central actions of nicotine

Central Actions of Nicotine

Mario D. Aceto and Billy R. Martin Drpnrlmrnf of Pknrmnrology, Mrd i rn l Collrgr of Virginin. Virginin Commonwrnlfh Uniwrsity. MCV

Stnlion, Box 6 1 3 , Rirhmond, Virginin 2 3 2 9 8

I . Introduction ............................ . . . . . . . . . . . . . . . . . . 43 11. Nicotinic Acetylcholine Receptor ......................................... 44

A. Historical Aspects .................................................... 44 B. Peripheral Nicotinic Cholinergic Receptor .............................. 45 C. Central Nicotinic Cholinergic Receptor ................................ 46

Ill. C N S Distribution Studies ................................................ 51 A. Nicotine Localization ................................................. 51 B. Neurotoxin Localization .............................................. 54

IV. Neurotransmitter Studies ............................................... 55 A. Noradrenaline ....................................................... 55 B. Dopamine ........................................................... 56 C . Serotonin ........................................................... 57

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I. INTRODUCTION

Although nicotine has no recognized therapeutic uses, millions of people are chronically exposed to this alkaloid. However, it has served as an invaluable research tool and has been widely studied. No doubt many of the studies have been prompted by the association of nicotine with tobacco. The chronic use of tobacco has been casually linked with a large number of serious diseases including coronary artery disease, cerebro- vascular disease, Buerger’s disease, and cancer of the lung, oral cavity, esophagus, bladder, and pancreas to name a few. Although these diseases may be related more to other constituents of tobacco and tobacco smoke, nicotine is believed to play a prominent role in the maintenance of the smoking habit. Nicotine has also been studied because it mimics certain actions of one of the major neurohormones.. namely acetylcholine. During the past 30 years, much progress has been made in our understanding of the mechanism of action of this alkaloid at the neuromuscular junction and on autonomic ganglia. The peripheral nicotinic receptor has actually been isolated and reconstituted and studies on the autoimmune response of this receptor in myasthenia gravis are leading to a greater understanding of many of the basic aspects of this disease and other dystrophies as well.

During the past two decades, much research has been conducted on the

Medicinal Research Reviews, Vol. 2, No. 1, pp. 43-62 (1982) Q 1982 by John Wiley & Sons, Inc. Ccc 0198-6325/82/010043-20$02.00

44 ACETO AND MARTIN

action of nicotine on the central nervous system. The work is important because ultimately it may lead to ou r understanding of the mechanism of tolerance and dependence to nicotine and of the functional role of the nicotinic cholinergic nervous system. Although these objectives are only goals at this time, certain patterns are emerging from the work reported in the literature. While preparing this review, we concentrated on nicotine’s central effects and focused primarily on nicotinic receptors and nicotine’s effects on the monoamine neurohormones.

11. NICOTINIC ACETYLCHOLINE RECEPTOR

A. Historical Aspects

The development of the concept of a nicotinic acetylcholine receptor (nAChR) evolved through a number of stages. In 1889, Langley and Dickinson’ demonstrated that one of the sites upon which nicotine acted was the ganglion. Later, Langley expressed the idea of a “receptive substance” for this alkaloid.’ He also reported that curare competitively blocked the action of n i ~ o t i n e . ~ During this period Dale demonstrated the nicotinic and muscarinic properties of acetylcholine using the respective alkaloids.4 It is also noteworthy that studies by Gaddum’ and Clark” with acetylcholine played a role in the development of a theoretical basis for receptors. During the period of 1921-1926 Loewi and his coworkers

Mario D. Aceto was born in Providence, R l in 1930. He rereived his Bachelor of Scienre (Pharmacyl degree from the University of Rhode Island, his Master of Scienre dcgree (Pharmarologyl from the Uniuersity of Maryland in 1955 and his Dortor of Philosophy degree (Pharmacologylfrom the University of Cannerticut in 1959 . He was involzvd i n teaching and research at the Universityof Pittsburghfrom 1 9 5 8 - 1 9 6 2 and was employed by Sterling Winthrop Research Institute where he was Sertion Head of CNSpharmacology until 1973 . Since then he has been teaching and conducting researrh at the Mediral College of Virginia, Virginia Commonwealth Unizwsity as Associate Professor. His research interests inrlude mechanisms a/ action of analgesics and psychothrrapeutir agents and drug dependenre.

Billy R. Martin was born in Winston Salem, N C in 7 943. He receioed an AB degree in rhemistryfrom the Unioersity of North Carolina (Chapel Hill l in 1 9 6 5 anda Ph.D. degree in pharmacology from the same institution in 1 9 7 4 . He received a postdortoralfellowshipfrom the Swedish Medical Research Council andspent one year a t the Unioersity of Uppsala, Uppsala, Sweden. He then receizwd a fellowship /lorn the WeIlrome Trust Foundation for researrh at the Unizwsi ty of Oxford, Oxford, Enxland. In 19 7 6 , he joined thefaculty at the Mediral College of Virginia where he is rurrently an Assistant Professor. His researrh interest inrlude the pharmacokinetics and pharmaro- dynamics of agents that affect the central nervous system.

