A. Nontraditional Ligands
There are several ligands that interact with nAChRs at sites other than the agonist-binding domains, yielding either potentiation or depression of receptor activity. This section will focus on studies of exogenous nAChR modulators that found their way to the clinics and on studies of endogenous ligands that physiologically regulate the activity of specific nAChR subtypes.
At the neuromuscular junction, nicotinic function is enhanced by inhibition of acetylcholinesterase (AChE), the enzyme that metabolizes the endogenous neurotransmitter ACh. However, unlike muscle nAChRs, some neuronal nAChRs, particularly the α7 nAChRs, recognize both ACh and its metabolite choline as full agonists (371
). Therefore, AChE inhibition may not necessarily enhance functions mediated by these nAChRs. In fact, as described above, AChE inhibitors do not affect α7 nAChR-mediated synaptic transmission evoked by low-frequency stimulation of cholinergic fibers in chick ciliary ganglia (522
An alternative means to increase nicotinic functions in the brain is to sensitize the nAChRs to activation by the endogenous agonist(s) using the so-called nicotinic allosteric potentiating ligands (APLs), which include drugs such as physostigmine and galantamine, a drug currently approved for the treatment of AD. Studies from the early 1980s provided evidence that the cholinesterase (ChE) inhibitor physostigmine could interact directly with nAChRs at the frog neuromuscular junction and induce nicotinic single-channel currents (428
). In the early 1990s, galantamine, an alkaloid originally extracted from the bulbs and flowers of the wild Caucasian snowdrop Galanthus nivalis
and other related Amaryllidacea species, was found to act like physostigmine on muscle and neuronal nAChRs (370
). Surprisingly, however, activation of nAChRs by galantamine or physostigmine was insensitive to blockade by competitive nAChR antagonists, was detected even when the receptors were desensitized by high agonist concentrations, and was inhibited by the monoclonal antibody FK1 (350
). The agonistic activity of physostigmine and galantamine, initially referred to as noncompetitive agonists (NCAs; Ref. 450
), was found to result from their binding to a site close to, but distinct from, the ACh-binding site on nAChR α subunits (4
). The region flanking the amino acid Lys-125 on the nAChR α subunits contains essential elements of the physostigmine site and is highly conserved across species (372
The nicotinic NCA action is not common to all ChE inhibitors, since, for example, the ChE inhibitor pyridostigmine is unable to induce nicotinic single-channel currents by directly interacting with nAChRs (39
). Conversely, a drug does not have to be a ChE inhibitor to be a nicotinic NCA. For instance, studies carried out in PC12 cells demonstrated that codeine, a drug with no significant effect on ChE, can activate nicotinic single-channel currents and that this nicotinic agonist effect is sensitive to inhibition by FK1 while unaffected by classical nAChR antagonists (450
Even though NCAs induce opening of nAChR single channels in numerous neuronal and nonneuronal preparations, the probability of channel openings by these compounds is so low that the single-channel currents they activate do not give rise to macroscopic responses (4
). In different systems, however, NCAs have been shown to potentiate the activation of most nAChRs by subsaturating concentrations of classical nAChR agonists. The nicotinic potentiating action of these drugs is also sensitive to inhibition by the FK1 antibody, and, thereby, likely to result from their interactions with the physostigmine-binding site on nAChRs (414
). A recent study performed in HEK293 cells stably expressing muscle nAChRs, however, revealed that galantamine acts as a nicotinic NCA but not as a nicotinic APL (4
). Thus the possibility cannot be ruled out that the NCA and the APL sites share some common elements, but are in fact distinct from one another in the nAChRs. As described below, a recent study using the AChBP isolated from the mollusk Aplysia californica
shed some light onto this puzzle.
