Cardiac sarcoplasmic reticulum Ca2+ pump as a target for benzoquinones.

1. Three benzoquinones (p-benzoquinone, 2,5-dimethyl-p-benzoquinone and tetramethyl-1,4-benzoquinone), differing for their electrophilicity, were tested on Ca2+ ATPase activity of cardiac sarcoplasmic reticulum membrane vesicles. 2. Only p-benzoquinone and 2,5-dimethyl-p-benzoquinone inhibited Ca2+ ATPase activity in a time-and concentration-dependent way, tetramethyl-1,4-benzoquinone being ineffective. 3. p-Benzoquinone and 2,5-dimethyl-p-benzoquinone decreased in a concentration-dependent way the total--SH groups of cardiac sarcoplasmic reticulum vesicles. 4. The results suggest that the most electrophilic benzoquinones interact directly with some critical--SH groups of Ca2+ pump inhibiting the enzymatic activity.

Metabolism of simple quinones in guinea pig and rat cardiac tissue.

1. The metabolism of the benzoquinone 2,5-dimethylbenzoquinone and of the naphthoquinone 2,3-dimethoxy-1,4-naphthoquinone was studied in subcellular fractions isolated from cardiac tissue of guinea pig and rat. 2. In both species the benzoquinone was mainly metabolized through the mitochondrial NADH-ubiquinone-oxidoreductase, whereas the naphthoquinone was metabolized to approximately equal extents by mitochondrial reductase and by soluble DT-diaphorase. 3. Guinea pig heart metabolized 3 times more naphthoquinone than rat heart. 4. As a consequence of quinone metabolism, marked amounts of O2- center dot - were generated; naphthoquinone-induced O2- center dot - generation was about 4-fold higher in guinea pig than in rat heart.

Structure-activity relationship for the inhibition of cardiac sarcoplasmic reticulum Ca2+ ATPase activity by naphthoquinones.

Four naphthoquinones (5-OH-1,4-naphthoquinone (juglone), 5-OH-2-CH3-1,4-naphthoquinone (plumbagine), 2-CH3-1,4-naphthoquinone (menadione) and 2,3-(OCH3)2-1,4-naphthoquinone (2,3diOmeNQ)), differing for the presence of electrophilic groups in orto position in respect of quinone mojety and for hydroxylation in C5, were tested on Ca2+ ATPase activity of cardiac sarcoplasmic reticulum membrane vesicles. The 2-unsubstituted quinone, juglone, was a potent inhibitor of Ca2+ ATPase activity, while the 2-methyl-substituted quinones, plumbagine and menadione, inhibited the enzyme activity only after a sufficiently long preincubation time 2,3DiOMeNQ did not affect Ca2+ ATPase activity at all. Hydroxylation in C5 was responsible for the type of inhibition, making it irreversible. A direct interaction of the electrophilic naphthoquinones with -SH groups of the enzyme is suggested.

Chemical and pharmacological characterization of Erythrophleum lasianthum alkaloids.

Two alkaloids 1 and 2 were isolated from the seeds of Erythrophleum lasianthum. Their structures were assigned by spectroscopic and chemical means as 3 beta-hydroxynorerythrosuamine (1) and its 3-O-beta-D-glucopyranoside (2). In spontaneously beating atria, both compounds 1 and 2 showed a marked and concentration-dependent positive inotropic activity and a weak negative chronotropic activity. The positive inotropic effect induced by 1 and 2 was not modified by propranolol, prazosin, carbachol, and ranitidine plus pyrilamine. Both 1 and 2 were very active in inhibiting the Na+/K(+)-ATPase isolated from bovine cardiac sarcolemmal vesicles.

Pharmacological characterization of a new milrinone analogue.

