Subtypes of muscarinic receptors in insect nervous system

Comp. Biochem. Physiol. Vol. 9OC, No.

I, pp. 275-280,

1988

Printed in Great Britain

0

SUBTYPES

OF MUSCARINIC NERVOUS M.

Department

of Zoophysiology,

RECEPTORS SYSTEM

0306-4492/88 $3.00 + 0.00 1988 Pergamon Press plc

IN INSECT

and H. BRRER*

KNIPPER*

University of Osnabrtick, D-4500 Telephone (0541) 608-2877

Osnabriick,

West Germany.

(Received 24 June 1987) Abstract-l. High affinity binding sites with muscarinic characteristics have been identified in the nervous system of Locusta migrutoriu and analyzed with regard to molecular size and modulation by ions or guanylnucleotides. 2. Putative subtypes of muscarinic cholinergic receptors, as defined by differential affinity for pirenzipine, an antagonist for M,-subtypes, have been detected in locusts nervous tissue. 3. Muscarinic binding sites in synaptosomal membranes displayed a low affinity for pirenzipine, indicating that in the nerve terminals, M,-subtypes of muscarinic receptors predominate. 4. This is supported by a significant attenuation of CAMP formation in locusts synaptosomes by oxotremorine.

using locust synaptosomes have shown that the release of ACh from isolated nerve terminals is inhibited via presynaptically located muscarinic auto1984). These receptors (Breer and Knipper, observations suggest different physiological functions of muscarinic receptors in insect ganglia. In mammalian brain most of the cholinergic receptors are of the muscarinic type and have been categorized, due to its different affinity for the novel muscarinic antagonist pirenzipine, into two pharmacologically distinct subpopulations, M, and M, (Hammer and Giachetti, 1982; Birdsall and Hulme, 1983). In the nervous tissue of insect, only 10% of all cholinergic binding sites are muscarinic (Salvaterra and Foders, 1979; Breer, 198la; Lummis and Sattelle, 1985); with regard to the receptor function and insect pharmacology, it is now of considerable interest if different subtypes can be distinguished.

INTRODUCTION

In the nervous system of arthropods, acetylcholine (ACh) is supposed to be the major excitatory neurotransmitter which mediates signal transmission between sensory neurons and specific interneurons (Florey, 1973). Accordingly, it was found in recent ligand binding experiments that the nervous tissue of insects contains very high concentrations of putative acetylcholine receptors, and two pharmacologically distinct populations of binding sites, muscarinic and nicotinic, have been identified (Schmidt-Nielsen et al., 1977; Dudai, 1978; Eldefrawi and Eldefrawi, 1980; Breer, 198la; Lummis and Sattelle, 1985; Meyer and Reddy, 1985). It is of considerable interest that the relative levels of nicotinic and muscarinic receptors in vertebrate brain and insect ganglia are reversed; the significance of the differences, however, has to be evaluated. Up to now, efforts to evaluate physiological and molecular properties of cholinergic receptors in insects were mainly concentrated on the predominent nicotinic receptor type (Sattelle, 1985; Breer et al., 1985; Hanke and Breer, 1986, 1987) but recently interest has also turned to the muscarinic receptors in insects (Dudai and Ben-Barak, 1977; Jones and Sumikawa, 1981; Shaker and Eldefrawi, 1981; Aguilar and Lunt, 1984). Very little is known still about specific functions of the muscarinic receptor in the nervous system of insects, but recent deafferentation experiments suggest that a considerable proportion is located postsynaptically on neuronal elements (Meyer and Reddy, 1985) on the other hand, perfusion studies Abbreviufions: ACh, acetylcholine; AChR, acetylcholine receptor; QNB, quinuclidinyl benzilate; PrBCM, proovlbenzilvlcholine mustard; GPP(NH)P, S-auanvlimidodiphosphate; GTP-y-S,. guanosine’ 5’-(3-&thib)triphosphate. *Present address: Department of Zoophysiology, University of Stuttgart, D-7000 Stuttgart 70, West Germany. Telephone (0711) 459 2266.

