Publications : Books : Nicotine Addiction in Britain :

2 Physical and pharmacological effects of nicotine

2.1 Nicotine receptors and subtypes

Introductory perspective: general features of nicotinic acetylcholine receptors

Although nicotine was first used by Langley at the turn of the century in his classic experiments that gave rise to the concept of a 'receptive substance', and hence 'receptor', it was many years before receptors for nicotine in the brain were identified. Langley, and later Dale (who distinguished the nicotinic and muscarinic actions of acetylcholine), examined nicotine on skeletal muscle or autonomic ganglia. The utility of muscle preparations for electrophysiological analyses, together with the model system of the electric organ from the marine ray Torpedo that provides a rich source of receptor protein, have resulted in the muscle nicotinic acetylcholine receptors (nAChR) being the best characterised ligand-gated ion channel to date.1

Briefly, the nAChR is a pentamer composed of five homologous membrane-spanning subunits around a central pore or ion channel. Two a subunits and one each of b, g and d subunits (with a change during development from g to e) are arranged in the order agadb. The two a subunits are the primary agonist binding subunits, and the co-operative binding of two molecules of acetylcholine is required to open the channel through an allosteric mechanism. The ion channel of the muscle nAChR is primarily permeable to Na+ and K+. In normal physiological conditions, opening of the channel results in an inward flux of Na+ producing local depolarisation that can lead to muscle contraction. The nAChR channel remains open for only a brief period before undergoing a series of conformational changes that produce a desensitised state (Fig 2.1). In this configuration, the channel is closed to ions and is refractory to activation by agonist, although agonist can still bind to the receptor with enhanced affinity. Low concentrations of agonist can push the receptor into the desensitised state without going through the open state. These properties have implications for the functional effects of nicotine during tobacco use.


Fig 2.1. Conformational states of the nicotinic acetylcholine receptor.

 

Heterogeneity of neuronal nicotinic acetylcholine receptors

Neuronal nAChRs share the same overall structural and functional features of the muscle nAChR prototype, but generally comprise two a and three b subunits, and also involve different subunits to those expressed in muscle. The number of neuronal nAChR subunit genes identified to date (9 in mammals, including humans) could generate a vast number of pentameric combinations. Certain combinations are not viable, but there is nevertheless considerable heterogeneity (for review, see Ref 2). The different pharmacological and biophysical properties of these subtypes will have a number of implications for the actions of nicotine (Table 2.1), although the subunit composition and physiological function of any nAChR subtype in the brain is only poorly delineated (see below). Hypothetically, a smoker's average plasma level of nicotine sustained throughout the day may be sufficient to desensitise (partially or fully) a population of nAChR with high affinity for nicotine (also making it unresponsive to the natural ligand, acetylcholine), while the raised nicotine levels from a bolus of cigarette smoke will activate/desensitise other, less sensitive nAChR subtypes to varying degrees. The perceived effect of smoking (stimulation/relaxation) will be the balance between these processes.

Table 2.1. Implications of nicotinic acetylcholine receptor (nAChR) heterogeneity.

nAChR subtypes may vary with respect to:

  • anatomical localisation (pathways)
  • cellular localisation (presynaptic/nerve terminals or cell soma/dendrites)
  • proximity to sites of ACh release (postsynaptic or extrasynaptic)
  • sensitivity to nicotine (activation vs desensitisation)
  • rate of desensitisation
  • Ca2+ permeability (cell regulation, long-term potentiation)
  • regulation (allosteric/phosphorylation)

Nomenclature of nicotinic acetylcholine receptor subunits

The nine neuronal nAChR subunit genes cloned to date from mammals and shown to be expressed in neurones are designated a (a2-alpha7) or b (b2-b4), with muscle a and b subunits being the first in the series. a subunits are characterised by the presence of a pair of adjacent cysteine residues close to the acetylcholine binding site in the N-terminal domain, and shown to be important for agonist binding. Hence, a subunits have also been referred to as 'agonist binding subunits', whereas b subunits have been called 'structural subunits' on the presumption that they contribute to the formation of the nAChR channel but play no role in agonist recognition. These distinctions are incorrect. The a5 subunit appears to be incapable of binding agonist because it lacks another key residue - a tyrosine found in all other a subunits upstream of the pair of cysteine residues mentioned above.3 Moreover, b subunits do influence nAChR pharmacology: the agonist binding site is thought to reside at the interface between an a and the adjacent subunit. Thus, exchanging b4 for b2 in an ab hetero-oligomer expressed experimentally in a system such as Xenopus oocytes resulted in an increase in sensitivity to nicotine.4 The naming of nAChR subunits and subtypes has recently been reviewed by a subcommittee of the International Union of Pharmacology Nomenclature Committee.5

Prevalence and distribution of nicotinic acetylcholine receptor subunits

Evidence from in situ hybridisation and immunocytochemistry indicates that the various nicotinic subunits have different, but overlapping distributions in the brain (the anatomical distribution in the human brain is reviewed by Gotti et al6). They differ in abundance, with b2 the most widely expressed subunit. The loss of nicotine self-administration behaviour in knock-out mice lacking this subunit suggests that it contributes to nAChR relevant to nicotine dependence.7 alpha7 and alpha4 are also widely expressed, but with complementary distributions: for example, there are high levels of alpha7 in hippocampus, but low expression of a4. The other subunits have more restricted distributions: for example, a6 and b3 gene transcripts are limited to areas containing dopamine and noradrenaline cell bodies,8 and the a6 subunit has been localised to dopamine neurones in the ventral tegmentum (VTA).9 Lack of coincident expression prevents certain subunit combinations from occurring, but the distribution patterns are compatible with enormous heterogeneity of nAChR subtypes. For example, the VTA expresses a3, a4, a5, alpha7 and b2 in addition to a6 and b3. Heterologous expression systems have demonstrated restrictions in the assembly of subunits to create functional nAChR (summarised briefly in Table 2.2).

Neuronal nicotinic subunits have also been reported to be expressed in certain non-neuronal cells, including lymphocytes, skin, epithelial cells and small-cell lung carcinoma.6 The novel alpha9 subunit is expressed in sensory end organs, notably the outer hair cells of the auditory system.10

Subunit composition of native nicotinic acetylcholine receptors

The question then arises whether these nAChR formed in experimental systems represent native nAChR occurring in the brain. This is a much less tractable problem, with few definitive answers so far. alpha7-like nAChRs have pharmacological and biophysical properties and high Ca2+ permeability, comparable to homomeric alpha7 nAChR in expression systems, but there is debate about the possible inclusion of additional types of subunit.11 More recently, data in chicken12 and in rat cardiac ganglia13 support variants of alpha7-like nAChR. Although alpha7 nAChRs are not particularly sensitive to nicotine (EC50 human alpha7 ~ 40 µM), it has been demonstrated in vitro that this subtype can enhance glutamate release from hippocampal nerve endings in response to 500 nM nicotine. This is interpreted as an ability to respond to the levels of nicotine in smokers.14

Table 2.2. Nicotinic acetylcholine receptor (nAChR) subtypes: evidence from heterologous expression systems.

