Inhibition of surfactant release from isolated type II cells by compound 4880

Release of [³H]phosphatidylcholine from pulmonary Type II epithelial cells was stimulated by terbutaline, forskolin and cytochalasin D. Compound $\text{48}\text{80}$ inhibited both basal and agonist-stimulated release of [³H]PC. The IC₅₀ for inhibition by compound $\text{48}\text{80}$ was 1–2 μg/ml, and was similar for inhibition of both basal and stimulated release of [³H]phosphatidylcholine. Inhibitory effects of $\text{48}\text{80}$ were noted following a 1 h exposure to compound $\text{48}\text{80}$ and persisted up to 3 h. The inhibitory effect of compound $\text{48}\text{80}$ was entirely reversed by removing compound $\text{48}\text{80}$ from the external milieu. Compound $\text{48}\text{80}$ had no effect on cytosolic cyclic AMP levels or lactate dehydrogenase release. Inhibition of surfactant release produced by compound $\text{48}\text{80}$ was unaffected by changes in extracellular calcium concentrations. Compound $\text{48}\text{80}$ is a non-toxic inhibitor of phosphatidylcholine release from Type II epithelial cells.     

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.     

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.     

Albumin stimulates the release of lysophosphatidylcholine from cultured rat hepatocytes.

The effect of albumin on the release of [3H]lysophosphatidylcholine from cultured rat hepatocytes prelabelled with [Me-3H]choline was studied. In the absence of serum and albumin from the medium, the cells released essentially no [3H]lysophosphatidylcholine. Albumin stimulated this process dramatically, and it reached a plateau at 2 mg/ml. After an initial lag of 30 min, the release of [3H]lysophosphatidylcholine was linear for at least 4 h. At low concentrations, albumin slightly stimulated [3H]phosphatidylcholine release. The albumin had no measurable effect on the metabolism of cellular [3H]phosphatidylcholine, [3H]lysophosphatidylcholine or [3H]glycerophosphocholine. In addition, albumin did not alter the release of 3H-labelled water-soluble compounds, including [3H]glycerophosphocholine, into the medium. The possibility that the [3H]lysophosphatidylcholine was arising from catabolism of [3H]phosphatidylcholine in the medium by secreted enzymes was excluded. The effect on [3H]lysophosphatidylcholine secretion was also observed when the cells were incubated with alpha-cyclodextrin, a cyclic polysaccharide that has the ability to bind lysophosphatidylcholine. The albumin-released lysophosphatidylcholine was enriched in unsaturated fatty acids. Alteration of the fatty acid composition of cellular phosphatidylcholine gave rise to parallel changes in phosphatidylcholine and lysophosphatidylcholine in the medium. It is concluded that phosphatidylcholine is constantly being degraded in the rat hepatocyte to lysophosphatidylcholine which is released into the medium only when a suitable acceptor is present.     

Hydrolysis of phosphatidylcholine by phospholipase D is a common response to mitogens which stimulate inositol lipid hydrolysis in Swiss 3T3 fibroblasts

The stimulated hydrolysis of inositol lipids and phosphatidylcholine (PtdCho) by bombesin, [Arg8]vasopressin ([Arg8]Vp) and prostaglandin F2 alpha (PGF2 alpha) was analysed in Swiss 3T3 cells pre-labelled to isotopic equilibrium with either [methyl-3H]choline, myo-[2-3H]inositol or [9,10 (n)-3H]palmitic acid. All three agonists activated the phospholipase D-catalysed hydrolysis of PtdCho as determined by the release of [3H]choline (Cho) and the formation of [3H]phosphatidylbutanol (PtdBut). The release of [3H]choline by each agonist exhibited similar sensitivity to prolonged pre-exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The release of [3H]choline exhibited the same dose dependency as the production of total inositol phosphates for each mitogen suggesting that the two responses might be mediated through identical receptors. Acute pre-treatment with TPA allowed the dissociation of inositol lipid hydrolysis from PtdCho breakdown, since it inhibited inositol phosphate accumulation but stimulated choline generation. The loss of mitogen stimulated choline release in cells pre-treated with the phorbol ester for 48 h was not due to loss of stimulated inositol phosphate production which was reproducibly enhanced in these 'down-regulated' cells.     

