L. Soliakov

Presynaptic nicotinic receptors modulating dopamine release in the rat striatum

The modulation of striatal dopamine release by presynaptic nicotinic acetylcholine receptors is well documented for both synaptosomes and slices. Because the latter retain local anatomical integrity, we have compared [3H]dopamine release evoked by the nicotinic receptor agonists (-)-nicotine and (+/-)-anatoxin-a from striatal synaptosome and slice preparations in parallel. At higher agonist concentrations, mecamylamine-sensitive [3H]dopamine release was greater from slices, indicative of an additional component, and this increase was abolished by glutamate receptor antagonists. To begin to examine the localisation of specific nicotinic acetylcholine receptor subtypes in the striatum, immunogold electron microscopy was undertaken with the beta2-specific monoclonal antibody 270. In striatal sections, gold particles were associated with symmetric synapses (dopaminergic) but were absent from asymmetric synapses (glutamatergic). Surface labelling of striatal synaptosomes with gold particles was also demonstrated. Taken together, these results are consistent with dopamine release mediated by beta2-containing nicotinic acetylcholine receptors on dopamine terminals, while non-beta2-containing nicotinic acetylcholine receptors may enhance dopamine release indirectly by releasing glutamate from neighbouring terminals.

Nicotine activates the extracellular signal-regulated kinase 1/2 via the α7 nicotinic acetylcholine receptor and protein kinase A, in SH-SY5Y cells and hippocampal neurones

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate many cellular mechanisms, such as cell survival and memory processing, which are also influenced by the serine/threonine protein kinases ERK1/2. In SH‐SY5Y cells and hippocampal neurones, nicotine (100 µm) increased the activity of ERK1/2. This effect was Ca2+ dependent, and prevented by the α7 nAChR antagonist α‐bungarotoxin (α‐Bgt) and an inhibitor (PD98059) of the upstream kinase MEK. To determine the intervening steps linking Ca2+ entry to MEK‐ERK1/2 activation, inhibitors of Ca2+‐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 nicotine‐dependent 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 significantly 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 α7 nAChR to the activation of ERK1/2, in a Ca2+ and PKA dependent manner.

Differential Inhibition by α‐Conotoxin‐MII of the Nicotinic Stimulation of [3H]Dopamine Release from Rat Striatal Synaptosomes and Slices

Abstract: 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.

Voltage‐Sensitive Ca2+ Channels Involved in Nicotinic Receptor‐Mediated [3H]Dopamine Release from Rat Striatal Synaptosomes

Abstract: The potent nicotinic agonist anatoxin‐a elicits mecamylamine‐sensitive [3H]dopamine release from striatal synaptosomes, and this action is both Na+ and Ca2+ dependent and is blocked by Cd2+. This suggests that stimulation of presynaptic nicotinic receptors results in Na+ influx and local depolarisation that activates voltage‐sensitive Ca2+ channels, which in turn provide the Ca2+ for exocytosis. Here we have investigated the subtypes of Ca2+ channels implicated in this mechanism. [3H]Dopamine release evoked by anatoxin‐a (1 µM) was partially blocked by 20 µM nifedipine, whereas KCl‐evoked release was insensitive to the dihydropyridine. However, a 86Rb+ efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L‐type channels. The N‐type Ca2+ channel blocker ω‐conotoxin GVIA (1 µM) 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 KCl was inhibited by only 30%. The P‐type channel blocker ω‐agatoxin IVA (90 nM) also inhibited KCl‐evoked release by ∼30%, whereas anatoxin‐a‐evoked release was insensitive. The Q‐type channel blocker ω‐conotoxin MVIIC (1 µM) 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 Ca2+ channels. In contrast, KCl‐evoked [3H]dopamine release appears to involve both N‐type and P‐type channels.

