Juan V. Esplugues

NO as a signalling molecule in the nervous system

The discovery that nitric oxide (NO) functions as a signalling molecule in the nervous system has radically changed the concept of neural communication. Indeed, the adoption of the term nitrergic for nerves whose transmitter function depends on the release of NO or for transmission mechanisms brought about by NO (Moncada et al., 1997) emphasizes the specific characteristics of this mediator. The physical properties of NO prevent its storage in lipid-lined vesicles and metabolism by hydrolytic degradatory enzymes. Therefore, unlike established neurotransmitters, NO is synthesized on demand and is neither stored in synaptic vesicles nor released by exocytosis, but simply diffuses from nerve terminals. The distance of this NO diffusion (40 – 300 μm in diameter) implies that structures in the vicinity of the producing cell, both neuronal and non-neuronal, are influenced following its release. This suggests that, as well as acting as a neurotransmitter, NO has a neuromodulatory role (Garthwaite & Boulton, 1995). In addition, it diffuses into rather than binds with protein receptors on adjacent cells, and most of its known actions are the consequence of interplay with intracellular targets that would usually be regarded as secondary messengers. The activity of conventional neurotransmitters is terminated either by re-uptake mechanisms or enzymatic degradation while inactivation of NO follows reaction with a substrate. There are multiple points at which biological control can be exerted over the production and activity of conventional neurotransmitters. However, control of the synthesis of NO is the key to regulating its activity. Endothelial NOS (eNOS) and inducible NOS (iNOS) are present in the nervous system and will be duly addressed here. However, neuronal NOS (nNOS) is the principal isoform present in said system and will be the main focus of this review. All nNOS positive neurones exhibit α-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity, which has become the histochemical marker of nitrergic neurones. However, early results demonstrating this may have been limited by inappropriate fixation procedures and should be viewed with caution (Wolf, 1997). The original cloning of full-length nNOS produced what is now designated as nNOSα, and which accounts for the majority of nNOS activity in nervous tissue (Bredt et al., 1991). In addition, four splice variants have recently been identified (nNOSβ, nNOSγ, nNOSμ and nNOS-2) and these appear to exhibit distinct cellular and tissue locations (Gibson, 2001; Nakane et al., 1993; Silvagno et al., 1996; Alderton et al., 2001). In particular, there is growing evidence that nNOS biosynthesis in excitable tissues is not restricted to neurones while substantial amounts of this enzyme have been identified in skeletal muscle, where it is involved in the regulation of metabolism and muscle contractility (Stamler & Meissner, 2001). The magnitude of literature dealing with the role of NO in the nervous system is so great that it would be impossible to include in this review the entirety of the research carried out. For logistical reasons, only groundbreaking references have been quoted, but when necessary, recent reviews dealing with specific areas within the field have been included and are intended to act as a guideline for further reading.

The vasodilator role of endogenous nitric oxide in the rat gastric microcirculation.

The role of endogenous nitric oxide (NO) in the gastric microcirculation of the anaesthetised rat was investigated using the selective inhibitor of NO synthesis, NG-monomethyl-L-arginine (L-NMMA). L-NMMA (12.5-50 mg kg-1 i.v.) induced a dose-dependent increase in systemic arterial blood pressure (BP) and fall in resting gastric mucosal blood flow (MBF), as estimated by hydrogen-gas clearance. The effects of L-NMMA on BP and MBF were abolished by concurrent administration of L-arginine. The enantiomer D-NMMA had no effect on resting BP or MBF. These findings indicate that endogenous NO, derived from L-arginine, plays a local vasodilator role in the gastric mucosal microvasculature.

Angiotensin II induces leukocyte-endothelial cell interactions in vivo via AT1 and AT2 receptor-mediated P-selectin upregulation

