VASCULAR EFFECTS OF ARACHIDONATE IN LUNGS/Wicks et al. 2. Scheuer J, Stezoski SW: Effect of high energy phosphate depletion and repletion on the dynamics and electrocardiogram of isolated rat hearts. Circ Res 23: 519 530, 1968 3. Rovelto M J, Whitmer JT, Neely J R: Comparison of the effects of anoxia and whole heart ischemia on carbohydrate utilization in isolated working rat hearts. Circ Res 32: 699-711, 1973 4. Williamson JR: Glycolytic control mechanisms. II. Kinetics of intermediate changes during the aerobic-anoxic transition in perfused rat heart. J Biol Chem 241: 5026-5036, 1966 5. Scheuer J, Stezoski SW: Protective role of increased myocardial glycogen stores in cardiac anoxia in the rat. Circ Res 27: 835-849, 1970 6. Hewitt RL, Lolley DM, Adrouny GA, Drapanas T: Protective effect of glycogen and glucose on the anoxic arrested heart. Surgery 75: 1-10, 1974 7. Cornblath M, Randle PJ, Parmeggiani A, Morgan HE: Regulation of glycogenolysis in muscle; effects of glucagon and anoxia on lactate production, glycogen content and phosphorylase activity in the perfused isolated rat heart. J Biol Chem 238: 1592-1597, 1963 8. George WJ, Poison JB, OToole AG, Goldberg ND: Elevation of guanosine 3',5'-cyclic phosphate in rat hearts after perfusion with acetylcholine. Proc Natl Acad Sci USA 66: 398-403, 1970 9. George WJ, Wilkerson RD, Kadowitz PJ: Influence of acetylcholine on contractile force and cyclic nucleotide levels in the isolated perfused rat heart. J Pharmacol Exp Ther 184: 228-235, 1973 10. Wollenberger A, Babskii EB, Krauss EG, Genz S, Blohm D, Bogdanova EV: Cyclic changes in levels of cyclic AMP and cyclic GMP in frog myocardium during the cardiac cycle. Biochem Biophys Res Commun 55: 446-452, 1973 11. Busuttil RW, Paddock RJ, George WJ: Effects of glucagon on cyclic nucleotide levels and cardiac performance in isolated perfused rat hearts following hypoxia (abstr). Fed Proc 33: 344, 1974. 12. Goldberg ND, O'Dea RF, Haddox MK: Cyclic GMP. Adv Cyclic Nucleotide Res 3: 155-223, 1973 13. Oilman AG: A protein binding assay for adenosine 3',5'-cyclic mono-
167
phosphate. Proc Natl Acad Sci USA 67: 305-312, 1970 14. Steiner AL, Parker CW, Kipnis DM: Radioimmunoassay for cyclic nucleotides. J Biol Chem 247:1106-1113, 1972 15. Marbach EP, Weil MH: Rapid enzymatic measurement of blood lactate and pyruvate. Clin Chem 13: 314-325, 1967 16. Glick G, Parmley WW, Wechsler AS, Sonnenblick EH: Glucagon—its enhancement of cardiac performance in the cat and dog and persistence of its inotropic action despite beta-receptor blockage with propranolol. Circ Res 22: 789-799, 1968 17. Regan TJ, Lehan PH, Henneman DH, Behar A, Hellems HK: Myocardial metabolic and contractile response to glucagon and epinephrine. J Lab Clin Med 63: 638-647, 1964 18. Nobel-Allen N, Kirsch M, Lucchesi BR: Glucagon; its enhancement of cardiac performance in the cat with chronic heart failure. J Pharmacol Exp Ther 187: 475-481, 1974 19. Hearse DJ, Chain EB: Effect of glucose on enzyme release from, and recovery of, the anoxic myocardium. In Recent Advances in Studies on Cardiac Structure and Metabolism, vol 3, edited by N S Dhalla. Baltimore, University Park Press, 1972, pp 763-772 20. Cooper EG, Booker WM, Johnson JB: Pharmacological interventions in experimental myocardial infarction. In Recent Advances in Studies on Cardiac Structure and Metabolism, vol 3, edited by NS Dhalla. Baltimore, University Park Press, 1972, pp 831-847 21. Sutherland EW, Robison GA, Butcher RW: Some aspects of the biological role of adenosine 3',5'-monophosphate (cyclic AMP). Circulation 37: 279-306, 1968 22. Mayer SE, Namm DH, Rice L: Effect of glucagon on cyclic 3',5'-AMP, phosphorylase activity, and contractility of heart muscle of the rat. Circ Res 26: 225-233, 1970 23. Brooker G: Change in myocardial adenosine 3',5'-cyclic monophosphate during the contraction cycle. In Recent Advances in Studies on Cardiac Structure and Metabolism, vol 3, edited by NS Dhalla. Baltimore, University Park Press, 1973, pp 207-212
Vascular Responses to Arachidonic Acid in the Perfused Canine Lung THOMAS C. WICKS, P H . D . , JOHN C. ROSE, M.D.,
MALCOLM JOHNSON,
PH.D.,
PETER W. RAMWELL, P H . D . , AND PETER A. K O T , M.D.
SUMMARY We compared the effects of arachidonic acid (AA), the bisenoic prostaglandin precursor, with those of prostaglandin F to (PGF*,) and norepinephrine (NE) on pulmonary vascular resistance in the isolated (in situ), perfused canine lung lobe. The isolated lobe was perfused with autologous blood or an artificial perfusate under conditions of constant flow. Lobar artery and venous pressures were constantly monitored after bolus injections of AA, PGF IO , and NE into the inflow cannula. AA (100 /ig/kg) produced a significant increase in the pressure gradient (93.3 ± 8.4%, SE) in the lobe. Similarly, PGF*, (1 M g/kg) significantly increased the pressure gradient (41.2 ± 6.5%), as did NE (1 M g/ kg, 41.6 ± 3.2%). Aspirin (25 mg/kg) completely blocked the pul-
monary vascular effect of AA, but did not affect the response to PGFfe, Linoleic acid, a control fatty acid, did not produce pulmonary vasoconstriction. The pressor effect of AA was not blocked by pretreatment with phentolamine, propranolol, cyproheptadine, or atropine. The use of an artificial perfusate free of cellular elements did not prevent the vasoconstrictor action of AA. The times to onset of action of the three agents were similar, and short. These results suggest that AA is converted into vasoactive intermediates or a prostaglandin, and the vasoactive intermediates or the prostaglandin act directly on precapillary pulmonary vascular smooth muscle rather than through platelet, plasma, adrenergic, or cholinergic mechanisms.
