Stimulation of dopamine release by nicotinic acetylcholine receptor ligands in rat brain slices correlates with the profile of high, but not low, sensitivity α4β2 subunit combination
Graphical abstract
Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) belong to the family of pentameric acetylcholine-gated cation channels and are assembled from twelve subunits, α2–α10 and β2–β4. These subunits combine to form homomeric or heteromeric nAChRs that mediate a wide range of physiological and pharmacological effects in the nervous system. All are thought to be pentameric, comprised of a mixture of alpha-subunits (α2–α6) and beta-subunits (β2–β4) with exceptions of homomers of α7–α9 subunits, or heteromers of α9–α10 subunits [1]. A recent report provided evidence for the existence of a functional α7β2 nAChR in rodent basal forebrain [2]. The α4β2-containing (α4β2*) nAChRs account for 90% of the high affinity nicotine binding sites and are widely distributed throughout the brain including in the cortex, hippocampus, substantia nigra, and ventral tegmental area (reviewed in [3]). The last two regions are particularly important since they are rich in dopaminergic neurons whose function is regulated by various nicotinic subunits including α4β2* subtype and thought to participate in modulating the reinforcing effects of nicotine and other addictive substances (reviewed in [4]) and in CNS disorders such as Parkinson's disease.
Initially, heteromeric α4β2 subunits were thought to exist as a single subunit entity exhibiting high affinity to nicotine [5]. However, a variety of combinations and stoichiometries are possible as evidenced by injecting varying ratios of α4 and α2 cRNA or cDNA [6], [7], [8], [9].
Combination of excess α4 with respect to β2 subunit is thought to generate predominantly (α4)3(β2)2 pentamer with low sensitivity (LS) to ACh (EC50 ∼ 100 μM), whereas the reverse ratio is thought to favor the (α4)2(β2)3 expression with high sensitivity (HS) to ACh (EC50 ∼ 1 μM). This concept is supported by studies using linked α4β2 subunits assembled with β2 subunits as (α4β2)2β2 pentamers and with α4 subunits as (α4β2)2α4 pentamers to address the stochiometry of functional receptor assemblies [10]. Other compounds exhibiting differential profiles at HS and LS profiles include a variety of agonists (nicotine, epibatidine, cytisine, 3-Br-cytisine, 3-[2(S)-azetidinylmethoxy]pyridine (A-85380), 5-ethoxy-metanicotine (TC-2559), A-163554 and A-168939) and some antagonists, particularly chlorisondamine [9], [11]. The LS form is thought to be more permeable to Ca2+ than the HS form whereas the HS form is more sensitive to upregulation by chronic nicotine and reduced ambient temperature [12]. Other combinations with various degrees of sensitivities and selectivities for nAChR ligands can be formed with the substitution of other subunits such as α2, α3, α6 or β4 [3]. Like α4β2, HS and LS forms of α3β2 also have been demonstrated, suggesting this may be a general feature of β2-containing nAChR [9]. The α5 and β3 subunits do not participate in the formation of acetylcholine binding sites, but may have accessory or regulatory roles in the receptor affecting the properties of the expressed pentamer. For instance, (α4β2)2 α5 nAChRs were reported to be more permeable to Ca2+ than either (α4β2)2β2 and (α4β2)2α4 pentamers [13]. One brain region that has been demonstrated to be especially rich in the variety of nAChRs present is the striatum with up to six nAChR combinations proposed [14]. Through the use of null mutant mice, immunoprecipitation, and inhibition with α-conotoxin MII, it has been ascertained that there likely exist α4β2, α4α5β2, α6β2, α4α6β2β3, and α6β2β3 combinations as well as the homomeric α7 in the striatum.
However, HS and LS forms of β2-containing nAChR are defined mainly by kinetic (EC50) parameters. Whether both forms are expressed in the CNS and, if so, whether they have different functional roles is not yet fully elucidated. In mouse thalamic synaptosomes, responses to HS α4β2 nAChR selective agonists such as A-163554 and A-168939 suggest that at least the HS form is expressed functionally in that brain region [9]. Whether LS α4β2 nAChR also is expressed and whether the pattern may vary among brain regions remain open questions.
In this study, we hypothesized that there may be differential contributions of HS, (α4)2(β2)3, and LS, (α4)3(β2)2, nAChRs in neurotransmitter release. We addressed the contribution of HS and LS α4β2 to nAChR-mediated [3H]-dopamine release from slices of rat prefrontal cortex and striatum by using a set of five agonists, and compared the pharmacological profiles of transmitter release with those of HS and LS α4β2 nAChRs expressed in Xenopus oocytes and α4β2* ([3H]-cytisine) binding in rat brain. Additionally, since multiple receptor subtypes are involved in nAChR-mediated [3H]-dopamine release in the striatum, α-conotoxin MII was used to distinguish the role of α4β2* from α6β2* nAChRs. Our results suggest that the HS, but not the LS, form of α4β2 nAChR is primarily involved in nicotine-stimulated dopamine release.
Section snippets
Materials
Membranes for radioligand binding studies were prepared from frozen rat brains (PelFreez, Rogers, AR, USA). For neurotransmitter release studies, male Sprague–Dawley rats were used from Charles River Laboratories (Portage, MI, USA, 250–400 g). Oocytes were obtained from adult female Xenopus laevis frogs (Blades Biological Ltd., Cowden, Edenbridge, Kent, UK). Animals were cared for in accordance with the Institutional Animal Care Committee guidelines that meet the guidelines of the National
Results
Binding affinities of nAChR ligands at α4β2* nAChRs were assessed by displacement of [3H]-cytisine binding to rat brain membranes (Table 1). Varenicline had a Ki value of 0.08 nM, which was 10-fold higher than (−)-nicotine (Ki = 0.94 nM). The other three compounds also had relatively high affinity with Ki values of 2.7 nM for AZD3480, 11.4 nM for dianicline, and 14.1 nM for ABT-089.
Changes in the stoichiometry of the nAChR pentameric structure can be influenced by the ratio of human subunit cRNAs
Discussion
Previous studies have demonstrated that α4β2 nAChRs can exist in high sensitivity (HS) and low sensitivity (LS) states dependent on stoichiometry [6], [7], [8], [9]. However, the contributions of high and low sensitivity combinations to functional effects mediated by nAChR ligands, as for example dopamine release, remain unclear. Release of [3H]-dopamine from rat brain slices evoked by nicotine and nAChR agonists is mediated by multiple receptor subtypes, including α4β2-containing and
Acknowledgement
This work was supported by Abbott Laboratories.
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Present address: Dept. Neuroscience, Rosalind Franklin University, 3333 Green Bay Road, North Chicago, IL 60064, USA.