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Xerox University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 77-2473 O ’NEILL, Patrick Joseph, 1949- ACETALDEHYDE AND NEUROAMINE-DERIVED TETRAHYDROISOQUINOLINE ALKALOIDS: ROLE IN ALCOHOL TOXICITY AND DEPENDENCE.
The Ohio State University, Ph.D., 1976 Pharmacology
Xerox University Microfilms, Ann Arbor, Michigan 48106 ACETALDEHYDE AND NEUROAMINE-DERIVED TETRAHYDROISOQUINOLINE
ALKALOIDS: ROLE IN ALCOHOL TOXICITY AND DEPENDENCE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
by
Patrick Joseph O'Neill, B.S
*****
The Ohio State University
1976
Reading Committee: Approved By
Dennis R. Feller Duane D. Miller Ralf G. Rahwan Harold H. Wolf Adviser College of Pharmacy ACKNOWLEDGMENTS
Dr. Ralf G. Rahwan, for providing direction and inspiration
throughout my graduate career, both in the laboratory
and in the classroom.
My wife, Janie, for her constant support, encouragement,
and understanding.
My parents, for encouraging academic achievement.
Drs. Dennis R. Feller, Duane D. Miller, and Harold H. Wolf,
for helpful suggestions during this work.
The American Foundation for Pharmaceutical Education, for
financial support during certain phases of this work.
ii VITA
July 12, 1949 Born-Columbus, Ohio
1972...... B.S. with distinction in Pharmacy, The Ohio State University, Columbus, Ohio
1972-1974...... Teaching Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio
1974-197 5...... Research Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio
1975-197 6 ...... Abe Plough Citation Fellow of The American Foundation for Pharmaceutical Education
PUBLICATIONS
"Pharmacological Investigations with Chlorpheniramine Isomers." B.S. Degree with Distinction, The Ohio State University, 1972.
"Differential Secretion of Catecholamines and Tetrahydro- isoquinoline Alkaloids from the Bovine Adrenal Medulla." Fed. Proc. 3^:497, 1974.
"Differential Secretion of Catecholamines and Tetra- hydroisoquinoline Alkaloids from the Bovine Adrenal Medulla." Life Sci. 14:1927-1938, 1974.
"Stereoisomers of an Antihistamine and the Pharmacologic Receptors of Rabbit Aorta." Pharmacological Res. Comm. 7:273-279, 1975.
"Experimental Evidence for Calcium-independent Catechol amine Secretion from the Bovine Adrenal Medulla." Fed. Proc. 3£:739' 1975.
"Experimental Evidence for Calcium-independent Catechol amine Secretion from the Bovine Adrenal Medulla." J. Pharmacol. Exp. Ther. 193:513-522, 1975. iii "Protection Against Acute Toxicity of Acetaldehyde in Mice." Res. Conun. Chem. Path. Pharmacol. 13:125- 128, 1976.
"Absence of Formation of Brain Salsolinol During Chronic Ethanol Administration to Mice." Pharmacologist. In press, 1976.
"A Modified Gas Chromatographic/Electron Capture Assay Method for Salsolinol in Brain Tissue." Submitted for publication.
"Absence of Formation of Brain Salsolinol in Ethanol- dependent Mice." Submitted for publication.
FIELDS OF STUDY
Major Field: Pharmacology
Pharmacology of Ethanol and Acetaldehyde
Mechanisms of Stimulus-Secretion Coupling
Toxicology TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... H
VITA ...... ii;i-
LIST OF TABLES...... viii
LIST OF FIGURES...... **
Chapter
I. INTRODUCTION
1.1 Interaction of Ethanol with Neuro- transmitter Systems...... 1
1.2 The Tetrahydroisoguinoline Alkaloid Hypotheses...... 5
1.3 Biological Formation and Inhibition of Formation of Tetrahydroisoquinoline Alkaloids...... 13
1.4 Pharmacology of the Tetrahydroiso- quinolines...... 21
1.5 Statement of the Problem...... 27
II. METHODS AND MATERIALS
M .1 Synthesis and Release of Tetrahydro isoquinoline Alkaloids from the Bovine Adrenal Medulla...... 31
M.2 Role of Intracellular Calcium in Acetaldehyde-induced Catecholamine Release from the Adrenal Medulla 36
M.3 Protection Against Acute Toxicity of Acetaldehyde in Mice...... 3!)
M.4 Chronic Alcohol Administration Experiments...... v Page M.5 Chemical Determinations
M.5.1 Measurement of Catechol amines and TIQs by Thin-layer Chromatography...... 44
M.S.2 Measurement of Catechol amines by Colorimetry...... 46
M.S.3 Measurement of Protein in Sub- cellular Fractions of Bovine Adrenal Medulla...... 47 45 M.S.4 Determination of Ca in Sub- cellular Fractions of Bovine Adrenal Medulla...... 48
M.5.5 Measurement of Mitochondrial Succinic Cytochrome c Reductase in Sucrose Density Gradient Fractions of Bovine Adrenal Medulla...... 49
M.5.6 Determination of Ethanol in Blood and Chamber Air...... 50
M.S.7 Analysis of Whole Brain Salsolinol...... 51
M.5.8 Analysis of Salsolinol in Brain Parts...... „...... 55
III. RESULTS
R.l Synthesis and Release of Tetrahydro- isoquinoline Alkaloids from the Bovine Adrenal Medulla...... 56
R.2 Role of Intracellular Calcium in Acet- aldehyde-induced Catecholamine Release from the Adrenal Medulla...... 5 2
R.2.1 Concentration-Effect Relation ships...... 62
R.2.2 Effect of Magnesium...... 65
R.2.3 Release of Catecholamines...... 65
R.2.4 Release of ^ C a ...... 58
vi Page R.2.5 Subcellular Distribution of Mitochondria and Chromaffin Gran ules . . . 71
R.2.6 Subcellular Distribution of 45Ca ...... 76
R.3 Protection Against Acute Toxicity of Acetaldehyde in Mice...... 78
R.4 Absence of Formation of Brain Salsol inol in Ethanol-dependent Mice...... 83
R.4.1 Chamber Alcohol/Blood Alcohol Relationship...... 83
R.4.2 Establishment of Physical Dependence...... 90
R.4.3 Analysis of Single Whole Brains for Salsolinol...... 93
R.4.4 Analysis of Pooled Whole Brains for Salsolinol...... 109
R.4.5 Analysis of Pooled Brain Parts for Salsolinol...... 109
IV. DISCUSSION
D.l Synthesis and Release of Tetrahydro- isoquinoline Alkaloids from the Bovine Adrenal Medulla...... 116
D.2 Role of Intracellular Calcium in Acetaldehyde-induced Catecholamine Release from the AdrenalMedulla ...... 118
D.3 Protection Against Acute Toxicity of Acetaldehyde in Mice...... 123
D .4 Absence of Formation of Brain Salsol inol in Ethanol-dependent Mice...... 125
BIBLIOGRAPHY 128 LIST OF TABLES
Table Page
1. Scoring System for Convulsions Elicited by Lifting a Mouse by theTail ...... 45
2. Enzyme Distribution in Sucrose Density Gradient Fractions of Bovine Adrenal Medulla...... 72
3. Effect of Acetaldehyde on Subcellular Distribution of 45ca in Isolated Bovine Adrenal Medulla...... 77
4. Effect of Protecting Agents on Acute Toxicity of Acetaldehydein Mice and Rats... 81
5. Retention Times and Recoveries for Tyramine, Dopamine and Salsolinol...... 103
6. Summary of Chronic Alcohol Administration Studies (single brain experiments)...... 10 4 LIST OF FIGURES
Figure Page
1. Structures and Routes of Formation of the Tetrahydroisoquinoline Alkaloids...... 8
2 . Schematic Representation of the Apparatus Used to Deliver Alcohol Vapors to the Test Mice...... 42
3. Flowsheet for Extraction of Tyramine, Dopamine and Salsolinol...... 53
4. Cumulative Concentration-effect Curves for Acetaldehyde-induced Catecholamine Secretion from Isolated Bovine Adrenal Glands in Presence and Absence of Extracellular Calcium...... 58
5. Thin-layer Chromatograms of Catechol Compounds Released from Bovine Adrenal Medullae...... 61
6 . Cumulative Concentration-effeet Curves for Acetaldehyde-evoked Catecholamine Secretion from the Isolated Bovine Adrenal Medulla...... 64
7. Catecholamine Secretion from the Isolated Bovine Adrenal Medulla Evoked by Acetaldehyde...... 67 8 . Effect of Acetaldehyde on ^ C a Washout from Isolated Bovine Adrenal Medulla..... 70
9. Effect of Acetaldehyde on Subcellular Distribution of Catecholamines in the Isolated Bovine Adrenal Medulla...... 74
10. Effect of Acetaldehyde on Subcellular Distribution of ^ C a in the Isolated Bovine Adrenal Medulla...... 80
ix Figure Page
11. Relationship Between Alcohol Concentration in the Inspired Air and Blood Ethanol Levels...... 85
12. Relationship Between Alcohol Concentration in the Inspired Air and Blood Ethanol Levels...... 87
13. Relationship Between Alcohol Concentration in the Inspired Air and Blood Ethanol Levels...... 89
14. Mean Withdrawal Score of Mice Following 5 days of Continuous Inhalation of Ethanol.. 9 2
15. Chromatogram of a Single Control Mouse Brain Without Added Salsolinol...... 95
16. Chromatogram of a Single Control Mouse Brain With Added Salsolinol...... 97
17. Chromatogram of a Single Control Mouse Brain With Added Salsolinol...... 99
18. Chromatogram of a Single Control Mouse Brain With Added Salsolinol...... 101
19. Chromatogram of a Single Experimental Mouse Brain...... 106
20. Chromatogram of a Single Experimental Mouse Brain...... 108
21. Chromatogram of 2 Pooled Whole Brains from Experimental Mice...... Ill
22. Chromatogram of 3 Pooled Whole Brains from Experimental Mice...... 113
23. Chromatogram of the Pooled Brain Stem and Corpus Striatum of 5 Experimental Mice...... 115
x INTRODUCTION
I.1 Interaction of Ethanol with Neurotransmitter Systems
While much is known about ethanol and its effects in vivo, elucidating its mechanism of action and understand ing the underlying cause of physical dependence on this agent are problems which have thus far defied solution.
Until recently, a major obstacle in studies of alcohol dependence has been the lack of availability of a credible animal model. Goldstein (1975) and Mello (1973) have reviewed the various methods for producing addiction to alcohol. According to Goldstein (1975), the ideal situation (i.e. to mimic human alcoholism) would be to have animals ingest ethanol voluntarily (by mouth) to an extent capable of producing intoxication, tolerance and physical dependence; furthermore, they would learn to avoid with drawal reactions by taking more alcohol, and would voluntarily return to drinking alcohol after a period of abstinence. Thus far, a practical model which meets all of these criteria has not been developed. Other systems, such as offering alcohol as the only fluid source or addition of alcohol to an entirely liquid diet have been attempted; the former fails since most of the drinking is done at night, so the animal is not intoxicated the majority of the time. The latter has met with some success, as has schedule-induced polydipsia and self- administration by injection (Goldstein, 1975, 1976).
While some of the aforementioned systems are meaning ful from a behavioral standpoint (for example, studying the reinforcing effects of ethanol), they suffer from the standpoint that the experimenter has limited control over the dose of alcohol received by the subject. Although accurate dose control is achieved with forced administra tion (by injection or intubation), this technique has been found unsuitable for producing continuous intoxication and withdrawal in mice and rats, due to rapid metabolism of the alcohol. The alcohol inhalation system has been successfully used to induce intoxication and physical dependence on alcohol in mice by Goldstein (Goldstein and
Pal, 1971; Goldstein, 1972) and Griffiths et al. (1973).
In this model, virtually continuous intoxication is maintained, physical dependence develops rapidly, and the degree of dependence can be quantitated by seizure suscep tibility. Furthermore, the dose can be controlled by the experimenter, and no additional pharmacological treatments or alterations in the nutritional status (such as starva tion or cold stress, both of which increase the animal's caloric need and induce drinking) of the animal is needed
(Goldstein, 1975). Many studies have examined central nervous system neurotransmitter mechanisms as possible targets for the action of ethanol, but have contributed little to the elucidation of the cause of addiction or withdrawal. It has been demonstrated that acute administration of ethanol to rats caused no change in brain norepinephrine levels, but decreased turnover (Pohorecky, 19 74); similarly, with chronic treatment, no change in steady-state levels was noted. Several laboratories have reported that the with drawal period following chronic alcohol administration is associated with an increased turnover rate for norepineph rine (Ahtee and Svartstrom-Fraser, 19 75; Hunt and
Majchrowicz, 1974a; Pohorecky, 1974; Pohorecky et a l .,
1974). Whether this change is a cause or a consequence of the abstinence syndrome is uncertain at this time.
However, Goldstein (1973) has shown that reserpine, alpha- methyl-p>-tyrosine, phentolamine and propranolol increase the severity of the withdrawal reaction, and these findings have been confirmed in other laboratories (Griffiths et al. ,
1974; Blum and Wallace, 1974). This suggests that the increased norepinephrine turnover may in fact be a compensatory mechanism to counteract the events associated with withdrawal, since interference with these pathways increases the severity of the abstinence syndrome. During withdrawal, Griffiths et al. (1974) reported increased dopamine levels, and Hunt and Majchrowicz (19 74a) noted decreased dopamine turnover; however, a more recent report (Ahtee and Svartstrom-Fraser, 19 75) did not confirm either of these findings. Neither inhibiting the cholinergic system (with atropine) nor enhancing its activity (with physostigmine) affects the severity of the withdrawal syndrome in mice (Goldstein, 1973). Serotonin levels and turnover rate in the rat are unaffected during withdrawal (Hunt and Majchrowicz, 1974b); this is interest ing in light of the finding that inhibition of serotonin synthesis (with £-chlorophenylalanine) has been shown to decrease the rate of tolerance development to ethanol in rats (Frankel et al., 1975). Aminooxyacetic acid (a GABA- transaminase inhibitor), which elevates GABA levels in the
CNS, reduces the severity of withdrawal (Goldstein, 1973), but chronic ethanol treatment does not alter the turnover rate of GABA (Sutton and Simmonds, 1973).
The pharmacological activity of acetaldehyde, the primary metabolite of ethanol (Hawkins and Kalant, 19 72), has led to speculation regarding its role in alcohol toxicity and dependence (Ortiz et al., 1974; Rahwan, 1974).
Acetaldehyde is a sympathomimetic agent in vivo (Egle, 1972
Egle et al^. , 1973; Walsh et al^. , 1969) and releases endogenous catecholamines In vitro (Greenberg and Cohen,
1973; Lai and Hudgins, 1975; Schneider, 1971, 1974a,b) and in vivo (Walsh and Truitt, 1968). The mechanism of this sympathomimetic action is interesting, since it appears not to utilize the normal physiological mechanism (exocytosis) for catecholamine release (Schneider, 1971) . [For a discussion of exocytosis, see Rahwan and Borowitz (1973) and Rubin (1970, 1974).] Acetaldehyde also alters the metabolic disposition of norepinephrine in vivo, resulting in a greater utilization of the reductive pathway, pre sumably due to inhibition of aldehyde dehydrogenase
(Walsh et al., 1970). Acetaldehyde is 300 times more potent than ethanol in depressing behavior in mice
(Holtzman and Schneider, 19 74), and has been shown to cause physical dependence and withdrawal in this species
(Ortiz et al., 1974). Furthermore, acetaldehyde condenses with catecholamines under physiological conditions (neutral pH and ambient temperature) to form unstable Schiff bases which cyclize to form tetrahydroisoquinolines (TIQs)(Whaley and Govindachari, 1951). This fact has led to the hypothe sis that these TIQs or other related abnormal catecholamine metabolites may be involved in the toxic or addictive properties of ethanol (vide infra).