T H E CARDIOVASCULAR actions of the bisenoic prosta-
peripheral vasoconstricting agent. Both compounds have a
glandins, prostaglandin E, (PGE,) and F t o (PGF*,), have
direct positive inotropic effect on the heart. 2 ' 3 The precursor
been investigated intensively in several species of animals. 1
of the bisenoic prostaglandins, arachidonic acid (AA), 4 is
P G E , produces systemic vasodilajion and a depressor re-
released from cell phospholipids and other lipid sources,6"7
sponse in the dog, whereas P G F ^ is a moderately active
and is converted into endoperoxide intermediates and even-
From the Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, D.C. Supported by grants from the U.S. Public Health Service (HE-05527), the Washington Heart Association, and the Educational Foundation of America. Received August 15, 1975; accepted for publication December 1, 1975.
that AA (300 Mg/kg) produces a systemic depressor re-
tually into PGE 2 and PGF,,,.* Recent studies have shown sponse in dogs which is equivalent to that caused by a dose of 5 Mg/kg of P G E , . ' Information about the selective action of A A on different
CIRCULATION RESEARCH
168
segments of the circulatory system is lacking. The specific aims of this investigation were: (1) to determine the effects of AA on the canine pulmonary circulation; (2) to determine whether the response represented a direct action of A A; and (3) to assess the role of the platelet in the cardiovascular actions of AA in light of studies which describe the platelet-aggregating potential of AA10"12 and the release of vasoactive substances from platelets by AA. 13 Studies were performed on the isolated, perfused canine left lower lung lobe under conditions of constant flow. Methods Twenty-eight mongrel dogs, unselected as to sex or weight, were anesthetized with intravenous sodium pentobarbital (30 mg/kg) and maintained on positive-pressure ventilation (room air) by endotracheal intubation. Ventilation rate was 15/min and the tidal volume was adjusted for each dog within a range of 225-300 ml. Expiration was passive. A thoracotomy was performed in the 4th or 5th left intercostal space. We isolated the circulation to the left lower lung lobe from the remainder of the pulmonary circulation by cannulating the lobar artery and vein with polyethylene catheters. The bronchus was left intact to maintain ventilation of the lobe. The isolated lobe was perfused with autologous citrated (10% vol/vol of 3.8% sodium citrate) blood by means of a peristaltic pump. Pulmonary venous blood was drained into an open, siliconized reservoir maintained at 37°C and was recirculated through the isolated lobe. The volume of the system was approximately 200 ml. Mean lobar arterial and venous pressures were monitored at the inflow and outflow cannulas, respectively. Flow rate through the lobe was adjusted manually to maintain mean lobar artery pressure between 12 and 16 mm Hg. This was accomplished with a flow rate between 6 and 7 ml/kg per min. The height of the outflow cannula was adjusted to produce a mean lobar venous pressure of 0-5 mm Hg. We recorded airway pressure, which was used as an index of airway resistance, by inserting a needle into the endotracheal tube. Systemic arterial pressure was monitored through an indwelling femoral arterial catheter. All pressures were recorded continuously with a multichannel direct-writing recorder. Additional anesthetic agent, when needed, was administered through a femoral venous catheter. Measurements of Po a , Pco 2 , and pH were made at intervals to ensure that ventilation and perfusion were adequate. The ranges of these were: Po 2 , 118-123 mm Hg; Pco,, 22-29 mm Hg; and pH, 7.40-7.47. Arachidonic acid (5,8,11,14-eicosatetraenoic acid, >99% pure and obtained from porcine liver) and linoleic acid ( > 9 9 % pure) were obtained from Sigma. The sodium salts of these acids were prepared by dissolving them in 100 mM sodium carbonate with constant stirring under nitrogen, in the absence of light. The solutions were prepared daily and were used only if they were clear. Prostaglandins (tromethamine salts) were supplied by Upjohn. Saline solutions of the prostaglandins were made daily from stock ethanol solutions. The sodium salt of
VOL. 38, No.
3, MARCH
1976
acetylsalicylic acid (Merck) was prepared in modified Tyrode's solution and the pH was adjusted to 7.35 with sodium hydroxide. Norepinephrine (norepinephrine bitartrate, Levophed) was diluted in normal saline (75 /ig/ml). a-Adrenergic blockade was achieved with phentolamine (Regitine) at doses of 100-200 /ig/kg (7.5-15 /ig/ml of blood in the system). /3-Adrenergic antagonism was achieved with propranolol hydrochloride (Inderal), 100 /ig/kg (7.5 jig/ml of blood in the system). Cyproheptadine (Periactin), an antihistamine and antiserotonin agent, was used at a dose of 55 jig/kg (4 /ig/ml of blood in the system). Atropine sulfate (200 /ig/kg or 15 /ig/ml of blood) was used as an anticholinergic agent. Blockades were tested with the appropriate challenge drugs in doses of 1-5 jig/kg. Challenge drugs and test substances were administered as bolus injections directly into the inflow cannula just proximal to the point of its insertion in the lobar artery. Injection volumes were 0.1-0.4 ml. Blocking agents were administered directly into the reservoir. Sufficient time was permitted for blockade to become effective. The system was allowed to return to control conditions after a test injection (approximately 5 minutes) before another agent was administered. In the first series of experiments (17 animals), the pulmonary vascular effects of P G F ^ (1 /ig/kg), norepinephrine (NE)(1 M g/kg)and AA (100/ig/kg) were studied in the blood-perfused, isolated lobe. In the second group (nine animals), the effects of these agents on pulmonary vascular reactivity were studied in blood-perfused lobes pretreated with aspirin (25 mg/kg). In the third group (five animals) pulmonary vascular responses to these compounds were first studied in the blood-perfused lobe and then immediately after replacement of the blood with a dextran-based artificial perfusate (Perfudex, Pharmacia) containing 2.5 mM CaCl2 and 25 mM NaHCO 3 . For statistical analysis of the data obtained, we used Student's /-test. Significance was set at the 0.05 level. Results RESPONSES OF BLOOD PERFUSED LOBES TO AA Dose-response relationships for AA were established in the blood-perfused lung. The pressor response to AA showed a nearly linear relationship up to a dose of 150 /ig/kg. The threshold dose for this system was between 1 and 10 jig/kg. On the basis of these results, a dose of 100 /ig/kg of AA was used in all subsequent studies on isolated lobes. This dose resulted in a reproducible pressor response, from which recovery occurred within 5 minutes. Through trial, we selected a dose of PGF t o of 1 /ig/kg as one which elicited a reproducible pulmonary pressor response. Since a dose of NE of 1 /ig/kg produced an equipressor response, this dose was continued throughout this study. Recovery from the pressor responses to both agents was complete in 5 minutes. Figure IA demonstrates the pulmonary vascular response to a dose of AA of 100 /ig/kg. A A produced a mean increase of 93.3 ± 8.4% (SE) in lobar artery pressure at the peak of the response. Lobar vein and airway pressures did not change in any experiments. Systemic arterial pressure also
VASCULAR EFFECTS OF ARACHIDONATE IN LUNG/Wicks etal.
