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Inhibition of hypoxic pulmonary vasoconstriction by nifedipine
Inhibitionof Hypoxic Pulmonary Vasoconstriction By Nlfedipine THOMAS KENNEDY, MD, MPH and WARREN SUMMER, MD
Nifediplne is a potent slow channel calcium antagonist and systemic vasodilator recently reported to attenuate hypoxic pulmonary vasoconstriction in man. Other systemic vasodilators have also been shown to attenuate hypoxic pulmonary vasoconstriction, but their effects in some species may be mediated by reflex beta-adrenergic discharge. We evaluated the effect of nifedipine on the relation between pulmonary arterial pressure and blood flow during hyperoxia (inspired partial pressure of oxygen [PO,] 200 mm Hg) and hypoxia (inspired PO* 50 mm Hg) in denervated ventilated pig lungs perfused in situ with the animal’s own blood. Ten lungs were
ventilated with alternating 15 minute periods of hyperoxia and hypoxia. Hypoxia shifted the pulmonary artery pressure (x axis)-blood flow (y axis) relationship to the right and decreased its slope, indicating vasoconstriction. Ntfedipine, given as a 0.1, 1, or 10 pg/kg bolus into the pulmonary artery, caused a dose-dependent reduction of hypoxic pulmonary vasoconstriction. It is concluded that nifedipine is a potent pulmonary vasodilator acting locally within the lung and that it might be useful in the therapy of hypoxic pulmonary hypertension from chronic lung disease in man.
Pulmonary hypertension and resulting car pulmonale account for 7 to 10% of all heart disease in the United States.l Probably the most important cause of car pulmonale is hypoxic pulmonary hypertension associated with disorders such as chronic bronchitis and emphysema, bronchiectasis, cystic fibrosis, kyphoscoliosis, and chronic mountain sickness. The recommended therapy for hypoxic pulmonary hypertension is sufficient oxygen to relieve alveolar hypoxia and systemic hypoxemia.2 However, supplemental oxygen is not always effective in relieving the elevated pulmonary artery pressure in these disorders3 and is unquestionably expensive and inconvenient. Recently, Simonneau et a1.4 demonstrated that nifedipine, a potent slow channel calcium antagonist and systemic vasodilator, inhibits hypoxic pulmonary vasoconstriction in patients with acute respiratory failure without deleterious effects on arterial oxygen
saturation or delivery. Muramoto et al.5 reported similar findings in a group of 9 patients with clinically stable chronic obstructive pulmonary disease. In their study subjects experienced an improvement in cardiac output and pulmonary vascular resistance at rest and during exercise, with a shift in pressure-flow curves to the right, indicating a decrease in pulmonary pressure at similar levels of cardiac output occurring when subjects were treated with nifedipine as opposed to placebo. Arteriovenous oxygen content decreased whereas arterial oxygen saturation did not change. These results indicate that nifedipine is potentially useful as a vasodilator to reduce the afterload of the right ventricle in car pulmonale resulting from hypoxic pulmonary hypertension, but shed no light on whether nifedipine acts locally within the lung or indirectly through the sympathetic nervous system. Minoxidil reduces hypoxic pulmonary hypertension, but its effect in hypoxic cattle is prevented by beta-adrenergic blockade.6 Nifedipine also stimulates a reflex sympathetic discharge.T,s To determine whether nifedipine locally or reflexly inhibits hypoxic pulmonary hypertension, we investigated the ability of increasing doses of nifedipine to reverse the pulmonary hypoxic response in isolated perfused pig lungs void of any sympathetic innervation.
From the Departments of Medicine, Anesthesiology and Critical Care Medicine, and Environmental Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland. Manuscript received December 15, 1981; revised manuscript received March 31, 1982, accepted April 16,1982. Address for reprints: Thomas Kennedy, MD, The Johns Hopkins School of Hygiene and Public Health, 615 N. Wolfe Street, Room 7032, Baltimore, Maryland 21205.
884
October 1992
The American Journal of CARDIOLOGY
Volume 50
PULMONARY
HYPERTENSION-KENNEDY
AND SUMMER
4
STANDPIPE
( [FA,
RESPIRATOR
, j,
phi+
j
=EOOmmHq
I
PT1
FIGURE 2. Typical pulmonary pressure (Ppa)-flow (6) curves during hyperoxia (P102 = 200 mm Hg) and hypoxia (Plop = 50 mm Hg).
FIGURE 1. Diagram of perfusion apparatus for pig lung. F = flow; FM = flow meter; HE = heat exchanger; PLA = left atrial pressure; PPA = pulmonary artery pressure; PT = tracheal pressure.
