The Pharmacology of Cholinoceptors on the Somatic Muscle Cells of the Parasitic Nematode Ascaris Suum

/. exp. Bwl. 158, 509-530 (1991) 509 Printed in Great Britain © The Company of Biologists Limited 1991

THE PHARMACOLOGY OF CHOLINOCEPTORS ON THE SOMATIC MUSCLE CELLS OF THE PARASITIC NEMATODE ASCARIS SUUM

BY L. COLQUHOUN, L. HOLDEN-DYE* AND R. J. WALKER Department of Physiology and Pharmacology, University of Southampton, Bassett Crescent East, Southampton SO9 3TU

Accepted 28 March 1991

Summary 1. Acetylcholine (ACh) elicited depolarization and an increase in input conductance of the somatic muscle cells of the parasitic nematode Ascaris suum. 2. The relative potency of nicotinic and muscarinic agents was studied in this preparation. The order of potency of these compounds was metahydroxyphenylpropyltrimethylammonium (HPPT)> 1,1 dimethyl-4-phenylpiperazinium (DMPP)> ACh> carbachol> nicotine> tetramethylammonium (TMA+)> muscarone> furtrethonium> arecoline. Decamethonium was also a weak agonist. McN-A-343 elicited a very weak depolarization at concentrations above 1 mmoll"1. Bethanechol and methacholine were without effect up to 1 mmoll"1. Pilocarpine and muscarine elicited a slight hyperpolarization of up to 3 mV with a threshold for the response of around SOOianoll"1. Oxotremorine (1 mmoll"1) was without effect. 3. The nitromethylene insecticide 2(nitromethylene)tetrahydro 1,3-thiazine (NMTHT), an agonist at insect nicotinic receptors, was without effect on Ascaris muscle cells up to lmmolP1. 4. Mecamylamine and benzoquinonium were the most potent antagonists of the acetylcholine response. The order of potency of the other antagonists was tetraphenylphosphonium (TPP) > quinacrine > pancuronium, curare > trimethaphan > atropine > chlorisondamine, decamethonium > hexamethonium > dihydro-/3-ery throidine. 5. The agonist profile of the Ascaris muscle cell ACh receptor clearly indicates that it is nicotinic. The potency of ganglionic and neuromuscular nicotinic receptor antagonists in Ascaris does not enable a further subclassification of this nicotinic receptor. The Ascaris nicotinic receptor seems to possess some of the pharmacological properties of each type of vertebrate nicotinic receptor. The pharmacology of the Ascaris nicotinic receptor is discussed in relation to that of nicotinic receptors in other invertebrate preparations and in vertebrate preparations.

Introduction The presence of acetylcholine (ACh) (Mellanby, 1955), the excitatory action of *To whom reprint requests should be addressed.

^sy words: Ascaris suum, cholinoceptors, nicotinic, nematode. 510 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER

ACh on the muscle cells (Del Castillo et al. 1963) and the presence of cholinesterase in the nervous system (Lee, 1962; Knowles and Casida, 1966) have led to the suggestion that ACh is the excitatory transmitter at the neuromuscular junction in Ascaris suum. The nematodes are probably the lowest phylum of the evolutionary tree to use ACh as a transmitter (see Venter et al. 1988, for a review). The receptor for ACh on Ascaris muscle cells is, therefore, of particular interest from a phylogenetic standpoint. It has also been demonstrated that the primary site of action of the anthelmintics pyrantel, morantel and levamisole is the Ascaris ACh receptor (Aubry et al. 1970; Harrow and Gration, 1985). Thus, an understanding of the relationships between structure and function at this receptor is important for the development of anthelmintics. Most of the early studies on the Ascaris ACh receptor have used a muscle strip preparation. The receptor has not been characterised by electrophysiological means. Previous studies on the pharmacology of this receptor using the muscle strip preparation have given a preliminary basis for its classification. The nicotinic agonists nicotine and dimethylphenylpiperazinium (DMPP) are good at eliciting muscle contraction, whereas muscarinic agonists are weak or ineffective (Baldwin and Moyle, 1949; Natoff, 1969; Rozhkova et al. 1980) and, on the basis of these studies, it has been proposed that the receptor in Ascaris is of the nicotinic type. Tubocurare is an antagonist, though of varying potency (Del Castillo et al. 1963; Baldwin and Moyle, 1949; Natoff, 1969; Rhozkova et al. 1980). The selective muscarinic antagonist atropine (Baldwin and Moyle, 1949; Natoff, 1969; Rozhkova et al. 1980) and the ganglion blocker hexamethonium are weak antagonists of the ACh-elicited contraction (Natoff, 1969; Rhozhkova et al. 1980). In this study we have used intracellular recordings of Ascaris muscle cells to look at the direct effect of various compounds and to provide a more complete picture of the pharmacology of the cholinoceptor in this preparation. We highlight some important differences between mammalian, insect and Ascaris nicotinic receptors.

Materials and methods Ascaris suum were obtained from a local abattoir on a weekly basis. They were transported to the laboratory in a flask containing artificial perienteric fluid (APF) the composition of which was (inmmolP1) NaCl, 67; sodium acetate, 67; KC1, 3; MgCl2, 15.7; CaCl2, 3; Tris base, 5; adjusted to pH 7.6 with glacial acetic acid. In the laboratory they were maintained in APF with SmmolP1 glucose at 37°C in a water bath. The worms remained healthy for up to 1 week, as indicated by their appearance and the resting membrane potentials (—30 mV) and input conductances (2-3 (tS) of the somatic muscle cells.