In a new series of milrinone analogues (esters of 2-substituted 5-acetyl-1,6-dihydro-6-oxo-3-pyridinecarboxylic acids), ethyl 5-acetyl-1,6-dihydro-6-oxo-2-phenyl-3-pyridinecarboxylate (compound 2f) has been found to be more potent and more effective than milrinone as a positive inotropic agent while affecting only marginally the frequency rate of guinea-pig isolated atria. This finding prompted us to study the mechanism of cardiac action of compound 2f in electrically driven left atrium from reserpine-treated guinea pigs. Compound 2f induced a statistically significant increase in the contractile force at a concentration as low as 1 microM, while the minimum effective concentration of milrinone was 10 microM. The beta-blocker propranolol (0.1 microM) caused a marked inhibition of the inotropic effect of compound 2f. Adenosine deaminase (1 and 2 U/ml) inhibited significantly and in a concentration-dependent manner the increase in inotropism induced by compound 2f and the adenosine deaminase-resistant response was abolished by 0.1 microM propranolol. In the presence of 0.1 microM propranolol, compound 2f (5 to 30 microM) antagonised in competitive manner the negative inotropic effect induced by N6-(R-phenylisopropyl) adenosine (R-PIA) (0.01-1.0 microM), a stable adenosine receptor agonist. Schild regression analysis gave in fact a slope of 1.02 +/- 0.06 and the pA2 value for compound 2f was 5.41 +/- 0.28. Compound 2f also inhibited phosphodiesterase (PDE) III isolated from calf heart, this inhibition being quantitatively significant only at the highest concentrations tested (0.5 M to 1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of rat liver microsomal cGMP-inhibited cAMP phosphodiesterase (PDE III) by phospholipase C and D.

The specific activity of cGMP-inhibited cAMP phosphodiesterase (PDE III) of rat liver microsomal membranes is increased in a concentration-dependent way by adding phospholipase C from Clostridium perfringens or phospholipase D from Streptomyces chromofuscus. The effect depends on an increase in Vmax of the enzyme. Treatment of microsomal membranes with phospholipase C causes a marked increase in the relative amounts of phosphatidylserine and phosphatidylinositol, and mild stimulation of PDE III activity. Treatment with phospholipase D increases phosphatidic acid and strongly increases PDE III activity. These data suggest that phosphatidic acid is the most important regulator of membrane-bound PDE III activity in liver.

One- and two-electron reduction of menadione in guinea-pig and rat cardiac tissue.

1. In both guinea-pig and rat heart, mitochondrial NADH-ubiquinone-reductase and soluble DT-diaphorase accounted for 49-50% and 48-50% of menadione metabolism, respectively. Microsomal NADPH-cytochrome P450-reductase was responsible for less than 1% of menadione reduction. 2. Menadione was a high-affinity substrate for all reductases (Km values from 1 to 10 microM). 3. Marked amounts of O2-. (superoxide anion) were generated as a consequence of cardiac metabolism of menadione. 4. Menadione-induced O2-. generation was about 3-fold higher in guinea-pig than in rat heart. 5. All results were compared with data obtained on guinea-pig and rat liver.

Antagonism towards endogenous adenosine and inhibition of cGI-PDE in the cardiac effects of amrinone, milrinone and related analogues.

1. The effect of amrinone, milrinone and of three milrinone analogues was tested on spontaneous chronotropic and inotropic activity of guinea-pig isolated atria, on the activity of cGMP-inhibited phosphodiesterase (cGI-PDE) from guinea-pig heart and on specific binding of N6-cyclohexyl[3H]adenosine ([3H]CHA) to Ri adenosine receptors in guinea-pig atria. 2. The Ki-values towards [3H]CHA binding to Ri receptors were linearly related to the EC50S for the increase in force of contraction but not to the EC50S for the increase in frequency of the atria. The Ki values towards cGI-PDE were linearly related to the EC50S for the positive chronotropic effect.

The catecholamine-mediated positive inotropic effect of simple quinones is related to superoxide anion generation.

