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Pharmacology of the isolated foregut of the locust Schistocerca gregaria—IV. Characterization of a muscarinic acetylcholine receptor
Camp.B&hem. Physiol.Vol. 103C,No. 3, pp. 527-534,1992 Printed in Great Britain
0306-4492/92$5.00+ 0.00 0 1992Pergamon Press Ltd
PHARMACOLOGY OF THE ISOLATED FOREGUT OF THE LOCUST SCHISTOCERCA GREGARIA-IV. CHARACTERIZATION OF A MUSCARINIC ACETYLCHOLINE RECEPTOR S. J. WOOD,* R. H. OsBoRNE,*t S. GOMEZ” and P. J. JEWESS: Division of Biological Sciences, Faculty of Applied Sciences, Bristol Polytechnic, Coldharbour Lane, Frenchay, Bristol BS16 lQY, U.K. [Tel. (0272) 6562611; @hell Research Ltd, Sittingbourne, Kent (Received 10 April 1992; acceptedfor publication 15 May 1992) Abstract-l.
Acetylcholine (ACh; 10e6 M-7 x lo-5 M), in the presence of neostigmine (10e5 M), caused contraction of the locust isolated foregut. 2. The effect of ACh was mimicked by carbachol, propionylcholine (PCh), butyrylcholine (BCh), nicotine, SD35651, oxotremorine and muscarine. 3. The contractions caused by ACh, BCh and carbachol were abolished by atropine (10e6 M) and reduced by d-tubocurarine (10m5M) and decamethonium (5 x 10e5 M). Hexamethonium and a-bungarotoxin had no effect on contractions caused by the above agonists. 4. None of the antagonists used in this study blocked the contractile effects of nicotine. 5. It is concluded that the foregut contains a neuronal nicotinic receptor which, when activated, causes release of ACh which acts on a neuromuscular muscarinic receptor.
INTRODUCTION Although acetylcholine (ACh) has been identified as an important transmitter substance in invertebrate
tissues, its role in the control of insect gut function has not been defined clearly. Reports of specific cholinergic responses and cholinergic receptors in insect alimentary tissues are few and contradictory. For example, whole intestine preparations of the cockroach Periplaneta americana were stimulated to contract in the presence of ACh or eserine and the ACh-induced contractions were potentiated in the presence of this cholinesterase inhibitor (Kooistra, 1950). Similarly, both innervated and denervated preparations of the foregut of the cockroach Blaberus giganteus were contracted by ACh and this effect was potentiated by eserine (Cook et al., 1969). Conversely, Freeman (1966) was unable to record a response to ACh in either the fore- and hindgut of Locusta migratoria or the hindgut of Periplaneta americana. Cook and Holman (1979) reviewed the putative transmitter candidates in insect visceral muscle and dismissed ACh as a major transmitter in the gut. More recent work by Banner et al. (1987a) suggested that ACh may be a neuromuscular transmitter in the locust foregut. In contrast to previous reports that ACh was a contractant of gut tissue, the isolated foregut of Schistocerca gregaria was relaxed by ACh in the presence of the anticholinesterase neostigmine. This effect was mimicked by nicotine and antagonized by d-tubocurarine while the muscarinic compound arecoline was an ineffective agonist. Consequently, it was concluded ACh-induced relaxtTo whom correspondence
should be addressed.
ation of the locust foregut was achieved via activation of nicotinic ACh receptors. The experiments reported in this paper represent an attempt to establish clearly a physiological role for ACh in the isolated foregut of the locust Schistocerca greguriu. Furthermore, the use of a wide range of cholinergic pharmacological tools permitted the characterization of a muscarinic receptor mediating the contractile actions of ACh in this tissue.
MATERIALS AND METHODS The details of the experimental apparatus and procedures have been reported elsewhere (Banner et al., 1987a). Isolated locust foreguts (oesophagus to proventriculus) were incubated in Clarke Insect Ringer at room temperature (18 f 2°C) for 30 min prior to commencing experiments. All drug solutions were made up in Clarke Insect Ringer and applied to the tissue in a 6 min dose cycle with two washes. Dose-response curves were constructed for acetylcholine (ACh), propionylcholine (PCh), butyrylcholine (BCh), carbachol and nicotine in the absence and presence of the antagonists atropine (5 x lo-‘M to 10m6M) and dtubocurarine (d-TC) (10v5 M to 10m4M). In addition the antagonist effects of decamethonium (5 x 10m6M to 5 x 10e5M), a-bungarotoxin (lo-*M to 5 x lo-‘M) and hexamethonium (10°6M to lO-5 M) on the tissue’s responses to applied ACh and nicotine were investigated. The foregut was incubated with antagonist for 20 min prior to retesting the effects of an agonist. ACh-induced responses were recorded in the presence of the cholinesterase inhibitor neostigmine (lo-’ M). The agonist effects of oxotremorine, muscarine, arecoline, bethanechol, acetyl-b-methylcholine, choline, tetramethylammonium and the nitromethylene heterocycle SD3565 l_were also investigated. _ SD35651 was a eift from Shell Research Ltd whilst all other drugs and reagents were obtained from the Sigma Chemical Company. 527
S.
528
J. WOOD
et al.