Methods Experimental preparation: The pig was chosen as the experimental animal because its pulmonary circulation responds vigorously to hypoxia.g Pigs weighing 20 to 40 kg were anesthetized with intravenous pentobarbital sodium and ventilated through a tracheal cannula with room air by a respirator. After mid-sternal thoracotomy the animal was given 10,000 U of heparin intravenously, the left atrium was cannulated, and the animal was then bled a volume of approximately 1,000 ml into a Plexiglas” reservoir (Fig. 1). This volume of blood was supplemented with 6% dextran in normal saline solution to yield a total perfusate volume of 1,500 ml. The pulmonary artery was then cannulated and the root of the aorta was tied off. During perfusion, blood entered the left atrium, drained by gravity into the reservoir, and was then pumped with a roller pump (Sarns model 3500) through a heat exchanger, blood filter, and electromagnetic flow probe to the pulmonary artery. Except during pressure-flow determinations, blood flow was constant at 1.0 liter/min. Pulmonary artery and left atrial pressure was measured with strain gauges zero-referenced to the level of the right atrioventricular valve. Left atria1 pressure was maintained at -20 mm Hg by adjusting the level of the reservoir. After perfusion was begun, the lungs were ventilated with a normoxic normocapnic gas mixture at a rate of 6 to 7 breaths/min and a tidal volume of 400 ml. End-expiratory tracheal pressure was kept at 3 to 4 cm of water to prevent alveolar collapse. Airway oxygen and carbon dioxide tensions were measured with gas analyzers (Beckman). Blood temperature was kept between 38.5 and 39.5’C. Blood gas tensions and pH were measured by standard electrode techniques (Radiometer BMS3 MK2). Blood PCOz was 35 to 40 mm Hg and pH 7.35 to 7.45. Hyperoxia and hypoxia: After a stabilization period of 60 minutes, the lungs were exposed alternately to hyperoxic and hypoxic gas mixtures. During hyperoxia the inspired gas consisted of 38.2% oxygen, 5.4% carbon dioxide, and 66.4% nitrogen. During hypoxia it consisted of 7% oxygen, 5.4%
carbon dioxide, and 87.6% nitrogen. This level of hypoxia was chosen because in the pig it causes maximal steady state pulmonary vasoconstriction.g When pulmonary artery pressure stabilized, the pulmonary artery pressure-flow relation was determined by stopping ventilation in end-expiration, increasing pump flow to fill the standpipe (Fig. 1) and, when pulmonary artery pressure was stable, suddenly stopping the pump to allow blood to flow by gravity from the standpipe through the lungs into the reservoir until zero flow was achieved. During this procedure, an x-y recorder was used to record the relation between pulmonary artery pressure (x) and flow (y). Measurement of the relation between pulmonary artery pressure and blood flow has previously been used by a number of investigators1c-12 to demonstrate the state of pulmonary vascular resistance over a wide range of possible blood flows, thus avoiding possible pitfalls of measuring a single value of pulmonary artery pressure at constant flow. Figure 2 illustrates typical curves recorded during normoxia and hypoxia. During hypoxia, the curve is shifted to the right and decreased in slope, indicating vasoconstriction. Changes in these pressure-flow relations were quantified by determining the pulmonary artery pressure at a flow of 1 liter/min directly from the record curve. Nifedipine administration: Two groups of animals (n = 5 in each) were studied. All lungs underwent 300 minutes of perfusion. Pulmonary artery pressure-flow relations were determined at the end of each period of hyperoxia and hypoxia. After determination of a curve, the inspired gas mixture was changed and time was allowed for stabilization of pulmonary artery pressure before another curve was recorded. Generally, stabilization occurred within 15 minutes. The experimental group received nifedipine into the pulmonary artery in injections of 0.1, 1, and 10 pg/kg at perfusion times of 120, 180, and 240 minutes. Nifedipine in a concentration of 100 pg/ml was prepared under conditions of reduced light in a vehicle consisting of 14 ml of polyethylene glycol, 22 ml of absolute ethanol, and 64 ml of water, and was diluted with appropriate volumes of vehicle to achieve an injection volume of 0.1 ml/kg. The control group received injections of 0.1 ml/kg of vehicle alone into the pulmonary artery at perfusion times of 120,160, and 240 minutes. Blood pH was checked after each administration of drug to confirm that it had not changed.
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100 100
150
200
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300
150 Mmutes
203
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xx1
of Perfusm
Mm&sof PerfusIon FIGURE 3. Pulmonary artery pressure of control animals (n = 5) at a blood flow of 1 literlmin (Ppa,) during alternating periods of hyperoxia (P102 = 200 mm Hg) and hypoxia (PlOs = 50 mm Hg) ventilation. Open circles represent mean PPa f 1 standard error of the mean.
FIGURE 4. Pulmonary artery pressure of nifedipine-treated animals (n = 5) at a blood flow of 1 liter/min (Ppa,) during alternating periods of hyperoxia (PlOs = 200 mm Hg) and hypoxia (PlOs = 50 mm Hg) ventilation. Open circles represent mean PPa f 1 standard error of the mean.
Values for pulmonary artery pressure at a flow of 1 literlmin (Ppar) during hyperoxia and hypoxia for control and experimental animals and differences between Ppar for each hyperoxic and its subsequent hypoxic value for control and experimental animals were compared using a Z-way analysis of variance. When F ratios achieved a 0.05 level of significance, a protected least significant difference was computed according to the method of Fisher.r3
nifedipine-treated animals are shown in Figure 4. A decrease in mean pulmonary artery preasure at a flow of 1 liter/min was not seen until after the 1.0 pg/kg dose of nifedipine. After the 10 pglkg dose there was nearly complete abolition of hypoxic vasoconstriction with some transient decrease in mean Ppar during hyperoxia as well. Mean inhibition of the hypoxic pressor response (mean APpaI) as a function of dose ofnifedipine (Fig. 5). For each animal, APpal was determined as the dif-
Results Control versus nifedipine: Results for control animals are shown in Figure 3, in which mean values of pulmonary artery pressure at a blood flow of 1 liter/min (Ppal) for the 5 animals are plotted for each set of hyperoxic and hypoxic curves determined during the 300 minute perfusion. Both hypoxic and hyperoxic responses tended to reach a relative plateau after 1‘20 minutes. Mean values of pulmonary artery pressure at a flow of 1 liter/min during hyperoxia and hypoxia for
ference between pulmonary artery pressure at a blood flow of 1 liter/min during hyperoxia at perfusion times of 90, 150,210, and 270 minutes, and the subsequent stable pulmonary artery pressure at 1 liter/min flow during hypoxia at perfusion times of 105,165,225, and 285 minutes, respectively, for control and increasing doses of nifedipine. Thus APpal corresponds to the difference between the last hyperoxic and hypoxic response during each study period before subsequent administration of the next larger dose of drug and was chosen to allow the respective doses of drug to circulate within the perfusion system so that drug effect could stabilize. The mean APpar of control animals remained stable over all 4 study periods. The mean APpal for nifedipine-treated animals decreased progressively with logarithmically increasing doses of drug, so that at a dose of 1 pg/kg there was 50% inhibition of the hypoxic response, and at a dose of 10 pg/kg nearly complete inhibition occurred. Discussion
I
o-?
