Adenosine Is a Pulmonary Vasodilator in Newborn Lambs'?
G. GANESH KONDURI, LAINE L. WOODARD, ARINDAM MUKHOPADHYAY, and DEVENDRA R. DESHMUKH
Introduction
Persistent pulmonary hypertension of the newborn is a condition associated with high morbidity and mortality, The affected infants have an increased pulmonary vascular resistance and hypoxemia secondary to right to left shunting of blood across fetal channels (1). However, the factors that maintain the pulmonary vascular tone in the newborn or the factors that regulate the pulmonary vascular response to hypoxia in the newborn are not wellunderstood. Adenosine, a purinergic nucleoside, has been shown to be a potent vasodilator in the coronary, cerebral, and splanchnic vascular beds in adult animals and is regarded as an endogenous modulator of blood flow to these organs (2-4). Tissue levels of adenosine increase during hypoxia, and adenosine is known to cause reactive hyperemia and hypoxia-induced vasodilation in systemicvascular beds (5). However, hypoxia constricts pulmonary vessels, a response that is different from that of systemicvessels. Weproposed that pulmonary vasoconstriction in response to hypoxia is in part mediated by decreased release of adenosine into the pulmonary circulation and that infusion of exogenous adenosine into pulmonary circulation will decrease the pulmonary vascular resistance to baseline values. Because adenosine appears to be rapidly metabolized by vascular endothelium (6), wealso proposed that intravenous infusion of adenosine in newborn animals would produce a selective pulmonary vasodilation and that this effect would be mediated by theophylline-sensitive P 1 purinergic receptors, as described in other vascular beds (3, 4). The objective of our experiments, therefore, was to investigate the pulmonary vascular effects of adenosine in newborn lambs during conditions of normoxemia and acute pulmonary hypertension induced by alveolar hypoxia. We measured the changes in plasma adenosine levels in pulmonary artery and left atrium with alveolar hypoxia and during the infusion of exogenous adenosine. We also investigated the effect of ami670
SUMMARY We Investigated the systemic and pulmonary vascular effects of adenosine and determined plasma adenosine levels In pulmonary circulation In 12 newborn lambs during normoxla and during alveolar hypoxia (10% O2 , 5% C0 2t and 85% N2) . Lambs were Instrumented at 7 days of age with catheters In the descending aorta, main pulmonary artery, and right and left atria, and a flow transducer around the main pulmonary artery, and were studied following a 3-day recovery. Adenosine or an equal volume of normal saline (control) was Infused Into the right atrial line In doses ranging from 0.01 to 2.5 Ilmollkg/mln. In normoxlc lambs, adenosine caused a significant decrease In pUlmonary vascular resistance and Increase In heart rate In doses of 0.15 to 2.5 Ilmollkg/mln and a decrease In systemic vascular resistance, with Increase In cardiac output In doses of 0.3 to 2.5 Ilmollkg/mln. Baseline plasma adenosine levels In pulmonary artery and left atrium decreased significantly during alveolar hypoxia. Adenosine Infusion In hypoxic lambs caused decreases In pulmonary artery pressure and pulmonary vascular resistance at all the doses tested. Aortic pressure and systemic vascular resistance decreased, and heart rate and cardiac output Increased at doses ~ 0.3 Ilmollkg/mln In hypoxic lambs during adenosine Infusion. The pulmonary vascular effects of adenosine in hypoxic lambs were attenuated by prior treatment of animals with aminophylline. Thus, adenosine appears to be an Important regUlator of pulmonary vascular response to hypoxia In newborn lambs. Its vasodilator effects were specific for pulmonary circulation when It was Infused In doses"," 0.15 Ilmollkg/mln Into the right atrium and appear to be mediated by PI purlnerglc receptors. AM REV RESPIR DIS 1992; 146:670-676
nophylline on the pulmonary vascular effects of adenosine in lambs during hypoxia. We chose newborn lamb as a model because its pulmonary circulation has been studied extensively in the past, and its response to hypoxia has been documented well (7). We used chronically instrumented animals for our studies because it allows us to study the pulmonary circulation without the effects of surgery or anesthesia. Methods Eighteen lambs were studied between the ages of 10and 16 days. 1\velveof these lambs were studied during conditions of normoxia and hypoxia to determine the effects of incremental doses of adenosine. Six additional lambs were studied during hypoxia to determine the effect of aminophylline on the response to adenosine. Lambs were brought into our laboratory at 1 day of age and housed in separate Plexiglas cages. They were trained to feed ad libitum from an artificial feeding system that contained goat's milk (Irish Hills Farm natural goat's milk, Mason, MI). Lambs adjusted well to the feeding system and gained weight at the rate of 50 to 75 g/kg/day prior to surgery.
Surgical Procedure Each lamb underwent one sterile surgical procedure at 7 days of age. Lambs were given preanesthetic medication consisting of atro-
pine sulfate (0.01 mg/kg SC) and ketamine HCI (10 mg/kg intramuscular). We then intubated each animal's trachea with a cuffed endotracheal tube and maintained anesthesia by ventilating the lungs with 1to 2070 isoflurane and oxygen. We monitored rectal temperature and electrocardiogram continuously and arterial blood gases periodically during surgery and maintained the core temperature at or close to 39° C with a heating pad. We made an incision in the left groin and inserted catheters into the descending aorta and inferior vena cava via the femoral artery and femoral vein. We then performed a left lateral thoracotomy and inserted catheters by direct puncture into the main pulmonary artery and right and left atria. We implanted an ultrasonic flow transducer (size 12S; 'fransonic Systems Inc., Ithaca, NY) around the main pulmonary artery to measure the cardi-
(Received in original form March 27, 1991 and in revised form February 18, 1992) 1 From the Departments of Pediatrics, Children's Hospital of Michigan, Hutzel Hospital, and Wayne State University School of Medicine, Detroit, Michigan. 2 Supported by a grant from the American Heart Association of Michigan. 3 Correspondence and requests for reprints should be addressed to G. Ganesh Konduri, M.D., Department of Pediatrics, Hutzel Hospital, 4707 St. Antoine Boulevard, Detroit, MI 48201.
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ADENOSINE AND PULMONARY VASODILATION
ac output. We identified and ligated the ductus arteriosus and tunneled the catheters and flow transducer cable to the lamb's back. We allowed the animal to recover from surgery for at least 3 days before experiments were done. Cardiovascular function and oxygen consumption were shown to return to normal by 3 days after thoracotomy in lambs (8). Antibiotics (procaine penicillin 50,000 U/kg and gentamicin 5 mg/kg intramuscular) were administered on the day of surgery and on each postoperative day until the studies were completed.
Experimental Protocol Wesecured each lamb in a sling and connected the vascular catheters to strain gauge manometers (model P23XL; Spectramed Inc., Oxnard, CA) and the flow transducer to an ultrasonic transit time blood flowmeter (Transonic Systems Inc., Ithaca, NY). The aortic, pulmonary arterial, left and right atrial pressures, and pulmonary blood flow wererecorded on a Grass model7D polygraph (Grass Instruments, Quincy, MA). From these data, we calculated the pulmonary vascular resistance as the difference between mean pulmonary arterial and left atrial pressures divided by cardiac output in L/kg of body weight. Similarly, systemic vascular resistance was calculated as the difference between mean aortic and right atrial pressures divided by cardiac output in L/kg. We studied each lamb during conditions of normoxia and hypoxia. For both normoxia and hypoxia studies, data were obtained with an infusion of normal saline to serve as a control for the data obtained with the infusion of adenosine. Each lamb, therefore, had four experiments done: control and adenosine studies during both normoxia and hypoxia. Each of the four experiments was done on a different day, and the order of these experiments was randomized for each animal.