Immunoprecipitation studies provide good evidence for the occurrence of alpha4beta2 hetero-oligomers.15 This nAChR has the highest sensitivity to nicotine (EC50 ~ 0.1-1 µM), but its functional status in smoking is equivocal because it can be desensitised by lower concentrations.16 Other nAChR subtypes/subunits are less abundant in the brain:

  • a3a5b4/b2 nAChR mediate synaptic transmission in sympathetic ganglia
  • a3/a4+b4/b2+a5 nAChR occur in the brain (for review, see Ref 2)
  • nAChR containing alpha3 and b2 subunits can modulate dopamine release in striatum.17

The expression of a6 and b3 subunits predominantly in catecholaminergic nuclei, including the VTA, makes them candidate subunits of nAChR operative in the Reward pathway. Transgenic animals deficient in (or overexpressing) particular subunits will help to clarify their roles. To date, b2, alpha7, b3 and alpha4 knock-out mice have been reported, but so far only the b2 knock-out has been characterised with respect to rewarding and other behaviours.7,18

Pharmacological distinctions

There is a relative lack of specific ligands for studying nAChR. Nature has provided the most selective and potent tools, although the burgeoning interest in nAChR as a target by the pharmaceutical industry is generating promising new compounds for research purposes.

Radioligands. Radiolabelled ligands are useful for:

  • quantifying and mapping the distribution of nAChR
  • determining pharmacological profiles
  • assaying nAChR during biochemical procedures such as purification.

Radioligands have defined two major populations of nAChR. The first is alpha7-type nAChR. The snake toxin abungarotoxin (aBgt) is a specific and almost irreversible alpha7 antagonist. [125I] or [3H]aBgt labels this subtype with an affinity of about 1 nM. Following the use of [125I]aBgt to label muscle nAChR, [125I]aBgt binding sites were characterised in mammalian brain in the 1970s, but their receptor status was controversial at that time. Cloning and expression of the alpha7 subunit in 1990 confirmed that [125I]aBgt labels an nAChR.19 A tritiated version of the Delphinium toxin methyllycaconitine (MLA) has recently been developed,20 which also selectively labels alpha7-type nAChR with an affinity of about 1 nM. However, its binding is reversible and it can discriminate between muscle and alpha7 nAChR.

The second defined population is alpha4beta2 nAChR. Tritiated nicotine was first reported to bind to brain tissue by Romano and Goldstein in 1980.21 Since then, [3H]nicotine binding has been extensively characterised, and the binding sites appear identical to those labelled with other agonists, namely [3H]acetylcholine, [3H]cytisine, [3H]methylcarbamylcholine and [3H]ABT418. Immunoprecipitation experiments indicate that these sites correspond to alpha4beta2 nAChR.15 Consistent with this, [3H]nicotine binding is absent in b2 knock-out mice.18

These radiolabelled agonists bind to brain tissue with affinities of about 1-10 nM. Affinities of this order are required for the successful use of a radioligand so, although by definition, all nAChR bind nicotine, only the alpha4beta2 type appears to be sensitive enough for this binding to be measurable in an assay. It should be noted that the nanomolar binding affinities of these agonists are lower than their EC50 values for activating alpha4beta2 nAChR (0.1-10 µM), and reflect binding to the high affinity, desensitised state of the nAChR (Fig 2.1).16 While competition binding assays with such agonists can provide information on the relative potencies of competing ligands, the results can be misleading with respect to receptor activation. Binding assays do not distinguish agonists from antagonists, and full inhibitors of binding can be partial agonists with respect to nAChR function. [3H]nicotine has also been used for positron emission tomography studies in humans to examine the distribution and changes in numbers of nAChR.22

The portfolio of tritiated agonists was recently extended by the advent of [3H]epibatidine, secreted from the skin of a South American frog. It is the most potent nicotinic agonist to date and binds to brain tissue with an affinity of about 10-100 pM.23 As well as labelling the same nAChR as the other tritiated agonists discussed above (alpha4beta2), [3H]epibatidine also identifies one or more additional nAChR.24 The persistence of [3H]epibatidine labelling of the medial habenula and interpeduncular nucleus in b2 knock-out mice is interpreted in favour of an nAChR containing alpha3 and b4 subunits. [3H]epibatidine labels a3-containing nAChR in sympathetic cell lines lacking the alpha4 subunit. Thus, [3H]epibatidine is useful for labelling additional subtypes, but this can be problematic in tissue with a heterogeneous population of nAChR such as brain, where multiple subtypes are present.

The minor snake venom component, known as neuronal bungarotoxin (nBgt), has been iodinated and used to label brain nAChR. In the presence of aBgt to block its binding to alpha7-type nAChR, [125I]nBgt labels a smaller population of sites tentatively equated with a3-containing nAChR.25 nBgt is not commercially available, and concerns about its purity and stability, together with loss of activity on iodination, have limited its utility.

Antibodies have also been valuable tools for histological localisation (eg Ref 9), isolation,3 purification11 and quantitation26 of specific neuronal nAChR subtypes and subunits.

Functional studies: agonists. Although all nAChR, by definition, respond to nicotine, they differ with respect to the nicotine concentrations required for activation or desensitisation. Selected EC50 values for nicotine and acetylcholine of heterologously expressed human nAChR subtypes are compared in Table 2.3.

Table 2.3. EC50 values (µM) for nicotine and acetylcholine at nicotinic acetylcholine receptor subtypes.6.

  alpha4beta2 alpha3beta2 alpha3beta4 alpha7
Nicotine 5 132 80 40
Acetylcholine 68 440 203 79

No agonists are truly specific for a particular subtype of nAChR, but may differ in potency by one or two orders of magnitude. The tobacco alkaloid anabasine and its synthetic derivative GTS-21 (also known as DMXB) have diminished potency and efficacy at alpha4beta2 nAChR in Xenopus oocytes, relative to alpha7 nAChR at which they are highly efficacious. Thus GTS-21 has been referred to as functionally selective for alpha7.27 This ignores its possible interactions with minor subtypes of nAChR. Choline is reputedly an alpha7-selective agonist.28 Although it is not very potent (EC50 1.6 mM), micromolar concentrations that might occur in the brain readily desensitise the alpha7 nAChR. Thus, actions of nicotine must be viewed against a backdrop not only of endogenous acetylcholine but also of its principal metabolite. With regard to efficacy, it seems that the alpha4beta2 nAChR is particularly prone to submaximal activation/partial agonism.29

Table 2.4. Selectivity and potency of some nicotinic receptor antagonists.

Antagonist

Source

Subtype selectivity

Potency IC50

aBgt

Bungarus multicinctus

alpha7

1 nM

MLA

Delphinium sp

alpha7 (>alpha3>alpha4)

1 nM

alphaconotoxin IMI

Conus imperialis

alpha7 (>alpha9>alpha1)

220 nM

alphaconotoxin MII

Conus magnus

alpha3beta2

~1 nM

alphaconotoxin AuIB

Conus aulicus

alpha3beta4

~1 µM

DHbE

Erythrina

alpha4 (>alpha3>alpha7)

0.1 µM

mecamylamine

synthetic

alpha3 (>alpha4>alpha7)

0.1 µM

aBgt = abungarotoxin; DHbE = dihydro-b-erythroidine; MLA = methyllycaconitine. 

Competitive antagonists. Competitive antagonists (Table 2.4) compete with agonists for binding to the same or overlapping sites (see Ref 6 for a more comprehensive account). They can exhibit a high degree of nAChR subtype selectivity. Competitive antagonists acting at alpha4beta2 nAChR displace [3H]nicotine binding, while those acting at alpha7 nAChR displace [125I]aBgt binding.