Preparation and electrophysiological techniques An anterior section (approximately 1 cm) of the worm was excised and slit along one lateral line. The section was pinned out, cuticle side down, in a Pharmacology of Ascaris cholinoceptors 511 perfusion chamber (volume 2 ml). The intestine was carefully removed using fine forceps, revealing the muscle bag cells underneath. The preparation was continu- ously perfused with APF at lOmlmin"1. The temperature in the bath was 32-33 °C. The muscle bag cells (diameter 50-200//m) were impaled with two glass microelectrodes (10-30 MQ), filled with 4 moll"1 potassium acetate and lOmmoir1 KC1 and connected to the headstage of an Axoclamp 2A (Axon Instruments) by Ag/AgCl wire. The reference electrode was a 3 moll"1 KCl/agar bridge. Membrane potential was routinely monitored with the Axoclamp in bridge mode and current pulses (10-40 nA, 500ms pulse width, 0.1 Hz) were passed through the second microelectrode using a Grass stimulator and Grass stimulus isolation unit. The true current being passed through the current electrode was constantly monitored and permanent records of both the current stimulus and the muscle cell membrane potential were obtained on a Gould two-channel chart recorder. Membrane potential was monitored on a Gould digital storage oscillo- scope. Smaller cells near the nerve cord were chosen for voltage-clamp experiments (approx. diameter 50 ,um). These experiments were performed with the Axoclamp in two-electrode voltage-clamp mode.

Drug application Drugs were applied directly to the area of the cell from which a recording was being made, via a fine-bore tube, at a rate of 10 ml min"1. Switching from APF to the drug (by moving the tubing from one beaker to the other) introduced a bubble into the tubing, which separated the two solutions. To minimise mechanical disturbance, the bubble was allowed to escape from the tubing via a small slit just prior to entry to the bath. Temperature was maintained at a constant 32-33°C during drug application by keeping the drug solution in the same water bath as the APF. In addition, temperature was monitored by a fine temperature probe placed near to the cell under study. This method of drug application enabled the concentration of drug around the cell to be changed rapidly and completely, allowing fast on and off switching of responses. Agonists were applied for up to 45 s, with the duration of application being consistent for each individual experiment. Antagonists were applied for 2min prior to the application of the agonist and concurrent with the application of the agonist.

Experiments in calcium-free artificial perienteric fluid Application of agonists to the muscle preparation causes muscle cell depolarization, which in many cases results in contraction and subsequent displacement of the recording electrode from the cell. To minimise this problem and to enable a full dose-response relationship for ACh to be studied, we investigated the action of higher concentrations of ACh in Ca2+-free APF in some experiments. (The composition of this was identical to that of normal APF except that CaCl2 was omitted and lmmoll"1 EGTAwas added.) The protocol of these experiments was as follows. The cell was impaled and the effect of ACh (1-10//moll"1) in the ^2+-containing APF was determined. The perfusing medium was then changed 512 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER to Ca2+-free APF. The cell was bathed in this medium for 15min, which was probably sufficient to deplete extracellular Ca2+ levels (as indicated by the inhibition of the Ca2+-mediated spontaneous activity; see Fig. 2). The dose- response relationship for 10~6-10~3moll~1 ACh was then determined. The relative potency of the agonist nicotine compared to ACh was also studied in Ca2+- free APF.

Analysis of results The potency of agonists was expressed relative to that of ACh in the following manner: the response, either conductance increase or depolarization, to increasing concentrations (1-30/anoH"1) of ACh was determined and plotted as a dose-response curve. This was repeated for the compound being assessed. Relative potency of the agonist compared to ACh was determined by taking the ratio of the concentration of ACh to the concentration of agonist that produced equivalent responses from approximately parallel portions of the dose-response curves. The sensitivity of the cells to ACh did not change appreciably during the course of the experiment, as tested by the application of ACh at the end of each experiment, though the depolarization sometimes diminished slightly. The potency of the antagonists was determined as an IC50 value. The IC50 value was the concentration of antagonist that reduced the response to a submaximal concentration of ACh (5-lO^moll"1) by 50 % and was determined using at least four concentrations of antagonist. In order to pool the data for agonists the results were normalized with respect to the response to 5//moll"1 ACh for each cell. All results are expressed as the mean±s.E.M. for N experiments.

Drugs Drugs were obtained from the following sources; acetylcholine bromide, nicotine, trimethylammonium, bethanechol, methacholine, pilocarpine, quinacrine, curare, atropine, hexamethonium, neostigmine and physostigmine from Sigma; DMPP, TPP and decamethonium from Aldrich; carbachol from Koch- Light; muscarone and muscarine from Ciba-Geigy; arecoline from BDH; McN-A- 343 from Research Biochemicals. We are grateful to the following for their generous gifts of compounds: Smith Kline & French for furtrethonium; Merck Sharp & Dohme for mecamylamine; Ciba-Geigy for chlorisondamine; Organon for pancuronium; Roche Products Ltd for trimethaphan; Dr M. Caulfield (University College London) for HPPT (metahydroxyphenylpropyltrimethylammonium); Bayer for benzoquinonium; and Merck & Co. for dihydro-^S-erythroidine.

