Purinoceptor agonists stimulate phosphatidylcholine secretion in primary cultures of adult rat type II pneumocytes

We examined the effect of purinoceptor agonists on phosphatidylcholine secretion in primary cultures of type II pneumocytes from adult rats. Surfactant is a major product of the type II cell and phosphatidylcholine is its principal component. Adenosine, AMP, ADP and ATP stimulated phosphatidylcholine secretion in a concentration-dependent manner. At the optimum concentration (1 mM), adenosine and AMP stimulated phosphatidylcholine secretion more than 2-fold, while ATP stimulated 5-fold and ADP almost 7-fold. Because of the magnitude of the response it is tempting to speculate that secretion of surfactant may be under purinoceptor regulation. None of these agents influenced cellular phosphatidylcholine synthesis or lactate dehydrogenase release into the medium, so the effects were primarily on secretion and were not secondary to effects on synthesis or cell damage. Non-metabolizable analogs of adenosine, 5'-N-ethyl-carboxyamidoadensoine (NECA) and L-N6-phenylisopropyladenosine (L-PIA), stimulated secretion to the same extent as adenosine and the effect of NECA was antagonized by 8-phenyltheophylline, suggesting a P1 purinoceptor-mediated mechanism. The stimulatory effect of ATP was diminished by alpha, beta-methylene ATP but only slightly by 8-phenyltheophylline, suggesting that, although part of the ATP effect could be explained by catabolism to adenosine, the P2 purinoceptor may also be involved in regulation of surfactant secretion.     

Stimulation of pulmonary surfactant secretion by activating neutrophils in rat type II pneumocytes culture

The influence of activating neutrophils on the secretion of phosphatidylcholine (PC), the predominant component of pulmonary surfactant, was examined using primary culture of rat type II pneumocytes. Simultaneous addition of neutrophils and opsonized zymosan, but not neutrophils or opsonized zymosan alone, to type II pneumocytes caused a significant increase in PC secretion without affecting the release of lactate dehydrogenase, a marker of cytotoxicity. The increase in PC secretion was dependent on the number of activating neutrophils. In addition, pretreatment of culture with the combination of superoxide dismutase and catalase inhibited the increase in PC secretion. These findings indicate that activating neutrophils stimulate the secretion of pulmonary surfactant and that the stimulation is mediated by oxygen radicals.     

Role of platelet-activating factor in phosphatidylcholine secretion in primary cultures of rat type II pneumocytes

This study was designed to investigate the effect of platelet-activating factor (PAF) in the secretory response of type II pneumocytes, that are involved in the synthesis and secretion of the pulmonary surfactant. PAF increased phosphatidylcholine secretion in a concentration-dependent manner in the 10(-5) - 10(-10) M range, with a maximum phosphatidylcholine secretion of up to 3.3 fold the basal values (3.4 +/- 0.3% phosphatidylcholine secreted). This effect was prevented by the synthetic PAF-receptor antagonist WEB 2086. A study of the mechanism through which PAF exerts its stimulatory effect was carried out adding different agents that are well known stimulants of phosphatidylcholine secretion. Thus, PAF increased the TPA- and terbutaline-stimulated phosphatidylcholine secretion, that are PKC and PKA activators respectively, suggesting the involvement of both protein kinases in the process. This involvement was further supported by the use of inhibitors of protein kinases and by the stimulation of cAMP production in type II pneumocytes incubated with PAF.     

Arachidonic acid metabolites stimulate phosphatidylcholine secretion in primary cultures of type II pneumocytes

There is evidence from whole animal and intact lung studies that prostaglandins are involved in the regulation of surfactant secretion. To explore this further we examined the effect of arachidonic acid on secretion of phosphatidylcholine in primary cultures of adult rat type II pneumocytes. Arachidonic acid stimulated phosphatidylcholine secretion and this effect was dependent on concentration in the range 1-8 microM. Arachidonic acid (8 microM) stimulated secretion by 79% from a basal rate of 1.17% total cellular phosphatidylcholine secreted in 90 min to 2.09%. We examined the effects of inhibitors of arachidonic acid metabolism on the stimulatory effect. Nordihydroguairaretic acid (0.1 microM), a lipoxygenase inhibitor, reduced the stimulatory effect by 64%. The same concentration of cyclooxygenase inhibitors had no effect. We conclude that arachidonic acid metabolites stimulate surfactant secretion in type II cells. Whether this effect is mediated by leukotrienes or other products remains to be established.     

