Redox agents affecting drug actions (in excitable tissues)

Gen, Pharmac.. Vol. I I. pp. 409 to 418 © Pergamon Press Ltd 1980. Printed in Great Britain

0306-3623/80/0901-0409502.00/0

REVIEW REDOX AGENTS AFFECTING D R U G ACTIONS (IN EXCITABLE TISSUES) A. PtJpPi, M. DELY and P. PRAGER Central Laboratory of Animal Research, University Medical School, P~cs, Hungary (Received 2 January 1980)

acetylcholine receptor is a muscarlnic type, the specific S-S reducing and sulfhydryl oxidizing agents were without effect. From all of these observations in (f) follows, that at least specific SH, S-S reagents act exclusively on nicotinergic acetylcholine receptor. (g) On the other hand there are numerous data, having shown that non-SH S-S specific oxidants and reductants affect not only the nicotinergic acetylcholine systems, but also other structures involved in excitatory and inhibitory processes. It was described (Puppi et al., 1969) on isolated frog heart and ileum, that if the oxidative character of the medium is increased [by using methylene-blue (0.1 mM) as oxidant], the acetylcholine (ACh) effects were excitatory (depolarizing), while the adrenaline actions inhibitory (hyperpolarizing) in character. At the same time if the redox character of the medium had been increased towards the reducing side (by using ascorbate in 0.1 mM), this resulted in an inhibitory (hyperpolarizing) type of ACh effect and a positive excitatory (depolarizing) type of adrenaline action. Later it was established on frog (Puppi et al., 1976) by simultaneous recording of myograms, cardiomyograms and tissue RSP, that in tissues with higher RSP levels (+ 170 mV or more; rectus muscle, smooth muscle of stomach) ACh exerted a depolarizing (exhibitory) type of action, while in the frog heart ventricule (with RSP value lower than + 170 mV), this mediator triggered a hyperpolarizing type ACh effect. Since following an appropriate adjustment of RSP values by exogenous redox agents the depolarizing and/or hyperpolarizing ACh effects can be modelled on any organs, independenily of the original type of effect of ACh, it may be assumed that the existing redox state in general plays an important role in the determination of the depolarizing and/or hyperpolarizing effect of ACh. Because the negative ino- and chrono-tropic action of the ACh on heart can be inverted by using oxidants of different types (phenazine-methosulphate, thionine, methylene-blue, oxidized neutral-red, oxidized safranine, indigotetrasulphonate) and increased by reductants (such as thiamine, ascorbate, methol) it refers to the fact, that non-SH, S-S specific redox agents act also, on systems having muscarinergic receptors (Puppi et al., 1979). This conclusion is strengthened by another observation as well, namely that on frog stomach smooth muscles an oxidation by methylene-blue in-

I. I N T R O D U C T I O N

The acid-base reactions, which involve proton transfer are considered in every experiment and clinical work, when problems of excitation and inhibition are considered. At the same time the significance of biochemical redox reactions, which involve electrontransfer are reckoned with scarcely ever in this respect. Considerations of the significance of redox balance in excitatory processes is a far less familiar topic in spite of the accumulating data in favour of the importance of the actual redox state potential (RSP) in regulation of excitatory and inhibitory events. It is, in effect, not possible to worry about the protons only and let the electrons take care of themselves, because: (a) Between the redox potentials (at pH 7 = E~) of the medium and the electrolyte distribution of the extra and/or intracellular spaces a close correlation exists (Conway & Mullaney, 1960). (b) Ascorbic acid, as a potent reducing agent significantly influences the effects of sympathetic and parasympathetic actions (Ungar, 1938). (c) Cystein inhibits the effect of acetylcholine (Laborit, 1965). (d) On the arterial blood pressure hydrochinon exerts a hypertensive, while parachinon--a hypotensive action (Laborit, 1965). (e) According to Karlin & Bartels (1966) the membrane effect of acetylcholine in the electroplax of electric eels is inhibited by a reduction of disulphyde groups by dithiothreitol and the action of this reduction can be reversed by the oxidizing agent, 5'-5'-dithio-bis (2-nitrobenzoate). (f) Similar observations were made later on other nicotinergic objects by numerous authors too: on rat denervated muscle (Albuquerque et al., 1968); on frog sartorius (Del Castillo et al., 1971; Ben-Haim et al., 1973); frog rectus (Mittag & Tormay, 1970); chick biventer cervix (Rang & Ritter, 1971); cultured chick embryonic skeletal muscles (Harvey & Dryden, 1974); on acetylcholine enriched membrane vesicles from Torpedo californica (Schiebler et al., 1977); and also on leech muscle (Ross & Triggle, 1972). In intestinal muscles (Kuhnen-Clausen, 1975) and on Aplysia ganglion cells (Sato et al., 1976), where the Abbreviations used: DTT, Dithiothreitol; PCMB, parachloromercuribenzoate. o.P 115

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In all of the many sided phenomenon described in (a)-(i) there is a common feature. Elevating the RSP by exogenous oxidants, or decreasing it by applying exogenous reductants, the effect of the cited drugs changes with inverse sign. Starting from the principle that the RSP value of various tissues differs from oneanother and that the RSP level shows great variations during different physiological and biochemical events (we will come back to this question later) the following consequences seem to be justified. In excitable systems there exists beyond those already known--a fairly complicated regulatory mechanism which consists of; 1. possible and plausible variations in the E; value of exogenous or metabolical origin in the cytoplasma, the membrane and in the intercellular spaces and; 2. working sites which are sensitive to the variations above. (These sites of action will be discussed later on.)

