Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with pi GST.

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, can protect cells against membrane oxidation through glutathione (GSH)-dependent reduction of phospholipid hydroperoxides to corresponding alcohols. However, purified native or recombinant enzyme in vitro generally lacks GSH peroxidase (GPx) activity because of oxidation of its single conserved cysteine. Reduction of the resultant oxidized cysteine is difficult because of its protected location within the homodimer formed by the oxidized protein monomers. Partial purification of 1-cysPrx from bovine lung revealed the presence of pi GST in an active preparation, while purification to homogeneity yielded enzyme that inactivated with time. We show that heterodimerization of 1-cysPrx with GSH-saturated pi GST results in glutathionylation of the oxidized cysteine in 1-cysPrx followed by subsequent spontaneous reduction of the mixed disulfide and restoration of enzymatic activity. Maximum activation of 1-cysPrx occurred with a 1:1 molar ratio of GSH-saturated pi GST and a 2:1 molar ratio of GSH to 1-cysPrx. Liposome-mediated delivery of oxidized recombinant enzyme into NCI-H441 cells that lack 1-cysPrx but express pi GST resulted in 1-cysPrx activation, whereas activation in MCF7 cells required co-delivery of pi GST. Our data indicate a physiological mechanism for glutathionylation of the oxidized catalytic cysteine of 1-cysPrx by its heterodimerization with pi GST followed by its GSH-mediated reduction and enzyme activation.

Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic.

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.

1-Cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase A2 activities.

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca(2+)-independent phospholipase A(2) (aiPLA(2)). The cDNA encoding aiPLA(2) was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH(2) terminus, did not exhibit aiPLA(2) activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H(2)O(2) at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA(2) activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA(2) activity. Mutation of Ser(32) to Ala abolishes aiPLA(2) activity, yet the NSGPx activity remains unaffected; a Cys(47) to Ser mutant is devoid of peroxidase activity but aiPLA(2) activity remains intact. These results suggest that Ser(32) in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA(2), while Cys(47) in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.

Transcriptional activation and protein binding by two regions of the rat surfactant protein A promoter.

Surfactant protein A (SP-A) is expressed in lung alveolar type II cells and bronchiolar Clara cells. We have identified two active regions in the promoter of the rat SP-A gene by deletion analysis of a plasmid containing 163 bp before the start of transcription (-163 bp), linked to a reporter gene. Constructs were transfected into lung cell lines derived from each of the cell types that produces SP-A. We found a novel region of promoter activity at approximately 90 bp before the transcriptional start (SP-A(-90)). Mutation of four nucleotides in SP-A(-90) that are highly conserved among species (-92 to -89 bp) decreased expression of the SP-A construct by approximately 50% in both cell lines. Electrophoretic mobility shift analysis showed specific binding to SP-A(-90) by nuclear proteins from the cell lines, as well as from rat lung and liver. The electrophoretic mobility of the bands shifted by lung nuclear proteins changed late in fetal development. Although in the Clara cell line no reduction of promoter activity was seen on deletion of the region upstream of SP-A(-90), in the type II cell line, deletion of residues -163 to -133 did reduce activity by approximately 50%. This region contains a recognition element for thyroid transcription factor-1 (TTF-1). Endogenous TTF-1 binding activity was substantially higher in the type II cell line than in the Clara cell line, but cotransfection of a TTF-1 expression plasmid enhanced expression of the SP-A construct better in the Clara cell line than in the type II cell line. These results suggest that the recognition element for TTF-1 has varying activity in the lung cell lines of different origin due to the availability of TTF-1.

Phospholipid hydroperoxides are substrates for non-selenium glutathione peroxidase.

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl- or sn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM(-1) min(-1)) were k(1) = 1-3 x 10(5) and k(2) = 4-11 x 10(4). In the presence of 0.36 mM GSH, apparent K(m) was 120-130 microM and apparent V(max) was 1.5-1.6 micromol/min/mg of protein. Assays with H(2)O(2) and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7-8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K(i) 4 miroM. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.

Cloning and expression of rat lung acidic Ca(2+)-independent PLA2 and its organ distribution.

