THE PHARMACOLOGY OF CHOLINOCEPTORS ON THE SOMATIC … · (DMPP)> ACh> carbachol> nicotine>...

/. exp. Bwl. 158, 509-530 (1991) 5 0 9Printed in Great Britain © The Company of Biologists Limited 1991

THE PHARMACOLOGY OF CHOLINOCEPTORS ON THESOMATIC MUSCLE CELLS OF THE PARASITIC NEMATODE

ASCARIS SUUM

BY L. COLQUHOUN, L. HOLDEN-DYE* AND R. J. WALKERDepartment 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 inputconductance of the somatic muscle cells of the parasitic nematode Ascaris suum.

2. The relative potency of nicotinic and muscarinic agents was studied in thispreparation. The order of potency of these compounds was metahydroxy-phenylpropyltrimethylammonium (HPPT)> 1,1 dimethyl-4-phenylpiperazinium(DMPP)> ACh> carbachol> nicotine> tetramethylammonium (TMA+)> mus-carone> furtrethonium> arecoline. Decamethonium was also a weak agonist.McN-A-343 elicited a very weak depolarization at concentrations above1 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 athreshold for the response of around SOOianoll"1. Oxotremorine (1 mmoll"1) waswithout effect.

3. The nitromethylene insecticide 2(nitromethylene)tetrahydro 1,3-thiazine(NMTHT), an agonist at insect nicotinic receptors, was without effect on Ascarismuscle cells up to lmmolP 1 .

4. Mecamylamine and benzoquinonium were the most potent antagonists of theacetylcholine response. The order of potency of the other antagonists wastetraphenylphosphonium (TPP) > quinacrine > pancuronium, curare > trimetha-phan > atropine > chlorisondamine, decamethonium > hexamethonium >dihydro-/3-ery throidine.

5. The agonist profile of the Ascaris muscle cell ACh receptor clearly indicatesthat it is nicotinic. The potency of ganglionic and neuromuscular nicotinic receptorantagonists in Ascaris does not enable a further subclassification of this nicotinicreceptor. The Ascaris nicotinic receptor seems to possess some of the pharmaco-logical properties of each type of vertebrate nicotinic receptor. The pharmacologyof the Ascaris nicotinic receptor is discussed in relation to that of nicotinicreceptors in other invertebrate preparations and in vertebrate preparations.

IntroductionThe 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. H O L D E N - D Y E AND R. J. WALKER

ACh on the muscle cells (Del Castillo et al. 1963) and the presence ofcholinesterase in the nervous system (Lee, 1962; Knowles and Casida, 1966) haveled to the suggestion that ACh is the excitatory transmitter at the neuromuscularjunction in Ascaris suum.

The nematodes are probably the lowest phylum of the evolutionary tree to useACh as a transmitter (see Venter et al. 1988, for a review). The receptor for AChon Ascaris muscle cells is, therefore, of particular interest from a phylogeneticstandpoint. It has also been demonstrated that the primary site of action of theanthelmintics pyrantel, morantel and levamisole is the Ascaris ACh receptor(Aubry et al. 1970; Harrow and Gration, 1985). Thus, an understanding of therelationships between structure and function at this receptor is important for thedevelopment of anthelmintics.

Most of the early studies on the Ascaris ACh receptor have used a muscle strippreparation. The receptor has not been characterised by electrophysiologicalmeans. Previous studies on the pharmacology of this receptor using the musclestrip preparation have given a preliminary basis for its classification. The nicotinicagonists nicotine and dimethylphenylpiperazinium (DMPP) are good at elicitingmuscle contraction, whereas muscarinic agonists are weak or ineffective (Baldwinand Moyle, 1949; Natoff, 1969; Rozhkova et al. 1980) and, on the basis of thesestudies, 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 selectivemuscarinic antagonist atropine (Baldwin and Moyle, 1949; Natoff, 1969;Rozhkova et al. 1980) and the ganglion blocker hexamethonium are weakantagonists of the ACh-elicited contraction (Natoff, 1969; Rhozhkova et al. 1980).

In this study we have used intracellular recordings of Ascaris muscle cells to lookat the direct effect of various compounds and to provide a more complete pictureof the pharmacology of the cholinoceptor in this preparation. We highlight someimportant differences between mammalian, insect and Ascaris nicotinic receptors.