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Fig. 1. Relations between cardiomyogram, myogram and tissue redox state potentials (RSP). Record from top: RSP of frog ileum (continuous line) and heart ventricle (dotted line): cardiomyogram of isolated frog heart: myogram of isolated frog ileum, after acetylcholine (ACH) 1 tlg/ml methylene blue as oxidant (MB) 30/ag/ml, and ascorbate as reductant (ASC) 30/~g/ml treatment. 1": washing out (Puppi et aL. 19767. creases, but a reduction by ascorbate decreases the effect of ACh contractures. (h) On giant neurones of Lymnea stagnalis it was established that: 1. the depolarizing effect of ACh on D-type cells was enhanced by exidizing agents (methylene-blue, thionine) and at the same time H-type cells, become frequently D-type ones after the application of oxidants; 2. redox catalysts of the reducing type, such as ascorbate, hydrochinon, p-methyl-amino-phenolsulphate caused an expressed inhibition of ACh triggered depolarization on D-type cells. In some cases, the D-type character of cells was converted into H-type one (Puppi & Kiss, 1973). In the same object with the voltage clamp method it was observed that AV~ch values were increased by the extracellular oxidants and decreased by reductants. Following intracellular application of redox agents the reductant effect occured inversely (Puppi & Kazachenko, 1978). (i) According to Sobrino and Del Castillo (1972), potassium chlorate and permanganate, potassium and sodium iodate, dehydroascorbic acid and oxidized

DRUG

REDOX

ACTIONS

AFFECTED

INFLUENCES

Though it is a rough-and-ready rule that the significance of the redox balance is hardly considered in pharmacological literature, however the best investigated drug in this respect is acetylcholine. The most important items in the case of ACh were already cited before. Now let us look at the sporadic data in relation to redox regulation also of other drugs playing a role in excitatory and inhibitory events. Catecholamines

It has long been known that Na-pyruvate antagonizes the effect of adrenaline and that the hypertensive action of adrenaline is increased on the effect of reductants (hydroquinone and pirocatechin) but decreased by using the oxidant, such as chlorpromazine (Laborit, 1965). In respect of the correlations beetween the adrenaline effect and the influence of redox agents a detailed analysis was made on isolated frog heart and on ileum (Puppi et al., 1972). In these experiments it was established that following oxidant pre-treatment (methylene-blue, 0.1 mM) on ileum ACh contraction the effect of adrenaline rose by 16%, but in the heart the positive ino- and chronotropic action of epinephrine decreased by 53%. After ascorbate (0.1 mM) pretreatment (reductant) the inhibitory character of this catecholamine on ileum ACh contractions was totally eliminated, but at the same time on the heart--after reductant pretreatment--the positive ino- and chronot'ropic action of adrenaline was augmented by 48°/0. On comparing the redox effects on acetylcholine and adrenaline actions, the following hypothesis was proposed by the authors: an increment of the electron acceptor character in tissues favours the cholinergic exhibitory and adrenergic inhibitory processes, at the same time an increase of the electron donor properties in the tissues results in a cholinergic inhibitory and adrenergic exhibitory effect. If this hypothesis is verified, the problems of shifts from the equilibrium of the sympathetic and parasympathetic nervous influences, observed following irradiations, air poilu-