Nitric oxide decreases surfactant protein gene expression in primary cultures of type II pneumocytes

Inhaled nitric oxide (NO) is a selective pulmonary vasodilator effective in treating persistent pulmonary hypertension in newborns and in infants following congenital heart disease surgery. Recently, multiple in vivo and in vitro studies have shown a negative effect of NO on surfactant activity as well as surfactant protein gene expression. Although the relationship between NO and surfactant has been studied previously, the data has been hard to interpret due to the model systems used. The objective of the current study was to characterize the effect of NO on surfactant protein gene expression in primary rat type II pneumocytes cultured on a substratum that promoted the maintenance of type II cell phenotype. Exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP), decreased surfactant protein (SP)-A, (SP)-B, and (SP)-C mRNA levels in type II pneumocytes in a time- and dose-dependent manner. The effect was mediated in part by an increase in endothelin-1 secretion and a decrease in the intracellular messenger, phosphorylated ERK1/2 mitogen-activated protein kinases (MAPK). Exposing type II pneumocytes to endothelin-1 receptor antagonists PD-156707 or bosentan before exposure to SNAP partially prevented the decrease in surfactant protein gene expression. The results showed that NO mediated the decrease in surfactant protein gene expression at least in part through an increase in endothelin-1 secretion and a decrease in phosphorylated ERK1/2 MAPKs.     

Verapamil: a novel probe of surfactant secretion from rat type II pneumocytes

Surfactant from type II pneumocytes prevents the alveolar atelectasis found in both the neonatal and adult forms of respiratory distress syndrome. We have found that verapamil, a phenylalkene with calcium channel and alpha 1-receptor binding properties, has a multiphasic concentration effect on surfactant secretion from [3H]choline-labeled rat type II pneumocytes in culture. Verapamil (10(-8) M) caused a 24% stimulation of surfactant secretion, whereas an 8% inhibition was found at 10(-6) M and a 70% stimulation was found at 10(-4) M. Lactate dehydrogenase release occurred at 5 x 10(-4) M verapamil. Verapamil (10(-4) M) also produced a 100% increase in adenosine 3',5'-cyclic monophosphate (cAMP) in comparison with concentrations of less than or equal to 10(-6) M, an effect that could not be blocked by propranolol (10(-4) M). Verapamil (10(-6) M) increased the total formation of inositol phosphates (IP) by 23% in comparison with IP formation in control cells. Calcium influx was inhibited 15% by 10(-8) M verapamil and 37% by 10(-4) M verapamil. Calcium efflux was stimulated 44% by 10(-5) M verapamil. In combination with 50% effective concentrations (EC50) of terbutaline, phorbol ester, and ATP, the respective effects of verapamil (10(-4) M) on surfactant secretion were approximately additive. We conclude that verapamil has a novel multiphasic concentration effect on surfactant secretion, which appears to involve several signal transduction pathways including cAMP formation, IP formation, inhibition of calcium influx, and stimulation of calcium efflux.     

