Inhibition of pulmonary surfactant function by phospholipases.

Inhibition of pulmonary by phospholipases BRUCE A. HOLM, LISA KEICHER, AND GORAN ENHORNING

surfactant function

MINGYAO

LIU,

JUNE

Departments of Gynecology/Obstetrics, Pediatrics, and Pharmacology State University of New York, Buffalo, New York 14222

SOKOLOWSKI, and Therapeutics,

rate of 50-70% (10, 12, 21, 22). Much of the surfactant dysfunction in ARDS appears to be related to the inhibifuctunt function byphospholipases. J. Appl. Physiol. 7l( 1): 317- tion of surfactant activity by plasma-derived proteins (5, 321,1991.-Previous studieshave shownthat respiratory fail- 7-11, 13, 23, 25). However, there is also the possibility ure associated with disorders such as acute pancreatitis that proteases or phospholipases may detrimentally afcorrelates well with increased levels of phospholipase A, fect surfactant function in lung injury. (PLAs) in lung lavagesand that intratracheal administration of Previous studies have shown that the respiratory failPLA, generatesan acute lung injury. In addition, bacteria such ure associated with acute pancreatitis correlates well as Pseudomonashave been shown to secrete phospholipaseC (PC) levels and in(PLC). We studied the effects of these phospholipaseson pul- with decreased phosphatidylcholine A, (PLA,) activity in bronchoalmonary surfactant activity using a pulsating bubble surfacto- creased phosphalipase meter. Concentrations >O,l unit/ml PLA, destroyed veolar lavage (20, 27, 28). It is also known that a variety surfactant biophysical activity, increasing surface tension at of bacteria, including Pseudomonas, secrete phospholiminimum bubble size from 4 to 15 mN/m. This surfactant pase C (PLC) (24). inactivation was predominantly related to the effect of lysoThe studies presented here were designed to determine phosphatidylcholine on the surface film, although the fatty the in vitro effects of phospholipases on pulmonary suracidsreleasedwith higher PLA, concentrations alsohad a detrifactant biophysical activity. In addition, these studies mental effect on surfactant function. Similarly, as little as 0.1 also examine the effects of the phospholipase hydrolysis unit PLC increasedthe surface tension at minimal size of an oscillating bubble from 4 to 15 mNfm, an effect that could be products on surfactant function in the absence of enmimicked by the addition of dipalmitin to surfactant in the zyme, allowing for independent analyses of quantitative alterations. absenceof PLC. Moreover, lower, noninhibitory concentra- and functional phospholipid tions (0.01 unit/ml) of PLA, and PLC increasedthe sensitivity HOLM, BRUCE A., LISA KEICHER,MINGYAO LIU, JUNESOKOL~~SKI,ANDG~RANENH~RNING.~~~~~~~~~~O~~L~L~O~~~~ SUT-

of surfactant to other inhibitory agents,suchasalbumin. Thus, MATERIALS AND METHODS relatively low concentrations of PLC and PLA, can causesevere breakdown of surfactant function and may contribute sigPLA, (Naja naja venom, 1,000 U/mg protein), PLC nificantly to someforms of lung injury. (CZostridium perfringens, 15 Wmg protein), dipalmitin, phospholipase A,; surfactant inactivation; adult respiratory distresssyndrome

PULMONARY SURFACTANT is a complex mixture of phospholipids and specific apoproteins that acts to lower the surface tension of the alveolar liquid lining layer, thereby decreasing the work of breathing and promoting alveolar stability (1820). A quantitative surfactant deficiency in the underdeveloped lungs of the premature newborn is responsible for the neonatal respiratory distress syndrome (RDS) (1). RDS is a leading cause of infant morbidity and mortality that has been successfully treated with exogenous surfactant supplementation therapy (14). More recently, a variety of studies have shown that surfactant system dysfunction may also play a significant role in the development and progression of the adult respiratory distress syndrome (ARDS) (6, 8, 10, 21), an acute form of respiratory failure that results from a variety of etiologies and may carry an associated mortality

