European Journal of Pharmacology, 216 (1992) 9-16
9
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52450
N-0861 selectively antagonizes adenosine A 1 receptors in vivo R i c h a r d J. B a r r e t t , D a v i d A. D r o p p l e m a n a n d K a t h r y n F. W r i g h t Department of Pharmacology, Whitby Research, Inc., P.O. Box 27426, Richmond, VA 23261-7426, USA Received 12 December 1991, revised MS received 7 February 1992, accepted 25 February 1992
Experiments were performed to determine the antagonistic actions of (_+)N6-endonorbornan-2-yl-9-methyladenine (N-0861) at A 1 and A 2 adenosine receptors in vivo, and to evaluate the pharmacodynamics of the observed responses. The selectivity of antagonism of A 1 vs. A 2 receptors by N-0861 was evaluated by generating dose-response curves to adenosine-induced bradycardia (A 1 effect), and vasodilation in the in situ constant-flow perfused rat hindquarter vasculature (A 2 effect). N-0861, at doses >/1 /~mol/kg + 0.04 p~mol/kg per min, i.v. produced dose-related rightward shifts of the A 1 dose-response curve, but had no effect on the A 2 dose-response curve at doses as high as 100/zmol/kg, i.v. In contrast, the non-selective A 1 / A 2 adenosine receptor antagonist 8-phenyltheophylline antagonized both A~ and A 2 receptor-mediated responses to adenosine. The minimum effective i.v. dose and the duration of action of N-0861 were determined by evoking bradycardic responses to i.v. adenosine (A 1 effect) in anesthetized, vagotomized,/3-blocked rats before and after single bolus doses of vehicle or N-0861 (0.3, 0.6, 1.0, 3.0 or 10.0 ~mol/kg). The lowest i.v. dose of N-0861 to antagonize A1 receptor-mediated bradycardia was 0.3 ~mol/kg i.v.; the duration of effect ranged from 1 min (following 0.3-1 /~mol/kg) to approximately 2.5 h (following 10 /zmol/kg). N-0861 is a selective (by >/333-fold) antagonist of adenosine A 1 receptors. Adenosine; Adenosine A 1 receptors; Adenosine A 1 receptor antagonists
1. Introduction
Stimulation of adenosine A~ and A2 receptors with the non-selective agonist adenosine has found direct clinical utility in the treatment of supraventricular tachycardia (A 1 effect) (Belardinelli et al., 1989), and simulation of exercise during myocardial perfusion imaging studies ( A 2 effect) (Verani et al., 1990). In spite of these beneficial clinical applications, inappropriate stimulation of A 1 a n d / o r A 2 receptors may be undesirable, and may even be pathogenic in cardiac conduction disorders (Belardinelli et al., 1989) and renal failure (Bohle et al., 1990). The only clinically approved adenosine receptor antagonists are the xanthines, most of which antagonize both A~ and A 2 receptors, and also inhibit phosphodiesterases and adenosine uptake (Rall, 1990). Selective agonists and antagonists that do not inhibit phosphodiesterase activity or adenosine uptake may have therapeutic utility in a number of disorders; recent progress in this area has been reviewed (Jacobson et al., 1991).
Correspondence to: R.J. Barrett, Department of Pharmacology, Whitby Research, Inc., P.O. Box 27426, Richmond, VA 23261-7426, U.S.A. Tel. 1.804.254 4072, fax 1.804.254 4038.
In a series of in vitro assays, N-0861 (( _+)N 6-endo norbornan-2-yl-9-methyladenine) was identified as a selective A~ adenosine receptor antagonist that has little affinity for other receptors, and little effect on phosphodiesterases and adenosine uptake (May eta[., 1991; Belardinelli et al., 1990). Studies were performed to characterize the adenosine A 1 vs. A z subtype specificity of N-0861 in vivo, and to determine the preclinical pharmacodynamics of this novel adenosine antagonist.
2. Materials and methods
2.1. General Drug-naive, male Sprague-Dawley rats (Hilltop Farms, Scottdale, PA) were anesthetized with urethane (1.5 g / k g i.p.), and kept on a Harvard heated operating board to maintain the body t e m p e r a t u r e at 37°C. A ventral cervical incision was made, and the trachea, jugular vein, carotid artery, and both vagus nerves were isolated. Polyethylene catheters were inserted into the trachea, carotid artery and jugular vein, and the vagi were sectioned. Systemic blood pressure was measured from the carotid arterial catheter with a Statham Gould
10 P23XL or Sorenson Transpac II pressure transducer. H e a r t rate was derived from the blood pressure signal with a Grass 7P4 tachograph. Signals were displayed and recorded on a Grass Model 7E or P79D polygraph. The rats were then subjected to one of two experimental protocols designed to determine (1) the selectivity of antagonism of A~ vs. A 2 adenosine receptors, or (2) the dose relation and duration of adenosine A~ receptor antagonism by N-0861.
2.2. Selectit,ity of adenosine A 1 us. A 2 receptor antagonism 2.2.1. Surgical procedure After completing the general surgical preparation described above, 29 rats (mean _+ S.E.M. weight 387 _+ 5.0 g) were further p r e p a r e d by methods adapted from Merrick and Holcslaw (1981). A midline abdominal incision was made, and a 2.5-3.0 cm segment of the abdominal aorta between the left renal artery and the posterior aortic bifurcation was exposed and dissected free from the vena cava. Three ligatures were placed around the aorta, and Na + heparin (125 U i.v.) was administered. The center ligature was tightened, and the distal aorta was cannulated with one end of the perfusion tubing. In order to minimize the time ( < 1 min) that the hindquarter vasculature was deprived of blood, the carotid arterial cannula was attached via a t-tube to allow arterial blood to perfuse the hindquarters. The proximal aorta was then cannulated, the perfusion tubing was unclamped, and blood was allowed to flow through the tubing. At this time, with autoperfusion to the hindquarters restored, the carotid arterial cannula was disconnected from the circuit. The abdominal wound was closed with stainless steel wound clips. The perfusion tubing was filled with 2.0 ml heparinized (50 U / m l ) 6% dextran 70. A small length of latex tubing and a plastic t-tube were introduced distal to the p u m p to allow intra-arterial injection of drugs and m e a s u r e m e n t of hindquarter perfusion pressure (mmHg), respectively. Hindquarter perfusion pressure was measured with a Statham Gould P23XL or Sorenson Transpac II pressure transducer, and the signal was displayed as described above. 2. 2. 2. Experimental procedure Respiration was maintained (70 s t r o k e s / m i n , 2.5 ml) with a Harvard rodent ventilator. The perfusion tubing was inserted into an Isco W I Z peristaltic p u m p (Lincoln, NE) and blood was directed distally into the hindquarter vascular bed. The flow rate was set between 4 and 6 m l / m i n and norepinephrine (3-19 n m o l / m i n ; mean ( ± S . E . M . ) 11.0 _+ 1.0 n m o l / m i n ) w a s infused directly into the hindquarter vasculature via the injection tubing to maintain perfusion pressure at
115-135 mmHg. The effect of norepinephrine was limited to the hindquarters; no effects on systemic blood pressure or heart rate were observed. Once set, the p u m p rate and norepinephrine infusion rate were not altered during the course of the experiment. When perfused at constant flow, vasodilation in an isolated vascular bed is manifested as a decrease in perfusion pressure. The maximal vasodilatory capacity of the hindquarter vascular bed of each rat was established by evoking maximal endothelium-dependent and endothelium-independent vasodilatory responses with simultaneous i.a. injection of acetylcholine (0.06/xmol) and nitroglycerin (0.06 txmol). A d e n o s i n e A2 receptor-mediated vasodilatory responses were produced by i.a. injections of adenosine (0.006-0.6 tzmol in 20 p.l). These (A 2) responses were expressed as a percent of the maximal vasodilatory capacity. Adenosine A~ receptor-mediated bradycardic responses were evoked by injections of adenosine (0.1-6 #tool in 100 ill) into the jugular vein. These (A 1) responses were expressed as percents change from basal heart rate preceeding the adenosine challenge. D o s e - r e s p o n s e curves to adenosine were obtained in this manner prior to administration of N-0861. Because of its short duration of action, three of the four doses of N-0861 were administered as bolus followed by infusion (1 / x m o l / k g + 0.04 / x m o l / k g per rain, 10 t x m o l / k g + 0 . 4 / ~ m o l / k g per min or 30 t z m o l / k g + 1.2 i x m o l / k g per min). Only one dose of N-0861 was administered to each animal. D o s e - r e s p o n s e curves for the A1 and A 2 responses were again generated during the infusion of N-0861. The highest dose of N-0861 tested, 100 i z m o l / k g i.v. was injected slowly as a bolus (over 2 min) and was not followed by infusion; o n l y A 2 responses were evoked in these animals. In order to demonstrate that adenosine-induced vasodilation in the in situ perfused hindquarters was indeed due to stimulation of adenosine receptors and that both A~ and A 2 receptor-mediated responses could be antagonized in the preparation, two additional groups of rats were treated with the non-selective adenosine receptor antagonist 8-phenyltheophylline (8-PT, 5 or 40 p.mol/kg, i.v. bolus (Collis et al., 1985)). Each animal received only one dose of antagonist. D o s e - r e s p o n s e curves to adenosine were generated before and > 5 rain following injection of 8-PT.