Involvement of calcium in the stimulation of phosphatidylcholine secretion in primary cultures of rat type II pneumocytes by Escherichia coli lipopolysaccharide

The purpose of this study was to evaluate the mechanism by which Escherichia coli lipopolysaccharide stimulates the secretion of phosphatidylcholine in primary cultures of rat type II pneumocytes. The stimulatory effect of lipopolysaccharide on phosphatidylcholine secretion was additive to those of terbutaline and TPA (protein kinase A and C activators respectively) and this effect was not suppressed by inhibitors of both protein kinases. On the other hand, lipopolysaccharide did not modify the increase on phosphatidylcholine secretion induced by the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin, and enhanced slightly the calcium-ionophore A23187 stimulated phosphatidylcholine secretion. In addition, the stimulatory effect of lipopolysaccharide was suppressed by BAPTA, an intracellular Ca²⁺ chelator, and KN-62, a specific inhibitor of Ca²⁺-calmodulin-dependent protein kinase. These results, together with the lipopolysaccharide-mediated increase in the cytosolic [Ca²⁺], suggest that stimulation of phosphatidylcholine secretion by lipopolysaccharide in type II pneumocytes occurs by a calcium-dependent transduction mechanism via Ca²⁺-calmodulin-dependent protein kinase activation.     

The proliferative effects of retinoic acid on primary cultures of adult rat type II pneumocytes depend upon cell density

Retinoic acid (RA) is important for maintaining integrity of alveolar epithelial cells, but the mechanism has not been defined. We cultured type II pneumocytes at confluent, high cell density (10⁴ cells/mm²) and found that RA (10⁻⁶ M) inhibited thymidine incorporation to 60% of control, despite a dose-dependent increase in epidermal growth factor receptor (EGFR) levels. However, at lower, subconfluent density (10² cells/mm²), RA stimulated thymidine incorporation to 280% of control. EGF increased thymidine incorporation at concentrations as low as 0.1 ng/mL, but no further increase was observed at higher concentrations up to 100 ng/mL. In subconfluent cells co-treated with EGF (100 ng/mL) and increasing concentrations of RA (10⁻⁸ M–10⁻⁵ M RA), thymidine incorporation was significantly greater at all concentrations than RA alone, with greatest increases observed at 10⁻⁷ (422% of control) and 10⁻⁶ (470% of control) M RA. In summary, the effects of RA on thymidine incorporation are sensitive to changes in cell density. RA inhibits thymidine incorporation at high cell density and stimulates thymidine incorporation at low density. RA increases EGFRs in cultured type II pneumocytes, and EGF stimulates thymidine incorporation independent of the cultured cell density. These data may help to explain how RA mediates lung repair in vivo.     

Survival signaling in type II pneumocytes activated by surfactant protein-A

Surfactant-associated protein-A (SP-A) is a component of pulmonary surfactant that acts as a cytokine through interaction with a cell-surface receptor (SPAR) on lung epithelial cells. SP-A regulates important physiological processes including surfactant secretion, gene expression, and protection against apoptosis. Tyrosine kinase and PI3K inhibitors block effects of SP-A, suggesting that SPAR may be a receptor tyrosine kinase and activate the PI3K-PKB/Akt pathway. Here we report that SP-A treatment leads to rapid tyrosine-specific phosphorylation of several important proteins in lung epithelial cells including insulin receptor substrate-1 (IRS-1), an upstream activator of PI3K. Analysis of anti-apoptotic signaling species downstream of IRS-1 showed activation of PKB/Akt but not of MAPK. Phosphorylation of IkappaB was minimally affected by SP-A as was NFkappaB gel shift activity. However, FKHR was rapidly phosphorylated in response to SP-A and its DNA-binding activity was significantly reduced. Since FKHR is pro-apoptotic, this may play an important role in signaling the anti-apoptotic effects of SP-A. Therefore, we have characterized survival-enhancing signaling activated by SP-A leading from SPAR through IRS-1, PI3K, PKB/Akt, and FKHR. The activity of this pathway may explain, in part, the resilience of type II cells to lung injury and their survival to repopulate alveolar epithelium after peripheral lung damage.     

