Pulmonary surfactant metabolism in infants lacking surfactant protein B.

Infants with inherited deficiency of pulmonary surfactant protein (SP) B develop respiratory failure at birth and die without lung transplantation. We examined aspects of surfactant metabolism in lung tissue and lavage fluid acquired at transplantation or postmortem from ten infants born at term with inherited deficiency of SP-B; comparison groups were infants with other forms of chronic lung disease (CLD) and normal infants. In pulse/chase labeling studies with cultured deficient tissue, no immunoprecipitable SP-B was observed and an approximately 6-kD form of SP-C accumulated that was only transiently present in CLD tissue. SP-B messenger RNA (mRNA) was approximately 8% of normal in deficient specimens, and some intact message was observed after, but not before, explant culture. Transcription rates for SP-B, assessed by nuclear run-on assay using probes for sequences both 5' and 3' of the common nonsense mutation (121ins2), were comparable in all lungs examined. The minimal surface tension achieved with lavage surfactant was similarly elevated in both deficient and CLD infants (26-31 mN/m) compared with normal infants (6 mN/m). Both SP-B-deficient and CLD infants had markedly decreased phosphatidylglycerol content of lavage and tissue compared with normal lung, whereas synthetic rates for phospholipids, including phosphatidylglycerol, were normal. We conclude that the mutated SP-B gene is transcribed normally but produces an unstable mRNA and that absence of SP-B protein blocks processing of SP-C. Chronic infant lung disease, of various etiologies, reduces surfactant function and apparently alters phosphatidylglycerol degradation.

Nitric oxide participates in early events associated with NNMU-induced acute lung injury in rats.

In this study, the biochemical mechanisms by which N-nitroso-N-methylurethane (NNMU) induces acute lung injury are examined. Polymorphonuclear neutrophil infiltration into the lungs first appears in the bronchoalveolar lavage (BAL) fluid 24 h after NNMU injection (10.58 +/- 3.00% of total cells; P < 0.05 vs. control animals). However, NNMU-induced elevation of the alveolar-arterial O2 difference requires 72 h to develop. Daily intraperitoneal injections of the inducible nitric oxide (. NO) synthase (iNOS)-selective inhibitor aminoguanidine (AG) initiated 24 h after NNMU administration improve the survival of NNMU-treated animals. However, AG administration initiated 48 or 72 h after NNMU injection does not significantly improve the survival of NNMU-treated animals. These results suggest that. NO participates in events that occur early in NNMU-induced acute lung injury. BAL cells isolated from rats 24 and 48 h after NNMU injection produce elevated. NO and express iNOS during a 24-h ex vivo culture. AG attenuates. NO production but does not affect iNOS expression, whereas actinomycin D prevents iNOS expression and attenuates. NO production by BAL cells during this ex vivo culture. These results suggest that NNMU-derived BAL cells can stimulate iNOS expression and. NO production during culture. In 48-h NNMU-exposed rats, iNOS expression is elevated in homogenates of whole lavaged lungs but not in BAL cells derived from the same lung. These findings suggest that the pathogenic mechanism by which NNMU induces acute lung injury involves BAL cell stimulation of iNOS expression and. NO production in lung tissue.

Recombinant SP-D carbohydrate recognition domain is a chemoattractant for human neutrophils.

Human pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin with high binding specificity to alpha-D-glucosyl residues. It is composed of four regions: a short NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain ("neck" region), and a COOH-terminal carbohydrate recognition domain (CRD). Previous studies demonstrated that SP-D is chemotactic for inflammatory cells. To test which domain of SP-D might play a role in this function, a mutant that contains only neck and CRD regions was expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose. A 17-kDa recombinant SP-D CRD was identified by two antibodies (antisynthetic SP-D COOH-terminal and neck region peptides) but not by synthetic SP-D NH2-terminal peptide antibody. The recombinant SP-D CRD was confirmed by amino acid sequencing. Gel-filtration analysis found that 84% of CRD was trimeric and the rest was monomeric. Analysis of the chemotactic properties of the trimeric CRD demonstrated that the CRD was chemotactic for neutrophils (polymorphonuclear leukocytes), with peak activity at 10(-10) M equal to the positive control [formyl-Met-Leu-Phe (fMLP) at 10(-8) M]. The chemotactic activity was abolished by 20 mM maltose, which did not suppress the chemotactic response to fMLP. The peak chemotactic activity of the CRD is comparable to the activity of native SP-D, although a higher concentration is required for peak activity (10(-10) vs. 10(-11) M). The chemotactic response to CRD was largely prevented by preincubation of polymorphonuclear leukocytes with SP-D, and the response to SP-D was prevented by preincubation with CRD. These preincubations did not affect chemotaxis to fMLP. These results suggest that trimeric CRD accounts for the chemotactic activity of SP-D.

