R. Santi

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.

On the mechanism of spasmolytic effect of papaverine and certain derivatives

Abstract The authors have compared the spasmolytic activity and the effect on oxidative phosphorylation of papaverine and some of its derivatives (dihydropapaverine, ethaverine and eupaverin). With guinea pig ileum and rabbit duodenum, papaverine, dihydropapaverine and ethaverine faithfully mimic the effect of anoxia, cyanide, 2,4-DNP or other enzyme inhibitors, by suppressing the “tonic phase”, of acetylcholine, histamine, BaCl 2 -induced contraction, without affecting the “spike phase” according to West et al . 1 Papaverine, dihydropapaverine, ethaverine strongly inhibit the oxygen uptake of rat liver mitochondria oxidizing glutamate under phosphorylative conditions. This effect is not reversed by 2,4-DNP. With succinate as substrate the oxygen uptake is unaffected by these drugs. The results suggest that the inhibition takes place in the electron-transfer reactions chain between nicotinamide-adenine dinucleotide and cytochrome b. Papaverine and ethaverine show the greatest activity both in experiments with isolated gut and with rat liver mitochondria. Eupaverin demonstrated a peculiar behaviour, because it failed to give an “anoxia-like” effect on the isolated gut and its mechanism of action on rat liver mitochondria is rather different. Possible relations between these biochemical effects of the drugs and their spasmolytic activity are discussed.

Pharmacological properties of tetrahydropapaveroline.

Tetrahydropapaveroline (thp) exerts β‐sympathomimetic effects similar to those of isoprenaline. On guinea‐pig isolated atria, thp elicits positive inotropic and chronotropic activities which are not abolished by previous reserpinization of the animals; on isolated mammalian heart these effects are associated with an increase in coronary flow. In the dog, thp increases myocardial contractile force and rate, elicits a hypotensive effect and stimulates respiratory activity in normal and reserpinized animals; when injected intra‐arterially the drug causes vasodilatation. All the effects are prevented by the β‐adrenergic blocking agents propranolol, dichloroisoprenaline and pronethalol. Structure‐activity relationships between tetrahydroisoquinoline derivatives and their open‐ring phenylethylamine congeners, which are closely related to sympathomimetic drugs, are discussed.

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.

Liberation of cyanide from succinonitrile

Abstract Release of cyanide from succinonitrile (DNS) was investigated in vivo and in vivo . Administration of DNS to rabbit and rat resulted in a conspicuous increase of thiocyanate excretion in the urine. It was estimated that approximately 60 per cent of DNS μmole administered was transformed to CN − in the animal body and excreted as SCN − . Liberation of CN − from DNS was not found in experiments either with homogenates of rat or rabbit liver or with isolated mitochondrial, microsomal and soluble fractions. By contrast it was demonstrated that rat and rabbit liver slices could catalyze this reaction. Addition of the non-ionic detergent Triton X-100 to the incubation medium almost completely abolished the cyanide liberation from DNS. Moreover it was observed that liver slices from rat pretreated with CCI 4 , had lost their ability to release CN − from DNS. These data suggest that the integrity of cellular membrane is essential for release of cyanide from DNS.

PHARMACOLOGICAL PROPERTIES OF TETRAHYDROPAPAVEROLINE AND THEIR RELATION TO THE CATECHOLAMINES.

