Fernando Henao

Fluorescence measurements of steady state peroxynitrite production upon SIN-1 decomposition: NADH versus dihydrodichlorofluorescein and dihydrorhodamine 123.

The production of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition can be continuously monitored, with a sensitivity ≤ 0.1 μM, from the kinetics of NADH fluorescence quenching in phosphate buffers, as well as in buffers commonly used with cell cultures, like Lockes buffer or Dulbeccos modified Eagles medium (DMEM-F12). The half-time for peroxynitrite production during SIN-1 decomposition ranged from 14–18 min in DMEM-F12 (plus and minus phenol red) to 21.5 min in Lockes buffer and 26 min in DMEM-F12 supplemented with apotransferrin (0.1 mg/mL). The concentration of peroxynitrite reached a peak that was linearly dependent upon SIN-1 concentration, and that for 100 μM SIN-1 amounted to 1.4 ± 0.2 μM in Lockes buffer, 3.2–3.6 μM in DMEM-F12 (plus and minus phenol red) and 1.8 μM in DMEM-F12 supplemented with apotransferrin. Thus, the maximum concentration of peroxynitrite ranged from 1.2 to 3.6% of added SIN-1. NADH was found to be less sensitive than dihydrorhodamine 123 and 2′,7′-dichlorodihydrofluorescein diacetate to oxidation by H2O2, which is produced during SIN-1 decomposition in common buffers. It is shown that peroxynitrite concentration can be controlled (±5%) during predetermined times by using sequential SIN-1 pulses, to simulate chronic exposure of cells or subcellular components to peroxynitrite.

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/ nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES‐containing Lockes buffer (pH 7.4) to 50–100 µm SIN‐1 during 2 h decreased the steady‐state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 ± 33 nm to 60 ± 10 nm, whereas exposure to ≥ 0.3 mm SIN‐1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 ± 10 nm (due to inactivation of L‐type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN‐1‐induced necrotic cell death. Both effects of SIN‐1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4‐benzoic acid)porphyrin chloride. Supplementation of Lockes buffer with catalase before addition of 0.5–1 mm SIN‐1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60–120 min exposure to SIN‐1 and also protected against SIN‐1‐induced necrotic cell death. α‐Tocopherol, the potent NMDA receptor antagonist (+)‐MK‐801 and the N‐ and P‐type Ca2+ channels blocker ω‐conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN‐1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60–120 min exposure to 0.5–1 mm SIN‐1. It is concluded that L‐type Ca2+ channels are a primary target of SIN‐1‐induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5–1 μm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.

Synaptosomal plasma membrane Ca2+ pump activity inhibition by repetitive micromolar ONOO− pulses

A sustained increase of intracellular free [Ca(2+)] ([Ca(2+)](i)) has been shown to be an early event of neuronal cell death induced by peroxynitrite (ONOO(-)). In this paper, chronic exposure to ONOO(-) has been simulated by treatment of rat brain synaptosomes or plasma membrane vesicles with repetitive pulses of ONOO(-) during at most 50 min, which efficiently produced nitrotyrosine formation in several membrane proteins (including the Ca(2+)-ATPase). The plasma membrane Ca(2+)-ATPase activity at near-physiological conditions (pH 7, submicromolar Ca(2+), and millimolar Mg(2+)-ATP concentrations), which plays a major role in the control of synaptic [Ca(2+)](i), can be more than 75% inhibited by a sustained exposure to micromolar ONOO(-) (e.g., to 100 pulses of 10 microM ONOO(-)). This inhibition is irreversible and mostly due to a decreased V(max), and to the 2-fold increase of the K(0.5) for Ca(2+) stimulation and about 5-fold increase of the K(M) for Mg(2+)-ATP. [Ca(2+)](i) increases to >400 nM when synaptosomes are subjected to this treatment. Reduced glutathione can afford only partial protection against the inhibition produced by micromolar ONOO(-) pulses. Therefore, inhibition of the plasma membrane Ca(2+)-pump activity during chronic exposure to ONOO(-) may account by itself for a large and sustained increase of intracellular [Ca(2+)](i) in synaptic nerve terminals.

The NADH oxidase activity of the plasma membrane of synaptosomes is a major source of superoxide anion and is inhibited by peroxynitrite

Plasma membrane vesicles from adult rat brain synaptosomes (PMV) have an ascorbate‐dependent NADH oxidase activity of 35–40 nmol/min/(mg protein) at saturation by NADH. NADPH is a much less efficient substrate of this oxidase activity, with a Vmax 10‐fold lower than that measured for NADH. Ascorbate‐dependent NADH oxidase activity accounts for more than 90% of the total NADH oxidase activity of PMV and, in the absence of NADH and in the presence of 1 mm ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 ± 0.5 nmol AFR/min/(mg protein). NADH‐dependent ·O2− production by PMV occurs with a rate of 35 ± 3 nmol/min/(mg protein), and is a coreaction product of the NADH oxidase activity, because: (i) it is inhibited by more than 90% by addition of ascorbate oxidase, (ii) it is inhibited by 1 µg/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the KM(NADH) of the plasma membrane NADH oxidase activity and of NADH‐dependent ·O2− production are identical. Treatment of PMV with repetitive micromolar ONOO– pulses produced almost complete inhibition of the ascorbate‐dependent NADH oxidase and ·O2− production, and at 50% inhibition addition of coenzyme Q10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5‐fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 µm ONOO– pulses lowers the K0.5 for cytochrome c stimulation from 6 ± 1 (control) to 1.5 ± 0.5 µm. Thus, the ascorbate‐dependent plasma membrane NADH oxidase activity can act as a source of neuronal ·O2−, which is up‐regulated by cytosolic cytochrome c and down‐regulated under chronic oxidative stress conditions producing ONOO–.