palmitic acid, lysophosphatidylcholine (LPC), and bovine serum albumin (fraction V; 96-99% albumin) were purchased from Sigma Chemical, St. Louis, MO. Natural lung surfactant (LS) was obtained by bronchoalveolar lavage of lungs from freshly killed calves as previously described (11, 18). Briefly, the lungs were lavaged three times with 1 liter of 0.15 M NaCl infused and withdrawn intermittently with a vacuum line. The lavage fluid was centrifuged at 200 g for 6 min to remove cells and cellular debris. The supernatant was then spun at 12,000 g to pellet phospholipids and associated proteins. This material was 86% phospholipid, 5% neutral lipid, and 9% protein. The phospholipid class distribution of this material determined by thin-layer chromatography was rich in PC and disaturated PC as typical for pulmonary surfactant (17). This material also contained all surfactant apoproteins. A calf lung surfactant extract (CLSE) was obtained from the LS material by extraction with chloroform and methanol as previously described (2) and was subsequently stored in chloroform at -2O’C. Before experiments, CLSE was dried from chloroform under nitrogen

0161-7567/91 $1.50 Copyright 0 1991 the American Physiological Society

317

Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on October 22, 2018. Copyright © 1991 American Physiological Society. All rights reserved.

318 n

SURFACTANT O-0

25

A-A

m-I +-+

CONTROL

401 UNITS PL42 .1 UNfTS PlA2 1 UNITPlA2

TIME (minutes) FIG. 1. Effects of phospholipase AZ (PLA,) on surfactant activity. Surface tension at minimum bubble radius is plotted as a function of time after bubble creation for calf lung surfactant extract (CLSE, 3 mg/ml) plus increasing concentrations of PLA,. Values are means -t SE; n = 5. All values for 0.1 and 1.0 unit PLA, are significantly different from control, P < 0.001.

and resuspended in 0.15 M NaCl by mechanical mixing (18). CLSE has a phospholipid composition identical to LS but contains only the low-molecular-weight hydrophobic surfactant proteins (29) Preliminary studies indicated that the effects of phospholipases on the LS and CLSE preparations were virtually identical. Therefore, the experiments presented here were performed with the CLSE mixture, which was prepared and stored under sterile conditions, thereby eliminating potential artifactual effects related to contaminating bacteria. In all studies, 30 mg/ml stock preparations of CLSE were diluted to 3 mg/ml working solutions with 5 mM tris(hydroxymethyl)aminomethane buffer, pH 7.4, containing 5 mM CaC1,. Previous studies have shown that 3 mg/ml CLSE rapidly lowers surface tension on an oscillating bubble surfactometer and is relatively resistant to inhibition by plasma proteins such as albumin. The pH and calcium concentrations were held constant based on previous studies showing that these conditions provided for optimum activity of the phospholipases (24, 27). Incubations with varying concentrations of PLA, or PLC were then carried out for 30 min at 37V. Composition analyses of surfactant-phospholipase mixtures were accomplished by thin-layer chromatography using the solvent system of Touchstone et al. (26) and the microphosphorus assay of Chen et al. (3). Surface tension measurements under dynamic film compression were carried out at 37OC and 100% humid(Electronetics, ity on a pulsating bubble surfactometer Amherst, NY) as described by Enhorning (4). An air bubble was formed in a 25~1 subphase containing the test and mixture and was then pulsed between maximum minimum areas of ’ 3.6 and 1.8 mm2 (50% area compression) at a rate of 20 cycles/min. The pressure difference across the air-liquid interface was monitored continuously, and surface tension values were calculated by the law or Young and Laplace for spherical surfaces AP = 27/R where AP is the pressure difference across the bubble surface, y is the surface tension, and R is the bubble radius. l

