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Biomedical subjects

R M Dawson

Publications and source records attributed to R M Dawson.

At least 19 recordsLinked to original sources
PubMed1998

The toxicology of microcystins.

Microcystins are a family of more than 50 structurally similar hepatotoxins produced by species of freshwater cyanobacteria, primarily Microcystis aeruginosa. They are monocyclic heptapeptides, characterised by some invariant amino acids, including one of unusual structure which is essential for expression of toxicity. Microcystins are chemically stable, but suffer biodegradation in reservoir waters. The most common member of the family, microcystin-LR (L and R identifying the 2 variable amino acids, in this case leucine and arginine respectively) has an LD50 in mice and rats of 36-122 microg/kg by various routes, including aerosol inhalation. Although human illnesses attributed to microcystins include gastroenteritis and allergic/irritation reactions, the primary target of the toxin is the liver, where disruption of the cytoskeleton, consequent on inhibition of protein phosphatases 1 and 2A, causes massive hepatic haemorrhage. Microcystins are tight-binding inhibitors of these protein phosphatases, with inhibition constants in the nanomolar range or lower. Uptake of microcystins into the liver occurs via a carrier-mediated transport system, and several inhibitors of uptake can antagonise the toxic effects of microcystins. The most effective of these is the antibiotic rifampin (a drug approved for clinical use), which protects mice and rats against microcystin-induced lethality when given prophylactically and, in some cases, therapeutically.

Animals↗
PubMed1995

Oxime effects on the rate constants of carbamylation and decarbamylation of acetylcholinesterase for pyridostigmine, physostigmine and insecticidal carbamates.

The effects of the oximes 2-pyridine aldoxime methiodide (PAM), HI-6, HS-6, toxogonin and TMB-4 on the rate of carbamylation of membrane-bound bovine erythrocyte acetylcholinesterase were studied. The second-order rate constant of carbamylation (ki) and the first-order rate constant of decarbamylation (k3) were calculated from the proportion of free acetylcholinesterase at equilibrium and the rate of approach to equilibrium. Twenty insecticidal carbamates plus physostigmine and pyridostigmine were studied. The oximes increased ki for several carbamates, with HI-6 causing an increase in the most number of cases (12) and PAM the least (3). HI-6 was also a potent accelerator of decarbamylation (increase in k3) in all cases, whereas PAM caused a significant decrease in k3 in 15 cases and a nonsignificant decrease in the other 7. Toxogonin and TMB-4 increased k3 or had no significant effect. The results were generally consistent with a proposal in the literature that there is a correlation between increased ki and increased toxicity of the carbamate in the presence of an oxime.

Acetylcholinesterase↗
PubMed1994

Rate constants of carbamylation and decarbamylation of acetylcholinesterase for physostigmine and carbaryl in the presence of an oxime.

Membrane-bound bovine erythrocyte acetylcholinesterase was inhibited with physostigmine or carbaryl, and the rate constants of carbamylation and decarbamylation were determined from the proportion of inhibited acetylcholinesterase in the steady state, and the rate of approach to the steady state. The oximes 2-PAM, HI-6, HS-6, TMB-4 and toxogonin, at 0.1 mM, all decreased the rate of carbamylation by physostigmine, but increased the rate of carbamylation by carbaryl. TMB-4 and toxogonin were the most effective oximes in potentiating carbamylation by carbaryl, with an enhancement of the second-order rate constant of 54- and 17-fold respectively. The greatest reduction in the rate constant for carbamylation by physostigmine (3.7-fold) was caused by HI-6. HS-6 and HI-6 increased the rate of decarbamylation, while 2-PAM reduced the rate of decarbamylation if physostigmine was the carbamate. 2-PAM and HI-6 were also studied with soluble bovine erythrocyte acetylcholinesterase, and similar results were obtained. The results extend those in a recent report by other authors who studied the half-life of carbamylation for acetylcholinesterase and butyrylcholinesterase in an attempt to understand the mechanism by which oximes increase the toxicity of carbaryl in vivo. These authors proposed binding of the oximes to an allosteric site on the enzyme. While not discounting this possibility, the present results, taken with other reports in the literature, suggest that binding of the oximes to the anionic subsite of the active site of the enzyme is also feasible. The present results also offer an explanation for another recent report, in which anomalous results were presented for decarbamylation of physostigmine-inhibited and carbaryl-inhibited erythrocyte acetylcholinesterase in the presence of 2-PAM or HI-6.