Regulation of surfactant secretion from isolated Type II pneumocytes by substance P

Substance P, an eleven amino acid neuropeptide, significantly inhibited release of [3H]phosphatidylcholine from pulmonary Type II epithelial cells in vitro. Basal release and release in response to the beta-adrenergic agonist, terbutaline and 12-O-tetradecanoylphorbol 13-acetate (TPA) were significantly decreased in the presence of substance P. Inhibitory effects of substance P were noted following a 1 h exposure of primary cultures of Type II cells in vitro and persisted up to 3 h in the presence of the secretagogues, TPA and terbutaline. The IC50 values for substance P inhibition of [3H]PC release were 10 microM for basal release, 40 microM for TPA-induced release and 50 microM for terbutaline-induced release. The related neuropeptide, physalaemin and the stable active analog of substance P, [pGlu5, MePhe8, MeGly9]substance P [5-11], had no significant inhibitory effects on surfactant release whether in the presence or absence of TPA or terbutaline. These data support the hypothesis that NH2-terminal basic groups of substance P are necessary for inhibition of surfactant secretion from isolated Type II cells and support the concept that an inhibitory system contributes to mediation of surfactant secretion from Type II epithelial cells.     

Developmental regulation of phospholipid secretion by fetal type II pneumocytes

Surfactant sufficiency is dependent upon adequate synthesis and secretion of surfactant by the type II alveolar epithelium. Our laboratory has previously shown that basal secretion of surfactant phospholipid by differentiated fetal type II cells is lower than the basal secretion by adult cells. The purposes of this study were to determine if undifferentiated fetal type II cells can secrete phosphatidylcholine, to determine if terbutaline, a β-adrenergic agonist, stimulates secretion of surfactant phospholipids by undifferentiated fetal cells and to examine the effects of differentiation on secretion of surfactant phospholipids by fetal cells. Constitutive (basal) secretion of phosphatidylcholine increased linearly as a function of time in both undifferentiated and differentiated cells, but the rate of secretion was greater in differentiated cells than the rate of secretion in undifferentiated cells. Terbutaline caused a concentration-dependent increase in secretion in both undifferentiated and differentiated cells. Maximal effective concentration and EC₅₀ were similar for undifferentiated (10⁻⁶ M, 0.2 μM) and differentiated (10⁻⁵ M, 0.3 μM) cells. The relative stimulation of secretion above control values was greater for undifferentiated cells. The kinetics of terbutaline stimulation varied significantly with cellular differentiation. Terbutaline resulted in 230% stimulation of secretion in undifferentiated cells at 30 min followed by a decline in the response to terbutaline at 60 to 120 min. In contrast, terbutaline stimulated secretion by differentiated cells showed a sustained linear increase from 0 to 120 min. This regulation of stimulated secretion is not present in undifferentiated cells. We conclude that undifferentiated type II cells are capable of the secretion of phosphatidylcholine and that terbutaline stimulates secretion by undifferentiated cells. Furthermore, basal secretion increases as a function of differentiation of type II cells and the regulation of stimulated secretion seen in differentiated cells is not developed in undifferentiated cells. The developmental regulation of the secretion of surfactant is complex and probably involves both excitatory as well as inhibitory mechanisms which develop at different stages of differentiation of the type II cell.     

Natural protection from apoptosis by surfactant protein A in type II pneumocytes

Surfactant-associated protein A (SP-A) is a component of pulmonary surfactant that binds to a specific receptor (SPAR) on the surface of type II alveolar cells of the lung and regulates gene expression and surfactant secretion. Previously we have shown that activation of SPAR by SP-A binding initiates a signal through pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of phosphatidylinositol 3-kinase (PI3K). In other cell types, cytokines that activate the PI3K signaling pathway promote cell survival. Therefore we investigated whether there was an effect of SP-A on apoptosis as measured by DNA laddering, FACS analysis, TUNEL assay, and annexin V binding. SP-A protected primary cultures of rat type II alveolar cells against the apoptotic effects of etoposide and UV light and also protected the H441 human Clara lung tumor cell line against staurosporine-induced apoptosis. The protective effects of SP-A were abrogated by inhibition of either tyrosine-specific protein kinase activity or PI3K. SP-A/SPAR interaction thus initiates a signaling pathway that regulates apoptosis in type II cells. These findings may be important in understanding the pathogenesis of acute lung injury and pulmonary tumorigenesis and may suggest new therapeutic options.     

