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NEUROPHARMACOLOGY
3/6β2* Nicotinic Receptors in Monkey StriatumThe Parkinson's Institute, Sunnyvale, California (K.O., N.P., M.Q.); and Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M.)
Received January 18, 2008; accepted February 25, 2008.
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Abstract |
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4β2* (nAChR containing the 4 and β2 subunits, but not 3 or 6) and 3/6β2* (nAChR composed of the 3 or 6 subunits and β2) nAChRs. Competition binding studies showed that cotinine interacts with both 4β2* and 3/6β2* nAChR subtypes in the caudate, with cotinine IC50 values for inhibition of 5-[125 I]iodo-3-[2(S)-azetinylmethoxy]pyridine-2HCl ([125I]A-85380) and 125I--conotoxinMII binding in the micromolar range. This interaction at the receptor level is of functional significance because cotinine stimulated both 4β2* and 3/6β2* nAChR [3H]dopamine release from caudate synaptosomes. Our results unexpectedly showed that nicotine evokes [3H]dopamine release from two 3/6β2* nAChR populations, one of which was sensitive to cotinine and the other was not. This cotinine-insensitive subtype was only present in the medial caudate and was preferentially lost with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal damage. In contrast, cotinine and nicotine elicited equivalent levels of 4β2* nAChR-mediated dopamine release. These data demonstrate that cotinine functionally discriminates between two 3/6β2* nAChRs in monkey striatum, with the cotinine-insensitive 3/6β2* nAChR preferentially vulnerable to nigrostriatal damage.
). These observations raise the question of whether cotinine is pharmacologically active in vivo, thereby contributing to the overall effects of tobacco exposure. Indeed, numerous studies indicate that cotinine influences autonomic functions including heart rate, blood pressure, respiration, and hormone regulation (Borzelleca et al., 1962; Dominiak et al., 1985; Andersson et al., 1993; Buccafusco et al., 2007), and it also affects behavioral and cognitive task performance (Risner et al., 1985; Buccafusco and Terry, 2003; Terry et al., 2005).
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and β subunits. Cotinine displaces binding of various radiolabeled nAChR ligands to rat whole brain, hippocampal, and cortical membrane preparations, with a potency 
100-fold less than that of nicotine (Sloan et al., 1984; Anderson and Arneric, 1994; Vainio and Tuominen, 2001). This interaction is of functional relevance because cotinine has been shown to both activate and desensitize nAChRs. For instance, cotinine stimulates striatal dopamine (DA) release and noradrenaline release from bovine chromaffin cells (Vainio et al., 1998b; Dwoskin et al., 1999). On the other hand, cotinine pre-exposure leads to a subsequent inhibition of nAChR-mediated responses, an effect attributed to nAChR desensitization (Vainio et al., 1998a; Koh et al., 2003). These combined data suggest that, in addition to nicotine, cotinine may contribute to the overall effects of tobacco exposure.
nAChRs are important modulators of dopaminergic function in the striatum, with several subtypes implicated in DA release. This includes the 4β2* (non-3/6-containing) receptor population (* denotes the possible presence of additional subunits), which is present on DA terminals and other neurons in the striatum, and the 3/6β2* subtype, which is located exclusively on the nigrostriatal dopaminergic terminals (Le Novère et al., 1996; Champtiaux et al., 2002; Perry et al., 2002; Zoli et al., 2002; Salminen et al., 2004). As mentioned, cotinine evokes [3H]dopamine release from rat striatal slices via nAChR activation (Dwoskin et al., 1999). However, the subtypes through which cotinine elicits dopamine release are not yet known. Such knowledge is important because accumulating studies indicate that the 4β2* and 3/6β2* subtypes are differentially modified after nigrostriatal damage, nicotine administration, and other drug treatments (Quik and McIntosh, 2006). In addition, there seems to be a unique regulation of 3/6β2* receptor subtypes with nicotine exposure and nigrostriatal degeneration (Bordia et al., 2007; Khwaja et al., 2007; Perry et al., 2007). Therefore, the present experiments were done to examine the nAChR subtype specificity of cotinine using functional [3H]DA release assays and ligand-binding autoradiography in monkey striatum. Monkeys were used because they have similar levels of 4β2* and 3/6β2* nAChRs in striatum, unlike rodents that express low levels of 3/6β2* receptors, making it difficult to study this subtype. To distinguish between the 3/6β2* and 4β2* nAChR subtypes, we have used the nAChR antagonist -conotoxinMII. Accumulating evidence shows that this toxin is selective for 3β2* and 6β2* nAChRs across species, including rodents, monkeys, and humans (Champtiaux et al., 2002; Zoli et al., 2002; Quik and McIntosh, 2006; Bordia et al., 2007). Our results show that cotinine can discriminate between two 3/6β2* nAChR subtypes, one of which is particularly vulnerable to nigrostriatal damage.