Materials and methods

Ascaris suum were obtained from a local abattoir on a weekly basis. They weretransported 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. Inthe laboratory they were maintained in APF with SmmolP1 glucose at 37°C in awater bath. The worms remained healthy for up to 1 week, as indicated by theirappearance and the resting membrane potentials (—30 mV) and input conduc-tances (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 alongone 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 fineforceps, revealing the muscle bag cells underneath. The preparation was continu-ously perfused with APF at lOmlmin"1. The temperature in the bath was32-33 °C. The muscle bag cells (diameter 50-200//m) were impaled with two glassmicroelectrodes (10-30 MQ), filled with 4 moll"1 potassium acetate andlOmmoir1 KC1 and connected to the headstage of an Axoclamp 2A (AxonInstruments) by Ag/AgCl wire. The reference electrode was a 3 moll"1 KCl/agarbridge. Membrane potential was routinely monitored with the Axoclamp in bridgemode and current pulses (10-40 nA, 500ms pulse width, 0.1 Hz) were passedthrough the second microelectrode using a Grass stimulator and Grass stimulusisolation unit. The true current being passed through the current electrode wasconstantly monitored and permanent records of both the current stimulus and themuscle cell membrane potential were obtained on a Gould two-channel chartrecorder. Membrane potential was monitored on a Gould digital storage oscillo-scope. Smaller cells near the nerve cord were chosen for voltage-clamp exper-iments (approx. diameter 50 ,um). These experiments were performed with theAxoclamp in two-electrode voltage-clamp mode.

Drug application

Drugs were applied directly to the area of the cell from which a recording wasbeing made, via a fine-bore tube, at a rate of 10 ml min"1. Switching from APF tothe drug (by moving the tubing from one beaker to the other) introduced a bubbleinto the tubing, which separated the two solutions. To minimise mechanicaldisturbance, the bubble was allowed to escape from the tubing via a small slit justprior to entry to the bath. Temperature was maintained at a constant 32-33°Cduring drug application by keeping the drug solution in the same water bath as theAPF. In addition, temperature was monitored by a fine temperature probe placednear to the cell under study. This method of drug application enabled theconcentration of drug around the cell to be changed rapidly and completely,allowing fast on and off switching of responses. Agonists were applied for up to45 s, with the duration of application being consistent for each individualexperiment. Antagonists were applied for 2min prior to the application of theagonist 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 depolariz-ation, which in many cases results in contraction and subsequent displacement ofthe recording electrode from the cell. To minimise this problem and to enable afull dose-response relationship for ACh to be studied, we investigated the actionof higher concentrations of ACh in Ca2+-free APF in some experiments. (Thecomposition of this was identical to that of normal APF except that CaCl2 wasomitted and lmmoll"1 EGTAwas added.) The protocol of these experiments wasas 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


to Ca2+-free APF. The cell was bathed in this medium for 15min, which wasprobably sufficient to deplete extracellular Ca2+ levels (as indicated by theinhibition of the Ca2+-mediated spontaneous activity; see Fig. 2). The dose-response relationship for 10~6-10~3moll~1 ACh was then determined. Therelative 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 followingmanner: the response, either conductance increase or depolarization, to increas-ing concentrations (1-30/anoH"1) of ACh was determined and plotted as adose-response curve. This was repeated for the compound being assessed.Relative potency of the agonist compared to ACh was determined by taking theratio of the concentration of ACh to the concentration of agonist that producedequivalent responses from approximately parallel portions of the dose-responsecurves. The sensitivity of the cells to ACh did not change appreciably during thecourse of the experiment, as tested by the application of ACh at the end of eachexperiment, though the depolarization sometimes diminished slightly.

The potency of the antagonists was determined as an IC50 value. The IC50 valuewas the concentration of antagonist that reduced the response to a submaximalconcentration of ACh (5-lO^moll"1) by 50 % and was determined using at leastfour concentrations of antagonist.

In order to pool the data for agonists the results were normalized with respect tothe response to 5//moll"1 ACh for each cell. All results are expressed as themean±s.E.M. for N experiments.