INHIBITION RESULTS

Figure 1 shows surface tension vs. time plots for mixtures of surfactant and PLA, analyzed with a pulsating bubble surfactometer. The graph depicts the surface tension (at minimum bubble size) of a 3-mg/ml CLSE solution plotted as function of time after expansion of the bubble and its pulsation in the presence of increasing amounts of PLA,. The data indicate that PLA, causes a dose-dependent decrease in pulmonary surfactant activity, with’observable effects occurring with as little as 0.01 unit/ml of PLA,. When the surfactant was incubated with 0.1 and 1 unit/ml PLA,, the resultant surface tensions at minimum bubble size of 12 and 17 mN/m, respectively, were clearly incompatible with a physiologically active surfactant. The data in Table 1 indicate that the primary effect of incubating surfactant with PLA, was to decrease the total PC content, with a concomitant increase in the LPC content. If the surfactant-inhibitory effects of PLA, were predominately related to the decreased concentration of the principal surface active component, dipalmitoylphosphatidylcholine (DPPC), one would expect that the effect of lower PLA, concentrations (0.1 unit/ml) would at most result in a prolongation in the time required to achieve a low surface tension on the bubble rather than the dramatic increase in surface tension that was observed in these studies. Therefore, further experiments were performed to determine whether the hydrolysis products generated by the phospholipase action were capable of inhibiting surfactant function in the absence of enzyme. Figure 2 shows the effects of LPC and palmitic acid (PA), the hydrolysis products of the PLA,-surfactant reaction, on the biophysical activity of 3 mg/ml surfactant in the absence of PLA,. These results are also compared with the effect of simply decreasing the surfactant concentration in the absence of phospholipases. The data indicate that the abnormal surface activity of surfactant preparations incubated with PLA, is at least in part related to the inhibitory effects of LPC on surfactant function. The results in Fig. 2 indicate that adding increasing concentrations of LPC to surfactant causes a progressive increase in minimum surface tension, which mimics the effects of equivalent LPC levels generated by the addition of PLA, in Fig. 1. Figure 2 also shows that, at low levels, PA has relatively little effect on surface tension at minimum bubble size, although an increase to 8 mN/m is observed for higher PA concentrations. TABLE 1. Percent distribution of surfuctunt phospholipids ufter phospholipase A, incubation Phospholipase Phospholipid

Component

Lysophosphatidylcholine Sphingomyelin Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol Others

A2 Concentration,

units/ml

0

0.01

0.1

1.0

10.0

4t1 421

13t3 4t2

33t5

54t,2

77t5

3t1

4tl

2tl

76t2

63,t4

45t3

24t4

8t3

2t1 4t1 5-tl

3tl 3&l 621

4tl 2tl 5tl

3tl 6-tl 7tl

3tl lOt2 6tl

Values are means t SE; n = 4.


SIJRFACTANT 0 DECREASED m INCREASED CSI INCREASED

319

INHIBITION

PC CONCENTRATKWJ LPC CONCENTRATION PA CONCENTRATION

0 @MXES

OF PC HYDROLYZED:

0.15

0.3

0.6

10%

20%

0

0.01

0.1

1.0

10

UNITS OF PHOSPHOUPASE C ADDED 5%

PERCENT OF PC HYDROLYZED:

FIG. 2. Effects of PLA, hydroIysis products on surfactant activity. Hydrolysis products caused by addition of PLAz to surfactant [lysophosphatidylcholine (LPC) and palmitic acid (PA)] were incubated with 3 mg/ml surfactant in the absence of PLA, before surface tension measurements. Effect of decreasing surfactant concentration (in absence of enzyme) on surface tension at minimum bubble size is also shown. Values are means t SE after 5 min of pulsation; n = 4. LPC effects from 10 and 20% hydrolysis are significantly different from control, P < 0.005.

