An unusual excitatory action of adenosine on the ventricular muscle of the south african clawed toad (Xenopus laevis)

European Journal of Pharmacology, 89 (1983) 251-258

251

Elsevier Biomedical Press

AN U N U S U A L EXCITATORY ACTION OF A D E N O S I N E ON T H E V E N T R I C U L A R M U S C L E OF T H E S O U T H AFRICAN CLAWED TOAD (Xenopus laevis) PARVIZ MEGHJI and G E O F F R E Y B U R N S T O C K

*

Department of Anatomy and Embryology and Centre for Neuroscience, University College London, Gower Street, London WCIE 6BT, U.K. Received 11 October 1982, revised MS received 18 January 1983, accepted 3 February 1983

P. MEGHJI and G. BURNSTOCK, An unusual excitatory action of adenosine on the ventricular muscle of the South African clawed toad (Xenopus laevis), European J. Pharmacol. 89 (1983) 251-258. The effects of adenyl compounds and some of their analogues were examined on atrial and ventricular muscle of Xenopus laevis. In contrast to all heart muscle preparations studied previously, adenosine produced excitation of the ventricle of Xenopus. 2-Chloroadenosine, ATP,/3,~,-methylene ATP (APPCP) and a,/3-methylene ATP (APCPP) also elicited excitation of Xenopus ventricles; the order of potency being 2-chloroadenosine > ATP >/adenosine > APPCP = APCPP. The excitatory effects of 2-chloroadenosine, adenosine, ATP and APPCP were antagonised by 8-phenyltheophylline (8-PT). A small excitatoty response to ATP persisted in the presence of 8-PT. The effects of adenosine were not potentiated by dipyridamole. Propranolol antagonised the excitatory responses to adrenaline, phenylephrine, 5-hydroxytryptamine (5-HT) and dopamine. High concentrations of histamine were needed to produce even small excitatory responses. Neither propranolol nor phentolamine affected the responses to adenosine and ATP. Adenosine, ATP and APPCP elicited inhibition of Xenopus atria which was antagonised by 8-PT but was not potentiated by dipyridamole. APCPP, which is resistant to degradation, did not produce a similar inhibitory response. It is concluded that excitation, mediated largely by excitatory Pl- and possibly by some P2-purinoceptors, occurs in the Xenopus ventricle and that there are inhibitory Pl-purinoceptors in the Xenopus atrium. ATP

Adenosine

Purinoceptor

Heart

Xenopus

1. Introduction The effects of exogenous ATP and adenosine on the frog heart have been well documented. ATP is excitatory (Cook et al., 1958; Versprille, 1963; Flitney et al., 1977; Goto et al., 1977; Burnstock and Meghji, 1981) whereas adenosine has been reported to be inhibitory or inactive (Cook et al., 1958; Flitney et al., 1977; Burnstock and Meghji, 1981) in the heart of many species of frogs. It has been proposed (Burnstock, 1978), mainly on the basis of the relative potencies of adenosine, AMP, A D P and ATP, that receptors for purine nucleotides and nucleosides can be classified into

* To whom all correspondence should be addressed. 0 0 1 4 / 2 9 9 9 / 8 3 / 0 0 0 0 - 0 0 0 0 / $ 0 3 . 0 0 © 1983 Elsevier Biomedical Press

two types: Pl-purinoceptors, which are most sensitive to adenosine, are competitively blocked by methylxanthines, and occupation of which leads to changes in cyclic adenosine Y,5'-monophosphate (cyclic AMP) accumulation; and P2-purinoceptors which are most sensitive to ATP and which are blocked (although not competitively) by quinidine, 2-substituted imidazolines, 2,2'-pyridylisatogen, apamin (by blockade of potassium channels) and arylazido aminopropionyl ATP (ANAPP3) which has been claimed to be a specific P2-purinoceptor antagonist (Hogaboom et al., 1980; Fedan et al., 1981). The P~-purinoceptor agonist, adenosine, produces inhibitory effects in all tissues studied to date except for trout arterial gill vessels, where it causes vasoconstriction (Colin and Leray, 1979;