As mentioned in section I, the AChBP is a soluble homopentamer that resembles the extracellular domain of the nAChRs and has the ligand-binding elements that make up the sites for classical agonists and competitive antagonists (206
). Crystallographic analysis of the AChBP-galantamine complex revealed that galantamine associates with elements present at the interface between two AChBPs (207
). As expected from the pharmacological studies described above, no significant interaction was observed between galantamine and the vicinal cysteine residues that are essential for binding of classical nicotinic agonists and competitive antagonists (207
; see also ). Elements that appear essential for binding of galantamine to the AChBP include the tryptophan residues 147 and 149, the tyrosine residue 55 or 93, and to a lesser extent the tyrosine residue 195. It has also been proposed that the dipole between the carbonyl group of the tryptophan residues and the protonated nitrogen of galantamine may be strengthened by the anionic side chain of the residue aspartate-89 (207
). These findings are in agreement with our earlier suggestion that the region including and surrounding the residue Lys-125 on the nAChR α subunits, which spans the nAChR epitope against which the antibody FK1 was raised, contains elements that are essential for the binding of galantamine to the nAChRs (372
). However, the crystallographic study of the AChBP-galantamine complex also revealed that some of the residues that contact galantamine in the complex are conserved among non-α nAChR subunits, suggesting that galantamine may bind to both α- and non-α interfaces (207
). Therefore, it is tempting to speculate that, depending on the subunit composition of the nAChRs, differential interactions of galantamine with α- or non-α interfaces can favor its action as an NCA or an APL.
FIG. 7 Regulation of nAChRs by nontraditional ligands. In this illustration, an agonist-binding α subunit (light blue) and a structural β subunit (dark blue) are shown with a solid surface looking from the extracellular side with either nicotine (more ...)
The exact mechanism by which nicotinic APLs sensitize nAChRs to activation by classical agonists is still poorly understood. There are reports that nicotinic APLs increase the probability of nAChR channel openings in duced by ACh in outside-out patches from PC12 cells and enhance the apparent potency, but not the efficacy of nicotinic agonists in activating different nAChR subtypes (406
). These results support the notion that APLs enhance the binding affinity of agonists and/or the frequency of channel openings for a given level of receptor occupancy as long as receptor activation is still submaximal.
The nicotinic APL action is not common to all ChE inhibitors; for instance, donepezil and rivastigmine are devoid of this action (405
). To date, all compounds characterized as nicotinic APLs have a nitrogen that is cationic at physiological pH and is located at a fixed distance from a phenolic group (372
). The few drugs identified so far as nicotinic APLs increase with similar potencies the activity of different nAChR subtypes and have a bimodal effect on these receptors (406
). Therefore, it has so far not been possible to pinpoint the pharmacophore that will make a compound to act exclusively as a nicotinic APL in a given nAChR subtype.
The discovery of galantamine as a nonconventional nicotinic ligand of exogenous origin led to the suggestion that an endogenous galantamine-like ligand might exist. Initial attempts to identify such endogenous compound(s) were focused on the concept that a given substance can control synaptic activity in the brain by acting as the primary agonist in one neurotransmitter system and as a modulator in another system. Glycine is a classical example of such an endogenous substance. Whereas in glycin-ergic synapses glycine activates glycine-gated channels, in the glutamatergic system glycine acts as a coagonist at the NMDA-receptor channels. Since some studies have indi cated that indolamines can interact with the ChEs found in the plaques of patients with AD (505
), and since some ChE inhibitors, including galantamine, act as nicotinic APLs, the neurotransmitter serotonin (5-HT) was tested for its ability to modulate ACh-evoked nicotinic currents in PC12 cells (414
). As consistently reported in other systems (e.g., Refs. 172
), 5-HT at micromolar concentrations was also found to inhibit agonist-induced nAChR activity in PC12 cells (414
). However, at submicromolar concentrations, 5-HT sensitized the nAChRs to classical agonists, an effect that could be blocked by FK1 (414
). Thus the possibility exists that 5-HT acts as an endogenous nicotinic APL.
It remains unclear whether under normal physiological conditions, endogenous galantamine-like modulators of nAChR activity would be stored together with ACh in the cholinergic terminals or would have a paracrine action. Considering that in many CNS areas, tryptaminergic and cholinergic synapses are colocalized (327
), it is tempting to speculate that 5-HT released from its terminal could diffuse away and act as a nicotinic APL on closely located nAChRs. The finding that submicromolar concentrations of 5-HT are sufficient to potentiate nicotinic responses is in agreement with a paracrine action of 5-HT on nAChRs. It also lends support to the concept that brain functions are regulated by complex neuronal and chemical networks.
