Surfactant as an Airway Smooth Muscle Relaxant - ATS Journals

Surfactant as an Airway Smooth Muscle Relaxant Rommy Koetzler, Mahmoud Saifeddine, Zhenguo Yu, F. Samuel Schu ¨ rch, Morley D. Hollenberg, and Francis H. Y. Green Department of Pathology and Laboratory Medicine, Department of Pharmacology & Therapeutics and Department of Medicine, and Department of Physiology and Biophysics, University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada

A variety of clinical and experimental evidence indicates that surfactant may be important in the pathogenesis and treatment of asthma. The purpose of this study was to determine the pharmacologic effect of pulmonary surfactant and its major lipid and protein constituents on bronchial smooth muscle. First-generation bronchi from male Sprague-Dawley rats were contracted with methacholine and exposed to two kinds of surfactant: whole rat surfactant and two bovine surfactant extracts in clinical use. The latter lack the hydrophilic surfactant-associated proteins (SP)-A and SP-D. All the surfactants relaxed the rat bronchi in a concentration-dependent manner; however, whole rat surfactant was more potent than the bovine extracts. Both surfactant lipids and SP-A contributed to the bronchial relaxation. The relaxation response produced by the highest concentration (0.5 mg/ml) of whole rat surfactant was equivalent to that caused by substance P (5 ␮M) and approximately half of that caused by 1 ␮M isoproterenol. The relaxation response was epithelium-dependent and blocked by indomethacin but not by N-␻-nitro-L-arginine methyl ester. We conclude that surfactant can relax airway smooth muscle directly via a prostanoid-mediated, epithelium-dependent process that does not involve nitric oxide synthase. Keywords: asthma; nitric oxide; prostaglandins; smooth muscle; surfactant

Pulmonary surfactant is a complex mixture of phospholipids, neutral lipids, and specific proteins. Phospholipids account for ⵑ 80% by mass of surfactant. The most abundant of these is phosphatidylcholine (PC), mainly in the disaturated dipalmitoylphosphatidylcholine (DPPC) form. Other phospholipids include phosphatidylglycerol (PG), phosphatidylethanolamine (PEA), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and lysophosphatidylcholine (LPC) (1, 2). In most mammals cholesterol comprises 8–10% by weight of whole surfactant (3); the four surfactant-specific proteins (SP), SP-A, -B, -C, and -D, make up the balance (1, 4). The most important role of surfactant is considered to be stabilization of alveolar walls and prevention of alveolar collapse at low lung volumes (1). Surfactant also lines the narrow conducting airways of the tracheobronchial tree, where it forms a continuous film with an estimated surface tension of 15 mJ/m2 in the intrapulmonary airways (5). Here it helps preserve bronchiolar patency during normal and forced respiration (1). It has a similar composition to alveolar surfactant, from which it is largely derived (2).

(Received in original form June 23, 2005 and in final form December 9, 2005) This work was supported in part by the Alberta Lung Association and the Canadian Institutes for Health Research. Correspondence and requests for reprints should be addressed to Francis H. Y. Green, M.D., Professor, Department of Pathology & Laboratory Medicine, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, AB T2N 4N1 Canada. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 34. pp 609–615, 2006 Originally Published in Press as DOI: 10.1165/rcmb.2005-0228OC on January 13, 2006 Internet address: www.atsjournals.org

