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Effects of adenosine on norepinephrine and acetylcholine release from guinea pig right atrium: Role of A1-receptors
~
Pergamon
Neurochem. Int. Vol. 27, No. 4/5, pp. 345-353, 1995
0197-0186(95)00016-X
Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 01974)186/95 $9.50+ 0.00
EFFECTS OF ADENOSINE ON NOREPINEPHRINE A N D ACETYLCHOLINE RELEASE FROM GUINEA PIG RIGHT ATRIUM" ROLE OF A1-RECEPTORS H. N A K A T S U K A j'2'3, O. N A G A N O 1'3, F. F. F O L D E S l, H. N A G A S H I M A 1 and E. S. VIZI 2. LDepartment of Anesthesiology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, N.Y. 10467, U.S.A. 21nstitute of Experimental Medicine, Hungarian Academy of Sciences, 1450-Budapest, P.O. Box 67, Hungary 3Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama 700, Japan (Received I October 1994 ; accepted 21 December 1994)
Almtraet--The effect of adenosine or its stable analogues (2-chloroadenosine, CADO: Y-N-ethylcarboxamidoadenosine, NECA; and Nf-cyclopentyladenosine, CPA) and a non-selective A~ and A2receptor antagonist, 8-phenyltheophylline (8-PT), or an Al-receptor antagonist, 8-cyclopentyl-l,3-dipropylxanthine (DPCPX), on the stimulation-evoked release of [3H]norepinephrine ([3H]NE) and [3H]acetylcholine ([3H]ACh) from the isolated guinea pig right atrium was investigated. Adenosine and its stable analogues (CADO, NECA and CPA) inhibited the stimulation-evoked release of [aH]NE in a concentrationdependent manner. The order of potencies was CPA > NECA > CADO > adenosine. CGS 21680 (30 nM), an A2a receptor agonist, failed to affect the release. The inhibitory effect of adenosine and CADO on [3H]NE release was competitively antagonized by 8-PT. DPCPX also prevented the effect of adenosine (Kd = 5.2 nM) and CADO (Kd = 3.3 nM). The Kd value of 8-PT was 0.40/tM for the antagonism of CADO and 0.51 #M for the antagonism of adenosine. When the negative feedback modulation of NE release was inhibited by idazoxan, the inhibitory effect of adenosine and CADO on [3H]NE release was more pronounced. Under this condition DPCPX (10 nM) prevented the inhibitory effect of CADO, indicating that A~-purinoceptors are involved in this action. The release of [3H]NE is tonically modulated by ACh released from the vagal nerve endings, as evidenced by the finding that 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), a M3-subtype selective muscarinic receptor antagonist, and atropine significantly enhanced the release of NE. Adenosine, its stable analogues (CADO and NECA), and 8-PT did not have any effect on the stimulation-evoked release of [3H]ACh. Even when the muscarinic autoinhibition was eliminated by atropine, adenosine and CADO did not have any effect on [3H]ACh release. Quinpirole, a selective D2-receptor agonist, and neuropeptide Y (NPY) failed to affect the release of ACh. However, atropine and 4-DAMP, a selective M3-receptor antagonist, significantly enhanced the stimulation-evoked release of [3H]ACh. These findings indicate that there are no presynaptic heteroceptors (adenosine, D2, and NPY) on the vagal nerve endings of the guinea pig right atrium. It is concluded that the sympathetic nerve endings of the guinea pig right atrium are equipped with A~-, subclass of purinoceptors and ctzB-,and muscarinic (M3)-receptors. Cholinergic vagal nerve endings in the heart are only equipped with muscarinic autoreceptors. Therefore, adenosine liberated during hypoxia inhibits NE release from the cardiac sympathetic nerve and thereby protects against tachyarrhythmia caused by myocardial hypoxia. In contrast, adenosine does not inhibit the vagal innervation of the right atrium.
Several studies have shown that autonomic modulation of the heart rate is determined by the interaction of sympathetic and vagal innervation at the effector level (sinus node) (cf. Urthaler et al., 1986). Increased
vagal-sympathetic antagonism, i.e. augmentation of the vagal action in the presence of prevailing sympathetic activity, is of primary importance for the control of the heart rate (cf. Muscholl, 1990). This interaction could be manifested either on the sinus cells or as it was recently suggested (cf. Muscholl,
* Author to whom all correspondence should be addressed. 345
346
H. Nakatsuka et al.
1990; M a n a b e et al., 1991 ; V i z i e t al., 1991) at the presynaptic terminals o f the postganglionic sympathetic a n d p a r a s y m p a t h e t i c nerves. Whereas noradrenergic axon terminals are equipped with inhibitory muscarinic receptors (cf. Muscholl, 1980, 1990; M a n a b e et al., 1991), so that A C h released from the postganglionic vagal nerve is able to tonically control the release o f NE, the postganglionic vagal nerve terminals are not equipped with ~2-adrenoceptors ( M a n a b e et al., 1991), a n d N E does not influence the release o f ACh. Since adenosine is released in excess during hypoxia a n d accumulates in the myocardium in m i c r o m o l a r concentrations (Berne, 1980) and it is also used in m a n y clinical situations as a vasodilator, antiaggregatory, a n d a n t i a r r h y t h m i c agent (cf. Sollevi, 1986), its effect on the a u t o n o m i c innervation o f heart is of physiological, pharmacological, and clinical importance. While the inhibitory effect of adenosine on N E release from the sympathetic nerve endings of the heart has been well d o c u m e n t e d (Richardt et al., 1987; W e n n m a l m et aL, 1988), no attempt has been made so far to classify the subtype of these receptors and no i n f o r m a t i o n has been m a d e available on the effect o f adenosine o n the release of A C h from the vagal nerve endings. Since the release of N E is also subject to presynaptic m o d u l a t i o n t h r o u g h muscarinic heteroceptors by A C h released from the vagus (cf. Muscholl, 1980, 1990 ; F61des et al., 1989 ; M a n a b e et al., 1991), and t h r o u g h ~2-autoreceptors by N E released from the sympathetic nerve endings, we also made an a t t e m p t to classify the subtype o f ~2 and muscarinic receptors located on postganglionic axon terminals using subtype-selective antagonists. In this study, using a radiochemical technique we measured directly the effect o f adenosine a n d its stable analogues (2-chloroadenosine, C A D O ; 5'-N-ethylc a r b o x a m i d o a d e n o s i n e , N E C A ; and N~-cyclopen tyladenosine, CAP), the AdA2-receptor antagonist 8-phenyltheophylline (8-PT), a n d the A~-receptor antagonist 8-cyclopentyl-l,3-dipropylxanthine ( D P C P X ) on the stimulation-evoked release of N E and A C h from guinea pig right atria. EXPERIMENTAL PROCEDURES Norepinephrine reh, ase Male guinea pigs of 300-500 g body weight, lightly anesthetized with enflurane, were killed by a blow to the head, and the right atria were dissected out and incubated for 40 min at 3T'C in modified Krebs' solution (F61des, 1981) containing 370 kBq/ml 1-(7,8-3H)-norepinephrine ([3H]NE) (specific activity 1.14 kBq/mmol). The solution was aerated with 95% 02 5% CO2. To facilitate the uptake of [3H]NE,
during incubation the preparations were continuously stimulated at 1 Hz with supramaximal (35 V/cm) impulses of 1 ms duration, through two platinum electrodes placed above and below the suspended atria (field stimulation). To remove excess [3H]NE after incubation the atria were transferred to 1.5 ml organ baths and were superfused at a rate of I ml/min for 90 min with Krebs' solution. To prevent non-enzymatic breakdown on NE, 27 pM Na2EDTA and 300/tM ascorbic acid were added to the perfusion solution (McCulloch et al., 1974). After the 90-min washout period, superfusion was continued at the rate of 1 ml/min and 3-min fractions of the superfusate were collected throughout the experiment. Starting at the beginning of the 10th (S~), 28th ($2), 46th ($3), and 64th ($4) min, the preparations were stimulated for 2 min at 2 Hz (240 stimuli) (Fig. 1). Except for the stimulation rate, the method of stimulation was the same as during incubation. Compounds to be investigated were added to the perfusing solution 6 min after $2 and kept until the end of experiments. The spontaneous (resting) release of 3H was determined from the average 3H content of two non-stimulated 3-min fractions before and after each stimulation period. The computer-derived regression line of the resting release was determined with an exponential curve-fitting program. The evoked release of 3H was determined by subtracting the resting release from the total release measured in the consecutive fractions collected following the start of stimulation. Using HPLC combined with radiochemieal detection (Manabe et al., 1991) 87.4_+4.3% of the total radioactivity released by electrical stimulation was due to [3H]NE. This is in agreement with our earlier findings (Manabe et al., 1991). In control experiments the ratios of amounts of 3H released during consecutive stimulation periods (i.e. $2/S~, $3/$2, $4/$3) were similar (Fig. 1). Therefore any increase or decrease of these
1,6
~ 0
-
1.4
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1.2
-
1.o
-
0.8
-
J
o
,
15
IIIIII $2
I S3
I 30
I 45
m $4
i 60
I 75
Time (min) Fig. 1. Fractional rele~e of radioactivity from isolated right atrium loaded with [3H]NE in response to electrical field stimulation (2 Hz, 240 shocks). Average of four identical experiments. Note that four stimulations (S~, $2, $3 and $4) as indicated resulted in almost identical release of radioactivity. Mean + SEM is indicated.
347
Norepinephrine and acetylcholine release ratios caused by the addition of drugs could be attributed to the effect on the compound used on NE release. The IC20of adenosine and its analogues was also calculated from the concentration-response curves plotted in Fig. 2. IC20 indicates the concentration needed to reduce the release of radioactivity by 200. The ratio of the amount of radioactivity released by two consecutive stimulations ($3/$2) in the absence of drugs (0.92+0.03, n = 6) was taken as the control release ratio.
