Glucocorticoids regulate expression of the fatty acid synthase gene in fetal rat type II cells.

Fatty acids are integral components of pulmonary surfactant, a mixture of phospholipids and specific proteins that lines the alveolar surface and is essential for normal lung function. There are developmental increases in fatty acid biosynthesis and surfactant production in late-gestation fetal lung, and both processes are accelerated by glucocorticoids. Fatty acid synthase (FAS) is a key enzyme in de novo fatty acid biosynthesis, and increased FAS activity is responsible for the developmental and hormone-induced increases in fatty acid biosynthesis in fetal lung. Using cultured fetal lung explants, it has been reported that dexamethasone (Dex) increases FAS activity, protein content, mRNA content and rate of transcription. However, FAS expression has not been measured in isolated type II cells, the cellular source of surfactant within the lung. In the present study we measured parameters of FAS expression in type II cells isolated from the lungs of Dex-treated rats. Pregnant rats were injected with Dex or saline on days 18 and 19 of gestation and the fetuses delivered on day 20. Type II cells and fibroblasts were then isolated from the fetal lungs. Dex increased FAS activity, protein content, mRNA content and rate of transcription in the type II cells but not in the fibroblasts. Increased FAS expression in fetal type II cells in response to Dex is consistent with a critical role for FAS in the biosynthesis of lung surfactant.

Regulation of SP-B and SP-C secretion in rat type II cells in primary culture.

Secretion of lung surfactant phospholipids is a highly regulated process. A variety of physiological and pharmacological agents stimulate surfactant phospholipid secretion in isolated type II cells. Although the lipid and hydrophobic protein components of surfactant are believed to be secreted together by exocytosis of lamellar body contents, regulation of surfactant protein (SP) B and SP-C secretion has not previously been examined. To address the question of whether secretion of SP-B and SP-C is stimulated by the same agonists that stimulate phospholipid secretion, we measured secretion of all four SPs under the same conditions used to measure phosphatidylcholine secretion. Freshly isolated rat type II cells were cultured overnight and exposed to known surfactant phospholipid secretagogues for 2.5 h, after which the amounts of SP-A, SP-B, SP-C, and SP-D in the medium were measured with immunoblotting. Secretion of SP-B and SP-C was stimulated three- to fivefold by terbutaline, 5'-(N-ethylcarboxyamido)adenosine, ATP, 12-O-tetradecanoylphorbol 13-acetate, and ionomycin. Similar to their effects on phospholipid secretion, the stimulatory effects of the agonists were abolished by Ro 31-8220. Secretion of SP-A and SP-D was not stimulated by the secretagogues tested. We conclude that secretion of the phospholipid and hydrophobic protein components of surfactant is similarly regulated, whereas secretion of the hydrophilic proteins is regulated differently.

Transcriptional regulation of the lung fatty acid synthase gene by glucocorticoid, thyroid hormone and transforming growth factor-beta 1.

Fatty acid synthase (FAS) is a key enzyme in the biosynthesis of lung surfactant. FAS expression in fetal lungs is increased by glucocorticoids and this effect is largely due to increased transcription. The stimulatory effect of glucocorticoid on FAS expression is antagonized by thyroid hormone and transforming growth factor-beta 1 (TGF-beta 1). To determine the glucocorticoid responsive regions of the FAS gene we employed deletion analysis and reporter gene assays. A549 cells were transfected with various FAS gene constructs ligated to the firefly luciferase gene and cultured with dexamethasone (Dex) for 24 h after which luciferase activity was measured. Dex increased luciferase expression in response to a fragment in the promoter and 5'-flanking region of the FAS gene, from -1592 to +65 bp. This increase was antagonized by triiodothyronine (T(3)) and TGF-beta 1. Serial deletions showed that the full response to Dex and T(3) were retained in the 89 bp -33/+56 bp fragment whereas the response to TGF was mediated by the immediately upstream -104/-34 bp sequence. The Dex responsive region of the FAS gene could not be separated from the minimal promoter showing that they are intimately associated. The extents of Dex stimulation and antagonism by T(3) and TGF in A549 cells were similar to those noted on parameters of FAS expression in fetal lung explants. These data show that the effects of Dex, T(3) and TGF on FAS expression are mediated by DNA sequences in the promoter region of the gene.