Another important phenomenon observed in these studies was the potentiating effect of noninhibitory levels of PLA, on the sensitivity of surfactant to plasma protein-induced inactivation. As shown in Fig. 3, addition of 0.01 unit of PLA, or 5 mg/ml albumin to pulmonary surfactant at 3 mg/ml had minimal effects on the surface tension achieved when the bubble surface area was maximally compressed. However, when these low fevels of PLA, and albumin were added together, the surface tension attained by 3 mg/ml surfactant &d not fall below 10 mN/m. Studies were also performed to assess the effects of PLC on surfactant function. The data in Fig. 4A show that as little as 0.1 unit of this enzyme, which acts to cleave the polar head group from the diacylglycerol portion of the phospholipid, causes a dramatic increase in the minimum surface tension generated by 3 mg/ml puln

e-m A-A U---a 4-4

25

E

\ 2 E 5 3 z k

20

CONTF?OL 41 UNITS PLAZ 5 mgfmt ALBUMIN .Ol UNITS PlA2 + 5 mg/ml ALBUMIN

15 10

w 0 E QI 3 tA

5 0 0

i

j

TIME (minutes) 3. Effects of PLA, on surfactant sensitivity to inhibition by albumin. Surface tension at minimum bubble radius is plotted as a function of time for 3 mg/ml CLSE incubated with 0.01 unit PLA,, 5 mg/ml albumin, or both. Values are means t SE; n = 4. Values for PLA, plus albumin were significantly different from control, P < 0.005. FIG.

0

10

20

40

50

% DiPALMtTlN ADDED (by weight of DPPC) FIG. 4. Effects of phospholipase C (PLC) and its hydrolysis products on surfactant activity. A: surface tension at minimum bubble radius after 10 min of pulsation for 3 mg/ml CLSE incubated with increasing concentrations of PLC. Results represent means k SE; n = 4. Values for incubations with ~0.1 unit/ml PLC were significantly different from control, P < 0.05. B: surface tension at minimum bubble radius after 10 min of pulsation for 3 mg/ml CLSE incubated with increasing concentrations of PLC hydrolysis product dipalmitin in absence of enzyme. Values are means t SE; n = 4. Values for incubations with >20% dipalmitin were significantly different from control, P < 0.05.

monary surfactant. As noted for the PLA, results, this high surface tension cannot be explained by the decreased phospholipid concentration alone. Thin-layer chromatographic analysis of mixtures of surfactant with PLC showed that all surfactant phospholipids were affected by the enzyme (data not shown). However, because DPPC is the most abundant phospholipid in pulmonary surfactant, a much larger amount of DPPC was hydrolyzed at each PLC concentration. The addition of the PLC-surfactant hydrolysis product dipalmitin to 3 mg/ml surfactant caused a concentration-dependent increase in the surface tension achieved on a pulsating bubble surfactometer, as shown in Fig. 4B. Finally, further experiments indicated that low concentrations of PLC were also capable of potentiating the inhibitory effects of albumin on surfactant activity, similar to the results shown above for PLA,. These data are presented in Fig. 5.


320

SURFACTANT *-a

CONTROL

A-A D-D 4-4

,Ol UNITS PLC 5 mg/ml ALBUMIN .Ol UNffS PLC + 5 mg/mi ALBUMIN

6

TIME (minutes) 5. Effects of PLC on surfactant sensitivity to inhibition by albumin. Surface tension at minimum bubble radius is plotted as a function of time for 3 mg/ml CLSE incubated with 0.01 unit PLC, 5 mg/ml albumin, or both. Values are means -+ SE; n = 4. Values for PLC plus albumin were significantly different from control, P < 0.005. FIG.