  • alphaBgt, as already noted, binds specifically to alpha7-type nAChR and is diagnostic for the presence of this subunit (although it will also label nAChR composed of the alpha8 subunit, found only in chickens, and the alpha9 and muscle nAChR). Low nanomolar concentrations of aBgt block alpha7 responses. The blockade is reversible only very slowly, but its large size and slow kinetics can be a disadvantage. aBgt does not block other mammalian neuronal nAChR subtypes.
  • MLA is alpha7-selective rather than specific. 1-10 nM concentrations will reversibly block alpha7-type nAChR, while 100 nM MLA begins to block a3-type receptors.30 Concentrations above 1 µM are required to antagonise alpha4beta2 responses.
  • Dihydro-beta-erythroidine (DHbE) preferentially blocks alpha4beta2 nAChR at submicromolar concentrations that do not impair the function of alpha7 nAChR.
  • alphaConotoxins (Table 2.4), peptides with nicotinic antagonist activity, are produced by marine cone snails. They have high potency and selectivity, and are proving very useful for in vitro studies.31

Non-competitive antagonists. Non-competitive antagonists do not bind at the agonist binding site. In many cases, they act by occluding the ion channel of the nAChR. If they enter the channel, their action will be voltage-dependent. Compounds which block the channel may lack specificity and also block non-nicotinic receptor channels.

  • Mecamylamine is the classical nicotinic antagonist, much used for in vivo studies because it accesses the brain freely; it has also been used for smoking cessation in humans. It preferentially inhibits a3- and then a4-type nAChR.6 Its mechanism of action is not understood.
  • Chlorisondamine is a ganglionic blocker that produces a remarkably long lasting (>2 weeks) blockade of nAChR.32
  • The N-methyl-D-aspartate (NMDA) channel blockers MK 801 and PCP will inhibit nicotinic responses,33 albeit at higher concentrations than for NMDA receptor blockade.
  • Bupropion, a dopamine uptake inhibitor recently marketed for smoking cessation, has been found to be a non-competitive inhibitor of nAChR responses.34
  • Agonist molecules (including nicotine and acetylcholine) at high concentration will block the nicotinic channel,35 but it is unlikely that this will have functional significance in vivo.

Conclusions

In summary, nAChR are heterogeneous ligand gated ion channels, but in almost all cases the precise subunit composition of any native nAChR has not been unambiguously assigned. nAChR expressed artificially in cell lines or Xenopus oocytes may not therefore give a true reflection of the properties of nAChR existing in the brain.36 The propensity of nAChR to desensitise will confound the interpretation of the effects of nicotine in vivo. Binding assays with radiolabelled or competing agonists will reflect the desensitised state of the nAChR, and the ability to bind to the receptor does not necessarily reflect the functional properties of the ligand. New drugs and toxins with nAChR subtype selectivity are emerging, and will help to unravel the contributions of nAChR subtypes to normal brain function and nicotine dependence.

2.2 Pharmacology and pharmacokinetics of nicotine

Chemistry of nicotine in tobacco smoke

Cigarette smoke is composed of volatile and particulate phases. Some 500 gaseous compounds including nitrogen, carbon monoxide (CO), carbon dioxide, ammonia, hydrogen cyanide and benzene, have been identified in the volatile phase which accounts for about 95% of the weight of cigarette smoke; the other 5% is accounted for by particulates. There are about 3,500 different compounds in the particulate phase, of which the major one is the alkaloid nicotine. Other alkaloids include nornicotine, anatabine, and anabasine.37 The particulate matter without its alkaloid and water content is called tar. Many carcinogens, including polynuclear aromatic hydrocarbons, N-nitrosamines and aromatic amines, have been identified in cigarette tar.

Nicotine is a tertiary amine consisting of a pyridine and a pyrrolidine ring. There are two stereoisomers of nicotine: (S)-nicotine is the active isomer which binds to nicotinic cholinergic receptors and is found in tobacco. During smoking, some racemisation takes place, and small quantities of (R)-nicotine, a weak agonist of cholinergic receptors, are found in cigarette smoke.

Absorption of nicotine from tobacco products

Nicotine is distilled from burning tobacco, and small droplets of tar containing nicotine are inhaled and deposited in the small airways and alveoli. Nicotine is a weak base, and thus its absorption across cell membranes depends on the pH. The pH of smoke from most American cigarettes (blonde tobacco) is acidic (pH 5.5). At this pH, nicotine is mostly ionised and does not freely cross cell membranes. Consequently, nicotine from the blonde tobacco cigarette smoke is not absorbed through the buccal mucosa. However, the pH of smoke from tobacco in pipes and cigars is alkaline (pH 8.5), at which pH nicotine is mostly unionised and well absorbed from the mouth.

When nicotine from cigarette smoke reaches the small airways and the alveoli of the lung, it is buffered to physiological pH and rapidly absorbed into the pulmonary alveolar capillary and venous circulation, and hence directly into systemic arterial blood. From here, nicotine is distributed quickly throughout the body. It takes about 10-19 seconds for nicotine to reach the brain. The arterial blood perfusing the brain contains levels of nicotine following cigarette smoking which exceed venous levels by a factor of two- to sixfold.38,39 Levels of nicotine in the plasma as well as in the brain decline rapidly as a result of distribution to peripheral tissues, and of excretion and elimination. Since no current nicotine replacement therapy (NRT) formulation uses the pulmonary route of absorption, none can mimic either the extremely high and rapidly acquired arterial nicotine concentrations which occur when tobacco products are inhaled, or the rapid pharmacological effect that this produces. The typical time course of the increase in nicotine levels in venous blood after smoking a cigarette is also faster than after most nicotine replacement products (Fig 2.2)40 (see Chapter 7.2 for further discussion of NRT).


Fig 2.2. Schematic diagram showing rise in venous blood nicotine levels after smoking a
cigarette and after using different nicotine replacement therapy products, following
overnight abstinence from cigarettes (data adapted from Ref 40) .

 

When smokers smoke multiple cigarettes during the day, there are oscillations between peak and trough plasma nicotine levels. However, because of its half-life of two hours, nicotine accumulates over 6-8 hours, reaching levels in the plasma typically ranging from 20-40 ng/ml, which then fall progressively during the night.41 There is considerable variation between people both in their plasma nicotine levels and in their intake of nicotine from a cigarette.42,43 The smoker can manipulate the intake of nicotine from different cigarettes to achieve and maintain the desired level of nicotine (see Chapter 6) by changing puff volume, the number of puffs per cigarette, the intensity of puffing, the depth of inhalation, and by blocking ventilation holes in the filter.44 No other nicotine product provides this degree of control over the rate and quantity of nicotine absorption.

Nicotine from chewing tobacco and snuff is absorbed through the oral and/or nasal mucosa. Plasma nicotine concentrations rise more slowly with these products than with cigarettes, reaching plateau levels by about 30 minutes, declining slowly over approximately two hours. Nicotine is continually released throughout the time of exposure.

The rapid absorption of nicotine from cigarette smoking, and the high arterial levels which reach the brain as a result, allow for rapid behavioural reinforcement from smoking. The falling nicotine levels between the smoking of individual cigarettes allow time for the brain nicotinic receptors to become somewhat resensitised between the cigarettes. Rapid delivery of nicotine to the brain also allows the smoker to manipulate and titrate the dose of nicotine from a cigarette to achieve a desired effect. Tolerance to the toxic effects of nicotine such as nausea rapidly develops and persists, while the reinforcing effects of nicotine are renewed with each cigarette. Thus, what is typically a noxious pharmacological experience for the novice smoker becomes an addictive pharmacological experience for the experienced smoker.

