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Effects of purine compounds on cholinergic nerves. Specificity of adenosine and related compounds on acetylcholine release in electrically stimulated guinea pig ileum
European Journal of Pharmacology, 48 (1978) 297--307 © Elsevier/North-Holland Biomedical Press
297
EFFECTS OF PURINE COMPOUNDS ON CHOLINERGIC NERVES. SPECIFICITY OF ADENOSINE AND RELATED COMPOUNDS ON ACETYLCHOLINE RELEASE IN ELECTRICALLY STIMULATED GUINEA PIG ILEUM EIICHI HAYASHI, M O T O K U N I MORI, SHIZUO Y A M A D A
and M A S A R U K U N I T O M O
Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, 2-2-10shiha, Shizuoka, 422 Japan Received 9 August 1977, revised MS received 14 October 1977, accepted 6 December 1977
E. HAYASHI, M. MORI, S. YAMADA and M. KUNrrOMO, Effects ofpurine compounds on cholinergic nerves. Specificity o f adenosine and related compounds on acetylcholine release in electrically stimulated guinea pig ileum, European J. Pharmacol. 48 (1978) 297--307. The action of 21 purine compounds on the twitch response of the electrically stimulated guinea pig isolated ileum has been investigated. Adenosine and related compounds produced a dose-dependent depression of the response. Adenosine was the most potent and 2S-deoxyadenosine had one hundredth the potency of adenosine. Adenine, hypoxanthine, inosine, IMP, ITP, xanthine, xanthosine, XMP, XTP, guanine, GMP and GTP were ineffective at concentrations less than 1 mM. Adenosine (30 p_M) reduced the electrically induced ACh output from the ileal strips. The dose---depression curve for adenosine (0.1--30/2M) was shifted to the right in the presence o f xanthine derivatives and of these, theophylline was the most p o t e n t inhibitor of adenosine. On the other hand, dipyridamole (0.1--1 pM) and hexobendine (0.1--1 ~tM) shifted the curve to the left. They markedly inhibited 3H-adenosine uptake into the ileum. Theophylline (0.1 mM), dipyridamole (0.3 /2M) and hexobendine (0.3 p_M) did not affect tetrodotoxin-, adrenaline-, strychnine- and morphine-induced inhibition of the twitch response. The present investigations have revealed that adenosine and related compounds reduce ACh release from the intramural cholinergic nerves in the guinea pig ileum possibly in a specific manner (or through a specific receptor site) different from that of other inhibitors such as morphine. Inhibition o f twitch response
Adenosine
Theophylline
1. Introduction Various actions of purine nucleotides in gut have been described: Burnstock (1972) has Suggested that adenosine triphosphate (ATP) might be a transmitter liberated by non~holinergic, non-adrenergic intramural inhibitory nerves of mammalian intestine, suggesting the existence of "purinergic nerves". McDougal and Borowitz (1972) reported that adenosine selectively inhibited the contractile response of guinea pig ileum to indirectly acting agonists. Adenosine and adenine nucleotides have been shown to depress the twitch response of transmurally stimulated guinea pig ileum (Takagi and
ACh release
Purine compounds
Takayanagi, 1972; Gintzler and Musacchio, 1975; Sawynok and Jhamandas, 1976). In earlier experiments, we found that adenosine antagonized non-competitively the contractile response of the guinea pig ileum to nicotine and markedly reduced the twitch response which was probably mediated through acetylcholine (ACh) release from the intramural cholinergic nerves (Hayashi et al., 1977). Thus adenosine and adenine nucleotides have been suggested to have an inhibitory effect on the cholinergic nerves in the guinea pig ileum. In an a t t e m p t to determine the actions of adenosine-related compounds in cholinergic nerves, we examined the actions of 21 purine compounds on the twitch response of the
298
electrically stimulated guinea pig isolated ileum. Adenosine and some related compounds had an inhibitory effect on the cholinergic nerves. Adenosine was the most potent inhibitor and in addition its effect was selectively antagonized by xanthine derivatives such as theophylline and enhanced by dipyridamole and hexobendine. Present investigations have shown that adenosine and related compounds reduce ACh release from the intramural cholinergic nerves in the guinea pig ileum possibly in a specific manner different from that of other inhibitors such as morphine. Preliminary accounts of this work have been presented to the VIth International Congress of Pharmacology (Mori et al., 1975) and the Japanese Pharmacological Society (Mori et al., 1973; Hayashi et al., 1975).
2. Materials and methods
2.1. General The experiments were carried out on ileal strips (3--4 cm, unstretched) of male guinea pigs weighing 250--350 g. Preparations were suspended in Tyrode solution gassed with a mixture of 95% 02 and 5% CO2. The capacity of the organ bath was 10 ml and it was kept at 37°C. The composition of the Tyrode solution has been described previously (Hayashi and Yamada, 1975b). The strips were subjected to 1.0 g resting tension. Transmural electrical stimulation of the ileum was carried out according to the method of Paton (1955, 1957). The electrodes were made of platinum and the intramural electrode was the anode. The preparations were stimulated with rectangular pulses lasting 0.4msec with a frequency of 0.1 Hz and sufficient strength to produce a supramaximal response. The supramaximal response was used because the response to this stimulation was more constant than that to submaximal stimulation. The preparation was allowed to stabilize for 60 min before an experiment. The response of
E. HAYASHI ET AL.
the ileum was recorded isometrically on a recticorder (Nihon Kohden, RJG-3024) with a force-displacement transducer (Nihon Kohden). Purine compounds were cumulatively added to the organ bath and the other drugs were used as pretreatment for 5--10 min at 2--4 dose levels.
2.2. Assay of ACh released An ileal strip was suspended in an organ bath containing 2 ml of Tyrode solution (37°C) gassed with the gas mixture. The strip was electrically stimulated for 1 min in the absence or presence of adenosine and other drugs, and the samples were collected. The released ACh was assayed on another ileal strip suspended in 5 ml Tyrode solution (37°C) containing physostigmine (0.1/~M) and morphine (1/~M) (Paton and Vizi, 1969). The estimate of ACh content of samples was based on control dose-response curves for ACh. Ileal strips were incubated for 60 min in Tyrode solution containing 14C-choline (30 pM, 0.2/~Ci/ml, specific activity 56.85 mCi/ mmole), and then superfused with Tyrode solution for 60 min. The strip was electrically stimulated for 1 min in the absence and presence of adenosine and other drugs; the radioactivity in the samples was measured with an Aloka liquid scintillator and expressed as dpm/100 mg wet weight tissue/min.
2.3. Measurement of uptake of 3H-adenosine Ileal strips were pretreated with dipyridamole and hexobendine for 10 min and then incubated for 30 min in Tyrode solution conraining 3H-adenosine (10/zM, 2/~Ci/ml, specific activity 23.2 Ci/mmole). After the incubation, the strips were rinsed with the chilled Tyrode solution, blotted, weighed and digested with soluene 350 (Parkad). Radioactivity was measured and expressed as dpm/ g wet weight tissue/30 min.
SPECIFICITY OF ADENOSINE ON ACH RELEASE
2.4. Measurement o f the activity o f enzymes
The activity of the acetylcholinesterase and adenosine deaminase of guinea pig ileum was determined by the methods of Guenther and Klaus (1970) and of Kalckar (1947) respectively. The activity of adenosine deaminase (purified deaminase of pig intestine, Seikagaku Kogyo) was measured spectrophotometrically (265 p). The following drugs were used: acetylcholine chloride (Daiichi), adrenaline hydrochloride (Daiichi), tetrodotoxin (Sankyo), morphine hydrochloride (Takeda), strychnine sulphate (Merck), chlorthiazide (Takeda), dipyridamole (Tanabe), hexobendine (Toyo jozo), nicotinic acid (Wako), physostigmine sulphate (Merck), adenosine triphosphate (Kyowa), adenosine diphosphate (Kyowa), 3',5'-cyclic AMP (Kyowa), NS-dibutyryl-3',5 '-
299
cyclic AMP (Kyowa), other purine compounds (Kohjin). Xanthine and derivatives were gifts from Prof. M. Sekiya of this college. 3HAdenosine and 14C-choline were obtained from New England Nuclear, Boston. 3. Results
3.1. Effects o f purine compounds on twitch response
In order to investigate the structural features of the adenine derivatives associated with their inhibitory effects on the cholinergic nerves, we examined the action of 21 purine compounds on the twitch response of guinea pig isolated ileum to electrical stimulation. Adenosine and related compounds produced a dose-dependent depression of twitch
TABLE 1 Inhibitory actions of adenosine and related compounds on the twitch response of the electrically stimulated guinea pig ileum, pD2 values were calculated from the dose--depression curves. The number of experiments is indicated in parentheses. Compound
Structure I
ATP ADP 5'-AMP 2'-AMP 3',5'-c-AMP 2',3'-c-AMP Dibu-c-AMP 3
pD 2 value (mean _+S.E.M.)