252 Colin et al., 1979). This appears to be a direct effect of adenosine which is antagonised by theophylline, a P~-antagonist (Colin and Leray, 1979; Colin et al., 1979). Adenosine also produces vasoconstriction in some other tissues; this is not a direct effect on purinoceptors but is via the release of 5-hydroxytryptamine (5-HT) in the femoral vascular bed of the rat (Sakai and Akima, 1977; 1978; Sakai, 1978; Sakai et al., 1979) and in the rat tail artery (Brown and Collis, 1981), and via angiotensin II in dog kidney (Osswald et al., 1979). In the present study, investigation of the effects of adenosine, ATP and their analogues has been extended to the atria and ventricles of Xenopus laevis as this has not been done previously. Although Xenopus laevis is usually referred to as ' T h e South African Clawed Toad', it belongs to the frog family Pipidae (Deuchar, 1975).

2. Materials and methods

Frogs (Xenopus laevis) were stunned by a blow to the back of the head, decapitated and pithed. The hearts were removed and placed in modified oxygenated Ringer solution (Gambhir and Tripathi, 1978). Atria, together with the sinus venosus, and ventricular strips were set up in 10 ml organ baths at room temperature (17-20°C). A resting tension of 0.5 g was applied to the preparations. Ventricular strips were stimulated at 0.5 Hz using 5 ms pulses of twice threshold voltage. Atria were usually allowed to beat spontaneously, but some preparations were electrically driven at 1 Hz using 5 ms pulses of twice threshold voltage. The mechanical activity was recorded isometrically by means of a Grass FT 03C force transducer and a Grass model 79D polygraph. The preparations were allowed to equilibrate for 60 rain before addition of drugs. The bathing solution was changed every 15 min during the equilibration period. Cumulative log concentration-response curves to adenosine, ATP, 2-chloroadenosine, /~,7-methylene A T P (APPCP), a,/~-methylene A T P (APCPP), inosine and guanosine were obtained in the ventricle. In some experiments, responses to single concentrations of adenosine, ATP, adrenaline, phenylephrine, dopamine, 5-hydroxy-

tryptamine (5-HT) and histamine were compared in the absence and in the presence of an c~-adrenoceptor antagonist, phentolamine, a /~-adrenoceptor antagonist, propranolol, and a P~-purinoceptor antagonist, 8-phenyltheophylline (8-PT). In the atria, log concentration-response curves were constructed from responses obtained to single additions of ATP, adenosine, APPCP and APCPP because the responses obtained were sometimes biphasic. In experiments where 8-PT, dipyridamole and propranolol were used, they were added to the organ baths 20 rain before their effects were investigated. In experiments where indomethacin was used, the tissues were incubated with indomethacin for 1 h before its effect was examined.

2.1. Drugs and solvents Adenosine, adenosine 5'-triphosphate, /LYmethylene adenosine 5'-triphosphate (APPCP), c~,/~-methylene adenosine 5'-triphosphate (APCPP), 2-chloroadenosine, guanosine, inosine, acetylcholine, adrenaline, dopamine, histamine, 5-hydroxytryptamine (5-HT), indomethacin, phenylephrine and propranolol were obtained from Sigma. Dipyridamole was obtained from Boehringer Ingelheim, 8-phenyltheophylline (8-PT) from Calbiochem and phentolamine mesylate from Ciba. Indomethacin was made up in 0.2 M sodium carbonate solution. Ascorbic acid (100 /~M) was added to solutions of adrenaline and dopamine to prevent their rapid oxidation. A stock solution of 10 mM 8-PT was made up in 80% v / v methanol containing 0.2 M N a O H . Addition of 8-PT stock solution (or an equivalent concentration of solvent) to a final concentration of 10 5 M 8-PT altered the pH of the Ringer solution from 7.1 to 7.4. The solvent alone did not exert a direct effect on the heart preparations nor did it affect the responses produced by adenosine. All other drugs were dissolved in distilled water.

2.2. Analysis of results Responses were measured as the percentage changes from basal levels. Means and standard errors of the mean were calculated for each group. Statistical significance was evaluated by Student's

253

t-test for unpaired samples, and P values of 0.05 or less were considered to be significant.