Regulation of surfactant secretion.

Lung surfactant is synthesized in the alveolar type II cell. Its lipids and hydrophobic proteins (SP-B and SP-C) are stored in lamellar bodies and secreted by regulated exocytosis. In contrast, the hydrophilic proteins (SP-A and SP-D) appear to be secreted independently of lamellar bodies. Regulation of surfactant secretion is mediated by at least three distinct signaling mechanisms: activation of adenylate cyclase with formation of cAMP and activation of cAMP-dependent protein kinase; activation of protein kinase C; and a Ca(2+)-regulated mechanism that likely results in the activation of Ca(2+)-calmodulin-dependent protein kinase. These signaling mechanisms are activated by a variety of agonists, some of which may have a physiological role. ATP is one such agent and it activates all three signaling mechanisms. There is increasing information on the identity of several of the signaling proteins involved in surfactant secretion although others remain to be established. In particular the identity of the phospholipase C, protein kinase C and phospholipase D isomers expressed in the type II cell and/or involved in surfactant secretion has been established. Distal steps in the secretory pathway beyond protein kinase activation as well as the physiological regulation of surfactant secretion, are major issues that need to be addressed.

Glucocorticoid enhances the response of type II cells from newborn rats to surfactant secretagogues.

There is a developmental increase in agonist-induced surfactant secretion in type II cells. The response to the P2Y(2) agonist UTP is negligible in early newborn cells but increases with age. The response to terbutaline, N-ethylcarboxyamidoadenosine (NECA), and ATP also increases with age. As glucocorticoids are known to accelerate several aspects of lung maturation we examined the effect of dexamethasone (Dex) on the response of 1-day-old rat type II cells to surfactant secretagogues. Freshly isolated cells were cultured +/-10(-6) M Dex for 18--20 h after which phosphatidylcholine secretion was measured. Dex slightly decreased the basal secretion rate. However, it significantly increased the response to terbutaline, NECA, ATP and UTP. This effect was dependent on Dex concentration (EC(50)=2-6 x 10(-9) M) and blocked by the glucocorticoid receptor antagonist RU-486. It is unlikely to be due to increased receptor content as Dex had no effect on adenylate cyclase, phospholipase C or phospholipase D activation and the response to cAMP, forskolin and phorbol ester, secretagogues acting downstream from receptors, was also increased by Dex. These data show that Dex acts directly on the type II cell to enhance the response to surfactant secretagogues, that the effect of the hormone is mediated by the glucocorticoid receptor and suggest induction of a common downstream signaling step(s). Regulation of surfactant secretion may be an important function of glucocorticoids in the developing lung.

Surfactant secretagogue activation of protein kinase C isoforms in cultured rat type II cells.

Several lung surfactant secretagogues are known to activate protein kinase C (PKC) in type II cells. Such agents include 12-O-tetradecanoylphorbol 13-acetate (TPA) and cell-permeable diacylglycerols that directly activate PKC. Other agents include ATP and UTP, which act at P2Y(2) receptors coupled to phosphoinositide-specific phospholipase C, activation of which leads to formation of diacylglycerols and consequent activation of PKC. Activation of PKC is associated with redistribution of enzyme from a cytosolic to a membrane fraction of the cell. We examined the PKC isomers that are translocated by ATP, UTP, TPA, and dioctanoylglycerol in cultured type II cells isolated from adult rats. PKC isoforms were identified by Western blotting using isoform-specific antibodies. Treatment of type II cells with ATP, UTP, TPA, and dioctanoylglycerol resulted in a significant redistribution of PKC-mu from cytosol to membrane. TPA and dioctanoylglycerol also activated PKC-alpha, -betaI, -betaII, -delta, and -eta, but those isoforms were not activated by ATP or UTP. The effects of TPA and dioctanoylglycerol on PKC-mu were more pronounced than those of the P2Y(2) agonists, and the effect of TPA was also more rapid than that of ATP. The data show that direct activators and agents that generate endogenous diacylglycerols have different PKC activation patterns. Because it is activated by different types of secretagogues, PKC-mu may have an important role in the physiological regulation of surfactant secretion.