GTP-related effects and binding by differentiating fetal rabbit type II alveolar cells in vitro.

Type II alveolar cells were isolated from fetal rabbit lungs and used to determine the effect of GTP-binding protein activation on release of surfactant-related material. Cells were prelabelled with [3H]choline for 24 hours. NaF, a G-protein activator and GTP gamma S, a nonhydrolysable analogue of GTP, both loaded by hypoosmotic shock treatment, stimulated release of radioactive disaturated phosphatidylcholine. Localization of the cellular binding of [alpha-32P]GTP in fetal type II cells which were induced to differentiate by exposure to fetal lung fibroblast conditioned medium showed that two proteins of apparent molecular weights of 39.6 kd and 17.9 kd bound [alpha-32P]GTP. These proteins were detected only in the cells exposed to the conditioned medium. These results suggest GTP-binding proteins are involved in DSPC secretion and differentiation of fetal type II cells is accompanied by changes in GTP binding characteristics.     

Differential activation of membrane phospholipid turnover by compound 48/80 and ionophore A23187 in rat mast cells

Membrane phospholipid turnover was investigated during histamine release from rat mast cells. Addition of calcium ionophore A23187 (0.5 microgram/ml) to mast cells prelabeled with [3H]glycerol induced the rapid and progressive increase in phosphatidic acid (PA) and 1,2-diacylglycerol (DG), which was concomitant with the small rise in phosphatidylinositol (PI). Loss of the level in triacylglycerol (TG) was very marked. Polyamine compound 48/80 (5 micrograms/ml) was shown to cause rises in PA, 1,2-DG, and PI without any significant changes in TG. Both stimuli increased incorporation of exogenous [3H]glycerol into phospholipids, indicating the involvement of de novo synthesis in phospholipid metabolism. Studies with [3H]arachidonic acid-labeled mast cells showed an enhanced liberation of radioactive arachidonate and metabolites upon histamine release. There were associated decreases of radioactivity in phosphatidylcholine (PC) and TG when exposed to A23187, while phosphatidylethanolamine (PE) was degraded as a result of 48/80 activation. The transient increases of [3H]arachidonoyl-1,2-DG and PA were caused by 48/80, while A23187 showed a gradual rise in the radioactivity in these two lipid fractions. These findings reflect activation of phospholipase C. When mast cells were activated by low concentrations of A23187 (0.1 microgram/ml) and 48/80 (0.5 microgram/ml), different behaviors of PI metabolism were observed. An early degradation of PI and a subsequent formation of 1,2-DG and PA suggest that the lower concentrations of these agents stimulate the PI cycle initiated by PI breakdown rather than de novo synthesis. These results demonstrate that marked and selective changes in membrane phospholipid metabolism occur during histamine release from mast cells, and that these reactions seem to be controlled by the coordination of degradation and biosynthesis, depending on the type and the concentration of stimulants. A23187 stimulates arachidonate release perhaps via the cleavages of PC and TG, whereas 48/80 liberates arachidonate from PE.     

Properties of freshly isolated type II alveolar epithelial cells

The biochemical characteristics of type II alveolar epithelial cells dissociated from adult rabbit lung by instillation of low concentrations of an elastase trypsin mixture are reported. Cells studied immediately (within 4 h) after isolation were found to incorporate the radioactively labelled precursors [U-¹⁴C]glucose, [methyl-³H]choline and [³H]palmitate into cellular phosphatidylcholine at rates 2–10-fold higher than previously reported for cells not subject to short-term cell culture. Secretion of phosphatidylcholine was stimulated by beta-adrenergic agonists. Measurement of specific activities of enzymes of phospholipid biosynthesis in subcellular fractions of isolated lung cells showed a significant enrichment of acyl coenzyme A-lysophosphatidylcholine acyltransferase, an enzyme believed to be involved in pulmonary surfactant phosphatidylcholine remodeling, in the endoplasmic reticulum of type II cells. These observations support the utility of freshly isolated type II cells as a model system for the study of the functions of the alveolar epithelium.     