2.3. Pharmacodynamics of bolus doses of N-0861 2.3.1. Experimental procedure After they had stabilized following the surgical preparation described above (2.1.), 38 rats (mean_+ S.E.M. weight 356 _+ 4.4 g) were subjected to the following experimental protocol. Adenosine was administered through one jugular vein catheter, and a second catheter (in the same vein) was used for injection of
11 isoproterenol, atenolol and N-0861. Heart rate responses to i.v. isoproterenol (0.07 nmol) were measured before and periodically after atenolol (37.5 > m o l / k g i.v.) to ensure the endurance of /3-blockade. Adenosine A l receptor-mediated changes in heart rate evoked by repeated doses of 0.6 p~mol adenosine, i.v., were measured several times before and periodically after N-0861 or vehicle. N-0861 (0.3, 0.6, 1, 3 or 10 /.tmol/kg) or vehicle (pH 2.42 Na + phosphate buffer) was administered i.v. (over 30 s) at time 0; the adenosine challenges were continued for 30 min following 0.3, 0.6 and 1 /.tmol/kg N-0861, and for 3 h following administration of 3 and 10 # m o l / k g N-0861. Only one dose of N-0861 was administered to each animal. Data have been expressed as % change from the basal heart rate immediately prior to injection of adenosine.
Davis, Morris Plains, N J; all other compounds, Sigma, St. Louis, MO.
2.5. Statistics In the adenosine A l vs. A 2 selectivity experiments, the negative chronotropic and hindquarter vasodilatory responses to individual doses of adenosine generated prior to and after N-0861 were paired, and were therefore analyzed with a paired, 2-tailed t-test. The d o s e response curves representing the A l and A 2 responses to adenosine evoked in each animal before vs. after N-0861 or 8-PT were analyzed with a computerized non-linear regression analysis program (GraphPad InPlot 3.1; SanDiego, CA). Fitting the data to sigmoid curves facilitated calculation of EDs0 values for both the A I responses (the dose of adenosine required to reduce heart rate 50% from control) and the A 2 responses (the dose of adenosine required to produce 50% of the maximum vasodilatory response to adenosine). The EDs0 values for the A l and A 2 responses obtained in each animal prior to antagonist were compared with those obtained after antagonist treatment with a paired, 2-tailed t-test. To determine if the control (pre-antagonist) A I and A 2 responses evoked in rats from all four N-0861-treated groups could be pooled for illustration, the data were subjected to one-way analysis of variance. In the pharmacodynamic experiments, the percents change in heart rate evoked by adenosine before N0861 or vehicle treatment between the six groups were subjected to one-way analysis of variance. The percents change in heart rate evoked by adenosine at each time point in each treatment group were analyzed by one way analysis of variance and Dunnett's test, comparing
2.4. Drugs N-0861 was dissolved in 0.1 M N a H 2 P O 4 buffer acidified with 1-1.5 mEq of HC1 for each mEq of N-0861. The vehicle was prepared by adjusting the pH of a 0.1 M N a H z P O 4 buffer solution to 2.42, the pH of the most acidic solution of N-0861. Adenosine, atenolol, isoproterenol, norepinephrine, acetylcholine and nitroglycerin were dissolved in 0.9% NaC1; stock solutions of adenosine and atenolol required slight acidification for dissolution. 8-PT was dissolved in 50% (v/v) P E G / 0 . 1 N N a O H (pH 12); pilot studies showed that the vehicles did not influence the magnitude of the responses to adenosine. Sources of compounds: N-0861, Whitby Research, Inc., Richmond, VA; 8-PT, Research Biochemicals, Natick, MA; nitroglycerin, Parke PERCENT DECREASEFROM CONTROL HEART RATE
IO0
PERCENT MAXIMUM VASODILATORY CAPACITY
100 /
80
Control o 1 lamol/kg + • 0.04 lamol/kg per min
60
10 tamol/kg + ' 0.4 ~mol/kg per rain
40 20
30 gtmol/kg + •
~
1.2 gmol/kg per m
i
\ ~
.-r
.~'"
~'~" m"~
60
~1'...,/
40
10
~ 20
0
I 9
8
1-
I
7
6
DOSE ADENOSINE (-log mol)
0 5
~7 9
8
I
I
7
6
5
DOSE ADENOSINE (-log tool)
Fig. 1. The effect of N-0861 on the bradycardic (A1) responses to i.v. adenosine and vasodilatory (A2) responses to i.a. adenosine. Data are expressed as means+S.E.M. Control curves represent data pooled from all treatment groups prior to administration of N-0861. Left panel. Bradycardic responses are expressed as percent decrease from resting heart rate evoked by i.v. adenosine. Each A~ dose-response curve was significantly different from its paired control curve. Right panel. Vasodilatory responses evoked by i.a. adenosine are expressed as percent of the maximal vasodilatory capacity of the perfused hindquarters. N-0861 had no significant effect on A 2 responses to adenosine. Symbols are as illustrated in left panel; A l responses were not evoked in rats treated with 100/xmol/kg N-0861.
12 r e s p o n s e s from each of the five N-0861 t r e a t m e n t groups to responses of v e h i c l e - t r e a t e d rats at the same time point. I n all analyses, statistical significance was assigned w h e n P < 0.05.
3. Results 3.1. Selectivity o f adenosine A 1 t,,s. A 2 receptor antagonism Vasodilatory r e s p o n s e s to i.a. a d e n o s i n e were expressed as a p e r c e n t of the m a x i m u m vasodilatory capacity of the h i n d q u a r t e r s . I n the 29 rats tested, nitroglycerin + acetylcholine d e c r e a s e d p e r f u s i o n pressure from ( m e a n + S.E.M.) 126.8 _+ 1.70 to 70.7 _+ 1.61 m m H g , yielding a m e a n m a x i m u m vasodilatory capacity of 56.0 _+ 1.94 m m H g . W h e n injected i.a. into the p e r f u s i o n tubing, a d e n o sine p r o d u c e d an i m m e d i a t e drop in h i n d q u a r t e r perfusion pressure; b r a d y c a r d i a was d e t e c t e d only at doses of a d e n o s i n e >1 0.6 ~ m o l , a n d only after a n obvious delay of 1 0 - 1 5 s. I.v. a d m i n i s t e r e d a d e n o s i n e evoked an i m m e d i a t e bradycardic (A~) response, while the vasodilatory ( A 2) response was expressed in the perfused h i n d q u a r t e r s only after a 1 0 - 1 5 s delay. T h e bradycardic ( A 1) a n d vasodilatory ( A 2) responses to a d e n o s i n e in rats with in situ p e r f u s e d h i n d q u a r t e r s were dose-responsive a n d r e p r o d u c i b l e (figs. 1, 2). Doses of 0 . 1 - 3 p.mol (i.v.) a d e n o s i n e were r e q u i r e d to construct A 1 r e c e p t o r d o s e - r e s p o n s e curves, while only 0.006-0.6 ~ m o l (i.a.) a d e n o s i n e were r e q u i r e d to o b t a i n d o s e - r e s p o n s e curves for the A 2 r e c e p t o r - m e d i a t e d vasodilation. Both the bradycardic a n d vasodilatory r e s p o n s e s to a d e n o s i n e o b t a i n e d before N-0861 t r e a t m e n t were similar in all groups of rats ( c o m p a r e EDs0 values in table 1), a n d have b e e n pooled for illustration. T h e bradycardic (A~) a n d vasodilator (A 2) responses to a d e n o s i n e evoked before
100' 80'
TABLE 1 EDs0 values (means_+S.E.M.) for the A~ (bradycardic) and A 2 (vasodilatory) receptor-mediated responses to adenosine before (control) and following N-0861. Treatment group
A Eresponse EDs0 ( - log tool)
A 2 response EDs0
Control (n = 5) N-0861. 1 /xmol/kg+ 0.04/xmol/kg per min
6.55 + 0.063 6.16+0.060 P < 0.05 ~'
7.29 + 0.102 7.42+0.107 ns
Control (n = 4) N-0861, 10 #mol/kg+ 0.4/~mol/kg per rain
6.42+_0.119 5.79_+0.008 P < 0.05 "
7.31 +0.121 7.18_+0.044 ns
Control (n = 4) N-0861, 30/xmol/kg + 1.2 p.mol/kg per min
6.63 _+0.030 5.68+0.051 P < 0.05 a
7.19 _+0.077 7.11 +0.061 ns
Control (n = 5) N-0861, 100/xmol/kg
NT NT
7.37 + 0.054 7.43_+0.057 ns
Pooled controls
6.53 _+0.005 (n = 13)
7.29 _+0.044 (n = 18)
a VS. EDso values derived from responses evoked before N-0861 (paired, two-tailed t-test); ns = no significant difference; NT = not tested).