A clone for a rat acidic Ca(2+)-independent phospholipase A2 (aiPLA2) was isolated from a cDNA library prepared from rat granular pneumocytes with a probe based on the human aiPLA2 sequence (T.S. Kim, C.S. Sundaresh, S. I. Feinstein, C. Dodia, W. R. Skach, M. K. Jain, T. Nagase, N. Seki, K. Ishikawa, N. Nomura, and A. B. Fisher. J. Biol. Chem. 272: 2542-2550, 1997). In addition, a consensus sequence for mouse aiPLA2 was constructed from several mouse cDNA clones in the GenBank and dbEST databases. Each sequence codes for a 224-amino acid protein with 88% identity of the amino acids among the three species and conservation of a putative lipase motif (GDSWG). Translation of mRNA produced from the rat clone in a wheat germ system resulted in expression of PLA2 activity with properties similar to those of the human enzyme, i.e., acidic pH optimum and Ca2+ independence. The localization of aiPLA2 in rat tissues was studied with the human cDNA probe, polyclonal and monoclonal antibodies, and aiPLA2 activity. aiPLA2 is present in the lung as evidenced by high levels of mRNA and protein expression and by enzymatic activity that is inhibited by anti-PLA2 antibody and by the transition state analog 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Immunocytochemistry showed the presence of aiPLA2 in alveolar type II cells, alveolar macrophages, and bronchiolar epithelium. In the brain, heart, liver, kidney, spleen, and intestine, aiPLA2 mRNA content was < 50% of that in the lung, immunoreactive protein was not detectable, and enzymatic activity was not inhibited by MJ33 or aiPLA2 antibody. These results show marked enrichment of aiPLA2 in the lung compared with the other organs and suggest translational control of aiPLA2 expression.

Influence of epidermal growth factor and transforming growth factor beta-1 on patterns of fetal mouse lung branching morphogenesis in organ culture.

Transforming growth factor-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation, and epidermal growth factor (EGF), a mitogenic polypeptide that binds to cell surface receptors, are important regulators of cell differentiation; however, their distinct role(s) in lung development and their mechanisms of action are not well understood. We evaluated the effects of these factors on lung morphogenesis in murine fetal lungs at gestational day 14 (time:zero) and again after 7 days in culture. Baseline controls were cultured after tracheal transection in supplemented BGJb medium, and other tracheally transected lungs were cultured following addition of EGF (10 ng/ml BGJb), TGF-beta1 (2 ng/ml BFJb), or with both in combination added to the medium. The control lungs in culture had poorly developed airways and an absence of defined acinar structures. The addition of EGF resulted in hyperplasia of primary airways with stunted outgrowths, monopodial branching, and absence of distinct acinar structures. Addition of TGF-beta1 alone, led to significant elongation of primary airways, without normal airway branching; however, terminal dipodial branching was seen and the prospective pulmonary acini were well defined. Combination of these growth factors (GF) resulted in a more normal branching pattern and differentiation, suggesting their epigenetic role in lung morphogenesis and mutual interactive mechanisms that regulate lung development. These lungs had more abundant and larger lamellar bodies than those after other treatments. Control lungs remained immature with prominent glycogen aggregates with occasional dense lamellar bodies. The total protein and DNA contents were highest with EGF treatment, followed by combination treatment; these observations were supported by immunohistochemical localization of proliferating cell nuclear antigen, an indication of the proliferative state of tissues. All the surfactant proteins were relatively unaltered and their messages were up-regulated for SP-A, but down-regulated for SP-B and SP-C in the lungs treated with growth factors. In conclusion, we have demonstrated enhanced biochemical and structural development of lungs treated in vitro with GF, and propose that further research in this area may lead to therapeutic uses of GF alone or in combination with other agents for the treatment of newborn respiratory distress due to lung immaturity or hypoplastic lung development.

An antisense oligodeoxyribonucleotide to m-calpain mRNA inhibits secretion from alveolar epithelial type II cells.

We investigated the effect of translational suppression of m-calpain on [3H]-phosphatidylcholine (PC) secretion utilising an antisense oligodexoyribonucleotide (oligo) directed against mRNA encoding m-calpain catalytic subunit. Two types of oligo, sense (S) and antisense (AS), to a portion of exon 12 of rat m-calpain catalytic subunit mRNA were tested. Constitutive secretion was decreased by 23% by AS-oligo (1 microM) treatment, while S-oligo (1 microM) had no effect. TPA-stimulated secretion was inhibited about 50-60% by AS-oligo (1-3 microM) and the inhibition was concentration-dependent, while S-oligo (1 microM) only inhibited about 10% of TPA-stimulated secretion. Northern and Western blot analyses revealed that the AS-oligo treatment reduced m-calpain mRNA and protein levels by 32% and 78%, respectively. The data indicate that antisense strategy is effective in suppressing calpain expression and type II cell secretion.