Acetylcholine release After dissection, the atria were incubated for 40 min at 37°C in Krebs' solution containing 185 kBq/ml methyl[3H]choline (specific activity 2.89 TBq/mmol). The solution was aerated with 95% 02-5% CO2 throughout the experiment. During incubation, the incorporation of [3H]choline into the ACh pool of the nerve terminal was facilitated by electrical field stimulation. The method of stimulation was the same as in the NE release studies. After incubation, the atria were transferred to 1.5 ml-organ baths and were superfused at a rate of 1 ml/min for 60 min. Subsequently the preparations were superfused for another 30 min with Krebs' solution containing 50 #M hemicholinium-3 to prevent re-uptake of the [3H]choline liberated by the hydrolysis of [3H]ACh. After washout, superfusion with Krebs' solution containing 50 #M hemicholinium-3 was continued at the rate of 1 ml/min and 30 min fractions of superfusate were collected throughout the experiment. Starting at the beginning of the 10th (SO, 28th ($2), 46th ($3), and 64th ($4) min, the preparations were stimulated for 2 min at 2 Hz. Compounds to be investigated are added to the superfusing Krebs' solution 6 min after $2. It was reported that 9 2 o of the radioactivity released in response to the stimulation was due to [3H]ACh
O e~e v~v s~1 @~ 0
1.0 0.9 0.8
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0.7
0.6 0.5
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Measurement o f radioact&ity A l-ml aliquot of each collected fraction was transferred to scintillation vials containing 7 ml of scintillation fluid (Echoscinti, National Diagnostics). The radioactivity of the samples was measured in a scintillation beta spectrophotometer (TRI-CARB 4530, Packard Instrument Co.). At the end of each experiment, the residual 3H content of the atria was determined. The tissue was kept overnight in 1 ml of tissue solubilizer (Soluvable, NEN Research Product) at room temperature and the 3H content of the sample was measured. The radioactivity of the samples was expressed in terms of disintegrations per gram wet weight of tissue (Bq/g). To normalize the quantity of radioactivity released, the release of [3H]NE of [3H]ACh was expressed as a percentage of the total radioactivity present in the tissue at the onset of stimulation (fractional release). The fractional release was calculated by a computer program taking into account the content of radioactivity at the end of experiment and the release of radioactivity in the samples. Cah'ulations In some experiments the apparent dissociation constant (Kd) for antagonists was determined by the dose-ratio method. The following equation was used to relate the dissociation constant to the dose-ratio and the antagonist concentration
K~-
a
DR-- 1
Where DR is the concentration-ratio, i.e. the EC20 value for agonist in the presence of the antagonist divided by the EC20 value in the absence of antagonist and a is the concentration of antagonist. Four different concentrations of agonists were applied to establish a concentration-response curve. In some experiments the Lineweaver-Burk plot was used and the Kd value was calculated (pA2 = - log Kd). The means ___SEM of data are presented. One way analysis of variance followed by Dunnett's test was used. A value of P < 0.05 was considered to be significant.
Control Adenosine GADO NECA CPA
O
"~
(Manabe et al., 1991). The stimulation-evoked release of ACh was calculated as described in the NE release studies.
] 1000
Agonist concentration (IJ.mol/L) Fig. 2. Effect of different adenosine receptor agonists [adenosine, 2-chloroadenosine (CADO), 5'-ethyl-carboxamidoadenosine (NECA), Nr-cyclopentyladenosine (CPA)] on the stimulation-evoked release of radioactivity from isolated right atrium loaded with [3H]NE. Drugs were added into the organ bath 6 min after $2. Note that adenosine and its stable analogues concentration-dependently reduced the release of radioactivity evoked by electrical field stimulation (2 Hz, 240 shocks). Each point represents 4 or 6 experiments. Mean ___SEM is indicated.
Materials Adenosine, 2-chloroadenosine (CADO), 5'-N-ethylcarboxamidoadenosine (NECA), N~-cyclopentyladenosine (CPA), 8-phenyltheophylline (8-PT), 2-[4-(2-carboxyethyl) phenethyl amino]-5'-N-ethylcarboxamino adenosine HCI (CGS 21680), 8-cyclopentyl- 1,3-dipropylxanthine (DPCPX), 4-diphenylacetoxy-N-methylpiperidine methiodide (4DAMP), quinpirole, and prazosin hydrochloride were purchased from RBI (Natick, MA); ascorbic acid, hemicholinium-3, atropine, adazoxane, and neuropeptide Y, from Sigma Chemical Co. (St. Louis, Mo.); EGTA and Na2EDTA, from Fisher Scientific (Pittsburgh, Pa) ; [3H]norepinephrine (45 Ci/mmol) and methyl-[3H]choline (76 Ci/ mmol), from Amersham (Arlington Heights I1). The composition of the modified Krebs' solution was (mM): NaC1, 113; CaC12, 1.4; KCI, 4.7; KH2PO4, 1.2; NaHCO3, 25 ; MgSO4 0.9 ; and glucose, 11.5 (F61des, 1981).
348
H. Nakatsuka et al. RESULTS
Norepinephrine release After loading with [3H]NE and washout, the tissue contained 1.08 × 106-+5.0 × 10 4 Bq/g radioactivity (n = 22). The resting release in the first 3-min collection period was 6640 + 240 Bq/g (0.64 + 0.04% of the total radioactivity, n = 22) and it was relatively constant from one collection period to the next throughout the experiment (Fig. 1). Radioactivity released by the first stimulation (Sj) was 16,800 + 750 Bq/g, which represents 1.62 +0.07% (n = 22) of the radioactivity present at the start of stimulation. The ratios of 3H released during consecutive stimulation periods, $2/S~, $3/$2, and $4/S~, were 0.93_+0.02, 0.92 + 0.03 and 0.99_+ 0.03 (n = 6), respectively. This indicates that the amount of fractional release in response to consecutive stimulations were similar and the fractional release during consecutive periods remained relatively constant (Fig. I). When [CaZ+],, was removed and EGTA (1 raM) was added the stimulation failed to release [3H]NE. The resting release was not affected. These findings indicate that the release of[3H]NE associated with axonal activity (field stimulation) is [Ca2+]o-dependent. EJ]ects o[adenosine analo,ques and adenosine receptor antagonists Adenosine, CADO, NECA and CPA concentration-dependently decreased the evoked release of [3H]NE (Fig. 2) in a concentration-dependent manner. As indicated by the change of the $3/$2 ratios the maximum inhibition that could be achieved by adenosine, CADO, NECA and CPA was 35, 34 36 and 35% of control, respectively. IC20 values of adenosine, CADO, NECA and CPA were 10.4, 1.24, 0.33 and 0.16/tM, respectively. The relative potencies of adenosine, CADO, NECA and CPA were 1, 8.4, 32 and 64. 8-Phenyltheophylline antagonized the inhibitory effect of adenosine or CADO on [3H]NE release. 8Phenyltheophylline (1 /~M) shifted the log concentration-response curves of adenosine and CADO to the right (data not shown). These parallel shifts and the Lineweaver-Burk plots indicate that the antagonism is competitive. The Ko values of 8-PT, calculated from the Lineweaver-Burk plot were 0.40 #M for CADO and 0.51 #M for adenosine. Neither adenosine receptor agonists nor antagonists influenced the spontaneous release of radioactivity (data not shown). When the drugs (agonists and antagonists) were added to the perfusion fluid 6 min after $2, the $3/S~ ratio in the presence of l or 10/~M 8-PT alone was not significantly different from control. DPCPX, a
selective A~-purinoceptor antagonist, did not enhance the release, but at concentration of 100 nM it significantly reduced the inhibitory effect of adenosine o1 CADO (Table l). The K~ value of DPCPX was 5.2 nM for the antagonism of adenosine and 3.3 nM for the antagonism of CADO. In five experiments the A2, agonist, CGS 21680 (30 nM), had no effect on [3H]NE release. Idazoxan, a specific ~2-adrenoceptor antagonist (Doxley et al., 1983), increased the evoked release of [~H]NE in a concentration-dependent manner (Table 2). In the presence ofidazoxan, i.e. when the negative feedback modulation was excluded and thereby the release was significantly higher, the inhibitory effects of adenosine and CADO were augmented. In the absence of idazoxan, CADO 10 /~M caused about 34% inhibition, in the presence of 10 ~tM idazoxan CADO (10/~M) caused 58% inhibition. In the presence of idazoxan (10 /~M), DPCPX (10 nM) significantly antagonized the inhibitory effect of CADO (10 pM) (Table 2). 4-DAMP, an M~-receptor selective antagonist, and prazosin, an ~2B-adrenoceptor antagonist, significantly enhanced the release of [3H]NE evoked by field stimulation (Table 2). This finding indicates that the negative feedback modulation of NE release is mediated via ~2~ subtype of adrenoceptors. None of these antagonists changed the spontaneous release of [3H]NE (data not shown). Modulation ~)[acelylcholine release Alter loading with [3H]choline and washout, the tissue contained 3.19x 105+2.46x l 0 4 Bq/g radioactivity (n = 18). The resting release in the first 3-min collection period was 755 + 65 Bq/g (0.23 + 0.02% of the total radioactivity, n = 18). The radioactivity released by S~ was 926+89 Bq/g. This represents a fractional release of 0.30 + 0.02% (n = 18). When the stimulation was repeated with intervals of 18 rain, S~/S~, $3/$2 and 84/83 ratio were 0.88+0.03, 0.86 + 0.02, and 0.80 + 0.04 (n = 6), respectively. The Arpurinoceptor agonists adenosine, CADO, and NECA or the A~/A2-purinoceptor antagonist, 8PT had no effect on the evoked release of [3H]ACh. The $3/$2 ratios in the presence of adenosine (100 itM), CADO (10 ~tM), NECA (1 /~M), or 8-PT (10 /~M) were not different from the control value (Table 3). Atropine, a specific muscarinic, but not subtype selective receptor antagonist, and 4-DAMP, a M 3muscarinic subtype selective receptor antagonist, significantly increased the evoked release of [3H]ACh. When the negative feedback modulation was blocked
349
N o r e p i n e p h r i n e a n d acetylcholine release Table 1. Effects of adenosine analogues and antagonists on [3H]NE release evoked by electrical stimulation from guinea-pig right atrium
Compound Control (no drug) Adenosine 100 #M CADO 10 #M NECA 3 #M CPA 1 #M 8-PT 10 gM 8-PT 1 #M 8-PT 1/xM +Adenosine 100 #M 8-PT 1 gM + C A D O 10/~M DPCPX, 100 nM DPCPX 10 nM DPCPX 100 nM +Adenosine 100 #M DPCPX 100 nM + C A D O 10 #M
S, (fractional release, %)
$2/S,
$3/$2 (in the presence of drugs)
1.59 -+_0.16 1.21 _+0.26 1.68 _+0.12 1.40+_0.05 1.72_+0.15 1.55 _.%0.03 _ 1.34 _+0.09
0.93 _+0.02 0.92 _+0.03 0.91 _+0.02 0.93 _+0.01 0.92_+0.02 0.97 _+0.02 0.93 _+0.02
0.92 _+0.03 0.60 _+0.03 * 0.61 _+0.03* 0.59_+0.03* 0.61 +0.04* 0.99 + 0.04 0.94 -+0.02
1.63 _+0.16
0.91 _ + 0 . 0 1
0.88_+0.02*5.
