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The effect of adenyl compounds on the heart of the dogfish, Scyliorhinus canicula
Camp. Liiochem. Physiol. Vol. 7X, No. 2, pp. 295-300, 1984
0306~4492/84$3.00+ 0.00 0 1984 Pergamon Press Ltd
Printed in Great Britain
THE EFFECT OF ADENYL COMPOUNDS ON THE HEART OF THE DOGFISH, SCYLIORHINUS CANICULA Department
PARVIZ MEGHJI and GEOFFREY BURNSTOCK* of Anatomy and Embryology and Centre for Neuroscience, University College London, Gower Street, London WCIE 6BT, UK. Telephone: 01-387-7050 (Received 29 June 1983)
Abstract-l.
The effects of adenyl compounds were examined on dogfish atria and ventricles. 2. Adenosine, ATP, b, y-methylene ATP (APPCP) and 2-chloroadenosine produced negative inotropic and chronotropic effects on the dogfish atrium, which were antagonized by 8-phenyltheophylline, a P,-purinoceptor antagonist. 3. tr-fi-Methylene ATP (APCPP), which is resistant to degradation, did not produce a similar inhibitory response in the dogfish atrium. 4. Atropine did not affect the responses to adenosine, indicating that adenosine did not produce its effects indirectly by the release of acetyicholine. 5. The effects of adenosine and ATP were not potentiated by dipyridamole, which blocks adenosine uptake; and 2-chloroadenosine, which is reported lo be resistant to uptake and deamination, was equipotent with adenosine; this suggests the absence of an adenosine uptake system. 6. Dogfish ventricles were insensitive to adenyl compounds. Adrenaline, noradrenaline and acetylcholine produced positive inotropic effects on the ventricle. 7. It is concluded that inhibitory P,-purinoceptors are present in the dogfish atrium. However, adenyl compounds had no direct action on the contractility of the dogfish ventricle.
INTRODIJCTION
(1978) proposed that receptors for adenyl compounds could be classified into two types: P,-purinoccptors, which are most sensitive to adenosine, are competitively blocked by methylxanthines, and occupation of which leads to changes in cyclic adenosine 3’, S-monophosphate (cyclic AMP) accumulation; and P,-purinoceptors, which are most sensitive to ATP, do not appear to act via an adenylate cyclase system and are blocked (although not competitively or specifically) by quinidine, 2-substituted imidazolines, 2,2’-pyridylisatogen and apamin (by blockade of potassium channels). More recently arylazido aminopropionyl ATP (ANAPP,) has been claimed to be a specific P,-purinoceptor antagonist (Hogaboom et al., 1980; Fedan et al., 1981). Since the studies of Drury and Szent-Gyiirgyi (1929) the inhibitory effects of adenyl compounds on the heart have been described in many species. Studies on both mammalian and amphibian atria have shown that adenosine and adenine nucleotides have an overall effect similar to that of acetylchohne: they cause the atrium to beat more slowly and also to contract less forcibly (Hollander and Webb, 1957; De Gubareff and Sleator, 1965; James. 1965: Hartzell. 1979; Burnstock and Meghji, 1981: Niedergerke and Page. 1981). These inhibitorv effects are mediated via P,-purinoceptors (Burnstock, 1980; Burnstock and Meghji, 1981; Collis and Pettinger, 1982). In addition to the inhibitory effects, an initial positive inotropic effect to ATP, but not to adenosine, has been reported in the amphibian atrium (Goto et al., 1977;
Niedergerke and Page, 1981; Burnstock and Meghji, 1981), and it is likely that these effects are mediated via P,-purinoceptors. Adenyl compounds do not have a direct effect on the action potentials or contractile properties of mammalian ventricles (Johnson and McKinnon, 1956; Lammerant and Becsei, 1973; Shah et al., 1974, Schrader et al., 1979; Endoh and Yamashita, 1980). ATP is excitatory in the frog ventricle (Flitney et at., 1977; Burnstock and Meghji, 1981) and adenosine has been reported to be inhibitory or inactive in the frog ventricle (Cook er nl., 1958; Fhtney ct al., 1977; Singh and Flitney, 1980; Burnstock and Meghji, 1981). Although there are reports in the literature on the effects of adenyl compounds on teleost fish heart (Cohen et af., 1981; Rotmensch ef al., 1981), the effects of these compounds on the heart of elasmobranch fish have not been studied. Thus the aim of the present study was to examine whether purinoceptors are present in the dogfish heart.
Burnstock
*To whom all correspondence
should be addressed.
MATERIALS AND METHODS Adult spotted dogfish Scyiiorhinus canicula (50-70 cm in length, N = 19) were obtained from the Marine Biologicai Association, Plymouth, and were kept in a circulating sea-water aquarium at lC-12°C. The fish were decapitated and pithed. The hearts were removed and placed in elasmobranch fish Ringer of the following composition (mM): NaCl. 280; KCl, 3; CaCl,. 4; MaCl,. 0.5: NaHCO,. 3: Hepes, 5; Urea, 360 and Glucose, iO,which was bubbled with oxygen. Strips of ventricle, which were mounted on platinum strip electrodes (separated by 7mm), and atria, together with the sinus venosus, were transferred to 1Oml overtlow organ baths at room temperature (1%22°C). A resting tension of 0.5g was applied to the preparations. Atria were allowed to beat spontaneously. Ventricular strips
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PARVIZ MEGHJI and
were stimulated at 0.5 Hz using 5 msec pulses of twice threshold voltage using a Grass SD9 stimulator. The mechanical activity was recorded isometrically by means of a Grass FT 03C force transducer and displayed on a Grass model 79D polygraph. The preparations were allowed to equilibrate for 60min. The bathing solution was changed every I5 min during that time. In the atria cumulative concentration-response curves were obtained to adenosine. ATP, p.y-methylene ATP (APPCP), a,p-methylene ATP (APCPP) and 2-chloroadenosine. Cumulative concentration-response curves to adenosine, ATP and APPCP were also obtained in the presence of 8%PT. Cumulative concentrationresponse curves to adenosine and ATP were obtained in the
absence and in the presence of dipyridamole. In some experiments, responses to single concentrations of adenosine and acetylcholine were compared in the absence and in the presence of atropine. In experiments where 8-PT. dipyridamole and atropine were used, they were added to the organ bath 20min before their effects were investigated. cumulative concentration-response In the ventricle, curves were obtained to adenosine, ATP, APPCP, APCPP, adrenaline, noradrenaline and acetylcholine. Responses were measured as percentage changes of basal levels. The mean and standard error of the mean were calculated for each group. Statistical analysis was carried out using Student’s t-test for paired and unpaired samples. A probability of P < 0.05 was considered to be significant.
