The University of Western Ontario, Department of Pharmacology, London, Ontario, Canada.

* This research was supported in part by a Defence Research Board of Canada grant. A.M. is a recipient of an Alcoholism and Drug Addiction Foundation Research scholarship.

 

INTRODUCTION

 

Very few of the more than sixty naturally-occurring isoquinoline alkaloids have been screened for pharmacological activity, although certain members of this class have known hallucinogenic and cardiovascular activity. Recently the suggestion that biosynthetic catecholamine-derived tetrahydroisoquinolines are involved in the etiology of alcoholism has stimulated research into their activity. Both salsolinol and its desmethyl analogue, 6,7-dihydroxytetrahydroisoquinoline, have been shown to exacerbate ethanol withdrawal convulsions in mice. However, the action of such isoquinolines in altering the behavioural expression of acute ethanol intoxication has not been reported. The results presented here demonstrate the ability of salsolinol and 3-carboxysalsolinol and their non-cyclised amine precursors, dopamine and L-DOPA respectively, to increase the duration of ethanol-induced narcosis in mice. The carboxylated isoquinoline was the most potent of the four compounds tested. Pretreatment with the alcohol dehydrogenase inhibitor pyrazole significantly lengthened the narcosis produced by ethanol and by both of the isoquinolineethanol combinations. Disulfiram, an inhibitor of both aldehyde dehydrogenase and dopamine-beta-hydroxylase, led to an increased sleeping time after either ethanol or L-DOPAethanol treatments, but had no effect on the isoquinoline ethanol narcosis. When carbidopa, an inhibitor of peripheral L-amino-acid decarboxylase, was administered, a significant increase in duration of narcosis following either L-DOPA or 3-carboxysalsolinol with ethanol was obtained. The results show that simple isoquinolines such as salsolinol and 3-carboxysalsolinol potentiate the narcotic effect of a single injection of ethanol and suggest that in vivo formation of such compounds may play a significant role in both acute and chronic alcoholism.

 

PHARMACOLOGICAL ACTIONS OF NATURAL AND SYNTHETIC ISOQUINOLINES

 

Isoquinoline alkaloids occur widely in nature. They are to be found in many families of plants, including species of Anonaceae, Berberidaceae, Cactaceae, Chenopodiaceae, Combretaceae, Fumaraceae, Hernandiaceae, Lauraceae, Leguminoseae, Magnoliaceae, Menispermaceae, Monimiaceae, Nymphaceae, Papaveraceae, Ranunculaceae, Rhamnaceae and Rutaceae (1).

On structural grounds this group of natural products can be divided into two major categories: the simple isoquinolines which possess only one aromatic nucleus and the benzylisoquinolines which contain two such nuclei (Figure 1). Both categories can be expanded to include phenol-ether linked dimers and trimers. Many other groups of alkaloids are structurally related to the benzylisoquinolines. These include the protoberberines, protopines, pavines, aporphines and the phthalide-isoquinolines (1). In some plants the isoquinolines are present in combination with biosynthetic precursor phenethylamines (2,3); in others, isoquinolines are primogenitors of more complex alkaloids (1).

 

While the isoquinolines are the most numerous of all the groups of alkaloids, very few of the more than sixty natural isoquinolines have been screened for pharmacological activity. Sporadic investigations into their actions have taken place for more than one hundred years (4), with results accruing from signs and symptoms during toxicity and effects on respiration, the cardiovascular system and visceral smooth muscle.

 

Trioxytetrahydroisoquinolines present in the peyote cactus Anhalonium lewinii Britton and Rose [Lophophara williamsii (Lemaire) Coulter] were among the isoquinolines tested by Heffter in 1898 (5). These substances are central nervous system stimulants. The most potent base, lophophorine, produced hyperexcitability and accelerated respiration in doses of 7 mg/kg in rabbits. An increase in blood pressure was elicited by 2.5 mg/kg with large doses causing a decrease. There was no effect on the heart (5). Another isoquinoline, anhalonidine, did not produce significant symptoms in mammals (5). A more recent study has confirmed these results for Brossi et al. (6) have demonstrated that anhalamine, anhalonidine and pellotine have little activity as sedatives, anticonvulsants, or tranquilizers.

 

Mescaline is the major alkaloid present in the peyote cactus. The ability of this phenethylamine to cause hallucinations is, without doubt, the reason for the use of sacramental mescal buttons in the religious ceremonies of the Native American Church (7). Co-existing tetrahydroisoquinolines may not have hallucinogenic properties (6) and are not considered to contribute to the psychotogenicity of peyote (8).