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Materials and Methods |
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) and were approved by the Institutional Animal Care and Use Committee at the Parkinson's Institute.
Tissue Preparation. The brains were rapidly removed and sectioned along the midline (McCallum et al., 2005). One half was placed in a Plexiglas mold and sliced into 6-mm-thick blocks, which were quick-frozen on glass slides in isopentane on dry ice, and stored at –80°C for future use. These blocks were sectioned (20 µm) on a cryostat (Leica Microsystems Inc., Deerfield, IL), mounted, air-dried on Superfrost Plus slides (Fisher Scientific Co., Pittsburgh, PA), and stored at –80°C for autoradiography experiments. The second half of the brain was sliced into 2-mm blocks. The medial and lateral caudate were dissected from the blocks at level A15 to A13.5 mm anterior to bregma (Emmers and Akert, 1963) and used immediately for DA release assays.
[3H]DA Release. Striatal synaptosomes were prepared and [3H]DA release assays were performed as described previously (McCallum et al., 2005). Fresh striatal tissue (15 mg per region) was homogenized in 2 ml of cold homogenization buffer (0.32 M sucrose and 5 mM HEPES, pH 7.5) and centrifuged at 12,000g for 20 min. P1 pellets were resuspended in 0.8 ml uptake buffer (128 mM NaCl, 2.4 mM KCl, 3.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM HEPES, pH 7.5, 10 mM glucose, 1 mM ascorbic acid, and 0.01 mM pargyline) and incubated for 10 min at 37°C. [3H]DA (4 µCi for a final concentration of 100 nM DA; PerkinElmer Life Analytical Sciences, Waltham, MA) was then added, and the tissue was incubated for another 5 min. Eighty-microliter aliquots of synaptosomes (approximately 0.5–2 mg of tissue) was placed on 5-mm diameter A/E glass-fiber filters (Gelman Instrument Co., Ann Arbor, MI) and perfused with uptake buffer containing 0.1% bovine serum albumin and 10 µM nomifensine for 10 min at a rate of 1 ml/min before fraction collection was initiated. Tissue was then stimulated with either (–)-nicotine hydrogen tartrate salt (0.03–30 µM), (–)-cotinine (3 µM–3 mM), or 20 mM K+ buffer for 18 s. When -conotoxinMII (50 nM) (J. M. McIntosh) was used, tissue was pre-exposed to the antagonist for 3 min before agonist exposure (Cartier et al., 1996). For each tissue aliquot, 15 fractions (18 s) were collected, including basal release before and after stimulation. Econosafe scintillation cocktail (Research Products International, Mt. Prospect, IL) was added to the fractions, and radioactivity was counted on a Beckman Coulter LS6500 scintillation counter (Fullerton, CA).
[125I]RTI-121 Autoradiography. [125I]RTI-121 (PerkinElmer Life Analytical Sciences) was used to measure binding to the DA transporter as described previously (McCallum et al., 2005). Twenty-micrometer-thick striatal sections were thawed and preincubated for 2 x 15 min in 50 mM Tris-HCl buffer, pH 7.4, containing 120 mM NaCl and 5 mM KCl at room temperature. Sections were then incubated for 2 h in the same buffer plus 50 pM [125I]RTI-121, 0.025% bovine serum albumin, and 1 µM fluoxetine. Addition of 100 µM nomifensine to the incubation buffer was used to define nonspecific binding. Incubation was followed by 4 x 15-min washes in 4°C preincubation buffer. Sections were then dipped in ice-cold water, air dried, and exposed to Kodak BioMax MR film (VWR, West Chester, PA) with 125I microscale standards (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
125I--ConotoxinMII Autoradiography. 125I--ConotoxinMII (J. M. McIntosh) was synthesized and radiolabeled as described previously (Whiteaker et al., 2000), and binding was performed as reported previously (McCallum et al., 2005). HEPES buffer (20 mM), containing 144 mM NaCl, 1.5 mM KCl, 2 mM CaCl2, and 1 mM MgSO4, pH 7.5, was used throughout the experiment. Thawed sections were preincubated at room temperature for 15 min in buffer plus 0.1% bovine serum albumin and 1 mM phenylmethylsulfonyl fluoride. Cotinine (0.1 nM–10 mM) was included in the preincubation and incubation buffers for the competition studies. Nicotine (100 µM) was used to define nonspecific binding and was added to both preincubation and incubation buffers, when used. Sections were then incubated for 1 h in buffer that included 0.5 nM 125I--conotoxinMII, 0.5% bovine serum albumin, 5 mM EDTA, 5 mM EGTA, and 1 µl/ml each of aprotinin, leupeptin, and pepstatin A. Sections were washed with buffer for 10 min at room temperature and then at 4°C, followed by a 2 x 10-min wash in 4°C 0.1x buffer, and dipped in 4°C water twice. They were air-dried and exposed to Kodak BioMax MR film with 125I standards for several days.