120

100

IC

3. Results "6

3. l. Ventricular strips

80 b

60

Adenosine, 2-chloroadenosine, ATP, APPCP and APCPP produced positive inotropic effects in Xenopus ventricle (fig. 1A). ATP was equipotent with adenosine over the concentration range 3-100 /~M; however, ATP produced a significantly larger response at a concentration of 300/~M (P < 0.05). The order of potency of these compounds in producing positive inotropic effects was 2-chloroadenosine > ATP >~ adenosine > APPCP = APCPP. High concentrations of inosine and guanosine (100-300 ffM) produced increases of less than 5% in the contractile force of the ventricle (fig. 1A). The excitatory effects of 2-chloroadenosine, adenosine, ATP and APPCP were antagonised by 8-PT (fig. 1B). The responses to APCPP were not affected by 8-PT. 8-PT completely antagonised the excitatory responses to adenosine (300 ffM) reverting the positive inotropic effect to a small inhibitory response; however, a small excitatory response to ATP (300 ffM) persisted in the presence of 8-PT. Excitatory responses to 2-chloroadenosine (100-300 ~M) also persisted in the presence of 8-PT. Dipyridamole (0.5 I~M) did not potentiate the responses to adenosine. Adrenaline (1-100 nM) produced excitatory responses of a size comparable to those produced by adenosine (300 ~M) and ATP (300 ~M). Propranolol (0.5 ffM) completely antagonised the responses to adrenaline without affecting the responses produced by ATP or adenosine (fig. 2A,C). 8-PT (10 /xM) antagonised the effects of ATP and adenosine but did not affect the responses produced by adrenaline (fig. 2B, C). Phentolamine (3 ffM) did not antagonise the effects of adrenaline, ATP nor adenosine. Incubation with indomethacin (20 ffM) for 60 min did not affect the responses to adenosine, ATP nor adrenaline. The effects of phenylephrine, 5-HT, histamine and dopamine were also examined on the Xenopus ventricle. 5-HT (300 ~M), dopamine (100/~M) and

a

40

E & 20 e

fi 0

I 10

;

1~}0

i

1000

Concentration (,uM)

B

60

E_ o

40

"5 ~'~ ~

20 0

~

a

*

*

*

*

~J

-20

i

i

10

100

i

1000

Concent ration Cu M )

Fig. 1. (A) Log concentration-response curves of electrically driven Xenopus ventricular strips to the inotropic effects of (a) adenosine, (b) ATP, (c) 2-chloroadenosine, (d) fl, y-methylene ATP (APPCP), (e) a,fl-methylene ATP (APCPP), (f) guanosine and (g) inosine. (B) Log concentration-response curves of electrically driven Xenopusventricular strips to the inotropic effects of (a) adenosine, (b) ATP, (c) 2-chloroadenosine and (d) APPCP in the presence of 10 t~M 8-phenyltheophylline (8-PT). Each point is the mean of at least 6 observations from at least 3 animals. Vertical bars show S.E.M. Asterisks represent the significance of the differences between control responses and responses obtained in the presence of 8-PT. * P < 0.05; ** P < 0.01; *** P < 0.001.

phenylephrine (10-30 /~M) produced excitatory responses of a magnitude that was comparable to that produced by ATP (300 #M) and adenosine (300 ffM). However, in 2 out of 6 preparations tested, an inhibitory response was produced by

254 A*

_A adrenaline 30 nM

adenosine 300 #M

ATP 300 gM

adenosine 300 9M

kTP

b + propranolol

m adrenaline 30 nM

300 #M

05g

Ba

lmin

dopamine. In the presence of propranolol (0.5 ffM), dopamine (100 ffM) produced an inhibitory response. The excitatory responses to 5-HT and phenylephrine were antagonised by propranolol (0.5 ffM). High concentrations (100-300 ffM) of histamine produced an increase in ventricular contractile force of less than 10%. Acetylcholine (0.1 300 ffM) produced a concentration-dependent negative inotropic effect. 3.2. Atria

b+8-PT

adrenaline 30 nM

adenosine 300 #M

ATP 300pM

Adenosine, ATP and APPCP reduced the rate and force of contraction of spontaneously beating adrenaline 30 nM

l°°ff

adenos, ne 300 pM

adrenaline (10-30 nM)

adenosine (300 ~M)