Acetylcholinesterase↗
PubMed1993

Effects of the nerve agents soman and tabun on the uptake and release of GABA and glutamate in synaptosomes of guinea pig cerebral cortex.

1. Crude and purified synaptosomes were prepared from the cerebral cortex of the rat or the guinea pig and used to study the uptake and release of [3H]GABA and [3H]glutamate. 2. Baclofen at 10(-5) M inhibited stimulated release of [3H]GABA from crude rat and guinea pig synaptosomes, but not from purified rat synaptosomes. 3. 1-2 mM tabun decreased the uptake of [3H]GABA and increased the uptake of [3H]glutamate by purified guinea pig synaptosomes. 4. Soman and tabun at 10(-6) M and 10(-5) M inhibited basal release of [3H]GABA and [3H]glutamate from crude guinea pig synaptosomes. Tabun at 10(-5) M decreased stimulated release of [3H]GABA while soman had no effect. 5. The results do not sustain the possibility that nerve agents cause convulsions by affecting the uptake or release of GABA or glutamate. However indirect evidence was obtained that soman and tabun inhibit catabolism of GABA and glutamate.

Animals↗
PubMed1992

Assessment of a model for the opposing effects of N-ethylmaleimide on the affinity of muscarinic agonists for M2 receptors.

1. Homogenates of guinea pig right atrium (M2 receptors) were treated with N-ethylmaleimide, after which the ability of carbachol to inhibit binding of [3H]quinuclidinyl benzilate was studied. 2. At 37 degrees C, 10(-4) M and 10(-3) M, but not 10(-5) M, N-ethylmaleimide increased the affinity of carbachol for the receptor. At 2 degrees C, 10(-5) M and 10(-4) M, but not 10(-3) M, N-ethylmaleimide decreased carbachol affinity. At 2 degrees C, after the homogenate had been incubated at 37 degrees C without N-ethylmaleimide, 10(-5) M N-ethylmaleimide decreased carbachol affinity, as at 2 degrees C without preincubation, but 10(-3) M N-ethylmaleimide increased carbachol affinity, as at 37 degrees C. 10(-4) M N-ethylmaleimide was without effect. 3. The results are discussed with respect to a previously proposed model in which N-ethylmaleimide interacts with two sites, causing an increase or decrease in agonist affinity respectively.

Animals↗
PubMed1991

The interaction of tacrine with benzodiazepine and GABA binding sites of guinea pig brain.

Tacrine (1,2,3,4-tetrahydro-9-acridinamine) inhibited binding of [3H]flunitrazepam to benzodiazepine receptors of guinea pig hippocampus with an inhibition constant of 46 microM at 2 degrees C and 37 degrees C. gamma-Aminobutyric acid (GABA) decreased the affinity of tacrine for the receptor, suggesting that tacrine may act as an inverse agonist. A Hill coefficient less than 1 was observed under all conditions. Allosteric interactions may explain this behaviour, since 100 microM tacrine increased the rate of dissociation of [3H]flunitrazepam from the receptor. Tacrine inhibited the binding of 11 nM [3H]GABA to GABA receptors of guinea pig cerebral cortex with I50 = 188 microM. Bicuculline methiodide was 4 times as potent (I50 = 49 microM). The interaction of tacrine with GABA or benzodiazepine binding sites is unlikely to be of clinical significance.

Animals↗
PubMed1990

Reversibility of the inhibition of acetylcholinesterase by tacrine.