CENTRAL ACTIONS OF NICOTINE 45

discovered the neurotransmitter "Vagusstoff" and presented evidence that it was ace ty l~ho l ine .~ ,~

B. Peripheral Nicotinic Cholinergic Receptor

The next stage involved the use of radiolabeled nicotinic agonist and antagonists. In the late 1950s, attempts were made to identify cholinergic receptors in eel electric tissue (Elertrophorus electricus), which was known to contain high concentrations of acetylcholinesterase, using [" Clgal- lamine, a nicotinic antagonist." The binding that was observed was later shown to be an interaction with an acidic mucopolysaccharide." l 2 A protein isolated from electric tissue was also considered to be a candidate for the receptor until it was shown to possess nonspecific binding proper tie^.'^ ''

(14C]Acetylcholine and ['4C]dimethyl-~-tubocurarine were reported to bind to a ribonucleoprotein in human skeletal muscle^.'^ Binding of ACh was reduced but no change in binding was observed with D-tubocurarine. However, other workers suggested that high concentrations of cholinesterase in the preparation metabolized ACh and that nonspecific ["C]- choline binding probably occurred.'6

The chemical theory of synaptic transmission predicts that subsyn- aptic membranes contain postsynaptic receptors upon which a trans- mitter acts. This receptor is likely to be protein in nature and intimately bound to the lipoprotein structure of the plasma membrane. Thus, attempts were made to isolate the AChR from synaptosomal fractions of the central nervous system, electric organ, and muscle. A proteolipid with apparent receptor properties was isolated from these t i s s ~ e s ' ~ - ~ ' but it was shown later that the chloroform-methanol solvent system used did not extract affinity-labeled, AChR." 23 A loss of bungarotoxin-binding activity occurred when the proteolipid was emulsified in 1% Triton X-100 solution. The implication was that the binding activity noted was not associated with the protein fraction of the proteolipid extract. Other workers noted that this proteolipid contained a large amount of triphosphoinositol which bound cholinergic ligands.'" 25

The use of I- and H-labeled neurotoxins such as a-bungarotoxin (a-BTX) and cobratoxin which bind with the nAChR with a high affinity and form stable complexes greatly facilitated the study of this receptor. Other important advances involved the use of peroxidase-labeled toxins and the use of high-affinity labeling reagents such as 4-(N-maleimide)- benzyltrimethylammonium iodide which forms a covalent bond with the thiol group of the receptor after exposure to the disulfide bond reducing agent dithiothreitol.

It is now known that the receptor which has been isolated from the electric organs of seven species of fish, and human skeletal muscle is nicotinic. I t has also been established that this receptor belongs to a class of membrane proteins which will respond to the neurotransmitter or

46 ACETO AND MARTIN

other stimuli by opening pores permitting Na' and K' ions to flow and ultimately, leading to muscle contraction. It is also known that the functional unit or ACh regulator consists of at least two distinct sites. The first is the ACh receptor site which binds ACh ligands including snake venoms such as a-BTX. The other site or ACh ionophore is concerned with ion translocation. The receptor has also been reconstituted in an artificial planar lipid bilayer. As a result of the application of a monoclonal antibody technique, a greater understanding of some of the fundamental aspects of myasthenia gravis have been made. This disease, which is characterized by weakness and fatigue of skeletal muscle, is now believed to be an autoimmune disorder resulting in loss of nAChr in the postsynaptic membrane of muscle. Details of all these accomplishments are beyond the scope of this article and have been comprehensively reviewed or published.'"-"'

C. Central Nicotinic Cholinergic Receptor Studies

1. Muscarone and Neurotoxin Binding

Research on the nature and function of this receptor in the central nervous system has been slow compared with that conducted on specialized peripheral organs. The brain is much more complex since it contains receptors for many putative neurotransmitters and drugs such as opiates, benzodiazepines, neuroleptics, etc. Furthermore, cholinergic receptors in the brain may not be the same as those in the periphery since the synapses in the central nervous system are between neurons whereas those in peripheral tissues are between neurons and organs or glands.

The first at tempts to study the binding of cholinergic ligands to brain tissues were reported in 1967 by Azcurra and DeRobertis." Biochemical studies of subcellular fractions of rat brain cortex demonstrated that three cholinergic antagonists bound preferentially to acetylcholinesterase-enriched synaptic membranes. Extending these studies using cat cerebral cortex, these workers" reported that the receptor properties for D-tubocurarine might be in a type of proteolipid present in the nerve ending membrane. Further studies indicated that the receptor, which was extracted using chloroform-methanol, was a protolipid protein but was not the AChR.'R,4R

The availability of I3 Hlmuscarone, a cholinergic agonist ligand with potent muscarinic and nicotinic properties, offered new opportunities for study. This keto analog of muscarine is not metabolized by cholinesterase. Since the central nervous system of insects is very rich in ACh and cholinesterase;' and since housefly brain (Musca dornrnestica) contains a concentration of cholinesterase as high as that found in electric tissue" it was believed that study of the AChR in the CNS would be greatly facilitated by use of this radioligand. I t was found that the supernatant of housefly brain bound muscarone reversibly. Furthermore, 17 cholinergic