DISCUSSION

A variety of recent studies has shown that surfactant abnormalities may contribute to the development and pathophysiology of ARDS (6,8). Although some ARDS lung injury models are characterized by decreased alveolar surfactant levels related to type II pneumocyte metabolic dysfunction (8), most of the surfactant abnormalities in ARDS have been linked to the plasma proteinderived inhibition of surfactant biophysical activity (8, 10,25). There have also been some reports of changes in surfactant phospholipid composition in experimental and clinical ARDS (6). Thin-layer chromatographic analyses of pulmonary surfactant recovered by lung lavage from animal models of shock lung (27) or acute pancreatitis (20) have demonstrated decreased levels of DPPC with corresponding increases in LPC. These results are consistent with the theory that PLG, may be hydrolyzing surfactant DPPC in some forms of lung injury. This possibility was supported by subsequent findings of 6- to 2O-fold increases in lavage PLA, levels in ARDS patients and animal models (27,28). Studies by Niewoehner et al. (16) have shown that intratracheal instillation of the PLA, hydrolysis product, LPC, induces acute pulmonary injury in hamsters. These authors and others note that LPC readily inserts into phospholipid bilayers and may cause cell membrane instability, resulting in increased alveolar permeability. However, it is also possible that phospholipase-induced surfactant dysfunction may have played a role in the development and pathophysiology of these lung injuries. The in vitro studies presented here clearly show that incubation of pulmonary surfactant with PLA, causes a concentration-dependent decrease in surfactani biophysical activity. Composition analyses indicated that PLA, action was primarily on DPPC, the major phospholipid component of pulmonary surfactant and the agent responsible for surfactant’s surface tension-lowering ability. However, the decreased biophysical activity caused by PLA, could not be explained by the decreased DPPC

INHIBITION

content alone and was actually simulated by the addition of LPC to surfactant in the absence of enzyme. Similarly, PLC also caused decreased surfactant function that appeared to be related to the inhibitory effects of hydrolysis products on the surfactant film. This enzyme is secreted by P. aeruginosa and may be one of the virulence factors of this bacteria (24). The mechanisms by which phospholipase hydrolysis products inhibit surfactant function cannot be definitively determined from the studies presented here, although several possibilities exist. Previous studies by this laboratory have indicated that plasma proteins such as albumin seem to inhibit surfactant function by competitively preventing surfactant adsorption to an airliquid interface (7). Although this mechanism may also occur with phopholipase hydrolysis products, it is likely that more complex interactions are also involved, such as intercalation of agents like LPC into the formed surfactant film. Such a mechanism may be inferred based on the combined findings that LPC readily inserts into cell membranes (16) and that surfactant inhibition by fluid membrane lipids is stoichiometrically different from the inhibition caused by plasma proteins (9). Our previous mechanistic studies using preformed films could not provide definitive information about surfactant inhibition by membrane lipids (7). However, the recent development of a method for introducing agents into the hypophase surrounding an oscillating bubble during the process of dynamic cycling should provide such information in future studies. Another important finding from these studies was that low noninhibitory levels of phospholipases actually increased the sensitivity of pulmonary surfactant to biophysical inactivation by plasma proteins. This suggests that a low-grade cellular and/or surfactant injury resulting from small increases in alveolar concentrations of phospholipases could escalate into a more severe injury. Thus physiologically relevant levels of phospholipases (27) can cause significant decreases in the biophysical function of pulmonary surfactant. Moreover, this surfactant dysfunction seems to be caused, at least in part, by the inhibitory effects of DPPC hydrolysis products released by the phospholipases and may contribute significantly to the pathophysiology of some forms of acute lung injury. This work was supported in part by grants from the Women and Children’s Health Research Foundation (B. A. Helm and G. Enhorning) and National Heart, Lung, and Blood Institute Grants HL-40896 (G. Enhorning and 3. Holm) and HL-45170 (B. A. Holm). L. Keicher was the recipient of a medical student summer research fellowship from the State University of New York at Buffalo. Address for reprint requests: B. A. Holm, Perinatal Center, Children’s Hospital of Buffalo, 219 Bryant St., Buffalo, NY 14222. Received 30 August 1990; accepted in final form 4 March 1991. REFERENCES

M. E., AND J. MEAD. Surface properties in relation to atelectasis and hyaline membrane disease. Am. J. Dis. Child. 97: 517-523,

1. AVERY, 1959.

2. BLIGH, E. G., AND W. J. DYER. A rapid method of total lipid extraction and purification. Can. J. Biuchem. Fhysid. 37: 911-917,1959. 3. CHEN, P. S., T. Y. TORIBARA, AND W. HUBER. Microdetermination of phosphorus. Anal. Chem. 28: 1756-1768,1956.


