Control of Lung Surfactant by Ventilation, Adrenergic Mediators, and Prostaglandins in the Rabbif4
MANUEL J. OYARZUN5 and JOHN A. CLEMENTS6
SUMMARY ________________________________________________________ In a previous study, we showed that increasing minute ventilation (VE) in rabbit lung by adding a dead space augmented pulmonary surfactant in the airspaces by a cholinergically mediated mechanism. Using the same model in the present study of 148 rabbits, we found that increasing VE augmented airspace phospholipid, the main component of surfactant, from 2.50 ± 0.61 (mean ± SD) mg per g of lung during normal VE to 3.15 ± 1.22 (mean ± SD) mg per g of lung during increased VE (P = 0.02). Both blocking beta-adrenergic receptors with propranolol or sotalol and inhibiting prostaglandin synthetase with indomethacin or sodium meclofenamate prevented the expected increase in phospholipid during increased VE (P < 0.05). The beta-2 agonist, terbutaline, increased phospholipid by 43 per cent during normal \IE (P < 0.01), and propranolol blocked this increase (P < 0.05). Isoproterenol, arachidonic acid, prostaglandins E1 , E2, F21%' and a cyclic endoperoxide analog of prostaglandin H2 (U-46619) injected during normal VE failed to increase phospholipid. We concluded that acetylcholine (previous study), beta-adrenergic mediators, and prostaglandins are involved in controlling alveolar surfactant during increased VE.
Introduction Many investigators have shown by studies in vivo and in vitro that increased ventilation can affect lung surfactant. Faridy and co-workers (1) and McClenahan and Urtnowski (2) found that (Received in original form October 10, 1977 and in revised form January 23, 1978) 1 From the Cardiovascular Research Institute, School of Medicine, University of California, San Francisco, Calif. 2Supported by Grant No. HL-06285 from the National Heart, Lung and Blood Institute. aPresented in part at the 61st Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, Ill., April 1977. 4 Requests for reprints should be addressed to Dr. J. A. Clements, Cardiovascular Research Institute, 1315 M, University of California, San Francisco, Calif. 94143. 5A Fellow of the American Heart Association during a part of this research. 6A Career Investigator of the American Heart Association.
increased ventilation of isolated rat and dog lungs, respectively, decreased compliance. The effect was attributed to a decrease in surface activity and was consistent with surface properties of lung extracts examined in a modified Wilhelmy balance. This alteration was directly related to tidal volume, ventilation rate, and duration of ventilation. Similar results in mechanically hyperventilated guinea pigs were reported by Forrest (3). However, because his work did not rule out the presence of pulmonary edema and because no quantitative chemical analysis of surfactant was made, it is difficult to conclude whether hyperventilation inactivated or decreased pulmonary surfactant, or perhaps had both effects. Using radioactive precursors of lecithin in open-chested cats whose lungs were hyperventilated for 3 hours, Wyszogrodski and co-workers (4) found increased elastic recoil (air pressurevolume curves). increased minimal surface tension of lung extracts, and increased radioactivity in alveolar lecithins. These authors suggested
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 117, 1978
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that hyperventilation prompted release of lecithins from the tissue to the alveoli and inactivated the released material. Faridy (5) reported an increase in surface activity and lecithin content in the saline washings of "extra pulmonary" airways of rats (trachea and major bronchi) after increased ventilation. The greater amount of lecithin found in the upper airways after hyperventilation may have originated from the alveoli, because Young and Tierney (6) found that the disaturated lecithin obtained by lavage of the trachea and major bronchi of normally ventilated rats was less than 4 per cent of that obtained by lavage of the whole lung. In another study, of excised lower lobes of dog lungs, Faridy (7) reported an increase in the lecithin content in the pulmonary lavage after the lungs were inflated with air. Hildebran and co-workers (8) also reported that air inflation of excised lungs to maximal volume increased the amount of phospholipid recovered from the air spaces. Recently, they reported that atropine blocked this effect (9). Several morphologic and biochemical observations indicate that the autonomic nervous system or its mediators control pulmonary surfactant. Tooley and associates (10) showed that vagotomy for 2 to 4 hours increased surface'tension of lung extracts in guinea pigs. Bolande and Klaus (II) obtained similar results, which they attributed to the presence of pulmonary edema. Goldenberg and co-workers (12) reported atelectasis and a decreased number of secretory granules in alveolar epithelial type 2 cells in vagotomized rats. In a subsequent paper (13) they reported that pilocarpine caused expulsion of secretory granules from type 2 cells into the alveoli. Morgan and Morgan (14) reported earlier and greater incorporation of carbon-14 palmitate into disaturated lecithin in the alveolar lavage fluid from rats treated with pilocarpine than in control animals. Massaro (15) observed that pilocarpine stimulated the release of radioactive protein into a surface active fraction after administration of carbon-14 leucine. This effect was blocked by atropine. One hour after administration of a single injection of pilocarpine or isoproterenol, Olsen (16) found a decrease in the number of lamellar bodies in alveolar epithelial cells and an increase of phospholipids in lung lavage fluid. These effects of pilocarpine and isoproterenol were blocked by atropine and propranolol, respectively.