8-PT were also similar in rats that received 5 or 40 ~ m o l / k g 8-PT; these control data also have b e e n p o o l e d (table 2, fig. 2). T h e stability a n d reproducibility of both A I (bradycardic) a n d A 2 (vasodilatory) responses is obvious in the similarities of the control curves in figs. 1 a n d 2 a n d in their respective EDso values (tables 1, 2). D o s e - r e s p o n s e curves to both the A 1 a n d A 2 recept o r - m e d i a t e d actions of a d e n o s i n e were c o n s t r u c t e d in three groups of rats prior to, a n d following N-0861; only A 2 responses were elicited in the r e m a i n i n g group of rats t r e a t e d with 100 / x m o l / k g N-0861. T h e c h r o n o t r o p i c (A 1) responses to individual i.v. doses of
PERCENT DECREASE FROM CONTROl HEART RATE
PERCENT MAXIMUM VASODILATORY CAPACITY
100"T Control 0 80 Jr 5 I.imollkg [] ~
Control o5 lamol/kg [] 40 Iamol/kg 0
60
60" 40'
( - log tool)
-~ \
t 40 gmol/kgO ~ ~ S s~ 5 " ~ , ~ , ~
40 1
20 0
9
i 8
7
DOSE A DENOSINE (-log mol)
6
5
0
9
8
t
7
i
6
5
DOSE ADENOSINE (-log tool)
Fig. 2. The effect of 8-phenyltheophylline (8-PT) on the bradycardic (A 1) responses to i.v. adenosine and vasodilatory (A 2) responses to i.a. adenosine. Data are expressed as means_+S.E.M. Control curves represent data pooled from both treatment groups prior to administration of 8-PT. Left panel. Bradycardic responses are expressed as percent decrease from resting heart rate evoked by i.v. adenosine. Both doses of 8-PT significantly attenuated the A I responses to adenosine. Right panel. Vasodilatory responses evoked by i.a. adenosine are expressed as percent of the maximal vasodilatory capacity of the perfused hindquarters. Unlike N-0861, 8-PT significantlyantagonized the A 2 responses to adenosine.
13 TABLE 2 EDs0 v a l u e s ( m e a n s i S . E . M . ) for the A I ( b r a d y c a r d i c ) a n d A 2 (vasodilatory) r e c e p t o r - m e d i a t e d r e s p o n s e s to a d e n o s i n e b e f o r e (control) and following 8 - p h e n y l t h e o p h y l l i n e (8-PT). Treatment group
A l response EDs0 ( - log mol)
A 2 response EDs0 ( - log tool)
C o n t r o l (n = 5) 8-PT, 5/~ m o l / k g
6.5 _+0.05 5.9 ± 0.03 P < 0.01 ~'
7.3 + 0.09 6.5 ± 0.24 P < 0.05 "
C o n t r o l (n = 6) 8-PT, 4 0 / z m o l / k g
6.5 ± 0.07 5.5 + 0.07 P < 0.01 '~
7.4 +_0.06 5.8 +_0.21 P < 0.05 "
P o o l e d controls (n = 11 )
6.5 + 0.04
7.3 ± 0.05
significant, dose-related, rightward shifts of the d o s e response curves representing both the A I (bradycardic) and A 2 (vasodilatory) responses to adenosine (fig. 2, table 2). Antagonism of A~ receptor-mediated bradycardia by 8-PT appeared to be competitive, but the data do not permit such a conclusion for the A 2 receptor-mediated responses.
3.2. Pharmacodynamics of bolus doses of N-0861
~' vs. ED50 v a l u e s d e r i v e d from r e s p o n s e s e v o k e d b e f o r e 8-PT (paired, t w o - t a i l e d t-test).
0.1, 0.3, 0.6 and 1 /xmol adenosine were significantly attenuated (P < 0.05) by all doses of N-0861, while the response to 3 / z m o l i.v. adenosine was attenuated only in the group treated with the 30 / x m o l / k g + 1.2 /zmol/kg per min dose of N-0861. N-0861 produced significant, dose-related, rightward and parallel shifts of the adenosine A~ (bradycardic) dose-response curves, but had no effect on the adenosine A 2 (vasodilatory) dose-response curves (fig. 1). The EDs0 values derived for the adenosine dose-response curves were significantly changed by each dose of N-0861 (table 1). These results provide in vivo evidence for the adenosine A 1 receptor selectivity of N-0861, and suggest that N-0861 antagonizes A I receptor-mediated bradycardia in a competitive manner. In contrast to animals treated with N-0861, treatment with the non-selective A t / A 2 receptor antagonist 8-PT (5 /zmol/kg or 40 /zmol/kg, i.v.) produced
In order to evoke a bradycardic response that would be due to only adenosine A I receptor stimulation, autonomic control of the heart was ablated by bilateral vagotomy and the/3~-adrenoceptor-selective antagonist atenolol. The mimimum effective i.v. dose and the duration of A~ receptor antagonism by N-0861 was evaluated by comparing the bradycardic responses to adenosine (A~ effect) in rats treated with N-0861 to adenosine responses evoked in rats treated with vehicle. Separate groups of five to seven rats were treated with the vehicle or with single bolus doses of N-0861. Basal heart rates and responses to adenosine (0.6/xmol i.v.) evoked 5 times prior to treatment with vehicle or N-0861 were similar in all groups (table 3). Neither the vehicle, N-0861, nor repeated administration of adenosine altered the basal heart rate. Before administration of vehicle or N-0861, adenosine reduced heart rate by approximately 44%, from 327 to 185 beats/rain (average data from table 3). These adenosine A~ receptormediated bradycardic responses were stable over 3 h in vehicle-treated rats (fig. 3). The very slight enhancement of the bradycardic responses seen after vehicle injection was not statistically significant. The duration and degree of adenosine A j receptor
TABLE 3 R e s t i n g h e a r t r a t e s and r e s p o n s e s to i.v. a d e n o s i n e prior to N-0861 or vehicle. T h e basal ( c o n t r o l ) h e a r t r a t e s and r e s p o n s e s to a d e n o s i n e w e r e similar in each t r e a t m e n t g r o u p p r i o r to a d m i n i s t r a t i o n of vehicle or N-0861. T h e effects of five successive a d e n o s i n e injections (0.6 p~mol i.v.) a d m i n i s t e r e d 3 min a p a r t have b e e n a v e r a g e d . D a t a in ' % c h a n g e ' c o l u m n are r e p r e s e n t e d as ' P r e - N - 0 8 6 1 ' in fig. 3. Treatment group
Basal (beats/min)
Adenosine (beats/rain)
Change (beats/min)
% change
n
Vehicle
Mean ± S.E.M.
311.5 11.95
177 27.5
- 134.5 24.07
- 43.7 7.9
6
0.3/xmol/kg
Mean ±S.E.M.
308 14.26
190.4 27.22
- 117.8 14.72
- 39.3 6.2
5
0.6/xmol/kg
Mean ± S.E.M.
337.8 5.47
182.5 18.37
- 155.33 17.76
- 46.1 5.3
6
1 /zmol/kg
Mean ± S.E.M.
339.6 6.29
177 20.06
- 162.14 15.05
- 48.2 5.0
7
3/zmol/kg
Mean ± S.E.M.
345.7 9.25
181.1 15.79
- 117.14 49.95
- 47.4 4.5
7
10/xmol/kg
Mean ± S.E.M.