SURFACTANT 4. ENHORNING, G. Pulsating bubble technique for evaluating pulmonary surfactant. J. Appl. Physiol. 43: 198-203, 1977. 5. FUCHIMUKAI, T., T. FUJIWAFW, A. TAKAHASHI, AND G. ENHORNING. Artificial pulmonary surfactant inhibited by proteins. J. AppZ. Physiol. 57: 1134-1142, 1987. 6. HALLMAN, M., R. SPFUGG, J. H. HARRELL, AND K. M. MOSER. Evidence of lung surfactant abnormality in respiratory failure. J. Clin. Invest. 70: 673-683, 1982. 7. HOLM, B. A., G. ENHORNING, AND R. H. NOTTER. A mechanism by which plasma proteins inhibit surfactant function. Chem. Phys. Lipids 49: 49-E&1988. 8. HOLM, B. A., AND S. MATALON. Role of pulmonary surfactant in the development and treatment of adult respiratory distress syndrome. Anesth. An&. 69: 805~818,1989. 9, HOLM, B. A., AND R. H. NOTTER. Effects of hemoglobin and red blood cell membrane lipids on the biophysical and physiological activity of pulmonary surfactant. J. AppZ. Physiol. 63: 1434-1442, 1987. 10. HOLM, B. A., AND R. H. NOTTER. Surfactant therapy in ARDS and lung injury. In: Surfuctant Replacement Therapy, edited by D. L. Shapiro and R. H. Notter. New York: Liss, 1989, p. 273-304. 11. HOLM, B. A., R. H. NOTTER, AND 3. N. FINKELSTEIN. Surface property changes from interactions of albumin with natural lung surfactant extracted lung lipids. Chem. P!zys. Lipids 38: 287-298,1985, 12. HOPEKELL, P. C., AND J. MURRAY. The adult respiratory distress syndrome. In: Respiratory Emergencies, edited by E. M. Shibel and K. M. Moser. St. Louis, MO: Mosby, 1977, p. 101-128. 13. IKEGAMI, M., A. JOBE, H. JACOBS, AND R. LAM. A protein from airways of premature lambs that inhibits surfactant function. J. Appl. P/z&d. 57: 1134-1142, 1984. 14. JOBE, A., AND M. IKEGAMI. State of the art: surfactant for the treatment of respiratory distress syndrome. Am. Rev. Respir. Dis. 36: 1256-1275,1987. 15. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 132: 265-275,195l. 16. NLEWOEHNER, D. E., K. RICE, A. A. SINM, AND D. WANGENSTEEN. Injurious effects of lysophosphatidylcholine on barrier properties of alveolar epithelium. J. Appl, Physbl. 63: 1979-1986, 1987.

