John A. Clements

Surface Tension of Lung Extracts

Summary Saline-extractable surface-active material has been found in the lungs of rat. cat. and dog. This material, probably mucoprotein. imparts large hysteresis and characteristic elasticity to the fluid surface. Its effect on lung mechanics has been studied. Its possible influence on diffusion across the alveolar barrier remains to be elucidated.

Subfractionation of lung surfactant. Implications for metabolism and surface activity.

Because previous studies have suggested that lung surfactant is not a simple compartment of homogeneous material, we subfractionated lamellar bodies and components of alveolar lavage from male New Zealand white rabbits, according to differences in sedimentability. We recovered two lamellar body populations at different densities in discontinuous sucrose density gradients; we separated six subfractions of alveolar lavage by differential centrifugation. To determine whether or not precursor-product relationships existed among the subfractions, we injected radioactive palmitate intravenously, killed the rabbits 1-72 h later, and measured phospholipid specific activities. The two populations of lamellar bodies had similar phospholipid composition, fatty acyl composition of phosphatidylcholine and phosphatidylglycerol, and surface activity. Light lamellar bodies had a higher ratio of phospholipid to protein, and labelled with tracer later in time than dense ones. For alveolar lavage subfractions, later labelling with tracer, lower adsorption rate and lower total protein and phosphatidylglycerol content seemed to correlate with decreasing average density and particle size as well as with the disappearance of tubular myelin structure and appearance of predominantly vesicular structure. The subfractions appear to be in a metabolic sequence in which heavier, more dense material is a precursor to lighter, less dense material. The results suggest that subfractions of surfactant are extensively recycled.

Alveolar epithelial cell mitochondria as source of the surface-active lung lining.

We propose that the surface-active lining of the mammalian lung is formed in the mitochondria of the alveolar epithelial cells. Findings supporting this hypothesis are the presence of strong surfactant uniquely in the washed mitochondrial fraction of mammalian lung, almost complete loss of mitochondrial lamellar forms accompanying loss of lung surface activity after vagotomy, and the absence of strong surface activity from the lung extracts of animals whose alveolar lining cells show no lamellar forms.

Pulmonary surfactant and evolution of the lungs.

Pulmonary surfactant has been looked for and found in 11 representatives of four vertebrate classes. The amount of surfactant, estimated by quantitative spreading as a surface film, correlates well with alveolar surface area and with the amount of saturated, mainly dipalmitoyl, phosphatidylcholine in the lung parenchyma. The quantities of other phospholipids do not correlate well with alveolar surface area.

Surfactant apoprotein A modifies the inhibitory effect of plasma proteins on surfactant activity in vivo.

ABSTRACT: Surfactant apoprotein A (SP-A) reduces the inhibitory effects of plasma proteins on the surface tension lowering properties of pulmonary surfactant in vitro. To test the effects of SP-A in vivo we administered a complete natural dog lung surfactant (DLS) containing apoproteins SP-A, SP-B, and SP-C, a butanol extract of DLS (DLSE) containing only apoproteins SP-B and SP-C, and DLSE supplemented with SP-A intratracheally to prematurely delivered rabbit pups in the presence of increasing amounts of human plasma. In the absence of plasma DLS and DLSE (100 mg/kg phospholipid) had comparable effects on lung mechanics (compliance during ventilation with a tidal volume of 6–7 mL/kg and quasi-static pressure-volume behavior) in this surfactant deficiency model. Plasma proteins in increasing amounts to a maximum protein concentration of 62.5 mg/mL had no effect on the response of the pups to DLS. In contrast, plasma added to DLSE in concentrations above 20 mg/mL significantly increased the peak inspiratory pressure (PIP) required to ventilate the pups with a tidal volume of 6–7 mL/kg, reduced the calculated total lung compliance, and decreased the deflation lung volumes. The inhibitory effects of plasma on DLSE were significantly less when SP-A was added to DLSE (5:1, phospholipid:SP-A, wt:wt). The addition of SP-A to DLSE in plasma restored the activity of the extract to levels comparable to complete DLS. These results suggest that plasma can interfere with surfactant function and that SP-A has a significant protective effect for surfactant against the inhibitory effects of plasma in vivo.

Precursor-product relationship between rabbit type II cell lamellar bodies and alveolar surface-active material: Surfactant turnover time

To estimate the turnover time of alveolar surfactant in New Zealand white rabbits, we injected [9,10-(3)H] palmitic acid and [2-(14)C] glycerol intravenously. From 1-48 h after injection, wer killed the animals, lavaged the lungs for alveolar surfactant with saline, and isolated the lamellar bodies by homogenization and sucrose density gradient centrifugation. Lamellar bodies and alveola surfactant had comparable phospholipid composition and surface activity. Lamellar bodies contained little DNA, no mitochondrial enzyme activity and less than 5% contaminating phospholipids from microsomal and Golgi-enriched fractions. We measured radioactivity of phosphatidylcholine, saturated phosphatidylcholine and phosphatidylglycerol for each isotope in lamellar bodies and surfactant at each time point. The plot of the integral with respect to time of the difference between lamellar body and surfactant specific activity against surfactant specific activity has a slope determined by the turnover time, and a shape which tests the precision of the precursor-product relationship. This analysis does not assume a pulse label and allows for possible recycling of tracer from surfactant to lamellar bodies. We obtained turnover times of 4-11 h. We detected an imprecise precursor-product relationship between lamellar bodies and alveolar surfactant, which is not due to experimental variability or to contamination of lamellar bodies by other subcellular fractions but may reflect imperfect mixing within surfactant compartments.

