In asthma, inflammation-mediated surfactant dysfunction contributes to increased airway resistance, but the mechanisms for dysfunction are not understood. To test mechanisms that alter surfactant function, atopic asthmatics underwent endobronchial antigen challenge and bronchoalveolar lavage (BAL). BAL fluids were sequentially separated into cells, surfactant, and supernatant, and multiple end points were analyzed. Each end point's unique relationship to surfactant dysfunction was determined. Our results demonstrate that minimum surface tension (gamma(min)) of surfactant after antigen challenge was significantly increased with a spectrum of responses that included dysfunction in 6 of 13 asthmatics. Antigen challenge significantly altered the partitioning of surfactant phospholipid measured as a decreased ratio of large surfactant aggregates (LA) to small surfactant aggregates (SA), LA/SA ratio. Phosphatidylglycerol (PG) was significantly reduced in the LA of the dysfunctional asthmatic BALs. There was a corresponding significant increase in the ratio of phosphatidylcholine to PG, which strongly correlated with both increased gamma(min) and decreased LA/SA. Altered surfactant phospholipid properties correlated with surfactant dysfunction as well or better than either increased eosinophils or protein. Secretory phospholipase activity, measured in vitro, increased after antigen challenge and may explain the decrease in surfactant PG. In summary, alteration of phospholipids, particularly depletion of PG, in the LA of surfactant may be an important mechanism in asthma-associated surfactant dysfunction.

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. This has been caused, in large part, by the lack of adequate assay systems in which such processes can be meaningfully measured. The purpose of this and the companion paper [E. E. McKee, B. L. Grier, G. S. Thompson, A. C. F. Leung, and J. D. McCourt. Am. J. Physiol. 258 [Endocrinol. Metab. 21):E503-E510, 1990] is to describe a system in which the quantitative regulation of mitochondrial protein synthesis in rat heart can be investigated. In this report the conditions for mitochondrial isolation and labeling are described, and the importance of isolating intact, tightly coupled mitochondria in obtaining high and reliable rates of protein synthesis is demonstrated. The highest levels of protein synthesis are obtained in mitochondria isolated from hearts perfused and homogenized in the presence of subtilisin, conditions in which the fastest rates of state 3 respiration and the highest respiratory control ratios are also observed. Analysis of the free amino acid pools indicates that isolated heart mitochondria have a negligible level of endogenous methionine as well as other amino acids. As a result, the concentration and specific radioactivity of the [35S]methionine pool serving protein synthesis could be easily determined. Optimal translation occurred at 30 degrees C at a pH of 7.0-7.2 and required the addition of methionine (20 microM), the other 19 amino acids (0.1 mM each), K+ (60-90 mM), Cl- (30-90 mM), Mg2+ (0.5-5 mM), and bovine serum albumin (1 mg/ml). As shown in the companion paper, adenine nucleotide (0.5-4.0 mM) and oxidizable substrate (10-20 mM glutamate) are also required for isolated heart mitochondrial protein synthesis. Analysis of labeled mitochondrial translation products demonstrated that bona fide mitochondrial peptides were synthesized.

Secretory A(2) phospholipases (sPLA(2)) hydrolyze surfactant phospholipids cause surfactant dysfunction and are elevated in lung inflammation. Phospholipase-mediated surfactant hydrolysis may disrupt surfactant function by generation of lysophospholipids and free fatty acids and/or depletion of native phospholipids. In this study, we quantitatively assessed multiple mechanisms of sPLA(2)-mediated surfactant dysfunction using non-enzymatic models including supplementation of surfactants with exogenous lysophospholipids and free fatty acids. Our data demonstrated lysophospholipids at levels >or=10 mol% of total phospholipid (i.e., >or=10% hydrolysis) led to a significant increase in minimum surface tension and increased the time to achieve a normal minimum surface tension. Lysophospholipid inhibition of surfactant function was independent of the lysophospholipid head group or total phospholipid concentration. Free fatty acids (palmitic acid, oleic acid) alone had little effect on minimum surface tension, but did increase the maximum surface tension and the time to achieve normal minimum surface tension. The combined effect of equimolar free fatty acids and lysophospholipids was not different from the effect of lysophospholipids alone for any measurement of surfactant function. Surfactant proteins did not change the percent lysophospholipids required to increase minimum surface tension. As a mechanism that causes surfactant dysfunction, depletion of native phospholipids required much greater change (equivalent to >80% hydrolysis) than generation of lysophospholipids. In summary, generation of lysophospholipids is the principal mechanism of phospholipase-mediated surfactant injury in our non-enzymatic models. These models and findings will assist in understanding more complex in vitro and in vivo studies of phospholipase-mediated surfactant injury.

Background Secretory phospholipases A2 (sPLA2) hydrolyze phospholipids in cell membranes and extracellular structures such as pulmonary surfactant. This study tests the hypothesis that sPLA2 are elevated in human lungs during acute respiratory distress syndrome (ARDS) and that sPLA2 levels are associated with surfactant injury by hydrolysis of surfactant phospholipids. Methods Bronchoalveolar lavage (BAL) fluid was obtained from 18 patients with early ARDS (<72 hours) and compared to samples from 10 healthy volunteers. Secreted phospholipase A2 were measured (enzyme activity and enzyme immunoassay) in conjunction with ARDS subjects’ surfactant abnormalities including surfactant phospholipid composition, large and small aggregates distribution, and surface tension function. Results BAL sPLA2 enzyme activity was markedly elevated in ARDS samples relative to healthy subjects when measured by ex vivo hydrolysis of both phosphatidylglycerol (PG) and phosphatidylcholine (PC). Enzyme immunoassay identified increased PLA2G2A protein in the ARDS BAL fluid, which was strongly correlated with the sPLA2 enzyme activity against PG. Of particular interest, we demonstrated an average depletion of 69% of the PG in the ARDS sample large aggregates relative to the normal controls. Furthermore, the sPLA2 enzyme activity against PG and PC ex vivo correlated with the BAL recovery of in vivo PG and PC, respectively, and also correlated with the altered distribution of the large and small surfactant aggregates. Conclusions These results support the hypothesis that sPLA2-mediated hydrolysis of surfactant phospholipid, especially PG by PLA2G2A, contributes to surfactant injury during early ARDS.

The lethal chicken mutation nanomelia leads to severe skeletal defects because of a deficiency of aggrecan, which is the largest aggregating chondroitin sulphate proteoglycan of cartilage. In previous work, we have demonstrated that nanomelic chondrocytes produce a truncated aggrecan precursor that fails to be secreted, and is apparently arrested in the endoplasmic reticulum (ER). In this study, we investigated the biosynthesis and extent of processing of the abnormal aggrecan precursor. The truncated precursor was translated directly in cell-free reactions, indicating that it does not arise post-translationally. Further studies addressed the processing capabilities of the defective precursor. We found that the mutant precursor was modified by N-linked, mannose-rich oligosaccharides and by the addition of xylose, but was not further processed; this is consistent with the conclusion that it moves no further along the secretory pathway than the ER. Using brefeldin A we demonstrated that the defective precursor can function as a substrate for Golgi-mediated glycosaminoglycan chains, but does not do so in the nanomelic chondrocyte because it fails to be translocated to the appropriate membrane compartment. These studies illustrate how combined cell biological/biochemical and molecular investigations may contribute to our understanding of the biological consequences and molecular basis of genetic diseases, particularly those involving errors in large, highly modified molecules such as proteoglycans.