In our own studies of control of pulmonary surfactant in the rabbit (17), we found that increasing minute ventilation (VE) approximatly 100 per cent by augmenting dead space for periods of I to 4 hours produced more alveolar phospholipid than in control rabbits with normal ventilation. Because this increase of phospholipid was obtained both with acetylcholine and with electrical cervical vagus stimulation, we concluded that the amount of alveolar surfactant can be increased by an acetylcholine-mediated mechanism. However. the results obtained in this study (17) did not rule out the possibility that other mechanisms, including sympathetic excitation and release of humoral substances such as adrenergic mediators and prostaglandins, are also involved in the response to increased ventilation. In addition to Olsen's study (16), 2 other studies have shown an influence of beta-adrenergic receptors on surfactant. Wyszogrodski and coworkers (18) suggested that isoxuprine injections in fetal rabbits 3 hours before delivery promote surfactant release into alveolar spaces. Recently, Dobbs and Mason (19) found that terbutaline promotes secretion of disaturated phosphatidylcholine from isolated pulmonary alveolar type 2 cells. The addition of propranolol inhibited the stimulatory effect of terbutaline. Beckman and Mason (20) have suggested that alpha-adrenergic receptors influence lung surfactant because they found that 30-sec electrical stimulation of the stellate ganglia in openchested cats caused a decrease in dynamic lung compliance. They attributed this result to an alteration of the pulmonary surfactant. The effect was prevented by phenoxybenzamine or phentolamine, alpha-adrenergic blocking agents, or by reserpine, a catecholamine-depleting agent (21). However, Wyszogrodski and Taeusch (22) were unable to demonstrate that prolonged intermittent stimulation of the stellate ganglia in adult cats under mechanical ventilation affected pressure-volume relationships and surface tension properties of lung extracts. The role of prostaglandins in controlling surfactant is suggested by several studies of prostaglandins. For example. in a study of isolated and perfused guinea pig lungs. increased ventilation released prostaglandin E2 (23). Similarly, mechanical hyperinflation of isolated and perfused lower lobes of dog lungs released prostaglandins (24). Because this effect was inhibited after aspirin was infused into the lung. the authors concluded that stretching of the lung in-
CONTROL OF LUNG SURFACTANT IN RABBIT
creased the synthesis of prostaglandins and their release into the circulation. In addition, mechanical disturbance of transformed mouse fibroblasts stimulated the production of both prostaglandin E2 and prostaglandin F 2 a.' and indomethacin inhibited this effect (25). Both prostaglandins E2 and F 2.. stimulated the incorporation of carbon-l4 palmitate and hydrogen-3 choline into phosphatidylcholine in transformed cells from human lung carcinoma (line A 549), suggesting that prostaglandins are able to stimulate phosphatidylcholine biosynthesis (26) in these cultured cells. However, it is difficult to extrapolate this result to type 2 alveolar cells, because A 549 cells apparently lack some important morphologic and biochemical characteristics of type 2 cells isolated from adult rat lung (27). To sum up, our previous study of surfactant regulation showed that increased ventilation can augment the amount of air space surfactant through a cholinergically mediated mechanism, but it did not rule out the participation of other pathways. The other papers cited previously suggested that adrenergic mechanisms may control the amount of pulmonary surfactant and that the formation of prostaglandins in the lungs is enhanced during increased ventilation. Therefore, in this study we explored the role of alphaand beta-adrenergic receptors and the role of prostaglandins in the mediation of the response of the surfactant system to increased ventilation. We deliberately kept the experimental protocols as uniform as possible throughout this series of tests to facilitate comparison of different agonists and antagonists. Materials and Methods General procedures. The methods used were similar to those described in our previous paper (17). We used New Zealand white male rabbits weighing 2.3 to 3.8 kg. Rabbits were anesthetized intravenously with 30 mg of sodium pentobarbital per kg of body weight (Diabutal®, Diamond Labs Inc.) and light anesthesia was maintained with small doses (9 to 12 mg) of sodium pentobarbital throughout the experiment. All observations were made while the rabbits were breathing spontaneously. To measure airflow and breathing frequency, we inserted a cannula into the trachea and attached a Fleisch pneumotachygraph to it. Airflow was integrated electrically to give tidal volume. Arterial blood pressure was measured in a femoral artery by a Statham P23DC pressure transducer (Statham Instruments, Oxnard, Calif.). Airflow, tidal volume, and arterial blood pressure were recorded continuously on a Grass polygraph. Every 30 or 60 min we de-
881
termined P02 , Pc02 , and pH in systemic arterial blood samples by electrode measurement (Radiometer). We used these general procedures in all of the experiments. Experimental Protocols Every experiment began with a one-hour control period and was followed by one of 6 experimental protocols. For control animals, we used rabbits with normal ventilation or with ventilation increased by about 100 per cent for 2 hours. Ventilation was increased by adding dead space to the tracheal cannula. Beta-adrenergic blockade during normal and increased ventilation. We used 2 different beta-adrenergic blockers during increased ventilation in 2 different series of experiments. Because propanolol in high doses can have membrane-stabilizing properties, we also used sotalol, even though it is 10 times less powerful than propranolol, because it is only known to block beta-adrenergic receptors. In one series of experiments we intravenously injected I mg of propranolol hydrochloride per kg of body weight (Inderal®, Ayerst) for 6 min into 4 rabbits in which normal ventilation was maintained for 2 hours, and into 4 rabbits in which ventilation was increased by about 100 per cent for 2 hours by adding a dead space to the tracheal cannula. Into 5 other rabbits, we intravenously injected 10 mg of sotalol hydrochloride per kg of body weight (Regis Chemical Co.) for 6 min. Beta-adrenergic blockade induced by both propranolol and sotalol was sufficient to block the cardiovascular effects of isoproterenol hydrochloride in test doses of 0.5 p,g per kg of body weight (Elkins-Sinn Inc.) in 1 ml injected intravenously. Alpha-adrenergic blockade during increased ventilation. Alpha-adrenergic receptors were blocked by injecting 3 mg of phenoxybenzamine hydrochloride per kg of body weight at the rate of 0.5 mg per min (Dibenzyline®, generously supplied by Ms. Eileen Gallagher of Smith, Kline and French Laboratories) into 9 rabbits in which ventilation was increased by about 100 per cent for 2 hours. Alpha-adrenergic blockade induced by phenoxybenzamine was sufficient to block the cardiovascular effects of an intravenous bolus of 1.3 p,g of L-norepinephrine bitartrate (Levophed®, Winthrop). Beta-adrenergic stimulation during normal ventilation. In 5 rabbits, beta-adrenergic receptors were stimulated by injecting 15 p,g of isoproterenol hydrochloride (Elkins-Sinn Inc.) per ml in 0.9 per cent NaCI at a rate of approximately 0.1 ml per min via a femoral vein, enough to decrease the aortic pressure by 25 per cent. This decrease was maintained during 2 hours of normal ventilation. Into 11 other rabbits, the beta-2 adrenergic agonist, terbutaline sulfate (Bricanyl Sulfate®, kindly provided by Mr. Richard J. Esper of Astra Pharmaceuticals) was continuously injected into a femoral vein (at a rate of 150 p,g per
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OYARZUN AND CLEMENTS