Anatoxin-a-evoked [3H]dopamine release from rat striatal synaptosomes

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. Using rat striatal synaptosomes loaded with [3H]dopamine, we have characterized the action of the selective nicotinic agonist, (+/-)anatoxin-a, with respect to [3H]dopamine release, in order to explore the mechanisms coupling nicotinic receptor activation to exocytosis. Anatoxin-a evoked [3H]dopamine release in a concentration-dependent and mecamylamine-sensitive manner, EC50 = 0.11 microM. The maximum [3H]dopamine release elicited by anatoxin-a was only 20% of the maximum elicited by KCl depolarization; there was no additivity between anatoxin-a and sub-maximal concentrations of KCl. Both agents stimulated Ca(2+)-dependent release that was equally sensitive to inhibition by 200 microM Cd2+. This result suggests that anatoxin-a-stimulated exocytosis is mediated by Ca2+ influx via voltage-sensitive Ca2+ channels, with little contribution from Ca2+ entering directly through the nicotinic receptor channel. This view is supported by the abolition of anatoxin-a-evoked [3H]dopamine release in Na(+)-depleted medium. A partial (40%) inhibition by tetrodotoxin was observed. These data suggest that activation of presynaptic nicotinic acetylcholine receptors by anatoxin-a results in an influx of Na+, producing sufficient local depolarization to open voltage-sensitive Ca2+ and Na+ channels. The latter may then amplify the response, activating further Ca2+ channels. The particular voltage-sensitive Ca2+ channels involved remain to be determined.

Tetrodotoxin-sensitivity of nicotine-evoked dopamine release from rat striatum

Recent observations from synaptosome preparations have questioned the tetrodotoxin (TTX) insensitivity of nicotine-evoked release in the striatum, a characteristic previously considered diagnostic of presynaptically located nicotinic acetylcholine receptors (nAChRs). Therefore, we have undertaken a comparison of nicotine-evoked dopamine release in the presence of TTX from the rat striatum in vitro, using synaptosomes and brain slices, and in vivo, using microdialysis. In P2 and Percoll-purified synaptosome preparations, 1.5 microM TTX partially inhibited nicotine-evoked [3H]dopamine release by 54% and 37%, respectively, whereas in more intact preparations (brain slices and microdialysis) TTX completely inhibited mecamylamine-sensitive nicotine-stimulated dopamine release. These results suggest that caution should be exercised in the interpretation of TTX sensitivity of nicotine-evoked responses with regard to the location of nAChRs.

Presynaptic nicotinic acetylcholine receptors in the brain

Nicotine, acting through nicotinic acetylcholine receptors (nAChR) located on nerve terminals, can evoke the release of various neurotransmitters in the brain. The presynaptic nicotinic stimulation of acetylcholine release, demonstrated in cortical and hippocampal preparations, may reflect a positive feedback mechanism via autoreceptors. This site is a target for novel nicotinic agonists in the symptomatic treatment of Alzheimers disease. Nicotinic heteroreceptors can modulate the release of catecholamines and amino acid transmitters in diverse brain regions. Differences in agonist potency and efficacy and in antagonist sensitivities between different transmitter pathways suggests heterogeneity of subtypes of presynaptic nAChR (notably between α3‐ and α4‐containing nAChR). While neurochemical studies have failed to find any evidence for the involvement of α7‐type nAChR in the presynaptic modulation of transmitter release, recent electrophysiological studies have disclosed this as a possibility with respect to glutamate transmission.

Involvement of protein kinase C in the presynaptic nicotinic modulation of [3H]-dopamine release from rat striatal synaptosomes

Presynaptic nicotinic ACh receptors modulate transmitter release in the brain. Here we report their interactions with protein kinase C (PKC) with respect to [3H]‐dopamine release from rat striatal synaptosomes, monitored by superfusion. Two specific PKC inhibitors, Ro 31‐8220 (1 μM) and D‐erythro‐sphingosine (10 μM) significantly reduced (by 51 and 26% respectively) [3H]‐dopamine release stimulated by anatoxin‐a (AnTx), a potent and selective agonist of nicotinic ACh receptors. The inactive structural analogue of Ro 31‐8220, bisindolylmaleimide V (1 μM) had no effect. Two phorbol esters, PDBu (1 μM) and PMA (1 μM) potentiated AnTx‐evoked [3H]‐dopamine release by 50 – 80%. This was Ca2+‐dependent and prevented by PKC inhibitors. In the absence of nicotinic agonist, phorbol esters enhanced basal release through a PKC‐independent mechanism. A 86Rb+ efflux assay of nicotinic ACh receptor function confirmed that Ro 31‐8220 has no nonspecific effect on presynaptic nicotinic ACh receptors. These results suggest that PKC is activated by nicotinic ACh receptor stimulation and mediates a component of AnTx‐evoked [3H]‐dopamine release. In addition, independent activation of PKC can further amplify the response, offering a potential mechanism for receptor crosstalk.