Life Sciencee,Vol. 22, pp. 1413-1420 Printed in the U.S.A.
Pergemon Press
ADPase ACTIVITY OF ISOLATED PERFUSED RAT LUNG D. J.
Crutchley,
T. E. Eling and H. W. Anderson
Prostaglandin Section Laboratory of Pulmonary Function and Toxicology National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709
deceived in final form February 21, 1978) Sumnary More than 90% of 3H-ADP was metabolized following bolus injection into rat isolated perfused lungs. The major metabolite was inosine, with lesser amounts of adenosine and AMP. The mean pulmonary transit time for the radioactivity associated with ADP and its metabolites was the same as that for the vascular marker 14C-dextran, indicating that ADP is metabolized by enzymes in the pulmonary vessel walls. The metabolism of 3H-ADP was apparently unaffected by the simultaneous injection of prostacyclin or by continuous infusion of indomethacin or aspirin. 3H-ADP was similarly metabolized by the lung following continuous infusion, although relatively higher amounts of adenosine were observed. The metabolism of ADP by the lung represents biological inactivation since over 95% of the platelet-aggregatory activity of ADP was lost on passage through the lung. The importance of ADP as a primary mediator of platelet aggregation is well established. Aggregatory agents such as thrombin, collagen, endotoxin, epinephrine and fatty acids release ADP from platelet granules, and may exert their pro-aggregatory action via this mechanism; ADP itself aggregates all mammalian platelets tested (for review, see ref. 1). The aggregation produced by ADP in platelet-rich plasma is reversible at low concentrations, whilst at higher concentrations of ADP irreversible aggregation of platelets together with the release of further amounts of ADP occurs (2). Enzymes affecting the breakdown of ADP in the vicinity of platelets may, therefore, play an important role in limiting platelet aggregation and arterial thrombus formation. ADP in blood can be de raded in several ways. Erythrocytes contain high levels of adenylate kinase 93), while plasma contains specific ADPases (4). However, the kinetics of these enzymes suggest that they will be relatively ineffective in controlling the explosive release of ADP that occurs in aggregation (5). Recent1 , a specific ADPase has been discovered in human aortic intima extracts (5,6r , in rabbit aortic homogenates and aortic cell preparations (7), and in rabbit aortic rings (8). The degradation of ADP by arterial ADPase has been suggested as a possible explanation of the ability of healthy arteries to resist platelet adhesion. The lungs are strategically placed in the circulation, and are known to control the concentration of various biological1 active substances entering the arterial circulation (for review, see ref. 9J . Circulating platelets and microemboli become trapped in the pulmonary circulation (10). If'an ADPase were Present in the pulmonary vasculature, the concentration of ADP entering the
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Copyright @ 1978 Pergmon Press
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systemic arterial circulation could be reduced, and a mechanism for preventing the growth of trapped microemboli would exist. Thus it seemed,important to determine whether ADPase activity was present in the lung. Materials and Methods Materials Rabbits, New Zealand White, were purchased from Dutchland Laboratory Animals Inc., Denver, PA. Rats, Sprague-Dawley CD, were purchased from Charles River, Wilmington, MA. The following radioactive substances were obtained from the Amersham/Searle Corp., Arlington Heights, IL: (2-3H)-ADP, 19 Ci/mnole; (8-14C)-AMP,-ADP.-ATP,-adenosine,-inosine,and -adenine, 61 mCi/mnole. "Aquasol" scintillation cocktail and 14C-dextran m.wt. s 70,000 were obtained from New England Nuclear, Boston, MA. Blue dextran, m.wt. 1~ 2 x lo6 was purchased from Phannacia Fine Chemicals, Uppsala Sweden. Acetylsalicylic acid (aspirin), indomethacfn and unlabeled ADP were purchased from Sigma Chemical Co., St. Louis, MO. Silica gel G thin-layer chromatography plates, 25011,were obtained from Analab Inc., Wilmington, DE. All solvents used were of analytical grade or the best grade obtainable. Methods i). Perfusion of lungs. This was performed by a method described in previously (11). Briefly, female rats weighing 250-3509. were anesthetized with halothane. The trachea was cannulated and the lungs were removed and perfused via the pulmonary artery at 30 ml/min with Krebs-Ringer medium containing 5 NM glucose and 4.5% bovine serum albumin. The medium was adjusted to pH 7.4, equilibrated with 95% oxygen, 5% carbon dioxide, and maintained at 37°C. Perfusion rate was monitored by a flow transducer. The lungs were ventilated with an alternating negative pressure. Before initiation of the experiment, perfusion success was determined by the injection of blue dextran. India ink was similarly injected at the end of the experiment. Lack of perfusion was noted by absence of color. Lungs perfused less than 90% were rejected. 