GEOFFREY BURNSTWK
Incubation with dipyridamole (0.5 PM), an adenosine uptake blocker, for 20 min did not significantly potentiate the negative inotropic and chronotropic effects of adenosine (Fig. 4). Similar results were obtained for ATP. 2-Chloroadenosine (I-300 PM) produced negative inotropic and chronotropic effects which were not significantly different from those produced by adenosine at any of the concentrations tested (Fig. 4). Ventricle
ATP, adenosine, APPCP and APCPP produced changes in the force of contraction of less than 10% which were not significant (Fig. 5). Adrenaline, noradrenaline and acetylcholine produced concentration-dependent positive inotropic effects (Fig. 5). Adrenaline was 170 times more potent than noradrenaline at producing these effects; the EC,, values of adrenaline and noradrenaline being 0.025 _t 0.008 and 4.2 k 1.O PM respectively.
Drugs and SOILWIS Adenosine, adenosine 5’-triphosphate, fl,;~-methylene adenosine 5’-triphosphate (APPCP), r,a-methylene adenosine 5’-triphosphate (APCPP), 2-chloroadenosine, acetylcholine, adrenaline and noradrenaline were obtained from Sigma. Dipyridamole was obtained from Boehringer Ingelheim, I-phenyltheophylline (8-PT) from Calbiochem and atropine from Antigen Ltd. Ascorbic acid (I 00 p M) was added to solutions of adrenaline and noradrenaline to prevent their rapid oxidation. A stock solution of IOmM 8-PT was made up in 80% v/v methanol containing 0.2 M NaOH. All other drugs were dissolved in distilled water.
RESULTS Atrium
Adenosine, ATP and APPCP (l-300 PM) reduced the force and rate of contraction of dogfish atria in a concentration-dependent manner (Fig. 1). The effects of adenosine, ATP and APPCP were not significantly different from each other at any of the concentrations tested. APCPP (lo-300 PM) produced changes of less than 5% in the force and rate of contraction (Fig. 1). These changes were not significant. Examples of the effects of adenosine, ATP, APPCP and APCPP are shown in Fig. 2a. Both negative inotropic and chronotropic effects of adenosine, ATP and APPCP were antagonized by S-PT (Fig. 2), the concentration-response curves to adenosine and ATP being shifted 20-fold to the right (Fig. 1). The effects of APPCP were antagonized to a greater extent than the effects of adeno:ine and ATP (Fig. 1). Acetylcholine (10 p M) produced a negative inotropic and chronotropic effect (Fig. 3). The responses to adenosine (10 PM) were not affected by a concentration of atropine (1.4 PM) that significantly antagonized the responses to 10pM acetylcholine (Fig. 3).
Fig. I Log concentration-response curves of the spontaneously beating dogfish atrium to (a) the intropic and (b) the chronotropic effects of (0) adenosine, (m) ATP, (A) fl,y-methylene ATP (APPCP) and (v) cc,b-methylene ATP (APCPP) in the absence of R-phenyltheophylline (I-PT) and (0) adenosine, (0) ATP and (A) APPCP in the presence of IOpM 8-PT. Each point is the mean of observations from at least six preparations. Vertical bars show SEM. Asterisks indicate the significance of the differences between control responses and responses obtained in the presence of 8-PT using the unpaired t-test. *P < 0.05; **P
Adenyl compounds on the dogfish
a
b
Adenosine
291
heart
+8-PT
Adenosine
(10 PM)
(10 PM)
ii
ii
1
I
ATP
ATP
clO~M)
0.59
(10pM’
l%ec
APPCP
APPCP
(10 PM)
(10pM)
iv
iv
APCPP
APC PP
(100 PM>
(100
PM’
Fig. 2. Responses of the spontaneously beating dogfish atrium to (i) adenosine, (ii) ATP, (iii) j,y-methylene ATP (APPCP) and (iv) r,b-methylene ATP (APCPP) (a) in the absence and (b) in the presence of 10 p M 8-phenyltheophylline (8%PT).
ii
, Acetylcholine b + Atropine
B
(10 PM)
ii
10.29 1Osec
I
1
Acetylcholine
Adenosine
(10 PM)
00 PM)
Acetylcholine
Adenosine
(10 PM)
Adenosine
b
ns
Fig. 3.(A) Responses of the spontaneously beating dogfish atrium to (i) acetylcholine and (ii) adenosine (a) in the absence and (b) in the presence of 1.4~ M atropine. (B) The effects of acetylcholine (10 p M) and adenosine (1OpM) in the absence (unshaded) and in the presence of 1.4pM atropine (shaded). Vertical columns, which show the percentage change in contractile force (a) and rate (b) of the atria1 preparations were constructed from the means of observations from four preparations. Vertical bars show SEM. Asterisks represent the significance of the difference between control responses and responses in the presence of atropine using the paired t-test. ns, not significant; *P < 0.05; **P -c 0.01.
PARVIZ MECHJI and
GEOFFREY BURNSTOCK DISCUSSION
The results of the present study have demonstrated the negative inotropic and chronotropic effects of adenosine, ATP and APPCP in the dogfish atrium. The responses of dogfish atria to ATP differ from
1oc
/-
8C
/-
6C
40
20
0 1
100
10 Concentrat,on
1000
(FM)
Fig. 4. Log concentration-response curves of the spontaneously beating dogfish atrium to (a) the inotropic and (b) the chronotropic effects of(m) 2-chloroadenosine (N = 4), (0) adenosine (N = 8) in the absence of and adenosine (N = 8) in the presence of 0.5 PM dipyridamole (0). N = the number of observations from which the means were calculated. Vertical bars show SEM. Control responses to adenosine were not significantly different from responses obtained in the presence of dipyridamole or to those obtained to 2-chloroadenosine, using the unpaired t-test.
