Introduction
Acetylcholine (ACh) is the main neurotransmitter in the peripheral
nervous system of vertebrates and humans. In particular, it is
responsible for the transmission of signals from the motor nerve to
skeletal muscle (Del Castillo & Katz, 1957; Ciani & Edwards, 1963).
Since neuromuscular junction (NMJ) is a key linker in the initiation of
any motor act (from voluntary movement of the limbs to breathing and
contraction of the vocal cords), investigation of the regulation of
neuromuscular transmission is of great importance for both fundamental
neurobiology and applied medicine.
Since the midst of the 20th century, the data began to
accumulate indicating that ACh, released in the synaptic cleft from the
nerve endings, activates presynaptic cholinergic receptors, thus
exerting a modulatory effect on the neurotransmission process by
changing the amount and/or dynamics of subsequent portions of
neurotransmitter release (Ciani & Edwards, 1963; Starke et al., 1989;
Bowman et al., 1990; Prior et al., 1995; Miller, 1998; Nikolsky et al.,
2004). Initially pharmacologically, and later by other methods it has
been shown that both ionotropic nicotinic and metabotropic muscarinic
cholinergic receptors are present in the motor nerve terminal, and
activation of these receptors can lead to autoregulation of ACh release
(Bowman et al., 1990; Miller, 1990; Santafé et al., 2004).
When studying autoregulation mediated by muscarinic cholinergic
receptors, it was found that activation of the
M1-subtype receptors led to facilitation of the release.
In contrast, activation of the M2-subtype caused
inhibition of the ACh quanta release (Oliveira et al., 2002; Santafé et
al., 2003). Both M1‐ and M2‐mediated
mechanisms depend on calcium influx (Santafé et al., 2003; Slutsky et
al., 2003; Khaziev et al., 2016; Zhilyakov et al., 2019).
Studies of the mechanisms of autoregulation of ACh release mediated by
nicotinic cholinergic receptors are complicated by the fact that the
predominant population of these proteins is located in the postsynaptic
membrane. Their activation is accompanied by depolarization of
sarcolemma and subsequent generation of action potential, which
ultimately leads to muscle contraction. The data collected by to date
indicate that activation of presynaptic nicotinic cholinergic receptors
leads to inhibition of the process of ACh release (Van der Kloot, 1993;
Prior & Singh, 2000; Balezina et al., 2006).
Also, experimental evidence was obtained indicating possible involvement
of voltage-gated calcium channels (VGCCs) of L-type
(Cav1) in modulation of neurotransmission (Prior &
Singh, 2000). Meanwhile, the results of a number of studies demonstrate
that neither the Cav1 type nor the N-type (Cav2.2) VGCCs participates in
the evoked release of ACh in mammalians mature neuromuscular contacts
(Penner & Dreyer, 1986; Atchison, 1989; Protti et al., 1991; Bowersox
et al., 1995).
Thus, the question on the role of calcium channels in the mechanisms of
regulation of ACh release, mediated by nicotinic cholinergic receptors,
remains open as of now.
In the present study, using a pharmacological approach,
electrophysiological techniques and the method of optical registration
of changes in the calcium level in the motor nerve ending, we made the
following observations. An agonist of nicotinic receptors (at a
concentration not significantly affecting the state of the postsynaptic
membrane) leads to a decrease in the amount of released ACh quanta. This
effect is accompanied not by a decrease, but by an increase of calcium
ions entry into the motor nerve terminal. Our data suggest that
nicotinic cholinergic receptors responsible for the mechanism of ACh
release autoregulation are the receptors of neuronal type. Activation of
these receptors leads to upregulation of Cav1 type of
VGCCs, resulting the enhancement of Ca2+ entry into
the nerve ending.