3H-ADP and the vascular marker 14C-dextran were injected either as a bolus or were administered to the lung at a constant arterial concentration for 3 min. The effluent from the lungs was collected for 1.14 or 6.0 sec. intervals. Aliquots were mixed with "Aquasol" scintillation cocktail and the radioactivity was measured. The remaining effluent was acidified to pH 3.5 with 10% perchloric acid, and suitable aliquots were applied to silica gel plates and analyzed by thin-layer chromatography in the solvent system isobutyl alcohol-amyl alcoholethylene glycol monoethyl ether-ammonia-water(45:30:90:45:60) (12). Authentic 14C-labeled AMP, ADP, ATP, adenosine, inosine and adenine were also applied to the plates as standards. After development, the plates were scraped into 0.4 cm sections from the origin to the solvent front, and the radioactivity in each section was determined and corrected for spillover and quenching. . The effect of prostacyclin (PGI2), aspirin and indomethacin on the metabolism of ADP was examined. Mixtures of 3H-ADP. dextran and PG12 were injected, and compared with identical mixtures of JH-ADP, dextran and PGI -vehicle injected immediately beforehand. Lungs were also perfused for f0 min with medium containing indomethacin 50 IJM,and 3H-ADP and dextran bolus injections were made during the infusion. The results from these injections were compared with identical injections made itmnediatelybefore commencement of the infusions. (ii). Platelet aqqregation. Rabbits of either sex, weighing 3-4 kg. were anesthetized with pentobarbitone sodfun. 40 mg/kg intravenously. Blood was obtained by carotid artery cannulation and coiiected into 0.1 vol. 3.8% w/v trisodium citrate. Blood was centrifuged at 250 xg for 15 mfn. at 22-24"C, and
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a portion of the resulting platelet-rich plasma (PRP) was further centrifuged at 1.000 xg for 20 min. to give platelet-poor plasma which was used for calibration of the aggregometer. Platelet aggregation was measured by a turbidometric method (13) using a Chrono-log aggregometer. 1 ml samples of PRP were added to the cuvette and warmed to 37°C for 3 min. Then lo-200 pl alfquots of lung perfusion medium containing ADP. collected either immediately before or after passage through the lung, were added. Venous and arterial samples were adjusted to the same volume with lung perfusion medium before addition to PRP. The effect of 200 ~1 lung perfusion medium itself was also examined. Results Bolus injection of 3H-ADP. A total of 12 bolus injections, ranging from 0.1 to 10.0 nmols ADP. were made in 6 patrs of lungs. Radioactivity emerged in the effluent within 3 sec. after injection. In each case the peak of tritfum corresponded exactly with that of dextran (Fig. 1A). Neither tritiun nor dextran were retained by the lungs, indicating that ADP remains in the pulmonary vasculature following bolus injection. Analysis of the tritium in the effluent from the lungs revealed that only a small percentage of the radioactivity was in the form of ADP. The majority existed as inosine, with lesser amounts of adenosine (Fig. la). ATP and adenine were not found. The extent and pattern of the metabolism of ADP appeared to be independent of the dose fnjected over the range studies (Table 1). 3H-ADP was not altered by passage through the apparatus alone. The metabolism of 1.0 nmol ADP was not affected by continuous infusion of indaaethacin. 50 uM. The metabolism of 5.0 mnol ADP was simflarly unaffected by continuous infusion of aspirin 550 PM. The metabolism of 1.0 nmol ADP was apparently unaffected by the simultaneous injection of 2.0 or 10.0 nmol PG12.
b
FIG. 1A
t
@P * :! f
P
Efflux of 3H (0) and 14C (0) following bolus injection into rat isolated perfused lungs of a mixture of 1 nmol (3H)-ADP and the vascular marker (14C)-dextran. Fractions were collected imnediately after injection, at 1.14 sec. intervals.
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FIG. 18 Thin-layer chrunatographic analysis of 3H in fractions 5-15 of above experiment. B--m ADP; O--O AMP; M adenosine; CI inosine.