In addition to maintaining small airway patency, surfactant lipids and proteins play important roles in physiologic homeostasis and innate immunity. Several of these are likely to be beneficial in asthma, including scavenging oxygen radicals, an ability to limit water loss from the airway surface (thus maintaining a constant periciliary fluid environment), enhancement of mucociliary clearance, and downregulation of inflammation (6–8). There is considerable evidence that surfactant function is impaired in asthma (1, 7, 9). Alterations in phospholipid composition (10) and decreased synthesis of the hydrophobic surfactant protein SP-B (11) are associated with declines in pulmonary function. Surfactant pool size is decreased, and the surface activity of surfactant recovered from lungs from a guinea pig model of asthma is impaired (12). Furthermore, the biophysical and anti-inflammatory properties of surfactant are inhibited by several factors released during acute asthma, such as oxygen free radicals and plasma proteins (13), inflammatory mediators such as eosinophil cationic protein (14), and LPC (15). The addition of SP-A and DPPC can reverse some of these effects (15). Surfactant treatment has been successfully used to relieve allergen-induced bronchoconstriction in animal models (16), but has achieved mixed success in treating human asthma (17–19). One of these studies using natural porcine surfactant in human subjects with asthma showed surprisingly that surfactant pretreatment augmented eosinophilic inflammation after allergen challenge (19). These anomalous results might be due to facilitation of allergen presentation to lung epithelial cells by exogenous surfactant (8). The studies described above reveal a broad range of potentially beneficial effects of surfactant in asthma as well as some potentially harmful ones. A mechanism of surfactant action that has not been studied is a direct pharmacologic effect on bronchial smooth muscle. The objective of this study was to investigate the effect of surfactant on precontracted rat airway smooth muscle. We report that natural rat surfactant, two commercially available bovine surfactants, and some individual components of surfactant have potent smooth muscle relaxant properties in vitro, and that these effects require an intact epithelium and are mediated by cyclooxygenase-dependent pathways.

MATERIALS AND METHODS Materials Methacholine, substance P, isoproterenol hydrochloride, indomethacin, N-␻-nitro-L-arginine methyl ester (L-NAME) hydrochloride, sodium nitroprusside dehydrate, dimethyl sulfoxide (DMSO), cholesterol, unsaturated PC (uPC), DPPC, PG, LPC, and human albumin were purchased from Sigma-Aldrich (Oakville, ON, Canada). L-arginine free base was obtained from Calbiochem (La Jolla, CA). Perfluorodecalin was obtained from F2 Chemicals Ltd (Preston, Lancashire, UK). Native human SP-A was obtained through the courtesy of Dr. Nades Palaniyar (Oxford, UK). Lipopolysaccharide (LPS) was obtained courtesy of Dr. Margaret Kelly and polymyxin B courtesy of Dr. Brent Winston (both at the University of Calgary, Calgary, Alberta, Canada). A suspension of LPC was prepared by adding 100 ␮l of DMSO and 900 ␮l of distilled water to 5 mg of LPC (L-␣-lysophosphatidylcholine from bovine brain). The concentration of the suspension was 5 mg/ml.

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For the LPC experiments, DMSO was used as vehicle control. The final concentration of DMSO in the organ bath was ⭐ 0.1% (vol/vol). Cholesterol was dissolved in a 1:1:0.9 ratio by volume of chloroform, methanol, and distilled water, respectively. The fluids were vortexed and the top phase of water and methanol was discarded. The bottom phase of chloroform and cholesterol was then transferred into a glass tube and dried under N2. The cholesterol was then resuspended in 0.9% saline. The concentration of the solution was 2.7 mg/ml. PG (in chloroform-methanol 98:2) was dried under N2 and resuspended in HEPES. The concentration of the solution was 30 mg/ml. A solution of 1:1 ratio of methanol and chloroform by volume was made and DPPC or uPC was added. The solution was dried under N2. DPPC or uPC was resuspended in HEPES. The concentration of the solutions was 30 mg/ml. DPPC was also added to a solution of PG (in chloroform-methanol 98:2) and the mixture was dried under N2. The DPPC:PG (7:3) was resuspended in HEPES. The concentration of the solution was 21 mg/ml of DPPC and 9 mg/ml of PG. Albumin was dissolved in distilled water for a concentration of 30 mg/ml. SP-A was dissolved in 2 mM EDTA ⫹ 20 mM Tris (pH 7.4).

Extraction and Purification of Natural Rat Surfactant For lung lavage, male and female Sprague-Dawley rats (ⵑ 250–350 g) were anaesthetized with halothane and killed by exsanguination. The lungs were lavaged with ⵑ 30 ml/kg of HEPES buffer three times. After removal of cells, surfactant was extracted from the primary supernatant by centrifugation into two fractions: a heavier, large aggregate fraction (pellet) and a lighter, small aggregate fraction (supernatant) (20). The small aggregate fraction (supernatant) was saved and used for control studies. The large aggregate pellets were resuspended in HEPES buffer and phospholipid analysis was performed based on a modified version of Bartlett’s method (21).