ACh
;+.>flQ lo-5M
5x1o-6
(b)
cjlL_v 10-e M
M
2x10-5
M
L/d JJ
5~10-~
M
2x10-5M
G+P
(cl
5x10-5M
5x1o-~rvl
2x10-5
M
5x10-5
5x10-5
M
M
Fig. 1. The effect of ACh (10m6M to 5 x lo-’ M) on the in citro locust foregut in three different tissues; (a) sustained contraction (b) “spasm-like” spontaneous contractile activity (c) rapid, short duration contraction. Calibration 10 mm and 30 set applies to all traces. Arrows show the point of drug addition. RESULTS
In the presence of the cholinesterase inhibitor (10-j M), ACh caused dose-dependent contraction of the isolated foregut over the concentration range 10m6M to 7 x 10-j M (Fig. 1). The contraction response observed in different tissues was variable, in 50% of the tissues the time to onset of contraction was typically 10 set and the response was sustained throughout the 2 min drug-tissue contact neostigmine
time (Fig. la). Other tissues investigated exhibited stepped “spasm-like” contractions (Fig. lb) or rapid, short duration contractions (Fig. lc). In contrast to the sustained contraction, the time to onset of this rapid response was typically 6 set and the duration of the contraction response was less than 1 min. The ACh dose-response curve showed non-competitive inhibition in the presence of increasing concentrations of the muscarinic antagonist atropine (5 x lo-* M to 5 x lo-‘M; Fig. 2). The maximum
-‘0
-‘4 to0 ,.
concentration
ACh M
Fig. 2. Acetylcholine (ACh) dose-response curve in the presence of neostigmine IO-’ M (0) and atropine 5 x 10-r M (0) IO-‘M (0) and 5 x IO-‘M (m). Each point is the mean of 6 replicates (-&SEM).
ACh receptors in locust foregut
529
concentration ‘OQw
ACh M
Fig. 3. Acetylchohne dose-response curve in the presence of neostigmine IO-’ M (0) and d-tubocurarine IO-‘M (II) and 10m4M (a). Each point is the mean of 5 replicates (&SEM).
tissue response was reduced progressively by 21%, 62% and 75% with the ACh EDGE value 4.8 If: 0.6 x 1W6 M being increased CorrespondingIy (5 x lo-‘M: 2.0 f 1.0 x lo-‘M, n = 6, not significant; 10S7 M: 3.5 + 0.9 x 10e6 M, n = 6, P < 0.025; 5 x 1O-7 M: 3.8 f 0.9 x lo-’ M, n = 6, P < 0.025). At lo-* M atropine abolished completely the response to ACh. Similarly, the nicotinic antagonist d-TC (lo-’ M to 10m4M) antagonized non-competitively ACh-induced tissue contraction (Fig. 3). At a concentration of iO_’ M the antagonist reduced the maximum response by 53% and the tqS value was increased significantly in the presence of lo-’ M d-TC (from a value of 2.9 f 0.8 x 10e6 M to 2.6 + 0.9 x 10e5 M; P c 0.05, n = 5). However, increasing the dose of d-TC did not further reduce the ACh-induced response. Increasing concentrations of decamethonium, 5 x 10W6M, 2 x lo-‘M and 5 x lo-‘M, reduced the maximum response to ACh by 16%, 16% and 32% (Fig. 4) with the ACh EDGEvalues being increased from 2.0 2 0.9 x 10m6M to 2.7 & 0.6 x 10m6M (n = 6, not significant); 1.2 + 0.3 x 1O-5M (n = 8,
100
P < 0.005) and 3.5 + 0.6 x 10e6 M (n = 6, not significant) respectively. The close analogues of ACh, PCh and BCh induced tissue contraction that was rapid in onset and of only short duration. In order to compare directly their agonist action the response to BCh was plotted as a percentage of the maximum contractile response caused by PCh thereby showing that the intrinsic activity of BCh was 0.83 compared with the value of 1.0 for PCh (Fig. 5). BCh responses occurred at concentrations ranging from lo-’ M to 10e3 M with a maximum response at 5 x 10m4M and an EDDY value of 2.1 + 0.2 x 10W4M (n = 7). By comparison PCh showed reduced affinity initiating contractions at 5 x 10m5M with the maximum response occurring 9.1 k 2.4 x 10W4M, at 5 x IO-‘M (EDGE value n = 7). Atropine was shown to be an effective antagonist of BCh (Fig. 6) whilst having no effect on the PCh dose-response curve. The dose-response curve for BCh was shifted significantly to the right in the presence of atropine (5 x lo-’ M) and the EDGEvalue correspondingly increased from 1.7 + 0.1 x 1O-4 M to 2.9 + 0.4 x 10e4 M (P < 0.05, n = 7) whilst the
4
oonowtratlon ‘OQ $0
ACh M
Fig. 4. Acetylcholine dose-response curve in the presence of neostigmine lo-’ M (0) and decamethonium S x lo-” M (V), 2 x 10-r M (D) and 5 x 10e5 M (a). Each point is the mean of 4-g replicates. SEM omitted in the interests of ctarity but typical values are l&20% of the response.
S.
530
100
J.
WOOD et al.
-
I
z : L
E p
50-
;; 2 ac
?
r
-6
-4 tw ,.
-‘3
concentration
8goni(lt
M
Fig. 5. Dose-response curves for propionylcholine (PCh 0) and butyrylcholine (BCh 1) induced tissue contraction of the isolated locust foregut. The response to BCh is plotted as a percentage of the maximum response attained by PCh on the same tissue. Each point is the mean of 7 replicates (+SEM).
ml
10
conoantratlon
BCh
M
Fig. 6. Butyrylcholine (BCh) dose-response curve in the absence (Of and presence of atropine 5 x IO-’ M (m). Each point is the mean of 4 replicates (+SEM). maximum response was reduced by 45%. In the presence of 10m5M d-TC the reduction of the maximum response achieved by BCh was not so marked (Fig. 7), nevertheless the increase in the EDGE value
I
from 1.6kO.3 x 10m4M to 4.6& 1.2 x 10e4M was significant (P < 0.05, n = 4). Surprisingly, d-TC had no inhibitory effects on the contractile activity of PCh.
I
-‘6
I
-5
-4
‘OS10
concmtratlon
BCA
M
Fig. 7. Butyrylcholine (BCh) dose-response curve in the absence (a) and presence of d-tub~urarine fOef M (m). Each point is the mean of 4 replicates (*SEM).
ACh receptors in locust foregut
-‘6
43 ‘OT 0
concentration
531
-‘4 carbachol
M
Fig. 8. Carbachol dose-response curve in the absence (0) and presence of atropine 5 x 1W’ M (I). Each point is the mean of 7 replicates (FSEM).