Expression of laminin alpha3, alpha4, and alpha5 chains by alveolar epithelial cells and fibroblasts.

Laminins are principal components of basement membranes. Eleven laminin isoforms are known, each a heterotrimer composed of polypeptide chains designated alpha, beta, and gamma. Five alpha chains have been identified to date: alpha1, alpha2, alpha3, alpha4, and alpha5. Recent studies of fetal and adult mouse lung show prominence of alpha3, alpha4, and alpha5 in alveolar tissue, and point to differences in the cellular expression of these alpha chains in the developing alveolus. We examined isolated rat alveolar type II cells and lung fibroblasts for expression of laminins alpha3, alpha4, and alpha5. We found that laminin alpha3 was expressed only by alveolar epithelial cells, that laminin alpha4 was expressed only by lung fibroblasts, and that laminin alpha5 was expressed primarily by alveolar epithelial cells. Metabolic labeling and immunoprecipitation confirmed the production of laminin alpha4 by fibroblasts and laminin alpha5 by alveolar epithelial cells in culture. These studies indicate that different alveolar cell types contribute different laminin alpha chains to the laminin isoforms in alveolar basement membranes. Immunohistochemistry showed colocalization of these laminin alpha chains with the laminin beta1, beta2, and gamma1 chains, indicating the likelihood that laminins 6 to 11 are present in alveolar basement membranes.

Bovine surfactant therapy for patients with acute respiratory distress syndrome.

Lung surfactant is deficient in patients with acute respiratory distress syndrome (ARDS). We performed a randomized, prospective, controlled, open-label clinical study of administration of a bovine surfactant to patients with ARDS to obtain preliminary information about its safety and efficacy. Patients received either surfactant by endotracheal instillation in addition to standard therapy or standard therapy only. Three different groups of patients receiving surfactant were studied: patients receiving up to eight doses of 50 mg phospholipids/kg, those receiving up to eight doses of 100 mg phospholipids/kg, and those receiving up to four doses of 100 mg phospholipids/kg. Outcome measures included ventilatory support parameters, arterial blood gases, organ system failures, bronchoalveolar lavage (BAL) analyses, immunologic analyses, survival, and adverse events during the 28-d study period. Fifty-nine study patients were evaluable; 43 in the surfactant group and 16 in the control group. The FI(O2) at 120 h after treatment began was significantly decreased only for patients who received up to four doses of 100 mg phospholipids/kg surfactant as compared with control patients (p = 0.011). Mortality in the same group of patients was 18.8%, as compared with 43.8% in the control group (p = 0.075). The surfactant instillation was generally well tolerated, and no safety concerns were identified. This pilot study presents preliminary evidence that surfactant might have therapeutic benefit for patients with ARDS, and provides rationale for further clinical study of this agent.

Inhibition of nitric oxide synthase attenuates NNMU-induced alveolar injury in vivo.

The purpose of this study was to determine if the acute alveolar injury induced by subcutaneous injections of N-nitroso-N-methylurethane (NNMU) in rats is mediated by nitric oxide (NO.). We show that intraperitoneal injections of the NO. synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine significantly attenuate the NNMU-induced alveolar injury as assessed by 1) normalization of the alveolar-arterial O2 difference, 2) attenuation of the lowered phospholipid-to-protein ratio in the crude surfactant pellet (CSP), 3) attenuation of the elevated minimal surface tension of the CSP, and 4) attenuation of polymorphonuclear neutrophilic infiltration into the alveolar space. Injections of N omega-nitro-D-arginine methyl ester, the inactive stereoisoform of L-NAME, did not affect the acute lung injury. Western blot analysis of whole lung homogenates demonstrate an elevated expression of transcriptionally inducible, Ca(2+)-independent NOS (iNOS) in NNMU-injected rats compared with control saline-injected rats. NOS inhibitors did not affect NNMU-induced iNOS expression. These investigations demonstrate that the inhibition of NOS attenuates NNMU-induced acute lung injury, suggesting a role for NO. in the progression of acute respiratory distress syndrome.

