Veterinary pharmacology, ion-channels and anthelmintics

Abstract

In this collection of papers modes of action of anthelmintic and anaesthetic drugs used in Veterinary Practice are investigated using electrophysiological techniques. New preparations and analytical techniques for investigation are described along with properties of ion-channel target sites. The pharmacology of piperazine, levamisole, pyrantel, morantel, oxantel, avermectins, cyclic depsipeptides, ketamine, metomidate, alphaxalone and xylazine are studied. This information is reviewed in formats suitable for graduate and undergraduate study.Two-micropipette current-clamp and voltage-clamp techniques for recording effects of GABA agonists and antagonists on nematode muscle are developed using Ascaris suum. Piperazine, an anthelmintic, is shown to act as a GABA agonist of low potency and, like GABA, to mediate an increase in CI conductance by an action on extrasynaptic receptors. Diethylcarbamazine, a piperazine derivative, did not to act as a GABA agonist but blocked a voltage-activated K current. The agonist profile of the Ascaris GABA receptor was similar to vertebrate GABAa receptors but the antagonist profile was very different, indicating the presence of a distinctive type of receptor (GABAN). Novel arylaminopyridazine derivatives were synthesised and tested as competitive antagonists on the GABAN receptor. The KB for NCS 281-93, was 4.7 pM. A preparation suitable for patch-clamp studies was developed and GABA receptors activated by GABA and piperazine. Both agonists activated channels with a mean single-channel conductance of 22 pS but GABA mean open-times were longer (32 ms) than those of piperazine (14 ms).The effects of ivermectin on muscle GABA receptors in Ascaris and on 19 pS CI channels were observed using cell-attached and isolated inside-out patches. Ivermectin locked open the small CI channels when applied to the outside membrane. A novel fluorescent and active bodipy ivermectin analogue was synthesised and the movement of the probe followed in vesicle membranes using the FRAP (fluorescence recovery after photobleaching) technique. Quenching experiments showed that the ivermectin probe did not move through the cell membrane in the time scale of the experiment (30 min). A novel two-microelectrode current-clamp preparation of the Ascaris pharynx was used to show that an ivermectin analogue potentiated a glutamate gated CI channel at low concentrations and produced and increase in the CI conductance at a higher concentration. The actions of potent and novel potential anthelmintic cyclic depsipeptides were investigated using conventional parasitological techniques and the mode of action investigated in Ascaris muscle using current-clamp. It was found that these compounds did not act directly as a GABA agonist or acetylcholine antagonist. The actions of the anthelmintic praziquantel are reviewed and a tegumental preparation of Schistosoma mansoni developed for single-channel recording.Extrasynaptic nicotinic acetylcholine receptors were found to be located on the bag region of Ascaris suum muscle with iontophoresis experiments. Acetylcholine gated non-selective cation channels which were blocked by high concentrations of diethylcarbamazine. A novel muscle vesicle preparation was developed and used to record single-channel currents activated by acetylcholine, and the anthelmintics levamisole, pyrantel, oxantel and morantel. Mean open-times of the channels opened by these agonist were in the range 0.5 to 3ms and channels with two different conductances were detected. The anthelmintics levamisole, pyrantel, oxantel and morantel all produced open-channel block. Morantel was the most effective blocker and its action modelled using a novel two-ion block model. Effects of levamisole on another parasitic nematode, Oesophagostomum dentatum, were studied using the muscle vesicle preparation. The different conductances and mean open-times of channels showed that levamisole receptors were not homogeneous but that there are at least two major types, G35 and G45.During patch-clamp experiments on Ascaris muscle, a novel high-conductance Ca-dependent CI channel was observed. The channel was found to be permeable to mono-carboxylic and di-carboxylic acids with a pore size estimated to be 6.6A0. The channel was sensitive to intracellular pH and is hypothesised to be involved in the excretion of carboxylic acids from anaerobic metabolism. The distinctive features of the channel suggest it plays a role in regulating the membrane potential of the cell and is a possible drug target site. A two microelectrode voltage-clamp was used to identify a voltage-activated Ca current and two K currents in somatic muscle of the Ascaris. A Kostyuk voltage-clamp preparation to record from an isolated bag preparation was developed. A slow non-selective cation current was identified which was increased in amplitude by acetylcholine through a nonnicotinic receptor. The FaRP peptide PF1 had an inhibitory effect on the current showing that there were peptide receptors on the isolated muscle.Properties of zona-free oocytes and voltage-activated Na and K currents in a human neuroblastoma cell line and K and CI currents in rat bone marrow derived mast cells were examined by using the patch-clamp technique. New analytical techniques for single ion-channel current behaviour were developed. To detect periods of non-stationary - drift in experimental conditions - a cusum technique was used to separate out stationary periods. Multiple channel openings of in a single patch recording could be described by a generalized binomial of Poisson.