Drug Preparation Adenosine (Sigma Chemical Co., St. Louis, MO) was obtained in a powder form, and the solution for intravenous administration was prepared each day by mixing it with sterile normal saline with a final concentration of 4 umol/ml of adenosine.
Assay for Adenosine Adenosine was assayed by a method described by Von Borstel and coworkers (9). Blood samples were collected in volumes of 0.5 ml and transferred immediately to tubes containing 0.5 ml of trichloroacetic acid kept at 0 to 4 0 C. The samples were immediately centrifuged, and the supernatant was frozen at - 20 0 C. Adenosine in supernatant was assayed by high-pressure liquid chromatography (HPLC) (Waters 820 system equipped with an autosampler and maxima software), using a Waters micro-Bondapak C-18reversed-phase column. An isocratic method was used to elute adenosine (10070 methanol in 10mM potassium phosphate, pH 5.8) at a flow rate of 1.6
ml/min. Adenosine was detected by UV absorbance at 254 nm (Waters 481 UV detector). The plasma concentration of adenosine was expressedas nmol/ml. The intraassay and interassaycoefficients of variation determined for adenosine standard solutions of 100, 500, and 1,000nm/ml were 0.3070 and 0.5% respectively. The smallest detectable amount of adenosine was 1 nmol/ml.
Protocol for Normoxia Experiments For each experiment, we placed the animal in a sling and recorded the hemodynamic variables when the animal was awake and resting quietly. Wegave an infusion of adenosine into the right atrial line starting at a dose of 0.01 umol/kg/min with a Harvard infusion pump (Harvard Apparatus, South Natick, MA). We measured the variables after the animals receivedthe infusion for 10min. Then we doubled the infusion rate for 10 min to give the animal 0.02, 0.04, 0.08, 0.15, 0.30, 0.60, 1.20, and 2.50 umol/kg/min of adcnosine. Wemeasured hemodynamic variables at the end of each 1O-min infusion. The halflife for adenosine in blood was shown to be less than 10 s (10), so that 10 min would be adequate time to achieve steady-state levels of the nucleotide in plasma. Control experiments were done on a different day with infusion of equal volumes of normal saline.
Protocol for Hypoxia Experiments Wemeasured hemodynamic variables, and arterial blood gases during normoxia. We then induced alveolar hypoxia by placing a loosely fitting plastic bag over the animal's head and allowing it to breathe a gas mixture of 10% 0 1 , 5% Cal' and 85% N 1 • Lambs frequently hyperventilate during hypoxia and lower their Paco1 , which tends to attenuate the pulmonary vascular response to hypoxia (11). We added 5% Cal to the gas mixture to maintain the Paco1 stable and at or close to 40 mm Hg (5.3 kPa). Variables were measured after the animals had stable hypoxemia for 10 min. Adenosine was then infused into the right atrial line, in doses of 0.01 to 2.5 umol/ kg/min, as described previously for normoxia experiments. Each infusion rate was maintained for 10 min, and variables were measured at the end of each 10 min period. Samples of arterial blood were drawn at each infusion rate for determination of pH and blood gas tensions. Blood samples for plasma adenosine levels were drawn from pulmonary artery and left atrium during normoxia, hypoxia, and at each infusion rate of adenosine in eight out of 12 lambs that were studied. Control experiments were done on a different day with infusion ofequal volumes of saline. The plasma adenosine levels were measured only during studies done with infusion of adenosine. The volume of blood withdrawn at each time point was 0.2 ml for the control experiment and 1.2 ml for the adenosine experiment. The total amount of blood withdrawn, therefore, was 15 to 16 ml for both experiments and represented only 3%
of the blood volume taken over a 2-day period. (The weight of the study animals was 5.4 ± 0.8 kg, and blood volume was assumed to be 85 ml/kg.)
Protocol for Experiments to Investigate the Mechanism of the Effects of Adenosine Six lambs were studied twice each on separate days, and the order of the experiments was randomized for each animal. For each experiment, baseline variables were recorded when the animal was breathing room air. Alveolar hypoxia was induced by allowing the animals to breathe a gas mixture of 10% 0 1 , 5% CO 2 , and 85% N1 • The animals werethen given a bolus dose of either aminophylline 7 mg/kg or an equal volume of saline into the right atrial line, and variables wererecorded 30 min after the bolus, with ongoing hypoxia. Adenosine was then infused into the right atrial line in doses of 0.01 to 2.5 umol/kg/min, as described previously for other experiments. Each infusion rate was maintained for 10 min and variables were recorded at the end of each lO-min period.
Statistical Analysis All data are expressed as mean ± SD. The data obtained during normoxia and during hypoxia alone from different days on each lamb were compared by single factor analysis of variance (ANOVA) for repeated measures to determine if baseline variables and the response to hypoxia were similar on different days. Data obtained during control and adenosine experiments were compared by twoway ANOVA for repeated measures - the two factors affecting the outcome being adenosine versus saline and the dose of adenosine that was infused. When significant differences, defined as a p value of < 0.05 were found, a Student-Newman-Keuls multiplerange test was done to determine which means were different (12). The data obtained during hypoxia studies and studies done to determine the mechanism of the hemodynamic effects of adenosine were analyzed similarly using two-way ANOVA for repeated measures and a Student-Newman-Keuls test.
Results
Baseline variables measured on different days werecomparable and showed no significant differences. Normoxic lambs had no significant changes in the hemodynamic variables during the control experiments done with infusion of saline (table 1). Adenosine caused a significant decrease in pulmonary vascular resistance in doses of 0.15to 2.5 umol/kg/min and systemic vascular resistance in doses of 0.3 to 2.5 umol/kg/min. Pulmonary arterial, and left and right atrial pressures did not change during adenosine infusion, and the aortic pressure decreased with infusion rates of 0.6 to 2.5 umol/kg/
672
KONDURI, WOODARD, MUKHOPADHYAY, AND DESHMUKH
TABLE 1 HEMODYNAMIC VARIABLES MEASURED DURING NORMOXIA AND INFUSION OF ADENOSINE (AD) OR NORMAL SALINE (NS) Infusion Rates of AD or NS (JJmol/kg/min) Drug
Baseline
0.01
0.04
0.08
0.15
0.30
0.60
2.50
Heart rate, bpm
NS AD
179 ± 13 178 ± 17
176 ± 16 184 ± 13
174 ± 17 182 ± 19
174 ± 21 203 ± 21
181 ± 21 211 ± 22*
190 ± 19 236 ± 20*
188 ± 17 241 ± 19*
180 ± 22 220 ± 21*
Cardiac output, mllkg/min
NS AD
192 ± 22 198 ± 24
196 ± 24 203 ± 19
189 ± 26 204 ± 26
190 ± 22 217 ± 22
192 ± 17 227 ± 20
200 ± 16 272 ± 19*
204 ± 17 277 ± 31*
207 ± 19 294 ± 34*
Aortic pressure, mm Hg
NS AD
69 ± 7 70 ± 6
71 ± 8 68 ± 7
70 ± 6 66 ± 5
68 ± 7 68 ± 6
66 ± 7 65 ± 6
70 ± 6 64 ± 7
66 ± 5 55 ± 6*
68 ± 6 50 ± 5*
Right atrial pressure, mm Hg
NS AD
3 ± 1 2 ± 1
3 ± 1 3 ± 2
3 ± 1 2 ± 1
4 ± 1 3 ± 1
3 ± 2 3 ± 1
2 ± 1 3 ± 1
4 ± 1 3 ± 2
5±2 3 ± 1
Pulmonary artery pressure, mm Hg
NS AD
13 ± 3 14 ± 4
12 ± 2 12 ± 3
13 ± 2 14 ± 2
14 ± 2 13 ± 2
13 ± 2 12 ± 2
13 ± 3 12 ± 2
14 ± 2 11 ± 4
13 ± 3 12 ± 2
Left atrial pressure, mm Hg
NS AD
3 ± 1 2 ± 1
2 ± 1 2 ± 1
3 ± 2 3 ± 1
2 ± 1 2 ± 1
2 ± 1 2 ± 2
3 ± 1 3 ± 1
3 ± 2 4 ± 1
3 ± 1 4±2
Systemic vascular resistance, mm Hg L-l kg-1 mirr"
NS AD
340 ± 41 344 ± 46
352 ± 49 328 ± 44
360 ± 51 316 ± 51
340 ± 44 309 ± 48
325 ± 42 290 ± 44
340 ± 46 229 ± 42*
330 ± 36 190 ± 41*
320 ± 41 160 ± 38*
Pulmonary vascular resistance, mm Hg L-l kg-1 mlrr"
NS AD
58 ± 10 60 ± 11
52 ± 9 50 ± 8
54 ± 8 52 ± 9
61 ± 11 52 ± 9
58 ± 9 43 ± 8*
51 ± 8 35 ± 7*
55 ± 9 25 ± 8*
50 ± 7 26 ± 6*
Variable
Data are mean ± SO for n = 12. • P < 0.05 from baseline and normal saline (NS).