Phorbol ester down-regulation of lung surfactant protein B gene expression by cytoplasmic trapping of thyroid transcription factor-1 and hepatocyte nuclear factor 3.

The lung-specific surfactant protein B (SP-B) is essential for surfactant function and normal respiration. We investigated the role of thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor 3 (HNF3) in the down-regulation of SP-B gene expression by phorbol ester in pulmonary adenocarcinoma H441 cells. Responsiveness to 12-O-tetradecanoylphorbol-13-acetate (TPA) localized to the SP-B proximal promoter (-140/-65 bp) and specifically to binding sites for TTF-1 and HNF3, which act as cell-specific enhancers of SP-B expression. Treatment of cells with TPA (10 nM) caused a time-dependent decrease in both TTF-1 and HNF3 in nuclear extracts and accumulation of both factors in the cytoplasm as assessed by electromobility shift, Western, Southwestern, and immunofluorescence assays. Treatment did not alter the mRNA content or DNA binding activity for either transcription factor. We conclude that down-regulation of SP-B gene expression by phorbol ester involves cytoplasmic trapping and loss of TTF-1 and HNF3 from the nucleus. This mechanism of action is independent of AP-1 and other transcription factors known to be influenced by phorbol ester.

Identification of a human cDNA clone for lysosomal type Ca2+-independent phospholipase A2 and properties of the expressed protein.

A Ca2+-independent phospholipase A2 (PLA2) maximally active at pH 4 and specifically inhibited by the transition-state analogue 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) was isolated from rat lungs. The sequence for three internal peptides (35 amino acids) was used to identify a 1653-base pair cDNA clone (HA0683) from a human myeloblast cell line. The deduced protein sequence of 224 amino acids contained a putative motif (GXSXG) for the catalytic site of a serine hydrolase, but showed no significant homology to known phospholipases. Translation of mRNA produced from this clone in both a wheat germ system and Xenopus oocytes showed expression of PLA2 activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA2 with dipalmitoylphosphatidylcholine as substrate were Km = 0.25 mM and Vmax = 1.89 nmol/h. Activity with alkyl ether phosphatidylcholine as substrate was decreased significantly compared with diacylphosphatidylcholine. Significant lysophospholipase, phospholipase A1, or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase activity was not observed. Enzyme activity was insensitive to p-bromophenacyl bromide, bromoenol lactone, trifluoromethylarachidonoyl ketone, mercaptoethanol, and ATP, but was inhibited by MJ33 and diethyl p-nitrophenyl phosphate, a serine protease inhibitor. SDS-polyacrylamide gel electrophoresis with autoradiography of the translated [35S]methionine-labeled protein confirmed a molecular mass of 25.8 kDa, in good agreement with the enzyme isolated from rat lung. By Northern blot analysis, mRNA corresponding to this clone was present in both rat lung and isolated rat granular pneumocytes. These results represent the first molecular cloning of a cDNA for the lysosomal type Ca2+-independent phospholipase A2 group of enzymes.

Sequence of rat surfactant protein A gene and functional mapping of its upstream region.

Surfactant protein A (SP-A) is the major pulmonary surfactant protein. We have isolated a rat SP-A genomic clone and determined the sequence from 2.9 kilobases upstream of the transcriptional start through the termination of translation. The exon-intron structure of the rat gene has been determined and compared with the mouse, rabbit, and human genes. We have localized the major transcriptional start site in adult rat lung to nucleotide 30 downstream from the start of the TATA box. Functional mapping indicates that a DNA fragment containing 163 nucleotides upstream of the transcriptional start (-163) can function as a promoter of transcription of a reporter gene in both lung and nonlung derived cell lines. However, the function of this element is weaker in cells of nonlung origin. DNA elements located between -2902 and -163 silence the promoter activity in both lung and nonlung cells. Because the SP-A gene promoter region exhibits limited tissue specificity, the results suggest the existence of other DNA elements which overcome the silencer and confer further lung specificity.

Transcriptional regulation of the apolipoprotein B-100 gene: identification of cis-acting elements in the first nontranslated exon of the human apolipoprotein B-100 gene.

