Metrifonate induces cholinesterase inhibition exclusively via slow release of dichlorvos

Metrifonate, a long-acting cholinesterase (ChE) inhibitor with very low toxicity in warm-blooded animals, inhibits rat brain and serum cholinesterase (ChE) in vitro through its hydrolytic degradation product, dichlorvos. This conclusion is based on the finding that metrifonate-induced ChE inhibition showed the same pH dependence as its reported dehydrochlorination to dichlorvos. The ChE inhibition induced by dichlorvos was not pH dependent. It was mediated by a competitive drug interaction with the catalytic site of the enzyme, which led to irreversible inhibition within several minutes of incubation. After this time, addition of further substrate to the inhibited enzyme was not able to promote drug dissociation and hence enzyme reactivation. Similar characteristics of inhibition, i.e. interaction with the substrate binding site and time-dependent switch to non-competitive inhibition were observed with the reference compound, physostigmine. However, the physostigmine-induced inhibition of ChE could be readily reversed by further substrate addition. Another reference compound, tetrahydroaminoacridine (THA), also induced a reversible inhibition of rat brain and serum cholinesterase, but with a mechanism of action different from that of both dichlorvos and physostigmine in that enzyme inhibition occurred rapidly upon drug addition at an allosteric site on the enzyme surface. It is suggested that the unique slow release plus the slow inhibition of ChE by dichlorvos is responsible for the lower toxicity of metrifonate compared to that of directly acting ChE inhibitors.     

Characterization of a pseudocholinesterase purified from surgeonfish tissues confirms the atypical nature of this enzyme

The sialated, presumed-globular form of an atypical pseudocholinesterase (pseudo-ChE) previously described from surgeonfish tissues (Leibel: Comparative Biochemistry and Physiology 1988) has been purified to apparent homogeneity using a combination of salt fractionation along with ion-exchange and concanavalin A-Sepharose affinity chromatographic techniques. An overall 1,400-fold purification has been achieved with a 24% final yield of a cholinesterase (ChE) whose final specific activity is 50 mumol/min-mg. The purified enzyme was subjected to detailed biochemical and physical analysis. The purified pseudo-ChE is a sialated, globular, tetrameric enzyme with an apparent sedimentation coefficient of 11.5 S (+/- 0.5 S) and a molecular weight of 250 kilodaltons. The monomers are apparently not secured by disulfide bridges. The enzyme preferentially hydrolyzes acetyl(thio)choline but also hydrolyzes propionyl(thio)choline at reduced but comparable rates along with a wide variety of other noncholine esters. As such, it demonstrates the relative nonspecificity associated with classical pseudo-ChEs. However, the enzyme exhibits limited, but real, substrate inhibition with all choline esters as does true acetylcholinesterase (AChE). The enzyme is insensitive to the AChE inhibitor BW 284C51, sensitive to one (RO2-0683) of two (RO2-1250) pseudo-ChE inhibitors, and particularly sensitive to paraoxon inhibition (10(3)-10(4)-fold more so than AChE). It exhibits the short thermal half-life characteristic of pseudo-ChEs but not the expected ionic activation/inhibition profile. It is clear from this and other studies of atypical extrasynaptic cholinesterase activities occurring in other vertebrates that the orthodox categorization of cholinesterase as either "true" ("specific"; E.C. 3.1.1.7) or "pseudo" ("nonspecific"; E.C. 3.1.1.8) is inadequate to accommodate the increasing instances of ChE activities that exhibit atypical, intermediate properties.     

Anticholinesterase activity of a new carbamate, heptylphysostigmine, in view of its use in patients with Alzheimer-type dementia

The anticholinesterase activity of a new carbamate, heptylphysostigmine, was studied in vitro. This compound is a competitive inhibitor of acetylcholinesterase (or true cholinesterase) having Ki = (1 +/- 0.5) X 10(-7) M. The inhibition was instantaneous at the onset and did not diminish with prolonged incubation of the drug and enzyme.     

New findings about Ellman's method to determine cholinesterase activity

The original Ellman's spectrophotometrical method for cholinesterase activity determination uses 5,5'-dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent) as a chromogen and records the level of cholinesterase activity as an increase of absorbance at 412 nm. Although this procedure usually poses no problem, exceptions arise when the concentration of DTNB is far higher than the concentration of acetylthiocholine (ATCH). It was found that the ratio of concentrations of DTNB/ATCH is an important parameter for the ATCH hydrolysis course: high excess of DTNB decreases the hydrolysis rate resulting in a lower measured enzyme activity. Our experiments indicate that this influence of DTNB concentration can be explained by the inhibition of ATCH hydrolysis by DTNB.     