01
IO
IO
Doseof Nrfedrpne(,ug/kg)
FIGURE 5. Hypoxic pressor response (APpa,) in control (n = 5) and nifedipine-treated (n = 5) animals as a function of dose of nifedipine. Each open circle represents mean APpa, f 1 standard error of the mean.
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Hypoxia is an effective and consistent stimulus to pulmonary hypertension. I4 However, debate still exists as to whether hypoxia exerts its effects directly on pulmonary vascular smooth muscle or indirectly through mediator release within the lung.r5 The results of our study indicate that nifedipine is a potent inhibitor of the pulmonary hypoxic response, independent of the
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reflex adrenergic discharge. However, we cannot conclude where within the lung itself nifedipine’s action is located. Effect of slow channel calcium antagonists on pulmonary hypoxic response: Blockade of the pulmonary hypoxic response by slow channel calcium antagonists was first reported in 1976 by McMurtry et al.,le who used verapamil to inhibit hypoxic pulmonary hypertension in isolated rat lungs. These investigators suggested that hypoxia might act directly to depolarize pulmonary vascular smooth muscle, and that verapamil inhibited the transmembrane flow of calcium into the cell and prevented activation of the contractile machinery. Where and how this supposed inhibition of transmembrane calcium flux works in vascular smooth muscle is unclear. It is even unclear whether verapamil and nifedipine affect calcium flux in the same way. In cardiac muscle, where contraction is associated with changes in calcium flux initiated by cellular membrane depolarization, verapamil alters the kinetics of calcium flux, whereas nifedipine affects total calcium conductance, presumably by differential effects on the two gates of the calcium channel of cardiac tissue.17 In vascular smooth muscle, however, contraction was recently found to be dependent on pharmacomechanical coupling, that is, contraction that is induced by the binding of an exogenous agent such as norepinephrine to a receptor, precipitating changes in calcium flux not necessarily associated with changes in transmembrane potentia1.l” We are not aware of any work conclusively pointing to differential sites of action of nifedipine and verapamil on calcium flux in vascular smooth muscle. Until such work is available, the demonstration that both nifedipine and verapamil inhibit the pulmonary hypoxic response does not add to our basic understanding of the potential subcellular mechanisms in smooth muscle that might be operative in hypoxic pulmonary vasoconstriction. Site and mechanism of inhibition of pulmonary hypoxic response: It is not even certain that slow channel calcium antagonists are working only at the level of vascular smooth muscle to inhibit the pulmonary hypoxic response. It was previously suggested that the pulmonary hypoxic response is induced by mast cell release of vasoactive substances,1g,20 and mast cells are strategically dispersed along the course of the pulmonary resistance vessels.21 The observation that both nifedipine and verapamil inhibit pulmonary hypertension in cat lobes infused with a stable prostaglandin endoperoxidez” indicates that these agents are able to block pharmacomechanical coupling in pulmonary vascular smooth muscle. In preliminary in vitro experiments, some investigators2” claimed that nifedipine blocks the release of platelet-activating factors and slow reactive substances, and reduces release of acid phosphatase and glucuronidase from human polymorphonuclear leukocytes induced by ionophore, zymozan, or zymozan-complement complexes, If these results are confirmed and shown true for mast cells as well, it is possible that nifedipine might be blocking the calcium-dependent release of some vasoactive substance
HYPERTENSION-KENNEDY
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released locally within the lung in response to hypoxia. Clinical implications: Regardless of the mechanism of subcellular action, the demonstration that a vasodilator works locally within the lung to attenuate hypoxic pulmonary hypertension may be considerably important in predicting any ultimate benefit to patients with pulmonary hypertension given the agent as therapy. Nitroglycerin was explored as a vasodilator in patients with pulmonary hypertension from chronic obstructive pulmonary disease, and was demonstrated to decrease mean pulmonary artery pressure.24 However, nitroglycerin accomplishes this only by altering venous capacitance to decrease right ventricular preload, without affecting pulmonary vascular resistance. Thus, a decrease in mean pulmonary artery pressure is achieved at the expense of a decrease in cardiac output and oxygen delivery. Bishop et a1.25 recently reported that both nifedipine and minoxidil inhibit the pulmonary hypoxic response in intact dogs. However, minoxidil was previously demonstratedfi to lose its effectiveness as a pulmonary vasodilator in hypoxic cattle after beta-adrenergic blockade. Despite the fact that nifedipine also elicits a reflex beta-adrenergic discharge,7g8 our results clearly document that pulmonary vasodilation from nifedipine in a denervated pig lung is void of any potential for reflex sympathetic activity. Thus, if the human circulation responds similarly to the hypoxic bovine pulmonary circulation, subjects with lung disease might not have pulmonary vasodilation from minoxidil if they are also receiving cardioselective beta-blockers or centrally acting antihypertensive agents that decrease sympathetic outflow, but their response to nifedipine might be expected to remain intact. To be useful in a clinical setting, nifedipine would have to be proved efficacious in producing a decrease in pulmonary vascular resistance without markedly altering redistribution of blood to poorly ventilated regions so as to not worsen hypoxemia. We were unable to address this question because the perfusate reservoir of our preparation was open to air, but in a recent report, Bishop et a1.25 found that nifedipine increased venous admixture in intact dogs. However, Simonneau et al.4 found only a slight (45 f 2 to 42 f 2 mm Hg) although statistically significant change in arterial PO2 in a group of 13 patients with acute respiratory failure given nifedipine, and Muramoto et al.” show no change in arterial PO:, either at rest or during exercise in 9 subjects with chronic obstructive pulmonary disease given the agent. This lack of clinically significant desaturation after the administration of nifedipine in man might be explained on the basis of an increase in cardiac output and a decrease in arteriovenous oxygen content, as occurred in Muramoto’s patients,” were it not for the fact that mixed venous PO2 did not change in subjects studied by Simonneau et a1.4 Thus, the ventilationperfusion relations that follow nifedipine administration may be unusually complex. Nevertheless, these agents offer promise in the treatment of patients with car pulmonale in which low cardiac output is a factor limiting clinical well-being.