[125I]A-85380 Autoradiography. [125I]A-85380 binding was performed as described previously (Kulak et al., 2002a). In brief, thawed sections were incubated for 60 min at room temperature in 50 mM Tris buffer (120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, and 1 mM MgCl2, pH 7.5) containing 200 pM [125I]A-85380. Cotinine (1 nM–1 mM) was included in the incubation buffer for the competition studies. Nicotine (100 µM) was used to define nonspecific binding. Sections were washed for 2 x 5 min in 4°C buffer on ice and 1 x 10 s in ice-cold ddH2O. They were air-dried and exposed to Kodak BioMax MR film with 125I standards for several days.
Data Analysis. [3H]DA release was quantified as described previously (McCallum et al., 2005). Release was plotted as cpm versus fraction number using a curve-fitting algorithm in SigmaPlot 5.0 for MS-DOS (SPSS Inc., Chicago, IL). Fractions before and after the peak were selected to calculate basal release by plotting values as a single exponential decay function. Baseline was subtracted out, and fractions above 10% of baseline were added to obtain evoked release. This value was then normalized to wet tissue weight per filter to obtain cpm/mg tissue. 4β2* and 3/6β2* nAChR components of release were discriminated by the addition of -conotoxinMII to perfusion buffer. Release remaining in the presence of -conotoxinMII was mediated by 4β2* nAChRs. The 3/6β2* nAChR-mediated component was determined by subtraction of the 4β2* component from total release. Rmax and EC50 values for dose-response curves were calculated by nonlinear regression equations in GraphPad Prism (GraphPad Software Inc., San Diego, CA).
The ImageQuant program (GE Healthcare) was used to obtain optical density values from autoradiographic films. Specific binding was calculated by subtracting background tissue levels from total binding, and these values were converted to femtomole per milligram of tissue using standard curves generated from 125I radioactivity standards. Sample optical density readings were within the linear range of the film.
Statistical Analysis. Statistical comparisons were conducted using two-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests. A level of p < 0.05 was considered significant. All analyses were performed using GraphPad Prism. Data represent the mean ± S.E.M. of the indicated number of animals. Values for each animal represent the average from two or more independent experiments.
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Results |
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3/6β2* and 4β2* nAChRs in Caudate. To confirm that cotinine is interacting with striatal nAChRs and to determine the subtypes that bind cotinine, we characterized cotinine inhibition of 125I--conotoxinMII and [125I]A-85380 binding in the medial and lateral caudate. We used these two radioligands because 125I--conotoxinMII binds to 3/6β2* nAChRs, whereas [125I]A-85380 binds to both 3/6β2* and 4β2* receptors (Kulak et al., 2002a,b), the major nAChR subtypes in the striatum.
Cotinine blocked 125I--conotoxinMII binding in a concentration-dependent manner in both striatal regions, with complete inhibition observed at 10–3 M, suggesting that cotinine binds to all 3/6β2* receptor subtypes (Fig. 2A). The IC50 values for cotinine inhibition of 125I--conotoxinMII binding were similar in the medial [3.588 (1.08–4.51) µM] and lateral [3.101 (1.11–2.68) µM] caudate (Hill slopes =–0.88 and –0.87, respectively). Thus, cotinine is nearly 1000-fold less potent than nicotine (IC50 = 5.7 ± 0.4 nM) at striatal 125I--conotoxinMII binding sites (Quik et al., 2001).
