Relationships between in vivo and in vitro inhibition of acetylcholinesterase (AChE) and impairment of neuromuscular transmission in the rat phrenic-nerve diaphragm by a tertiary anticholinesterase and its quaternary analogue

Abstract

The tertiary anticholinesterase agent pinacolyl S-(2-dimethylaminoethyl) methylphosphonothioate (compound I) and its quaternary analogue (compound II), were administered subcutaneously to atropinized rats. The animals were killed 30 min later and the tension of the isolated phrenic nerve-diaphragm preparation, stimulated indirectly at 100 Hz for 10 sec, was compared with that of preparations from control animals. A dose of 1.5 μmole/kg of the quaternary compound II, which has a bimolecular rate constant of inhibition twice that of the tertiary compound I, reduced the tetanic response of the diaphragm by 50 per cent; the equivalent dose of the tertiary compound I was 5.2 μmole/kg. At these dose levels, compound II inhibited 65 per cent of the diaphragm acetylcholinesterase (AChE) activity, determined on homogenates, and compound 192 per cent. When the phrenic nerve-diaphragm preparation from untreated animals was incubated for 20 min with various concentrations of compound I or II, the rate of enzyme inhibition conformed approximately to first order kinetics and equimolar concentrations of the inhibitors reduced tetanic tension to 50 per cent in the same time. There was no discontinuity in the plot of per cent AChE inhibition vs logarithm of the concentration of compound I or II but the slope was much less with the quaternary compound II. The results provide additional evidence that quaternary compounds can reach all sites with AChE activity only in vitro and that their distribution when applied in vitro is not the same as that in vivo.