Results Resting properties of the muscle cells The typical resting membrane potential of the somatic muscle cells was — 30 mV Pharmacology of Ascaris cholinoceptors 513

Experiments were only performed on those cells with resting input conductances of less than 3/zS and a resting membrane potential greater than — 25 mV, as this was taken to indicate that a reasonably healthy impalement of the cell had been achieved. Some cells, particularly those near the nerve cord, exhibited spontaneous activity, which took the form of either 'slow' waves or action potentials of an amplitude up to 40 mV. Acetylcholine depolarized the cells with a threshold of around 1 /xmoll"1. The response to ACh did not show any signs of desensitization at the concentrations used in these studies. Cells varied in the amount of rectification that they exhibited. Generally, the cells showed little rectification in the hyperpolarizing direction, particularly if the applied hyperpolarizing current was below 30 nA (Fig. 1A,B). However, current- voltage plots indicate rectification in the depolarizing direction in some cells and, as we have not been able routinely to clamp the membrane potential of these cells, it is important to bear this in mind when using the increase in input conductance as a measure of the activation of the ACh receptor. For example, in one cell ACh (10/zmol I"1) elicited a depolarization of 10 mV and an increase in conductance of 0.82 yS. When this cell was stepped by current injection alone to the same potential to which it depolarized in the presence of ACh, the input conductance increased by 0.27 ^S. Thus, 33 % of the increase in input conductance apparently elicited by ACh could have been secondary to the ACh-induced depolarization. For this reason, data on the effect of ACh on membrane potential have been included in this paper.

-LOr -10 r

/(nA)

+40 -40 30 > > B B

-60 -60

Fig. 1. Current-voltage plots for Ascaris suum muscle cells illustrating a cell that shows no rectification (A) and one that does (B). Depolarizing or hyperpolarizing current pulses of 500 ms pulse width were injected into the cell, which had a resting membrane potential indicated by the origin of each graph. 514 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER

Dose-response relationships for acetylcholine Typical responses to ACh and their dose-response relationships are shown in Fig. 2. ACh produced a dose-dependent and reproducible depolarization and increase in input conductance of the muscle cells. In Ca2+-free APF, the EC50 (the concentration that produced a half-maximal response) for ACh to elicit a depolarization was 10±2/xmoll (N=S; Fig. 2B). A value could not be obtained in Ca2+-containing APF as the recording was not stable when high concentrations

10 mV 10 n A

liiiiiiiiiiifiiiiii:

10 100 1000 [ACh] (/rniolP1) 5mV[ [ACh] (/mioi

Fig. 2. Dose-response relationships for acetylcholine (ACh). (A) An example of an intracellular recording from an Ascaris muscle cell showing the response to increasing concentrations of ACh. The first set of responses to 1-lOjanoir1 ACh were in the presence of Ca2+. The arrow indicates the switching of the perfusion medium to Ca2+- free artificial perienteric fluid (APF). The dose-response relationship between l^moll"1 and lmmolP1 ACh was then recorded. The downward deflections are electrotonic potentials caused by current injection (0.1 Hz, 500 ms pulse width). The magnitudes of the current pulses are indicated in the lower trace. The depolarizing potentials (indicated by the broken arrow) are the result of spontaneous activity in the muscle cell. Note that they decrease in magnitude and frequency and eventually disappear in Ca2+-free APF. The bars indicate the durations of applications of ACh. The resting membrane potential was — 25 mV. (B) The dose-response relationship for the depolarization caused by ACh in the presence (•) and absence (O) of Ca2+; 7V=8; values are mean±s.E.M. (C) The dose-response relationship for the ACh-elicited increase in input conductance in the presence (•) and absence (O) of Ca2+; N=8; values are mean±s.E.M. (D) An example of the inward current recorded from a muscle cell clamped at resting membrane potential (-30 mV) and perfused with ACh (concentrations as indicated) for 15 s. Pharmacology of Ascaris cholinoceptors 515 of ACh were applied in this medium. The EC50 for the ACh-elicited conductance increase could not be estimated either, as it continued to increase even at lmmoir1 ACh (Fig. 2C). At the lower concentrations of ACh, the dose- response curves in the presence and absence of Ca2+ were not significantly different (Fig. 2B,C). Voltage-clamp experiments indicated that ACh elicited an inward current of between 7 and 29 nA (Figs 2D, 11) when the cells were voltage-clamped at resting membrane potential.

The effects of agonists The relative potencies of the agonists studied are given in Table 1 and Figs 3 and 4. For all compounds except nicotine these were calculated in Ca2+-containing APF only. Nicotine mimicked the effect of ACh, though with a lower potency. The relative potency of nicotine compared to ACh was studied in the presence and absence of Ca2+. The values in the presence of Ca2+ are given in Table 1. The

Table 1. The relative potency of agonists for the Ascaris acetylcholine receptor compared to acetylcholine Relative potency

Agonist Depolarization Conductance HPPT 6.5 9 DMPP 3 2 Acetylcholine 1 1 Carbachol 0.4 0.5 Nicotine 0.2 0.3 TMA+ 0.04 0.05 Muscarone 0.01 0.006 Furtrethonium 0.007 0.007 Arecoline 0.001 0.002 NMTHT No effect McN-A-343 Weak depolarization No effect Bethanechol No effect Methacholine No effect Pilocarpine Weak hyperpolarization (5 out of 7 cells) Muscarine Weak hyperpolarization Oxotremorine Without effect

