Butyrylcholinesterase and acetylcholinesterase activity and quantal transmitter release at normal and acetylcholinesterase knockout mouse neuromuscular junctions

Abstract

1 The present study was performed to evaluate the presence and the physiological consequences of butyrylcholinesterase (BChE) inhibition on isolated phrenic-hemidiaphragm preparations from normal mice expressing acetylcholinesterase (AChE) and BChE, and from AChE-knockout mice (AChE(-/-)) expressing only BChE. 2 Histochemical and enzymatic assays revealed abundance of AChE and BChE in normal mature neuromuscular junctions (NMJs). 3 In normal NMJs, in which release was reduced by low Ca(2+)/high Mg(2+) medium BChE inhibition with tetraisopropylpyrophosphoramide (iso-OMPA) or bambuterol decreased ( approximately 50%) evoked quantal release, while inhibition of AChE with fasciculin-1, galanthamine (10, 20 micro M) or neostigmine (0.1-1 micro M) increased (50-80%) evoked quantal release. Inhibition of both AChE and BChE with galanthamine (80 micro M), neostigmine (3-10 micro M), O-ethylS-2-(diisopropylamino)ethyl-methylphosphono-thioate (MTP) or phospholine decreased evoked transmitter release (20-50%). 4 In AChE(-/-) NMJs, iso-OMPA pre-treatment decreased evoked release. 5 Muscarinic toxin-3 decreased evoked release in both AChE(-/-) and normal NMJs treated with low concentrations of neostigmine, galanthamine or fasciculin-1, but had no effect in normal NMJs pretreated with iso-OMPA, bambuterol, MTP and phospholine. 6 In normal and AChE(-/-) NMJs pretreatment with iso-OMPA failed to affect the time course of miniature endplate potentials and full-sized endplate potentials. 7 Overall, our results suggest that inhibition or absence of AChE increases evoked quantal release by involving muscarinic receptors (mAChRs), while BChE inhibition decreases release through direct or indirect mechanisms not involving mAChRs. BChE apparently is not implicated in limiting the duration of acetylcholine action on postsynaptic receptors, but is involved in a presynaptic modulatory step of the release process.

Figure 1

Presence of BChE and AChE activity at the NMJ of mature mouse skeletal muscle fibres. (A) Control untreated Triangularis sterni muscle stained for cholinesterases. (B) Preparation treated successively with 10 μM BW284C51 and 100 μM iso-OMPA to inactivate AChE and BChE. (C) Muscle treated with 100 μM iso-OMPA to reveal AChE. (D) Preparation treated with 10 μM BW284C51 to reveal BChE activity. Scale bar in D (20 μm) applies to all images.

Figure 2

Dose-dependent effect of various cholinesterase inhibitors on the mean quantal content (m_o) of EPPs in wild-type NMJs bathed in a physiological solution containing 0.4 mM Ca²⁺ and 6 mM Mg²⁺. Effects of inhibitors are given as per cent of the corresponding control m_o values (determined before the application of drugs). Each point represents the mean±s.e.mean of n=5–9 separate experiments. Asterisks indicate significant changes in m_o (^*P<0.05; ^**P<0.03, ^***P<0.001, Student's t-test) versus control values.

Figure 3

Effect of different concentrations of MT-3 on the mean quantal content (m_o) of EPPs, in wild-type (A), and AChE^−/− (B) NMJs bathed in a physiological solution containing 0.4 mM Ca²⁺ and 6 mM Mg²⁺. Note that MT-3 had a significant effect only in AChE^−/− NMJs. Values are expressed as a percentage of the respective controls, and they represent the mean±s.e.mean, calculated from 4–9 separate experiments. Asterisks denote values significantly different from control values (^*P<0.05, Student's t-test).

Figure 4

Effect of various concentrations of MT-3 on the mean quantal content (m_o) of EPPs recorded in wild-type NMJs treated, or pretreated with various cholinesterase inhibitors (see Methods). Experiments were performed in standard saline containing 0.4 mM Ca²⁺ and 6 mM Mg²⁺. Values are expressed as percentage of the respective controls (determined before drug application). Note the absence of MT-3 effect on m_o when BChE, and both BChE and AChE are inactivated. Each column represents the mean±s.e.mean, calculated from 4–9 separate experiments. Asterisks indicate significant changes in m_o (^*P<0.05; ^**P<0.03, Student's t-test) with respect to values obtained with the corresponding inhibitor alone.

Figure 5

Examples of EPPs recorded in wild-type (A,C) and AChE^−/− (B,D) NMJs bathed in normal physiological saline containing 2.2 μM μ-conotoxin GIIIB. EPPs evoked by single nerve stimulation (A,B), and during a train of 100 ms duration at 100 Hz (C,D). The decay time constant (τ) of EPPs is indicated for comparison in A and B. Data in A,C and B,D are from the same NMJs. The resting membrane potential during recordings were −65 mV (A,C) and −67 mV (B,D). (E) Quantal content of full-sized EPPs evoked at 0.1 Hz and recorded in a wild-type junction bathed in standard physiological solution containing μ-conotoxin GIIIB (2.2 μM) before, and after the addition (arrow) of 150 nM bambuterol to the medium (filled circles). For comparison data obtained in another junction during the same time period under control conditions (open circles) are also shown. Each circle represents the data computed during 1 min. The insets show averaged traces of EPPs and MEPPs recorded in the same junction before (a), and 5 (b), 10 (c), and 20 (d) min after the addition of bambuterol.

Figure 6

(A) Representative repetitive EPPs evoked during 1 s nerve stimulation at 100 Hz and recorded in wild type and AChE^−/− NMJs bathed in normal physiological saline containing 3 μM (+)-tubocurarine. EPPs are shown at the beginning (left) and at the end of the train (right). Note the slight enhancement of EPP amplitudes at the beginning of stimulation and their reduction at the end of the train, and the fact that this pattern was unaffected by pretreatment with iso-OMPA (100 μM). Also note the inability of AChE^−/− NMJs to sustain evoked EPPs at the end of the train. (B) Changes in EPP amplitudes during nerve stimulation, under control conditions (open bars) and after pretreatment with 100 μM iso-OMPA (filled bars) in wild-type and AChE^−/− NMJs. Nerve stimuli were delivered at 10, 20 and 40 Hz for 10 s, and at 100 Hz for 1 s. For each set of data, the first and last EPPs in each train were computed, and results are expressed as per cent of the 1st EPP. Note that the depression of EPPs was unaffected by iso-OMPA pretreatment. Bars represent the mean±s.e.mean. (n=3–9).

Figure 7

(A) Representative spontaneous MEPPs and G-MEPPs (arrows) recorded in an AChE^−/− NMJ bathed in standard physiological solution. (B) Amplitude-distribution histograms and a cumulative plot of spontaneous events recorded in wild-type and AChE^−/− NMJs. Note the different cumulative distribution of events in AChE^−/− when compared to wild-type NMJs (P<0.001, Kolmogorov-Smirnov test) which is due to the presence of G-MEPPs.

Figure 8

Effect of pretreatment with iso-OMPA (100 μM) on amplitude (A), half-decay time (B) and frequency (C) of MEPPs recorded in wild-type (WT) and AChE^−/− NMJs. G-MEPPs were excluded for the calculation of the three parameters. Data were obtained from 21 fibres (six different WT muscles) and from 11 fibres (four different AChE^−/− muscles) and represent the mean±s.e.mean (^*P<0.05, Student's t-test). Resting membrane potential of the fibres was comprised between −70 and −67 mV for WT and AChE^−/− NMJs.