PKC isoforms and other signaling proteins involved in surfactant secretion in developing rat type II cells.

We previously reported that there is a developmental increase in surfactant secretion in response to P2Y2 purinoceptor agonists. UTP does not stimulate secretion in type II cells from 1- or 2-day-old rats; there is a small response to UTP in cells from 4-day-old animals, and the response increases with increasing age thereafter. Second messenger formation in response to P2Y2 agonists has a similar developmental pattern. We have investigated whether the failure to respond to P2Y2 agonists is due to a deficiency in the P2Y2 receptor or in downstream signaling factors. We compared type II cells from adult and 1- to 2-day-old rats with respect to expression of the P2Y2 receptor gene and the levels of phospholipase C-beta (PLC-beta) and protein kinase C (PKC) isomers and of the alpha-subunit of the GTP-binding protein Gq. We measured gene expression by reverse transcriptase-polymerase chain reaction and protein levels by immunoblotting. We identified PKC-alpha, -betaI, -betaII, -delta, -eta, -zeta, -theta, and -mu, PLC-beta3, and Gqalpha in adult and newborn type II cells. PKC-epsilon, -gamma, and -lambda and PLC-beta1, -beta2, and -beta4 were not present in adult or newborn type II cells. Expression of the P2Y2 receptor gene was essentially the same in newborn and adult cells. However, the levels of PKC-alpha, -betaI, -betaII, and -zeta in newborn type II cells were only 43-57% those of adult cells. The level of PKC-theta also tended to be lower in the newborn cells. There was little difference between newborn and adult type II cells in the levels of PKC-delta, -eta, and -mu, PLC-beta3, and Gqalpha. These data suggest that the lack of response of early newborn type II cells to P2Y2 agonists is not due to a lack of expression of the receptor gene but possibly to insufficient amounts of one or more of the alpha, betaI, betaII, or zeta PKC isoforms.

Early increase in expression of surfactant protein A gene in type II cells from silica-treated rats.

Silica is known to cause an increase in lung surfactant and to promote type II cell hypertrophy and hyperplasia. Two populations of type II cells can be isolated from silica-treated rats: type IIA cells that are similar to normal type II cells and type IIB cells that are larger, contain more surfactant phospholipids, and have increased rates of phospholipid biosynthesis. As much less is known about the influence of silica on the amounts of surfactant proteins (SPs) in type II cells, we examined expression of the genes for all four SPs in types IIA and IIB cells isolated from rats 1, 3, and 7 days after a single intratracheal injection of silica. There was a rapid increase in expression of the SP-A gene in type II cells from the silica-treated animals. SP-A mRNA content was 8- to 10-fold greater in types IIA and IIB cells isolated 1 day after silica injection than in type II cells from saline-injected animals. SP-A mRNA levels were also elevated in the cells isolated on days 3 and 7 after silica injection, but the extent of the increase was less than in the cells isolated on day 1 and declined with time after injection. SP-B, SP-C, and SP-D mRNA levels were 2.5- to 4-fold greater in type IIA cells on day 3 after silica injection than in control type II cells. However, those mRNA levels were not significantly increased in the type IIA cells isolated on days 1 and 7 or in type IIB cells at any time point. These data show that silica causes a rapid and substantial increase in expression of the SP-A gene in type II cells.