drugs demonstrated strong blockade. Since these drugs included nicotine, succinycholine, decamethonium, atropine, and pilocarpine, the authors concluded that the receptor in fly brain had both nicotinic and muscarinic characteristic^.^' These studies were extended and [3 Hlnicotine was shown to bind to fly brain in a fashion similar to muscarone. Although it had a binding constant of 3.2 pM, concentrations of 100 pM caused only 15-74% blockade of binding of other ligands such as dimethycurarine and muscarone. These results suggested that multiple binding sites were involved. In addition, i t was noted that the receptor material was a protein.52 Other investigations showed that binding in rat brain differed from that in fly brain.53 Muscarone and atropine but not decamethonium, dimethylcurare or nicotine bound reversibly to a mitochondria1 fraction. These results were interpreted to mean that the higher-affinity site for atropine binding is the site more likely related to the AChR. Other attempts5' to isolate and characterize the AChR from guinea pig cerebral cortex were made using [' Hlacetyl-a-BTX to label the receptor. It was shown that D-tubocurarine, ACh, carbachol but not atropine, gallamine, decamethonium o r hexamethonium inhibited binding. The receptor was characterized as a protein but its high molecular weight suggested that i t was different from the AChR isolated from electroplax. Other workers suggested that the a-BTX had not been acetylated and that the binding noted was due to free i3 H I a ~ e t a t e . ~ ~ Additional evidence that this binding was unrelated to AChR was provided by studies on eel electroplax and hog brain56 and rat and guinea pig brains57 in which ["'I]-a-BTX was used. Moreover, in 1974, another report appeared whose results agreed with those of the last two cited publications. Eterovic and Benne tP reported that binding of [3H]-a-BTX was more specific and reliable than '251-labeled-a-BTX for brain AChR. In addition, their work showed that nicotine decreased tritiated-toxin binding in the rat brain crude mitochondrial fraction by 80% at 1.3 X I P M . The components binding [' HI- a-BTX were found to be present in low concentrations (pmoleslg). This is in general agreement with that reported in two studies by Moore and L o Y , ~ ~ and Salvaterra and Moore.57

The localization of binding sites for ["'I]-a-BTX was investigated in subcellular fractions of rat brain areas." Toxin binding activity was found to be highest in synaptosomal fractions and showed a distribution similar to that of acetylcholinesterase, choline acetyltransferase and Na' and K'-activated ATPase. Toxin binding was high in olfactory lobes, cerebral cortex, thalamic region, caudate nucleus and brainstem and lower in hippocampus and lowest in cerebellum. These investigators also indicated that they did not encounter difficulties using I]-a-BTX reported by Eterovic and Bennett.5R In another study, the binding of ["'I]-a-BTX to the crude membrane preparation of rat cortex was characterized.6a At least two saturable sites were found. One binds 50 fmol toxinlmg protein (high affinity) and the other 120 fmollmg. For the high affinity site, the K,, was found to be 9.2 X 10-'OM.

48 ACETO AND MARTIN

The successful extraction of the receptor protein from rat brain represented another important advance. In one study by Moore et al.," the AChR extracted with 1% Emulphogene yielded a soluble fraction which specifically bound ["*I]-a-BTX (K,, = 5 X 10-9M). Nicotine at concentrations of i 0 P M blocked the toxin-receptor complex whereas tubocurarine reduced this interaction by 40%. Eserine and atropine had no effect a t 1 P M . The nAChR was also solubilized from rat brain by extracting with Triton-X-100 detergent by Salvaterra and Mahler.62 Lowy and his coworkers believed that this toxin-binding receptor resembled the peripheral n AChR as regards to toxin binding kinetics, solubility, isoelectric point, and hydrodynamic proper tie^.'^

Central nicotinic receptors were also identified by Speth et a1.,64 using [1251]-a- to~in~ from naja naja siamensis (K, , of 0.11 nM). The receptor sites showed specific, saturable and high-affinity binding which was blocked by nicotine (y = 0.23 p M ) . The number of binding sites correlated well with those calculated for [I2' I]-a-BTX. The regional distribution of this receptor also correlated well with those previously reported except for the corpus striatum.

The binding of [3H]-a-BTX to a striatal synaptosomal preparation of rat and sheep revealed specific binding sites (7.5-11.2 fmolelmg protein). It was shown that acetylcholine stimulated the release of dopamine from synaptosomes. In the presence of muscarinic antagonists, acetylcholine stimulated dopamine release whereas in the presence of nicotinic antagonists, acetylcholine had the opposite effect. These authors suggested that presynaptic excitatory nicotinic and inhibitory muscarinic receptors were responsible for modulating dopamine

In a study involving a-BTX binding on a clonal rat sympathetic nerve cell line no correlation could be shown between the ability of cholinergic ligands to affect cholinergic function and to inhibit a-BTX binding.66 In another study reported by the same a ~ t h o r s , ' ~ the results distinguished a ganglionic nicotinic receptor from the a-BTX binding component of the cells studied. Studies by other workers" on chick sympathetic gangion also suggested that a-BTX binding in nervous tissue was to membrane components other than the nAChR.