Cholinesterases from plant tissues: I. Purification and characterization of a cholinesterase from mung bean roots

A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 mum for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 mum). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis.     

A versatile equation to describe reversible enzyme inhibition and activation kinetics: modeling beta-galactosidase and butyrylcholinesterase

Current treatments for Alzheimer's disease involve inhibiting cholinesterases. Conversely, cholinesterase stimulation may be deleterious. Homocysteine is a known risk factor for Alzheimer's and vascular diseases and its active metabolite, homocysteine thiolactone, stimulates butyrylcholinesterase. Considering the opposing effects on butyrylcholinesterase of homocysteine thiolactone and cholinesterase inhibitors, understanding how these molecules alter this enzyme may provide new insights in the management of dementia. Butyrylcholinesterase does not strictly adhere to Michaelis-Menten parameters since, at higher substrate concentrations, enzyme activation occurs. The substrate activation equation for butyrylcholinesterase does not describe the effects of inhibitors or non-substrate activators. To address this, global data fitting was used to generate a flexible equation based on Michaelis-Menten principles. This methodology was first tested to model complexities encountered in inhibition by imidazole of beta-galactosidase, an enzyme that obeys Michaelis-Menten kinetics. The resulting equation was sufficiently flexible to permit expansion for modeling activation or inhibition of butyrylcholinesterase, while accounting for substrate activation of this enzyme. This versatile equation suggests that both the inhibitor and non-substrate activator examined here have little effect on the substrate-activated form of butyrylcholinesterase. Given that butyrylcholinesterase inhibition can antagonize stimulation of this enzyme by homocysteine thiolactone, cholinesterase inhibition may have a role in treating Alzheimer and vascular diseases related to hyperhomocysteinemia.     

The mechanism of anticholinesterase action of acetylene organophosphorus inhibitors]

Introduction of the triple bond in the leaving group of the organophosphorus inhibitor molecule gives a sharp raise of the inhibitor activity but does not change principal characteristics of the cholinesterase inhibition mechanism. The reactivation experiments suggest that inactivation of cholinesterases by these compounds occurs due to phosphorylating of the serine hydroxyl by the corresponding phosphoric acid. A close similarity was shown between acetylenic and saturated organophosphorus inhibitors in altering ka upon change of pH and tetraalkylammonium ions action. It is demonstrated that S-alkynyl esters of thioacetic acid are slowly hydrolyzed by acetylcholinesterase and cholinesterase without irreversible inhibition of the enzymes.     

Structural insights into cholinesterases inhibition by harmane β-carbolinium derivatives: A kinetics – molecular modeling approach

The natural indole alkaloids, the β-carbolines, are often associated with cholinesterase inhibition, especially their quaternary salts, which frequently have higher activity than the free bases. Due to lack of information explaining this fact in the literature, the cholinesterase inhibition by the natural product harmane and its two β-carbolinium synthetic derivative salts (N-methyl and N-ethyl) was explored, together with a combination of kinetics and a molecular modeling approach. The results, mainly for the β-carbolinium salts, demonstrated a noncompetitive inhibition profile, ruling out previous findings which associated cholinesterase inhibition by β-carbolinium salts to a possible mimicking of the choline moiety of the natural substrate, acetylcholine. Molecular modeling studies corroborate this kind of inhibition through analyses of inhibitor/enzyme and inhibitor/substrate/enzyme complexes of both enzymes.     

Cholinesterases from Plant Tissues: II. Inhibition of Bean Cholinesterase by 2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine Carboxylate Methyl Chloride (AMO-1618)

A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 μm for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 μm). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis.     

Effect of temperature and pH on carbamoylation and phosphorylation of serum cholinesterases. Theoretical interpretation of activation energies in complex reactions

1. The effect of temperature and pH was studied on the kinetics of inhibition of horse serum and human serum cholinesterase by four organophosphorus compounds and five carbamates. 2. For all compounds, and at each pH and temperature, the inhibition followed the kinetics of a bimolecular reaction with the inhibitor in excess, and with a negligible concentration of the Michaelis complex. 3. The second-order rate constants (k(a)) for inhibition of human serum cholinesterase by one organophosphate and one carbamate increased from 5 degrees to 40 degrees C with an apparent activation energy of 46kJ/mol (11kcal/mol). 4. The k(a) constant for inhibition of horse serum cholinesterase increased with temperature from 5 degrees to 30 degrees C, and then decreased from 30 degrees to 40 degrees C. The theoretical interpretation of such an unusual effect of temperature is derived. 5. The increase of k(a) with pH (human serum cholinesterase) followed the dissociation curve for a single group on the enzyme (pK7.5). 6. Rate constants for decarbamoylation (k(+3)) were determined, and the time-course of inhibition was calculated from the k(a) and k(+3) constants.     

