Francisco Fernandez-Belda

On the Inhibition Mechanism of Sarcoplasmic or Endoplasmic Reticulum Ca2+-ATPases by Cyclopiazonic Acid

Ca2+-ATPase inhibition by stoichiometric and substoichiometric concentrations of cyclopiazonic acid was studied in sarcoplasmic reticulum preparations from rabbit fast-twitch muscle. The apparent affinity of the nonphosphorylated enzyme for ATP showed a Kd of ∼3 μM in the absence of cyclopiazonic acid and ∼28 μM in the presence of the drug. Fractional saturation of the enzyme by cyclopiazonic acid was accompanied by the appearance of two ATP-binding populations (enzyme with and without drug) and a progressive increase in the half-maximal concentration for saturating the ATP-binding sites. Enzyme turnover in the presence of stoichiometric concentrations of cyclopiazonic acid displayed lower apparent affinity for ATP and lower maximal hydrolytic activity than in the absence of the drug. When cyclopiazonic acid is in the substoichiometric range, the observed kinetic parameters will correspond to the simultaneous contribution of two different reaction cycles sustained by the enzyme with and without drug. The inhibition could be elicited by adding ATP to allow the enzyme turnover when cyclopiazonic acid was preincubated with the enzyme in the presence of Ca2+. The onset of inhibition during enzyme cycling was observed over a period of seconds, revealing the existence of a low inhibition rate constant. It is concluded that cyclopiazonic acid decreases enzyme affinity for ATP in non-turnover conditions by approximately one order of magnitude. This allows enzyme cycling after drug binding, provided that a high ATP concentration is used. Cyclopiazonic acid and ATP do not compete for the same binding site.

Clomipramine and Related Structures as Inhibitors of the Skeletal Sarcoplasmic Reticulum Ca2+ Pump

The Ca2+-pumping activity of skeletal sarcoplasmic reticulum vesicles is half-maximallyinhibited by 120 μM clomipramine, 250 μM desipramine, and 500 μM imipramine or trimipramine.The inhibition is attributed to the dihydrodibenzazepine moiety, since3-(dimethylamino)propionitrile, reproducing the aliphatic amine chain, has no inhibitory action. The inhibitionis shown as a marked decrease of Ca2+ binding at equilibrium in theabsence of ATP and asa reduction of phosphorylation of the Ca2+-free conformation byinorganic phosphate. Therefore,the drug effect is consistent with preferential interaction of tricyclic antidepressants withthe Ca2+-free conformation of the nonphosphorylated enzyme. An additional decrease in theapparent rate constant of enzyme dephosphorylation, i.e., in the release of phosphate fromATP during enzyme cycling was also noticed.

Doxorubicin-induced oxidative stress: The protective effect of nicorandil on HL-1 cardiomyocytes

The primary cardiotoxic action of doxorubicin when used as antitumor drug is attributed to the generation of reactive oxygen species (ROS) therefore effective cardioprotection therapies are needed. In this sense, the antianginal drug nicorandil has been shown to be effective in cardioprotection from ischemic conditions but the underlying molecular mechanism to cope with doxorubicin-induced ROS is unclear. Our in vitro study using the HL-1 cardiomyocyte cell line derived from mouse atria reveals that the endogenous nitric oxide (NO) production was stimulated by nicorandil and arrested by NO synthase inhibition. Moreover, while the NO synthase activity was inhibited by doxorubicin-induced ROS, the NO synthase inhibition did not affect doxorubicin-induced ROS. The inhibition of NO synthase activity by doxorubicin was totally prevented by preincubation with nicorandil. Nicorandil also concentration-dependently (10 to 100 μM) decreased doxorubicin-induced ROS and the effect was antagonized by 5-hydroxydecanoate. The inhibition profile of doxorubicin-induced ROS by nicorandil was unaltered when an L-arginine derivative or a protein kinase G inhibitor was present. Preincubation with pinacidil mimicked the effect of nicorandil and the protection was eliminated by glibenclamide. Quantitative colocalization of fluorescence indicated that the mitochondrion was the target organelle of nicorandil and the observed response was a decrease in the mitochondrial inner membrane potential. Interference with H+ movement across the mitochondrial inner membrane, leading to depolarization, also protected from doxorubicin-induced ROS. The data indicate that activation of the mitochondrial ATP-sensitive K+ channel by nicorandil causing mitochondrial depolarization, without participation of the NO donor activity, was responsible for inhibition of the mitochondrial NADPH oxidase that is the main contributor to ROS production in cardiomyocytes. Impairment of the cytosolic Ca2+ signal induced by caffeine and the increase in lipid peroxidation, both of which are indicators of doxorubicin-induced oxidative stress, were also prevented by nicorandil.