 

The suaharo cactus, Carnegie gigantea (Engelmann) generates two isoquinolines, carnegine and gigantine.

 

Carnegine has pharmacological properties that are similar to the isoquinolines in peyote, provoking convulsions in mammals (9). The more recently discovered alkaloid gigantine (10) is of particular interest. This tetrahydroisoquinoline is an isomer of pellotine, differing only in the transposition of a phenolic hydroxy group (11). Gigantine is considered to be an hallucinogen in squirrel monkeys and cats (10).

 

Species of Chenopodiaceae elaborate the 6,7-dioxytetrahydroisoquinolines salsoline and salsolidine. Both alkaloids are vasodilators and respiratory stimulants (12). These effects may be mediated by sympatholytic actions for the alkaloids are reported to be antagonists to pressor responses induced by adrenaline (12). Salsoline has antihistaminic activity (13), a mild antidiuretic effect mediated, in part, by an action of the compound on the hypothalamic-hypophyseal system (14) and the unusual property of increasing the rate of coagulation of whole or oxalated blood (15). It is one of the few isoquinolines of therapeutic value, being used in the U.S.S.R. in the therapy of hypertension and cerebral angiospasms (16).

 

Cotarnine, an isoquinoline associated with, and possibly derived from, the phthalide-isoquinoline narcotine has sympatholytic (17,18) and analgesic effects (19). It, and the related isoquinolines hydrastinine and hydrastine are less effective inhibitors of the cough reflex than narcotine (20,21).

 

Still fewer naturally occuring benzylisoquinolines have been tested pharmacologicallly. Laudanosine, given intravenously, elicits convulsions in dogs that are prevented by intraspinal anesthesia (22). Its effects on peripheral structures are qualitatively similar to papaverine (23,24). This latter alkaloid is well-established as a smooth muscle relaxant (25). As recent studies have shown an accumulation of cAMP in smooth muscle treated with papaverine, it is probable that the general relaxant properties reflect an ability to inhibit phosphodiesterase (26). The spasmolytic activity shown by papaverine can be extended to many synthetic analogues (27), some of which are of therapeutic utility (25, 27).

 

Papaverine is a weak analgesic and, unlike laudanosine, depresses the central nervous system (28). The disparity in central activity has been observed with other benzylisoquinolines which differ in containing aromatic or reduced isoquinoline nuclei (29).

 

The pharmacological properties of a large number of synthetic simple isoquinolines have been investigated by Hjort et al. (30-33). Like their natural alkaloid analogues these substances have central nervous system stimulant or depressant qualities. Variant effects are also produced on the cardiovascular system and on isolated smooth muscle. In the main, compounds that contained a catecholamine moiety were found to be predominantly pressor in action; there was evidence of tachyphylaxis, and there was a potentiation of adrenaline. The analogous ethoxy and methoxy derivatives were more toxic, elicited either biphasic depressor-pressor, or solely depressor effects and frequently inhibited the cardiovascular response to adrenaline (32).

 

ISOQUINOLINE ALKALOIDS AND ETHANOL

 

The isoquinolines formed in plants are biosynthesised from precursor phenethylamines. These probably condense with keto-acids or aldehydes to yield the cyclised products (1). Analogous reactions occur, in vitro, under pseudophysiological conditions (34).

 

Ethanol is oxidatively metabolised, in vitro, to acetaldehyde and acetate. The primary enzymes involved are alcohol and aldehyde dehydrogenase (35). These require as co-factor, nicotine adenine dinucleotide, NAD. During oxidation the co-factor is reduced to its protonated form, NADH. The process of ethanol oxidation can alter the biochemic redox potential of the body (36), which can lead to aberrant metabolism of endogenous monoamines. It has been shown that the excretion of acidic metabolites of 5-hydroxytryptamine, noradrenaline and adrenaline are depressed with concommitant increases in reduced catabolites, following ethanol consumption (37,38). A significant factor in the alteration of normal monoamine metabolism is considered to be competitive inhibition, by acetaldehyde of aldehyde dehydrogenase (39,40). This effect prolongs the half life of intermediate aldehyde metabolites (40). Several investigators have considered the possibility that such reactive intermediates could condense with endogenous neuroamines to generate isoquinolines (41,43).