Glucocorticoids increase fatty-acid synthase mRNA stability in fetal rat lung.

Fatty-acid synthase (FAS) is a critical enzyme in surfactant biosynthesis. In fetal lung, glucocorticoids increase synthesis of phosphatidylcholine, the principal lipid component of surfactant, and there is evidence that this effect is mediated by increased expression of the FAS gene. Dexamethasone increases FAS activity, mass, mRNA content, and rate of transcription in cultured explants of fetal rat lung. As previous experiments with actinomycin D suggested that dexamethasone may also increase FAS mRNA content by a posttranscriptional mechanism, we examined the effect of the hormone on FAS mRNA stability. Explants of 19-day fetal rat lungs were cultured for 44 h with and without 100 nM dexamethasone. Some explants were harvested at that point, and others were cultured further with 60 microM 5,6-dichlororibofuranosylbenzimidazole (DRB), an inhibitor of transcription. RNA was then extracted, and FAS mRNA levels were measured by Northern analysis, mRNA stability was assessed by comparing the amount remaining after culture with DRB with the initial level before addition of the inhibitor. The apparent half-life of FAS mRNA was 4 h in control explants cultured without hormone. FAS mRNA stability was increased 84% in the explants cultured with dexamethasone for 44 h and by 40% in those cultured with the hormone for 5 h. We conclude that glucocorticoids enhance expression of the FAS gene in fetal lung by increasing mRNA stability in addition to stimulating transcription.

Influence of the protein kinase C inhibitor 3-[1-[3-(amidinothio) propyl]-1H-indoyl-3-yl]-3-(1-methyl-1H-indoyl-3-yl) maleimide methane sulfonate (Ro-318220) on surfactant secretion in type II pneumocytes.

We have investigated the influence of 3-[1-[3-(amidinothio)propyl]-1H-indoyl-3-yl]-3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate (Ro-318220), a potent and selective inhibitor of protein kinase C, on phosphatidylcholine secretion in response to surfactant secretagogues in rat type II cells. Freshly isolated cells were cultured overnight with [3H]choline to label the phosphatidylcholine pool and were preincubated for 30 min in fresh medium with or without Ro-318220. Secretagogues were then added, and the incubation was continued for 90 min after which [3H]phosphatidylcholine secretion was measured. Ro-318220 (10 microM) almost completely abolished the stimulatory effects of 36 microM terbutaline, 10 microM ATP, and 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) and significantly antagonized the effects of 10 microM 5'-(N-ethylcarboxyamido) adenosine (NECA), 100 microM dioctanoylglycerol, and 0.05 microM ionomycin. The effect of Ro-318220 was dependent on concentration. The IC50 values for Ro-318220 inhibition of the effects of terbutaline, NECA, TPA, and ionomycin were not significantly different. The IC50 value for Ro-318220 inhibition of the effect of TPA in the type II cell (0.05 microM) was similar to that reported for inhibition of protein kinase C in vitro (0.08 microM). We conclude that Ro-318220 antagonizes the effects of the different surfactant secretagogues by antagonizing a step common to the different signaling pathways. It remains to be established if it is protein kinase C or another step that is inhibited.

Adenylate cyclase-coupled ATP receptor and surfactant secretion in type II pneumocytes from newborn rats.