Hysteretic behaviour and GSSG substrate inhibition shown by glutathione reductase from Phycomyces blakesleeanus

Phycomyces blakesleeanus glutathione reductase shows hysteretic behaviour under experimental conditions, when GSSG substrate inhibition is observed. The progress curves for the reaction show an acceleration phase. The degree of hysteresis varied inversely as the enzyme concentration. It increased when GSSG or NADPH concentration increased, whereas the addition of GSH or NADP+ to the initial reaction mixture prevented it from occurring. In addition, hysteresis was dependent on pH, ionic strength and temperature, decreasing as any of these parameters increased. The parallel effects of pH and ionic strength on the GSSG substrate inhibition and hysteretic behaviour suggest a relationship between these two mechanisms. From the overall results reported in this paper, we propose that the hysteretic behaviour shown by Phycomyces glutathione reductase could be due to a process of time-dependent accumulation of reaction products rather than to a slow conformational change.     

Metrifonate and dichlorvos: Effects of a single oral administration on cholinesterase activity in rat brain and blood

Cholinesterase activities in rat forebrain, erythrocytes, and plasma were assessed after a single oral administration of metrifonate or dichlorvos. In 3-month-old rats, the dichlorvos (10 mg/kg p.o.)-induced inhibition of cholinesterase reached its peak in brain after 15–45 min and after 10–30 min in erythrocytes and plasma. Cholinesterase activity recovered rapidly after the peak of inhibition, but did not reach control values in brain and erythrocytes within 24 h after drug administration. The recovery of plasma cholinesterase activity, in contrast, was already complete 12 h after dichlorvos treatment. Metrifonate (100 mg/kg p.o.) had qualitatively similar inhibition kinetics as dichlorvos, albeit with a slightly delayed onset. Peak values were attained 45–60 min (brain) and 20–45 min (blood), after drug administration. Apparently complete recovery of cholinesterase activity was noted in both tissues 24 h after treatment. The dose-dependence of drug-induced inhibition of cholinesterase in rat blood and brain was determined at the time of maximal inhibition, i.e., 30 min after dichlorvos treatment and 45 min after metrifonate treatment. The oral ED₅₀ values obtained for dichlorvos were 8 mg/kg for brain and 6 mg/kg for both erythrocyte and plasma cholinesterase. The corresponding oral ED₅₀ values for metrifonate were 10 to 15 times higher, i.e., 90 mg/kg in brain and 80 mg/kg in erythrocytes and plasma. In rats deprived of food for 18 h before drug treatment, the corresponding ED₅₀ values for metrifonate were 60 and 45 mg/kg, respectively, indicating an about two-fold higher sensitivity of fasted rats to metrifonate-induced cholinesterase inhibition compared to non-fasted rats. Compared to 3-month-old rats, 19-month-old rats showed a higher sensitivity towards metrifonate and dichlorvos. At the time of maximal inhibition, there was a strong correlation between the degree of cholinesterase inhibition in brain and blood. These results demonstrate that single oral administration of metrifonate and dichlorvos induces an inhibition of blood and brain cholinesterase in the conscious rat in a dose-dependent and apparently fully reversible manner. While the efficiency of a given dose of inhibitor may vary with the satiety status or age of the animal, the extent of brain ChE inhibition can be estimated from the level of blood ChE activity.     

Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation

In ethanol fermentation, instantaneous biomass yield of the yeast Saccharmoyces cerevisiae was found to decrease (from 0.156 to 0.026) with increase in ethanol concentration (from 0 to 107 g/L), indicating a definite relationship between biomass yield and product inhibition. A suitable model was proposed to describe this decrease which incorporates the kinetic parameters of product inhibition rather than pure empirical constants. Substrate inhibition was found to occur when substrate concentration is above 150 g/L. A similar definite relationship was observed between substrate inhibition and instantaneous biomass yield. A simple empirical model is proposed to describe the declines in specfic growth rate and biomass yield due to substrate inhibition. It is observed that product inhibition does not have any effect on product yield whereas substrate inhibition significantly affects the product yield, reflecting a drop in overall product yield from 0.45 to 0.30 as the initial substrate concentration increases from 150 to 280 g/L. These results are expected to have a significant influence in formulating optimum fermentor design variables and in developing an effective control strategy for optimizing ethanol producitivity.     