Redox agents affecting drug actions

411

tions and alterations in air ionization might be laid nea ganglions, and with non-specific redox agents on on redox basis, the latter is dependent on the former. M ytilus muscles. In relation to the effects of redox agents on adrenergic mechanisms the following objection would also Cyclic A M P be set up: Adrenaline (and other catecholamines as The effect of cyclic AMP and aminophylline on well) has a reducing character. sodium transport through the toad bladder epitheThis means that an appropriate oxidant in tissues lium was correlated with protein sulfhydryl-di-sulfide can oxidize them, causing a decrease in their concen- equilibrium (Farah et al., 1969). tration, while a reductant may reduce the enzymatiIn view of the high correlation between the redox cally oxidized adrenochrome back into adrenaline, state potential level in the biophase and the bioelectriincreasing the concentration of the active amine. This cal activity (triggered by acetylcholine and influenced explanation might indeed be valid with respect to by cAMP) in skeletal muscles would suggest that the change of adrenaline action on the heart but fails to redox state potential level influences the effect not account for the inversion of the inhibitory action of only of the acetylcholine, but also the actions of epinephrine on ileum. cAMP (Puppi, unpublished results). The following observations might refer also to the According to some authors NADH inhibits adenysignificance of RSP in adrenergic systems: On the late cyclase activity in isolated membranes and cAMP rabbit sinoatrial nodal pacemaker fibers, two possibi- is inversely correlated to NADH levels in whole cells. lities are proposed for the mechanisms of the inhibi- Furthermore, atebrin, which inhibits the plasma tory effects of Cd2÷: the first is that a blockade of membrane dehydrogenase, inhibits cyclase at the membrane sulfhydryl groups by Cd 2+ may inhibit same concentrations (LSw et al., 1978). binding of norepinephrine with adrenoceptive recepLewin (1973) assumed a close correlation between tors and the second that the effect of norepinephrine the ascorbic-dehydroascorbic acid redox system and on the permeability of membranes for Na + may the formation of cAMP. Data of Mukherjee & Lynn appear effective, when sulfhydryl groups are not (1979) on adipocytes clearly indicate that the catalytic greatly inhibited (Toda, 1973). component of adenylate cyclase is active only in the Dithiothreitol depressed the contractile response to reduced state of its key sulfhydryl groups and inactive norepinephrine and potassium chloride of isolated rat in the oxidized (disulfide) state. Since this site is in the aortic strips, while dithio-bio-2-nitrobenzoic acid a cytoplasmic face of the plasma membrane, its physiosulfhydryl oxidizing agent, restored the responsiveness logical regulation may be coupled with intracellular of strips to these contractile agents (Mushlin et al., redox potential. The contradiction between the observations of L/Sw and Mukherjee might be solved by 1978). The dopamine receptors on H-cells of Aplysia considering the following facts: In Mukherjee's work ganglion cells (Sato, 1976), and dopamine, epineph- it is noted, that H202 in low concentration (0.1 mM) rine and norepinephrine receptors on Mytilus catch in some cases increased rather than decreased the acmuscles (Twarog et al., 1977) seemed to be not tivity of adenylate cyclase. This inversion in action of the oxidant may be due to the possible involvement affected by sulfhydryl reagents. and availability of an intermediate redox component Indolamines which, when oxidized, may in turn reduce and actiIn experiments carried out on giant neurones of vate the catalytic site (an overshoot of endogenous Lymnea stugnalis, it was observed (Puppi & Kiss, redox feedback systems). On the other hand this 1973) that a shift into the oxidative and/or reductive explanation seems to be opposed to the observations side of the redox state of the biophase with non-SH, of Puppi et al. (1976) that following the application of S-S specific redox agents did not influence the depo- oxidants also in low concentrations (0.1 mM) there is larizing action of serotonine so equivocally than after an elevation of tissue redox state potential. In spite of these difficulties in analysis of events, the application of ACh. Two possibilities might be proposed for the explanation to the lack of signifi- however, remains the fact, that the RSP plays an important role in the regulation also in cAMP triggered cance of redox effect in this object: The giant neurones do not have redox sensitive physiological events. sites influenced by serotonin actions, or 5-HT, being a fairly strong reducing agent in itself, acts not only as Gamma-aminobutyric acid transmitter, but also as a redox catalysator on giant Ben-Haim, working on crab neuromuscular juncneurones. We prefer the second alternative, because it tion concluded that dithiothreitol probably had no is known on another mollusc (Mytilus) that effect on the specific receptors for GABA and gluta5 x 1 0 - 4 M mersalyl (SH-reagent) reversibly blocked mate in this preparation (Ben-Haim et al., 1973). Simithe relaxing action of serotonin. 1 0 - a M p-chloro- lar observations were also made by Sato et al. (1976) merculibenzoate also blocked serotonin (Muneoka & in Aplysia ganglion cells. From this data it seems that Twarog, 1972), and that the blocking action of mersa- there is no effect of specific SH, S-S reagents on lyl is reversed by exposing the anterior byssus retrac- GABA triggered events. tor muscle to 1 0 - 2 M dithiothreitoi for 8 min. This reversal is strong evidence that mersalyl has inacti- Other drugs vated the relaxing receptor by combining with sulfThe membrane stabilizing action of quinidine can hydryl groups at or near the receptor, which are re- be antagonized by pyruvate (Laborit, 1965). We have stored by DTT. For dissolving the difference in results also data showing that methylene-blue (oxidant) debetween the two species, experiments have to be car- creased but ascorbate (reductant) greatly increased ried out with SH, S-S specificreagents also on Lyre- the inhibition caused by atropine (1.44.10 -4 mM) in

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ACh contractures of frog smooth muscles. The dose dependent curves of the redox agent actions have not shown the signs of competition and it is hardly to be supposed that these non-specific redox agents acted only in ACh receptor (Dely et al., 1976). Methylene-blue (as oxidant) is capable of increasing the K-strophantoside contracture of rectus muscle of frog (by 71°,), while ascorbate and dithiothreitol (as reductants) counteract this contracture (by 49 and 19",, respectively/. Methylene-blue intensifies the ACh + K-strophantoside contracture as well (Dely et al., 19801. 3. ROLE OF E N D O G E N O U S REDOX AGENTS EFFECTING DRUG ACTIONS