Bovine Surfactant Bovine lipid extract surfactant (BLES) was obtained from BLES Biochemicals Inc. (London, ON, Canada). Survanta was obtained from Abbot Laboratories Ltd (Saint-Laurent, PQ, Canada). BLES was supplied in 0.6% NaCl and 1.5 mM CaCl2 with a phospholipid concentration of 27 mg/ml. Survanta was supplied in 0.9% NaCl with a phospholipid concentration of 25 mg/ml.

Preparation of Rat Bronchial Tissues for Bioassay Male Sprague-Dawley rats (ⵑ 250–350 g) were cared for in accordance with recommendations of the Canadian Council on Animal Care and killed by decapitation. The lungs were removed and bronchial rings (2 mm ⫻ 3 mm) were dissected free from surrounding tissue. Left and right first generation bronchial rings were mounted in plastic organ baths containing 4 ml Krebs-Henseleit buffer (pH 7.4), and gassed (95% O2 and 5% CO2) at 37⬚C. Four tissues from two animals were mounted for each experiment. Tissues were allowed to equilibrate for 60 min before being exposed to agonists. Changes in isometric tension were measured with a Statham force-displacement transducer (Gould Statham Instruments Inc., Cleveland, OH) (22).

Measurement of the Relaxation Response to Natural Rat Surfactant and Bovine Surfactant In a typical experiment, after 60 min of equilibration at ⵑ 0.5 g of resting tension, tissues were tested for responsiveness by exposure to KCl (50 mM) followed by tissue wash. A period of 20–30 min between the addition of agonists was allowed for the tissues to recover. Tissues were precontracted with methacholine (1 ␮M) and substance P (5 ␮M) added to the bath to monitor presence of viable epithelium. Bronchial rings were then precontracted with methacholine (1 ␮M) and after ⵑ 5 min (when stable contraction was reached), single increasing concentrations of natural rat surfactant or bovine surfactant (0.000115–1 mg/ml) diluted in HEPES or saline were added to the organ bath. The maximal relaxation response was measured over 5 min. This was done because the response to surfactant was found to be gradual. Controls were done on precontracted tissues using either HEPES, the small aggregate fraction in the bronchoalveolar lavage fluid supernatant, or saline at the same volumes as used for the natural rat surfactant or bovine surfactant. Isoproterenol (1 ␮M) was used as a positive control.

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When surfactant alone was added to the tissue bath containing Krebs-Henseleit buffer aerated with 95% O2 and 5% CO2, stable bubbles were formed that affected the isometric force. To avoid this problem, perfluorodecalin (ⵑ 1 ml), a fluorocarbon liquid with a higher density and O2–CO2 solubility than water, was added to the bottom of the bath. The oxygen was bubbled through the perfluorodecalin, which coated it, thus reducing the interfacial tension between the gas bubbles and buffer and reducing foam formation. As LPS is a known inducer of airway hyperresponsiveness (23) and a potential contaminant of the surfactant preparations, we examined the relaxant response to BLES (1 mg/ml) in the presence of the LPS antagonist polymyxin B (10 and 20 ng/ml). We also examined the BLES response in the presence of added LPS (1 and 10 ␮g/ml) and the effects of LPS alone on baseline tension and on methacholine precontracted bronchi at concentrations of 1 and 10 ␮g/ml.

Surfactant Lipids and Proteins To determine the constituent(s) of surfactant that may be responsible for the relaxant effect, bronchial tissues were precontracted with methacholine (1 ␮M) as described above and exposed in separate experiments to uPC (0.2 mg/ml), DPPC (0.5 mg/ml), PG (0.15 mg/ml), DPPC:PG (0.65 mg/ml), cholesterol (0.1 mg/ml), LPC (0.005 mg/ml), SP-A (0.01 mg/ml), and albumin (0.1 mg/ml). The doses of the surfactant components were based on their estimated concentration in a 1 mg/ml sample of whole surfactant.