Functional impairment of bronchoalveolar lavage phospholipids in early Pneumocystis carinii pneumonia in rats.

Surfactant abnormalities may contribute to the impairment of gas exchange observed in Pneumocystis carinii pneumonia. Analysis of rat bronchoalveolar lavage (BAL) lipid extracts from normal controls, steroid controls, trimethaprim-sulfamethoxazole (TMP-SMX) controls, TMP-SMX/P. carinii pneumonia controls, and P. carinii pneumonia animals reveal similar total phospholipid and total protein levels. However, there was a marked reduction in phosphatidylglycerol (PG) from the BAL of P. carinii pneumonia rats as compared with control animals, with a decrease from 4.91 +/- 1.29 nmol/mg protein to 0.46 +/- 0.57 nmol/mg protein (p<0.05) and a decrease, as a percent of total phospholipids, from 7.7% +/- 0.88% to 0.91% +/- 0.59% (p<0.001). Furthermore, in vitro surface activities of BAL lipid extracts from control and P. carinii pneumonia rats revealed minimum surface tension increases from 9.38 +/- 1.71 mN/m in controls to 16.36 +/- 0.83 mN/m in P. carinii pneumonia rats (p<0.05) and likewise maximum surface tension increases from 22.14 +/- 4.34 mN/m to 38.57 +/- 2.07 mN/m (p<0.01). Of interest, the surface activity of PG-deficient P. carinii pneumonia BAL lipid extracts is completely restored to that of normal controls by the addition of exogenous PG. These findings suggest that a functionally abnormal surfactant occurs in P. carinii pneumonia and that this may account, in part, for the impairment of gas exchange observed in this disorder.

Entactin expression by rat lung and rat alveolar epithelial cells.

Although the composition of the subepithelial basement membrane of the alveolar septum has been studied in detail, there is relatively little information about which cells produce it. We examined intact rat lung and isolated rat alveolar type II cells for the expression of entactin, an integral basement membrane component that binds laminin and type IV collagen. By Northern analysis, late gestation and early neonatal rat lungs expressed high levels of entactin mRNA whereas lungs from adult animals had only minimal levels of entactin mRNA. These latter findings were confirmed by in situ hybridization, which showed prominent signal for entactin mRNA in cells in the alveolar walls of neonatal animals and no signal for entactin mRNA in the alveolar walls of lungs from adult animals. The entactin mRNA throughout the alveolar walls of neonatal animals was not limited to cells that expressed surfactant-associated protein C mRNA, a marker of alveolar type II cells. Freshly harvested adult alveolar type II cells and alveolar type II cells in culture for < 6 days expressed none to minimal entactin mRNA or protein. However, with longer periods in culture, both entactin mRNA and entactin protein synthesis were evident and progressively increased. In situ hybridization indicated that >60% of the alveolar epithelial cells expressed entactin mRNA with increasing time in culture. When cultured on Engelbreth-Holm-Swarm matrix, alveolar type II cells showed the same time course of entactin mRNA expression as cells cultured on plastic. Neonatal lung mesenchymal cells produced abundant entactin in culture, consistent with the likelihood that these cells are the principal source of entactin in alveolar walls in the developing lung. These results indicate that entactin production in the normal alveolar wall occurs primarily during lung development and that mesenchymal cells are probably the principal source of production. However, because adult alveolar epithelial cells synthesize entactin in culture, it is possible that alveolar epithelium contributes to the entactin in the alveolar subepithelial basement membrane.

Partial deficiency of surfactant protein B in an infant with chronic lung disease.