Drugs

Drugs were obtained from the following sources; acetylcholine bromide,nicotine, trimethylammonium, bethanechol, methacholine, pilocarpine, quina-crine, curare, atropine, hexamethonium, neostigmine and physostigmine fromSigma; 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 theirgenerous gifts of compounds: Smith Kline & French for furtrethonium; MerckSharp & Dohme for mecamylamine; Ciba-Geigy for chlorisondamine; Organonfor pancuronium; Roche Products Ltd for trimethaphan; Dr M. Caulfield(University College London) for HPPT (metahydroxyphenylpropyltrimethylam-monium); Bayer for benzoquinonium; and Merck & Co. for dihydro-^S-erythroi-dine.

Results

Resting properties of the muscle cells

The typical resting membrane potential of the somatic muscle cells was — 30 mV


Experiments were only performed on those cells with resting input conductancesof less than 3/zS and a resting membrane potential greater than — 25 mV, as thiswas taken to indicate that a reasonably healthy impalement of the cell had beenachieved. Some cells, particularly those near the nerve cord, exhibited spon-taneous activity, which took the form of either 'slow' waves or action potentials ofan amplitude up to 40 mV. Acetylcholine depolarized the cells with a threshold ofaround 1 /xmoll"1. The response to ACh did not show any signs of desensitizationat the concentrations used in these studies.

Cells varied in the amount of rectification that they exhibited. Generally, thecells showed little rectification in the hyperpolarizing direction, particularly if theapplied 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 asa 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 of0.82 yS. When this cell was stepped by current injection alone to the samepotential to which it depolarized in the presence of ACh, the input conductanceincreased by 0.27 ^S. Thus, 33 % of the increase in input conductance apparentlyelicited by ACh could have been secondary to the ACh-induced depolarization.For this reason, data on the effect of ACh on membrane potential have beenincluded in this paper.

-LOr

-60

-10 r

>B

/(nA)

+40 -40

-60

>B

30

Fig. 1. Current-voltage plots for Ascaris suum muscle cells illustrating a cell thatshows no rectification (A) and one that does (B). Depolarizing or hyperpolarizingcurrent pulses of 500 ms pulse width were injected into the cell, which had a restingmembrane 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 inFig. 2. ACh produced a dose-dependent and reproducible depolarization andincrease in input conductance of the muscle cells. In Ca2+-free APF, the EC50 (theconcentration 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 obtainedin Ca2+-containing APF as the recording was not stable when high concentrations

10 mV10 n A

liiiiiiiiiiifiiiiii:

10 100

[ACh] (/rniolP1)

1000

5mV[

[ACh] (/mioi

Fig. 2. Dose-response relationships for acetylcholine (ACh). (A) An example of anintracellular recording from an Ascaris muscle cell showing the response to increasingconcentrations of ACh. The first set of responses to 1-lOjanoir1 ACh were in thepresence of Ca2+. The arrow indicates the switching of the perfusion medium to Ca2+-free artificial perienteric fluid (APF). The dose-response relationship betweenl^moll"1 and lmmolP1 ACh was then recorded. The downward deflections areelectrotonic potentials caused by current injection (0.1 Hz, 500 ms pulse width). Themagnitudes of the current pulses are indicated in the lower trace. The depolarizingpotentials (indicated by the broken arrow) are the result of spontaneous activity in themuscle cell. Note that they decrease in magnitude and frequency and eventuallydisappear 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 forthe 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-elicitedincrease 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 musclecell clamped at resting membrane potential (-30 mV) and perfused with ACh(concentrations as indicated) for 15 s.


of ACh were applied in this medium. The EC50 for the ACh-elicited conductanceincrease could not be estimated either, as it continued to increase even atl m m o i r 1 ACh (Fig. 2C). At the lower concentrations of ACh, the dose-response curves in the presence and absence of Ca2+ were not significantlydifferent (Fig. 2B,C).

Voltage-clamp experiments indicated that ACh elicited an inward current ofbetween 7 and 29 nA (Figs 2D, 11) when the cells were voltage-clamped at restingmembrane potential.