min. Adenosine infusion resulted in significant increases in heart rate at doses of 0.15to 2.5 umol/kg/min and cardiac output at doses of 0.3 to 2.5 umol/kg/min. The response of hemodynamic and blood gas variables to hypoxia alone was
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similar for both control and adenosine studies done on different days. Alveolar hypoxia resulted in significant increases in heart rate, pulmonary artery pressure, and pulmonary vascular resistance (figures 1 and 2). Infusion of normal saline did not produce further changes in these variables. Adenosine caused a significant decrease in pulmonary artery pressure in doses of 0.01, 0.04, 0.15, 0.60, and 2.5
umol/kg/min, Pulmonary vascular resistance decreased with all the infusion rates tested (figure 1). Aortic pressure and systemic vascular resistance decreased significantly during adenosine infusion in doses of 0.3 to 2.5 umol/kg/min, along with a significant increase in heart rate (figure 2). Cardiac output increased with infusion rates of 0.60 to 2.5 umol/kg/min of adenosine.
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675
ADENOSINE AND PULMONARY VASODILATION
may be taken up into the endothelial cell (6), where it may be phosphorylated to AMP, a vasoactive nucleotide, byadenosine kinase or deaminated to inosine, an inactive metabolite. Adenosine kinase (6) appears to have a much higher affinity for adenosine compared with adenosine deaminase, so that most of the adenosine transported into the cell is normally phosphorylated to AMP, which may be further converted to cyclic AMP. Although the nature of receptors for adenosine on the endothelial cells has not been delineated, Burnstock (21)proposed that the effects of purine nucleotides are mediated by two types of purinergic receptors. P t receptors are more sensitive to adenosine, act via the adenylate cyclase system, and are antagonized by methylxanthines in lower concentrations than are needed to inhibit phosphodiesterase. P, receptors are more sensitive to ATP and are not antagonized by methylxanthines. We have shown that aminophylline attenuates the pulmonary vasodilation caused by 0.01 to 0.15 umol/kg/min infusion rates of adenosine. Aminophylline did not block the pulmonary and systemic vasodilation caused by higher infusion rates of adenosine, indicating a reversible antagonism between the two drugs at the receptor level. These data indicate that the effects of adenosine are mediated at least in part by the stimulation of P t purinergic receptors. Adenosine may be converted to ATP by adenosine kinase, and ATP may cause pulmonary vasodilation by stimulation of P, receptors. However, the high affinity of adenosine kinase for adenosine is expected to result in conversion of adenosine to ATP even at doses of < 0.30 umol/kg/min, causing some vasodilation in aminophylline-treated animals. Therefore, we do not consider it a likely explanation for vasodilation seen with adenosine at higher doses. Based on receptor affinity and radioligand binding studies, P t receptors have been further classified into At and A, subtypesthe At receptors inhibit adenylate cyclase, and the A, receptors stimulate adenylate cyclase and mediate the vasodilator effects of adenosine. The specific receptors that mediate the effects of adenosine in pulmonary arteries of lambs remain to be elucidated. Although purine nucleotides such as ATP and ADP are capable of stimulating prostacyclin synthesis by the endothelial cells, Pearson and colleagues (22) have demonstrated that adenosine does not increase prostaglandin synthesis by the cultured porcine endothelial cells. Neely and coworkers (16) have shown that meclofenamate, a pros-
taglandin synthesis inhibitor, does not alter the effects of adenosine on the feline pulmonary artery. Therefore, this mechanism is unlikely to explain the pulmonary vascular effects of adenosine. Recently, Abman and associates (23) have shown that endothelium-derived relaxing factor (EDRF) is an important vasodilator in perinatal lamb. We have preliminary data to show that nitro-L-arginine, an inhibitor of EDRF synthesis, attenuates the pulmonary vasodilation caused by adenosine in fetal lambs (24), suggesting a role for this mechanism. The increase in Pan, seen in hypoxic lambs at the highest infusion rate of adenosine is primarily due to an increase in mixed venous POz during adenosine infusion at 1.2 and 2.5 umol/kg/min, The mixed venous POz increased from 20 ± 4 during hypoxia alone to 23 ± 5 at 1.2 umol/kg/min and 27 ± 4 (p < 0.05 from hypoxia alone) at 2.5 umol/kg/min infusion rates. The increase in arterial Po z was accounted for by the increase in mixed venous POz, which was associated with a significant increase in cardiac output. The increase in Paco, and decrease in pH during hypoxia were seen equally in both control and adenosine experiments. They are unlikely to account for vasodilation seen with adenosine, because acidosis has been shown to constrict rather than dilate pulmonary vessels (25). Adenosine has been shown to exert vasodilator effects in cerebral, coronary, and mesenteric vascular beds (2-4). Adenosine has also been shown to playa significant role in causing reactive hyperemia in skeletal muscle following exercise (5). Increased plasma and interstitial fluid levels of adenosine have been observed in systemic vascular beds in response to decreased oxygen supply or increased oxygen demand (5). Adenosine release has been implicated as a mechanism for coronary vasodilation during exerciseor hypoxia (2). We did not measure the adenosine levels in interstitial fluid or lymph from lung during hypoxia. However, the plasma adenosine levels in pulmonary circulation in our experiments showed a striking decrease during alveolar hypoxia. Pulmonary vessels usually constrict in response to hypoxia in contrast to systemic vessels, which usually dilate during mild-to-moderate hypoxia. These data and the dilation of pulmonary vessels during hypoxia in response to exogenous adenosine suggest that a decreased release of adenosine is an important mechanism for the pulmonary vasoconstriction during hypoxia. The role of adenosine in maintaining the pulmonary vessels