Only the actions of exogenously applied redox agents have been dealt with. However in excitable tissues there are plenty of endogenous redox agents and redox enzymes. In contrast to the commonly held view that the quantity of these redox agents and enzymes and the proportionality of oxidized and reduced forms of redox metabolites o r t h e activity of various redox enzymes are maintained within a narrow range in an organism, we have a mass of evidence to criticise this attitude, because: (a) In vitamin C deficient guinea-pigs (such deficiency frequently occurs also in man) the quantity of cytochrome p-450 and cytochrome h 5 and the activity of NADPH cytochrome p-450 reductase and NADPH cytochrome c reductase decreased significantly (Zannoni et al., 1972). (b) Membrane depolarization in guinea-pig cortical slices induced by electrical pulses leads to a biphasic change in nicotinamide nucleotide fluorescence (Lipton. 1973). This change indicates an early nucleotide oxidation of the nicotinamide nucleotide. The early nicotinamide nucleotide oxidation following stimulation have been noted in other preparations: for instance in skeletal muscle cells (Zinchenko, 1972), and on frog dorsal root ganglia (Rodriguez-Estrada, 1967). (cl At the same time as burst activity in the cat's cortex, redox state potential level oscillations were also observed (Sanchez & Ramos, 1969). (d) Suggestions have been made that much of the damage done to tissues by ionizing radiations is due to free radical oxidations (Bacq & Alexander, 1964: Gerschman, 1964) principally by peroxide radicals formed during irradiation. The tissue RSP as measured by changes in sulfhydryl disulfide has been reported to rise following exposure to radiation (Ord and Stocken, 1963). (e) Acetylcholine (Puppi et al., 1976) and indirect stimulation of frog skeletal muscles evoke a rise in redox state potential. Following stimulation of the left gastrocnemius the RSP will also grow not only in the appropriate right muscle but even in the liver, presumably by the way of the metabolites transported through the blood. (f) The reactivity of structural proteins as well as (redox) enzymes may be alerted by changes in numerous factors in their molecular environment, among which are temperature, pressure, pH, ionic strength, metabolites, etc. In power of the circumstance that many of these factors vary in relatively

broad range, it is conveivable that the RSP level does fluctuate. (g) It is also known that between the different organs of the same species (and individuals) there significant differences exist in their mean RSP values; the highest in frog liver (about 200 mV), lower in skeletal muscles (170mV) and in frog stomach smooth muscles (165 mV) and the lowest in heart ventricle (about 110 mV) (Puppi et al., 1976). Data also indicates that tissue RSP as measured by fluorimetry of the pyridine nucleotide system or as calculated from sulfhydryl and disulfide concentrations, is elevated in brain, lung and other tissues exposed to elevated concentrations of oxygen (Jamieson & Chance, 1966; Jamieson et al., 1963). The redox state of cytosol is sensitive to changes in intensity of the individual reactions for glycolysis, and the RSP of mitochondria is sensitive to the reactions of the Krebs cycle. These complex reactions in turn depend on the levels of carbohydrate, inorganic phosphate, ADP and oxygen (Bucher, 1970; Kaplan, 1966; Krebs & Veech, 1970). Because the concentration of the latter metabolites may alter in rather great ranges in different organs, it would reflect redox differences. It is also known that the allosteric interactions of 2,3-DPG (2,3-diphosphoglyceric acid) with haemoglobin facilitate oxygen transfer to the tissues (Chanutin and Curnish, 1967; Miller et al., 1970). According to Shapiro (1972) this 2,3-DPG-haemoglobin interaction can regulate the oxygenation and redox state in the tissues, depending on where such a regulation is needed. It is also known that concentrations of redox buffer systems, such as ascorbic acid andglutathion (red/ox proportion) are different in various tissues (Titaev, 1960). The specific type of metabolism in different organs and/or tissues also determines a definite redox state in the organs and/or tissues. (h) Stresses of different types decrease glutathion content and total non-protein sulfhydryl in various tissues (Varma, 1968). Other studies with perfused organs have demonstrated lowering of the tissue RSP in fasting, or under conditions of inadequate nutrition (Krebs, 1967). Starting from the fact that the exogenously applied redox agents influence and regulate the action of many drugs--as it was presented in Section 1 and 2 we have strong evidence so suggest that their endogenous analogues do this as well. If it is the case in every experimental and therapeutic use of drugs that the level of RSP (similar to the pH values) has to be standardized or measured, or at least reckoned with, in order to obtain a reliable basis for the possible elucidation of various observations and for comparison between different experimental data. For better orientation in the biochemical machinery of endogenous redox systems affecting excitability, the reader is referred to the excellent review of Dikstein (1971). 4. D R U G S ALTERING REDOX STATE P O T E N T I A L IN TISSUES

Redox state potential levels in the various tissues of an organism may change not only in consequence of the unavoidable influence of natural components or