kg of body weight per min) during 2 hours of normal ventilation. In 5 other rabbits, beta-adrenergic receptors were first blocked by intravenously injecting I mg of propranolol hydrochloride per kg of body weight and were stimulated 30 min later by injecting 150 p.g of terbutaline per kg of body weight per min during 2 hours of normal ventilation. Betaadrenergic blockade induced by propranolol was sufficient to block the cardiovascular effects of test doses of 0.5 p.g of isoproterenol hydrocholoride per kg of body weight, administered intravenously. Prostaglandin-synthetase inhibition during normal and increased venti/ation. To inhibit prostaglandin synthetase, we administered 2 different drugs: 15 mg of indomethacin per kg of body weight (Merck, Sharp and Dohme), which we gave to 8 rabbits, and 5 mg of sodium medofenamate per kg of body weight (generously provided by Parke, Davis and Co.), which we gave to 5 other rabbits. Each drug was injected intravenously 30 min before ventilation was increased by about 100 per cent for 2 hours. We gave 8 other rabbits 5 mg of sodium medofenamate per kg of body weight during the control period and then allowed these rabbits to breath normally for 2 hours. Both indomethacin and meclofenamate irreversibly inactivate prostaglandin synthetase, and replacement of enzyme protein by de novo synthesis is said to be necessary to terminate the action of these drugs, which lasts many hours (28). The reason we used 2 different inhibitors of prostaglandin synthetase was that in addition to interfering with the prostaglandin synthetase, aspirin-like drugs may inhibit a variety of other enzymes and cell systems. However, the concentrations of drugs required to inhibit the prostaglandin synthetase are generally much smaller than the concentrations that inhibit other enzymes (28). Because Block and co-workers (29) found that treatment with 10 mg of indomethacin per kg of body weight blocked the release of prostaglandins from New Zealand rabbit heart, we used 15 mg of indomethacin per kg of body weight to ensure that we would be able to induce the effect. In several different prostaglandin-synthetase preparations, meclofenamic acid is about 3 times more potent an inhibitor than is indomethacin (28), so we used 5 mg of medofenamate per kg of body weight. Prostaglandin injection during normal ventilation. For prostaglandin injection, a polyethylene catheter was placed into the pulmonary artery via the femoral vein. The position of the catheter tip was followed by recording blood pressure and observing the pulse contour. Proper position was confirmed at autopsy. Prostaglandin El was continuously injected into 5 rabbits. Six other rabbits received prostaglandin E 2, and 6 others received prostaglandin F 20;' All 3 compounds (kindly provided by Upjohn Co.) were injected at a dose rate of 0.4 p.g per kg of body weight per min, sufficient to produce a change of approximately 15 per cent in both pulmonary and aortic
blood pressures for 2 hours during normal ventilation. Injection of a prostaglandin precursor and a prostaglandin intermediate during normal ventilation. Arachidonic acid (Supelco, Inc.), a precursor of endogenous prostaglandins, was injected intravenously into II rabbits for 15 min during normal ventilation at a rate of 200 p.g per kg of body weight per min, sufficient to decrease arterial blood pressure by about 20 per cent. As control animals, 6 rabbits were injected intravenously with oleic acid (Calbiochem) for 15 min during normal ventilation at a rate of 200 p.g per kg of body weight per min. Instead of using natural cyclic endoperoxides, which are very unstable, we used a stable, biologically active endoperoxide analog of prostaglandin H 2, [(15S)-hydroxy 110;,90;-(epoxymethano)prosta-5Z,13E-dienoic acid] (U-46619, synthesized and generously provided by Dr. G. L. Bundy of Upjohn Co.). This analog was injected for 15 min during normal ventilation into one femoral vein in each of 6 rabbits at a rate of 0.03 p.g per kg of body weight per min, sufficient to increase pulmonary arterial blood pressure by 30 per cent. Analysis of materials. At the end of each experiment, the rabbits were killed with an intravenous dose of sodium pentobarbital. The lungs were immediately removed, weighed, and separated by inserting cannulas into the left and right major bronchi. Each lung was degassed and washed 3 times with 0.9 per cent NaCl at room temperature at a volume of 10 ml per g of lung. To avoid distending the lungs further during the second and third washings, the isotonic saline residl:le (5 per cent of the total lavage fluid used) was taken into account to determine the volume to be injected. No difference in retention of lavage fluid was found between the lungs in the control and experimental series. In control animals we recovered 94.29 ± 2.07 per cent (mean ± SD, n = 29) of the lavage fluid, and this percentage was the same in the experimental series (by variance analysis). The lavage fluids from the 3 washings were pooled, lipids were extracted from a sample of the wellmixed alveolar lavage fluid by Bligh and Dyer's method (30), and the concentration of phosphorus values in the lipids was determined by Bartlett's method (31). Lipid phosphorus values were multiplied by 25 (phospholipid = 4 per cent lipid phosphorus) to give phospholipid content. The phospholipid content from each lung was analyzed in triplicate, and the values were averaged; the resulting averages for the 2 lungs (right and left) were then averaged to give a value for the whole rabbit. In separate experiments (17), we found that the first 3 lung lavages removed 65 per cent of the amount of phospholipid removable with 12 washes; this percentage was the same for the control and experimental series. In 30 samples from 6 different series of experiments, the amount of disaturated phosphatidylcholine in the lavage fluid was determined
CONTROL OF LUNG SURFACTANT IN RABBIT
by using osmium tetroxide according to the method described by Mason and associates (32). Paired and unpaired "Student's" t tests, analysis of variance, and Dunnett's test were used for the statistical analysis of the results (33). When analysis of variance gave a P value of less than 0.05, we used Dunnett's test to compare the means of several experimental series with a control value. Criteria for acceptance of the data. We used data only when they came from rabbits that met the fol· lowing 4 criteria: (1) normal arterial blood gases and pH, (2) normal lung appearance (Lungs with macroscopic lesions, hemorrhagic zones, pneumonia, and/or foam in the airways were discarded.), (3) normal ratio of lung weight to body weight and normal ratio of lung wet weight to lung dry weight (In control animals, these ratios were 0.00364 ± 0.00030 [mean ± SD, n = 29] and 4.72 ± 0.36 [mean ± SD, n = 29], respectively. When these ratios for experimental data differed from those of the control series by more than 2 units of SD, the experiment was discarded. Pulmonary edema was evident when the ratio of lung weight to body weight was 7 to II units of SD greater than mean [17].), (4) lavage fluid lactate dehydrogenase (LDH·L) activity, protein content, and number of cells within ranges previously determined for normal rabbits (LDH-L activity was analyzed with Calbiochem-LDH-L reagents according to the method of Wacker and coworkers [34]. The method is sensitive enough to detect approximately 0.3 per cent of the total LDH-L activity present in the lavage fluid from the whole
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Fig. 1. Effect of beta-adrenergic blockade during normal and increased ventilation. In control groups, phospholipid content was significantly greater with increased ventilation than with normal ventilation (P = 0.02 by unpaired "Student's" t test). In rabbits in which beta-adrenergic receptors were blocked, phospholipid content was significantly less than in the respective control animals (P < 0.01 by Dunnett's test). mg PL/g lung = mg of phospholipid in lavage fluid per g of lung; SE = standard error of the mean; N = number of experiments; V = minute ventilation.
