Levamisole receptors

Muscle-vesicle for recording single-channel properties of levamisole receptors... 

Fig. 21.4. Vesicle formation and patch-clamp techniques used to record levamisole receptor channel currents from Ascaris muscle. (A) Muscle membrane vesicles bud-off from the bag membrane following a 10 min collagenase treatment and incubation for 1 h at 37°C in Ascaris saline. (B) Levamisole is applied to the outside surface of the membrane to activate receptor channels cell-attached patches are usually used but it is also possible to make inside-out and outside-out patch recordings.

The advantage of the patch-clamp technique for recording from single levamisole receptors is that biophysical properties of individual receptors... 

One test that was carried out when trying to compare levamisole receptor properties in SENS and LEVR isolates was to count the numbers of patches of recorded membranes that contained active channels. Figure 21.7 shows the proportion of patches from SENS and LEVR isolates that contained active patches at different concentrations of levamisole. It was... 

The different subtypes of levamisole receptor (receptor heterogeneity) were illustrated by the presence, in some patches, of channels with different conductances (Fig. 21.9). The different conductances were seen as channel currents opening to different levels producing different peaks in the scatter... 

Fig. 21.10. Schematic diagram of the presence of the four levamisole receptor subtypes present in O. dentatum in the SENS isolate. The four subtypes are shown to be present in one muscle cell but they may not all occur in the same muscle or animal. In the LEVR isolate three subtypes are shown as present, with the G35 missing. The G40 and G45 are shown as modified because the data on the probability of opening showed that they behaved in a significantly different manner to that ofthe G40 and G45 subgroups of the SENS isolates.

It has already been pointed out that resistant isolates have on average fewer active channels per patch than the sensitive isolates. In addition, the average proportion of time that the levamisole receptors were open (P-open) in the LEVR isolates was significantly less than the proportion of time that the levamisole receptors were open in the SENS isolates (Fig. 21.11). Thus, two factors can be identified that lead to a reduction in the current carried across the membrane by levamisole receptors in the resistant isolate when compared with the sensitive isolate. The factors are, again, a reduced density of active channels in LEVR isolates, and a reduced proportion of time that the active channels in the resistant isolate are open. These factors combine so that there is a tenfold reduction in the current carried across the membrane at 30 pM. 

The distribution of levamisole receptor conductances from the SENS isolate in this study was skewed towards G45 and fitted by the sum of four Gaussian distributions, suggesting the presence of four main conductance subtypes of fevamisofe receptor that are referred to as G25, G35, G40 and G45. In addition to the variation in the conductance of the levamisole receptors a large variation was noted between the P-open values of individual... 

Caenorhabditis elegans is a soil nematode that has been subjected to detailed genetic analysis. The possible structure of the nematode levamisole receptor, based on the pentameric structure of the nicotinic channel of the Torpedo electric organ (Devillers-Thiery et al, 1993), is shown in Fig. 21.12. 

Martin, R.J., Robertson, A.P., Bjorn, H. and Sangster, N.C. (1997) Heterogeneous levamisole receptors a single-channels study of nictotinic acetylcholine receptors from Oesophagostomum dentatum. European Journal of Pharmacology 322, 249-257. 

Fig. 21.3. Two-micropipette current-clamp recording and effect of maintained application of 30 pM levamisole, which produces a 15 mV depolarization (upward movement of trace). The downward transients are the result of injected current pulses used to measure membrane conductance. The trace gets narrower as the input conductance increases from 2.35 pS to 4.35 pS as the levamisole ion channels open. The peak amplitude of the membrane potential response and change in input conductance are used as an assay of the number and activity of the levamisole ion channel receptors present in the muscle cell membrane. The response was fully reversible on washing (not shown).

Figure 21.11A illustrates representative vesicle-attached recordings from a sensitive isolate (SENS), a pyrantel-resistant isolate (PYRR) and a levamisole-resistant isolate (LEVR) produced by 30 pM levamisole. There are differences in mean P-open values of receptors between sensitive and resistant isolates that would produce, on average, a reduction in the effect of the anthelmintic. However, one of the puzzling, but very consistent, observations is that there is a big range in the P-open values observed in different patches under the same experimental conditions in the same isolate (Fig. 21.1 IB). For example, the P-open values of the sensitive isolate were observed to vary between 0.090 and 0.003 at -50 mV with 30 pM levamisole, a 30-fold difference between the biggest and the smallest P-open...