OBJECTIVE: To evaluate components of pulmonary surfactant and identify mutations in the surfactant protein B gene (SP-B) of a term infant with severe respiratory distress and chronic lung disease. PATIENT AND TESTING: Respiratory distress developed in an infant delivered at term, and he required extracorporeal bypass support for 2 weeks. Until his unexpected death at 9.5 months, he was ventilator and oxygen dependent and required continual dexamethasone therapy. Tracheobronchial lavage samples were analyzed for content of surfactant proteins (SPs), and DNA from blood samples were sequenced and analyzed by polymerase chain reaction restriction analysis for the presence of SP-B gene mutations. Surfactant lipid composition and function, the contents of SPs and their messenger RNAs (mRNAs), and the immunostaining pattern for SPs were determined in postmortem lung tissue. RESULTS: The lavage sample contained SP-A but not SP-B, and DNA restriction analysis indicated that the patient and his mother were heterozygous for the previously described 121ins2 mutation of SP-B. Postmortem lung tissue contained normal levels of SP-A and its mRNA, a low but detectable level of SP-B, and near normal content of SP-B mRNA. SP-C was abundant on staining, and some 6-kd precursor was present in tissue. A surfactant fraction was deficient in phosphatidylglycerol and was not surface active. On DNA sequencing, a point mutation was found in exon 7 of the patient's SP-B gene allele without the 121ins2 mutation, resulting in a cysteine for arginine substitution, and the father was a carrier for the same mutation. CONCLUSIONS: We describe a patient who is a compound heterozygote with a new mutation and only a partial deficiency of SP-B. Some forms of inherited SP-B deficiency may have low expression of immunoreactive and possibly functional SP-B with milder lung disease and longer survival. These infants may benefit from glucocorticoid therapy and may not develop antibodies to SP-B after either lung transplant or gene therapy.

Sublethal hydrogen peroxide inhibits alveolar type II cell surfactant phospholipid biosynthetic enzymes.

Alveolar type II cell injury by phagocytic cell-derived reactive oxygen metabolites represents a potential mechanism for the altered surfactant metabolism found in patients with the adult respiratory distress syndrome (ARDS). Previous studies demonstrated altered surfactant phospholipid metabolism after sublethal oxidant exposure. In this study, we measured intracellular ATP levels and the activities of several enzymes involved in surfactant phospholipid biosynthesis after sublethal H2O2 exposure of cultured rat alveolar type II cells. Intracellular ATP levels were reduced by 46.6% after exposure to 75 microM H2O2. The activity of CTP:phosphorylcholine cytidyltransferase was unchanged after H2O2 exposure when measured in whole cell homogenates. However, when measured in the microsomal fraction, cytidyltransferase activity significantly fell after exposure of type II cells to 75 microM H2O2. Activity in the cytosolic fractions remained unchanged. Similarly, microsomal cholinephosphotransferase was reduced after H2O2 exposure. We conclude that H2O2 decreases surfactant phosphatidylcholine biosynthesis independently of its ability to deplete intracellular ATP content. These deleterious effects may partially explain the diminished alveolar surfactant observed in patients with ARDS.

Carbonic anhydrase II expression in rat type II pneumocytes.

Pulmonary carbonic anhydrase (CA) activity plays important roles in carbon dioxide exchange, fluid secretion, and pH regulation. This study reports the use of molecular and immunologic techniques to characterize expression of the high-activity cytosolic isoenzyme CA II in rat lung tissue. Northern blot analysis of RNA isolated from various rat tissues revealed that the lung is a site of abundant tissue-specific CA II gene expression. The cell type primarily responsible for CA II expression in the lung was identified by immunohistochemistry as the alveolar type II pneumocyte. RNA blot and immunoblot analyses of isolated rat type II cells in culture confirmed CA II expression by this cell type. Little immunoreactive CA I and no CA IV was detected in these cells. Inhibition studies confirmed that the majority of CA activity in isolated type II cells is attributable to CA II. CA II expression was found to continue in these cells beyond 72 h in culture, a timeframe during which these cells had dedifferentiated. The ontologic pattern of CA II expression in the lung was found by RNA blot analysis to be disparate from that of the surfactant-associated proteins. These observations suggest roles for CA II in alveolar pneumocytes independent of (or in addition to) participation in surfactant biology. Such roles may include the regulation of fluid secretion or facilitation of carbon dioxide elimination.

The major glycolipid recognized by SP-D in surfactant is phosphatidylinositol.