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Cotinine also inhibited [125I]A-85380 binding, with a complete block at 10–3 M. These data indicate that cotinine interacts with both 3/6β2* and 4β2* receptor subtypes (Fig. 2B). The IC50 for cotinine inhibition of [125I]A-85380 binding was 78.7 µM (48.7–127.1 µM) in the medial caudate and 64.8 µM (32.4–129.5 µM) in the lateral caudate, approximately 10,000-fold less potent than nicotine (7.53 nM) (Kulak et al., 2002a). The Hill slope for the medial caudate was –0.65, possibly suggesting negative cooperativity or a heterogeneous population of receptors. However, a nonlinear regression one-site versus two-site comparison analysis indicated the presence of only one binding site. The Hill slope in the lateral caudate was –0.99.
Cotinine Selectively Activates Only a Subpopulation of 3/6β2* nAChRs in the Medial and Lateral Caudate. We next examined the functional effects of cotinine on nAChR-evoked [3H]DA release from medial and lateral caudate synaptosomes. The 3/6β2*-specific antagonist -conotoxinMII was used to discriminate between 3/6β2* and 4β2* nAChR-evoked release. Release remaining in the presence of -conotoxinMII was defined as that mediated by 4β2* nAChRs, whereas 3/6β2* nAChR-mediated release was defined as the difference between total and -conotoxin-MII-resistant (4β2*) release.
Cotinine and nicotine dose-response curves revealed that cotinine was less potent than nicotine at 3/6β2* receptors, with cotinine EC50 values 200-fold greater than those for nicotine in both the medial and lateral caudate (Table 1; Fig. 3). Cotinine was also less efficacious than nicotine in the medial although not lateral caudate. Cotinine-stimulated 3/6β2* nAChR-mediated [3H]DA release was only approximately half that of nicotine in the medial caudate (p < 0.001; Fig. 3, A and C; Table 3). In contrast, cotinine and nicotine yielded similar levels of release in the lateral caudate, with a maximal dose of each ligand (Rmax) eliciting 6000 cpm/mg of tissue [3H]DA release (5659 ± 681.4 cotinine, 6669 ± 533.9 nicotine) (Fig. 3B; Table 3). It is noteworthy that this value is comparable to maximal cotinine-evoked [3H]DA release in the medial caudate (7612 ± 1422 cpm/mg tissue) (Fig. 3, A and C).
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These combined data suggest that there are two 3/6β2* nAChR subpopulations that mediate DA release, only one of which is activated by cotinine. The cotinine-insensitive subtype seems to be present only in the medial caudate, whereas the cotinine-sensitive population mediates DA release in both the medial and lateral caudate.
Cotinine Does Not Discriminate between 4β2* nAChR Subtypes Mediating [3H]DA Release in the Medial or Lateral Caudate. As with 3/6β2* receptors, cotinine was less potent than nicotine in eliciting 4β2* subtype-mediated [3H]DA release in the medial and lateral caudate (Table 1). However, in both regions, cotinine and nicotine stimulated similar levels of [3H]DA release (Fig. 4; Table 4). Consistent with previous studies, greater release was observed in the medial compared with lateral caudate (McCallum et al., 2005, 2006).
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The Cotinine-Insensitive 3/6β2* nAChR Subpopulation in the Medial Caudate Is Preferentially Lost with MPTP Lesioning. We subsequently evaluated the effects of nigrostriatal damage on 3/6β2* nAChR-mediated [3H]DA release. MPTP administration resulted in a significant decrease in DA transporter values in the medial caudate (Table 2). This was accompanied by a significant reduction in nicotine-stimulated [3H]DA release, with a 44% decrease in Rmax (p < 0.05; Figs. 5A and 6B; Table 3), similar to previously reported results (McCallum et al., 2006). In contrast, cotinine-evoked release was similar to control after nigrostriatal damage (Figs. 5B and 6B; Table 3). It is noteworthy that the nicotine-stimulated release was reduced to a level similar to cotinine-evoked release in the control and MPTP-lesioned conditions (Figs. 5, A and B, and 6B; Table 3). These data suggest that dopaminergic terminals in the medial caudate expressing the cotinine-insensitive 3/6β2* nAChR subtype are preferentially lost with MPTP-induced nigrostriatal degeneration. This loss of cotinine-insensitive release was accompanied by a 36% decrease in 125I--conotoxinMII binding. These results suggest that approximately one third of the 3/6β2* nAChRs in the medial caudate are selectively vulnerable to nigrostriatal damage (Fig. 6, A and B). However, although this 3/6β2* nAChR subtype binds cotinine, it does not seem to be involved in cotinine-mediated DA release because release is similar in the control and lesioned medial caudate.