in dilated state during normoxia is not clear. If adenosine regulates va.scular tone during normoxia, adenosine antagonists should increase the pulmonary vascular resistance during normoxia and accentuate the hypoxic pulmonary vasoconstriction. However,aminophylline had no effect on pulmonary circulation during normoxia in newborn lambs in our study and did not increase the pulmonary vascular resistance in hypoxic lambs. Biaggioni and associates reported that aminophylline causes a small decrease in baseline pulmonary artery pressure in adult sheep. Aminophylline has opposing effects on cyclic AMP by preventing its stimulation by adenosine and by increasing cyclicAMP via phosphodiesterase inhibition. Further experiments should be done evaluating the effects of more specific P. receptor blockers such as 8-phenyltheophylline. The significance of our findings is that we demonstrated the presence of adenosine responsive P t purinergic receptors in pulmonary vessels of newborn lambs. We also documented the normal changes in plasma adenosine levels in neonatal pulmonary circulation during hypoxia. The selective pulmonary vasodilation caused by adenosine in lower doses may be useful in the management of infants with pulmonary hypertension. The wide range of doses that appears to be selective for the pulmonary circulation may facilitate better titration of the dose needed for individual patients. The short halflife and rapid clearance of adenosine by endothelium may allow us to use this drug in infants with compromised renal function, a situation in which tolazoline therapy appears to be less desirable (26). However, the presence of right-to-left shunts at the level of foramen ovale or ductus arteriosus in these infants may allow adenosine to enter systemic circulation even in low doses and result in systemic vasodilation. In addition, the pulmonary artery pressure in our lambs was increased by alveolar hypoxia, whereas infants with persistent pulmonary hypertension usually have supersystemic pulmonary artery pressures inspite of receiving high PIo z. The response of pulmonary vessels in infants with pulmonary hypertension may also be different from that of newborn lambs with normal pulmonary circulation because of their structural (27,28) and possibly functional differences. These differences should be considered while extrapolating our results to infants with persistent pulmonary hypertension. Further studies are required to evaluate fully the role of purinergic receptors in cardiopulmonary
676
KONDURI, WOODARD, MUKHOPADHYAY, AND DESHMUKH
changes seen during the perinatal period and the usefulness of adenosine as a therapeutic agent in the management of newborn infants with pulmonary hypertension. References 1. Fox WW, Gewitz MH, Dinwiddie R, Drummond WH, Peckham GJ. Pulmonary hypertension in the perinatal aspiration syndromes. Pediatrics 1977; 59:205-11. 2. Berne RM. The role of adenosine in the regulation of coronary blood flow. Circ Res 1980; 47:807-13. 3. Morii S, Ngai AC, Ko KR, Winn HR. Role of adenosine in regulation of cerebral blood flow: effects of theophylline during normoxia and hypoxia. Am J Physiol 1987; 253:HI65-75. 4. Ezzat WR, Lautt WW.Hepatic arterial pressureflow autoregulation is adenosine mediated. Am J Physiol 1987; 252:H836-45. 5. Berne RM, Knabb RM, Ely SW, Rubio R. Adenosine in the local regulation of blood flow: a brief review. Fed Proc 1983; 42:3136-42. 6. Dieterle Y,Ody C, Ehrensberger A, Stalder H, Junod AF. Metabolism and uptake of adenosine triphosphate and adenosine by porcine aortic and pulmonary endothelial cellsand fibroblasts in culture. Circ Res 1978; 42:869-76. 7. Lock JE, Hamilton F, Olley PM, Coceani F. The effect of alveolar hypoxia on pulmonary vascular responsiveness in the conscious newborn lambs. Can J Physiol Pharmacol 1980; 58:153-9. 8. Sidi D, Kuipers JR, Heymann MA, Rudolph AM. Recovery of cardiovascular function in newborn lambs after thoracotomy. Pediatr Res 1982; 16:705-10.
9. Von Borstel RW, Wurtman RJ, Conlay LA. Chronic caffeine consumption potentiates the hypotensive action of circulating adenosine. Life Sci 1983; 32:1151-8. 10. Klabunde RE. Dipyridamole inhibition of adenosine metabolism in human blood. Eur J Pharmacol 1983; 93:21-6. 11. Malik AB, Kidd BSL. Time course of pulmonary vascular response to hypoxia in dogs. Am J Physiol 1973; 224:1-6. 12. Winer BJ. Statistical principles in experimental design. 2nd ed. New York: McGraw-Hill Inc., 1971; 191-204, 514-603. 13. Biaggioni I, King LS, Enayat N, Robertson D, Newman JH. Adenosine produces pulmonary vasoconstriction in sheep. Evidence for thromboxane Az/prostaglandin endoperoxide-receptor activation. Circ Res 1989; 65:1516-25. 14. Silver PJ, Walus K, Disalvo J. Adenosinemediated relaxation and activation of cyclicAMPdependent protein kinase in coronary arterial smooth muscle. J Pharmacol Exp Ther 1984; 228: 342-7. 15. Shaul P, Moore M, Buja LM. Developmental changes in pulmonary artery adenylate cyclase activity (abstract). Pediatr Res 1989; 25:317A. 16. Neely CF, Kadowitz PJ, Lippton H, Neiman M, Hyman A. Adenosine does not mediate the pulmonary vasodilator response of adenosine 5'-triphosphate in the feline pulmonary vascular bed. J Pharmacol Exp Ther 1989; 250:170-6. 17. Custer JR, Hales CA. Influence of alveolar oxygen on pulmonary vasoconstriction in newborn lambs versus sheep. Am Rev Respir Dis 1985; 132: 326-31. 18. Rudolph AM, Kurland MD, Auld PAM, Paul MH. Effects of vasodilator drugs on normal and serotonin-constricted pulmonary vesselsof the dog. Am J Physiol 1959; 197:617-23.
19. Konduri GO, Woodard LL. Selective pulmonary vasodilation by low dose infusion of adenosine triphosphate (ATP) in newborn lambs. J Pediatr 1991; 119:94-102. 20. Fineman JR, Crowley MR, Soifer SJ. Selective pulmonary vasodilation with ATP·MgClz during pulmonary hypertension in lambs. J Appl Physiol 1990; 69:1836-42. 21. Burnstock G. Overview: Purinergic mechanisms. In: Dubyak OR, Fedan JS, eds. Biological actions of extracellular ATP. New York: New York Academy of Sciences 1990; 603:1-18. 22. Pearson JD, Slakey LL, Oordon JL. Stimulation of prostaglandin production through purinoceptors on cultured porcine endothelial cells. Biochem J 1983; 214:273-6. 23. Abman SH, Chatfield BA, Hall SL, McMurtry IF. Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth. Am J Physiol 1990; 259:HI921-7. 24. Konduri 00, Theodorou AA. Nitro-Larginine attenuates the pulmonary vasodilation caused by adenosine in fetal Iambs. Pediatr Res 1992; (In Press). 25. Rudolph AM, Yuan S. Response of the pulmonary vasculature to hypoxia and H+ ion concentration changes. J Clin Invest 1966;45:399-411. 26. Ward RM, Kendig JW, Daniel CH. Potentially lethal accumulation of tolazoline in neonates with oliguria (abstract). Pediatr Res 1982; 16:313A. 27. Haworth SO, Reid L. Persistent fetal circulation: newly recognized structural features. J Pediatr 1976; 88:614-20. 28. Murphy JD, Rabinovitch M, Goldstein JD, Reid L. The structural basis of persistent pulmonary hypertension of the newborn infant. J Pediatr 1981; 98:962-7.