Although there is evidence that a-BTX binding sites in the central nervous system are identical with the nAChR, the use of a-BTX as a marker has been questioned: (1) at present, there is no physiological evidence that a-BTX binds to a postsynaptic receptor in the CNS. In fact, attempts to do so were unsuccessful. In one attempts to block the nicotinic acetylcholine mediated synaptic transmission in the spinal cord of the frog using a-BTX failed. Cholinergic antagonists and other dendrotoxins blocked this pathway. Another similar attempt using the cat also failed." ( 2 ) Although a-BTX binds specifically and with high affinity to receptors in the outer and inner plexiform layer of the pigeon retina, nicotine only blocked the binding at the inner plexform layer.71 In another study involving a-BTX binding in rat brain crude membrane


fractions, high affinities were found for nicotinic ligands, including nicotine. In fact, nicotine ranked just below tubocurarine. Also, the ganglionic stimulating drugs bound more strongly than the

Additional evidence that the a-BTX binding sites in the CNS may be the nAChR was recently provided by a study in which solubilized hypothalamic nicotinic receptors from the rat brain were found to be immunologically similar to the receptor from torpedo p l a ~ . ~ ~ Pharmaco- logical studies utilizing nicotine, decamethonium and atropine also indicated that a-BTX binding in the goldfish optic tectum is similar to that reported at central or peripheral a-BTX sites in other specie^.^'

Biochemical and autoradiographic studies of [Iz5 I]-a-BTX binding to homogenates of various rat brain areas revealed that specific binding occurred in the hypothalamus, hippocampus, pons, cingulate cortex, medulla, septum, thalamus, caudate, and cerebellum. The greatest amount occurred in the hypothalamus and the least in the cerebellum. The saturable binding was prevented by nicotine (10-“M) and other nicotine ligands but not by atropine or o x o t r e m ~ r i n e . ~ ~ O n the other hand, the basal nuclei, caudate, and putamen contained the highest concentration of muscarinic receptors in the brain; the cerebral neocortex and hippocampus contain relatively high binding sites and the thalamus and hypothalamus contain the fewest binding Nicotinic receptors may predominate in the limbic system whereas muscarinic receptor predominate in the extrapyramidal areas.

2 . Nirotine Binding

The development of the concept of a nicotinic receptor has relied heavily upon radioligand-binding studies. However, few of the early studies utilized radiolabeled nicotine, the reasons for this are numerous. Nicotine apparently has a rather low affinity for the receptor and there appears to be a limited number of receptors in the CNS. These difficulties were magnified by the lack of radiolabeled nicotine with high specific activity and by the limited availability of optically pure (+)-nicotine, the unnatural isomer.

Schleifer and E l d e f r a ~ i ’ ~ carried out one of the first studies on the binding of [ 3 Hlnicotine. They found that nicotine bound reversibly to mitochondria1 and synaptosomal preparations from mouse brain, the K,, for total binding being 7.2 nM. No distinction was made between saturable and nonsaturable binding. The nicotine binding was blocked by ACh and anabasine but not by pilocarpine or atropine. No further attempts were made to evaluate the specificity of binding. However, the authors did provide evidence that suggested [Iz5 I]-a-BTX was binding to sites other than the nAChR. Abood et al.” failed to show specific nicotine binding to brain homogenates but they were able to show that nicotine bound to brain slices and glass-fiber filters (K,, = I P M and 2 X IO-’M, respectively). This nicotine binding to brain slices and filters was also stereospecific in that the unnatural (+)-nicotine was approximately ten

50 ACETO AND MARTIN

times less effective than (-)-nicotine in displacing ( - ) - [ 3 Hlnicotine. A variety of cholinergic agents had no effect on nicotine binding to brain slices which prompted the authors to postulate the existence of a noncholinergic nicotine receptor. The authors7” also showed a correlation between the behavioral activity of nicotine and piperidine derivatives and their ability to displace nicotine in the binding assay. However, there was not a good correlation between the pharmacological effect of (+)-nicotine (100 times less active than (-)-nicotine) and its ability to displace (-)- [” Hlnicotine (10 times less active than (-)-nicotine).

Nicotine has also been reported to bind to synaptosomes from rat brains which appear to be cholinergic in nature. Regional differences in the brain showed that specific nicotine binding was predominate in hypothalamus, hippocampus, and thalamus. The authors claimed that in these experiments the f3H]nicotine concentrations was 0.2 pM (one mL incubations); however, the radioactivity would not be detectable with the low specific activity (250 mCilmmole) of their [ 3 Hlnic~t ine .~‘

Studies were undertaken in our laboratories several years ago to investigate the binding sites of nicotine in the central nervous system. One of our primary objectives was to correlate pharmacological stereospecificity with stereospecific binding. Ample supplies of (+)-nicotine were obtained from the resolution of a racemic mixture of nicotine. The pharmacological stereospecificity was relatively small as measured by lethality, blood pressure, heart rate”’ and behavioral activity.” These results were in agreement with previous studies which showed that (+)-nicotine was 1-30 times less potent than (-)-nicotine in the central nervous system except for these noted in the EEG studies reported by Abood et aL7’

In preliminary studies, the binding of ( - ) - [ 3 Hlnicotine (50 mCilmmo1) was characterized as low-affinity binding that exhibited a slight degree of stereospecificity.R2 However, the synthesis of ( - ) - [ 3 Hlnicotine with higher specific activity (5 Cilmmol) allowed for the identification of a high-affinity binding site that was sensitive to incubation conditions such as ion concentrations, time, pH, temperature, e t ~ . ” ~ The (-)- [ 3 Hlnicotine binding apparently lacked stereospecificity since it was displaced by both (+)- and (-)-nicotine. ( + ) - 3 H-Nicotine was then synthesized so that the&’s of (+)-and (-)-3H-nicotine could be compared directly. Scatchard plots of the saturable binding of (+)-[’H]nicotine to the crude mitochondria1 fraction of whole rat brains revealed a of 2 .2 X 10-7M which was approximately three times greater than that obtained for (-)-[3H]nicotine (& = 6 . 3 X IOPM).”‘ This small degree of stereospecificity in binding does not account for all of nicotine’s pharmacological stereospecificity but it does account for a portion.R4 It was also found that the saturable binding of (-)-I3 Hlnicotine was greatest in hypothalamus and hippocampus and least in c e r e b e l l ~ r n . ~ ~