R1
R2
(~(~2 ®~® ~ -OH
-OH -OH -OH --OH ~
--OH (~
R3 -OH -OH -OH ~ -OH ~ ' ~ -OC(CH2 )2CH3
5.33 5.52 5.72 5.81 5.18 5.44 4.43
_+0.04 +- 0.09 + 0.11 _+0.09 -+ 0.04 + 0.08 _+0.09
(7) (7) (7) (8) (8) (7) (8)
4 4
4 4 4
O Adenosine 2'-Deoxyadenosine 1
--OH -OH
-OH -OH
-OH --H
NH2
R2 R3
2 (~ = phosphate. 3
NH2 . . . .
NH(CH2)2CH3.
The levels o f significance for adenosine and other compounds are shown by 4(p
5.83 -+ 0.04 (42) 3.80 + 0.09 (7) 4
300
E. HAYASHI ET AL.
•
Ade
7
• 20
• 70
• 200
• 700
,I,~F
xlO nM W W
l
j2g 1 rain
Fig. 1. Inhibitory action of adenosine on the twitch response of the electrically stimulated guinea pig ileum. Adenosine (Ade 0.03--10 pM) was cumulatively added to the organ bath. Dots (o) indicate the addition of adenosine and the arrows (~LW) washing of the preparation.
response; the pD2 values (Schild, 1949; Ari~ns and Van Rossum, 1957) are shown in table 1. Adenosine was the most potent and 2'-deoxyadenosine had one hundredth the potency of adenosine. The order of potency was adenosine > 2'-AMP = 5'-AMP > 2',3'-cyclic AMP > ATP > 3',5'-cyclic AMP > dibutyryl-cyclic AMP > 2'-deoxyadenosine. The difference in pD2 values between adenosine and related compounds except 2'- and 5'-AMP was significant at the 1% level (t-test). On the other hand, adenine, hypoxanthine, inosine, IMP, ITP, xanthine, xanthosine, XMP, XTP, guanine, GMP and GTP produced no
inhibition at concentrations less than 1 mM. The depressant effect Of adenosine was investigated further. Low concentrations of adenosine (0.03--10 #M) produced a dosedependent depression of the twitch response (fig. 1). The effect was relatively rapid in onset with maximal depression always developing within 1 min. The inhibitory effect disappeared rapidly (3--5 min) after washing the preparation. Thus adenosine reduced the response by 6.1 + 0.7% (mean + S.E.M.) (n = 42) at 0.1/~M and by 98.7 + 0.5% (n = 42) at 30 #M (pD2 = 5.83 + 0.04). Adenosine in these concentrations antagonized the effect of nicotine non-competitively without affecting the direct muscle response to ACh (Hayashi et al., 1977). The effect o f adenosine was compared with that of tetrodotoxin (Ogura et al., 1966; Gershon, 1967), adrenaline (Paton and Vizi, 1969; Kosterlitz et al., 1970), strychnine (Takagi and Takayanagi, 1966) and morphine (Paton and Vizi, 1969; Kosterlitz et al., 1970) which are known to inhibit the twitch response. These agents reduced the twitch response but recovery o f t h e response was much slower ( 3 0 - 6 0 min). The pD2 values for tetrodotoxin, adrenaline, strychnine and morphine were 7.78 + 0.01 (n =5), 7 . 0 9 + 0 . 0 6 ( n = 1 5 ) , 5 . 0 7 + 0 . 1 0 (n = 8) and 7.07 + 0.08 (n = 7) respectively.
TABLE 2 Inhibitory actions of adenosine (30 p_M), morphine (1 ~M) and tetrodotoxin (0.1 p_M) on electrically induced ACh and 14 C output from the guinea pig ileum preincubated with 14 C-choline. Ileal strips were electrically stimulated for 1 min in the absence and presence of these drugs, and the released ACh was assayed on another ileal strip. Resting ACh output was subtracted. Ileal strips were also incubated for 60 rain in Tyrode solution containing 14 C-choline and then electrically stimulated for 1 min in its absence and presence. The 14 C output was measured. The number of experiments is indicated in parentheses. Drugs
None Adenosine Morphine Tetrodotoxin
. Concentration (/~M)
30 1 0.1
ACh o u t p u t (ng/min) (mean + S.E.M.)
14 C output (dpm/lO0 mg wet weight tissue/min) (mean +- S.E.M.)
Electrical stimulation
Resting
Electrical stimulation
54.2 + 4.0 (10) 13.1 + 1.5 (8) 13.3 -+ 2.6 (8)
589.2 _+58.8 (10)
1378.8 676.0 590.2 644.0
+_ 82.2 (10) + 93.7 (8) + 65.6 (8) + 101.7 (8)
SPECIFICITY OF ADENOSINE ON ACH RELEASE 3.2. Effect
of adenosine on ACh release
The effect o f adenosine on ACh release from the electrically stimulated ileum was examined. This was carried o u t by assaying the ACh o u t p u t and by determining the 14C o u t p u t from 14C-choline-incubated ileum in the absence and presence of adenosine. Adenosine (30 pM) reduced the electrically induced ACh o u t p u t and the 14C o u t p u t by 70--80% and by 90--100% respectively (table 2). Morphine (1/~M) and t e t r o d o t o x i n (0.1 /~M) exerted a similar depression. 3.3. Effects of some drugs on adenosineinduced inhibition o f the twitch response 3.3.1. Chlorthiazide and nicotinic acid Adenosine has been shown to stimulate the formation of cyclic AMP in guinea pig cerebral cortex slices (Sattin and Rall, 1970; Shimizu and Daly, 1970; Huang et al., 1972). In order to investigate the possibility of cyclic AMP involvement in the adenosine-induced inhibi-
301 tion, the effect of chlorthiazide, an inhibitor and of nicotinic acid, an activator of phosphodiesterase respectively (Senft et al., 1968; Krishna et al., 1966) on the inhibition was examined. The dose-depression curve for adenosine was unchanged in the presence of chlorthiazide (10/~M) and nicotinic acid (1 mM). 3.3.2. Xanthine derivatives Caffeine antagonized the depressant action of adenosine and of the nucleotides on the contraction and action potentials of mammalian atrial muscle (De Gubareff and Sleator, 1965). Aminophylline inhibited the coronary vasodilatory action o f adenosine in dogs (Afonso, 1970). The effects on the adenosineinduced inhibition of 10 xanthine derivatives which had been found inactive in terms of an inhibitory effect on the twitch response were investigated. Xanthine and the methyl-substituted analogs at concentrations of 10 #M-I mM shifted the dose--depression curve for adenosine to the right without depressing
TABLE 3 Effects of xanthine derivatives on adenosine-induced inhibition o f twitch response. Pretreatment with xanthine derivatives at 2--4 dose levels was for 5 rain. The dose--depression curve for adenosine was obtained in the absence and presence o f these c o m p o u n d s and PA2 values were calculated. The number o f experiments is indicated in parentheses.
Compound
Structure I
pA2 value (mean + S.E.M.)
RI
R2
R3
H H
H H
H H
---
Xanthine
H
1-Methylxanthine
H H
7-Methylxanthine 1,3-Dimethylxanthine
CH3 H H CHs
3,7-Dimethylxanthine
H
1,7-Dimethylxanthine 1,3,7-Trimethylxanthine
CH3 CH3
H H H CH3 H CH3 CH3 CH3
3.67 4.25 4.07 3.96 4.93 4.07 4.35 4.18
Hypoxanthine *
Uric acid ** 3-Methylxanthine
o
RI"-N" ~ ' ~
R2
N-R3 N
N
CH 3
H CH3 CH3 H
CH3
(5) (5)
+ 0.24 (8) + 0.06 (8) + 0.06 (8) + 0.08 (8) +-0.05 (26) + 0.11 (8) + 0.13 (8) + 0.07 (8)
302
E. HAYASHI ET AL. Adr
Nor
2g
J
(a)
Ade
l rain
Theo
j/
100-
50,
Fig. 2. Effect of theophylline on adenosine-induced inhibition of the twitch response. (a) Adenosine (Ade 10 p-M)-induced inhibition of twitch response and its reversal by an addition of theopbylline (Theo 0.1 raM). (b) The effect of theophyUine (Theo 0.1 raM) pretreatment on adenosine (Ade 10 /IM)induced inhibition. Dots ($) indicate the addition o f adenosine or theophylline, and the arrows (~W) washing of the preparation.
the maximal effect and significantly decreased the pD2 values, although hypoxanthine and uric acid were only slightly active. The pA2 values (table 3) show that 1,3-dimethylxanthine (theophylline) was the most potent 100
J
Fig. 4. Effect o f theophylline on the dose--depression curves for tetrodotoxin, adrenaline, strychnine and morphine. (o) Control, in the presence of theophylline (e) 0.1 raM. Ordinates: average % o f inhibition of the response produced by t e t r o d o t o x i n (TTX), adrenaline (Adr), strychnine (Str) and morphine (Mor). Abscissae: concentration (log M) of the drugs. Each point represents the mean (±S.E.M.) of at least 5 experiments.