G. GANESH KONDURI, LAINE L. WOODARD, ARINDAM MUKHOPADHYAY, and DEVENDRA R. DESHMUKH
Introduction
Persistent pulmonary hypertension of the newborn is a condition associated with high morbidity and mortality, The affected infants have an increased pulmonary vascular resistance and hypoxemia secondary to right to left shunting of blood across fetal channels (1). However, the factors that maintain the pulmonary vascular tone in the newborn or the factors that regulate the pulmonary vascular response to hypoxia in the newborn are not wellunderstood. Adenosine, a purinergic nucleoside, has been shown to be a potent vasodilator in the coronary, cerebral, and splanchnic vascular beds in adult animals and is regarded as an endogenous modulator of blood flow to these organs (2-4). Tissue levels of adenosine increase during hypoxia, and adenosine is known to cause reactive hyperemia and hypoxia-induced vasodilation in systemicvascular beds (5). However, hypoxia constricts pulmonary vessels, a response that is different from that of systemicvessels. Weproposed that pulmonary vasoconstriction in response to hypoxia is in part mediated by decreased release of adenosine into the pulmonary circulation and that infusion of exogenous adenosine into pulmonary circulation will decrease the pulmonary vascular resistance to baseline values. Because adenosine appears to be rapidly metabolized by vascular endothelium (6), wealso proposed that intravenous infusion of adenosine in newborn animals would produce a selective pulmonary vasodilation and that this effect would be mediated by theophylline-sensitive P 1 purinergic receptors, as described in other vascular beds (3, 4). The objective of our experiments, therefore, was to investigate the pulmonary vascular effects of adenosine in newborn lambs during conditions of normoxemia and acute pulmonary hypertension induced by alveolar hypoxia. We measured the changes in plasma adenosine levels in pulmonary artery and left atrium with alveolar hypoxia and during the infusion of exogenous adenosine. We also investigated the effect of ami670
SUMMARY We Investigated the systemic and pulmonary vascular effects of adenosine and determined plasma adenosine levels In pulmonary circulation In 12 newborn lambs during normoxla and during alveolar hypoxia (10% O2 , 5% C0 2t and 85% N2) . Lambs were Instrumented at 7 days of age with catheters In the descending aorta, main pulmonary artery, and right and left atria, and a flow transducer around the main pulmonary artery, and were studied following a 3-day recovery. Adenosine or an equal volume of normal saline (control) was Infused Into the right atrial line In doses ranging from 0.01 to 2.5 Ilmollkg/mln. In normoxlc lambs, adenosine caused a significant decrease In pUlmonary vascular resistance and Increase In heart rate In doses of 0.15 to 2.5 Ilmollkg/mln and a decrease In systemic vascular resistance, with Increase In cardiac output In doses of 0.3 to 2.5 Ilmollkg/mln. Baseline plasma adenosine levels In pulmonary artery and left atrium decreased significantly during alveolar hypoxia. Adenosine Infusion In hypoxic lambs caused decreases In pulmonary artery pressure and pulmonary vascular resistance at all the doses tested. Aortic pressure and systemic vascular resistance decreased, and heart rate and cardiac output Increased at doses ~ 0.3 Ilmollkg/mln In hypoxic lambs during adenosine Infusion. The pulmonary vascular effects of adenosine in hypoxic lambs were attenuated by prior treatment of animals with aminophylline. Thus, adenosine appears to be an Important regUlator of pulmonary vascular response to hypoxia In newborn lambs. Its vasodilator effects were specific for pulmonary circulation when It was Infused In doses"," 0.15 Ilmollkg/mln Into the right atrium and appear to be mediated by PI purlnerglc receptors. AM REV RESPIR DIS 1992; 146:670-676
nophylline on the pulmonary vascular effects of adenosine in lambs during hypoxia. We chose newborn lamb as a model because its pulmonary circulation has been studied extensively in the past, and its response to hypoxia has been documented well (7). We used chronically instrumented animals for our studies because it allows us to study the pulmonary circulation without the effects of surgery or anesthesia. Methods Eighteen lambs were studied between the ages of 10and 16 days. 1\velveof these lambs were studied during conditions of normoxia and hypoxia to determine the effects of incremental doses of adenosine. Six additional lambs were studied during hypoxia to determine the effect of aminophylline on the response to adenosine. Lambs were brought into our laboratory at 1 day of age and housed in separate Plexiglas cages. They were trained to feed ad libitum from an artificial feeding system that contained goat's milk (Irish Hills Farm natural goat's milk, Mason, MI). Lambs adjusted well to the feeding system and gained weight at the rate of 50 to 75 g/kg/day prior to surgery.
Surgical Procedure Each lamb underwent one sterile surgical procedure at 7 days of age. Lambs were given preanesthetic medication consisting of atro-
pine sulfate (0.01 mg/kg SC) and ketamine HCI (10 mg/kg intramuscular). We then intubated each animal's trachea with a cuffed endotracheal tube and maintained anesthesia by ventilating the lungs with 1to 2070 isoflurane and oxygen. We monitored rectal temperature and electrocardiogram continuously and arterial blood gases periodically during surgery and maintained the core temperature at or close to 39° C with a heating pad. We made an incision in the left groin and inserted catheters into the descending aorta and inferior vena cava via the femoral artery and femoral vein. We then performed a left lateral thoracotomy and inserted catheters by direct puncture into the main pulmonary artery and right and left atria. We implanted an ultrasonic flow transducer (size 12S; 'fransonic Systems Inc., Ithaca, NY) around the main pulmonary artery to measure the cardi-
(Received in original form March 27, 1991 and in revised form February 18, 1992) 1 From the Departments of Pediatrics, Children's Hospital of Michigan, Hutzel Hospital, and Wayne State University School of Medicine, Detroit, Michigan. 2 Supported by a grant from the American Heart Association of Michigan. 3 Correspondence and requests for reprints should be addressed to G. Ganesh Konduri, M.D., Department of Pediatrics, Hutzel Hospital, 4707 St. Antoine Boulevard, Detroit, MI 48201.
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ADENOSINE AND PULMONARY VASODILATION
ac output. We identified and ligated the ductus arteriosus and tunneled the catheters and flow transducer cable to the lamb's back. We allowed the animal to recover from surgery for at least 3 days before experiments were done. Cardiovascular function and oxygen consumption were shown to return to normal by 3 days after thoracotomy in lambs (8). Antibiotics (procaine penicillin 50,000 U/kg and gentamicin 5 mg/kg intramuscular) were administered on the day of surgery and on each postoperative day until the studies were completed.
Experimental Protocol Wesecured each lamb in a sling and connected the vascular catheters to strain gauge manometers (model P23XL; Spectramed Inc., Oxnard, CA) and the flow transducer to an ultrasonic transit time blood flowmeter (Transonic Systems Inc., Ithaca, NY). The aortic, pulmonary arterial, left and right atrial pressures, and pulmonary blood flow wererecorded on a Grass model7D polygraph (Grass Instruments, Quincy, MA). From these data, we calculated the pulmonary vascular resistance as the difference between mean pulmonary arterial and left atrial pressures divided by cardiac output in L/kg of body weight. Similarly, systemic vascular resistance was calculated as the difference between mean aortic and right atrial pressures divided by cardiac output in L/kg. We studied each lamb during conditions of normoxia and hypoxia. For both normoxia and hypoxia studies, data were obtained with an infusion of normal saline to serve as a control for the data obtained with the infusion of adenosine. Each lamb, therefore, had four experiments done: control and adenosine studies during both normoxia and hypoxia. Each of the four experiments was done on a different day, and the order of these experiments was randomized for each animal.