I.2 The tetrahydroisoguinoline alkaloid hypotheses
The TIQ alkaloids are structurally related to numerous naturally occurring psychoactive compounds found in the plant kingdom (Shamma, 1972). Almost simultaneously, two hypotheses evolved which proposed a role for compounds of this class in the toxic or addictive effects of ethanol
(Cohen and Collins, 1970; Davis and Walsh, 1970; Davis et al., 19 70), although an earlier communication alluded to such a possibility (Collins and Cohen, 1968). Both of these hypotheses place acetaldehyde in a pivotal role in the genesis of the proposed tetrahydroisoquinoline alkaloids. Acetaldehyde is primarily produced by the action of liver alcohol dehydrogenase (Hawkins and Kalant,
1972). The relative contributions of the microsomal ethanol oxidizing system and catalase in vivo are uncertain
(Majchrowicz, 1975). A small portion of the acetaldehyde escapes from the liver into the blood for distribution throughout the entire body, including the brain (Ortiz et al. , 1974) . In addition, brain tissue has been reported to possess alcohol dehydrogenase activity (Raskin, 1973;
Raskin and Sokoloff, 1968), thus providing for local production of acetaldehyde during alcohol ingestion. The two hypotheses, and the role of acetaldehyde in each, will be considered separately.
THE SIMPLE TIQ ALKALOIDS
Cohen and Collins (1970) proposed the direct conden sation of acetaldehyde with endogenous catecholamines to form a series of substituted l-methyl-6,7-dihydroxytetra- hydroisoquinolines (Fig. la). The authors noted the Figure 1. Structures and routes of formation of the tetrahydroisoquinoline alkaloids (TIQs). The simple
TIQs (a) are formed by the direct condensation of acetaldehyde with a catecholamine. The complex alka loids (b) are derived from the condensation of dopa mine with its deaminated metabolite, 3,4-dihydroxy- phenylacetaldehyde (dopaldehyde). DA (dopamine),
DOPAC (dihydroxyphenylacetic acid), THP (tetrahydro- papaveroline). 8
HO
HO
Sal*otlno1 NE-TIO E-TIQ
R1 - H Rj -O H R l-O H R2 - H R2-CH3
HO HO AldDH CHO COOH HOICO,
DA Dop T HON HOx O HOv HOX O THP Figure 1 structural similarity of the TIQs to a number of psycho active plant alkaloids, and suggested that these TIQs may be responsible for some of the behavioral effects of alcohol. Furthermore, the inherent structural similarity between the TIQs and the parent catecholamines suggested to Cohen and Collins (1970) that these new abberant metabolites might interact with adrenergic systems in vivo in such a way as to interfere with the normal functions of the catecholamines. It was envisioned that this inter ference might occur on a variety of levels: (1) The TIQs could inhibit catecholamine storage (2) These alkaloids might be stored and released into the area of the receptor and act as agonists or antagonists (3) They may influence the enzymes responsible for the synthesis or breakdown of catecholamines. By any of these actions, it was felt that the simple TIQs might in some way contribute to the pharmacologic response to ethanol and/or to the development of dependence on this agent. The findings lending support to this hypothesis were that the TIQs were, in fact, formed in bovine adrenal medullae (a model of a catecholamine storage site) perfused with acetaldehyde {Collins and Cohen, 1968; Cohen and Collins, 1970). Later findings regarding the simple TIQ alkaloids are discussed in sections 1.3 and 1.4. 10 THE COMPLEX TIQ ALKALOIDS In contrast to the proposal of Cohen and Collins (1970), Davis et al. (1970) invoked acetaldehyde in a different role. It had been previously reported that the addition of dopamine to MAO preparations (Holtz et al^. , 1963, 1964) led to the formation of a novel biotransforma tion product, tetrahydropapaveroline (Fig. lb), as a result of the condensation of dopamine with its deaminated metabolite, 3,4-dihydroxyphenylacetaldehyde (dopaldehyde). While this is not generally regarded as a normal pathway of dopamine metabolism (there is, in fact, no evidence to suggest that it is), Davis et al. (19 70) proposed that during heavy and prolonged ethanol consumption, acetal dehyde, by competitively inhibiting aldehyde dehydrogenase, might elevate dopaldehyde levels, and that under these conditions tetrahydropapaveroline could conceivably be synthesized. In vivo evidence for the requisite shift to a more reductive pathway in catecholamine metabolism (rather than the normal oxidative pathway) during ethanol ingestion was presented by Davis ejt ad. (1967) . The fact that tetrahydropapaveroline is an intermediate in the synthesis of morphine by the opium poppy prompted this group to propose that perhaps a similar set of coupling reactions could occur in mammalian systems, and lead to the formation of an addictive morphine-like alkaloid in vivo following alcohol ingestion, thus forming a common basis for addiction. This hypothesis has been strongly criticized (Goldstein and Judson, 1971; Halushka and Hoffman, 1970; Seevers, 1970). Halushka and Hoffman (1970) discounted the in vivo importance of tetrahydropapaveroline, since they were earlier only able to demonstrate tetrahydro papaveroline synthesis (following exogenous dopamine administration) in the liver (Haluska and Hoffman, 1968). Seevers (1970) based his criticism of the hypothesis on the symptomatological differences between alcohol and morphine dependence, the fact that cross dependence does not occur between alcohol and narcotics, and that other addicting compounds (barbiturates, for example) which exhibit cross tolerance with alcohol cannot be converted to morphine-like compounds. Goldstein and Judson (1971) provided indirect evidence against in vivo formation of opiates, since administration of naloxone did not precipi tate withdrawal in alcohol-addicted mice. It should be noted that considerable evidence has accumulated which demonstrates that some of the coupling reactions required to satisfy the hypothesis can indeed take place in mammalian systems (Cashaw et aJL. , 1974 ; Davis et al. , 1975 Kametani et al^. , 1972; Meyerson and Davis, 1975; see section 1.3), and similar diversions to reductive pathways of catecholamine metabolism have been observed for the 12 barbiturates {Davis et al., 19 74). An interesting finding regarding possible common denominators between alcohol, TIQs and narcotics has been the work of Ross and his coworkers (Ross et a_l., 1974; Ross and Cardenas, 1975) showing that the cerebral calcium- depleting actions of morphine, ethanol and salsolinol are all blocked by naloxone. Furthermore, these investigators demonstrated that naloxone had no effect on the calcium depleting effect of phenobarbital and reserpine, nor on that of t-butanol or isopropanol. The latter two compounds cannot result in the formation of TIQs since they are not metabolized to intermediate aldehydes. While this is worthy of note, it should be viewed in light of the fact that t-butanol and isopropanol are capable of inducing physical dependence (Goldstein, 1976) despite the inability to generate aldehydes or TIQs, and exhibit cross-tolerance with ethanol (LeBlanc and Kalant, 19 75). TETRAHYDRO-beta-CARBOLINES A similar route of investigation with regard to the interaction of alcohol (via acetaldehyde) with tryptamines was pursued by Mclsaac (1961a), who demonstrated in vivo formation of tetrahydro-beta-carbolines following adminis tration of ethanol, disulfuram and iproniazid to rats. The formation of these compounds proceeds by a condensation reaction similar to that for the TIQs. The tetrahydro- beta-carbolines are structurally related to the hallucino genic harmala alkaloids, and thus, Mclsaac (1961b) advanced a general biochemical concept of mental disease focusing on this class of compounds. Dajani and Saheb (1973) con firmed and extended the findings of Mclsaac (1961a), and proposed a role for compounds of the tetrahydro-beta- carboline group in alcoholism. 1.3 Biological formation and inhibition of formation of tetrahydroisoquinoline alkaloids______ Holtz et al. (1963, 1964) first noted that 3,4- dihydroxyphenylacetaldehyde (the oxidative deamination product of dopamine) could condense with its parent compound (dopamine) to yield tetrahydropapaveroline when the latter was incubated with guinea pig liver monoamine oxidase. Following the suggestion that tetrahydropapaver oline may mediate some of the effects of dopamine iri vivo (Holtz, 1966; McNay and .Goldberg, 1966), Halushka and Hoffman (196 8) attempted to correlate the pharmacological response (hypotension) to injected dopamine with in vivo synthesis of tetrahydropapaveroline. Upon finding only trace amounts of this compound (principally in the liver) at the time of peak response to dopamine, they concluded that the small quantity of tetrahydropapaveroline which might be produced in vivo would be incapable of exerting 14 a significant pharmacologic effect in the overall response to dopamine. It should be noted, however, that intravenous administration of dopamine precludes the possibility of tetrahydropapaveroline formation in the brain, where the capacity to oxidize aldehydes is inferior to that in the periphery (Deitrich, 1966). In the central nervous system, therefore, there could be a higher level of the 3,4- dihydroxyphenylacetaldehyde precursor, and an increased likelihood for tetrahydropapaveroline synthesis. Walsh et a_l. (19 70b) later demonstrated that t'etra- hydropapaveroline is the major metabolite of dopamine in homogenates of brain stem or liver when NAD availability is limited (which would therefore limit oxidative metabolism); addition of this cofactor abolished tetra hydropapaveroline formation in the liver homogenate (metabolism proceeded to the acid or neutral metabolites of dopamine), but tetrahydropapaveroline remained the major (about 70%) product formed in the brain stem preparation, thus suggesting that formation of this compound in brain tissue might be of importance in vivo. Furthermore, Davis et aJL. (1970) and Yamanaka et. al_. (1970) demonstrated that addition of ethanol or acetaldehyde to the brain stem homogenates facilitated the formation of tetrahydropapaveroline and in addition, led to salsolinol formation. It was on the basis of these findings that 15 Davis et a_l. (1970) formulated the hypothesis for a role of abberant catecholamine metabolites and their possible conversion to morphine-like compounds in alcohol dependence (vide supra). Robbins (1968) suggested that during alcohol ingestion, acetaldehyde may condense directly with biogenic amines to form pharmacologically active simple tetrahydroisoquinoline alkaloids. At approximately the same time, Cohen and Barrett(1969) and Collins and Cohen (1970) demonstrated the formation of the simple formaldehyde-derived TIQs in rat adrenal glands following methanol administration. Collins and Cohen (1969) and Cohen and Collins (1970) further showed the formation of EPI-TIQs and NE-TIQs following the perfusion of isolated bovine adrenal glands with either acetaldehyde or formaldehyde, and these findings formed the basis for their hypothesis (vide supra) for a possible role for simple TIQs in ethanol dependence. Because of the question of the high concentrations (100 yg/ml) of acetaldehyde used in the earlier studies of Cohen and Collins (1970), Cohen (1971b) proceeded to demonstrate the in situ formation of EPI-TIQ and NE-TIQ 14 following the perfusion of bovine adrenals with C-acetyl- dehyde at levels of 1 yg/ml, a concentration seen in man following consumption of alcohol (Majchrowicz and Mendelson, 1970) . Later work showed that these adrenal 16 TIQs are stored within the chromaffin granules (Greenberg and Cohen, 1972; Schneider, 1974b) and share some of the properties of the catecholamines with regard to their mechanism of release from the adrenal medulla (Greenberg and Cohen, 19 73). Sourkes (19 71) suggested that tetrahydropapaveroline (or its metabolites) may mediate some of the actions of L-dopa. The first evidence that any of these TIQs may form in man was presented by Sandler et a^L. (1973) . The latter group reported that significant quantities of both tetrahydropapaveroline and salsolinol appeared in the urine of parkinsonian patients receiving L-dopa therapy both before and after alcohol ingestion. Furthermore, ethanol administration caused a significant enhancement in salsolinol excretion. The fact that salsolinol was demonstrated in the urine of these patients even before alcohol ingestion implies an endogenous source of ethanol or acetaldehyde. There is in fact evidence that such a source of ethanol exists in rat, rabbit, and man (Blomstrand, 1971; Krebs and Perkins, 1970; McManus et al., I960). There have also been several recent reports that formaldehyde may be produced endogenously from 5-methyl- tetrahydrofolic acid (Hsu and Mandell, 1975; Laduron and Leysen, 1975; Rosengarten et al., 1975) which could conceivably participate in TIQ biosynthesis (Collins and 17 Cohen, 1970) . It is important to carefully consider the limitations of the findings of Sandler et a_l. (1973) . First, the patients were ingesting 3-4 g of L-dopa daily, which would provide a larger than normal peripheral dopamine pool; the TIQs may well have originated outside the CNS and been excreted in the urine. Secondly, the TIQs may have formed in the bladder, and thus would have no opportunity to exert any systemic pharmacological effects. Third, since the urine samples were allowed to stand for 3 hours prior to acidification [a condition which inhibits the condensa tion of aldehydes and catecholamines (Cohen and Collins, 19 70)] there is the possibility that the TIQs were formed extracorporeally. Finally, Cohen (1976), commenting on the findings of Sandler et al. (197 3), reported that the salsolinol recovered from the urine of patients prior to ingestion of alcohol was apparently a function of reagent contamination by acetaldehyde, and was not excreted after formation in the body as reported. Cashaw et al^. (1974) demonstrated that metabolites of tetrahydropapaveroline (tetrahydroprotoberberine alkaloids) are present in the urine of parkinsonian patients ingesting L-dopa; this necessitates formation and biotransformation of tetrahydropapaveroline in the body prior to excretion, since the tetrahydroprotoberber- ines do not form non-enzymatically. Indeed, Meyerson and . 