Interstitial adenosine with dipyridamole: effect of adenosine receptor blockade and adenosine deaminase TAO WANG, ROBERT M. MENTZER,
JR., AND DAVID
G. L. VAN WYLEN
Departments of Surgery and Physiology, School of Medicine and Biomedical State University of New York at Buffalo, Buffalo, New York 14215 Wang, Tao, Robert M. Mentzer, Jr., and David Van Wylen. Interstitial adenosine with dipyridamole:
G. L.
effect
of adenosine receptor blockade and adenosine deaminase. Am. 1992.J. Physiol. 263 (Heart Circ. Physiol. 32): H&2-H558, Dipyridamole is proposed to increase coronary blood flow (CBF) by inhibition of adenosine uptake into cells, resulting in an increase in interstitial fluid (ISF) adenosine and an adenosine-mediated vasodilation. The purpose of this study was to determine the changes in CBF and ISF adenosine, inosine, and hypoxanthine during dipyridamole infusion in the absence or presence of adenosine receptor blockade or adenosine deaminase. To sample cardiac ISF, cardiac microdialysis probes were implanted in the left ventricular myocardium of chloralose-urethan-anesthetized dogs and perfused with Krebs-Henseleit buffer. The metabolite concentration in the effluent dialysate was used as an index of intramyocardial ISF metabolite concentration. In response to dipyridamole, CBF and dialysate adenosine concentration increased 4.4-fold and 2.2-fold, respectively, whereas dialysate inosine was unchanged and dialysate hypoxanthine decreased 50%. Adenosine receptor blockade, achieved by intracoronary 8(p-sulfophenyl)theophylline infusion, attenuated the increase in CBF induced by dipyridamole without changing dialysate adenosine concentration. Adenosine deaminase fully attenuated the dipyridamole-induced increases in CBF and dialysate adenosine. These results demonstrate that dipyridamole increases ISF adenosine in the dog and suggest that adenosine is the sole mediator of dipyridamole-induced coronary vasodilation. dog; coronary blood flow; 8(p-sulfophenyl)theophylline; dialysis; inosine; hypoxanthine
micro-
IS A WELL-KNOWN andpotentcoronary vasodilator (1,2,7,15). Based on evidence from myocyte cell cultures (8, 24,25), isolated perfused hearts (5,6,10, 11, 16, 23), and intact blood-perfused hearts (15, 17), it has been suggested that the mechanism of action of dipyridamole is due to the inhibition of adenosine uptake into cells, leading to an increase in interstitial fluid (ISF) adenosine levels and subsequent vasodilation. If this is the mechanism of action of dipyridamole, then 1) dipyridamole-induced increases in coronary blood flow (CBF) should be associated with increased ISF adenosine; 2) exogenously administered adenosine deaminase, the enzyme that converts adenosine to the vasoinactive metabolite inosine, should attenuate the vascular dilation induced by dipyridamole in proportion to the reduction of ISF adenosine; and 3) adenosine receptor blockade should attenuate the dipyridamole-induced increase in CBF without necessarily altering ISF adenosine. To test these hypotheses, we determined the changes in ISF adenosine, adenosine metabolites, and CBF during the administration of dipyridamole in the absence or presence of intracoronary infusion of either 8- (p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist, or adenosine deaminase. Cardiac ISF was sampled with the cardiac microdialysis technique, a DIPYRIDAMOLE
H552
0363-6135/92
$2.00 Copyright
Sciences,
recently developed technique that allows intramyocardial ISF to be sampled in the intact dog heart (32). METHODS Animal preparation. Adult mongrel dogs of either sex weighing 20-30 kg were premeditated with 3 mg/kg morphine and then anesthetized with 20 mg/kg thiamylal iv, followed by 70 mg/kg cu-D-chloralose and 200 mg/kg urethan iv. The animals were intubated and ventilated with a mixture of 100% oxygen and room air (model 613 dog respirator, Harvard Apparatus, South Natick, MA). Tidal volume, respiratory rate, and percent oxygen in the inspired air were adjusted to maintain normal blood gas and pH values (model 170 pH/blood gas analyzer, Ciba Corning Diagnostics, Medfield, MA). Core body temperature was monitored with an esophageal temperature probe and maintained with a heating pad at ~38’C. The right femoral artery was cannulated for the measurement of arterial blood pressure and the determination of arterial blood gases and pH. The right femoral vein was cannulated for infusion of fluid and anesthetic supplement when necessary. A thoracotomy was performed through the left fifth intercostal space, and the pericardium was incised to expose the left ventricle. The left anterior descending coronary artery (LAD) and the left circumflex coronary artery (LC) were dissected, and ultrasonic doppler flow probes (model T201 flowmeter, Transonics Systems, Ithaca, NY) were placed around each of the arteries for the measurement of CBF. For intracoronary infusions, a 22-gauge angiocatheter was inserted into the LAD proximal to the flow probe. The angiocatheter was positioned such that the tip was at least 1 cm distal to the branch point of the left main coronary artery. Cardiac microdialysis technique. The cardiac microdialysis technique has been described in detail elsewhere, including a discussion of the advantages, disadvantages, and limitations of this technique and a comparison of microdialysis to other techniques for sampling cardiac ISF (32). Briefly, each microdialysis probe consisted of a single 300-pm dialysis fiber, a platinum wire within the fiber, and two hollow silica tubes inserted, adjusted, and sealed within the dialysis fiber such that the distance between the ends of the silica tubes was 2 cm. In each animal, two microdialysis probes were implanted into the left ventricular myocardium, one in the region perfused by the LAD and the other in the region perfused by the LC. After insertion of the microdialysis probes, the inflow silica tube of each probe was connected via a larger silica tube to a gastight glass syringe filled with Krebs-Henseleit buffer and perfused at 2 pl/min (model CMAIOO microinjection pump, Bioanalytical Systems, West Lafayette, IN). The Krebs-Henseleit buffer consisted of (in mM) 118.0 NaCl, 25.0 NaHC03, 11.0 glucose, 4.7 KCl, 1.25 CaCl,, I.2 MgS04.7HZ0, and 1.2 KH,PO, and was bubbled with 5% CO,-95% N, before filling the syringe. This gave the Krebs-Henseleit buffer a PO,, Pco~, and pH of approximately 20 mmHg, 40 mmHg, and 7.35, respectively. The low PO, approximates tissue PO, levels, thus minimizing the supply or removal of oxygen locally by the microdialysis probes. The effluent, referred to as the dialysate, was collected from the outflow silica tube in small plastic tubes and frozen (-SOOC) until biochemical analysis.
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
ISF ADENOSINE
DURING
The theory of cardiac microdialysis is that as the KrebsHenseleit solution passes through the dialysis fiber, diffusion occurs between the fluid within the fiber and the ISF surrounding the fiber. The dialysate concentration is therefore used as an index of intramyocardial ISF concentration. Local myocardial blood flow (MBF) was measured from the area surrounding the dialysis fiber by the hydrogen clearance technique, using the platinum wire within the microdialysis probe to polarographically detect tissue hydrogen (36). MBF was calculated from the time required for the hydrogen levels to fall from 90% maximum to 40% using the formula MBF (ml. mine1 100 g-l) = [ln(40/90)]/time] X 100. Arterial blood pressure, heart rate, LAD and LC flow, and the hydrogen clearance curves from the LAD and the LC region were recorded on an eight-channel recorder (model 3800, Gould, Cleveland OH). Protocols. For all protocols, a minimum of 90 min was allowed for recovery from microdialysis probe implantation before initiation of the protocols, a time that has been shown to allow stabilization of dialysate adenosine (32). A preliminary group of animals (n = 4) was used to determine whether dipyridamole produced a sustained increase in dialysate adenosine. After control samples were obtained, a dipyridamole infusion (0.5 mg. kg-l h-l iv) was initiated. After a stable response to dipyridamole was achieved, six IO-min dialysate samples were collected. LAD flow was measured in two of these animals. Two series of experiments were performed (Fig. 1). The purpose of series 1 (Fig. IA) was to determine the effect of adenosine receptor blockade on ISF adenosine during dipyridamoleinduced increases in CBF. In these animals (n = 6), two IO-min dialysate samples were collected from each microdialysis probe during the control period. A dipyridamole infusion (0.5 mg*kg-l*h-l iv) was then started and continued throughout the remainder of the experiment. After a stable increase in CBF was achieved (- 10 min after beginning the dipyridamole infusion), three successive 5-min dialysate samples were collected. An intracoronary infusion into the LAD of 1 ml/min of either l
l
A SERIES
1
i;..:..l
+1 0’+10’-+5q-5’q-5’+
1 o-t-1
o’+lo~+l
Dialysate B SERIES
o*+lo’+
2a
Dipyridamole
- 0.5 mg/kg/hr
Dialysate C SERIES
oq+l
Samples
i.v.