TABLE 4
Effect of dipyridamole, hexobendine and theophylline on 3 H-adenosine uptake into the ileal strips. Ileal strips were pretreated with dipyridamole, hexobendine and theophylline for 10 rain and then incubated for 30 min in Tyrode solution containing 3 H-adenosine. The radioactivity in the tissues was measured. The number of experiments is indicated in parentheses. Drugs
0
-7
-;
-;
-'4
Fig. 3. Effect of theophylline on the dose--depression curve for adenosine. (o) Control; in the presence of theophylline, (o) 10 p_M, (A) 30 pM, (A) 100 /~M, (D) 300 #M. Ordinate: average % of inhibition of the response produced by adenosine. Abscissa: concentration (log M) of adenosine. Each point represents the mean (±S.E.M.) of 26 experiments.
None Dipyridarnole
Conc. (pM)
0.1 1
Hexobendine
Theophylline
0.03 0.1 0.3 1 100
Uptake of 3 H-adenosine (dpm/g wet weight tissue/ 30 rain) (mean + S.E.M.) × 10 s 6.95 ± 0.49 (12) 4.43 _+0.44 (7) 1.32 +0.12 (7) 6.67 ± 0.51 (7) 4.44 ± 0.48 (7) 2.90 ± 0.48 (7) 1.45 ± 0 . 2 6 (7) 6.42 ± 0.30 (5)
SPECIFICITY OF ADENOSINE ON ACH RELEASE inhibitor of adenosine. The order of p o t e n c y is 1,3-dimethyl ~ 1,7~iimethyl ~ 1,3,7-trimethyl ~ 3,7-dimethyl ~ 1-methyl ~ 3-methyl 7-methyl,-xanthine ~ xanthine ~ hypoxanthine = uric acid. Fig. 2 shows the antagonistic effect o f theophyUine (0.1 raM) o f the adenosine-induced inhibition. The antagonism was reversible and surmountable b y increasing the concentration of adenosine (fig. 3), suggesting competitive antagonism such as was reported for the adenosine effect in heart (Bringer et al., 1975), kidney (Osswald, 1975) and intestine (Sawynok and Jhamandas, 1976). Although tehophylline itself had very little effect on the twitch response of the ileum at concentrations less than 0.1 mM, it potentiated the response at higher concentrations (0.3--1 raM) b y 20--30% as previously reported b y Moritoki et al. (1976) and Kazic (1977). On the other hand, tetrodotoxin-, adrenaline-, strychnine- and morphineinduced inhibition was not antagonized b y theophylline (0.1 raM) (fig. 4). Concentrations of theophylline less than 1 mM had little effect on the activity of enzymes such as acetylcholinesterase and adenosine deaminase and on the uptake of 3H-adenosine into the ileum (table 4).
3.3.3. Dipyridamole and hexobendine to
Dipyridamole and hexobendine are k n o w n potentiate the inhibitory effects of
DP
100
-0
-7
-5
-S
-8
-7
-6
-5
Fig. 5. Effect of dipyridamole (DP, left) and hexobendine (HB, right) on the dose--depression curves for adenosine. (o) Control; in the presence of dipyrldamole, (e) 0.1 p2Vl, (A) 0.3 p~I, (A) 1 p_M.Ordinates: average % of inhibition of the response produced by adenosine. Abscissa: concentration (log M) of adenosine. Each point represents the mean (±S.E.M.) of at least 7 experiments.
303
r
HB
Ad.
TTX
Iq0r
r
TTX
HB
Str
Fig. 6. Effect of hexobendine on adenosine-, tetrodotoxin-, adrenaline-, strychnine- and morphine-
induced inhibition. Dots (e) indicate the addition of adenosine (Ade 0.03 p_M),tetrodotoxin (TTX 0.01 p~M), adrenaline (Adr 0.03 p_M), strychnine (Str 0.1 p-M) and morphine (Mot 0.01 ~M). At the arrows (J'HB), hexobendine (0.3 p~M) pretreatment for 10 min.
adenosine and of the nucleotides in coronary artery (Stafford, 1966; McInnes and Parratt, 1969) and taenia coli (SatcheU et al., 1972). The dose--depression curves for adenosine were shifted to the left in the presence of dipyridamole and hexobendine at low concentrations (0.1--1 ~M) which had little effect on t h e twitch response (fig. 5); the pD2 values were increased significantly (pD2 for adenosine: control, 5.95 ± 0.11, in the presence of dipyridamole (0.1/~M) 6.21 -+ 0.10, (0.3 ~M) 6.51 ± 0.10, (1/~M) 6.69 ± 0.10, n = 7; control, 5.83 ± 0.04, in the presence of hexobendine (0.1 #M) 6.41 ± 0.11, (0.3 #M) 6.95 ± 0.10, (1 ~M) 7.24 ± 0.15, n = 7). On the other hand, these agents did not affect tetrodotoxin-, adrenaline-, strychnine- and morphine-induced inhibition (fig. 6). Dipyridamole and hexobendine at higher concentrations ( 3 - - 1 0 0 ~ M ) reduced the contractile response to ACh as well as that to electrical stimulation. L o w concentrations (0.1--1 ~M) of dipyridamole and hexobendine inhibited the uptake of 3H-adenosine into the ileum b y 40-80% (table 4). Higher concentrations (30-100 ~M) inhibited the uptake of 3H-adenosine b y 90--100% and also adenosine deaminase activity b y 20 60% (inhibition rate: dipyridamole (100 ~M) 18.4 -+ 2.0%, n = 4; hexobendine (30 #M) 17.3 ± 1.8%, n-- 4; (100 /~M) 63.0 ± 3.4%, n = 5).
304
4. Discussion The effects of 21 purine compounds on the twitch response of guinea pig isolated ileum to electrical stimulation were examined. The adenosine derivatives with a 6-amino group {adenosine derivatives caused a dose-dependent depression of the twitch response, whereas the other compounds were ineffective. Adenosine was the most potent and 2'~leoxyadenosine had only approximately one hundredth the potency of adenosine. Adenosine has been reported as having approximately one hundredth the potency of ATP with respect to the inhibitory effect on the gut (Burnstock, 1972). However, the inhibitory effect on the twitch response (cholinergic nerves} tended to decrease gradually with the addition of phosphate to the ribose group. Structure--activity studies suggested that the 6-amino group, the 2'hydroxy group and the ribose group play an important role in the depression of the twitch response by purines. Adenosine reduced the electrically induced ACh output from the ileal strips by 70--80% and the 14C output from 14C-choline-incubated strips by 90--100%. The 14C output evoked appears to be largely ~4C-ACh since it was sensitive to tetrodotoxin blockade. These findings show that the adenosine-induced inhibition of the twitch response is due to a reduced release of ACh from the intramural cholinergic nerves in the guinea pig ileum. On the other hand, the depression of ACh release by adenosine could not be shown at other cholinergic synapses, such as t h e neuromuscular junction of motor nerves or autonomic ganglia. Similar results with morphine have been reported by Paton (1957). The inhibitory action of adenosine on the cholinergic nerves was further characterized. The antagonism of methylxanthines against adenosine and adenine nucleotides has been demonstrated in heart {De Gubareff and Sleatot, 1965; Afonso, 1970; Bringer et al., 1975), kidney (Osswald, 1975), brain (Sattin and RaU, 1970; Shimizu and Daly, 1970; Huang
