Inhibition of serum cholinesterase by trialkylphosphorothiolates

The kinetics of inhibition of horse serum cholinesterase (EC 3.1.1.8) by six trialkylphosphorothiolates was studied (25° C, pH 7.4). The compounds were: OOS-trimethylphosphorothiolate (OOS-Me), OSS-trimethylphosphorodithiolate (OSS-Me), SSS-trimethylphosphorotrithiolate (SSS-Me) and their corresponding ethyl analogues (OOS-Et, OSS-Et, SSS-Et). The second order rate constants of inhibition ranged from 7.2 to 2128 mol^–1 1 min^–1, and the enzyme/inhibitor dissociation constants from 0.079 to 1.5 mM. The ethyl esters were better inhibitors than their methyl analogues and the OSS-compounds were better inhibitors than the OOS-or SSS-compounds. The same structure-activity relationship is known to hold for the reaction of the compounds with acetylcholinesterase (EC 3.1.1.7).This work is taken in part from the BSc Thesis of L. F. (University of Zagreb)     

Inhibition of the activity of human serum cholinesterase by aminoalkyl benzenesulfonyl fluorides]

Aminoalkyl benzenesulfonyl fluorides, like organophosphates, act as irreversible inhibitors of serine proteinases by splitting off hydrogen fluoride to form an enzyme-inhibitor complex, stable in the physiological pH region. Several of these compounds are characterized by a higher rate of inhibition when trypsin is used and the second order rate constants are compared with those of organophosphates. On the other hand, upon inhibition of human serum cholinesterase by DFP and 4-nitrophenyl diethyl phosphate, some orders of magnitude higher than that of benzenesul fonyl fluorydes are observed. As shown by an oral toxicity study in mice similar differences exist with respect to LD50 values.     

Determination of some local anesthetics in human serum by gas chromatography with solid-phase extraction

A method of analysis based on solid-phase extraction coupled with capillary gas chromatographic system for determination of mepivacaine, bupivacaine and lidocaine from human serum was developed. As extraction sorbents were used Chromosorb 103, Tenax-GC and Chromosorb T. The best extraction sorbent proved to be Chromosorb 103. Their recoveries ranged from 91 to 94% at the target concentrations of approx. 1.5 microgml(-1) in serum. Relative standard deviation of the recoveries ranged from 3.11 to 5.30 at these concentrations. As internal standard was used lidocaine. The chromatographic analysis was performed on a gas chromatograph equipped with a capillary column, HP-Innowax, and flame ionisation detector. Samples were injected in splitless mode. This method was applied in a stomatological clinic to healthy volunteers to whom superior-posterior alveolar nerve block anesthesia with mepivacaine was administered.     

Inhibition of wild-type and mutant neuronal nicotinic acetylcholine receptors by local anesthetics.

Inhibition of neuronal nicotinic receptors can be regulated by the presence of specific amino acids in the beta subunit second transmembrane domain (TM2) domain. We show that the incorporation of a mutant beta4 subunit, which contains sequence from the muscle beta subunit at the TM2 6' and 10' positions of the neuronal beta4 subunit, greatly reduces the sensitivity of receptors to the local anesthetic [2-(triethylamino)-N-(2,6-dimethylphenyl)acetamide] (QX-314). Although differing in potency, the inhibition of both wild-type alpha3beta4 receptors and alpha3beta4(6'F10'T) receptors by QX-314 is voltage-dependent and noncompetitive. Interestingly, the potency of the local anesthetic tetracaine for the inhibition of alpha3beta4 and alpha3beta4(6'F10'T) receptors seems unchanged when measured at -50 mV. However, whereas the onset of inhibition of wild-type alpha3beta4 receptors is voltage-dependent and noncompetitive, the onset of inhibition of alpha3beta4(6'F10'T) receptors by tetracaine is unaffected by membrane voltage, and at concentrations < or = 30 microM seems to be competitive with acetylcholine. This may be due to either direct effects of tetracaine at the acetylcholine binding site or preferential block of closed rather than open channels in the mutant receptors. Further analysis of receptors containing the 6' mutation alone suggests that although the 6' mutation is adequate to alter the voltage dependence of tetracaine inhibition, both point mutations are required to produce the apparent competitive effects.     