Redox agents affecting drug actions physical and chemical repercussions, but also by taking drugs. The drugs, in many cases influence their own effect by altering simultaneously the RSP in the target tissues. In dog skeletal muscles following adrenaline infusion (1.5 #g/kg/min) there is an increase in lactate/pyruvate, but decrease in NAD/ NADH quotient and in RSP (Fiorentini and Camoni, 1968). The evoked redosis thus in turn increases the depolarizing, but decreases the hyperpolarizing type adrenaline actions (Puppi et al., 1972). On the other hand a shift to redosis decreases the depolarizing type acetylcholine effects (Puppi et al., 1968) and increases the activity of (Na ÷ & K ÷) transport ATP-ase (Dick et al., 1969; Dikstein, 1971; Wald et al., 1972; Puppi et al., 1975), influences the cAMP level (L6w et al., 1978; Mukherjee, 1979) and so on. It is clear, even from this instance, that applying a drug, we have a complex mass of side effects of redox origin. Many drugs used are strong oxidants or reductants. The study of effects of various drugs and chemicals on tissue RSP is a first step towards an improved understanding of some of their long-term actions. Many of the vitamins (e.g. ascorbic acid, thiamine, riboflavine etc.) have respectable redox catalyst features, and because many coenzymes participating in the redox balance of metabolism are synthetized from vitamins, it appears likely that hypo- and hypervitaminoses will be found to be accompanied by changes in RSP. Acetylcholine is capable to evoke the inhibition of the pentose-phosphate shunt (Laborit, 1965) thus elevating the RSP. Theophylline increases but imidazole decreases the redox state potential in frog skeletal muscles (Puppi--unpublished results). DibutyrylcAMP in adipose tissue inhibits 6-phosphogluconate dehydrogenase, thus causing glutathion oxidation (Bray, 1967). Thyroxine has been found to alter the redox state of the pyridine nucleotides in perfused rat liver (Hassinen et al., 1970). There is some data in the literature demonstrating in perfused organs, the lowering of the tissue RSP following administration of ethanol (Hassinen et al., 1970). The carbon monoxide inhaled by city dwellers increases the NADH/NAD ÷ quotient (Whereat, 1970). Not only organic drugs influence tissue RSP, but also simple ions can also do so; for example, it is known that the oxygen consumption of rat small intestine, in vitro is influenced by the chloride and bicarbonate, as well as by sodium (Jackson & Kurcher, 1977). In rat cerebral cortex slices, longer term increases in the steady state redox potential were observed at higher potassium concentrations (Bull & Cummins, 1973). Inspite of the scarce data, the view of a complicated regulatory system, consisting of the controlling effect of the actual redox state potential on drug actions on the one hand, and the RSP adjusting action of different drugs on the other becomes more distinct. These complicated interrelationships must be taken into consideration both in experiments and in therapy.

413

raise the question which points can the redox agents act through? Building upon recent knowledge of data in excitable tissues, the most important site of actions would be as follows: 1. 2. 3. 4. 5. 6. 7.

Drug receptors Transmitter mobilization Inactivation of drugs Ionic channels Active transport Excitation-contraction coupling Contractile elements.

1. Druo receptors

The most widely investigated subject in this respect is the acetylcholine receptor, mostly by using specific SH, S-S reagents; Working on isolated eel electroplax preparation, Karlin & Bartels (1966) established that following a treatment with dithiothreitol (DTT)--an agent specifically reducing disulfide bonds--the sensitivity to acetylcholine is strongly decreased. This action could be eliminated with oxidants: potassiumferrycyanide or di-thio-bis-(2-nitrobenzoate). After DTT addition of n-ethylmaleimide, specifically alkylating the SH-groups makes the block irreversible. None of these agents affected the excitable membrane and the activity of acetylcholinesterase. Reduction of sulfhydryl groups, altered the character of the effect of bis-ammonium derivatives; depolarizing action of decamethonium was increased, hexamethoniumwhich is normally an ACh antagonist--becomes a receptor activator. From these observations it seems that S-S groups play an important role in cholino-receptive processes evolving changes of conductance following ACh actions. Building upon this, Karlin elaborated a model of nicotinic cholinoreceptor; According to this model, disulfide bonds and the surrounding hydrophobic region are located 1.2 nm from the anionic site of the resting cholinoreceptor. For activation, it is requisite to change conformation so that this distance would shrink to 0.9-1 nm. Fixation of the cholinoreceptor into one of these conformations is dependent on the structure of the drug linking with it, that is from the distance between the cationic head and hydrophobic parts of the molecule (acyl radicals of choline-esters, phenyltrimethylammonium, hydrophobic regions of cholinolytics etc.). After reduction of disulfide link the hydrophobicity of this region of the cholinoreceptor is significantly diminished. This counteracts the capability for interconnections between the cholinoreceptor and the acyl-radicals of choline-esters, excitation fails. In this (at first sight very attractive) hypothesis there are some weaknesses. The distance between the anionic site and the sulfhydryl group is determined by the authors by using choline-esters of different molecular length. However, there is no experimental proof that these molecules react with the same SH, or S-S groups. Then, Karlin assumed that for the transformation of the cholinoreceptor to active conformation, hydrophobic connections between the activator 5. SITE OF ACTIONSOF REDOX AGENTS molecules and the region of cholinoreceptor encircAFFECTING DRUG ACTIONS ling the site of disulfide linking are requisite. At the Treating the problem of the regulatory influences of same time hydrophobicity of the acyl-groups containredox agents on drug actions unthinkingly, would ing regions of the molecules of bromo-acetylcholine

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A. PuPPI, M. DELYand P. PRAGER