883
lung. We discarded experiments with the LDH-L activity detectable in the lavage fluid. Protein content was determined by the method of Lowry and associates [35], and the number of cells was determined with a Spencer hemocytometer and methyl violet. In control rabbits [n = 29], the lavage fluid had 4.64 ± 1.7 mg of protein per g of lung [mean ± SD] and 5.5 ± 2.0 X 106 cells per g of lung [mean ± SD].). On the basis of these criteria, 37 of 185 experiments were prospectively discarded. In the experiments that we accepted, the ratio of lung weight to body weight, the ratio of lung wet weight to lung dry weight, and the number of cells in the lavage fluid were not significantly different (analysis of variance) in the control and experimental series. Protein content was not significantly different in the control and experimental series, with the exception of prostaglandins El (10.97 ± 5.2 mg of protein per g, mean ± SD) and F2a (8.86 ± 2.6 mg of protein per g, mean ± SD), which showed a significant increase (Dunnett's test. P
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Fig. 2. Effect of alpha- and beta-adrenergic blockade during increased ventilation. In rabbits in which alphaadrenergic receptors were blocked by administration of 3 mg of phenoxybenzamine per kg of body weight, phospholipid content was not significantly different from the control value (Dunnett's test). In rabbits in which beta-adrenergic receptors were blocked (propranolol and 8Otalol results pooled), phospholipid content was significantly less than the control value (P < 0.01 by Dunnett's test). For definition of abbreviations, see figure 1.
885
CONTROL OF LUNG SURFACTANT IN RABBIT
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2 (U-46619) did not have a significant effect on alveolar phospholipid content (2.11 ± 0.62 mg of phospholipid per g, mean ± SD; n 5, P 0.22 by unpaired "Student's" t test), but it produced a 33 per cent increase in pulmonary arterial blood pressure and a small increase (1.5 cm H 20) in the amplitude of transpulmonary pressure swings. Ventilation (VE, frequency, and tidal volume), Pao2, Paco2' and arterial pH remained within normal limits in these 3 series of experiments.
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phospholipid content, but the difference between these 2 effects was not significant (figure 5). Prostaglandin El and prostaglandin E2 each decreased pulmonary arterial mean pressure by an average of 13 and 20 per cent, respectively, and prostaglandin F24 increased this pressure by 13 per cent. Neither prostaglandin El nor prostaglandin E2 changed the pattern of breathing for 2 hours, but prostaglandin F 24 significantly increased both the respiratory rate and VE (P < 0.05 by paired "Student's" t test) (table 3).
samples of pulmonary lavage fluid, selected randomly from 6 different groups. Synthetic carbon14 dipalmitoylphosphatidylcholine was used as an internal standard in all determinations, and its average recovery was 85.6 ± 7.5· per cent (mean ± SD). There was no significant difference in recoveries between the control and experimental series. DSPC accounted for 55.1 ± 11.5 per cent (mean ± SD) of the total phospholipid in the lavage fluid. There was no significant difference in the ratio of DSPC to total phospholipid between the control and experimental series (table 4). Discussion
Our first concern in assessing the significance of our results was the reliability of the methods. In these series of experiments we used the phospholipid content in the pulmonary lavage
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886
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MANUEL J. OYARZUN5 and JOHN A. CLEMENTS6
SUMMARY ________________________________________________________ In a previous study, we showed that increasing minute ventilation (VE) in rabbit lung by adding a dead space augmented pulmonary surfactant in the airspaces by a cholinergically mediated mechanism. Using the same model in the present study of 148 rabbits, we found that increasing VE augmented airspace phospholipid, the main component of surfactant, from 2.50 ± 0.61 (mean ± SD) mg per g of lung during normal VE to 3.15 ± 1.22 (mean ± SD) mg per g of lung during increased VE (P = 0.02). Both blocking beta-adrenergic receptors with propranolol or sotalol and inhibiting prostaglandin synthetase with indomethacin or sodium meclofenamate prevented the expected increase in phospholipid during increased VE (P < 0.05). The beta-2 agonist, terbutaline, increased phospholipid by 43 per cent during normal \IE (P < 0.01), and propranolol blocked this increase (P < 0.05). Isoproterenol, arachidonic acid, prostaglandins E1 , E2, F21%' and a cyclic endoperoxide analog of prostaglandin H2 (U-46619) injected during normal VE failed to increase phospholipid. We concluded that acetylcholine (previous study), beta-adrenergic mediators, and prostaglandins are involved in controlling alveolar surfactant during increased VE.