Recently, Romano and Goldstein“ reported that the racemic mixture of [3H]nicotine bound to rat brain membranes and that (-)-nicotine was


63 times more effective than (+)-nicotine in displacing the (+)-['HI- nicotine. Interestingly, the stereospecificity in the binding assay was greater than the pharmacological stereospecificity.Rs The K,! of the racemic mixture was 4.3 X 10-'MM. The pharmacological agents that displaced the radiolabeled nicotine supported the notion that the receptor was cholinergic.

In summary, the evidence accummulated thus far in several laboratories, including our own, suggests that there are central nicotinic receptors and that nicotine, rather than agonists and antagonists, may serve as the radioligand. The receptor is stereospecific and unevenly distributed throughout the central nervous system. Most studies show that the highest population of binding sites are in hypothalamus and hippocampus. One of the remaining questions to be answered is whether the central nicotinic receptor is cholinergic or noncholinergic.

111. CNS DISTRIBUTION STUDIES

A. Nicotine

The distribution of nicotine in the CNS has been studied by a number of investigators who were attempting to identify its locus and mechanism of action.

The first study employing nicotine [ I 4 Clmethyl in mice and rats was reported by Hansson and S~hmit ter low."~ Soon after intravenous administration of nicotine, whole-body autoradiography revealed that high concentrations of radioactivity appeared in whole brain. However, hexobarbital pretreatment was required in order to protect the animals from an otherwise lethal dose of nicotine (3 .3 mglkglIV). This high dose was required due to the low specific activity of nicotine. These studies were extendedRR to provide a more detailed analysis of the distribution in the brains of mice and cats. The cats were pretreated with pentobarbital; the mice were not, although they received 400pg of nicotinelkg IV, a dose equivalent to the LD,, :' In the cat, a high concentration of radioactivity was observed in thegrey matter whereas the lowest concentration was in the white matter at 5 min. By 15 min, a high concentration was also observed in the hippocampus. Autoradiograms of the mice also showed high concentrations of nicotine in the brain. Other studies reported later" confirmed and extended these findings. It was shown that nicotine and its metabolites leave the CNS fairly rapidly. The pattern of nicotine localization in the brain was not governed solely by blood supply and the major portion of radioactivity in brain was due to unchanged nicotine and cotinine, the major metabolite of nicotine. Conspicuous differences between the distribution of nicotine and cotinine were found."' The highest concentration of [3H]-(-)-cotinine in the brain was seen in the dense cell area of the cerebellum. The apparent failure of cotinine to reach brain areas normally associated with nicotine activity may explain the weak pharmacological activity of cotinine compared with nicotine.

52 ACETO AND MARTIN

These investigators concluded that the difference in distribution could be due to differences in their lipid solubility or to the lack of affinity of cotinine for the nicotinic receptor. However, it was later pointed out, that although some generalizations could be made from these studies, tritium is not ideally suited for whole-body autoradiography because energies higher than that of tritium are needed to expose x-ray film.9' The distribution of [ I * Clnicotine (methyl labeled) and [ I " Clcotinine were studied in four strains of male and female mice by whole-body autoradiography. The animals received doses of nicotine in the range of 0.2-1.3mglkg IV. Large amounts of radioactivity were found in the brain up to 6 min and after that time the amount rapidly decreased with residual activity persisting primarily in the hippocampus. The only difference among the four strains was that the pigmented strains revealed intense radioactivity in melanin in the eyes, hair and meninges of the brain. The distribution of [ I 4 Clcotinine was remarkably uniform.

Because whole-body autoradiography does not distinguish between parent drug and its metabolites, extraction procedures were used by Yamamoto et al.92 to determine the regional and subcellular distribution of labeled nicotine at a dose of 5 mglkg intraperitoneally. This dose produces convulsions in 20% of the animals. The maximum level of ["Clnicotine in the whole brain was found to be 1.0pglkg. Nearly three times that amount was found in the liver. In brain cortex, brain stem and cerebellum, the range found was 0.5-1.0 pglkg. The incidence of tremor and clonic convulsions increased with increasing level^.^' These workers also studied the subcellular distribution of [ I * Clnicotine in mouse brain following the same dose used There was an accumulation of radioactivity in the nuclear, mitochondrial, nerve endings, and mic- rosomal fraction. The greater part of the ['"Clnicotine was found in the soluble fraction; the radioactivity associated with microsomes and nerve endings, was ten times less than that in the soluble fraction. No attempts were made to determine what component of the subcellular distribution of nicotine was relevant to the pharmacological action. According to another report,g4 one might expect mecamylamine to alter the subcellular distribution of nicotine in accordance with bloackade of nicotine-induced tremors.