1.48_+0.22 1.14-+0.08 1.09_+0.08
0.91 +0.06 0.94_+0.06 1.01 +0.01
0.70+_0.02** 1.01 _+0.04 0.90_+0.01
1.35+__0.14
0.96-+0.02
0.81 _+0.04*5"
1.39_+0.10
1.00+0.01
0.87_+0.03*§
Mean_+ SEM (n = 4-6). Three stimulations (2 Hz, 240 shocks) were applied. The drugs were added to the perfusion fluid 6 min after $2 and kept throughout the experiments, therefore any effect of drugs is shown on $3/$2 ratio. * Significant difference from control (P < 0.01). t Significant difference from adenosine 100 #M (only P < 0.01). :~Significant difference from CADO 10/zM (P < 0.05). § Significant difference from CADO 10 #M (P < 0.01).
by atropine (1 #M), adenosine (100 #M) and CADO (10/aM) still failed to affect the release (Table 3). Since it was suggested that the D2-receptor agonist quinpirole (Roquebert et al., 1991) and NPY (Potter, 1987) might be able to attenuate the cardiac vagal action, we made an attempt to study their effect on ACh release under condition, in which the post-
ganglionic fibers were surely stimulated. Both quinpirole 0.5 #M and NPY 0.1 /~M failed to affect the release of [3H]ACh evoked by field stimulation (2 Hz, 240 shocks) (Table 3). Idazoxan 1 #M also did not increase the release of radioactivity and xylazine 1 /~M, an c~2-adrenoceptor agonist, did not reduce the release (data not shown).
Table 2. Effects of CADO, DPCPX, idazoxan, 4-DAMP and prazosin on [3H]NE release evoked by electrical stimulation from guinea-pig right atrium
Compound Control (no drug) ldazoxan 0.1/~M ldazoxan 1 #M Idazoxan 10/zM ldazoxan 10 gM + C A D O 10 #M Idazoxan 10 #M + C A D O 10 #M + D P C P X 10 nM ldazoxan 10 #M + C A D O 10 #M + DPCPX 100 nm 4-DAMP 0.5 #M Prazosin 1/~M
S, (fractional release, %)
$2/S,
$3/$2 (in the presence of drugs)
1.59 + 0.16 1.49 + 0.26 1.72_+0.18 1.59 _+0.15
0.93 + 0.02 0.97 + 0.02 0.94_+0.03 0.93 _+0.02
0.92 + 0.03 1.33 _+0.06* 1.93_+0.08" 2.53 _+0.09*
1.41 _ + 0 . 1 7
0.94_+0.13
1.07_+0.05,
1.22_+0.10
0.92+_0.01
1.37+_0.10"§
1.39+_0.12 1.21 +0.16 1.42_+0.12
0.96-+0.06 0.95-+0.01 0.93-+0.02
2.14_+0.12"* 1.32-+0.12]" 2.98_+0.16"
Mean + SEM (n = 4-6). The drugs were added to the perfusion fluid 6 rain after $2 and kept throughout the experiments. * Significant difference from control (P < 0.01). t Significant difference from control (P < 0.05). * Significant difference from CADO 10 #M (P < 0.01). § Significant difference from idazoxan 10 #M (P < 0.05).
350
H. Nakatsuka et al. Table 3. Effects of adenosine analogues, 8-PT, atropine, quinpirole, NPY and 4-DAMP on [)H]ACh release evoked by electrical stimulation from guinea-pig right atrium
Compound Control (no drug) Adenosine 100 tim C A D O I 0 #M N ECA 1 /IM 8-PT l0 I~M Atropine I ,uM Atropine I itM + Adenosine 100 ItM Atropine I ltM + C A D O 10 ltM 4-DAMP 0.5 pM Quinpirole 0.5 itM NPY 0.1 ItM
SI (fractiomd release, '!4,)
SjSr
SdS: (in the presence of drugs)
(I.28 ± 0.09 0.30 -+ 0.10 0.34-+ 0.06 0.33 ± 0.09 0.27+0.01 0.22 ± 0.(17
0.88 ± 0.03 0.85 -+ 0.03 0.88 + 0.04 (I.88 ± 0.1)4 0.90+0.04 0.88 _+0.08
0.86_+0.02 O.89+O.05 0.83_+0.03 0.87+0.01 0.87_+0.03 .69_+0.16"
0.28 ± 0.04
0.86 -+ 0.05
.72+0.19"
0.27 ~ (/.04 0.45-+0.06 0.36-+ 0.(t2 0.30_+0.03
0.83 -+ 0.04 (I.90±0.01 0.89 + 0.08 0.89±0.02
.68-+0.18" .12-+0.03"I0.92-+0.12 0.94_4_0.05
Mean_+SEM (n = 4 6). The drugs were added to the per±us±on fluid 6 rain after $2 and kept throughout the experiments. * Significant difference from control (P < 0.01) 4-Significant difference from control (P < 0.05).
DISCUSSION
The ability of adenosine and adenosine triphosphate (ATP) to modulate cardiovascular activity has been recognized for more than 60 years (Drury and Szent-Gy6rgyi, 1929). The depressant effect of adenosine on synaptic transmission was first observed at the neuromuscular junction, by using an electrophysiological method (Ginsborg and Hirst, 1972). With this preparation neurochemical evidence was also obtained (Somogyi et al., 1987; Nagano et al., 1992) that adenosine inhibits the release of ACh. It was also reported that the evoked release of labeled NE (Hedqvist and Fredholm, 1976) and of endogenous (Vizi and Knoll, 1976) and labeled (Manabe et al., 1991) ACh was reduced by adenosine derivatives in several different tissues. In the myocardium adenosine is derived from the breakdown of ATP or that of S-adenosyl homocysteine (Bardenheur and Schrader, 1986). During tissue hypoxia or increased sympathetic discharge, formation of adenosine from ATP increased dramatically (Fredholm and Sollevi, 1986; Sparks and Bardenheuer, 1986). Therefore, it seemed interesting to study the effect of adenosine on NE and ACh release in response to neuronal activity from the sympathetic and vagal nerve terminals in the right atrium using electrical field stimulation and neurochemical methods. With this technique, it was possible to differentiate between the effect of compounds on the stimulation-evoked, [Ca- ],,-dependent quantal and [Ca2+]o-independent non-quantal release of neurotransmitters (Vizi, 1984; Viziet al., 1985, 1991 ; Vizi and Lfibos, 1991).