Inhibition by 4-hydroxynonenal (HNE) of Ca2+ transport by SERCA1a: Low concentrations of HNE open protein-mediated leaks in the membrane

Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition of the maximal ATPase activity and Ca(2+) transport ability of SERCA1a, the Ca(2+) pump in these membranes. The concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca(2+) binding to the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also reacted with Lys(515) within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the steady-state ability of SR vesicles to sequester Ca(2+) and the ATPase activity of SR membranes in the absence of added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNE-treated SR vesicles to Ca(2+), an increase in permeability that did not arise from alteration of the lipid component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent increase in permeability probably arose from modification of proteins of about 150-160kDa, present in very low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae). HNE-induced promotion, via these proteins, of Ca(2+) leakage pathways might be involved in the general toxic effects of HNE.

Inhibition of the sarcoplasmic reticulum (Ca2+ + Mg2+-ATPase by Zn(II)

Micromolar Zn 2+ concentrations have been found to inhibit the (Ca 2+ + Mg 2+ )-ATPase of sarcoplasmic reticulum vesicles solubilized with deoxycholate or Triton X-100 or made leaky with A23187 and of the purified enzyme. Kinetic studies on the dependence of this activity upon Zn 2+ concentrations have been carried out at 25°C and 37°C under a variety of experimental conditions, such as absence of Mg 2+ in the assay medium, different total Ca 2+ , total Mg 2+ and total ATP concentrations and at various total membrane protein concentrations. The concentrations of relevant kinetic species (free divalent cations, free ATP and ATP complexes) have been computed for all these assay conditions. As a result, we have found that the inhibition of the (Ca 2+ + Mg 2+ )-ATPase activity is produced by free Zn 2+ , with a value of the inhibition constant K i of 8 ± 2 μM. In addition, Zn 2+ · ATP can be used as an alternative substrate by this ATPase with a K M value of 30 μM and with V max of (2.0 ± 0.2) μmol ATP hydrolyzed per min per mg protein at 37°C. In conclusion, our results suggest the existence of sites in the ATPase distinct to the high-affinity Ca 2+ binding sites and to the Mg 2+ subside in the catalytic center, to which binding of Zn 2+ produces inhibition and a shift of the E 1 /E 2 conformational equilibrium.

Thymbra capitata Essential Oil Prevents Cell Death Induced by 4-Hydroxy-2-Nonenal in Neonatal Rat Cardiac Myocytes

An interdisciplinary experimental investigation on the antioxidant activity of Thymbra capitata essential oil was made. This plant is a Mediterranean culinary herb, whose essential oil antioxidant power has recently been demonstrated in vitro as one of the highest in nature. We tested if this in vitro antioxidant capacity was reproducible on biological systems using as model system primary cultures of neonatal rat cardiomyocytes treated with the lipid peroxidation product 4-hydroxy-2-nonenal. The composition and the in vitro antioxidant activity of the T. capitata essential oil were also assessed. Cell viability, mitochondrial membrane potential, and reactive oxygen species level were measured in cells treated with pathophysiologic doses of 4-hydroxy-2-nonenal (< 10 µM) or vehicle after being pre-incubated with small concentrations of the T. capitata essential oil, and the ability of small doses (< 40 ppm) to prevent the death of neonatal rat cardiomyocytes proved very remarkable. Long-term pre-incubation (12 h) with 20 ppm prevented 4-hydroxy-2-nonenal-induced cell death and avoided mitochondrial membrane potential loss and reactive oxygen species generation caused by 4-hydroxy-2-nonenal. A deleterious effect was shown at doses higher than 40 ppm. The results of this study pave the way to further analysis in animal models to achieve a deeper understanding of the in vivo antioxidant power of T. capitata essential oil.

Projection Map of Covalently Phosphorylated Ca-ATPase from Tubular Crystals

F. DELAVOIE,a D. MCINTOSH,b F. HENAO,c G. PERANZI,a P. CHAMPEIL,d D. STOKES,e AND J-J. LACAPEREa, aINSERM U410, 75870 Paris, France bDepartment of Chemical Pathology, Universtiy of Cape Town Medical School, Cape Town, South Africa cBioquimica y Biologia Molecular, Universidad de Extremadura, 06080 Badajoz, Spain dCNRS U2096, SBFM/DBJC, CEA-Saclay, 91191 Gif-sur-Yvette, France eSkirball Institute, Cell Biology, New York University, New York, New York 10016, USA

Fluorescnece anisotropy of fluorescein phosphatidylethanolthiocarbamide in lipid bilayers and in Ca2+-ATPase/lipid reconstituted systems

Abstract In this communication it is reported that the anisotropy of the fluorescence of fluorescein phosphatidylethanolthiocarbamide (FPE) is higher in reconstituted membranes of sarcoplasmic reticulum Ca2+-ATPase than in liposomes of phosphatidylcholine or of phosphatidylcholine-phosphatidylserine, and is dependent on the lipid to protein ratio. The dependence of the fluorescence anisotropy of FPE on the molar fraction of annular lipids does not fit the theoretical prediction for random distribution of FPE between annular and bulk lipids. The experimental results are fitted, however, by introducing the effect of preferential binding of FPE over unlabelled lipids to annular sites. The theoretical simulations allow us to conclude that FPE binds to the annular Ca2+-ATPase sites with 3- to 7-fold higher affinity than phosphatidylcholine or phosphatidylserine. This study shows the utility of the fluorescence anisotropy of lipids labelled at the polar head group to monitor lipid binding to annular sites of sarcoplasmic reticulum Ca2+-ATPase. The methods developed in this paper can also be applied to other reconstituted membranes and to decide whether or not a given fluorescent lipid derivative should be used for measurements of interfacial physical-chemical properties of the lipid bilayer of native biological membranes.