Fig. 2. Conformations of the cholinoreceptor as supposed by Karlin & Winnik (1968): l--native cholinoreceptor resting position; ll--non-reduced cholinoreceptor, activated by acetylcholine; Ill--reduced cholinoreceptor alkylated by [N-maleimido]~t-benzyltrimethylammonium (irreversible block); I V reduced cholinoreceptor, alkylated by bromo-acetylcholine (irreversible activation). and carbachol is very weak and in the tetramethyl- competition, but applying the ACh + atropine ammonium ion there is no hydrophobic region at all together with oxidants or reductants the curves will (corresponding with the localization of acyl-regions of not show the signs of competition (Dely et al., 1976). choline-esters); all of these agents can activate native The observation, however, that N ÷ group on redox cholinoreceptor and their action is dependent on the agents presumably plays the role of "site director for redox receptors", because oxidants having no N ÷ (e.g. receptor reduction/oxidation. It seems probable that reduction of disulfide in- 5',5',7',7' indigo-tetrasulfonate, p-benzo-quinone) act hibits not so much the association of cholinomimetics rather weakly on heart, refers to a very short distance with the receptors, as throws difficulties in the ways of between the ACh and redox receptors also on mustransformation of cholinoreceptor protein to the acti- carinic systems (Puppi et al., 1979). vated conformation. This latter view seems strengthened by the data of Hamilton et al. (1977) that the 2. T r a n s m i t t e r mobilization There are many exogenous and endogenous receptor in Torpedo electric tissue membrane is a mixture of monomer and dimer and that monomers are influences and drugs that can liberate neurotransmitters. Among these are agents whose mechanism of linked in dimer by disulfide bonds. Disregarding some weaknesses in the theory of action can be due to their redox feature. In the presKarlin et al. (1966), the following facts seem to be well ence of uncouplers of the mitochondrial oxidative established. In the vicinity of the nicotinic cholinore- phosphorylation (a change in RSP) an increase in ceptor there exists a reversibly reducable or reoxid- miniature end-plate potentials could be observed in able S-S, SH group. The activability of the receptor nerve-muscle preparations (Glagoleva et al., 1970). Moreover, on analyzing these effects, a regulatory depends on the presence of S-S bonds. In the long run, therefore, as a site of action of redox agents system emerges, where oxidation increases but reduc(mostly specific SH, S-S reagents) the conformational tion decreases the liberation of acetylcholine. Accordalterations of nicotinergic receptors have to be taken ing to Carlen et al. (1976) both diamide and DIP act to increase transmitter release by the intracellular oxiinto consideration. On the other hand, the question of muscarinic cho- dation of glutathione. On the other hand adenosine linoreceptors as a site of action of redox agents has (which augments the pentose phosphate shunt, thus not been elucidated so far. As far as the role of causing decrease in RSP) decreases the amount of specific SH, S-S reagents are concerned the stand- transmitter released from nerve endings at the neuropoint is uniform: there are no specific SH, S-S redox muscular junction in the rat diaphragm (Ginsborg & receptors! (Albuquerque et al., 1968; Karlin & Win- Hirst, 1972). The analysis of redox effects of such types, as the nik, 1968; Del Castillo et al., 1971; Ben-Haim et al., 1973; Mittag & Tormay, 1970; Ross & Triggle, 1972; observed increment in ACh liberation following the Kuhnen-Clausen, 1975; Sato et al., 1976). On the application of the sulfhydryl reagent, p-chloromerother hand some work shows that non SH, S-S curibenzoate (Paton et al., 1971), seems to be more specific redox agents, such as: methylene-blue, thio- complicated. In such cases the tissue RSP measurenine, phenazine-methosulphate, oxidized neutral-red, ments might give explanations. There is no room for oxidized safranine, indigo-tetrasulphonate as oxi- doubt that alterations in RSP may influence the liberdants, and ascorbate, thiamine, methol, as reductants, ation of neurotransmitters but it would be a sweeping are effective in the same direction also in isolated frog generalization to deduce all the redox actions to this heart and ileum (Puppi et al., 1968; Puppi et al., 1976; mechanism , because: 1. There are many arguments proving the manyPuppi et al., 1979; Dely et al., 1976). Here, the question is not resolved as to whether these agents act sidedness of the site of effects of redox agents. 2. Some redox effects even contradict the supposimilarly in ACh receptors or in other sites of action (e.g. ionic channels, transport enzymes etc.) where the sition that the only mechanism is a regulation of specific SH, S-S agents are ineffective. The sensitive transmitter liberation. For instance, oxidants in frog dependency in redox effect from the extracellular con- heart do not increase the negative ino- and chronocentrations of Ca 2÷ and Na ÷ ions on heart at least tropic action of ACh, on the contrary they reverse the indicates a channel interaction. The same conclusion effect to a positive ino- and chronotropic type would follow from the following observation. The influence (Puppi et al., 1976). An increase only in the dose dependent ACh, and ACh + atropine curves of ACh liberation would have simply increased the negasmooth muscle contractures demonstrate a strict tive actions.