Carbachol caused sustained tissue contraction over the concentration range 5 x 10e6 M to 2 x 10V4M with an EDGE value of 1.5 + 0.2 x lo-’ M (n = 15). The agonist action of carbachol was inhibited by both a&opine 5 x lo-‘M (Fig. 8) and d-TC IO-‘M (Fig. 9). In both cases the maxims response was reduced by 50% while the EDGE value increased to 2.8 rf: 0.6 x lo-’ M (P < 0.01, n = 7) in the presence of atropine and 2.4 4 0.3 x 10-j M (P < 0.05, n = 8) in the presence of d-TC. Nicotine (5 x lo-‘M to 2 x 1O-3 M) and SD35651 (1O-6 M to 5 x 10e4 M) also caused tissue contraction (Fig. 10). Unlike the response to nicotine, a dose of SD35651 induced a rapid and brief contraction. Figure 11 shows the dose-response curve for nicotine with the maximum response being achieved at a dose of 2 x low3 M with ED,, and EDGE values of 2.6 f 0.2 x 10m4M and 4.6 f 0.3 x 10m4M respectively (n = 43). While the contractile effects of nicotine were not inhibited by atropine (5 x lo-’ M), the effects of d-TC on nicotine-induced contraction were less clear. Thus, Fig. 1I shows insignificant inhibition of nicotine-stimulated tissue contraction in the presence of 5 x lO-‘j M d-TC. The maximum response
was reduced by 12% and the EDDY value increased from 3.3 + 0.4 x 10m4M to 5.2 i 1.6 x toe4 M (n = ll), although these differences were not significant. Of the 11 tissues, studied, d-TC potentiated (n = 4), inhibited (n = 4) or was without effect (n = 3) on nicotine-induced responses. Increasing the antagonist dose above 5 x 10e6 M did not cause inhibition of nicotine-induced contraction. Decamethonium (2 x IO-‘M and 5 x lo-‘M) had no significant antagonist effect on the actions of nicotine. The muscarinic agonists arecoline, bethanechol, acetyl-fi-methylcholine, oxotremorine and muscarine were each tested on 6 tissues over the concentration range 5 x 10m6M to 10e3 M. Muscarine and oxotr~morine were the most powerful of these agonists eliciting contractions at concentrations greater than 5 x lo-’ M (Fig. 12). Dose-response curves were not plotted for these agonists as the responses were inconsistent and difficult to measure due to the occurrence of “spasm-like” activity. Arecoline and bethanechol caused only very slight contraction at concentrations in excess of 5 x 10e4 M, whilst acetylj?-methylcholine was without effect. Finally, tetramethylammonium and choline were also ineffective as
-b
-k ‘O@lO
concontratlon
-‘4 cerbmhol
M
Fig. 9. Carbachol dose-response curve in the absence (0) and presence of d-tubocurarine Each point is the mean of 8 replicates (ItSEM).
low5 M (0).
S. J. WOOD et al.
532 Nicotine
LY-fl~ t t 5 10-3
M
SD3565
M
n
2x10-3rd
1
Fig. IO. The effects of nicotinic agonists on the in vitro locust foregut. The records shown are from two different tissues. Nicotine (10e4 M to 2 x 10-j M) induced a sustained tissue contraction whilst SD35651 (10e6 M to 5 x 1O-4 M) induced a rapid, short duration contractile response. Calibration 10 mm and 30sec applies to all traces. Arrows show the point of drug addition.
agonists in this preparation over the same range of concentrations. The relative potency of the major agonists used in this study is shown in Table 1. Of the other antagonists studied hexamethonium (low6 M to 10e5 M) and a-bungarotoxin (lo-* M to 5 x lo-‘M) failed to cause significant inhibition of either ACh- or nicotine-induced responses. Furthermore, at the concentrations used, none of the antagonists investigated elicited a direct agonist response. DISCUSSION
It is well documented that ACh is an important neurotransmitter in insect central nervous transmission although a role for ACh in the periphery, particularly in visceral muscle, is less well established. The demonstration that, after preincubation with an anticholinesterase, ACh is a powerful contractant of the isolated foregut of Schistocerca gregaria is one of only a few reports of a specific cholinergic response
loI3
in the insect gut. In addition the demonstration of an ACh response is in contrast with a previous report by Freeman (1966) that ACh was without effect on foreand hindguts of Locusta migratoria. However, these experiments were conducted without making allowances for the action of cholinesterase within the tissue. ACh, in the presence of neostigmine, and nicotine have been reported previously to cause relaxation of the isolated locust foregut (Banner et al., 1987a). However, the magnitude of the relaxation reported is very small by comparison with the amplitude of the ACh-induced contraction observed in this study. Thus, less than 1% of the tissues used in this study were relaxed by ACh in the presence of the anticholinesterase neostigmine (Wood, 1992). The observation of ACh-induced contractile activity in the locust foregut also concurs with previous reports by both Kooistra (1950) and Cook et al. (1969) that ACh caused contraction of the cockroach gut.
10
concentration
nicotine
M
Fig. Il. Nicotine dose-response curve in the absence (0) and presence of d-tubocurarine 5 x 10m6 M (V) and 10e5 M (m). Each point is the mean of 11replicates. SEM omitted in the interests of clarity but typical values are l(r20% of the response.
ACh receptors in locust foregut
533
Oxotremorine
@7->-r/d/ 10-5
M
5x10-5
M
2x10-4
10_4M
M
5x10-4
M
Muscarine
L
I
+-+-J-+++/ IO-~M
5,10--t
I 10-3M
Fig. 12. The effects of muscarinic agonists on the in uitro locust foregut. The records shown are from two different tissues. Oxotremorine (IO-’ M to 5 x 10m4M) and muscarine (5 x 1O-5 M to 10-j M) induced “spasm-like” spontaneous contractile activity. Calibration 10 mm and 30 set applies to all traces. Arrows show the point of drug addition.
