0306-4492/92$5.00+ 0.00 0 1991Pergamon Press pk
Camp. Biochem. Physid. Vol. IOIC, No. I, pp. 175-181, 1992
Printed in Great Britain
EFFECTS OF ADENINE NUCLEOSIDES AND NUCLEOTIDES ON THE ISOLATED HEART OF THE SNAIL HELIX ASPERSA AND THE SLUG ARION ATER GILLIAN E. KNIGHT, CHARLESH. V. HOYLE and CEOFFIUZYBURNSTOCK* Department of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, Gower Street, London WCIE 6BT, U.K. (Telephone: 071-387-7050; Fax: 071-380-7349) (Received 6 March 1991) Abstract-l. Adenine nucleosides and nucleotides were examined for pharmacological activity in hearts isolated from the snail Helix mpersa and the slug Arion ater. 2. Adenosine, AMP, ADP and ATP (above 100 FM) produced either an excitation or an inhibition in the isolated hearts of the snail and slug. 3. 2-Chloroadenosine, CI,/I-methylene ATP and 2-methylthio ATP were inactive at concentrations up to 1 mM. 4. Responses were not blocked by any commonly accepted vertebrate purinoceptor antagonists, indicating that these purinoceptors are dissimilar to vertebrate purinoceptors and cannot be classified according to accepted purinoceptor classifications. 5. Electrical field stim~ation of the snail heart produced frequency-de~ndent responses: I-4Hz produced predominantly excitation, 8-32 Hz predominantly inhibition. These responses were unaffected by the purines up 3 mM.
~RODU~ON Actions of purine nucleotides and nucleosides on mammalian tissues have been known for many years
(Drury and Szent-Gyorgyi, 1928; Gaddum and Holtz, 1933) and these actions have been extensively investigated since that time (see Burnstock, 1990). The responses to purine com~unds have been classified according to their activity, such that P,-purinoceptors are preferentially activated by adenosine and AMP, and P,-purinoceptors are preferentially activated by ADP and ATP (Burnstock, 1978). In addition to this, both types of receptors have been subclassified: Pr-Purinoceptors into A,- and A,subclasses and P*-pu~no~ptors into Prx- and PzYsubclasses (Bumstock and Buckley, 1985; Burnstock and Kennedy, 1985). Following a greater understanding of the importance of purinergic neurotransmission in vertebrate species (Burnstock, 1972,198l; Baer and Drummond, 1979; Stone, 1981; Daly et al., 1983), the actions of purine compounds in invertebrate species are beginning to be investigated. For example, olfactory and cerebral purinoceptors have been investigated in crustaceans (Carr and Thompson, 1983; Carr et al., 1987; Derby et al., 1987) and phagostimulant purinoceptors of bloodsucking insects have also been examined, (Galun et al., 1963, 1984, 1985; Friend and Smith, 1977; Smith and Friend, 1976; Ellgaard ef al., 1987). A number of isolated tissues, both muscular and neuromuscular, from species from several different invertebrate phyla were investigated *To whom correspondence
should be addressed.
for their responsiveness to adenylyl compounds (Hoyle and Greenberg, 1988) and this and other studies have helped to indicate the diverse array of invertebrate species that respond to purine compounds, varying from Bacteria (Azam and Hudson, 1977), Protozoa (Pothier et al., 1987), Crustacea (Carr and Thompson, 1983; Lindgren and Smith, 1986), Coelenterata (Hoyle ei af., 1989), Echinodermata (Knight et al., 1990) and Mollusca (Aikawa and Ishida, 1966). The aim of this study was to compare the actions of purine compounds on the hearts of two molluscs, the land snail Helix uspersa and the large black slug Arion afer, and to attempt to provide a classification of pu~no~pto~ by the use of specific blocking agents. Purine compounds have already been shown to have various actions, including cardioexcitation in the clam Katefysiu rhytiphora (Sathananthan and Bumstock, 1976) and the pond snail Lymnuea stagnalis, and cardioinhibition in the oyster Crassostrea nippona (Aikawa and Ishida, 1966; S-R&a,
1969) and the freshwater mussel Anu~on~~ (Nistratova, 1980).
cygneu
MRTHODS AND MATRRIALS
General procedures
All experiments were performed at room temperature (23 f 2%). Isolated tissues were mounted in 5 ml organ baths containing either continuously aerated Mena’s snail Ringer solution%f the following composition (mM): NaCl, 59.0: KCl. 5.8: NaHCO,. 13.1: MxC1,.6H,O. 16.3 and CaCl, 47.9; or &ion afer sal&e based-on* hae-molymph analysis (Roach, 1963) of the following composition (mM): NaCl, 43.1; KCl, 3.5; MgSO,, 4.06, NaH,PO,, 0.19; NaHCO,, 4.05; Na,SO,, 6.2; ghmose, 63.8 and CaCl,, 3.0.
GILLIANE. KNIGHTet al.
