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The effect of serum, glucocorticoids, gender and gestational age on the inactivation, synthesis and secretion of surfactant phospholipids by cultured foetal rat type II pneumocytes

Damas, Jolanta Elzbieta (2010) The effect of serum, glucocorticoids, gender and gestational age on the inactivation, synthesis and secretion of surfactant phospholipids by cultured foetal rat type II pneumocytes. PhD thesis, Murdoch University.

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Type II pneumocytes are responsible for the synthesis and secretion of pulmonary surfactant, which lines the alveoli of the lungs. Its presence as a monolayer at the airliquid interface in the alveoli diminishes surface tension, thus reducing the tendency of alveoli to collapse during expiration. For this effect to be sustained, however, the integrity of the surfactant components needs to be maintained. In this study it has been demonstrated that when cultured type II pneumocytes are exposed to lipoprotein-free serum (LFS) the level of lyso-phosphatidylcholine (lyso-PC) in the secreted surfactant phospholipids is elevated. The lyso-PC content of secreted phospholipids in serumsupplemented cultures increased 5.4-fold with a concomitant decline in the level of phosphatidylcholine (PC) when compared to cells grown in absence of LFS. This effect is without doubt the result of hydrolysis of surfactant phosphatidylcholine by serum phospholipase A2 (PLA2)-like activity. Given that many cases of respiratory trauma are associated with leakage of serum into the alveoli, this enzyme may be a significant contributor to surfactant inactivation and the onset of adult respiratory distress syndrome (ARDS). Subjecting human LFS to DEAE-Sepharose chromatography demonstrated that the enzyme, which stimulates the production of lyso-PC, co-elutes with albumin and is biochemically distinct from the secretory form of PLA2. Testing of commercially purified human serum albumin (HSA) also showed the presence of this enzymatic activity, indicating that the serum factor not only co-elutes with albumin but is possibly bound to it. This is supported by the fact that, when subjected to polyacrylamide gel electrophoresis, the activity associated with LFS and human serum albumin (HSA) has an almost identical elution profile. Furthermore, when partially purified preparations of these were subjected to IEF electrophoresis, both yielded a similar double protein-banding pattern. Although the predominant band had similar migratory properties to that of albumin (pI of approximately 4.5) it was not possible, given that the IEF involves a denaturing gel, to assign the PLA2-like activity to either of these two bands. The present study has also revealed that exposure of type II pneumocytes to recombinant human serum albumin (rHSA) stimulates the secretion of phospholipids and is almost as effective as LFS in enhancing the level of lyso-PC in the media. The latter observation implies that rHSA is able to directly generate lyso-PC and suggests that PLA2 activity may be an innate activity of albumin.

Neonatal respiratory distress syndrome (NRDS) is a significant cause of mortality in preterm human infants. Because underdevelopment of the lungs is known to contribute to the pathogenesis of NRDS, corticosteroid treatment using synthetic glucocorticoids has been widely used to accelerate maturation of the type II alveolar cells and to prematurely initiate synthesis of surfactant. Dexamethasone has no effect upon the rate of synthesis of surfactant lipids when applied directly to cultured foetal type II cells. However, if type II cells are exposed to medium previously conditioned by cultured lung fibroblasts in the presence of dexamethasone, then there is a significant elevation (17.3% increase) in the rate of surfactant lipid synthesis. This indirect effect of dexamethasone, presumably acting via the production of fibroblast pneumocyte factor (FPF), is significantly greater if both the fibroblasts and type II cells are derived from female foetal rats (47.6% increase in PC synthesis) as compared to those derived from male foetuses (19.0% increase). The present study has confirmed that there are sexspecific and gestational age differences in the glucocorticoid receptor level of lung fibroblasts but not of type II cells, thus, regulation of the appearance of the v glucocorticoid receptor in foetal lungs in the later stages of gestation appears to be both sex- and cell-specific. The interaction of glucocorticoids with their specific receptor has also been shown to indirectly affect the rate of surfactant secretion. This study has demonstrated that -adrenergic receptors are present and functional in cultures enriched with foetal type II pneumocytes. The -adrenoreceptor activity is elevated if the type II cells are exposed to glucocorticoids but, as has been shown in this study, the elevation of receptor activity is significantly greater if the type II cells are exposed to media that had been previously conditioned by fibroblasts in the presence of glucocorticoids. Both of these effects are more pronounced if the type II cells were derived from female foetuses rather than from males, despite the cells being grown under similar conditions. The observed elevation in -adrenergic receptor activity in type II cells exposed to either glucocorticoids or fibroblast-conditioned media has been shown to lead to a greater physiological response to the -agonist, (—)-isoproterenol, as measured by surfactant lipid secretion. Indeed, there is a very strong correlation (r = 0.962) between the -adrenergic receptor activity and the ability of agonists to stimulate phospholipid secretion from type II cells. Given that the enhanced -adrenergic receptor activity of type II cells and the resultant cellular response to -agonists is higher if the cells are treated with FCM rather than with dexamethasone, it is suggested that these glucocorticoid-induced responses may be indirect via an increased release of FPF from lung fibroblasts.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Biological Sciences and Biotechnology
Supervisor(s): Cake, Max
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