F. Carpenedo

Effect of quercetin on membrane-linked activities

Abstract The flavonoid quercetin inhibited the Mg ++ -dependent, Na + and K + stimulated ATPase.† The effect was dependent on the concentration of Na + and K + . Quercetin, like other known inhibitors which act on both transport ATPase and oxidative phosphorylation, inhibited the mitochondrial ATPase activity. Mitochondrial electron transport was also affected. Sulfhydryl compounds partially reversed the effect of quercetin on ATPase activities and completely eliminated that on electron transport. It is concluded that quercetin shows affinity for membrane-dependent cellular activities.

New derivatives of methyl-xanthines: Effect of thiocaffeine thiotheophylline and 8-phenyltheophylline on lipolysis and on phosphodiesterase activities

The effects of some old and new methylxanthines and 6-thioxanthines, i.e. theophylline (TH), caffeine (CAFF), thiotheophyl line (S-TH), thiocaffeine (S-CAFF), 8-phenyltheophylline (8-PT), 3-isobutyl-1-methyl-xanthine (IBMX) were compared at the level of adipose tissue on spontaneous and norepinephrine-induced lipolysis as well as on fat cell phosphodiesterases. These agents stimulated lipolysis. 8-PT was the most potent; thiocaffeine and thiotheophylline the least potent; IBMX and theophylline had intermediate potencies. The order of potency of the same drugs in potentiating norepinephrine-stimulated lipolysis was: IBMX greater than 8-PT greater than S-CAFF greater than greater than S-TH greater than CAFF greater than TH. The rank order of potency to inhibit cAMP phosphodiesterases was: IBMX greater than S-TH and S-CAFF greater than TH much greater than 8-PT (uneffective). Thus (a) thiocaffeine and thiotheophylline were more potent than the parent compound theophylline in inhibiting cAMP-PDE, although their per se stimulating effect on lipolysis was much lower. This indicates that in the thioxanthines the stimulus per se on lipolysis can be dissociated from their effectiveness as inhibitors of PDE. (b) In contrast, 8-PT (a blocker of adenosine receptors) has a potent lipolytic action per se and a potentiating effect on norepinephrine-induced lipolysis, even if deprived of effect on PDE. This indicates that the potentiating power of methylxanthines on norepinephrine-stimulated lipolysis is not strictly dependent on their anti-PDE activities. These results suggest that in adipose tissue (more similar than adipocytes to the in vivo conditions) the anti-adenosine potency of methylxanthines is the prominent factor in stimulating basal lipolysis. Both anti-adenosine and anti-PDE activities are involved in modulating hormone-induced lipolysis.

Increase of Na+K+ ATPase activity in intact rat brain synaptosomes after their interaction with phosphatidylserine vesicles

Abstract Intact synaptosomes prepared from rat brain were incubated with phosphatidylserine vesicles. The synaptosomes incorporated the phospholipid in proportion to its concentration in the preincubation medium. The activity of membrane-bound enzyme Na + K + ATPase increased proportionally after treatment with phosphatidylserine liposomes. When breaking phosphatidylserine-enriched synaptosomes by osmotic shock or by sonication and when preparing synaptosomal membranes, the expected increase of Na + K + ATPase activity was not seen. Therefore, cellular integrity was fundamental in order to see the effect of phosphatidylserine on Na + K + ATPase activity.

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

Summary1. In electrically driven guinea pig left atria, micromolar concentrations (2 μmol/l to 80 μmol/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 ochlorobenzamil but did not contribute at all to the effect of 3′,4′-dichlorobenzamil. When tested in the presence of the inhibitor of phosphodiesterase isobutylmethylxanthine, ochlorobenzamil 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 μmol/l to 30 μmol/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. 4. The results suggest that 3′,4′-dichlorobenzamil is a quite unspecific compound and its cardiac effects are the result of an interference with several enzymatic and transport systems. In contrast, both the inhibition of the Na-Ca-exchange carrier and cAMP-dependent phosphodiesterase can contribute to the increase in the force of contraction induced by o-chlorobenzamil. Finally, the antagonism of o-chlorobenzamil against the cardiac effects of ouabain can be explained by the inhibition of the Na-Ca-exchange carrier.

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.