Attempts to classify the cholinergic receptor mediating the contractions induced by ACh revealed a complex pharmacological profile. Thus ACh-induced contraction is mimicked by PCh, BCh, carbachol, the nicotinic compounds nicotine and SD3565 1, and the muscarinic agonists muscarine and oxotremorine. Furthermore, ACh-induced contraction is sensitive to the muscarinic antagonist atropine, the nicotinic antagonist d-TC and the putative mixed nicotinic/muscarinic receptor ligand (Harris et al., 1981) decamethonium. As would be expected the endogenous ligand, acetylcholine, has the greatest affinity for the cholinergic receptor(s) in this tissue. The relative potency of agonists on this tissue the being ACh > carbachol > SD35651 > BCh > oxotremorine > nicotine > muscarine > PCh whilst the most potent antagonist is atropine. The agonist/antagonist profile described is obviously neither wholely nicotinic nor wholely muscarinic. The fact that BCh is a better agonist than PCh is an unexpected finding as in mammalian tissues the opposite is true (R. H. Osborne, unpublished observation). The experiments were conducted in the absence of cholinesterase inhibitors and it is therefore possible to speculate that PCh is more vulnerable to Table
I,
Comparison of the EDGEand affinity values of cholinergic agonists on the locust foregut qO
value (M)
9.0 2.4 4.2 1.0 2.1 4.6 5.0 9.1
x x x x x x x x
Agonist Acetylcholine Carbachol SD35651 Oxotremorine* Butyrylcholine Nicotine Muscarine’ Propionylcholine
10-e IO-’ IO..’ 10-4 10-4 10-d 10-4 10-4
Affinity (moles) I.1 4.2 2.4 I.0 4.8 2.2 2.0 I.1
x x x x x x x x
105 IO’ IO’ 104 IO’ IO’ IO’ 103
estimated from traces rather than *Apparent ED~ values dose-response curves because of difficulties of data interpretation caused by “spasm-like” contractions.
degradation by the tissue cholinesterases than BCh, being somewhat closer in structure to ACh than is BCh. Alternatively, it may be that BCh is a better agonist at the cholinergic receptor in this tissue. The nitromethylene heterocycles (NMHs) have been proposed to act at nicotinic cholinergic receptors (Benson, 1989). In the foregut muscle, nicotine has a lower affinity for the cholinergic receptor than the NMH SD35651 which is a potent muscle contractant. The observed effect suggests that the toxicity of NMHs may in part be a result of direct agonist action on the gut. There are two commonly accepted cholinergic receptor types: the nicotinic and the muscarinic receptors as well as in invertebrates the variously described “mixed binding protein” or “mixed nicotinic-muscarinic receptor”. The latter was proposed over a decade ago (Harris et al., 1981) before the availability of very high affinity ligands and genetically cloned receptors. However, Cartaud et al. (1978) showed that nicotinic receptors are ligand gated ion channels composed of five subunits arranged around a central pit while muscarinic receptors were shown subsequently to be G-protein linked (Haga et al., 1986). Consequently, it is very unlikely that an ACh receptor with mixed nicotinic-muscarinic properties exists in this or other tissues. The mixed pharmacological profile suggests that more than one cholinergic receptor exists in the foregut musculature. Thus two distinct receptors, one nicotinic and one muscarinic, may mediate the contractile actions of ACh and the range of reported agonist effects. However, if separate neuromuscular nicotinic and muscarinic receptors existed then it would be unlikely that the cholinergic antagonist atropine (10e6 M) could block completely the tissue response to applied ACh. Interestingly the same effect could not be demonstrated with concentrations of d-TC as high as 10m4 M, thereby giving rise to speculation that there might be a neuronal nicotinic ACh receptor which when activated causes release of
S. J. Wool
534
ACh which acts on a muscarinic neuromuscular receptor. Unexpectedly, none of the cholinergic antagonists used in this study blocked nicotine-induced responses, an observation which calls into question the nature of the receptor site at which the alkaloid is exerting its effects. Acknowledgements-We are grateful for financial support from Shell Research Ltd. We thank Dr I. Duce for his helpful comments made during the preparation of this manuscript. REFERENCES
Banner S. E., Osborne R. H. and Cattell K. J. (1987a) The pharmacology of the isolated foregut of the locust Schistocerca gregaria--I. The effect of a range of putative neurotransmitters. Comp. Biochem. Physiol. 88C, 131-138. Benson J. A. (1989) Insect nicotinic acetylcholine receptors as targets for insecticides. BCPC Mono. 43. Progress and prospects in insect control, pp. 59-70 (N. R. McFarlane Ed.). Cartaud J., Benedetti E. L., Sobel A. and Changeux J.P. (1978) A morphological study of the cholinergic receptor
et al. protein from Torpedo marmorata in its membrane environment and in its detergent-extracted purified form. J. Cell Sci. 29, 313-337. Cook B. J., Eraker J. and Anderson G. R. (1969) The effect of various biogenic amines on the activity of the foregut of the cockroach, Blaberus giganteus. J. Insect. Physiol. 15, 44-455. Cook B. J. and Holman G. M. (1979) The pharmacology of insect visceral muscle. Comp. Biochem. Physiol. 64C, 183-190. Freeman M. A. (1966) The effect of drugs on the alimentary canal of the African migratory locust, Locusta migratorii. Coma. Biochem. Phvsiol. 17. 755-764. Haga K., Haga T. and Ichiyama A. (1986) Reconstruction of the muscarinic acetylcholine receptor. J. biol. Chem. 261, 10,133310,140. Harris R., Cattell K. J. and Donnellan J. F. (1981) The purification and molecular characterisation of a putative nicotinic-muscarinic acetylcholine receptor from housefly heads. Insect Biochem. 11, 371-385. Kooistra G. (1950) Contribution to the knowledge of the action of acetylcholine in the intestine of Periplaneta americana. Physiol. Comp. 2, 75-80. Wood S. J. (1992) Pharmacology of proctolin, FMRFamide and acetylcholine in the foregut of the locust Schistocerca gregaria. Ph.D Thesis, Bristol Polytechnic.