_c o 60~~ c

~o

ATP 300 ,uM ATP b (300 JJM) ns

a ns

40

20

Fig. 2. (A) Responses of Xenopus ventricular strips to adrenaline, adenosine and ATP (a) in the absence and (b) in the presence of propranolol (0.5 p,M). (B) Responses of Xenopus

ventricular strips to adrenaline, adenosine and ATP (a) in the absence and (b) in the presence of 8-phenyltheophylline (8-PT) (10/*M). (C) The effects of adrenaline, adenosine and ATP in the absence (a) and in the presence of (b) propranolol (0.5 ffM), (c) 8-PT (10 t-tM) and (d) propranolol (0.5 ffM) and 8-PT (10 ffM). Vertical columns, which show the percentage change in contractile force of the Xenopus ventricular strips, are constructed from the means of at least 2 observations from at least 2 animals. Vertical bars show S.E.M. Asterisks represent the significance of the differences between control responses and responses in the presence of 8-PT a n d / o r propranolol, ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

8

A

$ 7°I 60 £ 5 o 50

~ 6o E o o

5O

Aden 5

~P 40

o)

2 m

40

APPCP

APPCP 30

g ~ 2o

"~ 20

g_

5_

/5 10

to

..c

c

N

o

1'

1'o Concentration

lOO ' (uM)

o

i

lO Concentration (:jaM)

i

1dO

Fig. 3. Log concentration-response curves of spontaneously beating atria of Xenopus to (A) the negative chronotropic and (B) the negative inotropic effects of adenosine (Aden), ATP and fl,y-methylene ATP (APPCP). Each point is the mean of observations from at least 4 preparations. Vertical bars show S.E.M.

255

Xenopus atria in a concentration-dependent

4° F c 35

o

~ 3o

Aden 1

ATP

×/¢

~' , 20

~

15

c 10

jjf

~o

Z/f/- ) .J

I

I

10 100 Concentration [taM )

V

APPCPJ

Aden -

T **ATP I

o-o.

I

1OOO

Fig. 4. Log concentration-response curves of electrically driven

manner (fig. 3). The effects of adenosine, ATP and APPCP on Xenopus atria were not significantly different from each other. In 6 out of 14 preparations the negative inotropic response was followed by a positive inotropic effect when concentrations of 30/~M and above were tested. Adenosine, ATP and APPCP also produced concentration-dependent reductions in the force of contraction of electrically driven Xenopus atria (fig. 4). These compounds were not significantly different in potency. After incubation with 10/~M 8-PT for 20 min, the concentration-response curves to adenosine, ATP and APPCP were shifted to the right (fig. 4). However, dipyridamole (0.5 ~M) did not potentiate the responses to adenosine, ATP, nor to APPCP. APCPP (1-10 /~M) produced small inhibitory responses. Higher concentrations of A P C P P (30-100 /~M) produced excitatory responses (fig. 5). The responses to APCPP were not affected by 8-PT.

Xenopus atria to the negative inotropic effects of adenosine (Aden), ATP and #,y-methylene ATP (APPCP) in the absence (control) and in the presence of 10 /~M 8-phenyltheophylline (8-PT). Each point is the mean of observations from at least 8 preparations. Vertical bars show S.E.M. Asterisks represent the significance of the differences between control responses and responses in the presence of 8-PT. * P < 0.05; ** P < 0.01; • ** P < 0.001.

= 20

o

8 "6 ~ 10 "~ 5 .I3

._

g

0

"~-5

i

~b

~6o

Concentration fJJM3

Fig. 5. Log concentration-response curve of electrically driven Xenopus atria to the inotropic effects of a,#-methylene ATP (APCPP). Each point is the mean of observations from at least 7 preparations. Vertical bars show S.E.M.