H553
DIPYRIDAMOLE
normal saline or SPT, an adenosine receptor antagonist, was then initiated. Each SPT infusion, designated in Fig. 1A as SPTl (0.5 x 10m3 M), SPTz (1.0 x 10m3 M), and SPT, (5.0 x IO-” M), consisted of a greater concentration of the antagonist. Ten-minute dialysate samples were collected during the intracoronary SPT or normal saline infusion. The purpose of series 2 (Fig. 1, B and C) was to determine the effect of adenosine deaminase on the relationship between ISF adenosine and CBF during dipyridamole administration. As in series 1, dipyridamole (0.5 mg. kg-l h-l) was given intravenously after the collection of two lo-min control dialysate samples. After the stable response of CBF to dipyridamole was established and three 5-min dialysate samples were collected, adenosine deaminase was administered into the LAD. In series 2a (Fig. IB, n = 6), adenosine deaminase was given for -8 min at a dose of 5 U kg-l. min-l, the dose most frequently used by other investigators (22, 30). Because this dose produced a sustained CBF response in both LAD and LC regions, in series 2b (Fig. lC, n = 6), adenosine deaminase was infused at 0.5 for -60 min. In both series 2a and 2b, two 5-min U kg-l min-l dialysate samples followed by five IO-min dialysate samples were collected from each microdialysis probe after the initiation of the adenosine deaminase infusion. The dilution of adenosine deaminase was adjusted to deliver 0.5 or 5 U kg-l min-l at an infusion rate Cl.0 ml/min. AnaZytical procedures. The dialysate samples were analyzed for adenosine, inosine, and hypoxanthine using reverse-phase high-performance liquid chromatography (models 600E solvent delivery system, 484 detector, 712 injector, and 820 MAXIMA data analysis system; Waters Division of Millipore, Milford, MA). Separation of the compounds was achieved using a Supelcosil LC-18 column (Supelco, Bellefonte, PA) and a 1 (pH 5.3) to 25% (pH 5.58) methanol in 100 mM KH,PO, gradient. Adenosine, inosine, and hypoxanthine were detected by an absorbance change at 254 nm and were identified and quantified by comparing retention times and peak heights with known standards. Materials. The adenosine deaminase used was type VIII calf intestinal adenosine deaminase, prepared as a suspension in 3.2 M (NH&SOd, pH 6.0 (Sigma Chemical, St. Louis, MO). The adenosine deaminase was diluted in 0.14 M NaCl containing 0.012 M tris(hydroxymethyl)aminomethane (Tris) HCl (pH 7.5) to neutralize the (NH&SO4 and filtered (20 pm) before infusion into the LAD. SPT (Research Biochemicals, Natick, MA) was dissolved in normal saline, and dipyridamole (Sigma Chemical, St. Louis, MO) was dissolved in methanol (IO mg/ml). Statistical analysis. Mean t SE values were calculated for all data. For comparison of values obtained during control and different treatments, differences between mean values were determined by one-way analysis of variance followed by Duncan’s or Dunnett’s multiple range test for repeated measures. P < 0.05 was accepted as indication of a statistically significant difference. l
l
l
l
l
l
Samples
RESULTS
2b
Dipyridamole-induced changes in dialysate metabolites and CM?. Because the changes in dialysate metabolites
LAD Adenosine deaminase 0.5 U/kg/min
Dipyridamole 5+5’+5+5’+5’+1
- 0.5 mg/kg/hr O’-+l
Dialysate
o’+l
i.v.
O’-+
Samples
Fig. 1. Experimental protocols for series 1 and 2. SPT, 8-(p-sulfophenyl)theophylline; NS, normal saline; LAD, left anterior descending artery; ADA, adenosine deaminase. SPT concentrations: 0.5 x IO-” (SPT1), 1.0 x lo-” (SPT2), and 5.0 x 10e3 M (SPT3).
and CBF in response to dipyridamole were determined in all animals of series 1 and 2 (n = I@, these data have been pooled and are shown in Fig. 2. The dialysate sample chosen for graphic presentation in Fig. 2 is the middle of the three dialysate samples taken from the LAD-perfused region after initiation of the dipyridamole infusion and therefore represents changes during the steady-state response to dipyridamole. Dipyridamole, which caused an
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
ISF ADENOSINE
0 m
DURING
DIPYRIDAMOLE
CONTROL DIPYRIDAMOLE
* p < 0.05 n=18
vs. Control
-I-
IN0
ADO
CBF
CBF
HYPO
o!
Fig. 2. Changes in coronary blood flow (CBF) and dialysate adenosine (HYPO) with dipyridamole (ADO), inosine (INO), and hypoxanthine infusion (n. = 18). Data are from the LAD-perfused region. Values are means t SE; * P < 0.05 compared with control.
0
IO
20
30
40
50 60 (minutes)
70
80
E
40 50 60 TIME (minutes)
70
80
90
TIME
B
-4.4-fold increase in LAD flow, was associated with a 2.2fold increase in dialysate adenosine, no change in dialysate inosine, and a decrease in dialysate hypoxanthine. In these animals, mean arterial pressure decreased from 133 t 4 to 127 t 4 mmHg during dipyridamole infusion, while heart rate increased from 113 t 6 to 141 t 7 beats/ min. In the four animals in which dialysate adenosine levels were determined during 60 min of sustained dipyridamole, dialysate adenosine increased from a control level of 0.57 t 0.17 to 0.96 t 0.26,0.94 t 0.21,l.Ol & 0.24, 1.00 t 0.25, 0.95 t 0.24, and 0.88 t 0.21 PM at 5, 15, 25, 35,45, and 55 min of dipyridamole infusion, respectively. CBF increased in response to dipyridamole and remained elevated throughout the dialysate collection period in the two animals in which CBF was measured. Series 1. The hemodynamic data from series 1 experiments are shown in Table 1. Although mean arterial blood pressure was stable during dipyridamole infusion in series 1, heart rate increased significantly. Neither mean arterial pressure nor heart rate was altered during intracoronary infusion of SPT or normal saline. The CBF and dialysate metabolite changes from series 1 are shown in Table 1 and Fig. 3. CBF increased during dipyridamole infusion, as indicated both by the changes in LAD and LC flow (Fig. 3) and by hydrogen clearance
measurements of myocardial perfusion (Table 1). Although the changes of regional blood flow measured by hydrogen clearance paralleled qualitatively the changes in LAD and LC flow, the hydrogen clearance measurements of myocardial perfusion indicated a smaller increase in
Table 1. Changes in hemodynamic
blood flow, and dialysate
parameters,
myocardial
140,
3 I20 3 k loo 0 OF 80 $ *E $
60
2 o 5 CJ
40 2o
*
p < 0.05
vs. Control
nt “0
IO
30
20
Fig. 3. CBF and ADO results (means t SE) for series 1. All intracoronary infusions were at 1.0 ml/min into the LAD. A: data from LADperfused region. B: data from left circumflex artery (LC)-perfused region. See Fig. 1 for SPT1, SPT2, and SPT3 concentrations.
Series 1: Intracoronary
inosine
and hypoxanthine
SPT
Variable Control
Dipyridamole
SPTl
NS
SPT2
NS
SPT3
12128 119*7 120t7 121k7 120&8 132~17 124t7 MAP, mmHg 165tl2* 170t9* 170&B* 168tlO* 130t13 174t8* 175*7* HR, beats/min MBF, ml min-l 100 g-l 6426 111216 94t18 82t14 76t5 141&20* 109t23 LAD region 102t22 128tll* 127t7* 137t16* 140t17* 67t9 152t6* LC region Dialysate Ino, PM 0.57t0.11 0.77t0.10 0.59t0.13 0.98t0.34 0.65t0.26 0.65t0.20 0.58t0.11 LAD region 0.46t0.06 0.49kO.10 0.42t0.06 0.41t0.08 0.40t0.06 0.48t0.12 0.52t0.19 LC region Dialysate Hypo, PM 1.78t0.81 1.67t0.82 1.39rto.52 1.08t0.51 1.59kO.67 1.68t0.71 3.9321.15 LAD region 0.77t0.34 0.76kO.29 0.91t0.35 0.81t0.36 0.71t0.27 0.79t0.34 2.09t1.09 LC region Values are means t SE. SPT, 8-(p-sulfophenyl)theophylline; NS, normal saline; MAP, mean arterial pressure; HR, heart rate; MBF, myocardial blood flow; LAD, left anterior descending artery; LC, left circumflex artery; Ino, inosine; Hypo, hypoxanthine; ADA, adenosine deaminase. Concentrations of SPT: 0.5 x 10B3 (SPT1), 1.0 X 10e3 (SPT2), and 5.0 X low3 M (SPT3). * P < 0.05, compared with control. l
l
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
ISF ADENOSINE
DURING
H555
DIPYRIDAMOLE
blood flow than that in the LAD and LC probably because of a diffusion limitation of hydrogen at high flow. The dipyridamole-induced increase in CBF in the LAD-perfused region was attenuated by intracoronary SPT infusion, being almost totally suppressed by the highest concentration of SPT (Fig. 3A). The decline in LAD flow during the intervening intervals of normal saline infusion and the gradual decrease in LC flow after the initiation of SPT infusion into the LAD (Fig. 3B) likely reflect the systemic accumulation of SPT. Despite the attenuation of dipyridamole-induced increase in CBF by SPT, the elevated dialysate adenosine levels were not altered by intracoronary SPT infusion (Fig. 3), nor were there significant changes in dialysate inosine and hypoxs, thine concentrations during the SPT infusion (Tab’ 1). Series 2. The ht,_lodynamic parameters from series 2 experiments are shown in Table 2. In series 2a and 2b, mean arterial blood pressure and heart rate were not significantly altered during dipyridamole or during the subsequent adenosine deaminase infusion, although heart rate tended to increase after dipyridamole infusion. As shown in Fig. 44, adenosine deaminase, given for 4 min at 5 Us kg-l min-l (series 2a), completely and rapidly attenuated the dipyridamole-induced increase in LAD flow and sustained this attenuation up to 45 min after the adenosine deaminase infusion was stopped. In response to the LAD infusion of adenosine deaminase, the dipyridamole-induced increase of flow in the LC was also decreased to control levels (Fig. 4B). In both the LAD- and LC-perfused regions, the attenuation of the dipyridamole-induced increase in CBF was mirrored by reductions in dialysate adenosine concentra-
tion (Fig. 4). As shown in Table 2, dialysate inosine concentration increased after the start of the adenosine deaminase infusion, indicating that there was indeed enhanced deamination of adenosine. Dialysate hypoxanthine, which was decreased in response to dipyridamole, was not altered by adenosine deaminase infusion. In series 2b, in which adenosine deaminase was infused into the LAD at 0.5 U kg-l emin-l, the dipyridamoleinduced increases in CBF and dialysate adenosine were gradually attenuated in both the LAD- and LC-perfused regions (Fig. 5 and Table 2). Although dialysate inosine levels tended to increase during the administration of adenosine deaminase, these changes were not statistically significant. Dialysate hypoxanthine levels were stable during adenosine deaminase infusion (Table 2). Based on the assumption that dialysate adenosine concentration is a reflection of intramyocardial ISF adenosine levels, the major findings of the present study were 1) dipyridamole induced a concomitant increase in ISF adenosine and CBF, 2) adenosine deaminase administration attenuated the dipyridamole-induced increase in CBF simultaneous with a reduction in ISF adenosine, and 3) adenosine receptor blockade attenuated the dipyridamole-induced increase in CBF without altering ISF adenosine levels. The dipyridamole-induced increase in ISF adenosine we observed with cardiac microdialysis is consistent with that seen with other techniques to estimate ISF adenosine levels. Using the pericardial infusate technique, Knabb et al. (17) observed almost a threefold increase in pericardial infusate adenosine levels after dipyridamole
Table 2. Changes in hemodynamic
blood flow, and dialysate
DISCUSSION
l
Variable
Control
parameters,
myocardial
Dipyridamole
Series 2a: intracoronary
MAP, mmHg HR, beats/min MBF, ml mine1 . 100 g-l LAD region LC region Dialysate Ino, PM LAD region LC region Dialysate Hypo, PM LAD region LC region
