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PD 116,948, a highly selective A1 adenosine receptor antagonist
Life Sciences, Vol. 40, pp. 555-561 Printed in the U.S.A.
Pergamon Journals
PD 116,948, A HIGHLY SELECTIVE Al ADENOSINE RECEPTORANTAGONIST. S.J. Haleen, R.P. Steffen, and H.W. Hamilton + + Department of Pharmacology and Chemistry Warner-Lambert/Parke-Davis Pharmaceutical Research Ann Arbor, Michigan 48105 (Received in final form November 3, 1986)
Summary (R)-N-(1-Methyl-2-phenylethyl)adenosine (R-PIA), an adenosine receptor agonist has both negative chronotropic a c t i v i t y and coronary vasodilator a c t i v i t y . These actions of R-PlA are proposed to be mediated by subtypes (A~ and A2) of adenosine receptors. PD 116,948 is a xanthine d e r i v a t i v e which is a highly selective A I adenosine receptor ligand. In t h i s study PD 116,948 s e l e c t i v e l y antagonized the negative chronotropic a c t i v i t y of R-PIA in the isolated rat heart. These results are consistent with, and add f u r t h e r support to the hypothesis that adenosine receptor agonists mediate t h e i r negative chronotropic a c t i v i t y via A~ receptors and t h e i r vasodilator a c t i v i t y via A2 receptors. Key Words: Adenosine, AI Adenosine Receptor Antagonist, Heart Rate, Coronary Flow, Isolated Rat Heart, (R)-N-(1-Methyl-2phenylethyl)adenosine, 8 - C y c l o p e n t y l - 3 , 7 - d i h y d r o - l , 3 - d i p r o p y l 1H-purin-2,6-dione. Two subclasses of adenosine receptors have been distinguished based on receptor mediated i n h i b i t i o n (Az receptors) or stimulation (A2 receptors) of i n t r a c e l l u l a r cAMP accumulation ( i ) . Biological responses to adenosine receptor agonists have been put f o r t h as A~ or A2 mediated based on comparisons between receptor binding a f f i n i t i e s and rank order of biological potencies (2-4). Reliance on this technique was necessary due to the lack of an appropriate adenosine receptor antagonist. An appropriate antagonist would be free of i n t r i n s i c a c t i v i t y ( i n a c t i v e in the absence of an adenosine receptor agonist) and would be highly potent and selective for one of the adenosine receptor subtypes. PD 116,948, 8 - c y c l o p e n t y l - 3 , 7 - d i h y d r o - l , 3 - d i p r o p y l - l H - p u r i n - 2 , 6 - d i o n e , (FIG. 1) has been shown to be a highly selective A~ receptor ligand in rat brain (5,6). I t is a xanthine d e r i v a t i v e , the prototype structure for adenosine antagonists. This compound follows known s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s for xanthine binding to adenosine receptors (7). For example, this 1,3-dipropyl xanthine exhibits greater a f f i n i t y than the corresponding 1,3-dimethyl analog, 8-cyclopentyltheophylline (Az receptor ICso of 0.69 nM vs ICso of 20 nM, and A2 receptor ICso of 502 nM vs IC5o of 2000 nM) (values provided by Bruns et al, (5)).
Copyright
0024-3205/87 $3.00 + .00 (c) I987 Pergamon Journals Ltd.