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Nicotine- and Cotinine-Evoked 3/6β2* nAChR-Mediated [3H]DA Release in the Lateral Caudate Is Reduced with Nigrostriatal Damage. Nicotine- and cotinine-evoked 3/6β2* nAChR-mediated [3H]DA release from lateral caudate synaptosomes were both reduced to approximately the same extent by MPTP treatment (Figs. 5, C and D, and 6D; Table 3). Rmax values were decreased by 49% for nicotine (p < 0.001) and 47% for cotinine (p < 0.01) (Fig. 6D; Table 3). These data suggest that DA terminals in the lateral caudate that express cotinine-sensitive 3/6β2* nAChRs are vulnerable to nigrostriatal damage, unlike in the medial caudate where they remain unaffected by MPTP treatment. This decrease in DA release is accompanied by a similar loss in the dopamine transporter and 125I--conotoxinMII binding sites in the lateral caudate, which are reduced to 42 and 33% of control levels, respectively (Table 2; Fig. 6C).
MPTP Lesioning Differentially Affects Nicotine- and Cotinine-Evoked 4β2* nAChR-Mediated [3H]DA Release in Lateral but Not Medial Caudate. In the medial caudate, both nicotine- and cotinine-elicited release mediated by 4β2* nAChRs remained unchanged after MPTP administration (Fig. 7, A and B; Table 4). In contrast, although nicotine- and cotinine-stimulated [3H]DA release is equivalent in the control lateral caudate, cotinine-stimulated release is completely lost after nigrostriatal damage (Fig. 7D; Table 4), whereas nicotine-evoked release is only reduced by 46% (p < 0.01, Rmax values) (Fig. 7C).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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3/6β2* nAChR populations on nigrostriatal dopamine terminals in the nonhuman primate striatum. The presence of these two 3/6β2* subtypes was region-specific, with both the cotinine-sensitive and cotinine-insensitive receptors present in the medial caudate but only the cotinine-sensitive population in the lateral caudate. Lesion studies suggest that the dopaminergic terminals that express these cotinine-insensitive receptors are preferentially vulnerable to nigrostriatal damage.
Evidence for the presence of two pharmacologically distinct 3/6β2* nAChRs in the medial caudate stems from the results of our functional studies. These demonstrated that cotinine and nicotine differentially stimulate 3/6β2* nAChR-mediated DA release in the medial caudate, with cotinine-evoked release only 50% of the nicotine-evoked release. In contrast, in the lateral caudate, these two agonists were equally efficacious, and the level of release was similar to cotinine-stimulated release in the medial caudate. These combined data suggest that a cotinine-insensitive subpopulation of 3/6β2* nAChRs is expressed in the medial but not lateral caudate. Our previous data demonstrated a higher expression of 3/6β2* nAChRs in monkey medial compared with the lateral caudate (Quik et al., 2001; McCallum et al., 2005, 2006). These cotinine-insensitive receptors may at least partly account for the higher levels of 3/6β2* nAChR binding sites and nicotine-evoked [3H]DA release in monkey medial caudate.
The use of selective lesioning is frequently used as a means to identify the specific cellular locations of molecular structures. Such studies have contributed to the finding that striatal 3/6β2* nAChRs are present exclusively on DA terminals, whereas 4β2* nAChRs are on both dopaminergic terminals and other neurons in the striatum (Schwartz et al., 1984; Clarke and Pert, 1985; Quik et al., 2001; McCallum et al., 2005, 2006). Therefore, we determined 3/6β2* nAChR expression and function in the striatum of monkeys lesioned with the selective dopaminergic neurotoxin MPTP. The results showed that nicotine-evoked 3/6β2* nAChR-mediated DA release in the medial caudate was decreased after MPTP lesioning as previously observed (McCallum et al., 2005, 2006). However, unexpectedly, cotinine-evoked release was not affected by nigrostriatal damage. It is interesting to note that the lesion reduced nicotine-stimulated release in the medial caudate to a level similar to cotinine-evoked release in the control and MPTP-lesioned conditions. These data provide further support for the presence of two distinct 3/6β2* nAChRs on dopaminergic terminals in monkey medial caudate. In addition, these data indicate that cotinine-insensitive 3/6β2* nAChRs are selectively lost with nigrostriatal damage. One possible explanation for these results is that DA terminals expressing these nAChRs are particularly vulnerable to nigrostriatal degeneration. If this interpretation is correct, this unique subtype could serve as a selective marker for these nigrostriatal fibers in the medial caudate. Alternatively, because striatal dopaminergic measures have been shown to recover after an acute lesion, a lesion-induced decline in cotinine-sensitive 3/6β2* nAChRs may have been masked by axonal sprouting and a selective re-expression of this subtype. However, this seems unlikely given the fact that the cotinine-sensitive subtype was unaffected at both postlesion time points tested (2 and 6 weeks).