Rosecrans and Schechter9' attempted to correlate [ I 4 Cl nicotine to brain area distribution with changes in spontaneous activity in different strains of male and female rats. Nicotine did not alter spontaneous activity in males but the increases noted in the females of both strains were significant. Brain area nicotine levels were higher in the females. In general, correlations between brain nicotine levels and behavior were not significant between strains; but were significant with regard to the sex of the animals. However, the behavioral test used may not have been sufficiently sensitive. Another report by the same laboratoryg6 noted few differences in brain levels between "high"or "1ow"activity rats following 400 pglkg (SC) of nicotine. Again, females in both groups contained


higher brain concentrations. Also, repeated doses increased brain area nicotine levels. Later, it was showng7 that the discriminative stimulus property of nicotine is directly related to brain levels. In this study, doses of 400 and 200 pglkg (SC) were used.

MansneroR showed a correlation between ['" C] nicotine brain levels and antinociception. Another correlation was also claimed between nicotine brain levels and tremor and antidiuresis.""

Tissue distribution of 3H-nicotine in dogs and rhesus monkeys following an IV injection of small doses (IOOpglkg) was reported.loO Five minutes after injection, high concentrations of nicotine were found in the adrenal medulla and cerebral cortex. Nicotine concentrations in various areas of CNS of both species arranged in descending order were as follows: hypophysis, cerebral cortex, cerebellum, thalamus, hypothalamus, medulla, pons, spinal cord, and cerebral white matter. These results are in agreement with those reported for the cat."' These authors speculated that the differences in concentration in the CNS could be due to difference in cell and vascular density.

['"C]Nicotine levels in blood and whole brain were measured as a function of sex and age in two strains of mice and compared to convulsive behavioral responses.'o' The results indicated that blood levels of nicotine do not accurately predict brain levels or correlate with convulsive behavior whereas brain levels correlated with convulsive behavior. The older males of both strains concentrated the drug in the brain more than females but there were no corresponding differences in LD,,'s o r ED50'~.102 It is difficult to reconcile these results with those reported by Rose~rans ."~

The distribution of intravenously injected [ l * Clnicotine in mice and rabbit was studied.lo3 Most of the radioactivity appeared in the lung and brain. Radioactivity in brain dropped rapidly. Similar results were seen in the pentobarbital-anesthetized rabbit.

Part of the [''Clnicotine taken up in rat striatum, hypothalamus, cortex, and cerebellum in rat"" and monkey brain slices'os accumulates intracellularly. It is dependent on extracellular pH and is independent of the membrane pH gradient. Mecamylamine, procaine, lobeline, physostigmine, and metanephrine compete with [ I " Clnicotine for the binding sites within the intracellular space.'06 An ionic basis for this phenomenon was p r~v ided . "~ It appears that the degree of binding is dependent upon the K' concentration.

Despite the limitations and differences in the experimental design of these studies, certain generalizations can be made. Nicotine is concentrated in brain tissue of many species and it leaves the CNS fairly rapidly. Cotinine, the main metabolite is found in the brain. Most of the radioactivity seems to be associated with gray areas of the brain. The highest concentration of nicotine is found in the hippocampus and the lowest amount is found in the cerebellum. Nicotine accumulates in a variety of intracellular and subcellular regions. Finally, blood levels do

54 ACETO AND MARTIN

not always predict brain levels and brain levels do not always correlate with the various parameters studied, especially those concerned with behavior.

B. Neurotoxin Localization

A number of investigators have studied the in-uitro distribution of a-BTX in the CNS. Autoradiographic localization of a-BTX revealed binding sites in distinct areas of the rat and chick brain.'"' Highest concentration of binding sites occur in the optic tectum, parabigeminal isthmic nuclei, dorsal motor nucleus of the vagus, and nucleus gen- iculatus. Lower values were found for thalamus, inferior colliculi, and reticular formation of rat and chick and neocortex of rat and the dorsal ventricular ridge of the chick. Lowest values were seen in the cerebellum, caudate putamen, and certain mamillo-thalamic and pyramidal tracts. In another regional and subcellular distribution study in rat brain, I]-a- BTX binding, acetylcholinesterase and choline acetyltransferase activ- ities were relatively high in olfactory lobes, cerebral cortex, thalamic region, caudate nucleus, and brain stem, lower in hippocampus and lowest in cerebellum. Primary fractions showing the highest specific activity were crude mitochondria and nerve endings and the subfraction exhibiting the greatest recovery was the nerve ending.'" The distribution of [' HI-a-BTX in rat brains after intraventricular injection revealed autoradiographic binding in portions of the hypothalamus and amygdala only. Aparently there is limited diffusion from the ventricles. D- Tubocurarine, but not atropine pretreatment, reduced this binding.'" In another laboratory, the highest concentration of ['" I ] -a-BTX toxin binding was consistently found in the hypothalamus.'" High levels were also found in the hippocampus, inferior colliculus, and cortex. The lowest levels were in the cerebellum.

Using procedures in which a-BTX was conjugated with horseradish peroxidase which permits the localization of toxin binding at high resolution with the electron microscope, investigators were able to demonstrate nAChR sites in synapses of the reticular formation of the midbrain and preoptic nucleus of the hypothalamus.'" The relatively small number of nicotinic synapses demonstrated by this technique was disappointing. Another electron microscopic autoradiographic localization study demonstrated a strong association between a - B T X and nAChR within the hippocampal f ~ r m a t i o n . " ~

on the binding patterns of a-BTX in the CNS of the rat indicated that the toxin bound (autoradiography) to various nuclei of the hippocampus and hypothalamus. It was suggested that the limbic forebrain and midbrain structures a s well as sensory nuclei are the main nicotinic cholinoceptors of the brain. Toxin binding sites were associated with sensory input areas (olfactory bulbs and substantia gelatinosa) and with limbic areas of brain.