In this study it was demonstrated that while adenosine and its stable analogues inhibited the evokedrelease of [3H]NE from the sympathetic nerve ending of guinea pig right atrium, they did not inhibit the evoked release [3H]ACh from the vagal nerve endings. The order of potencies for the inhibition of NE release was : CPA > NECA > CADO > adenosine and the fact that DPCPX, a selective A,-receptor antagonist, inhibited the effects of adenosine and CADO strongly indicates that their inhibitory effect on NE release was mediated via the A~ receptors. This is consistent with the observation (Richardt et al., 1987; Sch~tz et al., 1991) that cyclohexyladenosine also inhibited the release of endogenous NE from rat heart. In corroboration with others (Manabe et al., 1991 ; Smith et al., 1992) we also observed that the release of [3H]NE was subject to negative feedback modulation, i.e. NE released from the axon terminal reduced its own release. When the release of NE was freed from ~2-receptor-mediated control, using idazoxan (I #M), the inhibition achieved by A,-purinoceptor stimulation with CADO (10/~M) was higher (54%). This finding indicates that all sympathetic axon terminals are also equipped with A,-receptors and the different modulatory receptors are not located in different populations of axon terminals. Similar observations were made by Allgainer et al. (1987) on the influence of adenosine and CADO on the release of NE from cerebral cortex slices. 8-Phenyltheophylline competitively antagonized the inhibitory effect of adenosine or CADO, but 8-PT alone had no effect on NE release. It was reported that ATP is released from the sympathetic nerve endings
351
Norepinephrine and acetylcholine release together with NE. ATP is readily metabolized to adenosine, and adenosine is metabolized to inosine or is taken up by the tissue. Therefore, it is probable that under our experimental conditions, the concentration of endogenous adenosine released is not high enough to decrease NE release ; however, during ischemia its concentration is much higher and adenosine may create regional autonomic imbalance and also may protect the ischemic myocardium from excessive catecholamine stimulation (Miyazaki and Zipes, 1990). The finding that adenosine and its congeners had no effect on the release of [3H]ACh associated with neuronal activity in the guinea pig right atria, even when the muscarinic autoinhibition of ACh release was prevented by atropine, indicates that the vagal nerve endings in the guinea pig right atria are not equipped with A~-purinoceptors. This indicates that cholinergic axon terminals in the Auerbach plexus (Vizi and Knoll, 1976 ; Somogyi and Vizi, 1988 ; Milusheva et al., 1990) and hippocampus (Duntr-Engstr6m and Fredholm, 1988; Cunha et al., 1994) are different from right atria: the stimulation of these receptors resulted in inhibition of stimulation-evoked release of ACh. Adenosine and ATP have been used clinically to induce hypotension during anesthesia (Sollevi, 1986) or to stop paroxysmal supraventricular tachycardia (Rankin et al., 1990; Sollevi, 1986). Furthermore, it was reported (Pitarys et al., 1991) that intravenous infusion of adenosine reduced myocardial reperfusion injury in dogs. It is well known that a large amount of adenosine is liberated from myocardium during hypoxia (Fredholm and Sollevi, 1986; Sparks and Bardenheuer, 1986). Taking into account our data, it is conceivable to suggest that adenosine liberated during hypoxia inhibits NE release from the cardiac sympathetic nerve without inhibiting ACh release and thereby offers protection against tachyarrhythmia caused by myocardial hypoxia. It was suggested by Roquebert et al. (1991) that vagal nerves are endowed with a pharmacologically relevant population of D:-receptors and therefore D2receptor agonists are able to attenuate the vagalinduced but not the ACh-induced bradcardia in pithed rats. However, in their experiments the site of action of quinpirole, a selective D?receptor agonist, was not precisely located. It could have been on the ganglion and/or on the postganglionic axon terminals. In our recent experiments adenosine ; quinpirole, a selective D2-receptor agonist, and NPY did not influence the release of ACh in response to field stimulation. Using field stimulation both pre- and postganglionic fibers are stimulated. And previously using the same prep-
aration we showed (Kobayashi et al., 1987) that ~2adrenoceptor agonist also failed to affect the release. However, there are several observations (Richardt et al., 1987; Miyazaki and Zipes, 1990; Roquebert et aL, 1991 ; Potter, 1987 ; Revington et aL, 1990) that NE, adenosine, dopamine and NPY attenuate the responses of the heart to preganglionic vagal stimulation. Even if it was suggested that their site of action is presynaptic, the preganglionic vagal nerve was also stimulated in their experiments, and in our experiment field stimulation was used, i.e. the postganglionic fibers were also stimulated. Therefore the discrepancy can be easily explained that the inhibitory effect observed by others may be mediated via an effect at the ganglionic level (Fig. 3), In corroboration with Sympathetic
Doparnine
(quinpirol¢)
Adenosin~ I I I
~2-1t
N
It,, t', \\ \
NE.~"
~NE
Vagal
/
~anglion
-~. t
",//
ACh ---
~ r i a l ~-ATP
pacem ake_r~""-~.~ (myocardialcell)
Adenosine
Fig. 3. Scheme of the possible interaction between vagal and sympathetic innervation of the heart. Note that Arreceptors are located on both myocardial cells and sympathetic axon terminals and adenosine released from the tissue and/or decomposed from ATP in increased amounts during ischemia and hypoxia may have an inhibitory effect on both sites. While the sympathetic activation results in tachycardia and encourages the generation of arrhythmias under conditions of ischemia, adenosine may exert a protective effect by inhibiting the release of NE and by counteracting the effect of catecholamines on myocardial cells. The noradrenergic axon terminals are also equipped with inhibitory muscarinic receptors. Thus, vagal activation reduces ventricular vulnerability ; this is the result of a combination of inhibition of NE release and direct effect on myocardial cells. Since the axon terminals of postganglionic vagal fibers are not equipped with inhibitory A~-receptors therefore adenosine released during ischemia inhibits only the release of NE, but not that of ACh. The imbalance in autonomic innervation produced by adenosine may result in a vagal tone. The negative feedback modulation of NE and ACh release from the postganglionic fibers is mediated via ~2- and muscarinic-receptors, respectively, The myocardial cells are equipped with muscarinic-receptors. The ganglionic transmission is modulated presynaptically through D2-, ~2-(the subtype is not yet specified)and Arreceptors, It is very likely that neuropeptide Y is also able to inhibit transmission in the parasympathetic ganglion.
352
H. Nakatsuka et al.
this, it was s h o w n (Dawes a n d Vizi, 1973) that N E inhibits the release o f A C h from the ganglion. In o u r case, by using field stimulation, the site o f action was tested also on the axon terminals next to the effector cells. O u r data suggest t h a t the vagal postganglionic nerve endings are not equipped with receptors sensitive to NE, adenosine, quinpirole, or NPY. In summary, the heart with its dual a u t o n o m i c innervation is a site where there is a only one-sided interaction between the two nerves at the presynaptic level o f postganglionic neurons. Sympathetic axon terminals are quipped with 72B-autoreceptors a n d muscarinic ( M 0 a n d Aj-heteroceptors. Thus the release of N E is subject to negative feedback m o d u l a t i o n via stimulation of ~26-adrenoceptors and presynaptic inhibitory m o d u l a t i o n by A C h released from the vagal nerve a n d by adenosine. However, the vagal postganglionic nerve endings are not equipped with 72- and A~-receptors, but there is a muscarinic-autoreceptormediated negative feedback modulation. Thus N E released during sympathetic activity and adenosine released in excess from the heart muscle during hypoxia do not have any effect on the postganglionic vagal axon terminals, but they might have an inhibitory effect at the level of p a r a s y m p a t h e t i c ganglia (Fig. 3). The imbalance in a u t o n o m i c i n n e r v a t i o n produced by adenosine may result in a vagal tone. This work was partly supported by Hungarian Research Fund (OTKA) and Medical Research Council (ETT). Acknowledgements
REFERENCES
Allgainer C., Hertting G. and Kagelgen O. V. (1987) The adenosine receptor-mediated inhibition of noradrenaline release possibly involves a N-protein and is increased by c~:-autoreceptor blockade. Br, J. Pharmae. 90, 403-412. Bardenheuer H. and Schrader J. (1986) Supply-to-demand ratio for oxygen determines formation of adenosine by the heart. Am. J. Physiol. 250, HI73 180. Berne R. M. (1980) Role of adenosine in the regulation of coronary blood flow. Circ. Res. 47, 807-813. Cunha R. A., Milusheva E., Vizi E. S., Ribeiro .l.A. and Sebastiao A. M. (1994) Different excitatory and inhibitory effects of adenosine on the electrically-evoked [SH]acetylcholine release from different areas of the rat hippocampus. J. Neurochem. 63, 207---214. Dawes P. M. and Vizi E. S. (1973) Acetylcholine release from the rabbit isolated superior cervical ganglion preparation. Br. J. Pharmac. 47, 765-777. Doxley J. C., Roach A. G. and Smith C. F. C. (1983) Studies of RX781094 : a selective potent and specific antagonist of 72-adrenoceptors. Br. J. Pharmac. 78, 489-505. Drury A. N. and Szent-Gy/Srgyi A. (1929) The physiological activity of adenosine compounds with especial reference