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Acknowledgment We gratefully acknowledge the assistance of Jimmie Sylvester, MD, in the design and analysis of this study, David Poorvin, MD, of Pfizer Laboratories for providing nifedipine, and Janet Hupp and Ernestine Mikeal for help in the preparation of the manuscript.
References 1. IngramRH, Grossman 2.
3. 4. 5.
6.
7.
8.
9.
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GP. Chronrc car pulmonale In. Hurst JW, Logue RB, Schlant RC, Wenger NK, eds The Heart. New York. McGraw-Hill, 1974 1278. Nocturnal Oxygen Therapy Tnal Group Continuous or nocturnal oxygen therapy in hypoxemlc chronic obstructive lung disease: a clinlcal trial Ann Intern Med 1980;93:391-398 Levine BE, Slgelow DB, Hamstra RD, et al. Role of long-term continuous oxygen administration in patients with chronic airway obstruction and hypoxemia. Ann Intern Med 1967;66.639-650 Slmonneau G, Escourrou P, Duroux P, Lockhart A. lnhtblhon of hypoxic pulmonary vasoconstriction by nifedlpine N Engl J Med 1981;304: 1582-1585. Muramoto A, Caldwell J, Lakshminarayan S, Albert RK, Butler J. Nifedlplne reduces pulmonary artery pressure at a comparable cardiac output in patients with chronic obstructive pulmonary disease (COPD) (abstr). Clrculabon 1981;64:Suppl IV:IV-179 Weir EK, Chidsey CA, Weil JU, Grover RF. Minoxidil reduces pulmonary vascular resistance in dogs and cattle. J Lab Clin Med 1976.88:885894. Moses J, Wertheimer JH, Bodenheimer MM, Banka VS. Feldman M, Helfant RH. Efficacy of nifedipine in rest angina refractory to propranolol and nitrates in patients with otistructive coronary artery disease Ann Intern Med 1981;94:425-429. Seer N, Gallegos I, Cohen A, Klein N, Sonnenblick E, Frishman W. Efficacy of sublingual nifedipine in the acute treatment of systemic hypertension Chest 1981:79:571-574. Sylvester i, Harabin AL, Leake MD, Frank RS. Vasodilator and constrictor
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responses to hypoxia In Isolated pig lungs J Appl Physiol 1980;49:820825 IO. Hall P. Effects of anoxia on postarteriolar pulmonary vascular resistance Circ Res 1953.1.238-241 11. Dawes G. Pulmanarv circulahon m the fetus and newborn. Br Med Bull 1966;22:61-65 ’ 12. Sylvester JT, McGowan C. The effects of agents that bind to cytochrome P-450 on hypoxlc pulmonary vasoconstrtctlon Circ Res 1978;43.429-
._.
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13. Kirk RE. ExperImental Design, Procedures for the BehavIoral Sciences Belmont, CA- Brooks/Cole, 1968:67 14. Harris P, Heath D. The Human Pulmonary Circulation New York: Churchill Llvlngstone. 1977 452-455 15. Fishman AP. Hypoxia on the pulmonary circulation- how and where it acts Circ Res 1976;38 221-231 16. McMurtry I, Davidson A, Reeves J, Grover R. InhIbItIon of hypoxic pulmonary vasoconstriction by calcium antagonists tn isolated rat lungs. Circ Res 1976.38:99-104 17. Casteels R. Electra- and pharmacomechanlcal coupling in vascular smooth muscle Chest 1980:78:150-156 18. Zelis R, Flaim S. “Calcium influx blockers” and vascular smooth muscle: do we really understand the mechanisms? Ann Intern Med 1981;94: 124-126. 19. Lloyd TC Jr. Hypoxlc pulmonary vasoconstnctron: role of penvascular tissue J Appl Physlol 1968.25:560-565. 20. Haugue A. Role of histamine In hypoxlc pulmonary hypertension in the rat I Blockade or potentlatlon of endogenous amines, klmns. and ATP Clrc Res 1968,22 371-383 21. Haas F. Beraofskv EH. Role of the mast cell in the oulmonary pressor response’to hyboxla J Chn Invest 1972;51:3154-3882 . 22. Kadowltz PJ, Hyman AL. Vasodilator actions of nifedlplne and verapamil In the pulmonary vascular bed (abstr) Fed Proc 1981,40:590. 23. Cerrina J, Denjean A, Alexandre G, Lockhart A, Duroux P. Inhibition of exercise-induced asthma by a calcium antagonist, nifedipine Am Rev Resp Dis 1981:123:156-160. 24. Matihay kA, Brent BN, Berger JH, Mahler DA, Langou R, Zarel BL. Hemodynamlc effects of mtroglycerin in patients with chronic obstructive pulmonary disease. pulmonary hypertension. and decreased right ventncular function (abstr) Am Rev Resp Dis 1981,123 (SuppI): 25. Bishop MJ, Gronka R, Cheney FW. Minoxldll and nifedipine Inhibit hypoxlc pulmonary vasoconstriction (abstr). Chest 1981;80:388
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Nifediplne is a potent slow channel calcium antagonist and systemic vasodilator recently reported to attenuate hypoxic pulmonary vasoconstriction in man. Other systemic vasodilators have also been shown to attenuate hypoxic pulmonary vasoconstriction, but their effects in some species may be mediated by reflex beta-adrenergic discharge. We evaluated the effect of nifedipine on the relation between pulmonary arterial pressure and blood flow during hyperoxia (inspired partial pressure of oxygen [PO,] 200 mm Hg) and hypoxia (inspired PO* 50 mm Hg) in denervated ventilated pig lungs perfused in situ with the animal’s own blood. Ten lungs were
ventilated with alternating 15 minute periods of hyperoxia and hypoxia. Hypoxia shifted the pulmonary artery pressure (x axis)-blood flow (y axis) relationship to the right and decreased its slope, indicating vasoconstriction. Ntfedipine, given as a 0.1, 1, or 10 pg/kg bolus into the pulmonary artery, caused a dose-dependent reduction of hypoxic pulmonary vasoconstriction. It is concluded that nifedipine is a potent pulmonary vasodilator acting locally within the lung and that it might be useful in the therapy of hypoxic pulmonary hypertension from chronic lung disease in man.