In 1970, Davis et al. and Cohen and Collins reported the in vitro formation of tetrahydroisoquinolines after exposure of biological, systems to acetaldehyde or ethanol (42-45). The generated isoquinolines are salsolinol derivatives of catecholamines and acetaldehyde, and tetrahydropapaveroline formed by the condensation of dopamine with its own intermediate aldehyde, 3,4-dihydroxyphenylacetaldehyde (Figure 2). The benzylisoquinoline, tetrahydropapaveroline, can be further metabolized by mammalian systems to isoquinoline-based protoberberines (46,47). Others have subsequently identified salsolinol and tetrahydropapaveroline in vivo following ethanol administration (48-50). Similar reactions occur with biogenic tryptamines, generating triple ring structures called harmans or 13-carbolines. Dajani and Saheb have identified 13-carbolines in urine after ethanol administration to rats (51).

 

On kinetic grounds the condensation of acetaldehyde with biogenic phenethylamines is most likely to occur with dopamine, followed by L-DOPA and then noradrenaline (52).

 

A further series of endogenous simple tetrahydroisoquinolines may form from condensations of catecholamines with a 1-carbon unit, the prerequisite carbon atom being transferred by way of 5-methyltetrahydrofolate. Vandenheuvel et al. have shown that 6,7-dihydroxy-1,2,3,4-tetrahydrosioquinoline is formed when dopamine is incubated with the 1-carbon unit donor (53). The additional carbon atom required for elaboration of the protoberberine skeleton from tetrahydropapaveroline may also involve 5-methyltetrahydrogolate (46,47). The intermediacy of N-methyl groups in this cyclisation process is known to occur in plants (54-56).

 

Recent reviews of the pharmacological actions of the mammalian-based isoquinolines have been published (57-60). Table 1 summarizes the smooth-muscle, central nervous system and biochemical pharmacological activities possessed by these compounds.

 

The formation of "abnormal" metabolites of central neuroamines following ethanol administrations is of biochemical interest. If such substances interact with the consumed ethanol to alter the acute or chronic effects of the alcohol, then they are of pharmacological importance and have to be regarded as more than metabolic curiosities (109).

 

Interactions of ethanol with the alcohols derived by reductive catabolism of dopamine, tryptamine and 5-hydroxytryptamine have been demonstrated (110,111). Feldstein et al. in 1970 (110), found tryptophol and 5-hydroxytryptophol to be central nervous system depressants, inducing sleep in mice. When co-administered with ethanol there was a potentiation of ethanol-induced sleeping time. In related studies, Blum et al. (111), showed that tryptophol and 3,4-dihydroxyphenylethanol acted synergistically with ethanol, prolonging ethanol sleep-time. This interaction did not seem to be related to an alteration of ethanol metabolism. In a parallel series of experiments, the neuroamines dopamine and 5-hydroxytryptamine themselves, in much lower doses,' protracted ethanol-induced depression, confirming the earlier results of Rosenfeld (112).

 

These investigations have revealed that one series of Abnormal metabolites can alter an acute action of ethanol. However, the neuroamine-derived alcohols cannot be considered to be inert biochemically. It is not improbable that the administered biogenic alcohols could be oxidised by an alcohol dehydrogenase to reactive aldehydes. These products could then react with endogenous neuroamines. Similarly, the neuroamines, co-administered with ethanol, could yield isoquinoline or 0-carboline condensates, by reacting with acetaldehyde.

 

EFFECTS OF ISOQUINOLINE ALKALOIDS ON ETHANOL-INDUCED NARCOSIS

 

A series of experiments were performed to see if ethanol sleeping time could be influenced by isoquinoline bases. Ethanol was given alone or in combination with salsolinol or the related compound, 3-carboxysalsolinol. Comparative experiments were conducted with dopamine and L-DOPA. Some studies were repeated after pretreatment of animals with drugs known to alter the metabolic disposition of ethanol or suppress the peripheral decarboxylation of the amino-acid compounds.

 

MATERIALS AND METHODS

 

Swiss-Webster albino male mice (24-32 g) were used in the experiments. The sleeping time protocol employed was that developed by Kakihana et al. (113). All experiments were conducted during the late morning. After drug administration the time at which each mouse lost the righting-reflex was noted. The narcosis duration endpoint was considered to occur when the subject has regained the reflex twice within a thirty second interval. Artifactual rightings, caused by intermittent leg jerks during the narcosis, were negated by this method. A value of 150 minutes was arbitrarily recorded if the subject was still without the righting reflex after that time.