Inhibition of the Na+/Ca2+ exchange in cardiac sarcolemmal vesicles by amiloride

The pyrazine diuretic amiloride inhibits the Na+/Ca2+ exchange activity of cardiac sarcolemmal vesicles in a concentration-dependent way. A good relationship between the uptake of amiloride by the vesicles and the inhibition of the exchanger has been found. Kinetic analyses indicate that the inhibition of Na+/Ca2+ exchange activity by amiloride is non-competitively removed by Ca2+ and competitively overcome by an outwardly directed Na+ gradient.

Nephrotoxic Effect of Atractyloside in Rats

Abstract1.In male albino rats Atractyloside (50 mg/kg i.p.), well-known as inhibitor of oxidative phosphorylation, produces a tubular nephrosis 180 min after its administration. The nephrosis is characterized by a deep lesion in the cells of distal portion of proximal convoluted tubule and is accompanied by an increase of water excrection and by proteinuria, glicosuria, ketonuria, and kaliuria. The glomerulus appears intact; this is confirmed by the creatiniue and urea tests. The renal alteration subsides after 2 days of its onset. Similar effects are observed in mice but not in guinea pigs and rabbits.2.Carboxyatractyloside (2 mg/kg i.p.), an analogue of atractyloside with a second carboxylic group in position 4, more active inhibitor of oxidative phosphorylation and more toxic, is devoid of any effect on rat kidney.3.No functional or morphological alterations are observed in liver and heart of rats injected with these drugs.4.The oxidative phosphorylation in homogenates of kidney and liver of rats pretreated with these compounds is inhibited to different extents. The possible relationship between nephrotoxic effect of atractyloside and the inhibition of oxidative phosphorylation is discussed.Zusammenfassung1.Atractyloside (50 mg/kg e. p.), das wie bekannt, die oxydative Phosphorilation hemmt, bewirkt bei der Ratte eine Nierenschädigung im Tubulusbereich 150 min nach seiner Verabreichung. Die Schädigung ist gekennzeichnet durch schwere Läsion des distalen Bereiches des proximalen Tubulus und geht mit einer vermehrten Wasserausscheidung, Proteinurie, Glykosurie, Ketonurie und Kaliurie einher. Der Glomerulus bleibt intakt wie aus der Kreatininclearance hervorgeht. Der Nierenschaden beginnt sich 2 Tage nach seine n Auftreten rückzubilden. Ähnliche Schäden beobachtet man bei der Maus, aber nicht beim Meerschweinchen oder beim Kaninchen.2.Carboxyatractyloside (2 mg/kg e.p.), mit einer zweiten Carboxylgruppe in Position 4, ist als Phosphorilationshemmer aktiver und besitzt eine größere Allgemeintoxicität, bewirkt aber keine Nierenschäden bei der Ratte.3.Bei Ratten, denen diese Substanzen verabreicht wurden, konnten weder morphologische noch functionelle Leber-oder Herzschäden beobachtet werden.4.Die oxydative Phosphorilation wird bei Nieren- und Leberhomogenaten von Ratten, denen diese Stoffe verabreicht wurden, in verschiedenem Ausmaß gehemmt. Es wird der mögliche Zusammenhang zwischen nephrotoxischem Effekt des Atractylosides und der Phosphorilationshemmung diskutiert.

Effects of papaverine and eupaverin on calcium uptake by isolated sarcoplasmic vesicles

Papaverine and eupaverin increase the rate of uptake of calcium by sarcoplasmic vesicles isolated from rabbit white skeletal muscle. The degree of activity of the above drugs is clearly affected by changes of ATP, oxalate and Ca2+ concentrations. The results are discussed in view of present knowledge about the effects of papaverine‐like drugs upon muscular contraction.

Calcium and papaverine interaction with soluble cardiac phosphodiesterase

Abstract The c AMP phosphodiesterase (PDEase), found in the 105,000 g supernatant fraction of calf heart homogenate, was regulated by Ca 2+ , being inhibited by low and stimulated by high calcium concentrations. Such a sensitivity to Ca 2+ was maintained during the purification of the enzyme through a DEAE-cellulose column. EGTA demonstrated through indirect evidence that the PDEase-inhibiting effect of papaverine was not Ca. 2+ -dependent. Moreover, the inhibitory effect of papaverine was not removed by imidazole. An increase in cAMP content and synthesis induced by noradrenaline was observed in the presence of papaverine. From these observations the effect of papaverine on cAMP accumulation could be ascribed to its inhibitory effect on PDEase, through both a direct interaction with the enzyme and an altered availability of Ca 2+ .

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.