Nicotine metabolism

Nicotine is extensively metabolised, primarily in the liver but also to a small extent in the lung and brain. About 70-80% of nicotine is metabolised to cotinine via C-oxidation, and another 4% to nicotine N'-oxide.45,46 There is considerable interindividual variability in the rate of metabolism of nicotine to cotinine,43 but it is also established that regular smokers metabolise nicotine more slowly than non-smokers.47,48 Several cytochrome P450 enzymes, as well as flavin mono-oxygenases, are purported to play a role in nicotine metabolism, but CYP2A6 appears to be the principal enzyme involved in converting nicotine to cotinine, via an intermediary metabolite nicotine D1(5')-iminium ion.49,50 Cotinine is further metabolised to trans-3'-hydroxycotinine, the major nicotine metabolite found in urine.51 CYP2A6 is also the enzyme thought to be responsible for the oxidation of cotinine.52 Cotinine has a much longer half-life than nicotine (14-20 hours), and consequently is used as a marker of nicotine intake.53-55 Nicotine, cotinine, and trans-3'-hydroxycotinine are further metabolised by glucuronidation.45 When using tobacco or nicotine medications, the strongest predictor of an individual's plasma nicotine levels is nicotine clearance. Cotinine levels are most strongly correlated with nicotine dose and, to a lesser extent, fractional conversion of nicotine to cotinine and cotinine clearance.42 Renal clearance of nicotine depends on urine pH, being higher in acidic urine and lower in alkaline urine, and accounts for 2-35% of total nicotine clearance.56

Ethnic differences in nicotine metabolism have recently been demonstrated. African-Americans have been shown in several studies to have higher levels of cotinine, normalised for cigarettes smoked per day.57,58 Recently, Perez-Stable et al administered deuterium-labelled nicotine and cotinine to African-American and Caucasian smokers, and found that the former metabolised cotinine more slowly than Caucasians.59 African-Americans appear to do this both by slower oxidation to trans-3'-hydroxycotinine and by slower N-glucuronidation. They were also shown to take in 20% more nicotine per cigarette, which means an intake of 20% more tobacco smoke per cigarette. This may be related to the fact that the majority of African-Americans smoke mentholated cigarettes, whereas relatively few Caucasians smoke such cigarettes. Menthol cools the airways and might be associated with a greater volume or depth of inhalation. Taking a greater dose of nicotine per cigarette and also metabolising cotinine more slowly explain the higher cotinine levels per cigarette in African-Americans than in Caucasians. Greater smoke intake per cigarette might also explain the higher lung cancer risks, normalised for cigarette consumption, observed in African-Americans.60

Based on the idea that smokers regulate levels of nicotine in their bodies by adjusting how many cigarettes they smoke or how they smoke them, it is reasonable to speculate that smokers who metabolise nicotine more rapidly may need to take in more cigarette smoke, and vice versa. Pursuing this idea with respect to nicotine addiction, Pianezza et al61 have studied the prevalence of mutant genes for CYP2A6, the liver enzyme primarily responsible for nicotine metabolism. They have reported that the presence of a CYP2A6*v1 mutant allele, presumably reflecting slower than normal nicotine metabolism, is associated with a lower risk of progression from experimental to addictive smoking. This study was in a relatively small group of smokers, and the effect of the CYP2A6*v1 alleles on the rate of nicotine metabolism has not yet been demonstrated, so the findings remain speculative.

Cardiovascular, endocrine and metabolic effects of nicotine

Nicotine effects on the cardiovascular system are mediated by sympathetic neural stimulation associated with an increase in the levels of circulating catecholamines. Nicotine causes sympathetic stimulation through central and peripheral mechanisms. Central nervous system (CNS)-mediated mechanisms include activation of peripheral chemoreceptors, particularly the carotid chemoreceptor, and direct effects on the brain stem and spinal cord.62 Peripheral mechanisms include release of catecholamines from the adrenal glands and vascular nerve endings. These effects of nicotine result in an acute increase in heart rate and blood pressure when nicotine is delivered via cigarette smoking, chewing gum, nasal spray or intravenous (IV) infusion.43,63,64 Transdermal nicotine causes less intense changes.65 Substantial, but incomplete, tolerance develops to the cardiovascular effects of nicotine, with a short half-life of approximately 35 minutes; there is a persistent effect of nicotine, which is about 20% of the predicted effect if tolerance did not exist.66 After brief dosing, there is no acute development of tolerance when the arterial plasma nicotine levels are determined. The difference in the observations between venous and arterial plasma levels may be because the levels of nicotine in arterial blood reflect the concentration of nicotine at the receptors, whereas the concentrations of nicotine in the venous blood reflect the levels of nicotine after distribution to the tissues. There is a lag time between the decline in venous compared to arterial levels, which could account for 'pseudotolerance', as assessed by venous blood level-response curves.38,67

Nicotine differentially affects blood flow to different organs, causing vasoconstriction in some vascular beds (eg skin) and vasodilatation in others (eg skeletal muscle). Cutaneous vasoconstriction results in a decrease in the fingertip temperature.43

Nicotine induces vasoconstriction in coronary arteries, as evidenced by a lack of increased blood flow in response to increased oxygen demand,68 and by direct observation, particularly in atherosclerotic arteries.69-71 Coronary vasoconstriction appears to be mediated by catecholamines, and can be abolished by the a-adrenergic blocker phentolamine.70

Nicotine affects the metabolic rate, and smokers weigh on average 4 kg less than non-smokers.72 The lower weight is maintained by an increase in metabolic rate, with concomitant appetite suppression.72 Both cigarette smoking and IV nicotine increase the metabolic rate.73 Quitting cigarette smoking is associated with an increase in appetite and caloric intake, with a subsequent weight gain over 6-12 months. Thereafter, both caloric intake and weight return to baseline.74

Nicotine has a variety of endocrine effects, including release of ACTH and cortisol,75 and of b-endorphin,76 and has been shown to have analgesic effects.77

2.3 Pathophysiological effects and toxicity of nicotine

Nicotine has a number of toxic or adverse effects, some of which are also potentially relevant in disease pathogenesis. For the purposes of this discussion, these are categorised as:

  • acute systemic effects
  • local toxic effects
  • chronic systemic toxicity.

Acute systemic effects

Acute systemic toxic effects of nicotine include:78

  • CNS effects: headache, dizziness, insomnia, abnormal dreams, nervousness
  • gastrointestinal (GI) distress: dry mouth, nausea, vomiting, dyspepsia, diarrhoea
  • musculoskeletal symptoms: arthralgias, myalgia.

In general, in relation to NRT for smokers, these effects tend to be mild. Interpretation of the CNS effects of nicotine in smokers who have recently quit smoking is complicated by the potential emergence of nicotine withdrawal symptoms that can be similar to some of the toxic effects of nicotine.

Local toxic effects

Local toxic effects of nicotine include:

  • sore mouth and mouth ulcers from nicotine gum
  • cutaneous sweating, itching, burning, erythema from patch application
  • nasal irritation with burning, itching, sneezing
  • watery eyes with nicotine nasal spray.

The mechanism of these effects is complex, but it appears to include activation of local afferent neurones and axon reflexes, with release of vasodilators such as bradykinin, substance P and histamine.79 Local reactions from skin patches generally resolve within 24-48 hours. Nasal irritation with the use of nicotine nasal spray usually resolves with the development of tolerance over 2-3 days.

Chronic systemic toxicity

The main areas of concern over chronic systemic toxic effects of nicotine relate to effects on:

  • cardiovascular disease, particularly coronary heart disease and stroke
  • the aggravation of hypertension
  • delayed wound healing
  • peptic ulcer disease
  • pregnancy (discussed further in Section 2.4) and reproductive health.