those of frog atria where ATP has been reported to be excitatory (Goto et al., 1977; Yatani et al., 1978; Burnstock and Meghji, 1981), but are similar to those of mammalian atria (Hollander and Webb, 1957; De Gubareff and Sleator, 196.5; James, 1965; Burnstock and Meghji, 198 1) and teleostean atria (Cohen et al., 1981; Rotmensch et al., 1981). Reptilian atria have been reported to be insensitive to adenyl compounds (Meghji and Burnstock, 1983a). In the present study, the inhibitory effects of adenosine, ATP and APPCP were antagonized by %PT, a P,-purinoceptor antagonist, indicating that these effects are mediated via P,-purinoceptors. It is not clear why the responses to APPCP were affected to a greater extent than those to ATP and adenosine. It is probable that ATP is metabolized to adenosine and AMP before acting on the P,-purinoceptors, since APCPP, which is resistant to degradation (Satchel1 and Maguire, 1975; Maguire and Satchell, 1979), was without effect on the dogfish atrium. However, it is possible that APCPP has a lower affinity for the P,-purinoceptor than has ATP The possibility that adenine compounds may be producing their inhibitory effects indirectly by releasing acetylcholine from nerve terminals was negated since a concentration of atropine which significantly antagonized the effects of acetylcholine had no effect on the responses to adenosine. Dipyridamole did not potentiate the effects of ATP or adenosine in the dogfish atrium. The failure of
I-
T )-
,-
)-
, -
1-
, -
I-
0 01
0.1
1
10
Concentration
(PM)
100
1000
Fig. 5. Log concentration-response curves of electrically driven dogfish ventricular strips to the inotropic effects of: (0) adenosine, (W) ATP, (A) fi,y-methylene ATP (APPCP), (v) a,S-methylene ATP (APCPP), (0) adrenaline, (0) noradrenaline and (a) acetylcholine. Each point is the mean of observations from at least six preparations. Vertical bars show SEM. Asterisks indicate a significant response, using the paired f-test. *P -c 0.05; **P < 0.01; ***P < 0.001.
Adenyl compounds on the dogfish dipyridamole to potentiate adenosine effects has also been observed in rat atria (Stafford, 1966; Kolassa et al., 197 1; Burnstock and Meghji, 1983) and Xenopus laeuis atria (Meghji and Burnstock, 1983b). Hopkins and Goldie (1971) and Kolassa et al. (1971) have shown, with the use of 14C-adenosine, that the adenosine uptake process in rats was not blocked by dipyridamole and it is likely that these observations account for the inability of dipyridamole to potentiate adenosine actions in the rat heart. In the present study, 2-chloroadenosine which is reported to be resistant to uptake and deamination (Clarke et ul., 1952; Rockwell and Maguire, 1966; Muller and Paton, 1979) was not significantly different from adenosine at producing negative inotropic and chronotropic effects. However, 2-chloroadenosine was about 100 times more potent than adenosine at producing a negative chronotropic effect in rat atria (Paton and Kurahashi, 1981). The lack of potentiation of the adenosine effect in the presence of dipyridamole, together with chloro substitution at the C2 position of the purine moiety, failing to result in a more potent analogue in the present study, may indicate an absence of an adenosine uptake system in dogfish atria. In the present study, the ventricle of the dogfish was shown to be insensitive to the effects of adenosine, ATP, APPCP and APCPP. This is consistent with reports on the hearts of mammals where adenosine does not have a direct effect on the action potentials or contractile properties of ventricles (Johnson and McKinnon, 1956; Lammerant and Becsei, 1973, Shah et ul., 1974; Schrader et ul., 1979; Endoh and Yamashita, 1980). The reptilian ventricle has also been reported to be insensitive to adenyl compounds (Meghji and Burnstock, 1983a). However, in frog ventricles ATP has been reported to be excitatory (Flitney et al., 1977; Burnstock and Meghji, 1981) while adenosine is inhibitory or inactive (Cook et al., 1958; Flitney et al., 1977; Singh and Flitney, 1980; Burnstock and Meghji, 1981). There are no reports at present of the responses of the teleostean ventricle to adenyl compounds. inotropic Positive effects of catecholamines were observed on the isolated ventricular strips in the present study. The order of potency of these catecholamines for their inotropic effects (adrenaline ) noradrenaline) was the same as that which has been previously demonstrated in teleost (Falck et al., 1966; Gannon, 1971) and elasmobranch fish hearts (ijstlund, 1954; Capra and Satchell, 1977). Acetylcholine also produced a positive inotropic effect in dogfish ventricle. A lack of negative inotropic effect to cholinergic agonists is also found in ventricles of teleost fish (Gannon, 1971; Holmgren, 1977). In the elasmobranch fish heart, acetylcholine is reported to sometimes cause an initial inhibitory effect followed by a positive inotropic and chronotropic effect (ijstlund, 1954). This adrenaline-like effect of acetylcholine is also seen in the ventricles of higher animals (McDowall, 1944; Hoffman et at., 1945; Burn and Rand, 1965). It is concluded that inhibitory P,-purinoceptors are present in the dogfish atrium. However, adenyl compounds had no direct the dogfish ventricle.
action
on the
contractility
of
299
heart
Acknowledgements-We
are grateful to the Department of Biophysics, University College London for providing aquarium facilities, to Dr Stephanie Clark, Judith Hills and Lorna Meldrum for their helpful comments on the manuscript and to Mary Sheridan for the typing. REFERENCES
Burn J. H. and Rand M. J. (1965) Acetylcholine in adrenergic transmission. A. Reo. Pharmac. 5, 163-182. Burnstock G. (I 978) A basis for distinguishing two types of purinergic receptor. In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach (Edited by Straub R. W. and Bolis, L.), pp. 1077118. Raven Press. New York. Burnstock G. (1980) Purinergic receptors m the heart. Circulation Res. 46, Suppl I, 175-182. Burnstock G. and Meghji P. (1981) Distribution of P,- and P1-purinoceptors in the guinea-pig and frog heart. Br. J. Pharmuc. 73, 8799885. Burnstock G. and Meghji P. (1983) The effect of adenyl compounds on the rat heart. Br. J. Pharmac. 79,211~218. Capra M. F. and Satchel1 G. H. (1977) Adrenergic and cholinergic responses of the isolated saline-perfused heart of the elasmobranch fish Squalus acanthias. Gen. Pharmat. 8, 59-65. Clarke D. A., Davoll J., Philips F. S. and Brown G. B. (I 952) Enzymatic deamination and vasodepressor effects of adenosine analogs. J. Pharmac. exp. Ther. 106, 291-302. Cohen S., Rotmensch H. H., Rubinstein R. and Lass Y. (1981) Lack of uptake or degradation of adenosine in the termination of its action in the beating carp atrium. Biochem. Pharmac. 30, 89&893. Collis M. G. and Pettinger S. J. (1982) Can ATP stimulate P,-receptors in guinea-pig atrium without conversion to adenosine? Eur. J. Pharmac. 81, 521-529. Cook M. H., Greene E. A. and Lorber V. (1958) Effect of purine and pyrimidine ribosides on an isolated frog ventricle preparation. Circulation Res. 6. 7355739. De Gubarelf T: and Sleator W. (I 965) Effects of caffeine on mammalian atrial muscle and its interaction with adenosine and calcium. J. Pharmac. exp. Ther. 148,202-214. Drury A. N. and Szent-Gyorgyi A. (1929) The physiological activity of adenine compounds with special reference to their action upon the mammalian heart. J. Physiol., Land. 68, 213.-237. Endoh M. and Yamashita S. (1980) Adenosine antagonizes the inotropic action mediated via /I-. but not a-adrenoceptors in the rabbit papillary muscle. Eur. J. Pharmac. 65, 445448. Falck B., Von Mecklenburg C., Myhrberg H. and Persson H. (1966) Studies on adrenergic and cholinergic receptors the isolated hearts of Lampetra Jtuviatilis kyclostomata) and Pleuronectes Piatessa (Teleostei). Acta physiol. stand. 68, 6471. Fedan J. S., Hogaboom G. K., O’Donnell J. P., Colby J. and Westfall D. P. (1981) Contribution by purines to the neurogenic response of the vas deferens of the guinea-pig. Eur. J. Pharmar. 69, 41-53. Flitney F. W., Lamb J. F. and Singh J. (1977) Effects of ATP on the hypodynamic frog ventricle. J. Physiol., Lond. 273, 5&52P. Cannon B. J. (1971) A study of the dual innervation of the teleost heart by a field stimulation technique. Camp. gen. Pharmac. 2, 175-l 83. Goto M., Yatani A. and Tsuda Y. (1977) An analysis of the action of ATP and related compounds on membrane current and tension components in bullfrog atria1 muscle. Jap. J. Physiol. 27, 81-94. Hartzell H. C. (1979) Adenosine receptors in frog sinus venosus. slow inhibitory potentials produced by adenine compounds and acetylcholine. J. Physiol., Lond. 293, 23-49.