18 Davis (1975) identified a benzyltetrahydroisoquinoline methyltransferase enzyme in rat liver which could convert tetrahydropapaveroline to tetrahydroprotoberberines. Furthermore, Cashaw et al^. (19 74) demonstrated the in vitro conversion of tetrahydropapaveroline to tetra hydroprotoberberines by rat brain homogenates, attesting to the ability of this tissue to effect the enzymatic conversion. However, whether the alkaloids recovered from human urine (Cashaw et al., 1974) were formed centrally or peripherally is not known. Direct evidence for the iri vivo formation of tetra hydropapaveroline and salsolinol did appear, however. In 19 74, Turner et al., reported the formation of tetrahydro papaveroline (8 ng/g) in the brains of rats following 8 days consumption of L-dopa and Ro 4-4602 (an inhibitor of peripheral dopa-decarboxylase) in their drinking water. Addition of ethanol (10% w/v) to the fluid resulted in an increase in tetrahydropapaveroline to 10-25 ng/g of brain tissue. No tetrahydropapaveroline was detectable when ethanol alone was given (Turner et cal. , 1974). Collins and Bigdeli (1975a,b) found salsolinol (17 ng/g) in the catecholamine-rich areas of rat brain following acute administration of ethanol (9 g/kg in 3 i.p. doses over a 7 hour period) to pyrogallol pretreated rats. This pretreatment causes a tenfold elevation of the levels 19 of acetaldehyde following ethanol administration (Collins et al., 1974) and reduces TIQ metabolism by inhibition of COMT (Rubenstein and Collins, 1973). Collins and Bigdeli (1975b) also found that under these same conditions, EPI-TIQ was synthesized in the adrenal- medulla to the extent of 4 ug/g of tissue. When ethanol alone was given, no TIQs were detected in brain or adrenal tissue. The levels of salsolinol and EPI-TIQ reported both reflect about 1% conversion of catecholamine precursor to the respective TIQ (Collins and Bigdeli, 1975a,b). There is evidence that if formed iii vivo, these TIQs would likely be subjected to biotransformation. Salsolinol is known to be a good substrate for COMT (Collins et al^. , 1973), as are the TIQs derived from norepinephrine and acetaldehyde or formaldehyde, and that derived from dopamine and formaldehyde (Creveling et ad. , 19 72; Rubenstein and Collins, 1973). Tetrahydropapaveroline is not only O-methylated by liver COMT (Collins et ad., 1973), but is also cyclized (with the i'nsertion of a methylene bridge) iri vitro by rat liver benzyltetrahydroisoquinoline methyltransferase to form complex tetrahydroprotoberberine- type alkaloids (Meyerson and Davis, 19 75) . Kametani et al. (1972) have also shown that reticuline (a substituted TIQ) is cyclized to coreximine (a morphine precursor) in vivo by rats. There is no evidence to suggest that either 20 salsolinol or tetrahydropapaveroline is a substrate for MAO, although they are inhibitors of this enzyme (Collins et al., 1973). If these TIQs are synthesized in vivo following alco hol ingestion, and mediate some of the addictive or toxic actions of ethanol and/or acetaldehyde (vide supra), it would be of interest to establish methods for blocking their formation as a means of preventing these undesirable effects. Several investigators (Blum et a_l. , 1974; Sprince et al., 1974, 1975; Ward et al., 1972) have shown that numerous compounds (cysteine, ascorbate, thioctic acid, homocysteine, glutathione, lysine, glycine, serine) afford protection against acetaldehyde and alcohol toxicity and lethality in rats, and Nagasawa et al. (19 75) have shown that sulfhydryl amino acids are capable of complexing the acetaldehyde produced i_n vivo following ethanol admin istration. Similarly, Alivasatos et a^l. (19 73) demonstrated that some of these same compounds are capable of inhibiting TIQ formation in rat brain homogenates, and suggested that their inhibitory action is via aldehyde displacement, complexation or reduction. These findings may have some therapeutic implications if acetaldehyde or TIQs have a role in the effects of ethanol; it should be noted, however, that alternative explanations for some of these iri vivo findings have been proposed (Blum et al., 1974; Sprince et al., 1974, 1975). 21 I.4 Pharmacology of the tetrahydroisoquinolines. Characterization of the pharmacological activity of some of the TIQs has been hampered by the lack of avail ability of pure compounds for study. Collins and Kernozek (1972) have successfully synthesized 4,6,7-trihydroxy- tetrahydroisoquinoline (the norepinephrine-formaldehyde derivative), and salsolinol and tetrahydropapaveroline are available in pure form, but the norepinephrine-acetaldehyde and epinephrine-acetaldehyde TIQs have never been prepared in pure form (Cohen, 19 76). The direct condensation of catecholamines in aqueous solution with acetaldehyde will yield their respective TIQs, but several additional products are also formed (Cohen, 19 71a; Cohen and Collins, 1970; King et ad. , 1974; Osswald et a_l. , 1975) . The simple TIQs have received more attention than the complex alkaloids with respect to their activity in biological systems, and most of the studies focus on their role as potential false adrenergic neurotransmitters (Cohen and Collins, 1970; Cohen, 1973). Heikkila e_t ad. (1971) demonstrated that the acetaldehyde-derived TIQs of dopamine (salsolinol) and norepinephrine (NE-TIQ), in _ 3 concentrations of approximately 10 M, release endogenous catecholamines from rat brain slices. In addition, they are taken up into the synaptosomal fraction of rat brain homogenates, though the uptake of TIQs is clearly not as effective as that for norepinephrine and dopamine; the 22 tissue/medium ratio in these studies for dopamine and sal- “ 8 solinol (both at 10 M), for example, were 33 7 and 8, respectively. Furthermore, over a 3 log unit concentration _ Q _g range (10 to 10 M), the relative uptake of salsolinol remained the same, while that of dopamine declined. This suggests saturation of a specific accumulating mechanism by dopamine, whereas the uptake for salsolinol is relatively unsaturable in the concentrations tested. Heikkila et al_. (1971) also noted that a major fraction of the salsolinol uptake could occur at 0° C, while that for the catecholamines was virtually abolished. Other studies have also examined the in vivo uptake and release of exogenously administered TIQs (Mytilineou et al., 1974; Cohen et al., 1972). Using the formaldehyde- derived TIQs of norepinephrine and dopamine, these investigators demonstrated (by fluorescence microscopy) that the compounds were accumulated in the adrenergic nerve plexus of the mouse iris following reserpinization or pretreatment with alpha-methy1-p-tyrosine methyl ester; furthermore, this accumulation was blocked by desmethyl- imipramine. However, the fluorescence was rather short lived (approximately 2 hours), which suggests that either the compounds are not tightly bound or they are metabolized. When these experiments were repeated in reserpinized rats, and the preganglionic fibers to the iris were stimulated. 23 the iris was shown to release the TIQ, as demonstrated both by fluorescence assay and by the protrusion of the eyeball, retraction of the eyelid, and contraction of the iris, all responses indicative of alpha-adrenergic receptor activa tion (Mytilineou et a_l. , 1974). Other investigators have also studied the uptake and storage of TIQs into adrenergic tissues (Locke et al., 1973 ; Tennyson et al., 1973) and have reported similar findings. Salsolinol has been found to be a weak inhibitor of norepinephrine and dopamine uptake (Alpers et a^. , 19 75; Cohen et aT., 1974; Tuomisto and Tuomisto, 1973). Similar findings were reported by Heikkila and Cohen (1974) for 6,7-dihydroxytetrahydroisoquinoline as an inhibitor of serotonin accumulation into rat brain slices. Further elucidation of the pharmacological activity of these compounds was provided by Brezenoff and Cohen (1973), who showed that the intraventricular administration of 3.5-350 yg of 6,7-dihydroxytetrahydroisoquinoline caused a profound hypothermia in rats, similar to that produced by norepinephrine. Pretreatment with 6-hydroxy- dopamine abolished the TIQ response, suggesting that the TIQ (at these doses) acts entirely through endogenous norepinephrine (Brezenoff and Cohen, 1973). 24 Simpson (1975) conducted a thorough investigation of the sympathomimetic action of 6,7-dihydroxytetrahydro- isoquinoline in the pithed rat, and found that this compound is approximately 3 orders of magnitude less potent than norepinephrine on alpha- or beta-receptor mediated responses. More recently, Baird-Lambert and Cohen (1975) investigated the effects of several catechol amine-derived TIQs on the hypogastric nerve-vas deferens preparation; only when the tissue was superfused with 6,7-dihydroxytetrahydroisoguinoline was a significant response observed, i.e. a potentiation of the second phase of the twitch response following nerve stimulation. This response was attributed to either inhibition of uptake of the released norepinephrine (Cohen et al., 19 74; Heikkila et al., 1971; Tuomisto and Tuomisto, 1973) or inhibition of MAO (Cohen and Katz, 1975). On beta-adrenergic systems, Lee et_ al. (1974) found that neither isomer of salsolinol was very effective as a stimulant of lipolysis in rat adipose tissue, though the S(-) isomer was slightly more potent (ED50 approximately 10 ^ M) than the R(+) isomer; neither isomer was capable of eliciting the maximal response seen with norepinephrine. Further work with salsolinol demonstrated that it is extremely weak as an agonist on both the guinea pig trachea and spontaneously beating right atrium (Feller et al^. , 1975); 50% of maximal response to isoproterenol was not 25 reached despite the use of concentrations approximating - 3 10 M. It is equally ineffective on the dopamine- sensitive adenylate cyclase of rat caudate nucleus (Sheppard and Burghardt, 1974). Collins et al. (1973) demonstrated that salsolinol is a substrate for rat liver COMT (Km approximately equal to that for norepinephrine and dopamine). Furthermore, it is also a weak competitive inhibitor of the oxidation of serotonin by rat brain MAO (Ki = .14 mM) and the O-methylation of dopamine by rat liver COMT (Ki = .13 mM) (Collins et aul. , 1973). In the only experiments reported thus far with the epinephrine-acetaldehyde TIQ, Osswald et al.,(1975) observed that a crude preparation of this compound had approximately 1/1000th the activity of norepinephrine on several preparations. Following a 1 mg/kg i.p. dose, it did, however, cause a marked decrease in the norepinephrine content of the heart, hypothalamus and aorta of the guinea pig (Osswald et al., 1975). Tetrahydropapaveroline's activity is primarily beta- adrenergic agonist in nature. Santi et aJL. (1964, 1967) reported that this compound had positive inotropic and chronotropic actions in vivo and in vitro (approximately l/50th the potency of isoproterenol) which were blocked by dichloroisoproterenol; similarly, an increase in hind limb blood flow was seen following administration of this compound into the femoral artery. This agent also caused a lipolytic response in vivo and ±ri vitro (Santi et al. , 1964, 1967). Lee et al_. (1974) extended this finding to an evaluation of the isomers of tetrahydropapaveroline on lipolysis, and found that the S(-) isomer is more active than the R(+), though neither was capable of achieving -5 the maximal response attained with 2 x 10 M norepineph rine despite the use of concentrations of tetrahydro papaveroline 2 orders of magnitude greater. Similar results were reported on the isolated guinea pig trachea and right atrium (Feller et ad., 1975). Tetrahydropapaveroline has also been tested for its effect on catecholamine uptake. Alpers et aL. (1975) and Cohen et al. (19 74) both report weak (Ki approximately -5 -4 10 to 10 M) inhibition of uptake of dopamine and norepinephrine by this TIQ. Alpers et al. (1975) also tested two potential metabolites of tetrahydropapaveroline, tetrahydroxyberbine and tetramethoxyberbine, and found these to be equally weak inhibitors of catecholamine accumulation. Tetrahydropapaveroline has been reported to have negligible inhibitory action on brain phosphodiesterase activity (Furlanut et al., 1973), and to be ineffective as a stimulant of the dopamine-sensitive adenylate cyclase 27 of rat striatum (Miller et al^. , 1974; Sheppard and Burghardt, 1974). It is also a weak inhibitor of MAO (Ki = 0.2 mM), but slightly more potent as a COMT inhibitor (Ki = 0.02 mM) (Collins et al. , 1973) . I.5 Statement of the problem. Interest in the pharmacological and toxicological actions of acetaldehyde has been stimulated by recent speculations regarding its role in ethanol toxicity and dependence. The former agent has been suggested to be responsible for addiction both directly (Ortiz et al^. , 1974) and indirectly via its participation in the production of neuroamine-derived tetrahydroisoquinoline alkaloids (Cohen and Collins, 1970; Davis et a_l. , 1970). Some aspects of the mechanisms whereby acetaldehyde exerts its pharmacological and toxicological effects have been investigated (Egle, 1972; Schneider, 1971, 1974a,b; Walsh et ad., 1969; and see INTRODUCTION), but more experimentation into the intimate mechanisms of acetalde- hyde's sympathomimetic effects and the factors controlling the synthesis and release of the TIQs from their storage sites was needed to better understand how this agent functions in biological systems. In addition, although the in vivo biosynthesis of TIQs had been demonstrated following alcohol administration to laboratory animals (Collins and Bigdeli, 1975a,b; Turner et al., 1974) and 28 humans (Sandler et al., 1973), these studies have failed to show any relationship between TIQ synthesis and alcohol dependence, and have employed highly artificial experimental conditions. The purpose of the following studies was, therefore, four-fold: (1) Previous studies have shown that exposure of the bovine adrenal medulla (a model of a catecholamine storage site) to acetaldehyde results in the release of catechol amines (Schneider, 1971, 1974a,b) and the synthesis of granule-bound TIQs (Greenberg and Cohen, 19 72) which are releaseable from the gland by acetylcholine (Cohen and Collins, 1970; Greenberg and Cohen, 1973). It was unknown to what extent the presence or absence of calcium--a cellular constituent necessary for the physiological cate cholamine release process (Rahwan and Borowitz, 1973; Rubin, 1970, 1974)— might influence the synthesis of these TIQs and/or their secretion from the medulla when acetalde hyde was used both as a TIQ precursor and a secretagogue. Therefore, studies were undertaken to elucidate the mech anism of synthesis and release of the TIQs, using the bovine adrenal medulla as an experimental model. (2) It was of interest to study in detail the intimate cellular mechanism whereby acetaldehyde releases catecholamines from their storage sites. Extracellular or intracellular calcium is a critical intermediate in the 29 stimulus-secretion coupling process for adrenomedullary catecholamine release (Rahwan and Borowitz, 19 73; Rubin, 1970, 1974), and thus this cation served as a focal point for these studies. (3) Various compounds have been shown to inhibit in vitro TIQ biosynthesis in brain homogenates {Alivasatos et al., 1973) and to complex acetaldehyde iri vivo following ethanol administration (Nagasawa et al^. , 1975) . These same, or closely related, compounds have also been demon strated to protect rats from ethanol- and acetaldehyde- induced toxicity and lethality in vivo (Blum et al^. , 1974; Sprince et al. , 1974, 1975; Ward et a^L. , 1972) . It was, therefore imperative to test these compounds for their ability to antagonize the effects of acetaldehyde in our test species, the mouse. It was the goal of these experiments to find a compound(s) which^might be useful as an inhibitor of in vivo TIQ biosynthesis for future experiments directed at modifying the addiction process. (4) Finally, none of the reported experiments demonstrating in vivo biosynthesis of TIQs have attempted to relate the levels of these compounds to any behavioral effects (i.e. addiction or withdrawal), and all were performed under conditions which elevated TIQ precursors or inhibited the metabolic degradation of these alkaloids (Collins and Bigdeli, 1975a,b; Sandler et al., 1973; Turner et al., 1974). It was the goal, therefore, of the present study to determine the parameters necessary to establish physical dependence on alcohol in mice [using the ethanol vapor inhalation method (Goldstein, 19 72)], and to explore the relationship between the severity of the alcohol abstinence syndrome (convulsions on handling) and the levels of TIQ alkaloids in the brains of addicted mice. If the TIQs are causally related to alcohol dependence, there should presumably be an inverse relation ship between TIQ levels and the susceptibility to with drawal seizures. Finally, using the protectant compounds (TIQ synthesis inhibitors), it was the aim of these studies to attempt to alter the addiction/withdrawal syn drome by preventing iri vivo TIQ biosynthesis. CHAPTER II METHODS AND MATERIALS M.l Synthesis and release of tetrahydroisoquinoline alkaloids from the bovine adrenal medulla (Rahwan, O'Neill and Miller, 1974).