319.7 6.64
199.9 17.32
- 120.00 14.97
-37.7 4.8
7
14
l~mol/kg
%
PreN-0861
1
3
6
9
12
15 3 0 4 5
60
90
t20
150
~BO
90
t20
150
180
90
:l;~O
150
1BO
90
120
150
180
90
~120
150
180
TIME (min)
0.6 ,mo,/kg
%
PreN-0861
1
PPeN-OB6:I
1
3
6
9
t2
15 3 0
45 60
TIME (rain)
%
3
6
9
t2
15 3 0
45
60
TIME (min)
3 #mol/kg
%
PreN-0861
:t
PreN - 0 8 6 :l
1
3
6
9
12
15 3 0 4 5
60
TIME (min)
3
6
9
12
15 3 0 4 5
50
TIME (min)
Fig. 3. Duration of antagonism by N-0861 of A t bradycardic responses to i.v. adenosine. Bradycardic responses (expressed as percents decrease from basal heart rate) evoked by i.v. administration of 0.6 ~mol adenosine before and at the indicated times after bolus doses of N-0861 (0.3, 0.6, 1, 3 or 10 # m o l / k g i.v., shaded bars) in anesthetized, vagotomized, /3-blocked rats (n = 5-7/group). All data are expressed as means_+ S.E.M. Data from vehicle-treated rats (n = 6) is represented by open bars in each panel. Note that, for illustration's sake, S.E.M. for the vehicle-treated group is presented only in the bottom panel, and that the abscissa is not linear over time. ~' P < 0.01; b p < 0.05, Dunnett's test vs. vehicle-treated rats at the same time period.
a n t a g o n i s m b y N - 0 8 6 1 is p r e s e n t e d T h e l o w e s t i.v. d o s e o f N - 0 8 6 1 t o nize the bradycardic response to /~mol/kg. The three lowest doses
g r a p h i c a l l y in fig. 3. significantly antagoa d e n o s i n e w a s 0.3 o f N - 0 8 6 1 (0.3, 0.6
a n d 1.0 ~ m o l / k g ) w e r e e f f e c t i v e f o r o n l y 1 m i n , w h i l e t h e h i g h e r d o s e s (3 a n d 10 > m o l / k g ) significantly a t t e n u a t e d t h e b r a d y c a r d i c ( A ~ ) r e s p o n s e s f o r 12 a n d 150 m i n , r e s p e c t i v e l y .
15 4. Discussion
Adenosine reduces systemic blood pressure by simultaneously activating both A I and A 2 adenosine receptor-dependent vasodepressor mechanisms: direct relaxation of vascular smooth muscle, reduction of cardiac rate and output, inhibition of renin release and attenuation of sympathetic drive (Collis, 1989; Belardinelli et al., 1989; Jackson, 1991). Because of these diverse and interrelated actions, we considered it essential to separate completely the vasodilatory responses to adenosine A 2 receptor stimulation from other concurrent adenosine-induced changes in hemodynamics. This was achieved in rats with in situ perfused hindquarters by injecting adenosine i.v. to evoke A l receptor-mediated bradycardic responses, and i.a. to evoke A 2 receptor-mediated vasodilatory responses. This is the first report to demonstrate that stimulation of adenosine A 2 receptors causes vasodilation in the in situ perfused rat hindquarters preparation. Sakai et al. (1979) reported that i.a. injection of adenosine into a cross-perfused isolated rat hindlimb preparation caused dose-related vasoconstriction that was attributable to release of 5-hydroxytryptamine. In these studies in the in situ perfused rat hindquarters, adenosine and specific A 2 receptor agonists consistently produced only vasodilation - never vasoconstriction. Adenosine may cause vasoconstriction in vivo in the kidney (Jackson, 1991) and skin (Proctor and Stojanov, 1990), but stimulation of adenosine A 2 receptors causes only vasodilation in skeletal muscle of the dog (Horn and Lokhandwala, 1981), rat (Mohrman and Heller, 1984), rabbit (Gustafsson et al., 1988), hamster (Proctor, 1984) and man (Sollevi, 1986). It is likely that the vasodilatory responses to i.a. adenosine in the present studies were mediated by A 2 receptors in the vasculature supplying the skeletal muscle of the hindquarters. The non-selective adenosine receptor antagonist 8-PT (Collis et al., 1985) antagonized both the bradycardic (A~) and the vasodilatory (A 2) responses to adenosine in rats with in situ perfused hindquarters, indicating that both responses were indeed evoked by adenosine receptor stimulation, and that antagonism of both A l and A 2 receptor-mediated responses could be reliably detected in this preparation. The A~ (bradycardic) d o s e - r e s p o n s e curves to adenosine had similar slopes and maxima before and after 8-PT, suggesting - as had been observed in vitro - that the nature of A~ antagonism by 8-PT was competitive. While the vasodilatory (A 2) d o s e - r e s p o n s e curves were parallel, the maxima achieved after 8-PT were less than those reached before the antagonist; thus the nature of the antagonism by 8-PT at A 2 receptors in vivo cannot be inferred from this data. Most importantly, the results of the present experiments clearly demonstrated that the novel adenosine
AI receptor-selective antagonist N-0861 attenuated only adenosine A~ receptor-mediated bradycardia, and preserved adenosine A 2 receptor-mediated vasodilation. The A 2 receptor-mediated vasodilatory responses were fully expressed even after the highest dose of N-0861 (100 / ~ m o l / k g i.v.). Thus, the A I receptorselective antagonistic action of N-0861 originally established in vitro (May et al., 1991; Belardinelli et al., 1990) was also apparent in vivo. When tested in vitro, N-0861 was a competitive antagonist at A 1 receptors (May et al., 1991). In the present in vivo studies, Hill coefficients and maxima were similar in the A I d o s e response curves obtained before and after N-0861. While recognizing the limitations of kinetic analyses made with in vivo data, we suggest that N-0861 competitively antagonized A~ adenosine receptor-mediated bradycardia in vivo as well. Having demonstrated the selectivity of N-0861 for adenosine A~ receptors, only antagonism of A j receptor-mediated bradycardia was measured when examining the pharmacodynamics of N-0861. Although repeated administration of sub-threshold doses (1 p~g/kg i.v.) of adenosine to anesthetized rats was reported to potentiate subsequent adenosine-induced bradycardia and hypotension (Vidrio et al., 1987), we consistently found that the bradycardic responses repeatedly evoked by a near maximal dose of adenosine (0.6 # m o l i.v.) remained unchanged over 3 h. That low doses of N-0861 (0.3-1 # m o l / k g i.v.) antagonized adenosine A~ receptor-mediated bradycardic responses for only 1 rain suggests that the compound may be metabolically inactivated a n d / o r excreted very rapidly. The dramatic prolongation of effect (from 12 to 150 rain) over a 10-fold increase in dose (from 1 to 10 / s m o l / k g ) suggests that N-0861 may not follow linear distribution a n d / o r excretion kinetics, or that it may be biotransformed to a longer-acting metabolite. Additional studies on the disposition, metabolism, and excretion of N-0861 are in progress. Since the lowest dose of N-0861 that antagonized Aj receptor-mediated bradycardia was 0.3 /~mol/kg, and no antagonism of A 2 receptors was evident following 100 ~ m o l / k g i.v., we conclude that N-0861 has at least 333-fold selectivity for A ~vs. A 2 receptors in vivo.