E. HAYASHI ET AL.
et al., 1972; Schultz and Daly, 1973; Kuroda and Kobayashi, 1975) and intestine (Sawynok and Jhamandas, 1976; Mckenzie et al., 1977; Okwuasaba et al., 1977). We tested the effects of 10 xanthine derivatives on the adenosineinduced inhibition and found the action of adenosine to be competitively antagonized by all of them. Theophylline was the most potent and hypoxanthine and uric acid were weak antagonists of adenosine. These data suggest that the carbonyl group and the 1-, 3- and 7methyl groups are important for determining the potency of inhibition. TheophyUine had no effect on the activity of enzymes such as acetylcholinesterase and adenosine deaminase of the ileum and on uptake of adenosine into the tissue. Although theophyUine is a well known inhibitor of phosphodiesterase, the concentrations of theophylline used in the present study were significantly lower than those needed to inhibit the activity of phosphodiesterase (Sheppard et al., 1972). Thus the effect of theophylline on phosphodiesterase can be ruled out. Because theophylline only antagonized the adenosine-induced inhibition without affecting the response to tetrodotoxin, adrenaline, strychnine and morphine, it is suggested that they compete at adenosinespecific receptor sites. The existence of such receptors has been demonstrated in other tissues (Burnstock et al., 1970; Sattin and Rall, 1970; Spedding et al., 1975; Spedding and Weetman, 1976; Mckenzie et al., 1977), It was suggested that there is a link between morphine and adenine derivatives actions in the electrically stimulated guinea pig ileal longitudinal strips since theophyUine (0.2-0.5 mM) antagonized the morphine-induced inhibition as well as the adenosine- and adenine nucleotides-induced inhibition of the twitch response (Sawynok and Jhamandas, 1976). However, in the present experiments, theophyUine did not alter the morphineinduced inhibition when used in the concentrations (10--100 ~M) which antagonized the adenosine-induced inhibition; at the higher concentrations {0.3--1.0 raM) used by Sawynok and Jhamandas, theophylline poten-
SPECIFICITY OF ADENOSINE ON ACH RELEASE
tiated the twitch response in agreement with the findings of Moritoki et al. (1976) and Kazic (1977). Therefore it appears unlikely that morphine-induced inhibition is mediated through a c o m m o n mechanism with adenosine as suggested by Sawynok and Jhamandas (1976). Dipyridamole and hexobendine have been shown to potentiate the coronary vasodilating (Stafford, 1966; McInnes and Parratt, 1969; Kolassa et al., 1970) and negative chronotropic (Kolassa et al., 1971) effects of adenosine as well as the inhibitory effects of adenosine and adenine nucleotides in the intestine (Satchell et al., 1972; Satchell and Burnstock, 1975; Okwuasaba et al., 1977) and trachea (Coleman, 1976). The adenosine-induced inhibition of acetylcholine release was effectively enhanced in the presence of low concentrations of dipyridamole and hexobendine, whereas tetrodotoxin-, adrenaline-, strychnineand morphine-induced inhibitions were not. Degradation by adenosine deaminase and the uptake into the tissue have been considered to be an inactivation process of adenosine. Dipyridamole and hexobendine in concentrations which enhanced the effect of adenosine prevented adenosine uptake into the tissue, while at higher concentrations t h e y also reduced adenosine deaminase activity. Therefore the potentiation of adenosine action by dipyridamole and hexobendine appears to be exerted mainly through the blockade of adenosine uptake. This has also been demonstrated for heart (Kolassa e t al., 1971; Hopkins, 1973), lungs ( C o l e m a n , 1976) and taenia coli (Satchell et al., 1972). Present investigations have revealed that adenosine and adenine nucleotides reduce ACh release from the intramural cholinergic nerves in the guinea pig ileum possibly in a specific manner (or through a specific receptor site) different from that of tetrodotoxin, adrenaline, strychnine and morphine. It is proposed that endogenous adenine derivatives may have a physiological role as modulators which control ACh release in the guinea pig ileum in agreement with the suggestions of
305
other workers (Berne et al., 1971; McIlwain, 1974; Su, 1 9 7 5 ) .
Acknowledgements The authors wish to thank Prof. M. Sekiya, Shizuoka College of Pharmaceutical Sciences for kindly supplying xanthine derivatives and also Kobjin Co., Ltd., for the generous gift of purine compounds.
References Afonso, S., 1970, Inhibitio n of coronary vasodilating action of dipyridamole and adenosine by aminophylline in the dog, Circulation Res. 26,743. Ari~ns, E.J. and J.M. Van Rossum, 1957, PDx, pAx and pD~ values in the analysis of pharmacodynamics, Arch. Intern. Pharmacodyn. 1i0,275. Berne, R.M., R. Rubio, J.D. Dobson and R.R. Cornish, 1971, Adenosine and adenine nucleotides as possible mediators of cardiac and skeletal muscle blood flow regulation, Circulation Res. 28 Suppl. 1,115, Biinger, R., F.J. Haddy and E. Oerlach, 1975, Coronary responses to dilating substances and competitive inhibition by theophylline in the isolated perfused guinea-pig heart, Pfluegers Arch. 358, 213. Burnstock, G., 1972, Purinergic nerves, Pharmacol. Rev. 24,509. Buihstock, G., G. Campbell, D. Satchell and A. Smythe, 1970, Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut, Brit. J. Pharmacol. 40, 668. Coleman, R.A., 1976, Effects of some purine derivafives on the guinea-pig trachea and their interaction with drugs that block adenosine uptake, Brit. J. Pharmacol. 57, 51. De Gubareff, T. and W. Sleator, 1965, Effects of caffeine on mammarian atrial muscle and its interaction with adenosine, J. Pharmacol. Exptl. Therap. 148,202. Fennessy, M.R, R.L.H. Heimans and M.J. Rand, 1969, Comparison of effect of morphine-like analgesics on transmurally stimulated guinea-pig ileum, Brit. J. Pharmacol. 37,436. Gershon, M.D., 1967, Effects of tetrodotoxin on innervated smooth muscle preparations, Brit. J. Pharmacol. 29, 259.
306 Gintzler, A.R. and J.M. Musacchio, 1975, Interactions of morphine, adenosine, adenosine triphosphate and phosphodiesterase inhibitors on the fieldstimulated guinea-pig ileum, J. Pharmacol. Exptl. Therap. 194, 575. Guenther, V. and S. Klaus, 1970, Red cell and plasma cholinesterase activities in microsamples of human and animal blood determined simultaneously by a modified acetylthiocholine/DTNB procedure, Toxicol. Appl. Pharmacol. 16, 764. Hayashi, E. and S. Yamada, 1975, Pharmacological studies on surugatoxin, the toxic principle from Japanese ivory mollusc (Babylonia japonica), Brit. J. Pharmacol. 53, 207. Hayashi, E., M. Mori, S. Yamada and Y. Shirasaki, 1975, Effect of purine nucleotides on parasympathetic nerves. (6). Effect of adenosine and morphine on 14C efflux from the guinea-pig ileum strips incubated with 14C-choline, Jap. J. Pharmacol. 25 suppl., 147 p. Hayashi, E., S. Yamada and M. Mori, 1977, Comparative studies on anti-nicotinic action of hexamethonium, mecamylamine and adenosine in the guinea pig isolated ileum, Jap. J. Pharmacol. 27, 659. Hopkins, S,V., 1973, The potentiation of the action of adenosine on the guinea-pig heart, Biochem. Pharmacol. 22, 341. Huang, M., H. Shimizu and J.W. Daly, 1972, Accumulation of cyclic adenosine monophosphate in incubated slices of brain tissue. 2. Effects of depolarizing agents, membrane stabilizers, phosphodiesterase inhibitors and adenosine analogues, J. Med. Chem. 15,462. Kalckar, H.M., 1947, Differential spectrophotometry of purine compounds by means of specific enzymes, J. Biol. Chem. 167,429. Kazic, T., 1977, Action of methylxanthines and imidazole on the contractility of the terminal ileum of the guinea pig, European J. Pharmacol. 41, 103. Kolassa, N., K. Pfleger and W. Rummel, 1970, Specificity of adenosine uptake into the heart and inhibition of dipyridamole, European J. Pharmacol. 9, 265. Kolassa, N., K. Pfleger and M. Tr~n, 1971, Species differences in the action and elimination of adenosine after dipyridamole and hexobendine, European J. Pharmacol. 13,320. Kosterlitz, H.W., R.J. Lyden and A.J. Watt, 1970, The effects of adrenaline, noradrenaline and isoprenaline on inhibitory ~- and ~-adrenoceptors in the longitudinal muscle of the guinea-pig ileum, Brit. J. Pharmacol. 39,398. Krishna, G., P. Weiss, J.I. Davis and S. Hynie, 1966, Mechanism of nicotinic acid inhibition of hormone-induced lipolysis, Federation Proc. 25, 719. Kuroda, Y. and K. Kobayashi, 1975, Effects of ade-