BackgroundAngiotensin II (Ang II) plays a critical role in the development of vascular lesions in hypertension, atherosclerosis, and several renal diseases. Because Ang II may contribute to the leukocyte recruitment associated with these pathological states, the aim of the present study was to assess the role of Ang II in leukocyte–endothelial cell interactions in vivo. Methods and ResultsIntravital microscopy of the rat mesenteric postcapillary venules was used. Sixty minutes of superfusion with 1 nmol/L Ang II induced a significant increase in leukocyte rolling flux (83.8±20.7 versus 16.4±3.1 cells/min), adhesion (11.4±1.0 versus 0.8±0.5 cells/100 &mgr;m), and emigration (4.0±0.7 versus 0.2±0.2 cells/field) without any vasoconstrictor activity. These effects were not mediated by mast cell activation. Intravenous pretreatment with AT1 (losartan) or AT2 (PD123,319) receptor antagonists significantly reduced Ang II–induced responses. A combination of both receptor antagonists inhibited the leukocyte rolling flux, adhesion, and extravasation elicited by Ang II at 60 minutes. Pretreatment of animals with fucoidin or an adhesion-blocking anti–rat P-selectin monoclonal antibody abolished Ang II–induced leukocyte responses. Furthermore, rat platelet P-selectin expression was not affected by Ang II stimulation. ConclusionsAng II induces significant leukocyte rolling, adhesion, and emigration, which may contribute not only to hypertension but also to the onset and progression of the vascular damage associated with disease states in which plasma levels of this peptide are elevated.

Endogenous Nitric Oxide as a Mediator of Gastric Mucosal Vasodilatation During Acid Secretion

The role of the endothelium-derived vasodilator, nitric oxide, as a mediator of the increase in gastric mucosal blood flow and as a modulator of the acid secretory response induced by pentagastrin was investigated in the anesthetised rat. Intravenous administration of the selective inhibitor of endogenous nitric oxide synthesis, NG-monomethyl-L-arginine (12.5 and 50 mg/kg), which dose-dependently increased systemic arterial blood pressure, did not affect resting acid output. However, NG-monomethyl-L-arginine significantly reduced resting gastric mucosal blood flow at the higher dose, as determined by hydrogen gas clearance. Infusion of pentagastrin (80 micrograms kg-1.h-1) stimulated gastric acid secretion and elevated gastric mucosal blood flow. Pretreatment with NG-monomethyl-L-arginine (12.5 mg/kg IV) did not affect this stimulation of acid output but substantially attenuated (by 65% +/- 10%; P less than 0.01) the associated increase in gastric mucosal blood flow. Pretreatment with NG-monomethyl-L-arginine (50 mg/kg IV) induced a minor inhibition of pentagastrin-stimulated acid secretion but abolished the increase in gastric mucosal blood flow. When administered during pentagastrin infusion, NG-monomethyl-L-arginine (50 mg/kg IV) did not affect the acid secretory response but induced a 76% +/- 8% inhibition (P less than 0.05) of the elevated gastric mucosal blood flow. The effects of NG-monomethyl-L-arginine on blood pressure, acid secretion, and gastric mucosal blood flow were abolished by pretreatment with the precursor for nitric oxide synthesis, L-arginine (300 mg/kg IV). These findings in the rat suggest that endogenous nitric oxide, synthesized from L-arginine, does not directly modulate the acid secretory response induced by pentagastrin but makes a substantial contribution to the mucosal vasodilatation associated with the stimulation of gastric acid secretion.

Inhibition of mitochondrial respiration by endogenous nitric oxide: A critical step in Fas signaling

We have found that activation of human adult T cell leukemia (Jurkat) cells with anti-Fas Ab leads, in a concentration-dependent manner, to an early burst of production of nitric oxide (NO), which inhibits cell respiration. This results in mitochondrial hyperpolarization, dependent on the hydrolysis of glycolytic ATP by the F1Fo-ATPase acting in reverse mode. During this early phase of activation, there is a transient release of superoxide anion. All these processes can be prevented by an inhibitor of NO synthase. Approximately 2 h after stimulation with anti-Fas Ab, a distinct second phase can be detected. This comprises a concentration-dependent collapse in mitochondrial membrane potential, a second wave of free radical production, and activation of caspase-8 leading to apoptosis. This second phase is abolished by an inhibitor of caspase activation. In contrast, inhibition of NO synthesis leads to an enhancement and acceleration of these latter processes, suggesting that the early NO-dependent phase represents a protective mechanism. The significance of the two phases in relation to cell survival and death remains to be studied.

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.

Enhanced oxidative stress and increased mitochondrial mass during Efavirenz-induced apoptosis in human hepatic cells

Efavirenz (EFV) is widely used in the treatment of HIV‐1 infection. Though highly efficient, there is growing concern about EFV‐related side effects, the molecular basis of which remains elusive.