176
Mechanical activity was recorded with the tissues attached to a rigid support in the organ bath and connected to a Grass FTO3C force-displacement transducer. Mechanical activity was displayed on a Grass ink-writing oscillograph. Electrical field stimulation (EFS) was fa~litated by 2 platinmn wire rings 2.5 mm in diameter and I cm apart, through which the preparations were threaded. An initial load of 100-250 mg was applied to the tissue which was then allowed to equilibrate for not less than 30 min. Dissections
Helix aspersa. Specimens of the garden snail Helix aspersa were anaesthetised by being placed on ice for at least 30 min prior to dissection. The snail shell was removed and the specimen was then pinned to a dissecting board. An incision was made along the junction of the lung and the kidney to reveal the heart in the pericardial cavity. Silk ligatures were applied to the conducting vessels each side of the atrium and the ventricle prior to dissecting it out of the mdlusc. Arion ater. The slugs were an~stheti~d as described for the snail, and were secured to the dissecting board. An incision was made across the mantle from the pneumostome to beyond the midline, and down the midline from the tail to the mantle. The body wall flaps were firmly pinned back to reveal the viscera. The reproductive organs were removed and the alimentary tract displaced to reveal the floor of the mantle cavity with the heart, pericardium and kidney. Silk
ligatures were applied to the ventricle and atrium prior to dissecting it out of the mollusc. The isolated tissues were then secured in the organ bath via the silk ligatures, which had one end attached to the rigid support and the other to the fork-displacement transducer. Concentration -response curves
In order to examine the action of the purine compounds, single doses of the compound were added to the organ bath until a maximum response had been observed; contact time was approximately 2min, the drug was then washed out. After wash-out, tone was allowed to return to baseline for at least 5min before a subsequent application of purine compound. When apparently inactive compounds were tested for antagonistic activity, the compound was allowed to equilibrate for at least 20 min before the agonist was applied. Concentration-response curves, when possible, were constructed by expressing the response of a given dose of purine compound, as a percentage of the ma~mum response, or in some cases, of the maximum obtainable response. Drugs used
Adenosine hemisulphate, adenosine 5’-monophosphate sodium salt (AMP), adenosine S-diphosphate sodium salt (ADP), adenosine S&phosphate sodium salt (ATP), u,~-methyiene ATP lithium salt, Zchloroadenosine,
400mg
A
I 2min
Ado
O.&M ATP
Ado !mtvl
Ado !mM
ADP trnb.4
AMP :rnM.
ADP :mM
ATP :mM
ATP ?mM
Fig. I. Actions of purine compounds on the isolated, spontaneously beating heart of the snail Helix manner. B: Several different actions of purine compounds (Ado, ADP and ATP 3 mM), showing a range of their actions on different specimens. C: Actions of one concentration of purine compounds (1 mM), on a single preparation, all having similar responses both in nature and magnitude.
aspersa. A: Actions of adenosine (Ado) and ATP (0.1-3 mM) in a concentration-dependent
177
Purine effects on two molhrscan hearts
200mg
Ach I:M Fig. 2. Action of acetylcholine on the spontaneously beating heart of Helix aspersn, showing a concentration-dependent cardioinhibition. chlorobut~ol, Reactive Blue 2, theophyl~ne, q~ni~ne and ivermectin were all obtained from Sigma Chemical Company. 2-Methylthio ATP and 8-phenyltheophylline were obtained from RBI, glibenclamide from H. N. Norton & Co., physostigmine sulphate from BDH, atropine from Antigen Pharmaceuticals and phentolamine from Ciba Laboratories. Stock solutions were made up in distilled water, and the volume added to the organ bath to produce the final bath concentration was not in excess of 50~1. Chlorobutanol (10% w/v) was made up in ethanol.
RESULTS
E~ogeno~~~ applied purine &ompou~d~ Helix aspersa. In the isolated, spontaneously beating heart (ventricle and atrium), adenosine, AMP, ADP and ATP all produced concentration-dependent effects, with the threshold for all the compounds being above 100 PM. The nature of the response, however, differed from preparation to preparation, whilst being consistent for any particular preparation. The effect of the various put-me compounds varied from cardioexcitation to cardioinhibition, both of which were concentration-dependent (Fig. 1A and B). For any given preparation, the type of response obtained, either excitatory or inhibitory, was also consistent between the purine compounds themselves, so that each compound produced a very similar response both in nature and in amplitude (Fig. 1C). In contrast, in all preparations the response to acetylcholine was cardioinhibitory (Fig. 2). Similarly,
physosti~ine (10 FM) caused a proionged inhibition of the heart, which was reversible upon washing. The responses of the heart to the purine compounds, either excitatory or inhibitory, were not affected by any of the putative antagonists or blocking agents investigated. These included 8-phenyltheophylline (10 FM), Reactive Blue 2 (100 PM), gli~nclamide (10 p M), quinidine (10 p M), ivermectin (1 FM), phentolamine (1 PM) and atropine (1 PM). Stable analogues of the purine compounds, a,/%methylene ATP, 2-methylthio ATP and 2-chloroadenosine (up to a concentration of 1 mM), had no direct action on the beating hearts in either an excitatory or inhibitory manner, nor did they all&t responses to applied adenosine or ATP. Arion ater. The isolated heart of the slug on no occasion beat spontaneously. Occasional bursts of activity were observed in 2 out of 16 preparations, so in order to observe the effects of the purine compounds on an actively beating heart, it was necessary to drive the heart. This was achieved by the application of 5hydroxytryptamine at a concentration sufficient to evoke constant beating. The mean concentration to produce this activity was 190 k 80pM (N = 16). Application of the purine compounds produced similar responses to those observed in the snail, in that the responses were either cardioexcitatory or cardioinhibitory with threshold concentrations for each purine compound above 100 p M. The responses were consistent within a preparation but varied between specimens of Arion (Fig. 3).
AMP &nM
Ado &Y&I
ADP imM ATP !mM Fig. 3. Action of purine compounds on isolated, driven heart of the slug him ater. Individual preparations having different rates and patterns of activity, are expressed in the variety of individual responses seen. Adenosine (Ado) * = 48 set, ** = 20 mg; AMP * = 24 set, ** = 100 mg; ADP * = 24 set, **=200mg;ATPt=24sec,**=40mg).