Mechanism of Natural Rat Surfactant– and Bovine Surfactant–Induced Relaxation Experiments with and without epithelium. In a typical experiment, substance P (5 ␮M) was added to the bath to monitor presence of viable epithelium (presence of relaxation on a precontracted tissue). Rings were then precontracted with methacholine (1 ␮M) and exposed to a single concentration of natural rat surfactant (0.06 mg/ml) or bovine surfactant (BLES, 1 mg/ml). Tissues were taken out of the bath, their epithelium removed by rubbing, and the tissues were remounted in the bath and allowed to equilibrate. They were precontracted again with methacholine (1 ␮M) and exposed to natural rat surfactant (0.06 mg/ml) or BLES (1 mg/ml). The absence of an intact epithelium in the preparation was ascertained by showing an absent relaxant response to substance P (5 ␮M) and by histology using formaldehyde fixed tissues obtained at the conclusion of the experiment. Repeated exposure of precontracted tissues to natural rat surfactant (0.06 mg/ml) or BLES (1 mg/ml) was used to assess reproducibility. Experiments with indomethacin. Bronchial tissues were precontracted with methacholine (1 ␮M) and exposed to a single concentration of natural rat surfactant (0.06 mg/ml) or BLES (1 mg/ml). The tissues were washed three times and indomethacin (10 ␮M) added to the bath at baseline. After 20–30 min of incubation with indomethacin, the rings were again precontracted with methacholine (1 ␮M) and exposed to natural rat surfactant (0.06 mg/ml) or BLES (1 mg/ml). The activity of indomethacin was validated by demonstrating its ability to abrogate relaxation in response to 5 ␮M substance P. Isoproterenol (1 ␮M) was also added to the indomethacin-treated precontracted rings to validate tissue responsiveness. Experiments with L-NAME. After exposing the precontracted tissues to natural rat surfactant (0.06 mg/ml) or BLES (1 mg/ml), tissues were washed and L-NAME (100 ␮M) was added at baseline tension. After 20–30 min, the rings were precontracted with methacholine (1 ␮M) and exposed to natural rat surfactant (0.06 mg/ml) or BLES (1 mg/ml). A relaxation response on adding L-arginine served as a pharmacologic index of functional inducible nitric oxide synthase induction (24).

Statistical Analysis Paired t test was used for a comparison of “before and after” experiments. If the normality assumption failed, signed rank test was performed. The effects of both the type and the concentration of surfactant on the degree of tissue relaxation were compared using the two-way ANOVA. Student-Newman-Keuls method was used for post hoc comparisons. Results are given as mean ⫾ SEM. A P value ⬍ 0.05 was considered to indicate a significant difference. SigmaStat, version 2.03 (SPSS Inc., Chicago, IL), was used for the statistical analysis.

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RESULTS

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The relaxant responses to either natural rat surfactant or BLES were reproducible when the following cycle of tissue exposure

was repeated at least three times: contraction with methacholine, addition of the surfactants to the organ bath to monitor the relaxant response, and washing the tissue free of surfactants and other reagents with buffer to re-equilibrate the tissue at baseline tension before the next exposure to methacholine. Thus, the responses of the tissues to surfactant before and after treatment of the tissues by inhibitors could be compared and the effects of any putative inhibitor could be readily interpreted. The relaxation response caused by either natural rat surfactant or BLES was dependent on the presence of an intact epithelium (Figure 3), with a significant inhibition of the response for both surfactants observed in epithelium-denuded preparations (P ⬍ 0.001 for natural rat surfactant and P ⫽ 0.004 for BLES). Further, the relaxant response to both surfactants was inhibited by pretreatment of the tissues with the cyclooxygenase inhibitor, indomethacin (Figure 4). Representative tracings of the response kinetics to the surfactants and other agonists in the bioassay experiments as well as the effects of removing the epithelium are shown in Figure 5. In contrast with the surfactant-mediated effects, relaxation due to the ␤-adrenergic agonist, isoproterenol, known to affect the bronchial smooth muscle directly, rather than by acting via the epithelium, was maintained either in the presence or absence of an intact epithelium (Figure 3). Only a small reduction in relaxation was observed upon denuding the tissue of the epithelium. As expected, indomethacin had no effect on relaxation caused by isoproterenol (Figure 4). The actions of the surfactants mirrored the well-known relaxant actions of substance P, which were absent (P ⬍ 0.001) in tissues denuded of an intact epithelium (Figure 3) and were inhibited by indomethacin (P ⬍ 0.001) (Figure 4). In essence, a tissue’s relaxant response or lack thereof upon exposure to substance P served as an index of the presence (relaxation) or absence (lack of substance P–mediated relaxation) of an intact epithelium in preparations used to test the actions of the surfactants. The nitric oxide synthase inhibitor, L-NAME, had no effect on the natural rat surfactant–induced relaxation (19 ⫾ 2% and