Redox agents affecting drug actions 3. Inactivation of drugs In spite of the data of Zahavi et al. (1972) that in Myzon persicae (Aphidae) the acetylcholinesterase has sulfhydryl groups which are sensitive t o S H reagents, in our experiments we have found (working with human serum cholinesterase), that reducing agents, such as ascorbate, hydroquinone, p-methyl-aminophenosulphate, and CuCI2 did not influence the enzyme activity, while from among oxidants only the reagents having N ÷ groups (competition!) were efficacious. This question remains to be solved. 4. Ionic channels Ionic channels in excitable membrane consist of two functional units: a gating unit, which regulates the opening and the closing of the channels and an ionophore, which con~ ins the selectivity filter and determines which ion can pass the channel at what maximum rate. It is not known if these functional units are different molecular structures. At the postsynaptic membrane gating may be achieved (a) by transmitter molecules (transmitter channels); (b) depolarizing electrically the membrane (voltagedependent channels); and (c) through leakage channels. On analyzing the redox effects on these pores the following can be stated: 1. According to Karlin & Bartels (1966), Ban-Haim et al. (1973) and Harvey & Dryden (1974) the action of the specific S-S reducing agent dithiothreitol is related exclusively to the ACh receptor itself, rather than to other elements taking part in excitatory processes. At the same time we have some data indicating a direct channel effect in the case of non SH, S-S specific redox agents. For instance, drugs which act on the ACh receptor site, such as d-tubucurarine, depress the peak amplitude of the end-plate current, but do not appreciably alter its time course, nor the dependence of peak amplitude and half-decay time of membrane potential. In contrast, agents which act on the channels, such as some local anaesthetics and the histrionicotoxins, alter both the end-plate current, amplitude and time course and modify the influence of membrane potential on the peak amplitude and half decay time (Albuquerque et al., 1978). Puppi & Kazachenko (1977) analyzing the voltage/ampere characteristics of the ion flux through cholinergic channels of giant neurones of Lymnea stagnalis following the extracellular application of methylene-blue (oxidant) and ascorbate (reductant) found that these redox agents changed both the end-plate current, amplitude and time course and even the equilibrium potential of acetylcholine. Similar changes were found with other oxidants (thionine and para-aethoxychrosoidine) and reductants (p-methyl-amino-phenosulphate, hydroquinone) (Puppi & Kazachenko, 1978). The authors assumed that in cholino-receptive channels there is a potential barrier. In this case, if the redox agents due to their action on the molecular structure of the membrane (which was described by Webb, 1966) lead to an alteration in symmetry and in the height of the potential barrier, a change in Ee will occur. Thus, when discussing the effect of redox agents on Ee, besides the speculation about the alterations of the proportionality of ion fluxes, it can be suggested, that they act also on the height and sym-

415

metry of the potential barriers of the cholinoreceptive channels in the membranes. Furthermore decreasing the extracellular concentration of Na ÷. K +, Ca 2÷ or CI- ions does not proportionally change in the redox effect as would be expected supposing only a receptor targetted site of action of redox agents. In frog skeletal muscles the methylene-blue effect on increasing the ACh contractures was not only diminished proportionally with the diminution of extracellular concentration of Na ÷ions, but also shows an inversion of action in the case of 50~,,, Na ÷. Similar observations were made in Lymnea stagnalis giant neurones (Puppi & Kiss, 1975). All this data indicates a direct channel influence of redox agents on transmitter evoked pores. 2. We have data showing the direct influence of the change in RSP on non-transmitter specific pathways of permeability through membranes; According to Shapiro et al. (1970) p-chloromercuribenzoate and p-chioro-mercuri-benzene-sulfonate react with at least three classes of sulfhydryles, two of which are associated with the sodium-potassium barrier and, when altered, result in potassium loss, and sodium accumulation in human erythrocyte membrane. On the basis of the observations of Kitigawa et al. (1961) and Tolberg and Macey (1972), it seemed possible that radiation (RSP alterations!) and/or SH reagents might affect cation permeability of red cells by altering the calcium binding characteristics of the red cell membrane. Toda (1973a, b) suggested that membrane sulfhydryl groups may relate closely to the permeability of atrial cell membranes to Na ÷ and Ca 2. during excitation, and as a consequence, in the maintenance of the spontaneous pacemaker activity. In frog skin Lindemann (1978) described that the SH reagent p-chloro-mercury-phenylsulphonate, increases Na + current, because the number of open pores can be stabilized close to their maximal value by treating the membrane with this SH reagent. Working with voltage-clamp technique on isolated giant neurones of Lymnea stagnalis, Puppi and Kazachenko (1978) described that the slope resistance of the potential-dependent K ÷ channels--both in the case of extra- and intracellular application of various redox agents--displays a monotonic decrement in parallel with the increasing RSP. This means that the outflux of K ÷ ions is directly proportional to the redox state potential. In the case of N a ÷ - C a 2÷ input current, there are peak-type correlations between conductance and the existing RSP in the biophase. In atrial trabeculae fibres of the frog (Rana esculenta) it was established (Puppi et al., 1979) that after 3 min of application of methylene-blue (oxidant) to the fibers, on electrical excitation of the membranes, the N a ÷ - C a 2÷ input diminished but the K÷-effiux increased, compared with controls. In contrast to this, following the use of ascorbate (reductant) the inverse process could be observed. In contrast to the commonly held view that leakage current channels are not influenced by exogenous modificators, Puppi & Kazachenko (1978) described, that after extracellular application of redox agents the resistance of the leakage channels through membranes diminished almost linearly with increasing RSP in the reducing range. In the oxidizing range