4. Discussion

4.1. Ventricles Adenosine, ATP, 2-chloroadenosine, APPCP and APCPP produced excitatory responses in the ventricle of Xenopus. 2-Chloroadenosine, which is reported to be resistant to uptake and deamination (Clarke et al., 1952; Rockwell and Maguire, 1966; Muller and Paton, 1979), was more potent than adenosine at producing this effect. The responses elicited by adenosine in Xenopus ventricle differ from those produced in the hearts of all other vertebrates that have been studied so far. Adenosine has been reported to be inhibitory in Rana temporaria ventricle (Flitney et al., 1977; Singh and Flitney, 1980). In Rana pipiens ventricle, adenosine either produced a transient excitation followed by an inhibition, an inhibition only (Cook et al., 1958) or was inactive (Burnstock and Meghji, 1981). Adenosine did not modify the contractile properties of the ventricle of guinea-pig (Schrader et al., 1979), cat (Shah et al., 1974), dog (Lammerant and Becsei, 1973) or rabbit (Endoh and Yamashita, 1980). Adenosine exerted a depressant

256

effect on the spontaneous firing rate of ventricular pacemaker cells of the guinea-pig (Szentmikl6si et al., 1980). Adenosine increased the left ventricular contractile force in Langendorff rabbit hearts secondary to the release of noradrenaline (Buckley, 1970a;b) and in the dog ventricle it produced a small negative inotropic effect followed by a marked positive inotropic effect (Chiba and Himori, 1975). In the present study, it seems likely that adenosine may act directly via Prpurinoceptots to produce excitatory responses in the Xenopus ventricle, although the possibility that adenosine is acting indirectly cannot be completely excluded. It is likely that adenosine, ATP, APPCP and 2-chloroadenosine elicit excitation via Pl-purinoceptors since they were antagonised by a concentration of 8-PT (10 /zM) which did not affect the responses to adrenaline. In the present study, excitatory responses to ATP were antagonised by 8-PT; however, in Rana pipiens ventricles, excitatory responses to ATP were not antagonised by theophylline (Burnstock and Meghji, 1981), another Pl-purinoceptor antagonist. These findings suggest that the mode of action of ATP in the Xenopus ventricle is different from that in Rana pipiens, i.e. ATP acts via excitatory P2-purinoceptors in Rana pipiens but via excitatory Pl-purinoceptors in Xenopus. At high concentrations (300 /~M), ATP produced a larger excitatory response than did adenosine, and a small excitatory response to ATP persisted in the presence of 10 /~M 8-PT, a concentration which completely blocked the excitatory effect of adenosine. These results suggest that ATP may also have some action which is independent of its metabolism to adenosine and action on P~-purinoceptors and which is possibly due to the involvement of P2-purinoceptors; a view substantiated by the finding that the excitatory responses to APCPP were not antagonised by 8-PT. An excitatory response to 2-chloroadenosine also persisted in the presence of a concentration of 8-PT (10/~M) which completely blocked the excitatory effect of adenosine. The resistance of 2chloroadenosine to uptake and deamination (Clarke et al., 1952; Rockwell and Maguire, 1966; Muller and Paton, 1979) may account for this observation.

Since it has been reported that some effects of ATP are mediated by prostaglandins (Burnstock et al., 1975), the effect of indomethacin, a prostaglandin synthetase inhibitor (Vane, 1971), was examined. Prostaglandins PGE I and PGE 2 produce excitatory effects on Rana temporaria ventricles (Flitney and Singh, 1978) and indomethacin has been shown to reduce the positive inotropic effect of ATP on the Rana temporaria heart (Flitney and Singh, 1980). In the study reported here, indomethacin failed to antagonise the excitatory actions of ATP, adenosine and adrenaline and therefore it is unlikely that these excitatory responses can be accounted for by stimulation of prostaglandin synthesis. The effect of adrenaline, which is the sympathetic neurotransmitter in amphibian hearts (Loewi, 1921; 1936; Von Euler, 1946), was also examined. In general, adenosine has been reported to be inhibitory in the heart and therefore it is possible that excitatory responses produced by adenosine in Xenopus ventricles are secondary to the release of a myocardial neurotransmitter. In the present study, excitatory responses to ATP and adenosine were not modified by a concentration of propranolol (0.5 /xM) that completely antagonised the excitatory responses to adrenaline and phenylephrine, an c~-adrenoceptor agonist. Phentolamine, an a-adrenoceptor antagonist, did not antagonise the effects of adenosine or ATP. These findings indicate that excitatory responses to adenosine and ATP are not mediated by the action of endogenously released adrenaline on a- or /~-adrenoceptors. Dopamine and 5-HT were found to be excitatory in Xenopus ventricle but their responses were completely blocked by propranolol, thus showing that they produce their effects via interaction with fl-adrenoceptors. Since the responses to adenosine and ATP were resistant to propranolol, their effects cannot be ascribed to the release of dopamine or 5-HT from endogenous stores. High concentrations of histamine were needed to produce even small excitatory responses, and therefore it is unlikely that the excitatory responses to adenosine and ATP are mediated via histamine. In this study, both inosine and guanosine, which lack the 6-amino group of adenosine, were found to be inactive in the ventricle of Xenopus. How-

