No abstract

Organophosphates, such as tetraethyl pyrophosphate (TEPP) and diisopropyl phosphorofluoridate (DFP), which have a dialkylphosphato group, form with human plasma cholinesterase (also called pseudo-or butyro-cholinesterase) two types of phosphorylated enzyme which possess different stabilities of the enzyme-phosphorus linkage (Hobbiger, 1955). Only phosphorylated enzyme J, which is formed initially, can be readily reactivated by nucleophilic reagents such as nicotinhydroxamic acid methiodide (NHAM). Phosphorylated enzyme I then changes into phosphorylated enzyme II, most probably by transphosphorylation. With DFP or diisopropyl p-nitrophenylphosphate (D 600) the rate of transphosphorylation is faster than with TEPP or diethyl p-nitrophenylphosphate (E 600) and at 370 C. and a pH of 7.45 approximately 90% of the inhibited enzyme is converted into phosphorylated enzyme II within 1 hour.Since the hydrolysis of acetylcholine in vivo is mainly accomplished by true cholinesterase (also called aceto-cholinesterase) the formation of a phosphorylated true cholinesterase which cannot be reactivated by nucleophilic reagents could seriously affect the therapeutic usefulness of NHAM and other more potent substances. The experiments on human plasma cholinesterase were therefore extended to human and bovine true cholinesterases which had been inhibited by an organophosphate containing either a diethyl-or diisopropylphosphato group and the findings are reported in this paper. In addition to NHAM, ammonium molybdate and pyridine-2-aldoxime methiodide, a potent reactivator of phosphorylated electric eel cholinesterase and phosphorylated human and rat true ch6lin-esterases (Wilson and Ginsburg, 1955;and Childs, Davies, Green, and Rutland, 1955), were used for enzyme reactivation. The results obtained with all these reactivators show that the organophosphates form two types of phosphorylated enzyme with human and bovine true cholinesterases which thus behave like plasma cholinesterase. Only the initial form (phosphorylated enzyme l) can readily be reactivated by the nucleophilic reagents. Transphosphorylation-formation of the truly irreversible phosphorylated enzyme Il-occurs both in vitro and in vivo, and the rate of transphosphorylation is dependent upon pH. METHODSAU experiments were carried out in the Warburg apparatus.Cholinesterase activity was determined by the manometric technique using 0.01 M-acetylcholine chloride as substrate for human and bovine true cholinesterases and 0.02 M-(±)-acetyl-f-methylcholine chloride as substrate for the true cholinesterases of whole rabbit blood. The procedures adopted for determination of enzyme activity and reactivation were the same as those described in an earlier publication (Hobbiger, 1955).Except when otherwise stated, the gas phase consisted of 95% N2+5% CO2 and a solution containing 0.025 M-NaHCO3, 0.075 M-NaCl, 0.075 M-KCl, 0.04 M-MgCl2 and 0.1% bovine plasma albumin (Armour Laboratories) was used for all dilutions and as the medium during inhibition, reactivation and determination ...

No abstract

The effect of 18 pyridinium aldoximes on diethylphosphoryl-acetocholinesterase in vitro and the protection against lethal poisoning by ethyl pyrophosphate (TEPP) in mice pretreated with 0.095 m.mole/kg. of these oximes was investigated. Monoximes and dioximes of polymethylenebispyridinium compounds were studied in greater detail since they were up to 22 times more potent than pyridine-2-aldoxime methiodide (2-hydroxyiminomethyl-N-methylpyridinium iodide) in reactivating diethylphosphoryl-acetocholinesterase in vitro and protected mice against lethal poisoning by up to 15 LD100 of ethyl pyrophosphate. These oximes were also up to 52 times more potent than pyridine-2-aldoxime methiodide in reactivating di-isopropylphosphorylacetocholinesterase in vitro and were effective in preventing lethal poisoning by dyflos (di-isopropyl phosphorofluoridate). The antidotal action against diethyl phosphostigmine (Ro 3-0340) was even greater than that against ethyl pyrophosphate. Some of the most effective oximes had antidotal actions in poisoning by ethyl pyrophosphate, diethyl phosphostigmine and dyflos when given in 0.0095 m.mole/kg. and this effect was enhanced by 1 mg./kg. atropine sulphate. In vivo reactivation of diethylphosphoryl-acetocholinesterases by 0.0095 or 0.095 m.mole/kg. of oximes of polymethylenebispyridinium compounds was demonstrated in blood but not in brain. Atropine-like and neuromuscular blocking activities were studied on isolated organs and protection against lethal doses of neostigmine and related anticholinesterases were also investigated. Some of the oximes of polymethylenebispyridinium compounds have, relative to pyridine-2-aldoxime methiodide, a higher therapeutic ratio in mice and considerably greater water-solubility. The possible advantages to be gained from their use in preference to pyridine-2-aldoxime methiodide are discussed.The interaction between anticholinesterases of the organophosphate type and acetocholinesterase (also often referred to as true cholinesterase or acetylcholinesterase) yields phosphorylated acetocholinesterase which is enzymatically inactive and in many instances has a half-life ranging from several days to weeks. While cholinesterase is in a phosphorylated form acetylcholine accumulates and thus the administration of organophosphates is followed by muscarinic and nicotinic symptoms of acetylcholine poisoning.

1. The actions on the taenia of 4-(m-chlorophenylcarbamoyloxy)-2..butynyltrimethylammonium chloride (McN-A-343), Nabenzyl-3-pyrrolidyl acetate methobromide (AHR-602), tetramethylammonium (TMA) and choline phenyl ether have been examined and compared with the actions of acetylcholine, nicotine and 1,1-dimethyl-4-phenylpiperazinium (DMPP). 2. Responses of the taenia to these agonists are quantitatively and often qualitatively dependent on the tone of the preparation. A method is described which makes allowance for the effect of tone on heig!hts of contractions. 3. Acetylcholine, McN-A-343 and AHR-602 produced only contractions; TMA produced contractions or biphasic responses; and choline phenyl ether, nicotine and DMPP produced contractions, relaxations or biphasic responses. 4. The mode of action of these compounds has been analysed by means of hyoscine, ganglionlblocking drugs, tetrodotoxin and local anaesthetics. 5. It is concluded that acetylcholine, McN-A-343, AHR-602, TMA and choline phenyl ether act on muscarinic receptors in the smooth muscle. Choline phenyl ether has an additional action on nicotinic receptors of cholinergic neurones. Nicotine and DMPP act on nicotinic receptors of cholinergic neurones and of inhibitory neurones. An action on the latter is sometimes also seen with TMA and choline phenyl ether. 6. With nicotine or DMPP, and TMA or choline phenyl ether in the presence of hyoscine, part of the contraction phase of biphasic responses (in which a contraction follows relaxation) is best explained as being triggered by the initial relaxation-that is, as a "redbound contraction". 7. None of the compounds tested appeared to exert an atropine-sensitive action on neurones.8. In the presence of hyoscine high concentrations of agonists can act on sites not involved with lower concentrations.