In guinea pig and rat cardiac tissue, redox cycling benzoquinones (2,5-dimethyl-p-benzoquinone and duroquinone) and naphthoquinones (menadione and 2,3-dimethoxy-1,4-naphthoquinone) generated superoxide anion (O2-.) both through one- and two-electron reductions, the generation being significantly greater in guinea pig than in rat tissue. In electrically driven left atria isolated from guinea pig and rat, menadione and 2,5-dimethyl-p-benzoquinone but not duroquinone caused a concentration-dependent positive inotropic effect. Unlike guinea pig, 2,3-dimethoxy-1,4-naphthoquinone had no effect in rat tissue. Naphthoquinones and 2,5-dimethyl-p-benzoquinone were more active in guinea pig than in rat tissue, their effect being dependent on the release of catecholamines from adrenergic stores. A linear relationship (r = 0.90) between the amount of O2-. generated by benzo- and naphthoquinones in guinea pig and rat heart and the extent of catecholamine-dependent positive inotropic effect was evident. An amount of O2-. higher than 600 nmol/g of tissue per min was calculated to be necessary to determine the catecholamine-mediated increase in contractility. Lipid peroxidation was not involved in quinone-induced catecholamine release.

Phosphatidylserine vesicles increase Ca2+ uptake by rat brain synaptosomes.

Phosphatidylserine (PS) vesicles incorporated into rat brain synaptosomes increased total Ca2+ uptake. Total Ca2+ uptake was resolved in three components: K+ depolarization-induced Ca2+ uptake, Na+/Ca2+ exchange, and passive Ca2+ entry, which were differently affected by PS depending on the amount of incorporated phospholipid. K+ depolarization-induced Ca2+ uptake was stimulated by 0.05-0.10 mumol PS/mg protein while 0.10-0.30 mumol PS/mg protein increased Na+/Ca2+ exchange activity and passive Ca2+ entry but not K+ depolarization-induced Ca2+ uptake. High amounts of incorporated PS also increased passive Rb+ uptake.

Modifications of cardiac contractility by redox cycling alkylating and mixed redox cycling/alkylating quinones.

The effects of redox cycling, alkylating and mixed redox cycling/alkylating benzo- and naphthoquinones were examined in electrically driven guinea pig left atria. Cardiac microsomal and mitochondrial NAD(P)H-dependent metabolism of the quinones and consequent generation of superoxide anion (O2.-) were also measured. Mixed redox cycling/alkylating 2-methyl-1,4-naphthoquinone, redox cycling 2,3-dimethoxy-1,4-naphthoquinone and alkylating p-benzoquinone determined concentration-dependent positive inotropic responses, whereas redox cycling 2,3,5,6-tetramethyl-p-benzoquinone had no effect. The positive inotropic effect of 2,3-dimethoxy-1,4-naphthoquinone was completely catecholamine-mediated, that of 2-methyl-1,4-naphthoquinone was approximately 70% adrenergic and 30% direct. p-Benzoquinone acted directly on heart muscle. In time, quinones with alkylating properties caused increases in the resting force of atria, whereas redox cycling quinones did not produce toxic effects. Mitochondrial NADH-oxidoreductase accounted for 90 to 95% of the metabolism of all quinones, whereas the contribution of the microsomal pathway was negligible. Considerable amounts of O2.- were produced by mitochondrial biotransformation of 2-methyl-1,4-naphthoquinone and 2,3-dimethoxy-1,4-naphthoquinone but not of 2,3,5,6-tetramethyl-p-benzoquinone and p-benzoquinone, suggesting a kind of relation between O2.- generation and the release of catecholamines.

Inhibition of rat liver monooxygenase activities by 2-methyl-1,4-naphthoquinone (menadione).

