Effects of nicotine on the electrophysiological parameters of the neuromuscular junction
Using the intracellular microelectrode technique, we recorded the resting membrane potential (RMP) of the muscle fiber, amplitude of miniature endplate potentials (mEPPs), frequency of occurrence of mEPPs and the amplitude of the evoked potentials of the end plate (EPPs).
Alterations in RMP and MEPP amplitude indicate the postsynaptic action of the pharmacological agent. While changes in the frequency of occurrence of mEPPs suggest presynaptic action of the drug. The EPP amplitude, in turn, can vary due to changes at both pre- and post-synaptic levels. Therefore, it was necessary to assess the effect of nicotine on every parameter mentioned above to determine the optimal effective concentration of nicotine to study the autoregulation.
Control RMP value of muscle fibers was -71.48 ± 0.77 mV (n = 5, 30 NMJs). Application of nicotine at concentrations of 0.1 μM, 1 and 5 μM did not affect the RMP significantly, providing the value of -69.72 ± 0.90 mV (n = 5, 30 NMJs), -70.91 ± 0.78 mV (n = 5, 30 NMJs), and -70.85 ± 0.87 mV (n = 5, 30 NMJs), respectively (Figure 1A). A slight significant depolarization was observed when nicotine concentration was increased to 10 μM (-67.04 ± 0.79 mV; n = 5, 30 NMJs); at a concentration of 50 μM a more pronounced depolarization was observed and the mean RMP value decreased to -56.94 ± 1.29 mV (n = 5, 30 NMJs; Figure 1A).
Another sign of postsynaptic action of nicotine was the change in the amplitude of mEPP. The mean value of the amplitude of the spontaneous signal in control was 0.88 ± 0.05 mV (n = 5, 30 NMJs). Application of nicotine in concentration up to 10 μM did not affect mEPP amplitude significantly; the value was: 0.83 ± 0.04 mV (n = 5, 30 NMJs) for 0.1 μM, 0.84 ±0.05 mV (n = 5, 30 NMJs) for 1 μM, 0.96 ± 0.06 mV (n = 5, 30 NMJs) for 5 μM, and 0.83 ± 0.05 mV (n = 5, 30 NMJs) for 10 μM (Figure 1B). Significant decrease in the mEPPs amplitude to 0.55 ±. 0.04 mV (n = 5, 30 NMJs) was observed only with 50 μM nicotine (Figure 1B).
In contrast to the amplitude of mEPPs, the effect of nicotine on the frequency of occurrence of spontaneous signals was detected at significantly lower concentrations. That is, the average value of the frequency of mEPPs upon application of 0.1 and 1 μM nicotine was 1.64 ± 0.15 Hz (n = 5, 30 NMJs) and 1.22 ± 0.12 Hz (n = 5, 30 NMJs), respectively, and did not differ from the control value of 1.57 ± 0.14 Hz; n = 5, 30 NMJs (Figure 1С). After application of 5 μM nicotine the frequency significantly decreased to 1.07 ± 0.08 Hz (n = 5, 30 NMJs) and inhibition was further enhanced to 0.98 ± 0.07 Hz, n = 5, for 10 μM and 0.57 ± 0.06 Hz, n = 5, for 50 μM ( Figure 1С).
The amplitude of the EPP, which reflects the level of evoked ACh release and depends on changes in the sensitivity of the postsynaptic membrane in the area of the neuromuscular contact, was 32.80 ± 1.02 mV (n = 5, 30 NMJs) in control. Application of nicotine at concentrations of 0.1 μM, 1 and 5 μM did not alter the average amplitude of EPP, which was equal to 31.93 ± 1.21 mV (n = 5, 30 NMJs), 32.43 ± 1.11 mV (n = 5, 30 NMJs), and 32.33 ± 1.18 mV (n = 5, 30 NMJs), respectively (Figure 1D). However, nicotine produced a decrease in EPPs amplitude, starting at the concentration of 10 μM (28.36 ± 1.27 mV; n = 5, 30 NMJs), while at 50 μM the amplitude decreased almost twofold to 16.89 ± 1.09 mV (n = 5, 30 NMJs; Figure 1D).
Thus, for further investigations of the ACh release autoregulation mechanisms, concentration of 10 μM nicotine was chosen. When using nicotine at this concentration, a decrease in the EPP amplitude was observable, while there were no changes in the mEPP amplitude (with only a slight depolarization of the sarcolemma).