Inhibition of bovine erythrocyte acetylcholinesterase (AChE) by 1,2,3,4-tetrahydro-9-acridinamine (tacrine) was independent of time of incubation and was partially reversed by dilution and by increased substrate concentration. It was fully reversed by dialysis. Similar results were obtained with AChE from other sources. The results are consistent with some reports in the literature, but not with others; none of these reports examined all four criteria of reversibility. The results do not explain the prolonged inhibition of AChE in vivo or the ability of tacrine to protect animals against the lethal effects of organophosphate anticholinesterases.

Acetylthiocholine↗
PubMed1990

Opposing effects of N-ethylmaleimide on the affinity of carbachol for muscarinic cholinoceptors of guinea-pig atrium.

1. Inhibition of the binding of [3H]quinuclidinyl benzilate to homogenates of guinea pig right atrium (M2 receptors) by varying concentrations of carbachol was studied. 2. Pretreatment of membranes with 5 x 10(-5) M N-ethylmaleimide at 2 degrees C shifted the carbachol inhibition curve to the right, indicating decreased affinity of the receptor for carbachol. However pretreatment at 37 degrees C moved the curve to the left. 3. The ability of guanyl-5'-yl imidodiphosphate to reduce agonist affinity was largely eliminated by treatment with N-ethylmaleimide at both temperatures. 4. Conflicting reports in the literature and the present results can be explained by invoking a model in which N-ethylmaleimide has a high affinity for a heat-labile site and a lower affinity for a heat-insensitive site. Reaction with the first site decreases agonist affinity, but at 37 degrees C this site is largely inactivated and reaction with the second site, which leads to increased agonist affinity, predominates.

Animals↗
PubMed1989

Tacrine slows the rate of ageing of sarin-inhibited acetylcholinesterase.

Bovine erythrocyte acetylcholinesterase was inhibited by the organophosphate sarin, and the rate of ageing (the time-dependent decrease in the ability of an oxime to reactivate the enzyme) was studied. At pH 7.0 and 37 degrees C, 10(-5) M or 10(-6) M tacrine (tetrahydroaminoacridine) decreased the rate of ageing in low ionic strength buffer. Tacrine at 10(-5) M also significantly decreased the rate of ageing in 150 mM NaCl. The results indirectly demonstrated that the inhibition of substrate hydrolysis by tacrine is reversible, and that tacrine does not prevent reactivation of sarin-inhibited acetylcholinesterase. Both these observations, which were also made for rat brain acetylcholinesterase, are in contrast with reports in the literature.

Acetylcholinesterase↗
PubMed1989

Factors influencing the calculation of results from studies of the release of tritiated neurotransmitters from superfused slices of guinea pig striata.

Slices of guinea pig striata were incubated with tritiated choline, dopamine, or serotonin and the release of radioactive transmitter was studied in a superfusion system. Some experiments were also done on the release of [3H]acetylcholine from rat striatal slices. For analysis of the results, various parameters of the system were determined in order to establish the most reliable method of assessing drug effects on transmitter release. Peaks of radioactivity (S1 and S2) above basal release of radioactivity (B1 and B2) were observed after two depolarizations of the nerve endings with high K+ buffer. Each stimulation was for 2 min, and S2 occurred 20 min after S1. There was a highly significant correlation between S1 and the protein content of the slices for acetylcholine release from guinea pig striata. Basal release of radioactivity, and the ratio S2/S1, were not sensitive to minor changes in the experimental conditions. It was concluded that S2/S1, rather than S1 or S1/B1, should be used as a measure of drug effects on release. The experiments also demonstrated that re-uptake of released neurotransmitter is operative in the superfusion system for dopamine, but not for serotonin. Differences were observed between the rat and the guinea pig with respect to the release of [3H]acetylcholine.

Acetylcholine↗
PubMed1989

Inhibition constants and GABA-shifts at 2 degrees C and 37 degrees C for a spectrum of ligands acting on the benzodiazepine receptors of guinea pig hippocampus.