Drug Preparation Adenosine (Sigma Chemical Co., St. Louis, MO) was obtained in a powder form, and the solution for intravenous administration was prepared each day by mixing it with sterile normal saline with a final concentration of 4 umol/ml of adenosine.
Assay for Adenosine Adenosine was assayed by a method described by Von Borstel and coworkers (9). Blood samples were collected in volumes of 0.5 ml and transferred immediately to tubes containing 0.5 ml of trichloroacetic acid kept at 0 to 4 0 C. The samples were immediately centrifuged, and the supernatant was frozen at - 20 0 C. Adenosine in supernatant was assayed by high-pressure liquid chromatography (HPLC) (Waters 820 system equipped with an autosampler and maxima software), using a Waters micro-Bondapak C-18reversed-phase column. An isocratic method was used to elute adenosine (10070 methanol in 10mM potassium phosphate, pH 5.8) at a flow rate of 1.6
ml/min. Adenosine was detected by UV absorbance at 254 nm (Waters 481 UV detector). The plasma concentration of adenosine was expressedas nmol/ml. The intraassay and interassaycoefficients of variation determined for adenosine standard solutions of 100, 500, and 1,000nm/ml were 0.3070 and 0.5% respectively. The smallest detectable amount of adenosine was 1 nmol/ml.
Protocol for Normoxia Experiments For each experiment, we placed the animal in a sling and recorded the hemodynamic variables when the animal was awake and resting quietly. Wegave an infusion of adenosine into the right atrial line starting at a dose of 0.01 umol/kg/min with a Harvard infusion pump (Harvard Apparatus, South Natick, MA). We measured the variables after the animals receivedthe infusion for 10min. Then we doubled the infusion rate for 10 min to give the animal 0.02, 0.04, 0.08, 0.15, 0.30, 0.60, 1.20, and 2.50 umol/kg/min of adcnosine. Wemeasured hemodynamic variables at the end of each 1O-min infusion. The halflife for adenosine in blood was shown to be less than 10 s (10), so that 10 min would be adequate time to achieve steady-state levels of the nucleotide in plasma. Control experiments were done on a different day with infusion of equal volumes of normal saline.
Protocol for Hypoxia Experiments Wemeasured hemodynamic variables, and arterial blood gases during normoxia. We then induced alveolar hypoxia by placing a loosely fitting plastic bag over the animal's head and allowing it to breathe a gas mixture of 10% 0 1 , 5% Cal' and 85% N 1 • Lambs frequently hyperventilate during hypoxia and lower their Paco1 , which tends to attenuate the pulmonary vascular response to hypoxia (11). We added 5% Cal to the gas mixture to maintain the Paco1 stable and at or close to 40 mm Hg (5.3 kPa). Variables were measured after the animals had stable hypoxemia for 10 min. Adenosine was then infused into the right atrial line, in doses of 0.01 to 2.5 umol/ kg/min, as described previously for normoxia experiments. Each infusion rate was maintained for 10 min, and variables were measured at the end of each 10 min period. Samples of arterial blood were drawn at each infusion rate for determination of pH and blood gas tensions. Blood samples for plasma adenosine levels were drawn from pulmonary artery and left atrium during normoxia, hypoxia, and at each infusion rate of adenosine in eight out of 12 lambs that were studied. Control experiments were done on a different day with infusion ofequal volumes of saline. The plasma adenosine levels were measured only during studies done with infusion of adenosine. The volume of blood withdrawn at each time point was 0.2 ml for the control experiment and 1.2 ml for the adenosine experiment. The total amount of blood withdrawn, therefore, was 15 to 16 ml for both experiments and represented only 3%
of the blood volume taken over a 2-day period. (The weight of the study animals was 5.4 ± 0.8 kg, and blood volume was assumed to be 85 ml/kg.)
Protocol for Experiments to Investigate the Mechanism of the Effects of Adenosine Six lambs were studied twice each on separate days, and the order of the experiments was randomized for each animal. For each experiment, baseline variables were recorded when the animal was breathing room air. Alveolar hypoxia was induced by allowing the animals to breathe a gas mixture of 10% 0 1 , 5% CO 2 , and 85% N1 • The animals werethen given a bolus dose of either aminophylline 7 mg/kg or an equal volume of saline into the right atrial line, and variables wererecorded 30 min after the bolus, with ongoing hypoxia. Adenosine was then infused into the right atrial line in doses of 0.01 to 2.5 umol/kg/min, as described previously for other experiments. Each infusion rate was maintained for 10 min and variables were recorded at the end of each lO-min period.
Statistical Analysis All data are expressed as mean ± SD. The data obtained during normoxia and during hypoxia alone from different days on each lamb were compared by single factor analysis of variance (ANOVA) for repeated measures to determine if baseline variables and the response to hypoxia were similar on different days. Data obtained during control and adenosine experiments were compared by twoway ANOVA for repeated measures - the two factors affecting the outcome being adenosine versus saline and the dose of adenosine that was infused. When significant differences, defined as a p value of < 0.05 were found, a Student-Newman-Keuls multiplerange test was done to determine which means were different (12). The data obtained during hypoxia studies and studies done to determine the mechanism of the hemodynamic effects of adenosine were analyzed similarly using two-way ANOVA for repeated measures and a Student-Newman-Keuls test.
Results
Baseline variables measured on different days werecomparable and showed no significant differences. Normoxic lambs had no significant changes in the hemodynamic variables during the control experiments done with infusion of saline (table 1). Adenosine caused a significant decrease in pulmonary vascular resistance in doses of 0.15to 2.5 umol/kg/min and systemic vascular resistance in doses of 0.3 to 2.5 umol/kg/min. Pulmonary arterial, and left and right atrial pressures did not change during adenosine infusion, and the aortic pressure decreased with infusion rates of 0.6 to 2.5 umol/kg/
672
KONDURI, WOODARD, MUKHOPADHYAY, AND DESHMUKH
TABLE 1 HEMODYNAMIC VARIABLES MEASURED DURING NORMOXIA AND INFUSION OF ADENOSINE (AD) OR NORMAL SALINE (NS) Infusion Rates of AD or NS (JJmol/kg/min) Drug
Baseline
0.01
0.04
0.08
0.15
0.30
0.60
2.50
Heart rate, bpm
NS AD
179 ± 13 178 ± 17
176 ± 16 184 ± 13
174 ± 17 182 ± 19
174 ± 21 203 ± 21
181 ± 21 211 ± 22*
190 ± 19 236 ± 20*
188 ± 17 241 ± 19*
180 ± 22 220 ± 21*
Cardiac output, mllkg/min
NS AD
192 ± 22 198 ± 24
196 ± 24 203 ± 19
189 ± 26 204 ± 26
190 ± 22 217 ± 22
192 ± 17 227 ± 20
200 ± 16 272 ± 19*
204 ± 17 277 ± 31*
207 ± 19 294 ± 34*
Aortic pressure, mm Hg
NS AD
69 ± 7 70 ± 6
71 ± 8 68 ± 7
70 ± 6 66 ± 5
68 ± 7 68 ± 6
66 ± 7 65 ± 6
70 ± 6 64 ± 7
66 ± 5 55 ± 6*
68 ± 6 50 ± 5*
Right atrial pressure, mm Hg
NS AD
3 ± 1 2 ± 1
3 ± 1 3 ± 2
3 ± 1 2 ± 1
4 ± 1 3 ± 1
3 ± 2 3 ± 1
2 ± 1 3 ± 1
4 ± 1 3 ± 2
5±2 3 ± 1
Pulmonary artery pressure, mm Hg
NS AD
13 ± 3 14 ± 4
12 ± 2 12 ± 3
13 ± 2 14 ± 2
14 ± 2 13 ± 2
13 ± 2 12 ± 2
13 ± 3 12 ± 2
14 ± 2 11 ± 4
13 ± 3 12 ± 2
Left atrial pressure, mm Hg
NS AD
3 ± 1 2 ± 1
2 ± 1 2 ± 1
3 ± 2 3 ± 1
2 ± 1 2 ± 1
2 ± 1 2 ± 2
3 ± 1 3 ± 1
3 ± 2 4 ± 1
3 ± 1 4±2
Systemic vascular resistance, mm Hg L-l kg-1 mirr"
NS AD
340 ± 41 344 ± 46
352 ± 49 328 ± 44
360 ± 51 316 ± 51
340 ± 44 309 ± 48
325 ± 42 290 ± 44
340 ± 46 229 ± 42*
330 ± 36 190 ± 41*
320 ± 41 160 ± 38*
Pulmonary vascular resistance, mm Hg L-l kg-1 mlrr"
NS AD
58 ± 10 60 ± 11
52 ± 9 50 ± 8
54 ± 8 52 ± 9
61 ± 11 52 ± 9
58 ± 9 43 ± 8*
51 ± 8 35 ± 7*
55 ± 9 25 ± 8*
50 ± 7 26 ± 6*
Variable
Data are mean ± SO for n = 12. • P < 0.05 from baseline and normal saline (NS).