GILLIANE. KNIGHTet al.
178
ATP itlmtvl
I
0.3rkM
1Oomg
48sec
Ado b3mM
lit-&$
Fig. 4. Action of purine compounds on the isolated, quiescent heart of Arion ater. Top panel shows concentration-dependent contractions of ATP (0.1-10 mM). Bottom panel shows concentrationdependent contractions due to adenosine (Ado) (0.3-3 mM). On quiescent hearts purine compounds produced inconsistent results. In 4 out of 7 preparations, the purine compounds had no effect, up to a concentration of 1 mM. In 3 out of 7 preparations concentration-dependent contractions were observed. However, the concentration of purine compound required to produce the result was in excess of 100pM (Fig. 4). As seen in the snail heart preparation, responses to purine compounds were not affected by the various blocking agents tested. These included: atropine (1 FM), theophylline (100 PM) and S-phenyltheophylline (10 PM). Neither octopamine (1 mM) nor the stable purine analogues a,/?-methylene ATP,
Gs
2PPS
16pps
2-methylthio ATP and 2-chloroadensosine (up to a concentration of 1 mM) had any effect as direct agonists nor did they modify the effects of adenosine or ATP. Electrical field stimulation (EFS)
Helix aspersa. The response to EFS (OSmsec, 100 V, 30 see), on the spontaneously beating heart of the snail varied with the frequency of stimulation. Generally, at low frequencies (l-4 Hz) the response was excitatory with an increase in the force of contraction and the number of beats. However, higher frequencies (16-32 Hz) had a direct inhibitory action which was followed by a period of excitation once the
4i;bs
32pf;s _1 2min
400mg
Fig. 5. Isolated spontaneous activity of the snail Helix aspersu.Responses due to EFS (0.5 msec duration, 150 V, stimulus time 30 set, 1-32 Hz). Responses to l-4 Hz were usually excitatory, while those of the higher frequencies (8-32Hz) were inhibitory, arresting the heart in diastole. Eight Hz produced an intermediary response with an initial excitation rapidly followed by inhibition.
179
Purine effects on two molluscan hearts
J
40mg
12sec
0.lpps Fig. 6. Action of EFS on the isolated heart of Arion ater. Top panel shows the effect of increasing the pulse width from 0.1 to 0.5 msec. Bottom panel shows an electrically field-stimulated heart (0, 0.1 pps, 150V, 0.5 msec). stimulus had ceased. The response to 8 Hz exhibited an intermediary response with an initial excitation rapidly followed by an inhibition (Fig. 5). The response to EFS was unaffected by any of the purine compounds or by their stable analogues, up to a concentration of 1 mM. Similarly, the local anaesthetic chlorobutanol (100 PM) had no effect on the responses to EFS in either the snail or the slug heart. Arion ater. Single pulses of EFS on the slug heart produced prolonged contractions on the quiescent heart. On other occasions, short bursts of activity were observed, the size of which depended on the duration of the stimulus (Fig. 6). As in the snail heart, responses to EFS were not affected by any of the purine compounds at concentrations up to 1 mM. DISCUSSION
The isolated beating hearts of both the snail Helix aspersa and the slug Arion ater were responsive to the purine compounds adenosine, AMP, ADP and ATP. The type of response observed, however, varied greatly, with any of the purine compounds causing either cardioexcitation or cardioinhibition to any given preparation. The high concentrations of purine that were necessary to produce the observed effects (above 100 PM) were by no means unusual. Adenosine is observed to arrest the heart of the oyster Crassostrea nippona in systole at a concentration of 10 mM (Aikawa and Ishida, 1966), and cardioinhibition of the crabs Muia squinado and Carcinus maenas was only observed at concentrations of 1 mM and above (Welsh, 1939). Similarly adenosine, ADP and ATP all cause an increase in tone of the circular muscle from the pedal disc of Actinia equina above a concentration of 100pM (Hoyle et al., 1989).
Since all the purine compounds had very similar responses with respect to the type of activity and also the magnitude of the response, it would appear that the heart of neither species had the ability to distinguish between any of the purine compounds. It would also appear that the nucleotides were not dissociating into adenosine to elicit the response because no single compound was significantly more potent than any of the others. The inability of the synthetic and stable analogues of adenosine and ATP, i.e. 2-chloroadenosine, u,jmethylene ATP and 2-methylthio ATP, to have an effect on the hearts would seem to suggest that an intact and unchanged purine nucleus is necessary, as well as an easily hydrolysable phosphate chain, if a phosphate chain is present, for the compound to have an effect on the heart in either an excitatory or inhibitory manner. The inactivity of these analogues and the conventional vertebrate purinoceptor blockers tested, would also suggest that the receptors activated by the purine compounds in these two molluscan hearts are not comparable to the purinoceptors of vertebrate systems (Burnstock, 1978; Burnstock and Buckley, 1985; Burnstock and Kennedy, 1985), where adenosine and AMP preferentially activate P,-purinoceptors and ADP and ATP preferentially activate Pr-purinoceptors. The receptor that is, in this case, being activated can only be described as a non-specific purinoceptor and since the responses were not able to be blocked there is little correlation between this invertebrate purinoceptor and that of vertebrates. This is the same as found in the heart of the marine gastropods, Busycon contrarium and Melongena corona, and bivalve mollusc, Atrina serrata (Hoyle and Greenberg, 1988). The hearts from Arion ater and Helix aspersa had very similar responses in terms of both type of response and magnitude. The only real difference
GILLIAN E. KNIGHTet al.