556
Selective A 1 Adenosine Antagonist
Vol. 40, No. 6, 1987
0
FIG. i PD 116,948 8-cyclopentyl-3,7-dihydro-l,3-dipropyl-lH-purin-2,6-dione Adenosine receptor agonists are known to decrease heart rate and to increase coronary flow, effects which have been proposed to be A~ and A2 receptor mediated r e s p e c t i v e l y (8). In the present study, (R)-N-(1-methyl-2phenylethyl)adenosine (R-PIA) had near equimolar potency at decreasing heart rate and increasing coronary flow in the isolated r a t heart. I f the heart rate responses to R-PIA were A~ mediated, then PD 116,948 would be expected to block the heart rate response of R-PIA but not the coronary flow response. We tested t h i s hypothesis in the isolated Langendorff perfused r a t heart. Methods Sprague-Dawley normotensive rats were anesthetized with Na Pentobarbital (50 mg/kg, IP) and heparinized (2000 u n i t s , IP) to prevent blood c l o t t i n g . Hearts were r a p i d l y isolated and perfused by the Langendorff method (FIG. 2) with a modified Krebs Henseleit bicarbonate buffer of the following composition in mM concentration: NaCl, 127; NaHCO~, 25; dextrose, 5.5; Na Pyruvate, 2.0; KCl, 4.7; MgSO~, 1.1; KH 2PO~, 1.2; CaCl2.2H~O, 2.5; CaNa2 EDTA, 0.05. Perfusion pressure was maintained constant at 70 mmHg via a microcomputer controlled servo mechanism and the hearts were permitted to beat spontaneously. Because coronary perfusion pressure (CPP) was maintained constant, changes in coronary flow reflected changes in coronary resistance, The microcomputer controls CPP and R-PIA concentration. The level at which CPP and R-PIA concentration were maintained could be changed independently by commands communicated through the microprocessor keyboard. The adenosine receptor antagonist (PD 116,948) was added to the main oxygenation reservoir at a fixed concentration. R-PIA and PD 116,948 concentrates were prepared in NaOH solutions at pH 10 and 11 respectively. O n l y minute volumes were added to the perfusion medium which did not affect the pH (7.36-7.42) of the buffered solution. Control R-PIA dose response curves were compared to R-PIA dose response curves following pretreatment with PD 116,948 at concentrations of lx10-8, lx10-~, or lx10-6 molar. Heart rate was determined from the electrocardiogram and coronary flow was determined by calibrating pumps 1 and 2. Analog data was d i g i t i z e d and averaged with a microcomputer Buxco data analyzer. S t a t i s t i c a l analysis was performed by the Warner-Lambert/Parke-Davis Pharmaceutical Research Biometrics Department. Paired t tests were used to determine any s i g n i f i c a n t differences between control and treatment groups for basal heart rate and coronary flow values and for any differences in heart
Vol. 40, No. 6, 1987
Selective A 1 Adenosine Antagonist
557
T~
.
; o oo~ PC
L
" ....
f!
=. . . . ilill
I
=
MICROPROCESSOR
-
RECORDER
D~A ANALYZER
Isolated
Abbreviations:
PC,
Proportioning
FIG. 2 Langendorff
Circuit:
Heart
P1,
Model
Coronary
Perfusion
Pump;
P2,
Coronary Effluent Pump; P3, Drug Infusion Pump; T~, Perfusion Pressure Transducer; T2, Heart Chamber Pressure Transducer; DC, Drug Concentrate; M, Mixing Valve; OXY, Oxygenator; and ECG, Electrocardiogram Electrodes. Both hearts are perfused in parallel by PI which is controlled by the microprocessor to maintain coronary perfusion pressure constant independent of change in coronary resistance. Tz provides the pressure input signal to the microprocessor. Total coronary flow for both hearts is measured by recording the calibrated output signal from PI. Coronary flow for heart B is measured by recording the output signal from P2 which removes coronary effluent from the heart chamber at a rate equal to coronary flow via the servo mechanisms. T~ provides the column height pressure signal input to the microprocessor. Coronary flow for heart A is calculated from the difference between total and heart B coronary flow rates. PC provides an input signal to the microprocessor which is equal to the proportionate pumping rates for P~ and P~. The microprocessor then controls P3 to maintain the drug infusion rate proportional to total coronary flow. The M value mixes the DC with the physiological salt solution thereby assuring equal drug concentration for each heart which receives the treatment in parallel.
558
Selective A 1 Adenosine Antagonist
Vol. 40, No. 6, 1987
rate and coronary flow values between pre and post PD 116,948 treatment. To determine s i g n i f i c a n t differences between control R-PIA dose response curves and R-PIA dose response curves following treatment with PD 116,948 a common slope model was f i t to a range of responses over which the curves appeared to be p a r a l l e l . From the model, r e l a t i v e potency estimates were calculated with 95% confidence intervals for each PD 116,948 pretreated group r e l a t i v e to the R-PIA control. Results Basal heart rate and coronary flow values for each treatment group and the effects of PD 116,948 on heart rate and coronary flow are shown in Table I .
TABLE I Basal Heart Rate and Coronary Flow Parameters and the Effects of PD 116,948 on Heart Rate and Coronary Flow.