Pulmonary hypertension and resulting car pulmonale account for 7 to 10% of all heart disease in the United States.l Probably the most important cause of car pulmonale is hypoxic pulmonary hypertension associated with disorders such as chronic bronchitis and emphysema, bronchiectasis, cystic fibrosis, kyphoscoliosis, and chronic mountain sickness. The recommended therapy for hypoxic pulmonary hypertension is sufficient oxygen to relieve alveolar hypoxia and systemic hypoxemia.2 However, supplemental oxygen is not always effective in relieving the elevated pulmonary artery pressure in these disorders3 and is unquestionably expensive and inconvenient. Recently, Simonneau et a1.4 demonstrated that nifedipine, a potent slow channel calcium antagonist and systemic vasodilator, inhibits hypoxic pulmonary vasoconstriction in patients with acute respiratory failure without deleterious effects on arterial oxygen
saturation or delivery. Muramoto et al.5 reported similar findings in a group of 9 patients with clinically stable chronic obstructive pulmonary disease. In their study subjects experienced an improvement in cardiac output and pulmonary vascular resistance at rest and during exercise, with a shift in pressure-flow curves to the right, indicating a decrease in pulmonary pressure at similar levels of cardiac output occurring when subjects were treated with nifedipine as opposed to placebo. Arteriovenous oxygen content decreased whereas arterial oxygen saturation did not change. These results indicate that nifedipine is potentially useful as a vasodilator to reduce the afterload of the right ventricle in car pulmonale resulting from hypoxic pulmonary hypertension, but shed no light on whether nifedipine acts locally within the lung or indirectly through the sympathetic nervous system. Minoxidil reduces hypoxic pulmonary hypertension, but its effect in hypoxic cattle is prevented by beta-adrenergic blockade.6 Nifedipine also stimulates a reflex sympathetic discharge.T,s To determine whether nifedipine locally or reflexly inhibits hypoxic pulmonary hypertension, we investigated the ability of increasing doses of nifedipine to reverse the pulmonary hypoxic response in isolated perfused pig lungs void of any sympathetic innervation.
From the Departments of Medicine, Anesthesiology and Critical Care Medicine, and Environmental Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland. Manuscript received December 15, 1981; revised manuscript received March 31, 1982, accepted April 16,1982. Address for reprints: Thomas Kennedy, MD, The Johns Hopkins School of Hygiene and Public Health, 615 N. Wolfe Street, Room 7032, Baltimore, Maryland 21205.
884
October 1992
The American Journal of CARDIOLOGY
Volume 50
PULMONARY
HYPERTENSION-KENNEDY
AND SUMMER
4
STANDPIPE
( [FA,
RESPIRATOR
, j,
phi+
j
=EOOmmHq
I
PT1
FIGURE 2. Typical pulmonary pressure (Ppa)-flow (6) curves during hyperoxia (P102 = 200 mm Hg) and hypoxia (Plop = 50 mm Hg).
FIGURE 1. Diagram of perfusion apparatus for pig lung. F = flow; FM = flow meter; HE = heat exchanger; PLA = left atrial pressure; PPA = pulmonary artery pressure; PT = tracheal pressure.