5-Min Post-ADA
ADA
ADA,
5 U- kg-l.
inosine
and hypoxanthine
15Min Post-ADA
35-Min Post-ADA
55-Min Post-ADA
13Ok7 125tl9
min-l
126t7 137210
129t6 135tl4
12827 129k17
130t7 127tl9
130t7 127tl8
881tl4 79&B
197k28* 194tl2*
161227 15ltl6*
117tl9 12ltl4
103k24 102kll
105tlO 102tll
0.99t0.40 l.lBt0.28
0.94t0.28 1.06kO.16
3.lOtl.35* 1.68t0.46
2.17kO.30 2.76&1.13*
1.841kO.51 1.36kO.56
1.26kO.36 1.3lt0.48
1.16t0.32 0.621kO.32
2.3220.71 2.81k0.82
1.03t0.22* 1.15kO.19
l.llt0.35 0.9lt0.18*
1.26kO.40 1.53rizO.86
l.lOt0.26 1.76rtO.76
1.06t0.21 1.19kO.54
0.9lt0.17 0.7OkO.17
132t8 112tl2
l
Variable
Control
Dipyridamole
Series 2b: intracoronary
5-Min
ADA,
ADA
0.5 U. kg-l
15-Min l
ADA
35-Min
ADA
97k22 86klO
55-Min
ADA
rnin-l
MAP, mmHg 135k6 13Ok4 128t3 129t4 130t5 HR, beats/min 98tl2 113t8 109t7 107&B 107t9 MBF, ml - min-l 100 g-l LAD region 120tl4 187&22* 153224 12ltl6 113tlO 226tlO* LC region 92tl8 165tl4* 108tlO 96tl3 Dialysate Ino, I,~M LAD region 0.75t0.18 0.7lkO.12 0.85&O. 19 1.04t0.17 0.9lrtO.06 0.6520.28 0.63t0.05 0.66&O. 10 0.75&O. 13 0.90~0.10 LC region Dialysate Hypo, PM LAD region 2.28k0.34 1 .OBtO. 14* 0.92&O. 18* 1.07t0.32* 0.84&O. 18* LC region 2.01t0.73 0.85&O. 14* 0.74t0.13* 0.74t0.17* 0.67&O. 14* Values are means t SE. See Table 1 for abbreviations and text for further details. * P < 0.05 compared with control.
130t5 105t9
l
113tlO 83kl2 0.89t0.10 0.95t0.01 O.Blt0.16* 0.72t0.17*
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
H556
ISF ADENOSINE
1 *
0
20
40 60 TIME (minutes)
p < 0.05
DURING
DIPYRIDAMOLE
vs. Control
80
100
40 60 TIME (minutes)
0
80
100
B 180 240
II
17 U/kg/mi;l
160
*
140 3 0ii
120
E
100
F d-g zg 2 2 0 0 0
80 60 40 20
P 0
20
40 60 TIME (minutes)
80
100
0
20
40 TIME (minutes)
60
80
Fig. 4. CBF and ADO results (means t SE) for series 2~. ADA was infused into the LAD at 5 U kg-l l rein-? A: data from LAD-perfused region; B: data from LC-perfused region. * P < 0.05 compared with control.
Fig. 5. CBF and ADO results (means 2 SE) for series Zb. ADA was infused into the LAD at 0.5 Us kg-l. min-l. A: data from LAD-perfused region; B: data from LC-perfused region. * P < 0.05 compared with control.
administration in the anesthetized dog. Based on epicardial exudate adenosine levels in isolated perfused guinea pig hearts, Decking et al. (10) and Mohrman and Heller (23) have estimated a 2- to lo-fold increase in ISF adenosine during dipyridamole infusion. Finally, using a multiple-indicator dilution technique to extrapolate from venous adenosine levels to ISF adenosine concentration in the isolated perfused guinea pig heart, Wangler et al. (35) calculated that in response to dipyridamole, ISF adenosine increased from 6.5 to 191 nM during constantpressure perfusion and to 88 nM during constant-flow perfusion. Taken together with our data, these reports leave little doubt that dipyridamole does indeed lead to an increase in cardiac ISF adenosine concentration. Despite the consistency with which dipyridamole has been shown to be associated with enhanced ISF adenosine levels, the precise mechanism by which dipyridamole leads to elevated ISF adenosine levels is not known with certainty. Several lines of evidence document the ability of dipyridamole to inhibit the uptake of exogenous adenosine into cells. In cultured myocytes, dipyridamole virtually eliminates adenosine uptake from the medium (24, 25), whereas in isolated perfused hearts and intact hearts,
dipyridamole inhibits the uptake of exogenous adenosine from the vascular space into the myocardium (2, 9, 18). Interestingly, whereas guinea pig, dog, and human are very responsive to dipyridamole, the rat is relatively unresponsive to dipyridamole (7, 15, 19). The more difficult issue relates to the action of dipyridamole on endogenous adenosine transport. The major unresolved issue is whether dipyridamole acts as a symmetrical inhibitor of adenosine transport, in which case it would inhibit both adenosine release and uptake, or whether dipyridamole selectively inhibits adenosine uptake into cells without having a major effect on adenosine release. Based on experiments by Mustafa et al. (24, 25) on cultured myocytes, it was suggested that dipyridamole leads to enhanced ISF adenosine by inhibiting the uptake of adenosine into the myocyte without affecting release. This concept received additional support from Bukoski and Sparks (8), who showed that dipyridamole led to enhanced accumulation of adenosine in the medium of isolated adult cardiac myocytes. However, the idea that dipyridamole selectively inhibits adenosine uptake was recently challenged by Meghji et al. (21), who found that
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
ISF ADENOSINE
DURING
dipyridamole reduced the efflux of adenosine from primary cultures of chick ventricular myocytes by 70-90%, leading to an increase in the amount of adenosine trapped inside the cell. If dipyridamole inhibits the release of adenosine from cells in vivo, then it is difficult to reconcile with the increase in CBF and with the data demonstrating increased ISF adenosine during dipyridamole. The resolution of the mechanism of dipyridamole action is likely related to another unsettled issue, namely whether adenosine is produced intracellularly by a cytosolic 5’-nucleotidase (28) or by a membrane-bound ecto5’-nucleotidase, which has been suggested to be an enzyme carrier complex (12). It is likely that, in vivo, adenosine is produced both intracellularly by dephosphorylation of intracellular adenosine 5’-monophosphate (AMP) and extracellularly by either the enzyme carrier complex or by the degradation of extracellularly released ATP (29). In this context, dipyridamole could inhibit the transport of intracellularly formed adenosine out of the cell and yet, by inhibiting the uptake of extracellularly formed adenosine, lead to enhanced ISF adenosine. However, the details of dipyridamole action have yet to be definitively resolved in the intact heart. Although the increase in ISF adenosine during dipyridamole was expected, we anticipated that this would be associated with reductions in the ISF concentrations of both inosine and hypoxanthine, indicative of the reduced availability of intracellular adenosine for catabolism to inosine and hypoxanthine. However, our data indicated that while ISF hypoxanthine was reduced during dipyridamole, ISF inosine did not change. It is possible that this reflects differences in sites and biochemical pathways for production of inosine and hypoxanthine. Hypoxanthine is produced from inosine by nucleoside phosphorylase, an enzyme believed to be located predominantly in the endothelial cells (20). Because dipyridamole inhibits inosine uptake into cells (I@, the lower ISF hypoxanthine may be a result of reduced uptake of inosine from the ISF into endothelial cells. The production of inosine, however, is more complex. Inosine can not only be produced from adenosine by adenosine deaminase, but also by the dephosphorylation of inosine 5’monophosphate (IMP). Furthermore, there is currently some debate as to whether adenosine deaminase is restricted to pericytes and endothelial cells (27, 31) or whether it also exists in myocytes (4) and the ISF (3). Therefore, the lack of change in ISF inosine during dipyridamole may be the result of alternate biochemical pathways for inosine production as well as multiple compartments where adenosine deaminase is located. Nevertheless, our data in the intact heart do differ from results of isolated cells, where dipyridamole causes a reduction in extracellular inosine (25). The ability of adenosine receptor blockade and adenosine deaminase to completely reverse the dipyridamoleinduced increase in CBF has been observed by others (5, 15). Our data confirm these observations and in addition show that the attenuation caused by SPT occurs without altering ISF adenosine, while the reduction in the dipyridamole-induced increase in CBF brought about by adenosine deaminase occurs simultaneously with the re-
H557
DIPYRIDAMOLE
duction in ISF adenosine. These observations are consistent with the data of Becker et al. (5), who demonstrated in the isolated perfused guinea pig heart that theophylline blocked the increase in CBF induced by dipyridamole without preventing the enhanced release of adenosine into the venous effluent, whereas adenosine deaminase prevented both the increased CBF and increased venous adenosine concentration associated with dipyridamole. These data and our data seem to leave little doubt that adenosine is the sole mediator of the dipyridamole-induced increase in CBF. It has recently been shown that the vasodilatory response to exogenous adenosine occurs without an increase in ISF adenosine (13, 14, 34). This and data from isolated vessels have introduced an important role of the vascular endothelium in mediating the vasodilatory response to intravascular adenosine (26). If dipyridamole increases plasma adenosine levels, it is possible that the vascular relaxation in response to dipyridamole is mediated in part by the vascular endothelium. Because plasma adenosine was not measured in the present study, we are not able to evaluate the potential contribution of the vascular endothelium to the response to dipyridamole. However, it is important to note that our results with adenosine receptor blockade and adenosine deaminase do not distinguish between elevated plasma adenosine acting on the endothelium or elevated ISF adenosine acting directly on the vascular smooth muscle. It is also of interest that dipyridamole has been shown to relax isolated coronary artery strips (33). Thus it is possible that dipyridamole may also act on a pool of adenosine produced locally within the vasculature itself. In summary, with the use of the cardiac microdialysis technique to sample intramyocardial ISF in the intact dog heart, the results of the present study indicate that dipyridamole-induced increases in CBF are accompanied by increased ISF adenosine. In addition, the observation that adenosine receptor blockade attenuated the CBF response to dipyridamole without altering ISF adenosine, whereas adenosine deaminase reduced both the CBF response and ISF adenosine, demonstrates that adenosine is the sole mediator of dipyridamole-induced coronary vasodilation. We gratefully acknowledge the technical assistance of Roy Fiorella and Vickie Armenia. This work was presented at the 74th Annual Meeting of the Federation of American Societies for Experimental Biology, Washington, DC, April 1-5, 1990. This work was supported by National Heart, Lung, and Blood Institute Grants HL-40878, HL-34579, and HL-46027. Address for reprint requests: D. G. L. Van Wylen, Dept. of Physiology, State Univ. of New York at Buffalo, 462 Grider St., Buffalo, NY 14215. Received
3 June 1991; accepted in final form 31 March
1992.