The relative potency was determined for each cell by taking the ratio of the concentration of ACh to the concentration of agonist that produced the same response from parallel portions of the dose-response curve (therefore a relative potency greater than 1 indicates a compound more potent than ACh). This was done both for the depolarizing response to ACh and for the conductance increase elicited by ACh; N=3-6. HPPT, metahydroxyphenylpropyltrimethylammonium; DMPP, l,l-dimethyl-4-phenylpiperazinium; TMA+, tetramethylammonium; NMTHT, 2(nitromethylene)tetrahydro 1,3,- [hiazine. 516 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER values in the absence of Ca2+ were 0.08 for the depolarization and 0.008 for the conductance increase. Both these values are lower than the relative potency for nicotine obtained in the presence of Ca2+. In all seven cells studied (four in the presence and three in the absence of Ca2+) it was found that the response to ACh was reduced after the cell had been exposed to nicotine (Fig. 5).

10 100 1000 [Agonist] (/imolP1)

Fig. 3. The relative potencies of nicotinic and muscarinic agonists at the Ascaris muscle cell ACh receptor compared to the potency of ACh. ACh O; carbachol •; nicotine •; TMA+ ; muscarone A; furtrethonium •; arecoline +. The responses were normalized with respect to the response to S^moll"1 ACh. iV>3; vertical bars indicate ±S.E.M.

Fig. 4. The most potent agonists at the Ascaris receptor were nicotinic ganglionic agonists. Responses are normalized with respect to the response to SJOTIOIF1 ACh. HPPT •; DMPP •; ACh O. 7V=4-5; vertical bars indicate ±S.E.M. Pharmacology of Ascaris cholinoceptors 517

10/anoir' ACh 1 mmol T1 nicotine O1 Ch

Fig. 5. Evidence to indicate that nicotine desensitizes the Ascaris muscle cell response to ACh. The artificial perienteric fluid for this experiment was Ca2+-free. These are consecutive recordings from an Ascaris muscle cell. The downward deflections are caused by current injection (0.1 Hz, 500 ms, the amplitude of the current pulse is shown in the lower trace). The resting membrane potential was —25 mV. The bars indicate the durations of applications of drug at the concentration indicated on the figure. The depolarization and the increase in input conductance in response to ACh were reduced after the cell had been exposed to nicotine. The same effect was seen in six other cells.

For the other compounds studied, the relative potencies determined from the depolarization and from the conductance increase are comparable (Table 1). HPPT and DMPP were the only compounds that were more potent than ACh (Fig. 4). TMA+ was less potent than ACh. The duration of the response to carbachol was greater than that to ACh (Fig. 6B). Muscarone was 100 times less potent than ACh. Arecoline and furtrethonium both elicited depolarizations and conductance increases with about one-thousandth of the potency of ACh. McN-A- 343 was virtually inactive, requiring a concentration of greater than 1 mmol I"1 to elicit a depolarization of about 2 mV. Bethanechol and methacholine were without effect. Pilocarpine and muscarine caused a slight (up to 3mV) hyperpolarization with a threshold for the response of SOOjanoir1. The muscarinic agonist oxotremorine (1 mmol I"1) had no effect on two cells. NMTHT was without effect at concentrations up to lmmolP1 on three cells. The effects of two anticholinesterases were investigated. Neither physostigmine nor neostigmine had any significant effect on resting membrane potential or input conductance when applied on their own at concentrations up to lO/imoll"1. Physostigmine had no effect on the response to ACh at low concentrations; however, at concentrations greater than lO^moll"1 it blocked the response to ACh in a reversible manner (N=3). Neostigmine potentiated the response to ACh at concentrations of l-lO/anoll"1 (Fig. 6A,C).

The effects of antagonists The potencies of the antagonists at blocking the depolarization and the conductance increase in response to ACh are given in Table 2. For most compounds these are comparable; however, for both atropine and chlorisondamine the IC50 values calculated from the conductance change are lower than the IC50 values calculated from the block of the depolarization. For example, the IC50 IJetermined for atropine from the conductance response was 6.7±2.1^moll~1 518 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER

[ACh](/rnioir')

10 /anol 1 carbachol

C Control 20 mv 20s 1/anoll ' neostigmine

10/imoll ' neostigmine f

Fig. 6. Evidence for the presence of cholinesterase in the Ascaris muscle strip preparation. (A) The potentiation of the response to ACh by neostigmine (N=3; values are mean±s.E.M.). Control conductance increase in response to ACh O; conductance increase in the presence of lO^mol I"1 neostigmine •. (B) An example of the response to ACh and carbachol. (C) An example of the potentiation of the response to ACh by neostigmine (1 and lO^moll"1) in a muscle cell. The resting membrane potential of this cell was -30 mV. The downward deflections are caused by the injection of current (0.1 Hz, 500 ms pulse width, 32 nA). The bars in C indicate the application of lO^imoll"1 ACh. Pharmacology o/Ascaris cholinoceptors 519

Table 2. Antagonists at the Ascaris acetylcholine receptor ICso^oir1)