Methods Experimental preparation: The pig was chosen as the experimental animal because its pulmonary circulation responds vigorously to hypoxia.g Pigs weighing 20 to 40 kg were anesthetized with intravenous pentobarbital sodium and ventilated through a tracheal cannula with room air by a respirator. After mid-sternal thoracotomy the animal was given 10,000 U of heparin intravenously, the left atrium was cannulated, and the animal was then bled a volume of approximately 1,000 ml into a Plexiglas” reservoir (Fig. 1). This volume of blood was supplemented with 6% dextran in normal saline solution to yield a total perfusate volume of 1,500 ml. The pulmonary artery was then cannulated and the root of the aorta was tied off. During perfusion, blood entered the left atrium, drained by gravity into the reservoir, and was then pumped with a roller pump (Sarns model 3500) through a heat exchanger, blood filter, and electromagnetic flow probe to the pulmonary artery. Except during pressure-flow determinations, blood flow was constant at 1.0 liter/min. Pulmonary artery and left atrial pressure was measured with strain gauges zero-referenced to the level of the right atrioventricular valve. Left atria1 pressure was maintained at -20 mm Hg by adjusting the level of the reservoir. After perfusion was begun, the lungs were ventilated with a normoxic normocapnic gas mixture at a rate of 6 to 7 breaths/min and a tidal volume of 400 ml. End-expiratory tracheal pressure was kept at 3 to 4 cm of water to prevent alveolar collapse. Airway oxygen and carbon dioxide tensions were measured with gas analyzers (Beckman). Blood temperature was kept between 38.5 and 39.5’C. Blood gas tensions and pH were measured by standard electrode techniques (Radiometer BMS3 MK2). Blood PCOz was 35 to 40 mm Hg and pH 7.35 to 7.45. Hyperoxia and hypoxia: After a stabilization period of 60 minutes, the lungs were exposed alternately to hyperoxic and hypoxic gas mixtures. During hyperoxia the inspired gas consisted of 38.2% oxygen, 5.4% carbon dioxide, and 66.4% nitrogen. During hypoxia it consisted of 7% oxygen, 5.4%
carbon dioxide, and 87.6% nitrogen. This level of hypoxia was chosen because in the pig it causes maximal steady state pulmonary vasoconstriction.g When pulmonary artery pressure stabilized, the pulmonary artery pressure-flow relation was determined by stopping ventilation in end-expiration, increasing pump flow to fill the standpipe (Fig. 1) and, when pulmonary artery pressure was stable, suddenly stopping the pump to allow blood to flow by gravity from the standpipe through the lungs into the reservoir until zero flow was achieved. During this procedure, an x-y recorder was used to record the relation between pulmonary artery pressure (x) and flow (y). Measurement of the relation between pulmonary artery pressure and blood flow has previously been used by a number of investigators1c-12 to demonstrate the state of pulmonary vascular resistance over a wide range of possible blood flows, thus avoiding possible pitfalls of measuring a single value of pulmonary artery pressure at constant flow. Figure 2 illustrates typical curves recorded during normoxia and hypoxia. During hypoxia, the curve is shifted to the right and decreased in slope, indicating vasoconstriction. Changes in these pressure-flow relations were quantified by determining the pulmonary artery pressure at a flow of 1 liter/min directly from the record curve. Nifedipine administration: Two groups of animals (n = 5 in each) were studied. All lungs underwent 300 minutes of perfusion. Pulmonary artery pressure-flow relations were determined at the end of each period of hyperoxia and hypoxia. After determination of a curve, the inspired gas mixture was changed and time was allowed for stabilization of pulmonary artery pressure before another curve was recorded. Generally, stabilization occurred within 15 minutes. The experimental group received nifedipine into the pulmonary artery in injections of 0.1, 1, and 10 pg/kg at perfusion times of 120, 180, and 240 minutes. Nifedipine in a concentration of 100 pg/ml was prepared under conditions of reduced light in a vehicle consisting of 14 ml of polyethylene glycol, 22 ml of absolute ethanol, and 64 ml of water, and was diluted with appropriate volumes of vehicle to achieve an injection volume of 0.1 ml/kg. The control group received injections of 0.1 ml/kg of vehicle alone into the pulmonary artery at perfusion times of 120,160, and 240 minutes. Blood pH was checked after each administration of drug to confirm that it had not changed.
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100 100
150
200
250
300
150 Mmutes
203
250
xx1
of Perfusm
Mm&sof PerfusIon FIGURE 3. Pulmonary artery pressure of control animals (n = 5) at a blood flow of 1 literlmin (Ppa,) during alternating periods of hyperoxia (P102 = 200 mm Hg) and hypoxia (PlOs = 50 mm Hg) ventilation. Open circles represent mean PPa f 1 standard error of the mean.
FIGURE 4. Pulmonary artery pressure of nifedipine-treated animals (n = 5) at a blood flow of 1 liter/min (Ppa,) during alternating periods of hyperoxia (PlOs = 200 mm Hg) and hypoxia (PlOs = 50 mm Hg) ventilation. Open circles represent mean PPa f 1 standard error of the mean.
Values for pulmonary artery pressure at a flow of 1 literlmin (Ppar) during hyperoxia and hypoxia for control and experimental animals and differences between Ppar for each hyperoxic and its subsequent hypoxic value for control and experimental animals were compared using a Z-way analysis of variance. When F ratios achieved a 0.05 level of significance, a protected least significant difference was computed according to the method of Fisher.r3
nifedipine-treated animals are shown in Figure 4. A decrease in mean pulmonary artery preasure at a flow of 1 liter/min was not seen until after the 1.0 pg/kg dose of nifedipine. After the 10 pglkg dose there was nearly complete abolition of hypoxic vasoconstriction with some transient decrease in mean Ppar during hyperoxia as well. Mean inhibition of the hypoxic pressor response (mean APpaI) as a function of dose ofnifedipine (Fig. 5). For each animal, APpal was determined as the dif-
Results Control versus nifedipine: Results for control animals are shown in Figure 3, in which mean values of pulmonary artery pressure at a blood flow of 1 liter/min (Ppal) for the 5 animals are plotted for each set of hyperoxic and hypoxic curves determined during the 300 minute perfusion. Both hypoxic and hyperoxic responses tended to reach a relative plateau after 1‘20 minutes. Mean values of pulmonary artery pressure at a flow of 1 liter/min during hyperoxia and hypoxia for
ference between pulmonary artery pressure at a blood flow of 1 liter/min during hyperoxia at perfusion times of 90, 150,210, and 270 minutes, and the subsequent stable pulmonary artery pressure at 1 liter/min flow during hypoxia at perfusion times of 105,165,225, and 285 minutes, respectively, for control and increasing doses of nifedipine. Thus APpal corresponds to the difference between the last hyperoxic and hypoxic response during each study period before subsequent administration of the next larger dose of drug and was chosen to allow the respective doses of drug to circulate within the perfusion system so that drug effect could stabilize. The mean APpar of control animals remained stable over all 4 study periods. The mean APpal for nifedipine-treated animals decreased progressively with logarithmically increasing doses of drug, so that at a dose of 1 pg/kg there was 50% inhibition of the hypoxic response, and at a dose of 10 pg/kg nearly complete inhibition occurred. Discussion
I
o-?
