Uptake of orotate and thymidine by normal and regenerating rat livers

1. At 1h after operation livers from partially hepatectomized rats showed a 60-100% increase in the capacity to concentrate (3)H radioactivity from orotate, thymidine or uridine with respect to the radioactivity in plasma. Uptake of [(3)H]cytidine into liver was unaffected, as was entry of any precursor studied into any tissue other than liver. 2. This increase in intracellular radioactivity was detectable 10min after operation with both orotate and thymidine. With orotate the augmentation had disappeared by 3 days, but with thymidine it was still evident 8 days after partial hepatectomy, when [(3)H]thymidine incorporation into DNA was no longer increased. Competition studies established that orotate was not entering the liver by the same mechanism as thymidine. 3. In the soluble fraction of the liver all the (3)H radioactivity from orotate was present as uridine nucleotides. Thymidine was not phosphorylated, and was believed to be catabolized.     

Ultrastructural morphology of the air-blood barrier and pulmonary surfactant in pulmonary inflammation in experimental alcoholic intoxication

Combined investigation of ultrastructure of components of air-haematic barrier and surface-active properties of surfactant in 21 guinea pigs' lungs with simulated pneumonia against a background of alcoholic intoxication was carried out. It was established, that alcoholic intoxication aggravates a deficiency of pulmonary surfactant occurred in pneumonia because of its high phagocytosis with alveolar macrophages. The increase of mobilization of alveolar macrophages in alcoholic intoxication may be connected with the rise of surfactant secretion by hyperfunctional pneumocytes of the 2nd type. Stopping of alcoholic intoxication may lead to normalization of qualitative composition of surfactant phospholipids.     

Studies on lung tumours. IV. Correlation between [3H]thymidine labelling of lung and liver cells and tumour formation in GRS/A and C3Hf/A male mice following administration of dimethylnitrosamine.

Male mice of the inbred strain GRS/A are highly susceptible to lung tumour but refractory to liver tumour formation, whereas the opposite relation holds for C3Hf/A male mice. Liver and lung cells of these 2 mouse strains were studied autoradiographically after intraperitoneal injection of [3H]dimethylnitrosamine (DMN) and of [3H]thymidine at days 1--14 after administration of unlabelled DMN. Corresponding cell types in the lungs or livers of these 2 mouse strains bound similar amount of [3H]DMN. Among the various types of lung cells only the alveolar Type II cells, from which the lung adenomas derive, showed a strain-specific difference in [3H]thymidine labelling indices, much more cells becoming labelled in the case of the GRS/A than of the C3Hf/A strain at days 3--7 after carcinogen administration. Opposite thymidine labelling indices were exhibited by the parenchymal liver cells of the 2 strains, with C3Hf/A now showing a greater response than did GRS/A males. Thus thymidine-labelling and tumourigenic responses of target lung and liver cells to carcinogen in the 2 strains coincided. Sulphur dioxide and carbon tetrachloride mimicked the effects of DMN on the thymidine labelling indices of, respectively, the lung alveolar Type II and the thymidine labelling indices of, respectively, the lung alveolar Type II and the liver parenchymal cells of the 2 strains. The nature of the differential effect of carcinogen on the [3H]thymidine labelling of the cells and the correlation of these patterns with susceptibility to tumour formation, are briefly discussed.     

Surfactant protein A regulates surfactant phospholipid clearance after LPS-induced injury in vivo

Previous in vitro studies have suggested that surfactant protein A (SP-A) may play a role in pulmonary surfactant homeostasis by mediating surfactant secretion and clearance. However, mice made deficient in SP-A [SP-A (-/-) animals] have relatively normal levels of surfactant compared with wild-type SP-A (+/+) animals. We hypothesize that SP-A may play a role in surfactant homeostasis after acute lung injury. Bacterial lipopolysaccharide was instilled into the lungs of SP-A (-/-) mice and SP-A (+/+) mice to induce injury. Surfactant phospholipid levels were increased 1.6-fold in injured SP-A (-/-) animals, although injury did not alter [3H]choline or [14C]palmitate incorporation into dipalmitoylphosphatidylcholine (DPPC), suggesting no change in surfactant synthesis/secretion 12 h after injury. Clearance of [3H]DPPC from the lungs of injured SP-A (-/-) animals was decreased by approximately 40%. Instillation of 50 microg of exogenous SP-A rescued both the clearance defect and the increased phospholipid defect in injured SP-A (-/-) animals, suggesting that SP-A may play a role in regulating clearance of surfactant phospholipids after acute lung injury.     