Control Heart Rate (beats/min)
278±11
IO-SM
IO-TM
Pre
Post
266±14
286±18
Pre 279±7
IO-~M Post
308±7*
Pre 251±11
Post 315±7"
Coronary Flow 14.2±0.8 14.1±1.1 14.3±1.2 14.8±1.4 14.6±1.2 11.6±1.1 12.4±0.8 (ml/min) Data represents the Mean ± SEM (p < 0.05, N=6) * S i g n i f i c a n t l y d i f f e r e n t from pretreatment There were no s i g n i f i c a n t d i f f e r e n c e s among treatment groups f o r e i t h e r basal heart rate or coronary f l o w values. PD 116,948 did not s i g n i f i c a n t l y a f f e c t basal coronary f l o w at any of the c o n c e n t r a t i o n s t e s t e d . Heart rate was s i g n i f i c a n t l y increased by PD 116,948 at lxlO-~M and 1xlO-6M. R-PlA caused dose dependent increases i n coronary f l o w and dose dependent decreases in heart rate ( c o n t r o l R-PIA curves FIG. 3). At 3x10-gM, R-PIA decreased heart rate s i g n i f i c a n t l y by 10% and at lx10-SM s i g n i f i c a n t l y increased coronary f l o w by 16%. However, at 3x10-SM R-PlA was about e q u a l l y potent on heart rate and coronary f l o w . At lxlO-~M, R-PIA had decreased heart rate by an average of 80% w i t h v e n t r i c u l a r a s y s t o l e o c c u r r i n g in one of the s i x hearts and a t r i a l a s y s t o l e o c c u r r i n g in two of the remaining f i v e hearts. The dose response curve was ended at ixlO-~M due to the above a r r h y t h m i a s . Coronary f l o w had increased an average of 59±8% at ixlO-TM R-PIA. Heart rate dose response to R-PIA was s i g n i f i c a n t l y s h i f t e d to the r i g h t by PD 116,948 in a dose r e l a t e d manner (ixlO-~M was s i g n i f i c a n t l y s h i f t e d to the r i g h t o f lx10 -7 and 1x10-SM). Comparing the amount of s h i f t at the 20% change from c o n t r o l l e v e l , PD 116,948 s h i f t e d the curve by about 1.5 log u n i t s at lxlO-SM, by 2.5 log u n i t s at 1x10-TM and by over 3.5 log u n i t s at lxlO-6M. Again using the 20% change from a c o n t r o l l e v e l , c a l c u l a t i o n s from a S c h i l d p l o t (9) give a PA2 of 9.44 w i t h a k i of 3.63xlO-Z°M and a slope equal to
Vol. 40, No. 6, 1987
Selective A 1 Adenosine Antagonist
I
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i
I
I
!
I
CORONARY
60 -
;
I
559
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FLOW
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-
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o o rr-" h
-
b
.
.
-
~
10-9 10-8 10-7 IG6 R-PIA (molar) t0-9
oo
10-8
,.
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,i~
10-7
10-6
10-5 t0-5
li
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-
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FIG. 3 Effects of adenosine receptor blockade by PD 116,948 on the R-PIA coronary flow (upper panel) and heart rate (lower panel) dose response curves (control - o) (PD 116,948 pretreated - =, ixlO-SM; A, ix10-~M; e, ixZO-6M). Points are mean values (N=6) (* s i g n i f i c a n t l y d i f f e r e n t from control, + s i g n i f i c a n t l y d i f f e r e n t from 1x10-SM and ixlO-TM PD 116,948, p < 0.05).