Drug Conductance Current Depolarization Benzoquinonium 0.29±0.07 0.46±0.04 Mecamylamine 0.33±0.(M 0.41±0.04 Chlorisondamine 1.74±0.33 65±33 TPP - 1.90±0.30 - Quinacrine - 2.05±0.60 - Curare 3.10±0.40 5.26±1.14 Pancuronium 3.20±0.30 4.49±1.80 Trimethaphan 4.27±1.44 17±10 Atropine 6.70±2.10 52±4 Decamethonium 14±3 96 Hexamethonium 43 ±6 329 Dihydro-/J-erythroidine >100 >100

The ICJO is the concentration of antagonist that was required to reduce the response to ACh by 50 %. Values are the mean±s.E.M. for 3-4 experiments. TPP, tetraphenylphosphonium.

ino-i

50-

10 30 50 100 [Atropine] (/(moll"1)

Fig. 7. The blockade by atropine of the depolarization (O) and the conductance (•) increases in response to ACh. The control response was the response to lO^moll"1 ACh. JV=4; values are mean±s.E.M. Significant difference was determined using the paired Student's f-test; * P<0.05.

(N=A, determined by extrapolation) compared to 5214/zmoll"1 (N=4) determined from the ability to block the depolarization (Fig. 7). Pancuronium and curare antagonised the ACh response. The antagonism by 520 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER

Aiii

10 mV L 10 s

Fig. 8. The blockade of the ACh response by curare and mecamylamine. Examples are from a single cell for each case. (A) Curare. The resting membrane potential was —26mV. The downward deflections are caused by current injection (10-30 nA; routinely, current injection was 38 nA). The bars indicate the duration of application of lOfimoll"1 ACh. (i) Control response to lO^moll"1 ACh; (ii) response to ACh after the cell had been exposed to 10 /itnol I"1 curare for 2 min; (iii) response to ACh after a 5min wash. (B) Mecamylamine. The resting membrane potential was - 30mV. The downward deflections are caused by the injection of current (0.1 Hz, 500 ms pulse width, 10nA). The bars indicate applications of ACh. (i) Control response to 10/anoll~L ACh; (ii) response to lO^moll"1 ACh after the cell had been exposed to O.S^moir1 mecamylamine for 2 min; (iii) response to lO^moll"1 ACh after a 5 min wash.

curare was readily reversible, with 100% recovery in 5-15min (Fig. 8A). The block by atropine and pancuronium lasted longer, requiring a wash of 20 min for a recovery of greater than 50 %. Mecamylamine was a potent antagonist of the ACh response (Table 2; Fig. 8B). The block of the ACh response was reversed slowly. After a 5-15 min wash it reversed by about 30 % in six cells (Fig. 8B). The block reversed completely in one cell washed for longer than 30min. The other ganglion blockers tested in this study, trimethaphan and chlorisondamine, also antagonised the ACh response (Table 2), though less potently than mecamylamine. The relative potencies of hexamethonium and decamethonium as blockers of the ACh response were studied in three cells. Decamethonium was found to be more potent than hexamethonium (Fig. 9; Table 2). It also had a direct, depolarizing effect on membrane potential (N=3, Fig. 10). Quinacrine blocked the inward current elicited by ACh with an IC50 of Pharmacology of Ascaris cholinoceptors 521

10 100 1000 [Antagonist] (/rnioll"1)

Fig. 9. The inhibition of the ACh response by hexamethonium (O) and decamethonium (•) in the same cells. The control response was the response to lO/imoll"1 ACh in the absence of antagonist; N=3. Vertical bars show ±S.E.M.

10 mV

30s

100 /imoll decamethonium

30/nnoll decamethonium 300^moll decamethonium Wash

Fig. 10. The direct effects of decamethonium on an Ascaris muscle cell. The bars indicate applications of lO^moll"1 ACh. Resting membrane potential was — 30 mV throughout. Arrows indicate the addition of decamethonium to the perfusate. The downward deflections are caused by the injection of current (0.1 Ffz, 500 ms pulse width, 20nA).

2.05±0.60/imoir1 (N=3, Fig. 11). TPP also blocked the current elicited by ACh with an IC*, of 1.9±0.3/xmoir1 (N=3; Fig. 12). Dihydro-/J-erythroidine (lmmoll"1) did not block the action of ACh on the muscle cells. However, the other neuromuscular blocking agent, benzoquinonium, produced a potent, reversible block of the ACh response (N=3; Table 2).

Discussion Responses to acetylcholine These results confirm the excitatory nature of ACh on Ascaris muscle cells. It is 522 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER

[Quinacnne] 1 (//moir ) 0.1 0.3 0.5 10 30 Wash

3nA

1 min

Fig. 11. Currents elicited by perfusion of 5 //rnol I"1 ACh, in a cell voltage-clamped at resting membrane potential (—30 mV), were blocked by quinacrine (concentration as indicated). The 30 mV calibration refers to the top trace, which is the voltage recording from the cell. Quinacrine was applied for 2min prior to and concurrent with the ACh application. The wash period was 5 min. The block of the response to ACh was partially reversed following the wash period.