min. Adenosine infusion resulted in significant increases in heart rate at doses of 0.15to 2.5 umol/kg/min and cardiac output at doses of 0.3 to 2.5 umol/kg/min. The response of hemodynamic and blood gas variables to hypoxia alone was
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similar for both control and adenosine studies done on different days. Alveolar hypoxia resulted in significant increases in heart rate, pulmonary artery pressure, and pulmonary vascular resistance (figures 1 and 2). Infusion of normal saline did not produce further changes in these variables. Adenosine caused a significant decrease in pulmonary artery pressure in doses of 0.01, 0.04, 0.15, 0.60, and 2.5
umol/kg/min, Pulmonary vascular resistance decreased with all the infusion rates tested (figure 1). Aortic pressure and systemic vascular resistance decreased significantly during adenosine infusion in doses of 0.3 to 2.5 umol/kg/min, along with a significant increase in heart rate (figure 2). Cardiac output increased with infusion rates of 0.60 to 2.5 umol/kg/min of adenosine.
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675
ADENOSINE AND PULMONARY VASODILATION
may be taken up into the endothelial cell (6), where it may be phosphorylated to AMP, a vasoactive nucleotide, byadenosine kinase or deaminated to inosine, an inactive metabolite. Adenosine kinase (6) appears to have a much higher affinity for adenosine compared with adenosine deaminase, so that most of the adenosine transported into the cell is normally phosphorylated to AMP, which may be further converted to cyclic AMP. Although the nature of receptors for adenosine on the endothelial cells has not been delineated, Burnstock (21)proposed that the effects of purine nucleotides are mediated by two types of purinergic receptors. P t receptors are more sensitive to adenosine, act via the adenylate cyclase system, and are antagonized by methylxanthines in lower concentrations than are needed to inhibit phosphodiesterase. P, receptors are more sensitive to ATP and are not antagonized by methylxanthines. We have shown that aminophylline attenuates the pulmonary vasodilation caused by 0.01 to 0.15 umol/kg/min infusion rates of adenosine. Aminophylline did not block the pulmonary and systemic vasodilation caused by higher infusion rates of adenosine, indicating a reversible antagonism between the two drugs at the receptor level. These data indicate that the effects of adenosine are mediated at least in part by the stimulation of P t purinergic receptors. Adenosine may be converted to ATP by adenosine kinase, and ATP may cause pulmonary vasodilation by stimulation of P, receptors. However, the high affinity of adenosine kinase for adenosine is expected to result in conversion of adenosine to ATP even at doses of < 0.30 umol/kg/min, causing some vasodilation in aminophylline-treated animals. Therefore, we do not consider it a likely explanation for vasodilation seen with adenosine at higher doses. Based on receptor affinity and radioligand binding studies, P t receptors have been further classified into At and A, subtypesthe At receptors inhibit adenylate cyclase, and the A, receptors stimulate adenylate cyclase and mediate the vasodilator effects of adenosine. The specific receptors that mediate the effects of adenosine in pulmonary arteries of lambs remain to be elucidated. Although purine nucleotides such as ATP and ADP are capable of stimulating prostacyclin synthesis by the endothelial cells, Pearson and colleagues (22) have demonstrated that adenosine does not increase prostaglandin synthesis by the cultured porcine endothelial cells. Neely and coworkers (16) have shown that meclofenamate, a pros-
taglandin synthesis inhibitor, does not alter the effects of adenosine on the feline pulmonary artery. Therefore, this mechanism is unlikely to explain the pulmonary vascular effects of adenosine. Recently, Abman and associates (23) have shown that endothelium-derived relaxing factor (EDRF) is an important vasodilator in perinatal lamb. We have preliminary data to show that nitro-L-arginine, an inhibitor of EDRF synthesis, attenuates the pulmonary vasodilation caused by adenosine in fetal lambs (24), suggesting a role for this mechanism. The increase in Pan, seen in hypoxic lambs at the highest infusion rate of adenosine is primarily due to an increase in mixed venous POz during adenosine infusion at 1.2 and 2.5 umol/kg/min, The mixed venous POz increased from 20 ± 4 during hypoxia alone to 23 ± 5 at 1.2 umol/kg/min and 27 ± 4 (p < 0.05 from hypoxia alone) at 2.5 umol/kg/min infusion rates. The increase in arterial Po z was accounted for by the increase in mixed venous POz, which was associated with a significant increase in cardiac output. The increase in Paco, and decrease in pH during hypoxia were seen equally in both control and adenosine experiments. They are unlikely to account for vasodilation seen with adenosine, because acidosis has been shown to constrict rather than dilate pulmonary vessels (25). Adenosine has been shown to exert vasodilator effects in cerebral, coronary, and mesenteric vascular beds (2-4). Adenosine has also been shown to playa significant role in causing reactive hyperemia in skeletal muscle following exercise (5). Increased plasma and interstitial fluid levels of adenosine have been observed in systemic vascular beds in response to decreased oxygen supply or increased oxygen demand (5). Adenosine release has been implicated as a mechanism for coronary vasodilation during exerciseor hypoxia (2). We did not measure the adenosine levels in interstitial fluid or lymph from lung during hypoxia. However, the plasma adenosine levels in pulmonary circulation in our experiments showed a striking decrease during alveolar hypoxia. Pulmonary vessels usually constrict in response to hypoxia in contrast to systemic vessels, which usually dilate during mild-to-moderate hypoxia. These data and the dilation of pulmonary vessels during hypoxia in response to exogenous adenosine suggest that a decreased release of adenosine is an important mechanism for the pulmonary vasoconstriction during hypoxia. The role of adenosine in maintaining the pulmonary vessels