Other reports by these


It should be noted that a-BTX and cobra neurotoxin failed to label one of the few central nicotinic receptors whose functional role has been established, namely the synapse between the motorneuron axon col- lateral and Renshaw cell."5

Only broad generalizations can be made when comparing the nicotine distribution studies with the a-BTX studies. Most of the distribution was associated with grey brain area and hippocampal areas and the lowest distribution occurred in the cerebellum.

IV. NEUROTRANSMITTER STUDIES

A. Noradrenaline (NA)

In the 1950s, nicotine was shown to release NA from the adrenal gland."' NA has also been released by nicotine from atrial sympathetic nerve terminal^"^-"^ and from rabbit and guinea pig and from rabbit ear artery.Iz3 Nicotine facilitory presynaptic receptors have also been proposed on noradrenergic nerve endings.""

That nicotine's central effects might be related in part to NA release was first suggested by Vogt,I2' and Burn.I2' A number of reports have failed to note changes in brain NA concentration after acute o r chronic administration of However, a significant reduction in catecholamine stores was demonstrated by microspectrofluorometrical evaluation in the medial palisade zone of the median eminence.'" Nicotine injected into the lateral ventricle of the rat brain in doses as low as 10 pg was shown to release [13H]NA131 and to increase the turnover rate of this amine.'32 O n e of these workers also showed that chronic nicotine treatment increased the rate of disappearance of intraven- tricularly administered H-NA which suggested an increase in the synthesis and utilization of NA. An increase in turnover rate was also reported later by other workers.'29 This alkaloid has also been shown to release [3H]-NA from slices of rat hypothalamus, cortex, and cere- b e l l ~ m . ' ~ ~ The effect was dependent on extracellular calcium and was inhibited by both hexamethonium and ACh.

In another study involving brain slices from hippocampus and striatum, nicotine produced a significant increase in catecholamines release, the effect being more pronounced in the striatum. The time course of efflux radioactivity released by nicotine was unlike that produced by potassium but similar t o that produced by tyramine and the process was independent of extracellular calcium. It was proposed that the mechanism of action involved the displacement of catecholamine from storage vesicle^.'^' B a l f o ~ r ' ~ ~ showed that high doses M ) of nicotine inhibited the uptake and retention of [I4 CINA in rat brain homogenates from the hypothalamus and hippocampus. Lower concentrations and lo-' M ) caused an increase in the initial uptake of this amine and a decrease in retention, especially in the hypothalamus. These results were

56 ACETO AND MARTIN

interpreted to mean that nicotine's effect was different from that of amphetamine since one of the major properties of the latter drug involved the inhibition of NA ~ p t a k e . ' ~ ~ - ' ~ ~ Nicotine and amphetamine have been cited as having many similar behavioral effect^.'^^"^^ For example, amphetamine also released NA from rat brain.'40,'41 Nicotine was also shown to release ['HINA from synaptosomes from the h y p ~ t h a l a m u s . ' ~ ~ These authors concluded that nicotine receptors in brain synaptic regions play a role in the hypothalamus by releasing NA. Furthermore, the release mechanism was considered to be unlike that of reserpine or tyr.amine and the receptors were thought to be presynaptic in 01-igin.l~'

The important NA-containing nucleus locus coeruleus (LC) is known to innervate much of the brain and which may function as part of a reward m e c h a n i s n ~ ' ~ ~ - ~ ~ ~ and may play a role in behavioral extinction,'"' in a t t e n t i ~ n , ' ~ ~ and in the expression of a n ~ i e t y . ' ~ ' , ' ~ ~ The LC, which also contains cholinergic receptors, has been activated by systemically administered doses of nicotine and muscarinic agonists such as physostigmine and oxotremorine. However, nicotine applied microiontro- phoretically to the LC had no e f f e ~ t . ' ~ ~ , ' ~ ' Scopolamine applied micro- iontrophoretically on the LC neurons antagonized the stimulatory effects of systemically injected physostigmine but not that of nicotine. The author suggested that stimulation of the LC by cholinergic drugs involved muscarinic receptors; the activation by nicotine is indirect and probably results from ACh-induced release of NA and mediated by noncholinergic LC input.'50

Many of nicotine's central effects could be explained by its effects on the LC. This structure projects to many other brain areas such as the hypothalamus, medulla, spinal cord, limbic, and cortical areas. In addition, afferents from some of these areas can activate the LC. Nicotine's effects on memory, the skeletal muscle, relaxant effects, the decreases in irritability and appetite could be related to its effects in the LC. In addition, some of the withdrawal signs associated with the cessation of smoking such as drowsiness, irritability, and increased appetite could reflect a hypersensitivity of the LC. If this is true, then drugs which suppress LC activity may alleviate these withdrawal signs. However, experimental proof must be provided. Obviously, much remains to be done in this area.

