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.