Cardiovascular disease. As described in Section 2.2, nicotine exerts cardiovascular effects primarily by activating the sympathetic nervous system, resulting in an increase in heart rate, blood pressure and cardiac contractility, thereby increasing myocardial oxygen consumption and demand for blood flow. Nicotine may also limit coronary blood flow by constricting coronary arteries, an effect more prominent in individuals with underlying coronary atherosclerosis.80 Nicotine has also been associated with coronary spasm.81 Other important cardiovascular toxins in cigarette smoke include CO, which reduces oxygen delivery to the heart, and oxidant gases, which may be responsible for endothelial dysfunction and platelet activation. Effects on endothelial function and platelets, mediated by oxidant gases, may be responsible for the thrombosis and/or coronary vasoconstriction that further restricts blood flow to the heart.82

Nicotine per se, at least when administered transdermally, does not activate platelets and probably does not contribute to thrombosis.83 Prostacyclin is an endothelial-derived vasodilator and inhibitor of platelet aggregation. Nicotine has been shown to inhibit prostacyclin synthesis in vitro,84 but studies of smokers and of smokeless tobacco and nicotine patch users found no evidence of decreased prostacyclin production.85,86

Alterations in the lipid profile, with an increase in very low-density lipoprotein (VLDL) and low-density lipoprotein and a decrease in high-density lipoprotein (HDL) cholesterol, are believed to be important mechanisms in smoking-induced atherosclerosis. Nicotine, via release of catecholamines, increases lipolysis and releases free fatty acids which are then taken up by the liver.87 This might be expected to promote the synthesis of VLDL and decrease the synthesis of HDL, consistent with the changes seen in smokers,88 but such abnormalities have not been found in people undergoing NRT.89,90

Studies of the effects of smokeless tobacco provide evidence regarding the cardiovascular safety of nicotine. Smokeless tobacco users are exposed to the same levels of nicotine in the body as cigarette smokers, but are not exposed to tar, CO and oxidant gases.91 There is a significant pharmacokinetic difference in the rate of absorption, with cigarette smoking producing much higher transient arterial blood concentrations than smokeless tobacco; this must be kept in mind as a caveat in comparing nicotine exposures from the two routes.

Snuff use results in acute cardiovascular effects similar to those with cigarette smoking: that is, an increase in heart rate and blood pressure.92 Cigarette smoking has been shown to affect platelet activation, as evidenced by increased thromboxane (TX) A2 metabolite excretion, and to impair endothelial function, primarily by reducing the release of nitric oxide (NO), which has antiplatelet activity and is a vasodilator.85,86,93,94 None of the effects on TXA2 or NO is seen with snuff users, suggesting that the effects of smoking on these physiological functions are not mediated by nicotine.95 However, results from epidemiological studies of cardiovascular disease in snuff users are conflicting. One case-control study found no increased risk of myocardial infarction (MI) in snuff users,96 whereas another cohort study reported an increased risk.97 The reason for the discrepancies between these two studies is unclear.

Clinical trials of nicotine medication in patients with coronary artery disease provide another important source of information. Two controlled clinical trials of transdermal nicotine to aid smoking cessation in patients with cardiovascular disease have found no evidence that nicotine is injurious.98,99 Importantly, many of the subjects in these studies continued to smoke while using transdermal nicotine, resulting in plasma nicotine levels that might have been higher than those seen with smoking alone. A study by Mahmarian et al100 examined quantitative thallium myocardial perfusion defect size in smokers with coronary heart disease prescribed nicotine patches to aid smoking cessation. When these subjects used 21-mg nicotine patches, their blood nicotine and cotinine levels were twice those seen with smoking alone, but their expired CO levels were reduced by about 50% because they smoked fewer cigarettes. The total and reversible thallium perfusion defect sizes were significantly reduced during the patch use, despite the high nicotine levels. This study suggests that components of cigarette smoke other than nicotine are responsible for acute ischaemia. Finally, the large Lung Health Study in patients with chronic obstructive pulmonary disease (COPD) found no increase in cardiovascular disease in smokers using nicotine gum for as long as five years.101 Thus, the clinical trial data to date support the idea that nicotine medication is not a significant risk factor for cardiovascular events, even in patients with coronary heart disease.

Sporadic case reports have been published describing patients with acute cardiovascular events during the use of NRT. They include descriptions of patients who developed atrial fibrillation, acute MI or stroke.102-104 Some of these patients were smoking at the same time as they were using transdermal nicotine. There was no consistent pattern with respect to how long these individuals had been using nicotine, time of day of the adverse event, or any other factor clearly identifying these events as related to the pharmacological effects of NRT. It must be recognised that cardiovascular disease is common in the age group of smokers undergoing smoking cessation therapy, and that some adverse cardiovascular events are expected to occur by chance in any 1-3 month period (the duration of most courses of NRT). A US Food and Drug Administration advisory committee reviewed the cases of MI in people using nicotine patches in 1992 and judged the events not to be causally related to their use.

Aggravation of hypertension. Although cigarette smoking and nicotine per se acutely increase blood pressure, cigarette smoking is not a risk factor for chronic hypertension.105 Conceivably, factors such as lower body weight or differences in dietary intake in smokers might confound any blood pressure elevation due to nicotine.

Progression of chronic hypertension to accelerated or malignant hypertension, however, is much more likely in cigarette smokers.106,107 Nicotine may contribute to the acceleration of hypertension by aggravating vasoconstriction. Animal studies indicate that nicotine may reduce renal blood flow which, in a patient with marginal renal blood flow due to hypertensive vascular disease, could cause renal ischaemia and aggravate hypertension.108 There is therefore concern about using nicotine therapy in patients with severe hypertension.

Delayed wound healing. A prominent cardiovascular effect of nicotine is to reduce skin blood flow and subcutaneous tissue oxygen.109,110 Adequate blood flow to the skin is important in the process of wound healing. Animal and human studies indicate that exposure to cigarette smoke or nicotine impairs the healing of skin flaps after plastic surgery.111-113 It is likely that nicotine exposure in people will also delay wound healing after surgical procedures, although few clinical data are available on this issue.

Acid peptic disease. There is evidence that cigarette smoking aggravates gastro-oesophageal reflux, an effect documented in humans using transdermal nicotine.114,115 Cigarette smoking is a strong risk factor for the development, delayed healing and relapse of peptic ulcer disease.116 Nicotine could contribute both to reflux disease and gastric ulcer by provoking reflux of bile,115 and to duodenal ulceration by decreasing bicarbonate secretion, an effect that may be related to depression of prostaglandin synthesis.117-119 However, the results of the Lung Health Study do not support a role for nicotine per se in causing peptic disease. This study found no evidence that nicotine gum used for several years increased the risk of peptic ulcer disease, but rather that gum use had a borderline protective effect.101

Cancer. Because of the strong causal link between tobacco use and cancer, there has been concern as to whether nicotine contributes to cancer aetiology. Nicotine has not been shown to be carcinogenic in animals. There are theoretical concerns about nicotine and cancer related to metabolic activation or to stimulation of nicotinic cholinergic receptors that regulate release of lung tumour growth factors.120,121 Nicotine could also contribute to cancer if it is nitrosated to form carcinogenic tobacco-specific nitrosamines.122 Tobacco-specific nitrosamines are found in tobacco itself, resulting from the reaction of nitrites and alkaloids in the cigarette tobacco curing process. Nitrosamines can also be formed in the GI tract after oral administration of secondary amines and nitrites. Human exposure to nitrites occurs in the diet, and nicotine enters the GI tract both by swallowing products such as nicotine from nicotine medications and also by diffusion of nicotine from the bloodstream and ionic trapping by the acidic gastric fluid. Studies of urinary concentrations of nicotine-derived nitrosamines in humans exposed to nicotine are underway. It is likely that some nitrosation occurs, but the unresolved question is whether the amount of nicotine-derived nitrosamines is sufficient to contribute to cancer.

