Calcium signalling mediated through α7 and non‐α7 nAChR stimulation is differentially regulated in bovine chromaffin cells to induce catecholamine release

Abstract

BACKGROUND AND PURPOSE Ca2+ signalling and exocytosis mediated by nicotinic receptor (nAChR) subtypes, especially the a7 nAChR, in bovine chromaffin cells are still matters of debate. EXPERIMENTAL APPROACHWe have used chromaffin cell cultures loaded with Fluo-4 or transfected with aequorins directed to the cytosol or mitochondria, several nAChR agonists (nicotine, 5-iodo-A-85380, PNU282987 and choline), and the a7 nAChR allosteric modulator PNU120596. KEY RESULTS Minimal [Ca 2+]c transients, induced by low concentrations of selective a7 nAChR agonists and nicotine, were markedly increased by the a7 nAChR allosteric modulator PNU120596. These potentiated responses were completely blocked by the a7 nAChR antagonist a-bungarotoxin (a7-modulated-response). Conversely, high concentrations of the a7 nAChR agonists, nicotine or 5-iodo-A-85380 induced larger [Ca 2+ ]c transients, that were blocked by mecamylamine but were unaffected by a-bungarotoxin (non-a7 response). [Ca 2+ ]c increases mediated by a7 nAChR were related to Ca 2+ entry through non-L-type Ca 2+ channels, whereas non-a7 nAChR-mediated signals were related to L-type Ca 2+ channels; Ca [Ca 2+ ]c and catecholamine release mediated by a7 nAChRs required an allosteric modulator and low doses of the agonist. At higher agonist concentrations, the a7 nAChR response was lost and the non-a7 nAChRs were activated. Catecholamine release might therefore be regulated by different nAChR subtypes, depending on agonist concentrations and the presence of allosteric modulators of a7 nAChRs. [Ca 2+ ]c, cytosolic concentration of calcium; BGT, a-bungarotoxin; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; nAChR, nicotinic acetylcholine receptor; VOCC, voltage operated calcium channel BJP British Journal of Pharmacology CONCLUSIONS AND IMPLICATIONS Abbreviations

Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: Advantages and limitations

“…Additional evidence of D i states is found in a recent study measuring calcium currents evoked by nicotine, PNU-282987, and PNU-120596 in bovine chromaffin cells [150]. In this study, the authors were only able to detect an α-bungarotoxin (α-btx)-sensitive and PNU-120596-dependent increase in calcium response when low (1 μM) concentrations of nicotine were used to stimulate the cells.…”

“…In this study, the authors were only able to detect an α-bungarotoxin (α-btx)-sensitive and PNU-120596-dependent increase in calcium response when low (1 μM) concentrations of nicotine were used to stimulate the cells. At concentrations of ≥ 3 μM nicotine, neither PNU-120596 nor α-btx had an effect on the response, indicating that the α7 component was lost by the higher nicotine concentrations [150]. To isolate and more closely examine the disappearing α7 component, varying concentrations of the α7 agonist PNU-282987 with 1 μM PNU-120596 were applied for 5 minutes to evoke a calcium response.…”

“…Any fluorescence increases that were insensitive to blockade by α-btx were subtracted from the total response. Interestingly, as stimulating agonist (in this case PNU-282987) concentrations increased, the α7-dependent calcium responses decreased, indicating greater D i as agonist concentrations increased in the presence of a fixed concentration of PNU-120596 [150]. …”

Investigation of the Molecular Mechanism of the α7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulator PNU-120596 Provides Evidence for Two Distinct Desensitized States

“…Fourth, an ␣-bungarotoxinsensitive and PNU-120596-dependent increase in calcium response from bovine chromaffin cells was only observed when low (1 M) concentrations of nicotine were used to stimulate the cells. At concentrations of Ն3 M nicotine, neither PNU-120596 nor ␣-bungarotoxin had an effect on the response, indicating that the ␣7 component was lost by the higher nicotine concentrations (del Barrio et al, 2011). In addition, when the ␣7 component of the total calcium response was isolated, increased concentrations of the ␣7 agonist N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide (PNU-282987) with a fixed PNU-120596 concentration resulted in decreased ␣7-dependent calcium responses (del Barrio et al, 2011).…”

The effect of α7 nicotinic receptor activation on glutamatergic transmission in the hippocampus

“…The source of the calcium increase would be mainly comprised of the calcium influx directly through the α7 nAChR itself (Fayuk & Yakel, 2007a; Gilbert et al , 2009), and any calcium-induced calcium release from intracellular calcium stores (Weisskopf & Nicoll, 1995; Sharma & Vijayaraghavan, 2003; Sharma et al , 2008). Although calcium influx through voltage-gated calcium channels (VGCC) previously was shown not to contribute significantly to the α7 nAChR-mediated presynaptic effect (Dickinson et al , 2008), other studies reported that calcium influx through VGCCs could contribute to some degree (Barrantes et al , 1995; Fayuk & Yakel, 2007b; del Barrio et al , 2011). In addition, recent studies showed that α7 nAChR activation enhanced NMDAR-mediated whole-cell currents and glutamate release from hippocampal synaptosomes (Zappettini et al , 2010; Li et al , 2013).…”