References
Belardinelli, L., J. Linden and R.M. Berne, 1989, The cardiac effects of adenosine, Prog. Cardiovasc. Dis. 32, 73. Belardinelli, L., H. Coleman and M. Boykin, 1990, A novel nonxanthine adenosine A~ receptor antagonist attenuates the negative dromotropic effects, but not the coronary dilation caused by adenosine and hypoxia in guinea pig hearts, Circulation 82, II]-150. Bohle, A., J. Christensen, F. Kokot, H. Osswald, B. Schubert, H. Kendziorra, H. Pressler and J. Marcovic-Lipkovski, 1990, Acute
16 renal failure in man: new aspects concerning pathogenesis, Am. J. Nephrol. 10, 374. Collis, M.G., 1989, The vasodilator role of adenosine, Pharmacol. Ther. 41, 143. Collis, M.G., D.B. Palmer and V.L. Saville, 1985, Comparison of the potency of 8-phenyltheophylline as an antagonist at A~ and A z adenosine receptors in atria and aorta from the guinea pig, J. Pharm. Pharmacol. 37, 278. Gustafsson, L.E., M. Persson, A. Ohlen and P. Hedqvist, 1988, Purines are regulators of skeletal muscle vascular tone, in: Adenosine and Adenine Nucleotides, Physiology and Pharmacology, ed. D.M. Paton (Taylor and Francis, New York) p. 289. Horn, G.J. and M.F. Lokhandwala, 1981, Presynaptic inhibition of vascular sympathetic neurotransmission by adenosine, Eur. J. Pharmacol. 69, 101. Jackson, E.K., 1991, Adenosine: A physiological brake on renin release, Ann. Rev. Pharmacol. Toxicol. 31, 1. Jacobson, K.A., B.K. Trivedi, P.C. Churchill and M. Williams, 1991, Novel therapeutics acting via purine receptors, Biochem. Pharmacol. 10, 1399. May, J.M., P.L. Martin and J.R. Miller, 1991, N-0861: A selective A I adenosine receptor antagonist, FASEB J. 5, A1573. Merrick, B.A. and T.L. Holcslaw, 1981, Peripheral vascular effects of atropine and methylatropine in the rat autoperfused hindlimbs preparation, J. Pharmacol. Exp. Ther. 218, 771. Mohrman, D.E. and L.J. Heller, 1984, Effect of aminophylline on
adenosine and exercise dilation of rat cremaster arterioles, Am. J. Physiol. 246, H592. Proctor, K.G., 1984, Reduction of contraction-induced artcriolar vasodilation by adenosine deaminase or theophylline, Am. J. Physiol. 247, H195. Proctor, K.G. and I Stojanov, 1990, Adenosine-mediated vasoconstriction in the skin microcirculation, in: Purines in Cellular Signaling, Targets for New Drugs, eds. K.A. Jacobson, J.W. Daly and V. Manganiello (Springer-Verlag, New York) p. 108. Rail, T.W., 1990, Drugs used in the treatment of asthma, in: The Pharmacological Basis of Therapeutics, 8th edn., eds. A.G. Gilman, T.W. Rail, A.S. Nies and P. Taylor (Pergamon, New York) p. 618. Sakai, K., M. Akima and H. Matsushita, 1979, Femoral vascular response to purine and pyrimidine derivatives: release of 5-hydroxytryptamine by purine derivatives in isolated, cross-circulated rat hindlimb, Jap. J. Pharmacol. 29, 243. Sollevi, A., 1986, Cardiovascular effects of adenosine in man; possible clinical implications, Prog. Neurobiol. 27, 319. Verani, M.S., J.J. Mahmarian, J.B. Hixson, T.M. Boyce and R.A. Staudacher, 1990, Diagnosis of coronary artery disease by controlled coronary vasodilation with adenosine and thallium-201 scintigraphy in patients unable to exercise, Circulation 82, 80. Vidrio, H., F. Garcia-Marquez and G.A. Magos, 1987, Repeated administration of adenosine increases its cardiovascular effects in rats, Eur. J. Pharmacol. 133, 341.
9
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52450
N-0861 selectively antagonizes adenosine A 1 receptors in vivo R i c h a r d J. B a r r e t t , D a v i d A. D r o p p l e m a n a n d K a t h r y n F. W r i g h t Department of Pharmacology, Whitby Research, Inc., P.O. Box 27426, Richmond, VA 23261-7426, USA Received 12 December 1991, revised MS received 7 February 1992, accepted 25 February 1992
Experiments were performed to determine the antagonistic actions of (_+)N6-endonorbornan-2-yl-9-methyladenine (N-0861) at A 1 and A 2 adenosine receptors in vivo, and to evaluate the pharmacodynamics of the observed responses. The selectivity of antagonism of A 1 vs. A 2 receptors by N-0861 was evaluated by generating dose-response curves to adenosine-induced bradycardia (A 1 effect), and vasodilation in the in situ constant-flow perfused rat hindquarter vasculature (A 2 effect). N-0861, at doses >/1 /~mol/kg + 0.04 p~mol/kg per min, i.v. produced dose-related rightward shifts of the A 1 dose-response curve, but had no effect on the A 2 dose-response curve at doses as high as 100/zmol/kg, i.v. In contrast, the non-selective A 1 / A 2 adenosine receptor antagonist 8-phenyltheophylline antagonized both A~ and A 2 receptor-mediated responses to adenosine. The minimum effective i.v. dose and the duration of action of N-0861 were determined by evoking bradycardic responses to i.v. adenosine (A 1 effect) in anesthetized, vagotomized,/3-blocked rats before and after single bolus doses of vehicle or N-0861 (0.3, 0.6, 1.0, 3.0 or 10.0 ~mol/kg). The lowest i.v. dose of N-0861 to antagonize A1 receptor-mediated bradycardia was 0.3 ~mol/kg i.v.; the duration of effect ranged from 1 min (following 0.3-1 /~mol/kg) to approximately 2.5 h (following 10 /zmol/kg). N-0861 is a selective (by >/333-fold) antagonist of adenosine A 1 receptors. Adenosine; Adenosine A 1 receptors; Adenosine A 1 receptor antagonists
1. Introduction
Stimulation of adenosine A~ and A2 receptors with the non-selective agonist adenosine has found direct clinical utility in the treatment of supraventricular tachycardia (A 1 effect) (Belardinelli et al., 1989), and simulation of exercise during myocardial perfusion imaging studies ( A 2 effect) (Verani et al., 1990). In spite of these beneficial clinical applications, inappropriate stimulation of A 1 a n d / o r A 2 receptors may be undesirable, and may even be pathogenic in cardiac conduction disorders (Belardinelli et al., 1989) and renal failure (Bohle et al., 1990). The only clinically approved adenosine receptor antagonists are the xanthines, most of which antagonize both A~ and A 2 receptors, and also inhibit phosphodiesterases and adenosine uptake (Rall, 1990). Selective agonists and antagonists that do not inhibit phosphodiesterase activity or adenosine uptake may have therapeutic utility in a number of disorders; recent progress in this area has been reviewed (Jacobson et al., 1991).
Correspondence to: R.J. Barrett, Department of Pharmacology, Whitby Research, Inc., P.O. Box 27426, Richmond, VA 23261-7426, U.S.A. Tel. 1.804.254 4072, fax 1.804.254 4038.
In a series of in vitro assays, N-0861 (( _+)N 6-endo norbornan-2-yl-9-methyladenine) was identified as a selective A~ adenosine receptor antagonist that has little affinity for other receptors, and little effect on phosphodiesterases and adenosine uptake (May eta[., 1991; Belardinelli et al., 1990). Studies were performed to characterize the adenosine A 1 vs. A z subtype specificity of N-0861 in vivo, and to determine the preclinical pharmacodynamics of this novel adenosine antagonist.
2. Materials and methods
2.1. General Drug-naive, male Sprague-Dawley rats (Hilltop Farms, Scottdale, PA) were anesthetized with urethane (1.5 g / k g i.p.), and kept on a Harvard heated operating board to maintain the body t e m p e r a t u r e at 37°C. A ventral cervical incision was made, and the trachea, jugular vein, carotid artery, and both vagus nerves were isolated. Polyethylene catheters were inserted into the trachea, carotid artery and jugular vein, and the vagi were sectioned. Systemic blood pressure was measured from the carotid arterial catheter with a Statham Gould
10 P23XL or Sorenson Transpac II pressure transducer. H e a r t rate was derived from the blood pressure signal with a Grass 7P4 tachograph. Signals were displayed and recorded on a Grass Model 7E or P79D polygraph. The rats were then subjected to one of two experimental protocols designed to determine (1) the selectivity of antagonism of A~ vs. A 2 adenosine receptors, or (2) the dose relation and duration of adenosine A~ receptor antagonism by N-0861.