The effects of agonists

The relative potencies of the agonists studied are given in Table 1 and Figs 3 and4. For all compounds except nicotine these were calculated in Ca2+-containingAPF only. Nicotine mimicked the effect of ACh, though with a lower potency. Therelative potency of nicotine compared to ACh was studied in the presence andabsence 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 receptorcompared to acetylcholine

Agonist

HPPTDMPPAcetylcholineCarbacholNicotineTMA+

MuscaroneFurtrethoniumArecolineNMTHTMcN-A-343BethanecholMethacholinePilocarpine

MuscarineOxotremorine

Relative potency

Depolarization

6.5310.40.20.040.010.0070.001No effectWeak depolarizationNo effectNo effectWeak hyperpolarization

(5 out of 7 cells)Weak hyperpolarizationWithout effect

Conductance

9210.50.30.050.0060.0070.002

No effect

The relative potency was determined for each cell by taking the ratio of the concentration ofACh to the concentration of agonist that produced the same response from parallel portions ofthe dose-response curve (therefore a relative potency greater than 1 indicates a compound morepotent than ACh). This was done both for the depolarizing response to ACh and for theconductance increase elicited by ACh; N=3-6.

HPPT, metahydroxyphenylpropyltrimethylammonium; DMPP, l,l-dimethyl-4-phenyl-piperazinium; TMA+, tetramethylammonium; NMTHT, 2(nitromethylene)tetrahydro 1,3,-[hiazine.


values in the absence of Ca2+ were 0.08 for the depolarization and 0.008 for theconductance increase. Both these values are lower than the relative potency fornicotine obtained in the presence of Ca2+. In all seven cells studied (four in thepresence and three in the absence of Ca2+) it was found that the response to AChwas reduced after the cell had been exposed to nicotine (Fig. 5).

o

10 100

[Agonist] (/imolP1)

1000

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

o

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


10/anoir' ACh 1 mmol T1 nicotine O 1 Ch

Fig. 5. Evidence to indicate that nicotine desensitizes the Ascaris muscle cell responseto ACh. The artificial perienteric fluid for this experiment was Ca2+-free. These areconsecutive recordings from an Ascaris muscle cell. The downward deflections arecaused by current injection (0.1 Hz, 500 ms, the amplitude of the current pulse is shownin the lower trace). The resting membrane potential was —25 mV. The bars indicatethe durations of applications of drug at the concentration indicated on the figure. Thedepolarization and the increase in input conductance in response to ACh were reducedafter 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 thedepolarization 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 tocarbachol was greater than that to ACh (Fig. 6B). Muscarone was 100 times lesspotent than ACh. Arecoline and furtrethonium both elicited depolarizations andconductance 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 toelicit a depolarization of about 2 mV. Bethanechol and methacholine were withouteffect. Pilocarpine and muscarine caused a slight (up to 3mV) hyperpolarizationwith a threshold for the response of SOOjanoir1. The muscarinic agonistoxotremorine (1 mmol I"1) had no effect on two cells. NMTHT was without effectat concentrations up to l m m o l P 1 on three cells.

The effects of two anticholinesterases were investigated. Neither physostigminenor neostigmine had any significant effect on resting membrane potential or inputconductance 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 toACh in a reversible manner (N=3). Neostigmine potentiated the response to AChat concentrations of l-lO/anoll"1 (Fig. 6A,C).

The effects of antagonists

The potencies of the antagonists at blocking the depolarization and theconductance increase in response to ACh are given in Table 2. For mostcompounds these are comparable; however, for both atropine and chlorisonda-mine the IC50 values calculated from the conductance change are lower than theIC50 values calculated from the block of the depolarization. For example, the IC50IJetermined for atropine from the conductance response was 6.7±2.1^moll~1


[ACh](/rnioir')

10 /anol 1 carbachol

C Control

1/anoll ' neostigmine

20 mv20s

10/imoll ' neostigmine

f

Fig. 6. Evidence for the presence of cholinesterase in the Ascaris muscle strippreparation. (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 ofthe response to ACh and carbachol. (C) An example of the potentiation of theresponse to ACh by neostigmine (1 and lO^moll"1) in a muscle cell. The restingmembrane potential of this cell was -30 mV. The downward deflections are caused bythe injection of current (0.1 Hz, 500 ms pulse width, 32 nA). The bars in C indicate theapplication of lO^imoll"1 ACh.

Pharmacology o/Ascaris cholinoceptors

Table 2. Antagonists at the Ascaris acetylcholine receptor

519

Drug

BenzoquinoniumMecamylamineChlorisondamineTPPQuinacrineCurarePancuroniumTrimethaphanAtropineDecamethoniumHexamethoniumDihydro-/J-erythroidine

Conductance

0.29±0.070.33±0.(M1.74±0.33

--

3.10±0.403.20±0.304.27±1.446.70±2.10

14±343 ±6>100

I C s o ^ o i r 1 )

Current

1.90±0.302.05±0.60

Depolarization

0.46±0.040.41±0.04

65±33--

5.26±1.144.49±1.80

17±1052±4

96329

>100

The ICJO is the concentration of antagonist that was required to reduce the response to AChby 50 %.

