No abstract

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate many cellular mechanisms, such as cell survival and memory processing, which are also in¯uenced by the serine/ threonine protein kinases ERK1/2. In SH-SY5Y cells and hippocampal neurones, nicotine (100 lM) increased the activity of ERK1/2. This effect was Ca 2+ dependent, and prevented by the a7 nAChR antagonist a-bungarotoxin (a-Bgt) and an inhibitor (PD98059) of the upstream kinase MEK. To determine the intervening steps linking Ca 2+ entry to MEK-ERK1/2 activation, inhibitors of Ca 2+ -dependent kinases were deployed. In SH-SY5Y cells, selective blockers for PKC (Ro 31±8220), CaM kinase II (KN-62) or PI3 kinase (LY 294002) failed to inhibit the nicotine-evoked increase in ERK1/2 activity. In contrast, two structurally different inhibitors of PKA (KT 5720 and H-89) completely prevented the nicotinedependent increase in ERK1/2 activity. Inhibition of the nicotine-evoked increase in ERK1/2 activity by H-89 was also observed in hippocampal cultures. Down stream of PKA, the activity of B-Raf was signi®cantly decreased by nicotine in SH-SY5Y cells, as determined by direct measurement of MEK1 phosphorylation or in vitro kinase assays, whereas the modulation of MEK1 phosphorylation by Raf-1 tended to increase. Thus, this study provides evidence for a novel signalling route coupling the stimulation of a7 nAChR to the activation of ERK1/2, in a Ca 2+ and PKA dependent manner. Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated cation channels permeable to Na + and Ca 2+ and therefore capable of increasing intracellular Ca 2+ concentrations per se and by activation of voltage-operated Ca 2+ channels (VOCC). As a result, they have the capacity to in¯uence a variety of neuronal activities, such as neurotransmitter release (Wonnacott 1997), cell survival (Donnelly Roberts and Brioni 1999), synaptic plasticity (Ji et al. 2001) and memory processing (Levin and Simon 1998). Numerous subtypes of nAChR occur in the brain, including the two major subtypes comprised of a7 and a4b2 subunits, respectively. The a7 nAChR, in particular, has been implicated in several cellular processes, including long-term potentiation (Mansvelder and McGehee 2000;Matsuyama et al. 2000), neuroprotection (Donnelly Roberts et al. 1996;Dajas-Bailador et al. 2000;Kihara et al. 2001) and learning and memory (Levin et al. 1999). Although the Ca 2+ dependence of some of these a7 nAChR-mediated actions has been demonstrated, the downstream mechanisms that follow nAChR activation, and those attributed to a7 in particular, have not been fully elucidated.One candidate implicated in many of the cellular processes also modulated by nAChR stimulation is the mitogen activated/extracellular signal-regulated protein kinase Abbreviations used: a-Bgt, a-bungarotoxin; CaM, kinase II, Ca 2+ calmodulin dependent kinase II; CREB, cyclic AMP response element binding protein; DMEM, Dulbecco's modi®ed Eagle's medium; ECL, electrochemiluminescence; ERK1/2, extracellular-signal regulated pro...

The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, α‐conotoxin‐MII has been reported to inhibit potently and selectively the rat α3/β2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (±)‐anatoxin‐a‐evoked release of [3H]dopamine from rat striatal synaptosomes and slices. α‐Conotoxin‐MII (112 nM) completely blocked acetylcholine‐evoked currents of α3β2 nAChRs expressed in Xenopus oocytes (IC50 = 8.0 ± 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nMα‐conotoxin‐MII. On perfused striatal synaptosomes and slices, α‐conotoxin‐MII dose‐dependently inhibited [3H]dopamine release evoked by 1 µM (±)‐anatoxin‐a with IC50 values of 24.3 ± 2.9 and 17.3 ± 0.1 nM, respectively. The dose‐response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by α‐conotoxin‐MII (112 nM) was 44.9 ± 5.4% for synaptosomes and 25.0 ± 4.1% for slices, compared with an inhibition by 10 µM mecamylamine of 77.9 ± 3.7 and 88.0 ± 2.1%, respectively. These results suggest the presence of presynaptic α3β2‐like nAChRs on striatal dopaminergic terminals, but the incomplete block of (±)‐anatoxin‐a‐evoked [3H]dopamine release by α‐conotoxin‐MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an α‐conotoxin‐MII‐insensitive nAChR.

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha3beta2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha4 and beta2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha4beta2* and alpha3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [(3)H]-dopamine release from striatal synaptosomes with EC(50) values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)-UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of (86)Rb(+) efflux from thalamic synaptosomes, a model of an alpha4beta2* nAChR response, (+/-)-UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha4beta2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)-UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca(2+) in SH-SY5Y cells, a functional assay for native alpha3-containing nAChR. These data support the involvement of alpha4beta2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.

The potent nicotinic agonist anatoxin-a elicits mecamylamine-sensitive [3H]dopamine release from striatal synaptosomes, and this action is both Naãnd Ca2d ependent and is blocked by Cd2~. This suggests that stimulation of presynaptic nicotinic receptors results in Naĩnflux and local depolarisation that activates voltagesensitive Ca2~channels, which in turn provide the Ca2f or exocytosis. Here we have investigated the subtypes of Ca2~channels implicated in this mechanism.[3H]-Dopamine release evoked by anatoxin-a (1 tiM) was partially blocked by 20~.tMnifedipine, whereas KCI-evoked release was insensitive to the dihydropyridine. However, a 56Rb~efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L-type channels. The Ntype Ca2~channel blocker w-conotoxin GVIA (1~tM) blocked anatoxin-a-evoked [3H]dopamine release by 60% but had no significant effect on 86Rb~efflux; release evoked by both 15 and 25 mM KCI was inhibited by only 30%. The P-type channel blocker w-agatoxin IVA (90 nM) also inhibited KCI-evoked release by~30%, whereas anatoxin-a-evoked release was insensitive. The Q-type channel blocker w-conotoxin MVIIC (1~tM)had no effect on either stimulus. These results suggest that presynaptic nicotinic receptors on striatal nerve terminals promote [3H]dopamine release by activation of N-type Ca2channels. In contrast, KCI-evoked [3H]dopaminerelease appears to involve both N-type and P-type channels. Key Words: Presynaptic nicotinic acetylcholine receptor-Voltage-sensitive calcium channels-Calcium channel blockers-Dihydropyridines-[3H] Dopamine release-86Rb~efflux.