A.R. Contessa

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.

Digitoxin and prostaglandin ei as inhibitors of catecholamine-stimulated lipolysis and their interaction with Ca2+ in the process

Abstract The antagonistic action of digitoxin and prostaglandin E 1 on the norepinephrine-stimulated lipolysis has been studied qualitatively and quantitatively. The actions of the two drugs are somewhat similar: (a) their antagonism appears, from the log dose-effect curves, to be of a mixed type; (b) Ca 2+ concentration in the medium clearly influences their antagonistic effect. A relation between the action of these drugs on lipolysis and active ion transport or oxidative phosphorylation cannot be entirely excluded. Calcium movements appear more directly involved. The importance of Ca 2+ in the process studied is pointed out.

Activation by phospholipids of particulate mitochondrial ATPase from rat liver

Abstract 1. 1. The activation by phospholipids of particulate mitochondrial ATPase from rat liver was studied on a preparation extracted from submitochondrial particles with cholate. 2. 2. Lysophosphatidylcholine and the highly acidic phospholipids diphosphatidylglycerol, phosphatidylinositol and phosphatidylserine stimulated the ATPase at low concentration (50–60 μg/mg protein); phosphatidylethanolamine was less effective whereas phosphatidylcholine exhibited the lowest activity. 3. 3. The saturation curves of phospholipids acting at low concentration showed in most cases a deviation from normal Michaelis-Menten kinetics. This devation was absent or less manifest with phosphatidylethanolamine and phosphatidylcholine. Competition between phospholipids and oligomycin was seen with acidic phospholipids or mixtures of neutral and acidic phospholipids. 4. 4. Similarly to oligomycin, dicyclohexylcarbodiimide, tributyltin and chlorpromazine competitively inhibited the activation produced by phosphatidylserine, phosphatidylinositol and diphosphatidylglycerol. 5. 5. It is concluded that both the hydrophobic-hydrophilic balance and the negative charge of phospholipids are important in the interaction between phospholipids and protein at the level of particulate mitochondrial ATPase. The competition between phospholipids and inhibitors of mitochondrial ATPase suggests that the activation induced by phospholipids is involved in the inhibitory effect.

Solubilization of mitochondrial ATPase by phospholipids

Summary Brief incubation of submitochondrial particles from bovine-heart with the acidic phospholipid, diphosphatidylglycerol (cardiolipin), resulted in the solubilization of mitochondrial ATPase (F 1 ). Little or no effect was evident with submitochondrial particles from rat-liver. The solubilized ATPase was oligomycin insensitive, cold labile and showed the same electrophoretic pattern as pure F 1 . The specific activity and the polyacrylamide gel electrophoresis indicated that mitochondrial ATPase is extracted in a considerable degree of purity. Other phospholipids partially reproduced the effect of diphosphatidylglycerol whereas some anionic or non ionic detergents were ineffective under the same conditions.

Role of phosphatidylserine and phosphatidylcholine in the dicyclohexylcarbodiimide-induced inhibition of mitochondrial ATPase

Abstract Mixed phospholipids, purified phosphatidylcholine and phosphatidylserine restored the ATPase activity of submitochondrial particles pretreated with dicyclohexylcarbodiimide (DCCD). Pretreatment of mixed phospholipids with DCCD resulted in preparations which were able to transfer the inhibitor to submitochondrial particles. Phosphatidylcholine alone was fully effective. Phosphatidylserine, which apparently forms a stable bond with DCCD, was inactive. These findings suggest different functions of individual phospholipids in the DCCD-induced inhibition. The observation that phosphatidylserine displays unusual high activity in the restoration of ATPase activity in phospholipid-depleted submitochondrial particles indicates that it plays an important role in the activation of mitochondrial ATPase and deserves attention as costituent of the final receptor site for DCCD.

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.

The binding of atractyloside and oligomycin to liver mitochondria

Abstract 1. 1. Intact liver mitochondria bind atractyloside in amount corresponding to that required for maximal inhibitionof phosphate uptake stimulated by phosphate acceptor. The capacity to bind atractyloside is lost if the mitochondria are damaged by pretreatment with Pi or deoxycholate. 2. 2. Both intact and damaged mitochondria bind oligomycin in amount several times higher than that required for maximal inhibition of phosphate uptake. 3. 3. The 2,4-dinitrophenol-stimulated ATPase of intact mitochondria is inhibited by atractyloside, whereas the Mg2+-stimulated ATPase of Pi or deoxycholate-pretreated mitochondria is scarcely affected. In contrast, oligomycin inhibits the ATPase stimulated by either 2,4-dinitrophenol or Mg2+. 4. 4. It is suggested that the binding of atractyloside is dependent on the structural organization of the mitochondria, and that the lack of effect of atractyloside in respect to some mitochondrial and submitochondrial preparations may be explained by the loss of the capacity to bind the inhibitor.

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

Different effects of oligomycin and dicyclohexylcarbodiimide on ATPases from mammalian cells

1. 1. The effects of oligomycin and dicyclohexylcarbodiimide on particulate mitochondrial ATPase from rat liver and on several preparations with Na+,K+-stimulated ATPase activity were compared. 2. 2. In contrast to oligomycin, dicyclohexylcarbodiimide produced a weak inhibition of Na+,K+-stimulated ATPase from rabbit kidney, calf heart and human erythrocyte ghosts. Moreover, it was unable to reproduce the stimulating effect of oligomycin on p-nitrophenylphosphatase activity in the presence of limiting amounts of K+. 3. 3. The Na+,K+-stimulated ATPase preparations were found to be more sensitive to oligomycin than generally assumed; the levels for half-maximal inhibition were only 4–40 times higher than those needed for mitochondrial ATPase. 4. 4. Similarly to oligomycin, dicyclohexylcarbodiimide scarcely inhibited particulate mitochondrial ATPase if phospholipids or triolein were included in the incubation medium. 5. 5. It is concluded that oligomycin in a comparable range of concentrations is able to influence several mammalian ATPases associated with membranes, whereas the effect of dicyclohexylcarbodiimide is much more specific for mitochondrial ATPase. Inhibition of other ATPase systems from mammalian cells is produced at concentrations about 1000 times higher. On the basis of the antagonism by phospholipids it is proposed that the effect of both inhibitors is made possible by the presence of phospholipids in these structurally organized ATPase systems.