The effects of insulin and hyperglycemia on surfactant phospholipid synthesis in organotypic cultures of type II pneumocytes

Organotypic cultures of fetal type II epithelial cells were incubated in media containing insulin at concentrations ranging from 10 to 400 microunits/ml. Exposure to insulin resulted in increased glucose uptake from the media and in the rate of glucose conversion to CO2. Furthermore, both glucose uptake and CO2 production were dependent on the glucose concentration in the media. Surfactant and residual phosphatidylcholine fractions were isolated from the organotypic cultures by sucrose density centrifugation. The presence of low doses of insulin (10-25 microunits/ml) caused a significant increase in the incorporation of glucose into both surfactant and residual phosphatidylcholine. Insulin at levels of 100 microunits/ml or higher resulted in a significant decrease in glucose incorporation into both phosphatidylcholine fractions. Increasing the media glucose concentration from 5.6 to 20 mM caused a 2- to 2.5-fold increase in glucose utilization for surfactant and residual phospholipid synthesis, but did not produce any significant changes in choline incorporation into either surfactant or residual phosphatidylcholine. The addition of 400 microunits/ml of insulin to media containing 20 mM glucose, however, resulted in a 20% decrease in choline incorporation into surfactant phosphatidylcholine but had no effect on choline incorporation into residual phosphatidylcholine. These results suggest that insulin is an important hormone regulating fetal lung maturation and that hyperinsulinemia may be responsible for the delayed lung development in infants of diabetic mothers.     

Leukotrienes stimulate phosphatidylcholine secretion in cultured type II pneumocytes

We previously reported that arachidonic acid stimulates secretion of phosphatidylcholine in cultures of type II pneumocytes and, based on studies with cyclooxygenase and lipoxygenase inhibitors, suggested that this effect was mediated by lipoxygenase products of arachidonic acid metabolism (Gilfillan, A.M. and Rooney, S.A. (1985) Biochim. Biophys. Acta 833, 336-341). We have now examined the effect of leukotrienes on phosphatidylcholine secretion in type II cells as well as the effect of a leukotriene antagonist, FPL55712, on the stimulatory effect of arachidonic acid. Leukotrienes C4, D4 and E4 stimulated phosphatidylcholine secretion and this effect was dependent on concentration in the range 10(-12)-10(-6) M. Leukotriene E4 was the most stimulatory, followed by D4 and C4. Leukotriene B4 had no effect. Incubation of the cells with 10(-7) M leukotriene E4 for 90 min resulted in a 107% increase in the rate of phosphatidylcholine secretion. Incubation with 10(-6) M leukotrienes D4 and C4 for the same period resulted in 81% and 63% stimulation, respectively. The leukotrienes had no effect on cellular phosphatidylcholine synthesis or on lactate dehydrogenase release. The stimulatory effects of leukotrienes E4 and D4 were abolished by FPL55712. Similarly, the stimulatory effect of 6 X 10(-6) M arachidonic acid on phosphatidylcholine secretion was reduced from 74% to 25% by 10(-5) M FPL55712. Thus, the stimulatory effect of arachidonic acid on surfactant phospholipid secretion in type II cells is mediated at least in part by leukotrienes.     

Stimulation of phosphatidylcholine synthesis by fatty acids in fetal rabbit type II pneumocytes