 

Each of the four test compounds, namely L-DOPA (60 and 1000 1.04/kg), dopamine (60 pM/kg), salsolinol (60, 460 and 920 pM/kg) and 3-carboxysalsolinol (7.5, 15, 30 and 60 pM/kg) was prepared in saline. The test compounds were given (in a 5 pl/g body weight volume) immediately prior to ethanol administration. Delivery of ethanol was made in the form of a 25% (v/v) solution in saline, in a dose of 4 g/kg. The route of administration for all drugs was intraperitoneal, unless otherwise stated. Pretreatments with enzyme inhibitors included administration of pyrazole (34 mg/kg) thirty minutes prior to the above drugs; carbidopa (25 mg/kg) given orally in a gum acacia suspension one hour before other substances; and disulfiram (75 mg/kg) given intraperitoneally in a suspension in acacia twenty-four hours before other treatments. Each pretreatment series had a saline-treated control. The means and standard errors of the means for experimentally determined sleep-times were calculated. Student's t-tests were used to determine levels of significance.

 

The two isoquinolines used in these experiments were synthesised in our laboratory. The synthesis developed by Buck (32) was used as a basis for salsolinol preparations (66). Its carboxylated analogue, 3-carboxysalsolinol was synthesised by the method of Brossi et al. (114). L-DOPA and dopamine were supplied by Sigma Chemical Co., St. Louis, Missouri; carbidopa was generously donated by Merck and Co., West Point, Pennsylvania.

 

The four test compounds were injected either alone or in combination with ethanol. A reference sleeping time was obtained upon the administration of saline (5 pl/g body weight) with ethanol. Several combination experiments were repeated, incorporating the pretreatments indicated above. Tests were conducted on groups containing at least ten mice per group.

 

RESULTS

 

The experimental values for durations of sleep-time with the various treatments and combinations of treatments are shown in Table 2. Times of onset of sleep for experimental and ethanol-saline treated control mice were not significantly different. Dopamine, L-DOPA, salsolinol and 3-carboxysalsolinol, alone and after inhibitory drug pretreatments, were without obvious effect in the absence of ethanol. Several treatment combinations protracted ethanol induced sleeping time. Significant prolongations (p < 0.05) occurred when ethanol was given at the same time as 1 mM L-DOPA, 60 pM dopamine, 460 pM salsolinol, or 15 pM 3-carboxysalsolinol. Higher doses of the isoquinoline compounds further increased the sleeping times.

 

Pretreatments with agents that alter the metabolism of ethanol increased the central depressant effect. Both suppression of alcohol dehydrogenase by pyrazole (115-117) and aldehyde dehydrogenase by disulfiram (118) significantly prolonged ethanol narcosis. The pyrazole-ethanol sleeping-time was not altered by the addition of 1 mM L-DOPA, 60 pM dopamine, or 60 pM salsolinol, although it was elevated by 60 tip of 3- carboxysalsolinol.

 

As mentioned, disulfiram pretreatment increased ethanol sleep-time. This was further increased when 60 pM L-DOPA was given at the same time as ethanol. Co-administrations with doses of dopamine, salsolinol or 3-carboxysalsolinol had no influence on the basal disulfiram-ethanol sleep-time.

 

In contrast, inhibition of L-amino acid decarboxylase by carbidopa did not alter ethanol-induced narcosis. Sleep-times were increased in carbidopa-pretreated animals receiving injections of ethanol and 250 UM or 1 mM of L-DOPA or 7.5 pM of 3-carboxysalsolinol.

 

DISCUSSION

 

POSSIBLE MECHANISMS OF ISOQUINOLINE ETHANOL-INTERACTIONS

 

The isoquinoline alkaloids found in plants show many pharmacological properties in common with those derived following alcohol administrations to mammals. While they differ appreciably, in that the plant alkaloids show more variable levels of oxidation and the majority of oxygen substituents exist as methoxy or methylenedioxy moieties, both groups produce effects on smooth muscle and in the central nervous system. The non-mammilian alkaloids have not been exposed to the definitive levels of biological investigation that have centered on the ethanol-related isoquinolines (Table 1). Nonetheless, many of the effects exerted by these compounds may be mediated through the same direct or indirect mechanisms demonstrated for those of mammalian origin.

