Biochemical and Biophysical Research Communications
Regular ArticleFunctional Modulation of Human "Ganglionic-like" Neuronal Nicotinic Acetylcholine Receptors (nAChRs) by L-Type Calcium-Channel Antagonists
Abstract
Recent studies suggest that the neuronal nicotinic acetylcholine receptors present on chromaffin cells contain a 1,4-dihydropyridine-sensitive site whose occupation blocks membrane depolarization (1). In the present study, several L-type Ca2+ channel blockers inhibited the activation of the nAChRs present in the human neuroblastoma cell line, IMR 32, in a dose-dependent manner with IC50 values ranging from 0.8-3 μM. In contrast, ω-Conotoxin GVIA and ω-Agatoxin IVA had no effect up to 100 μM. Furthermore, the inorganic channel blocker, cadmium, had no effect either alone or on the modulatory role of the L-type antagonists, suggesting that the effects of these agents on nAChRs are not mediated via an interaction with calcium channels but possibly via a direct interaction with the nAChR ionophore.
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α3* and α7 nAChR-mediated Ca<sup>2+</sup> transient generation in IMR-32 neuroblastoma cells
2010, Neurochemistry Internationalα3-Containing (α3*) and α7 nicotinic acetylcholine receptors (nAChRs) are expressed in human IMR-32 neuroblastoma cells and implicated in Ca2+ signaling. In this study, we investigated the intracellular Ca2+ transient generation evoked by selective activation of α3* (agonist potency rank order: epibatidine > varenicline > nicotine ≈ cytisine) and α7 (rank order in the presence of α7 positive allosteric modulator or PAM: A-795723 > NS6784 ≈ PNU-282987) using, respectively, varenicline and NS6784 (+α7 PAM) by Ca2+ imaging. Effects of inhibitors of nAChRs (MLA and mecamylamine), ER Ca2+ ATPase pump (CPA and thapsigargin), Ca2+-induced Ca2+ release (ryanodine and dantrolene), Ca2+ channels (nitrendipine, diltiazem, and Cd2+), and removal of extracellular Ca2+ were examined. α7 PAMs, when tested in the presence of NS6784, were more active when added first, followed by the agonist, than in the reverse order. Removal of extracellular Ca2+ – but not CPA, thapsigargin, ryanodine, dantrolene, nitrendipine, diltiazem, or Cd2+ – diminished the α7 agonist-evoked Ca2+ transients. In contrast, only diltiazem and nitrendipine and removal of extracellular Ca2+ inhibited the α3*-mediated Ca2+ transients. The differential effect of diltiazem and nitrendipine versus Cd2+ was due to direct inhibition of α3* nAChRs as revealed by Ca2+ imaging in HEK-293 cells expressing human α3β4 nAChRs and patch clamp in IMR-32 cells. In summary, this study provides evidence that α3* and α7 nAChR agonist-evoked global Ca2+ transient generation in IMR-32 cells does not primarily involve voltage-dependent Ca2+ channels, intracellular Ca2+ stores, or Ca2+-induced Ca2+ release. These mechanisms may, however, be still involved in other forms of nAChR-mediated Ca2+ signaling.
The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: Intracellular calcium increase, calpain/caspase 3 activation, and functional upregulation
2010, Toxicology and Applied PharmacologyPrevious work by our group demonstrated that homomeric α7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca2+ increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific α7 agonist PNU 282987 with IC50 values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human α7 but not with α4β2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and α-bungarotoxin but not by dihydro-β-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on α7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca2+ release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca2+ levels and induced an increase in α-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and α7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca2+-dependent enzymes such as protein kinase C and nitric oxide synthase, which are involved in the generation of ROS and the blockade of the dopamine transporter. This, together with caspase 3 activation, must play a role in MDMA-induced cytotoxicity.