ATP stimulation of surfactant secretion in type II cells is mediated by both a P2Y2 receptor coupled to phospholipase C and a receptor coupled to adenylate cyclase. UTP also activates the P2Y2 receptor but does not stimulate adenosine 3',5'-cyclic monophosphate (cAMP) formation. We have examined surfactant secretion and signaling parameters in response to ATP and UTP in type II cells from newborn rats. There was a developmental increase in the response to both agonists. However, whereas ATP increased secretion as early as day 1, the effect of UTP did not become significant until 4 days after birth. ATP increased cAMP formation as early as day 1 but did not promote diacylglycerol formation or phospholipase D activation until day 4. Thus the adenylate cyclase-coupled ATP signaling mechanism is functional early in development but the P2Y2 pathway is not. We therefore used type II cells from 1- to 2-day-old rats to investigate the adenylate cyclase-coupled mechanism in the absence of interactions with the P2Y2 system. Effects of ATP and 5'-(N-ethylcarboxamido)adenosine (NECA) on surfactant secretion and cAMP formation were not additive, and their effects on secretion were antagonized by the same adenosine receptor antagonists. Overnight culture of the cells with NECA almost completely abolished the subsequent increase in cAMP formation in response to NECA, adenosine, and ATP but not to terbutaline. These data suggest that ATP, NECA, and adenosine activate the same receptor. Effects of ATP were not decreased by adenosine deaminase, showing that they are not mediated by adenosine acting directly at adenosine receptors. We suggest that ATP directly activates an adenosine receptor on the type II cell.

Glucocorticoid stimulation of fatty-acid synthase gene transcription in fetal lung: antagonism by retinoic acid.

Glucocorticoid hormones are known to stimulate the rate of fatty acid biosynthesis and to increase the activity and mRNA level of fatty-acid synthase (FAS) in late gestation fetal lung. We have now examined the effect of dexamethasone on FAS transcription in fetal rat lung. Explants of 19-day fetal rat lung cultured for 48 h in serum-free medium were exposed to dexamethasone (10(-7) M) for various time periods. Nuclei were isolated, and the rate of [32P]UTP incorporation into FAS and gamma-actin RNA transcripts was measured by transcription-elongation assay. Dexamethasone increased FAS transcription but had no effect on that of actin. The maximum effect of the hormone, approximately threefold increase, was observed 1-2 h after addition of the hormone but was still apparent up to 48 h. FAS transcription but not that of actin was inhibited by cycloheximide and puromycin in both control and dexamethasone-treated cultures. However, the stimulatory effect of the hormone was not significantly reduced by the inhibitors. Retinoic acid antagonized the stimulatory effects of dexamethasone on FAS activity, mRNA content as measured by Northern analysis, mass as measured by Western blotting, and rate of transcription. The effect of retinoic acid was dependent on concentration in the relatively narrow range of 5 x 10(-6) to 5 x 10(-4) M. These data show that glucocorticoids stimulate transcription of the FAS gene in late gestation fetal rat lung, that normal transcription of the FAS gene is dependent on ongoing protein synthesis, and that glucocorticoid stimulation of FAS gene expression is antagonized by retinoic acid.

P2u purinoceptor stimulation of surfactant secretion coupled to phosphatidylcholine hydrolysis in type II cells.

ATP is known to stimulate surfactant phospholipid secretion in type II cells, and there is evidence that this effect is mediated by a P2 purinoceptor. At least five subtypes of the P2 receptor have been reported, but it is not clear which one exists on the type II cell. To determine whether it is the P2u subtype, at which UTP is equipotent with ATP, we have compared the effects of ATP and UTP on phosphatidylcholine secretion and second messenger formation in primary cultures of rat type II cells. ATP and UTP were equally potent in stimulating phosphatidylcholine secretion and phospholipase D activation. The potency order, UTP = ATP > ADP > 2-methylthio-ATP, was the same as that reported for the P2u receptor. UTP stimulated diacylglycerol and phosphatidic acid formation to the same extent as ATP. ATP also increased choline formation. Formation of diacylglycerol was biphasic, and the first peak in response to ATP was previously shown to be associated with inositol trisphosphate formation. Northern analysis showed that the P2u receptor gene was expressed to a greater extent in type II cells than in whole lung. These data suggest that ATP and UTP act via a P2u receptor that is coupled to phosphoinositide-specific phospholipase C with subsequent activation of phospholipase D acting on phosphatidylcholine. ATP has also been reported to act at an additional type II cell receptor coupled to adenylate cyclase. In contrast, UTP did not promote adenosine 3',5'-cyclic monophosphate formation and therefore does not act at that receptor.