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Hoffmann F., Hoffmann E. J., Middleton S. and Talesnik J. (1945) The stimulating effect of acetylcholine on the mammalian heart and the liberation of an epinephrinelike substance by the isolated heart. Am. J. Physiol. 144, 189-198. Hogaboom G. K., O’Donnell J. P. and Fedan J. S. (1980) Purinergic receptors: photoaffinity analog of adenosine triphosphate is a specific adenosine triphosphate antagonist. Science 208, 1273-1276. Hollander P. B. and Webb J. L. (1957) Effects of adenine nucleotides on the contractility and membrane potentials of rat atrium. Circulation Res. 5, 349-353. Holmgren S. (1977) Regulation of the heart of a teleost, Gadus morhua, by autonomic nerves and circulating catecholamines. Acta physiol. stand. 99, 62-74. Hopkins S. V. and Goldie R. G. (I 97 I) A species difference in the uptake of adenosine by heart. Biochem. Pharmac. 20, 3359-3365. James T. N. (1965) The chronotropic action of ATP and related compounds studied by direct perfusion of the sinus node. J. Pharmac. exp. Ther. 149, 233-247. Johnson E. A. and McKinnon M. G. (1956) Effect of acetylcholine and adenosine on cardiac cellular potentials. Nature, Land. 178, 1174-1175. Kolassa N., Pfleger K. and Tram M. (1971) Species differences in action and elimination of adenosine after dipyridamole and hexobendine. Eur. J. Pharmac. 13, 320-325. Lammerant J. and Becsei I. (1973) Left ventricular contractility and developed tension in the intact dog submitted to an intracoronary infusion of adenosine. J. PhysioL, Lond. 229, 41-49. Maguire M. H. and Satchel1 D. G. (1979) The contribution of adenosine to the inhibitory actions of adenine nucleotides on the guinea-pig taenia coli: studies with phosphate-modified adenine nucleotide analogs and dipyridamole. J. Pharmac. exp. Ther. 211, 626631. McDowall R. J. S. (1944) The stimulating action of acetylcholine on the heart. J. Physiol., Land. 103, 33P. Meghji P. and Burnstock G. (1983a) Absence of purinoceptors in the heart of the turtle (Emys orbicularis). Comp. Biochem. Physiol. 76C, 255-257. Meghji P. and Burnstock G. (1983b) An unusual excitatory action of adenosine on the ventricular muscle of the South African clawed toad (Xenopus laevis). Eur. J. Pharmac. 89, 251-258.
GEOFFREY BURNSTOCK Muller M. J. and Paton D. M. (1979) Presynaptic inhibitory actions of 2-substituted adenosine derivatives on neurotransmission in rat vas deferens: effects of inhibitors of adenosine uptake and deamination. NaunynSchmiedeberg’s Arch. Pharmac. 306, 23-28. Niedergerke R. and Page S. (1981) Two physiological agents that appear to facilitate calcium discharge from the sarcoplasmic reticulum in frog heart cells: adrenalin and ATP. Proc. R. Sot. Lond. B. 213, 325-344. dstlund E. (1954) The distribution of catecholamines in lower animals and their effect on the heart. Acta. physiol. stand. 31, Suppl. 112, l-67. Paton D. M. and Kurahashi K. (1981) Structure-activity relations for negative chronotropic action of adenosine in isolated rat atria: evidence for an action on A, receptors. IRCS Med. Sci. 9, 447. Rockwell M. and Maguire M. H. (1966) Studies on adenosine deaminase. I. Purification and properties of ox heart adenosine deaminase. Molec. Pharmac. 2, 574584. Rotmensch H. H., Cohen S., Rubinstein R. and Lass Y. (1981) Effects of adenosine in the isolated carp atrium. Israel J. Med. Sci. 11, 393. Satchel1 D. G. and Maguire M. H. (1975) Inhibitory effects of adenine nucleotide analogs on the isolated guinea-pig taenia coli. J. Pharmac. exp. Ther. 195, 54&548. Schrader J., Gerlach E. and Baumann G. (1979) Sites and mode of action of adenosine in the heart. II. Ventricular myocardium. In Physiological and Regulatory Functions of Adenosine and Adenine Nucleotides (Edited by Baer H. P. and Drummond G. I.), pp. 137-144. Raven Press, New York. Shah A., Kechejian S. J., Kavaler F. and Fisher V. J. (1974). Effects of adenine nucleotides on contractility of normal and postischemic myocardium. Am. Heart. J. 87, 740-749. Singh J. and Flitney F. W. (1980) Adenosine depresses contractility and stimulates 3’,5’ cyclic nucleotide metabolism in the isolated frog ventricle. J. molec. cell. Cardiol. 12, 285-297. Stafford A. (1966) Potentiation of adenosine and the adenine nucleotides by dipyridamole. Br. J. Pharmac. Chemother. 28, 2 18-227. Yatani A., Goto M. and Tsuda Y. (1978) Nature of catecholamine-like actions of ATP and other energy rich nucleotides on the bullfrog atrial muscle. Jap. J. Physiol. 28, 47-6 I.