Fig. 21.11. (Opposite) (A) Representative vesicle-attached patch recordings from SENS, PYRR and LEVR isolates 30 pM levamisole in the patch-pipette, -50 mV patch potential. Note that the SENS isolate recording contains more openings than the other two patches and so will carry more current across the membrane. (B) Log10 plot of the proportion of open-time against the isolate type. Open circles represent individual receptor channels from different preparations at -50 mV with 30 pM levamisole as the agonist. Note that there is a wide spread of greater than tenfold difference between the maximum and minimum values observed for each of the isolates. There is overlap between values of the isolates but the mean (closed square) for SENS is greater than for LEVR and PYRR.

Fig. 21.12. Schematic diagram of the putative structure of a levamisole nicotinic receptor. (A)...

Fleming, J.T., Squire, M.D., Barnes, T.M., Tornoe, C., Matsuda, K., Ahnn,J., Fire, A., Sulston, J.E., Barnard, E.A., Sattelle, D.B. and Lewis, J.A. (1997) Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor svlbxmixs,. Journal of Neuroscience 17, 5843-5857. 

Hoekstra, R., Visser, A., Wiley, L.J., Weiss, A.S., Sangster, N.C. and Roos, M.H. (1997) Characterization of an acetylcholine receptor gene of Haemonchus contortus in relation to levamisole resistance. Molecular and Biochemical Parasitology 84, 179—187. 

The substituted imidazo-thiazole, dexamisole, has antidepressant properties and its isomer, levamisole, possesses anthelmintic and immunostimulant properties. Enantiomers of HA-966 (3-amino-l-hydroxypyrrolid-2-one) exhibit distinct central nervous system effects (+)-HA-966 is a selective glycine/A-methyl-o-aspartate receptor antagonist, but (-)-HA-966 is a potent i-butyrolactone-Uke sedative." A comparison of (+) and (-)-3-methoxycypro-heptadine shows that aU of the anticholinergic activity of the ( )-3-methoxycyproheptadine resides solely in the dextrorotatory enantiomer, while the antiserotonin activity resides in the levorotatory enantiomer." ... 

We have shown that the anthelmintics pyrantel, morantel and levamisole, activate acetylcholine (Ach) receptors on the muscle cell membranes of Ascaris suum and also that the avermectin 22,23-dihydroavermectin Bla (DHAVM) blocks the function of muscle cell gamma-aminobutyric acid (GABA) receptors. In experiments using micro-iontophoresis, the cholinergic anthelmintics were found to act directly on the muscle bag membrane, evoking a depolarizing response. 

Action of Morantel, Pyrantel and Levamisole on the Function of Muscle Ach Receptors... 

Exposure of nematode parasites to morantel, pyrantel and levamisole causes spastic paralysis (21 ) consistent with the activation of excitatory Ach receptors. In experiments on A suum, Aubry et al. (60 showed that muscle contractions evoked by pyrantel are sensitive to block by the cholingeric antagonist, d-tubocurarine. In our experiments we used current or voltage clamped muscle bag preparations to investigate directly the mechanism of the paralyzing action of morantel, pyrantel and levamisole. 

Previous studies to characterize the pharmacology of Ach receptors on A. suum muscle show that they possess a nicotinic profile (22, 23). Of all the agents reported to have agonist activity at these receptors, our results show that morantel and pyrantel are the most potent agonists described to date. We have also shown that levamisole is an agonist at the muscle Ach receptor of A. suum, but is much less potent than the pyrimidines. 

Levamisole causes rapid muscle contractions that lead to spastic paralysis of nematodes. It has been suggested that these contractions are caused by levamisole acting as a nerve ganglion stimulant ( , 24). The results of our study show that levamisole also acts directly on Ach receptors on the muscle cells of A. suum. Both the increases in input conductance and the depolarization of muscle cells were recorded from preparations bathed in a calcium-free, high magnesium saline under these conditions transmission at chemical synapses is completely blocked. We have also obtained further evidence for the direct action of levamisole on the muscle cell membrane by recording depolarizing responses evoked by the iontophoretic delivery of levamisole to the muscle bag membrane. 

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