560
Selective A 1 Adenosine Antagonist
Vol. 40, No. 6, 1987
1.02. In contrast, PD 116,948 did not s i g n i f i c a n t l y s h i f t the coronary flow dose response. Discussion PD 116,948, a xanthine analogue, has selective adenosine Az receptor binding a f f i n i t y and s e l e c t i v i t y in causing dose dependent s h i f t s of the chronotropic effect of R-PIA to the right. These results show PD 116,948 to be a competitive and selective adenosine A~ receptor antagonist. PD 116,948 increased heart rate in a non-dose dependent manner by about 9% at i x l O -8 and IxlO-~M. This small e f f e c t could be due to the blockade of endogeneous adenosine which would be expected to have a negative c h r o n o t r o p i c effect. The e x p l a n a t i o n f o r the 25% increase in heart rate observed f o l l o w i n g 1x10-6M of PD 116,948 is not apparent and may be due to some n o n - s p e c i f i c e f f e c t s of the compound, i . e . , PDE i n h i b i t i o n , or release of catecholamines. PD 116,948 alone had no s i g n i f i c a n t e f f e c t on coronary f l o w . The a f f i n i t y of PD 116,948 for the A2 receptor in rat brain is about 5xlO-~M (5) and i t is possible that 1x10-6M was an i n s u f f i c i e n t concentration to e f f e c t i v e l y block any endogeneous adenosine effect in coronary arteries. R-PIA was an appropriate agonist to use for these studies because i t had nearly equal potency for both Az and A2 type a c t i v i t i e s in the isolated rat heart in which selective antagonism could be demonstrated within a single experiment. R-PIA s i g n i f i c a n t l y decreased heart rate at 3x10-gM and s i g n i f i c a n t l y increased coronary flow at 1xlO-SM. In rat brain the ICso a f f i n i t i e s of A~ and A2 for R-PIA are 1.2x10-gM and 1.2x10-TM respectively ( i 0 ) . Based on the binding results, i t might be expected that an Az mediated effect ( i . e . , heart rate) versus an A2 mediated effect ( i . e . , coronary flow) by R-PIA would be separated by a two log dose concentration difference. The greater than expected potency of R-PIA at increasing coronary flow has also been observed in guinea pig hearts (11) whereas in rabbit heart (8) the potency separation between heart rate and coronary flow effects are more closely correlated with receptor binding results (10). I t must be pointed out that the receptor binding a f f i n i t i e s are measured in purified rat brain microsomes and tissue differences in binding a f f i n i t i e s and access to the receptors may d i f f e r in intact tissue. PD 116,948 is greater than 700 fold A~ selective in rat brain tissue (5) and is about 1000 fold more effective at antagonizing the heart rate effect of R-PIA than R-PIA's coronary flow effect. Based on these results, i t is clear that the negative chronotropic action of R-PIA is mediated by A~ adenosine receptors. Furthermore the r e l a t i v e l y poor A2 a f f i n i t y of PD 116,948 and i t s impotent antagonism of the vasodilator a c t i v i t y of R-PIA suggest that the adenosine receptor mediating coronary vasodilation is of the A2 subtype. I t should be pointed out that previous studies have indicated that the AI adenosine receptor was probably responsible for mediating negative chronotropic a c t i v i t y (2,8,11). Howeverthese results were based on rank orders of potency of selective adenosine agonists. This study demonstrates that the negative chronotropic a c t i v i t y of adenosine receptor activation can be selectively antagonized by the selective A~ adenosine antagonist, PD 116,948. In this study PD 116,948 was demonstrated to be: 1) free of i n t r i n s i c cardiac or vascular a c t i v i t y at effective adenosine antagonist concentrations; 2) to be a very potent functional Az adenosine receptor antagonist, and f i n a l l y ; 3) to be highly selective for the A~ adenosine receptor subtype. These findings indicate that PD 116,948 could be a valuable pharmacologic tool
Vol. 40, No. 6, 1987
Selective A I Adenosine Antagonist
561
for determining the role of adenosine and i t s m u l t i p l e receptor subtypes in physiologic and pathophysiologic responses. Acknowledgement The authors wish to thank H. Haber for the s t a t i s t i c a l analysis and R.F. Bruns f o r his assistance and c r i t i c a l review during the preparation of t h i s manuscript. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
C. LONDOS, D. COOPER and J. WOLFF. Proc. Natl. Acad. Sci. USA 77:25512554 (1980). D.B. EVANS and J.H. SCHENDEN. Life Sci. 3__1:2425-2432 (1982). L. EDVENSSON and B.B. FREDHOLM. Br. J. Pharmac. 80:631-637 (1983). S. KUSACHI. D. ROBERT, R.D. THOMPSON and R.A. OLSSON. J. Pharmac. Exper. Thera. 227:316-321 (1983). J.H. FERGUS, R.F. BRUNS, E.W. BADGER, J.A. BRISTOL. J.D. HARTMAN, L.A. SANTAY, S.J. HAYS and C.C. HUANY. Fed. Proc. 45:800 (1986). K.S. LEE and M. REDDINGTON. Brain Res. 368:394-398 (1968). R.F. BRUNS, J.W. DALY and S.H. SNYDER. Proc. Natl. Acad. Sci. USA 80:2077 (1983). S.J. HALEEN and D.B. EVANS. Life Sci. 36:127-137 (1985). O. ARUNLAKSHANA and H.O. SCHILD. B r i t . J. Pharmacol. 14:48-58 (1959). R.F. BRUNS, G.H. LU and T.A. PUGSLEY. Mol. Pharmacol. 2__9:331-346 (1986). R.P. STEFFEN, S.J. HALEEN and D.B. EVANS. Physiologist 41:A-44 (1983).