Surfactant protein D (SP-D), a multimeric calcium-dependent lectin isolated from pulmonary alveolar lavage, has been previously shown to interact reversibly with crude surfactant [Persson et al. (1990) J. Biol. Chem. 265, 5755-5760]. In this study, SP-D is shown to interact reversibly with a preparation of organelles enriched in lamellar bodies, in a manner inhibited by calcium-chelating agents and by competing saccharides. An interaction with an endogenous glycoprotein could not be identified by electrophoresis of surfactant or lamellar body-associated proteins followed by electrotransfer of the separated proteins to nitrocellulose and then probing with radioiodinated SP-D via lectin overlay. Separation of the surfactant or lamellar body lipids on two-dimensional thin-layer chromatography (2D-TLC) followed by probing with radioiodinated SP-D via lectin overlay demonstrated binding to a single lipid. This interaction was dependent on the presence of calcium and was inhibited by competing saccharides. By assaying column fractions for the ability to bind radioiodinated SP-D after TLC, the glycolipid was purified to homogeneity and identified as phosphatidylinositol (PI). Identification was confirmed by mass spectrometry. We further demonstrate the ability of radiolabeled SP-D to bind to PI presented in a lipid bilayer through separation of free SP-D from liposome-bound SP-D on density gradients of Percoll. The interaction of SP-D with PI is dependent on calcium and inhibited by competing saccharides. SP-D binds with similar efficiency to liposomes with mole fractions of PI ranging from 2.5% to 30%, thereby demonstrating the lectin's ability to recognize mole fractions of PI available in surfactant.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects on experimental acute lung injury 24 hours after exogenous surfactant instillation.

Subcutaneous injection of N-nitroso-N-methylurethane (NNNMU) produces an acute lung injury mimicking the adult respiratory distress syndrome. NNNMU-injured rats treated with intratracheal Survanta, 100 mg phospholipid/kg body weight, air, or normal saline were observed for 24 h. Twenty-four hours after treatment survival among Survanta-treated rats was significantly greater than for air- and saline-treated rats (9/15 vs. 2/15 and 3/15, respectively). The alveolar-to-arterial O2 gradient was lower in Survanta-treated than in either air- or saline-treated rats during the 24-h period. Analysis of bronchoalveolar lavage fluid revealed a higher phospholipid: protein ratio (1.73 +/- 0.31 Survanta-treated, 0.20 +/- 0.05 air control, and 0.41 +/- 0.17 saline control) and a more normal phospholipid composition among treated than control rats. Minimum dynamic surface tension was significantly lower among treated rats (10.9 +/- 2.9 dyn/cm) than air and saline control rats (36.0 +/- 0.6 and 35.8 +/- 1.0 dyn/com, respectively). In vitro mixing of surfactant with pulmonary edema proteins significantly raised the minimum surface tension of surfactant from a group of Survanta-treated, NNNMU-injured rats (8.7 +/- 3.5 dyn/cm before and 32.0 +/- 0.5 dyn/cm after mixing). Intratracheal Survanta shows a beneficial effect on physiologic parameters and biochemical and functional characteristics of alveolar surfactant for 24 h in rats with NNNMU-induced acute lung injury.

Surfactant chemical composition and biophysical activity in acute respiratory distress syndrome.

Acute Respiratory Distress Syndrome (ARDS) is characterized by lung injury and damage to the alveolar type II cells. This study sought to determine if endogenous surfactant is altered in ARDS. Bronchoalveolar lavage was performed in patients at-risk to develop ARDS (AR, n = 20), with ARDS (A, n = 66) and in normal subjects (N, n = 29). The crude surfactant pellet was analyzed for total phospholipids (PL), individual phospholipids, SP-A, SP-B, and minimum surface tension (STmin). PL was decreased in both AR and A (3.48 +/- 0.61 and 2.47 +/- 0.40 mumol/ml, respectively) compared to N (7.99 +/- 0.60 mumol/ml). Phosphatidylcholine was decreased in A (62.64 +/- 2.20% PL) compared to N (76.27 +/- 2.05% PL). Phosphatidylglycerol was 11.58 +/- 1.21% PL in N and was decreased to 6.48 +/- 1.43% PL in A. SP-A was 123.64 +/- 20.66 micrograms/ml in N and was decreased to 49.28 +/- 21.68 micrograms/ml in AR and to 29.88 +/- 8.49 micrograms/ml in A. SP-B was 1.28 +/- 0.33 micrograms/ml in N and was decreased to 0.57 +/- 0.24 micrograms/ml in A. STmin was increased in AR (15.1 +/- 2.53 dyn/cm) and A (29.04 +/- 2.05 dyn/cm) compared to N (7.44 +/- 1.61 dyn/cm). These data demonstrate that the chemical composition and functional activity of surfactant is altered in ARDS. Several of these alterations also occur in AR, suggesting that these abnormalities occur early in the disease process.