In rat liver microsomes, 2-methyl-1,4-naphthoquinone (menadione) inhibits cytochrome P450 (cyt P450)-mediated aniline-p-hydroxylation and aminopyrine-N-demethylation with Ki values of 12 and 14.5 microM, respectively. The inhibitions of aniline-p-hydroxylation and aminopyrine-N-demethylation are mixed uncompetitive-noncompetitive and mixed competitive-noncompetitive, respectively. NADP antagonizes the inhibitory effect of menadione on aniline-p-hydroxylase activity but not that on aminopyrine-N-demethylase activity. Menadione does not give rise to any spectral change of cyt P450, but modifies the type I binding spectrum induced by aminopyrine. In contrast, menadione does not change the type II binding spectrum induced by aniline. These results indicate that menadione may inhibit aniline-p-hydroxylase activity by acting as a substrate for NADPH-cyt P450 reductase in the place of cyt P450 and inhibit aminopyrine-N-demethylase activity by impairing the binding of aminopyrine to cyt P450.

Acidic phospholipids and lysophospholipids stimulate cAMP phosphodiesterase of rat liver microsomal membranes.

Acidic phospholipids and lysophospholipids modify cAMP phosphodiesterase activity of rat liver microsomal membranes to different extents, depending on the cAMP concentrations employed. At low concentrations, they activate the hormone-sensitive low-Km form of the enzyme through an increase of Vmax (diphosphatidylglycerol greater than phosphatidylglycerol greater than phosphatidic acid = lysophosphatidylserine greater than phosphatidylserine greater than lysophosphatidylcholine). At high concentrations, only lysophospholipids activate the high-Km form of phosphodiesterase through a marked increase in both Vmax and apparent Km for the cAMP.

Inhibition of cardiac sarcoplasmic reticulum Ca2+-ATPase activity by menadione.

2-Methyl-1,4-naphthoquinone (menadione) inhibits Ca2+-ATPase activity of cardiac sarcoplasmic reticulum membrane vesicles in a time- and concentration-dependent way; after 60 min of preincubation an apparent Ki value of 33.5 microM was calculated. Inhibition is not reversible in that it persists even after the drug is removed and Ca2+-ATPase activity is assayed in a menadione-free medium. GSH (2 mM), but not DTT, is able to prevent and reverse the inhibition of Ca2+-ATPase by menadione. The relative importance of menadione metabolism in the inhibition of Ca2+-ATPase was studied in cell-free systems composed of vesicles and subcellular fractions containing metabolizing enzymes. Under these experimental conditions, 105,000g supernatants isolated from heart or liver that biotransform menadione through DT-diaphorase reduce the inhibition of Ca2+-ATPase activity determined by menadione. Also liver microsomes that biotransform menadione through NADPH-cytochrome P450 reductase decrease the inhibition by menadione. By contrast, cardiac microsomes that do not biotransform the drug do not influence the effect of menadione. These results indicate that, under the experimental conditions used for this study, menadione does not require metabolism to inhibit cardiac sarcoplasmic reticulum Ca2+-ATPase activity.

Effects of 2-methyl-1,4-naphthoquinone (menadione) on myocardial contractility and cardiac sarcoplasmic reticulum Ca-ATPase.

(1) In electrically driven guinea-pig left atria, menadione (2-methyl-1,4-naphthoquinone) (1 to 20 mumol/l) and menadione sodium bisulfite (30 to 200 mumol/l) produced marked positive inotropic effects. Endogenously released catecholamines and histamine contributed to 80-85% of the effect, the residual 15-20% appearing as a direct effect. (2) In electrically driven guinea-pig ventricular strips, low micromolar concentrations of menadione (0.05 to 0.3 mumol/l) exerted a catecholamine-mediated small positive inotropic effect. (3) In both myocardial preparations, the increase in force of contraction was followed by a non-reversible rise of resting force. In its effects on cardiac contractility menadione resembled the thiol group blocking agent p-chloromercuribenzoate and H2O2. Pretreatment of atria with glutathione prevented the increase in resting force, while dithiothreitol only slightly delayed it. By contrast, the pretreatment with the NAD(P)H-quinone reductase (DT-diaphorase) inhibitor, dicumarol, markedly increased the rate of appearance of the toxic effect of menadione. (4) Among enzymatic and transport systems involved in the onset and control of cardiac contractility, sarcoplasmic reticulum Ca-ATPase was significantly inhibited by menadione after a long contact time. The inhibition was concentration-dependent and persistent, and was antagonized by addition of glutathione. (5) On the basis of these results, the increase in resting force caused by menadione appears to be related to an impairment of the thiol groups of proteins (Ca-ATPase), presumably caused by the drug per se.