Molecular and cellular processing of lung surfactant.

Pulmonary surfactant, a complex material that lines the alveolar surface of the lung, is synthesized in the type II pneumocyte. Surfactant consists largely of phospholipids, of which phosphatidylcholine is by far the most abundant component, and is mainly responsible for surface activity. Surfactant also contains four unique proteins, surfactant protein (SP)-A, SP-B, SP-C, and SP-D, which are synthesized in a lung-specific manner. SP-A and SP-D are glycoproteins (M(r) approximately 30,000-40,000) whereas SP-B and SP-C are small (M(r) approximately 5,000-18,000), extremely hydrophobic proteolipids released from large precursors by proteolysis. Synthesis of surfactant lipids and proteins is developmentally regulated in fetal lung and can be accelerated by glucocorticoids and other hormones. Developing fetal lung in vivo and in organ culture has been used extensively to study regulation of surfactant synthesis and gene expression. Glucocorticoids stimulate the rate of fetal lung phosphatidylcholine biosynthesis and the activity of the rate-regulatory enzyme, cholinephosphate cytidylyltransferase (CYT). The hormone, however, does not increase the amount of CYT; there is evidence that the increase in activity is mediated by increased fatty biosynthesis due to enhanced expression of the fatty acid synthase gene. Glucocorticoids also regulate expression of the SP-A, SP-B, and SP-C genes in the late gestation fetal lung. Hormone response elements and other cis-acting regulatory elements have been identified in the 5'-flanking regions of the SP-A, SP-B, and SP-C genes. Surfactant phospholipids are stored in lamellar bodies, secretory granules in the type II cell, and secreted by exocytosis. Lamellar bodies are also rich in SP-B and SP-C but there are conflicting data on the cellular distribution of SP-A. Secretion of SP-A may be constitutive and occur independently of lamellar bodies. Phosphatidylcholine secretion is a regulated process, and in isolated type II cells it can be stimulated by physiological and other agents that act via at least three signal-transduction mechanisms. After secretion, surfactant is transformed into tubular myelin, and the lipid and protein components are separated as the lipid is inserted into a monolayer at the air-liquid interface. The majority of surfactant is removed from the alveolar space by reuptake into the type II cell by mechanisms that may include receptor-mediated endocytosis. Some components of surfactant are directly recycled into new surfactant whereas other components are degraded.

Fatty-acid synthase activity and mRNA level in hypertrophic type II cells from silica-treated rats.

Silica instillation causes a massive increase in lung surfactant. Two populations of type II pneumocytes can be isolated from rats administered silica by intratracheal injection: type IIA cells similar to type II cells from normal rats and type IIB cells, which are larger and contain elevated levels of surfactant protein A and phospholipid. Activities of choline-phosphate cytidylyltransferase, a rate-regulatory enzyme in phosphatidylcholine biosynthesis, and fatty-acid synthase (FAS) are increased in type IIB cells isolated from rats 14 days after silica injection. In the present study, we examined the increase in FAS and cytidylyltransferase activities in type IIB cells as a function of time after silica administration. FAS activity increased rapidly, was approximately threefold elevated 1 day after silica administration and has reached close to the maximum increase by 3 days. Cytidylyltransferase activity was not increased on day 1, was significantly increased on day 3 but was not maximally increased until day 7. Inhibition of de novo fatty-acid biosynthesis, by in vivo injection of hydroxycitric acid and inclusion of agaric acid in the type II cell culture medium, abolished the increase in cytidylyltransferase activity on day 3 but not FAS and had no effect on activities of two other enzymes of phospholipid synthesis. FAS mRNA levels were not increased in type IIB cells isolated 1-14 days after silica injection. These data show that the increase in FAS activity in type IIB cells is an early response to silica, that it mediates the increase in cytidylyltransferase activity, and that it is not due to enhanced FAS gene expression.