Uptake and reutilization of surfactant phospholipids by type II cells of isolated perfused lung.

To investigate the role of type II alveolar cells in surfactant uptake and reutilization while cells are in situ, the combination of isolated perfused rat lung and type II cell isolation has been used. After an equilibration period, isolated perfused rat lungs received an intratracheal injection of labeled rat surfactant or a labeled bovine-derived preparation, and the perfusion continued. At time intervals less than or equal to 3 h, lungs were lavaged and type II cells isolated. Freshly isolated cells from perfused lungs were highly viable as judged by trypan blue exclusion and by linear incorporation of labeled leucine into trichloroacetic acid precipitable protein during perfusion. After cell isolation, total phospholipid or phosphatidylcholine was extracted from cells. Incorporation of surfactant phosphatidylcholine label into cellular phosphatidylcholine was shown to be linear with time whether lungs received natural rat surfactant or bovine-derived surfactant preparation. Uptake of natural surfactant from alveoli into type II cells was approximately three times greater than uptake of bovine-derived material. Results of administration of 0.5 mumol surfactant phospholipid or 2.0 mumol were similar except that administration of 2.0 mumol of natural rat surfactant caused uptake to become saturated at 1 h of perfusion. An increase in cell-associated phosphatidylcholine occurred whether results were expressed on basis of cellular DNA or specific activity of cellular phosphatidylcholine. Majority of administered surfactant label remained lavage fluid associated at 3 h, whereas remainder was associated with isolated cells or debris from cell preparation. Little label was detected in perfusion medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of exogenous surfactant instillation on experimental acute lung injury.

Pulmonary surfactant replacement has previously been shown to be effective in the human neonatal respiratory distress syndrome. The value of surfactant replacement in models of acute lung injury other than quantitative surfactant deficiency states is, however, uncertain. In this study an acute lung injury model using rats with chronic indwelling arterial catheters, injured with N-nitroso-N-methylurethane (NNNMU), has been developed. The NNNMU injury was found to produce hypoxia, increased mortality, an alveolitis, and alterations in the pulmonary surfactant system. Alterations of surfactant obtained by bronchoalveolar lavage included a reduction in the phospholipid-to-protein ratio, reduced surface activity, and alterations in the relative percentages of the individual phospholipids compared with controls. Treatment of the NNNMU-injured rats with instilled exogenous surfactant (Survanta) improved oxygenation; reduced mortality to control values; and returned the surfactant phospholipid-to-protein ratio, surface activity, and, with the exception of phosphatidylglycerol, the relative percentages of individual surfactant phospholipids to control values.

Glycerol as a substrate for phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats.

Glycerol and glucose utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozotocin-diabetic rats. In cells from diabetic rats, incorporation of [1,3-14C]glycerol into total phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) occurred to a greater degree by the glycerol 3-phosphate pathway as opposed to the dihydroxyacetone phosphate pathway. Total incorporation of glycerol into each of the major cellular phospholipids was increased up to 6-fold in cells from diabetic rats, while the total incorporation of glucose into the same lipids was decreased 2-fold. While the percentage of both glucose and glycerol carbons incorporated into the backbone of DSPC was increased in cells from diabetic rats, the percentage of carbons from both substrates incorporated into the fatty acid moieties was decreased. As a measure of DSPC synthesis, choline incorporation into DSPC was significantly decreased in type II cells from diabetic animals if the cells were incubated in the presence of glucose, palmitate and choline but not glycerol. Addition of 0.1 or 0.3 mM glycerol to the incubation medium restored choline incorporation to the control value in cells from diabetic rats, but did not affect the rate of choline incorporation into DSPC in cells from normal rats. These results suggest that exogenous glycerol can compensate for reduced glucose metabolism in type II cells of diabetic animals to maintain a constant rate of DSPC synthesis.