Effects of single or repeated dermal exposure to methyl parathion on behavior and blood cholinesterase activity in rats

The effects of a single or repeated dermal administration of methyl parathion on motor function, learning and memory were investigated in adult female rats and correlated with blood cholinesterase activity. Exposure to a single dose of 50 mg/kg methyl parathion (75% of the dermal LD(50)) resulted in an 88% inhibition of blood cholinesterase activity and was associated with severe acute toxicity. Spontaneous locomotor activity and neuromuscular coordination were also depressed. Rats treated with a lower dose of methyl parathion, i.e. 6.25 or 12.5 mg/kg, displayed minimal signs of acute toxicity. Blood cholinesterase activity and motor function, however, were depressed initially but recovered fully within 1-3 weeks. There were no delayed effects of a single dose of methyl parathion on learning acquisition or memory as assessed by a step-down inhibitory avoidance learning task. Repeated treatment with 1 mg/kg/day methyl parathion resulted in a 50% inhibition of blood cholinesterase activity. A decrease in locomotor activity and impairment of memory were also observed after 28 days of repeated treatment. Thus, a single dermal exposure of rats to doses of methyl parathion which are lower than those that elicit acute toxicity can cause decrements in both cholinesterase activity and motor function which are reversible. In contrast, repeated low-dose dermal treatment results in a sustained inhibition of cholinesterase activity and impairment of both motor function and memory.     

Cholinesterase Newfoundland: a new succinylcholine-sensitive variant of cholinesterase at locus 1.

A family from Newfoundland was found to have a new rare variant for plasma cholinesterase (E.C.3.1.1.8) recognized by a high-percentage inhibition by dibucaine (DN), particularly when succinyldithiocholine was used as substrate (DNSDTC) but also somewhat high when benzoylcholine was substrate (DNBZCH). The family data demonstrated that the variant is determined by an allele of the usual and atypical alleles at locus 1, and the new allele is designated CHE1*NFLD. The proband who was heterozygous for the Newfoundland and atypical alleles had shown sensitivity to succinylcholine. It is postulated that cholinesterase Newfoundland (NFLD) has a reduced affinity for succinylcholine. Samples selected for high DNs with a benzoylcholine from 200 Canadian Caucasians and 70 Newfoundlanders did not have the variant, and, therefore, it is assumed that the remainder of the samples did not have the variant.     

A versatile equation to describe reversible enzyme inhibition and activation kinetics: Modeling β-galactosidase and butyrylcholinesterase

Current treatments for Alzheimer's disease involve inhibiting cholinesterases. Conversely, cholinesterase stimulation may be deleterious. Homocysteine is a known risk factor for Alzheimer's and vascular diseases and its active metabolite, homocysteine thiolactone, stimulates butyrylcholinesterase. Considering the opposing effects on butyrylcholinesterase of homocysteine thiolactone and cholinesterase inhibitors, understanding how these molecules alter this enzyme may provide new insights in the management of dementia. Butyrylcholinesterase does not strictly adhere to Michaelis–Menten parameters since, at higher substrate concentrations, enzyme activation occurs. The substrate activation equation for butyrylcholinesterase does not describe the effects of inhibitors or non-substrate activators. To address this, global data fitting was used to generate a flexible equation based on Michaelis–Menten principles. This methodology was first tested to model complexities encountered in inhibition by imidazole of β-galactosidase, an enzyme that obeys Michaelis–Menten kinetics. The resulting equation was sufficiently flexible to permit expansion for modeling activation or inhibition of butyrylcholinesterase, while accounting for substrate activation of this enzyme. This versatile equation suggests that both the inhibitor and non-substrate activator examined here have little effect on the substrate-activated form of butyrylcholinesterase. Given that butyrylcholinesterase inhibition can antagonize stimulation of this enzyme by homocysteine thiolactone, cholinesterase inhibition may have a role in treating Alzheimer and vascular diseases related to hyperhomocysteinemia.     

Partial nicotinic receptor blockade unmasks a modulatory role of nitric oxide on urethral striated neuromuscular transmission.