to their action upon the mammalian heart. J. Physiol. Lond. 68, 213 237. Dun6r-EngstrOm M. and Fredholm B. B. (1988) Evidence that prejunctional adenosine receptors regulating acetylcholine release from rat hippocampal slices are linked to an N-ethylmaleimide-sensitive G-protein, but not to adenylate cyclase or dihydrophyridine-sensitive Ca 2+channels. Acta Physiol. Scand. 134, 119-126. F61des F. F. (1981) The significance of physiological Ca 2~ and Mg 2+ for in vitro experiments on synaptic transmission. L([e Sci. 28, 1585 1590. F01des F. F., Kobayashi O., Kinjo M., Harsing L. G. Jr, Nagashima H., Duncalf D., Goldiner P. L. and Vizi E. S. (1989) Presynaptic effect of muscle relaxants on the release of ~H-norepinephrine controlled by endogenous acetylcholine in guinea pig atrium. J. Neural Trans. 76, 169180. Fredholm B. B. and Sollevi A. (1986) Cardiovascular effects of adenosine. ~Tin. Physiol. 6, 1-21. Ginsborg B. L. and Hirst G. D. S. (1972) The effect of adenosine on the release of the transmitter from the phrenic nerve of the rat. J. Physiol, Lond. 224, 629-645. Hedqvist P. and Fredholm B. B. (1976) Effects of adenosine on adrenergic neurotransmission ; prejunctional inhibition and postjunctional enhancement. Naunyn-Schmiedebery 3' Arch. Pharmac. 293, 217 223. Kobayashi O., Nagashima H., Duncalf D., Chaudhry 1. A., Harsing L. G. Jr, F61des F. F., Goldiner P. L. and Vizi E. S. (1987) Direct evidence that pancuronium and gallamine enhance the release of norepinephrine from the atrial sympathetic nerve by inhibiting prejunctional muscarinic receptors. J. Auton. Nerr. Syst. 18, 55-60. Manabe N., F61des F. F., T6r6csik A., Nagashirna H., Goldiner P. L. and Vizi E. S. (1991) Presynaptic interaction between vagal and sympathetic innervation in the heart: modulation of acetylcholine and noradrenaline release. J. Auton. Nerv. Syst. 32, 233 242. McCulloch M. W., Rand M. J. and Story D. F. (1974) Resting and stimulation-induced etttux of tritium from guinea pig atria incubated with 3H-noradrenaline. Clin. exp. Pharmac. Physiol. 1,275-289. Milusheva E., Sperl~igh B., Kiss B., Szporny L., Pfisztor E.. Papasova M. and Vizi E. S. (1990) Inhibitory effect of hypoxic condition on acetylcholine release is partly due to the effect of adenosine released from the tissue. Brain Res. Bull. 24, 369 373. Miyazaki T. and Zipes D. P. (1990) Presynaptic modulation of efferent sympathetic and vagal neurotransmission in the canine heart by hypoxia, high K +, low pH, and adenosine. Circ. Res. 66, 289 301. Muscholl E. (1980) Peripheral muscarinic control of norepinephrine release in the cardiovascular system. Am. J. Physiol. 239, H713-720. Muscholl E. (1990) The role of vagus activity in the presynaptic control of norepinephrine release from rabbit atria. Neurochem. Int. 17, 189--195. Nagano O.. FNdes F. F., Nakatsuka H., Reich D., Ohta Y., Sperlagh B. and Vizi E. S. (1992) Presynaptic A c purinoceptor-mediated inhibitory effects of adenosine and its stable analogues on the mouse hemidiaphragm preparation. Naunyn-Sehmiedeberq's Arch. Pharmac. 346, 197 202. Pitarys C. J., Virmani R., Vildibill H. D., Jackson E. K, and Forman M. B. (1991) Reduction of myocardial reper-
Norepinephrine and acetylcholine release fusion injury by intravenous adenosine administered during the early reperfusion period. Circulation 83, 237-247. Potter E. K. (1987) Presynaptic inhibition of cardiac vagal postganglionic nerves by neuropeptide Y. Neurosci. Lett. 83, 101-106. Rankin A. C., Oldroyd K. G., Chong E., Dow J. W., Rae A. P. and Cobbe S. M. (1990) Adenosine or adenosine triphosphate for supraventricular tachycardias? Comparative double-blind randomized study in patients with spontaneous or inducible arrhythmias. Am. Heart J. 119, 316-323. Revington M., Potter E. K. and McCloskey D. I. (1990) Prolonged inhibition of cardiac vagal action following sympathetic stimulation and galanin in anaesthetized cats. J. Physiol. 431, 495--503. Richardt G., Waas W., Kranzh6fer R., Mayer E. and Sch6mig A. (1987) Adenosine inhibits exocytotic release of endogenous noradrenaline in rat heart: a protective mechanism in early myocardial ischemia. C&c. Res. 61, 117-123. Roquebert J., Moran A., Demichel P. and Sauvage M-F. (1991) Pharmacological characterization of dopamine receptors on parasympathetic innervation of rat heart. Eur. J. Pharmac. 200, 59~63. Schtltz W., St6her M., Freissmuth M., Valenta B. and Singer E. A. (1991) Adenosine receptors mediate a pertussis toxininsensitive prejunctional inhibition of noradrenaline release on a papillary muscle model. Naunyn-Schmiedebery's Arch. Pharmac. 343, 311-316. Smith K., Connaughton S. and Docherty J. R. (1992) Investigations of prejunctional ~2-adrenoceptors in rat atrium, vas deferens and submandibular gland. Eur. J. Pharmac. 211, 251-256. Sollevi A. (1986) Cardiovascular effects of adenosine in man ; possible clinical implications. Pro9. Neurobiol. 27, 319349. Somogyi G. T. and Vizi E. S. (1988) Evidence that cholinergic
353
axon terminals are equipped with both muscarinic and adenosine receptors. Brain Res. Bull. 21, 575-579. Somogyi G. T., Vizi E. S., Chaudhry I. A., Nagashima H., Duncalf D. and F61des F. F. (1987) Modulation of stimulation-evoked release of newly formed acetylcholine from mouse hemidiaphragm preparation. Naunyn-Schmiedeberg's Arch. Pharmac. 336, 11-15. Sparks H. V. and Bardenheuer H. (1986) Regulation of adenosine formation by the heart. Circ. Res. 58, 193-201. Urthaler F., Neely B. H. and Hageman G. R. (1986) Differential interaction of adrenergic and cholinergic effects on AV junctional automaticity and AV conduction. Am. Heart J. 112, 765-774. Vizi E. S. (1984) Non-synaptic interactions between neurons : modulation of neurochemicals transmission. Pharmacological and Clinical Aspects. Wiley, New York. Vizi E. S. and Knoll J. (1976) The inhibitory effect of adenosine and related nucleotides on the release of acetylcholine. Neuroscience I, 391-398. Vizi E. S. and L~ibos E. (1991) Nonsynaptic interactions at presynaptic level. Progr. Neurobiol. 37, 145-163. Vizi E. S. Kiss J. and Elenkov I. J. (1991) Presynaptic modulation of cholinergic and noradrenergic neurotransmission: interaction between them. News Physiol. Sci. 6, 119-123. Vizi E. S., Ono K., Adam-Vizi V. and F61des F. F. (1984) Presynaptic inhibitory effect of Met-enkephalin on 14Cacetylcholine release from the myenteric plexus and its interaction with muscarinic negative feedback inhibition. J. Pharmac. Exp. Ther. 230, 493499. Vizi E. S., Somogyi G. T., Harsing L. G. Jr and Zimanyi I. (1985) External Ca-independent release of norepinephrine by sympathomimetics and its role in negative feedback modulation. Proc. natn. Acad. Sci. U.S.A. 82, 8775-8779. Wennmalm M., Fredholm B. B. and Hedqvist P. (1988) Adenosine as a modulator of sympathetic nerve-stimulation-induced release of noradrenaline from the isolated rabbit heart. Acta Physiol. Scand. 132, 487-494.
Pergamon
Neurochem. Int. Vol. 27, No. 4/5, pp. 345-353, 1995
0197-0186(95)00016-X
Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 01974)186/95 $9.50+ 0.00
EFFECTS OF ADENOSINE ON NOREPINEPHRINE A N D ACETYLCHOLINE RELEASE FROM GUINEA PIG RIGHT ATRIUM" ROLE OF A1-RECEPTORS H. N A K A T S U K A j'2'3, O. N A G A N O 1'3, F. F. F O L D E S l, H. N A G A S H I M A 1 and E. S. VIZI 2. LDepartment of Anesthesiology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, N.Y. 10467, U.S.A. 21nstitute of Experimental Medicine, Hungarian Academy of Sciences, 1450-Budapest, P.O. Box 67, Hungary 3Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama 700, Japan (Received I October 1994 ; accepted 21 December 1994)
Almtraet--The effect of adenosine or its stable analogues (2-chloroadenosine, CADO: Y-N-ethylcarboxamidoadenosine, NECA; and Nf-cyclopentyladenosine, CPA) and a non-selective A~ and A2receptor antagonist, 8-phenyltheophylline (8-PT), or an Al-receptor antagonist, 8-cyclopentyl-l,3-dipropylxanthine (DPCPX), on the stimulation-evoked release of [3H]norepinephrine ([3H]NE) and [3H]acetylcholine ([3H]ACh) from the isolated guinea pig right atrium was investigated. Adenosine and its stable analogues (CADO, NECA and CPA) inhibited the stimulation-evoked release of [aH]NE in a concentrationdependent manner. The order of potencies was CPA > NECA > CADO > adenosine. CGS 21680 (30 nM), an A2a receptor agonist, failed to affect the release. The inhibitory effect of adenosine and CADO on [3H]NE release was competitively antagonized by 8-PT. DPCPX also prevented the effect of adenosine (Kd = 5.2 nM) and CADO (Kd = 3.3 nM). The Kd value of 8-PT was 0.40/tM for the antagonism of CADO and 0.51 #M for the antagonism of adenosine. When the negative feedback modulation of NE release was inhibited by idazoxan, the inhibitory effect of adenosine and CADO on [3H]NE release was more pronounced. Under this condition DPCPX (10 nM) prevented the inhibitory effect of CADO, indicating that A~-purinoceptors are involved in this action. The release of [3H]NE is tonically modulated by ACh released from the vagal nerve endings, as evidenced by the finding that 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), a M3-subtype selective muscarinic receptor antagonist, and atropine significantly enhanced the release of NE. Adenosine, its stable analogues (CADO and NECA), and 8-PT did not have any effect on the stimulation-evoked release of [3H]ACh. Even when the muscarinic autoinhibition was eliminated by atropine, adenosine and CADO did not have any effect on [3H]ACh release. Quinpirole, a selective D2-receptor agonist, and neuropeptide Y (NPY) failed to affect the release of ACh. However, atropine and 4-DAMP, a selective M3-receptor antagonist, significantly enhanced the stimulation-evoked release of [3H]ACh. These findings indicate that there are no presynaptic heteroceptors (adenosine, D2, and NPY) on the vagal nerve endings of the guinea pig right atrium. It is concluded that the sympathetic nerve endings of the guinea pig right atrium are equipped with A~-, subclass of purinoceptors and ctzB-,and muscarinic (M3)-receptors. Cholinergic vagal nerve endings in the heart are only equipped with muscarinic autoreceptors. Therefore, adenosine liberated during hypoxia inhibits NE release from the cardiac sympathetic nerve and thereby protects against tachyarrhythmia caused by myocardial hypoxia. In contrast, adenosine does not inhibit the vagal innervation of the right atrium.
Several studies have shown that autonomic modulation of the heart rate is determined by the interaction of sympathetic and vagal innervation at the effector level (sinus node) (cf. Urthaler et al., 1986). Increased
vagal-sympathetic antagonism, i.e. augmentation of the vagal action in the presence of prevailing sympathetic activity, is of primary importance for the control of the heart rate (cf. Muscholl, 1990). This interaction could be manifested either on the sinus cells or as it was recently suggested (cf. Muscholl,