Secretion of surfactant protein A from rat type II pneumocytes.

Secretion of surfactant phosphatidylcholine has been extensively studied and there is evidence that it is a regulated process that can be influenced by a variety of physiological factors and pharmacological agents. In contrast, secretion of the major surfactant protein, surfactant protein A (SP-A), has been investigated to much lesser extent. It is not known whether SP-A secretion is constitutive or regulated and, if regulated, whether its regulation is similar to that of phosphatidylcholine. To address those questions we measured SP-A secretion in primary cultures of type II pneumocytes under conditions identical to those used to study phosphatidylcholine secretion. Freshly isolated cells from adult rats were cultured overnight, washed, and then incubated in fresh medium in the presence and absence of surfactant phospholipid secretagogues. As previously reported for phosphatidylcholine, SP-A secretion was linear with time for up to 4 h. However, the rate of SP-A secretion, approximately 6% of total SP-A (cells+medium) released into the medium per hour, was more than sixfold greater than that of the lipid. Although freshly isolated cells contained 70% more SP-A than cells that were cultured overnight, the rate of SP-A secretion was not significantly different. Secretion of SP-A by freshly isolated or cultured type II cells was not increased by a combination of ATP, terbutaline, the adenosine A2 receptor agonist 5'(N-ethylcarboxyamido)adenosine, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin at concentrations that optimally stimulated phosphatidylcholine secretion. We conclude that secretion of the major lipid and protein components of surfactant are independently regulated.

Glucocorticoid regulation of fatty acid synthase gene expression in fetal rat lung.

There are developmental and glucocorticoid-induced increases in the rate of fatty acid biosynthesis and in the activity of fatty acid synthase in late gestation fetal lung. We have now measured mRNA levels of fatty acid synthase and of two other enzymes of fatty acid biosynthesis, ATP citrate lyase and acetyl-CoA carboxylase, in developing fetal and postnatal rat lung and in fetal lung explants cultured with and without dexamethasone. There was a developmental increase in the mRNA for fatty acid synthase with the maximum level being reached on fetal day 21 (term is fetal day 22). This profile was similar to that reported for de novo fatty acid synthesis and fatty acid synthase activity. There was a similar but less pronounced developmental increase in the mRNA for ATP citrate lyase and a corresponding increase in its activity. There was no developmental change in the mRNA for acetyl-CoA carboxylase. Dexamethasone increased the level of fatty acid synthase mRNA approximately threefold but had no effect on those for ATP citrate lyase and acetyl-CoA carboxylase. The effect of dexamethasone on fatty acid synthase mRNA was rapid, biphasic, and partly inhibited by actinomycin D and cycloheximide. We conclude that glucocorticoids increase expression of the gene for fatty acid synthase in fetal lung. The effect of the hormone appears to be due to increased transcription and post-transcriptional events and is dependent on protein synthesis.

Signal-transduction mechanisms of ATP-stimulated phosphatidylcholine secretion in rat type II pneumocytes: interactions between ATP and other surfactant secretagogues.