01
IO
IO
Doseof Nrfedrpne(,ug/kg)
FIGURE 5. Hypoxic pressor response (APpa,) in control (n = 5) and nifedipine-treated (n = 5) animals as a function of dose of nifedipine. Each open circle represents mean APpa, f 1 standard error of the mean.
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Hypoxia is an effective and consistent stimulus to pulmonary hypertension. I4 However, debate still exists as to whether hypoxia exerts its effects directly on pulmonary vascular smooth muscle or indirectly through mediator release within the lung.r5 The results of our study indicate that nifedipine is a potent inhibitor of the pulmonary hypoxic response, independent of the
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reflex adrenergic discharge. However, we cannot conclude where within the lung itself nifedipine’s action is located. Effect of slow channel calcium antagonists on pulmonary hypoxic response: Blockade of the pulmonary hypoxic response by slow channel calcium antagonists was first reported in 1976 by McMurtry et al.,le who used verapamil to inhibit hypoxic pulmonary hypertension in isolated rat lungs. These investigators suggested that hypoxia might act directly to depolarize pulmonary vascular smooth muscle, and that verapamil inhibited the transmembrane flow of calcium into the cell and prevented activation of the contractile machinery. Where and how this supposed inhibition of transmembrane calcium flux works in vascular smooth muscle is unclear. It is even unclear whether verapamil and nifedipine affect calcium flux in the same way. In cardiac muscle, where contraction is associated with changes in calcium flux initiated by cellular membrane depolarization, verapamil alters the kinetics of calcium flux, whereas nifedipine affects total calcium conductance, presumably by differential effects on the two gates of the calcium channel of cardiac tissue.17 In vascular smooth muscle, however, contraction was recently found to be dependent on pharmacomechanical coupling, that is, contraction that is induced by the binding of an exogenous agent such as norepinephrine to a receptor, precipitating changes in calcium flux not necessarily associated with changes in transmembrane potentia1.l” We are not aware of any work conclusively pointing to differential sites of action of nifedipine and verapamil on calcium flux in vascular smooth muscle. Until such work is available, the demonstration that both nifedipine and verapamil inhibit the pulmonary hypoxic response does not add to our basic understanding of the potential subcellular mechanisms in smooth muscle that might be operative in hypoxic pulmonary vasoconstriction. Site and mechanism of inhibition of pulmonary hypoxic response: It is not even certain that slow channel calcium antagonists are working only at the level of vascular smooth muscle to inhibit the pulmonary hypoxic response. It was previously suggested that the pulmonary hypoxic response is induced by mast cell release of vasoactive substances,1g,20 and mast cells are strategically dispersed along the course of the pulmonary resistance vessels.21 The observation that both nifedipine and verapamil inhibit pulmonary hypertension in cat lobes infused with a stable prostaglandin endoperoxidez” indicates that these agents are able to block pharmacomechanical coupling in pulmonary vascular smooth muscle. In preliminary in vitro experiments, some investigators2” claimed that nifedipine blocks the release of platelet-activating factors and slow reactive substances, and reduces release of acid phosphatase and glucuronidase from human polymorphonuclear leukocytes induced by ionophore, zymozan, or zymozan-complement complexes, If these results are confirmed and shown true for mast cells as well, it is possible that nifedipine might be blocking the calcium-dependent release of some vasoactive substance
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released locally within the lung in response to hypoxia. Clinical implications: Regardless of the mechanism of subcellular action, the demonstration that a vasodilator works locally within the lung to attenuate hypoxic pulmonary hypertension may be considerably important in predicting any ultimate benefit to patients with pulmonary hypertension given the agent as therapy. Nitroglycerin was explored as a vasodilator in patients with pulmonary hypertension from chronic obstructive pulmonary disease, and was demonstrated to decrease mean pulmonary artery pressure.24 However, nitroglycerin accomplishes this only by altering venous capacitance to decrease right ventricular preload, without affecting pulmonary vascular resistance. Thus, a decrease in mean pulmonary artery pressure is achieved at the expense of a decrease in cardiac output and oxygen delivery. Bishop et a1.25 recently reported that both nifedipine and minoxidil inhibit the pulmonary hypoxic response in intact dogs. However, minoxidil was previously demonstratedfi to lose its effectiveness as a pulmonary vasodilator in hypoxic cattle after beta-adrenergic blockade. Despite the fact that nifedipine also elicits a reflex beta-adrenergic discharge,7g8 our results clearly document that pulmonary vasodilation from nifedipine in a denervated pig lung is void of any potential for reflex sympathetic activity. Thus, if the human circulation responds similarly to the hypoxic bovine pulmonary circulation, subjects with lung disease might not have pulmonary vasodilation from minoxidil if they are also receiving cardioselective beta-blockers or centrally acting antihypertensive agents that decrease sympathetic outflow, but their response to nifedipine might be expected to remain intact. To be useful in a clinical setting, nifedipine would have to be proved efficacious in producing a decrease in pulmonary vascular resistance without markedly altering redistribution of blood to poorly ventilated regions so as to not worsen hypoxemia. We were unable to address this question because the perfusate reservoir of our preparation was open to air, but in a recent report, Bishop et a1.25 found that nifedipine increased venous admixture in intact dogs. However, Simonneau et al.4 found only a slight (45 f 2 to 42 f 2 mm Hg) although statistically significant change in arterial PO2 in a group of 13 patients with acute respiratory failure given nifedipine, and Muramoto et al.” show no change in arterial PO:, either at rest or during exercise in 9 subjects with chronic obstructive pulmonary disease given the agent. This lack of clinically significant desaturation after the administration of nifedipine in man might be explained on the basis of an increase in cardiac output and a decrease in arteriovenous oxygen content, as occurred in Muramoto’s patients,” were it not for the fact that mixed venous PO2 did not change in subjects studied by Simonneau et a1.4 Thus, the ventilationperfusion relations that follow nifedipine administration may be unusually complex. Nevertheless, these agents offer promise in the treatment of patients with car pulmonale in which low cardiac output is a factor limiting clinical well-being.