Values are the mean±s.E.M. for 3-4 experiments.TPP, tetraphenylphosphonium.

ino-i

50-

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

50 100

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 thepaired Student's f-test; * P<0.05.

(N=A, determined by extrapolation) compared to 5214/zmoll"1 (N=4) deter-mined from the ability to block the depolarization (Fig. 7).

Pancuronium and curare antagonised the ACh response. The antagonism by


Aiii

10 mVL10 s

Fig. 8. The blockade of the ACh response by curare and mecamylamine. Examplesare 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 oflOfimoll"1 ACh. (i) Control response to lO^moll"1 ACh; (ii) response to ACh afterthe cell had been exposed to 10 /itnol I"1 curare for 2 min; (iii) response to ACh after a5min wash. (B) Mecamylamine. The resting membrane potential was - 30mV. Thedownward deflections are caused by the injection of current (0.1 Hz, 500 ms pulsewidth, 10nA). The bars indicate applications of ACh. (i) Control response to10/anoll~L ACh; (ii) response to lO^moll"1 ACh after the cell had been exposed toO.S^moir1 mecamylamine for 2 min; (iii) response to lO^moll"1 ACh after a 5 minwash.

curare was readily reversible, with 100% recovery in 5-15min (Fig. 8A). Theblock by atropine and pancuronium lasted longer, requiring a wash of 20 min for arecovery 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 itreversed by about 30 % in six cells (Fig. 8B). The block reversed completely in onecell washed for longer than 30min. The other ganglion blockers tested in thisstudy, trimethaphan and chlorisondamine, also antagonised the ACh response(Table 2), though less potently than mecamylamine.

The relative potencies of hexamethonium and decamethonium as blockers ofthe ACh response were studied in three cells. Decamethonium was found to bemore 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


10 100

[Antagonist] (/rnioll"1)

1000

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

10 mV

30s

30/nnoll decamethonium

100 /imoll decamethonium

300^moll decamethonium Wash

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

2.05±0.60/imoir1 (N=3, Fig. 11). TPP also blocked the current elicited by AChwith 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 themuscle cells. However, the other neuromuscular blocking agent, benzoquino-nium, 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


[Quinacnne](//moir1) 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 atresting membrane potential (—30 mV), were blocked by quinacrine (concentration asindicated). The 30 mV calibration refers to the top trace, which is the voltage recordingfrom the cell. Quinacrine was applied for 2min prior to and concurrent with the AChapplication. The wash period was 5 min. The block of the response to ACh waspartially reversed following the wash period.

100 n

50-

0.01 0 1 1 10

|TPP] (//moir1)

100

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 cellswere clamped at resting membrane potential (approx. — 30mV). The same holdingpotential was used throughout for each individual experiment. The percentage of theresponse remaining as the cell was exposed to increasing concentrations of TPP wasdetermined and this is plotted against the TPP concentration. N=4; values aremean±s.E.M.

suspected that ACh is the excitatory transmitter at the Ascaris neuromuscularjunction. However, it should be noted that the responses to bath-applied ACh inthe preparation described in this paper will involve both the physiologicallyimportant synaptic ACh receptors and the extrasynaptic ACh receptors on themuscle bag cells. Pharmacological differences may exist between the synaptic andextrasynaptic ACh receptors.