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.     

Association of chlorphentermine with phospholipids in rat alveolar lavage materials, alveolar macrophages and type II cells

Administration of chlorphentermine to rats leads to an increase in the phospholipid content of pulmonary surfactant materials and alveolar macrophages. It is known that this drug binds to pure phospholipids and prevents their degradation by phospholipases. Therefore, experiments were carried out to determine if chlorphentermine binds to surfactant phospholipids in vitro and to measure the in vivo association of drug with phospholipids in alveolar lavage materials from rats injected with [14C]chlorphentermine. The presence of chlorphentermine in alveolar macrophages, type II cells and other small pneumocytes (a population of lung cells which does not include alveolar macrophages or type II cells) from treated animals was also assessed. Binding of the drug to surfactant phospholipids, as measured with the fluorescent probe, 1-anilino-8-naphthalene sulfonate, occurs in vitro and does not differ in various subfractions of alveolar lavage materials isolated by differential centrifugation. Following daily administration of chlorphentermine to rats for 3 days, the drug appears to be associated with surfactant phospholipids such that the molar ratio is 1:100 (chlorphentermine/phospholipid). Chlorphentermine is also associated with alveolar macrophages (molar ratio, 1:18) and type II cells (molar ratio, 1:33). Not much drug is associated with the population of other lung cells (molar ratio, 1:333). In alveolar macrophages, approx. 70% of the drug seems to be bound to phospholipid and/or sequestered in subcellular organelles. However, only 20% of the chlorphentermine is bound and/or sequestered in type II cells. The results of these experiments suggest that following chlorphentermine administration, the drug is associated with phospholipids in acellular pulmonary lavage materials, alveolar macrophages and type II cells. This drug-phospholipid interaction may impair phospholipid degradation and lead to a phospholipidosis in surfactant materials and alveolar macrophages.     

Type II pneumocyte changes during hyperoxic lung injury and recovery

Adult rabbits exposed to 100% O2 for 64 h and then returned to room air for up to 200 h, develop a lung injury characterized by decreased levels of alveolar surfactant followed by a rebound recovery. In the present study we isolated alveolar type II cells from rabbits at various times during hyperoxic exposure and recovery and measured rates of phosphatidylcholine (PC) synthesis, cellular lipid content, and the specific activity of glycerol 3-phosphate (G-3-P) acyltransferase, an enzyme that catalyzes one of the early reactions in phosphoglyceride biosynthesis. These biochemical parameters were compared with measurements of cell size and cell cycle phase by laser flow cytometry. Results showed that alterations in alveolar phospholipid levels in vivo correlated consistently with cellular lipid metabolic changes measured in isolated type II pneumocytes. In particular, alveolar pneumocytes isolated from lungs of rabbits exposed to 100% O2 for 64 h exhibited a 60% decrease in PC synthesis, cell lipid content, and G-3-P acyltransferase activity. All variables then followed a pattern of recovery to normal and ultimately supranormal levels beginning at approximately 3 days postexposure, at which point there was also a measured increase in the number of type II cells in S phase. These findings suggest that O2-induced changes in type II cell surfactant biosynthesis may account, at least in part, for observed changes in lung phospholipid levels in vivo.     