SIR,-Recent reports from this laboratory (Santi, Contessa & Ferrari, 1963 ; Santi, Ferrari & Contessa, 1964) have shown that papaverine is a powerful inhibitor of the aerobic oxidation of substrates linked to nicotinamide adenine dinucleotide (NAD) in rat liver mitochondria. The inhibition of oxidative phosphorylation which could be localised in the electron transfer step between NAD and cytochrome b might be important in understanding the mechanism of the spasmolytic effect of the drug. Since some effects of papaverine resemble those generally referred to stimulation of the so-called 8-receptors of adrenaline, Santi (1963) has put forward a working hypothesis based on the possibility that the adrenaline-like drugs produce an impairment of cellular energy sources. In this context, the conclusion by Holtz, Stock, & Westerman (1963) that a substance similar in structure to papaverine, tetrahydropapaveroline, could be formed by the condensation of the well-known precursor of adrenaline, dopamine, and dihydroxyphenylacetic aldehyde, is of interest. We have confirmed the results of Holtz & others (1963) and we believe that the pharmacological properties of tetrahydropapaveroline are in themselves very interesting in as much as this drug behaves in some respects like papaverine, in others like the catecholamines and particularly, isoprenaline. Some pharmacological properties of the drug were described several years ago by Laidlaw (1910). The spasmolytic activity of tetrahydropapaveroline, as seen on the isolated guinea-pig ileum, resembles that of eupaverin (1 -benzyl-3-ethyl-6,7-dimethoxyisoquinoline) rather than that of papaverine. It differs from papaverine in not inhibiting mitochondria1 respiration. Tetrahydropapaveroline stimulates the myocardium as was seen in vivo by measuring the contractile strength by means of the strain gauge technique described by Boniface, Brodie & Walton (1953), as well as in vitro on isolated guinea-pig atria. The latter effect is antagonised by dichloroisoprenaline (DCI). The action of tetrahydropapaveroline on the heart is presumably important in understanding its pharmacological activities. In the dog, 0.1 mg/kg injected intravenously greatly increases the contractile strength of the heart and also its frequency. At 0.02 pg/ml, it has a positive inotropic and chronotropic action; a similar effect may be shown on isolated atria of the previously reserpinised guinea-pig. In dogs and cats, tetrahydropapaveroline reduces blood pressure at concentrations 20-30 times lower than papaverine and 50 times greater than isoprenaline. The hypotensive effect mainly concerns diastolic pressure, whereas the systolic values remain unchanged, the differential increasing accordingly. The decrease of blood pressure must be presumed to be due to a peripheral vasodilatation, since when 5-10 pg of the drug was introduced into the femoral artery, a strong increase of blood flow was measured with a ShipleyWilson rotameter (1951). On the other hand, the vasodilator response of the blood vessels of the isolated rabbit ear according to the technique of Pissemski (1914) was present, although not intense. When administered intravenously to dogs and cats, the drug greatly stimulated respiration. Tetrahydropapaveroline, similarly to adrenaline (Ussing, 1960), and in contrast to papaverine, increases the short circuit current of the isolated frog skin as measured by the technique of Ussing & Zerahn (1951), modified by Vescovini & Marro (1960). Finally, the drug injected intraperitoneally produces an increase in the plasma level of free fatty acids in rats ; this effect is considered to be