169
ARACHIDONIC ACID (100 tig/kg) A. BEFORE P airway
„
Hg
B. AFTER ASPIRIN (25mg/kg)
-20 10 -0
Plob.art.
• 40
20 0
Plob.v. Psys
20
I
0
- 200 100 0
T FIGURE I A record of the response to one injection of arachidonic acid (100 ng/kg) into the isolated, blood-perfused canine pulmonary lobe before administration of aspirin (A) and after aspirin (B). Top trace, airway pressure; second trace, lobar artery pressure; third trace, lobar vein pressure; fourth trace, systemic arterial pressure. Arrow indicates time of injection. Time calibration = 30 seconds.
remained stable, indicating that there was no significant transfer of AA from the isolated lobe circulation into the systemic circulation. PGFj,, and NE also produced pressor responses in the isolated lobe: these were 41.2 ± 6.5% and 41.6 ± 3.2%, respectively, at a dose of 1 Mg/kg- Lobar vein, airway, and systemic arterial pressures were unchanged in both cases. The percentage changes in pulmonary artery pressure caused by AA, PGFjo and NE are summarized in Figure 2. ASPIRIN BLOCKADE The tracing shown in Figure IB demonstrates the effects of pretreatment with aspirin on the pulmonary vascular response to AA. Aspirin did produce a small reduction (1 mm Hg) in baseline perfusion pressure in many preparations. The pulmonary pressor response to AA was completely eliminated in every preparation pretreated with aspirin. In most cases a slight reduction (3-4 mm Hg) in pulmonary artery pressure occurred within 6 seconds after administration of AA. Comparable changes in pulmonary artery pressure were produced by the control fatty acid, linoleic acid; this finding suggests that this was a nonspecific effect of fatty acid in the presence of this anti-inflammatory agent. Responses to PGFa, and NE were not blocked by aspirin (Fig. 2). The response to PGFj,, was essentially unchanged (44.8 ± 7.4%) but the response to NE was diminished significantly (18.7 ± l.C
OTHER BLOCKING AGENTS
Phentolamine, propranolol, atropine, and cyproheptadine were tested and none blocked or attenuated the pulmonary vascular response to AA. ARTIFICIAL PERFUSION During perfusion of the isolated lobe with an artificial perfusate free of cellular elements, the pulmonary pressor response to AA was as great (98.4 ± 31.6%) as in the blood-perfused lobe. The pulmonary vascular response to NE, 1 /xg/kg, was also unaltered by substitution of the artificial perfusate but the pressor effect of PGFJQ was diminished significantly to 10.0 ± 0.95%. TIME OF ACTION RELATIONSHIPS The times to onset of action for the substances were quite similar and rapid (mean, 5.5-7 seconds). This time was significantly greater (mean, 12 seconds) only for PGF*, in the artificially perfused lobe. The time to peak effect of each substance was similar between groups (mean, 21-31 seconds) except for AA (mean, 59 seconds) and PGF t o (mean, 45 seconds) in the lobe pretreated with aspirin. Duration of response (not shown here) was variable. In all cases, this did not exceed 5 minutes. Discussion In contrast to its systemic hypotensive effect, AA produced a rapid and marked increase in pulmonary artery
170
CIRCULATION RESEARCH
VOL. 38, No. 3, MARCH
1976
100
75
o
50
^
25
-25
L
Before
After 25 mg/kg Aspirin BLOOD PERFUSED LOBE
ARTIFICIALLY PERFUSED LOBE
_L
J
FIGURE 2 Summary of the changes in lobar artery pressure after administration of arachidonic acid, 100 ng/kg (solid bars), prostaglandin ^20. ' fig/kg (cross-hatched bars), and norepinephrine, I fig/kg (open bars), to three groups of isolated perfused canine lung lobes: before (n = 17) and after (n = 9) administration of aspirin, 25 mg/kg, and during artificial perfusion (n = 5). Means expressed ± SE.