Pergamon Journals
PD 116,948, A HIGHLY SELECTIVE Al ADENOSINE RECEPTORANTAGONIST. S.J. Haleen, R.P. Steffen, and H.W. Hamilton + + Department of Pharmacology and Chemistry Warner-Lambert/Parke-Davis Pharmaceutical Research Ann Arbor, Michigan 48105 (Received in final form November 3, 1986)
Summary (R)-N-(1-Methyl-2-phenylethyl)adenosine (R-PIA), an adenosine receptor agonist has both negative chronotropic a c t i v i t y and coronary vasodilator a c t i v i t y . These actions of R-PlA are proposed to be mediated by subtypes (A~ and A2) of adenosine receptors. PD 116,948 is a xanthine d e r i v a t i v e which is a highly selective A I adenosine receptor ligand. In t h i s study PD 116,948 s e l e c t i v e l y antagonized the negative chronotropic a c t i v i t y of R-PIA in the isolated rat heart. These results are consistent with, and add f u r t h e r support to the hypothesis that adenosine receptor agonists mediate t h e i r negative chronotropic a c t i v i t y via A~ receptors and t h e i r vasodilator a c t i v i t y via A2 receptors. Key Words: Adenosine, AI Adenosine Receptor Antagonist, Heart Rate, Coronary Flow, Isolated Rat Heart, (R)-N-(1-Methyl-2phenylethyl)adenosine, 8 - C y c l o p e n t y l - 3 , 7 - d i h y d r o - l , 3 - d i p r o p y l 1H-purin-2,6-dione. Two subclasses of adenosine receptors have been distinguished based on receptor mediated i n h i b i t i o n (Az receptors) or stimulation (A2 receptors) of i n t r a c e l l u l a r cAMP accumulation ( i ) . Biological responses to adenosine receptor agonists have been put f o r t h as A~ or A2 mediated based on comparisons between receptor binding a f f i n i t i e s and rank order of biological potencies (2-4). Reliance on this technique was necessary due to the lack of an appropriate adenosine receptor antagonist. An appropriate antagonist would be free of i n t r i n s i c a c t i v i t y ( i n a c t i v e in the absence of an adenosine receptor agonist) and would be highly potent and selective for one of the adenosine receptor subtypes. PD 116,948, 8 - c y c l o p e n t y l - 3 , 7 - d i h y d r o - l , 3 - d i p r o p y l - l H - p u r i n - 2 , 6 - d i o n e , (FIG. 1) has been shown to be a highly selective A~ receptor ligand in rat brain (5,6). I t is a xanthine d e r i v a t i v e , the prototype structure for adenosine antagonists. This compound follows known s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s for xanthine binding to adenosine receptors (7). For example, this 1,3-dipropyl xanthine exhibits greater a f f i n i t y than the corresponding 1,3-dimethyl analog, 8-cyclopentyltheophylline (Az receptor ICso of 0.69 nM vs ICso of 20 nM, and A2 receptor ICso of 502 nM vs IC5o of 2000 nM) (values provided by Bruns et al, (5)).
Copyright
0024-3205/87 $3.00 + .00 (c) I987 Pergamon Journals Ltd.