2.2. Selectit,ity of adenosine A 1 us. A 2 receptor antagonism 2.2.1. Surgical procedure After completing the general surgical preparation described above, 29 rats (mean _+ S.E.M. weight 387 _+ 5.0 g) were further p r e p a r e d by methods adapted from Merrick and Holcslaw (1981). A midline abdominal incision was made, and a 2.5-3.0 cm segment of the abdominal aorta between the left renal artery and the posterior aortic bifurcation was exposed and dissected free from the vena cava. Three ligatures were placed around the aorta, and Na + heparin (125 U i.v.) was administered. The center ligature was tightened, and the distal aorta was cannulated with one end of the perfusion tubing. In order to minimize the time ( < 1 min) that the hindquarter vasculature was deprived of blood, the carotid arterial cannula was attached via a t-tube to allow arterial blood to perfuse the hindquarters. The proximal aorta was then cannulated, the perfusion tubing was unclamped, and blood was allowed to flow through the tubing. At this time, with autoperfusion to the hindquarters restored, the carotid arterial cannula was disconnected from the circuit. The abdominal wound was closed with stainless steel wound clips. The perfusion tubing was filled with 2.0 ml heparinized (50 U / m l ) 6% dextran 70. A small length of latex tubing and a plastic t-tube were introduced distal to the p u m p to allow intra-arterial injection of drugs and m e a s u r e m e n t of hindquarter perfusion pressure (mmHg), respectively. Hindquarter perfusion pressure was measured with a Statham Gould P23XL or Sorenson Transpac II pressure transducer, and the signal was displayed as described above. 2. 2. 2. Experimental procedure Respiration was maintained (70 s t r o k e s / m i n , 2.5 ml) with a Harvard rodent ventilator. The perfusion tubing was inserted into an Isco W I Z peristaltic p u m p (Lincoln, NE) and blood was directed distally into the hindquarter vascular bed. The flow rate was set between 4 and 6 m l / m i n and norepinephrine (3-19 n m o l / m i n ; mean ( ± S . E . M . ) 11.0 _+ 1.0 n m o l / m i n ) w a s infused directly into the hindquarter vasculature via the injection tubing to maintain perfusion pressure at
115-135 mmHg. The effect of norepinephrine was limited to the hindquarters; no effects on systemic blood pressure or heart rate were observed. Once set, the p u m p rate and norepinephrine infusion rate were not altered during the course of the experiment. When perfused at constant flow, vasodilation in an isolated vascular bed is manifested as a decrease in perfusion pressure. The maximal vasodilatory capacity of the hindquarter vascular bed of each rat was established by evoking maximal endothelium-dependent and endothelium-independent vasodilatory responses with simultaneous i.a. injection of acetylcholine (0.06/xmol) and nitroglycerin (0.06 txmol). A d e n o s i n e A2 receptor-mediated vasodilatory responses were produced by i.a. injections of adenosine (0.006-0.6 tzmol in 20 p.l). These (A 2) responses were expressed as a percent of the maximal vasodilatory capacity. Adenosine A~ receptor-mediated bradycardic responses were evoked by injections of adenosine (0.1-6 #tool in 100 ill) into the jugular vein. These (A 1) responses were expressed as percents change from basal heart rate preceeding the adenosine challenge. D o s e - r e s p o n s e curves to adenosine were obtained in this manner prior to administration of N-0861. Because of its short duration of action, three of the four doses of N-0861 were administered as bolus followed by infusion (1 / x m o l / k g + 0.04 / x m o l / k g per rain, 10 t x m o l / k g + 0 . 4 / ~ m o l / k g per min or 30 t z m o l / k g + 1.2 i x m o l / k g per min). Only one dose of N-0861 was administered to each animal. D o s e - r e s p o n s e curves for the A1 and A 2 responses were again generated during the infusion of N-0861. The highest dose of N-0861 tested, 100 i z m o l / k g i.v. was injected slowly as a bolus (over 2 min) and was not followed by infusion; o n l y A 2 responses were evoked in these animals. In order to demonstrate that adenosine-induced vasodilation in the in situ perfused hindquarters was indeed due to stimulation of adenosine receptors and that both A~ and A 2 receptor-mediated responses could be antagonized in the preparation, two additional groups of rats were treated with the non-selective adenosine receptor antagonist 8-phenyltheophylline (8-PT, 5 or 40 p.mol/kg, i.v. bolus (Collis et al., 1985)). Each animal received only one dose of antagonist. D o s e - r e s p o n s e curves to adenosine were generated before and > 5 rain following injection of 8-PT.
2.3. Pharmacodynamics of bolus doses of N-0861 2.3.1. Experimental procedure After they had stabilized following the surgical preparation described above (2.1.), 38 rats (mean_+ S.E.M. weight 356 _+ 4.4 g) were subjected to the following experimental protocol. Adenosine was administered through one jugular vein catheter, and a second catheter (in the same vein) was used for injection of
11 isoproterenol, atenolol and N-0861. Heart rate responses to i.v. isoproterenol (0.07 nmol) were measured before and periodically after atenolol (37.5 > m o l / k g i.v.) to ensure the endurance of /3-blockade. Adenosine A l receptor-mediated changes in heart rate evoked by repeated doses of 0.6 p~mol adenosine, i.v., were measured several times before and periodically after N-0861 or vehicle. N-0861 (0.3, 0.6, 1, 3 or 10 /.tmol/kg) or vehicle (pH 2.42 Na + phosphate buffer) was administered i.v. (over 30 s) at time 0; the adenosine challenges were continued for 30 min following 0.3, 0.6 and 1 /.tmol/kg N-0861, and for 3 h following administration of 3 and 10 # m o l / k g N-0861. Only one dose of N-0861 was administered to each animal. Data have been expressed as % change from the basal heart rate immediately prior to injection of adenosine.
Davis, Morris Plains, N J; all other compounds, Sigma, St. Louis, MO.
2.5. Statistics In the adenosine A l vs. A 2 selectivity experiments, the negative chronotropic and hindquarter vasodilatory responses to individual doses of adenosine generated prior to and after N-0861 were paired, and were therefore analyzed with a paired, 2-tailed t-test. The d o s e response curves representing the A l and A 2 responses to adenosine evoked in each animal before vs. after N-0861 or 8-PT were analyzed with a computerized non-linear regression analysis program (GraphPad InPlot 3.1; SanDiego, CA). Fitting the data to sigmoid curves facilitated calculation of EDs0 values for both the A I responses (the dose of adenosine required to reduce heart rate 50% from control) and the A 2 responses (the dose of adenosine required to produce 50% of the maximum vasodilatory response to adenosine). The EDs0 values for the A l and A 2 responses obtained in each animal prior to antagonist were compared with those obtained after antagonist treatment with a paired, 2-tailed t-test. To determine if the control (pre-antagonist) A I and A 2 responses evoked in rats from all four N-0861-treated groups could be pooled for illustration, the data were subjected to one-way analysis of variance. In the pharmacodynamic experiments, the percents change in heart rate evoked by adenosine before N0861 or vehicle treatment between the six groups were subjected to one-way analysis of variance. The percents change in heart rate evoked by adenosine at each time point in each treatment group were analyzed by one way analysis of variance and Dunnett's test, comparing
2.4. Drugs N-0861 was dissolved in 0.1 M N a H 2 P O 4 buffer acidified with 1-1.5 mEq of HC1 for each mEq of N-0861. The vehicle was prepared by adjusting the pH of a 0.1 M N a H z P O 4 buffer solution to 2.42, the pH of the most acidic solution of N-0861. Adenosine, atenolol, isoproterenol, norepinephrine, acetylcholine and nitroglycerin were dissolved in 0.9% NaC1; stock solutions of adenosine and atenolol required slight acidification for dissolution. 8-PT was dissolved in 50% (v/v) P E G / 0 . 1 N N a O H (pH 12); pilot studies showed that the vehicles did not influence the magnitude of the responses to adenosine. Sources of compounds: N-0861, Whitby Research, Inc., Richmond, VA; 8-PT, Research Biochemicals, Natick, MA; nitroglycerin, Parke PERCENT DECREASEFROM CONTROL HEART RATE
IO0
PERCENT MAXIMUM VASODILATORY CAPACITY
100 /
80
Control o 1 lamol/kg + • 0.04 lamol/kg per min
60
10 tamol/kg + ' 0.4 ~mol/kg per rain
40 20
30 gtmol/kg + •
~
1.2 gmol/kg per m
i
\ ~
.-r
.~'"
~'~" m"~
60
~1'...,/
40
10
~ 20
0
I 9
8
1-
I
7
6
DOSE ADENOSINE (-log mol)
0 5
~7 9
8
I
I
7
6
5
DOSE ADENOSINE (-log tool)
Fig. 1. The effect of N-0861 on the bradycardic (A1) responses to i.v. adenosine and vasodilatory (A2) responses to i.a. adenosine. Data are expressed as means+S.E.M. Control curves represent data pooled from all treatment groups prior to administration of N-0861. Left panel. Bradycardic responses are expressed as percent decrease from resting heart rate evoked by i.v. adenosine. Each A~ dose-response curve was significantly different from its paired control curve. Right panel. Vasodilatory responses evoked by i.a. adenosine are expressed as percent of the maximal vasodilatory capacity of the perfused hindquarters. N-0861 had no significant effect on A 2 responses to adenosine. Symbols are as illustrated in left panel; A l responses were not evoked in rats treated with 100/xmol/kg N-0861.
12 r e s p o n s e s from each of the five N-0861 t r e a t m e n t groups to responses of v e h i c l e - t r e a t e d rats at the same time point. I n all analyses, statistical significance was assigned w h e n P < 0.05.