pressure in the isolated blood-perfused dog lung lobe. This dose-related increase in pressure gradient occurred in the presence of constant flow and indicates that the rise in pressure was the direct result of an increase in pulmonary vascular resistance. The characteristics of this response were similar to those obtained with much smaller doses of PGF 2a and NE. The times to onset and peak action of each of the agents were very similar; this finding strongly suggests a direct action of both AA and P G F ^ . However, the pressor action of AA was blocked by aspirin; this indicates that biosynthetic conversion of AA to endoperoxides, prostaglandins, or other products is necessary for its vascular action. Hamberg and Samuelsson 8 demonstrated that conversion of AA into polar derivatives in a sheep seminal vesicle preparation was complete within 15 seconds, but formation of PGE 2 (measured indirectly) was complete only after 2 minutes. Moreover, Hamberg and Samuelsson 14 recently reported that administration of AA to the isolated artificially perfused guinea pig lung yielded only approximately 3% PGE 2 and 5% PGFto and more than 90% of other non-prostanoate compounds. In a later report 15 Hamberg and co-workers showed that biosynthesized endoperoxide prostaglandin intermediates had potent smooth muscle-stimulating abilities, both in vitro and in vivo. These data, in conjunction with the rapid onset of action of AA in the lung lobe, suggest that prostaglandin intermediates are the agents responsible for the pulmonary pressor
response. Conversion of AA into PGFfc, may contribute to this response, although the time for synthesis of P G F ^ seems to be greater than the 6-second time to onset of AA action. Our results with PGFj,, compare favorably with the results of studies by others on dogs and other species.1' *• Lonigro and Dawson16 reported that PGF t o exerts most of its effect on pulmonary vessels upstream to the capillaries and that degradation of P G F t o seems to be downstream from the site of action. In the present study, the times to onset of action of both AA and PGFj,, were identical, and strongly suggest that the sites of action are the small pulmonary arteries. The decreased pressor response to NE in the presence of aspirin cannot be adequately explained on the basis of our data. However, Kadowitz and others" 1 18 have shown that F series prostaglandins in subvasoactive doses increase vascular reactivity to NE. In addition, NE has been shown to cause the release of prostaglandins from several tissues.19 Either of these mechanisms might account for this decreased pressor response to NE, since generation of endogenous prostaglandins would be blocked by aspirin pretreatment with aspirin. Experiments with the other blocking agents demonstrate that a- and £-adrenergic, cholinergic, and serotonin receptors do not mediate the response to the active compounds synthesized from AA. Rather, other receptor sites are involved in the response to these vasoactive compounds.
VASCULAR EFFECTS OF ARACHIDONATE IN LUNG/ Wickset al. Silver and colleagues" demonstrated that a large dose of A A administered to rabbits intravenously produced death as a result of extensive blockage of the pulmonary circulation by platelet aggregates. No measurements of pulmonary artery pressure were made in these experiments. To rule out mechanical obstruction of small pulmonary vessels as the mechanism responsible for the pulmonary pressor response in our experiments, the isolated lobe was perfused with an artificial perfusate free of cellular elements. The pulmonary vascular responses to A A and NE were not significantly different when a perfusate free of cellular elements was used. Mechanical obstruction did not account for the rise in pulmonary artery pressure. Moreover, conversion of AA into active intermediates does not require the presence of platelets or other blood cellular elements. Other tissues, such as vascular endothelium,20 must be a source of prostaglandin synthetase for the conversion of AA into a cascade of vasoactive compounds. An adequate explanation for the diminished response to PGFfc, during use of an artificial perfusate is lacking. Binding of PGF20 to the dextran base, or absence of a necessary blood component for its action, could explain the reduced responsiveness in this artificial system. In addition, PGFjo may be metabolized by erythrocytes to the potent, smooth muscle-stimulating 15-keto PGFj,,.21- 22 In the absence of erythrocytes this prostaglandin metabolite may not be formed. Acknowledgments We thank T. Maish for the preparation of the solutions used in this study, and for her excellent technical advice.
References 1. Kadowitz PJ, Joiner PO, Hyman AL: Physiological and pharmacological roles of prostaglandins. Annu Rev Pharmacol 15: 285-306, 1975 2. Nakano J, McCurdy JR: Hemodynamic effects of prostaglandins E,, A,, and F*. in dogs. Proc Soc Exp Biol Med 128: 39-42, 1968 3. Nakano J: Cardiovascular actions of prostaglandins. /nThe Prostaglandins, vol. I, edited by PW Ramwell. New York, Plenum Publishing Corp., 1973, pp 238-316
171