556
Selective A 1 Adenosine Antagonist
Vol. 40, No. 6, 1987
0
FIG. i PD 116,948 8-cyclopentyl-3,7-dihydro-l,3-dipropyl-lH-purin-2,6-dione Adenosine receptor agonists are known to decrease heart rate and to increase coronary flow, effects which have been proposed to be A~ and A2 receptor mediated r e s p e c t i v e l y (8). In the present study, (R)-N-(1-methyl-2phenylethyl)adenosine (R-PIA) had near equimolar potency at decreasing heart rate and increasing coronary flow in the isolated r a t heart. I f the heart rate responses to R-PIA were A~ mediated, then PD 116,948 would be expected to block the heart rate response of R-PIA but not the coronary flow response. We tested t h i s hypothesis in the isolated Langendorff perfused r a t heart. Methods Sprague-Dawley normotensive rats were anesthetized with Na Pentobarbital (50 mg/kg, IP) and heparinized (2000 u n i t s , IP) to prevent blood c l o t t i n g . Hearts were r a p i d l y isolated and perfused by the Langendorff method (FIG. 2) with a modified Krebs Henseleit bicarbonate buffer of the following composition in mM concentration: NaCl, 127; NaHCO~, 25; dextrose, 5.5; Na Pyruvate, 2.0; KCl, 4.7; MgSO~, 1.1; KH 2PO~, 1.2; CaCl2.2H~O, 2.5; CaNa2 EDTA, 0.05. Perfusion pressure was maintained constant at 70 mmHg via a microcomputer controlled servo mechanism and the hearts were permitted to beat spontaneously. Because coronary perfusion pressure (CPP) was maintained constant, changes in coronary flow reflected changes in coronary resistance, The microcomputer controls CPP and R-PIA concentration. The level at which CPP and R-PIA concentration were maintained could be changed independently by commands communicated through the microprocessor keyboard. The adenosine receptor antagonist (PD 116,948) was added to the main oxygenation reservoir at a fixed concentration. R-PIA and PD 116,948 concentrates were prepared in NaOH solutions at pH 10 and 11 respectively. O n l y minute volumes were added to the perfusion medium which did not affect the pH (7.36-7.42) of the buffered solution. Control R-PIA dose response curves were compared to R-PIA dose response curves following pretreatment with PD 116,948 at concentrations of lx10-8, lx10-~, or lx10-6 molar. Heart rate was determined from the electrocardiogram and coronary flow was determined by calibrating pumps 1 and 2. Analog data was d i g i t i z e d and averaged with a microcomputer Buxco data analyzer. S t a t i s t i c a l analysis was performed by the Warner-Lambert/Parke-Davis Pharmaceutical Research Biometrics Department. Paired t tests were used to determine any s i g n i f i c a n t differences between control and treatment groups for basal heart rate and coronary flow values and for any differences in heart
Vol. 40, No. 6, 1987
Selective A 1 Adenosine Antagonist
557
T~
.
; o oo~ PC
L
" ....
f!
=. . . . ilill
I
=
MICROPROCESSOR
-
RECORDER
D~A ANALYZER
Isolated
Abbreviations:
PC,
Proportioning
FIG. 2 Langendorff
Circuit:
Heart
P1,
Model
Coronary
Perfusion
Pump;
P2,
Coronary Effluent Pump; P3, Drug Infusion Pump; T~, Perfusion Pressure Transducer; T2, Heart Chamber Pressure Transducer; DC, Drug Concentrate; M, Mixing Valve; OXY, Oxygenator; and ECG, Electrocardiogram Electrodes. Both hearts are perfused in parallel by PI which is controlled by the microprocessor to maintain coronary perfusion pressure constant independent of change in coronary resistance. Tz provides the pressure input signal to the microprocessor. Total coronary flow for both hearts is measured by recording the calibrated output signal from PI. Coronary flow for heart B is measured by recording the output signal from P2 which removes coronary effluent from the heart chamber at a rate equal to coronary flow via the servo mechanisms. T~ provides the column height pressure signal input to the microprocessor. Coronary flow for heart A is calculated from the difference between total and heart B coronary flow rates. PC provides an input signal to the microprocessor which is equal to the proportionate pumping rates for P~ and P~. The microprocessor then controls P3 to maintain the drug infusion rate proportional to total coronary flow. The M value mixes the DC with the physiological salt solution thereby assuring equal drug concentration for each heart which receives the treatment in parallel.
558
Selective A 1 Adenosine Antagonist
Vol. 40, No. 6, 1987
rate and coronary flow values between pre and post PD 116,948 treatment. To determine s i g n i f i c a n t differences between control R-PIA dose response curves and R-PIA dose response curves following treatment with PD 116,948 a common slope model was f i t to a range of responses over which the curves appeared to be p a r a l l e l . From the model, r e l a t i v e potency estimates were calculated with 95% confidence intervals for each PD 116,948 pretreated group r e l a t i v e to the R-PIA control. Results Basal heart rate and coronary flow values for each treatment group and the effects of PD 116,948 on heart rate and coronary flow are shown in Table I .
TABLE I Basal Heart Rate and Coronary Flow Parameters and the Effects of PD 116,948 on Heart Rate and Coronary Flow.