REFERENCES Afonso, S. Inhibition of coronary vasodilating action of dipyridamole and adenosine by aminophylline in the dog. Circ. Res. 26: 743-752, 1970. Afonso, S., and G. S. O’Brien. Mechanism of enhancement of adenosine action by dipyridamole and lidoflazine in dogs. Arch. Int. Pharmacodyn. Ther. 194: 181-186, 1971. Andy, R. J., and R. Kornfield. The adenosine binding protein of human skin fibroblasts is located on the cell surface. J. Biol. Chem. 257: 7922-7925, 1982.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
H558
ISF ADENOSINE
DURING
4. Arch, J. R. S., and E. A. Newsholme. The control of the metabolism and the hormonal role of adenosine. Essays Biochem. 14: 82-123, 1978. 5. Becker, B. F., H. Bardenheuer, I. Overhage de Reyes, and E. Gerlach. Effects of theophylline on dipyridamole induced coronary venous adenosine release and coronary dilation. In: Adenosine: Receptors and Modulation of Cell Function, edited by V. Stefanowich, K. Rudolphi, and P. Schubert. London: IRI Press Limited, 1985, p. 441-449. 6. Bellardinelli, L., R. A. Fenton, A. West, J. Linden, J. S. Althans, and R. M. Berne. Extracellular action of adenosine and the antagonism by aminophylline on the atrioventricular conduction of isolated perfused guinea pig and rat hearts. Circ. Res. 51: 569-579, 1982. 7. Brown, B. G., M. A. Josephson, R. B. Peterson, C. D. Pierce, M. Wong, H. S. Hecht, E. Bolson, and H. T. Dodge. Intravenous dipyridamole combined with isometric handgrip for near maximal acute increase in coronary flow in patients with coronary artery disease. Am. J. Cardiol. 48: 1077-1085, 1981. 8. Bukoski, R. D., and H. V. Sparks, Jr. Adenosine production and release by adult rat cardiocytes. J. Mol. Cell. Cardiol. 18: 595-605, 1986. 9. Bunag, R. D., C. R. Douglas, S. Imai, and R. M. Berne. Influence of a pyrimidine derivative on deamination of adenosine by blood. Circ. Res. 15: 83-88, 1984. 10. Decking, U. K. M., E. Juengling, and H. Kammermeier. Interstitial transudate concentration of adenosine and inosine in rat and guinea pig hearts. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): Hll25-Hl132, 1988. R. Rubio, and R. M. 11. Degenring, F. H., R. R. Curnish, Berne. Effect of dipyridamole on coronary blood flow in hypoxia and reactive hyperemia in the isolated perfused guinea pig heart. J. Mol. Cell. Cardiol. 8: 877-888, 1976. 12. Frick, G. P., and J. M. Lowenstein. Vectorial production of adenosine by 5’-nucleotidase in the perfused rat heart. J. Biol. Chem. 253: 1240-1244, 1978. 13. Gidday, J. M., R. Rubio, and R. M. Berne. Increases in coronary blood flow by intracoronary adenosine occur independent of changes in interstitial fluid adenosine. Physiologist 30: 187, 1987. 14. Heller, L. J., and D. E. Mohrman. Estimates of interstitial adenosine from surface exudates of isolated rat hearts. J. Mol. Cell. Cardiol. 20: 509-523, 1988. 15. Hintze, T. H., and S. F. Vatner. Dipyridamole dilates large coronary arteries in conscious dogs. Circulation 68: 1321-1327, 1983. 16. Hopkins, S. V. The potentiation of the action of adenosine on the guinea pig heart. Biochem. Pharmacol. 22: 341-348, 1973. 17. Knabb, R. M., J. M. Gidday, S. W. Ely, R. Rubio, and R. M. Berne. Effects of dipyridamole on myocardial adenosine and active hyperemia. Am. J. Physiol. 247 (Heart Circ. Physiol. 16): H804-H810, 1984. 18. Kolassa, N., K. Peleger, and W. Rummei. Specificity of adenosine uptake into the heart and inhibition by dipyridamole. Eur. J. Pharmacol. 9: 265-268, 1970. 19. Kolassa, N., K. Peleger, and M. Tran. Species differences in action and elimination of adenosine after dipyridamole and hexobendine. Eur. J. Pharmacol. 13: 320-325, 1971.
DIPYRIDAMOLE 20. Manfredi, J. P., and E. W. Holmes. Purine salvage pathways in myocardium. Annu. Rev. Physiol. 47: 691-705, 1985. 21. Meghji, P., R. Rubio, and R. M. Berne. Intracellular adenosine formation and its carrier-mediated release in cultured embryonic chick heart cells. Life Sci. 43: 1851-1859, 1988. 22. Merrill, G. F., H. F. Downey, and C. E. Jones. Adenosine deaminase attenuates canine coronary vasodilation during systemic hypoxia. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H579-H583, 1986. D. E., and L. J. Heller. Transcapillary adenosine 23. Mohrman, transport in isolated guinea pig and rat hearts. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H772-H783, 1990. 24. Mustafa, S. J. Effects of coronary vasodilator drugs on the uptake and release of adenosine in cardiac cells. Biochem. Pharmacol. 280: 2617-2634, 1979. 25. Mustafa, S. J., R. Rubio, and R. M. Berne. Adenosine metabolism in cultured chick-embryo heart cells. Am. J. Physiol. 228: 1474-1478, 1975. 26. Nees, S. Coronary flow increases induced by adenosine and adenine nucleotides are mediated by the coronary endothelium: a new principle of the regulation of coronary blood flow. Eur. Heart J. 10, Suppl. F: 28-35, 1989. 27. Nees, S., and E. Gerlach. Adenosine nucleotide and adenosine metabolism in cultured coronary endothelial cells: formation and release of adenosine compounds and possible functional implications. In: Regulatory Function of Adenosine, edited by R. M. Berne, T. W. Rall, and R. Rubio. The Hague: Martinus/Nijhoff, 1983, p. 347-355. 28. Newby, A. C., Y. Worku, and C. A. Holmquist. Adenosine formation: evidence for a direct biochemical link with energy metabolism. Adv. Cardiol. 6: 273-284, 1985. 29. Pelleg, A., and G. Burnstock. Physiological importance of ATP released from nerve terminals and its degradation to adenosine in humans. Circulation 82: 2269-2272, 1990. 30. Saito, D., C. R. Steinhart, D. G. Nixon, and R. A. Olsson. Intracoronary adenosine deaminase reduces canine myocardial reactive hyperemia. Circ. Res. 49: 1262-1267, 1981. 31 Schrader, W. P., and A. W. Clinton. Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit heart: implications for adenosine metabolism. Circ. Res. 66: 754762, 1990. D. G. L., J. Wills, J. Sodhi, R. J. Weiss, R. D. 32 Van Wylen, Lasley, and R. M. Mentzer, Jr. Cardiac microdialysis to estimate interstitial fluid adenosine and coronary blood flow. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): Hl642-1649, 1990. P., and E. Bassenge. Wirking von ATP, A-3,5-MP, 33. Walter, adenosin und dipyridamol an Streifenpraparaten der a. coronaria, a. renalis und der v. portae. Pfluegers Arch. 299: 52-65, 1968. T., R. M. Mentzer, Jr., and D. G. L. Van Wylen. 34. Wang, Changes in cardiac interstitial fluid adenosine during intracoronary adenosine infusion: effect of dipyridamole and erythro-9-(2hydroxy-3-nonyl)adenine (EHNA). FASEB J. 5: 1396, 1991. R. D., M. W. Gorman, C. Y. Wang, D. F. Dewitt, 35. Wangler, I. S. Chan, J. B. Bassingthwaighte, and H. V. Sparks. Transcapillary adenosine transport and interstitial adenosine concentration in guinea pig hearts. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H89-H106, 1989. 36. Young, W. Hz clearance measurement of blood flow: a review of techniaue and polarogranhic nrincinles. Stroke 11: 552-564. 1980.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
JR., AND DAVID
G. L. VAN WYLEN
Departments of Surgery and Physiology, School of Medicine and Biomedical State University of New York at Buffalo, Buffalo, New York 14215 Wang, Tao, Robert M. Mentzer, Jr., and David Van Wylen. Interstitial adenosine with dipyridamole:
G. L.
effect
of adenosine receptor blockade and adenosine deaminase. Am. 1992.J. Physiol. 263 (Heart Circ. Physiol. 32): H&2-H558, Dipyridamole is proposed to increase coronary blood flow (CBF) by inhibition of adenosine uptake into cells, resulting in an increase in interstitial fluid (ISF) adenosine and an adenosine-mediated vasodilation. The purpose of this study was to determine the changes in CBF and ISF adenosine, inosine, and hypoxanthine during dipyridamole infusion in the absence or presence of adenosine receptor blockade or adenosine deaminase. To sample cardiac ISF, cardiac microdialysis probes were implanted in the left ventricular myocardium of chloralose-urethan-anesthetized dogs and perfused with Krebs-Henseleit buffer. The metabolite concentration in the effluent dialysate was used as an index of intramyocardial ISF metabolite concentration. In response to dipyridamole, CBF and dialysate adenosine concentration increased 4.4-fold and 2.2-fold, respectively, whereas dialysate inosine was unchanged and dialysate hypoxanthine decreased 50%. Adenosine receptor blockade, achieved by intracoronary 8(p-sulfophenyl)theophylline infusion, attenuated the increase in CBF induced by dipyridamole without changing dialysate adenosine concentration. Adenosine deaminase fully attenuated the dipyridamole-induced increases in CBF and dialysate adenosine. These results demonstrate that dipyridamole increases ISF adenosine in the dog and suggest that adenosine is the sole mediator of dipyridamole-induced coronary vasodilation. dog; coronary blood flow; 8(p-sulfophenyl)theophylline; dialysis; inosine; hypoxanthine
micro-
IS A WELL-KNOWN andpotentcoronary vasodilator (1,2,7,15). Based on evidence from myocyte cell cultures (8, 24,25), isolated perfused hearts (5,6,10, 11, 16, 23), and intact blood-perfused hearts (15, 17), it has been suggested that the mechanism of action of dipyridamole is due to the inhibition of adenosine uptake into cells, leading to an increase in interstitial fluid (ISF) adenosine levels and subsequent vasodilation. If this is the mechanism of action of dipyridamole, then 1) dipyridamole-induced increases in coronary blood flow (CBF) should be associated with increased ISF adenosine; 2) exogenously administered adenosine deaminase, the enzyme that converts adenosine to the vasoinactive metabolite inosine, should attenuate the vascular dilation induced by dipyridamole in proportion to the reduction of ISF adenosine; and 3) adenosine receptor blockade should attenuate the dipyridamole-induced increase in CBF without necessarily altering ISF adenosine. To test these hypotheses, we determined the changes in ISF adenosine, adenosine metabolites, and CBF during the administration of dipyridamole in the absence or presence of intracoronary infusion of either 8- (p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist, or adenosine deaminase. Cardiac ISF was sampled with the cardiac microdialysis technique, a DIPYRIDAMOLE
H552
0363-6135/92
$2.00 Copyright
Sciences,
recently developed technique that allows intramyocardial ISF to be sampled in the intact dog heart (32). METHODS Animal preparation. Adult mongrel dogs of either sex weighing 20-30 kg were premeditated with 3 mg/kg morphine and then anesthetized with 20 mg/kg thiamylal iv, followed by 70 mg/kg cu-D-chloralose and 200 mg/kg urethan iv. The animals were intubated and ventilated with a mixture of 100% oxygen and room air (model 613 dog respirator, Harvard Apparatus, South Natick, MA). Tidal volume, respiratory rate, and percent oxygen in the inspired air were adjusted to maintain normal blood gas and pH values (model 170 pH/blood gas analyzer, Ciba Corning Diagnostics, Medfield, MA). Core body temperature was monitored with an esophageal temperature probe and maintained with a heating pad at ~38’C. The right femoral artery was cannulated for the measurement of arterial blood pressure and the determination of arterial blood gases and pH. The right femoral vein was cannulated for infusion of fluid and anesthetic supplement when necessary. A thoracotomy was performed through the left fifth intercostal space, and the pericardium was incised to expose the left ventricle. The left anterior descending coronary artery (LAD) and the left circumflex coronary artery (LC) were dissected, and ultrasonic doppler flow probes (model T201 flowmeter, Transonics Systems, Ithaca, NY) were placed around each of the arteries for the measurement of CBF. For intracoronary infusions, a 22-gauge angiocatheter was inserted into the LAD proximal to the flow probe. The angiocatheter was positioned such that the tip was at least 1 cm distal to the branch point of the left main coronary artery. Cardiac microdialysis technique. The cardiac microdialysis technique has been described in detail elsewhere, including a discussion of the advantages, disadvantages, and limitations of this technique and a comparison of microdialysis to other techniques for sampling cardiac ISF (32). Briefly, each microdialysis probe consisted of a single 300-pm dialysis fiber, a platinum wire within the fiber, and two hollow silica tubes inserted, adjusted, and sealed within the dialysis fiber such that the distance between the ends of the silica tubes was 2 cm. In each animal, two microdialysis probes were implanted into the left ventricular myocardium, one in the region perfused by the LAD and the other in the region perfused by the LC. After insertion of the microdialysis probes, the inflow silica tube of each probe was connected via a larger silica tube to a gastight glass syringe filled with Krebs-Henseleit buffer and perfused at 2 pl/min (model CMAIOO microinjection pump, Bioanalytical Systems, West Lafayette, IN). The Krebs-Henseleit buffer consisted of (in mM) 118.0 NaCl, 25.0 NaHC03, 11.0 glucose, 4.7 KCl, 1.25 CaCl,, I.2 MgS04.7HZ0, and 1.2 KH,PO, and was bubbled with 5% CO,-95% N, before filling the syringe. This gave the Krebs-Henseleit buffer a PO,, Pco~, and pH of approximately 20 mmHg, 40 mmHg, and 7.35, respectively. The low PO, approximates tissue PO, levels, thus minimizing the supply or removal of oxygen locally by the microdialysis probes. The effluent, referred to as the dialysate, was collected from the outflow silica tube in small plastic tubes and frozen (-SOOC) until biochemical analysis.
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
ISF ADENOSINE
DURING
The theory of cardiac microdialysis is that as the KrebsHenseleit solution passes through the dialysis fiber, diffusion occurs between the fluid within the fiber and the ISF surrounding the fiber. The dialysate concentration is therefore used as an index of intramyocardial ISF concentration. Local myocardial blood flow (MBF) was measured from the area surrounding the dialysis fiber by the hydrogen clearance technique, using the platinum wire within the microdialysis probe to polarographically detect tissue hydrogen (36). MBF was calculated from the time required for the hydrogen levels to fall from 90% maximum to 40% using the formula MBF (ml. mine1 100 g-l) = [ln(40/90)]/time] X 100. Arterial blood pressure, heart rate, LAD and LC flow, and the hydrogen clearance curves from the LAD and the LC region were recorded on an eight-channel recorder (model 3800, Gould, Cleveland OH). Protocols. For all protocols, a minimum of 90 min was allowed for recovery from microdialysis probe implantation before initiation of the protocols, a time that has been shown to allow stabilization of dialysate adenosine (32). A preliminary group of animals (n = 4) was used to determine whether dipyridamole produced a sustained increase in dialysate adenosine. After control samples were obtained, a dipyridamole infusion (0.5 mg. kg-l h-l iv) was initiated. After a stable response to dipyridamole was achieved, six IO-min dialysate samples were collected. LAD flow was measured in two of these animals. Two series of experiments were performed (Fig. 1). The purpose of series 1 (Fig. IA) was to determine the effect of adenosine receptor blockade on ISF adenosine during dipyridamoleinduced increases in CBF. In these animals (n = 6), two IO-min dialysate samples were collected from each microdialysis probe during the control period. A dipyridamole infusion (0.5 mg*kg-l*h-l iv) was then started and continued throughout the remainder of the experiment. After a stable increase in CBF was achieved (- 10 min after beginning the dipyridamole infusion), three successive 5-min dialysate samples were collected. An intracoronary infusion into the LAD of 1 ml/min of either l
l
A SERIES
1
i;..:..l
+1 0’+10’-+5q-5’q-5’+
1 o-t-1
o’+lo~+l
Dialysate B SERIES
o*+lo’+
2a
Dipyridamole
- 0.5 mg/kg/hr
Dialysate C SERIES
oq+l
Samples
i.v.