Apolipoprotein B-100, produced primarily in the human liver, is the sole protein component of low-density lipoprotein and serves as a ligand for the LDL receptor. Two cis-acting positive elements between -128 and -70 control hepatic cell-specific expression of the human apoB gene (H. K. Das, T. Leff, and J. L. Breslow, J. Biol. Chem. 263: 11452-11458, 1988). In this study, two apoB cis-acting elements (+20 to +40; +43 to +53) have been identified using DNase I footprint analysis. Through in vitro mutagenesis and transient transfection experiments in Hep G2 and HeLa cells, the element (+20 to +40) was observed to have a negative effect on transcription of the apoB gene. The element (+43 to +53) was found to have a strong positive effect on apoB gene transcription in Hep G2 cells and mild positive effect in HeLa cells. Therefore these two cis-acting elements mediate hepatic-cell specific expression of the apolipoprotein gene by interacting with trans-acting protein factors.

Correlation of expression of transcription factor C/EBP alpha and surfactant protein genes in lung cells.

C/EBP alpha is a transcription factor which can stimulate expression of genes in lipid-metabolizing epithelial cells. We have detected an increase in mRNA for C/EBP alpha in lungs of fetal rats between days 18 and 20 of gestation, correlating with events occurring during the maturation of the surfactant system, such as an increase in the amount of surfactant protein A mRNA. We have found that C/EBP alpha mRNA levels are substantially enriched in type II alveolar epithelial cells purified from adult lung and that the C/EBP alpha protein is present in type II cell nuclei. When the type II cells are removed from the lung and purified, the protein is rapidly lost. However, both surfactant protein gene expression and C/EBP alpha reappear when cells are plated on Matrigel. Levels of C/EBP alpha mRNA from purified cells decline much more slowly than the protein and are still detectable 48 h after cells have been plated on standard tissue culture plastic. We have also detected the C/EBP alpha protein in nuclear extracts of NCI-H441, a lung-derived cell line that expresses surfactant proteins A and B, but not in A549, a lung-derived cell line which does not express the surfactant proteins. Our data suggest that C/EBP alpha is involved in the development and maintenance of the surfactant system in lung type II cells.

Rabbit and rat liver nuclei both contain proteins which bind to the regions controlling apolipoprotein A-I gene expression.

We have tested the 5' flanking region of the apolipoprotein A-I (apo A-I) gene, which controls its expression in hepatic cells, for the ability to bind protein factors from rat and rabbit liver nuclei. We found that nuclear extracts from each species contain proteins which bind to three sites in the region which have been shown to be important for control of apo A-I gene transcription. These results contrast with a previous report [Dai, P. H., Lan, S. S. F., Ding, X. H. & Chao, Y. S. (1990) Eur. J. Biochem. 190, 305-310] that no rabbit liver nuclear protein could be detected which binds to the rat apo A-I upstream region and that this lack of binding could explain the failure of the rabbit liver to express the apo A-I gene. We have also shown that the low levels of apo A-I mRNA in the rabbit liver correlate with decreased transcription. Our data suggest that the lack of apo A-I gene expression in liver is a result of transcriptional control but cannot be due to simple lack of protein binding to this region of DNA.

Increased expression of apolipoprotein genes accompanies differentiation in the intestinal cell line Caco-2.

We have analyzed determinants of the synthesis and secretion of apolipoproteins including mRNA for apolipoproteins, in the human colon carcinoma cell line Caco-2 during differentiation in continuous culture. Significant increases in both cellular and secreted apolipoprotein A-I were observed early in the differentiation process. Increases in apolipoprotein B were limited to secreted protein and started later in the differentiation process. Levels of mRNA for apolipoproteins A-I, A-IV, B, C-III, and E increased significantly between the time cells reached confluence and 1 week postconfluence. The kinetics of mRNA accumulation were influenced by culture conditions. Nuclear extracts from postconfluent Caco-2 cells contained increased amounts of protein that bound to oligonucleotides containing the control regions of the apolipoprotein A-I and B genes. A competition experiment suggested that this protein recognized the control regions of both genes. We propose to name this protein DRIFT-1 (differentiation-related intestinal factor for transcription 1).

ATP and adenosine 3',5'-cyclic monophosphate stimulate the synthesis of surfactant protein A in rat lung.