0306-4492/92$5.00+ 0.00 0 1992Pergamon Press Ltd
PHARMACOLOGY OF THE ISOLATED FOREGUT OF THE LOCUST SCHISTOCERCA GREGARIA-IV. CHARACTERIZATION OF A MUSCARINIC ACETYLCHOLINE RECEPTOR S. J. WOOD,* R. H. OsBoRNE,*t S. GOMEZ” and P. J. JEWESS: Division of Biological Sciences, Faculty of Applied Sciences, Bristol Polytechnic, Coldharbour Lane, Frenchay, Bristol BS16 lQY, U.K. [Tel. (0272) 6562611; @hell Research Ltd, Sittingbourne, Kent (Received 10 April 1992; acceptedfor publication 15 May 1992) Abstract-l.
Acetylcholine (ACh; 10e6 M-7 x lo-5 M), in the presence of neostigmine (10e5 M), caused contraction of the locust isolated foregut. 2. The effect of ACh was mimicked by carbachol, propionylcholine (PCh), butyrylcholine (BCh), nicotine, SD35651, oxotremorine and muscarine. 3. The contractions caused by ACh, BCh and carbachol were abolished by atropine (10e6 M) and reduced by d-tubocurarine (10m5M) and decamethonium (5 x 10e5 M). Hexamethonium and a-bungarotoxin had no effect on contractions caused by the above agonists. 4. None of the antagonists used in this study blocked the contractile effects of nicotine. 5. It is concluded that the foregut contains a neuronal nicotinic receptor which, when activated, causes release of ACh which acts on a neuromuscular muscarinic receptor.
INTRODUCTION Although acetylcholine (ACh) has been identified as an important transmitter substance in invertebrate
tissues, its role in the control of insect gut function has not been defined clearly. Reports of specific cholinergic responses and cholinergic receptors in insect alimentary tissues are few and contradictory. For example, whole intestine preparations of the cockroach Periplaneta americana were stimulated to contract in the presence of ACh or eserine and the ACh-induced contractions were potentiated in the presence of this cholinesterase inhibitor (Kooistra, 1950). Similarly, both innervated and denervated preparations of the foregut of the cockroach Blaberus giganteus were contracted by ACh and this effect was potentiated by eserine (Cook et al., 1969). Conversely, Freeman (1966) was unable to record a response to ACh in either the fore- and hindgut of Locusta migratoria or the hindgut of Periplaneta americana. Cook and Holman (1979) reviewed the putative transmitter candidates in insect visceral muscle and dismissed ACh as a major transmitter in the gut. More recent work by Banner et al. (1987a) suggested that ACh may be a neuromuscular transmitter in the locust foregut. In contrast to previous reports that ACh was a contractant of gut tissue, the isolated foregut of Schistocerca gregaria was relaxed by ACh in the presence of the anticholinesterase neostigmine. This effect was mimicked by nicotine and antagonized by d-tubocurarine while the muscarinic compound arecoline was an ineffective agonist. Consequently, it was concluded ACh-induced relaxtTo whom correspondence
should be addressed.
ation of the locust foregut was achieved via activation of nicotinic ACh receptors. The experiments reported in this paper represent an attempt to establish clearly a physiological role for ACh in the isolated foregut of the locust Schistocerca greguriu. Furthermore, the use of a wide range of cholinergic pharmacological tools permitted the characterization of a muscarinic receptor mediating the contractile actions of ACh in this tissue.
MATERIALS AND METHODS The details of the experimental apparatus and procedures have been reported elsewhere (Banner et al., 1987a). Isolated locust foreguts (oesophagus to proventriculus) were incubated in Clarke Insect Ringer at room temperature (18 f 2°C) for 30 min prior to commencing experiments. All drug solutions were made up in Clarke Insect Ringer and applied to the tissue in a 6 min dose cycle with two washes. Dose-response curves were constructed for acetylcholine (ACh), propionylcholine (PCh), butyrylcholine (BCh), carbachol and nicotine in the absence and presence of the antagonists atropine (5 x lo-‘M to 10m6M) and dtubocurarine (d-TC) (10v5 M to 10m4M). In addition the antagonist effects of decamethonium (5 x 10m6M to 5 x 10e5M), a-bungarotoxin (lo-*M to 5 x lo-‘M) and hexamethonium (10°6M to lO-5 M) on the tissue’s responses to applied ACh and nicotine were investigated. The foregut was incubated with antagonist for 20 min prior to retesting the effects of an agonist. ACh-induced responses were recorded in the presence of the cholinesterase inhibitor neostigmine (lo-’ M). The agonist effects of oxotremorine, muscarine, arecoline, bethanechol, acetyl-b-methylcholine, choline, tetramethylammonium and the nitromethylene heterocycle SD3565 l_were also investigated. _ SD35651 was a eift from Shell Research Ltd whilst all other drugs and reagents were obtained from the Sigma Chemical Company. 527
S.
528
J. WOOD
et al.
ACh
;+.>flQ lo-5M
5x1o-6
(b)
cjlL_v 10-e M
M
2x10-5
M
L/d JJ
5~10-~
M
2x10-5M
G+P
(cl
5x10-5M
5x1o-~rvl
2x10-5
M
5x10-5
5x10-5
M
M
Fig. 1. The effect of ACh (10m6M to 5 x lo-’ M) on the in citro locust foregut in three different tissues; (a) sustained contraction (b) “spasm-like” spontaneous contractile activity (c) rapid, short duration contraction. Calibration 10 mm and 30 set applies to all traces. Arrows show the point of drug addition. RESULTS
In the presence of the cholinesterase inhibitor (10-j M), ACh caused dose-dependent contraction of the isolated foregut over the concentration range 10m6M to 7 x 10-j M (Fig. 1). The contraction response observed in different tissues was variable, in 50% of the tissues the time to onset of contraction was typically 10 set and the response was sustained throughout the 2 min drug-tissue contact neostigmine
time (Fig. la). Other tissues investigated exhibited stepped “spasm-like” contractions (Fig. lb) or rapid, short duration contractions (Fig. lc). In contrast to the sustained contraction, the time to onset of this rapid response was typically 6 set and the duration of the contraction response was less than 1 min. The ACh dose-response curve showed non-competitive inhibition in the presence of increasing concentrations of the muscarinic antagonist atropine (5 x lo-* M to 5 x lo-‘M; Fig. 2). The maximum
-‘0
-‘4 to0 ,.
concentration
ACh M
Fig. 2. Acetylcholine (ACh) dose-response curve in the presence of neostigmine IO-’ M (0) and atropine 5 x 10-r M (0) IO-‘M (0) and 5 x IO-‘M (m). Each point is the mean of 6 replicates (-&SEM).