180
observed was that while the heart of the snail was always spontaneously beating, that of the slug had to be driven by the application of 5hydroxytryptamine which has been reported to be the nemotransmitter in cardioexcitatory nerves of molluscs (Cottrell and Laverack, 1968). Morphologically, the atrium of the slug had very sparsely arranged muscle fibres. The atrium of the snail, however, had a very tight arrangement of muscle fibres forming a discrete chamber which was easily recognisable, unlike that of the slug. In the heart of both species, the response to acetylcholine was consistently cardioinhibitory. This agrees with previously reported findings that acetylcholine is the neurotransmitter in cardioinhibitory nerves of a variety of invertebrate hearts (Martin, 1974; Sathananthan and Bumstock, 1976; Nistratova, 1980; Smith and Hill, 1987). It may also be possible that since the reversible anticholinesterase, physostigmine, is said to resemble the structure of acetylcholine (Bowman and Rand, 1984) this may possibly account for its cardioinhibitory action. The responses to EFS may reflect the individual properties of these molluscan hearts, in that at low frequencies of stimulation, only excitatory nerves are stimulated, releasing 5-hydroxytryptamine and thereby causing cardioexcitation, whereas at higher frequencies of stimulation cardioinhibitory nerves are activated, releasing acetylcholine and thus inhibiting the heart in diastole. However, this remains to be studied in detail. The lack of effect of the purine compounds on responses evoked by EFS implies that they do not modulate release of the cardiac neurotransmitters. Although not previously described in an invertebrate heart, modulation of neurogenic responses by purine compounds has been described in several organs of various other invertebrate species (see Hoyle and Greenberg, 1988). In summary, this study has revealed that purine compounds (adenosine, AMP, ADP and ATP) have pharmacological activity on the isolated hearts of both Helix aspersa and Arion ater, causing both cardioexcitation and cardioinhibition but do not affect the responses elicited by EFS. Further, these responses appeared to be mediated via a purinoceptor that did not discriminate between these compounds and did not have the characteristics of a vertebrate purinoceptor. Acknowledgements-This work was supported by the SERC. The authors thank Philippa Charatan for assistance in the preparation of the manuscript. REFERENCES Aikawa T. and Ishida S. (1966) Effects of adenosine on the isolated heart auricle of the oyster, Crassostrea nippona Seki. Como. Biochem. Phvsiol. 18C. 779-800. Azam F. and Hudson R. E. (1977) Dissolved ATP in the sea and its utilisation by marine bacteria. Nature 267, 696-697.
Baer H. P. and Drummond G. I. (1979) (Editors) Physiological and Regulatory Functions of Adenosine and Adenine Nucleotides. Raven Press, New York. Bowman W. C. and Rand M. J. (1984) Textbook of Pharmacology, Ch. 10, pp. 34-35. Blackwell Scientific
Publications, Oxford.
Burnstock G. (1972) Purinergic nerves. Pharmac. Rev. 24, 509-560.
Burnstock G. (1978) A basis for distinguishing two types of purinergic receptor. In Cell Membrane Receptors for Drugs &d Hormones;
a Multidisciplinary Approach
(Edited bv Straub R. W. and Bolis L.). I, DD.107-118. __ Raven Press New York. Bumstock G. (1981) Neurotransmitters and trophic factors in the autonomic nervous system. J. Physiol., Lond. 313, l-35.
Bumstock G. (1990) Classification and characterisation of purinoceptors. In Purines in Cellular Signals: Targets for New Drugs (Edited by K.A. Jacobson, J. W. Daly and V. Maganiello), pp. 241-253. Springer, New York. Bumstock G. and Buckley N. J. (1985) The classification of receptors for adenosine and adenine nucleotides. In Methods Used in Adenosine Research-Methods in Pharmacology. (Edited by Paton D. M.), __ pp. 193-212. Raven Press; New York.Burnstock G. and Kennedy C. (1985) Is there a basis for distinguishing two types of P,-purinoceptor? Gen. Pharmac. 16, 433-440.
Carr W. E. S.. Ache B. W. and Gleeson R. A. (1987) Chemoreceptors of crustaceans: similarities to receptors for neuroactive substances in internal tissues. Environ. Healih Perspect. 71, 31-46.
Carr W. E. S. and Thompson H. (1983) Adenosine 5’monophospate, an internal regulatory agent, is a potent chemoattractant for a marine shrimp. J. camp. physiol. 153A, 47-53.
Cottrell G. A. and Laverack M. S. (1968) Invertebrate pharmacology. A. Rev. Pharmac. 8, 273-298. Daly J. W., Kuroda Y., Phillis J. W., Shimizu H. and Ui M. (1983) (Editors) Physiology and Pharmacology of Adenosine Derivatives. Raven Press, New York. Derby C. D., Ache B. W. and Carr W. E. S. (1987) Purinergic modulation in the brain of the spiny lobster. Brain Res. 421, 57-64.
Drury A. N. and Szent-Gydrgyi A. (1929) The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J. Physiol. 68, 213-237.
Ellgaard E. G., Capiola R. J. and Barker J. T. (1987) Preferential accumulation of Culex quinquefasciatus (Diptera: Culicidae) larvae in response to adenine nucleotides and derivatives. J. med. Entomol. 24, 633636.
Friend W. G. and Smith J. J. B. (1977) Factors affecting feeding by bloodsucking insects. A. Rev. Entomol. 22, 309-331.