Synthesis and secretion of surfactant protein A (SP-A) were studied in the isolated perfused rat lung using Trans35S-label (approximately 85% methionine, 15% cysteine) in the perfusate with or without 1 mM ATP or 0.1 mM 8-bromoadenosine 3',5',-cyclic monophosphate (8-BrcAMP) for up to 6 h of perfusion. By enzyme-linked immunosorbent assay, the SP-A content was 36 +/- 0.3% of total protein in extracellular surfactant and 10.8 +/- 1.9% of total protein in lamellar bodies of control lungs; these relativr proportions were maintained in the presence of ATP or 8-BrcAMP. Incorporation of [35S]methionine (cysteine) into the surfactant and lamellar body protein fraction could be detected at 4 h of perfusion. At 6 h, specific activity of total protein [disintegrations per minute (dpm)/micrograms)] was significantly increased in both the surfactant (54%) and lamellar body fractions (30%) under the influence of either secretagogue compared with control conditions. In the presence of ATP, there was a significant increase in the SP-A immunoprecipitable counts of 61 and 72% in extra- and intracellular compartments, respectively. However, no significant change was observed in the relative abundance of SP-A mRNA between control and secretagogue-treated lungs. This dissociation of SP-A mRNA abundance and label incorporation into protein indicates that alteration in translational efficiency or posttranslational factors may be involved in the secretagogue-induced stimulation of SP-A synthesis.

An antibody with specificity for surfactant protein C precursors: identification of pro-SP-C in rat lung.

Surfactant protein C (SP-C) is a lung-specific, hydrophobic peptide found in organic extracts of pulmonary surfactant. Alveolar SP-C (3.5 kD) is produced from proteolytic cleavage of a larger precursor molecule (pro-SP-C; 21 kD). While SP-C is synthesized by type II cells, the pathways for processing and secretion have remained elusive due, in part, to the lack of monospecific antibodies against SP-C or its precursors. This report describes production and characterization of a new antibody directed against pro-SP-C epitopes. Polyclonal antisera (anti-CPRO-SP-C) was prepared using a synthetic peptide corresponding to a portion of rat SP-C cDNA sequence (Ile26-Ser72). This contained amino acids 3-35 of mature SP-C plus additional C-terminal residues (His59-Ser72). On Western blots, anti-CPRO-SP-C competitively reacted to CPRO-SP-C but not to mature SP-C. Immunoblots of in vitro synthesized pro-SP-C confirmed that the antisera also recognized native protein. Immunocytochemistry with anti-CPRO-SP-C demonstrated staining for pro-SP-C peptides in isolated type II cells as well as in alveolar epithelial cells of rat lung sections. Pro-SP-C preferentially co-localized to cells that stained positive for Maclura pomifera antigen. Anti-CPRO-SP-C staining was not observed in lung interstitium, pulmonary vasculature, or several control tissues (brain, heart, and liver were negative). Western blotting of subcellular fractions demonstrated pro-SP-C peptides in plasma membrane (20 kD) and microsomal (20 and 21 kD) fractions with a 16 kD peptide present in lamellar bodies. No pro-SP-C peptides were detected in purified surfactant. These results demonstrate the use of a synthetic peptide to generate specific antiserum against more hydrophilic domains of pro-SP-C sequences and confirm that SP-C propeptides are unique to the lung.

cAMP increases synthesis of surfactant-associated protein A by perfused rat lung.

Synthesis and secretion of surfactant-associated protein were studied in isolated rat lungs perfused with [3H]phenylalanine or [35S]methionine in synthetic medium. Surfactant was isolated by lung lavage and density-gradient centrifugation followed by dialysis to remove unincorporated amino acid and extraction with ethanol-ether to yield a delipidated protein fraction. Incorporation of [3H]phenylalanine into the delipidated surfactant protein fraction showed a lag phase of approximately 3 h followed by progressive increase over the next 3 h at a rate of 1.6 nmol.mg protein-1.h-1. With 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 0.1 mM) added to the perfusate, the incorporation rate between 3 and 6 h was increased by 75%. 3H specific activity in a delipidated lamellar body-rich fraction isolated from lung homogenates was unchanged by 8-BrcAMP at 3 h but was increased by 45% at 6 h. The major peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surfactant and lamellar bodies corresponded to proteins of 27-36 kDa that were identified as surfactant protein A (SP-A) by immunoblot. In the presence of 8-BrcAMP during 6 h of perfusion, specific activity of 35S-labeled SP-A in immunoprecipitated protein was increased by 93% and the SP-A mRNA content of lung was increased 145%. These results show that isolated perfused lungs synthesize and secrete surfactant-associated proteins and that the presence of a permeable cAMP analogue in the lung perfusate leads to increased secretion followed by induction of synthesis for SP-A.