Reduced nAChR function/expression in the brain has been associated with the pathophysiology of catastrophic disorders, including AD and schizophrenia (discussed in later sections, and see Refs. 277
). In particular, the association of the α7 nAChR gene with a sensory gating deficit that is similar to attention deficits in patients with schizophrenia (157
), and the degree of α4β2 nAChR loss and altered α7 expresson correlate well with the magnitude of progressive cognitive decline in mild-to-moderate AD patients (46
). The nicotinic APL action of galantamine appears to be an important determinant of its clinical effectiveness (reviewed in Refs. 98
). Acting primarily as a nicotinic APL, galantamine improves synaptic transmission and decreases neurodegeneration, two effects essential for its cognitive-enhancing properties (40
). Of note is that in both of these catastrophic disorders, reduced nAChR activity/expression is accompanied by increased levels of kynurenic acid (KYNA), a tryptophan metabolite that in the brain is primarily produced and released by astrocytes (244
). The neuroactive properties of KYNA have long been attributed to the inhibition of NMDA receptors (329
). Electrophysiological studies, however, have demonstrated that physiologically relevant concentrations of KYNA block α7 nAChR activity noncompetitively and voltage independently (210
Biosynthesis and disposition of KYNA in the mammalian brain have been extensively investigated. KYNA is formed enzymatically by the irreversible transamination of l
-kynurenine, a major peripheral tryptophan metabolite with ready access to the brain. Immunohistochemical and lesion studies demonstrated that cerebral KYNA synthesis takes place almost exclusively in astrocytes (129
). Newly formed KYNA is rapidly liberated into the extracellular compartment for possible interaction with neurotransmitter receptors, including the α7 nAChRs and NMDA receptors (472
). Because of the absence of reuptake or degradation mechanisms, subsequent KYNA removal is accomplished exclusively by probenecid-sensitive brain efflux (330
). Interestingly, astrocytic KYNA production is regulated by neuronal activity (187
) and cellular energy metabolism (213
). This dependence of extracellular KYNA concentrations on the functional interplay between neurons and astrocytes is in line with the postulated neuromodulatory role of KYNA (418
) and adds to the complexity of the neurochemical networks in the brain. In the normal brain, >70% of KYNA formation is catalyzed by KAT II, one of the three cerebral KATs (199
). Systemic treatment of rats and mice with kynurenine leads to an elevation of brain levels of several neuroactive intermediates, including KYNA, the free radical generator 3-hydroxykynurenine, and the excitotoxic quinolinic acid (419
Because the overall effects of α7 nAChR and NMDA receptor antagonists on neuronal plasticity and viability are similar and resemble those of KYNA, a review of the neuroactive properties of KYNA in vivo and in vitro does not adequately resolve the question of whether the metabolite acts in vivo through α7 nAChRs or NMDA receptors. Mice with a null mutation in the gene that encodes KAT II became a unique tool to resolve this issue (31
). Low levels of KYNA in these mutant mice lead to α7 nAChR disinhibition in hippocampal CA1 SR interneurons, thereby increasing the activity of GABAergic interneurons impinging onto CA1 pyramidal neurons (31
). It is noteworthy that NMDA receptor activity in CA1 SR interneurons in hippocampal slices of mKat-2−/−
mice is not significantly different from that recorded from wild-type interneurons (31
). This constituted the first evidence that in the hippocampus endogenous levels of KYNA are sufficient to directly modulate the activity of α7 nAChRs, but not that of NMDA receptors (31
). Potential developmental and age-dependent adaptations to the elimination of KAT II, however, limit the usefulness of the mKat-2−/−
mice to the understanding of the pathological effects of KYNA in the mature brain. Thus brain levels of KYNA in 60-day-old mKat-2−/−
mice become comparable to those of agematched wild-type mice, and no phenotypic differences in hippocampal α7 nAChR activity or GABAergic transmission exist in these older animals (31
). Changes in the mechanisms that regulate the expression of KATs other than KAT-2 in the brain could represent adaptative responses to the elimination of the mKat-2 gene (517
).Therefore, a better understanding of how abnormal levels of brain KYNA contribute to the pathophysiology of disorders such as AD and schizophrenia will depend on pharmacological manipulations that induce selective fluctuations in brain KYNA levels at specific ages.
