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Amiodarone may induce lung damage by direct toxicity or indirectly through inflammation. To clarify the mechanism of direct toxicity, we briefly exposed rabbit alveolar macrophages to amiodarone and analyzed their morphology, synthesis, and degradation of dipalmitoylphosphatidylcholine (DPPC); distribution of lysosomal enzymes; and uptake of diphtheria toxin and surfactant protein (SP) A used as tracers of the endocytic pathway. Furthermore, in newborn rabbits, we studied the clearance of DPPC and SP-A instilled into the trachea together with increasing amounts of amiodarone. We found that in vitro amiodarone decreases the surface density of mitochondria and lysosomes while increasing the surface density of inclusion bodies, increases the incorporation of choline into DPPC, modifies the distribution of lysosomal enzymes, and does not affect the uptake and processing of diphtheria toxin but inhibits the degradation of SP-A. In vivo amiodarone inhibits the degradation of SP-A but not of DPPC. We conclude that the acute exposure to amiodarone perturbs the endocytic pathway acting after the early endosomes, alters the traffic of lysosomal enzymes, and interferes with the turnover of SP-A.

This paper examines the removal from the airways of Curosurf, a commercial surfactant derived from porcine lungs, administered at pharmacological concentrations to newborn or adult animals. Curosurf was labelled by the addition of radioactive dipalmitoyl phosphatidylcholine (DPPC) and administered intratracheally to newborn and adult rabbits at a dose of 200 mg x kg(-1) body weight. The disappearance of DPPC from the airways and its appearance in alveolar macrophages, lung parenchyma, lamellar bodies, serum, liver, kidneys and brain was then studied for 24-48 h. The in vitro degradation of Curosurf DPPC by alveolar macrophages was also studied. During the first 3 h after instillation, large amounts of Curosurf left the airways and became associated with tissue, indicating that it mixed rapidly with the endogenous pools of surfactant. A fraction of administered DPPC became associated with the lamellar bodies, suggesting that Curosurf can be recycled. Curosurf administration did not stop the secretion of endogenous surfactant. Very little intact radioactive DPPC could be recovered at any time in alveolar macrophages, however, macrophages have the ability, in vitro, to degrade Curosurf. Newborn rabbits lose Curosurf from the lungs at a slower rate than adult rabbits. One and two days after instillation, organic extracts from the liver, kidney, brain and serum contained small but measurable amounts of radioactivity. These results indicate that Curosurf rapidly enters the pathways of surfactant metabolism and that alveolar macrophages may play an important role in the catabolism of Curosurf.

Serum lipoproteins may enter the airways and appear in sputum (chyloptysis) when the lymphatic circulation is impaired by inflammation, neoplasia, or an abnormal proliferation of smooth muscle cells. While analyzing the bronchoalveolar lavage fluid of a patient with chyloptysis, we noticed that surfactant could not be separated from contaminating serum lipoproteins and speculated that lipoproteins might interact with surfactant components. To clarify this point we immobilized surfactant protein (SP) A on microtiter wells and incubated it with125I-labeled very low density lipoproteins (VLDLs), low-density lipoproteins, and high-density lipoproteins. We found that SP-A binds lipoproteins. Studying in greater detail the interaction of SP-A with VLDLs, we found that the binding is time and concentration dependent; is inhibited by unlabeled lipoproteins, phospholipids, and antibodies to SP-A; is increased by Ca2+; and is unaffected by methyl α-d-mannopyranoside. Whole surfactant is a potent inhibitor of binding. Furthermore, we found that SP-A increases the degradation of VLDLs by alveolar macrophages and favors the association of VLDLs with alveolar surfactant. We conclude that SP-A influences the disposal of serum lipoproteins entering the airways and speculate that binding to alveolar surfactant might represent an important step in the interaction between exogenous substances and the lung.