After 24 h exposure to 0.1 mM oleate or 0.1 mM palmitate there was a 2- and 1.7-fold increase, respectively, in the incorporation of choline into the lipids of type II pneumocytes. Palmitate increased the labeling of disaturated phosphatidylcholine (PC) from 23.0% of total labeled PC in control cultures to 56.6% and oleate decreased labeling of disaturated PC to 9.4%. The percentage of total cellular radioactivity found in the lipid fraction was also markedly higher in the fatty acid-treated cells (83.3% for oleate and 78.7% for palmitate) than in control cultures (64.0%). Radioactivity in water-soluble choline metabolites was correspondingly lower, with phosphocholine representing more than 95% of the label in both control and experimental cultures. After a 3 h pulse-chase period, oleate and palmitate significantly increased the percentage of total cellular radioactivity in PC and decreased the percentage in phosphocholine. Similar results were obtained by adding melittin (1–2 μg/ml) or phospholipase C (0.05 U/ml) to the culture medium. The stimulation of PC synthesis by fatty acids was demonstrated as early as 1 h after exposure to oleate or palmitate and at all concentrations from 0.025 to 0.25 mM. Cytidylyltransferase activity in total cell homogenates was also enhanced by long-term exposure to fatty acids and short-term addition of fatty acids or phospholipase C and melittin to the culture medium. A similar increase in Cytidylyltransferase activity was found in the 100 000 × g particulate fraction of type II cells exposed to fatty acids, whereas no differences were found between the cytosolic fractions of control and treated cells. These results support the concept that an increase in intracellular level of fatty acids either from an exogenous source or following the activation of endogenous phospholipases regulates PC synthesis in fetal type II pneumocytes.     

Pulmonary surfactant apoprotein A structure and modulation of surfactant secretion by rat alveolar type II cells

The pulmonary surfactant apoprotein with a reduced denatured molecular mass of 26-38 kDa (PSP-A) has recently been identified as an inhibitor of surfactant phospholipid secretion by isolated rat alveolar type II cells. We have investigated some of the structural determinants of PSP-A that are relevant to the inhibitory process. The PSP-A was isolated from rats given an intratracheal instillation of silica. The yield of PSP-A from silica-treated animals was 20-40-fold higher than that obtained from untreated animals. Reduction of PSP-A with 2-mercaptoethanol caused a reversible loss of biological activity that was restored by mild oxidation. Alkylation of the protein with excess iodoacetamide also led to inactivation, although titration with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that the protein initially contained no free sulfhydryl moieties. Neither alkylation nor reduction plus alkylation completely prevented the formation of oligomers as determined by gel permeation analysis. The apparent molecular mass of PSP-A at 4 degrees C in low ionic strength buffers was 1.6 megadaltons, and at 37 degrees C in normal ionic strength buffers was greater than 1.5 megadaltons. Removal of the oligosaccharide moiety with endoglycosidase F also had no effect upon biological activity. Five distinct monoclonal antibodies recognizing peptides epitopes on PSP-A were produced. All monoclonal antibodies exhibited similar affinity for PSP-A and recognized the delipidated and deglycosylated form. Four monoclonal antibodies reacted with epitopes on PSP-A that altered its function as an inhibitor. One monoclonal antibody was clearly ineffective at altering the activity of PSP-A. These results demonstrate that: 1) disulfide bonds are required for the activity of PSP-A, 2) disruption of disulfides does not prevent the formation of oligomeric forms of PSP-A, 3) the oligosaccharide moiety is not essential for biological activity, and 4) monoclonal antibodies can be used to map the epitopes responsible for biological activity.     

Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats

Pulmonary surfactant conventionally is prepared from material obtained by endobronchial lavage. Although it has been assumed that the components of surfactant are secreted by alveolar type II cells, direct proof of this assumption has not been available. Furthermore, it is possible that the final material obtained by lavage has been modified after secretion or altered during the isolation procedure. It has been shown previously that type II cells, after 1 day in primary culture, secrete saturated phosphatidylcholine, one of the lipid components of surfactant. Because saturated phosphatidylcholine is not unique to surfactant and because type II cells in culture lose differentiated characteristics over the first several days in culture, it has not previously been established how closely the secretory products of cultures of type II cells resemble surfactant as obtained by endobronchial lavage. We therefore studied the morphologic, physical and chemical characteristics of the material that type II cells secrete under basal conditions and after stimulation with terbutaline or 12-O-tetradecanoyl-13-phorbol acetate. The secreted material resembled surfactant obtained by lavage; it was similar morphologically to the lamellar material and tubular myelin seen in the fluid-filled alveoli of fetal rats, it lowered surface tension to 5 mN per meter, and it contained the 72000 dalton apolipoprotein of surfactant (as measured by the 'rocket' immunoelectrophoresis technique). When cells were incubated for 22 h with [1-(14)C]acetate, the distribution of radioactivity in the secreted material was very similar to the phospholipid composition of rat surfactant. We conclude that the material secreted by alveolar type II cells after 1 day in primary culture is similar to surfactant obtained by endobronchial lavage.     