______ Fresh bovine adrenal glands were obtained at a local slaughterhouse/ placed on ice during transport to the laboratory, and used about one hour post-mortem. Prior to all experiments, the glands were decorticated and the medullae were perfused retrograde through the adrenal vein with aerated Locke's solution at room temperature until a stable perfusion rate of 5 ml/min was achieved (Rahwan et al., 1973) using individual channel perfusion pumps (FMI, Inc., Oyster Bay, N.Y.). The composition of the Locke's solution was as follows: NaCl 154.0 mM, KCl 5.6 mM, CaCl2 *2H20 2.2 mM, N a 2HPC>4 •7H20 2.14 mM, NaH2P04 *H20 0.85 mM, glucose 10.0 mM. In order to study the role of extracellular calcium in acetaldehyde-evoked catecholamine secretion from the adrenal medulla, cumulative concentra- tion-effect curves were obtained by a modification of the method previously described by Rahwan et al. (19 73); the glands were perfused for 30 minutes with buffered isotonic Locke’s solution (containing 2.2 mM calcium) or calcium- free Locke's solution (isotonicity was maintained by the addition of NaCl) followed by infusion of increasing concentrations of acetaldehyde (J. T. Baker Chemical Co., Phillipsburg, N.J.) for 3 minute periods. The perfusate from the glands was collected in 3 minute fractions immediately before and during acetaldehyde perfusion. Catecholamine content of the perfusate was measured immediately after collection by the method of von Euler and Hamberg (1949)(see Chemical Determinations, M.5.2) to avoid the formation of tetrahydroisoquinoline alkaloids (Cohen and Collins, 1970) in the collecting vessel with the released amines and the excess acetaldehyde. Acetaldehyde itself does not interfere with this assay (Schneider, 1974a). Only total catecholamines were assayed, since Schneider (1971) has demonstrated that there is no prefer ential release of epinephrine or norepinephrine following stimulation with acetaldehyde. Evoked catecholamine release was corrected for resting secretion of amines, which was measured just prior to stimulation. The resting secretion rate of catecholamines is not affected by a change in perfusion medium from Locke’s solution containing 2.2 mM calcium to one containing no calcium (Rahwan et al., 1973), and a 30 minute perfusion with calcium-free Locke's solution has been demonstrated to be adequate to wash out extracellular calcium from the medullary tissue (Rahwan et al., 1973). 33 Concentrations of acetaldehyde as low as 2 3 *jM have been shown to induce the synthesis of TIQs in the bovine adrenal medulla (Cohen# 1971b). The lowest concentration of acetaldehyde capable of evoking measurable release of catecholamines from the bovine adrenal medulla is 1 mM (Schneider, 1971). Therefore# a concentration of 10 mM acetaldehyde was used to induce the _in situ synthesis of TIQs without causing appreciable depletion of adrenal catecholamines# whereas 32 mM acetaldehyde was used to evoke catecholamine and TIQ secretion from the glands. The higher concentrations of acetaldehyde were used in order to yield more readily detectable amounts of TIQs (Greenberg and Cohen# 19 73). Concentrations as high as 1 M have been shown not to cause tissue fixation or cellular damage (Schneider# 1971). In one set of experiments, bovine adrenal medullae were perfused with normal Locke's solution for 30 minutes, followed by a 60 minute perfusion with 10 mM acetaldehyde (prepared in normal Locke's solution). Extracellular acetaldehyde was then washed out by an additional 30 minute perfusion with acetaldehyde-free Locke's solution. The glands were then stimulated with 32 mM acetaldehyde (in normal Locke's solution) for 8 minutes, and 40 ml of perfusate were collected in chilled acidified flasks (Greenberg and Cohen# 19 73) in order to delay the oxida tion of catecholamines and TIQs, and to prevent the condensation of released catecholamines with the excess acetaldehyde in the perfusate in the collecting vessel. The catecholamines and TIQs were analyzed by thin-layer chromatography (see Chemical Determinations, M.5.1). Standard TIQs were prepared as previously published (Greenberg and Cohen, 19 73) by incubating solutions of epinephrine or norepinephrine (1 mg/ml) with an equal volume of 1 M acetaldehyde for 1 hour, and stored frozen. Serial dilutions of the TIQ standards were spotted in 4 yl quantities on silica gel thin-layer chromatoplates and developed as described in Chemical Determinations (M.5.1). The lowest concentration of EPI-TIQ and NE-TIQ that could be visualized was estimated at 4 8 ng/4 yl. In a second set of experiments, the adrenal medullae were perfused and treated as described above, except that calcium was omitted from the perfusion medium. Thus, the glands were perfused for 30 minutes with calcium-free Locke's solution, followed by a 60 minute perfusion with 10 mM acetaldehyde (prepared in calcium-free Locke's solution). A 40 ml fraction of the perfusate was collected during the first 8 minutes of this perfusion, and analyzed for TIQs and catecholamines (see Chemical Determinations, M.5.1). Perfusion was continued with acetaldehyde-free and calcium-free Locke's solution for an additional 30 minutes in order to wash out extracellular acetaldehyde. 35 The glands were then stimulated for 8 minutes with 32 mM acetaldehyde (prepared in calcium-free Locke's solution). The perfusate was collected and analyzed for catecholamines and TLQs. In a third set of experiments, adrenal medullae were perfused for 30 minutes with calcium-free Locke's solution, followed by a 60 minute perfusion with 10 mM acetaldehyde (prepared in calcium-free Locke's solution), and a 30 minute washout of residual acetaldehyde with calcium-free Locke's solution. Calcium (2.2 mM) was then reintroduced into the perfusion medium and perfusion continued for another 30 minutes to replenish the adrenal tissue with calcium. The glands were then stimulated for 8 minutes with 1.6 mM carbachol (Aldrich Chemical Co., Inc., Milwaukee, Wis.)(prepared in normal Locke's solution containing 2.2 mM calcium). The perfusate was collected and analyzed for catecholamines and TIQs. Reintroduction of calcium into the perfusion medium prior to stimulation with carbachol is essential, since this secretagogue is ineffective in the absence of extracellular calcium (Rubin, 1970). 36 M.2. Role of intracellular calcium in acetaldehyde- induced catecholamine release from the adrenal medulla. (O’Neill and Rahwan, 1975).______ Bovine adrenal glands were obtained and prepared as described above (vide supra, M.l). Cumulative concentra- tion-effect curves were obtained as described under M.l by perfusing decorticated adrenals with Locke's solution containing 2.2 mM calcium or calcium-free Locke's solution for 30 minutes prior to and during continuous infusion of increasing concentrations of buffered acetaldehyde for 3 minute periods. In some experiments, magnesium _3 (5 x 10 M) was added to the calcium-free medium 10 minutes prior to and during the concentration-effect studies with acetaldehyde, in order to block the action of intra cellular calcium (Kanno et al., 1973; Lastowecka and Trifaro, 19 74; Rahwan et al., 19 73). The perfusate from the glands was collected in 15 ml fractions immediately before and during stimulation with acetaldehyde, and analyzed for total catecholamines (epinephrine and norepinephrine) by the method of von Euler and Hamberg (1949)(see Chemical Determinations, M.S.2) immediately after collection in order to avoid condensation of the released amines with excess acetaldehyde to form tetra hydroisoquinoline alkaloids (vide supra; Cohen and Collins, 1970). 45 Experiments were performed with Ca in order to determine the effect, if any, of acetaldehyde on intra cellular calcium movements during evoked catecholamine secretion. After perfusion of decorticated adrenals for 20 minutes with calcium-free Locke's solution, labeling of the intracellular calcium pools was achieved by infusion 45 4 5 of Ca (1 yc of CaCl2 per ml; specific activity, 12,700 mc/g Ca)(International Chemical and Nuclear Corporation, Irvine, Ca.) for 2 minutes (Rahwan et al., 45 19 73). Extracellular Ca was washed out by a further 30 minute perfusion with calcium-free medium (Rahwan et al., 19 73). The glands were then stimulated for 10 minutes with 23 mM acetaldehyde dissolved in buffered calcium-free Locke’s solution. Control glands were not subjected to acetaldehyde stimulation, but were perfused with calcium-free medium for 4 0 minutes after labeling with radiocalcium. The perfusate was collected in 2 minute fractions. Immediately after acetaldehyde stimulation, each adrenal medulla (average weight 4 g) was minced and homogenized in ice-cold 0.32 M sucrose solution in an all glass conical tissue grinder (size 22; Kontes Glass Co., Vineland, N.J.). The number of up-and-down strokes was maintained at 8. From an aliquot of homogenate containing 2 g of medullary tissue, subcellular fractions were separated by differential and discontinuous sucrose density gradient centrifugation as described by Rahwan et al. (1973). Large cell fragments and nuclei were separated by centrifugation at 480 x g (2000 rpm) for 10 minutes in a Sorvall model RC2—B centrifuge (rotor no. SS 34; Ivan Sorvall, Inc., Newton, Conn.). Chromaffin granules and mitochondria were separated from microsomes and cell cytosol by centrifugation at 10,000 x g (9120 rpm) in a Sorvall model RC2-B centrifuge (rotor no. SS 34). Microsomes were separated from cytosol by further centrifugation at 86,000 x g (36,000 rpm) for 74 minutes in a Beckman model L preparative ultracentrifuge (rotor no. 40; Beckman Instruments, Inc., Palo Alto, Ca.). The chromaffin granules were separated from mitochondria on a discontinuous sucrose density gradient (1.25, 1.5, 1.75, and 2.0 M sucrose; 2.5 ml each) by centrifugation at 120,000 x g (28,500 rpm) for 48 minutes in a Beckman model L preparative ultracentrifuge with a type SW36 swinging bucket rotor. All separation steps were carried out at 4° C. The subcellular fractions were analyzed for protein by the biuret method (see Chemical Determinations, M.S.3). Total catecholamines in subcellular fractions were analyzed colorimetrically (see Chemical Determinations, M.5.2) after the proteins were precipitated with trichloroacetic acid. 45Ca in subcellular fractions and perfusate was counted 39 by liquid scintillation in a model 3375 Tri-Carb spectro meter (Packard Instrument Co., Downers Grove, 111.)(see Chemical Determinations, M.5.4). The efficiency of separation of mitochondria from chromaffin granules on the discontinuous density gradient was determined by measuring in each fraction the activity of the mitochrondrial marker enzyme succinic cytochrome c reductase (see Chemical Determinations, M.5.5), as well as by determining the distribution of catecholamines. M.3 Protection against acute toxicity of acetaldehyde in mice (O'Neill and Rahwan, 19 76)______ Male CD-I albino mice (Charles River, Syracuse, N.Y.) of various weights (28-40 g in different experiments, but uniform in individual experiments) were injected intraperitoneally (i.p.) with increasing doses of acetaldehyde (prepared in isotonic saline), and the ED90 for loss of righting reflex was determined by the method of Litchfield and Wilcoxon (1949). This dose, which was 415 mg/kg, was used in the subsequent experiments, and was verified periodically. In studies involving protecting agents, the following compounds were administered orally 30 minutes prior to the i.p. administration of the ED90 of acetaldehyde: L-cysteine HCl (3 mMoles/kg; 527 mg/kg), L-ascorbic acid (2 mMoles/kg; 352 mg/kg), DL-thioctic acid (2 mMoles/kg? 412 mg/kg), or 40 DL-homocysteine thiolactone (2 mMoles/kg; 307 mg/kg). All protectant compounds were dissolved in distilled water with the exception of thioctic acid, which was suspended in methylcellulose. M.4 Chronic alcohol administration experiments (O'Neill and Rahwan, submitted; Rahwan and O'Neill, 19 76) Groups of 20-25 male Swiss-Webster mice (Lab Supply, Indianapolis, Ind.) of various weights (20-40 g in different experiments, but uniform in individual experi ments) were housed in a clear plexiglas chamber measuring 52 x 34 x 27 cm; food and water were available ad libitum. The apparatus used to deliver alcohol vapors to the chamber is represented in Figure 2. Alcohol was dripped onto a filter paper wick using a Harvard model no. 9 31 infusion/withdrawal pump (Dover, Mass.); flow rates of ethanol were between 0.0382 and 0.1258 ml/min, depending upon the conditions desired. Air flow through the flask and into the chamber was held constant at 3 liters/min— a rate sufficient for the respiratory needs of 20-30 mice (Goldstein, ^972)— using a Harvard model no. 607 respirator. A small variable speed electric fan mounted within the chamber provided continuous circulation of air throughout the enclosed space. Thus, the chamber alcohol levels were controlled by altering the flow rate of ethanol and/or the fan speed within the chamber. 41 Figure 2. Schematic representation of the apparatus used to deliver alcohol vapors to the test mice. Alcohol was dripped onto a filter paper wick in the flask, and room air was pumped through the flask and into the chamber. A small variable speed electric fan within the chamber provided constant circulation of air; chamber alcohol was monitored by taking 0.5 ml samples of air from a port located in the lower 1/3 of the chamber at the same level as the mice. For details of alcohol and air delivery, see text. Alcohol Wick Sampling Porf Figure 2 to 43 Ethanol concentration in the inspired air was monitored spectrophotometrically (see Chemical Determina tions, M.S.6) in 0.5 ml samples of chamber air withdrawn from a port situated in the lower 1/3 of the chamber at the same level as the mice, using the method of Lundquist (1959). The chamber alcohol level on day 1 was approxi mately 14 mg/L; the concentration was gradually increased until 20-30 mg/L was reached on day 5-6. At this point, mice were either evaluated for withdrawal convulsions on handling (vide infra), or were sacrificed and their brains (either single or pooled in groups of 2 or 3) analyzed for the presence of salsolinol using a sensitive gas chromatographic/electron capture assay method (see Chemical Determinations, M.S.7). Blood alcohol levels in the mice were routinely monitored (4 mice/day were sampled and returned to the chamber) by taking 50 \i 1 blood samples via retro-orbital puncture, and determining ethanol content using the enzymatic method of Lundquist (19 59)(see Chemical Determinations, M.S.6). Similarly, a 50 yl blood sample was obtained from each mouse immediately prior to sacrifice. In a separate set of experiments, mice were housed under the above conditions, but at sacrifice, the brain stem and corpus striatum were removed, pooled in groups of 5, and analyzed for the presence of salsolinol (see M.S.8). Stress control mice were housed under identical 44 conditions in all experiments, with the notable exception that no alcohol was delivered to their chamber. Withdrawal Score: Mice were removed at the end of 5-6 days confinement in the alcohol chamber and placed in individual plastic cages (28 x 18 x 12 cm). They were then evaluated for susceptibility to seizures when lifted by the tail and gently turned (Goldstein and Pal, 1971; Goldstein, 1972). The procedure was repeated at 30-60 minute intervals for several hours, until the score began to decline. The scoring system is described in detail in Table 1. Mice that died during withdrawal, or those that were comatose at the end of the experiment were not included in the withdrawal analysis, though comatose mice were used in the biochemical determinations. M.5 Chemical Determinations M.5.1 Measurement of catecholamines and TIQs by thin-layer chromatography______ The catecholamines and TIQs in the perfusate from the bovine adrenal medulla were analyzed by adsorption onto, and elution from, freshly prepared A1(OH)^ (Greenberg and Cohen, 1973; Heikkila et al., 1971). The purified extract was concentrated down to 1 ml under a stream of nitrogen, and spotted in 4 yl quantities on silica gel plates (F-254, 0.25 mm Merck gel; Brinkmann Instruments, Inc., Westbury, N.Y.). The chromatograms were developed 45 Table 1 (Goldstein, 1972) Scoring system for convulsions elicited by lifting a mouse by the tail The characteristic tonic convulsion consists of tight ening of facial muscles (grimace), head thrown back, fore legs flexed and hind legs extended laterally, with general ized body tremor. A slight twist is used to evoke minimal signs. Score 1 = tonic convulsion when the mouse is lifted and given a gentle 180° turn. Score 2 = tonic-clonic convulsion elicited by the gentle spin, or tonic convulsions when lifted without turning. Score 3 = tonic-clonic convulsion not requiring any spin. Score 4 = violent tonic-clonic convulsion often con tinuing after release of the mouse. 