* Author to whom all correspondence should be addressed. 345
346
H. Nakatsuka et al.
1990; M a n a b e et al., 1991 ; V i z i e t al., 1991) at the presynaptic terminals o f the postganglionic sympathetic a n d p a r a s y m p a t h e t i c nerves. Whereas noradrenergic axon terminals are equipped with inhibitory muscarinic receptors (cf. Muscholl, 1980, 1990; M a n a b e et al., 1991), so that A C h released from the postganglionic vagal nerve is able to tonically control the release o f NE, the postganglionic vagal nerve terminals are not equipped with ~2-adrenoceptors ( M a n a b e et al., 1991), a n d N E does not influence the release o f ACh. Since adenosine is released in excess during hypoxia a n d accumulates in the myocardium in m i c r o m o l a r concentrations (Berne, 1980) and it is also used in m a n y clinical situations as a vasodilator, antiaggregatory, a n d a n t i a r r h y t h m i c agent (cf. Sollevi, 1986), its effect on the a u t o n o m i c innervation o f heart is of physiological, pharmacological, and clinical importance. While the inhibitory effect of adenosine on N E release from the sympathetic nerve endings of the heart has been well d o c u m e n t e d (Richardt et al., 1987; W e n n m a l m et aL, 1988), no attempt has been made so far to classify the subtype of these receptors and no i n f o r m a t i o n has been m a d e available on the effect o f adenosine o n the release of A C h from the vagal nerve endings. Since the release of N E is also subject to presynaptic m o d u l a t i o n t h r o u g h muscarinic heteroceptors by A C h released from the vagus (cf. Muscholl, 1980, 1990 ; F61des et al., 1989 ; M a n a b e et al., 1991), and t h r o u g h ~2-autoreceptors by N E released from the sympathetic nerve endings, we also made an a t t e m p t to classify the subtype o f ~2 and muscarinic receptors located on postganglionic axon terminals using subtype-selective antagonists. In this study, using a radiochemical technique we measured directly the effect o f adenosine a n d its stable analogues (2-chloroadenosine, C A D O ; 5'-N-ethylc a r b o x a m i d o a d e n o s i n e , N E C A ; and N~-cyclopen tyladenosine, CAP), the AdA2-receptor antagonist 8-phenyltheophylline (8-PT), a n d the A~-receptor antagonist 8-cyclopentyl-l,3-dipropylxanthine ( D P C P X ) on the stimulation-evoked release of N E and A C h from guinea pig right atria. EXPERIMENTAL PROCEDURES Norepinephrine reh, ase Male guinea pigs of 300-500 g body weight, lightly anesthetized with enflurane, were killed by a blow to the head, and the right atria were dissected out and incubated for 40 min at 3T'C in modified Krebs' solution (F61des, 1981) containing 370 kBq/ml 1-(7,8-3H)-norepinephrine ([3H]NE) (specific activity 1.14 kBq/mmol). The solution was aerated with 95% 02 5% CO2. To facilitate the uptake of [3H]NE,
during incubation the preparations were continuously stimulated at 1 Hz with supramaximal (35 V/cm) impulses of 1 ms duration, through two platinum electrodes placed above and below the suspended atria (field stimulation). To remove excess [3H]NE after incubation the atria were transferred to 1.5 ml organ baths and were superfused at a rate of I ml/min for 90 min with Krebs' solution. To prevent non-enzymatic breakdown on NE, 27 pM Na2EDTA and 300/tM ascorbic acid were added to the perfusion solution (McCulloch et al., 1974). After the 90-min washout period, superfusion was continued at the rate of 1 ml/min and 3-min fractions of the superfusate were collected throughout the experiment. Starting at the beginning of the 10th (S~), 28th ($2), 46th ($3), and 64th ($4) min, the preparations were stimulated for 2 min at 2 Hz (240 stimuli) (Fig. 1). Except for the stimulation rate, the method of stimulation was the same as during incubation. Compounds to be investigated were added to the perfusing solution 6 min after $2 and kept until the end of experiments. The spontaneous (resting) release of 3H was determined from the average 3H content of two non-stimulated 3-min fractions before and after each stimulation period. The computer-derived regression line of the resting release was determined with an exponential curve-fitting program. The evoked release of 3H was determined by subtracting the resting release from the total release measured in the consecutive fractions collected following the start of stimulation. Using HPLC combined with radiochemieal detection (Manabe et al., 1991) 87.4_+4.3% of the total radioactivity released by electrical stimulation was due to [3H]NE. This is in agreement with our earlier findings (Manabe et al., 1991). In control experiments the ratios of amounts of 3H released during consecutive stimulation periods (i.e. $2/S~, $3/$2, $4/$3) were similar (Fig. 1). Therefore any increase or decrease of these
1,6
~ 0
-
1.4
-
1.2
-
1.o
-
0.8
-
J
o
,
15
IIIIII $2
I S3
I 30
I 45
m $4
i 60
I 75
Time (min) Fig. 1. Fractional rele~e of radioactivity from isolated right atrium loaded with [3H]NE in response to electrical field stimulation (2 Hz, 240 shocks). Average of four identical experiments. Note that four stimulations (S~, $2, $3 and $4) as indicated resulted in almost identical release of radioactivity. Mean + SEM is indicated.
347
Norepinephrine and acetylcholine release ratios caused by the addition of drugs could be attributed to the effect on the compound used on NE release. The IC20of adenosine and its analogues was also calculated from the concentration-response curves plotted in Fig. 2. IC20 indicates the concentration needed to reduce the release of radioactivity by 200. The ratio of the amount of radioactivity released by two consecutive stimulations ($3/$2) in the absence of drugs (0.92+0.03, n = 6) was taken as the control release ratio.
Acetylcholine release After dissection, the atria were incubated for 40 min at 37°C in Krebs' solution containing 185 kBq/ml methyl[3H]choline (specific activity 2.89 TBq/mmol). The solution was aerated with 95% 02-5% CO2 throughout the experiment. During incubation, the incorporation of [3H]choline into the ACh pool of the nerve terminal was facilitated by electrical field stimulation. The method of stimulation was the same as in the NE release studies. After incubation, the atria were transferred to 1.5 ml-organ baths and were superfused at a rate of 1 ml/min for 60 min. Subsequently the preparations were superfused for another 30 min with Krebs' solution containing 50 #M hemicholinium-3 to prevent re-uptake of the [3H]choline liberated by the hydrolysis of [3H]ACh. After washout, superfusion with Krebs' solution containing 50 #M hemicholinium-3 was continued at the rate of 1 ml/min and 30 min fractions of superfusate were collected throughout the experiment. Starting at the beginning of the 10th (SO, 28th ($2), 46th ($3), and 64th ($4) min, the preparations were stimulated for 2 min at 2 Hz. Compounds to be investigated are added to the superfusing Krebs' solution 6 min after $2. It was reported that 9 2 o of the radioactivity released in response to the stimulation was due to [3H]ACh
O e~e v~v s~1 @~ 0
1.0 0.9 0.8
eq
¢~
0.7
0.6 0.5
I//] 0 t'0.01
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I 1
I 10
I 100
Measurement o f radioact&ity A l-ml aliquot of each collected fraction was transferred to scintillation vials containing 7 ml of scintillation fluid (Echoscinti, National Diagnostics). The radioactivity of the samples was measured in a scintillation beta spectrophotometer (TRI-CARB 4530, Packard Instrument Co.). At the end of each experiment, the residual 3H content of the atria was determined. The tissue was kept overnight in 1 ml of tissue solubilizer (Soluvable, NEN Research Product) at room temperature and the 3H content of the sample was measured. The radioactivity of the samples was expressed in terms of disintegrations per gram wet weight of tissue (Bq/g). To normalize the quantity of radioactivity released, the release of [3H]NE of [3H]ACh was expressed as a percentage of the total radioactivity present in the tissue at the onset of stimulation (fractional release). The fractional release was calculated by a computer program taking into account the content of radioactivity at the end of experiment and the release of radioactivity in the samples. Cah'ulations In some experiments the apparent dissociation constant (Kd) for antagonists was determined by the dose-ratio method. The following equation was used to relate the dissociation constant to the dose-ratio and the antagonist concentration
K~-
a
DR-- 1
Where DR is the concentration-ratio, i.e. the EC20 value for agonist in the presence of the antagonist divided by the EC20 value in the absence of antagonist and a is the concentration of antagonist. Four different concentrations of agonists were applied to establish a concentration-response curve. In some experiments the Lineweaver-Burk plot was used and the Kd value was calculated (pA2 = - log Kd). The means ___SEM of data are presented. One way analysis of variance followed by Dunnett's test was used. A value of P < 0.05 was considered to be significant.
Control Adenosine GADO NECA CPA
O
"~
(Manabe et al., 1991). The stimulation-evoked release of ACh was calculated as described in the NE release studies.
] 1000
Agonist concentration (IJ.mol/L) Fig. 2. Effect of different adenosine receptor agonists [adenosine, 2-chloroadenosine (CADO), 5'-ethyl-carboxamidoadenosine (NECA), Nr-cyclopentyladenosine (CPA)] on the stimulation-evoked release of radioactivity from isolated right atrium loaded with [3H]NE. Drugs were added into the organ bath 6 min after $2. Note that adenosine and its stable analogues concentration-dependently reduced the release of radioactivity evoked by electrical field stimulation (2 Hz, 240 shocks). Each point represents 4 or 6 experiments. Mean ___SEM is indicated.
Materials Adenosine, 2-chloroadenosine (CADO), 5'-N-ethylcarboxamidoadenosine (NECA), N~-cyclopentyladenosine (CPA), 8-phenyltheophylline (8-PT), 2-[4-(2-carboxyethyl) phenethyl amino]-5'-N-ethylcarboxamino adenosine HCI (CGS 21680), 8-cyclopentyl- 1,3-dipropylxanthine (DPCPX), 4-diphenylacetoxy-N-methylpiperidine methiodide (4DAMP), quinpirole, and prazosin hydrochloride were purchased from RBI (Natick, MA); ascorbic acid, hemicholinium-3, atropine, adazoxane, and neuropeptide Y, from Sigma Chemical Co. (St. Louis, Mo.); EGTA and Na2EDTA, from Fisher Scientific (Pittsburgh, Pa) ; [3H]norepinephrine (45 Ci/mmol) and methyl-[3H]choline (76 Ci/ mmol), from Amersham (Arlington Heights I1). The composition of the modified Krebs' solution was (mM): NaC1, 113; CaC12, 1.4; KCI, 4.7; KH2PO4, 1.2; NaHCO3, 25 ; MgSO4 0.9 ; and glucose, 11.5 (F61des, 1981).