0306~4492/84$3.00+ 0.00 0 1984 Pergamon Press Ltd
Printed in Great Britain
THE EFFECT OF ADENYL COMPOUNDS ON THE HEART OF THE DOGFISH, SCYLIORHINUS CANICULA Department
PARVIZ MEGHJI and GEOFFREY BURNSTOCK* of Anatomy and Embryology and Centre for Neuroscience, University College London, Gower Street, London WCIE 6BT, UK. Telephone: 01-387-7050 (Received 29 June 1983)
Abstract-l.
The effects of adenyl compounds were examined on dogfish atria and ventricles. 2. Adenosine, ATP, b, y-methylene ATP (APPCP) and 2-chloroadenosine produced negative inotropic and chronotropic effects on the dogfish atrium, which were antagonized by 8-phenyltheophylline, a P,-purinoceptor antagonist. 3. tr-fi-Methylene ATP (APCPP), which is resistant to degradation, did not produce a similar inhibitory response in the dogfish atrium. 4. Atropine did not affect the responses to adenosine, indicating that adenosine did not produce its effects indirectly by the release of acetyicholine. 5. The effects of adenosine and ATP were not potentiated by dipyridamole, which blocks adenosine uptake; and 2-chloroadenosine, which is reported lo be resistant to uptake and deamination, was equipotent with adenosine; this suggests the absence of an adenosine uptake system. 6. Dogfish ventricles were insensitive to adenyl compounds. Adrenaline, noradrenaline and acetylcholine produced positive inotropic effects on the ventricle. 7. It is concluded that inhibitory P,-purinoceptors are present in the dogfish atrium. However, adenyl compounds had no direct action on the contractility of the dogfish ventricle.
INTRODIJCTION
(1978) proposed that receptors for adenyl compounds could be classified into two types: P,-purinoccptors, which are most sensitive to adenosine, are competitively blocked by methylxanthines, and occupation of which leads to changes in cyclic adenosine 3’, S-monophosphate (cyclic AMP) accumulation; and P,-purinoceptors, which are most sensitive to ATP, do not appear to act via an adenylate cyclase system and are blocked (although not competitively or specifically) by quinidine, 2-substituted imidazolines, 2,2’-pyridylisatogen and apamin (by blockade of potassium channels). More recently arylazido aminopropionyl ATP (ANAPP,) has been claimed to be a specific P,-purinoceptor antagonist (Hogaboom et al., 1980; Fedan et al., 1981). Since the studies of Drury and Szent-Gyiirgyi (1929) the inhibitory effects of adenyl compounds on the heart have been described in many species. Studies on both mammalian and amphibian atria have shown that adenosine and adenine nucleotides have an overall effect similar to that of acetylchohne: they cause the atrium to beat more slowly and also to contract less forcibly (Hollander and Webb, 1957; De Gubareff and Sleator, 1965; James. 1965: Hartzell. 1979; Burnstock and Meghji, 1981: Niedergerke and Page. 1981). These inhibitorv effects are mediated via P,-purinoceptors (Burnstock, 1980; Burnstock and Meghji, 1981; Collis and Pettinger, 1982). In addition to the inhibitory effects, an initial positive inotropic effect to ATP, but not to adenosine, has been reported in the amphibian atrium (Goto et al., 1977;
Niedergerke and Page, 1981; Burnstock and Meghji, 1981), and it is likely that these effects are mediated via P,-purinoceptors. Adenyl compounds do not have a direct effect on the action potentials or contractile properties of mammalian ventricles (Johnson and McKinnon, 1956; Lammerant and Becsei, 1973; Shah et al., 1974, Schrader et al., 1979; Endoh and Yamashita, 1980). ATP is excitatory in the frog ventricle (Flitney et at., 1977; Burnstock and Meghji, 1981) and adenosine has been reported to be inhibitory or inactive in the frog ventricle (Cook er nl., 1958; Fhtney ct al., 1977; Singh and Flitney, 1980; Burnstock and Meghji, 1981). Although there are reports in the literature on the effects of adenyl compounds on teleost fish heart (Cohen et af., 1981; Rotmensch ef al., 1981), the effects of these compounds on the heart of elasmobranch fish have not been studied. Thus the aim of the present study was to examine whether purinoceptors are present in the dogfish heart.
Burnstock
*To whom all correspondence
should be addressed.
MATERIALS AND METHODS Adult spotted dogfish Scyiiorhinus canicula (50-70 cm in length, N = 19) were obtained from the Marine Biologicai Association, Plymouth, and were kept in a circulating sea-water aquarium at lC-12°C. The fish were decapitated and pithed. The hearts were removed and placed in elasmobranch fish Ringer of the following composition (mM): NaCl. 280; KCl, 3; CaCl,. 4; MaCl,. 0.5: NaHCO,. 3: Hepes, 5; Urea, 360 and Glucose, iO,which was bubbled with oxygen. Strips of ventricle, which were mounted on platinum strip electrodes (separated by 7mm), and atria, together with the sinus venosus, were transferred to 1Oml overtlow organ baths at room temperature (1%22°C). A resting tension of 0.5g was applied to the preparations. Atria were allowed to beat spontaneously. Ventricular strips
295
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PARVIZ MEGHJI and
were stimulated at 0.5 Hz using 5 msec pulses of twice threshold voltage using a Grass SD9 stimulator. The mechanical activity was recorded isometrically by means of a Grass FT 03C force transducer and displayed on a Grass model 79D polygraph. The preparations were allowed to equilibrate for 60min. The bathing solution was changed every I5 min during that time. In the atria cumulative concentration-response curves were obtained to adenosine. ATP, p.y-methylene ATP (APPCP), a,p-methylene ATP (APCPP) and 2-chloroadenosine. Cumulative concentration-response curves to adenosine, ATP and APPCP were also obtained in the presence of 8%PT. Cumulative concentrationresponse curves to adenosine and ATP were obtained in the
absence and in the presence of dipyridamole. In some experiments, responses to single concentrations of adenosine and acetylcholine were compared in the absence and in the presence of atropine. In experiments where 8-PT. dipyridamole and atropine were used, they were added to the organ bath 20min before their effects were investigated. cumulative concentration-response In the ventricle, curves were obtained to adenosine, ATP, APPCP, APCPP, adrenaline, noradrenaline and acetylcholine. Responses were measured as percentage changes of basal levels. The mean and standard error of the mean were calculated for each group. Statistical analysis was carried out using Student’s t-test for paired and unpaired samples. A probability of P < 0.05 was considered to be significant.