MATERIALS AND METHODS Adult Locustu migrutoriu migrutorioides were received from the Insektarium Dr Frieshammer, Jaderberg or Hiltner, Aurachtal. The insects were kept in special cages several days before use. (N-Me-‘H) choline chloride (spec. act. 80 Ci/mmoI) was obtained from New England Nuclear. 5-(‘rP) y-ATP (3000 Ci/mmol) ‘H-quinuclidinyl (phenyl-C “H) Benzilate (QNB) (30 Ci/mmol) were purchased from Amersham International. (W) Propylbenzilylcholine mustard (PrBCM) (33 Ci/mmol) were from New England Nuclear. Atropine, oxotremorine, octopamine, guanosine-Y-O(3-thiophosphate) (GTP-y-S), were received from Sigma. Pirenzipindihydrochloride-H,O was a gift from Dr Karl Thomae GmbH, Biberach a.d. Riss. Preparation of particulate fractions Synaptosomal fractions from the nervous tissue of locusts were prepared as described previously (Breer, 198 1b). Ganglia were sliced with a tissue chopper and subsequently carefully homogenized in 0.25 M sucrose (Tris-buffered, pH 7.4) using Pasteur pipettes of decreasing tip-diameter. The homogenate of ganglionic tissue was centrifuged at 800g for 10 min, the pellet P, rehomogenized and the combined supernatants centrifuged at 17,000 g for 20 min. resulting in a P, pellet.

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M. KNIPPERand H. BREER

For preparation of synaptosomes, the Pz pellet was resusoended in 0.25 M sucrose solution containing 23% Ficoi (w/v) and centrifuged at iO,~O~ for 45 mm in a JSl3-rotor. The resulting peilicle (synaptosomal fraction, PL) was collected in an appropriate volume of Tris-buffered sucrose unless states otherwise. Binding assay

P, or P, fraction of locust or mouse nervous tissue were resuspended in 50mM Na-P, buffer, pH 7.4 and aiiquots (5Opg protein) were incubated in a total volume of 1 ml of 50 mM Na-Pi buffer @H 7.4) containing 0.25 nM tritiated CINB fsn. act. 30Ci/mmol) and muscarinic ligands at indicated concentrations. The incubation was carried out at 37°C for 1 hr. In exoeriments where the effect of GTP-r-S on agonist binding was tested, the incubation medium contained 5 mM MgCl, , oxotremorine and GTP-y-S as indicated in figure legends and the incubation was carried out for 25min. In each case, the binding reaction was terminated by addition of 3 ml of ice-cold saline followed by a rapid filtration through Whatman GFjC filters. The filters were rinsed twice with 3 ml of ice-cold NaCl and the filter-bound radioactivity was determined by liquid scintillation counting. Assay for cyclic AMP

Aliquots of synaptosomal fractions P, and P, (5Opg protein) were incubated in a total volume of 100~1 of 15OmM NaCl, 5 mM KCl, 1OmM Tris, 10 mM glucose, 1 mM MgCl,, 1 mM CaCl, (pH 7.4) containing the receptor ligands at approximate concentrations. The incubation was carried out for 5 min at 37°C in a shaking water bath. The incubation was terminated by the addition of 200~1 of ice-cold ethanol and centrifugation at 17,000 g for 5 min. The pellet was rehomogenized in ethanol and the combined supematants dried: 100~1 of 50mM Tris-HCl, pH 7.4, 4mM EDTA were added to each sample. Cyclic AMP of 50 pi aliquots was determined by a CAMP rH) Radioassay Kit, (Amersham). Labelling of muscarinic receptor with propylbenzilylcholine mustard [(3H)PrBCM]

A method according to Dadi and Morris (1984) was used to label muscarinic AChR of insect s~aptosomal fractions. Locust ganglia were homogenized in 0.25 M sucrose, 10 mM HEPES, containing the following protease inhibitors at a final concentration: (1) EDTA, 1 mM; (2) bacitracin, 0.1 mg/ml; (3) leupeptin, 4 fig/ml; (4) pepstatin A, $g/ml;