INHIBITION

321

17. NOTTER, R. H. Surface chemistry of pulmonary surfactant: role of individual components. In: Pulmonary Surfactant, edited by B. Robertson, L. M. G. van Golde, and J. J. Batenburg. Amsterdam: Elsevier, 1984, p. 17-65. 18. NOTTER, R. H., E. A. EGAN, M. S. KWONG, B. A. HOLM, AND D. L. SHAPIRO. Lung surfactant replacement in premature lambs with extracted lipids from bovine lung lavage: effects of dose, dispersion technique, and gestational age. Pediutr. Res. 19: 569-577, 1985. 19. NOTTER, R, H., AND J. N. FINKELSTEIN. Pulmonary surfactant: an interdisciplinary approach. J. Appl. Physiol. 57: 1613-1624, 1984. 20. PASSI, R. B., AND F. POSSMAYER. Surfactant metabolism in acute pancreatitis. Progr. Respir. Res. 15: 136-140,1981. 21. PETTY, T. L., G. W. SILVERS, G. W. PAUL, AND R. E, STANFORD. Abnormalities in lung elastic properties and surfactant function in adult respiratory distress syndrome. Chest 75: 571-574,1979. 22. RINALDO, J. E., AND R. M. ROGERS. Adult respiratory distress syndrome: changing concepts of lung injury. N. Erzgl. J. 1Med. 15: 900909,1982. 23. SEEGER, W., G. STORH, H. R. D. WOLFE, AND H. NEUHOF. Alteration of surfactant function due to protein leakage: special interaction with fibrin monomer. J. AppZ. Physiol. 58: 326-338,1985. 24. STINSON, M. W., AND C. HAYDEN. Secretion of phsopholipase C by Pseudomonas aeruginosa. Infect. Immun. 25: 55%564,1979. 25. TIERNEY, D. F., AND R. P. JOHNSON. Altered surface tension of lung extracts and lung mechanics. J. AppZ. Physiol. 20: 1253-1260, 1965. 26. TOUCHSTONE, J. C., J. C. CHEN, AND K. M. BEAVER. Improved separation of phospholipids in thin layer chromatography. Lipids 15: 61-62,198O. 27. VADAS, P. Elevated plasma phospholipase AZ levels: correlation with the hemodynamic and pulmonary changes in gram-negative septic shock. J. Lab. Clin. 1Med, 104: 873-881, 1984. 28. VADAS, P., AND W. PRUZANSKI. Biology of disease: role of secretory phospholipases A2 in the pathobiology of disease. Lab. Invest. 55: 391-399,1986. 29. WHITSE~, J. A., B. L. OHNING, G. Ross, J. ME~TH, T. WEAVER, B. A. HOLM, D. L. S~PIRO, AND R. H. NOTIZR. Hydrophobic surfactant-associated protein in whole lung surfactant and its importance of biophysical activity in lung surfactant extracts used for replacement therapy. Pediutr. Res. 20: 460-467,1986.


Biophysical inhibition of pulmonary surfactant function by polymeric nanoparticles: role of surfactant protein B and C.

Long-chain Acylcarnitines Reduce Lung Function by Inhibiting Pulmonary Surfactant.

Pulmonary drug delivery by surfactant.

Function and regulation of expression of pulmonary surfactant-associated proteins.

Effect of single dose surfactant on pulmonary function.

Lysophosphatidylcholine sensitizes lipid extracts of pulmonary surfactant to inhibition by serum proteins.

Biophysical inhibition of synthetic vs. naturally-derived pulmonary surfactant preparations by polymeric nanoparticles.

Amiodarone- and desethylamiodarone-induced pulmonary phospholipidosis, inhibition of phospholipases in vivo, and alteration of [14C]amiodarone uptake by perfused lung.

Competitive inhibition of lipolytic enzymes. VI. Inhibition of two human phospholipases A2 by acylamino phospholipid analogues.

Inactivation of exogenous surfactant by pulmonary edema fluid.

The role of palmitic acid in pulmonary surfactant: enhancement of surface activity and prevention of inhibition by blood proteins.

Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline.

The unusual symmetric reopening effect induced by pulmonary surfactant.

Phospholipases introduced into the hypophase affect the surfactant film outlining a bubble.

Infant pulmonary function testing in chronic pneumonitis of infancy due to surfactant protein C mutation.

Structure-function relationships of bovine pulmonary surfactant proteins: SP-B and SP-C.

Regulation of pulmonary surfactant secretion and clearance.

An investigation of the carbon nanotube--Lipid interface and its impact upon pulmonary surfactant lipid function.

Clearance and recycling of pulmonary surfactant.

Inhibition of hypoxic pulmonary vasoconstriction by diphenyleneiodonium.

Structure-function relationships in pulmonary surfactant membranes: from biophysics to therapy.

Stalling autophagy: a new function for Listeria phospholipases.

Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

Pulmonary corpora amylacea contain surfactant apoprotein.