E. HAYASHI ET AL. nosine and adenine nucleotides on the postsynaptic potential and on the formation of cyclic adenosine 3',5r-monophosphate from radioactive adenosine triphosphate in guinea pig olfactory cortex slices, Proc. ,lap. Acad. 5 1 , 4 9 5 . McDougal, M.R. and J.L. Borowitz, 1972, The effect of adenosine and cold storage on the response of the guinea-pig ileum to various agonlsts, Arch. Intern. Pharmacodyn. 195, 177. McIlwain, H., 1974, Adenosine neurohumoral and regulatory roles in the brain, in: Central Nervous System, eds. E. Ganazzani and H. Hoiken (Springer-Verlag, Berlin) p. 3. McInnes, L. and J.R. Parratt, 1969, Studies on the mode of action of hexobendine, a prospective anti-anginal drug, Brit. J. Pharmacol. 37,272. Mckenzie, S.G., R. Frew and H.P. B~ir, 1977, Characteristics of the relaxant response of adenosine and its analogs in intestinal smooth muscle, European J. Pharmacol. 4 1 , 1 8 3 . Mori, M,, S. Yamada and E. Hayashi, 1975, Studies of depressant action of purine nucleotides on cholinergic nerves in guinea-pig ileum, Sixth International Congress on Pharmacology, Abstract, 187. Mori, M., S. Yamada, S. Takamura and E. Hayashi, .1973, Effect of purine nucleotides on acetylcholine output from cholinergic nerves in guinea-pig ileum, Jap. J. Pharmacol. 23 Suppl., 124. Moritoki, H., M. Morita and T. Kanbe, 1976, Effects of methylxanthines and imidazole on the contractions of guinea pig ileum induced by transmural stimulation, European J. Pharmacol. 35, 185. Ogura, Y., Y. Mori and Y. Watanabe, 1966, Inhibition of the release of acetylcholine from isolated guineapig ileum by crystalline tetrodotoxin, J. Pharmacol. Exptl. Therap. 154, 456. Okwuasaba, F.K., J.T. Hamilton and M.A. Cook, 1977, Antagonism by methylxanthines of purine nucleotide- and dipyridamole-induced inhibition of peristalic activity of the guinea pig ileum, European J. Pharmacol. 43, 181. Osswald, H., 1975, Renal effects of adenosine and their inhibition by theophylline in dogs, NaunynSchmiedeb. Arch. Pharmacol. 288, 79. Paton, W.D,M., 1955, The response of the guinea-pig ileum to electrical stimulation by coaxial electrodes, J. Physiol. London 127, 40 p. Paton, W.D.M., 1957, The action of morphine and related substances on contraction and on acetylcholine output of coaxially stimulated guineapig ileum, Brit. J. Pharmacol. 11,119. Paton, W.D.M. and E.S. Vizi, 1969, The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip, Brit. J. Pharmacol. 35, 10. Satchell, D.G. and G. Burnstock, 1975, Comparison
SPECIFICITY OF ADENOSINE ON ACH RELEASE
307
of the inhibitory effects on the guinea pig taenia coli of adenine nucleotides and adenosine in the presence and absence of dipyridamole, European J. Pharmacol. 32,324. Satchell, D.G., Lynch Angela, P.M. Bourke and G. Burnstock, 1972, Potentiation of the effects of exogenously applied ATP and purinergic nerve stimulation on the guinea-pig taenia coli by dipyridamole and hexobendine, European J. Pharmacol. 19, 343. Sattin, A. and T.W. Rall, 1970, The effect of adenosine and adenine nucleotides on cyclic adenosine 3',5'-phosphate content of guinea-pig cerebral cortex slices, Molecular Pharmacol. 6, 13. Sawynok, J. and K.H. Jhamandas, 1976, Inhibition of acetylcholine release from cholinergic nerves by adenosine, adenine nucleotides and morphine: antagonism by theophylline, J. Pharmacol. Exptl. Therap. 197,379. Schild, H.O., 1949, pAx and competitive drug antagonism, Brit. J. Pharmacol. 4,277. Schultz, J. and J.W. Daly, 1973, Cyclic adenosine 3t,5'-monophosphate in guinea-pig cerebral cortical slices. The role of phosphodiesterase activity in the regulation of levels of cyclic adenosine 3',5t-monophosphate, J. Biol. Chem. 248, 853. Senft, G., K. Munske, G. Schultz and M. Hoffmann, 1968, The influence of hydrochlorthiazide and other sulfamoyl diuretics on the activity of 3',5'AMP phosphodiesterase in rat kidney, NaunynSchmiedeb. Arch. Pharmacol. Exptl. Pathol. 259, 344. Sheppard, H., G. Wiggan and W.H. Tsien, 1972, Structure--activity relationship for inhibitors of phos-
phodiesterase from erythrocytes and other tissues, in: Advances in Cyclic Nucleotides Research, Vol. 1, eels. P. Greengard and G.A. Robinson (Raven Press, New York) p. 103. Shimizu, H. and J. Daly, 1970, Formation of cyclic adenosine 3f,5'-monophosphate from adenosine in brain slices, Biochim. Biophys. Acta, 222,465. Spedding, M., A.J. Sweetman and D.F. Weetman, 1975, Antagonism of adenosine 5'-triphosphateinduced relaxation by 2,2'-pyridylisatogen in the taenia of guinea-pig caecum, Brit. J. Pharmacol. 53, 575. Spedding, M. and D.F. Weetman, 1976, Identification of separate receptors for adenosine and adenosine 5'-triphosphate in causing relaxations of the isolated taenia of the guinea-pig caecum, Brit. J. Pharmacol. 57,305. Stafford, A., 1966, Potentiation of adenosine and the adenine nucleotides by dipyridamole, Brit. J. Pharmacol. 28, 218. Su, C., 1975, Neurogenic release of purine compounds in blood vessels, J. Pharmacol. Exptl. Therap. 195, 159. Takagi, K. and I. Takayanagi, 1966, Effects of strychnine, derivatives of phenyl acetate and catecholamines on contraction and acetylcholine output from the cholinergic nerve ending of guinea pig ileum, Jap. J. Pharmacol. 16, 211. Takagi, K. and I. Takayanagi, 1972, Effect of N, 2'o-dibutyryl 3',5'-cyclic adenosine monophosphate, 3r,5'-cyclic adenosine monophsphate and adenosine triphosphate on acetylcholine output from cholinergic neurons in the guinea-pig ileum, Jap. J. Pharmacol. 22, 33.
297
EFFECTS OF PURINE COMPOUNDS ON CHOLINERGIC NERVES. SPECIFICITY OF ADENOSINE AND RELATED COMPOUNDS ON ACETYLCHOLINE RELEASE IN ELECTRICALLY STIMULATED GUINEA PIG ILEUM EIICHI HAYASHI, M O T O K U N I MORI, SHIZUO Y A M A D A
and M A S A R U K U N I T O M O
Department of Pharmacology, Shizuoka College of Pharmaceutical Sciences, 2-2-10shiha, Shizuoka, 422 Japan Received 9 August 1977, revised MS received 14 October 1977, accepted 6 December 1977
E. HAYASHI, M. MORI, S. YAMADA and M. KUNrrOMO, Effects ofpurine compounds on cholinergic nerves. Specificity o f adenosine and related compounds on acetylcholine release in electrically stimulated guinea pig ileum, European J. Pharmacol. 48 (1978) 297--307. The action of 21 purine compounds on the twitch response of the electrically stimulated guinea pig isolated ileum has been investigated. Adenosine and related compounds produced a dose-dependent depression of the response. Adenosine was the most potent and 2S-deoxyadenosine had one hundredth the potency of adenosine. Adenine, hypoxanthine, inosine, IMP, ITP, xanthine, xanthosine, XMP, XTP, guanine, GMP and GTP were ineffective at concentrations less than 1 mM. Adenosine (30 p_M) reduced the electrically induced ACh output from the ileal strips. The dose---depression curve for adenosine (0.1--30/2M) was shifted to the right in the presence o f xanthine derivatives and of these, theophylline was the most p o t e n t inhibitor of adenosine. On the other hand, dipyridamole (0.1--1 pM) and hexobendine (0.1--1 ~tM) shifted the curve to the left. They markedly inhibited 3H-adenosine uptake into the ileum. Theophylline (0.1 mM), dipyridamole (0.3 /2M) and hexobendine (0.3 p_M) did not affect tetrodotoxin-, adrenaline-, strychnine- and morphine-induced inhibition of the twitch response. The present investigations have revealed that adenosine and related compounds reduce ACh release from the intramural cholinergic nerves in the guinea pig ileum possibly in a specific manner (or through a specific receptor site) different from that of other inhibitors such as morphine. Inhibition o f twitch response
Adenosine
Theophylline
1. Introduction Various actions of purine nucleotides in gut have been described: Burnstock (1972) has Suggested that adenosine triphosphate (ATP) might be a transmitter liberated by non~holinergic, non-adrenergic intramural inhibitory nerves of mammalian intestine, suggesting the existence of "purinergic nerves". McDougal and Borowitz (1972) reported that adenosine selectively inhibited the contractile response of guinea pig ileum to indirectly acting agonists. Adenosine and adenine nucleotides have been shown to depress the twitch response of transmurally stimulated guinea pig ileum (Takagi and
ACh release
Purine compounds
Takayanagi, 1972; Gintzler and Musacchio, 1975; Sawynok and Jhamandas, 1976). In earlier experiments, we found that adenosine antagonized non-competitively the contractile response of the guinea pig ileum to nicotine and markedly reduced the twitch response which was probably mediated through acetylcholine (ACh) release from the intramural cholinergic nerves (Hayashi et al., 1977). Thus adenosine and adenine nucleotides have been suggested to have an inhibitory effect on the cholinergic nerves in the guinea pig ileum. In an a t t e m p t to determine the actions of adenosine-related compounds in cholinergic nerves, we examined the actions of 21 purine compounds on the twitch response of the
298
electrically stimulated guinea pig isolated ileum. Adenosine and some related compounds had an inhibitory effect on the cholinergic nerves. Adenosine was the most potent inhibitor and in addition its effect was selectively antagonized by xanthine derivatives such as theophylline and enhanced by dipyridamole and hexobendine. Present investigations have shown that adenosine and related compounds reduce ACh release from the intramural cholinergic nerves in the guinea pig ileum possibly in a specific manner different from that of other inhibitors such as morphine. Preliminary accounts of this work have been presented to the VIth International Congress of Pharmacology (Mori et al., 1975) and the Japanese Pharmacological Society (Mori et al., 1973; Hayashi et al., 1975).