The lesion studies also revealed that the functional effects of the cotinine-insensitive 3/6β2* receptors are greater than would be expected based on their relative numbers in the medial caudate. The 50% decline in DA release attributed to a loss of cotinine-insensitive receptors was accompanied by only a 36% reduction in 125I--conotoxinMII binding, suggesting that approximately one third of all 3/6β2* nAChRs in the medial caudate are responsible for mediating half of the 3/6β2* nAChR-evoked DA release. In addition, all of these cotinine-insensitive receptors seem to be located on only 20% of DA terminals in the medial caudate, as demonstrated by the decline in dopamine transporter levels after nigrostriatal damage.
In contrast to the preservation of cotinine-sensitive 3/6β2* nAChR-mediated DA release in the medial caudate after lesioning, a decrease in release was observed in the lateral caudate. This decline may relate to the well known enhanced vulnerability of nigrostriatal dopaminergic neurons projecting to the dorsolateral striatum (Kish et al., 1988; Moratalla et al., 1992). The lack of cotinine-insensitive 3/6β2* nAChRs on dopaminergic terminals in the lateral caudate may somehow contribute to the increased susceptibility of these nigrostriatal projections to degenerative processes, relative to the medial caudate.
Combined, these data provide further evidence for the presence of a cotinine-insensitive 3/6β2* nAChR in the medial caudate. The possibility also exists that cotinine is acting as a partial agonist at nicotinic receptors. However, if this were the case, a decline in nicotine- and cotinine-evoked 3/6β2* nAChR-mediated [3H]DA release might be expected after nigrostriatal damage. Instead, only nicotine-stimulated release is reduced, with the level of release decreased to that of cotinine-evoked release in the control condition. A further argument against a partial agonist action stems from our results showing that cotinine is a full agonist in the lateral caudate, with cotinine and nicotine eliciting the same amount of [3H]DA release.
Cotinine also stimulates [3H]DA release via activation of 4β2* nAChRs. These results are in agreement with the receptor competition studies presented here and elsewhere showing that cotinine inhibits binding to both 3/6β2* and 4β2* nAChR sites (Sloan et al., 1984; Anderson and Arneric, 1994; Vainio and Tuominen, 2001). In the control condition, cotinine-evoked 4β2* nAChR-mediated release was similar to nicotine-mediated release in each of the caudate regions, indicating that cotinine does not discriminate between striatal 4β2* nAChR subpopulations. Nigrostriatal damage significantly decreased 4β2* nAChR-mediated DA release in the lateral but not medial caudate, consistent with previous studies showing an enhanced susceptibility of the dorsolateral striatum to MPTP treatment (McCallum et al., 2005, 2006). However, whereas nicotine-evoked release was reduced by approximately half, cotinine-stimulated release was completely abolished. One possible explanation for this discrepancy could be an MPTP-induced alteration in receptor stoichiometry, for instance, (4)2(β2)3 versus (4)3(β2)2. Recent work has shown that alternate stoichiometric arrangements of the 4β2 nAChR yield high- and low-affinity subforms that possess significantly different functional and pharmacological properties (Briggs et al., 2006; Moroni and Bermudez, 2006). Thus, a change in the ratio of 4 to β2 subunits with nigrostriatal damage may result in an 4β2* nAChR that is cotinine insensitive. Alternatively, the 4β2* receptors may have a different composition before and after lesioning, for instance, 4β2 versus 42β2, which both seem to be present in monkey striatum (Quik et al., 2005).