Carbon tetrachloride inhibits synthesis of pulmonary surfactant disaturated phosphatidylcholines and ATP production in alveolar type II cells

Other studies have shown that inhalation of carbon tetrachloride (CCl4) decreases the amount of pulmonary surfactant lining the alveolar surface. Therefore, we studied the effects of CCl4 on the synthesis of surfactant phosphatidylcholines (PCs) in rat alveolar type II cells in vitro. The rate of incorporation of choline, palmitate or glycerol into disaturated PC (DSPC) is decreased in a concentration-dependent manner. The CCl4 concentrations which cause maximal inhibition and 50% inhibition are similar for each substrate. The rate of incorporation of choline or glycerol into total PC is diminished to the same extent as their incorporation into DSPC. In addition, the rate of incorporation of glycerol into phosphatidylglycerol is decreased by the same extent as its incorporation into PC. All of these data suggest that there is a common site(s) at which CCl4 inhibits PC synthesis and that the inhibition occurs early in the biosynthetic pathway. However, individual enzymes involved in phospholipid synthesis do not seem to be affected by the solvent. Exposure of alveolar type II cells to CCl4 does cause a rapid and dramatic loss in cellular ATP, a cofactor required by some enzymes involved in PC synthesis. Studies with isolated lung mitochondria suggest that CCl4 inhibits the enzyme complex which catalyzes the synthesis of ATP from ADP. In addition, CCl4 causes a decrease in the amount of 3-O-methylglucose associated with type II cells, suggesting that glucose influx is impaired. This may also contribute to lower cellular ATP levels. The results of this study suggest that inhalation of CCl4 may impair surfactant phospholipid synthesis by decreasing ATP levels in alveolar type II cells.     

Stimulation of surfactant secretion by vasopressin in primary cultures of adult rat type II pneumocytes

The current study examined the effect of vasopressin on the secretion of phosphatidylcholine, the principal component of pulmonary surfactant, from adult rat alveolar type II pneumocytes in primary culture. Vasopressin stimulated secretion in a time- and dose-dependent manner. At a concentration of 10 nM, vasopressin stimulated release by 6-fold over the basal secretory rate. The concentration producing half the maximal response for vasopressin-induced secretion was 0.4 nM. The stimulation of phosphatidylcholine release by vasopressin was duplicated by the vasopressin fragment, amino acids 4 through 9. [Lys8]vasopressin and the selective vasopressin-2 agonist [deamino-8-D-Arg]vasopressin did not stimulate surfactant secretion effectively. The vasopressin- and fragment-induced secretion was inhibited by the vasopressin-1 receptor antagonist d(CH2)5TDAVP and the protein kinase C inhibitor, tetracaine, but not by the beta-adrenergic antagonist alprenolol. Vasopressin did not activate adenylate cyclase, which suggests that stimulation by vasopressin was independent of cyclic AMP. When vasopressin and isoproterenol were added concomitantly, the effects on phosphatidylcholine secretion were additive. This suggests that these two secretagogues operate via separate mechanisms.     

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.     

Cyclic stretch attenuates effects of hyperoxia on cell proliferation and viability in human alveolar epithelial cells

The treatment of severe lung disease often requires the use of high concentrations of oxygen coupled with the need for assisted ventilation, potentially exposing the pulmonary epithelium to both reactive oxygen species and nonphysiological cyclic stretch. Whereas prolonged hyperoxia is known to cause increased cell injury, cyclic stretch may result in either cell proliferation or injury depending on the pattern and degree of exposure to mechanical deformation. How hyperoxia and cyclic stretch interact to affect the pulmonary epithelium in vitro has not been previously investigated. This study was performed using human alveolar epithelial A549 cells to explore the combined effects of cyclic stretch and hyperoxia on cell proliferation and viability. Under room air conditions, cyclic stretch did not alter cell viability at any time point and increased cell number after 48 h compared with unstretched controls. After exposure to prolonged hyperoxia, cell number and [(3)H]thymidine incorporation markedly decreased, whereas evidence of oxidative stress and nonapoptotic cell death increased. The combination of cyclic stretch with hyperoxia significantly mitigated the negative effects of prolonged hyperoxia alone on measures of cell proliferation and viability. In addition, cyclic stretch resulted in decreased levels of oxidative stress over time in hyperoxia-exposed cells. Our results suggest that cyclic stretch, as applied in this study, can minimize the detrimental effects of hyperoxia on alveolar epithelial A549 cells.     