I
1 5
10 Fraction
I 15
number
Continuous infusion of 3H-ADP. A mixture of JH-ADP 30 UM and 14C-dextran was infused continuously into the lung for 3 mins. Tritium and dextran again emerged simultaneously (Fig. 2). However, only 80% of the tritium was recovered in the effluent from the lungs; recovery of dextran was essentially complete. Homogenization of the lungs revealed that a further 17% of tritium was retained by the lung. Thus it would appear that a significant proportion of ADP is taken up by the lung during a continuous infuslon, and is not returned to the circulation. The site of uptake and storage was not investigated further. Analysis of the effluent emerging from the lung during the infusion indicated that approximately 5% of the tritlum was in the form of ADP. The remainder was AMP, adenosine and inoslne (Fig. 2). Thus, the lung also effectively metabolizes ADP during a continuous infusion, although a relatively higher proportion of adenosine is formed than is seen following bolus injection. Platelet aggregation studies. ADP was infused into the lung at a concentration of 10-W. for 3 mins. Samples of the effluent were collected during the infusion and their platelet aggregatory activities compared with those of samples collected inmediately before passage through the lungs. Samples of the pulmonary arterial ADP solution showed marked platelet aggregatory activity; however, samples of effluent from the lung had undetectable activity Sn the amounts sampled. Lung perfusion nmdiun itself also had no aggregatory activity. Comparison between pulmonary arterial and venous samples showed that at least 95% of the platelet aggre atory effects of ADP were lost on a single passage through the lungs (Fig. 33 .
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of Rat Lung
FIG. 2 Metabolism of JH-ADP during continuous infusion into rat isolated perfused lung. A mixture of JH-ADP 30 uM and 14C-dextran were infused into the lung for the time shown (M). The concentration of 3H-ADP supplied to the lung is designated "arterial (3H)." The effluent from the lung was analyzed for total tritium (0) and total 14-carbon (0). Thinlayer chronmtographic analysis of the tritium emerging from the lun revealed unchanged ADP 4B---m) together with AMP (o---n), adenosine (A.+ ) and inosine (W ).
FIG. 3 Aggregation of rabbit plateletrich plasma (PRP) by samples of lung effluent. ADP, lo-4M was infused into rat isolated perfused lungs for 3 min. Arterial samples were collected itmsediately before, and venous samples were collected imnediately after, passage through the lungs. Suitable aliquots were added to 1 ml PRP prewarmed to 37Y for 3 min. Total additions to PRP were made up to 200 ul where appropriate by the addition of Krebs-Ringer medium containing 4.5% bovine serlrmalbumin.
F =v’
VENOUS
10
ARlERIAl
I
200
jll
ARTERIAL
0 ,:: MINUTES
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ADPaee Activity of Rat Lung
TABLE 1 Metabolism of 3H ADP following Bolus Injection into Isolated Perfused Rat Lung
ADP
'1: Product in Effluent Inosine Adenosine AMP
Control
0.7
3.4
80.7
14.2
1.0
II
5.0
4.1
81.7
6.3
1.0
I,
3.8
3.3
74.7
17.0
5.0
II
2.1
1.9
84.0
10.9
5.0
II
3.1
4.3
83.2
6.3
10.0
II
2.1
1.6
84.2
9.9
ADP
% Product in Effluent AMP Inosine Adenosine
Dose of ADP (nmol)
Treatment
0.1
Dose of ADP (nmol)
Treatment
1.0
Control
5.0
4.1
81.7
6.3
1.0
PG12 Pnmol
5.1
3.4
78.5
11.2
1.0
PG12 lOnmo1
2.6
0.7
76.1
18.3
1.0
Control
3.8
3.3
74.7
17.0
1.0
Indomethacin 50 PM
4.5
4.2
74.4
16.2
5.0
Control
2.1
1.9
84.0
10.9
5.0
Aspirin 550PM
2.5
1.7
74.4
19.6
Test bolus injections were made irunediatelyfollowing their respective control injections.