A further question that arises is the composition of the cotinine-sensitive and -insensitive 3/6β2* nAChR subpopulations. At least three different 3/6β2* nAChR populations have been identified by radioligand binding in monkey striatum, including 64β2β3, 6β2β3, and 3β2* nAChRs, with the 64β2β3 subtype particularly susceptible to nigrostriatal damage in all striatal subregions (Quik et al., 2005; Bordia et al., 2007). However, the present results show differential effects of cotinine on receptor-mediated function in the medial and lateral caudate. One interpretation of these seemingly discrepant results is that the 64β2β3 subtype is insensitive to cotinine in the medial, but it is sensitive in the lateral caudate. This may arise because of differential post-translational modifications and/or stoichiometric conformations of the receptors in the two regions. However, it may not be possible to correlate the present results derived from release assays with those from radioligand binding studies because the conditions for binding favor a desensitized state and the ligand may bind to both intracellular and extracellular receptors, whereas release assays involve only functional membrane-bound receptors. Further studies with more selective 3/6β2* nAChR agonists and antagonists are required to understand the specific subtypes involved. On the other hand, the differential experimental conditions of the binding and functional assays may point to an explanation for the greater differences in nicotine and cotinine potencies observed in the binding assays (10,000-fold differences in potency) compared with the functional assays (100–1000-fold). Nicotine may bind particularly well to desensitized receptors of one or more subtypes compared with cotinine. Alternatively, or as well, nicotine and cotinine may possess differential capacities for desensitization of the receptors and thus different affinities for the receptors.
The biological significance of cotinine is an important question because this nicotine metabolite has a very long half-life and is present at relatively high levels in plasma compared with nicotine. Behavioral studies have shown that cotinine improves performance on a number of cognitive tasks in rats (Buccafusco and Terry, 2003; Terry et al., 2005). In addition, cotinine protects against toxic insults in PC12 cells, with an 80% restoration of cell viability and a potency similar to that of nicotine (Buccafusco and Terry, 2003). Our results indicate that cotinine can stimulate striatal dopamine release in an in vitro preparation, suggesting that it may have functional effects in the nigrostriatal pathway. Admittedly, cotinine-mediated functional responses in striatal synaptosomes are observed at higher concentrations than those that are probably present in the brains of smokers based on plasma levels and the brain levels observed in rats (micromolar versus nanomolar concentrations) (Hatsukami et al., 1997; Ghosheh et al., 1999, 2001; Hukkanen et al., 2005). It is possible that cotinine exerts its effects at lower concentrations in intact brain compared with isolated synaptosomes. Alternatively, although brain cotinine concentrations are probably not high enough to activate nAChRs, persistent low levels of agonist are often associated with receptor desensitization (Giniatullin et al., 2005). Thus, at levels found in brains of smokers, cotinine may influence striatal function by chronically desensitizing nAChRs, thereby inhibiting acetylcholine-induced effects. This phenomenon has been demonstrated in a number of experimental systems including rats and bovine chromaffin cells (Vainio et al., 1998a, 2000; Koh et al., 2003; Buccafusco et al., 2007). Because excitotoxicity may be involved in neuronal loss in Parkinson's disease (PD), cotinine inhibition of nAChRs through desensitization may protect against nigrostriatal degeneration by decreasing dopaminergic terminal excitability (Egea et al., 2006). Therefore, cotinine may play a role in the neuroprotection against PD that has been observed in tobacco users, because smokers would presumably have sustained brain levels of cotinine. If cotinine does protect dopaminergic neurons by nAChR desensitization, the cotinine-insensitive receptors in the medial caudate may be more vulnerable to excitotoxic insult, thereby explaining their increased susceptibility to nigrostriatal damage. Therefore, the discrimination of cotinine between 3/6β2* nAChR subtypes may prove useful for the future development of PD therapies that are optimally beneficial with minimal side effects by specifically targeting this subtype.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; DA, dopamine; 4β2*, nAChR containing the 4 and β2 subunits, but not 3 or 6; 3/6β2*, nAChR composed of the 3 or 6 subunits and β2; DA, dopamine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; RTI-121, 3β-(4-iodophenyl) tropane-2β-carboxylic acid isopropyl ester; ANOVA, analysis of variance; A-85380, 5-[125I]iodo-3-[2(S)-azetinylmethoxy]pyridine-2HCl; PD, Parkinson's disease.
* denotes nicotinic receptors containing the indicated and β subunit and possible additional subunits. 
Address correspondence to: Dr. Maryka Quik, The Parkinson's Institute, 675 Almanor Ave., Sunnyvale, CA 94085-2934. E-mail: mquik{at}parkinsonsinstitute.org
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