 

The results of experiments incorporating dopamine and L-DOPA-derived isoquinolines illustrate that these compounds can interact, acutely, with ethanol. This phenomenon is supported by the results contained in Table 2. Salsolinol, and its more potent amino-acid analogue, demonstrated the capacity to prolong the ethanol-induced sleep-time. In confirmation of the results of others (112,119) this was also shown for dopamine and L-DOPA.

 

Preservation of the administered ethanol, by inhibiting alcohol dehydrogenase with pyrazole, a procedure employed by Goldstein and Pal (120), increased the ethanol sleep-time. A further prolongation was observed in animals treated additionally with 3-carboxysalsolinol, but not with the other tested compounds in the doses used. As the conversion of ethanol to acetaldehyde should be suppressed by the pyrazole pretreatment, there should be a corresponding decline in available acetaldehyde. Reduced quantities of this intermediate could attenuate the formation of abnormal metabolites from dopamine and L-DOPA (biogenic alcohols or derived isoquinolines) and, therefore, not allow synergistic interactions with ethanol to occur. Salsolinol may not have prolonged sleeping time after this treatment because of inadequate dosage. The 60 4I4 quantity of this compound with ethanol did not prolong sleeping time compared to the ethanol-treated control.

 

Disulfiram-treated animals slept longer after exposure to ethanol than control mice. This drug disrupts the facile conversion of acetaldehyde to acetate (118) as well as suppressing the conversion of dopamine to noradrenaline (121,122). The increased sleeping time observed may occur from reaction between elevated levels of acetaldehyde with endogenous neurotransmitter substances and further interactions of these with ethanol. Of significance was the observation that L-DOPA potentiated ethanol-induced sleep-time, in disulfiram-pretreated mice, in a dose that had no significant effect or non-pretreated animals. It is possible that the higher level of the aldehyde was sufficient to alter the metabolism of the amino acid, to yield abnormal metabolites from dopamine which interacted further with the ethanol.

 

The isoquinolines, salsolinol and 3-carboxysalsolinol, did not increase the sleeping time of ethanol-treated, disulfiram-pretreated mice. This would suggest that the isoquinolines are synergistic with ethanol rather than acetaldehyde. Disulfiram did not prolong the sleeping time associated with the co-administration of dopamine and ethanol. This could reflect the generation of an optimal quantity of the isoquinoline at lower acetaldehyde levels than ones induced by disulfiram. Acetaldehyde reacts more than twice as efficiently with dopamine than it does with L-DOPA to produce isoquinolines (52). Other doses of dopamine, ethanol and disulfiram are being examined to investigate this possibility.

 

Pretreatments with carbidopa increased sleeping times after ethanol and the amino-acids. The prevention of peripheral decarboxylation by this agent (123) implies that greater quantities of the administered L-DOPA or 3-carboxysalsolinol from biogenetic or synthetic sources, gain access to the central nervous system for interaction with ethanol. It is not known at this time if 3-carboxysalsolinol is a substrate for brain L-amino-acid decarboxylase and can be made into salsolinol in situ, but it is pertinent that inhibition of the peripheral and central enzyme by the less specific agent RO 4-4602 prevented L-DOPA from potentiating ethanol-induced sleep (124).

 

Isoquinolines can interact with other phases of ethanol intoxication. Amit and Sutherland (125) cite studies by Duby and Amit that show that ethanol-preferring rats, which are pretreated with a-methyl-p-tyrosine and then infused with an isoquinoline into the lateral hypothalamus, drink less ethanol than controls. This discovery is similar to results obtained by Geller et al. (126) who found that a 0-carboline, administered intraperitoneally, reduced voluntary alcohol consumption in rats.

Further interactions of isoquinolines and ethanol are reported by Blum et al. (82). An intracerebral injection of 50 ug of 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline increased the severity of withdrawal seizures in ethanol-dependent mice. Lower doses had no effect or reduced the severity of this withdrawal response.

 

In summary, isoquinolines, isolated from plants or produced in mammals after treatments that incorporate ethanol, are capable of eliciting diverse pharmacological effects. Some isoquinolines can increase the degree of central depression induced by ethanol and influence other aspects of ethanol intoxication. In view of the multiple mechanisms of action associated with these compounds it is not possible to reconcile the interactions of the isoquinolines with ethanol in a discrete manner. A definitive role for cyclised neuroamines, in alcoholism, awaits future clarification. However, the present state of knowledge suggests that these substances may be of critical importance in establishing a biochemical basis for this disorder.

 

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