Acting as an endogenous regulator of the α7 nAChR activity, astrocyte-derived KYNA can modulate synaptic transmission, synaptic plasticity, neuronal viability, and neuronal connectivity in different areas of the brain (). Activation of α7 nAChRs in somatodendritic and preterminal/terminal areas of interneurons in various strata of the CA1 region and in the dentate gyrus facilitates spontaneous quantal release of GABA (14
). Glutamate release from mossy fibers onto CA3 pyramidal neurons is also modulated by α7 nAChRs present in the mossy fiber terminals (190
). Furthermore, α7 nAChRs have been im plicated in “inhibitory” and “disinhibitory” circuits in the CA1 field of the hippocampus (19
; see also ). As mentioned above, under normal physiological conditions, endogenous levels of KYNA are sufficient to maintain a degree of inhibition of α7 nAChRs in CA1 SR interneurons that tunes down the intensity of the GABAergic transmission impinging onto CA1 glutamatergic neurons (15
FIG. 8 Role of astrocyte-derived kynurenic acid (KYNA) in regulating the activity of dopaminergic neurons in the ventral tegmental area. This simplified scheme illustrates the role of astrocyte-derived KYNA in modulating synaptic transmission between a cortical (more ...)
Activation of α7 nAChRs is known to contribute to the regulation of extracellular dopamine levels in the rat striatum (81
). Application via microdialysis of KYNA or α-BGT to the rat striatum significantly reduces the extracellular levels of dopamine, and the magnitude of the effect of either antagonist alone is comparable to that of both antagonists together (285
). In contrast, the NMDA receptor antagonist 7-chloro-KYNA has no significant ef fect on the extracellular levels of dopamine in the rat striatum (391
). As illustrated in , KYNA-induced reduction of extracellular dopamine levels can be explained by the inhibition of tonically active α7 nAChRs in the dopaminergic neurons within the VTA and/or in cortical glutamatergic terminals that synapse onto striatal neurons. VTA dopaminergic neurons represent the major dopaminergic input to the nucleus accumbens.
Disruption of reciprocal glia-neuron signaling mechanisms involving KYNA and nAChRs may be causally related to diseases such as AD and schizophrenia. Chronic α7 nAChR inhibition in the hippocampus by elevated levels of KYNA can contribute to auditory gating deficits, which appear to be associated with the development of schizophrenia (156
). It is also feasible that KYNA-induced inhibition of α7 nAChRs contributes to the cognitive impairment observed in patients with AD and schizophrenia (273
). Finally, the finding that KYNA, acting via α7 nAChRs, regulates striatal dopamine levels (; Refs. 285
) suggests that the interplay between astrocyte-derived KYNA and synaptic transmission can modify reward mechanisms implicated in the pathophysiology of drug abuse and neuropsychological disorders such as schizophrenia. Detailed knowledge of how KYNA, acting via α7 nAChRs, regulates synaptic transmission throughout the brain at different ages is essential for the understanding of the involvement of KYNA and α7 nAChRs in specific disease states.
The exact amino acids required for binding of KYNA to α7 nAChRs are yet to be identified. However, recent electrophysiological experiments have demonstrated a competitive interaction of galantamine and KYNA with α7 nAChRs in hippocampal neurons (285
). The finding suggested that KYNA-induced inhibition of α7 nAChRs is dependent on the interactions of the metabolite with the region on nAChRs that binds galantamine. Two questions were then raised: 1
) Why are the actions of KYNA and galantamine on α7 nAChRs opposite? 2
) Why does KYNA inhibit α7 nAChRs selectively, while galantamine acts more promiscuously as an APL on most nAChRs?
Superimposition of the lowest energy conformers of galantamine and KYNA shed some light on structural differences that could explain the opposite actions that result from the interactions of the two compounds with the APL-binding region on α7 nAChRs. Like galantamine, KYNA has an aromatic ring with a phenolic hydroxyl group. This group, which bears the same spatial orientation as the phenol group in galantamine, is located at a fixed distance from a pyridinic nitrogen. However, this nitrogen is largely unionized at physiological pH and is at a shorter distance from the phenolic group than the tertiary nitrogen is from the corresponding phenolic group in galantamine. The previous report that 7-chloro-KYNA does not inhibit α7 nAChRs (210
) suggests that the car-boxyl group contributes to interactions of KYNA with specific residues in the APL-binding region. The introduction of the electron-withdrawing chlorine in position 7 of the phenolic ring creates a dipole in the molecule that can weaken its potential interactions with positively charged residues in the APL region. The nAChR α7 subunit is the only mammalian nAChR α subunit that has a positively charged residue within the segment α118–140 of the putative APL-binding region. It is, therefore, tempting to speculate that the selectivity of KYNA for α7 nAChRs is encoded in the carboxyl group in position 2 of the pyridine ring.