Control Heart Rate (beats/min)
278±11
IO-SM
IO-TM
Pre
Post
266±14
286±18
Pre 279±7
IO-~M Post
308±7*
Pre 251±11
Post 315±7"
Coronary Flow 14.2±0.8 14.1±1.1 14.3±1.2 14.8±1.4 14.6±1.2 11.6±1.1 12.4±0.8 (ml/min) Data represents the Mean ± SEM (p < 0.05, N=6) * S i g n i f i c a n t l y d i f f e r e n t from pretreatment There were no s i g n i f i c a n t d i f f e r e n c e s among treatment groups f o r e i t h e r basal heart rate or coronary f l o w values. PD 116,948 did not s i g n i f i c a n t l y a f f e c t basal coronary f l o w at any of the c o n c e n t r a t i o n s t e s t e d . Heart rate was s i g n i f i c a n t l y increased by PD 116,948 at lxlO-~M and 1xlO-6M. R-PlA caused dose dependent increases i n coronary f l o w and dose dependent decreases in heart rate ( c o n t r o l R-PIA curves FIG. 3). At 3x10-gM, R-PIA decreased heart rate s i g n i f i c a n t l y by 10% and at lx10-SM s i g n i f i c a n t l y increased coronary f l o w by 16%. However, at 3x10-SM R-PlA was about e q u a l l y potent on heart rate and coronary f l o w . At lxlO-~M, R-PIA had decreased heart rate by an average of 80% w i t h v e n t r i c u l a r a s y s t o l e o c c u r r i n g in one of the s i x hearts and a t r i a l a s y s t o l e o c c u r r i n g in two of the remaining f i v e hearts. The dose response curve was ended at ixlO-~M due to the above a r r h y t h m i a s . Coronary f l o w had increased an average of 59±8% at ixlO-TM R-PIA. Heart rate dose response to R-PIA was s i g n i f i c a n t l y s h i f t e d to the r i g h t by PD 116,948 in a dose r e l a t e d manner (ixlO-~M was s i g n i f i c a n t l y s h i f t e d to the r i g h t o f lx10 -7 and 1x10-SM). Comparing the amount of s h i f t at the 20% change from c o n t r o l l e v e l , PD 116,948 s h i f t e d the curve by about 1.5 log u n i t s at lxlO-SM, by 2.5 log u n i t s at 1x10-TM and by over 3.5 log u n i t s at lxlO-6M. Again using the 20% change from a c o n t r o l l e v e l , c a l c u l a t i o n s from a S c h i l d p l o t (9) give a PA2 of 9.44 w i t h a k i of 3.63xlO-Z°M and a slope equal to
Vol. 40, No. 6, 1987
Selective A 1 Adenosine Antagonist
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FIG. 3 Effects of adenosine receptor blockade by PD 116,948 on the R-PIA coronary flow (upper panel) and heart rate (lower panel) dose response curves (control - o) (PD 116,948 pretreated - =, ixlO-SM; A, ix10-~M; e, ixZO-6M). Points are mean values (N=6) (* s i g n i f i c a n t l y d i f f e r e n t from control, + s i g n i f i c a n t l y d i f f e r e n t from 1x10-SM and ixlO-TM PD 116,948, p < 0.05).