348
H. Nakatsuka et al. RESULTS
Norepinephrine release After loading with [3H]NE and washout, the tissue contained 1.08 × 106-+5.0 × 10 4 Bq/g radioactivity (n = 22). The resting release in the first 3-min collection period was 6640 + 240 Bq/g (0.64 + 0.04% of the total radioactivity, n = 22) and it was relatively constant from one collection period to the next throughout the experiment (Fig. 1). Radioactivity released by the first stimulation (Sj) was 16,800 + 750 Bq/g, which represents 1.62 +0.07% (n = 22) of the radioactivity present at the start of stimulation. The ratios of 3H released during consecutive stimulation periods, $2/S~, $3/$2, and $4/S~, were 0.93_+0.02, 0.92 + 0.03 and 0.99_+ 0.03 (n = 6), respectively. This indicates that the amount of fractional release in response to consecutive stimulations were similar and the fractional release during consecutive periods remained relatively constant (Fig. I). When [CaZ+],, was removed and EGTA (1 raM) was added the stimulation failed to release [3H]NE. The resting release was not affected. These findings indicate that the release of[3H]NE associated with axonal activity (field stimulation) is [Ca2+]o-dependent. EJ]ects o[adenosine analo,ques and adenosine receptor antagonists Adenosine, CADO, NECA and CPA concentration-dependently decreased the evoked release of [3H]NE (Fig. 2) in a concentration-dependent manner. As indicated by the change of the $3/$2 ratios the maximum inhibition that could be achieved by adenosine, CADO, NECA and CPA was 35, 34 36 and 35% of control, respectively. IC20 values of adenosine, CADO, NECA and CPA were 10.4, 1.24, 0.33 and 0.16/tM, respectively. The relative potencies of adenosine, CADO, NECA and CPA were 1, 8.4, 32 and 64. 8-Phenyltheophylline antagonized the inhibitory effect of adenosine or CADO on [3H]NE release. 8Phenyltheophylline (1 /~M) shifted the log concentration-response curves of adenosine and CADO to the right (data not shown). These parallel shifts and the Lineweaver-Burk plots indicate that the antagonism is competitive. The Ko values of 8-PT, calculated from the Lineweaver-Burk plot were 0.40 #M for CADO and 0.51 #M for adenosine. Neither adenosine receptor agonists nor antagonists influenced the spontaneous release of radioactivity (data not shown). When the drugs (agonists and antagonists) were added to the perfusion fluid 6 min after $2, the $3/S~ ratio in the presence of l or 10/~M 8-PT alone was not significantly different from control. DPCPX, a
selective A~-purinoceptor antagonist, did not enhance the release, but at concentration of 100 nM it significantly reduced the inhibitory effect of adenosine o1 CADO (Table l). The K~ value of DPCPX was 5.2 nM for the antagonism of adenosine and 3.3 nM for the antagonism of CADO. In five experiments the A2, agonist, CGS 21680 (30 nM), had no effect on [3H]NE release. Idazoxan, a specific ~2-adrenoceptor antagonist (Doxley et al., 1983), increased the evoked release of [~H]NE in a concentration-dependent manner (Table 2). In the presence ofidazoxan, i.e. when the negative feedback modulation was excluded and thereby the release was significantly higher, the inhibitory effects of adenosine and CADO were augmented. In the absence of idazoxan, CADO 10 /~M caused about 34% inhibition, in the presence of 10 ~tM idazoxan CADO (10/~M) caused 58% inhibition. In the presence of idazoxan (10 /~M), DPCPX (10 nM) significantly antagonized the inhibitory effect of CADO (10 pM) (Table 2). 4-DAMP, an M~-receptor selective antagonist, and prazosin, an ~2B-adrenoceptor antagonist, significantly enhanced the release of [3H]NE evoked by field stimulation (Table 2). This finding indicates that the negative feedback modulation of NE release is mediated via ~2~ subtype of adrenoceptors. None of these antagonists changed the spontaneous release of [3H]NE (data not shown). Modulation ~)[acelylcholine release Alter loading with [3H]choline and washout, the tissue contained 3.19x 105+2.46x l 0 4 Bq/g radioactivity (n = 18). The resting release in the first 3-min collection period was 755 + 65 Bq/g (0.23 + 0.02% of the total radioactivity, n = 18). The radioactivity released by S~ was 926+89 Bq/g. This represents a fractional release of 0.30 + 0.02% (n = 18). When the stimulation was repeated with intervals of 18 rain, S~/S~, $3/$2 and 84/83 ratio were 0.88+0.03, 0.86 + 0.02, and 0.80 + 0.04 (n = 6), respectively. The Arpurinoceptor agonists adenosine, CADO, and NECA or the A~/A2-purinoceptor antagonist, 8PT had no effect on the evoked release of [3H]ACh. The $3/$2 ratios in the presence of adenosine (100 itM), CADO (10 ~tM), NECA (1 /~M), or 8-PT (10 /~M) were not different from the control value (Table 3). Atropine, a specific muscarinic, but not subtype selective receptor antagonist, and 4-DAMP, a M 3muscarinic subtype selective receptor antagonist, significantly increased the evoked release of [3H]ACh. When the negative feedback modulation was blocked
349
N o r e p i n e p h r i n e a n d acetylcholine release Table 1. Effects of adenosine analogues and antagonists on [3H]NE release evoked by electrical stimulation from guinea-pig right atrium
Compound Control (no drug) Adenosine 100 #M CADO 10 #M NECA 3 #M CPA 1 #M 8-PT 10 gM 8-PT 1 #M 8-PT 1/xM +Adenosine 100 #M 8-PT 1 gM + C A D O 10/~M DPCPX, 100 nM DPCPX 10 nM DPCPX 100 nM +Adenosine 100 #M DPCPX 100 nM + C A D O 10 #M
S, (fractional release, %)
$2/S,
$3/$2 (in the presence of drugs)
1.59 -+_0.16 1.21 _+0.26 1.68 _+0.12 1.40+_0.05 1.72_+0.15 1.55 _.%0.03 _ 1.34 _+0.09
0.93 _+0.02 0.92 _+0.03 0.91 _+0.02 0.93 _+0.01 0.92_+0.02 0.97 _+0.02 0.93 _+0.02
0.92 _+0.03 0.60 _+0.03 * 0.61 _+0.03* 0.59_+0.03* 0.61 +0.04* 0.99 + 0.04 0.94 -+0.02
1.63 _+0.16
0.91 _ + 0 . 0 1
0.88_+0.02*5.
1.48_+0.22 1.14-+0.08 1.09_+0.08
0.91 +0.06 0.94_+0.06 1.01 +0.01
0.70+_0.02** 1.01 _+0.04 0.90_+0.01
1.35+__0.14
0.96-+0.02
0.81 _+0.04*5"
1.39_+0.10
1.00+0.01
0.87_+0.03*§
Mean_+ SEM (n = 4-6). Three stimulations (2 Hz, 240 shocks) were applied. The drugs were added to the perfusion fluid 6 min after $2 and kept throughout the experiments, therefore any effect of drugs is shown on $3/$2 ratio. * Significant difference from control (P < 0.01). t Significant difference from adenosine 100 #M (only P < 0.01). :~Significant difference from CADO 10/zM (P < 0.05). § Significant difference from CADO 10 #M (P < 0.01).
by atropine (1 #M), adenosine (100 #M) and CADO (10/aM) still failed to affect the release (Table 3). Since it was suggested that the D2-receptor agonist quinpirole (Roquebert et al., 1991) and NPY (Potter, 1987) might be able to attenuate the cardiac vagal action, we made an attempt to study their effect on ACh release under condition, in which the post-
ganglionic fibers were surely stimulated. Both quinpirole 0.5 #M and NPY 0.1 /~M failed to affect the release of [3H]ACh evoked by field stimulation (2 Hz, 240 shocks) (Table 3). Idazoxan 1 #M also did not increase the release of radioactivity and xylazine 1 /~M, an c~2-adrenoceptor agonist, did not reduce the release (data not shown).
Table 2. Effects of CADO, DPCPX, idazoxan, 4-DAMP and prazosin on [3H]NE release evoked by electrical stimulation from guinea-pig right atrium
Compound Control (no drug) ldazoxan 0.1/~M ldazoxan 1 #M Idazoxan 10/zM ldazoxan 10 gM + C A D O 10 #M Idazoxan 10 #M + C A D O 10 #M + D P C P X 10 nM ldazoxan 10 #M + C A D O 10 #M + DPCPX 100 nm 4-DAMP 0.5 #M Prazosin 1/~M
S, (fractional release, %)
$2/S,
$3/$2 (in the presence of drugs)
1.59 + 0.16 1.49 + 0.26 1.72_+0.18 1.59 _+0.15
0.93 + 0.02 0.97 + 0.02 0.94_+0.03 0.93 _+0.02
0.92 + 0.03 1.33 _+0.06* 1.93_+0.08" 2.53 _+0.09*
1.41 _ + 0 . 1 7
0.94_+0.13
1.07_+0.05,
1.22_+0.10
0.92+_0.01
1.37+_0.10"§
1.39+_0.12 1.21 +0.16 1.42_+0.12
0.96-+0.06 0.95-+0.01 0.93-+0.02
2.14_+0.12"* 1.32-+0.12]" 2.98_+0.16"
Mean + SEM (n = 4-6). The drugs were added to the perfusion fluid 6 rain after $2 and kept throughout the experiments. * Significant difference from control (P < 0.01). t Significant difference from control (P < 0.05). * Significant difference from CADO 10 #M (P < 0.01). § Significant difference from idazoxan 10 #M (P < 0.05).
350
H. Nakatsuka et al. Table 3. Effects of adenosine analogues, 8-PT, atropine, quinpirole, NPY and 4-DAMP on [)H]ACh release evoked by electrical stimulation from guinea-pig right atrium
Compound Control (no drug) Adenosine 100 tim C A D O I 0 #M N ECA 1 /IM 8-PT l0 I~M Atropine I ,uM Atropine I itM + Adenosine 100 ItM Atropine I ltM + C A D O 10 ltM 4-DAMP 0.5 pM Quinpirole 0.5 itM NPY 0.1 ItM
SI (fractiomd release, '!4,)
SjSr
SdS: (in the presence of drugs)
(I.28 ± 0.09 0.30 -+ 0.10 0.34-+ 0.06 0.33 ± 0.09 0.27+0.01 0.22 ± 0.(17
0.88 ± 0.03 0.85 -+ 0.03 0.88 + 0.04 (I.88 ± 0.1)4 0.90+0.04 0.88 _+0.08
0.86_+0.02 O.89+O.05 0.83_+0.03 0.87+0.01 0.87_+0.03 .69_+0.16"
0.28 ± 0.04
0.86 -+ 0.05
.72+0.19"
0.27 ~ (/.04 0.45-+0.06 0.36-+ 0.(t2 0.30_+0.03
0.83 -+ 0.04 (I.90±0.01 0.89 + 0.08 0.89±0.02
.68-+0.18" .12-+0.03"I0.92-+0.12 0.94_4_0.05
Mean_+SEM (n = 4 6). The drugs were added to the per±us±on fluid 6 rain after $2 and kept throughout the experiments. * Significant difference from control (P < 0.01) 4-Significant difference from control (P < 0.05).