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chondria to the medium, while a subsequent addition of a reductant of the pyridine nucleotides causes reuptake of the released Ca 2+. The turn in this balance following changes of RSP may thus readily influence 5. Active transport the excitation-contraction coupling. Langer (1971) proposed that the increase in [-Na+]~ It was argued in Section 3 that the redox state of an organism may vary in a broad range depending on caused by digitalis induced pump inhibition produces various endo- or exogenic influences and that differ- a significant increase in Ca z + influx which is coupled to the Na + efflux (Na+-Ca 2+ exchange reaction). It is ences exist between the RSP values of various tissues known that oxidants or SH reagents also inhibit the (heart, skeletal and smooth muscles, liver) (Puppi et al., 1976). The differences in the actual RSP values active transport (Wilkers & Bader, 1969; Dick et al., may deeply influence the activity of various enzymes, 1969; Wald et al., 1972; Fromm & Fuhro, 1978: Skou because a principal function of a redox buffering sys- & Hilberg, 1965: Rothstein, 1970: Puppi et al., 1975, 1978), causing an increase in [,Na+]i. This, in turn, tem must be to keep proteins in the functional state. The reactivity of structural proteins as well as may evoke an increase in [Ca2+]~ by the way of enzymes, may be alerted by changes in numerous exchange-diffusion. In some cases, e.g. in the reversing factors in their molecular environment, among which effects of oxidants on ACh action in heart (Puppi et are: temperature, pressure, pH and ionic strength as al., 1976, 1979), this site of action as an explanation well as redox state. It was shown (Wilkers & Bader, might hold but in others, e.g. in the phenomenon that 1969: Dick et al., 1969: Wald et al., 1972) that oxidiz- oxidants decrease the amplitude and frequency of ing agents, such as N-methyl-phenazonium-metho- spontaneous heart contractures (Puppi et al.. 1978) is sulphate, Dithio-bis-nitrobenzoate, oxidized gluta- conflicting. thione, inhibit, but reductants, such as DTT and reduced glutathione stimulate the (Na + & K +)-ATP- 7. Contractile elements ase. (According to Dikstein [-1971] the inhibition by It is a fact that the contractile proteins contain SH, oxidants is not necessarily straightforward, but could S S groups, playing crucial role in their working mechanisms. If the physiologically determined probe mediated by transhydrogenases.) Data of Fromm & Fuhro (1978) suggest that oxi- portions of these groups are transformed (owing to dation of thiol groups sensitive to p-chloro-mercury- shifts in RSP) the functional machinery of contraction benzoate (PCMB) results in inhibition of active ion also changes. In other words, redox agents capable of transport processes through isolated gastric mucosa. reacting either with SH, or S-S groups might inIt has also been shown, that PCMB inhibits (Na + & fluence and regulate the contractures. According to K +) ATP-ase in vitro (Skou & Hilberg, 1965). Suif- Hartshorne & Daniel (1971) and Bailin & Bfirfiny hydryl blocking agents, such as p-chloro-mercury- (1973) the blocking of some SH groups of the myosin, benzene-sulfonic acid inhibits the active transport of results in contractile proteins which show a nonmonovalent cations across the cell membrane (Roths- regulated Ca 2+ insensitive ATP-ase, even in the presence of complete regulatory actin complex. tein, 1970). Working with short-circuit current, Puppi et al. Since all the sites of action of redox effects men(1975, 1978) showed that oxidants inhibit, but reduc- tioned above (from 1-7) seems to be plausible, they may be part of a complex regulatory system, and in a rants increase the active transport through isolated long run a result of these synergistic or antagonistic frog skin. The same authors using the chord-intercept effects will be observed. technique (Hong & Essig, 1976) observed, that the active Na + fluxes across isolated frog skin were diminished following oxidant (methylene-blue, thioCONCLUSION nine), but increased after application of ascorbate as Being familiar with the concept of redox regulation, reductant. The coefficient between the coupling can render great help for pharmacologist to underbetween metabolism and the transport showed a fall stand the mechanisms of action of drugs and for the following oxidants, but has been raised after reductant pretreatment. Redox changes influence not only clinician to intervene more expertly in the complithe transport enzyme itself, but also the metabolical cated clinical events. (Incidentally, for the consultation of the clinical significance of redox balance, the coupling supporting with energy of the former. We have data concerning the redox regulation of reader is referred to the excellent review of Shapiro active calcium pump: Dithiothreitol (10 raM) greatly (1972).) increased and dithio-bis-2-nitrobenzoic acid (100 pM) decreased the ATP dependent calcium pump activity of microsomes isolated from rat aortae (Mushlin et REFERENCES al., 1978). Summarizing, the influence of redox agents on ALBUQUERQUEE. X., SOKOLLM. D., SONESSONB. 8/, THESLEFFS. (1968) Studies on the Nature of the cholinergic active transport processes as a site of action have to receptor. Eur. J. Pharmac. 4, 4(Y46. be taken into consideration, because the (Na + & K +) ALBUQUERQUE E. X., ELDEFRAWIA. T., ELDEFRAWlH. 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