Inhibition of Na+/Ca2+ exchange by amiloride acting from opposite sides of cardiac sarcolemma.

Amiloride inhibited the Na+Ca2+ exchange activity of cardiac sarcolemmal vesicles with similar affinities at the cis and trans sides of the membrane, estimated apparent Ki on both sides of the sarcolemma being similar. The extent of amiloride inhibition on Na+/Ca2+ exchange activity was decreased by alkaline pH only when the drug was acting from the external side of the vesicle sarcolemma, whereas when vesicles were preincubated with the drug at different pH values, amiloride appeared to act as a weak permeant base, being a more effective inhibitor at alkaline pH values. In fact, a rise in the pH of the preincubation medium may favour the entry and consequently the effect of the drug on the exchanger. The pH dependence of the inhibition of Na+/Ca2+ exchange activity by either extravesicular or intravesicular amiloride was consistent with the hypothesis that in both cases the protonated drug was the active form. Evidence is presented that the pattern of interaction of amiloride on the Na+/Ca2+ exchange system strictly depended on the sidedness of drug action. In fact, while Na+ protected against inhibition by amiloride when it was acting on the same side of the vesicle membrane as the drug, it synergically interacted with amiloride to inhibit exchange activity when it was acting on the opposite side of the sarcolemma as the drug. Furthermore, only extravesicular amiloride removed the stimulation of Na+/Ca2+ exchange activity in Ca2+-treated vesicles.

Effects of N-chlorobenzyl analogues of amiloride on myocardial contractility, Na-Ca-exchange carrier and other cardiac enzymatic activities.

1. In electrically driven guinea pig left atria, micromolar concentrations (2 mumol/l to 80 mumol/l) of N-chlorobenzyl derivatives of amiloride (o-chlorobenzamil and 3',4'-dichlorobenzamil) produced quantitatively similar positive inotropic effects. Contracture developed with 3',4'-dichlorobenzamil. Endogenously released catecholamines contributed 30% to the positive inotropic effect of o-chlorobenzamil but did not contribute at all to the effect of 3',4'-dichlorobenzamil. When tested in the presence of the inhibitor of phosphodiesterase isobutylmethylxanthine, o-chlorobenzamil antagonized its positive inotropic effect, whereas 3',4'-dichlorobenzamil potentiated it. o-Chlorobenzamil also antagonized the positive inotropic effect of ouabain in that it shifted its concentration-effect curve to the right. Moreover, o-chlorobenzamil prevented the appearance of ouabain toxicity in terms of a rise in the resting force. 2. Also, in electrically driven guinea pig papillary muscle, micromolar concentrations (5 mumol/l to 30 mumol/l) of both N-chlorobenzyl derivatives of amiloride produced a positive inotropic effect. This effect was more marked with 3',4'-dichlorobenzamil than with o-chlorobenzamil and was associated for both compounds with lengthening of relaxation time. 3. o-Chlorobenzamil and 3',4'-dichlorobenzamil influenced, though not to the same extent, several systems involved in the onset and in the control of cardiac contractility. 3',4'-Dichlorobenzamil inhibited with the same potency Na-K-ATPase, sarcotubular Ca-ATPase, Na-Ca-exchange carrier, cAMP-dependent phosphodiesterase isolated from bovine heart and oxidative phosphorylation of mitochondria isolated from rat liver. Low micromolar concentrations of o-chlorobenzamil mainly inhibited Na-Ca-exchange carrier and cAMP-dependent phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)