Although there are some concerns, as noted above, the risk of nicotine-related cancer, if any, is likely to be small or insignificant in tobacco users who are exposed to high concentrations of many carcinogens.

2.4 Effects of nicotine on mother and fetus in pregnancy

An understanding of the effects of nicotine during pregnancy is important both in relation to how cigarette smoking produces its adverse effects and also in balancing the potential risks and benefits of NRT to aid smoking cessation in pregnant women. The injurious effects of smoking (and potentially nicotine) on pregnancy have been summarised in Section 1.4 and can be considered in two categories:

  • those that affect the pregnancy itself
  • those that specifically affect the fetus during and after pregnancy.

Pregnancy

It is still unclear whether the injurious effects of cigarette smoking in pregnancy are due to nicotine - and, if so, which effects. Numerous other toxins in cigarette smoke, such as CO, oxidant gases, and heavy metals including lead and cadmium, could adversely affect the placental circulation and/or fetal physiology and development. The best studied tobacco smoke toxins during pregnancy are nicotine and CO. CO binds tightly to maternal haemoglobin, and even more tightly to fetal haemoglobin, reducing oxygen carrying capacity and impairing oxygen release from blood to fetal tissues. The result of CO exposure in the range of 5-10% carboxyhaemoglobin (similar to that seen in smokers) is a significant reduction in oxygen delivery, with resulting fetal hypoxia.123 Animal studies show that CO exposure during pregnancy can reduce birth weight,124,125 produce functional and structural abnormalities in the fetal brain,126,127 and result in cognitive behavioural abnormalities in the newborn.128,129 Oxidant gases are believed to impair NO formation and release from endothelial cells, including those found in the placenta, and could contribute to placental vascular insufficiency.94,130 Lead is a well studied reproductive toxin which, among other effects, produces cognitive impairment in the newborn.131 Thus, any consideration of the risks and benefits of nicotine during pregnancy must consider the numerous other proven toxins that are part of tobacco smoke.

A major concern about the effects of nicotine per se during pregnancy is that nicotine could constrict placental blood vessels, producing a state of placental and fetal hypoperfusion. This phenomenon has been demonstrated in experimental animals receiving high doses of IV nicotine.132-134 However, the human placental circulation has considerable circulatory reserve, and studies in pregnant smokers either smoking cigarettes or receiving nicotine per se have not demonstrated evidence of placental hypoperfusion.135 Thus, the vascular insufficiency module of smoking related fetal injury can be questioned, and the role of nicotine in causing adverse obstetrical outcomes remains to be established.

The fetus

The other major concern is that nicotine may have direct adverse effects on the developing fetus. Experimental work by Slotkin136 and others has shown that exposure of pregnant rodents to nicotine results in impaired development of nicotinic cholinergic and other brain receptors in the offspring. Offspring so exposed have also been shown to have behavioural abnormalities, and to cope less well with hypoxic stress - the latter being a putative model for sudden infant death syndrome. Until otherwise demonstrated, it must therefore be assumed that nicotine has potential injurious effects on the developing fetus. However, since cigarette smoking exposes the individual to both nicotine and many other toxins, it seems clear that smoking is likely to be far more hazardous than nicotine obtained from alternative, cleaner sources such as nicotine replacement products.

The practical consequences of this for replacement therapy are discussed further in Chapter 7.

2.5 Animal self-administration and nicotine addiction

The concept of addiction has at its core the idea of compulsive use, as reflected in powerful drug-seeking and drug-taking behaviour. In IV self-administration (IVSA) experiments, animals learn to administer drugs to themselves. Typically, the animal has the opportunity to press a lever; when it does so, it receives an automatic IV infusion of a drug (through a chronically-indwelling venous catheter). Several animal species, notably rats and monkeys, will press levers to obtain injections of the 'classical' addictive drugs such as morphine, heroin, amphetamine, cocaine, barbiturates and benzodiazepines. Large amounts of these drugs can be self-administered in this way. Furthermore, animals will work very hard to obtain the drugs, for example, pressing a switch thousands of times, for hours on end, to obtain drugs. This drug-seeking and drug-taking behaviour can dominate the animals' behavioural repertoire to the detriment of normal behaviour, just as in cases of serious drug abuse in humans. In fact, under appropriate conditions, animals will administer to themselves most of the drugs abused by humans. IVSA in animals is therefore a suitable animal model for the study of drug dependence in humans.

It has been established that monkeys, dogs, rats and mice can all exhibit nicotine IVSA. Monkeys have pressed levers for nicotine at rates similar to those at which they pressed levers for cocaine.137 In these experiments, the nicotine or cocaine was paired (associated) with brief flashes of light, which in this model were functionally equivalent to the smell and taste stimuli associated with smoke inhalation. Both the nicotine and the light stimuli served as rewards for these animals, the latter by virtue of association with the nicotine. Nicotine IVSA has also been demonstrated in monkeys using simpler procedures without associated light stimuli, although rates and consistency of responding for the drug were less striking.138,139 Dogs have also learned to press pedals to activate IV injections of nicotine. Up to several hundred pedal presses were made to obtain a single injection of nicotine, indicating that its rewarding effect, although powerful, was less strong than that of cocaine.140 Nicotine IVSA has also been reported in mice both with an 'acute' procedure in which stress may be a confounding factor141,142 and in chronic IVSA experiments of the usual type.7,143 There is also some evidence for IV self-administration of pure solutions of nicotine in human subjects,144 but these studies do not seem to have been reported in full. The animals studied most extensively in nicotine self-administration experiments have been rats, and these results will be considered next.

Nicotine self-administration in rats

In 1989, Corrigall and Coen succeeded in developing a rat model for nicotine IVSA.145 The rats learnt to press levers to obtain IV infusions of nicotine, but did not press an inactive (control) lever in the same test chamber. The rate of lever pressing was related to the dose of nicotine, and the lever pressing ceased if nicotine was no longer available. As in the experiments in monkeys and dogs mentioned above, the lever-pressing produced nicotine and no other substance. The nicotine served as a goal object (positive reinforcer) for these animals, much in the same way as other drugs of abuse and natural rewards.
These observations have been reproduced and extended in numerous published experiments from many different laboratories.146-152 All these studies demonstrate that rats will self-administer solutions of pure nicotine in the absence of any other reward. The validity of the observation is supported by the finding that the plasma concentration of nicotine in rats during IVSA experiments can be close to that in heavy cigarette smokers who inhale.153

Some studies have failed to find robust nicotine IVSA. To understand these results, it is essential to recognise that the extent to which a drug is self-administered depends on a multitude of procedural, environmental and genetic factors. As discussed elsewhere,154 it was at one time difficult to demonstrate even the self-administration of opiate drugs. Some studies have failed to show nicotine IVSA,155 but these used a strain of rat shown subsequently to be particularly poor in performing this task;148 strain differences in animal studies may reflect genetic factors that influence human use of tobacco. Nearly all successful studies in rats used rapid 'bolus' injections, and data suggest that rapid infusions support self-administration more effectively than slow infusions.156 Other studies used relatively slow infusions of nicotine and obtained equivocal results.157 It is, however, apparent in most experiments that nicotine is a weaker reinforcer than cocaine, its self-administration is acquired more slowly and maintained under a narrower range of conditions. It is unclear whether this reflects on either the appropriateness of the animal procedures to model the richness of the human environment or on the importance of other reinforcers in human tobacco use. Non-pharmacological sources of reinforcement may be significant,158 and recent studies provide indirect support for the presence of other psychoactive substances in tobacco in addition to the nicotine.159,160