(5) antipain, 4pg/ml; (6) soy bean trypsin inhibitor, 5ygj ml; (7) iodoacetamide, 2 mM; (8) phenylmethylsulphonyl fluoride, 0.05 mM (made up freshly in absolute ethanol) (9) benzethonium chloride, 0.1 mM; (10) benzamidine, 1 mM. The homogenate of ganglionic tissue was centrifuged at 1OOgfor 10 min, the pellet P, rehomogenized in the same antiprotease buffer and the combined supematants centrifuged at 17,OOOg for 20 min. The resulting pellet was resuspended in a total volume of 100~1 phosphate buffer (10 mM) nH 7.5, containing the protease inhibitors I-6. kliquots (SO~1) were preincubated-at 4°C for 30 min either with or without 100 uM atronine. Precvclized (3H)PrBCM (31 nM) were added and membranes incubated for a further 15 min. Su~equendy, solid iodoacetamide and phenylmethylsuiphonyl fluoride were added to a final concentration of 2 mM and 0.5 mM. Membranes were centrifuged in 1OmM phosphate buffer containing protease inhibitors i-10. Labelled membranes were dissolved in sample buffer minus 2-mercaptoethanol. Dithiothreitol (1%) was added before boiling for 3 min, followed by alkylation with iodoa&amide. Samples were electrophoresed with the discontinuous gel system of Fairbanks et al. (1971) at 30 mA/gel for 5 hr. Gels were set up for fluorography using Amplify

(Amersham) and exposed at 20°C to Kodak X-Omat AR for 4 months. RESULTS In a recent study we have shown that the muscarinic antagonist quinuclidinyl benzilate (QNB) binds with high aflinity to an apparently homogenous population of binding sites, in the head and thoracic ganglia of locusts (Breer, 1981b). In refined binding tests using the pure L-isomer of labelled QNB, the number of putative muscarinic receptors in the nervous tissue of insect and mammals was estimated. It was confhmed that the number of QNB-binding sites is much higher in the mammalian brain tissue than in insect ganglia. Analysis of the subcellular distribution demonstrated an enrichment of receptors in the synaptosomal fraction. _In order to characterize the binding sites for muscarinic ligands in insect and to confirm the receptor nature, its properties were examined in different approaches and compared with the muscarinic receptors of the vertebrate nervous system. In a first approach the size of the muscarinic binding site was determined using an affinity labelling probe. It has been found that the muscarinic receptor from vertebrate brain can be covalently labelled using alkylating muscarinic antagonists, like tritiated propylbenzilylcholine mustard (3H-PrBCM) and subsequently identified on SDS-gel (Dadi and Morris, 1984). After incubation of neuronal membrane prep arations from locusts with 3H-PrBCM, followed by SDS-polyacrylamide gel electrophoresis and autoradiography almost all radioactivity was found in a single band migrating with an apparent M, of about 75,000 (Fig. 1). No labelling was observed after incubation in the presence of atropine (1O-4 M) confirming the specificity of the reaction and indicating that the muscarinic binding site in insect ganglia exists as a polypeptide with a molecular mass of 75,000 daltons. Thus, the molecular size of the iabelled polypeptide corresponds well with data reported for vertebrate muscarinic receptors (Dadi and Morris, 1984). It has been demonstrated that divalent cations, in particular Mgz+-ions, significantly increase the binding of muscarinic agonists (Schramm and Selinger, 1984). In Fig. 2 the competition of QNI3 binding by oxotremorine in the absence and presence of 5 mM MgCl, is displayed. The shift of the rc,,-value from 9 x 10m5 to 2 x 1W5 M indicates an increase in affinity for oxotremorine induced by Mg2+-ions. Furthermore, in mammalian preparations, it has been found that the affinity for muscarinic agonists is significantly reduced in the presence of guanyl nucleotides, especially by Gpp (NH)p, S’-guanylimidodiphosphate, or GTP-y-S, guanosine S(3-O-thio)t~phosphate, nonhydroIyzable analogs of GTP; the rationale for this observation is based on the concept that muscarinic receptors mediate cellular reactions via coupling to GTP-binding proteins. In experiments with membrane preparations it was found that GTP-y-S significantly reduced the ability of agonists, like oxotremorine and carbamylcholine, to displace tritiated QNB but did not effect the binding of the labelled antagonist itself. As shown in

Receptor subtypes

277

Fig. 2. Effect of Mg2+-ions on oxotremorine binding, evaluated in competition experiments with ‘H-QNB. Binding experiments were performed in the absence (0) or presence (0) of 5 mM MgClz. Maximal QNB-binding was 394fmol/mg protein. Data are the mean of 4 experiments + SD.