Identification of specific proteins synthesized by type II pneumocytes in primary culture

Proteins from primary cultures of type II granular pneumocytes have been examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to identify type II cell-specific proteins. The distribution of Coomassie Blue-stained bands in preparations of cellular proteins, culture medium, lavage and lamellar bodies have been compared. The most prominent stained band in the serum-free medium from type II cell cultures (HS1; Mr 39900) corresponds to a major protein in acellular sedimentable (20000 g for 30 min) crude surfactant obtained from rat lungs by saline (0.9% NaCl) lavage. A second protein (HS2; Mr 12000) is also found both in type II cell-conditioned medium and in lavage. Neither rat serum nor donor calf serum (used in the isolation of the type II cells) contains a protein co-migrating with HS1 or HS2 proteins. HS1 is also found in Coomassie Blue-stained gels of cellular proteins and of lamellar bodies isolated from whole lungs. Cultures of type II cells incorporate [14C]phenylalanine into HS1 and HS2 as shown by autoradiography of sodium dodecyl sulphate/polyacrylamide gels of culture medium. Rat lungs perfused in situ incorporate [35S]methionine into HS1 in the lamellar body fraction. A third protein (HS3; Mr 47000) is observed only in autoradiographs of cell culture medium; no corresponding Coomassie Blue-stained band can be identified in medium, in cells or in lung lavage. No protein bands corresponding to HS1, HS2 or HS3 are found in conditioned media from pulmonary alveolar macrophages, rat fibroblasts or bovine aorta endothelial cells. Two-dimensional gel electrophoresis of HS1 shows a single polypeptide with an isoelectric point of 6.3; HS3 appears as a chain of spots with a range of isoelectric points from 6.3 to 6.6. HS2 has not been identified on two-dimensional gels. The amino acid composition of HS1 does not differ significantly from that of surfactant apoproteins studied previously; however, HS1 is not detected by glycoprotein stains, nor does it appear to be a subunit of a thiol-linked multimer.     

Stimulation of DNA synthesis in primary cultures of adult rat hepatocytes by rat platelet-associated substance(s)

Summary Experiments in whole animals have shown that normally quiescent adult rat hepatocytes are induced to proliferate by blood borne substances, which we are now probing in primary monolayer cultures. Under our conditions, freshly isolated adult hepatocytes do not proliferate actively in a defined medium, but are stimulated to synthesize DNA — an essential first step — by either serum or an EGF-hormone combination. Stimulation of [³H]thymidine incorporation into hepatocyte DNA by addition of dialyzed mouse, human, horse, or bovine (fetal, newborn, or calf) serum, whose activities are all similar, is regularly surpassed by an EGF-insulin mixture without serum. This, in turn, is exceeded by dialyzed normal rat serum, which is several times more potent than the other sera tested. Removal of blood platelets reduces the activity of normal rat serum by over 50%. Heat inactivation (56° C) causes a similar loss, but heat treatment of platelet-poor serum fails to cause further reduction. The activity of mouse and human serum is not reduced by platelet removal. Serum from partially hepatectomized rats is not significantly more stimulatory than normal rat serum, and its activity is depressed in the same way by platelet deprivation and heat inactivation. Lack of enhancement by partial hepatectomy is not consonant with whole animal studies and requires further investigation. The heat-labile portion of the DNA synthesis-stimulating activity of rat serum appears to derive from platelets. This activity differs from the well-characterized heat-stable human PDGF. Its relation to other reported platelet-associated growth factors is still undetermined. This work was supported by USPHS Grants CA-02146 and AM-19435.