H553
DIPYRIDAMOLE
normal saline or SPT, an adenosine receptor antagonist, was then initiated. Each SPT infusion, designated in Fig. 1A as SPTl (0.5 x 10m3 M), SPTz (1.0 x 10m3 M), and SPT, (5.0 x IO-” M), consisted of a greater concentration of the antagonist. Ten-minute dialysate samples were collected during the intracoronary SPT or normal saline infusion. The purpose of series 2 (Fig. 1, B and C) was to determine the effect of adenosine deaminase on the relationship between ISF adenosine and CBF during dipyridamole administration. As in series 1, dipyridamole (0.5 mg. kg-l h-l) was given intravenously after the collection of two lo-min control dialysate samples. After the stable response of CBF to dipyridamole was established and three 5-min dialysate samples were collected, adenosine deaminase was administered into the LAD. In series 2a (Fig. IB, n = 6), adenosine deaminase was given for -8 min at a dose of 5 U kg-l. min-l, the dose most frequently used by other investigators (22, 30). Because this dose produced a sustained CBF response in both LAD and LC regions, in series 2b (Fig. lC, n = 6), adenosine deaminase was infused at 0.5 for -60 min. In both series 2a and 2b, two 5-min U kg-l min-l dialysate samples followed by five IO-min dialysate samples were collected from each microdialysis probe after the initiation of the adenosine deaminase infusion. The dilution of adenosine deaminase was adjusted to deliver 0.5 or 5 U kg-l min-l at an infusion rate Cl.0 ml/min. AnaZytical procedures. The dialysate samples were analyzed for adenosine, inosine, and hypoxanthine using reverse-phase high-performance liquid chromatography (models 600E solvent delivery system, 484 detector, 712 injector, and 820 MAXIMA data analysis system; Waters Division of Millipore, Milford, MA). Separation of the compounds was achieved using a Supelcosil LC-18 column (Supelco, Bellefonte, PA) and a 1 (pH 5.3) to 25% (pH 5.58) methanol in 100 mM KH,PO, gradient. Adenosine, inosine, and hypoxanthine were detected by an absorbance change at 254 nm and were identified and quantified by comparing retention times and peak heights with known standards. Materials. The adenosine deaminase used was type VIII calf intestinal adenosine deaminase, prepared as a suspension in 3.2 M (NH&SOd, pH 6.0 (Sigma Chemical, St. Louis, MO). The adenosine deaminase was diluted in 0.14 M NaCl containing 0.012 M tris(hydroxymethyl)aminomethane (Tris) HCl (pH 7.5) to neutralize the (NH&SO4 and filtered (20 pm) before infusion into the LAD. SPT (Research Biochemicals, Natick, MA) was dissolved in normal saline, and dipyridamole (Sigma Chemical, St. Louis, MO) was dissolved in methanol (IO mg/ml). Statistical analysis. Mean t SE values were calculated for all data. For comparison of values obtained during control and different treatments, differences between mean values were determined by one-way analysis of variance followed by Duncan’s or Dunnett’s multiple range test for repeated measures. P < 0.05 was accepted as indication of a statistically significant difference. l
l
l
l
l
l
Samples
RESULTS
2b
Dipyridamole-induced changes in dialysate metabolites and CM?. Because the changes in dialysate metabolites
LAD Adenosine deaminase 0.5 U/kg/min
Dipyridamole 5+5’+5+5’+5’+1
- 0.5 mg/kg/hr O’-+l
Dialysate
o’+l
i.v.
O’-+
Samples
Fig. 1. Experimental protocols for series 1 and 2. SPT, 8-(p-sulfophenyl)theophylline; NS, normal saline; LAD, left anterior descending artery; ADA, adenosine deaminase. SPT concentrations: 0.5 x IO-” (SPT1), 1.0 x lo-” (SPT2), and 5.0 x 10e3 M (SPT3).
and CBF in response to dipyridamole were determined in all animals of series 1 and 2 (n = I@, these data have been pooled and are shown in Fig. 2. The dialysate sample chosen for graphic presentation in Fig. 2 is the middle of the three dialysate samples taken from the LAD-perfused region after initiation of the dipyridamole infusion and therefore represents changes during the steady-state response to dipyridamole. Dipyridamole, which caused an
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
ISF ADENOSINE
0 m
DURING
DIPYRIDAMOLE
CONTROL DIPYRIDAMOLE
* p < 0.05 n=18
vs. Control
-I-
IN0
ADO
CBF
CBF
HYPO
o!
Fig. 2. Changes in coronary blood flow (CBF) and dialysate adenosine (HYPO) with dipyridamole (ADO), inosine (INO), and hypoxanthine infusion (n. = 18). Data are from the LAD-perfused region. Values are means t SE; * P < 0.05 compared with control.
0
IO
20
30
40
50 60 (minutes)
70
80
E
40 50 60 TIME (minutes)
70
80
90
TIME
B
-4.4-fold increase in LAD flow, was associated with a 2.2fold increase in dialysate adenosine, no change in dialysate inosine, and a decrease in dialysate hypoxanthine. In these animals, mean arterial pressure decreased from 133 t 4 to 127 t 4 mmHg during dipyridamole infusion, while heart rate increased from 113 t 6 to 141 t 7 beats/ min. In the four animals in which dialysate adenosine levels were determined during 60 min of sustained dipyridamole, dialysate adenosine increased from a control level of 0.57 t 0.17 to 0.96 t 0.26,0.94 t 0.21,l.Ol & 0.24, 1.00 t 0.25, 0.95 t 0.24, and 0.88 t 0.21 PM at 5, 15, 25, 35,45, and 55 min of dipyridamole infusion, respectively. CBF increased in response to dipyridamole and remained elevated throughout the dialysate collection period in the two animals in which CBF was measured. Series 1. The hemodynamic data from series 1 experiments are shown in Table 1. Although mean arterial blood pressure was stable during dipyridamole infusion in series 1, heart rate increased significantly. Neither mean arterial pressure nor heart rate was altered during intracoronary infusion of SPT or normal saline. The CBF and dialysate metabolite changes from series 1 are shown in Table 1 and Fig. 3. CBF increased during dipyridamole infusion, as indicated both by the changes in LAD and LC flow (Fig. 3) and by hydrogen clearance
measurements of myocardial perfusion (Table 1). Although the changes of regional blood flow measured by hydrogen clearance paralleled qualitatively the changes in LAD and LC flow, the hydrogen clearance measurements of myocardial perfusion indicated a smaller increase in
Table 1. Changes in hemodynamic
blood flow, and dialysate
parameters,
myocardial
140,
3 I20 3 k loo 0 OF 80 $ *E $
60
2 o 5 CJ
40 2o
*
p < 0.05
vs. Control
nt “0
IO
30
20
Fig. 3. CBF and ADO results (means t SE) for series 1. All intracoronary infusions were at 1.0 ml/min into the LAD. A: data from LADperfused region. B: data from left circumflex artery (LC)-perfused region. See Fig. 1 for SPT1, SPT2, and SPT3 concentrations.
Series 1: Intracoronary
inosine
and hypoxanthine
SPT
Variable Control
Dipyridamole
SPTl
NS
SPT2
NS
SPT3
12128 119*7 120t7 121k7 120&8 132~17 124t7 MAP, mmHg 165tl2* 170t9* 170&B* 168tlO* 130t13 174t8* 175*7* HR, beats/min MBF, ml min-l 100 g-l 6426 111216 94t18 82t14 76t5 141&20* 109t23 LAD region 102t22 128tll* 127t7* 137t16* 140t17* 67t9 152t6* LC region Dialysate Ino, PM 0.57t0.11 0.77t0.10 0.59t0.13 0.98t0.34 0.65t0.26 0.65t0.20 0.58t0.11 LAD region 0.46t0.06 0.49kO.10 0.42t0.06 0.41t0.08 0.40t0.06 0.48t0.12 0.52t0.19 LC region Dialysate Hypo, PM 1.78t0.81 1.67t0.82 1.39rto.52 1.08t0.51 1.59kO.67 1.68t0.71 3.9321.15 LAD region 0.77t0.34 0.76kO.29 0.91t0.35 0.81t0.36 0.71t0.27 0.79t0.34 2.09t1.09 LC region Values are means t SE. SPT, 8-(p-sulfophenyl)theophylline; NS, normal saline; MAP, mean arterial pressure; HR, heart rate; MBF, myocardial blood flow; LAD, left anterior descending artery; LC, left circumflex artery; Ino, inosine; Hypo, hypoxanthine; ADA, adenosine deaminase. Concentrations of SPT: 0.5 x 10B3 (SPT1), 1.0 X 10e3 (SPT2), and 5.0 X low3 M (SPT3). * P < 0.05, compared with control. l
l
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
ISF ADENOSINE
DURING
H555
DIPYRIDAMOLE
blood flow than that in the LAD and LC probably because of a diffusion limitation of hydrogen at high flow. The dipyridamole-induced increase in CBF in the LAD-perfused region was attenuated by intracoronary SPT infusion, being almost totally suppressed by the highest concentration of SPT (Fig. 3A). The decline in LAD flow during the intervening intervals of normal saline infusion and the gradual decrease in LC flow after the initiation of SPT infusion into the LAD (Fig. 3B) likely reflect the systemic accumulation of SPT. Despite the attenuation of dipyridamole-induced increase in CBF by SPT, the elevated dialysate adenosine levels were not altered by intracoronary SPT infusion (Fig. 3), nor were there significant changes in dialysate inosine and hypoxs, thine concentrations during the SPT infusion (Tab’ 1). Series 2. The ht,_lodynamic parameters from series 2 experiments are shown in Table 2. In series 2a and 2b, mean arterial blood pressure and heart rate were not significantly altered during dipyridamole or during the subsequent adenosine deaminase infusion, although heart rate tended to increase after dipyridamole infusion. As shown in Fig. 44, adenosine deaminase, given for 4 min at 5 Us kg-l min-l (series 2a), completely and rapidly attenuated the dipyridamole-induced increase in LAD flow and sustained this attenuation up to 45 min after the adenosine deaminase infusion was stopped. In response to the LAD infusion of adenosine deaminase, the dipyridamole-induced increase of flow in the LC was also decreased to control levels (Fig. 4B). In both the LAD- and LC-perfused regions, the attenuation of the dipyridamole-induced increase in CBF was mirrored by reductions in dialysate adenosine concentra-
tion (Fig. 4). As shown in Table 2, dialysate inosine concentration increased after the start of the adenosine deaminase infusion, indicating that there was indeed enhanced deamination of adenosine. Dialysate hypoxanthine, which was decreased in response to dipyridamole, was not altered by adenosine deaminase infusion. In series 2b, in which adenosine deaminase was infused into the LAD at 0.5 U kg-l emin-l, the dipyridamoleinduced increases in CBF and dialysate adenosine were gradually attenuated in both the LAD- and LC-perfused regions (Fig. 5 and Table 2). Although dialysate inosine levels tended to increase during the administration of adenosine deaminase, these changes were not statistically significant. Dialysate hypoxanthine levels were stable during adenosine deaminase infusion (Table 2). Based on the assumption that dialysate adenosine concentration is a reflection of intramyocardial ISF adenosine levels, the major findings of the present study were 1) dipyridamole induced a concomitant increase in ISF adenosine and CBF, 2) adenosine deaminase administration attenuated the dipyridamole-induced increase in CBF simultaneous with a reduction in ISF adenosine, and 3) adenosine receptor blockade attenuated the dipyridamole-induced increase in CBF without altering ISF adenosine levels. The dipyridamole-induced increase in ISF adenosine we observed with cardiac microdialysis is consistent with that seen with other techniques to estimate ISF adenosine levels. Using the pericardial infusate technique, Knabb et al. (17) observed almost a threefold increase in pericardial infusate adenosine levels after dipyridamole
Table 2. Changes in hemodynamic
blood flow, and dialysate
DISCUSSION
l
Variable
Control
parameters,
myocardial
Dipyridamole
Series 2a: intracoronary
MAP, mmHg HR, beats/min MBF, ml mine1 . 100 g-l LAD region LC region Dialysate Ino, PM LAD region LC region Dialysate Hypo, PM LAD region LC region