b’

a b Fig. 1 SDS-PAGE analysis of polypetides from locust neuronal membranes labelled with 3H-PrBCM. Membranes were prepared from locust head and tboracic ganglia in the

et ai., 1980) was employed in competition studies with tritiated QNB. Figure 4 shows the occupancyconcentration curves for pirenzipine and atropine to muscarinic receptors of neuronal membranes from locust and mouse, as determined in competition experiments. It is evident that the classical antimuscarinic drug, atropine, displays a high affinity, whereas the selective antagonist pirenzipine is much less potent; both ligands display about the same affinity for mouse and locust preparations, indicating that muscarinic receptor subtypes are present in the mouse brain as well as in locust ganglia. In recent purfusion experiments on synaptosomes it has been observed that the M,-type selective antagonist did not antagonize the muscarinic modulation of ACh release from locust synaptosomes, suggesting that the presynaptic autoreceptors, involved in negative feedback regulation at cholinergic nerve terminals, may represent the M,-subtype (in preparation). To localize putative M, and M, muscarinic receptor subtypes (as defined by differential affinity for

present of various protease inhibitors and tabelled with tritiated PrBCM. The autoradiograph shows the polypeptides labelled in the absence (b) or presence (a) of 100pM atropine. It is particularly clear from the densitometric evaluation of the autoradiographs, that polypeptides of an apparent molecular weight of about 75,OOG are specifically labelled.

Fig. 3, GTP-y -S in 50-100 PM concentrations altered the binding of oxotremorine to locust membranes; at a concentration of 10Y4M GTP-y-S the lqo for oxotremorine was decreased by about one order of magnitude. Thus, the molecular size, the increase of agonist affinity by Mg*+ and the decrease by GTP emphasize the view that binding sites for QNB in insect ganglia, in fact represent muscarinic AChRs. In order to reveal if subtypes of the muscarinic receptors in locust ganglia can be distinguished, the selective muscarinic antagonist pirenzipine (Hammer

Fig. 3. Reduction of agonist affinity by guanyl nucleotides. The binding affinity for oxotremorine was determined in competition experiments performed in the presence of different GTP-y-S concentrations. Each point represents the mean of 3 experiments done in duplicates.

M. KNIPPERand H.

278

L> Homopenute

muse

(A)

P, = Pericorya

r

loo

I

Homqena@

BREER

T

I

locust0 m

t - Synaptosomes

80

60

40

20

L

Fig. 4. Con~ntration~e~ndent competition of QNBbinding to mouse and locusts neuronal membranes by atropine and pirenzipine, respectively. Data represent the means + SD of 4 determinations.

pirenzipine) its distribution in subcellular fractions from mouse and locust nervous tissue has been studied. In mouse, only a slightly lower pirenzipine affinity was detected in the synaptosomal fraction; for locusts, the striking differences in afhnity for the fractions containing pericarya or nerve terminals, are immediately evident. The inhibitory constant for the PI-fraction (2 x lo-‘) is significantly different from the value determined for the synaptosomal fraction (3 x lo-‘), which is about two orders of magnitude higher and is quite similar to vatues reported for vertebrate smooth muscle and heart tissue. For the muscarinic antagonist atropine, superimposable binding curves and about the same &values were found in both fractions, Thus binding data suggest the presence of high a&&y receptors in the pericarya, whereas the nerve terminals contain receptors having low affinity for pirenzipine. Since it has been proposed that M, receptors are negatively linked to adenylate cyclase (Watson et al., 1984), the effect of muscarinic ligands on the production of cyclic adenosine 3’5’-monophosphate was studied. As can be seen in Fig. 6 the muscarinic agonist oxotremorine (20 PM) had no immediate effect on the basal level of CAMP in locust synof aptosomes, however, the large stimulation CAMP production induced by adding exogeneous octopamine (500 PM) was significantly attenuated by oxotremorine. In Fig. 7 it is shown that oxotremorine produced its maximal inhibitory effect at micromolar

(B) P, = Pericarya

PL =Synoptosomes T

_

20 -

I

[Ml Fig. 5. Comparison of atropine and pirenzipine binding, as monitored by competition of 3H-QNB binding, in sub.cellular fractions containing either cell bodies or nerve terminals as well from mouse brain (A) as from locust ganglia (B). The insect pericarya displayed a high affinity for pirenzipine (ICY= 2 x IO-’ M) whereas only tow afhnity binding (3 x IO-‘M) was found in synaptosomes. Data k SD are the mean of 3 experiments in which data points were measured in duplicates.