The objective of this study was to investigate the possible modulatory role of endogenous nitric oxide (NO) production on the urethral striated muscle (USM) function in the sheep urethra. Significant NO synthase (NOS) activity was measured in both the particulate and cytosolic fractions of USM homogenates. NOS activity was calcium-dependent and showed greater inhibition by NOS inhibitors selective of the neural NOS isoform (nNOS). nNOS immunoreactivity was present in intramural nerves as well as in the sarcolemma of some striated fibers, being denser at the neuromuscular junction (NMJ). Double immunolabeling showed co-localization of nNOS with both alpha-bungarotoxin and choline acetyltransferase, at the USM endplates. For the first time, functional data support a role of NO on the USM contractility "in vitro," which became evident following partial nicotinic receptor inactivation with low concentrations of D-tubocurarine. Only under D-tubocurarine (0.25 microM) treatment, different NOS inhibitors, specially N(G)-propyl-L-arginine, as well as the guanylate cyclase inhibitor ODQ, all showed a significant enhancing effect on contractions induced by electrical field stimulation of intrinsic somatic nerves. These data suggest that local production of NO at the urethral NMJ may modulate release and/or action of acetylcholine on motor endplates by cyclic GMP-mediated effects. This modulatory action could be especially relevant when neuromuscular transmission at the USM is impaired.     

Time-dependence of inhibition of carnitine palmitoyltransferase I by malonyl-CoA in mitochondria isolated from livers of fed or starved rats. Evidence for transition of the enzyme between states of low and high affinity for malonyl-CoA.

The degree of inhibition of CPT I (carnitine palmitoyltransferase, EC 2.3.1.21) in isolated rat liver mitochondria by malonyl-CoA was studied by measuring the activity of the enzyme over a short period (15s) after exposure of the mitochondria to malonyl-CoA for different lengths of time. Inhibition of CPT I by malonyl-CoA was markedly time-dependent, and the increase occurred at the same rate in the presence or absence of palmitoyl-CoA (80 microM), and in the presence of carnitine, such that the time-course of acylcarnitine formation deviated markedly from linearity when CPT I activity was measured in the presence of malonyl-CoA over several minutes. The initial rate of increase in degree of inhibition with time was independent of malonyl-CoA concentration. CPT I in mitochondria from 48 h-starved rats had a lower degree of inhibition by malonyl-CoA at zero time, but was equally capable of being sensitized to malonyl-CoA, as judged by an initial rate of increase of inhibition identical with that of the enzyme in mitochondria from fed rats. Double-reciprocal plots for the degree of inhibition produced by different malonyl-CoA concentrations at zero time for the enzyme in mitochondria from fed or starved animals indicated that the enzyme in the latter mitochondria was predominantly in a state with low affinity for malonyl-CoA (concentration required to give 50% inhibition, I0.5 congruent to 10 microM), whereas that in mitochondria from fed rats displayed two distinct sets of affinities: low (congruent to 10 microM) and high (less than 0.3 microM). Plots for mitochondria after incubation for 0.5 or 1 min with malonyl-CoA indicated that the increased sensitivity observed with time was due to a gradual increase in the high-affinity state in both types of mitochondria. These results suggest that the sensitivity of CPT I in rat liver mitochondria in vitro had two components: (i) an instantaneous sensitivity inherent to the enzyme which depends on the nutritional state of the animal from which the mitochondria are isolated, and (ii) a slow, malonyl-CoA-induced, time-dependent increase in sensitivity. It is suggested that the rate of malonyl-CoA-induced sensitization of the enzyme to malonyl-CoA inhibition is limited by a slow first-order process, which occurs after the primary event of interaction of malonyl-CoA with the mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS)     

Depression of potassium-evoked striatal acetylcholine release by delta-receptor activation: inhibition by cholinoactive agents

To examine the role of delta-opioid receptors in the modulation of striatal acetylcholine (ACh) release, the action of D-Pen2,L-Pen5-enkephalin, a selective delta-opioid receptor agonist, was tested on [3H]ACh release from slices of the rat caudate-putamen. Slices, incubated with [3H]choline, were superfused with a physiological buffer and stimulated twice by exposure to a high potassium (K+) concentration. In the absence of a cholinesterase inhibitor, 1 microM D-Pen2,L-Pen5-enkephalin produced a 46 and 35% decrease in the release of [3H]ACh evoked by 15 and 25 mM K+, respectively. The depressant action of the enkephalin analogue was concentration dependent, with a maximal effect on K+-evoked [3H]ACh release occurring at 1.0 microM, and was completely blocked in the presence of the delta-opioid receptor selective antagonist, ICI 174864 (1 microM). In the presence of the cholinesterase inhibitors physostigmine (10 microM) and neostigmine (10 microM), or the muscarinic receptor agonist oxotremorine (10 microM), D-Pen2,L-Pen5-enkephalin did not depress the K+-evoked release of [3H]ACh. Atropine (1 microM) blocked the inhibitory effect of physostigmine on the depressant action of D-Pen2,L-Pen5-enkephalin. The results of this study indicate that delta-opioid receptor activation is associated with an inhibition of striatal ACh release, but this opioid-cholinergic interaction is not apparent under conditions of presynaptic muscarinic receptor activation.