The EC50 for ACh at this receptor could not be determined exactly in all cells


because the recordings made after application of concentrations of ACh capable ofeliciting a maximal response in the presence of Ca2+ were unstable. However, itcan be estimated to be in the low micromolar range. From eight studies in theabsence of Ca2+, the EC50 for the depolarization was determined to belO/zmoll"1. The effect of ACh on the cell input conductance was not maximaleven at 1 mmoll"1. Harrow and Gration (1985) estimated that the EC50of ACh foreliciting a conductance increase was 110//moll"1. This may indicate that there area large number of 'spare' receptors for ACh in this preparation, i.e. the maximaldepolarization occurs at a concentration of ACh at which all the availablereceptors have not been activated. The methodological problems of measuring theconductance increase in a cell that is not voltage-clamped were dealt with in theMaterials and methods section. We measured both depolarization and conduc-tance 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 havealso found that the response is decreased when Na+ is partly replaced bygJucosamine in the external medium (L. Colquhoun, L. Holden-Dye and R. J.Walker, unpublished observations). Harrow and Gration (1985) have estimatedthat the reversal potential for the ACh current is about +10mV, which isconsistent with the receptor gating a cation channel through which Na+ is probablythe major charge carrier. Therefore, by analogy with other nicotinic receptors, theAscaris receptor seems to be a ligand-gated cation channel. The nicotinic nature ofthis 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 bythe anticholinesterase neostigmine, but not by physostigmine. Physostigmine(lOO/anoll"1) inhibited the maximum response to ACh by more than 50%. Thedifferent effects of these two anticholinesterases may be explained by theirdifferential affinity for the cholinesterase binding site and the nicotinic receptor. Ithas been shown in other preparations that these anticholinesterases can have adirect action at the ACh receptor site (Slater et at. 1986), and in Aplysia neuronesthe blockade of the receptor by physostigmine can occur at quite low concen-trations (Oyama etal. 1989). It would seem, therefore, that the duration of actionof ACh in the Ascaris muscle preparation is regulated by the presence of acholinesterase susceptible to blockade by neostigmine. The precise mechanism ofaction of physotigmine in this tissue will require further study. It is likely that itacts both as a cholinesterase inhibitor and as a receptor antagonist. Thephysiological relevance of the cholinesterase in terminating the action of synapticACh will have to be elucidated by looking at the possible effect of neostigmine onthe excitatory junction potentials.

The effects of agonists

We did not routinely employ a Ca2+-free medium to study the relative potencies


of agonists. The potency of nicotine on the Ascaris muscle preparation wasapparently lower in the absence of Ca2+. The significance of this has not beeninvestigated. The kinetics of the nicotine response also seemed to be much slowerthan that for ACh (Fig. 5). Nicotine was the only agonist that seemed to elicit aform of desensitization of the Ascaris muscle, in that the response to ACh wasreduced after exposure of the cell to nicotine (Fig. 5). Therefore, the action ofnicotine at the ACh receptor seems to possess some interesting features that wouldbe best investigated at the single-channel level using the patch-clamp technique.

The most effective cholinomimetic agents in Ascaris muscle were nicotinicagonists (Table 1). However, nicotine itself was not as potent as might beexpected. A similar low potency of nicotine, at an otherwise 'nicotinic' receptor, inan invertebrate preparation has been shown in Manduca sexta (Trimmer andWeeks, 1989). In the central nervous system of Manduca the potency of nicotine islow, with an ECso of around 20/imoll~1. This may be due to the presence inManduca 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 preparationshowing that the nicotinic ganglionic agonist DMPP was potent at eliciting Ascarismuscle strip contraction (Natoff, 1969; Rozhkova et al. 1980; Hayashi etal. 1980).In our preparation too, DMPP was one of the most potent agonists. This wouldsuggest that the Ascaris nicotinic receptor is like the nicotinic receptor atmammalian autonomic ganglia. However, it should also be noted that we foundthat 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 anagonist at frog ganglia (Lipscombe and Rang, 1988). Also, the slight stimulatoryaction of furtrethonium was surprising, as this compound is generally regarded as amuscarinic agonist. Thus, even from an agonist profile, the Ascaris ACh receptordoes 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 frogrectus muscle. In rat superior cervical ganglion it is 15 times more potent thanDMPP (Caulfield et al. 1990). In our preparation this compound was the mostpotent cholinomimetic tested, being at least twice as potent as DMPP.

The observation that pilocarpine and muscarine produced a weak hyperpolariz-ation is interesting, particularly in view of the fact that muscarinic receptors, oflower sensitivity than the nicotinic ACh receptors, are known to coexist withnicotinic receptors on other invertebrate cell types (Woodruff et al. 1971; Benson,1988). We have not pursued this possibility further in this study; however, thepotent muscarinic agonist, oxotremorine, was without effect on Ascaris musclecells.