Drugs currently approved to treat mild-to-moderate AD, including galantamine, donepezil, and rivastigmine, all inhibit AChE, the enzyme that hydrolyzes ACh (462
). As mentioned above, galantamine is unique in that it also acts as a nicotinic APL. Recently, these drugs have been evaluated as adjuvant therapies to decrease the cognitive impairment and negative symptoms of patients with schizophrenia. Data are still sparse and so far derived from small samples in open uncontrolled studies. However, a small randomized, double-blind trial showed positive outcomes when galantamine was administered as an add-on therapy to antipsychotics (417
). To date, no similarly promising clinical effects have been observed with donepezil or rivastigmine (310
). Since KYNA levels are significantly elevated in the brain of individuals with AD (49
) and schizophrenia (420
), it is possible that the antagonism of KYNA-induced inhibition of α7 nAChRs may be causally related to the effectiveness of galantamine in these catastrophic disorders.
Other endogenous ligands that impact on the activity of nAChRs noncompetitively and voltage independently include the amyloid β peptide 1–42 (Aβ1–42; Refs. 123
) and the canabinoid anandamide (356
). The Aβ1–42 peptide is one of the breakdown products of the proteolytic cleavage of the amyloid precursor protein by β- and γ-secretases. In biopsy samples of human brain tissue obtained from AD patients and in ectopic systems overexpressing either α7 nAChRs or APP, Aβ1–42 coimmunoprecipitates with α7 nAChRs (490
). The Aβ1–42 peptide also displaces binding of [3
H]MLA from α7 nAChRs in cerebral cortical and hippocampal synaptosomes (490
). More functional studies reported that while at picomolar concentrations Aβ1–42 activates α7 nAChRs ectopically expressed in Xenopus
), at nanomolar concentrations it inhibits α7 nAChRs present in different preparations (278
). The α7 nAChR inhibition by Aβ1–42 is noncompetitive with respect to the agonist, is voltage independent, and is therefore likely to be mediated by the interaction of the peptide with a site different from that for ACh on the nAChRs. Other studies have reported that α4β2 nAChRs are more sensitive than α7 nAChRs to inhibition by nanomolar concentrations of Aβ1–42 (506
). Factors that confer Aβ sensitivity to nAChRs include, but are not restricted to, nAChR subunit composition and stoichiometry, regional distribution of specific nAChR subtypes, neuronal compartmentalization of different nAChR subtypes, as well as neuronal and nonneuronal nAChR expression (122
). It is noteworthy that the α7 nAChR activity increases intracellular accumulation of Aβ in neurons (336
), and Aβ peptides, in addition to modulating nAChR activity, downregulate the expression of nAChRs (197
). Though poorly understood, reciprocal relationships might exist in vivo between endogenous levels of Aβ peptides and nAChR activity that are essential to the pathophysiology of AD.
Anandamide, a compound originally isolated from porcine brain extracts, is known to interact with canabinoid receptors 1 and 2 in the brain (120
). However, anandamide interacts with numerous other receptors, including voltage-gated Ca2+
), voltage-gated K+
), kainate receptors (3
), and nAChRs (356
). At nanomolar concentrations, anandamine blocks noncompetitively and voltage independently the activation of α7 nAChRs ectopically expressed in Xenopus
). It also inhibits the activity of α4β2 nAChRs expressed in SH-EP1 cells (443
). There is evidence that anandamide is produced by postsynaptic neurons in response to elevated intracellular Ca2+
levels. For instance, concomitant activation of α7 nAChRs and NMDA receptors triggers the production of anandamine in postsynaptic neurons (448
). Anandamine, then, functions as a retrograde messenger and regulates synaptic transmission by interacting with specific receptors in the presynaptic neurons/terminals (498
). It has been suggested that nAChRs may serve as potential targets for modulation of synaptic transmission by anandamide (356
). The mutual interactions between the endocannabinoid system and the nAChRs have led to the recent discovery of α7 nAChRs as potential targets for development of medical therapies for the treatment of cannabis addiction (440
Finally, bupropion (16
) and UCI-30002 (514
) are examples of synthetic compounds that act as noncompetitive inhibitors of different nAChRs, including those made up of the subunits α7, α4β2, or α3β4. Both compounds effectively decrease nicotine self-administration in rats (280
). Bupropion is presently approved as an adjunct therapy for smoking cessation. The sites that contribute to the inhibitory actions of these compounds are completely unknown.