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Acknowledgment We gratefully acknowledge the assistance of Jimmie Sylvester, MD, in the design and analysis of this study, David Poorvin, MD, of Pfizer Laboratories for providing nifedipine, and Janet Hupp and Ernestine Mikeal for help in the preparation of the manuscript.
References 1. IngramRH, Grossman 2.
3. 4. 5.
6.
7.
8.
9.
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GP. Chronrc car pulmonale In. Hurst JW, Logue RB, Schlant RC, Wenger NK, eds The Heart. New York. McGraw-Hill, 1974 1278. Nocturnal Oxygen Therapy Tnal Group Continuous or nocturnal oxygen therapy in hypoxemlc chronic obstructive lung disease: a clinlcal trial Ann Intern Med 1980;93:391-398 Levine BE, Slgelow DB, Hamstra RD, et al. Role of long-term continuous oxygen administration in patients with chronic airway obstruction and hypoxemia. Ann Intern Med 1967;66.639-650 Slmonneau G, Escourrou P, Duroux P, Lockhart A. lnhtblhon of hypoxic pulmonary vasoconstriction by nifedlpine N Engl J Med 1981;304: 1582-1585. Muramoto A, Caldwell J, Lakshminarayan S, Albert RK, Butler J. Nifedlplne reduces pulmonary artery pressure at a comparable cardiac output in patients with chronic obstructive pulmonary disease (COPD) (abstr). Clrculabon 1981;64:Suppl IV:IV-179 Weir EK, Chidsey CA, Weil JU, Grover RF. Minoxidil reduces pulmonary vascular resistance in dogs and cattle. J Lab Clin Med 1976.88:885894. Moses J, Wertheimer JH, Bodenheimer MM, Banka VS. Feldman M, Helfant RH. Efficacy of nifedipine in rest angina refractory to propranolol and nitrates in patients with otistructive coronary artery disease Ann Intern Med 1981;94:425-429. Seer N, Gallegos I, Cohen A, Klein N, Sonnenblick E, Frishman W. Efficacy of sublingual nifedipine in the acute treatment of systemic hypertension Chest 1981:79:571-574. Sylvester i, Harabin AL, Leake MD, Frank RS. Vasodilator and constrictor
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responses to hypoxia In Isolated pig lungs J Appl Physiol 1980;49:820825 IO. Hall P. Effects of anoxia on postarteriolar pulmonary vascular resistance Circ Res 1953.1.238-241 11. Dawes G. Pulmanarv circulahon m the fetus and newborn. Br Med Bull 1966;22:61-65 ’ 12. Sylvester JT, McGowan C. The effects of agents that bind to cytochrome P-450 on hypoxlc pulmonary vasoconstrtctlon Circ Res 1978;43.429-
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13. Kirk RE. ExperImental Design, Procedures for the BehavIoral Sciences Belmont, CA- Brooks/Cole, 1968:67 14. Harris P, Heath D. The Human Pulmonary Circulation New York: Churchill Llvlngstone. 1977 452-455 15. Fishman AP. Hypoxia on the pulmonary circulation- how and where it acts Circ Res 1976;38 221-231 16. McMurtry I, Davidson A, Reeves J, Grover R. InhIbItIon of hypoxic pulmonary vasoconstriction by calcium antagonists tn isolated rat lungs. Circ Res 1976.38:99-104 17. Casteels R. Electra- and pharmacomechanlcal coupling in vascular smooth muscle Chest 1980:78:150-156 18. Zelis R, Flaim S. “Calcium influx blockers” and vascular smooth muscle: do we really understand the mechanisms? Ann Intern Med 1981;94: 124-126. 19. Lloyd TC Jr. Hypoxlc pulmonary vasoconstnctron: role of penvascular tissue J Appl Physlol 1968.25:560-565. 20. Haugue A. Role of histamine In hypoxlc pulmonary hypertension in the rat I Blockade or potentlatlon of endogenous amines, klmns. and ATP Clrc Res 1968,22 371-383 21. Haas F. Beraofskv EH. Role of the mast cell in the oulmonary pressor response’to hyboxla J Chn Invest 1972;51:3154-3882 . 22. Kadowltz PJ, Hyman AL. Vasodilator actions of nifedlplne and verapamil In the pulmonary vascular bed (abstr) Fed Proc 1981,40:590. 23. Cerrina J, Denjean A, Alexandre G, Lockhart A, Duroux P. Inhibition of exercise-induced asthma by a calcium antagonist, nifedipine Am Rev Resp Dis 1981:123:156-160. 24. Matihay kA, Brent BN, Berger JH, Mahler DA, Langou R, Zarel BL. Hemodynamlc effects of mtroglycerin in patients with chronic obstructive pulmonary disease. pulmonary hypertension. and decreased right ventncular function (abstr) Am Rev Resp Dis 1981,123 (SuppI): 25. Bishop MJ, Gronka R, Cheney FW. Minoxldll and nifedipine Inhibit hypoxlc pulmonary vasoconstriction (abstr). Chest 1981;80:388
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