Introduction Many investigators have shown by studies in vivo and in vitro that increased ventilation can affect lung surfactant. Faridy and co-workers (1) and McClenahan and Urtnowski (2) found that (Received in original form October 10, 1977 and in revised form January 23, 1978) 1 From the Cardiovascular Research Institute, School of Medicine, University of California, San Francisco, Calif. 2Supported by Grant No. HL-06285 from the National Heart, Lung and Blood Institute. aPresented in part at the 61st Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, Ill., April 1977. 4 Requests for reprints should be addressed to Dr. J. A. Clements, Cardiovascular Research Institute, 1315 M, University of California, San Francisco, Calif. 94143. 5A Fellow of the American Heart Association during a part of this research. 6A Career Investigator of the American Heart Association.
increased ventilation of isolated rat and dog lungs, respectively, decreased compliance. The effect was attributed to a decrease in surface activity and was consistent with surface properties of lung extracts examined in a modified Wilhelmy balance. This alteration was directly related to tidal volume, ventilation rate, and duration of ventilation. Similar results in mechanically hyperventilated guinea pigs were reported by Forrest (3). However, because his work did not rule out the presence of pulmonary edema and because no quantitative chemical analysis of surfactant was made, it is difficult to conclude whether hyperventilation inactivated or decreased pulmonary surfactant, or perhaps had both effects. Using radioactive precursors of lecithin in open-chested cats whose lungs were hyperventilated for 3 hours, Wyszogrodski and co-workers (4) found increased elastic recoil (air pressurevolume curves). increased minimal surface tension of lung extracts, and increased radioactivity in alveolar lecithins. These authors suggested
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 117, 1978
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880
OYARZUN AND CLEMENTS
that hyperventilation prompted release of lecithins from the tissue to the alveoli and inactivated the released material. Faridy (5) reported an increase in surface activity and lecithin content in the saline washings of "extra pulmonary" airways of rats (trachea and major bronchi) after increased ventilation. The greater amount of lecithin found in the upper airways after hyperventilation may have originated from the alveoli, because Young and Tierney (6) found that the disaturated lecithin obtained by lavage of the trachea and major bronchi of normally ventilated rats was less than 4 per cent of that obtained by lavage of the whole lung. In another study, of excised lower lobes of dog lungs, Faridy (7) reported an increase in the lecithin content in the pulmonary lavage after the lungs were inflated with air. Hildebran and co-workers (8) also reported that air inflation of excised lungs to maximal volume increased the amount of phospholipid recovered from the air spaces. Recently, they reported that atropine blocked this effect (9). Several morphologic and biochemical observations indicate that the autonomic nervous system or its mediators control pulmonary surfactant. Tooley and associates (10) showed that vagotomy for 2 to 4 hours increased surface'tension of lung extracts in guinea pigs. Bolande and Klaus (II) obtained similar results, which they attributed to the presence of pulmonary edema. Goldenberg and co-workers (12) reported atelectasis and a decreased number of secretory granules in alveolar epithelial type 2 cells in vagotomized rats. In a subsequent paper (13) they reported that pilocarpine caused expulsion of secretory granules from type 2 cells into the alveoli. Morgan and Morgan (14) reported earlier and greater incorporation of carbon-14 palmitate into disaturated lecithin in the alveolar lavage fluid from rats treated with pilocarpine than in control animals. Massaro (15) observed that pilocarpine stimulated the release of radioactive protein into a surface active fraction after administration of carbon-14 leucine. This effect was blocked by atropine. One hour after administration of a single injection of pilocarpine or isoproterenol, Olsen (16) found a decrease in the number of lamellar bodies in alveolar epithelial cells and an increase of phospholipids in lung lavage fluid. These effects of pilocarpine and isoproterenol were blocked by atropine and propranolol, respectively.
In our own studies of control of pulmonary surfactant in the rabbit (17), we found that increasing minute ventilation (VE) approximatly 100 per cent by augmenting dead space for periods of I to 4 hours produced more alveolar phospholipid than in control rabbits with normal ventilation. Because this increase of phospholipid was obtained both with acetylcholine and with electrical cervical vagus stimulation, we concluded that the amount of alveolar surfactant can be increased by an acetylcholine-mediated mechanism. However. the results obtained in this study (17) did not rule out the possibility that other mechanisms, including sympathetic excitation and release of humoral substances such as adrenergic mediators and prostaglandins, are also involved in the response to increased ventilation. In addition to Olsen's study (16), 2 other studies have shown an influence of beta-adrenergic receptors on surfactant. Wyszogrodski and coworkers (18) suggested that isoxuprine injections in fetal rabbits 3 hours before delivery promote surfactant release into alveolar spaces. Recently, Dobbs and Mason (19) found that terbutaline promotes secretion of disaturated phosphatidylcholine from isolated pulmonary alveolar type 2 cells. The addition of propranolol inhibited the stimulatory effect of terbutaline. Beckman and Mason (20) have suggested that alpha-adrenergic receptors influence lung surfactant because they found that 30-sec electrical stimulation of the stellate ganglia in openchested cats caused a decrease in dynamic lung compliance. They attributed this result to an alteration of the pulmonary surfactant. The effect was prevented by phenoxybenzamine or phentolamine, alpha-adrenergic blocking agents, or by reserpine, a catecholamine-depleting agent (21). However, Wyszogrodski and Taeusch (22) were unable to demonstrate that prolonged intermittent stimulation of the stellate ganglia in adult cats under mechanical ventilation affected pressure-volume relationships and surface tension properties of lung extracts. The role of prostaglandins in controlling surfactant is suggested by several studies of prostaglandins. For example. in a study of isolated and perfused guinea pig lungs. increased ventilation released prostaglandin E2 (23). Similarly, mechanical hyperinflation of isolated and perfused lower lobes of dog lungs released prostaglandins (24). Because this effect was inhibited after aspirin was infused into the lung. the authors concluded that stretching of the lung in-