Gaddum J. H. and Holtz P. (1933) The localization of the actions of drugs on the pulmonary vessels of dogs and cats. J. Pharmac. 77, 139-158. Galun R., Ari-Dori Y. and Bar-Zeev M. (1963) Feeding response in Aedes aegypti: stimulation by adenosine triphosphate. Science 142, 1674-1675. Galun R., Koontz L. C., Gwadz R. W. and Ribeiro J. M. C. (1985) Effect of ATP analogues on the gorging response of Aedes aegypti. Physiol. Entomol. 10, 275-381. Galun R., Oren N. and Zecharaia M. (1984) Effect of plasma components on the feeding response of the mosquito Aedes aegypti to adenine nucleotides. Physiol. Entomol. 9, 403-408.
Hoyle C. H. V. and Greenberg M. J. (1988) Actions of adenylyl compounds in invertebrates from several phyla: evidence for internal purinoceptors. Comp. Biochem. Physiol. 9OC, 113-122. Hoyle C. H. V., Knight G. E. and Burnstock G. (1989) Actions of adenylyl compounds in the pedal disc of the Cnidarian Actinia equina. Comp. Biochem. Physiol. 94C, 111-114.
Knight G. E, Hoyle C. H. V. and Burnstock G. (1990) Glibenclamide antagonises the response to ATP, but
Purine effects on two molluscan hearts not adenosine or adrenalin, in the gastric ligament of the starfish Asterias rubens. Comp. Biochem. Physiol. 97C, 363-367.
Lindgren C. A. and Smith D. 0. (1986) Increased presynaptic ATP levels coupled to synaptic activity at the crayfish neuromuscular junction. J. Neurosci. 6, 26442652. Martin A. W. (1974) Circulation in invertebrates. A. Rev. Physiol. 36, 171-186. Nistratova S. N. (1980) Regulatory mechanisms of cholinergic transmission in the heart of the bivalve molluscs. In- Neurotransmitters. Comparative Aspects (Edited by Salanki J. and Turneav T. M.).,___ DD. 385-401, Akademiai Kiado, Budapest. _ Potheir F., Forget J., Sullivan R. and Couillard D. (1987) ATP and the contractile vacuole in Amoeba proteus: mechanism of action of exogenous ATP and related nucleotides. J. exp. 2001. 2.43, 379-387. Roach D. K. (1963) Analysis of the haemolymph of Arion ater L. (Gastropoda: Pulmonata). J. exp. Biol. 40, 613-623.
181
Sathananthan A. N. and Bumstock G. (1976) Evidence for a non-cholinergic, non-amine@ innervation of the Venus clam heart. Comp. Biochem. Physiol. SSC, 111-118.
Smith J. J. B. and Friend W. G. (1976) Potencies of combined doses of nucleotides as gorging stimulants for Rhodrius prolixus. J. Insect Physiol. 22, 1049-1052.
Smith P. J. S. and Hill R. B. (1987) Modulation of output from an isolated gastropod heart: effect of acetylcholine and FMRFamide. J. exp. Biol. 127, 105-120. S-R&a K. S. (1969) Theory of step-wise excitation in Gastropod hearts. In Comparative Physiology of the Heart: Current Trends (Edited by McCann F. V.), pp. 69-77. Birkhauser, Basel. Stone T. W. (1981) Physiological roles for adenosine and adenosine S-triphosphate in the nervous system. Neuroscience 6, 523-555.
Welsh J. H. (1939) Chemical mediation in crustaceans. 1: the occurrence of acetylcholine in nervous tissues and its action on the decapod heart. J. exp. Biol. 16, 198-219.
Camp. Biochem. Physid. Vol. IOIC, No. I, pp. 175-181, 1992
Printed in Great Britain
EFFECTS OF ADENINE NUCLEOSIDES AND NUCLEOTIDES ON THE ISOLATED HEART OF THE SNAIL HELIX ASPERSA AND THE SLUG ARION ATER GILLIAN E. KNIGHT, CHARLESH. V. HOYLE and CEOFFIUZYBURNSTOCK* Department of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, Gower Street, London WCIE 6BT, U.K. (Telephone: 071-387-7050; Fax: 071-380-7349) (Received 6 March 1991) Abstract-l. Adenine nucleosides and nucleotides were examined for pharmacological activity in hearts isolated from the snail Helix mpersa and the slug Arion ater. 2. Adenosine, AMP, ADP and ATP (above 100 FM) produced either an excitation or an inhibition in the isolated hearts of the snail and slug. 3. 2-Chloroadenosine, CI,/I-methylene ATP and 2-methylthio ATP were inactive at concentrations up to 1 mM. 4. Responses were not blocked by any commonly accepted vertebrate purinoceptor antagonists, indicating that these purinoceptors are dissimilar to vertebrate purinoceptors and cannot be classified according to accepted purinoceptor classifications. 5. Electrical field stim~ation of the snail heart produced frequency-de~ndent responses: I-4Hz produced predominantly excitation, 8-32 Hz predominantly inhibition. These responses were unaffected by the purines up 3 mM.