4. Anggard E, Samuelsson B: Biosynthesis of prostaglandins from arachidonic acid in guinea pig lung. J Biol Chem 240: 3518-3521, 1965 5. Bills TK, Silver MJ: Phosphotidylcholine is the primary source of arachidonic acid utilized by platelet prostaglandin synthelase (abstr). Fed Proc 34: 790, 1975 6. Danon A, Chang LCT, Sweelman BJ, Nies AS, Oates JA: Synthesis of prostaglandins by the rat renal papilla in vitro; mechanism of stimulation by angiotensin II. Biochim Biophys Acta 388: 71-83, 1975 7. Danon A, Heimberg M, Oates JA: Enrichment of rat tissue lipids with fatty acids that are prostaglandin precursors. Biochim Biophys Acta 388: 318-330, 1975 8. Hamberg M, Samuelsson B: Detection and isolation of an endoperoxide intermediate in prostaglandin biosynthesis. Proc Natl Acad Sci USA 70: 899-903, 1973 9. Rose JC, Johnson M, Ramwell PW, Kot PA: Effects of arachidonic acid on systemic arterial pressure, myocardial contractility and platelets in the dog. Proc Soc Exp Biol Med 147: 652-655, 1974 10. Hamberg M, Svensson J, Wakabayashi T, Samuelsson B: Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc Natl Acad Sci USA 71: 345-349, 1974 11. Silver MJ, Hoch W, Kocsis JJ, Ingerman CM, Smith JB: Arachidonic acid causes sudden death in rabbits. Science 183: 1085-1086, 1974 12. Furlow TW Jr, Bass NJ: Stroke in rats produced by carotid injection of sodium arachidonate. Science 187: 658-660, 1975 13. Vargaftig BB, Zirinis P: Platelet aggregation induced by arachidonic acid is accompanied by release of potential inflammatory mediators distinct from PGE, and PGF t o . Nature (New Biol) 244: 114-116, 1973 14. Hamberg M, Samuelsson B: Prostaglandin endoperoxides. VII. Novel transformations of arachidonic acid in guinea pig lung. Biochem Biophys Res Commun 61: 942-949, 1974 15. Hamberg M, Hedqvist P, Strandberg K, Svensson J, Samuelsson B: Prostaglandin endoperoxides. IV. Effects on smooth muscle. Life Sci 16: 451-462, 1975 16. Lonigro AJ, Dawson CA: Vascular responses to prostaglandin F*, in isolated cat lungs. Circ Res 36: 706-712, 1975 17. Kadowitz PJ: Effect of prostaglandin E,, E2 and A 2 on vascular resistance and responses to noradrenaline, nerve stimulation and angiotensin in the dog hindlimb. Br J Pharmacol 46: 395-400, 1972 18. Kadowitz PJ, Sweet CS, Brody MJ: Influence of prostaglandins on adrenergic transmission to vascular smooth muscle. Circ Res 31 (suppl 2): 36-50, 1972 19. Brody MJ, Kadowitz PJ: Prostaglandins as modulators of the autonomic ' nervous system. Fed Proc 33: 48-60, 1974 20. Gimbrone MA, Alexander RW: Angiotensin II stimulation of prostaglandin production in cultured human vascular endolhelium. Science 189: 219-220, 1975 21. Dawson W, Lewis RL, McMahon RE, Sweatman WJF: Potent bronchoconstrictor activity of 15-keto prostaglandin F*,. Nature 250: 331-332, 1974 22. Kaplan L, Lee S-C, Levine L: Partial purification and some properties of human erythrocyte prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase. Arch Biochem Biophys 167: 287-293, 1975
167
phosphate. Proc Natl Acad Sci USA 67: 305-312, 1970 14. Steiner AL, Parker CW, Kipnis DM: Radioimmunoassay for cyclic nucleotides. J Biol Chem 247:1106-1113, 1972 15. Marbach EP, Weil MH: Rapid enzymatic measurement of blood lactate and pyruvate. Clin Chem 13: 314-325, 1967 16. Glick G, Parmley WW, Wechsler AS, Sonnenblick EH: Glucagon—its enhancement of cardiac performance in the cat and dog and persistence of its inotropic action despite beta-receptor blockage with propranolol. Circ Res 22: 789-799, 1968 17. Regan TJ, Lehan PH, Henneman DH, Behar A, Hellems HK: Myocardial metabolic and contractile response to glucagon and epinephrine. J Lab Clin Med 63: 638-647, 1964 18. Nobel-Allen N, Kirsch M, Lucchesi BR: Glucagon; its enhancement of cardiac performance in the cat with chronic heart failure. J Pharmacol Exp Ther 187: 475-481, 1974 19. Hearse DJ, Chain EB: Effect of glucose on enzyme release from, and recovery of, the anoxic myocardium. In Recent Advances in Studies on Cardiac Structure and Metabolism, vol 3, edited by N S Dhalla. Baltimore, University Park Press, 1972, pp 763-772 20. Cooper EG, Booker WM, Johnson JB: Pharmacological interventions in experimental myocardial infarction. In Recent Advances in Studies on Cardiac Structure and Metabolism, vol 3, edited by NS Dhalla. Baltimore, University Park Press, 1972, pp 831-847 21. Sutherland EW, Robison GA, Butcher RW: Some aspects of the biological role of adenosine 3',5'-monophosphate (cyclic AMP). Circulation 37: 279-306, 1968 22. Mayer SE, Namm DH, Rice L: Effect of glucagon on cyclic 3',5'-AMP, phosphorylase activity, and contractility of heart muscle of the rat. Circ Res 26: 225-233, 1970 23. Brooker G: Change in myocardial adenosine 3',5'-cyclic monophosphate during the contraction cycle. In Recent Advances in Studies on Cardiac Structure and Metabolism, vol 3, edited by NS Dhalla. Baltimore, University Park Press, 1973, pp 207-212
Vascular Responses to Arachidonic Acid in the Perfused Canine Lung THOMAS C. WICKS, P H . D . , JOHN C. ROSE, M.D.,
MALCOLM JOHNSON,
PH.D.,
PETER W. RAMWELL, P H . D . , AND PETER A. K O T , M.D.
SUMMARY We compared the effects of arachidonic acid (AA), the bisenoic prostaglandin precursor, with those of prostaglandin F to (PGF*,) and norepinephrine (NE) on pulmonary vascular resistance in the isolated (in situ), perfused canine lung lobe. The isolated lobe was perfused with autologous blood or an artificial perfusate under conditions of constant flow. Lobar artery and venous pressures were constantly monitored after bolus injections of AA, PGF IO , and NE into the inflow cannula. AA (100 /ig/kg) produced a significant increase in the pressure gradient (93.3 ± 8.4%, SE) in the lobe. Similarly, PGF*, (1 M g/kg) significantly increased the pressure gradient (41.2 ± 6.5%), as did NE (1 M g/ kg, 41.6 ± 3.2%). Aspirin (25 mg/kg) completely blocked the pul-
monary vascular effect of AA, but did not affect the response to PGFfe, Linoleic acid, a control fatty acid, did not produce pulmonary vasoconstriction. The pressor effect of AA was not blocked by pretreatment with phentolamine, propranolol, cyproheptadine, or atropine. The use of an artificial perfusate free of cellular elements did not prevent the vasoconstrictor action of AA. The times to onset of action of the three agents were similar, and short. These results suggest that AA is converted into vasoactive intermediates or a prostaglandin, and the vasoactive intermediates or the prostaglandin act directly on precapillary pulmonary vascular smooth muscle rather than through platelet, plasma, adrenergic, or cholinergic mechanisms.