Protein composition of rabbit alveolar surfactant subfractions

The goal of this investigation was to characterize the proteins in subfractions of alveolar surfactant obtained by lung lavage and separated by differential centrifugation. It was previously demonstrated that the material in the more sedimentable fraction, which was enriched in tubular-myelin and was surface-active may be a precursor to the less sedimentable, vesicular, inactive material [1]. Separation of the proteins by polyacrylamide gel electrophoresis showed that the more sedimentable subfractions and rabbit surfactant isolated by conventional methods contained proteins with molecular weights comparable to those previously reported for alveolar surface active material (approximately 36 000 and 10 000). The less sedimentable subfractions contained less of these proteins. Immunoblots with anti-dog surfactant apoprotein antibodies, which cross-react with rabbit proteins, supported these observations. Immunoblots also showed that all of the subfractions contained serum proteins and secretory IgA, with the less sedimentable subfractions containing more secretory IgA. These results suggested that changes in protein composition may accompany functional changes in surfactant in the alveoli.

Effects of a protein-free, synthetic surfactant on survival and pulmonary function in preterm lambs

We have created a totally synthetic, protein-free surfactant (Exosurf) composed of dipalmitoylphosphatidylcholine, hexadecanol, and tyloxapol. We studied the effects of endotracheal instillation of Exosurf on survival and pulmonary function of preterm lambs delivered at 131 to 133 days gestation (term 148 days). Exosurf treatment was compared with instillation of surface-active material prepared from lung lavages of adult sheep and with no instillation. Lambs were delivered by cesarean section, paralyzed, and mechanically ventilated. The Exosurf group survived longer (80% alive at 11 hours) than did the no instillation group (30% alive at 11 hours) (P less than 0.05). There were no statistically significant differences between the Exosurf and sheep surfactant groups. We conclude that Exosurf, a synthetic surfactant, produces significant improvement in survival and pulmonary function in preterm lambs.

Pulmonary and Circulatory Effects of Fibrinopeptides

Bovine fibrinopeptide B and human fibrinopeptide A given in minute concentrations to rabbits, dogs, and lambs caused pulmonary hypertension, decreased effective pulmonary blood flow, decreased lung compliance, decreased ventilatory conductance, increased frequency and volume of ventilation, and increased differences in arterial and alveolar Pco2 and Po2. These effects appeared immediately after injection of the peptides into the lesser circulation, increased for 15 to 30 min, and were detectable for as long as 70 min. Mean aortic pressure, heart rate, stroke volume, left and right atrial pressures, and cardiac output showed no systematic changes after injection of these peptides. On the contrary, bovine fibrinopeptide A given in equimolar doses elicited none of the pulmonary responses but induced increases in heart rate, stroke volume, and cardiac output without consistent changes in mean pulmonary or aortic pressure. These responses appeared within 3 min, increased for 5 to 10 min, and disappeared within an hour. Some possible relationships of fibrinopeptides to clinical syndromes characterized by respiratory distress, hypoxemia, pulmonary vasoconstriction, and diminished lung compliance are briefly discussed. It is speculated that the lung may be an active site of fibrinopeptide catabolism and that accelerated fibrinogen-fibrin conversion may cause pulmonary failure through fibrinopeptide-induced vasoconstriction and reduction in compliance.

Hyaluronan Decreases Surfactant Inactivation In Vitro

Hyaluronan (HA) is an anionic polymer and a constituent of alveolar fluid that can bind proteins, phospholipids, and water. Previous studies have established that nonionic polymers improve the surface activity of pulmonary surfactants by decreasing inactivation of surfactant. In this work, we investigate whether HA can also have beneficial effects when added to surfactants. We used a modified pulsating bubble surfactometer to measure mixtures of several commercially available pulmonary surfactants or native calf surfactant with and without serum inactivation. Surface properties such as equilibrium surface tension, minimum and maximum surface tensions on compression and expansion of a surface film, and degree of surface area reduction required to reach a surface tension of 10 mN/m were measured. In the presence of serum, addition of HA dramatically improved the surface activities of all four surfactants and in some cases in the absence of serum as well. These results indicate that HA reduces inactivation of surfactants caused by serum and add evidence that endogenous HAs may interact with alveolar surfactant under normal and abnormal conditions.