DISCUSSION
The ability of adenosine and adenosine triphosphate (ATP) to modulate cardiovascular activity has been recognized for more than 60 years (Drury and Szent-Gy6rgyi, 1929). The depressant effect of adenosine on synaptic transmission was first observed at the neuromuscular junction, by using an electrophysiological method (Ginsborg and Hirst, 1972). With this preparation neurochemical evidence was also obtained (Somogyi et al., 1987; Nagano et al., 1992) that adenosine inhibits the release of ACh. It was also reported that the evoked release of labeled NE (Hedqvist and Fredholm, 1976) and of endogenous (Vizi and Knoll, 1976) and labeled (Manabe et al., 1991) ACh was reduced by adenosine derivatives in several different tissues. In the myocardium adenosine is derived from the breakdown of ATP or that of S-adenosyl homocysteine (Bardenheur and Schrader, 1986). During tissue hypoxia or increased sympathetic discharge, formation of adenosine from ATP increased dramatically (Fredholm and Sollevi, 1986; Sparks and Bardenheuer, 1986). Therefore, it seemed interesting to study the effect of adenosine on NE and ACh release in response to neuronal activity from the sympathetic and vagal nerve terminals in the right atrium using electrical field stimulation and neurochemical methods. With this technique, it was possible to differentiate between the effect of compounds on the stimulation-evoked, [Ca- ],,-dependent quantal and [Ca2+]o-independent non-quantal release of neurotransmitters (Vizi, 1984; Viziet al., 1985, 1991 ; Vizi and Lfibos, 1991).
In this study it was demonstrated that while adenosine and its stable analogues inhibited the evokedrelease of [3H]NE from the sympathetic nerve ending of guinea pig right atrium, they did not inhibit the evoked release [3H]ACh from the vagal nerve endings. The order of potencies for the inhibition of NE release was : CPA > NECA > CADO > adenosine and the fact that DPCPX, a selective A,-receptor antagonist, inhibited the effects of adenosine and CADO strongly indicates that their inhibitory effect on NE release was mediated via the A~ receptors. This is consistent with the observation (Richardt et al., 1987; Sch~tz et al., 1991) that cyclohexyladenosine also inhibited the release of endogenous NE from rat heart. In corroboration with others (Manabe et al., 1991 ; Smith et al., 1992) we also observed that the release of [3H]NE was subject to negative feedback modulation, i.e. NE released from the axon terminal reduced its own release. When the release of NE was freed from ~2-receptor-mediated control, using idazoxan (I #M), the inhibition achieved by A,-purinoceptor stimulation with CADO (10/~M) was higher (54%). This finding indicates that all sympathetic axon terminals are also equipped with A,-receptors and the different modulatory receptors are not located in different populations of axon terminals. Similar observations were made by Allgainer et al. (1987) on the influence of adenosine and CADO on the release of NE from cerebral cortex slices. 8-Phenyltheophylline competitively antagonized the inhibitory effect of adenosine or CADO, but 8-PT alone had no effect on NE release. It was reported that ATP is released from the sympathetic nerve endings
351
Norepinephrine and acetylcholine release together with NE. ATP is readily metabolized to adenosine, and adenosine is metabolized to inosine or is taken up by the tissue. Therefore, it is probable that under our experimental conditions, the concentration of endogenous adenosine released is not high enough to decrease NE release ; however, during ischemia its concentration is much higher and adenosine may create regional autonomic imbalance and also may protect the ischemic myocardium from excessive catecholamine stimulation (Miyazaki and Zipes, 1990). The finding that adenosine and its congeners had no effect on the release of [3H]ACh associated with neuronal activity in the guinea pig right atria, even when the muscarinic autoinhibition of ACh release was prevented by atropine, indicates that the vagal nerve endings in the guinea pig right atria are not equipped with A~-purinoceptors. This indicates that cholinergic axon terminals in the Auerbach plexus (Vizi and Knoll, 1976 ; Somogyi and Vizi, 1988 ; Milusheva et al., 1990) and hippocampus (Duntr-Engstr6m and Fredholm, 1988; Cunha et al., 1994) are different from right atria: the stimulation of these receptors resulted in inhibition of stimulation-evoked release of ACh. Adenosine and ATP have been used clinically to induce hypotension during anesthesia (Sollevi, 1986) or to stop paroxysmal supraventricular tachycardia (Rankin et al., 1990; Sollevi, 1986). Furthermore, it was reported (Pitarys et al., 1991) that intravenous infusion of adenosine reduced myocardial reperfusion injury in dogs. It is well known that a large amount of adenosine is liberated from myocardium during hypoxia (Fredholm and Sollevi, 1986; Sparks and Bardenheuer, 1986). Taking into account our data, it is conceivable to suggest that adenosine liberated during hypoxia inhibits NE release from the cardiac sympathetic nerve without inhibiting ACh release and thereby offers protection against tachyarrhythmia caused by myocardial hypoxia. It was suggested by Roquebert et al. (1991) that vagal nerves are endowed with a pharmacologically relevant population of D:-receptors and therefore D2receptor agonists are able to attenuate the vagalinduced but not the ACh-induced bradcardia in pithed rats. However, in their experiments the site of action of quinpirole, a selective D?receptor agonist, was not precisely located. It could have been on the ganglion and/or on the postganglionic axon terminals. In our recent experiments adenosine ; quinpirole, a selective D2-receptor agonist, and NPY did not influence the release of ACh in response to field stimulation. Using field stimulation both pre- and postganglionic fibers are stimulated. And previously using the same prep-
aration we showed (Kobayashi et al., 1987) that ~2adrenoceptor agonist also failed to affect the release. However, there are several observations (Richardt et al., 1987; Miyazaki and Zipes, 1990; Roquebert et aL, 1991 ; Potter, 1987 ; Revington et aL, 1990) that NE, adenosine, dopamine and NPY attenuate the responses of the heart to preganglionic vagal stimulation. Even if it was suggested that their site of action is presynaptic, the preganglionic vagal nerve was also stimulated in their experiments, and in our experiment field stimulation was used, i.e. the postganglionic fibers were also stimulated. Therefore the discrepancy can be easily explained that the inhibitory effect observed by others may be mediated via an effect at the ganglionic level (Fig. 3), In corroboration with Sympathetic
Doparnine
(quinpirol¢)
Adenosin~ I I I
~2-1t
N
It,, t', \\ \
NE.~"
~NE
Vagal
/
~anglion
-~. t
",//
ACh ---
~ r i a l ~-ATP
pacem ake_r~""-~.~ (myocardialcell)
Adenosine
Fig. 3. Scheme of the possible interaction between vagal and sympathetic innervation of the heart. Note that Arreceptors are located on both myocardial cells and sympathetic axon terminals and adenosine released from the tissue and/or decomposed from ATP in increased amounts during ischemia and hypoxia may have an inhibitory effect on both sites. While the sympathetic activation results in tachycardia and encourages the generation of arrhythmias under conditions of ischemia, adenosine may exert a protective effect by inhibiting the release of NE and by counteracting the effect of catecholamines on myocardial cells. The noradrenergic axon terminals are also equipped with inhibitory muscarinic receptors. Thus, vagal activation reduces ventricular vulnerability ; this is the result of a combination of inhibition of NE release and direct effect on myocardial cells. Since the axon terminals of postganglionic vagal fibers are not equipped with inhibitory A~-receptors therefore adenosine released during ischemia inhibits only the release of NE, but not that of ACh. The imbalance in autonomic innervation produced by adenosine may result in a vagal tone. The negative feedback modulation of NE and ACh release from the postganglionic fibers is mediated via ~2- and muscarinic-receptors, respectively, The myocardial cells are equipped with muscarinic-receptors. The ganglionic transmission is modulated presynaptically through D2-, ~2-(the subtype is not yet specified)and Arreceptors, It is very likely that neuropeptide Y is also able to inhibit transmission in the parasympathetic ganglion.
352
H. Nakatsuka et al.
this, it was s h o w n (Dawes a n d Vizi, 1973) that N E inhibits the release o f A C h from the ganglion. In o u r case, by using field stimulation, the site o f action was tested also on the axon terminals next to the effector cells. O u r data suggest t h a t the vagal postganglionic nerve endings are not equipped with receptors sensitive to NE, adenosine, quinpirole, or NPY. In summary, the heart with its dual a u t o n o m i c innervation is a site where there is a only one-sided interaction between the two nerves at the presynaptic level o f postganglionic neurons. Sympathetic axon terminals are quipped with 72B-autoreceptors a n d muscarinic ( M 0 a n d Aj-heteroceptors. Thus the release of N E is subject to negative feedback m o d u l a t i o n via stimulation of ~26-adrenoceptors and presynaptic inhibitory m o d u l a t i o n by A C h released from the vagal nerve a n d by adenosine. However, the vagal postganglionic nerve endings are not equipped with 72- and A~-receptors, but there is a muscarinic-autoreceptormediated negative feedback modulation. Thus N E released during sympathetic activity and adenosine released in excess from the heart muscle during hypoxia do not have any effect on the postganglionic vagal axon terminals, but they might have an inhibitory effect at the level of p a r a s y m p a t h e t i c ganglia (Fig. 3). The imbalance in a u t o n o m i c i n n e r v a t i o n produced by adenosine may result in a vagal tone. This work was partly supported by Hungarian Research Fund (OTKA) and Medical Research Council (ETT). Acknowledgements
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