Pharmacological properties and mechanism of action of atractyloside

Sm-During the past eight years, we have investigated the biochemical and pharmacological properties of a natural glycoside, atractyloside, extracted from the rhizome of a Mediterranean member of the Compositae, Atractylis gummifera L. and much information has been obtained. The toxic and therapeutic effects of this natural drug were well known in classical times, and are referred to in Dioscorides’ De Materia Medica under the name of “Chamaeleon”. Caccialanza & Landi (1960-1961) have confirmed that the use of this drug in skin diseases, as practiced in antiquity, was fully justified. The pharmacological action of the drug can be attributed to a glycoside formed by an aglycone which has the structure of a perhydrophenanthrene, one molecule of glucose, two of potassium sulphate, and one of isovaleric acid (Ajello, Piozzi, Quilico & Sprio, 1963). The relation between chemical structure and pharmacological action of the drug is of interest (Santi, Bruni, Contessa & Luciani, 1962). We now summarise the results of our in vivo and in vitvo investigations. In rats, dogs, mice and rabbits atractyloside induces hypoglycaemia, usually preceded by a phase of hyperglycaemia, depletion of muscular and hepatic glycogen reserves (myocardial glycogen remains unchanged or is even slightly increased), and inhibition of glycogen synthesis. An increase in the blood lactic acid level and a decrease in oxygen consumption have also been observed (see Santi, 1958). The effect elicited in vivo can be explained by the finding that the drug is a powerful inhibitor of oxidative phosphorylation in isolated mitochondria and in rat liver homogenate (see Santi, 1958). On this basis it may be presumed that the hypoglycaemic effect is connected with an inhibition in vivo of the Pasteur effect. The inhibition of glycogen synthesis as well as the inhibition of oxygen uptake and the increase in blood lactic acid are the consequence of the marked reduction in cellular oxidative and phosphorylation reactions. Subsequently, Bruni, Contessa & Luciani (1962) and Bruni & Luciani (1962) have shown that the atractyloside-induced inhibition of mitochondria1 oxygen uptake is reversed by the uncoupling effect of 2,4-dinitrophenol, and in general by conditions in which the respiratory processes are not controlled by phosphorylation. Among the partial reactions of the coupling system, 2-4-dinitrophenol-stimulated adenosine-triphosphatase (Awase), An-inorganic phosphate, and ATP-ADP exchanges are markedly inhibited by atractyloside, whereas Mg2+-stimulated ATPase is influenced only if the activity is elicited by a partial damage of mitochondria ; no effect whatever is induced if mitochondrial structure is severely disorganised. These results have led us to conclude that atractyloside inhibits oxidative phosphorylation by a process of specific interference with energy transfer reactions. Recently, the analysis of the mechanism of action of atractyloside on mitochondrial energy transfer has been brought a step forward by the finding

Liberation of cyanide from succinonitrile. 2. The effect of ethanol.

Abstract The influence of drug treatment on CN − release from DNS both in vivo and in vitro has been investigated. Treatment of rats with phenobarbital, 3–4 benzopyrene did not modify cyanide liberation from DNS.CC1 4 administration almost completely abolished the generation of CN − and consequently strongly decreased the acute toxicity of DNS. On the contrary, acute or chronic ethanol treatment significantly increased CN − formation from DNS in rat liver slices, increasing the toxicity of the drug. Thus ethanol administration to mice lowered the ld 50 i.p. from 63 mg/Kg to 38 mg/Kg. These results suggest that the considerable increase of DNS toxicity is due to a stimulation of cyanide release and a substrate limitation incurred in detoxification.

Dexamphetamine and lipid mobilization in obesity.

Four interesting points may be deduced from the Table. ( 1 ) Whereas with both samples of dextran given intraperitoneally or intravenously non-reactor rats failed to produce the anaphylactoid reaction, large intravenous doses of egg-white were effective. (2) The component in egg-white responsible for the reaction shown by reactor rats was not destroyed by heat. In contrast, boiled egg-white in non-reactors gave a less intense reaction than did fresh egg-white. (3) As egg-white was ineffective in non-reactor rats when given intraperitoneally but effective by the intravenous route, the active component when eggwhite is given intraperitoneally may not reach the blood stream in concentrations sufficient to produce a response or it may be modified before absorption from the abdominal cavity. (4) Whereas dextrin (Kerfoot) was ineffective by both routes in reactor and non-reactor rats, dextrin (Astra) when given intraperitoneally was equally effective in the two kinds of rat. However, the larger intravenous dose of dextrin (Astra) was less active in reactor rats than in non-reactors.

Anorexigenic drugs and lipid mobilisation

(10 mg/kg intravenously) abolished the inhibitory effect of adrenaline on the bronchial muscle but not that of ascorbic acid. Reserpinisation of the animals with four daily doses of reserpine, 2 mg/kg, intraperitoneally, did not modify the action of ascorbic acid on the spasmogen response. Dr. Collier & Mrs. Piper also tell us that adrenalectomy does not reduce the protective effect of ascorbic acid on bradykinin bronchospasm. The present results show that the inhibition of bronchospasm by ascorbic acid is not mediated by catecholamines, does not involve P-adrenergic receptors since its action is not prevented by pronethalol, and is probably a direct effect.