1. Dissociation constants and Hill coefficients were determined for 13 ligands inhibiting the binding of [3H]flunitrazepam to the benzodiazepine receptors of guinea pig hippocampus at 2 degrees C and 37 degrees C. 2. The ratio of I50 in the absence of gamma-aminobutyric acid (GABA) to that in the presence of 10(-5) M GABA (the GABA-shift) was found to vary from 0.4 to 2.5. 3. There was no correlation between the pharmacological activity of the ligand and the GABA-shift, or between the pharmacological activity and the magnitude of the increase in affinity of the ligand for the receptor as the temperature decreased from 37 degrees C to 2 degrees C. 4. A correlation was observed between the temperature-induced affinity change and the GABA-shift at 37 degrees C.

Animals↗
PubMed1988

Phenyldichlorophosphate as an aid in studies of decarbamylation of carbamylated acetylcholinesterase.

An improved method for assaying carbamylated acetylcholinesterase is described which has substantial benefits over current methods. Acetylcholinesterase was carbamylated with neostigmine and diluted extensively into buffer to allow decarbamylation to occur. At various times, phenyldichlorophosphate was added to the mixture of free and carbamylated enzyme, whereupon two very rapid, simultaneous reactions occurred: near total, and permanent, inactivation of free acetylcholinesterase by the organophosphate, and inactivation of phenyldichlorophosphate by hydrolysis. The carbamylated acetylcholinesterase was allowed to reactivate fully and then assayed for enzyme activity. The assay provided a measure of the amount of carbamylated enzyme present at the time of addition of phenyldichlorophosphate, thereby enabling the first-order rate constant for decarbamylation to be calculated. This new method of studying decarbamylation was applied to two systems of soluble acetylcholinesterase, where the half-life for decarbamylation was approximately 1/2 h or 4 min, respectively, and to membrane-bound acetylcholinesterase. The results agreed well with those determined by a conventional method; moreover, the standard error of the mean was lower for the new method. The advantages of the method using phenyldichlorophosphate over conventional methods are particularly evident when decarbamylation is rapid or when in vivo studies are being performed and it is not practical or desirable to run assays immediately on isolation of the tissue. The new method also has advantages over a published related technique using the organophosphate anticholinesterase soman.

Acetylcholinesterase↗
PubMed1988

Effects of guanyl nucleotides on the activation of cardiac muscarinic receptors.

Effects of guanyl nucleotides on the affinity of cholinergic agonists at the [3H]quinuclidinylbenzilate binding site have been compared with their effects on the intact left atrium. The binding of carbachol was described by a two-site model; guanyl nucleotides had little effect on the affinity at either site but increased substantially the proportion of low affinity sites. The negative inotropic effect of acetylcholine (ACh) was unaffected by exogenous guanyl nucleotides or variation in external Mg++ or Ca++. ACh (2 microM) shortened the action potential, reduced spike amplitude and usually increased the maximum diastolic potential. GTP and guanyl-5'-yl-imidodiphosphate in the presence of ACh increased action potential amplitude and duration and increased further the diastolic potential. Low external Mg++ had similar effects to guanyl-5'-yl-imidodiphosphate but their effects were not additive. It is suggested that these compounds delay the activation of a transient outward K+ current by ACh.

Acetylcholine↗
PubMed1987

Kinetic constants for the inhibition of eel and rabbit brain acetylcholinesterase by some organophosphates and carbamates of military significance.

The kinetics of the inhibition of eel and rabbit brain acetylcholinesterase by a number of organophosphates and carbamates has been studied. Five organophosphates (tabun, sarin, soman, GF, and VX) and two carbamates (neostigmine and physostigmine) were used. Small but significant differences have been found between the two enzyme preparations in both the dissociation constant and the rate constants for the irreversible step. The values of k2 ranged from 0.56 to 1.08 sec-1 for the eel enzyme and 0.19 to 0.73 sec-1 for the rabbit enzyme. Kd varied from 0.3 to 24.5 microM for the eel and 0.3 to 9.3 microM for the rabbit enzyme. The similarity between the enzymes is remarkable considering the differences in the species of origin.

Animals↗