The effect of human urogastrone on lung phospholipids in fetal rabbits

Previous in vivo studies have demonstrated that mouse epidermal growth factor (EGF) can enhance fetal lung maturation. We have examined the effect of urogastrone, the human equivalent of mouse EGF and a related growth factor, on the phospholipid profile of fetal rabbit lung lavage and its action on fetal rabbit Type II pneumocytes in culture. Urogastrone (1 or 8 micrograms) given i.p. to fetal rabbits on day 25 of gestation resulted in increased total phospholipid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine contents, increased phosphatidylinositol and phosphatidylethanolamine as a proportion of phospholipid and decreased sphingomyelin as a proportion of phospholipid in lung lavages on day 28. These changes were unaccompanied by alterations in body weight or lung weight, DNA or protein concentrations. Urogastrone (16 micrograms) resulted in increased fetal deaths. Phospholipid profiles on day 27 were unchanged after fetal administration of urogastrone (1 microgram) on day 25. Urogastrone (0.01 and 0.1 ng/ml) added to fetal rabbit Type II pneumocytes in culture for 24 h enhanced the incorporation of radiolabelled choline and thymidine into phosphatidylcholine and DNA respectively. These findings indicate that human urogastrone can alter the phospholipid composition of the rabbit lung in a similar manner to that which occurs during maturation of the lung surfactant system in late pregnancy. This effect can be achieved, at least in part, by a direct action on Type II pneumocytes.     

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.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.     

Differences in mRNA Expression,Protein Content,and Enzyme Activity of Superoxide Dismutases in Type II Pneumocytes of Acute and Chronic Lung Injury

The lung is protected against oxidative stress by a variety of antioxidants and type II pneumocytes seem to play an important role in antioxidant defense. Previous studies have shown that inhalation of NO₂ results in acute and chronic lung injury. How the expression and enzyme activity of antioxidant enzymes are influenced in type II cells of different inflammatory stages has yet not been studied. To elucidate this question, we exposed rats to 10 ppm NO₂ for 3 or 20 days to induce acute or chronic lung injury. From these and air-breathing rats, type II pneumocytes were isolated. The mRNA expression and protein content of CuZnSOD and MnSOD as well as total SOD-specific enzyme activity were determined. For the acute lung injury (3 d NO₂), the expression of CuZnSOD mRNA was significantly increased, while MnSOD expression was significantly reduced after 3 days of NO 2 exposure. For the chronic lung injury (20 d NO₂), CuZnSOD expression was still enhanced, while MnSOD expression was comparable to control. In parallel to CuZnSOD mRNA expression, the protein amount was significantly increased in acute and chronic lung injury however MnSOD protein content exhibited no intergroup differences. Total SOD enzyme activity showed a significant decrease after 3 days of NO₂ exposure and was similar to control after 20 days. We conclude that during acute and chronic lung injury in type II pneumocytes expression and protein synthesis of CuZnSOD and MnSOD are regulated differently.     

Differences in mRNA expression,protein content,and enzyme activity of superoxide dismutases in type II pneumocytes of acute and chronic lung injury

The lung is protected against oxidative stress by a variety of antioxidants and type II pneumocytes seem to play an important role in antioxidant defense. Previous studies have shown that inhalation of NO₂ results in acute and chronic lung injury. How the expression and enzyme activity of antioxidant enzymes are influenced in type II cells of different inflammatory stages has yet not been studied. To elucidate this question, we exposed rats to 10 ppm NO₂ for 3 or 20 days to induce acute or chronic lung injury. From these and air-breathing rats, type II pneumocytes were isolated. The mRNA expression and protein content of CuZnSOD and MnSOD as well as total SOD-specific enzyme activity were determined. For the acute lung injury (3 d NO₂), the expression of CuZnSOD mRNA was significantly increased, while MnSOD expression was significantly reduced after 3 days of NO 2 exposure. For the chronic lung injury (20 d NO₂), CuZnSOD expression was still enhanced, while MnSOD expression was comparable to control. In parallel to CuZnSOD mRNA expression, the protein amount was significantly increased in acute and chronic lung injury however MnSOD protein content exhibited no intergroup differences. Total SOD enzyme activity showed a significant decrease after 3 days of NO₂ exposure and was similar to control after 20 days. We conclude that during acute and chronic lung injury in type II pneumocytes expression and protein synthesis of CuZnSOD and MnSOD are regulated differently.     