The Ca2+ release channel in junctional sarcoplasmic reticulum: Gating and blockade by cations

1. By using a sarcoplasmic reticulum preparation containing feet structures and the 45Ca2+/filtration technique, the opening and closing response of the Ca(2+)-channel was studied. 2. Extravesicular Sr2+ can activate the channel even though this cation is less efficient than Ca2+ in stimulating the Ca2+ release. Higher Sr2+ concentrations display inhibitory action. 3. By studying the closing response high- and low-affinity cations can be distinguished, according to the concentration range required to exert their effect. 4. The synergistic behavior observed by combining high- and low-affinity blocking cations suggest that they interact through the same binding site. 5. The high-and low-affinity cations are noncompetitive blockers of the activating Ca2+ suggesting the existence of an inhibitory site which is different to the activating site.

Characterization of the palytoxin effect on Ca2+-ATPase from sarcoplasmic reticulum (SERCA).

The effect of palytoxin was studied in a microsomal fraction enriched in longitudinal tubules of the sarcoplasmic reticulum membrane. Half-maximal effect of palytoxin on Ca(2+)-ATPase activity yielded an apparent inhibition constant of approx. 0.4 microM. The inhibition process exhibited the following characteristics: (i) the degree of inhibition was dependent on membrane protein concentration; (ii) no protection was observed when the ATP concentration was raised; (iii) dependence on Ca(2+) concentration with a decreased maximum catalytic rate; (iv) it occurred in the absence of Ca(2+) ionophoric activity. Likewise, the inhibition mechanism was linked to: (i) rapid enzyme phosphorylation from ATP in the presence of Ca(2+) but lower steady-state levels of phosphoenzyme; (ii) more drastic effect on phosphoenzyme levels when the toxin was added to the enzyme in the absence of Ca(2+); (iii) decreased phosphoenzyme levels at saturating Ca(2+) concentrations; (iv) no effect on kinetics of phosphoenzyme decomposition. The palytoxin effect is related with lock of the enzyme in the Ca(2+)-free conformation so that progression of the catalytic cycle is impeded.

Testing the Versatility of the Sarcoplasmic Reticulum Ca2+-ATPase Reaction Cycle When p-Nitrophenyl Phosphate Is the Substrate

A detailed characterization ofp-nitrophenyl phosphate as energy-donor substrate for the sarcoplasmic reticulum Ca2+-ATPase was undertaken in this study. The fact that p-nitrophenyl phosphate can be hydrolyzed in the presence or absence of Ca2+ by the purified enzyme is consistent with the observed phenomenon of intramolecular uncoupling. Under the most favorable conditions, which include neutral pH, intact microsomal vesicles, and low free Ca2+ in the lumen, the Ca2+/Picoupling ratio was 0.6. A rise or decrease in pH, high free Ca2+ in the lumenal space, or the addition of dimethyl sulfoxide increase the intramolecular uncoupling. Alkaline pH and/or high free Ca2+ in the lumen potentiate the accumulation of enzyme conformations with high Ca2+ affinity. Acidic pH and/or dimethyl sulfoxide favor the accumulation of enzyme conformations with low Ca2+ affinity. Under standard assay conditions, two uncoupled routes, together with a coupled route, are operative during the hydrolysis of p-nitrophenyl phosphate in the presence of Ca2+. The prevalence of any one of the uncoupled catalytic cycles is dependent on the working conditions. The proposed reaction scheme constitutes a general model for understanding the mechanism of intramolecular energy uncoupling.