Figure 1. Concentration–response curves for natural rat surfactant (NRS), Survanta, and BLES. NRS was a more potent smooth muscle relaxant than either Survanta or BLES. Each concentration was tested on tissues from five or more animals; values are mean ⫾ SEM (n ⫽ 6–10). Two-way ANOVA indicated that surfactant-induced relaxation varied with concentration (P ⬍ 0.001) and surfactant type (P ⬍ 0.001). Student-Newman-Keuls test demonstrated that NRS-induced relaxation was greater than BLES-/Survanta-induced relaxation (P ⬍ 0.001). Note: (a ) ⫽ expressed as % of maximal contractional force (mN) induced by 1 ␮M methacholine.

Figure 2. Relaxant effect of the components of surfactant on rat bronchial tissues. The relaxant effect of uPC (0.2 mg/ml), DPPC (0.5 mg/ml), PG (0.15 mg/ml), DPPC:PG (0.65 mg/ml), cholesterol (chol., 0.1 mg/ml), LPC (0.005 mg/ml), SP-A (0.01 mg/ml), and albumin (alb., 0.1 mg/ml) was evaluated. The concentrations chosen were related to their concentrations in a sample of surfactant containing 1 mg/ml. Asterisks indicate significant difference (P ⬍ 0.05) from their respective vehicle controls. Experiments were done on tissues from five or more animals; values are mean ⫾ SEM (n ⫽ 6–17). Note: (a ) ⫽ expressed as % of maximal contractional force (mN) induced by 1 ␮M methacholine. NS ⫽ not significantly different from respective controls.

Concentration–Response Profiles for Three Different Surfactant Preparations

Natural rat surfactant and bovine surfactants (BLES and Survanta) all caused a concentration-dependent relaxation of precontracted bronchial tissues (Figure 1). The concentration– response relationship was approximately log-linear for all three surfactants. At all concentrations of surfactants tested, the relaxation response for natural rat surfactant was ⵑ 2-fold greater than was observed for either BLES or Survanta. Responses in the left and right bronchi from the same rat were not significantly different (data not shown). The relaxation response induced by the buffer used to dissolve the reagents (HEPES), the bronchoalveolar lavage fluid supernatant from which the surfactant was isolated, or from isotonic saline used as a diluent for some compounds was not different from zero. Perfluorodecalin, added to the organ bath to reduce foaming of the gassed solutions, had no measurable effect on the bioassay system. Preincubation with the LPS antagonist polymyxin B did not affect the relaxation response to BLES, nor did addition of LPS at 1 and 10 ng/ml with and without BLES affect either the baseline tension or relaxation response. Response Profiles for Surfactant Lipids and Surfactant Proteins

uPC, DPPC, PG, DPPC:PG, and SP-A all caused relaxation of precontracted bronchial tissues. The response to SP-A was greater than the response to any of the lipids. By contrast cholesterol, LPC and albumin did not relax precontracted rat bronchial tissue (Figure 2). Albumin alone had no relaxant effect on the precontracted tissue; however, when albumin was combined with BLES the relaxant effect of the BLES was slightly augmented (data not shown). Mechanism of Surfactant-Induced Relaxation

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Figure 3. Relaxant effect of substance P, NRS, BLES, and isoproterenol in rat bronchial tissues with [(⫹)EP] and without epithelium [(⫺)EP] precontracted with methacholine. A, B, and C are related to the experiments using NRS, and D, E, and F with the experiments using BLES. A, B, D, and E show that substance P–, NRS-, and BLES-induced relaxation is inhibited in (⫺)EP. In C and F, isoproterenol relaxation response is present in both (⫹)EP and (⫺)EP. Experiments were done on tissues from four or more animals; values are mean ⫾ SEM (n ⫽ 4–9). Note: (a ) ⫽ expressed as % of maximal contractional force (mN) induced by 1 ␮M methacholine.