~RODU~ON Actions of purine nucleotides and nucleosides on mammalian tissues have been known for many years
(Drury and Szent-Gyorgyi, 1928; Gaddum and Holtz, 1933) and these actions have been extensively investigated since that time (see Burnstock, 1990). The responses to purine com~unds have been classified according to their activity, such that P,-purinoceptors are preferentially activated by adenosine and AMP, and P,-purinoceptors are preferentially activated by ADP and ATP (Burnstock, 1978). In addition to this, both types of receptors have been subclassified: Pr-Purinoceptors into A,- and A,subclasses and P*-pu~no~ptors into Prx- and PzYsubclasses (Bumstock and Buckley, 1985; Burnstock and Kennedy, 1985). Following a greater understanding of the importance of purinergic neurotransmission in vertebrate species (Burnstock, 1972,198l; Baer and Drummond, 1979; Stone, 1981; Daly et al., 1983), the actions of purine compounds in invertebrate species are beginning to be investigated. For example, olfactory and cerebral purinoceptors have been investigated in crustaceans (Carr and Thompson, 1983; Carr et al., 1987; Derby et al., 1987) and phagostimulant purinoceptors of bloodsucking insects have also been examined, (Galun et al., 1963, 1984, 1985; Friend and Smith, 1977; Smith and Friend, 1976; Ellgaard ef al., 1987). A number of isolated tissues, both muscular and neuromuscular, from species from several different invertebrate phyla were investigated *To whom correspondence
should be addressed.
for their responsiveness to adenylyl compounds (Hoyle and Greenberg, 1988) and this and other studies have helped to indicate the diverse array of invertebrate species that respond to purine compounds, varying from Bacteria (Azam and Hudson, 1977), Protozoa (Pothier et al., 1987), Crustacea (Carr and Thompson, 1983; Lindgren and Smith, 1986), Coelenterata (Hoyle ei af., 1989), Echinodermata (Knight et al., 1990) and Mollusca (Aikawa and Ishida, 1966). The aim of this study was to compare the actions of purine compounds on the hearts of two molluscs, the land snail Helix uspersa and the large black slug Arion afer, and to attempt to provide a classification of pu~no~pto~ by the use of specific blocking agents. Purine compounds have already been shown to have various actions, including cardioexcitation in the clam Katefysiu rhytiphora (Sathananthan and Bumstock, 1976) and the pond snail Lymnuea stagnalis, and cardioinhibition in the oyster Crassostrea nippona (Aikawa and Ishida, 1966; S-R&a,
1969) and the freshwater mussel Anu~on~~ (Nistratova, 1980).
cygneu
MRTHODS AND MATRRIALS
General procedures
All experiments were performed at room temperature (23 f 2%). Isolated tissues were mounted in 5 ml organ baths containing either continuously aerated Mena’s snail Ringer solution%f the following composition (mM): NaCl, 59.0: KCl. 5.8: NaHCO,. 13.1: MxC1,.6H,O. 16.3 and CaCl, 47.9; or &ion afer sal&e based-on* hae-molymph analysis (Roach, 1963) of the following composition (mM): NaCl, 43.1; KCl, 3.5; MgSO,, 4.06, NaH,PO,, 0.19; NaHCO,, 4.05; Na,SO,, 6.2; ghmose, 63.8 and CaCl,, 3.0.
GILLIANE. KNIGHTet al.
176
Mechanical activity was recorded with the tissues attached to a rigid support in the organ bath and connected to a Grass FTO3C force-displacement transducer. Mechanical activity was displayed on a Grass ink-writing oscillograph. Electrical field stimulation (EFS) was fa~litated by 2 platinmn wire rings 2.5 mm in diameter and I cm apart, through which the preparations were threaded. An initial load of 100-250 mg was applied to the tissue which was then allowed to equilibrate for not less than 30 min. Dissections
Helix aspersa. Specimens of the garden snail Helix aspersa were anaesthetised by being placed on ice for at least 30 min prior to dissection. The snail shell was removed and the specimen was then pinned to a dissecting board. An incision was made along the junction of the lung and the kidney to reveal the heart in the pericardial cavity. Silk ligatures were applied to the conducting vessels each side of the atrium and the ventricle prior to dissecting it out of the mdlusc. Arion ater. The slugs were an~stheti~d as described for the snail, and were secured to the dissecting board. An incision was made across the mantle from the pneumostome to beyond the midline, and down the midline from the tail to the mantle. The body wall flaps were firmly pinned back to reveal the viscera. The reproductive organs were removed and the alimentary tract displaced to reveal the floor of the mantle cavity with the heart, pericardium and kidney. Silk
ligatures were applied to the ventricle and atrium prior to dissecting it out of the mollusc. The isolated tissues were then secured in the organ bath via the silk ligatures, which had one end attached to the rigid support and the other to the fork-displacement transducer. Concentration -response curves
In order to examine the action of the purine compounds, single doses of the compound were added to the organ bath until a maximum response had been observed; contact time was approximately 2min, the drug was then washed out. After wash-out, tone was allowed to return to baseline for at least 5min before a subsequent application of purine compound. When apparently inactive compounds were tested for antagonistic activity, the compound was allowed to equilibrate for at least 20 min before the agonist was applied. Concentration-response curves, when possible, were constructed by expressing the response of a given dose of purine compound, as a percentage of the ma~mum response, or in some cases, of the maximum obtainable response. Drugs used
Adenosine hemisulphate, adenosine 5’-monophosphate sodium salt (AMP), adenosine S-diphosphate sodium salt (ADP), adenosine S&phosphate sodium salt (ATP), u,~-methyiene ATP lithium salt, Zchloroadenosine,
400mg
A
I 2min
Ado
O.&M ATP
Ado !mtvl
Ado !mM
ADP trnb.4
AMP :rnM.