257

ever, inosine has been reported to be excitatory in the dog isolated ventricular muscle (Chiba et al., 1981). Both inosine and guanosine are excitatory in dog isolated atria (Chiba et al., 1979) and in the ventricle of Rana pipiens (Cook et al., 1958).

Acknowledgements The authors are grateful to Dr Stephanie Clark, Susan G. Griffith and Catherine J. Moody for their advice on the preparation of the manuscript and to Mary Sheridan for the typing.

4.2. Atria

References Adenosine, ATP and APPCP produced a negative inotropic and chronotropic effect on Xenopus atria. The inhibitory effects of adenosine, ATP and APPCP were not significantly different from each other. It is probable that ATP, adenosine and APPCP act via Pl-purinoceptors since their effects were antagonised by 8-PT. It appears that ATP is metabolised to adenosine and AMP before acting on the Pj-purinoceptor, since APCPP, which is resistant to degradation (Satchell and Maguire, 1975; Maguire and Satchell, 1979), did not produce responses similar to those to ATP. The mode of action of APCPP in producing small inhibitory responses at low concentrations and small excitatory responses at high concentrations is not known. The responses of Xenopus atria to ATP differ from those of Rana catesbeiana (Goto et al., 1977; Yatani et al., 1978) and Rana pipiens atria (Burnstock and Meghji, 1981) where ATP has been reported to be excitatory, but are similar to those of mammalian atria (Hollander and Webb, 1957; De Gubareff and Sleator, 1965; James, 1965; Burnstock and Meghji, 1981). The effects of adenosine were not potentiated by dipyridamole in either the ventricles or atria. The failure of dipyridamole in potentiating adenosine has also been observed in rat hearts (Stafford, 1966; Kolassa et al., 1971) where it was found to be due to the inability of dipyridamole to block adenosine uptake (Hopkins and Goldie, 1971; Kolassa et al., 1971). It is concluded that excitation, mediated largely by excitatory P1- and possibly also by P2-purinoceptors, occurs in the Xenopus ventricle and that there are inhibitory P~-purinoceptors in the Xenopus atrium.