in dilated state during normoxia is not clear. If adenosine regulates va.scular tone during normoxia, adenosine antagonists should increase the pulmonary vascular resistance during normoxia and accentuate the hypoxic pulmonary vasoconstriction. However,aminophylline had no effect on pulmonary circulation during normoxia in newborn lambs in our study and did not increase the pulmonary vascular resistance in hypoxic lambs. Biaggioni and associates reported that aminophylline causes a small decrease in baseline pulmonary artery pressure in adult sheep. Aminophylline has opposing effects on cyclic AMP by preventing its stimulation by adenosine and by increasing cyclicAMP via phosphodiesterase inhibition. Further experiments should be done evaluating the effects of more specific P. receptor blockers such as 8-phenyltheophylline. The significance of our findings is that we demonstrated the presence of adenosine responsive P t purinergic receptors in pulmonary vessels of newborn lambs. We also documented the normal changes in plasma adenosine levels in neonatal pulmonary circulation during hypoxia. The selective pulmonary vasodilation caused by adenosine in lower doses may be useful in the management of infants with pulmonary hypertension. The wide range of doses that appears to be selective for the pulmonary circulation may facilitate better titration of the dose needed for individual patients. The short halflife and rapid clearance of adenosine by endothelium may allow us to use this drug in infants with compromised renal function, a situation in which tolazoline therapy appears to be less desirable (26). However, the presence of right-to-left shunts at the level of foramen ovale or ductus arteriosus in these infants may allow adenosine to enter systemic circulation even in low doses and result in systemic vasodilation. In addition, the pulmonary artery pressure in our lambs was increased by alveolar hypoxia, whereas infants with persistent pulmonary hypertension usually have supersystemic pulmonary artery pressures inspite of receiving high PIo z. The response of pulmonary vessels in infants with pulmonary hypertension may also be different from that of newborn lambs with normal pulmonary circulation because of their structural (27,28) and possibly functional differences. These differences should be considered while extrapolating our results to infants with persistent pulmonary hypertension. Further studies are required to evaluate fully the role of purinergic receptors in cardiopulmonary
676
KONDURI, WOODARD, MUKHOPADHYAY, AND DESHMUKH
changes seen during the perinatal period and the usefulness of adenosine as a therapeutic agent in the management of newborn infants with pulmonary hypertension. References 1. Fox WW, Gewitz MH, Dinwiddie R, Drummond WH, Peckham GJ. Pulmonary hypertension in the perinatal aspiration syndromes. Pediatrics 1977; 59:205-11. 2. Berne RM. The role of adenosine in the regulation of coronary blood flow. Circ Res 1980; 47:807-13. 3. Morii S, Ngai AC, Ko KR, Winn HR. Role of adenosine in regulation of cerebral blood flow: effects of theophylline during normoxia and hypoxia. Am J Physiol 1987; 253:HI65-75. 4. Ezzat WR, Lautt WW.Hepatic arterial pressureflow autoregulation is adenosine mediated. Am J Physiol 1987; 252:H836-45. 5. Berne RM, Knabb RM, Ely SW, Rubio R. Adenosine in the local regulation of blood flow: a brief review. Fed Proc 1983; 42:3136-42. 6. Dieterle Y,Ody C, Ehrensberger A, Stalder H, Junod AF. Metabolism and uptake of adenosine triphosphate and adenosine by porcine aortic and pulmonary endothelial cellsand fibroblasts in culture. Circ Res 1978; 42:869-76. 7. Lock JE, Hamilton F, Olley PM, Coceani F. The effect of alveolar hypoxia on pulmonary vascular responsiveness in the conscious newborn lambs. Can J Physiol Pharmacol 1980; 58:153-9. 8. Sidi D, Kuipers JR, Heymann MA, Rudolph AM. Recovery of cardiovascular function in newborn lambs after thoracotomy. Pediatr Res 1982; 16:705-10.
9. Von Borstel RW, Wurtman RJ, Conlay LA. Chronic caffeine consumption potentiates the hypotensive action of circulating adenosine. Life Sci 1983; 32:1151-8. 10. Klabunde RE. Dipyridamole inhibition of adenosine metabolism in human blood. Eur J Pharmacol 1983; 93:21-6. 11. Malik AB, Kidd BSL. Time course of pulmonary vascular response to hypoxia in dogs. Am J Physiol 1973; 224:1-6. 12. Winer BJ. Statistical principles in experimental design. 2nd ed. New York: McGraw-Hill Inc., 1971; 191-204, 514-603. 13. Biaggioni I, King LS, Enayat N, Robertson D, Newman JH. Adenosine produces pulmonary vasoconstriction in sheep. Evidence for thromboxane Az/prostaglandin endoperoxide-receptor activation. Circ Res 1989; 65:1516-25. 14. Silver PJ, Walus K, Disalvo J. Adenosinemediated relaxation and activation of cyclicAMPdependent protein kinase in coronary arterial smooth muscle. J Pharmacol Exp Ther 1984; 228: 342-7. 15. Shaul P, Moore M, Buja LM. Developmental changes in pulmonary artery adenylate cyclase activity (abstract). Pediatr Res 1989; 25:317A. 16. Neely CF, Kadowitz PJ, Lippton H, Neiman M, Hyman A. Adenosine does not mediate the pulmonary vasodilator response of adenosine 5'-triphosphate in the feline pulmonary vascular bed. J Pharmacol Exp Ther 1989; 250:170-6. 17. Custer JR, Hales CA. Influence of alveolar oxygen on pulmonary vasoconstriction in newborn lambs versus sheep. Am Rev Respir Dis 1985; 132: 326-31. 18. Rudolph AM, Kurland MD, Auld PAM, Paul MH. Effects of vasodilator drugs on normal and serotonin-constricted pulmonary vesselsof the dog. Am J Physiol 1959; 197:617-23.
19. Konduri GO, Woodard LL. Selective pulmonary vasodilation by low dose infusion of adenosine triphosphate (ATP) in newborn lambs. J Pediatr 1991; 119:94-102. 20. Fineman JR, Crowley MR, Soifer SJ. Selective pulmonary vasodilation with ATP·MgClz during pulmonary hypertension in lambs. J Appl Physiol 1990; 69:1836-42. 21. Burnstock G. Overview: Purinergic mechanisms. In: Dubyak OR, Fedan JS, eds. Biological actions of extracellular ATP. New York: New York Academy of Sciences 1990; 603:1-18. 22. Pearson JD, Slakey LL, Oordon JL. Stimulation of prostaglandin production through purinoceptors on cultured porcine endothelial cells. Biochem J 1983; 214:273-6. 23. Abman SH, Chatfield BA, Hall SL, McMurtry IF. Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth. Am J Physiol 1990; 259:HI921-7. 24. Konduri 00, Theodorou AA. Nitro-Larginine attenuates the pulmonary vasodilation caused by adenosine in fetal Iambs. Pediatr Res 1992; (In Press). 25. Rudolph AM, Yuan S. Response of the pulmonary vasculature to hypoxia and H+ ion concentration changes. J Clin Invest 1966;45:399-411. 26. Ward RM, Kendig JW, Daniel CH. Potentially lethal accumulation of tolazoline in neonates with oliguria (abstract). Pediatr Res 1982; 16:313A. 27. Haworth SO, Reid L. Persistent fetal circulation: newly recognized structural features. J Pediatr 1976; 88:614-20. 28. Murphy JD, Rabinovitch M, Goldstein JD, Reid L. The structural basis of persistent pulmonary hypertension of the newborn infant. J Pediatr 1981; 98:962-7.
