Different sensitivities of rat skeletal muscles and brain to novel anti-cholinesterase agents, alkylammonium derivatives of 6-methyluracil (ADEMS)

Abstract

Background and purpose: The rat respiratory muscle diaphragm has markedly lower sensitivity than the locomotor muscle extensor digitorum longus (EDL) to the new acetylcholinesterase (AChE) inhibitors, alkylammonium derivatives of 6-methyluracil (ADEMS). This study evaluated several possible reasons for differing sensitivity between the diaphragm and limb muscles and between the muscles and the brain.

Experimental approach: Increased amplitude and prolonged decay time of miniature endplate currents were used to assess anti-cholinesterase activity in muscles. In hippocampal slices, induction of synchronous network activity was used to follow cholinesterase inhibition. The inhibitor sensitivities of purified AChE from the EDL and brain were also estimated.

Key results: The intermuscular difference in sensitivity to ADEMS is partly explained caused by a higher level of mRNA and activity of 1,3-bis[5(diethyl-o-nitrobenzylammonium)pentyl]-6-methyluracildibromide (C-547)-resistant BuChE in the diaphragm. Moreover, diaphragm AChE was more than 20 times less sensitive to C-547 than that from the EDL. Sensitivity of the EDL to C-547 dramatically decreased after treadmill exercises that increased the amount of PRiMA AChE(G4), but not ColQ AChE(A12) molecular forms. The A12 form present in muscles appeared more sensitive to C-547. The main form of AChE in brain, PRiMA AChE(G4), was apparently less sensitive because brain cholinesterase activity was almost three orders of magnitude more resistant to C-547 than that of the EDL.

Conclusions and implications: Our findings suggest that ADEMS compounds could be used for the selective inhibition of AChEs and as potential therapeutic tools.

Figure 1

Structures of ADEMS compounds.

Figure 2

Concentration-dependent effects of C-547 (A, B) and neostigmine (C, D) on amplitude (in nA; A, C) and decay time constant (τ in ms; B, D) of miniature endplate currents (MEPC) recorded in 10 experiments from diaphragm and extensor digitorum longus (EDL) muscles. CTR, control values. Note the significant 4.5 times increase in decay time in the EDL but not in the diaphragm after the application of 5 × 10⁻⁹M C -547 (B) and equivalent dose–response curves for neostigmine. Inset, time course of MEPC in single muscle fibre of diaphragm and EDL. For each record, 200 individual MEPC were pooled. While 5 nM of C-547 did not affect MEPC in the diaphragm, the drug increased the amplitude and τ (arrow) of the MEPC in EDL. This increase is typical when AChE is inhibited.

Figure 3

Concentration-dependent effects of C-857 (A, B), C-627 (C, D) on amplitude (in nA) and decay time constant (τ in ms) of MEPC recorded in 10 experiments from diaphragm and extensor digitorum longus (EDL) muscles. CTR, control values.

Figure 4

Effects of 5 nM (C, D) and 10 nM C-547 (E, F) on the amplitude (in nA, upper panel) and decay time constant τ (in ms, lower panel) of MEPC recorded in 10 diaphragm fibres, each from a different muscle. For each fibre, 200 MEPCs from diaphragm muscles, either control or pretreated with 50 µM iso-OMPA (a BuChE inhibitor) were averaged, pooled and expressed as mean ± SEM. *P < 0.05, significantly different from values without iso-OMPA.

Figure 5

Normalized dose–effect curves for C-547 on AChE activity in EDL and diaphragm homogenates.

Figure 6

Effects of 0.3 nM and 5 nM C-547 on amplitude (in nA, upper part) and decay time constant (τ in ms, lower part) of MEPC recorded in 6 experiments from EDL, either control or after treadmill exercises. *P < 0.05, significantly different from values without 5 nM C-547.

Figure 7

(A) 10% SDS-PAGE of one of the samples used during AChE isolation. Line 1 – ladder of markers, lines 2 and 3 – EDL and brain Coomassie Brilliant Blue labelled protein after purification by ultracentrifugation and gel filtration on Bio Gel A 50 column. Lines 4 and 5 show immunoblotting for EDL and brain, respectively, with E19 antibody to AChE. The main proteins in the samples are AChE monomers of 70–80 kDa. (B) Dose–effect curve for C-547 on purified brain and EDL AChE activity.

Figure 8

Field recordings of slow rhythmic activity in 70 mM ACh and neostigmine (A) and in 70 mM ACh and C-547 (B) in hippocampal cell body layer of CA3. Calibration for records 1–4 is bottom right, for extended bursts in the middle. Bottom records show disappearance of activity when atropine was added to the superfusing solution already containing 70 mM ACh and neostigmine (A4) or C-547 (B4).

Figure 9

Relative expression of invariable exon 2 (A) and read-through transcript (B) of 4th exon of AChE in the EDL and brain compared with diaphragm. Level in the diaphragm was taken as 1. *P < 0.05, significantly different from expression in diaphragm or brain.