5-Min Post-ADA
ADA
ADA,
5 U- kg-l.
inosine
and hypoxanthine
15Min Post-ADA
35-Min Post-ADA
55-Min Post-ADA
13Ok7 125tl9
min-l
126t7 137210
129t6 135tl4
12827 129k17
130t7 127tl9
130t7 127tl8
881tl4 79&B
197k28* 194tl2*
161227 15ltl6*
117tl9 12ltl4
103k24 102kll
105tlO 102tll
0.99t0.40 l.lBt0.28
0.94t0.28 1.06kO.16
3.lOtl.35* 1.68t0.46
2.17kO.30 2.76&1.13*
1.841kO.51 1.36kO.56
1.26kO.36 1.3lt0.48
1.16t0.32 0.621kO.32
2.3220.71 2.81k0.82
1.03t0.22* 1.15kO.19
l.llt0.35 0.9lt0.18*
1.26kO.40 1.53rizO.86
l.lOt0.26 1.76rtO.76
1.06t0.21 1.19kO.54
0.9lt0.17 0.7OkO.17
132t8 112tl2
l
Variable
Control
Dipyridamole
Series 2b: intracoronary
5-Min
ADA,
ADA
0.5 U. kg-l
15-Min l
ADA
35-Min
ADA
97k22 86klO
55-Min
ADA
rnin-l
MAP, mmHg 135k6 13Ok4 128t3 129t4 130t5 HR, beats/min 98tl2 113t8 109t7 107&B 107t9 MBF, ml - min-l 100 g-l LAD region 120tl4 187&22* 153224 12ltl6 113tlO 226tlO* LC region 92tl8 165tl4* 108tlO 96tl3 Dialysate Ino, I,~M LAD region 0.75t0.18 0.7lkO.12 0.85&O. 19 1.04t0.17 0.9lrtO.06 0.6520.28 0.63t0.05 0.66&O. 10 0.75&O. 13 0.90~0.10 LC region Dialysate Hypo, PM LAD region 2.28k0.34 1 .OBtO. 14* 0.92&O. 18* 1.07t0.32* 0.84&O. 18* LC region 2.01t0.73 0.85&O. 14* 0.74t0.13* 0.74t0.17* 0.67&O. 14* Values are means t SE. See Table 1 for abbreviations and text for further details. * P < 0.05 compared with control.
130t5 105t9
l
113tlO 83kl2 0.89t0.10 0.95t0.01 O.Blt0.16* 0.72t0.17*
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
H556
ISF ADENOSINE
1 *
0
20
40 60 TIME (minutes)
p < 0.05
DURING
DIPYRIDAMOLE
vs. Control
80
100
40 60 TIME (minutes)
0
80
100
B 180 240
II
17 U/kg/mi;l
160
*
140 3 0ii
120
E
100
F d-g zg 2 2 0 0 0
80 60 40 20
P 0
20
40 60 TIME (minutes)
80
100
0
20
40 TIME (minutes)
60
80
Fig. 4. CBF and ADO results (means t SE) for series 2~. ADA was infused into the LAD at 5 U kg-l l rein-? A: data from LAD-perfused region; B: data from LC-perfused region. * P < 0.05 compared with control.
Fig. 5. CBF and ADO results (means 2 SE) for series Zb. ADA was infused into the LAD at 0.5 Us kg-l. min-l. A: data from LAD-perfused region; B: data from LC-perfused region. * P < 0.05 compared with control.
administration in the anesthetized dog. Based on epicardial exudate adenosine levels in isolated perfused guinea pig hearts, Decking et al. (10) and Mohrman and Heller (23) have estimated a 2- to lo-fold increase in ISF adenosine during dipyridamole infusion. Finally, using a multiple-indicator dilution technique to extrapolate from venous adenosine levels to ISF adenosine concentration in the isolated perfused guinea pig heart, Wangler et al. (35) calculated that in response to dipyridamole, ISF adenosine increased from 6.5 to 191 nM during constantpressure perfusion and to 88 nM during constant-flow perfusion. Taken together with our data, these reports leave little doubt that dipyridamole does indeed lead to an increase in cardiac ISF adenosine concentration. Despite the consistency with which dipyridamole has been shown to be associated with enhanced ISF adenosine levels, the precise mechanism by which dipyridamole leads to elevated ISF adenosine levels is not known with certainty. Several lines of evidence document the ability of dipyridamole to inhibit the uptake of exogenous adenosine into cells. In cultured myocytes, dipyridamole virtually eliminates adenosine uptake from the medium (24, 25), whereas in isolated perfused hearts and intact hearts,
dipyridamole inhibits the uptake of exogenous adenosine from the vascular space into the myocardium (2, 9, 18). Interestingly, whereas guinea pig, dog, and human are very responsive to dipyridamole, the rat is relatively unresponsive to dipyridamole (7, 15, 19). The more difficult issue relates to the action of dipyridamole on endogenous adenosine transport. The major unresolved issue is whether dipyridamole acts as a symmetrical inhibitor of adenosine transport, in which case it would inhibit both adenosine release and uptake, or whether dipyridamole selectively inhibits adenosine uptake into cells without having a major effect on adenosine release. Based on experiments by Mustafa et al. (24, 25) on cultured myocytes, it was suggested that dipyridamole leads to enhanced ISF adenosine by inhibiting the uptake of adenosine into the myocyte without affecting release. This concept received additional support from Bukoski and Sparks (8), who showed that dipyridamole led to enhanced accumulation of adenosine in the medium of isolated adult cardiac myocytes. However, the idea that dipyridamole selectively inhibits adenosine uptake was recently challenged by Meghji et al. (21), who found that
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
ISF ADENOSINE
DURING
dipyridamole reduced the efflux of adenosine from primary cultures of chick ventricular myocytes by 70-90%, leading to an increase in the amount of adenosine trapped inside the cell. If dipyridamole inhibits the release of adenosine from cells in vivo, then it is difficult to reconcile with the increase in CBF and with the data demonstrating increased ISF adenosine during dipyridamole. The resolution of the mechanism of dipyridamole action is likely related to another unsettled issue, namely whether adenosine is produced intracellularly by a cytosolic 5’-nucleotidase (28) or by a membrane-bound ecto5’-nucleotidase, which has been suggested to be an enzyme carrier complex (12). It is likely that, in vivo, adenosine is produced both intracellularly by dephosphorylation of intracellular adenosine 5’-monophosphate (AMP) and extracellularly by either the enzyme carrier complex or by the degradation of extracellularly released ATP (29). In this context, dipyridamole could inhibit the transport of intracellularly formed adenosine out of the cell and yet, by inhibiting the uptake of extracellularly formed adenosine, lead to enhanced ISF adenosine. However, the details of dipyridamole action have yet to be definitively resolved in the intact heart. Although the increase in ISF adenosine during dipyridamole was expected, we anticipated that this would be associated with reductions in the ISF concentrations of both inosine and hypoxanthine, indicative of the reduced availability of intracellular adenosine for catabolism to inosine and hypoxanthine. However, our data indicated that while ISF hypoxanthine was reduced during dipyridamole, ISF inosine did not change. It is possible that this reflects differences in sites and biochemical pathways for production of inosine and hypoxanthine. Hypoxanthine is produced from inosine by nucleoside phosphorylase, an enzyme believed to be located predominantly in the endothelial cells (20). Because dipyridamole inhibits inosine uptake into cells (I@, the lower ISF hypoxanthine may be a result of reduced uptake of inosine from the ISF into endothelial cells. The production of inosine, however, is more complex. Inosine can not only be produced from adenosine by adenosine deaminase, but also by the dephosphorylation of inosine 5’monophosphate (IMP). Furthermore, there is currently some debate as to whether adenosine deaminase is restricted to pericytes and endothelial cells (27, 31) or whether it also exists in myocytes (4) and the ISF (3). Therefore, the lack of change in ISF inosine during dipyridamole may be the result of alternate biochemical pathways for inosine production as well as multiple compartments where adenosine deaminase is located. Nevertheless, our data in the intact heart do differ from results of isolated cells, where dipyridamole causes a reduction in extracellular inosine (25). The ability of adenosine receptor blockade and adenosine deaminase to completely reverse the dipyridamoleinduced increase in CBF has been observed by others (5, 15). Our data confirm these observations and in addition show that the attenuation caused by SPT occurs without altering ISF adenosine, while the reduction in the dipyridamole-induced increase in CBF brought about by adenosine deaminase occurs simultaneously with the re-
H557
DIPYRIDAMOLE
duction in ISF adenosine. These observations are consistent with the data of Becker et al. (5), who demonstrated in the isolated perfused guinea pig heart that theophylline blocked the increase in CBF induced by dipyridamole without preventing the enhanced release of adenosine into the venous effluent, whereas adenosine deaminase prevented both the increased CBF and increased venous adenosine concentration associated with dipyridamole. These data and our data seem to leave little doubt that adenosine is the sole mediator of the dipyridamole-induced increase in CBF. It has recently been shown that the vasodilatory response to exogenous adenosine occurs without an increase in ISF adenosine (13, 14, 34). This and data from isolated vessels have introduced an important role of the vascular endothelium in mediating the vasodilatory response to intravascular adenosine (26). If dipyridamole increases plasma adenosine levels, it is possible that the vascular relaxation in response to dipyridamole is mediated in part by the vascular endothelium. Because plasma adenosine was not measured in the present study, we are not able to evaluate the potential contribution of the vascular endothelium to the response to dipyridamole. However, it is important to note that our results with adenosine receptor blockade and adenosine deaminase do not distinguish between elevated plasma adenosine acting on the endothelium or elevated ISF adenosine acting directly on the vascular smooth muscle. It is also of interest that dipyridamole has been shown to relax isolated coronary artery strips (33). Thus it is possible that dipyridamole may also act on a pool of adenosine produced locally within the vasculature itself. In summary, with the use of the cardiac microdialysis technique to sample intramyocardial ISF in the intact dog heart, the results of the present study indicate that dipyridamole-induced increases in CBF are accompanied by increased ISF adenosine. In addition, the observation that adenosine receptor blockade attenuated the CBF response to dipyridamole without altering ISF adenosine, whereas adenosine deaminase reduced both the CBF response and ISF adenosine, demonstrates that adenosine is the sole mediator of dipyridamole-induced coronary vasodilation. We gratefully acknowledge the technical assistance of Roy Fiorella and Vickie Armenia. This work was presented at the 74th Annual Meeting of the Federation of American Societies for Experimental Biology, Washington, DC, April 1-5, 1990. This work was supported by National Heart, Lung, and Blood Institute Grants HL-40878, HL-34579, and HL-46027. Address for reprint requests: D. G. L. Van Wylen, Dept. of Physiology, State Univ. of New York at Buffalo, 462 Grider St., Buffalo, NY 14215. Received
3 June 1991; accepted in final form 31 March
1992.