Paradoxical nifedipine facilitation of <sup>45</sup>Ca uptake into rat hippocampal synaptosomes
2006, European Journal of PharmacologyNifedipine has a high incidence of neurologic adverse reactions as compared with other dihydropyridine Cav1 (L-type) channel blockers used for treating cardiovascular diseases. The mechanism mediating neuronal excitation by nifedipine is still in debate. Nifedipine caused a dual role on veratridine-induced 45Ca uptake by rat hippocampal synaptosomes. In the nanomolar range (0.001–0.3 μM), nifedipine decreased 45Ca uptake in a cadmium-sensitive manner. In contrast with nitrendipine (0.001–10 μM), nifedipine consistently facilitated 45Ca accumulation when used in low micromolar concentrations (0.3–10 μM). The cadmium-insensitive nifedipine facilitation became less evident upon increasing veratridine concentration from 5 to 20 μM and was not detected when the synaptosomes where depolarised with 30 mM KCl. Na+ substitution by N-methyl-d-glucamine (132 mM) or blockade of Na+ currents with tetrodotoxin (1 μM) both prevented nifedipine excitation. The Na+/Ca2+-exchanger inhibitor, KB-R7943 (3–50 μM), did not reproduce nifedipine actions. Data suggest that tetrodotoxin-sensitive Na+ channels may operate paradoxical nifedipine facilitation of 45Ca uptake by rat hippocampal synaptosomes.
Inhibition of type a GABA receptors by L-type calcium channel blockers
2004, NeuroscienceModulation of type A GABA receptors (GABAA) by L-type Ca++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat α1β2γ2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC50 for GABA current inhibition was lowest for nitrendipine (17.3±1.3 μM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC50 was 10-fold lower in α1β2 receptors. Nitrendipine's effect in recombinant human α1β2γ2 receptors was similar (IC50=23.0±1.3 μM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABAA receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (β2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist α-isopropyl-α-methyl-γ-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABAA receptor. Possible interaction of nitrendipine with the Zn++ site was also eliminated, as mutation of β2 H267 to A, which confers resistance to Zn++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABAA receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABAA receptor.
Cotinine and nicotine inhibit each other's calcium responses in bovine chromaffin cells
2000, Toxicology and Applied PharmacologyCotinine is the major metabolite of nicotine. It has some biological activity, but its pathophysiological effects are largely unclear. We studied whether cotinine initiates calcium transients or affects those induced by nicotine. In bovine adrenal chromaffin cells labeled with the fluorescent calcium indicator Fura 2, cotinine (0.32–3.2 mM) concentration-dependently increased the intracellular Ca2+ concentration ([Ca2+]i). The effect was abolished by omitting extracellular Ca2+ during the stimulations. Also nicotinic receptor channel blockers hexamethonium (10 μM–1 mM) and chlorisondamine (100 μM), as well as a competitive nicotinic receptor antagonist dihydro-β-erythroidine (10–100 μM), inhibited the response. Cotinine (0.32–3.2 mM) preincubation for 2 min inhibited both the nicotine-induced and the cotinine-induced increases in [Ca2+]i. Also nicotine (3.2–10 μM) inhibited the cotinine-induced increase in [Ca2+]i. Tetrodotoxin (1 μM) and thapsigargin (1 μM) pretreatments did not affect the responses to cotinine, while 300 nM nimodipine partially inhibited the cotinine-induced increase in [Ca2+]i. The results indicate that cotinine has nicotine-like effects on chromaffin cells. It may also desensitize the nicotinic cholinergic receptors, possibly by acting as a low-affinity agonist at these receptors.
Human neuronal nicotinic receptors
1997, Progress in NeurobiologyNicotine is a very widely used drug of abuse, which exerts a number of neurovegetative, behavioural and psychological effects by interacting with neuronal nicotinic acetylcholine receptors (NAChRs). These receptors are distributed widely in human brain and ganglia, and form a family of ACh-gated ion channels of different subtypes, each of which has a specific pharmacology and physiology. As human NAChRs have been implicated in a number of human central nervous system disorders (including the neurodegenerative Alzheimer's disease, schizophrenia and epilepsy), they are suitable potential targets for rational drug therapy.
Much of our current knowledge about the structure and function of NAChRs comes from studies carried out in other species, such as rodents and chicks, and information concerning human nicotinic receptors is still incomplete and scattered in the literature. Nevertheless, it is already evident that there are a number of differences in the anatomical distribution, physiology, pharmacology, and expression regulation of certain subtypes between the nicotinic systems of humans and other species.
This review will attempt to survey the major achievements reached in the study of the structure and function of NAChRs by examining the molecular basis of their functional diversity viewed mainly from pharmacological and biochemical perspectives. It will also summarize our current knowledge concerning the structure and function of the NAChRs expressed by other species, and the newly discovered drugs used to classify their numerous subtypes. Finally, the role of NAChRs in behaviour and pathology will be considered.