A kinetic study of the irreversible inhibition of an enzyme measured in the presence of coupled enzymes. Fluorescein isothiocyanate as inhibitor of the adenosinetriphosphatase activity from sarcoplasmic reticulum

A systematic procedure for the kinetic study of irreversible inhibition, when the enzymatic activity is measured in the presence of a coupled enzyme system, has been developed and analyzed. Simultaneous variation of the enzyme and inhibitor concentrations, maintaining a constant ratio between them, is recommended. The methodology is established to estimate the kinetic constants corresponding to the irreversible inhibitor. This approach is illustrated by the study of the inhibition of fluorescein isothiocyanate on the Ca2+-ATPase activity from sarcoplasmic reticulum measured in the presence of pyruvate kinase and lactate dehydrogenase as auxiliary enzymes. Treatment of the experimental data has been carried out by non-linear regression.

Mitochondrial ATPase inactivation by interaction with its substrate.

Abstract Purified F1-ATPase is slowly inactivated by interaction, in a preincubation medium, with its substrate MgATP. Interaction with Mg2+ before addition of ATP to the preincubation medium is essential to induce the inactivation. This inactivation is different from other Mg2+-induced inhibitions previously described. Free ATP concentration is implicated in the inactivation process and a linear relationship can be established between this concentration and the number of turnovers which are necessary for total inactivation. ITP, 2′-dATP, and GTP can also induce inactivation. Although ITP and GTP are hydrolyzed at a lower rate than ATP and 2′-dATP, they induce inactivation after a smaller number of turnovers than the latter. This process closely follows a kinetics of the type described for suicide enzymes. A reaction scheme is suggested and discussed.

Effect of the calcium-channel blockers on calcium accumulation in sarcoplasmic reticulum of skeletal muscle

Vesicular fragments of sarcoplasmic reticulum isolated from rabbit skeletal muscle were actively loaded with Ca2+ in the presence of ATP and an ATP-regenerating system using Arsenazo III as metallochromic indicator to monitor Ca2+ movements across the membrane. Once the Ca2+ release is triggered by the presence of tetraphenylboron in the reaction medium, the addition of verapamil or diltiazem gives rise to a net Ca2+ entry inside the vesicles. Preincubation in the presence of verapamil does not abolish the tetraphenylboron-induced Ca2+ release, the verapamil-induced Ca2+ accumulation being still observed. There appears to be a high-affinity site for verapamil titrated in the micromolar concentration range, whereas diltiazem demonstrates more complex behavior when its concentration is raised. This study suggests the existence of a Ca2+ pathway (putative channels) which is blocked by the drugs tested allowing Ca2+ accumulation inside the vesicles owing to the Ca2+-dependent ATPase activity.

Early oxidative damage induced by doxorubicin: Source of production, protection by GKT137831 and effect on Ca2+ transporters in HL-1 cardiomyocytes

In atrial-derived HL-1 cells, ryanodine receptor and Na(+)/Ca(2+)-exchanger were altered early by 5 μM doxorubicin. The observed effects were an increase of cytosolic Ca(2+) at rest, ensuing ryanodine receptor phosphorylation, and the slowing of Ca(2+) transient decay after caffeine addition. Doxorubicin triggered a linear rise of reactive oxygen species (ROS) with no early effect on mitochondrial inner membrane potential. Doxorubicin and ROS were both detected in mitochondria by colocalization with fluorescence probes and doxorubicin-induced ROS was totally blocked by mitoTEMPO. The NADPH oxidase activity in the mitochondrial fraction was sensitive to inhibition by GKT137831, and doxorubicin-induced ROS decreased gradually as the GKT137831 concentration added in preincubation was increased. When doxorubicin-induced ROS was prevented by GKT137831, the kinetic response revealed a permanent degree of protection that was consistent with mitochondrial NADPH oxidase inhibition. In contrast, the ROS induction by doxorubicin after melatonin preincubation was totally eliminated at first but the effect was completely reversed with time. Limiting the source of ROS production is a better alternative for dealing with oxidative damage than using ROS scavengers. The short-term effect of doxorubicin on Ca(2+) transporters involved in myocardiac contractility was dependent on oxidative damage, and so the impairment was subsequent to ROS production.