The nitromethylene insecticide NMTHT had no effect on the Ascaris musclecells. It has been shown to be cholinomimetic on cultured cockroach neurone^


where it opens channels with the characteristics of nicotinic receptor-gatedchannels 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. Ofthe ganglion blockers studied, mecamylamine was the most potent compound andboth chlorisondamine and trimethaphan were relatively weak antagonists.Although mecamylamine is widely regarded as a ganghon blocker (see Martin etal. 1989, for a review) at the concentrations that were effective in Ascaris, it hasalso been shown to block transmission at the frog neuromuscular junction in amanner characteristic of open-channel block (Varanda et al. 1985). Mecamylamineis one of the most potent antagonists of the nicotinic ACh response on thecockroach Df motoneurone and acts in a voltage-independent manner (David andSattelle, 1984). It has also recently been shown to be a potent antagonist of theACh response in Xenopus oocytes expressing the a-A and non-anicotinic receptorsubunits cloned from rat brain (Bertrand et al. 1990). Therefore, it can be seen thatour results with mecamylamine do not help us to classify the Ascaris musclenicotinic receptor as a neuromuscular junction type, ganglionic type or brain typeof nicotinic receptor. In future experiments it would be interesting to test thevoltage-dependency of mecamylamine action at the Ascaris ACh receptor, as itwould seem that a voltage-dependent action may be typical of the nicotinicneuromuscular receptor.

Two of the antagonists, chlorisondamine and atropine, show a markeddiscrepancy in their ability to block the conductance increase and the depolariz-ation elicited by ACh. Both compounds block the conductance increase morepotently than the depolarization (Table 2; Fig. 7). It has been shown that50 /anol I"1 atropine is required to block the ACh-stimulated contraction in Ascarismuscle strip (Baldwin and Moyle, 1949; Rozhkova et al. 1980; Natoff, 1969;Onuaguluchi, 1989; Hayashi et al. 1980). The relatively weak antagonism of thecontractile response and the muscle cell depolarization by atropine is compatiblewith the receptor being of a nicotinic rather than a muscarinic subtype. However,the receptor shows little discrimination between curare and atropine, with curareblocking the ACh-induced depolarization with an IC50 of about 5jm\o\\~l. Asimilar lack of discrimination by a nicotinic receptor between these two com-pounds 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 neuro-muscular blocking agents curare and pancuronium. Pancuronium is a steroidalnon-depolarising antagonist at the vertebrate neuromuscular junction of 10-foldgreater potency than curare (Buckett et al. 1968), but it has the same potency ascurare at the Ascaris neuromuscular junction.

Of the other neuromuscular junction blockers tested, benzoquinonium anddihydro-/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


dihydro-^-erythroidine is of interest as it is a nondepolarising neuromuscularjunction blocker, similar in mechanism of action to curare. However, unlike curareand other neuromuscular junction blockers, it is a tertiary rather than a quaternarycompound.

Tetraphenylphosphonium (TPP) was tested against the ACh response in Ascarisbecause 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 tothe extracellular beginning of the M2 transmembrane domain of the Torpedonicotinic receptor (Hucho et al. 1986). In Ascaris muscle cells voltage-clamped atthe resting membrane potential, TPP blocked the inward current elicited by AChin a dose-dependent manner (Fig. 12). Thus, the Ascaris nicotinic receptorchannel may have similar recognition sites to those that have been extensivelycharacterised on the Torpedo receptor at the molecular level.

Quinacrine (mepacrine) is another noncompetitive inhibitor of nicotinic recep-tors, although it is also known to have an action at the ACh recognition site (Tsai etal. 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 avermifuge, mainly for tapeworm infections. In this context, its potent blockade ofthe Ascaris nicotinic receptor is of some interest.

The bisquaternary methonium compounds hexamethonium and decametho-nium were both weak antagonists of the ACh response in Ascaris. However,decamethonium was more potent than hexamethonium (Fig. 9). At mammalianganglia, hexamethonium blocks transmission by entering the nicotinic channel(Gurney and Rang, 1984), whereas hexamethonium has little effect on trans-mission at the vertebrate neuromuscular junction (Milne and Byrne, 1981). Thus,the low potency of hexamethonium implies that the nicotinic channel in Ascarislacks a recognition site that is present in the channel of mammalian ganglionicnicotinic receptors. As mentioned previously, the weak agonist action of deca-methonium in Ascaris is similar qualitatively, if not quantitiatively, to its action atthe neuromuscular junction.