Inhibition by tertiary amine local anesthetics of phagocytosis in cultured mouse peritoneal macrophages

The tertiary amine local anesthetics, dibucaine, tetracaine and procaine, have been shown to exert a reversible inhibition of phagocytosis of opsonized sheep red blood cells by cultured mouse peritoneal macrophages. The observed inhibition was due primarily to a functional alteration of the macrophage. The potencies of the three local anesthetics for inhibition of phagocytic uptake were proportional to their lipophilic nature as measured by octanol-water partition. Inhibition of phagocytosis by tetracaine was antagonized by Ca²⁺. The above observations suggest that the general properties which characterize the action of tertiary amines as anesthetics, namely lipophilic nature and antagonism by Ca²⁺, are also those attributable to the inhibition of macrophage phagocytosis in vitro.     

Inhibition of cholinesterase by epinephrine and norepinephrine

Acetylcholinesterase and monoamine oxidase were found to be inhibited by each other's substrate. The inhibition of acetylcholinesterase by low concentration of epinephrine or norepinephrine was found to follow first-order reaction kinetics. The constants characterising this inhibition, the bimolecular rate ka (51.8 M-1 min-1 for epinephrine and 15.9 M-1 min-1 for norepinephrine) and the enzyme inhibitor dissociation constant (8.52 mM for epinephrine and 12.2 mM norepinephrine) were determined. The inhibition of acetylcholinesterase by epinephrine was found to be of the mixed type while its inhibition by norepinephrine was of the competitive type.     

Inhibition of cholinesterase activity by isocyanates

Exposure of workers to isocyanates may result in irritation and/or sensitization of the respiratory tract. An immunologic mechanism for sensitization has been presented previously. This investigation explored whether, as a possible mechanism for the irritation reaction, the toxic respiratory effect of isocyanates might be due to their ability to inhibit cholinesterases. Hexamethylene diisocyanate (HDI), hexyl isocyanate (HI), and 2,6-toluene diisocyanate (2,6-TDI) were found to completely inhibit purified human serum cholinesterase when added at molar ratios of 4:1 to 8:1 (isocyanate:enzyme). By contrast, molar ratios of 50:1 or greater were required for 50% enzyme inhibition by 2,4-toluene diisocyanate (2,4-TDI), phenyl isocyanate, or o-tolyl isocyanate. Enzyme inhibition was also achieved by exposure of purified cholinesterase to atmospheres containing 1 ppm isocyanates. Under these conditions, HDI and HI were again the most potent enzyme inhibitors with much less reactivity shown by 2,4-TDI and 2,6-TDI. Under more physiologic conditions, when whole human plasma was the source of cholinesterase, HDI and HI were still potent enzyme inhibitors. However, with the latter two isocyanates, the molar concentrations needed to effect 50% enzyme inhibition suggested affinity labeling by these reagents. The potent cholinesterase inhibition shown by HDI and HI may offer explanation for observed respiratory symptomatology noted upon exposure to these isocyanates.     

Inhibition of the Na,K-ATPase of canine renal medulla by several local anesthetics.

The ATPase activity of Na,K-ATPase-enriched membranes from canine renal medulla was determined in the absence of local anesthetic and in the presence of procaine, chloroprocaine, bupivacaine, mepivacaine, lidocaine, and two quaternary derivatives of lidocaine (QX-222 and QX-314) at 37( composite function)C. Chloroprocaine (IC(50)= 13 mM) had slightly greater potency than procaine (IC(50)= 17.7 mM). Bupivacaine (IC(50)= 6.7 mM) was more potent than its congener mepivacaine (IC(50)> 10 mM, the solubility limit). QX-222 (IC(50)> 600 mM) and QX-314 (IC(50)= 132 mM) had less potency than lidocaine (IC(50)= 30.4 mM). This study supports the interpretation that the uncharged forms of local anesthetics are much more potent inhibitors of Na,K-ATPase activity than the cationic forms.     