T H E CARDIOVASCULAR actions of the bisenoic prosta-
peripheral vasoconstricting agent. Both compounds have a
glandins, prostaglandin E, (PGE,) and F t o (PGF*,), have
direct positive inotropic effect on the heart. 2 ' 3 The precursor
been investigated intensively in several species of animals. 1
of the bisenoic prostaglandins, arachidonic acid (AA), 4 is
P G E , produces systemic vasodilajion and a depressor re-
released from cell phospholipids and other lipid sources,6"7
sponse in the dog, whereas P G F ^ is a moderately active
and is converted into endoperoxide intermediates and even-
From the Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, D.C. Supported by grants from the U.S. Public Health Service (HE-05527), the Washington Heart Association, and the Educational Foundation of America. Received August 15, 1975; accepted for publication December 1, 1975.
that AA (300 Mg/kg) produces a systemic depressor re-
tually into PGE 2 and PGF,,,.* Recent studies have shown sponse in dogs which is equivalent to that caused by a dose of 5 Mg/kg of P G E , . ' Information about the selective action of A A on different
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segments of the circulatory system is lacking. The specific aims of this investigation were: (1) to determine the effects of AA on the canine pulmonary circulation; (2) to determine whether the response represented a direct action of A A; and (3) to assess the role of the platelet in the cardiovascular actions of AA in light of studies which describe the platelet-aggregating potential of AA10"12 and the release of vasoactive substances from platelets by AA. 13 Studies were performed on the isolated, perfused canine left lower lung lobe under conditions of constant flow. Methods Twenty-eight mongrel dogs, unselected as to sex or weight, were anesthetized with intravenous sodium pentobarbital (30 mg/kg) and maintained on positive-pressure ventilation (room air) by endotracheal intubation. Ventilation rate was 15/min and the tidal volume was adjusted for each dog within a range of 225-300 ml. Expiration was passive. A thoracotomy was performed in the 4th or 5th left intercostal space. We isolated the circulation to the left lower lung lobe from the remainder of the pulmonary circulation by cannulating the lobar artery and vein with polyethylene catheters. The bronchus was left intact to maintain ventilation of the lobe. The isolated lobe was perfused with autologous citrated (10% vol/vol of 3.8% sodium citrate) blood by means of a peristaltic pump. Pulmonary venous blood was drained into an open, siliconized reservoir maintained at 37°C and was recirculated through the isolated lobe. The volume of the system was approximately 200 ml. Mean lobar arterial and venous pressures were monitored at the inflow and outflow cannulas, respectively. Flow rate through the lobe was adjusted manually to maintain mean lobar artery pressure between 12 and 16 mm Hg. This was accomplished with a flow rate between 6 and 7 ml/kg per min. The height of the outflow cannula was adjusted to produce a mean lobar venous pressure of 0-5 mm Hg. We recorded airway pressure, which was used as an index of airway resistance, by inserting a needle into the endotracheal tube. Systemic arterial pressure was monitored through an indwelling femoral arterial catheter. All pressures were recorded continuously with a multichannel direct-writing recorder. Additional anesthetic agent, when needed, was administered through a femoral venous catheter. Measurements of Po a , Pco 2 , and pH were made at intervals to ensure that ventilation and perfusion were adequate. The ranges of these were: Po 2 , 118-123 mm Hg; Pco,, 22-29 mm Hg; and pH, 7.40-7.47. Arachidonic acid (5,8,11,14-eicosatetraenoic acid, >99% pure and obtained from porcine liver) and linoleic acid ( > 9 9 % pure) were obtained from Sigma. The sodium salts of these acids were prepared by dissolving them in 100 mM sodium carbonate with constant stirring under nitrogen, in the absence of light. The solutions were prepared daily and were used only if they were clear. Prostaglandins (tromethamine salts) were supplied by Upjohn. Saline solutions of the prostaglandins were made daily from stock ethanol solutions. The sodium salt of
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acetylsalicylic acid (Merck) was prepared in modified Tyrode's solution and the pH was adjusted to 7.35 with sodium hydroxide. Norepinephrine (norepinephrine bitartrate, Levophed) was diluted in normal saline (75 /ig/ml). a-Adrenergic blockade was achieved with phentolamine (Regitine) at doses of 100-200 /ig/kg (7.5-15 /ig/ml of blood in the system). /3-Adrenergic antagonism was achieved with propranolol hydrochloride (Inderal), 100 /ig/kg (7.5 jig/ml of blood in the system). Cyproheptadine (Periactin), an antihistamine and antiserotonin agent, was used at a dose of 55 jig/kg (4 /ig/ml of blood in the system). Atropine sulfate (200 /ig/kg or 15 /ig/ml of blood) was used as an anticholinergic agent. Blockades were tested with the appropriate challenge drugs in doses of 1-5 jig/kg. Challenge drugs and test substances were administered as bolus injections directly into the inflow cannula just proximal to the point of its insertion in the lobar artery. Injection volumes were 0.1-0.4 ml. Blocking agents were administered directly into the reservoir. Sufficient time was permitted for blockade to become effective. The system was allowed to return to control conditions after a test injection (approximately 5 minutes) before another agent was administered. In the first series of experiments (17 animals), the pulmonary vascular effects of P G F ^ (1 /ig/kg), norepinephrine (NE)(1 M g/kg)and AA (100/ig/kg) were studied in the blood-perfused, isolated lobe. In the second group (nine animals), the effects of these agents on pulmonary vascular reactivity were studied in blood-perfused lobes pretreated with aspirin (25 mg/kg). In the third group (five animals) pulmonary vascular responses to these compounds were first studied in the blood-perfused lobe and then immediately after replacement of the blood with a dextran-based artificial perfusate (Perfudex, Pharmacia) containing 2.5 mM CaCl2 and 25 mM NaHCO 3 . For statistical analysis of the data obtained, we used Student's /-test. Significance was set at the 0.05 level. Results RESPONSES OF BLOOD PERFUSED LOBES TO AA Dose-response relationships for AA were established in the blood-perfused lung. The pressor response to AA showed a nearly linear relationship up to a dose of 150 /ig/kg. The threshold dose for this system was between 1 and 10 jig/kg. On the basis of these results, a dose of 100 /ig/kg of AA was used in all subsequent studies on isolated lobes. This dose resulted in a reproducible pressor response, from which recovery occurred within 5 minutes. Through trial, we selected a dose of PGF t o of 1 /ig/kg as one which elicited a reproducible pulmonary pressor response. Since a dose of NE of 1 /ig/kg produced an equipressor response, this dose was continued throughout this study. Recovery from the pressor responses to both agents was complete in 5 minutes. Figure IA demonstrates the pulmonary vascular response to a dose of AA of 100 /ig/kg. A A produced a mean increase of 93.3 ± 8.4% (SE) in lobar artery pressure at the peak of the response. Lobar vein and airway pressures did not change in any experiments. Systemic arterial pressure also
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ARACHIDONIC ACID (100 tig/kg) A. BEFORE P airway
„
Hg
B. AFTER ASPIRIN (25mg/kg)
-20 10 -0
Plob.art.
