed that a decrease of approximately 1% post-CPB AT activity may be expected per 3 years increase in patients age. Univariate analysis showed that post-CBP AT activity was inversely related to the need for transfusions, acute renal failure and occurrence of any complication (re-intervention, low cardiac output, arrhythmia, lung dysfunction, stroke, acute renal failure, mesenteric ischemia and re-hospitalization; P < 0.05). Multivariate analysis adjusted for age and pre-CPB AT did not show statistical significance. Odds ratio (OR) less than 1 was observed in most outcomes (0.8 on average), which suggested a reduction of the probability for an increase of 10% in post-CBP AT. Our results confirm the role of low postsurgery AT activity influencing outcomes in patients undergoing CPB.

Glucocorticoids as modulators of expression and activity of Antithrombin (At): Potential clinical relevance

INTRODUCTION An inverse relationship has been reported between decreased postoperative Antithrombin (AT) plasmatic levels and the incidence of complications. We hypothesized that Nuclear Hormone Receptors could modulate the expression of SERPINC1, encoding AT, through a Hormone Regulatory Element present in its promoter, and thus hormone analogs could be a pharmacological complement in surgical procedures to activate endogenous AT synthesis. MATERIALS AND METHODS The expression of SERPINC1 was analyzed in HepG2 cells by quantitative RT-PCR and Western Blot. Two studies were conducted with (a) patients submitted to cardiac surgery with cardiopulmonary bypass receiving (n =17) or not (n=321) glucocorticoids (GCs) as part of their pharmacological treatment, and (b) patients who received (n =20) or not (n=16) GCs as part of their surgery (exodontia or knee arthroscopic, respectively). AT activity in plasma was determined by Innovance Antithrombin Test on a BCS XP System hemostasis analyzer. RESULTS 13 nuclear hormone receptor ligands were assayed, being GW4064 (FXR ligand) the most potent activator. Retinoids, activating RXR, and GCs (Dexamethasone, cortisone and methylprednisolone) also resulted in increased AT expression. Chronic GC treatment mitigates the decreased AT activity observed after cardiac surgery. In patients who received two acute GC doses, pre-operative and post-operative AT activity was similar, whereas a significant decrease was observed after surgery in untreated patients. CONCLUSIONS Whereas retinoids and FXR ligands are investigational compounds, regulation of AT by GCS could have a higher potential for translation to clinical practice, pre-conditioning the patient against complications related to reduced AT levels. Larger prospective studies are needed to define the exact role of GCs and their potential clinical utility in cardiac surgery.

Changes in the expression of α1B-adrenoceptor in peripheral mononuclear cells correlates with blood pressure and plasmatic homocysteine

Human peripheral mononuclear cells (HPMC) have been suggested as a practical surrogate for myocardial or vascular cells. Present work analyses if changes in the expression of α1-adrenoceptors (ARs) in HPMC are related to the hypertensive state and its clinical consequences. Quantitative RT-PCR was employed to evaluate the mRNA levels of the three α1-ARs (α1A, α1B, α1D) in HPMC isolated from normotensive and hypertensive patients, and also in tissues from two animal models of hypertension: spontaneously hypertensive rats (SHR) and hypertension induced by chronic treatment with L-NAME. In patients, 24-h ambulatory blood pressure and serum biochemical profile were also recorded. We found that α1B-AR expression was higher in HPMC from hypertensive patients and correlated with blood pressure and plasmatic homocysteine. A rise in the α1B-AR expression in kidneys, but not in heart from hypertensive animal models was also found. α1D-AR did not change in HPMC, not in rat heart or kidney, but a significant correlation with plasmatic aldosterone was found. In conclusion, we have proved that α1-ARs mRNA expression in HPMC correlates with clinical variables and could be used as a potential biomarker in hypertensive patients.