Inhibition of Mitochondrial Function by Efavirenz Increases Lipid Content in Hepatic Cells

Efavirenz (EFV) is a non‐nucleoside reverse transcriptase inhibitor (NNRTI) widely used in human immunodeficiency virus (HIV) infection therapy. It has been associated with hepatotoxic effects and alterations in lipid and body fat composition. Given the importance of the liver in lipid regulation, we have evaluated the effects of clinically used concentrations of EFV on the mitochondria and lipid metabolism of human hepatic cells in vitro. Mitochondrial function was rapidly undermined by EFV to an extent that varied with the concentration employed; in particular, respiration and intracellular adenosine triphosphate (ATP) levels were reduced whereas reactive oxygen species (ROS) production increased. Results in isolated mitochondria suggest that the mechanism responsible for these actions was a specific inhibition of complex I of the respiratory chain. The reduction in energy production triggered a compensatory mechanism mediated by the enzyme adenosine monophosphate–activated protein kinase (AMPK), the master switch of cellular bioenergetics. Fluorescence and nuclear magnetic resonance demonstrated a rapid intracellular increase of neutral lipids, usually in the form of droplets. This was prevented by the AMPK inhibitor compound C and by removal of fatty acids from the culture medium. These effects were not reproduced by Nevirapine, another NNRTI. EFV is clinically coadministered with two nucleoside reverse transcriptase inhibitors. Evaluation of one of the most common combination, EFV/Lamivudine/Abacavir, revealed that the effects of EFV on ROS production were enhanced. Conclusion: Clinical concentrations of EFV induce bioenergetic stress in hepatic cells by acutely inhibiting mitochondrial function. This new mechanism of mitochondrial interference leads to an accumulation of lipids in the cytoplasm that is mediated by activation of AMPK. HEPATOLOGY 2010

Discrepancies Between Nitroglycerin and NO-Releasing Drugs on Mitochondrial Oxygen Consumption, Vasoactivity, and the Release of NO

It has been generally acknowledged that the actions of glyceryl trinitrate (GTN) are a result of its bioconversion into NO. However, recent observations have thrown this idea into doubt, with many studies demonstrating that NO is present only when there are high concentrations of GTN. We have explored this discrepancy by developing a new approach that uses confocal microscopy to directly detect NO. Intracellular levels of NO in the rat aortic vascular wall have been compared with those present after incubation with 3 different NO donors (DETA-NO, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine), endothelial activation with acetylcholine, or administration of GTN. We have also evaluated the relaxant effects of these treatments on isolated rings of aorta following activation of the enzyme soluble guanylyl cyclase and their inhibitory action on mitochondrial respiration, which is an index of the interaction of NO with the enzyme of the electron transport chain cytochrome C oxidase. In the case of the various NO donors and acetylcholine, we detected a concentration-dependent relationship in the intensity of vascular relaxation and degree of NO fluorescence and an increase in the Michaelis constant (Km) for O2. GTN did not produce similar effects, and although clinically relevant concentrations of this compound caused clear, concentration-related relaxations, there was neither any increase in NO-related fluorescence nor an augmented Km for O2. The nature of these differences suggests that these concentrations of GTN do not release free NO but probably a different species that, although it interacts with soluble guanylyl cyclase in vascular smooth muscle, does not inhibit O2 consumption by vascular mitochondria.

Oxidative Stress and Mitochondrial Dysfunction in Sepsis: A Potential Therapy with Mitochondria-Targeted Antioxidants

Sepsis and septic shock are the major causes of death in intensive care units. The prevalent hypothesis regarding the mechanisms of sepsis and septic shock indicates that this syndrome is caused by an excessive defensive and inflammatory response characterised by massive increases in reactive oxygen species (ROS), nitric oxide (NO) and inflammatory cytokines. The consequences of these syndromes are systemic damage to the vascular endothelium, impaired tissue and a compromised whole body respiration, glutathione depletion and mitochondrial respiratory dysfunction with diminished levels of ATP and O(2) consumption. In general, ROS are essential to the functions of cells and particularly immune cells, but adequate levels of antioxidant defenses are required to protect against the harmful effects of excessive ROS production. Mitochondrial oxidative stress damage and dysfunction contribute to a number of cell pathologies that manifest themselves in a range of conditions, including sepsis. This review considers the process of sepsis from a mitochondrial perspective, discussing strategies for the targeted delivery of antioxidants to mitochondria currently under development. We will provide a summary of the following areas: the cellular metabolism of ROS and its role in pathophysiological processes such as sepsis; currently available antioxidants and possible reasons for their efficacy and inefficacy in ameliorating oxidative stress-mediated diseases; and recent developments in antioxidants that target the matrix-facing surface of the inner mitochondrial membrane in order to protect against mitochondrial oxidative damage, and their therapeutic potential as a treatment for sepsis.