3. Results 3.1. Selectivity o f adenosine A 1 t,,s. A 2 receptor antagonism Vasodilatory r e s p o n s e s to i.a. a d e n o s i n e were expressed as a p e r c e n t of the m a x i m u m vasodilatory capacity of the h i n d q u a r t e r s . I n the 29 rats tested, nitroglycerin + acetylcholine d e c r e a s e d p e r f u s i o n pressure from ( m e a n + S.E.M.) 126.8 _+ 1.70 to 70.7 _+ 1.61 m m H g , yielding a m e a n m a x i m u m vasodilatory capacity of 56.0 _+ 1.94 m m H g . W h e n injected i.a. into the p e r f u s i o n tubing, a d e n o sine p r o d u c e d an i m m e d i a t e drop in h i n d q u a r t e r perfusion pressure; b r a d y c a r d i a was d e t e c t e d only at doses of a d e n o s i n e >1 0.6 ~ m o l , a n d only after a n obvious delay of 1 0 - 1 5 s. I.v. a d m i n i s t e r e d a d e n o s i n e evoked an i m m e d i a t e bradycardic (A~) response, while the vasodilatory ( A 2) response was expressed in the perfused h i n d q u a r t e r s only after a 1 0 - 1 5 s delay. T h e bradycardic ( A 1) a n d vasodilatory ( A 2) responses to a d e n o s i n e in rats with in situ p e r f u s e d h i n d q u a r t e r s were dose-responsive a n d r e p r o d u c i b l e (figs. 1, 2). Doses of 0 . 1 - 3 p.mol (i.v.) a d e n o s i n e were r e q u i r e d to construct A 1 r e c e p t o r d o s e - r e s p o n s e curves, while only 0.006-0.6 ~ m o l (i.a.) a d e n o s i n e were r e q u i r e d to o b t a i n d o s e - r e s p o n s e curves for the A 2 r e c e p t o r - m e d i a t e d vasodilation. Both the bradycardic a n d vasodilatory r e s p o n s e s to a d e n o s i n e o b t a i n e d before N-0861 t r e a t m e n t were similar in all groups of rats ( c o m p a r e EDs0 values in table 1), a n d have b e e n pooled for illustration. T h e bradycardic (A~) a n d vasodilator (A 2) responses to a d e n o s i n e evoked before
100' 80'
TABLE 1 EDs0 values (means_+S.E.M.) for the A~ (bradycardic) and A 2 (vasodilatory) receptor-mediated responses to adenosine before (control) and following N-0861. Treatment group
A Eresponse EDs0 ( - log tool)
A 2 response EDs0
Control (n = 5) N-0861. 1 /xmol/kg+ 0.04/xmol/kg per min
6.55 + 0.063 6.16+0.060 P < 0.05 ~'
7.29 + 0.102 7.42+0.107 ns
Control (n = 4) N-0861, 10 #mol/kg+ 0.4/~mol/kg per rain
6.42+_0.119 5.79_+0.008 P < 0.05 "
7.31 +0.121 7.18_+0.044 ns
Control (n = 4) N-0861, 30/xmol/kg + 1.2 p.mol/kg per min
6.63 _+0.030 5.68+0.051 P < 0.05 a
7.19 _+0.077 7.11 +0.061 ns
Control (n = 5) N-0861, 100/xmol/kg
NT NT
7.37 + 0.054 7.43_+0.057 ns
Pooled controls
6.53 _+0.005 (n = 13)
7.29 _+0.044 (n = 18)
a VS. EDso values derived from responses evoked before N-0861 (paired, two-tailed t-test); ns = no significant difference; NT = not tested).
8-PT were also similar in rats that received 5 or 40 ~ m o l / k g 8-PT; these control data also have b e e n p o o l e d (table 2, fig. 2). T h e stability a n d reproducibility of both A I (bradycardic) a n d A 2 (vasodilatory) responses is obvious in the similarities of the control curves in figs. 1 a n d 2 a n d in their respective EDso values (tables 1, 2). D o s e - r e s p o n s e curves to both the A 1 a n d A 2 recept o r - m e d i a t e d actions of a d e n o s i n e were c o n s t r u c t e d in three groups of rats prior to, a n d following N-0861; only A 2 responses were elicited in the r e m a i n i n g group of rats t r e a t e d with 100 / x m o l / k g N-0861. T h e c h r o n o t r o p i c (A 1) responses to individual i.v. doses of
PERCENT DECREASE FROM CONTROl HEART RATE
PERCENT MAXIMUM VASODILATORY CAPACITY
100"T Control 0 80 Jr 5 I.imollkg [] ~
Control o5 lamol/kg [] 40 Iamol/kg 0
60
60" 40'
( - log tool)
-~ \
t 40 gmol/kgO ~ ~ S s~ 5 " ~ , ~ , ~
40 1
20 0
9
i 8
7
DOSE A DENOSINE (-log mol)
6
5
0
9
8
t
7
i
6
5
DOSE ADENOSINE (-log tool)
Fig. 2. The effect of 8-phenyltheophylline (8-PT) on the bradycardic (A 1) responses to i.v. adenosine and vasodilatory (A 2) responses to i.a. adenosine. Data are expressed as means_+S.E.M. Control curves represent data pooled from both treatment groups prior to administration of 8-PT. Left panel. Bradycardic responses are expressed as percent decrease from resting heart rate evoked by i.v. adenosine. Both doses of 8-PT significantly attenuated the A I responses to adenosine. Right panel. Vasodilatory responses evoked by i.a. adenosine are expressed as percent of the maximal vasodilatory capacity of the perfused hindquarters. Unlike N-0861, 8-PT significantlyantagonized the A 2 responses to adenosine.
13 TABLE 2 EDs0 v a l u e s ( m e a n s i S . E . M . ) for the A I ( b r a d y c a r d i c ) a n d A 2 (vasodilatory) r e c e p t o r - m e d i a t e d r e s p o n s e s to a d e n o s i n e b e f o r e (control) and following 8 - p h e n y l t h e o p h y l l i n e (8-PT). Treatment group
A l response EDs0 ( - log mol)
A 2 response EDs0 ( - log tool)
C o n t r o l (n = 5) 8-PT, 5/~ m o l / k g
6.5 _+0.05 5.9 ± 0.03 P < 0.01 ~'
7.3 + 0.09 6.5 ± 0.24 P < 0.05 "
C o n t r o l (n = 6) 8-PT, 4 0 / z m o l / k g
6.5 ± 0.07 5.5 + 0.07 P < 0.01 '~
7.4 +_0.06 5.8 +_0.21 P < 0.05 "
P o o l e d controls (n = 11 )
6.5 + 0.04
7.3 ± 0.05
significant, dose-related, rightward shifts of the d o s e response curves representing both the A I (bradycardic) and A 2 (vasodilatory) responses to adenosine (fig. 2, table 2). Antagonism of A~ receptor-mediated bradycardia by 8-PT appeared to be competitive, but the data do not permit such a conclusion for the A 2 receptor-mediated responses.
3.2. Pharmacodynamics of bolus doses of N-0861
~' vs. ED50 v a l u e s d e r i v e d from r e s p o n s e s e v o k e d b e f o r e 8-PT (paired, t w o - t a i l e d t-test).
0.1, 0.3, 0.6 and 1 /xmol adenosine were significantly attenuated (P < 0.05) by all doses of N-0861, while the response to 3 / z m o l i.v. adenosine was attenuated only in the group treated with the 30 / x m o l / k g + 1.2 /zmol/kg per min dose of N-0861. N-0861 produced significant, dose-related, rightward and parallel shifts of the adenosine A~ (bradycardic) dose-response curves, but had no effect on the adenosine A 2 (vasodilatory) dose-response curves (fig. 1). The EDs0 values derived for the adenosine dose-response curves were significantly changed by each dose of N-0861 (table 1). These results provide in vivo evidence for the adenosine A 1 receptor selectivity of N-0861, and suggest that N-0861 antagonizes A I receptor-mediated bradycardia in a competitive manner. In contrast to animals treated with N-0861, treatment with the non-selective A t / A 2 receptor antagonist 8-PT (5 /zmol/kg or 40 /zmol/kg, i.v.) produced
In order to evoke a bradycardic response that would be due to only adenosine A I receptor stimulation, autonomic control of the heart was ablated by bilateral vagotomy and the/3~-adrenoceptor-selective antagonist atenolol. The mimimum effective i.v. dose and the duration of A~ receptor antagonism by N-0861 was evaluated by comparing the bradycardic responses to adenosine (A~ effect) in rats treated with N-0861 to adenosine responses evoked in rats treated with vehicle. Separate groups of five to seven rats were treated with the vehicle or with single bolus doses of N-0861. Basal heart rates and responses to adenosine (0.6/xmol i.v.) evoked 5 times prior to treatment with vehicle or N-0861 were similar in all groups (table 3). Neither the vehicle, N-0861, nor repeated administration of adenosine altered the basal heart rate. Before administration of vehicle or N-0861, adenosine reduced heart rate by approximately 44%, from 327 to 185 beats/rain (average data from table 3). These adenosine A~ receptormediated bradycardic responses were stable over 3 h in vehicle-treated rats (fig. 3). The very slight enhancement of the bradycardic responses seen after vehicle injection was not statistically significant. The duration and degree of adenosine A j receptor
TABLE 3 R e s t i n g h e a r t r a t e s and r e s p o n s e s to i.v. a d e n o s i n e prior to N-0861 or vehicle. T h e basal ( c o n t r o l ) h e a r t r a t e s and r e s p o n s e s to a d e n o s i n e w e r e similar in each t r e a t m e n t g r o u p p r i o r to a d m i n i s t r a t i o n of vehicle or N-0861. T h e effects of five successive a d e n o s i n e injections (0.6 p~mol i.v.) a d m i n i s t e r e d 3 min a p a r t have b e e n a v e r a g e d . D a t a in ' % c h a n g e ' c o l u m n are r e p r e s e n t e d as ' P r e - N - 0 8 6 1 ' in fig. 3. Treatment group
Basal (beats/min)
Adenosine (beats/rain)
Change (beats/min)
% change
n
Vehicle
Mean ± S.E.M.