Glycerol metabolism in type II pneumocytes isolated from streptozotocin-diabetic rats.

Glycerol utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozocinin-diabetic rats. With glucose in the incubation medium, incorporation of exogenous [1,3-14C]glycerol into disaturated phosphatidylcholine, total phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) was increased 4-fold in cells from diabetic rats. In the absence of glucose, glycerol incorporation was 5-fold greater than in its presence in cells from normal animals, but was further increased 2.2-fold in cells from diabetic rats. Insulin treatment of diabetic rats returned all incorporation rates to control values. The increased glycerol incorporation rates were not due to differences in either phospholipid turnover or the size of the glycerol 3-phosphate precursor pool. Kinetic analysis of glycerol entry into the acid-soluble cell fraction indicated that glycerol transport occurred largely by simple diffusion, and was not rate limiting for its entry into lipids. Glycerol entry into the total lipid fraction was saturable, reaching a Vmax of 48 pmol/micrograms DNA per h in normal cells and 120 pmol/micrograms DNA per h in cells from diabetic rats, with no change in the Km (0.31 mM). While glycerol oxidation was reduced 23% in cells from diabetic rats in the presence of glucose and by 44% in the absence of glucose, glycerol kinase activity in sonicates of cells from diabetic animals was increased 210% and was reversed by in vivo insulin treatment. These results suggest that glycerol utilization in type II pneumocytes is a hormonally regulated function of both glycerol oxidation and glycerol phosphorylation.

Matrix deposition and extracellular processing of newly synthesized collagens in the isolated perfused rat lung.

We have examined the matrix deposition and proteolytic processing of newly synthesized interstitial and basement membrane collagens in the isolated perfused adult rat lung. Isolated, perfused, and ventilated lungs were labeled for up to 4 h with radiolabeled proline. Collagens were partially purified from homogenates by salt fractionation and ion exchange chromatography and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The major collagenous species were identified as types I, III, and IV collagen by peptide mapping and indirect immunoprecipitation assays. Whereas extraction with neutral salts recovered radiolabeled types I and III collagen, extraction of the neutral salt residue with 2 M guanidine-HCl preferentially recovered types III and IV collagen. Reextraction of the guanidine-HCl residue in the presence of dithiothreitol selectively recovered type IV procollagen (PC) and covalently cross-linked aggregates of type IV chains. In pulse-chase experiments we observed extensive conversion of type I PC to collagen during a 4-h chase. Although type III PC was efficiently converted to p-collagen, only small amounts of fully processed chains were identified. Type IV PC did not undergo detectable proteolytic processing. The isolated perfused rat lung should prove useful for further studies of lung collagen metabolism.

Altered phospholipid secretion in type II pneumocytes isolated from streptozotocin-diabetic rats.

To study the effect of diabetes on pulmonary surfactant secretion, type II pneumocytes from adult streptozotocin-induced diabetic rats were placed in short-term culture. As opposed to a linear secretory rate by control type II cells, the secretory rate of type II cells from diabetic animals was biphasic reaching a minimum at 1.5 h. When exogenous surfactant containing radioactive phosphatidylcholine was added to the incubation media for 1.5 h, the cells from diabetic animals incorporated more exogenous phosphatidylcholine into lamellar bodies than control cells. This suggests that in the type II cell from diabetic animals, the rate of reutilization is greater than the rate of secretion until 1.5 h, at which time the rate of secretion becomes greater. The altered secretory pattern was reversed by in vivo insulin treatment 30 min prior to killing but not by the addition of insulin to the incubation media. When challenged by isoproterenol, a beta-adrenergic agonist, the secretory pattern of cells from diabetic animals was biphasic as observed with basal secretion; however, secretion was stimulated 30% as opposed to 100% increase in control cells. These data suggest that basal and stimulated secretion are altered in the cultured type II cell from diabetic animals and restored by in vivo but not in vitro insulin treatment.