GEOFFREY BURNSTWK
Incubation with dipyridamole (0.5 PM), an adenosine uptake blocker, for 20 min did not significantly potentiate the negative inotropic and chronotropic effects of adenosine (Fig. 4). Similar results were obtained for ATP. 2-Chloroadenosine (I-300 PM) produced negative inotropic and chronotropic effects which were not significantly different from those produced by adenosine at any of the concentrations tested (Fig. 4). Ventricle
ATP, adenosine, APPCP and APCPP produced changes in the force of contraction of less than 10% which were not significant (Fig. 5). Adrenaline, noradrenaline and acetylcholine produced concentration-dependent positive inotropic effects (Fig. 5). Adrenaline was 170 times more potent than noradrenaline at producing these effects; the EC,, values of adrenaline and noradrenaline being 0.025 _t 0.008 and 4.2 k 1.O PM respectively.
Drugs and SOILWIS Adenosine, adenosine 5’-triphosphate, fl,;~-methylene adenosine 5’-triphosphate (APPCP), r,a-methylene adenosine 5’-triphosphate (APCPP), 2-chloroadenosine, acetylcholine, adrenaline and noradrenaline were obtained from Sigma. Dipyridamole was obtained from Boehringer Ingelheim, I-phenyltheophylline (8-PT) from Calbiochem and atropine from Antigen Ltd. Ascorbic acid (I 00 p M) was added to solutions of adrenaline and noradrenaline to prevent their rapid oxidation. A stock solution of IOmM 8-PT was made up in 80% v/v methanol containing 0.2 M NaOH. All other drugs were dissolved in distilled water.
RESULTS Atrium
Adenosine, ATP and APPCP (l-300 PM) reduced the force and rate of contraction of dogfish atria in a concentration-dependent manner (Fig. 1). The effects of adenosine, ATP and APPCP were not significantly different from each other at any of the concentrations tested. APCPP (lo-300 PM) produced changes of less than 5% in the force and rate of contraction (Fig. 1). These changes were not significant. Examples of the effects of adenosine, ATP, APPCP and APCPP are shown in Fig. 2a. Both negative inotropic and chronotropic effects of adenosine, ATP and APPCP were antagonized by S-PT (Fig. 2), the concentration-response curves to adenosine and ATP being shifted 20-fold to the right (Fig. 1). The effects of APPCP were antagonized to a greater extent than the effects of adeno:ine and ATP (Fig. 1). Acetylcholine (10 p M) produced a negative inotropic and chronotropic effect (Fig. 3). The responses to adenosine (10 PM) were not affected by a concentration of atropine (1.4 PM) that significantly antagonized the responses to 10pM acetylcholine (Fig. 3).
Fig. I Log concentration-response curves of the spontaneously beating dogfish atrium to (a) the intropic and (b) the chronotropic effects of (0) adenosine, (m) ATP, (A) fl,y-methylene ATP (APPCP) and (v) cc,b-methylene ATP (APCPP) in the absence of R-phenyltheophylline (I-PT) and (0) adenosine, (0) ATP and (A) APPCP in the presence of IOpM 8-PT. Each point is the mean of observations from at least six preparations. Vertical bars show SEM. Asterisks indicate the significance of the differences between control responses and responses obtained in the presence of 8-PT using the unpaired t-test. *P < 0.05; **P
Adenyl compounds on the dogfish
a
b
Adenosine
291
heart
+8-PT
Adenosine
(10 PM)
(10 PM)
ii
ii
1
I
ATP
ATP
clO~M)
0.59
(10pM’
l%ec
APPCP
APPCP
(10 PM)
(10pM)
iv
iv
APCPP
APC PP
(100 PM>
(100
PM’
Fig. 2. Responses of the spontaneously beating dogfish atrium to (i) adenosine, (ii) ATP, (iii) j,y-methylene ATP (APPCP) and (iv) r,b-methylene ATP (APCPP) (a) in the absence and (b) in the presence of 10 p M 8-phenyltheophylline (8%PT).
ii
, Acetylcholine b + Atropine
B
(10 PM)
ii
10.29 1Osec
I
1
Acetylcholine
Adenosine
(10 PM)
00 PM)
Acetylcholine
Adenosine
(10 PM)
Adenosine
b
ns
Fig. 3.(A) Responses of the spontaneously beating dogfish atrium to (i) acetylcholine and (ii) adenosine (a) in the absence and (b) in the presence of 1.4~ M atropine. (B) The effects of acetylcholine (10 p M) and adenosine (1OpM) in the absence (unshaded) and in the presence of 1.4pM atropine (shaded). Vertical columns, which show the percentage change in contractile force (a) and rate (b) of the atria1 preparations were constructed from the means of observations from four preparations. Vertical bars show SEM. Asterisks represent the significance of the difference between control responses and responses in the presence of atropine using the paired t-test. ns, not significant; *P < 0.05; **P -c 0.01.
PARVIZ MECHJI and
GEOFFREY BURNSTOCK DISCUSSION
The results of the present study have demonstrated the negative inotropic and chronotropic effects of adenosine, ATP and APPCP in the dogfish atrium. The responses of dogfish atria to ATP differ from
1oc
/-
8C
/-
6C
40
20
0 1
100
10 Concentrat,on
1000
(FM)
Fig. 4. Log concentration-response curves of the spontaneously beating dogfish atrium to (a) the inotropic and (b) the chronotropic effects of(m) 2-chloroadenosine (N = 4), (0) adenosine (N = 8) in the absence of and adenosine (N = 8) in the presence of 0.5 PM dipyridamole (0). N = the number of observations from which the means were calculated. Vertical bars show SEM. Control responses to adenosine were not significantly different from responses obtained in the presence of dipyridamole or to those obtained to 2-chloroadenosine, using the unpaired t-test.
