Prejunctional effects of the nicotinic ACh receptor agonist dimethylphenylpiperazinium at the rat neuromuscular junction

Abstract

1. We have studied the effects of the nicotinic acetylcholine (ACh) receptor agonist dimethylphenylpiperazinium (DMPP) on the evoked release of ACh from motor terminals in the rat isolated hemidiaphragm using an electrophysiological approach. 2. DMPP (1-4 microM) had no effect on the rate of spontaneous quantal ACh release but increased the number of quanta of ACh released per impulse during 50 Hz stimulation. The DMPP-induced increase in evoked ACh release was dependent on the frequency of stimulation, being absent when it was reduced to 0.5 Hz, but was not Ca2+ dependent, being unaffected at 50 Hz by a 4-fold decrease in the extracellular Ca2+ concentration. 3. The facilitation of evoked ACh release at 50 Hz by 2 microM DMPP was abolished by 10 microM of the calmodulin antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride) and, in the presence of W7, 2 microM DMPP depressed evoked ACh release at 0.5 Hz. The ability of the nicotinic ACh receptor antagonist vecuronium (1 microM) to depress evoked ACh release at 50 Hz was also abolished by 10 microM W7. 4. The present findings demonstrate, using an electrophysiological technique, that DMPP can produce changes in the evoked ACh release from rat motor nerve terminals that are consistent with the existence of facilitatory nicotinic ACh receptors on the motor nerve endings. Further, they indicate a role for calmodulin-dependent systems in this facilitatory effect of the compound.

Figure 1. Representative effects of 2 μM DMPP on MEPCs and EPCs recorded from the rat hemidiaphragm preparation

Examples of MEPCs (A and B) and EPCs (C and D) recorded from the same muscle fibre in the absence (A and C) and presence (B and D) of 2.0 μM DMPP. Each trace shows a mean current signal assembled from 47 (A) or 43 (B) individual MEPCs or 80 (C and D) individual EPCs (evoked at 50 Hz) averaged after being aligned to the midpoint of their rising phases. Note that the lack of an effect of DMPP on EPC amplitude (C and D) accompanied by the depression in MEPC amplitude (A and B) implies a DMPP-induced increase in EPC quantal content. Respective calibration bars apply to both control and post-DMPP signals. [Ca²⁺]_o was 1.8 mM.

Figure 2. Effects of DMPP on MEPC amplitudes in the rat hemidiaphragm preparation

Each point is the mean ±s.e.m. of 6–8 individual measurements. For all three concentrations of DMPP studied there was a significant depression in MEPC amplitude (*P < 0.05). Values shown are MEPC amplitude in the presence of each concentration of DMPP expressed as a percentage of the matched control and indicate the concentration dependence of the effect of DMPP.

Figure 3. Effects of DMPP on EPC amplitudes (at 50 Hz) in the rat hemidiaphragm preparation

Data are shown for varying concentrations of DMPP with a fixed [Ca²⁺]_o of 1.8 mM (A) and for varying [Ca²⁺]_o with a fixed DMPP concentration of 2 μM (B). All points are means and s.e.m. of data from 6–8 individual determinations. *P < 0.05, DMPP versus control.

Figure 4. Effect of DMPP on EPC quantal content (at 50 Hz) in the rat isolated hemidiaphragm muscle preparation

Data are shown for varying concentrations of DMPP with a fixed [Ca²⁺]_o of 1.8 mM (A) and for varying [Ca²⁺]_o with a fixed DMPP concentration of 2 μM (B). Each point is the mean and s.e.m. of 6–8 individual determinations. Values shown are EPC amplitude in the presence of DMPP expressed as a percentage of the matched control. Note that the DMPP-induced increase in EPC quantal content is concentration dependent (A) but not [Ca²⁺]_o dependent (B). *P < 0.05 versus control.

Figure 5. Effect of W7 on the DMPP-induced changes in MEPCs and EPCs (at 50 Hz) in the rat hemidiaphragm muscle

Data show the effect of 2 μM DMPP on MEPC amplitude (A), EPC amplitude (B) and EPC quantal content (C) in the absence (-W7) and presence (+W7) of 10 μM of the calmodulin inhibitor W7. Each bar shows the mean and s.e.m. of 6 (-W7) or 10 (+W7) individual measurements. All EPCs were elicited at 50 Hz and [Ca²⁺]_o was 1.8 mM. Quantal content was calculated from the ratio of EPC and MEPC amplitudes. Note that in the absence of W7, DMPP depresses MEPC amplitudes more than EPC amplitudes. However, in the presence of W7 DMPP depresses EPC amplitudes more than MEPC amplitudes. Thus, 2 μM DMPP increases EPC quantal content in the absence of W7 but decreases this parameter in the presence of the compound. *P < 0.05 versus control.

Figure 6. Effect of W7 on the DMPP-induced changes in MEPCs and EPCs (at 0.5 Hz) in the rat hemidiaphragm muscle

Data show the effect of 2 μM DMPP on MEPC amplitude (A), EPC amplitude (B) and EPC quantal content (C) in the absence (-W7) and presence (+W7) of 10 μM of the calmodulin inhibitor W7. Each bar shows the mean and s.e.m. of 8 (-W7) or 6 (+W7) individual measurements. All EPCs were elicited at 0.5 Hz and [Ca²⁺]_o was 1.8 mM. Quantal content was calculated from the ratio of EPC and MEPC amplitudes. Note that in the absence of W7 DMPP similarly depresses EPC and MEPC amplitudes. However, in the presence of W7 DMPP depresses EPC amplitudes more than MEPC amplitudes. Thus, 2 μM DMPP has no effect on EPC quantal content in the absence of W7 but decreases this parameter in the presence of the compound. *P < 0.05 versus control.

Figure 7. Effect of W7 on the vecuronium-induced changes in MEPCs and EPCs (at 50 Hz) in the rat hemidiaphragm muscle

Data show the effect of 1 μM vecuronium on MEPC amplitude (A), EPC amplitude (B) and EPC quantal content (C) in the absence (-W7) and presence (+W7) of 10 μM of the calmodulin inhibitor W7. Each bar shows the mean and s.e.m. of 10 (-W7) or 8 (+W7) individual measurements. All EPCs were elicited at 50 Hz and [Ca²⁺]_o was 1.8 mM. Quantal content was calculated from the ratio of EPC and MEPC amplitudes. Note that in the absence of W7, vecuronium depresses EPC amplitudes more than MEPC amplitudes. However, in the presence of W7 DMPP similarly depresses both EPC and MEPC amplitudes. Thus, 1 μM vecuronium decreases EPC quantal content in the absence of W7 but has no effect on this parameter in the presence of the compound. *P < 0.05 versus control.