Inhibition of Hypoxic Pulmonary By Nlfedipine

Vasoconstriction

THOMAS KENNEDY, MD, MPH and WARREN SUMMER, MD

Nifediplne is a potent slow channel calcium antag- onist and systemic vasodilator recently reported to attenuate hypoxic pulmonary vasoconstriction in man. Other systemic vasodilators have also been shown to attenuate hypoxic pulmonary vasocon- striction, 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 pul- monary 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 pul- monale is hypoxic pulmonary hypertension associated with disorders such as chronic bronchitis and emphy- sema, 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 ni- fedipine, 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

From the Departments of Medicine, Anesthesiology and Critical Care Medicine, and Environmental Health Sciences, The Johns Hopkins Medical Institutions, Baltimore, Maryland. Manuscript received De- cember 15, 1981; revised manuscript received March 31, 1982, ac- cepted 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.

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. Arte- riovenous 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 sympa- thetic discharge.T,s To determine whether nifedipine locally or reflexly inhibits hypoxic pulmonary hyper- tension, 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.

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PULMONARY HYPERTENSION-KENNEDY AND SUMMER

STANDPIPE

PT1

( [FA, RESPIRATOR , j, j

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 re- sponds 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 ap- proximately 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 determina- tions, 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 ad- justing 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%

4

I phi+ =EOOmmHq

FIGURE 2. Typical pulmonary pressure (Ppa)-flow (6) curves during hyperoxia (P102 = 200 mm Hg) and hypoxia (Plop = 50 mm Hg).

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 pres- sure 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 deter- mining 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 hyp- oxia. After determination of a curve, the inspired gas mixture was changed and time was allowed for stabilization of pul- monary artery pressure before another curve was recorded. Generally, stabilization occurred within 15 minutes. The ex- perimental 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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201 1 L

50 100 150 200 250 300

Mm&s of 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.

Values for pulmonary artery pressure at a flow of 1 literlmin (Ppar) during hyperoxia and hypoxia for control and experi- mental animals and differences between Ppar for each hyp- eroxic and its subsequent hypoxic value for control and ex- perimental 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 ac- cording to the method of Fisher.r3

Results

Control versus nifedipine: Results for control ani- mals 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 hy- peroxic and hypoxic curves determined during the 300 minute perfusion. Both hypoxic and hyperoxic re- sponses 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

I o-? 01 IO IO

Dose of 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.

100 150 203 250 xx1

Mmutes of Perfusm

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) venti- lation. Open circles represent mean PPa f 1 standard error of the mean.

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- 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 re- sponse 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 in- hibition occurred.

Discussion

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

888 October 1982 The American Journal of CARDIOLOGY Volume 50

reflex adrenergic discharge. However, we cannot con- clude where within the lung itself nifedipine’s action is located.

Effect of slow channel calcium antagonists on pulmonary hypoxic response: Blockade of the pul- monary hypoxic response by slow channel calcium an- tagonists 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 ma- chinery. 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 conduc- tance, presumably by differential effects on the two gates of the calcium channel of cardiac tissue.17 In vascular smooth muscle, however, contraction was re- cently 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 under- standing 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 pulmo- nary 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 pulmo- nary resistance vessels.21 The observation that both nifedipine and verapamil inhibit pulmonary hyper- tension 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 exper- iments, some investigators2” claimed that nifedipine blocks the release of platelet-activating factors and slow reactive substances, and reduces release of acid phos- phatase and glucuronidase from human polymorpho- nuclear 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 cal- cium-dependent release of some vasoactive substance

PULMONARY HYPERTENSION-KENNEDY AND SUMMER

released locally within the lung in response to hyp- oxia.

Clinical implications: Regardless of the mechanism of subcellular action, the demonstration that a vasodi- lator works locally within the lung to attenuate hypoxic pulmonary hypertension may be considerably impor- tant 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, nitro- glycerin accomplishes this only by altering venous ca- pacitance to decrease right ventricular preload, without affecting pulmonary vascular resistance. Thus, a de- crease in mean pulmonary artery pressure is achieved at the expense of a decrease in cardiac output and oxy- gen delivery. Bishop et a1.25 recently reported that both nifedipine and minoxidil inhibit the pulmonary hypoxic response in intact dogs. However, minoxidil was previ- ously demonstratedfi to lose its effectiveness as a pul- monary vasodilator in hypoxic cattle after beta-adren- ergic 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 po- tential 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 de- crease sympathetic outflow, but their response to ni- fedipine 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 ni- fedipine, and Muramoto et al.” show no change in ar- terial 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 oc- curred 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 ventilation- perfusion relations that follow nifedipine administra- tion 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 Syl- vester, 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 prepa- ration of the manuscript.

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