those of frog atria where ATP has been reported to be excitatory (Goto et al., 1977; Yatani et al., 1978; Burnstock and Meghji, 1981), but are similar to those of mammalian atria (Hollander and Webb, 1957; De Gubareff and Sleator, 196.5; James, 1965; Burnstock and Meghji, 198 1) and teleostean atria (Cohen et al., 1981; Rotmensch et al., 1981). Reptilian atria have been reported to be insensitive to adenyl compounds (Meghji and Burnstock, 1983a). In the present study, the inhibitory effects of adenosine, ATP and APPCP were antagonized by %PT, a P,-purinoceptor antagonist, indicating that these effects are mediated via P,-purinoceptors. It is not clear why the responses to APPCP were affected to a greater extent than those to ATP and adenosine. It is probable that ATP is metabolized to adenosine and AMP before acting on the P,-purinoceptors, since APCPP, which is resistant to degradation (Satchel1 and Maguire, 1975; Maguire and Satchell, 1979), was without effect on the dogfish atrium. However, it is possible that APCPP has a lower affinity for the P,-purinoceptor than has ATP The possibility that adenine compounds may be producing their inhibitory effects indirectly by releasing acetylcholine from nerve terminals was negated since a concentration of atropine which significantly antagonized the effects of acetylcholine had no effect on the responses to adenosine. Dipyridamole did not potentiate the effects of ATP or adenosine in the dogfish atrium. The failure of
I-
T )-
,-
)-
, -
1-
, -
I-
0 01
0.1
1
10
Concentration
(PM)
100
1000
Fig. 5. Log concentration-response curves of electrically driven dogfish ventricular strips to the inotropic effects of: (0) adenosine, (W) ATP, (A) fi,y-methylene ATP (APPCP), (v) a,S-methylene ATP (APCPP), (0) adrenaline, (0) noradrenaline and (a) acetylcholine. Each point is the mean of observations from at least six preparations. Vertical bars show SEM. Asterisks indicate a significant response, using the paired f-test. *P -c 0.05; **P < 0.01; ***P < 0.001.
Adenyl compounds on the dogfish dipyridamole to potentiate adenosine effects has also been observed in rat atria (Stafford, 1966; Kolassa et al., 197 1; Burnstock and Meghji, 1983) and Xenopus laeuis atria (Meghji and Burnstock, 1983b). Hopkins and Goldie (1971) and Kolassa et al. (1971) have shown, with the use of 14C-adenosine, that the adenosine uptake process in rats was not blocked by dipyridamole and it is likely that these observations account for the inability of dipyridamole to potentiate adenosine actions in the rat heart. In the present study, 2-chloroadenosine which is reported to be resistant to uptake and deamination (Clarke et ul., 1952; Rockwell and Maguire, 1966; Muller and Paton, 1979) was not significantly different from adenosine at producing negative inotropic and chronotropic effects. However, 2-chloroadenosine was about 100 times more potent than adenosine at producing a negative chronotropic effect in rat atria (Paton and Kurahashi, 1981). The lack of potentiation of the adenosine effect in the presence of dipyridamole, together with chloro substitution at the C2 position of the purine moiety, failing to result in a more potent analogue in the present study, may indicate an absence of an adenosine uptake system in dogfish atria. In the present study, the ventricle of the dogfish was shown to be insensitive to the effects of adenosine, ATP, APPCP and APCPP. This is consistent with reports on the hearts of mammals where adenosine does not have a direct effect on the action potentials or contractile properties of ventricles (Johnson and McKinnon, 1956; Lammerant and Becsei, 1973, Shah et ul., 1974; Schrader et ul., 1979; Endoh and Yamashita, 1980). The reptilian ventricle has also been reported to be insensitive to adenyl compounds (Meghji and Burnstock, 1983a). However, in frog ventricles ATP has been reported to be excitatory (Flitney et al., 1977; Burnstock and Meghji, 1981) while adenosine is inhibitory or inactive (Cook et al., 1958; Flitney et al., 1977; Singh and Flitney, 1980; Burnstock and Meghji, 1981). There are no reports at present of the responses of the teleostean ventricle to adenyl compounds. inotropic Positive effects of catecholamines were observed on the isolated ventricular strips in the present study. The order of potency of these catecholamines for their inotropic effects (adrenaline ) noradrenaline) was the same as that which has been previously demonstrated in teleost (Falck et al., 1966; Gannon, 1971) and elasmobranch fish hearts (ijstlund, 1954; Capra and Satchell, 1977). Acetylcholine also produced a positive inotropic effect in dogfish ventricle. A lack of negative inotropic effect to cholinergic agonists is also found in ventricles of teleost fish (Gannon, 1971; Holmgren, 1977). In the elasmobranch fish heart, acetylcholine is reported to sometimes cause an initial inhibitory effect followed by a positive inotropic and chronotropic effect (ijstlund, 1954). This adrenaline-like effect of acetylcholine is also seen in the ventricles of higher animals (McDowall, 1944; Hoffman et at., 1945; Burn and Rand, 1965). It is concluded that inhibitory P,-purinoceptors are present in the dogfish atrium. However, adenyl compounds had no direct the dogfish ventricle.
action
on the
contractility
of
299
heart
Acknowledgements-We
are grateful to the Department of Biophysics, University College London for providing aquarium facilities, to Dr Stephanie Clark, Judith Hills and Lorna Meldrum for their helpful comments on the manuscript and to Mary Sheridan for the typing. REFERENCES
Burn J. H. and Rand M. J. (1965) Acetylcholine in adrenergic transmission. A. Reo. Pharmac. 5, 163-182. Burnstock G. (I 978) A basis for distinguishing two types of purinergic receptor. In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach (Edited by Straub R. W. and Bolis, L.), pp. 1077118. Raven Press. New York. Burnstock G. (1980) Purinergic receptors m the heart. Circulation Res. 46, Suppl I, 175-182. Burnstock G. and Meghji P. (1981) Distribution of P,- and P1-purinoceptors in the guinea-pig and frog heart. Br. J. Pharmuc. 73, 8799885. Burnstock G. and Meghji P. (1983) The effect of adenyl compounds on the rat heart. Br. J. Pharmac. 79,211~218. Capra M. F. and Satchel1 G. H. (1977) Adrenergic and cholinergic responses of the isolated saline-perfused heart of the elasmobranch fish Squalus acanthias. Gen. Pharmat. 8, 59-65. Clarke D. A., Davoll J., Philips F. S. and Brown G. B. (I 952) Enzymatic deamination and vasodepressor effects of adenosine analogs. J. Pharmac. exp. Ther. 106, 291-302. Cohen S., Rotmensch H. H., Rubinstein R. and Lass Y. (1981) Lack of uptake or degradation of adenosine in the termination of its action in the beating carp atrium. Biochem. Pharmac. 30, 89&893. Collis M. G. and Pettinger S. J. (1982) Can ATP stimulate P,-receptors in guinea-pig atrium without conversion to adenosine? Eur. J. Pharmac. 81, 521-529. Cook M. H., Greene E. A. and Lorber V. (1958) Effect of purine and pyrimidine ribosides on an isolated frog ventricle preparation. Circulation Res. 6. 7355739. De Gubarelf T: and Sleator W. (I 965) Effects of caffeine on mammalian atrial muscle and its interaction with adenosine and calcium. J. Pharmac. exp. Ther. 148,202-214. Drury A. N. and Szent-Gyorgyi A. (1929) The physiological activity of adenine compounds with special reference to their action upon the mammalian heart. J. Physiol., Land. 68, 213.-237. Endoh M. and Yamashita S. (1980) Adenosine antagonizes the inotropic action mediated via /I-. but not a-adrenoceptors in the rabbit papillary muscle. Eur. J. Pharmac. 65, 445448. Falck B., Von Mecklenburg C., Myhrberg H. and Persson H. (1966) Studies on adrenergic and cholinergic receptors the isolated hearts of Lampetra Jtuviatilis kyclostomata) and Pleuronectes Piatessa (Teleostei). Acta physiol. stand. 68, 6471. Fedan J. S., Hogaboom G. K., O’Donnell J. P., Colby J. and Westfall D. P. (1981) Contribution by purines to the neurogenic response of the vas deferens of the guinea-pig. Eur. J. Pharmar. 69, 41-53. Flitney F. W., Lamb J. F. and Singh J. (1977) Effects of ATP on the hypodynamic frog ventricle. J. Physiol., Lond. 273, 5&52P. Cannon B. J. (1971) A study of the dual innervation of the teleost heart by a field stimulation technique. Camp. gen. Pharmac. 2, 175-l 83. Goto M., Yatani A. and Tsuda Y. (1977) An analysis of the action of ATP and related compounds on membrane current and tension components in bullfrog atria1 muscle. Jap. J. Physiol. 27, 81-94. Hartzell H. C. (1979) Adenosine receptors in frog sinus venosus. slow inhibitory potentials produced by adenine compounds and acetylcholine. J. Physiol., Lond. 293, 23-49.