CONTROL OF LUNG SURFACTANT IN RABBIT
creased the synthesis of prostaglandins and their release into the circulation. In addition, mechanical disturbance of transformed mouse fibroblasts stimulated the production of both prostaglandin E2 and prostaglandin F 2 a.' and indomethacin inhibited this effect (25). Both prostaglandins E2 and F 2.. stimulated the incorporation of carbon-l4 palmitate and hydrogen-3 choline into phosphatidylcholine in transformed cells from human lung carcinoma (line A 549), suggesting that prostaglandins are able to stimulate phosphatidylcholine biosynthesis (26) in these cultured cells. However, it is difficult to extrapolate this result to type 2 alveolar cells, because A 549 cells apparently lack some important morphologic and biochemical characteristics of type 2 cells isolated from adult rat lung (27). To sum up, our previous study of surfactant regulation showed that increased ventilation can augment the amount of air space surfactant through a cholinergically mediated mechanism, but it did not rule out the participation of other pathways. The other papers cited previously suggested that adrenergic mechanisms may control the amount of pulmonary surfactant and that the formation of prostaglandins in the lungs is enhanced during increased ventilation. Therefore, in this study we explored the role of alphaand beta-adrenergic receptors and the role of prostaglandins in the mediation of the response of the surfactant system to increased ventilation. We deliberately kept the experimental protocols as uniform as possible throughout this series of tests to facilitate comparison of different agonists and antagonists. Materials and Methods General procedures. The methods used were similar to those described in our previous paper (17). We used New Zealand white male rabbits weighing 2.3 to 3.8 kg. Rabbits were anesthetized intravenously with 30 mg of sodium pentobarbital per kg of body weight (Diabutal®, Diamond Labs Inc.) and light anesthesia was maintained with small doses (9 to 12 mg) of sodium pentobarbital throughout the experiment. All observations were made while the rabbits were breathing spontaneously. To measure airflow and breathing frequency, we inserted a cannula into the trachea and attached a Fleisch pneumotachygraph to it. Airflow was integrated electrically to give tidal volume. Arterial blood pressure was measured in a femoral artery by a Statham P23DC pressure transducer (Statham Instruments, Oxnard, Calif.). Airflow, tidal volume, and arterial blood pressure were recorded continuously on a Grass polygraph. Every 30 or 60 min we de-
881
termined P02 , Pc02 , and pH in systemic arterial blood samples by electrode measurement (Radiometer). We used these general procedures in all of the experiments. Experimental Protocols Every experiment began with a one-hour control period and was followed by one of 6 experimental protocols. For control animals, we used rabbits with normal ventilation or with ventilation increased by about 100 per cent for 2 hours. Ventilation was increased by adding dead space to the tracheal cannula. Beta-adrenergic blockade during normal and increased ventilation. We used 2 different beta-adrenergic blockers during increased ventilation in 2 different series of experiments. Because propanolol in high doses can have membrane-stabilizing properties, we also used sotalol, even though it is 10 times less powerful than propranolol, because it is only known to block beta-adrenergic receptors. In one series of experiments we intravenously injected I mg of propranolol hydrochloride per kg of body weight (Inderal®, Ayerst) for 6 min into 4 rabbits in which normal ventilation was maintained for 2 hours, and into 4 rabbits in which ventilation was increased by about 100 per cent for 2 hours by adding a dead space to the tracheal cannula. Into 5 other rabbits, we intravenously injected 10 mg of sotalol hydrochloride per kg of body weight (Regis Chemical Co.) for 6 min. Beta-adrenergic blockade induced by both propranolol and sotalol was sufficient to block the cardiovascular effects of isoproterenol hydrochloride in test doses of 0.5 p,g per kg of body weight (Elkins-Sinn Inc.) in 1 ml injected intravenously. Alpha-adrenergic blockade during increased ventilation. Alpha-adrenergic receptors were blocked by injecting 3 mg of phenoxybenzamine hydrochloride per kg of body weight at the rate of 0.5 mg per min (Dibenzyline®, generously supplied by Ms. Eileen Gallagher of Smith, Kline and French Laboratories) into 9 rabbits in which ventilation was increased by about 100 per cent for 2 hours. Alpha-adrenergic blockade induced by phenoxybenzamine was sufficient to block the cardiovascular effects of an intravenous bolus of 1.3 p,g of L-norepinephrine bitartrate (Levophed®, Winthrop). Beta-adrenergic stimulation during normal ventilation. In 5 rabbits, beta-adrenergic receptors were stimulated by injecting 15 p,g of isoproterenol hydrochloride (Elkins-Sinn Inc.) per ml in 0.9 per cent NaCI at a rate of approximately 0.1 ml per min via a femoral vein, enough to decrease the aortic pressure by 25 per cent. This decrease was maintained during 2 hours of normal ventilation. Into 11 other rabbits, the beta-2 adrenergic agonist, terbutaline sulfate (Bricanyl Sulfate®, kindly provided by Mr. Richard J. Esper of Astra Pharmaceuticals) was continuously injected into a femoral vein (at a rate of 150 p,g per
882
OYARZUN AND CLEMENTS
kg of body weight per min) during 2 hours of normal ventilation. In 5 other rabbits, beta-adrenergic receptors were first blocked by intravenously injecting I mg of propranolol hydrochloride per kg of body weight and were stimulated 30 min later by injecting 150 p.g of terbutaline per kg of body weight per min during 2 hours of normal ventilation. Betaadrenergic blockade induced by propranolol was sufficient to block the cardiovascular effects of test doses of 0.5 p.g of isoproterenol hydrocholoride per kg of body weight, administered intravenously. Prostaglandin-synthetase inhibition during normal and increased venti/ation. To inhibit prostaglandin synthetase, we administered 2 different drugs: 15 mg of indomethacin per kg of body weight (Merck, Sharp and Dohme), which we gave to 8 rabbits, and 5 mg of sodium medofenamate per kg of body weight (generously provided by Parke, Davis and Co.), which we gave to 5 other rabbits. Each drug was injected intravenously 30 min before ventilation was increased by about 100 per cent for 2 hours. We gave 8 other rabbits 5 mg of sodium medofenamate per kg of body weight during the control period and then allowed these rabbits to breath normally for 2 hours. Both indomethacin and meclofenamate irreversibly inactivate prostaglandin synthetase, and