18 ⫾ 3% for untreated tissues and tissues treated with L-NAME, respectively; for n ⫽ 7 tissues from 7 animals; P ⫽ 0.771) or on the BLES-induced relaxation (18 ⫾ 3% and 18 ⫾ 4% for untreated tissues and tissues treated with L-NAME, respectively; for n ⫽ 6 tissues from 6 animals; P ⫽ 0.81). Positive controls using L-NAME were done to prove its nitric oxide synthase–inhibitory action in the preparation. As has been previously observed for other smooth muscle preparations (24), the addition of L-arginine to a precontracted tissue can cause tissue relaxation due to its enzymatic conversion to nitric oxide, a process that can be blocked by L-NAME. We confirmed that the precontracted bronchial preparation also displayed a reproducible relaxation upon adding L-arginine to the organ bath (data not shown). L-NAME was able to inhibit the L-arginine–induced relaxation of the bronchial preparations (21 ⫾ 3% and 9 ⫾ 3% relaxation for untreated tissues and tissues treated with L-NAME, respectively; for n ⫽ 9 tissues from 6 animals; P ⬍ 0.001). Thus, if the relaxant effects of the surfactants had been due to a release of nitric oxide, L-NAME would have attenuated the response.

DISCUSSION The main finding of our study was that several surfactant preparations and components of these preparations, not previously known to affect smooth muscle tissue function directly, were able to cause an epithelium-dependent relaxation of bronchial tissue by a cyclooxygenase (presumably prostanoid)-mediated nitric oxide–independent mechanism. In this respect, the actions of the surfactants mirrored the actions of substance P, which are also epithelium-dependent and prostanoid-mediated. These observations apply to normal rats. Additional studies would be required to determine if the relaxant response to sur-

factant is seen in atopic animals or in humans with asthma. The rats used in this study were not atopic and had no evidence of airway inflammation on histology. We also showed that these effects were not influenced by endotoxins, to which the rats would likely have been exposed to in low concentration and which are known to augment bronchial smooth muscle contractile responses in allergic airway inflammation (23). Our data provide further insights into the mechanism(s) whereby inhalation of surfactant relieves bronchoconstriction in humans with asthma (17) and in allergen-exposed animals (16). In addition to the anti-inflammatory and biophysical properties of surfactant that are beneficial in asthma, we demonstrate a pharmacologic role for surfactant in moderating airway tone. The response to methacholine in airway smooth muscle is mediated by both a direct effect on the muscle and by reflex neural mechanisms (25). Aerosolized surfactant inhibits the bronchoconstrictor response to inhaled (but not systemically given) acetylcholine in rats (26) and guinea pigs (15), possibly by binding to the epithelium and masking the activity of bronchial irritant receptors (27). These studies point to an epithelial– surfactant interaction that modulates airway hyperresponsiveness through locally acting afferent nerves. The relaxant effect of natural rat surfactant showed a loglinear concentration–response relationship that was moderately potent, producing more than 30% relaxation of precontracted bronchial smooth muscle at the highest concentration (0.5 mg/ml). This compared with a relaxation response of 50–60% produced by 1 ␮M isoproterenol. Natural rat surfactant was approximately twice as potent as the bovine-derived surfactants (Figure 1). The difference in potency may be related to the presence of surfactant proteins SP-A and SP-D in the natural rat surfactant (but not

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Figure 4. Effect of indomethacin (10 ␮M) on substance P–, NRS-, BLES-, and isoproterenol-induced relaxation in rat bronchial tissues with intact epithelium precontracted with methacholine. A, B, and C are related to the experiments using NRS, and D, E, and F with the experiments using BLES. A, B, D, and E show the inhibition of substance P–, NRS-, and BLES-induced relaxation by indomethacin. In C and F, isoproterenol relaxation response is present in both (⫺) and (⫹) indomethacin. Experiments were done on tissues from four or more animals; values are mean ⫾ SEM (n ⫽ 4–11). Note: (a ) ⫽ expressed as % of maximal contractional force (mN) induced by 1 ␮M methacholine.

