Ronald G. Salome

TNF-α increases ceramide without inducing apoptosis in alveolar type II epithelial cells

Ceramide is a bioactive lipid mediator that has been observed to induce apoptosis in vitro. The purpose of this study was to determine whether endogenous ceramide, generated in response to in vivo administration of tumor necrosis factor-alpha (TNF-alpha), increases apoptosis in primary rat alveolar type II epithelial cells. Intratracheal instillation of TNF-alpha (5 microgram) produced a decrease in sphingomyelin and activation of a neutral sphingomyelinase. These changes were associated with a significant increase in lung ceramide content. TNF-alpha concomitantly activated the p42/44 extracellular signal-related kinases and induced nuclear factor-kappaB activation in the lung. Hypodiploid nuclei studies revealed that intratracheal TNF-alpha did not increase type II cell apoptosis compared with that in control cells after isolation. A novel observation from separate in vitro studies demonstrated that type II cells undergo a gradual increase in apoptosis after time in culture, a process that was accelerated by exposure of cells to ultraviolet light. However, culture of cells with a cell-permeable ceramide, TNF-alpha, or a related ligand, anti-CD95, did not increase apoptosis above the control level. The results suggest that ceramide resulting from TNF-alpha activation of sphingomyelin hydrolysis might activate the mitogen-activated protein kinase and nuclear factor-kappaB pathways without increasing programmed cell death in type II cells.

Very low density lipoproteins stimulate surfactant lipid synthesis in vitro.

Surfactant synthesis is critically dependent on the availability of fatty acids. One fatty acid source may be circulating triglycerides that are transported in VLDL, and hydrolyzed to free fatty acids by lipoprotein lipase (LPL). To evaluate this hypothesis, we incubated immortalized or primary rat alveolar pre-type II epithelial cells with VLDL. The cells were observed to surface bind, internalize, and degrade VLDL, a process that was induced by exogenous LPL. LPL induction of lipoprotein uptake significantly increased the rates of choline incorporation into phosphatidylcholine (PC) and disaturated PC, and these effects were associated with a three-fold increase in the activity of the rate-regulatory enzyme for PC synthesis, cytidylyltransferase. Compared with native LPL, a fusion protein of glutathione S-transferase with the catalytically inactive carboxy-terminal domain of LPL did not activate CT despite inducing VLDL uptake. A variant of the fusion protein of glutathione S-transferase with the catalytically inactive carboxy-terminal domain of LPL that partially blocked LPL-induced catabolism of VLDL via LDL receptors also partially blocked the induction of surfactant synthesis by VLDL. Taken together, these observations suggest that both the lipolytic actions of LPL and LPL-induced VLDL catabolism via lipoprotein receptors might play an integral role in providing the fatty acid substrates used in surfactant phospholipid synthesis.

Identification of Sex-Specific Differences in Surfactant Synthesis in Rat Lung

Delayed lung maturation and lower levels of surfactant phosphatidylcholine have been previously identified in male fetuses compared with female fetuses in several species. We investigated the mechanisms for sex differences in surfactant content by examining parameters of phosphatidylcholine turnover and biosynthesis; the latter was evaluated by measuring metabolic steps within the biosynthetic pathway. Compared with male lung cells, freshly isolated lung cells from female fetuses contained higher levels of disaturated phosphatidylcholine, a marker of surfactant lipid. Female mixed monolayer cultures exhibited a 71% increase in choline incorporation into disaturated phosphatidylcholine compared with male cultures. Male cultures exhibited significantly greater release of [3H]-arachidonic acid into the medium compared with females, suggesting sex differences in phospholipase activity. However, pulse-chase studies showed no sex differences in degradation of disaturated phosphatidylcholine, which was confirmed by assays of phospholipase A2, phosphatidylcholine-specific phospholipase C, and phospholipase D. Female mixed lung cells, however, had greater rates of cellular choline transport and activity of cytidylyltransferase, the rate-regulatory enzyme for phosphatidylcholine synthesis. Separate studies showed that exposure of sex-specific pretype II cell cultures to cortisol-stimulated fibroblast-conditioned medium plus transforming growth factor-β–neutralizing antibody stimulated cytidylyltransferase activity to a greater extent in male cells compared with female cells. These studies indicate that sex differences in surfactant phospholipid content are not due to differences in phospholipid turnover, but rather differential regulation of specific metabolic steps within the surfactant biosynthetic pathway. The data also support a role for transforming growth factor-β as a negative regulator of a key surfactant biosynthetic enzyme within male lungs.

Epidermal Growth Factor is a Positive in Vivo Regulator of CTP: Cholinephosphate Cytidylyltransferase

CTP:cholinephosphate cytidylyltransferase (CT) is a key enzyme required for surfactant phosphatidylcholine synthesis, and its activity is regulated by lung lipids. This study evaluated the effect of epidermal growth factor (EGF) on the phospholipid content and the expression of CT in the lung following direct in vivo administration to the newborn rat. EGF caused an increase in cytidylyltransferase activity by 58% in lung cytosol. The increase in cytosolic activity was not mediated by a corresponding increase in enzyme mass. Further, these changes in cytidylyltransferase activity were associated with a significant increase in total lung phospholipid and phosphatidylcholine content. The results suggest that EGF may have important maturational effects on lung surfactant metabolism.