C. Nicotine Effects in Peripheral Systems and Inflammation
While the effects of nicotine in the CNS, including its addictive effects, remain a central focus of nAChR studies, as Langley and colleagues demonstrated over 100 years ago (259
), the alkaloid has dramatic effects on peripheral systems. This includes the ability of high nicotine concentrations to act on muscle receptors as well as to impart often more subtle effects through preganglionic receptors of the autonomic nervous system. Recent stud ies have identified nAChRs present in numerous nonneuronal cell types outside the nervous systems and investigated how these receptors participate in modifying a range of physiological processes. In fact, the relationship between tobacco abuse (including smokeless) and difficulty in healing, increased susceptibility to infection (especially oral), enhanced expression of indicators of skin aging, and increased cancer risk are all well-documented (383
). The recognition of the expression of nAChRs in adipose tissue (36
) provides a mechanistic rationalization for the long-standing observation that on average smokers appear thinner, and, yet, more prone to metabolic syndromes such as type II diabetes.
Probably the first written report of an interaction between nicotine and inflammation emerged over 150 years ago when the German physician Rudolph Virchow recognized that smoking could in some cases provide acute, and even long-term therapeutic relief to the symptoms of severe asthma. Modulation by nicotine of inflammatory responses in the intestines is much better reported. Early studies found that patients with ulcerative colitis who stopped smoking tobacco developed the disease or exhibited more severe disease progression, which was ameliorated by either returning to smoking (58
), or, in some cases, administering nicotine through transdermal patches (313
). In contrast, patients with Crohn’s disease experience much more severe disease when smoking (401
). Complicating this finding is that not all human subjects respond in this way. Recent studies of mice suggest that this may in part be related to specific nAChRs and their interactions with distinct inflammatory pathways (353
), which in turn are subject to individual genetics. Notably, mice with a null mutation in the gene that encodes the α5 nAChR subunit exhibit enhanced sensitivity to induction of inflammatory bowel disease relative to controls (353
). Despite increased sensitivity to disease initiation, administration of transdermal nicotine remains effective in attenuating the disease process. Therefore, again nicotine appears to impact on inflammatory processes with considerable specificity and tissue dependency. Understanding how these interactions proceed to pathology will require a much greater and detailed examination of the interaction between specific nAChR subtypes and inflammatory cytokines in different cell types, within the context of individual genetics.
There is current evidence that nAChRs present in skin cells modulate the responses triggered by inflammatory stimuli applied to the skin (354
). Smoking is a welldefined risk factor in delayed wound healing and possibly the development of premature facial wrinkling (226
). Distinct nAChRs are expressed in diverse cells that compose the skin (95
). For example, epithelial keratinocytes express functional nAChRs and, importantly, they also are capable of synthesizing the so-called cytotransmitter ACh (526
). Mechanistically, nicotine, acting through nAChRs, decreases keratinocyte migration (188
) and modifies the activity of PI3K/Akt, ERK, MEK, and JAK signaling pathways. Furthermore, pharmacological dissection of nicotine’s influence on cell cycle progression, apoptosis, and differentiation (43
) indicate that α7 nAChRs expressed in keratynocytes are important. Other receptors are clearly involved in this process, since atropine, a muscarinic and sometimes nAChR inhibitor (531
), reduces cell adhesion through decreasing desmoligein expression.
A relationship also exists between nAChRs and the normal physiology of adipose tissue. It has long been known that smokers tend to be leaner, and yet approximately four times more likely to become insulin resistant and develop type I diabetes (497
), a condition that is more commonly observed in obese patients. This correlation is of general biological relevance, because it also extends to certain mouse strains. For example, weight loss is observed when C57BL/6 and AKR mice, but not A/J, SJL, and NZW mice are exposed to cigarette smoking for 6 mo (198
). There is a genetic predisposition that may be linked to variable expression of nAChRs and individualizes the effect of nicotine on body weight. Although nAChRs are expressed in adipose tissue, their role in normal metabolism is not presently understood. Notably, nicotine pretreatment of rat adipocytes (279
) reduces the release of TNF-α as well as free fatty acids and the adipokine adiponectin (whose function is not known, although its levels change in metabolic syndrome). It remains to be determined whether the effects of nicotine on metabolism result from its direct interactions with specific nAChR subtypes in adipocytes controlling levels of proinflammatory cytokines and adipokines.