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Hoffmann F., Hoffmann E. J., Middleton S. and Talesnik J. (1945) The stimulating effect of acetylcholine on the mammalian heart and the liberation of an epinephrinelike substance by the isolated heart. Am. J. Physiol. 144, 189-198. Hogaboom G. K., O’Donnell J. P. and Fedan J. S. (1980) Purinergic receptors: photoaffinity analog of adenosine triphosphate is a specific adenosine triphosphate antagonist. Science 208, 1273-1276. Hollander P. B. and Webb J. L. (1957) Effects of adenine nucleotides on the contractility and membrane potentials of rat atrium. Circulation Res. 5, 349-353. Holmgren S. (1977) Regulation of the heart of a teleost, Gadus morhua, by autonomic nerves and circulating catecholamines. Acta physiol. stand. 99, 62-74. Hopkins S. V. and Goldie R. G. (I 97 I) A species difference in the uptake of adenosine by heart. Biochem. Pharmac. 20, 3359-3365. James T. N. (1965) The chronotropic action of ATP and related compounds studied by direct perfusion of the sinus node. J. Pharmac. exp. Ther. 149, 233-247. Johnson E. A. and McKinnon M. G. (1956) Effect of acetylcholine and adenosine on cardiac cellular potentials. Nature, Land. 178, 1174-1175. Kolassa N., Pfleger K. and Tram M. (1971) Species differences in action and elimination of adenosine after dipyridamole and hexobendine. Eur. J. Pharmac. 13, 320-325. Lammerant J. and Becsei I. (1973) Left ventricular contractility and developed tension in the intact dog submitted to an intracoronary infusion of adenosine. J. PhysioL, Lond. 229, 41-49. Maguire M. H. and Satchel1 D. G. (1979) The contribution of adenosine to the inhibitory actions of adenine nucleotides on the guinea-pig taenia coli: studies with phosphate-modified adenine nucleotide analogs and dipyridamole. J. Pharmac. exp. Ther. 211, 626631. McDowall R. J. S. (1944) The stimulating action of acetylcholine on the heart. J. Physiol., Land. 103, 33P. Meghji P. and Burnstock G. (1983a) Absence of purinoceptors in the heart of the turtle (Emys orbicularis). Comp. Biochem. Physiol. 76C, 255-257. Meghji P. and Burnstock G. (1983b) An unusual excitatory action of adenosine on the ventricular muscle of the South African clawed toad (Xenopus laevis). Eur. J. Pharmac. 89, 251-258.
GEOFFREY BURNSTOCK Muller M. J. and Paton D. M. (1979) Presynaptic inhibitory actions of 2-substituted adenosine derivatives on neurotransmission in rat vas deferens: effects of inhibitors of adenosine uptake and deamination. NaunynSchmiedeberg’s Arch. Pharmac. 306, 23-28. Niedergerke R. and Page S. (1981) Two physiological agents that appear to facilitate calcium discharge from the sarcoplasmic reticulum in frog heart cells: adrenalin and ATP. Proc. R. Sot. Lond. B. 213, 325-344. dstlund E. (1954) The distribution of catecholamines in lower animals and their effect on the heart. Acta. physiol. stand. 31, Suppl. 112, l-67. Paton D. M. and Kurahashi K. (1981) Structure-activity relations for negative chronotropic action of adenosine in isolated rat atria: evidence for an action on A, receptors. IRCS Med. Sci. 9, 447. Rockwell M. and Maguire M. H. (1966) Studies on adenosine deaminase. I. Purification and properties of ox heart adenosine deaminase. Molec. Pharmac. 2, 574584. Rotmensch H. H., Cohen S., Rubinstein R. and Lass Y. (1981) Effects of adenosine in the isolated carp atrium. Israel J. Med. Sci. 11, 393. Satchel1 D. G. and Maguire M. H. (1975) Inhibitory effects of adenine nucleotide analogs on the isolated guinea-pig taenia coli. J. Pharmac. exp. Ther. 195, 54&548. Schrader J., Gerlach E. and Baumann G. (1979) Sites and mode of action of adenosine in the heart. II. Ventricular myocardium. In Physiological and Regulatory Functions of Adenosine and Adenine Nucleotides (Edited by Baer H. P. and Drummond G. I.), pp. 137-144. Raven Press, New York. Shah A., Kechejian S. J., Kavaler F. and Fisher V. J. (1974). Effects of adenine nucleotides on contractility of normal and postischemic myocardium. Am. Heart. J. 87, 740-749. Singh J. and Flitney F. W. (1980) Adenosine depresses contractility and stimulates 3’,5’ cyclic nucleotide metabolism in the isolated frog ventricle. J. molec. cell. Cardiol. 12, 285-297. Stafford A. (1966) Potentiation of adenosine and the adenine nucleotides by dipyridamole. Br. J. Pharmac. Chemother. 28, 2 18-227. Yatani A., Goto M. and Tsuda Y. (1978) Nature of catecholamine-like actions of ATP and other energy rich nucleotides on the bullfrog atrial muscle. Jap. J. Physiol. 28, 47-6 I.