Brown, C.M. and M.G. Collis, 1981, Adenosine contracts the isolated rat tail artery by releasing endogenous 5-hydroxytryptamine, European J. Pharmacol. 76, 275. Buckley, N.M., 1970a, Cardiac effects of nucleosides after propranolol treatment of isolated hearts, Am. J. Physiol. 218, 1399. Buckley, N.M., 1970b, Role of endogenous norepinephrine in cardiac inotropic effects of nucleosides, Arch. Int. Pharmacodyn. Ther. 188, 92. Burnstock, G., 1978, A basis for distinguishing two types of purinergic receptor, in: Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, eds. R.W. Straub and L. Bolis (Raven Press, New York) p. 107. Burnstock, G., T. Cocks, B. Paddle and J. Staszewska-Barczak, 1975, Evidence that prostaglandin is responsible for the 'rebound contraction' following stimulation of non-adrenergic, non-cholinergic ('purinergic') inhibitory nerves, European J. Pharmacol. 31,360. Burnstock, G. and P. Meghji, 1981, Distribution of Pi- and P2-purinoceptors in the guinea-pig and frog heart, Br. J. Pharmacol. 73, 879. Chiba, S., Y. Furukawa and M. Kobayashi, 1979, Direct chronotropic and inotropic responses to guanosine and five different guanine nucleotides in isolated perfused dog atria, European J. Pharmacol. 57, 179. Chiba, S. and N. Himori, 1975, Different inotropic responses to adenosine on the atrial and ventricular muscle of the dog heart, Jap. J. Pharmacol. 25, 489. Chiba, S., H. Watanabe and Y. Furukawa, 1981, lnotropic responses of isolated atrial and ventricular muscle from the dog heart to inosine, guanosine and adenosine, Clin. Exp. Pharmacol. Physiol. 8, 171. Clarke, D.A., J. Davoll, F.S. Philips and G.B. Brown, 1952, Enzymatic deamination and vasodepressor effects of adenosine analogs, J. Pharmacol. Exp. Ther. 106, 291. Colin, D.A., R. Kirsch and C. Leray, 1979, Haemodynamic effects of adenosine on gills of the trout (Salmo gairdneri), J. Comp. Physiol. 130, 325. Colin, D.A. and C. Leray, 1979, Interaction of adenosine and its phosphorylated derivatives with putative purinergic receptors in the gill vascular bed of rainbow trout, Pfliigers Arch. 383, 35. Cook, M.H., E.A. Greene and V. Lorber, 1958, Effect of purine and pyrimidine ribosides on an isolated frog ventricle preparation, Circ. Res. 6, 735. De Gubareff.T. and W. Sleator, 1965, Effects of caffeine on

258 mammalian atrial muscle and its interaction with adenosine and calcium, J. Pharmacol. Exp. Ther. 148, 202. Deuchar, E.M., 1975, Xenopus: The South African Clawed Frog (John Wiley and Sons, New York). Endoh, M. and S. Yamashita, 1980, Adenosine antagonizes the positive inotropic action mediated via/3- but not c~-adrenoceptors in the rabbit papillary muscle, European J. Pharmacol. 65, 445. Fedan, J.S., G.K. Hogaboom, J.P. O'Donnell, J. Colby and D.P. Westfall, 1981, Contribution by purines to the neurogenic response of the vas deferens of the guinea-pig, European J. Pharmacol. 69, 41. Flitney, F.W., J.F. Lamb and J. Singh, 1977, Effects of ATP on the hypodynamic frog ventricle, 3. Physiol. (London) 273, 50P. Flitney, F.W. and J. Singh, 1978, Release of prostaglandins from the superfused frog ventricle during the development of the hypodynamic state, J. Physiol. (London) 285, 18P. Flitney, F.W. and J. Singh, 1980, Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides, J. Physiol. (London) 304, 21. Gambhir, S.S. and R.M. Tripathi, 1978, Dually innervated frog auricles for neuroeffector transmission studies, Pharmacol. Res. C o m m u n . 10, 137. Goto, M., A. Yatani and Y. Tsuda, 1977, An analysis of the action of ATP and related compounds on membrane current and tension components in bullfrog atrial muscle, Jap. J. Physiol. 27, 81. Hogaboom, G.K., J.P. O'Donnell and J.S. Fedan, 1980, Purinergic receptors: photoaffinity analog of adenosine triphosphate is a specific adenosine triphosphate antagonist, Science 208, 1273. Hollander, P.B. and J.L. Webb, 1957, Effects of adenine nucleotides on the contractility and membrane potentials of rat atrium, Circ. Res. 5, 349. Hopkins, S.V. and R.G. Goldie, 1971, A species difference in the uptake of adenosine by heart, Biochem. Pharmacol. 20, 3359. James, T.N., 1965, The chronotropic action of A T P and related compounds studied by direct perfusion of the sinus node, J. Pharmacol. Exp. Ther. 149, 233. Kolassa, N., K. Pfleger and M. Tr~im, 1971, Species differences in action and elimination of adenosine after dipyridamole and hexobendine, European J. Pharmacol. 13, 320. Lammerant, J. and I. Becsei, 1973, Left ventricular contractility and developed tension in the intact dog submitted to an intracoronary infusion of adenosine, J. Physiol. (London) 229, 41. Loewi, O., 1921, ldber humora|e IJbertragbarkeit der Herznervenwirkung. 1. Mitteilung, Pfltigers Arch. 189, 239. Loewi, O., 1936, Quantitative und qualitative Untersuchungen Ober den sympathicusstoff, Pfl0gers Arch. 237, 504. Maguire, M.H. and D.G. Satchell, 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. Pharmacol. Exp. Ther. 211,626. Muller, M.J. and D.M. Paton, 1979, Presynaptic inhibitory