Receptor subtypes 600 r

20 pLM Octopomlne SOOpM

Oxtiremorm

+ -

-

I*OpmtenerOl5oophl -

+ -

+ + -

+

+ +

Fig. 6. Inhibitory effect of oxotremorine on basal and actopamine induced CAMP-levels in locust synaptosomes. CAMP was determined in radioassays. The basal concentration was found to be 8.1 + 4.3 pmol/mg; incubating synaptosomes for 5 min with 500 PM octopamine raised the CAMP-level to 51.1 pmol/mg. Data are the mean of 34 experiments.

5

e

I

0

&I

Oxotremorin

[M]

Fig. 7. Concentration-dependent effect of oxotremorine on the CAMP accumulation in locust synaptosomal preparations induced by octopamine (1OOpM). CAMP concentrations were determined in radioassays. Data were the mean of 4-6 experiments + SD.

concentrations. The inhibitory effect of oxotremorine was almost completely eliminated by atropine. DISCUSSION

Results presented in this paper confirm and extend our recent findings (Breer, 1981b), that specific high affinity binding sites with muscarinic pharmacological profile can be detected in the nervous tissue of insects. In contrast to the vertebrate brain where the muscarinic receptor is the more abundant cholinergic receptor type, however, only a small proportion of the receptors for acetylcholine is of the muscarinic type (Breer, 1981b; Aguilar and Lunt, 1984; Lummis and Sattelle, 1985), although a large population of putative muscarinic receptors has been described for the terminal abdominal ganglion of crickets (Meyer and Reddy, 1985).

219

Subsequent to an affinity labelling with the alkylating muscarinic antagonist propylbenzilylcholine mustard, the muscarinic receptor migrated in SDSPAGE with an apparent M, of 75,000. This molecular size is quite similar to the number published for the muscarinic receptor from vertebrate brain tissue (Venter, 1983) and also found for insect muscarinic binding sites by different techniques (Lummis et af., 1984; Venter et al., 1984). Agonist binding of muscarinic receptors has been found to display a complex binding pattern controlled by ions (Fig. 2) and nucleotides (Fig. 3). It has been suggested that the effect of Mg2+ on the agonist binding is due to a conversion of high affinity (H) sites to superhigh affinity (SH) sites which is reflected in a lower Ic,,-value for agonists (Birdsall et al., 1978, 1979) and consequently the increase in IC, induced by guanine nucleotides is explained by a conversion of SH- and H-sites to low affinity (L) sites. The modulation of oxotremorine binding by Mg*+ and GTP-analogues, closely resemble the phenomena observed for mammalian muscarinic receptor (Nathanson, 1982); consequently, the modulatory effect of GTP-7-S suggests that in insect nervous tissue, too, muscarinic receptors display distinct transducing mechanisms involving guanine nucleotide binding protein. Using the selective muscarinic antagonist pirenzipine, M, and M,-receptor subtypes could be distinguished in the nervous tissue of locust, although only a small proportion of the receptors for acetylcholine in insect ganglia are muscarinic. Indications for muscarinic receptor subtypes in the insect nervous tissue obtained in different approaches have recently been reported (Trimmer and Berridge, 1985; Duggan and Lunt, 1986). In experiments to localize putative M, and M2 muscarinic receptor subtypes it was found that pirenzipine has only relatively low affinity for muscarinic binding sites in synaptosomal membranes, indicating that M, receptors are more abundant in nerve terminals, whereas M,-subtypes can preferentially be found on the cell body fraction (Fig. 5). Recently, it has been demonstrated that the rate of acetylcholine release from locust synaptosomes was reduced by the muscarinic agonist oxotremorine; thus it appears that muscarinic receptors are present in cholinergic terminals and involved in autoregulation of acetylcholine release. The differential distribution of muscarinic receptor subtypes suggest, that the modulation of acetylcholine release is brought about by M,-type receptors. Since M2 receptors are supposed to be negatively linked to adenylate cyclase (Watson et al., 1984) this concept is further supported by the findings that activation of muscarinic receptors significantly reduced the formation of cyclic adenosine monophosphate in locust synaptosomes. Thus, studying the receptor regulated acetylcholine release and using this process as a functional parameter may be a useful approach in exploring receptor heterogeneity in insect nervous tissue. Acknowledgements-The authors thank Miss G. Hinz for technical assistance and Mrs G. Moehrke for typing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB I7 I, C5).

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KNIPPER and

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H. BREER

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