ACh receptors in locust foregut
529
concentration ‘OQw
ACh M
Fig. 3. Acetylchohne dose-response curve in the presence of neostigmine IO-’ M (0) and d-tubocurarine IO-‘M (II) and 10m4M (a). Each point is the mean of 5 replicates (&SEM).
tissue response was reduced progressively by 21%, 62% and 75% with the ACh EDGE value 4.8 If: 0.6 x 1W6 M being increased CorrespondingIy (5 x lo-‘M: 2.0 f 1.0 x lo-‘M, n = 6, not significant; 10S7 M: 3.5 + 0.9 x 10e6 M, n = 6, P < 0.025; 5 x 1O-7 M: 3.8 f 0.9 x lo-’ M, n = 6, P < 0.025). At lo-* M atropine abolished completely the response to ACh. Similarly, the nicotinic antagonist d-TC (lo-’ M to 10m4M) antagonized non-competitively ACh-induced tissue contraction (Fig. 3). At a concentration of iO_’ M the antagonist reduced the maximum response by 53% and the tqS value was increased significantly in the presence of lo-’ M d-TC (from a value of 2.9 f 0.8 x 10e6 M to 2.6 + 0.9 x 10e5 M; P c 0.05, n = 5). However, increasing the dose of d-TC did not further reduce the ACh-induced response. Increasing concentrations of decamethonium, 5 x 10W6M, 2 x lo-‘M and 5 x lo-‘M, reduced the maximum response to ACh by 16%, 16% and 32% (Fig. 4) with the ACh EDGEvalues being increased from 2.0 2 0.9 x 10m6M to 2.7 & 0.6 x 10m6M (n = 6, not significant); 1.2 + 0.3 x 1O-5M (n = 8,
100
P < 0.005) and 3.5 + 0.6 x 10e6 M (n = 6, not significant) respectively. The close analogues of ACh, PCh and BCh induced tissue contraction that was rapid in onset and of only short duration. In order to compare directly their agonist action the response to BCh was plotted as a percentage of the maximum contractile response caused by PCh thereby showing that the intrinsic activity of BCh was 0.83 compared with the value of 1.0 for PCh (Fig. 5). BCh responses occurred at concentrations ranging from lo-’ M to 10e3 M with a maximum response at 5 x 10m4M and an EDDY value of 2.1 + 0.2 x 10W4M (n = 7). By comparison PCh showed reduced affinity initiating contractions at 5 x 10m5M with the maximum response occurring 9.1 k 2.4 x 10W4M, at 5 x IO-‘M (EDGE value n = 7). Atropine was shown to be an effective antagonist of BCh (Fig. 6) whilst having no effect on the PCh dose-response curve. The dose-response curve for BCh was shifted significantly to the right in the presence of atropine (5 x lo-’ M) and the EDGEvalue correspondingly increased from 1.7 + 0.1 x 1O-4 M to 2.9 + 0.4 x 10e4 M (P < 0.05, n = 7) whilst the
4
oonowtratlon ‘OQ $0
ACh M
Fig. 4. Acetylcholine dose-response curve in the presence of neostigmine lo-’ M (0) and decamethonium S x lo-” M (V), 2 x 10-r M (D) and 5 x 10e5 M (a). Each point is the mean of 4-g replicates. SEM omitted in the interests of ctarity but typical values are l&20% of the response.
S.
530
100
J.
WOOD et al.
-
I
z : L
E p
50-
;; 2 ac
?
r
-6
-4 tw ,.
-‘3
concentration
8goni(lt
M
Fig. 5. Dose-response curves for propionylcholine (PCh 0) and butyrylcholine (BCh 1) induced tissue contraction of the isolated locust foregut. The response to BCh is plotted as a percentage of the maximum response attained by PCh on the same tissue. Each point is the mean of 7 replicates (+SEM).
ml
10
conoantratlon
BCh
M
Fig. 6. Butyrylcholine (BCh) dose-response curve in the absence (Of and presence of atropine 5 x IO-’ M (m). Each point is the mean of 4 replicates (+SEM). maximum response was reduced by 45%. In the presence of 10m5M d-TC the reduction of the maximum response achieved by BCh was not so marked (Fig. 7), nevertheless the increase in the EDGE value
I
from 1.6kO.3 x 10m4M to 4.6& 1.2 x 10e4M was significant (P < 0.05, n = 4). Surprisingly, d-TC had no inhibitory effects on the contractile activity of PCh.
I
-‘6
I
-5
-4
‘OS10
concmtratlon
BCA
M
Fig. 7. Butyrylcholine (BCh) dose-response curve in the absence (a) and presence of d-tub~urarine fOef M (m). Each point is the mean of 4 replicates (*SEM).
ACh receptors in locust foregut
-‘6
43 ‘OT 0
concentration
531
-‘4 carbachol
M
Fig. 8. Carbachol dose-response curve in the absence (0) and presence of atropine 5 x 1W’ M (I). Each point is the mean of 7 replicates (FSEM).