actions of 2-substituted adenosine derivatives on neurotransmission in rat vas deferens: effects of inhibitors of adenosine uptake and deamination, Naunyn-Schmiedeb. Arch. Pharmacol. 306, 23. Osswald, H., J.A. Haas, W.S. Spielman and F.G. Knox, 1979, Localisation and mechanism of renal response to adenosine, Pflfigers Arch. 379, RI6. Rockwell, M. and M.H. Maguire, 1966, Studies on adenosine deaminase. 1. Purification and properties of ox heart adenosine deaminase, Mol. Pharmacol. 2, 574. Sakai, K , 1978, Tryptaminergic mechanism participating in induction of vasoconstriction by adenine nucleotides, adenosine, IMP and inosine in the isolated and blood-perfused hindlimb preparation of the rat, Jap. J. Pharmacol. 28, 579. Sakai, K. and M. Akima, 1977, Tryptaminergic vasoconstriction induced by adenosine in the femoral vascular bed of the rat, Jap. J. Pharmacol. 27, 908. Sakai, K. and M. Akima, 1978, Vasoconstriction after adenosine and inosine in the rat isolated hindlimb abolished by blockade of tryptaminergic mechanisms, Naunyn-Schmiedeb. Arch. Pharmacol. 302, 55. Sakai, K., M. Akima and H. Matsushita, 1979, Femoral vascular responses to purine and pyrimidine derivatives: release of 5-hydroxytryptamine by purine derivatives in isolated, cross-circulated rat hindlimb, Jap. J. Pharmacol. 29, 243. Satchell, D.G. and M.H. Maguire, 1975, Inhibitory effects of adenine nucleotide analogs on the isolated guinea-pig taenia coli, 3. Pharmacol. Exp. Ther. 195, 540. Schrader, J., E. Gerlach and G. Baumann, 1979, Sites and mode of action of adenosine in the heart. If. Ventricular myocardium, in: Physiological and Regulatory Functions of Adenosine and Adenine Nucleotides, eds. H.P. Baer and G.I. D r u m m o n d (Raven Press, New York) p. 137. Shah, A., S.J. Kechejian, F. Kavaler and V.J. Fisher, 1974, Effects of adenine nucleotides on contractility of normal and postischemic myocardium, Am. Heart J. 87, 740. Singh, J. and F.W. Flitney, 1980, Adenosine depresses contractility and stimulates 3',5' cyclic nucleotide metabolism in the isolated frog ventricle, J. Mol. Cell. Cardiol. 12, 285. Stafford, A., 1966, Potentiation of adenosine and the adenine nucleotides by dipyridamole, Br. J. Pharmacol. Chemother 28, 218. Szentmikl6si, A.J., M. N~meth, J. Szegi, J. Gy.Papp and L. Szekeres, 1980, Effect of adenosine on sinoatrial and ventricular automaticity of the guinea-pig, N a u n y n Schmiedeb. Arch. Pharmacol. 311, 147. Vane, J.R., 1971, Inhibition of prostaglandin synthesis as a mechanism of action of aspirin-like drugs, Nature (New Biol.) 231, 232. Versprille, A., 1963, The influence of uridine nucleotides upon the isolated frog heart, Pfliigers Arch. 277, 285. Von Euler, U.S., 1946, A specific sympathomimetic ergone in adrenergic nerve fibres (sympathin) and its relations to adrenaline and noradrenaline, Acta Physiol. Scand. 12, 73. Yatani, A., M. Goto and Y. Tsuda, 1978, Nature of catecholamine-like actions of ATP and other energy rich nucleotides on the bullfrog atrial muscle, Jap. J. Physiol. 28, 47.