46 in 2-butanol:formic acid:water (15:3:2) as a solvent (Greenberg and Cohen, 1973) and visualized by either placing the plates in an iodine chamber, which results in a brown coloration of the catecholamine and TIQ spots, or by the K3Fe(CN)g-FeCl^ spray procedure of Greenberg and Cohen (1973), which yields purple spots for these compounds. M.5.2 Measurement of catecholamines by colorimetry (von Euler and Hamberg, 1949)______ Catecholamine content in perfusates from adrenal glands was determined by taking a 4 ml aliquot of the perfusate, adding 1 ml of 1 M acetate buffer (pH 6.5), and 0.2 ml of 0.1 N iodine with shaking. After 3 minutes, 0.2 ml of 0.2 N sodium thiosulfate was added, shaken until the solution was decolorized, and the optical density at 529 nm immediately read in a Bausch and Lomb Spectronic 20 spectrophotometer (Bausch and Lomb, Inc., Rochester, N.Y.). The following subcellular fractions of adrenal medulla were obtained as described in M. 2. I-broken cell debris II-mitochondria III-mitochondria and lysosomes IV-lysosomes and chromaffin granules V-chromaffin granules Vl-chromaffin granules NUC-nuclei MIC-microsomes CYT-cytosol The volumes of fractions I-VI, NUC, and MIC were equalized with distilled water and an equal volume of 10% 47 trichloroacetic acid (TCA) was added to precipitate the proteins. The pellet resulting from centrifugation was washed twice with 0.5 ml of 5% TCA, and the acid super natants were combined. This supernatant fraction was extracted 3 times with diethyl ether {to remove the TCA) and the ether was discarded; the aqueous phase was gently warmed to remove any traces of ether. Aliquots (vide infra) of the aqueous phase were taken, and sufficient distilled water was added to make 4 ml: I, II, VI 1.0 ml III 0.5 ml IV 0.3 ml V 0.5 ml NUC 0.5 ml MIC 1.0 ml CYT 1.0 ml Analysis for catecholamines was performed as described for perfusates. M.5.3 Measurement of protein in subcellular fractions of bovine adrenal medulla (Layne, 1957)______ The proteins in the subcellular fractions of bovine adrenal medulla were precipitated with a volume of 10% trichloroacetic acid (TCA) equal to the volume of each fraction (see M.S.2). The pellet was washed twice with 0.5 ml of 5% TCA, and the acid supernatants were combined 4 5 for the determination of catecholamines (M.5.2) and Ca (M.5.4) in each subcellular fraction. The precipitated proteins were dissolved in 1 ml of 1 N NaOH with gentle heating, and an aliquot (1 ml for fractions I, IV, V, VI; 48 0.5 ml for II, III, NUC, MIC and SUP— plus an additional 0.5 ml of 1 N NaOH to these fractions) was added to 4 ml of biuret reagent. The mixture was allowed to stand at room temperature for 30 minutes, at which time the optical density at 550 nm was determined in a Bausch and Lomb Spectronic 20 spectrophotometer. Bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) was used to construct a standard curve. 45 M.5.4 Determination of Ca in subcellular fractions of bovine medulla (O'Neill and Rahwan, 1975).______ The acid supernatants obtained following protein precipitation and ether extraction (M.5.2) of subcellular 45 fractions were analyzed for Ca content by adding a 0.5 ml aliquot to a counting cocktail consisting of 0.6% 2,5- diphenyloxazole (PPO) and 0.01% 1,4-bis [2-(5-phenyl- oxazolylj]benzene (POPOP) in a 1:1 mixture of toluene and 2-ethoxyethanol. Radioactivity in perfusates was determined by taking a 0.1 ml aliquot from each fraction, and counting it in the above cocktail. Counting efficiency for perfusates and subcellular fractions was found to be 9 7-10 4% by the internal standard method. Recovery of radioactivity was essentially 100%. 49 M.5 . 5 Measurement of mitochondrial succinic cytochrome c reductase in sucrose density gradient fractions of bovine adrenal medulla (Borowitz, 1969) ___ The reaction mixture for the determination of succinic cytochrome c reductase consisted of the following* Na phosphate buffer {0.33 M, pH 7.5) 1.00 ml NaCN solution (0.01 M) 0.05 ml Na succinate solution {0.4 M) 0.15 ml Cytochrome c (horse heart derived; Sigma Chemical Co., St. Louis, Mo., 0.001 M) 0.50 ml Subcellular fraction 0.10 ml Distilled water qs 3.00 ml The subcellular tissue fraction (taken prior to the precipitation of proteins) was added at time 0, and allowed to react for 5 minutes at room temperature. At the end of 5 minutes, the optical density at 550 nm was determined in a Bausch and Lomb Spectronic 20. The blank contained an additional 0.15 ml of distilled water rather than the Na succinate solution. The activity of the enzyme was determined using the following formula: (average change in O.D./min) x (volume in cuvette) cytochrome c^ molar extinction coefficient (e = 19.5) Results were expressed as nmoles cytochrome c reduced/ min/g protein. 50 M.5.6 Determination of ethanol in blood and chamber air (Lundquist, 1959)______ The basis of this assay is the following reaction: Alcohol + NAD NADH + acetaldehyde alcohol dehydrogenase The stoichiometric appearance of NADH (absorption maximum 340 nm) is monitored as an index of alcohol in the sample. Reagents: Buffer solution (pH 8.6) Na pyrophosphate 10.0 g Semicarbazide HCl 2.5 g Glycine 0.5 g NaOH (2 N) 10.0 ml Water qs 300.0 ml Yeast alcohol Dehydrogenase 10 mg/ml (Sigma Chemical Co., St. Louis, Mo.) (in water) 8-NAD 10 mg/ml (Sigma Chemical Co., St. Louis, Mo.) (in water) Procedure: A 3 ml aliquot of a freshly prepared mixture of buffer, NAD, and enzyme (300:10:3) was placed in a test tube. The alcohol sample was added, and the optical density at 340 nm was read in a spectrophotometer (Bausch and Lomb Spectronic 20) after 30-60 minutes. Blood samples were prepared by deproteinization in 9 volumes of 3.4% perchloric acid. A 25 yl aliquot of the deproteinized filtrate was added to the 3 ml reaction mixture. 51 Air samples were measured by stoppering the reaction mixture tube with a rubber diaphragm, and injecting 0.5 ml of chamber air into the head space of the sealed tube. Analysis and quantitation was performed by comparing the optical density obtained with the blood or air sample to that of known amounts of ethanol. M.5.7 Analysis of whole brain salsolinol (O'Neill and Rahwan, submitted for publication)______ The extraction method from tissue is essentially that of Maruyama and Takemori (1972) with some modifications. The procedure is illustrated in Figure 3. The brain tissue (1, 2 or 3 whole brains) was added to an all glass grinding vessel (size 22; Kontes Glass Co., Vineland, N.J.) containing 1 ml of 0.05 N oxalic acid saturated with NaCl, and 3.5 ml of 25% n-butanol in isopropanol (25 BI), and was homogenized. Standards (vide infra) were added at this point, and mixed by passing the pestle through the homogenate. The homogenate was transferred to a conical test tube, centrifuged at 3000 x g for 5 minutes in a clinical centrifuge, and a 3 ml aliquot of the upper solvent phase was removed. Three ml of hexane and 0.5 ml of 0.5 M sodium phosphate buffer (pH 6.5) were added to this aliquot and the mixture was shaken for 5 minutes, followed by a 5 minute centrifugation. A 0.5 ml aliquot of the aqueous phase was saturated with NaCl and re-extracted Figure 3. Flowsheet for extraction of tyramine, dopamine and salsolinol. See text for details. 53 BRAIN 1mI 0.05 N OXALIC ACID (sat’d NaCl) 3.5ml 25 Bt homogenize 3 0 0 0 x g 5 min LOWER 3ml UPPER PHASE PHASE 3ml HEXANE discard 0.5ml PHOSPHATE BUFFER (0.5 Mi pH 6.5) shake UPPER 0.5m l LOWER PHASE PHASE 4 NaCl discard 0.5ml 25-BI 0.5 ml LOWER UPPER PHASE PHASE discard evaporate DERIVATIZE Figure 3 54 with 0.5 ml of 25 BI. One-half ml of the upper solvent phase was transferred to another tube and evaporated to dryness under nitrogen; the residue was used for deriv- atization. Derivatization of the compounds was carried out using a modification of the procedure of Bigdeli and Collins (1975). Acetonitrile (0.2 ml) and 0*05 ml of heptafluoro- butyric anhydride (HFBA; Eastman Kodak Co., Rochester, N.Y.) were added to the nitrogen-dried residue in the tube, and allowed to react at room temperature for 30 minutes. The samples were evaporated to dryness under nitrogen, and dissolved in 1 ml of benzene. Immediately prior to assay, each sample was washed with 5 ml of a saturated solution of sodium borate (SSSB)(Sams and Malspeis, 1976) by shaking, followed by a brief centrifugation to separate the aqueous and organic phases. A portion (2-5 yl) of the upper benzene layer was injected into the chromatograph. A Hewlett-Packard model 5713A gas chromatograph with 6 3 a Ni electron capture detector was used for the analyses. A 6 foot glass column (2 mm inner diameter) packed with 3% OV-17 on 100/120 Gas Chrom Q (Applied Science Labor atories, State College, Pa.) was used. Temperatures were: injector 250° C, oven 150° C, detector 300° C. The mobile phase was argonimethane (95:5) at a flow rate of 20 ml/min. 55 The peak height ratio method (Arnold and Ford, 19 73) was used to estimate quantities of salsolinol. Tyramine (500 ng; Eastman Kodak Co., Rochester, N.Y.) was added to each homogenate as the internal standard; various amounts of authentic salsolinol (4-400 ng; Aldrich Chemical Co., Milwaukee, Wis.) were also added to the brain homogenates of control animals. A standard curve was constructed from the control animal data by plotting the following: quantity of relative peak height of salsolinol authentic ______vs. salsolinol relative peak height of tyramine sample Regression lines were fitted by computer analysis using the method of least squares. M.5.8 Analysis of salsolinol in brain parts Each mouse brain was dissected freehand to remove the brain stem and corpus striatum; a stereotaxic atlas of the mouse brain (Montemurro and Dukelow, 19 72) was used as a guideline for the dissection procedure. The tissue from 5 mice was pooled and extracted as described in M.5.7; in these experiments, however, the assays were conducted on an instrument with an 8 foot column, gas flow 33 ml/min. CHAPTER III RESULTS R.l Synthesis and release of tetrahydroisoquinoline alkaloids from the bovine adrenal medulla (Rahwan, O'Neill and Miller, 1974) .______ Although the secretory effect of most drugs on the adrenal medulla is abolished by the removal of calcium from the perfusion medium (Rahwan and Borowitz, 1973; Rubin, 19 70, 1974), some secretagogues are only partially dependent upon extracellular calcium while others are totally independent of the presence of this cation in the extracellular fluid (Rahwan et al., 19 73). Figure 4 illustrates the cumulative concentration-effeet relation ship of acetaldehyde on catecholamine secretion under normal (4 glands) and calcium-free (8 glands) perfusion conditions. Acetaldehyde evoked the release of catecholamines from the isolated perfused bovine adrenal medulla in the presence and in the absence of calcium in the perfusion fluid. Extracellular calcium had no bearing on the secretory response of the adrenal medulla to acetaldehyde at concentrations of 1 to 64 mM. In experiments performed with normal Locke’s solution containing 2.2 mM calcium (7 glands), where the glands were perfused for 60 minutes with 10 mM acetaldehyde and 56 57 Figure 4. Cumulative concentration-effect curves for acetaldehyde-induced catecholamine secretion from isolated bovine adrenal glands in presence and in absence of extracellular calcium. Values are cor rected for resting secretion. Each point is the mean ± S.E.M. of 4 glands <•--- # ) or 8 glands ( o O ) • CATECHOLAMINE RELEASE |jg/3m in 300 200 250 100 150 0 5 . M Ca mM 0.0 . M Ca mM 2.2 OA CONCENTRATIONMOLAR OFACETALDEHYDE Figure 4 Figure 2 59 stimulated with 32 mM acetaldehyde, stimulation resulted in the release of EPI, NE, and their respective TIQs (Fig. 5, lane B). The values (expressed as mean ± S.E.M.) for the standard catecholamines and TIQs were EPI 0.29 ± 0.02 (n=6), NE 0.34 ± 0.02 (n=6), EPI-TIQ 0.20 ± 0.01 (n=7) and NE-TIQ 0.32 ± 0.02 (n=7). A rough estimate of the amounts of TIQs released from the glands upon stimulation can be obtained by comparing the color densities of the perfusate spots with those of a serial dilution of standards (Greenberg and Cohen, 1973). The average amount of EPI-TIQ or NE-TIQ in a 4 yl aliquot of purified perfusate (from 1 ml total volume) was approxi mately 480 ng, and the amount released was therefore calculated to be about 120 yg of each. This value is only slightly lower than that reported by Greenberg and Cohen (1973) for acetylcholine-induced TIQ secretion from bovine adrenals in similarly designed experiments. When the same experiments were repeated under calcium- free perfusion conditions, EPI and NE were released during perfusion with 10 mM acetaldehyde (4 glands) and after stimulation with 32 mM acetaldehyde (7 glands), whereas no TIQs were released in detectable amounts throughout these experiments (Fig. 5, lanes C and D). These findings indicate that omission of calcium from the perfusion medium may inhibit either the synthesis or the release of EPI-TIQ and NE-TIQ. 60 Figure 5. Thin-layer chromatograms of catechol compounds released from bovine adrenal medullae. Lane A, catecholamine and TIQ standards. Lane B, gland perfused with 10 mM acetaldehyde and stimulated by 32 mM acetaldehyde in the presence of calcium. Lane C, gland perfused with 10 mM acetaldehyde and, Lane D, stimulated with 32 mM acetaldehyde, in absence of calcium. Lanes E-G, glands perfused with 10 mM acetaldehyde in absence of calcium and stimulated by 1.6 mM carbachol in presence of calcium. POA, point of application. SF, solvent front. This figure illustrates the capricious nature of the assay system (see text) in the relative inadequacy of separation of NE and NE-TIQ (lane G) and the occasional loss of NE-TIQ on the chromatogram (lane E). 