2. Materials and methods
2.1. General The experiments were carried out on ileal strips (3--4 cm, unstretched) of male guinea pigs weighing 250--350 g. Preparations were suspended in Tyrode solution gassed with a mixture of 95% 02 and 5% CO2. The capacity of the organ bath was 10 ml and it was kept at 37°C. The composition of the Tyrode solution has been described previously (Hayashi and Yamada, 1975b). The strips were subjected to 1.0 g resting tension. Transmural electrical stimulation of the ileum was carried out according to the method of Paton (1955, 1957). The electrodes were made of platinum and the intramural electrode was the anode. The preparations were stimulated with rectangular pulses lasting 0.4msec with a frequency of 0.1 Hz and sufficient strength to produce a supramaximal response. The supramaximal response was used because the response to this stimulation was more constant than that to submaximal stimulation. The preparation was allowed to stabilize for 60 min before an experiment. The response of
E. HAYASHI ET AL.
the ileum was recorded isometrically on a recticorder (Nihon Kohden, RJG-3024) with a force-displacement transducer (Nihon Kohden). Purine compounds were cumulatively added to the organ bath and the other drugs were used as pretreatment for 5--10 min at 2--4 dose levels.
2.2. Assay of ACh released An ileal strip was suspended in an organ bath containing 2 ml of Tyrode solution (37°C) gassed with the gas mixture. The strip was electrically stimulated for 1 min in the absence or presence of adenosine and other drugs, and the samples were collected. The released ACh was assayed on another ileal strip suspended in 5 ml Tyrode solution (37°C) containing physostigmine (0.1/~M) and morphine (1/~M) (Paton and Vizi, 1969). The estimate of ACh content of samples was based on control dose-response curves for ACh. Ileal strips were incubated for 60 min in Tyrode solution containing 14C-choline (30 pM, 0.2/~Ci/ml, specific activity 56.85 mCi/ mmole), and then superfused with Tyrode solution for 60 min. The strip was electrically stimulated for 1 min in the absence and presence of adenosine and other drugs; the radioactivity in the samples was measured with an Aloka liquid scintillator and expressed as dpm/100 mg wet weight tissue/min.
2.3. Measurement of uptake of 3H-adenosine Ileal strips were pretreated with dipyridamole and hexobendine for 10 min and then incubated for 30 min in Tyrode solution conraining 3H-adenosine (10/zM, 2/~Ci/ml, specific activity 23.2 Ci/mmole). After the incubation, the strips were rinsed with the chilled Tyrode solution, blotted, weighed and digested with soluene 350 (Parkad). Radioactivity was measured and expressed as dpm/ g wet weight tissue/30 min.
SPECIFICITY OF ADENOSINE ON ACH RELEASE
2.4. Measurement o f the activity o f enzymes
The activity of the acetylcholinesterase and adenosine deaminase of guinea pig ileum was determined by the methods of Guenther and Klaus (1970) and of Kalckar (1947) respectively. The activity of adenosine deaminase (purified deaminase of pig intestine, Seikagaku Kogyo) was measured spectrophotometrically (265 p). The following drugs were used: acetylcholine chloride (Daiichi), adrenaline hydrochloride (Daiichi), tetrodotoxin (Sankyo), morphine hydrochloride (Takeda), strychnine sulphate (Merck), chlorthiazide (Takeda), dipyridamole (Tanabe), hexobendine (Toyo jozo), nicotinic acid (Wako), physostigmine sulphate (Merck), adenosine triphosphate (Kyowa), adenosine diphosphate (Kyowa), 3',5'-cyclic AMP (Kyowa), NS-dibutyryl-3',5 '-
299
cyclic AMP (Kyowa), other purine compounds (Kohjin). Xanthine and derivatives were gifts from Prof. M. Sekiya of this college. 3HAdenosine and 14C-choline were obtained from New England Nuclear, Boston. 3. Results
3.1. Effects o f purine compounds on twitch response
In order to investigate the structural features of the adenine derivatives associated with their inhibitory effects on the cholinergic nerves, we examined the action of 21 purine compounds on the twitch response of guinea pig isolated ileum to electrical stimulation. Adenosine and related compounds produced a dose-dependent depression of twitch
TABLE 1 Inhibitory actions of adenosine and related compounds on the twitch response of the electrically stimulated guinea pig ileum, pD2 values were calculated from the dose--depression curves. The number of experiments is indicated in parentheses. Compound
Structure I
ATP ADP 5'-AMP 2'-AMP 3',5'-c-AMP 2',3'-c-AMP Dibu-c-AMP 3
pD 2 value (mean _+S.E.M.)
R1
R2
(~(~2 ®~® ~ -OH
-OH -OH -OH --OH ~
--OH (~
R3 -OH -OH -OH ~ -OH ~ ' ~ -OC(CH2 )2CH3
5.33 5.52 5.72 5.81 5.18 5.44 4.43
_+0.04 +- 0.09 + 0.11 _+0.09 -+ 0.04 + 0.08 _+0.09
(7) (7) (7) (8) (8) (7) (8)
4 4
4 4 4
O Adenosine 2'-Deoxyadenosine 1
--OH -OH
-OH -OH
-OH --H
NH2
R2 R3
2 (~ = phosphate. 3
NH2 . . . .
NH(CH2)2CH3.
The levels o f significance for adenosine and other compounds are shown by 4(p
5.83 -+ 0.04 (42) 3.80 + 0.09 (7) 4
300
E. HAYASHI ET AL.
•
Ade
7
• 20
• 70
• 200
• 700
,I,~F
xlO nM W W
l
j2g 1 rain
Fig. 1. Inhibitory action of adenosine on the twitch response of the electrically stimulated guinea pig ileum. Adenosine (Ade 0.03--10 pM) was cumulatively added to the organ bath. Dots (o) indicate the addition of adenosine and the arrows (~LW) washing of the preparation.
response; the pD2 values (Schild, 1949; Ari~ns and Van Rossum, 1957) are shown in table 1. Adenosine was the most potent and 2'-deoxyadenosine had one hundredth the potency of adenosine. The order of potency was adenosine > 2'-AMP = 5'-AMP > 2',3'-cyclic AMP > ATP > 3',5'-cyclic AMP > dibutyryl-cyclic AMP > 2'-deoxyadenosine. The difference in pD2 values between adenosine and related compounds except 2'- and 5'-AMP was significant at the 1% level (t-test). On the other hand, adenine, hypoxanthine, inosine, IMP, ITP, xanthine, xanthosine, XMP, XTP, guanine, GMP and GTP produced no
inhibition at concentrations less than 1 mM. The depressant effect Of adenosine was investigated further. Low concentrations of adenosine (0.03--10 #M) produced a dosedependent depression of the twitch response (fig. 1). The effect was relatively rapid in onset with maximal depression always developing within 1 min. The inhibitory effect disappeared rapidly (3--5 min) after washing the preparation. Thus adenosine reduced the response by 6.1 + 0.7% (mean + S.E.M.) (n = 42) at 0.1/~M and by 98.7 + 0.5% (n = 42) at 30 #M (pD2 = 5.83 + 0.04). Adenosine in these concentrations antagonized the effect of nicotine non-competitively without affecting the direct muscle response to ACh (Hayashi et al., 1977). The effect o f adenosine was compared with that of tetrodotoxin (Ogura et al., 1966; Gershon, 1967), adrenaline (Paton and Vizi, 1969; Kosterlitz et al., 1970), strychnine (Takagi and Takayanagi, 1966) and morphine (Paton and Vizi, 1969; Kosterlitz et al., 1970) which are known to inhibit the twitch response. These agents reduced the twitch response but recovery o f t h e response was much slower ( 3 0 - 6 0 min). The pD2 values for tetrodotoxin, adrenaline, strychnine and morphine were 7.78 + 0.01 (n =5), 7 . 0 9 + 0 . 0 6 ( n = 1 5 ) , 5 . 0 7 + 0 . 1 0 (n = 8) and 7.07 + 0.08 (n = 7) respectively.