ADP :mM
ATP :mM
ATP ?mM
Fig. I. Actions of purine compounds on the isolated, spontaneously beating heart of the snail Helix manner. B: Several different actions of purine compounds (Ado, ADP and ATP 3 mM), showing a range of their actions on different specimens. C: Actions of one concentration of purine compounds (1 mM), on a single preparation, all having similar responses both in nature and magnitude.
aspersa. A: Actions of adenosine (Ado) and ATP (0.1-3 mM) in a concentration-dependent
177
Purine effects on two molhrscan hearts
200mg
Ach I:M Fig. 2. Action of acetylcholine on the spontaneously beating heart of Helix aspersn, showing a concentration-dependent cardioinhibition. chlorobut~ol, Reactive Blue 2, theophyl~ne, q~ni~ne and ivermectin were all obtained from Sigma Chemical Company. 2-Methylthio ATP and 8-phenyltheophylline were obtained from RBI, glibenclamide from H. N. Norton & Co., physostigmine sulphate from BDH, atropine from Antigen Pharmaceuticals and phentolamine from Ciba Laboratories. Stock solutions were made up in distilled water, and the volume added to the organ bath to produce the final bath concentration was not in excess of 50~1. Chlorobutanol (10% w/v) was made up in ethanol.
RESULTS
E~ogeno~~~ applied purine &ompou~d~ Helix aspersa. In the isolated, spontaneously beating heart (ventricle and atrium), adenosine, AMP, ADP and ATP all produced concentration-dependent effects, with the threshold for all the compounds being above 100 PM. The nature of the response, however, differed from preparation to preparation, whilst being consistent for any particular preparation. The effect of the various put-me compounds varied from cardioexcitation to cardioinhibition, both of which were concentration-dependent (Fig. 1A and B). For any given preparation, the type of response obtained, either excitatory or inhibitory, was also consistent between the purine compounds themselves, so that each compound produced a very similar response both in nature and in amplitude (Fig. 1C). In contrast, in all preparations the response to acetylcholine was cardioinhibitory (Fig. 2). Similarly,
physosti~ine (10 FM) caused a proionged inhibition of the heart, which was reversible upon washing. The responses of the heart to the purine compounds, either excitatory or inhibitory, were not affected by any of the putative antagonists or blocking agents investigated. These included 8-phenyltheophylline (10 FM), Reactive Blue 2 (100 PM), gli~nclamide (10 p M), quinidine (10 p M), ivermectin (1 FM), phentolamine (1 PM) and atropine (1 PM). Stable analogues of the purine compounds, a,/%methylene ATP, 2-methylthio ATP and 2-chloroadenosine (up to a concentration of 1 mM), had no direct action on the beating hearts in either an excitatory or inhibitory manner, nor did they all&t responses to applied adenosine or ATP. Arion ater. The isolated heart of the slug on no occasion beat spontaneously. Occasional bursts of activity were observed in 2 out of 16 preparations, so in order to observe the effects of the purine compounds on an actively beating heart, it was necessary to drive the heart. This was achieved by the application of 5hydroxytryptamine at a concentration sufficient to evoke constant beating. The mean concentration to produce this activity was 190 k 80pM (N = 16). Application of the purine compounds produced similar responses to those observed in the snail, in that the responses were either cardioexcitatory or cardioinhibitory with threshold concentrations for each purine compound above 100 p M. The responses were consistent within a preparation but varied between specimens of Arion (Fig. 3).
AMP &nM
Ado &Y&I
ADP imM ATP !mM Fig. 3. Action of purine compounds on isolated, driven heart of the slug him ater. Individual preparations having different rates and patterns of activity, are expressed in the variety of individual responses seen. Adenosine (Ado) * = 48 set, ** = 20 mg; AMP * = 24 set, ** = 100 mg; ADP * = 24 set, **=200mg;ATPt=24sec,**=40mg).
GILLIANE. KNIGHTet al.
178
ATP itlmtvl
I
0.3rkM
1Oomg
48sec
Ado b3mM
lit-&$
Fig. 4. Action of purine compounds on the isolated, quiescent heart of Arion ater. Top panel shows concentration-dependent contractions of ATP (0.1-10 mM). Bottom panel shows concentrationdependent contractions due to adenosine (Ado) (0.3-3 mM). On quiescent hearts purine compounds produced inconsistent results. In 4 out of 7 preparations, the purine compounds had no effect, up to a concentration of 1 mM. In 3 out of 7 preparations concentration-dependent contractions were observed. However, the concentration of purine compound required to produce the result was in excess of 100pM (Fig. 4). As seen in the snail heart preparation, responses to purine compounds were not affected by the various blocking agents tested. These included: atropine (1 FM), theophylline (100 PM) and S-phenyltheophylline (10 PM). Neither octopamine (1 mM) nor the stable purine analogues a,/?-methylene ATP,
Gs
2PPS
16pps
2-methylthio ATP and 2-chloroadensosine (up to a concentration of 1 mM) had any effect as direct agonists nor did they modify the effects of adenosine or ATP. Electrical field stimulation (EFS)
Helix aspersa. The response to EFS (OSmsec, 100 V, 30 see), on the spontaneously beating heart of the snail varied with the frequency of stimulation. Generally, at low frequencies (l-4 Hz) the response was excitatory with an increase in the force of contraction and the number of beats. However, higher frequencies (16-32 Hz) had a direct inhibitory action which was followed by a period of excitation once the
4i;bs
32pf;s _1 2min
400mg
Fig. 5. Isolated spontaneous activity of the snail Helix aspersu.Responses due to EFS (0.5 msec duration, 150 V, stimulus time 30 set, 1-32 Hz). Responses to l-4 Hz were usually excitatory, while those of the higher frequencies (8-32Hz) were inhibitory, arresting the heart in diastole. Eight Hz produced an intermediary response with an initial excitation rapidly followed by inhibition.