in BLES and Survanta), a species effect (the tissues were derived from rat not bovine sources), or a combination of factors. The potent relaxant effect of SP-A alone (Figure 2) would indicate that SP-A is important in this regard. The range of concentrations of the natural rat surfactant (0.006–0.5 mg/ml) that produced an effect in our in vitro system were similar to the concentrations (0.3–2 mg/ml) used in the bubble surfactometer to produce optimal surface tension changes (2, 13) and considerably less than recent studies showing that optimal surface properties require concentrations of phospholipids as high as 27 mg/ml (28). The advantage of using natural rat surfactant in this study was that all surfactant components were present and the surfactant was derived from the same species. Bovine surfactants, on the other hand, are more clinically relevant and are used in the management of neonatal respiratory distress syndrome (29). Despite their origin from a different species and lacking SP-A and SP-D, they nonetheless showed significant bronchial relaxant effects. BLES is isolated by organic solvent extraction of bovine lungs. The resulting surfactant preparation is composed of all the phospholipids of natural surfactant and the two small hydrophobic proteins, SP-B and SP-C (30, 31), but lacks SP-A and SP-D. Survanta is a modified natural surfactant also made from the organic solvent extracts of bovine lungs. It contains phospholipids, neutral lipids, fatty acids, and SP-B and SP-C, to which colfosceril palmitate, palmitic acid, and tripalmitin are added (30). It also lacks SP-A and SP-D. The relaxant effect of surfactant was mediated by some, but not all, of its constituent lipids, and by the hydrophilic surfactant protein SP-A. It required the presence of an intact epithelium. The airway epithelium expresses specific receptors that mediate the relaxation responses induced by substance P, ATP, and prostaglandin E2 (32). Epithelial cell membranes are rich in phosphatidate phosphohydrolases, such as PAP-2 (33), which are capable of hydrolyzing DPPC and PG to diacylglycerol. Diacylglycerol

is an important intracellular signaling molecule that activates protein kinase C (33). Protein kinases have many intracellular functions, including regulation of prostaglandin synthesis (34) and smooth muscle function (35). The recent demonstration that a synthetic surfactant containing DPPC and egg PG formulated as a dry powder could abolish the early asthmatic response to an inhaled allergen (17) is important, as these two surfactant lipids produced significant bronchial relaxation in this study (Figure 2). The preparation used in their study did not contain surfactant proteins; thus, surfactant proteins were not directly involved in the protective effect on FEV1 during the early phase response. However, it is possible that inhalation of surfactant lipids may have triggered the release of endogenous surfactant proteins widely expressed in bronchial epithelial cells (36). All of the phospholipids tested in this study, with the exception of LPC, showed bronchial relaxant effects. PG was the surfactant phospholipid with the greatest effect. Cholesterol, a normal lipid constituent of surfactant, had no effect on the rat bronchial tissues. LPC is present in small quantities in normal surfactant (1, 2). LPC is catabolized from PC by phospholipase A2 and is formed in excess during asthmatic inflammation (37). At high concentrations (⬎ 5 mg/ml), but not at lower concentrations, inhaled LPC has been shown to enhance bronchoconstriction in guinea pigs (15). In our study, using a low concentration of LPC (0.005 mg/ml), we observed no effect of LPC on the smooth muscle tension after methacholine challenge. Thus, under the conditions of our experiments, LPC did not appear to be playing a role. The more potent relaxant effect of natural rat surfactant compared with the commercially available surfactants, BLES and Survanta, indicated that the water-soluble surfactant proteins SP-A and SP-D may be playing a role. When we examined the individual components we found SP-A to be more potent than the surfactant lipids in causing bronchial relaxation (Figure 2).

