Neuronal nicotinic receptors alter calcium level in presynaptic terminal by gating L-type (Cav1) calcium channels
To identify the source of the increase in the calcium signal upon activation of presynaptic nNAChRs, a nonselective blocker of calcium-permeable channels, cadmium chloride at a concentration of 10 µM, was used. After application of cadmium chloride, a decrease in the amplitude of the calcium transient was observed by 54.5 ± 2.7% (n = 5, 9 NMJs). In the presence of cadmium the effect of nicotine on the alterations in calcium levels was completely abolished (101.4 ± 3.4%; n = 5, 9 NMJs; Figure 5). Therefore, the observed increase in the presynaptic calcium level upon activation of nNAChRs is mediated by proteins (channels) which are permeable for Ca2+. Further experiments were carried out to establish which type of VGCCs is involved in nicotine-induced increases in calcium transients.
Application of the specific P/Q-type (Cav2.1) VGCCs blocker ω-agatoxin IVA at a concentration of 40 nM that blocks only a certain proportion of channels (Protti & Uchitel, 1993) led to a significant decrease in the calcium transient by 67 ± 4.4 % (n = 5, 5 NMJs; Figure 5). In case of partial blockade of the main type of VGCCs Cav2.1, nicotine application (10 µM) led to an increase in the amplitude of the calcium transient significantly by 29.9 ± 3.8% (n = 5, 10 NMJs, Figure 5). Therefore, the effect of activation of nNAChRs on the intracellular calcium level is not mediated by Cav2.1 channels.
Cav1 calcium channel blockers such as verapamil (50 µM) and nitrendipine (25 µM), produced significant calcium transient decrease by 25 ± 4.4% (n = 5, 9 NMJs) and 18.8 ± 1.1% (n = 5, 17 NMJs), respectively (Figure 6). Application of nicotine after pretreatment by these blockers did not cause any changes in the calcium transient: the amplitudes were 101.8 ± 1.5% (n = 5, 9 NMJs) and 100.7 ± 1.2% (n = 5, 17 NMJs), respectively (Figure 6). These data allow us to conclude that observed increase in the calcium level in the nerve ending upon activation of nNAChRs by an exogenous agonist is due to mediation by Cav1 type VGCCs. Therefore, the phenomenon of endogenous activation of presynaptic cholinergic receptors discovered by us should also be mediated by calcium channels of this type. Indeed, the calcium transient-reducing effect of DHβE, when Cav1 channels were blocked by nitrendipine, was completely abolished (100.0 ± 0.8%; n = 5, 7 NMJs; Figure 6).
Thus, the results obtained allow us to conclude that activation of nNAChRs leads to an additional increase in the entry of Ca2+ into the nerve ending through VGCCs of the Cav1 type. Therefore, if this is the mechanism underlying the decrease in the quantal content upon activation of this type of cholinergic receptors, then it should be expected that the blockade of Cav1 type of calcium channels will eliminate the nicotine-induced decrease in the amount of released ACh quanta. Examining this assumption became the scope of the next step of the study.