Discussion The pivotal role of ADP as a primary mediator of platelet aggregation suggests that mechanisms limiting its concentration in the blood may play an important role in platelet function. ADP in plasma is broken down by adenylate kinase and by specific ADPases. but the plasma half-life of ADP (approx. 8 min.) suggests that these enzymes are relatively unimportant in controlling ADP concentrations in plasma (5). Recently, attention has been focused on the activity of specific ADPases located in healthy human artery walls (5,6) and rabbit
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aortas (7.8). The degradation of ADP by arterial ADPase has been suggested as one explanation of the ability of healthy artery walls to resist platelet adhesion. Furthermore, in atherosclerotic arteries, ADPase activity is impaired (5). The present study indicates that over 90% of ADP is metabolized in a single passage through rat lungs. The enzymes responsible for the degradation of ADP are apparently located in the pulmonary vessel walls, since the pulmonary transit time for the radioactivity associated with the ADP metabolites is identical to that for the vascular marker dextran. The absence of ATP among the metabolites would suggest that the metabolism is catalyzed by an ADPase rather than by aderlylatekinase. The apparent extravascular localization of these enzymes further supports this conclusion, as adenylate kinase is intracellular whereas arterial ADPase is located in the plasma membrane (7). The small amounts of AMP and larger amounts of adenosine found in the effluent from the lungs suggests that AMP formed as a result of the action of ADPase was further metabolized by 5'-nucleotidase. This enzymf!is localized in the vessel walls in the pulmonary vasculature of the rat (14). Adenosfne and AMP are the major metabolites of ADP produced by arterial ADPase (5.7). However, the major metabolite observed in the present study was inosine. Ryan and co-workers have shown that ATP is extensively metabolized in a single passage through rat lung. Although it is apparent that an ATPase is responsible for the initial degradation of ATP, it is not clear whether deamination also occurred (15.16). Inosine has little platelet aggregatory activity and adenosine is anti-aggregatory (see ref. l), hence the degradation of ADP to these products should result in biological inactivation. This is confirmed by the finding that ADP lost over 95% of its platelet aggregatory activity on a single passage through the pulmonary circulation. Arterial microscnnes (17) and rings (18) release the unstable PGI2 from prostaglandin endoperoxides. PGI has potent anti-aggregatory properties, and its production by arteries coul2 constitute another important mechanism in limiting platelet aggregation in healthy vessels (18). PG12 is apparently released within the lung since its stable end-product, 6-oxo-PGFla has been detected in the effluent from sensitized guinea-pig lungs following anaphylaxis (19). In the present study, PGI had little or no effect on the metabolism of ADP by rat lung. Moreover, tie non-steroid anti-inflanrnatoryagents indomethacin and aspirin, in concentrations which would inhibit cycle-oxygenase activity (20) and thus inhibit PGI2 production, also had no observable effects on the metabolism of ADP by rat lung. Thus, the two proposed anti-aggregatory mechanisms, the ADPase and PGI2 systems, may be capable of independent operation :,'tAe lung. Recent reports indicate that arterial ADPase is potentfated by non-steroid anti-inflammatory agents (8). The interaction between arterial ADPase and PGI2 has yet to be determined. The efficiency of the lung in metabolizing ADP in a single passage through the Pulmonary circulation suggests that it may play an important role as a first defense against ADP entering the systemic arterial circulation. Its efficiency in metabolizing bolus injections of ADP suggests that it may also play a physiological role in metabolizing ADP released explosively from platelets. In a recent report, 64% of lungs examined at autopsy showed evidence of trapped micro-embolf; this figure may be only a small proportion of the emboli trapped by the lung during life (21). The ability of the lung to protect itself against the dangerous growth of trapped platelet aggregates may be due in part to its ability to metabolize ADP.
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Acknowledgements We wish to thank Ms. Hollis J. Hawkins for her help in preparation of the isolated perfused lung and Mr. Jeff 8oyd for excellent technical assistance. We would also like to thank Or. John Westwick of the Royal College of Surgeons, London, England, for his helpful advice and criticism. We thank Or. K. C. Nicolaou of the University of Pennsylvania for the gift of prostacyclin. .We also wish to thank Ms. Janice Strother for the preparation of the manuscript. References
5. 6. 7. 8.
A. du P. HEYNS, C. J. EAOENH~nd~RE~roi&s. Haemostas., 37 429-435 (1977). K du P. HEYNS, 0. J. VAN DEN BERG, 6. M. POTGIETER. and F. P. RETIEF, Thromb. Oiathes. haemorrh., 32 417-431 (1974). G. E. LI-, 6. P. LEWISFand T. J. PETERS, Lancet, i 330-332 (1977). 6. P. LEWIS, G. E. LIEBERMAN, and J. WESTWICK, -8r. J. Pharmac., 61 449-450p (1977).
Y. S. EAKHLE, and J. R. VANE, Physiol. f&., 54 1007-1045 (1974). Invest,?3 843-855 (1964). R. H. ASTER and J. H. JANOL, J. m. 1:: 645-677 (1976). 11. M. W. ANDERSON and T. E. ELINE, Prostamnns;il 12.
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G. A. NORMAN, M. J. FOLLETT, and 0. A. HECTOR, ~~Chrunatog., ~105-111
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