replacement of enzyme protein by de novo synthesis is said to be necessary to terminate the action of these drugs, which lasts many hours (28). The reason we used 2 different inhibitors of prostaglandin synthetase was that in addition to interfering with the prostaglandin synthetase, aspirin-like drugs may inhibit a variety of other enzymes and cell systems. However, the concentrations of drugs required to inhibit the prostaglandin synthetase are generally much smaller than the concentrations that inhibit other enzymes (28). Because Block and co-workers (29) found that treatment with 10 mg of indomethacin per kg of body weight blocked the release of prostaglandins from New Zealand rabbit heart, we used 15 mg of indomethacin per kg of body weight to ensure that we would be able to induce the effect. In several different prostaglandin-synthetase preparations, meclofenamic acid is about 3 times more potent an inhibitor than is indomethacin (28), so we used 5 mg of medofenamate per kg of body weight. Prostaglandin injection during normal ventilation. For prostaglandin injection, a polyethylene catheter was placed into the pulmonary artery via the femoral vein. The position of the catheter tip was followed by recording blood pressure and observing the pulse contour. Proper position was confirmed at autopsy. Prostaglandin El was continuously injected into 5 rabbits. Six other rabbits received prostaglandin E 2, and 6 others received prostaglandin F 20;' All 3 compounds (kindly provided by Upjohn Co.) were injected at a dose rate of 0.4 p.g per kg of body weight per min, sufficient to produce a change of approximately 15 per cent in both pulmonary and aortic
blood pressures for 2 hours during normal ventilation. Injection of a prostaglandin precursor and a prostaglandin intermediate during normal ventilation. Arachidonic acid (Supelco, Inc.), a precursor of endogenous prostaglandins, was injected intravenously into II rabbits for 15 min during normal ventilation at a rate of 200 p.g per kg of body weight per min, sufficient to decrease arterial blood pressure by about 20 per cent. As control animals, 6 rabbits were injected intravenously with oleic acid (Calbiochem) for 15 min during normal ventilation at a rate of 200 p.g per kg of body weight per min. Instead of using natural cyclic endoperoxides, which are very unstable, we used a stable, biologically active endoperoxide analog of prostaglandin H 2, [(15S)-hydroxy 110;,90;-(epoxymethano)prosta-5Z,13E-dienoic acid] (U-46619, synthesized and generously provided by Dr. G. L. Bundy of Upjohn Co.). This analog was injected for 15 min during normal ventilation into one femoral vein in each of 6 rabbits at a rate of 0.03 p.g per kg of body weight per min, sufficient to increase pulmonary arterial blood pressure by 30 per cent. Analysis of materials. At the end of each experiment, the rabbits were killed with an intravenous dose of sodium pentobarbital. The lungs were immediately removed, weighed, and separated by inserting cannulas into the left and right major bronchi. Each lung was degassed and washed 3 times with 0.9 per cent NaCl at room temperature at a volume of 10 ml per g of lung. To avoid distending the lungs further during the second and third washings, the isotonic saline residl:le (5 per cent of the total lavage fluid used) was taken into account to determine the volume to be injected. No difference in retention of lavage fluid was found between the lungs in the control and experimental series. In control animals we recovered 94.29 ± 2.07 per cent (mean ± SD, n = 29) of the lavage fluid, and this percentage was the same in the experimental series (by variance analysis). The lavage fluids from the 3 washings were pooled, lipids were extracted from a sample of the wellmixed alveolar lavage fluid by Bligh and Dyer's method (30), and the concentration of phosphorus values in the lipids was determined by Bartlett's method (31). Lipid phosphorus values were multiplied by 25 (phospholipid = 4 per cent lipid phosphorus) to give phospholipid content. The phospholipid content from each lung was analyzed in triplicate, and the values were averaged; the resulting averages for the 2 lungs (right and left) were then averaged to give a value for the whole rabbit. In separate experiments (17), we found that the first 3 lung lavages removed 65 per cent of the amount of phospholipid removable with 12 washes; this percentage was the same for the control and experimental series. In 30 samples from 6 different series of experiments, the amount of disaturated phosphatidylcholine in the lavage fluid was determined
CONTROL OF LUNG SURFACTANT IN RABBIT
by using osmium tetroxide according to the method described by Mason and associates (32). Paired and unpaired "Student's" t tests, analysis of variance, and Dunnett's test were used for the statistical analysis of the results (33). When analysis of variance gave a P value of less than 0.05, we used Dunnett's test to compare the means of several experimental series with a control value. Criteria for acceptance of the data. We used data only when they came from rabbits that met the fol· lowing 4 criteria: (1) normal arterial blood gases and pH, (2) normal lung appearance (Lungs with macroscopic lesions, hemorrhagic zones, pneumonia, and/or foam in the airways were discarded.), (3) normal ratio of lung weight to body weight and normal ratio of lung wet weight to lung dry weight (In control animals, these ratios were 0.00364 ± 0.00030 [mean ± SD, n = 29] and 4.72 ± 0.36 [mean ± SD, n = 29], respectively. When these ratios for experimental data differed from those of the control series by more than 2 units of SD, the experiment was discarded. Pulmonary edema was evident when the ratio of lung weight to body weight was 7 to II units of SD greater than mean [17].), (4) lavage fluid lactate dehydrogenase (LDH·L) activity, protein content, and number of cells within ranges previously determined for normal rabbits (LDH-L activity was analyzed with Calbiochem-LDH-L reagents according to the method of Wacker and coworkers [34]. The method is sensitive enough to detect approximately 0.3 per cent of the total LDH-L activity present in the lavage fluid from the whole
mg PL g lung
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Fig. 1. Effect of beta-adrenergic blockade during normal and increased ventilation. In control groups, phospholipid content was significantly greater with increased ventilation than with normal ventilation (P = 0.02 by unpaired "Student's" t test). In rabbits in which beta-adrenergic receptors were blocked, phospholipid content was significantly less than in the respective control animals (P < 0.01 by Dunnett's test). mg PL/g lung = mg of phospholipid in lavage fluid per g of lung; SE = standard error of the mean; N = number of experiments; V = minute ventilation.