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Selective A 1 Adenosine Antagonist
Vol. 40, No. 6, 1987
1.02. In contrast, PD 116,948 did not s i g n i f i c a n t l y s h i f t the coronary flow dose response. Discussion PD 116,948, a xanthine analogue, has selective adenosine Az receptor binding a f f i n i t y and s e l e c t i v i t y in causing dose dependent s h i f t s of the chronotropic effect of R-PIA to the right. These results show PD 116,948 to be a competitive and selective adenosine A~ receptor antagonist. PD 116,948 increased heart rate in a non-dose dependent manner by about 9% at i x l O -8 and IxlO-~M. This small e f f e c t could be due to the blockade of endogeneous adenosine which would be expected to have a negative c h r o n o t r o p i c effect. The e x p l a n a t i o n f o r the 25% increase in heart rate observed f o l l o w i n g 1x10-6M of PD 116,948 is not apparent and may be due to some n o n - s p e c i f i c e f f e c t s of the compound, i . e . , PDE i n h i b i t i o n , or release of catecholamines. PD 116,948 alone had no s i g n i f i c a n t e f f e c t on coronary f l o w . The a f f i n i t y of PD 116,948 for the A2 receptor in rat brain is about 5xlO-~M (5) and i t is possible that 1x10-6M was an i n s u f f i c i e n t concentration to e f f e c t i v e l y block any endogeneous adenosine effect in coronary arteries. R-PIA was an appropriate agonist to use for these studies because i t had nearly equal potency for both Az and A2 type a c t i v i t i e s in the isolated rat heart in which selective antagonism could be demonstrated within a single experiment. R-PIA s i g n i f i c a n t l y decreased heart rate at 3x10-gM and s i g n i f i c a n t l y increased coronary flow at 1xlO-SM. In rat brain the ICso a f f i n i t i e s of A~ and A2 for R-PIA are 1.2x10-gM and 1.2x10-TM respectively ( i 0 ) . Based on the binding results, i t might be expected that an Az mediated effect ( i . e . , heart rate) versus an A2 mediated effect ( i . e . , coronary flow) by R-PIA would be separated by a two log dose concentration difference. The greater than expected potency of R-PIA at increasing coronary flow has also been observed in guinea pig hearts (11) whereas in rabbit heart (8) the potency separation between heart rate and coronary flow effects are more closely correlated with receptor binding results (10). I t must be pointed out that the receptor binding a f f i n i t i e s are measured in purified rat brain microsomes and tissue differences in binding a f f i n i t i e s and access to the receptors may d i f f e r in intact tissue. PD 116,948 is greater than 700 fold A~ selective in rat brain tissue (5) and is about 1000 fold more effective at antagonizing the heart rate effect of R-PIA than R-PIA's coronary flow effect. Based on these results, i t is clear that the negative chronotropic action of R-PIA is mediated by A~ adenosine receptors. Furthermore the r e l a t i v e l y poor A2 a f f i n i t y of PD 116,948 and i t s impotent antagonism of the vasodilator a c t i v i t y of R-PIA suggest that the adenosine receptor mediating coronary vasodilation is of the A2 subtype. I t should be pointed out that previous studies have indicated that the AI adenosine receptor was probably responsible for mediating negative chronotropic a c t i v i t y (2,8,11). Howeverthese results were based on rank orders of potency of selective adenosine agonists. This study demonstrates that the negative chronotropic a c t i v i t y of adenosine receptor activation can be selectively antagonized by the selective A~ adenosine antagonist, PD 116,948. In this study PD 116,948 was demonstrated to be: 1) free of i n t r i n s i c cardiac or vascular a c t i v i t y at effective adenosine antagonist concentrations; 2) to be a very potent functional Az adenosine receptor antagonist, and f i n a l l y ; 3) to be highly selective for the A~ adenosine receptor subtype. These findings indicate that PD 116,948 could be a valuable pharmacologic tool
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for determining the role of adenosine and i t s m u l t i p l e receptor subtypes in physiologic and pathophysiologic responses. Acknowledgement The authors wish to thank H. Haber for the s t a t i s t i c a l analysis and R.F. Bruns f o r his assistance and c r i t i c a l review during the preparation of t h i s manuscript. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
C. LONDOS, D. COOPER and J. WOLFF. Proc. Natl. Acad. Sci. USA 77:25512554 (1980). D.B. EVANS and J.H. SCHENDEN. Life Sci. 3__1:2425-2432 (1982). L. EDVENSSON and B.B. FREDHOLM. Br. J. Pharmac. 80:631-637 (1983). S. KUSACHI. D. ROBERT, R.D. THOMPSON and R.A. OLSSON. J. Pharmac. Exper. Thera. 227:316-321 (1983). J.H. FERGUS, R.F. BRUNS, E.W. BADGER, J.A. BRISTOL. J.D. HARTMAN, L.A. SANTAY, S.J. HAYS and C.C. HUANY. Fed. Proc. 45:800 (1986). K.S. LEE and M. REDDINGTON. Brain Res. 368:394-398 (1968). R.F. BRUNS, J.W. DALY and S.H. SNYDER. Proc. Natl. Acad. Sci. USA 80:2077 (1983). S.J. HALEEN and D.B. EVANS. Life Sci. 36:127-137 (1985). O. ARUNLAKSHANA and H.O. SCHILD. B r i t . J. Pharmacol. 14:48-58 (1959). R.F. BRUNS, G.H. LU and T.A. PUGSLEY. Mol. Pharmacol. 2__9:331-346 (1986). R.P. STEFFEN, S.J. HALEEN and D.B. EVANS. Physiologist 41:A-44 (1983).