100 n

50-

0.01 0 1 1 10 100 |TPP] (//moir1)

Fig. 12. The current elicited by ACh was blocked by tetraphenylphosphonium (TPP). The control response was the current elicited by 5 jumol I"1 ACh when the cells were clamped at resting membrane potential (approx. — 30mV). The same holding potential was used throughout for each individual experiment. The percentage of the response remaining as the cell was exposed to increasing concentrations of TPP was determined and this is plotted against the TPP concentration. N=4; values are mean±s.E.M. suspected that ACh is the excitatory transmitter at the Ascaris neuromuscular junction. However, it should be noted that the responses to bath-applied ACh in the preparation described in this paper will involve both the physiologically important synaptic ACh receptors and the extrasynaptic ACh receptors on the muscle bag cells. Pharmacological differences may exist between the synaptic and extrasynaptic ACh receptors. The EC50 for ACh at this receptor could not be determined exactly in all cells Pharmacology of Ascaris cholinoceptors 523 because the recordings made after application of concentrations of ACh capable of eliciting a maximal response in the presence of Ca2+ were unstable. However, it can be estimated to be in the low micromolar range. From eight studies in the absence of Ca2+, the EC50 for the depolarization was determined to be lO/zmoll"1. The effect of ACh on the cell input conductance was not maximal 1 even at 1 mmoll" . Harrow and Gration (1985) estimated that the EC50of ACh for eliciting a conductance increase was 110//moll"1. This may indicate that there are a large number of 'spare' receptors for ACh in this preparation, i.e. the maximal depolarization occurs at a concentration of ACh at which all the available receptors have not been activated. The methodological problems of measuring the conductance increase in a cell that is not voltage-clamped were dealt with in the Materials and methods section. We measured both depolarization and conductance increase in response to ACh application. We have not reported here on the ionic mechanism of this response. However, ACh elicits an inward current in voltage-clamped cells (Figs 2D, 11) and we have also found that the response is decreased when Na+ is partly replaced by gJucosamine in the external medium (L. Colquhoun, L. Holden-Dye and R. J. Walker, unpublished observations). Harrow and Gration (1985) have estimated that the reversal potential for the ACh current is about +10mV, which is consistent with the receptor gating a cation channel through which Na+ is probably the major charge carrier. Therefore, by analogy with other nicotinic receptors, the Ascaris receptor seems to be a ligand-gated cation channel. The nicotinic nature of this receptor has been confirmed by the effects of agonists, as described below.

Cholinesterase in Ascaris In this study the magnitude and duration of the ACh response were increased by the anticholinesterase neostigmine, but not by physostigmine. Physostigmine (lOO/anoll"1) inhibited the maximum response to ACh by more than 50%. The different effects of these two anticholinesterases may be explained by their differential affinity for the cholinesterase binding site and the nicotinic receptor. It has been shown in other preparations that these anticholinesterases can have a direct action at the ACh receptor site (Slater et at. 1986), and in Aplysia neurones the blockade of the receptor by physostigmine can occur at quite low concentrations (Oyama etal. 1989). It would seem, therefore, that the duration of action of ACh in the Ascaris muscle preparation is regulated by the presence of a cholinesterase susceptible to blockade by neostigmine. The precise mechanism of action of physotigmine in this tissue will require further study. It is likely that it acts both as a cholinesterase inhibitor and as a receptor antagonist. The physiological relevance of the cholinesterase in terminating the action of synaptic ACh will have to be elucidated by looking at the possible effect of neostigmine on the excitatory junction potentials.

The effects of agonists We did not routinely employ a Ca2+-free medium to study the relative potencies 524 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER of agonists. The potency of nicotine on the Ascaris muscle preparation was apparently lower in the absence of Ca2+. The significance of this has not been investigated. The kinetics of the nicotine response also seemed to be much slower than that for ACh (Fig. 5). Nicotine was the only agonist that seemed to elicit a form of desensitization of the Ascaris muscle, in that the response to ACh was reduced after exposure of the cell to nicotine (Fig. 5). Therefore, the action of nicotine at the ACh receptor seems to possess some interesting features that would be best investigated at the single-channel level using the patch-clamp technique. The most effective cholinomimetic agents in Ascaris muscle were nicotinic agonists (Table 1). However, nicotine itself was not as potent as might be expected. A similar low potency of nicotine, at an otherwise 'nicotinic' receptor, in an invertebrate preparation has been shown in Manduca sexta (Trimmer and Weeks, 1989). In the central nervous system of Manduca the potency of nicotine is low, with an ECso of around 20/imoll~1. This may be due to the presence in Manduca of a nicotine-resistant form of the receptor. Muscarone, a compound with slight nicotinic activity, was a very weak agonist. The muscarinic agonists bethanechol and methanechol were completely inactive. This agrees well with past results obtained from the muscle strip preparation showing that the nicotinic ganglionic agonist DMPP was potent at eliciting Ascaris muscle strip contraction (Natoff, 1969; Rozhkova et al. 1980; Hayashi etal. 1980). In our preparation too, DMPP was one of the most potent agonists. This would suggest that the Ascaris nicotinic receptor is like the nicotinic receptor at mammalian autonomic ganglia. However, it should also be noted that we found that decamethonium had a slight agonist action on Ascaris muscle (Fig. 10). Decamethonium is not an agonist at mammalian ganglia (Paton and Perry, 1953; Ascher et al. 1979; Gurney and Rang, 1984), though it has been shown to be an agonist at frog ganglia (Lipscombe and Rang, 1988). Also, the slight stimulatory action of furtrethonium was surprising, as this compound is generally regarded as a muscarinic agonist. Thus, even from an agonist profile, the Ascaris ACh receptor does not readily fit into a mammalian classification scheme. HPPT is one of a series of compounds synthesized by Barlow and Thompson (1969) and shown to be 50 times more potent than nicotine as an agonist in the frog rectus muscle. In rat superior cervical ganglion it is 15 times more potent than DMPP (Caulfield et al. 1990). In our preparation this compound was the most potent cholinomimetic tested, being at least twice as potent as DMPP. The observation that pilocarpine and muscarine produced a weak hyperpolarization is interesting, particularly in view of the fact that muscarinic receptors, of lower sensitivity than the nicotinic ACh receptors, are known to coexist with nicotinic receptors on other invertebrate cell types (Woodruff et al. 1971; Benson, 1988). We have not pursued this possibility further in this study; however, the potent muscarinic agonist, oxotremorine, was without effect on Ascaris muscle cells. The nitromethylene insecticide NMTHT had no effect on the Ascaris muscle cells. It has been shown to be cholinomimetic on cultured cockroach neurone^ Pharmacology of Ascaris cholinoceptors 525 where it opens channels with the characteristics of nicotinic receptor-gated channels at a low micromolar concentration (Buckingham et al. 1989).