TABLE 2 Inhibitory actions of adenosine (30 p_M), morphine (1 ~M) and tetrodotoxin (0.1 p_M) on electrically induced ACh and 14 C output from the guinea pig ileum preincubated with 14 C-choline. Ileal strips were electrically stimulated for 1 min in the absence and presence of these drugs, and the released ACh was assayed on another ileal strip. Resting ACh output was subtracted. Ileal strips were also incubated for 60 rain in Tyrode solution containing 14 C-choline and then electrically stimulated for 1 min in its absence and presence. The 14 C output was measured. The number of experiments is indicated in parentheses. Drugs
None Adenosine Morphine Tetrodotoxin
. Concentration (/~M)
30 1 0.1
ACh o u t p u t (ng/min) (mean + S.E.M.)
14 C output (dpm/lO0 mg wet weight tissue/min) (mean +- S.E.M.)
Electrical stimulation
Resting
Electrical stimulation
54.2 + 4.0 (10) 13.1 + 1.5 (8) 13.3 -+ 2.6 (8)
589.2 _+58.8 (10)
1378.8 676.0 590.2 644.0
+_ 82.2 (10) + 93.7 (8) + 65.6 (8) + 101.7 (8)
SPECIFICITY OF ADENOSINE ON ACH RELEASE 3.2. Effect
of adenosine on ACh release
The effect o f adenosine on ACh release from the electrically stimulated ileum was examined. This was carried o u t by assaying the ACh o u t p u t and by determining the 14C o u t p u t from 14C-choline-incubated ileum in the absence and presence of adenosine. Adenosine (30 pM) reduced the electrically induced ACh o u t p u t and the 14C o u t p u t by 70--80% and by 90--100% respectively (table 2). Morphine (1/~M) and t e t r o d o t o x i n (0.1 /~M) exerted a similar depression. 3.3. Effects of some drugs on adenosineinduced inhibition o f the twitch response 3.3.1. Chlorthiazide and nicotinic acid Adenosine has been shown to stimulate the formation of cyclic AMP in guinea pig cerebral cortex slices (Sattin and Rall, 1970; Shimizu and Daly, 1970; Huang et al., 1972). In order to investigate the possibility of cyclic AMP involvement in the adenosine-induced inhibi-
301 tion, the effect of chlorthiazide, an inhibitor and of nicotinic acid, an activator of phosphodiesterase respectively (Senft et al., 1968; Krishna et al., 1966) on the inhibition was examined. The dose-depression curve for adenosine was unchanged in the presence of chlorthiazide (10/~M) and nicotinic acid (1 mM). 3.3.2. Xanthine derivatives Caffeine antagonized the depressant action of adenosine and of the nucleotides on the contraction and action potentials of mammalian atrial muscle (De Gubareff and Sleator, 1965). Aminophylline inhibited the coronary vasodilatory action o f adenosine in dogs (Afonso, 1970). The effects on the adenosineinduced inhibition of 10 xanthine derivatives which had been found inactive in terms of an inhibitory effect on the twitch response were investigated. Xanthine and the methyl-substituted analogs at concentrations of 10 #M-I mM shifted the dose--depression curve for adenosine to the right without depressing
TABLE 3 Effects of xanthine derivatives on adenosine-induced inhibition o f twitch response. Pretreatment with xanthine derivatives at 2--4 dose levels was for 5 rain. The dose--depression curve for adenosine was obtained in the absence and presence o f these c o m p o u n d s and PA2 values were calculated. The number o f experiments is indicated in parentheses.
Compound
Structure I
pA2 value (mean + S.E.M.)
RI
R2
R3
H H
H H
H H
---
Xanthine
H
1-Methylxanthine
H H
7-Methylxanthine 1,3-Dimethylxanthine
CH3 H H CHs
3,7-Dimethylxanthine
H
1,7-Dimethylxanthine 1,3,7-Trimethylxanthine
CH3 CH3
H H H CH3 H CH3 CH3 CH3
3.67 4.25 4.07 3.96 4.93 4.07 4.35 4.18
Hypoxanthine *
Uric acid ** 3-Methylxanthine
o
RI"-N" ~ ' ~
R2
N-R3 N
N
CH 3
H CH3 CH3 H
CH3
(5) (5)
+ 0.24 (8) + 0.06 (8) + 0.06 (8) + 0.08 (8) +-0.05 (26) + 0.11 (8) + 0.13 (8) + 0.07 (8)
302
E. HAYASHI ET AL. Adr
Nor
2g
J
(a)
Ade
l rain
Theo
j/
100-
50,
Fig. 2. Effect of theophylline on adenosine-induced inhibition of the twitch response. (a) Adenosine (Ade 10 p-M)-induced inhibition of twitch response and its reversal by an addition of theopbylline (Theo 0.1 raM). (b) The effect of theophyUine (Theo 0.1 raM) pretreatment on adenosine (Ade 10 /IM)induced inhibition. Dots ($) indicate the addition o f adenosine or theophylline, and the arrows (~W) washing of the preparation.
the maximal effect and significantly decreased the pD2 values, although hypoxanthine and uric acid were only slightly active. The pA2 values (table 3) show that 1,3-dimethylxanthine (theophylline) was the most potent 100
J
Fig. 4. Effect o f theophylline on the dose--depression curves for tetrodotoxin, adrenaline, strychnine and morphine. (o) Control, in the presence of theophylline (e) 0.1 raM. Ordinates: average % o f inhibition of the response produced by t e t r o d o t o x i n (TTX), adrenaline (Adr), strychnine (Str) and morphine (Mor). Abscissae: concentration (log M) of the drugs. Each point represents the mean (±S.E.M.) of at least 5 experiments.
TABLE 4
Effect of dipyridamole, hexobendine and theophylline on 3 H-adenosine uptake into the ileal strips. Ileal strips were pretreated with dipyridamole, hexobendine and theophylline for 10 rain and then incubated for 30 min in Tyrode solution containing 3 H-adenosine. The radioactivity in the tissues was measured. The number of experiments is indicated in parentheses. Drugs
0
-7
-;
-;
-'4
Fig. 3. Effect of theophylline on the dose--depression curve for adenosine. (o) Control; in the presence of theophylline, (o) 10 p_M, (A) 30 pM, (A) 100 /~M, (D) 300 #M. Ordinate: average % of inhibition of the response produced by adenosine. Abscissa: concentration (log M) of adenosine. Each point represents the mean (±S.E.M.) of 26 experiments.
None Dipyridarnole
Conc. (pM)
0.1 1
Hexobendine
Theophylline
0.03 0.1 0.3 1 100
Uptake of 3 H-adenosine (dpm/g wet weight tissue/ 30 rain) (mean + S.E.M.) × 10 s 6.95 ± 0.49 (12) 4.43 _+0.44 (7) 1.32 +0.12 (7) 6.67 ± 0.51 (7) 4.44 ± 0.48 (7) 2.90 ± 0.48 (7) 1.45 ± 0 . 2 6 (7) 6.42 ± 0.30 (5)
SPECIFICITY OF ADENOSINE ON ACH RELEASE inhibitor of adenosine. The order of p o t e n c y is 1,3-dimethyl ~ 1,7~iimethyl ~ 1,3,7-trimethyl ~ 3,7-dimethyl ~ 1-methyl ~ 3-methyl 7-methyl,-xanthine ~ xanthine ~ hypoxanthine = uric acid. Fig. 2 shows the antagonistic effect o f theophyUine (0.1 raM) o f the adenosine-induced inhibition. The antagonism was reversible and surmountable b y increasing the concentration of adenosine (fig. 3), suggesting competitive antagonism such as was reported for the adenosine effect in heart (Bringer et al., 1975), kidney (Osswald, 1975) and intestine (Sawynok and Jhamandas, 1976). Although tehophylline itself had very little effect on the twitch response of the ileum at concentrations less than 0.1 mM, it potentiated the response at higher concentrations (0.3--1 raM) b y 20--30% as previously reported b y Moritoki et al. (1976) and Kazic (1977). On the other hand, tetrodotoxin-, adrenaline-, strychnine- and morphineinduced inhibition was not antagonized b y theophylline (0.1 raM) (fig. 4). Concentrations of theophylline less than 1 mM had little effect on the activity of enzymes such as acetylcholinesterase and adenosine deaminase and on the uptake of 3H-adenosine into the ileum (table 4).