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Figure 5. Representative responses of rat bronchial preparation (with and without epithelium) to NRS, BLES, and substance P. Tissues before rubbing the epithelium [(⫹) EP] and after rubbing [(⫺) EP] were precontracted with methacholine (MCh), 1 ␮M (triangles) and exposed to NRS, 0.06 mg/ml (circles) (tracings A); BLES, 1 mg/ml (squares) (tracings B), and substance P (SUB. P), 5 ␮M (filled diamonds) (tracings C). Isoproterenol (ISOP.), 1 ␮M (open diamonds) was used as a positive control. NRS, BLES, and substance P did not show a relaxant effect in the absence of epithelium.

We therefore compared the relaxant response of SP-A to albumin, which is also present in the alveolar lining fluid (1) and is considered an inhibitor of surfactant activity (38). Albumin had no independent relaxant effect on rat bronchial tissue (Figure 2); however, when combined with BLES, it enhanced the relaxant response. Wright (6) and Tino and Wright (39) have demonstrated specific binding of SP-A to alveolar type II cells. Binding of labeled SP-A was reduced by the presence of excess unlabeled SP-A and by heat treatment of the protein. Trypsin treatment of the type II cell surface reduced binding. The results suggest that type II cells have high-affinity binding sites for SP-A. It is possible that other respiratory epithelial cells have SP-A–binding sites; however, this remains to be established. Decreased levels of SP-A and SP-D have been reported in a murine model of asthma after allergen challenge (40), and SP-A is decreased in bronchoalveolar lavage fluid from patients with asthma (41). SP-A– and SP-D–deficient mice show in-

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creased inflammatory responses to infection (42), findings that illustrate the anti-inflammatory nature of these proteins. This is related in part to high-affinity binding of SP-A and SP-D for LPS (42). Indomethacin had a significant inhibitory effect on the substance P– and surfactant-induced relaxation in tissues precontracted with methacholine. This suggests that the surfactantinduced relaxation was dependent on prostanoid synthesis (32). Phospholipases are abundant on the luminal aspect of the airway epithelial surface and play important roles in lipid signaling. Phospholipase A2 hydrolyzes the sn-2 ester bond of cellular phospholipids, producing a free fatty acid and a lysophospholipid, both of which are lipid signaling molecules. The free fatty acid produced is frequently arachidonic acid, the precursor of the eicosanoid family of potent inflammatory mediators that includes prostaglandins, thromboxanes, leukotrienes, and lipoxins (43). Prostaglandin E2 is a potent smooth muscle relaxant and important in asthma (32). Phospholipase D is also an important effector enzyme in receptor-mediated signaling pathways in the lung. It catalyses the hydrolysis of the most abundant membrane phospholipid phosphatidylcholine (also abundant in surfactant), and generates choline and phosphatidic acid. Phosphatidic acid and its metabolites, lysophosphatidic acid and diacylglycerol, act as second messengers for a variety of cellular processes including prostaglandin synthesis (44). Nitric oxide, a known smooth muscle relaxant, did not seem to be involved, since the inhibitor of nitric oxide synthase, L-NAME, had no effect on the surfactantinduced relaxation. Our study is the first to show that surfactant is an airway smooth muscle relaxant in vitro. Previous studies done in vivo, while showing a bronchodilator effect in animal models of allergeninduced airway bronchoconstriction (16, 45), improvement in airflow in patients with asthma (17, 18), or attenuation of response to inhaled methacholine (26), were not able to distinguish between the biophysical properties of surfactant on airway opening pressure, its anti-inflammatory properties, or the pharmacologic effect that is shown in these studies. In summary, this study demonstrates a new role for surfactant. Both natural and bovine-derived surfactants used in clinical practice showed a relaxant effect on precontracted rat bronchial tissue. The surfactant-induced relaxation required both surfactantassociated lipids and proteins and was dependent on an intact epithelium and on the release of prostanoids, but not on the nitric oxide pathway. The findings in this study indicate a potential pharmacologic role for surfactant in the treatment of asthma. Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgments : The authors thank BLES Chemicals for providing the BLES and Dr. Nades Palaniyar for providing purified SP-A. They thank Artee Karkhanis and the late Stanley Cheng for technical support. They also thank Dr. Fred Possmayer for many helpful suggestions in writing the manuscript, and Hannah Park and Dr. Tamer El-Mays for manuscript preparation.

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