• 40
20 0
Plob.v. Psys
20
I
0
- 200 100 0
T FIGURE I A record of the response to one injection of arachidonic acid (100 ng/kg) into the isolated, blood-perfused canine pulmonary lobe before administration of aspirin (A) and after aspirin (B). Top trace, airway pressure; second trace, lobar artery pressure; third trace, lobar vein pressure; fourth trace, systemic arterial pressure. Arrow indicates time of injection. Time calibration = 30 seconds.
remained stable, indicating that there was no significant transfer of AA from the isolated lobe circulation into the systemic circulation. PGFj,, and NE also produced pressor responses in the isolated lobe: these were 41.2 ± 6.5% and 41.6 ± 3.2%, respectively, at a dose of 1 Mg/kg- Lobar vein, airway, and systemic arterial pressures were unchanged in both cases. The percentage changes in pulmonary artery pressure caused by AA, PGFjo and NE are summarized in Figure 2. ASPIRIN BLOCKADE The tracing shown in Figure IB demonstrates the effects of pretreatment with aspirin on the pulmonary vascular response to AA. Aspirin did produce a small reduction (1 mm Hg) in baseline perfusion pressure in many preparations. The pulmonary pressor response to AA was completely eliminated in every preparation pretreated with aspirin. In most cases a slight reduction (3-4 mm Hg) in pulmonary artery pressure occurred within 6 seconds after administration of AA. Comparable changes in pulmonary artery pressure were produced by the control fatty acid, linoleic acid; this finding suggests that this was a nonspecific effect of fatty acid in the presence of this anti-inflammatory agent. Responses to PGFa, and NE were not blocked by aspirin (Fig. 2). The response to PGFj,, was essentially unchanged (44.8 ± 7.4%) but the response to NE was diminished significantly (18.7 ± l.C
OTHER BLOCKING AGENTS
Phentolamine, propranolol, atropine, and cyproheptadine were tested and none blocked or attenuated the pulmonary vascular response to AA. ARTIFICIAL PERFUSION During perfusion of the isolated lobe with an artificial perfusate free of cellular elements, the pulmonary pressor response to AA was as great (98.4 ± 31.6%) as in the blood-perfused lobe. The pulmonary vascular response to NE, 1 /xg/kg, was also unaltered by substitution of the artificial perfusate but the pressor effect of PGFJQ was diminished significantly to 10.0 ± 0.95%. TIME OF ACTION RELATIONSHIPS The times to onset of action for the substances were quite similar and rapid (mean, 5.5-7 seconds). This time was significantly greater (mean, 12 seconds) only for PGF*, in the artificially perfused lobe. The time to peak effect of each substance was similar between groups (mean, 21-31 seconds) except for AA (mean, 59 seconds) and PGF t o (mean, 45 seconds) in the lobe pretreated with aspirin. Duration of response (not shown here) was variable. In all cases, this did not exceed 5 minutes. Discussion In contrast to its systemic hypotensive effect, AA produced a rapid and marked increase in pulmonary artery
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100
75
o
50
^
25
-25
L
Before
After 25 mg/kg Aspirin BLOOD PERFUSED LOBE
ARTIFICIALLY PERFUSED LOBE
_L
J
FIGURE 2 Summary of the changes in lobar artery pressure after administration of arachidonic acid, 100 ng/kg (solid bars), prostaglandin ^20. ' fig/kg (cross-hatched bars), and norepinephrine, I fig/kg (open bars), to three groups of isolated perfused canine lung lobes: before (n = 17) and after (n = 9) administration of aspirin, 25 mg/kg, and during artificial perfusion (n = 5). Means expressed ± SE.
pressure in the isolated blood-perfused dog lung lobe. This dose-related increase in pressure gradient occurred in the presence of constant flow and indicates that the rise in pressure was the direct result of an increase in pulmonary vascular resistance. The characteristics of this response were similar to those obtained with much smaller doses of PGF 2a and NE. The times to onset and peak action of each of the agents were very similar; this finding strongly suggests a direct action of both AA and P G F ^ . However, the pressor action of AA was blocked by aspirin; this indicates that biosynthetic conversion of AA to endoperoxides, prostaglandins, or other products is necessary for its vascular action. Hamberg and Samuelsson 8 demonstrated that conversion of AA into polar derivatives in a sheep seminal vesicle preparation was complete within 15 seconds, but formation of PGE 2 (measured indirectly) was complete only after 2 minutes. Moreover, Hamberg and Samuelsson 14 recently reported that administration of AA to the isolated artificially perfused guinea pig lung yielded only approximately 3% PGE 2 and 5% PGFto and more than 90% of other non-prostanoate compounds. In a later report 15 Hamberg and co-workers showed that biosynthesized endoperoxide prostaglandin intermediates had potent smooth muscle-stimulating abilities, both in vitro and in vivo. These data, in conjunction with the rapid onset of action of AA in the lung lobe, suggest that prostaglandin intermediates are the agents responsible for the pulmonary pressor
response. Conversion of AA into PGFfc, may contribute to this response, although the time for synthesis of P G F ^ seems to be greater than the 6-second time to onset of AA action. Our results with PGFj,, compare favorably with the results of studies by others on dogs and other species.1' *• Lonigro and Dawson16 reported that PGF t o exerts most of its effect on pulmonary vessels upstream to the capillaries and that degradation of P G F t o seems to be downstream from the site of action. In the present study, the times to onset of action of both AA and PGFj,, were identical, and strongly suggest that the sites of action are the small pulmonary arteries. The decreased pressor response to NE in the presence of aspirin cannot be adequately explained on the basis of our data. However, Kadowitz and others" 1 18 have shown that F series prostaglandins in subvasoactive doses increase vascular reactivity to NE. In addition, NE has been shown to cause the release of prostaglandins from several tissues.19 Either of these mechanisms might account for this decreased pressor response to NE, since generation of endogenous prostaglandins would be blocked by aspirin pretreatment with aspirin. Experiments with the other blocking agents demonstrate that a- and £-adrenergic, cholinergic, and serotonin receptors do not mediate the response to the active compounds synthesized from AA. Rather, other receptor sites are involved in the response to these vasoactive compounds.