179
Purine effects on two molluscan hearts
J
40mg
12sec
0.lpps Fig. 6. Action of EFS on the isolated heart of Arion ater. Top panel shows the effect of increasing the pulse width from 0.1 to 0.5 msec. Bottom panel shows an electrically field-stimulated heart (0, 0.1 pps, 150V, 0.5 msec). stimulus had ceased. The response to 8 Hz exhibited an intermediary response with an initial excitation rapidly followed by an inhibition (Fig. 5). The response to EFS was unaffected by any of the purine compounds or by their stable analogues, up to a concentration of 1 mM. Similarly, the local anaesthetic chlorobutanol (100 PM) had no effect on the responses to EFS in either the snail or the slug heart. Arion ater. Single pulses of EFS on the slug heart produced prolonged contractions on the quiescent heart. On other occasions, short bursts of activity were observed, the size of which depended on the duration of the stimulus (Fig. 6). As in the snail heart, responses to EFS were not affected by any of the purine compounds at concentrations up to 1 mM. DISCUSSION
The isolated beating hearts of both the snail Helix aspersa and the slug Arion ater were responsive to the purine compounds adenosine, AMP, ADP and ATP. The type of response observed, however, varied greatly, with any of the purine compounds causing either cardioexcitation or cardioinhibition to any given preparation. The high concentrations of purine that were necessary to produce the observed effects (above 100 PM) were by no means unusual. Adenosine is observed to arrest the heart of the oyster Crassostrea nippona in systole at a concentration of 10 mM (Aikawa and Ishida, 1966), and cardioinhibition of the crabs Muia squinado and Carcinus maenas was only observed at concentrations of 1 mM and above (Welsh, 1939). Similarly adenosine, ADP and ATP all cause an increase in tone of the circular muscle from the pedal disc of Actinia equina above a concentration of 100pM (Hoyle et al., 1989).
Since all the purine compounds had very similar responses with respect to the type of activity and also the magnitude of the response, it would appear that the heart of neither species had the ability to distinguish between any of the purine compounds. It would also appear that the nucleotides were not dissociating into adenosine to elicit the response because no single compound was significantly more potent than any of the others. The inability of the synthetic and stable analogues of adenosine and ATP, i.e. 2-chloroadenosine, u,jmethylene ATP and 2-methylthio ATP, to have an effect on the hearts would seem to suggest that an intact and unchanged purine nucleus is necessary, as well as an easily hydrolysable phosphate chain, if a phosphate chain is present, for the compound to have an effect on the heart in either an excitatory or inhibitory manner. The inactivity of these analogues and the conventional vertebrate purinoceptor blockers tested, would also suggest that the receptors activated by the purine compounds in these two molluscan hearts are not comparable to the purinoceptors of vertebrate systems (Burnstock, 1978; Burnstock and Buckley, 1985; Burnstock and Kennedy, 1985), where adenosine and AMP preferentially activate P,-purinoceptors and ADP and ATP preferentially activate Pr-purinoceptors. The receptor that is, in this case, being activated can only be described as a non-specific purinoceptor and since the responses were not able to be blocked there is little correlation between this invertebrate purinoceptor and that of vertebrates. This is the same as found in the heart of the marine gastropods, Busycon contrarium and Melongena corona, and bivalve mollusc, Atrina serrata (Hoyle and Greenberg, 1988). The hearts from Arion ater and Helix aspersa had very similar responses in terms of both type of response and magnitude. The only real difference
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180
observed was that while the heart of the snail was always spontaneously beating, that of the slug had to be driven by the application of 5hydroxytryptamine which has been reported to be the nemotransmitter in cardioexcitatory nerves of molluscs (Cottrell and Laverack, 1968). Morphologically, the atrium of the slug had very sparsely arranged muscle fibres. The atrium of the snail, however, had a very tight arrangement of muscle fibres forming a discrete chamber which was easily recognisable, unlike that of the slug. In the heart of both species, the response to acetylcholine was consistently cardioinhibitory. This agrees with previously reported findings that acetylcholine is the neurotransmitter in cardioinhibitory nerves of a variety of invertebrate hearts (Martin, 1974; Sathananthan and Bumstock, 1976; Nistratova, 1980; Smith and Hill, 1987). It may also be possible that since the reversible anticholinesterase, physostigmine, is said to resemble the structure of acetylcholine (Bowman and Rand, 1984) this may possibly account for its cardioinhibitory action. The responses to EFS may reflect the individual properties of these molluscan hearts, in that at low frequencies of stimulation, only excitatory nerves are stimulated, releasing 5-hydroxytryptamine and thereby causing cardioexcitation, whereas at higher frequencies of stimulation cardioinhibitory nerves are activated, releasing acetylcholine and thus inhibiting the heart in diastole. However, this remains to be studied in detail. The lack of effect of the purine compounds on responses evoked by EFS implies that they do not modulate release of the cardiac neurotransmitters. Although not previously described in an invertebrate heart, modulation of neurogenic responses by purine compounds has been described in several organs of various other invertebrate species (see Hoyle and Greenberg, 1988). In summary, this study has revealed that purine compounds (adenosine, AMP, ADP and ATP) have pharmacological activity on the isolated hearts of both Helix aspersa and Arion ater, causing both cardioexcitation and cardioinhibition but do not affect the responses elicited by EFS. Further, these responses appeared to be mediated via a purinoceptor that did not discriminate between these compounds and did not have the characteristics of a vertebrate purinoceptor. Acknowledgements-This work was supported by the SERC. The authors thank Philippa Charatan for assistance in the preparation of the manuscript. REFERENCES Aikawa T. and Ishida S. (1966) Effects of adenosine on the isolated heart auricle of the oyster, Crassostrea nippona Seki. Como. Biochem. Phvsiol. 18C. 779-800. Azam F. and Hudson R. E. (1977) Dissolved ATP in the sea and its utilisation by marine bacteria. Nature 267, 696-697.
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