ATP stimulates phosphatidylcholine secretion in type II cells, an effect that is mediated by both adenosine A2 receptors coupled to adenylate cyclase and P2 receptors coupled to phosphoinositide-specific phospholipase C. Activation of these effector enzymes leads to formation of cAMP, diacylglycerols and inositol trisphosphate (IP3). cAMP in turn activates cAMP-dependent protein kinase, diacylglycerols activate protein kinase C and IP3 promotes Ca2+ mobilization. To further investigate the signal-transduction mechanisms mediating the ATP effect, we examined its action in combination with that of other surfactant secretagogues: 5'(N-ethylcarboxyamido)adenosine (NECA), a A2 agonist that activates adenylate cyclase; TPA (12-O-tetradecanoylphorbol-13-acetate), a direct activator of protein kinase C; and ionomycin, an ionophore that increases intracellular Ca2+. The effects of NECA, TPA and ionomycin were additive and thus consistent with independent signaling mechanisms. However, the effects of all combinations of three or four secretagogues that contained ATP were 10-20% less than additive. This suggested that ATP and other secretagogues act via common mechanisms. Calmodulin antagonists decreased the effects of ionomycin and ATP by approx. 60% and 30%, respectively, but did not decrease the effects of NECA, terbutaline or TPA. Complete inhibition of the effect of ATP was achieved with a combination of a calmodulin antagonist, an A2 antagonist and a protein kinase C inhibitor. These and previous data suggest that the stimulatory effect of ATP on phosphatidylcholine secretion in type II cells is mediated by three signal-transduction mechanisms: activation of cAMP-dependent protein kinase; activation of protein kinase C; and a calmodulin-dependent mechanism.

Activation of phospholipase D in rat type II pneumocytes by ATP and other surfactant secretagogues.

Surfactant phospholipid secretion can be stimulated by a variety of agonists acting via a number of signal-transduction mechanisms. To determine whether phospholipase D has a role in surfactant secretion, we examined phosphatidylethanol formation in response to surfactant secretagogues in primary cultures of rat type II cells. Phosphatidylethanol formation was stimulated by ATP, 12-O-tetradecanoylphorbol-13 acetate (TPA), and dioctanoylglycerol, surfactant secretagogues that also activate protein kinase C. Surfactant secretagogues that act via other signaling mechanisms had no effect on phosphatidylethanol formation. The effect of ATP on phosphatidylethanol formation was dependent on time, with the maximum stimulation being achieved in approximately 10 min. It was also dependent on ATP concentration. The ATP concentration eliciting 50% of the maximum effect (EC50) was 2.45 x 10(-6) M. This was similar to the EC50 reported for ATP stimulation of surfactant secretion. ATP analogues also stimulated phosphatidylethanol formation with a potency order generally similar to that reported for surfactant secretion. The effects of ATP, TPA, and dioctanoylglycerol were antagonized by protein kinase C inhibitors. We speculate that activation of protein kinase C either directly by TPA and dioctanoylglycerol or indirectly subsequent to phosphoinositide-specific phospholipase C activation by ATP leads to initial stimulation of surfactant secretion as well as activation of phospholipase D. The action of phospholipase D on cellular phospholipids then leads to further generation of diacylglycerols, continued activation of protein kinase C, and sustained surfactant secretion.

Potentiation of A2 purinoceptor-stimulated surfactant phospholipid secretion in primary cultures of rat type II pneumocytes.

Surfactant secretion is mediated by a number of different signal-transduction mechanisms. Positive and negative interactions between different signaling pathways can have an important influence on the overall regulation of secretion. To examine interactions between the adenosine A2 receptor-mediated pathway and those involving activation of protein kinase C and a Ca++/calmodulin-dependent system, we examined the effect of the A2 agonist 5'-(N-ethylcarboxyamido) adenosine (NECA) in combination with 2 activators of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA) and dioctanoylglycerol, and the Ca++ ionophore ionomycin on phosphatidylcholine secretion in primary cultures of rat type II cells. The individual agonists increased secretion 3-5-fold over the rate in control cells. The stimulatory effects of NECA+TPA, NECA+dioctanoylglycoerol, and NECA+ionomycin were 44%, 20%, and 44% greater, respectively, than expected by addition of the effects of the individual agonists. NECA increased cAMP formation while the other agonists did not. However, the effect of NECA on cAMP formation was significantly enhanced by TPA and dioctanoylglycerol, while the duration of the increase in cAMP level was prolonged by dioctanoylglycerol and ionomycin. Although the possible involvement of other second messenger systems cannot be excluded, we speculate that the synergistic interaction between the agonists in stimulating phosphatidylcholine secretion is mediated by increased cAMP levels.