VASCULAR EFFECTS OF ARACHIDONATE IN LUNG/ Wickset al. Silver and colleagues" demonstrated that a large dose of A A administered to rabbits intravenously produced death as a result of extensive blockage of the pulmonary circulation by platelet aggregates. No measurements of pulmonary artery pressure were made in these experiments. To rule out mechanical obstruction of small pulmonary vessels as the mechanism responsible for the pulmonary pressor response in our experiments, the isolated lobe was perfused with an artificial perfusate free of cellular elements. The pulmonary vascular responses to A A and NE were not significantly different when a perfusate free of cellular elements was used. Mechanical obstruction did not account for the rise in pulmonary artery pressure. Moreover, conversion of AA into active intermediates does not require the presence of platelets or other blood cellular elements. Other tissues, such as vascular endothelium,20 must be a source of prostaglandin synthetase for the conversion of AA into a cascade of vasoactive compounds. An adequate explanation for the diminished response to PGFfc, during use of an artificial perfusate is lacking. Binding of PGF20 to the dextran base, or absence of a necessary blood component for its action, could explain the reduced responsiveness in this artificial system. In addition, PGFjo may be metabolized by erythrocytes to the potent, smooth muscle-stimulating 15-keto PGFj,,.21- 22 In the absence of erythrocytes this prostaglandin metabolite may not be formed. Acknowledgments We thank T. Maish for the preparation of the solutions used in this study, and for her excellent technical advice.
References 1. Kadowitz PJ, Joiner PO, Hyman AL: Physiological and pharmacological roles of prostaglandins. Annu Rev Pharmacol 15: 285-306, 1975 2. Nakano J, McCurdy JR: Hemodynamic effects of prostaglandins E,, A,, and F*. in dogs. Proc Soc Exp Biol Med 128: 39-42, 1968 3. Nakano J: Cardiovascular actions of prostaglandins. /nThe Prostaglandins, vol. I, edited by PW Ramwell. New York, Plenum Publishing Corp., 1973, pp 238-316
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4. Anggard E, Samuelsson B: Biosynthesis of prostaglandins from arachidonic acid in guinea pig lung. J Biol Chem 240: 3518-3521, 1965 5. Bills TK, Silver MJ: Phosphotidylcholine is the primary source of arachidonic acid utilized by platelet prostaglandin synthelase (abstr). Fed Proc 34: 790, 1975 6. Danon A, Chang LCT, Sweelman BJ, Nies AS, Oates JA: Synthesis of prostaglandins by the rat renal papilla in vitro; mechanism of stimulation by angiotensin II. Biochim Biophys Acta 388: 71-83, 1975 7. Danon A, Heimberg M, Oates JA: Enrichment of rat tissue lipids with fatty acids that are prostaglandin precursors. Biochim Biophys Acta 388: 318-330, 1975 8. Hamberg M, Samuelsson B: Detection and isolation of an endoperoxide intermediate in prostaglandin biosynthesis. Proc Natl Acad Sci USA 70: 899-903, 1973 9. Rose JC, Johnson M, Ramwell PW, Kot PA: Effects of arachidonic acid on systemic arterial pressure, myocardial contractility and platelets in the dog. Proc Soc Exp Biol Med 147: 652-655, 1974 10. Hamberg M, Svensson J, Wakabayashi T, Samuelsson B: Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc Natl Acad Sci USA 71: 345-349, 1974 11. Silver MJ, Hoch W, Kocsis JJ, Ingerman CM, Smith JB: Arachidonic acid causes sudden death in rabbits. Science 183: 1085-1086, 1974 12. Furlow TW Jr, Bass NJ: Stroke in rats produced by carotid injection of sodium arachidonate. Science 187: 658-660, 1975 13. Vargaftig BB, Zirinis P: Platelet aggregation induced by arachidonic acid is accompanied by release of potential inflammatory mediators distinct from PGE, and PGF t o . Nature (New Biol) 244: 114-116, 1973 14. Hamberg M, Samuelsson B: Prostaglandin endoperoxides. VII. Novel transformations of arachidonic acid in guinea pig lung. Biochem Biophys Res Commun 61: 942-949, 1974 15. Hamberg M, Hedqvist P, Strandberg K, Svensson J, Samuelsson B: Prostaglandin endoperoxides. IV. Effects on smooth muscle. Life Sci 16: 451-462, 1975 16. Lonigro AJ, Dawson CA: Vascular responses to prostaglandin F*, in isolated cat lungs. Circ Res 36: 706-712, 1975 17. Kadowitz PJ: Effect of prostaglandin E,, E2 and A 2 on vascular resistance and responses to noradrenaline, nerve stimulation and angiotensin in the dog hindlimb. Br J Pharmacol 46: 395-400, 1972 18. Kadowitz PJ, Sweet CS, Brody MJ: Influence of prostaglandins on adrenergic transmission to vascular smooth muscle. Circ Res 31 (suppl 2): 36-50, 1972 19. Brody MJ, Kadowitz PJ: Prostaglandins as modulators of the autonomic ' nervous system. Fed Proc 33: 48-60, 1974 20. Gimbrone MA, Alexander RW: Angiotensin II stimulation of prostaglandin production in cultured human vascular endolhelium. Science 189: 219-220, 1975 21. Dawson W, Lewis RL, McMahon RE, Sweatman WJF: Potent bronchoconstrictor activity of 15-keto prostaglandin F*,. Nature 250: 331-332, 1974 22. Kaplan L, Lee S-C, Levine L: Partial purification and some properties of human erythrocyte prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase. Arch Biochem Biophys 167: 287-293, 1975