B. Dopamine (DA)

BhagatI3' reported that nicotine increased the turnover rate of NA but found no change in the concentration or turnover rate of DA in rat brain homogenates. A reduced DA turnover in the nucleus caudatus was observed using a microspectrofluorimetrical evaluation of catecholamine fluorescence.129 W e ~ t f a l l ' ~ ~ who noted NA release by nicotine also demonstrated significant increases if [ 3 HI DA release after nicotine in


superfused rat striatal slices, a process that was calcium dependent. Acetylcholine, hexamethonium, and lidocaine inhibited this increased release while morphine was without effect and phenoxybenzamine reversed the effect. These results provided additional evidence for a postulated muscarinic inhibitory process in which ACh modulated the release of catecholamines from central neurons. A number of other studies involving the release of DA by ACh from nigrostriatal neurons have been p ~ b l i s h e d . ' ~ ~ - ' ~ ~ The results taken as a whole suggest the involvement of cholinergic presynaptic receptors of the nicotinic and muscarinic types.

it was demonstrated that low concentrations of nicotine ( I P M ) even in the presence of tetrodotoxin, would release newly synthesized ["IDA by its action on presynaptic receptors in nigrostriatal neurons. The process was calcium dependent and was prevented by tubocurarine and pempidine.

Other studies on the nicotine-induced release of catecholamines from striatal and hippocampal brain slices showed similar results for NA and DA.'34 Evidence that nicotinic receptors are not located on either DA o r NA nerve terminals was also ~ubmi t ted ."~

Nicotine has also been implicated in various neuroendocrine events .155-157 It was shown to reduce lutienizing hormone and prolactin secretion by selective activation of an inhibitory dopaminergic mechanism located inthe median eminence.

These results are very interesting but obviously much more work is required to determine DA's role in the action of nicotine.

In a more recent

C. 5-Hydroxytryptamine (5-HT)

Relatively few studies have been done on the effects of nicotine on 5-HT function. Bhaga~ '~ ' . ' ~ ' who studied nicotine's effect on storage and synthesis of NA in rat brain, also reported that endogenous concentrations of 5-HT were unaltered after chronic administration of nicotine (0.5 mglkg, SC) repeated 4 times a day, 5 days a week for 6 weeks. These results contrasted with those of previous workers'ZR who noted an increase concentration of this amine after chronic administration of nicotine. Others noted that repeated doses of nicotine reduced 5-HT.turnover, an effect which was not blocked by rne~amylamine . '~~ These results were consistent with those of Rosecrans'5q who showed that repeated doses of nicotine reduced accumulation of 5-HT after pargyline, a monoamine oxidase inhibitor.

Nicotine was also studied for its effects on the uptake and retention of [" C1-5-HT in rat hypothalamic and hippocampal synaptosomes. High (5 X ~ o - ~ M ) and low (5 X I P M ) concentrations of nicotine inhibited the uptake in both regions.'35 After studying the release of newly synthesized [3H]-5-HT from [3H]tryptophan in slices of rat hypothalamus, the author suggested that both muscarinic and nicotinic

58 ACETO AND MARTIN

receptors were involved in the control of 5-HT release. Muscarinic receptor stimulation resulted in inhibition of 5-HT release and nicotinic receptor stimulation activated 5-HT release."O

It appears that nicotine may also be involved in neuroendocrine function linked to 5-HT pathways. It is known that nicotine blocks the suckling-induced rise in prolactin in lactating rats,161 that it will inhibit the proestrus surge of prolactin in the rat,'62 and that p-chlorophenylalanine, a tryptophan hydroxylase inhibitor, will inhibit the suckling-induced rise,163 that it will enhance sexual behavior in female rats,164 and that 5-HT pathways have an inhibitory effect on sexual r e ~ e p t i v i t y . ' ~ ~ , ' ~ ~ Nicotine was shown to reduce 5-HT turnover and that these effects may in part, be attributable to the main metabolite ~ 0 t i n i n e . l ~ ~

V. SPECULATIONS

Nicotine has proven to be a valuable research tool. The pioneering experiments with this drug played a significant role in the development of ou r understanding of the peripheral cholinergic nervous system. The concept of a "receptive substance" for this toxic alkaloid led to the isolation and reconstitution of a nicotinic cholinergic receptor which in turn made it possible to study systemically the pathological processes that occur in myasthenia gravis and other skeletal muscle distrophies.

For a number of reasons already cited, progress in our understanding of the central cholinergic nervous system have not been as dramatic; although much remains to be learned about this system, certain patterns are now evident. Muscarinic receptors have been identified in brain by direct binding studies and they have been found to be more abundant in extrapyramidal structures and their levels appear to be much higher than that of nicotinic receptors. O n the other hand nicotinic receptors appear t o be associated more with limbic structures and sensory nuclei. It is probably not a coincident that muscarine produces Parkinsonlike effects by activation of receptors in the basal ganglia and that nicotine produces muscle relaxation, facilitation of memory and decreased irritability by an action on receptors in the LC and/or limbic system. Moreover, some of these actions may also involve noradrenergic, dopaminergic and ser- otonergic mechanisms.

Obviously much remains to be learned regarding the functional role of the central cholinergic nervous system in general and the central nicotinic cholinergic system in particular. Recent developments permit us to proceed in this direction. The availability of a stable form of nicotine with high specific activity should help to resolve the question as to whether or not the neurotoxins bind to nicotine's receptor.

Many thanks to Dr . William L. Dewey for his help and comments . Supported by NIDA Grant DA 02384.


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