311.5 11.95
177 27.5
- 134.5 24.07
- 43.7 7.9
6
0.3/xmol/kg
Mean ±S.E.M.
308 14.26
190.4 27.22
- 117.8 14.72
- 39.3 6.2
5
0.6/xmol/kg
Mean ± S.E.M.
337.8 5.47
182.5 18.37
- 155.33 17.76
- 46.1 5.3
6
1 /zmol/kg
Mean ± S.E.M.
339.6 6.29
177 20.06
- 162.14 15.05
- 48.2 5.0
7
3/zmol/kg
Mean ± S.E.M.
345.7 9.25
181.1 15.79
- 117.14 49.95
- 47.4 4.5
7
10/xmol/kg
Mean ± S.E.M.
319.7 6.64
199.9 17.32
- 120.00 14.97
-37.7 4.8
7
14
l~mol/kg
%
PreN-0861
1
3
6
9
12
15 3 0 4 5
60
90
t20
150
~BO
90
t20
150
180
90
:l;~O
150
1BO
90
120
150
180
90
~120
150
180
TIME (min)
0.6 ,mo,/kg
%
PreN-0861
1
PPeN-OB6:I
1
3
6
9
t2
15 3 0
45 60
TIME (rain)
%
3
6
9
t2
15 3 0
45
60
TIME (min)
3 #mol/kg
%
PreN-0861
:t
PreN - 0 8 6 :l
1
3
6
9
12
15 3 0 4 5
60
TIME (min)
3
6
9
12
15 3 0 4 5
50
TIME (min)
Fig. 3. Duration of antagonism by N-0861 of A t bradycardic responses to i.v. adenosine. Bradycardic responses (expressed as percents decrease from basal heart rate) evoked by i.v. administration of 0.6 ~mol adenosine before and at the indicated times after bolus doses of N-0861 (0.3, 0.6, 1, 3 or 10 # m o l / k g i.v., shaded bars) in anesthetized, vagotomized, /3-blocked rats (n = 5-7/group). All data are expressed as means_+ S.E.M. Data from vehicle-treated rats (n = 6) is represented by open bars in each panel. Note that, for illustration's sake, S.E.M. for the vehicle-treated group is presented only in the bottom panel, and that the abscissa is not linear over time. ~' P < 0.01; b p < 0.05, Dunnett's test vs. vehicle-treated rats at the same time period.
a n t a g o n i s m b y N - 0 8 6 1 is p r e s e n t e d T h e l o w e s t i.v. d o s e o f N - 0 8 6 1 t o nize the bradycardic response to /~mol/kg. The three lowest doses
g r a p h i c a l l y in fig. 3. significantly antagoa d e n o s i n e w a s 0.3 o f N - 0 8 6 1 (0.3, 0.6
a n d 1.0 ~ m o l / k g ) w e r e e f f e c t i v e f o r o n l y 1 m i n , w h i l e t h e h i g h e r d o s e s (3 a n d 10 > m o l / k g ) significantly a t t e n u a t e d t h e b r a d y c a r d i c ( A ~ ) r e s p o n s e s f o r 12 a n d 150 m i n , r e s p e c t i v e l y .
15 4. Discussion
Adenosine reduces systemic blood pressure by simultaneously activating both A I and A 2 adenosine receptor-dependent vasodepressor mechanisms: direct relaxation of vascular smooth muscle, reduction of cardiac rate and output, inhibition of renin release and attenuation of sympathetic drive (Collis, 1989; Belardinelli et al., 1989; Jackson, 1991). Because of these diverse and interrelated actions, we considered it essential to separate completely the vasodilatory responses to adenosine A 2 receptor stimulation from other concurrent adenosine-induced changes in hemodynamics. This was achieved in rats with in situ perfused hindquarters by injecting adenosine i.v. to evoke A l receptor-mediated bradycardic responses, and i.a. to evoke A 2 receptor-mediated vasodilatory responses. This is the first report to demonstrate that stimulation of adenosine A 2 receptors causes vasodilation in the in situ perfused rat hindquarters preparation. Sakai et al. (1979) reported that i.a. injection of adenosine into a cross-perfused isolated rat hindlimb preparation caused dose-related vasoconstriction that was attributable to release of 5-hydroxytryptamine. In these studies in the in situ perfused rat hindquarters, adenosine and specific A 2 receptor agonists consistently produced only vasodilation - never vasoconstriction. Adenosine may cause vasoconstriction in vivo in the kidney (Jackson, 1991) and skin (Proctor and Stojanov, 1990), but stimulation of adenosine A 2 receptors causes only vasodilation in skeletal muscle of the dog (Horn and Lokhandwala, 1981), rat (Mohrman and Heller, 1984), rabbit (Gustafsson et al., 1988), hamster (Proctor, 1984) and man (Sollevi, 1986). It is likely that the vasodilatory responses to i.a. adenosine in the present studies were mediated by A 2 receptors in the vasculature supplying the skeletal muscle of the hindquarters. The non-selective adenosine receptor antagonist 8-PT (Collis et al., 1985) antagonized both the bradycardic (A~) and the vasodilatory (A 2) responses to adenosine in rats with in situ perfused hindquarters, indicating that both responses were indeed evoked by adenosine receptor stimulation, and that antagonism of both A l and A 2 receptor-mediated responses could be reliably detected in this preparation. The A~ (bradycardic) d o s e - r e s p o n s e curves to adenosine had similar slopes and maxima before and after 8-PT, suggesting - as had been observed in vitro - that the nature of A~ antagonism by 8-PT was competitive. While the vasodilatory (A 2) d o s e - r e s p o n s e curves were parallel, the maxima achieved after 8-PT were less than those reached before the antagonist; thus the nature of the antagonism by 8-PT at A 2 receptors in vivo cannot be inferred from this data. Most importantly, the results of the present experiments clearly demonstrated that the novel adenosine
AI receptor-selective antagonist N-0861 attenuated only adenosine A~ receptor-mediated bradycardia, and preserved adenosine A 2 receptor-mediated vasodilation. The A 2 receptor-mediated vasodilatory responses were fully expressed even after the highest dose of N-0861 (100 / ~ m o l / k g i.v.). Thus, the A I receptorselective antagonistic action of N-0861 originally established in vitro (May et al., 1991; Belardinelli et al., 1990) was also apparent in vivo. When tested in vitro, N-0861 was a competitive antagonist at A 1 receptors (May et al., 1991). In the present in vivo studies, Hill coefficients and maxima were similar in the A I d o s e response curves obtained before and after N-0861. While recognizing the limitations of kinetic analyses made with in vivo data, we suggest that N-0861 competitively antagonized A~ adenosine receptor-mediated bradycardia in vivo as well. Having demonstrated the selectivity of N-0861 for adenosine A~ receptors, only antagonism of A j receptor-mediated bradycardia was measured when examining the pharmacodynamics of N-0861. Although repeated administration of sub-threshold doses (1 p~g/kg i.v.) of adenosine to anesthetized rats was reported to potentiate subsequent adenosine-induced bradycardia and hypotension (Vidrio et al., 1987), we consistently found that the bradycardic responses repeatedly evoked by a near maximal dose of adenosine (0.6 # m o l i.v.) remained unchanged over 3 h. That low doses of N-0861 (0.3-1 # m o l / k g i.v.) antagonized adenosine A~ receptor-mediated bradycardic responses for only 1 rain suggests that the compound may be metabolically inactivated a n d / o r excreted very rapidly. The dramatic prolongation of effect (from 12 to 150 rain) over a 10-fold increase in dose (from 1 to 10 / s m o l / k g ) suggests that N-0861 may not follow linear distribution a n d / o r excretion kinetics, or that it may be biotransformed to a longer-acting metabolite. Additional studies on the disposition, metabolism, and excretion of N-0861 are in progress. Since the lowest dose of N-0861 that antagonized Aj receptor-mediated bradycardia was 0.3 /~mol/kg, and no antagonism of A 2 receptors was evident following 100 ~ m o l / k g i.v., we conclude that N-0861 has at least 333-fold selectivity for A ~vs. A 2 receptors in vivo.
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