The effects of antagonists None of the antagonists that blocked the response to ACh was very potent. Of the ganglion blockers studied, mecamylamine was the most potent compound and both chlorisondamine and trimethaphan were relatively weak antagonists. Although mecamylamine is widely regarded as a ganghon blocker (see Martin et al. 1989, for a review) at the concentrations that were effective in Ascaris, it has also been shown to block transmission at the frog neuromuscular junction in a manner characteristic of open-channel block (Varanda et al. 1985). Mecamylamine is one of the most potent antagonists of the nicotinic ACh response on the cockroach Df motoneurone and acts in a voltage-independent manner (David and Sattelle, 1984). It has also recently been shown to be a potent antagonist of the ACh response in Xenopus oocytes expressing the a-A and non-anicotinic receptor subunits cloned from rat brain (Bertrand et al. 1990). Therefore, it can be seen that our results with mecamylamine do not help us to classify the Ascaris muscle nicotinic receptor as a neuromuscular junction type, ganglionic type or brain type of nicotinic receptor. In future experiments it would be interesting to test the voltage-dependency of mecamylamine action at the Ascaris ACh receptor, as it would seem that a voltage-dependent action may be typical of the nicotinic neuromuscular receptor. Two of the antagonists, chlorisondamine and atropine, show a marked discrepancy in their ability to block the conductance increase and the depolarization elicited by ACh. Both compounds block the conductance increase more potently than the depolarization (Table 2; Fig. 7). It has been shown that 50 /anol I"1 atropine is required to block the ACh-stimulated contraction in Ascaris muscle strip (Baldwin and Moyle, 1949; Rozhkova et al. 1980; Natoff, 1969; Onuaguluchi, 1989; Hayashi et al. 1980). The relatively weak antagonism of the contractile response and the muscle cell depolarization by atropine is compatible with the receptor being of a nicotinic rather than a muscarinic subtype. However, the receptor shows little discrimination between curare and atropine, with curare blocking the ACh-induced depolarization with an IC50 of about 5jm\o\\~l. A similar lack of discrimination by a nicotinic receptor between these two compounds has also been noted in leech neuropile glial cells (Ballanyani and Schlue, 1989) and cockroach Df motoneurone (David and Sattelle, 1984). The nicotinic receptor in Ascaris also fails to distinguish between the neuromuscular blocking agents curare and pancuronium. Pancuronium is a steroidal non-depolarising antagonist at the vertebrate neuromuscular junction of 10-fold greater potency than curare (Buckett et al. 1968), but it has the same potency as curare at the Ascaris neuromuscular junction. Of the other neuromuscular junction blockers tested, benzoquinonium and dihydro-/J-erythroidine, only benzoquinonium was a potent antagonist. Dihydro- ^erythroidine had no discernible effect even at 100 //mol 1~l. The lack of action of 526 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER dihydro-^-erythroidine is of interest as it is a nondepolarising neuromuscular junction blocker, similar in mechanism of action to curare. However, unlike curare and other neuromuscular junction blockers, it is a tertiary rather than a quaternary compound. Tetraphenylphosphonium (TPP) was tested against the ACh response in Ascaris because of its known potency in blocking nicotinic gated cation channels (reviewed in Heidmann et al. 1983; Spivak & Alburquerque, 1985). Trimethyl- phenylphosphonium, belonging to the same group of compounds as TPP, binds to the extracellular beginning of the M2 transmembrane domain of the Torpedo nicotinic receptor (Hucho et al. 1986). In Ascaris muscle cells voltage-clamped at the resting membrane potential, TPP blocked the inward current elicited by ACh in a dose-dependent manner (Fig. 12). Thus, the Ascaris nicotinic receptor channel may have similar recognition sites to those that have been extensively characterised on the Torpedo receptor at the molecular level. Quinacrine (mepacrine) is another noncompetitive inhibitor of nicotinic receptors, although it is also known to have an action at the ACh recognition site (Tsai et al. 1979). This compound was a potent inhibitor of the ACh response in Ascaris. As well as having a clinical use as an antimalarial, this drug is also used as a vermifuge, mainly for tapeworm infections. In this context, its potent blockade of the Ascaris nicotinic receptor is of some interest. The bisquaternary methonium compounds hexamethonium and decamethonium were both weak antagonists of the ACh response in Ascaris. However, decamethonium was more potent than hexamethonium (Fig. 9). At mammalian ganglia, hexamethonium blocks transmission by entering the nicotinic channel (Gurney and Rang, 1984), whereas hexamethonium has little effect on transmission at the vertebrate neuromuscular junction (Milne and Byrne, 1981). Thus, the low potency of hexamethonium implies that the nicotinic channel in Ascaris lacks a recognition site that is present in the channel of mammalian ganglionic nicotinic receptors. As mentioned previously, the weak agonist action of decamethonium in Ascaris is similar qualitatively, if not quantitiatively, to its action at the neuromuscular junction.