Carbachol caused sustained tissue contraction over the concentration range 5 x 10e6 M to 2 x 10V4M with an EDGE value of 1.5 + 0.2 x lo-’ M (n = 15). The agonist action of carbachol was inhibited by both a&opine 5 x lo-‘M (Fig. 8) and d-TC IO-‘M (Fig. 9). In both cases the maxims response was reduced by 50% while the EDGE value increased to 2.8 rf: 0.6 x lo-’ M (P < 0.01, n = 7) in the presence of atropine and 2.4 4 0.3 x 10-j M (P < 0.05, n = 8) in the presence of d-TC. Nicotine (5 x lo-‘M to 2 x 1O-3 M) and SD35651 (1O-6 M to 5 x 10e4 M) also caused tissue contraction (Fig. 10). Unlike the response to nicotine, a dose of SD35651 induced a rapid and brief contraction. Figure 11 shows the dose-response curve for nicotine with the maximum response being achieved at a dose of 2 x low3 M with ED,, and EDGE values of 2.6 f 0.2 x 10m4M and 4.6 f 0.3 x 10m4M respectively (n = 43). While the contractile effects of nicotine were not inhibited by atropine (5 x lo-’ M), the effects of d-TC on nicotine-induced contraction were less clear. Thus, Fig. 1I shows insignificant inhibition of nicotine-stimulated tissue contraction in the presence of 5 x lO-‘j M d-TC. The maximum response
was reduced by 12% and the EDDY value increased from 3.3 + 0.4 x 10m4M to 5.2 i 1.6 x toe4 M (n = ll), although these differences were not significant. Of the 11 tissues, studied, d-TC potentiated (n = 4), inhibited (n = 4) or was without effect (n = 3) on nicotine-induced responses. Increasing the antagonist dose above 5 x 10e6 M did not cause inhibition of nicotine-induced contraction. Decamethonium (2 x IO-‘M and 5 x lo-‘M) had no significant antagonist effect on the actions of nicotine. The muscarinic agonists arecoline, bethanechol, acetyl-fi-methylcholine, oxotremorine and muscarine were each tested on 6 tissues over the concentration range 5 x 10m6M to 10e3 M. Muscarine and oxotr~morine were the most powerful of these agonists eliciting contractions at concentrations greater than 5 x lo-’ M (Fig. 12). Dose-response curves were not plotted for these agonists as the responses were inconsistent and difficult to measure due to the occurrence of “spasm-like” activity. Arecoline and bethanechol caused only very slight contraction at concentrations in excess of 5 x 10e4 M, whilst acetylj?-methylcholine was without effect. Finally, tetramethylammonium and choline were also ineffective as
-b
-k ‘O@lO
concontratlon
-‘4 cerbmhol
M
Fig. 9. Carbachol dose-response curve in the absence (0) and presence of d-tubocurarine Each point is the mean of 8 replicates (ItSEM).
low5 M (0).
S. J. WOOD et al.
532 Nicotine
LY-fl~ t t 5 10-3
M
SD3565
M
n
2x10-3rd
1
Fig. IO. The effects of nicotinic agonists on the in vitro locust foregut. The records shown are from two different tissues. Nicotine (10e4 M to 2 x 10-j M) induced a sustained tissue contraction whilst SD35651 (10e6 M to 5 x 1O-4 M) induced a rapid, short duration contractile response. Calibration 10 mm and 30sec applies to all traces. Arrows show the point of drug addition.
agonists in this preparation over the same range of concentrations. The relative potency of the major agonists used in this study is shown in Table 1. Of the other antagonists studied hexamethonium (low6 M to 10e5 M) and a-bungarotoxin (lo-* M to 5 x lo-‘M) failed to cause significant inhibition of either ACh- or nicotine-induced responses. Furthermore, at the concentrations used, none of the antagonists investigated elicited a direct agonist response. DISCUSSION
It is well documented that ACh is an important neurotransmitter in insect central nervous transmission although a role for ACh in the periphery, particularly in visceral muscle, is less well established. The demonstration that, after preincubation with an anticholinesterase, ACh is a powerful contractant of the isolated foregut of Schistocerca gregaria is one of only a few reports of a specific cholinergic response
loI3
in the insect gut. In addition the demonstration of an ACh response is in contrast with a previous report by Freeman (1966) that ACh was without effect on foreand hindguts of Locusta migratoria. However, these experiments were conducted without making allowances for the action of cholinesterase within the tissue. ACh, in the presence of neostigmine, and nicotine have been reported previously to cause relaxation of the isolated locust foregut (Banner et al., 1987a). However, the magnitude of the relaxation reported is very small by comparison with the amplitude of the ACh-induced contraction observed in this study. Thus, less than 1% of the tissues used in this study were relaxed by ACh in the presence of the anticholinesterase neostigmine (Wood, 1992). The observation of ACh-induced contractile activity in the locust foregut also concurs with previous reports by both Kooistra (1950) and Cook et al. (1969) that ACh caused contraction of the cockroach gut.
10
concentration
nicotine
M
Fig. Il. Nicotine dose-response curve in the absence (0) and presence of d-tubocurarine 5 x 10m6 M (V) and 10e5 M (m). Each point is the mean of 11replicates. SEM omitted in the interests of clarity but typical values are l(r20% of the response.
ACh receptors in locust foregut
533
Oxotremorine
@7->-r/d/ 10-5
M
5x10-5
M
2x10-4
10_4M
M
5x10-4
M
Muscarine
L
I
+-+-J-+++/ IO-~M
5,10--t
I 10-3M
Fig. 12. The effects of muscarinic agonists on the in uitro locust foregut. The records shown are from two different tissues. Oxotremorine (IO-’ M to 5 x 10m4M) and muscarine (5 x 1O-5 M to 10-j M) induced “spasm-like” spontaneous contractile activity. Calibration 10 mm and 30 set applies to all traces. Arrows show the point of drug addition.
