Henry I. Yamamura

Recovery of central respiratory function following anticholinesterase intoxication

Spontaneous recovery of central respiratory function was studied in anesthetized guinea pigs intoxicated with pinacolyl methylphosphonofluoridate (Soman) or isopropyl methylphosphonofluoridate (Sarin). I.v adiministration of either agent produced an immediate disruption phrenic nerve activiity and resulting ventilatory failure. Animals were maintained on artifical respiration until spontaneous functional recovery was complete, as evidenced by the re-establishment of synchronized burst activity on the phrenic nerve and return of tracheal airflow. This usually occurred within 1 h. Animals were sacrificed at predetermined intervals after intoxication, and the brainstem homogenates were analyzed for AChE activity. Results showed no significant return of AChE activity after 1 h, although functional recovery of respiration was complete within this time. Additional doses of the agents were administered at various times after recovery from the respiratory blockade. Following spontaneous restoration of ventilatory function, subsequent injections of the organophosphorus compounds failed to reinstate central respiratory paralysis, although they further depressed brainstem AChE levels. These data suggest that spontaneous recovery of central respiratory function after intoxication with Soman or Sarin may not be related to the return of AChE activity.

Guinea pig brain acetylcholinesterase: partial purification by affinity chromatography.

The technique of affinity chromatography was used in the partial purification of guinea pig brain acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7). The enzyme from brain homogenates was solubilized by treatment with Triton X 100. 80% solubilization of the enzyme was achieved. The solubilized enzyme was passed through a small column of aminobutyl-Sepharose to which specific competitive inhibitors had been covalently attached. A high percentage of the nonspecific protein passed directly through the affinity column while the specific enzymatic protein remained bound to the gel. NaCl and choline chloride were used s eluants to release the bound enzymatic activity from the affinity support. m-Carboxyphenyl trimethylammonium iodide attached through 1,4-diaminobutane to Sepharose 4B proved to be more efficient in binding the enzymatic protein than its p-analog. A linear gradient elution utilizing choline chloride preceded by a NaCl wash proved to result in a rapid and efficient means of releasing the bound enzymatic activity. The results of the study showed that a specific activity of at least 10 mmoles of [14C]acetylcholine hydrolyzed/h per mg protein could be achieved, a 1000-fold purification. The partially purified enzyme had an apparent Km of 1.47·10−4 M using [14C]acetylcholine as the substrate.

Spontaneous recovery of central respiratory function after an irreversible AChE inhibitor

Abstract Spontaneous recovery of central respiratory function was studied in Soman-poisoned guinea pigs. Phrenic nerve activity and resulting ventilatory function were evident within an hour after intoxication. However, no significant return of brainstem AChE activity occurred during this time period. Repeated administrations of Soman after spontaneous recovery failed to reinstate central respiratory paralysis.