Comparison of Ascaris nicotinic receptors with those in other invertebratepreparations

Little is known of the pharmacology of the nicotinic receptors in othernematodes. Radioligand binding studies in the free-living nematode Caenor-habditis elegans identified a binding site with a high affinity for the levamisolegroup of compounds. This site also had a micromolar affinity for DMPP (Lewis etal. 1987).

In one species where electrophysiological studies on the pharmacology of themuscle cell receptors have been performed, Dipetalonema viteae, the AChresponse seems to be very different from that in Ascaris: both arecoline andpilocarpine were cholinomimetic (Rohrer et al. 1988) and the response was notblocked by curare (10/xmol I"1). It did resemble the Ascaris receptor, however, j ^


that the response was not blocked by hexamethonium. In the trematodeSchistosoma mansoni there is believed to be a cholinoceptor with similarpharmacology 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 ofreceptor are insensitive to hexamethonium, as in Ascaris. Decamethonium had apartial agonist action at the receptor on leech glia, but no direct action on leechmuscle. Thus, the receptor on the glia may most closely resemble the Ascarisreceptor in terms of recognition of the bisquaternary methonium compounds.

The nicotinic receptor in Ascaris differs from those of certain insect prep-arations in a number of ways. The Df motoneurone of the cockroach is insensitiveto DMPP (David and Sattelle, 1984), whereas it was the most potent agonist inAscaris. A further difference is indicated by the lack of sensitivity of the AscarisACh receptor to dihydro-/J-erythroidine, whereas this is one of the most potentantagonists on the Df neurone and is also a potent displacer of [125I]-o--bungarotoxin binding to locust ganglia (Macallan et al. 1988). Levamisole,pyrantel and morantel (Harrow and Gration, 1985) are more potent than ACh atthe Ascaris ACh receptor but are only weakly effective on the Df motoneurone(Pinnock et al. 1988). However, the receptors in insects and Ascaris share acommon sensitivity to mecamylamine and benzoquinonium, though, as mentionedearlier, 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 tohexamethonium. Dorsal unpaired median (DUM) cells that respond to ACh witha depolarization have been identified in the metathoracic ganglia of grasshoppers(Goodman & Spitzer, 1979). The depolarization was blocked by curare and byhigh (millimolar) concentrations of hexamethonium. The nicotinic receptor at anidentified synapse in Manduca sexta has some of the characteristics of the Ascarisnicotinic receptor (Trimmer and Weeks, 1989), as decamethonium was an agonistand hexamethonium did not block the ACh response. Mecamylamine and curarewere equipotent, though not very good, antagonists.

ACh receptors have also been described in molluscs. Three types of response toACh have been studied in Aplysia (Kehoe, 1972). The excitatory response isblocked by 50-100 ^mol I"1 curare (Kehoe, 1972; Ascher et al. 1978) and10-100^moll"1 hexamethonium. The studies on these receptors have concen-trated on the mechanism of action of antagonists rather than the pharmacologicalprofile of the receptor. It is interesting, however, that the receptor mediating theexcitatory response to ACh in Aplysia is blocked by hexamethonium withmoderate potency, as is the excitatory response to ACh in Limulus polyphemus(Walker and Roberts, 1982). Yavari et al. (1979) estimated pA2 values forcompounds that block the inhibitory and excitatory response to ACh in Helixaspersa. The excitatory response was weakly antagonised by hexamethonium,whereas the inhibitory response was completely unaffected. Curare was a weak.antagonist of both responses.


In conclusion, common features of the nicotinic receptors in invertebrateswould seem to be a poor recognition of the potent ganglion blocker hexameth-onium and an inability to discriminate well between curare and atropine. Therealso seem to be marked interspecies differences. In this study we have character-ised the somatic muscle cell ACh receptor in Ascaris. As with the receptors inother invertebrates, it cannot readily be classified according to mammaliannomenclature. The true identity of these receptors, and their relationship to thosein higher groups such as mammals, will not be elucidated until the nicotinicreceptor subunits for these species have been cloned and sequenced. Theinformation from the molecular biological approach, together with the pharmaco-logical data outlined above, will enable the relationships between structure andfunction to be determined for the invertebrate nicotinic receptors.

We gratefully acknowledge financial support from the SERC and JerseyGovernment. We are indebted to Chris Willis for a regular supply of healthyAscaris.

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