Attempts to classify the cholinergic receptor mediating the contractions induced by ACh revealed a complex pharmacological profile. Thus ACh-induced contraction is mimicked by PCh, BCh, carbachol, the nicotinic compounds nicotine and SD3565 1, and the muscarinic agonists muscarine and oxotremorine. Furthermore, ACh-induced contraction is sensitive to the muscarinic antagonist atropine, the nicotinic antagonist d-TC and the putative mixed nicotinic/muscarinic receptor ligand (Harris et al., 1981) decamethonium. As would be expected the endogenous ligand, acetylcholine, has the greatest affinity for the cholinergic receptor(s) in this tissue. The relative potency of agonists on this tissue the being ACh > carbachol > SD35651 > BCh > oxotremorine > nicotine > muscarine > PCh whilst the most potent antagonist is atropine. The agonist/antagonist profile described is obviously neither wholely nicotinic nor wholely muscarinic. The fact that BCh is a better agonist than PCh is an unexpected finding as in mammalian tissues the opposite is true (R. H. Osborne, unpublished observation). The experiments were conducted in the absence of cholinesterase inhibitors and it is therefore possible to speculate that PCh is more vulnerable to Table
I,
Comparison of the EDGEand affinity values of cholinergic agonists on the locust foregut qO
value (M)
9.0 2.4 4.2 1.0 2.1 4.6 5.0 9.1
x x x x x x x x
Agonist Acetylcholine Carbachol SD35651 Oxotremorine* Butyrylcholine Nicotine Muscarine’ Propionylcholine
10-e IO-’ IO..’ 10-4 10-4 10-d 10-4 10-4
Affinity (moles) I.1 4.2 2.4 I.0 4.8 2.2 2.0 I.1
x x x x x x x x
105 IO’ IO’ 104 IO’ IO’ IO’ 103
estimated from traces rather than *Apparent ED~ values dose-response curves because of difficulties of data interpretation caused by “spasm-like” contractions.
degradation by the tissue cholinesterases than BCh, being somewhat closer in structure to ACh than is BCh. Alternatively, it may be that BCh is a better agonist at the cholinergic receptor in this tissue. The nitromethylene heterocycles (NMHs) have been proposed to act at nicotinic cholinergic receptors (Benson, 1989). In the foregut muscle, nicotine has a lower affinity for the cholinergic receptor than the NMH SD35651 which is a potent muscle contractant. The observed effect suggests that the toxicity of NMHs may in part be a result of direct agonist action on the gut. There are two commonly accepted cholinergic receptor types: the nicotinic and the muscarinic receptors as well as in invertebrates the variously described “mixed binding protein” or “mixed nicotinic-muscarinic receptor”. The latter was proposed over a decade ago (Harris et al., 1981) before the availability of very high affinity ligands and genetically cloned receptors. However, Cartaud et al. (1978) showed that nicotinic receptors are ligand gated ion channels composed of five subunits arranged around a central pit while muscarinic receptors were shown subsequently to be G-protein linked (Haga et al., 1986). Consequently, it is very unlikely that an ACh receptor with mixed nicotinic-muscarinic properties exists in this or other tissues. The mixed pharmacological profile suggests that more than one cholinergic receptor exists in the foregut musculature. Thus two distinct receptors, one nicotinic and one muscarinic, may mediate the contractile actions of ACh and the range of reported agonist effects. However, if separate neuromuscular nicotinic and muscarinic receptors existed then it would be unlikely that the cholinergic antagonist atropine (10e6 M) could block completely the tissue response to applied ACh. Interestingly the same effect could not be demonstrated with concentrations of d-TC as high as 10m4 M, thereby giving rise to speculation that there might be a neuronal nicotinic ACh receptor which when activated causes release of
S. J. Wool
534
ACh which acts on a muscarinic neuromuscular receptor. Unexpectedly, none of the cholinergic antagonists used in this study blocked nicotine-induced responses, an observation which calls into question the nature of the receptor site at which the alkaloid is exerting its effects. Acknowledgements-We are grateful for financial support from Shell Research Ltd. We thank Dr I. Duce for his helpful comments made during the preparation of this manuscript. REFERENCES
Banner S. E., Osborne R. H. and Cattell K. J. (1987a) The pharmacology of the isolated foregut of the locust Schistocerca gregaria--I. The effect of a range of putative neurotransmitters. Comp. Biochem. Physiol. 88C, 131-138. Benson J. A. (1989) Insect nicotinic acetylcholine receptors as targets for insecticides. BCPC Mono. 43. Progress and prospects in insect control, pp. 59-70 (N. R. McFarlane Ed.). Cartaud J., Benedetti E. L., Sobel A. and Changeux J.P. (1978) A morphological study of the cholinergic receptor
et al. protein from Torpedo marmorata in its membrane environment and in its detergent-extracted purified form. J. Cell Sci. 29, 313-337. Cook B. J., Eraker J. and Anderson G. R. (1969) The effect of various biogenic amines on the activity of the foregut of the cockroach, Blaberus giganteus. J. Insect. Physiol. 15, 44-455. Cook B. J. and Holman G. M. (1979) The pharmacology of insect visceral muscle. Comp. Biochem. Physiol. 64C, 183-190. Freeman M. A. (1966) The effect of drugs on the alimentary canal of the African migratory locust, Locusta migratorii. Coma. Biochem. Phvsiol. 17. 755-764. Haga K., Haga T. and Ichiyama A. (1986) Reconstruction of the muscarinic acetylcholine receptor. J. biol. Chem. 261, 10,133310,140. Harris R., Cattell K. J. and Donnellan J. F. (1981) The purification and molecular characterisation of a putative nicotinic-muscarinic acetylcholine receptor from housefly heads. Insect Biochem. 11, 371-385. Kooistra G. (1950) Contribution to the knowledge of the action of acetylcholine in the intestine of Periplaneta americana. Physiol. Comp. 2, 75-80. Wood S. J. (1992) Pharmacology of proctolin, FMRFamide and acetylcholine in the foregut of the locust Schistocerca gregaria. Ph.D Thesis, Bristol Polytechnic.