883
lung. We discarded experiments with the LDH-L activity detectable in the lavage fluid. Protein content was determined by the method of Lowry and associates [35], and the number of cells was determined with a Spencer hemocytometer and methyl violet. In control rabbits [n = 29], the lavage fluid had 4.64 ± 1.7 mg of protein per g of lung [mean ± SD] and 5.5 ± 2.0 X 106 cells per g of lung [mean ± SD].). On the basis of these criteria, 37 of 185 experiments were prospectively discarded. In the experiments that we accepted, the ratio of lung weight to body weight, the ratio of lung wet weight to lung dry weight, and the number of cells in the lavage fluid were not significantly different (analysis of variance) in the control and experimental series. Protein content was not significantly different in the control and experimental series, with the exception of prostaglandins El (10.97 ± 5.2 mg of protein per g, mean ± SD) and F2a (8.86 ± 2.6 mg of protein per g, mean ± SD), which showed a significant increase (Dunnett's test. P
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isoproterenol per kg of body weight per min during 2 hours of normal ventilation did not change alveolar phospholipid significantly (figure 3). However, continuous intravenous injection of 150 ILg of the beta-2 adrenergic agonist, terbutaline sulfate, per kg of body weight per min dur-
Fig. 2. Effect of alpha- and beta-adrenergic blockade during increased ventilation. In rabbits in which alphaadrenergic receptors were blocked by administration of 3 mg of phenoxybenzamine per kg of body weight, phospholipid content was not significantly different from the control value (Dunnett's test). In rabbits in which beta-adrenergic receptors were blocked (propranolol and 8Otalol results pooled), phospholipid content was significantly less than the control value (P < 0.01 by Dunnett's test). For definition of abbreviations, see figure 1.
885
CONTROL OF LUNG SURFACTANT IN RABBIT
4
2 (U-46619) did not have a significant effect on alveolar phospholipid content (2.11 ± 0.62 mg of phospholipid per g, mean ± SD; n 5, P 0.22 by unpaired "Student's" t test), but it produced a 33 per cent increase in pulmonary arterial blood pressure and a small increase (1.5 cm H 20) in the amplitude of transpulmonary pressure swings. Ventilation (VE, frequency, and tidal volume), Pao2, Paco2' and arterial pH remained within normal limits in these 3 series of experiments.
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Fig. 3. Effect of beta-adrenergic stimulation during normal ventilation. In rabbits treated with 150 /oIg of terbutaline per kg of body weight per min, phospholipid content was significantly greater than the control value (p < 0.01 by Dunnett's test). Betaadrenergic blockade induced by administration of 1 mg of propranolol per kg of body weight prevented the increase in phospholipid expected with administration of terbutaline (p < 0.05 by Dunnett's test). In rabbits treated with 0.5 /oIg of isoproterenol per kg of body weight per min, phospholipid content was not significantly different from that in the control group (Dunnett's test). For definition of abbreviations, see figure 1.
phospholipid content, but the difference between these 2 effects was not significant (figure 5). Prostaglandin El and prostaglandin E2 each decreased pulmonary arterial mean pressure by an average of 13 and 20 per cent, respectively, and prostaglandin F24 increased this pressure by 13 per cent. Neither prostaglandin El nor prostaglandin E2 changed the pattern of breathing for 2 hours, but prostaglandin F 24 significantly increased both the respiratory rate and VE (P < 0.05 by paired "Student's" t test) (table 3).
samples of pulmonary lavage fluid, selected randomly from 6 different groups. Synthetic carbon14 dipalmitoylphosphatidylcholine was used as an internal standard in all determinations, and its average recovery was 85.6 ± 7.5· per cent (mean ± SD). There was no significant difference in recoveries between the control and experimental series. DSPC accounted for 55.1 ± 11.5 per cent (mean ± SD) of the total phospholipid in the lavage fluid. There was no significant difference in the ratio of DSPC to total phospholipid between the control and experimental series (table 4). Discussion
Our first concern in assessing the significance of our results was the reliability of the methods. In these series of experiments we used the phospholipid content in the pulmonary lavage
mg PL g lung
15
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886
OYARZUN AND CLEMENTS
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fluid as an index of pulmonary surfactant. We used this index because the phospholipid content in lavage fluid correlates well with surface ac· tivity (36, 37) and with disaturated phosphatidylcholine content, which accounts for 55.1 per cent of the total phospholipid content in the lavage flui~ of rabbits during different experimental conditions (table 4). This result very closely agreed with the DSPC reported in our previous study (17). In purified lung surfactant this ratio was 57 per cent (36). The mean value for the phospholipid pool size in this study was larger than that previously reported (17). Because the variability within the control group was sufficiently large (SD 25 per cent) that all the previous individual con· trol values fell within the range defined by the m!w mean, 2.5 mg of phospholipid per g, ± 2 SD; and because the percentage of lavage fluid recovered was the same as in the previous study, we believe that the difference was due to the in· dividual variation in surfactant pool size. Large individual variations have been found not only by phospholipid analysis, but also by radioimmunoassay of pulmonary surface active material (37). Although alveolar surfactant is undoubtedly the main contributor of phospholipid to the lung lavage fluid, there may be other sources, such as cellular debris, plasma lipids, alveolar macrophages, and mucus. We believe that our criteria for acceptance of the data permitted us to exclude these sources as significant contribu· tors to the effects on phospholipid content in
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CONTROL OF LUNG SURFACTANT IN RABBIT
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