3.3.3. Dipyridamole and hexobendine to
Dipyridamole and hexobendine are k n o w n potentiate the inhibitory effects of
DP
100
-0
-7
-5
-S
-8
-7
-6
-5
Fig. 5. Effect of dipyridamole (DP, left) and hexobendine (HB, right) on the dose--depression curves for adenosine. (o) Control; in the presence of dipyrldamole, (e) 0.1 p2Vl, (A) 0.3 p~I, (A) 1 p_M.Ordinates: average % of inhibition of the response produced by adenosine. Abscissa: concentration (log M) of adenosine. Each point represents the mean (±S.E.M.) of at least 7 experiments.
303
r
HB
Ad.
TTX
Iq0r
r
TTX
HB
Str
Fig. 6. Effect of hexobendine on adenosine-, tetrodotoxin-, adrenaline-, strychnine- and morphine-
induced inhibition. Dots (e) indicate the addition of adenosine (Ade 0.03 p_M),tetrodotoxin (TTX 0.01 p~M), adrenaline (Adr 0.03 p_M), strychnine (Str 0.1 p-M) and morphine (Mot 0.01 ~M). At the arrows (J'HB), hexobendine (0.3 p~M) pretreatment for 10 min.
adenosine and of the nucleotides in coronary artery (Stafford, 1966; McInnes and Parratt, 1969) and taenia coli (SatcheU et al., 1972). The dose--depression curves for adenosine were shifted to the left in the presence of dipyridamole and hexobendine at low concentrations (0.1--1 ~M) which had little effect on t h e twitch response (fig. 5); the pD2 values were increased significantly (pD2 for adenosine: control, 5.95 ± 0.11, in the presence of dipyridamole (0.1/~M) 6.21 -+ 0.10, (0.3 ~M) 6.51 ± 0.10, (1/~M) 6.69 ± 0.10, n = 7; control, 5.83 ± 0.04, in the presence of hexobendine (0.1 #M) 6.41 ± 0.11, (0.3 #M) 6.95 ± 0.10, (1 ~M) 7.24 ± 0.15, n = 7). On the other hand, these agents did not affect tetrodotoxin-, adrenaline-, strychnine- and morphine-induced inhibition (fig. 6). Dipyridamole and hexobendine at higher concentrations ( 3 - - 1 0 0 ~ M ) reduced the contractile response to ACh as well as that to electrical stimulation. L o w concentrations (0.1--1 ~M) of dipyridamole and hexobendine inhibited the uptake of 3H-adenosine into the ileum b y 40-80% (table 4). Higher concentrations (30-100 ~M) inhibited the uptake of 3H-adenosine b y 90--100% and also adenosine deaminase activity b y 20 60% (inhibition rate: dipyridamole (100 ~M) 18.4 -+ 2.0%, n = 4; hexobendine (30 #M) 17.3 ± 1.8%, n-- 4; (100 /~M) 63.0 ± 3.4%, n = 5).
304
4. Discussion The effects of 21 purine compounds on the twitch response of guinea pig isolated ileum to electrical stimulation were examined. The adenosine derivatives with a 6-amino group {adenosine derivatives caused a dose-dependent depression of the twitch response, whereas the other compounds were ineffective. Adenosine was the most potent and 2'~leoxyadenosine had only approximately one hundredth the potency of adenosine. Adenosine has been reported as having approximately one hundredth the potency of ATP with respect to the inhibitory effect on the gut (Burnstock, 1972). However, the inhibitory effect on the twitch response (cholinergic nerves} tended to decrease gradually with the addition of phosphate to the ribose group. Structure--activity studies suggested that the 6-amino group, the 2'hydroxy group and the ribose group play an important role in the depression of the twitch response by purines. Adenosine reduced the electrically induced ACh output from the ileal strips by 70--80% and the 14C output from 14C-choline-incubated strips by 90--100%. The 14C output evoked appears to be largely ~4C-ACh since it was sensitive to tetrodotoxin blockade. These findings show that the adenosine-induced inhibition of the twitch response is due to a reduced release of ACh from the intramural cholinergic nerves in the guinea pig ileum. On the other hand, the depression of ACh release by adenosine could not be shown at other cholinergic synapses, such as t h e neuromuscular junction of motor nerves or autonomic ganglia. Similar results with morphine have been reported by Paton (1957). The inhibitory action of adenosine on the cholinergic nerves was further characterized. The antagonism of methylxanthines against adenosine and adenine nucleotides has been demonstrated in heart {De Gubareff and Sleatot, 1965; Afonso, 1970; Bringer et al., 1975), kidney (Osswald, 1975), brain (Sattin and RaU, 1970; Shimizu and Daly, 1970; Huang
E. HAYASHI ET AL.
et al., 1972; Schultz and Daly, 1973; Kuroda and Kobayashi, 1975) and intestine (Sawynok and Jhamandas, 1976; Mckenzie et al., 1977; Okwuasaba et al., 1977). We tested the effects of 10 xanthine derivatives on the adenosineinduced inhibition and found the action of adenosine to be competitively antagonized by all of them. Theophylline was the most potent and hypoxanthine and uric acid were weak antagonists of adenosine. These data suggest that the carbonyl group and the 1-, 3- and 7methyl groups are important for determining the potency of inhibition. TheophyUine had no effect on the activity of enzymes such as acetylcholinesterase and adenosine deaminase of the ileum and on uptake of adenosine into the tissue. Although theophyUine is a well known inhibitor of phosphodiesterase, the concentrations of theophylline used in the present study were significantly lower than those needed to inhibit the activity of phosphodiesterase (Sheppard et al., 1972). Thus the effect of theophylline on phosphodiesterase can be ruled out. Because theophylline only antagonized the adenosine-induced inhibition without affecting the response to tetrodotoxin, adrenaline, strychnine and morphine, it is suggested that they compete at adenosinespecific receptor sites. The existence of such receptors has been demonstrated in other tissues (Burnstock et al., 1970; Sattin and Rall, 1970; Spedding et al., 1975; Spedding and Weetman, 1976; Mckenzie et al., 1977), It was suggested that there is a link between morphine and adenine derivatives actions in the electrically stimulated guinea pig ileal longitudinal strips since theophyUine (0.2-0.5 mM) antagonized the morphine-induced inhibition as well as the adenosine- and adenine nucleotides-induced inhibition of the twitch response (Sawynok and Jhamandas, 1976). However, in the present experiments, theophyUine did not alter the morphineinduced inhibition when used in the concentrations (10--100 ~M) which antagonized the adenosine-induced inhibition; at the higher concentrations {0.3--1.0 raM) used by Sawynok and Jhamandas, theophylline poten-
SPECIFICITY OF ADENOSINE ON ACH RELEASE
tiated the twitch response in agreement with the findings of Moritoki et al. (1976) and Kazic (1977). Therefore it appears unlikely that morphine-induced inhibition is mediated through a c o m m o n mechanism with adenosine as suggested by Sawynok and Jhamandas (1976). Dipyridamole and hexobendine have been shown to potentiate the coronary vasodilating (Stafford, 1966; McInnes and Parratt, 1969; Kolassa et al., 1970) and negative chronotropic (Kolassa et al., 1971) effects of adenosine as well as the inhibitory effects of adenosine and adenine nucleotides in the intestine (Satchell et al., 1972; Satchell and Burnstock, 1975; Okwuasaba et al., 1977) and trachea (Coleman, 1976). The adenosine-induced inhibition of acetylcholine release was effectively enhanced in the presence of low concentrations of dipyridamole and hexobendine, whereas tetrodotoxin-, adrenaline-, strychnineand morphine-induced inhibitions were not. Degradation by adenosine deaminase and the uptake into the tissue have been considered to be an inactivation process of adenosine. Dipyridamole and hexobendine in concentrations which enhanced the effect of adenosine prevented adenosine uptake into the tissue, while at higher concentrations t h e y also reduced adenosine deaminase activity. Therefore the potentiation of adenosine action by dipyridamole and hexobendine appears to be exerted mainly through the blockade of adenosine uptake. This has also been demonstrated for heart (Kolassa e t al., 1971; Hopkins, 1973), lungs ( C o l e m a n , 1976) and taenia coli (Satchell et al., 1972). Present investigations have revealed that adenosine and adenine nucleotides reduce ACh release from the intramural cholinergic nerves in the guinea pig ileum possibly in a specific manner (or through a specific receptor site) different from that of tetrodotoxin, adrenaline, strychnine and morphine. It is proposed that endogenous adenine derivatives may have a physiological role as modulators which control ACh release in the guinea pig ileum in agreement with the suggestions of
305
other workers (Berne et al., 1971; McIlwain, 1974; Su, 1 9 7 5 ) .
Acknowledgements The authors wish to thank Prof. M. Sekiya, Shizuoka College of Pharmaceutical Sciences for kindly supplying xanthine derivatives and also Kobjin Co., Ltd., for the generous gift of purine compounds.
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