61 SF NE NE-TIQ EPI EPI-TIQ O — POA Figure 5 62 Figure 5, lanes E , F and G represent the results of three of four experiments in which the adrenal medullae were perfused with 10 mM acetaldehyde under calcium-free conditions and stimulated with carbachol (1.6 mM) following reintroduction of calcium into the medium. Carbachol stimulation resulted in the release of EPI and NE, and one or both of their respective TIQ derivatives. These experiments indicate that the TIQs are indeed synthesized during perfusion with 10 mM acetaldehyde under calcium- free conditions. R. 2 Role of intracellular calcium in acetaldehyde- induced catecholamine release from the adrenal medulla (O'Neill and Rahwan, 19 75)______ R.2.1 Concentration-effect relationships. The cumulative concentration-effeet relationships of acetaldehyde on catecholamine secretion from the isolated bovine adrenal medulla under normal (2.2 mM) and calcium- free perfusion conditions are shown in Fig. 6. Acetalde hyde evoked the release of catecholamines in a dose-related fashion in the presence and in the absence of extracellular calcium, the magnitude of the responses under both conditions being the same. These results confirm the findings presented in section R.I., and indicate a lack of involvement of the extracellular calcium pool in the secretory action of acetaldehyde at concentrations of 1 to 64 mM. 63 Figure 6. Cumulative concentration-effeet curves for acetaldehyde-evoked catecholamine secretion from the isolated bovine adrenal medulla. Medullae were per fused (5 ml/min) with Locke's solution (containing 2.2 mM calcium) or calcium-free Locke's solution for 30 minutes prior to and during continuous infusion of increasing concentrations of acetaldehyde for 3 minute _ 3 periods. In some experiments, magnesium (5 x 10 M) was added to the calcium-free perfusion medium 10 min utes prior to and during acetaldehyde perfusion. Values are corrected for resting secretion determined immediately prior to acetaldehyde stimulation. Each point is the mean + S.E.M. (•— # , 5 glands; O O t 4 glands; A—A / 2 glands). The absence of calcium from, or the addition of magnesium to, the perfusion medium did not alter acetaldehyde-evoked catecholamine secretion. Resting secretion of catecholamines averaged 51 yg/3 min. CATECHOLAMINE RELEASE M 9/3m in 600 500 400 200 300 100 .0 1 0 — 0 * mM Ca 0*0 * m Ca+5x10”3MMg C mM0*0 * m Ca mM2*2 CTLEYE CONCENTRATIONACETALDEHYDE (mM) iue 6 Figure 10 100 65 R.2.2 Effect of magnesium. -3 Addition of magnesium (5 x 10 M, which blocks the action of intracellular calcium) to the calcium-free perfusion medium did not diminish the secretory response of the adrenal medulla to acetaldehyde (Fig. 6)/ suggesting a lack of involvement of intracellular calcium in acetaldehyde-evoked catecholamine secretion (see DISCUSSION). R.2.3 Release of catecholamines. To confirm that mobilization of intracellular calcium pools is not involved in the secretory effect of acetal dehyde on the adrenal gland, these intracellular pools were 45 labeled with Ca prior to acetaldehyde stimulation (see M.2) in order to facilitate the detection of small alterations in subcellular calcium distribution during stimulation (Rahwan et al., 1973). Figure 7 represents the release of catecholamines during stimulation with 23 mM acetaldehyde. Catecholamine release at every time interval during the 10-minute stimulation period was significantly greater than resting secretion (Pc.001 or better by Student's t^ test). The mean (± S.E.M.) catecholamine content of the adrenal medullae used in this study was 26,357 ± 970 pg. 66 Figure 7. Catecholamine secretion from the isolated bovine adrenal medulla evoked by acetaldehyde. Adrenal medullae were perfused (5 ml/min) with calcium-free Locke's solution for 20 minutes, followed by a 2- 4 5 minute infusion of Ca (1 yc/ml), and an additional 30 minute washout with calcium-free medium. The glands were then stimulated with 2 3 mM acetaldehyde for 10 minutes in calcium-free medium. Catecholamine 4 5 secretion xs shown in this figure, and Ca release is presented in Fig. 8. Each point is the mean ± S.E.M. of six glands. Acetaldehyde-evoked catechola mine secretion was significantly greater than resting secretion at all points (Pc.001 or better). CATECHOLAMINE RELEASE pg/2min 200 300 220 2 280 18 0 240 100 160 120 140 20 40 80 60 60 Control Acetaldehyde 2 IUE OF MINUTES iue 7 Figure 4 mulati n io t a l u im t s 6 8 10 68 R.2.4 Release of 45 C a . 45Ca released from the adrenal medullae during stimulation with 2 3 mM acetaldehyde is shown in Fig. 8 . 45 The Ca released from the stimulated glands was not significantly different from non-stimulated control glands at all time intervals (P>.05 to .5 by Student's t test), nor was the total amount of 45 Ca released during the 10-minute perfusion with acetaldehyde different from the spontaneous release of radio-calcium from the control glands (P = .1 by Student's t test). However, since the washout curves from the stimulated and control glands diverged slightly from each other, a more valid comparison 45 is that between the actual Ca washout during acetaldehyde stimulation and the extrapolated washout curve from the point just prior to acetaldehyde stimulation (interrupted line in Fig. 8 ). In this instance, it becomes evident that 4 5 no significant release of Ca occurs during acetaldehyde stimulation. Furthermore, if the prestimulation value of 45 Ca (the 30-minute time interval on the acetaldehyde curve of Fig. 8) is compared with each subsequent time interval 45 on the same curve, no significant increase in Ca release induced by acetaldehyde is observed (P>.3 to .5 by Student's 45 t test). The total adrenal medullary Ca content immediately prior to acetaldehyde stimulation was calcu lated to be 1,160,799 ± 247,820 cpm (mean ± S.E.M. of the 69 4 5 Figure 8 . Effect of acetaldehyde on Ca washout from the isolated bovine adrenal medulla. Adrenal medullae 45 were perfused, labeled with Ca, and stimulated with 23 mM acetaldehyde as described in the legend to Fig. 7. Each point is the mean ± S.E.M. of six glands. Acetaldehyde did not significantly alter (P>.05 to .5) 45 45 Ca washout when the actual Ca washout during stimulation (•—• ) was compared with either the extrapolated washout curve (interrupted line) or the 45 Ca washout from nonstimulated glands (O— O )• 95 90 • Ace+atdehyd 85 o Control 80 75 70 65 60 55 50 4 5 40 35 30 25 20 Sti mulation 15 10 5 8 16 24 32 4 0 TIME (mins) Figure 8 71 six glands which were subsequently stimulated with acetaldehyde). Of this amount, 391,831 cpm (or 33.76% of the glandular content) were lost during the 10-minute stimulation with acetaldehyde, of which 340,700 cpm (or 29.35% of the glandular content) represent the resting 45 release of Ca (calculated from the extrapolated curve in Fig. 8 ). Thus, acetaldehyde caused a net loss from the 45 medulla of 51,131 cpm (or 4.41% of the prestimulation Ca content of the glands). The possibility that this small 45 amount of Ca released during acetaldehyde stimulation may represent mobilized intracellular calcium which participates in pharmacomechanical stimulus-secretion coupling is ruled out by the finding that magnesium does not block the secretory effect of acetaldehyde (Fig. 6 ). 4 5 Therefore, it is likely that the Ca released from the gland under the influence of acetaldehyde corresponds to the amount of calcium which enters into the structure of the chromaffin granule complex (Borowitz, 19 70) and which is released along with the catecholamines during evoked secretion. R.2.5 Subcellular distribution of mitochondria and chromaffin granules.______ Table 2 and Fig. 9 show the effectiveness of separa tion of mitochondria from chromaffin granules on the discontinuous sucrose density gradient. In both the control and stimulated glands, mitochondria were mostly 72 Table 2 Enzyme distribution In sucrose density gradient fractions of bovine adrenal medulla Adrenal medullae were perfused (5 ml/min) for 20 minutes with calcium-free Locke's solution, followed by a 2-minute infusion of Ca (1 pc/ml) and a further 40-minute perfusion with calcium-free medium. Acetaldehyde (23 mM) was added during the last 10 minutes of perfusion. The medullae were homogenized, and subcellular fractions separated by differential and discontinuous density gradient centrifugation. Six fractions were separated on the gradient and assayed for succinic cytochrome c_ reductase activity. Each value is the mean ± S.E.M. of six glands. No significant differences in cytochrome jc reductase activity were observed between stimulated and nonstimulated glands. prac Succinic Cytochrome c^ Reductase (Mean ± S.E.M.) tion Acetaldehyde (23 mM) Control Umol cytochrome c reduced/min/g protein I 0.00 + 0.00 0.00 + 0.00 II 8.03 + 1.10 9.50 + 1.01 III 5.35 + 1.67 4.39 + 1.06 IV 0.49 ± 0.49 1.34 + 0.85 V 0.00 + 0.00 0.00 + 0.00 VI 0.27 + 0.27 0.36 + 0.26 73 Figure 9. Effect of acetaldehyde on subcellular distribution of catecholamines in the isolated bovine adrenal medulla. Adrenal medullae were perfused, 4 5 labeled with Ca, and stimulated with 23 mM acetal dehyde as described in the legend to Fig. 7. The medullae were then homogenized, and subcellular fractions were separated by differential and discontinuous density gradient centrifugation of an aliquot of homogenate containing 2 g of medullary tissue. Fraction I contains soluble constituents of disrupted organelles. Fractions II and III contain primarily mitochondria. Fractions IV, V and VI con tain primarily chromaffin granules. NUC, nuclei and cell debris; MIC, microsomes; CYT, cytosol. Each bar is the mean ± S.E.M. of six glands. There was significant (P<.02) mobilization of catecholamines from fraction VI of acetaldehyde-stimulated glands, which was accompanied by an average 39.4% increase in cytosolic catecholamine content in five out of the six glands. 240 J| | Acetaldehyde 220 □ Control z200 Ll J 0 180 cc £L ?160 E ?140 V) ^ 120 1 5 ioo o a 8 0 3 « 40 20 VI NUC MIC CYT FRACTION F ig u re 9 localized in the upper fractions (II and III) of the gradient as reflected by the succinic cytochrome c reductase activity (Table 2) , while the chromaffin granules were distributed in the lower fractions of the gradient (Fig. 9). Fraction II of the gradient contained almost exclusively mitochondria (Table 2) with little contamina tion by chromaffin granules (Fig. 9), whereas fractions IV, V and VI contained almost exclusively chromaffin granules (Fig. 9) with little contamination by mitochondria (Table 2). Fraction I at the top of the density gradient contains soluble material released from organelles damaged during density gradient centrifugation. Examination of the distribution of catecholamines in the subcellular fractions obtained from the adrenal medullae stimulated with 23 mM acetaldehyde and from non stimulated control glands (Fig. 9) reveals no significant alteration in the distribution of catecholamines in all but fraction VI, which contains epinephrine and norepinephrine granules (Borowitz, 1969). Moreover, the significant decrease (P<.02 by Student's t test) in catecholamine content of fraction VI of the stimulated glands was accompanied by an average 39.4% (range 2 3.6-7 8.0) increase in catecholamine content of the cytosolic fraction of these glands in five out of six experiments, with no change in the cytosolic fraction in one experiment. This discrete 76 translocation of catecholamines from a chromaffin granule fraction to the cytosol is indicative of a lack of structural damage to chromaffin granules induced by acetaldehyde, and also supports previous findings that acetaldehyde releases granule-bound catecholamines into the cytoplasm prior to their secretion from the adrenal medulla (Schneider, 1971, 1974b). 45 R.2.6 Subcellular distribution of Ca. 4 5 Whereas Ca labeling of whole (undecorticated) adrenal glands perfused retrogradely results in equal retention of radio-calcium within different glands during labeling and subsequent washout of radioactivity (Rahwan 45 et al., 19 73), the retention of Ca by decorticated adrenal medullae is unequal due to unequal loss of radio activity from the numerous exposed blood vessel apertures during the 2-minute labeling and subsequent washout. Thus, 45 for the purpose of calculating the Ca content of the subcellular fractions of stimulated and nonstimulated glands, it was necessary to normalize the data by expressing the values as the following ratio (Borowitz, 1969): 45 45 Ca in each fraction/total Ca in whole homogenate protein in each fraction/total protein in whole homogenate 45 Table 3 represents the actual Ca counts per minute and milligrams of protein in each subcellular fraction of one Table 3 45 Effect of acetaldehyde on subcellular distribution of Ca In isolated bovine adrenal medulla 45 Adrenal medullae were perfused, labeled with Ca, and stimulated with 23 mM acetaldehyde as described in the legend to figure 7, and subcellular fractions separated as described in the legend to figure 9. The data are shown for a representative experiment, and outlines the method for calculating the ratios presented in figure 10, FRACTION I II III IV V VI NUC MIC CYT Acetaldehyde-stimulated gland Protein (mg) in fraction 4.410 20.700 11.880 11.640 5.940 10.860 82.800 30.000 31.490 X of total protein in homogenate 2.103 9.870 5.664 5.550 2.832 5.178 39.481 14.305 15.015 ^ C a (CPM) in fraction 22,878 52,625 34,623 25,517 7,148 7,276 79,457 16,085 14,033 Z of total ^ C a in homogenate 8.440 19.410 12.770 9.410 2.640 2.680 29.310 5.930 5.180 Z total ^Ca/% total protein 4.013 1.967 2.255 1.695 0.932 0.518 0.742 0.415 0.345 Control gland Protein (mg) in fraction 2.580 21.720 13.980 10.350 5.670 12.930 84.960 38.280 38.450 X of total protein in homogenate 1.127 9.488 6.107 4.521 2.477 5.648 37.113 16.722 16.796 ^-*Ca (CPM) in fraction 15,817 72,952 63,781 25,564 6,439 10,431 101,287 34,153 22,664 1 of total ^5Ca in homogenate 4.480 20.661 18.064 7.240 1.B24 2.954 28.686 9.673 6.419 Z total ^Ca/% total protein 3.975 2.178 2.958 1.601 0.736 0.523 0.773 0,578 0.382 Total protein in homogenate (equivalent to 2 gm of medullary tissue): Acetaldehyde-stimulated gland *■ 209.72 mg; Control gland ■ 228.92 mg. Total ^ C a in homogenate (equivalent to 2 gm of medullary tissue): Acetaldehyde-stimulated gland * 271,092 CPM* ; Control gland ■ 353,088 CPM (* Corrected for acetaldehyde-induced ^ C a loss) 78 representative experiment. Fig 10 represents the mean normalized data of six experiments calculated as shown in Table 3. Examination of Fig. 10 reveals that stimulation of the adrenal medulla with 23 mM acetaldehyde did not 4 5 result in Ca mobilization from any of the intracellular 45 pools, since comparison of Ca content of subcellular fractions from stimulated and nonstimulated medullae showed no significant differences (P>.1 to .5 by Student's t test, with comparable values by the Mann-Whitney U test). This, 45 coupled with the absence of significant release of Ca from the glands during evoked secretion (Fig. 8) and the lack of inhibitory effect of magnesium on catecholamine secretion (Fig. 6 ), indicates that intracellular calcium is neither mobilized nor translocated during acetaldehyde- 45 induced catecholamine secretion. [The elevated Ca/protein ratios in fraction I (Fig. 10) at the top of the density gradient of stimulated and non-stimulated glands is a reflection of the very low protein content of this fraction.] R.3 Protection against acute toxicity of acetaldehyde in mice (O'Neill and Rahwan, 1976)______ The results of the experiments are summarized in Table 4 and are compared with previously published data on rats (Sprince et al^, 1974, 1975). Orally administered cysteine, ascorbic acid, or thioctic acid (for doses, see M.3) resulted in a significant [P<.05 by the chi-square 79 Figure 10. Effect of acetaldehyde on subcellular 45 distribution of Ca in the isolated bovine adrenal medulla. Adrenal medullae were perfused, labeled with 45 Ca, and stimulated with 2 3 mM acetaldehyde as des cribed in the legend to Fig. 7, and subcellular fractions separated as described in the legend to Fig. 9. Calculation of ratios is as described in Table 3. Each bar is the mean ± S.E.M. of six glands. 4 5 There was no significant mobilization of Ca by acetaldehyde from any subcellular fraction (P>.1 for fractions I and III; P>.2 for fraction II; P>.4 for MIC; P > .5 for fractions IV, V, VI, NUC and CYT). 9fo TOTAL 45Ca / % TOTAL PROTEIN 2 3 4 Ill o Figure10 IV n o i t c a r f v Control □ Acetaldehyde ■ VI NUC I CYT MIC o 03 81 Table 4 Effect of Protecting Agents on Acute Toxicity of Acetaldehyde in Mice and Rats MICE - RATS — % Loss of % Loss of Pretreatment — Number righting % Number righting % of mice reflex Death of rats reflex Death None 30 92 0 50 96 90 L-cysteine 47 63d 0 25 20 20 L-ascorbate 28 69d 0 15 53 27 DL-thioctic acid 48 70d 0 10 60 70 DL-homocysteine 36 79 0 10 60 60 — Mice were pretreated orally with the protecting agent 30 minutes prior to intraperitoneal administration of the ED90 (for loss of righting reflex) of acetaldehyde (415 mg/kg). — Data from Sprince et al. (1974, 1975). Rats (male albino, Carworth Farms, CFE, 365± 20 g) were pretreated orally with the protecting agent 30-4 5 minutes prior to oral administration of the 24-hour LD90 of acetaldehyde (79 3 mg/kg). — Doses of protecting agents were: L-cysteine, 3 mMoles/kg for mice and 2 mMoles/kg for rats; L-ascorbate, DL-thioctic acid, and DL-homocysteine, 2 mMoles/kg for both species. — Significantly different from control (P<.05). i i test (Schor, 1968)] protection against loss of righting reflex induced by the i.p. administration of the ED9 0 (415 mg/kg) of acetaldehyde. Conversely, the protection afforded by homocysteine was not statistically significant (P>.05). In an effort to increase the protectant actions of cysteine, for example, higher doses and repeated administration were attempted; these approaches did not enhance the protection against acetaldehyde, but in fact resulted in obvious signs of toxicity (ptosis and sedation) from cysteine. Likewise, higher doses or multiple admin istration of the other protectants did not enhance their protective properties against acetaldehyde-induced loss of righting reflex. These results differ from those of Sprince et al^ (1974, 1975) in rats. There are several possible reasons for this discrepancy other than the obvious one of species difference; these are discussed in detail in the DISCUSSION section. The results, however, indicated that these compounds are not very effective as protectants in this model system, and probably have limited value in the amelioration of the symptoms of acetaldehyde toxicity. 