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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 APPROACHIncreased 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 RESULTSThe 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 IMPLICATIONSOur findings suggest that ADEMS compounds could be used for the selective inhibition of AChEs and as potential therapeutic tools. AbbreviationsAChE, acetylcholinesterase; ADEMS, alkylammonium derivatives of 6

We compared the effects of the novel acetylcholinesterase (AChE) inhibitor C-547 on action potential configuration and sinus rhythm in the isolated right atrium preparation of rat with those of armin and neostigmine. Both armin (10(-7), 10(-6), and 10(-5) M) and neostigmine (10(-7), 10(-6), and 5 x 10(-6) M) produced a marked decrease in action potential duration and slowing of sinus rate. These effects were abolished by atropine and are attributable to the accumulation of acetylcholine in the myocardium. The novel selective AChE inhibitor C-547 (10(-9) to 10(-7) M), an alkylammonium derivative of 6-methyluracil, had no such effects. The inhibition constant of C-547 on cardiac AChE is 40-fold higher than that on extensor digitorum longus muscle AChE. These results suggest that C-547 might be employed to treat diseases such as myasthenia gravis or Alzheimer disease, without having unwanted effects on the heart.

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Selective labeling of barstar by the stable (15)N isotope of the valine residue with high selectivity of the label incorporation resulting from the process of gene expression in Escherichia coli BL21(DE3) has been optimized. We have shown that alpha-aminooxyacetic acid (AOAA) significantly reduces the isotope redistribution, thus increasing the selectivity of (15)N incorporation into the synthesized protein, as detected by 2D-NMR. Quantitative measurements were used to determine the selectivity for the incorporation of isotope-labeled valine residue, which was 96% in the case using AOAA. Studies of the dynamics of barstar synthesis showed that no suppression of barstar yield is observed under the regulation of the T7 polymerase expression system by isopropylthio-beta-D-galactoside (IPTG) and rifampicin using AOAA.

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