Inhibition of human and rabbit platelet aggregation by chlorophenoxyacid herbicides

Inhibition of human platelet aggregation by eight chlorophenoxyacid herbicides was studied in vitro. Thrombocyte aggregation in the platelet-rich plasma was induced by 1.0–32.0 M adenosine diphosphate (ADP), 0.32–32.0 M adrenaline or 7.5–30.0 g/ml collagen with and without chlorophenoxyacid (0.05–2.0 mg/ml). Platelet aggregation by each inducer was inhibited dose dependently by all the eight chlorophenoxyacids at concentrations between 0.1 and 2.0 mg/ml. Increasing the concentrations of ADP and collagen but not of adrenaline inhibited the antiaggregatory action of chlorophenoxy-acids. No essential differences in inhibitory effect were found between different chlorophenoxyacids varying in respect of their ring substituents and the length of the carboxylic side chain. In the platelet-rich plasma prepared from rabbits 2.5 h after subcutaneous injection of 2,4-dichlorophenoxyacetic acid or 4-chloro-2-methylphenoxy-acetic acid (100–150 mg/kg), platelet aggregation by ADP was inhibited 20–30%, compared to plasma taken from the rabbits before the chlorophenoxyacid treatment. The inhibition had disappeared by 20–23 h after administration. The results indicate that chlorophenoxyacid herbicides inhibit human platelet aggregation. Furthermore, the inhibition is probably involved in haemorrhages known to occur in various tissues of animals intoxicated by chlorophenoxyacid herbicides.     

Inhibition of cholinesterase by 5-hydroxytryptamine.

On the basis of structural relationships between 5-hydroxytryptamine (5-HT, serotonine) and eserine, the effect of 5-HT on partially purified brain cholinesterase (ChE) was studied. The addition of 5-HT to brain ChE in vitro resulted in its inhibition, and the constants characterizing this inhibition, namely, the inhibition rate ki (5.44 X 10(2) mol-1/l - min-1), the equilibrium constant K (1.86 X 10(-3), and the rate constant for spontaneous reactivation kr (1.01 min-1) were determined. The inhibition of AChE by 5-HT in vitro was found to be of the competitive type.     

Inhibition of human plasma cholinesterase by ingested dichlorvos: Effect of formulation vehicle

Human subjects were fed 0.9 mg of dichlorvos (2,2-dichlorovinyl dimethyl phosphate) three times a day for 21 days. No consistent cholinomimetic signs or symptoms were observed, nor was erythrocyte acetylcholinesterase inhibited. The only significant observation was a depression of plasma cholinesterase (ChE) activity. The magnitude of the depression in plasma cholinesterase was found to depend on the method by which dichlorvos was incorporated into the diet. Dichlorvos, formulated as a gelatin salad and consumed during the course of the meal, was only 64% as effective in inhibiting ChE activity as when administered as a premeal capsule containing cottonseed oil. The recovery of cholinesterase activity, after dosing was terminated, was due to the replacement of enzyme molecules. The half-life for this regeneration period was 13.7 days.     

Inhibition of cholinesterase activity induced by pyridaphenthion]

Inhibitory effects on cholinesterase (ChE) activity in blood of the rabbit induced by pyridaphenthion(PD), an organophosphorus compound, and effects of 2-pyridine aldoxime methiodide(PAM) on this inhibition were examined. 1) Experimental results within 24 hr: An oral administration of each dose of PD(100 approximately 750 mg/kg) gradually decreased ChE activity and ChE activity value decreased to 20.5% in the erythrocytes and 21.5% in the plasma 24 hr after administration of 500 mg/kg of PD. However, this value recovered remarkably with an intravenous injection of PAM. The ChE activity value was 55.5% in the erythrocytes and 41.4% in the plasma with a single injection, and respectively 67.8% and 59.1% when PAM was injected three times. 2) Experimental results for 14 days: Following an increase of the administered dose (100 approximately 400 mg/kg) of PD, a decrease in ChE activity was apparent and a recovery to normal values was delayed. This inhibition could be ameliorated quickly with an injection of PAM. Considering that PAM does not have remarkable detoxicative effects on organophosphorus compounds which have a low toxicity, PAM appears to be a promising antidote against PD.