Summary

IVSA is the primary animal model for studying drug-taking behaviour. The species thought to self-administer nicotine (and other drugs of abuse) in this way include mice, monkeys, dogs and rats. The combination of broad cross-species similarity of these animal data, plus the exceptionally high validity of drug self-administration procedures, generally strongly suggest that they are a reliable guide to the human condition. (Additional detail may be found in several reviews.154,161-164)
Studies have also shed light on the brain mechanisms underlying nicotine IVSA. Nicotinic receptors are found in many areas of mammalian brain, and their involvement in nicotine IVSA is supported by observations that the non-competitive nicotine antagonist mecamylamine can attenuate lever-pressing for nicotine. The competitive nicotinic antagonist DHbE blocks nicotine IVSA by an action on nicotinic receptors in the VTA of the mid-brain.165 Nicotine acts in the VTA to activate the ascending mesolimbic dopamine system.166,167 This neural pathway is also critically implicated in the reinforcing action of abused drugs such as amphetamine, cocaine and opioids. Like these substances, nicotine enhances the release of dopamine in some of the projection areas of the mesolimbic dopamine system, notably the nucleus accumbens.167-171 All these drugs, including nicotine, produce dopamine release mainly in the shell area of the nucleus accumbens rather than in the core.170,171

Dopamine antagonist drugs and selective neurotoxin-produced lesions of the dopamine-containing neurones of the nucleus accumbens strikingly attenuate nicotine IVSA.172,173 Recent studies suggest an impairment of both dopamine release and nicotine IVSA in transgenic mice lacking the b2 subunit of the nicotinic receptor;7 nicotinic receptors containing the alpha4beta2 subtype are the most prevalent in mammalian brain, and these important observations suggest that they may be required for nicotine IVSA. The roles of nicotinic receptors containing a and other b subunits have yet to be evaluated in a similar manner. Overall, the results to date suggest that the ascending mesolimbic dopamine system is essential for nicotine IVSA. This is also the major known neuroanatomical and neurochemical mechanism of reward for the classical addictive drugs. Therefore, both the behavioural effects and the mechanisms of action of nicotine in the IVSA model resemble those for classical drugs of abuse such as heroin and cocaine.

2.6 Nicotine neurochemistry: nicotine receptor and brain reward systems

Addictive drugs exhibit two important characteristics:

  1. They elicit effects within the brain which are pleasant or rewarding, and which reinforce self-administration of the drug in both experimental animals and human beings.
  2. Following a period of chronic exposure, withdrawal of the drug may elicit an abstinence syndrome which an addict may also seek to avoid by continuing to take the drug.

The primary objective of this section is to present the evidence that nicotine exerts effects within the brain which may account both for its positive reinforcing properties and for the presence of an abstinence syndrome following cessation of exposure.

The neurobiology underlying the positive reinforcing properties of nicotine

Studies of the mechanisms underlying the positive reinforcing properties of addictive drugs have been significantly influenced by experiments with the psychostimulant drugs, amphetamine and cocaine. These have shown that the ability of these compounds to act as locomotor stimulants and to reinforce self-administration in experimental animals depends critically upon the fact that they enhance neurotransmission at dopamine synapses in the mesolimbic system of the brain.174,175 This conclusion is supported most impressively by the fact that lesions of the mesolimbic pathway cause a marked attenuation of the locomotor stimulant properties of these drugs and their ability to serve as a reinforcer in a self-administration paradigm. Indeed, it has been suggested that the large increases in dopamine overflow in the nucleus accumbens evoked by cocaine and amphetamine result in such a powerful euphoriant effect that it alone accounts for the addiction to these drugs.174

The locomotor stimulant properties of nicotine and its ability to act as a reward in a self-administration paradigm also seem to depend upon the ability of nicotine to stimulate the dopamine-secreting neurones which innervate the principal terminal field of the mesolimbic system, the nucleus accumbens.165,173,176 In recent years, the effects of nicotine on dopamine release from these neurones have been studied extensively using the technique of in vivo microdialysis, a procedure which can be used to investigate the effects of drugs on neurotransmitter release from discrete areas of the brain in conscious, freely moving animals. These studies have shown clearly that nicotine preferentially stimulates dopamine release from the neurones which project to the nucleus accumbens.168 Mesolimbic dopamine neurones express the nicotinic receptors which are known to mediate the effects of nicotine within the brain.177 These receptors are located both on the nerve terminal membranes in the nucleus accumbens and on the membranes of the dopamine-secreting neurones in the mid-brain which innervate the nucleus accumbens.178 Although both groups of receptors may contribute to the effects of nicotine on dopamine release, there is convincing evidence that the responses to nicotine injections, given either IV or subcutaneously, are mediated at least predominantly by the receptors located on the cell bodies in the mid-brain.166,179 This implies that the effects of nicotine on the system depend upon its ability to influence the flow of impulses to the terminal field. In this respect, nicotine differs from cocaine and amphetamine which exert their effects by binding to the presynaptic dopamine transporter located on the nerve terminal membranes.

Studies on the neurobiology of drug addiction must take account of the fact that addiction is a consequence of chronic or repeated exposure to the drug. It is important to understand, therefore, the ways in which brain responses are influenced by chronic exposure to the drug since these may be crucial to our understanding of the neural mechanisms underlying addiction. In studies with experimental animals, repeated administration of amphetamine or cocaine results in sensitisation to their effects on dopamine release in the nucleus accumbens, measured using in vivo microdialysis.180 It has been suggested that this sensitisation may play a central role in the development of addiction, in particular that sensitisation of the pathway may facilitate the way in which behaviours associated with obtaining the drug are learned and with the process by which 'drug-liking' becomes 'drug-craving'.181 It is significant, therefore, that, like amphetamine and cocaine, repetitive injections of nicotine can also result in sensitisation of its effects on dopamine release in the accumbens.169 The mechanisms underlying sensitisation of the response remain to be established with certainty. However, they seem to involve costimulation of the NMDA receptor for glutamate, since both the development and expression of the sensitised dopamine response are attenuated or abolished by the administration of NMDA receptor antagonists.182,183 Costimulation of NMDA receptors has also been implicated in the mechanisms underlying sensitisation to other psychostimulant drugs of abuse, and is probably associated with an increase in the burst firing of the neurones.180,184 Thus, the effects of chronic nicotine on the pathway are similar in important respects to those of other psychostimulant drugs of addiction.

The conclusions concerning the role of mesoaccumbens dopamine pathways in nicotine addiction are almost entirely derived from studies with animal models. However, there is circumstantial evidence to suggest that the conclusions apply to the reinforcing effects of nicotine in tobacco smoke to the extent that the administration of a drug, haloperidol, which blocks the dopamine receptor in the brain, increases smoking in habitual smokers.185 This is the anticipated response if nicotine reward depends upon increased dopamine release in the brain since it reflects an attempt to overcome the blockade produced by the antagonist.

Other neural responses to nicotine which may contribute to its positive effects on smoking

Many neurones in the brain express the neuronal nicotinic receptors at which nicotine acts and, as a result, the drug stimulates other pathways which may be important to the development of addiction. These pathways include the noradrenaline-secreting neurones of the locus coeruleus which project to the forebrain, many of the acetylcholine-secreting neurones found in the hippocampus and cortex and terminals which secrete the excitatory amino acid, glutamic acid, and the inhibitory amino acid, g-aminobutyric acid.177,186,187 The psychopharmacological consequences of the effects of nicotine on these neurones remain to be established. However, it seems likely that stimulation of the receptor located on glutamate-secreting terminals facilitates release of the transmitter,186 and that stimulation of NMDA receptors located on the dopamine-secreting neurones in the VTA results in increased burst firing of the neurones, and thus an enhanced dopamine response to nicotine.188,189 It also seems likely that the effects of nicotine on acetylcholine-secreting neurones may be implicated in the increase in arousal and attention sometimes associated with smoking.190 In addition, the stimulatory effects on both acetylcholine and glutamate secretion in the hippocampus and cerebral cortex may mediate the improved cognitive function which has been reported for nicotine.191 Improved vigilance, attention and cognition have all been cited by smokers as reasons why they smoke.