83 R.4 Absence of formation of brain salsolinol in ethanol- dependent mice (O'Neill and Rahwan, submitted; Rahwan and O'Neill, 1976)______ R.4.1 Chamber alcohol/blood alcohol relationship. Preliminary experiments with groups of 20-25 mice were conducted to determine the conditions necessary to estab lish and maintain intoxicating levels of ethanol in the blood of mice.[Ortiz et al. (1974) have shown that brain alcohol levels are approximately equal to blood alcohol; therefore, brain ethanol concentrations were not determined.] A concentration of approximately 14 mg ethanoi/L of inspired air was selected as the starting point on day 1 . Figures 11, 12, and 13 represent three typical sets of blood/air alcohol for a 6 day period. When mice are exposed to alcohol inhalation, some variability in blood alcohol level to a given concentration of ethanol in the inspired air is encountered (see Fig. 11, 12, and 13; Goldstein, 1972). This problem may be minimized by the administration of daily doses of pyrazole, a compound shown to inhibit alcohol dehydrogenase in vitro (Theorell, 1969; Reynier, 1969) and to retard the elimination of ethanol in vivo (Goldberg and Rydberg, 1969). However, the use of pyrazole is controversial (see Rahwan, 1975); furthermore, it exerts additive central nervous system depressant effects with ethanol (LeBlanc and Kalant, 1973), induces weight loss and hypothermia (Littleton et al ., 1974), and 84 Figure 11. Relationship between alcohol concentration in. the inspired air and blood ethanol levels. Male Swiss-Webster mice were housed in a plexiglas chamber (52 x 34 x 27 cm) and exposed to increasing concentra tions of ethanol vapors. Concentration of ethanol in the inspired air (A— ▲ ) represents the average of two determinations daily from 0.5 ml samples of the chamber air. Blood alcohol levels (O— O) are the average of 3-4 randomly selected mice (shown individu ally by the dots). On the final day (6 ) all animals were assayed for blood alcohol. 6 5 20 Blood 4 Inspired Air Alcohol (m g/L) (m g/m l) 3 0 0 2 1 2 3 4 5 6 Day Figure 11 oq U1 86 Figure 12. Relationship between alcohol concentration in the inspired air and blood ethanol levels. See legend to Fig. 11. 24 Blood <16 Alcohol In Alcohol Inspired Air (mg /m l) 12 (mg/L) 0 0 4 1 2 3 4 Day Figure 12 88 Figure 13. Relationship between alcohol concentration in the inspired air and blood ethanol levels. See legend to Fig. 11. 6 24 5 20 16 Blood ^ Alcohol In Alcohol Inspired Air (m g/m l) 3 12 (mg/L) 0— 0 2 8 1 4 I J. 3 4 Doy Figure 13 00 VO 90 reduces brain norepinephrine levels {MacDonald et al., 1975). For these reasons, it was avoided in these studies. Blood alcohol levels ranged from approximately 1 mg/ml on day 1 (Fig. 11, 12 and 13) up to an average of as high as 5 mg/ml or more on day 6 (Table 6 ); some individual animals had blood ethanol levels approaching 7 mg/ml (these mice were, of course, comatose and very near death at that time). Mortality in these experiments was in excess of 50% for most groups; similar rates were reported by Goldstein (1972) and Ortiz et a^L. (1974). R.4.2 Establishment of physical dependence. In order to determine that the conditions employed were indeed sufficient to induce physical dependence on ethanol (and therefore, by definition, withdrawal), groups of mice were treated as described (M.4), and at the termination of the experiment evaluated for withdrawal convulsions on handling as described by Goldstein (1972). Figure 14 represents the mean withdrawal score (evaluated at 30-60 minute intervals) of 29 surviving mice following inhalation of ethanol vapors reaching 16-18 mg/L on day 5; the data for seizures in naive control mice are also shown for comparison, since this minimal convulsion is not a sign exclusive to the ethanol withdrawal syndrome, but can be occasionally observed in untreated mice (Goidstein, 1972; Littleton et al., 1974). One of the naive control 91 Figure 14. Mean withdrawal score of mice following 5 days of continuous inhalation of ethanol. Maximum chamber alcohol concentration reached was 18 mg/L. The score of the treated mice (•—•} is the mean of 12-29 mice; the control (0— 0) is the mean of 5 naive mice. The withdrawal scores of alcohol—treated mice are significantly higher (P<.05 by the median test) than controls between 2 and 4 hours after withholding ethanol. S.E.M. is presented as a measure of vari ation in seizure susceptibility. In experiments using comparable air ethanol concentrations, the blood alcohol level on day 5 was approximately 2.0-2.5 mg/ml. For details of the withdrawal scoring method, see text. 0 — ^ Alcohol Treated O """ O Control MEAN WITHDRAWAL SCORE TIME (hr) 10 to Figure 14 93 mice consistently gave a score of 1, which therefore accounts for the common score of 0.2 in this group. Seiz ures were observed in every alcohol-treated mouse. The time of maximum withdrawal score in non-pyrazole treated mice has been shown to occur at approximately 3-4 hours (Fig. 14; Littleton ejt ajL. , 1974). It should be noted that this is approximately 5 hours sooner than the time of peak withdrawal score in pyrazole treated animals after 6 days exposure to ethanol (Goldstein, 1972); similarly, pyrazole treated mice exhibit a higher peak in their withdrawal score time-course plot than non-pyrazole mice (Goldstein, 1972; Littleton et aJL. , 1974) . The scores presented here are in agreement with the findings of Littleton et al. (1974) in non-pyrazole treated mice. R.4.3 Analysis of single whole brains for salsolinol. Having determined that these conditions were adequate for the induction of physical dependence on alcohol, the analysis of brain tissue for salsolinol, the presumed in vivo biosynthetic product of endogenous dopamine and alcohol-derived acetaldehyde, was undertaken. Samples prepared from the brains of stress control mice with exogenously added salsolinol (see M.5.7) were extracted and analyzed in parallel with those from the brains of the alcohol-treated mice. Figures 15, 16, 17 and 18 represent the chromatograms obtained from the control samples; these figures represent single brains with 0, 40, 10 and 4 ng salsolinol added, respectively. Peaks are identified as 94 Figure 15. Chromatogram of a single control mouse brain without added salsolinol. Tyramine (TYR; 500 ng) was added to the homogenate prior to extraction. A 5 yl aliquot of the derivatized extract was injected at attenuation 512; following the appearance of the tyramine peak, the attenuation was changed to 256. The large arrow indicates the retention time of salsolinol (where it would have appeared had it been added to the homogenate). For details of the extraction and assay conditions, see text. iue 15 Figure DETECTOR RESPONSE 80 60 20 40 IE mn } (min TIME NE TYR DA 96 Figure 16. Chromatogram of a single control mouse brain with added salsolinol. Tyramine (TYR? 500 ng) and 40 ng salsolinol (SAL) were added to the homogenate prior to extraction. A 5 pi aliquot of the derivatized extract was injected at attenuation 512; following the appear ance of the tyramine peak, the attenuation was changed to 256. For details of the extraction and assay conditions, see text. 97 ao — TYR 60 — DA ui VO z O CL %/i Ot— U 40 — 20 — NE SAL ! 2 6 Figure 16 TIME (min) Figure 17. Chromatogram of a single control mouse brain with added salsolinol. Tyramine (TYR; 500 ng) and 10 ng salsolinol (SAL) were added to the homogen ate prior to extraction. A 5 yl aliquot of the derivatized extract was injected at attenuation 512; following the appearance of the tyramine peak, the attenuation was changed to 256. For details of the extraction and assay conditions, see text. 99 80 — TYR DA 60 — in oZ CL.m — LU oe tc O»— u “ 40---- 20 — SAL t 1 I 2 4 Figure 17 TIM E (mln) 100 Figure 18. Chromatogram ,of a single control mouse brain with added salsolinol. Tyramine (TYR; 500 ng) and 4 ng salsolinol (SAL) were added to the homogenate prior to extraction. A 5 pi aliquot of the derivatized extract was injected at attenuation 512; following the appearance of the tyramine peak, the attenuation was changed to 256. For details of the extraction and assay conditions, see text. 101 TYR 80 — 60 — in v t oZ a. DA co in ae oe O Ulu ul►- o 40 — 20 — NE SAL 1 l I t 2 4 6 Figure 18 TIME (min) 102 follows: TYR (tyramine), DA (dopamine), SAL (salsolinol); norepinephrine (NE) is neither well extracted by this procedure, nor is it as sensitive to detection by this method. Identity of the peaks was confirmed either by comparison of retention times of derivatized authentic standards, or by the addition of derivatized authentic standards to the injection solution. Recoveries and retention times for the compounds are shown in Table 5. Regression analysis indicated that this assay is linear (P<.05) over the tested range of 4-400 ng added salsolinol. Table 6 shows the results of the studies utilizing single whole brains of alcohol-exposed mice. Despite the high levels of ethanol in the blood of these mice (in some instances approaching 7 mg/ml at the time of sacrifice), there was no evidence of salsolinol formation in any of the samples. Figures 19 and 20 represent typical chromato grams from test mice; the blood ethanol level at sacrifice in these mice was 4.39 mg/ml and 5.40 mg/ml in Fig. 19 and 20 respectively. As noted previously (vide supra), the GC assay for salsolinol is sensitive to a level of 5-10 ng/g of brain tissue, and therefore can detect a conversion of as little as 1% of the endogenous dopamine pool (Maruyama and Takemori, 1972). It therefore appeared that under the most drastic of conditions (comatose, near death) of alcohol intoxication, detectable quantities of salsolinol were not being synthesized in vivo. 10 3 Table 5 Retention times and recoveries for tyramine, dopamine and salsolinol. Compound Retention Recovery3 time (min) (% ± S.E.M.) Tyramine 2.6 38.8 ± 1.13 Dopamine 3.5 68.5 ± 5.07 b Salsolinol 4.8 62.3 ± 2.14 a Mean of 6-16 determinations b Dopamine recovery is estimated on the basis of an average whole brain content of 3 50 ng (Maruyama and Takemori, 19 7 2). 104 Table 6 Summary of chronic alcohol administration studies (single brain experiments). Mice were housed for 6 days in an inhalation chamber, and exposed to ethanol vapors in con centrations from 14 mg/L (day 1) to 20-30 mg/1 (day 5-6). They were then sacrificed, and their brains analyzed individually for the presence of salsolinol by electron capture gas chromatography. Experiment na Mean blood Whole brain number ethanol level salsolinol at sacrifice (ng/g)b (mg/ml ± S.E.M.) 1 8 2.31 ± 0 . 35 none detectable 2 2 5. 54° none detectable 3 8 4.21 ± 0.59 none detectable 4 8 5.14 ± 0.45 none detectable a Twenty to 25 mice were used at the beginning of each experiment. The number under "n" does not represent the total number of surviving mice in every case, but only the number actually used for the salsolinol assay. b Level of sensitivity of the assay for salsolinol is 5-10 ng/g of brain tissue, or approximately 1% of the endogenous dopamine (Maruyama and Takemori, 1972) from which salsolinol would be derived. c The individual blood alcohol levels in this experiment were 5.50 mg/ml and 5.57 mg/ml. 105 Figure 19. Chromatogram of a single experimental mouse brain. Tyramine (TYR; 500 ng) was added to the homogenate prior to extraction. A 5 pi aliquot of the derivatized extract was injected at attenuation 512; the attenuation was changed to 256 following the appearance of the tyramine peak. The large arrow indicates the retention time of salsolinol (where it would have appeared had there been measurable quantities in the sample). The blood alcohol level at sacrifice in this mouse was 4.39 mg/ml. For details of the extraction and assay conditions, see text. 106 80 — TYR DA 60 — m z O o. in oc O►- O ui U l 40 Q 20 — I 2 4 Figure 19 TIME (mln) 107 Figure 20. Chromatogram of a single experimental mouse brain. Tyramine {TYR; 500 ng) was added to the homogenate prior to the extraction. A 5 yl aliquot of the derivatized extract was injected at attenuation 512; following the appearance of the tyramine peak, the attenuation was changed to 256. The large arrow indicates the retention time of salsolinol (where it would have appeared, had there been measurable quantities in the sample). The blood alcohol level at sacrifice in this mouse was 5.40 mg/ml. For details of the extraction and assay conditions, see text. 10 8 80 — TYR DA 60 — UJ VI Z O Q. I/I DC O UJ 40 Q 20 — 1 I 2 4 Figure 20 TIME (tnln) 109 R.4.4 Analysis of pooled whole brains for salsolinol. In an attempt to improve the chances for detecting salsolinol in brain tissue, a study was undertaken in which whole brains were pooled for the salsolinol analyses. In two samples in which 2 whole brains from intoxicated mice were extracted and assayed together, no evidence for salsolinol was seen (Fig. 21). Similar negative results were obtained when 3 whole brains were pooled for analysis (for a sample chromatogram, see Fig. 22). R.4,5 Analysis of pooled brain parts for salsolinol. Finally, brain parts were pooled from 5 mice as described in M.5.8. The corpus striatum and brain stem were separated from the rest of the brain and analyzed for a the presence of salsolinol by electron capture gas chromatography. Fig. 23 is a representative chromatogram from one of three such studies. No evidence for salsolinol formation in these combined brain areas was seen in any of the samples. 110 Figure 21. Chromatogram of 2 pooled whole brains from experimental mice. Tyramine (TYR; 500 ng) was added to the homogenate prior to extraction. A 5 pi aliquot of the derivatized extract was injected at attenuation 512; following the appearance of the dopamine (DA) peak, the attenuation was changed to 256. The large arrow indicates the retention time of salsolinol (where it would have appeared had there been measur able quantities in the sample). Blood alcohol levels in these mice at the time of sacrifice were 2.20 mg/ml and 4.40 mg/ml. For details of the extraction and assay conditions, see text. Figure 21 21 Figure DETECTOR RESPONSE 80 40 60 20 IE mln) (m TIME TYR DA 112 Figure 22. Chromatogram of 3 pooled whole brains from experimental mice. Tyramine (TYR; 500 ng) was added to the homogenate prior to extraction. A 5 yl aliquot of the derivatized extract was injected at attenuation 512; the attenuation was changed to 256 following the appearance of the dopamine (DA) peak. The large arrow indicates the retention time of salsolinol (where it would have appeared, had there been measurable quantities in the sample). Blood alcohol levels in these mice at the time of sacrifice were 1.72 mg/ml, 2.78 mg/ml and 4.78 mg/ml. For details of the extraction and assay conditions, see text. 113 TYR 80 — 114 Figure 23. Chromatogram of the pooled brain stem and corpus striatum of 5 experimental mice. Tyramine (TYR; 500 ng) was added to the homogenate prior to extraction. A 5 yl aliquot of the derivatized extract was injected at attenuation at 1024, and left unchanged. The large arrow indicates the retention time of salsolinol (where it would have appeared, had there been measur able quantities in the sample). Blood alcohol levels in these mice at the time of sacrifice were 2.28 mg/ml, 2.64 mg/ml, 3.24 mg/ml, 3.80 mg/ml and 5.72 mg/ml. For details of the extraction and assay conditions, see t ext . DA 80 TYR 60 ui 1 /1 z O a. UJ ec oc *-O (J I—U1 UJ 40 O 20 Figure 23 TIME (m in) CHAPTER IV DISCUSSION D.l Synthesis and release of tetrahydroisoquinoline alkaloids from the bovine adrenal medulla (Rahwan, O'Neill and Miller, 1974).