Alveolar macrophages degrade surfactant protein (SP) A and saturated phosphatidycholine [dipalmitoylphosphatidylcholine (DPPC)]. To clarify this process, using rabbit alveolar macrophages, we analyzed the effect of drugs known to affect phagocytosis, pinocytosis, clathrin-mediated uptake, caveolae, the cytoskeleton, lysosomal pH, protein kinase C, and phosphatidylinositol 3-kinase (PI3K) on the degradation of SP-A and DPPC. We found the following: 1) SP-A binds to the plasma membrane, is rapidly internalized, and then moves toward degradative compartments. Uptake could be clathrin mediated, whereas phagocytosis, pinocytosis, or the use of caveolae are less likely. An intact cytoskeleton and an acidic milieu are necessary for the degradation of SP-A. 2) Stimulation of protein kinase C increases the degradation of SP-A. 3) PI3K influences the degradation of SP-A by regulating both the speed of internalization and subsequent intracellular steps, but its inhibition does not prevent SP-A from reaching the lysosomal compartment. 4) The degradation of DPPC is unaffected by most of the treatments able to influence the degradation of SP-A. Thus it appears that DPPC is degraded by alveolar macrophages through mechanisms very different from those utilized for the degradation of SP-A.

Transformations of surfactant after secretion are incompletely understood. To clarify them, we lavaged lungs in fetuses and in newborn rabbits, fractionated the lavage fluid by differential and density gradient centrifugation, and analyzed the distribution of surfactant protein (SP) phospholipids, SP-A, SP-B, and SP-C. Furthermore, we administered into trachea of newborn rabbits labeled surfactant and compared the alveolar clearance of SP-A, SP-B, SP-C and saturated phosphatidylcholine. We found that, in the fetus, secreted lamellar bodies contain all components of surfactant, except a small amount of SP-A. As breathing starts and new surfactant subtypes are generated, the proteins are mostly associated with dense subtypes, but SP-B and SP-C are especially concentrated in dense materials that contain minor amounts of phospholipids and SP-A. Furthermore, we found that, during breathing, alveolar surfactant is degraded into more than one type of remnant, that the lavage fluid contains a pool of SP-A not associated with membranes, and that SP-A, SP-B, and SP-C are all turned over at a faster rate than saturated phosphatidylcholine.

To clarify the life cycle of surfactant protein C (SP-C), we obtained alveolar surfactant from 3-day-old rabbits killed 15 h after the tracheal administration of 32P and L-[35S]methionine. Small amounts of this surfactant were administered into the tracheas of 3-day-old rabbits, which were killed after 1 min and 2 and 4 h. We then analyzed the radioactivity associated with SP-C and with saturated phosphatidylcholine (DPPC) in lung lavage fluid and in a lamellar body-rich fraction isolated from the lung homogenate. We found that SP-C and DPPC are both taken up from the alveolar spaces and incorporated into lamellar bodies, but the time courses of reuptake are different. Our results indicate that in the alveoli SP-C is turned over faster than DPPC.

In patients with idiopathic alveolar proteinosis, the alveoli are filled with materials rich in surfactant components, especially surfactant protein A (SP-A). The anomaly could be caused by either increased secretion, decreased clearance, or both. To clarify this point, we studied five patients who underwent therapeutic lavage and then were ventilated mechanically for 24 h. During the first 8 h of mechanical ventilation, a surfactant-depleted lung was lavaged at selected intervals, and the bronchoalveolar lavage fluid was analyzed. We observed that, after lavage, various surfactant components accumulated in the airways with different time courses. We also observed that SP-A increased until the second hour and then dropped rapidly, suggesting the existence of an efficient mechanism of removal. These findings suggest that idiopathic alveolar proteinosis might be caused by a primary defect in a slow mechanism of removal or by the presence of factor(s) that interfere with the clearance of surfactant and that can be removed by lavage. It seems clear, however, that an increased secretion rate is unlikely to be the major cause of idiopathic alveolar proteinosis.