REFERENCES Afonso, S. Inhibition of coronary vasodilating action of dipyridamole and adenosine by aminophylline in the dog. Circ. Res. 26: 743-752, 1970. Afonso, S., and G. S. O’Brien. Mechanism of enhancement of adenosine action by dipyridamole and lidoflazine in dogs. Arch. Int. Pharmacodyn. Ther. 194: 181-186, 1971. Andy, R. J., and R. Kornfield. The adenosine binding protein of human skin fibroblasts is located on the cell surface. J. Biol. Chem. 257: 7922-7925, 1982.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
H558
ISF ADENOSINE
DURING
4. Arch, J. R. S., and E. A. Newsholme. The control of the metabolism and the hormonal role of adenosine. Essays Biochem. 14: 82-123, 1978. 5. Becker, B. F., H. Bardenheuer, I. Overhage de Reyes, and E. Gerlach. Effects of theophylline on dipyridamole induced coronary venous adenosine release and coronary dilation. In: Adenosine: Receptors and Modulation of Cell Function, edited by V. Stefanowich, K. Rudolphi, and P. Schubert. London: IRI Press Limited, 1985, p. 441-449. 6. Bellardinelli, L., R. A. Fenton, A. West, J. Linden, J. S. Althans, and R. M. Berne. Extracellular action of adenosine and the antagonism by aminophylline on the atrioventricular conduction of isolated perfused guinea pig and rat hearts. Circ. Res. 51: 569-579, 1982. 7. Brown, B. G., M. A. Josephson, R. B. Peterson, C. D. Pierce, M. Wong, H. S. Hecht, E. Bolson, and H. T. Dodge. Intravenous dipyridamole combined with isometric handgrip for near maximal acute increase in coronary flow in patients with coronary artery disease. Am. J. Cardiol. 48: 1077-1085, 1981. 8. Bukoski, R. D., and H. V. Sparks, Jr. Adenosine production and release by adult rat cardiocytes. J. Mol. Cell. Cardiol. 18: 595-605, 1986. 9. Bunag, R. D., C. R. Douglas, S. Imai, and R. M. Berne. Influence of a pyrimidine derivative on deamination of adenosine by blood. Circ. Res. 15: 83-88, 1984. 10. Decking, U. K. M., E. Juengling, and H. Kammermeier. Interstitial transudate concentration of adenosine and inosine in rat and guinea pig hearts. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): Hll25-Hl132, 1988. R. Rubio, and R. M. 11. Degenring, F. H., R. R. Curnish, Berne. Effect of dipyridamole on coronary blood flow in hypoxia and reactive hyperemia in the isolated perfused guinea pig heart. J. Mol. Cell. Cardiol. 8: 877-888, 1976. 12. Frick, G. P., and J. M. Lowenstein. Vectorial production of adenosine by 5’-nucleotidase in the perfused rat heart. J. Biol. Chem. 253: 1240-1244, 1978. 13. Gidday, J. M., R. Rubio, and R. M. Berne. Increases in coronary blood flow by intracoronary adenosine occur independent of changes in interstitial fluid adenosine. Physiologist 30: 187, 1987. 14. Heller, L. J., and D. E. Mohrman. Estimates of interstitial adenosine from surface exudates of isolated rat hearts. J. Mol. Cell. Cardiol. 20: 509-523, 1988. 15. Hintze, T. H., and S. F. Vatner. Dipyridamole dilates large coronary arteries in conscious dogs. Circulation 68: 1321-1327, 1983. 16. Hopkins, S. V. The potentiation of the action of adenosine on the guinea pig heart. Biochem. Pharmacol. 22: 341-348, 1973. 17. Knabb, R. M., J. M. Gidday, S. W. Ely, R. Rubio, and R. M. Berne. Effects of dipyridamole on myocardial adenosine and active hyperemia. Am. J. Physiol. 247 (Heart Circ. Physiol. 16): H804-H810, 1984. 18. Kolassa, N., K. Peleger, and W. Rummei. Specificity of adenosine uptake into the heart and inhibition by dipyridamole. Eur. J. Pharmacol. 9: 265-268, 1970. 19. Kolassa, N., K. Peleger, and M. Tran. Species differences in action and elimination of adenosine after dipyridamole and hexobendine. Eur. J. Pharmacol. 13: 320-325, 1971.
DIPYRIDAMOLE 20. Manfredi, J. P., and E. W. Holmes. Purine salvage pathways in myocardium. Annu. Rev. Physiol. 47: 691-705, 1985. 21. Meghji, P., R. Rubio, and R. M. Berne. Intracellular adenosine formation and its carrier-mediated release in cultured embryonic chick heart cells. Life Sci. 43: 1851-1859, 1988. 22. Merrill, G. F., H. F. Downey, and C. E. Jones. Adenosine deaminase attenuates canine coronary vasodilation during systemic hypoxia. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): H579-H583, 1986. D. E., and L. J. Heller. Transcapillary adenosine 23. Mohrman, transport in isolated guinea pig and rat hearts. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H772-H783, 1990. 24. Mustafa, S. J. Effects of coronary vasodilator drugs on the uptake and release of adenosine in cardiac cells. Biochem. Pharmacol. 280: 2617-2634, 1979. 25. Mustafa, S. J., R. Rubio, and R. M. Berne. Adenosine metabolism in cultured chick-embryo heart cells. Am. J. Physiol. 228: 1474-1478, 1975. 26. Nees, S. Coronary flow increases induced by adenosine and adenine nucleotides are mediated by the coronary endothelium: a new principle of the regulation of coronary blood flow. Eur. Heart J. 10, Suppl. F: 28-35, 1989. 27. Nees, S., and E. Gerlach. Adenosine nucleotide and adenosine metabolism in cultured coronary endothelial cells: formation and release of adenosine compounds and possible functional implications. In: Regulatory Function of Adenosine, edited by R. M. Berne, T. W. Rall, and R. Rubio. The Hague: Martinus/Nijhoff, 1983, p. 347-355. 28. Newby, A. C., Y. Worku, and C. A. Holmquist. Adenosine formation: evidence for a direct biochemical link with energy metabolism. Adv. Cardiol. 6: 273-284, 1985. 29. Pelleg, A., and G. Burnstock. Physiological importance of ATP released from nerve terminals and its degradation to adenosine in humans. Circulation 82: 2269-2272, 1990. 30. Saito, D., C. R. Steinhart, D. G. Nixon, and R. A. Olsson. Intracoronary adenosine deaminase reduces canine myocardial reactive hyperemia. Circ. Res. 49: 1262-1267, 1981. 31 Schrader, W. P., and A. W. Clinton. Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit heart: implications for adenosine metabolism. Circ. Res. 66: 754762, 1990. D. G. L., J. Wills, J. Sodhi, R. J. Weiss, R. D. 32 Van Wylen, Lasley, and R. M. Mentzer, Jr. Cardiac microdialysis to estimate interstitial fluid adenosine and coronary blood flow. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): Hl642-1649, 1990. P., and E. Bassenge. Wirking von ATP, A-3,5-MP, 33. Walter, adenosin und dipyridamol an Streifenpraparaten der a. coronaria, a. renalis und der v. portae. Pfluegers Arch. 299: 52-65, 1968. T., R. M. Mentzer, Jr., and D. G. L. Van Wylen. 34. Wang, Changes in cardiac interstitial fluid adenosine during intracoronary adenosine infusion: effect of dipyridamole and erythro-9-(2hydroxy-3-nonyl)adenine (EHNA). FASEB J. 5: 1396, 1991. R. D., M. W. Gorman, C. Y. Wang, D. F. Dewitt, 35. Wangler, I. S. Chan, J. B. Bassingthwaighte, and H. V. Sparks. Transcapillary adenosine transport and interstitial adenosine concentration in guinea pig hearts. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H89-H106, 1989. 36. Young, W. Hz clearance measurement of blood flow: a review of techniaue and polarogranhic nrincinles. Stroke 11: 552-564. 1980.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (132.210.236.020) on January 14, 2019.