Comparison of Ascaris nicotinic receptors with those in other invertebrate preparations Little is known of the pharmacology of the nicotinic receptors in other nematodes. Radioligand binding studies in the free-living nematode Caenor- habditis elegans identified a binding site with a high affinity for the levamisole group of compounds. This site also had a micromolar affinity for DMPP (Lewis et al. 1987). In one species where electrophysiological studies on the pharmacology of the muscle cell receptors have been performed, Dipetalonema viteae, the ACh response seems to be very different from that in Ascaris: both arecoline and pilocarpine were cholinomimetic (Rohrer et al. 1988) and the response was not blocked by curare (10/xmol I"1). It did resemble the Ascaris receptor, however, j^ Pharmacology of Ascaris cholinoceptors 527 that the response was not blocked by hexamethonium. In the trematode Schistosoma mansoni there is believed to be a cholinoceptor with similar pharmacology to that of the receptors in autonomic ganglia (Barker et al. 1966). Nicotinic receptors are present on leech muscle (Walker et al. 1970), neurones (Woodruff et al. 1971) and glial cells (Ballanyi & Schlue, 1989). All three types of receptor are insensitive to hexamethonium, as in Ascaris. Decamethonium had a partial agonist action at the receptor on leech glia, but no direct action on leech muscle. Thus, the receptor on the glia may most closely resemble the Ascaris receptor in terms of recognition of the bisquaternary methonium compounds. The nicotinic receptor in Ascaris differs from those of certain insect preparations in a number of ways. The Df motoneurone of the cockroach is insensitive to DMPP (David and Sattelle, 1984), whereas it was the most potent agonist in Ascaris. A further difference is indicated by the lack of sensitivity of the Ascaris ACh receptor to dihydro-/J-erythroidine, whereas this is one of the most potent 125 antagonists on the Df neurone and is also a potent displacer of [ I]-o-- bungarotoxin binding to locust ganglia (Macallan et al. 1988). Levamisole, pyrantel and morantel (Harrow and Gration, 1985) are more potent than ACh at the Ascaris ACh receptor but are only weakly effective on the Df motoneurone (Pinnock et al. 1988). However, the receptors in insects and Ascaris share a common sensitivity to mecamylamine and benzoquinonium, though, as mentioned earlier, neither of these antagonists was very potent on either preparation. Decamethonium is an agonist in Ascaris, whereas it is a weak antagonist in the Df motoneurone (David and Sattelle, 1984). Both preparations are insensitive to hexamethonium. Dorsal unpaired median (DUM) cells that respond to ACh with a depolarization have been identified in the metathoracic ganglia of grasshoppers (Goodman & Spitzer, 1979). The depolarization was blocked by curare and by high (millimolar) concentrations of hexamethonium. The nicotinic receptor at an identified synapse in Manduca sexta has some of the characteristics of the Ascaris nicotinic receptor (Trimmer and Weeks, 1989), as decamethonium was an agonist and hexamethonium did not block the ACh response. Mecamylamine and curare were equipotent, though not very good, antagonists. ACh receptors have also been described in molluscs. Three types of response to ACh have been studied in Aplysia (Kehoe, 1972). The excitatory response is blocked by 50-100 ^mol I"1 curare (Kehoe, 1972; Ascher et al. 1978) and 10-100^moll"1 hexamethonium. The studies on these receptors have concen- trated on the mechanism of action of antagonists rather than the pharmacological profile of the receptor. It is interesting, however, that the receptor mediating the excitatory response to ACh in Aplysia is blocked by hexamethonium with moderate potency, as is the excitatory response to ACh in Limulus polyphemus (Walker and Roberts, 1982). Yavari et al. (1979) estimated pA2 values for compounds that block the inhibitory and excitatory response to ACh in Helix aspersa. The excitatory response was weakly antagonised by hexamethonium, whereas the inhibitory response was completely unaffected. Curare was a weak .antagonist of both responses. 528 L. COLQUHOUN, L. HOLDEN-DYE AND R. J. WALKER

In conclusion, common features of the nicotinic receptors in invertebrates would seem to be a poor recognition of the potent ganglion blocker hexamethonium and an inability to discriminate well between curare and atropine. There also seem to be marked interspecies differences. In this study we have characterised the somatic muscle cell ACh receptor in Ascaris. As with the receptors in other invertebrates, it cannot readily be classified according to mammalian nomenclature. The true identity of these receptors, and their relationship to those in higher groups such as mammals, will not be elucidated until the nicotinic receptor subunits for these species have been cloned and sequenced. The information from the molecular biological approach, together with the pharmacological data outlined above, will enable the relationships between structure and function to be determined for the invertebrate nicotinic receptors.

We gratefully acknowledge financial support from the SERC and Jersey Government. We are indebted to Chris Willis for a regular supply of healthy Ascaris.

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