Surfactant-associated protein inhibits phospholipid secretion from type II cells

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.     

Lung surfactant in subacute pulmonary disease

Pulmonary surfactant is a surface active material composed of both lipids and proteins that is produced by alveolar type II pneumocytes. Abnormalities of surfactant in the immature lung or in the acutely inflamed mature lung are well described. However, in a variety of subacute diseases of the mature lung, abnormalities of lung surfactant may also be of importance. These diseases include chronic obstructive pulmonary disease, asthma, cystic fibrosis, interstitial lung disease, pneumonia, and alveolar proteinosis. Understanding of the mechanisms that disturb the lung surfactant system may lead to novel rational therapies for these diseases.     

Biochemical pathology of lung damage produced by chemicals.

Damage to the lung may be caused by chemicals that gain access to the alveolar zone by inhalation or via the pulmonary circulation. Several agents toxic to the lung have recently been found to bind covalently to pulmonary macromolecules or to disrupt certain metabolic reactions. However, it has also been observed that extensive chemical lung injury is not necessarily preceded by a depression of pulmonary metabolic reactions. One possible explanation for this might be that biochemical changes due to cell death are often masked and/or compensated for by changes associated with lung tissue repair. Substantial cell proliferation as a response to toxic lung damage is a common phenomenon in lung pathology. This makes it necessary to develop models that permit analysis of the biochemical events triggering and accompanying cell growth in lung. We have recently examined some aspects of cell proliferation in mouse lung. Intraperitoneal injection of the antioxidant butylated hydroxytoluene (BHT) produces within 3-5 days extensive hypertrophy, hyperplasia, and general disorganization of the cellular components of the lung. Total lung weight and total DNA per lung almost double within this time and are accompanied by proportional increases in protein and lipids. RNA accumulates at a faster rate than DNA. The changes in lung composition are accompanied by dose-dependent increases in the in vivo incorporation of thymidine into DNA and of leucine into protein. The activities of several enzymes (thymidine kinase, DNA polymerase, uridine kinase, glucose-6-phosphate dehydrogenase, and 5'-nucleotidase) increase substantially after BHT. Administration of BHT to mice seems to offer a convenient tool to study cell growth in the lungs of mice.     

Comparison of the enzyme activities of phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol synthesis in freshly isolated type II pneumocytes and whole lung from the adult rat

We compared the activities of enzymes of phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol synthesis in whole lung tissue and freshly isolated type II pneumocytes from adult rats. The activities of 1-acylglycerophosphocholine acyltransferase and CDPdiacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase were 2.9- and 4.4-fold higher, respectively, in type II cell sonicates than in whole lung homogenates. There was little difference between the type II cells and whole lung in the activities of choline kinase, choline-phosphate cytidyltransferase, cholinephosphotransferase, phosphatidate phosphatase, phosphatidate cytidylytransferase or CDPdiacylglycerol-inositol 3-phosphatidyltransferase. Since the type II cell is the source of pulmonary surfactant, and disaturated phosphatidylcholine and phosphatidylglycerol are major components of surfactant, it is of interest that this cell is enriched in the activities of enzymes exclusively involved in the synthesis of these lipids. In view of possible proteolytic damage during isolation we compared freshly isolated type II cells with those cultured for 1 day. The rates of incorporation of [methyl-3H]choline and [2-3H]glycerol into phospholipids, L-[U-14C]phenylalanine into protein and [methyl-3H]thymidine into DNA were the same in the freshly isolated and cultured cells. The composition of the phospholipids synthesized from [2-3H]glycerol and sodium [1-14C]acetate were also the same. The freshly isolated cells were at least 90% pure and did not release significant amounts of lactate dehydrogenase. Since use of freshly isolated cells avoids cell loss during culture they provide an attractive alternative, particularly in studies requiring large amounts of material.