Evidence for desensitisation of neuronal nicotinic receptors

Regular smoking results in the accumulation of nicotine in blood during the 'smoking day'. The nicotine level subsequently falls during sleep as the drug is metabolised and cleared from the body.192 Prolonged exposure to nicotine has been shown to cause desensitisation of many of the neuronal nicotinic receptors which mediate its effects in the brain. There is now good evidence that the plasma concentrations of nicotine commonly found in habitual smokers during the day are sufficient to desensitise the nicotinic receptors on the mesolimbic dopamine neurones which appear to mediate the rewarding properties of the drug which reinforce its self-administration.193 As a result, the administration of a nicotine bolus no longer causes increased dopamine release in the nucleus accumbens.194

These results have significant consequences for the 'dopamine hypothesis' of nicotine addiction. They imply that many smokers may continue smoking under conditions in which nicotine is unlikely to stimulate the mesolimbic dopamine neurones, and that other neural mechanisms must probably also contribute to the 'rewarding' properties of the drug which reinforce addiction.
In this context, it is important to remember that tobacco smoking habits are heterogeneous and that people smoke cigarettes at varying frequencies and in different ways. The plasma nicotine concentration is likely to remain fairly stable through the day for people who smoke frequently, whereas for those who smoke less frequently, significant peaks and troughs of nicotine may be observed.195 If the nicotine concentration in the trough falls below that required to desensitise the nicotinic receptors on mesolimbic dopamine neurones, each cigarette will be 'rewarded' with increased dopamine release; for these smokers, stimulation of dopamine release is probably the predominant mechanism underlying addiction to nicotine.

In contrast, it has been suggested that receptor desensitisation may be the response which is reinforced in frequent or heavy smokers.191 For example, the nicotinic receptors located on noradrenaline-secreting neurones are desensitised by nicotine concentrations similar to those found in the plasma of many smokers.196 This may contribute to the 'tranquillising' properties of tobacco smoke often reported by smokers exposed to environmental stressors.191 It is important to remember that nicotine exerts its effects in the brain by acting at a family of nicotine receptors. Thus, it is possible that other neural responses, mediated by receptors more resistant to desensitisation, may also play an important role in nicotine addiction.
In experimental animals, chronic exposure to nicotine, using regimens which cause desensitisation of the catecholamine responses to nicotine, often causes an increase in the density of the nicotine receptors which bind nicotine with high affinity.177 This increased density seems to reflect a decreased turnover of the receptor complex.197,198 The psychopharmacological significance of this effect remains unclear, although it appears to be associated with repeated or prolonged exposure to concentrations of nicotine which cause desensitisation of the receptors. It seems unlikely, however, that the increased receptor density accounts for the sensitisation to nicotine discussed above because upregulation of the receptors is not observed with dosing regimens which elicit the sensitised dopamine responses.199 The increase in receptor density may nevertheless be significant to the mechanisms underlying nicotine addiction since they are also observed in brain tissue taken from humans who have been habitual smokers.200

Effects on brain 5-hydroxytryptamine pathways

In experimental animals, chronic nicotine treatment causes a regionally-selective reduction in the concentration of 5-hydroxytryptamine (5-HT) in the hippocampus.201 Subsequent studies have shown this to be associated with a reduction in the formation and release of 5-HT in this region of the brain, and that tolerance to this effect does not develop in animals treated chronically with nicotine.202,203 The results suggest that chronic exposure to the drug causes repeated or prolonged reductions in the demand for 5-HT in the hippocampus by reducing the concentration and capacity to synthesise 5-HT in serotonergic terminals in the hippocampus. Other studies using human post-mortem tissue have shown that habitual smoking is also associated with a regionally-selective reduction in the concentration of 5-HT and its principal metabolite, 5-hydroxyindole acetic acid, in the hippocampus which is not observed in a majority of the other areas of the brain that have been studied (ie gyrus rectus, medulla oblongata, cerebellum).204

These data imply that the reduction in hippocampal 5-HT formation and release observed in experimental rats given nicotine also occurs in the hippocampus of habitual smokers. It therefore seems reasonable to suggest that the effect is mediated by the nicotine present in tobacco smoke. This conclusion is supported by the association of habitual smoking with an increase in the density of post-synaptic 5-HT1A receptors in the hippocampus since upregulation of these receptors is known to occur following treatments which elicit a chronic reduction of 5-HT release in the hippocampus.204

The psychopharmacological consequences of the changes in hippocampal 5-HT elicited by nicotine remain to be established. Experimental studies have shown that increased stimulation of 5-HT1A receptors in the hippocampus may be implicated in anxiety.205 It is possible, therefore, that the decrease in 5-HT release evoked by nicotine could mediate the reductions in anxiety often consistently reported by many smokers. There is some experimental evidence that nicotine has anxiolytic properties in some tests,206 although this is not a universal finding.207 If this hypothesis is correct, when habitual smokers first quit the habit, 5-HT release in this area of the brain will no longer be suppressed and, as a result, increased feelings of anxiety, mediated by the increased density of postsynaptic 5-HT1A receptors in the hippocampus, will be experienced. Thus, the increases in receptor density could also contribute to the symptoms often observed during the early stages of smoking cessation.208

Other reports suggest that the 5-HT projections to the hippocampus are involved more in adaptation to aversive stimuli, that impairments in the pathway contribute to the dysfunction of pituitary-adrenal activity seen in many patients who suffer from depression, and that these effects contribute to symptoms of the patients.209,210 There is a growing body of evidence to suggest that occupation of 5-HT receptors, including specifically 5-HT1A receptors, in the hippocampus plays a pivotal role in the expression of the glucocorticoid receptors which exert an important inhibitory effect on pituitary-adrenal activity.211 These receptors are thought to play a primary role in the mechanism by which we 'cope' with the stresses of daily life, especially those to which we are exposed repetitively. Experimental studies suggest that chronic treatment with nicotine inhibits the process underlying habituation of the adrenocortical response to an unavoidable stressor.212 This observation is entirely consistent with the fact that habituation of this response seems to depend upon increased expression of receptors which respond to glucocorticoids in the hippocampus, and that this increased receptor expression is highly dependent upon increased 5-HT release in this area of the brain.213,214

It seems reasonable to suggest, therefore, first, that one important consequence of the effects of chronic nicotine on hippocampal 5-HT function is to attenuate the mechanism which mediates adaptation to environmental stress; secondly, that this may explain some of the symptoms, such as anxiety and depression, observed when habitual smokers stop smoking,208 when they are no longer 'protected' from these symptoms by the effects of nicotine on other neural pathways within the limbic system of the brain which are also involved in responses to anxiogenic or stressful stimuli.

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Contents

Contributors, Foreword and Key Points
1  Tobacco smoking in Britain: an overview
2 Physical and pharmacological effects of nicotine
3 Psychological effects of nicotine and smoking in man
4 Is nicotine a drug of addiction?
5 The natural history of smoking: the smoker's career
6 Regulation of nicotine intake for smokers, and implications for health
7 The management of nicotine addiction
8 Regulatory approaches to tobacco products in Britain
9 Summary and recommendations

 

 


This page last updated on August 1, 2007