______ The bovine adrenal medulla has been previously used as a model of a catecholamine storage site, and for studying the synthesis and release of TIQs. Thus, EPI-TIQ and NE-TIQ can be formed by perfusion of bovine adrenal glands with concentrations of acetaldehyde comparable to those found in the blood of man (1 pg/ml) following alcohol consumption (Cohen, 19 71b). Furthermore, the TIQs formed in acetaldehyde-perfused glands are bound in the adrenal tissue within the chromaffin granules (Greenberg and Cohen, 1972), and are released together with the catecholamines upon stimulation of the adrenal medulla with acetylcholine or carbachol (Greenberg and Cohen, 1973) . Acetaldehyde- evoked catecholamine secretion does not take place by exocytosis or electromechanical coupling, since this aldehyde releases catecholamines from the perfused bovine adrenal medulla in the absence of extracellular calcium and without the simultaneous release of the soluble con stituents of the chromaffin granules (Schneider, 1971). 116 On the other hand, carbachol- or acetylcholine-induced release of preformed TIQs from the bovine adrenal medulla was reported to occur by exocytosis (Greenberg and Cohen, 1973), since omission of calcium from, or addition of tetracaine to, the perfusion medium abolished carbachol- induced TIQ secretion. However, since these conditions also abolished carbachol-induced catecholamine release (Greenberg and Cohen, 19 73), the use of carbachol, whose action is dependent upon the presence of extracellular calcium (Rubin, 1970)* does not provide conclusive evidence that the release mechanism of the TIQs essentially requires extracellular calcium (exocytosis), nor does it rule out the possibility that the release of catecholamines and their TIQ derivatives is coupled. The use of a secreta- gogue whose action does not necessarily require extra cellular calcium would therefore provide more conclusive information concerning the mechanism of release of the TIQs. Acetaldehyde, which was equally effective in releasing catecholamines in presence and in absence of extracellular calcium (Figs. 4 and 6), and whose action, therefore, does not require this particular calcium pool, was incapable of releasing detectable amounts of preformed TIQs from the adrenal medulla when calcium was omitted from the perfusion medium (Fig. 5, lanes C and D). This finding establishes a basic difference in the release mechanism of the catecholamines and their TIQ derivatives 118 based on extracellular calcium requirement, and provides evidence that the secretion of these substances can be uncoupled. Essentially, whereas the process of catechol amine release from the adrenal medulla can be achieved in presence or absence of extracellular calcium (Rahwan and Borowitz, 1973) depending upon the intrinsic properties (calcium requirement) of the secretagogue used (carbachol or acetaldehyde, respectively), the process of TIQ release appears to be quite different in that it requires the presence of extracellular calcium regardless of the secretagogue used (carbachol or acetaldehyde). D.2 Role of intracellular calcium in acetaldehyde- induced catecholamine release from the adrenal medulla (O'Neill and Rahwan, 1975).______ The purpose of these studies was to investigate the cellular mechanism of the catecholamine-releasing action of acetaldehyde. In the process, this aldehyde was demonstrated to be the first adrenomedullary secretagogue which is totally independent of calcium (extracellular or intracellular) for its effectiveness. It had been estab lished that acetaldehyde does not evoke catecholamine release via electromechanical coupling (Schneider, 1971), and Figures 4 and 6 confirm and extend these findings by demonstrating that the action of this aldehyde is indepen dent of calcium in a concentration range of 1-64 mM. Furthermore, whereas extracellular calcium may potentiate 119 the effect of certain secretagogues which are capable of evoking catecholamine release from the bovine adrenal medulla in a calcium-free environment {Rahwan et al., 1973), the secretory effect of acetaldehyde in concentrations ranging from 1 to 6 4 mM was found to be unaffected by the presence or absence of calcium in the perfusion medium (Fig. 6). Acetaldehyde does not evoke catecholamine secretion by physical disruption of cellular membranes, since a concentration of acetaldehyde as high as 100 mM does not cause a release of cytoplasmic enzymes from perfused bovine adrenals (Schneider, 1971), and a concen tration of 1 M acetaldehyde causes no release of soluble proteins from isolated bovine adrenomedullary chromaffin granules (Schneider, 1971). Furthermore, it appeared unlikely (although it was not established experimentally) that acetaldehyde evoked catecholamine secretion by pharmacomechanical mechanisms, since the work of Schneider (1971, 1974a,b) strongly suggests a direct interaction between the aldehyde and the chromaffin granule membrane which allows intragranular catecholamines to diffuse into the cytoplasm prior to release into the circulation. In the studies described in section R.2.1, acetalde hyde was used at a concentration of 23 mM. Previous studies on the mechanism of action of acetaldehyde on bovine adrenomedullary catecholamine secretion utilized 120 acetaldehyde concentrations ranging from 23 to 3 50 mM (Greenberg and Cohen, 1973; Schneider, 1971, 1974a,b). Although lower concentrations of acetaldehyde can release adrenal catecholamines (Cohen and Collins, 1970; Schneider, 1974a), prolonged periods of exposure of the glands to the aldehyde are required to achieve this effect (Schneider, 1974a), and justification of the use of the higher concen trations of acetaldehyde was discussed by Schneider (1974a). When the studies described in section R.2.1 demon strated the lack of involvement of the extracellular cal cium pool in acetaldehyde-evoked catecholamine secretion (vide supra), studies with magnesium (which blocks the action of intracellular calcium) were conducted (see R.2.2). The presence of magnesium in the calcium-free perfusion -3 medium, at a concentration (5 x 10 M) previously shown to block evoked catecholamine release significantly (Rahwan et al. , 1973; Rubin et a_l. , 1967), failed to alter acetaldehyde-evoked adrenomedullary secretion (Fig. 6). That magnesium interferes with an intracellular calcium receptor site in the adrenal chromaffin cell was first demonstrated by Rahwan et al^. (1973) and later confirmed by Lastowecka and Trifaro (1974). Kanno et al. (1973) likewise demonstrated by direct intracellular injections into mast cells the competition between magnesium and calcium for the intracellular calcium receptor site. An intracellular competition between calcium and magnesium 121 in vascular smooth muscle was also proposed by Turlapaty and Carrier (1973), and in nerve by Miledi (1973) and Krnjevic et a_l. (19 76) . From the foregoing, it appeared, therefore, that acetaldehyde-evoked adrenal catecholamine secretion was not mediated by intracellular calcium since the effect was not blocked by magnesium (Fig. 6). To confirm further the fact that acetaldehyde evoked catecholamine secretion by a calcium-independent mechanism and to establish the existence of such a mechanism in the adrenal medulla, the experiments with radiocalcium (section R.2.4 and R.2.6) were undertaken. Loss of calcium (as 45 monitored by Ca) from any intracellular pool, or trans location of calcium from one intracellular pool to another, would have implied a participation of intracellular calcium in the secretory action of acetaldehyde on the adrenal 4 5 medulla. However, no differences in Ca content of sub- cellular fractions from acetaldehyde-stimulated and control glands were observed (Fig. 10), nor was there a significant 45 efflux of Ca from the glands during acetaldehyde stim ulation (Fig. 8). These results indicate a lack of involve ment of intracellular calcium in the secretory presence of the adrenal medulla to acetaldehyde, and establish the existence of a calcium-independent secretory mechanism for adrenomedullary catecholamines. 122 4 5 Examination of the subcellular distribution of Ca (Fig. 10) and catecholamines (Fig. 9) from acetaldehyde- stimulated and nonstimulated glands adds additional insight into the mechanism of action of acetaldehyde. Although it was reported previously that acetaldehyde alters mito chondrial function (Rubin et ajL. , 1973; Yeh and Byington, 1973), the present studies indicate that calcium mobiliza tion from mitochondria is not induced by 23 mM acetalde hyde (Fig. 10). Furthermore, structural disruption of chromaffin granules was not evident in the present studies, since changes in subcellular distribution of catecholamines were very discrete. Thus, a significant mobilization of catecholamines from fraction VI (containing chromaffin granules) of the acetaldehyde-stimulated glands resulted in recovery of the released catecholamines in the cytosolic fraction (in all but one experiment) and in the effluent from the glands (Fig. 7). These results, therefore, support the observations that acetaldehyde localizes in and interacts with the chromaffin granules (Schneider, 1974b) resulting in the intracellular release of the catecholamines from the granules into the cytoplasm from where the hormones diffuse into the circulation (Schneider, 1974b). 123 D.3 Protection from acute toxicity of acetaldehyde in mice (O'Neill and Rahwan, 1976). Although statistically significant protection from acetaldehyde-induced loss of righting reflex was observed for cysteine, ascorbate and thioctic acid (Table 4), the results differ quantitively from the protection reported in rats by Sprince et al^. (1974, 1975; see Table 4). The use of the LD90 (loss of righting reflex) of acetaldehyde in the present study as opposed to the ID90 used in rats by Sprince et a]L. (1974, 1975) should have biased the results in our favor. However, the quantitively inferior protection observed in mice (Table 4) may be due to several reasons: First, and most obvious, the test species differ. It was imperative however to study reported protectants in the mouse, since this was the animal model of choice for future experiments on addiction, withdrawal and iii vivo TIQ biosynthesis. This variable, therefore, may account for the observed discrepancy. Second, a different route of acetaldehyde administra tion was used in the present study (i.p.) as compared to those of Sprince et aJL. (1974, 1975) (oral), although protectants were given orally in both studies. Since acetaldehyde does, in fact, form conjugates with some of the protectants tested (Alivasatos et al., 1973; Nagasawa et al. , 1975), a large part of the "protection" observed 124 by Sprince et al. (1974, 19 75) may have been due to complexation of the aldehyde in the gut (before absorption), and not due to inhibition of the toxic effects of circu lating acetaldehyde. By administering acetaldehyde i.p. and the protectants orally, it was hoped to avoid this problem in interpretation and more accurately assess the protection actually afforded by the latter compounds. However, under these conditions, the protection noted was inferior. Sprince et al. (1974, 1975) also noted decreased protection when protectants and acetaldehyde were not both given by the same route. Third, Sprince et al^. (1974, 1975) base some of their conclusions on data collected from a single test group of 10 animals. It was our experience that on occasion, substantial protection (70-80%) in a group of 10-12 mice might be observed, but when the experiment was repeated in a different group of the same size on a different day (all other factors being equal), minimal protection was observed. This phenomenon defied explanation, and points out the importance of using larger test groups in experiments of this type. The results shown in Table 4 do not lend strong support to the reports of other investigators (Blum et al., 1974; Sprince eb al.. , 1974, 1975; Ward et al^. , 1972), and suggest that these agents may have limited value as "acetaldehyde 125 antagonists," at least in the mouse. The protection might possibly be enhanced, however, under conditions where the introduction of acetaldehyde is not so abrupt, such as in vivo from the metabolism of ethanol. D.4 Absence of formation of brain salsolinol in ethanol- dependent mice {O'Neill and Rahwan, submitted; Rahwan and O'Neill, 19 76) The purpose of the studies described in section R.4 was to investigate the relationship, if any, between brain salsolinol and physical dependence on ethanol in mice. The assumption was made that if the TIQs are causally involved in ethanol dependence, maximal formation of these alkaloids would be expected to occur during alcohol consumption, and the decline in brain levels of TIQs (due to metabolism and/or excretion) below a critical level when alcohol is withheld would allow the withdrawal syndrome to appear. Consequently,^ an inverse relationship between brain TIQ levels and the severity of the withdrawal syndrome (as measured by withdrawal scores) would be expected. The availability of a convenient model system for the induction of alcohol dependence in mice (Goldstein, 1972) and the development of a rapid and sensitive assay for salsolinol (see M.5.7) allows a test of the validity of the above assumption. Salsolinol is a likely alkaloid to be 126 formed (by the condensation of acetaldehyde with dopamine) in mice during chronic ethanol intake for a variety of reasons: First, brain acetaldehyde levels under these conditions are reported to be high (Ortiz et al. , 1974). Second, dopamine is the predominant catecholamine in the mouse brain (Maruyama and Takemori, 1972). And third, the in vitro rate of formation of salsolinol is higher than that of the TIQ derived from the condensation of norepin ephrine and acetaldehyde (Robbins, 1968). Figures 19-23 indicate that there was no evidence for the in vivo synthesis of salsolinol in individual whole brains, pooled whole brains, or dopamine-rich areas of pooled brains obtained from mice rendered physically dependent on ethanol, even under conditions where blood ethanol levels reached 7 mg/ml. Since the analysis for brain salsolinol was performed immediately upon withholding ethanol from the mice, this would represent (according to the above assumption) the time of maximal TIQ formation. It may be argued that salsolinol could have escaped detection due to biotransformation and/or excretion prior to sacrifice and extraction. However, if salsolinol is causally involved in the development of physical dependence, and if its disappearance from the system is responsible for the withdrawal syndrome, then the latter syndrome should be observed with maximum intensity soon after withholding 127 alcohol, and not 3-4 hours later as demonstrated in this study (Fig. 14) and by Littleton et aJL. (19 74) . The fact that the GC/EC assay is capable of detecting levels of salsolinol of 8 ng/g of brain tissue (see R.4.3) indicates that a conversion of as little as 1% or more of the endogenous brain dopamine to salsolinol could not have escaped detection. Formation of salsolinol at levels below 8 ng/g brain tissue would be of questionable signif icance in view of the rather low pharmacological potency of this and other similar TIQ alkaloids (see 1.4). 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