Phorbol ester-stimulated hydrolysis of phosphatidylcholine and phosphatidylethanolamine by phospholipase D in HeLa cells. Evidence that the basal turnover of phosphoglycerides does not involve phospholipase D

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated the release of [3H]ethanolamine from HeLa cells prelabeled with [3H]ethanolamine within 2 min, and of [3H]choline from cells prelabeled with [3H]choline after a lag of 10-20 min. This result suggests that TPA activates phospholipase D. Propranolol alone or propranolol plus TPA stimulated phosphatidic acid (PA) labeling in cells prelabeled with [3H]hexadecanol. In the presence of ethanol, TPA stimulated the accumulation of labeled phosphatidylethanol (PEth); no PEth was formed in the absence of TPA. TPA-dependent PEth accumulation was not observed in cells pretreated with TPA to down-regulate protein kinase C, whereas propranolol-induced accumulation of PA was unaffected by TPA pretreatment. Incubation of prelabeled cells with propranolol alone caused a rapid loss of label and phospholipid mass from both phosphatidylethanolamine and phosphatidylcholine (PC) together with an accumulation of PA and phosphatidylinositol plus phosphatidylserine. When [3H]hexadecanol-prelabeled cells were pulse labeled with 32P to label nucleotide pools, propranolol induced the accumulation of both 3H- and 32P-labeled PA. When cells were prelabeled with lyso-PC double labeled with 3H and 32P, and incubated with propranolol, only 3H-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of 32P from the lipid. These results suggest that the basal turnover of phosphatidylethanolamine and PC involves the sequential actions of phospholipase C, diglyceride kinase, and PA phosphohydrolase.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Inhibition of non-cytotoxic histamine liberation from isolated rat mast cells by antihistaminic preparations]

Phenothiazines (chlorpromazine and promethazine) and antihistaminic quinuclidine derivatives [phencarol, quinuclidyl-3-di-(o-tolyl) carbinol, hydrochloride quinuclidyl-3-di-(o-methoxyphenyl) carbinol--HQMC] at concentrations preceding the histamine-releasing ones inhibited the compound 48/80-induced histamine release from the isolated rat mast cells. HQMC inhibited histamine release induced by selective liberators (compound 48/80, MCD-peptide, specific antigen), but potentiated histamine release induced by nonselective liberators (chlorpromazine, tryton X-100). The inhibition by HQMC of histamine release induced by compound 48/80 increased during 1 min and was reversible. The inhibitory effect of all the compounds tested was partially counteracted by glucose.     

The choline-depleted type II pneumonocyte. A model for investigating the synthesis of surfactant lipids.

When type II pneumonocytes from adult rats were maintained in a medium that lacked choline, the incorporation of [14C]glycerol into phosphatidylcholine was not greatly diminished during the period that the cells displayed characteristics of type II pneumonocytes. Cells that were maintained in choline-free medium that contained choline oxidase and catalase, however, became depleted of choline and subsequent synthesis of phosphatidylcholine by these cells was responsive to choline in the extracellular medium. Incorporation of [14C]glycerol into phosphatidylcholine by choline-depleted cells was stimulated maximally (approx. 6-fold) by extracellular choline at a concentration (0.05 mM) that also supported the greatest incorporation into phosphatidylglycerol. The incorporation of [14C]glycerol into other glycerophospholipids by choline-depleted cells was not increased by extracellular choline. When cells were incubated in the presence of [3H]cytidine, the choline-dependent stimulation of the synthesis of phosphatidylcholine and phosphatidylglycerol was accompanied by an increased recovery of [3H]CMP. This increased recovery of [3H]CMP reflected an increase in the intracellular amount of CMP from 48 +/- 9 to 76 +/- 16 pmol/10(6) cells. Choline-depleted cells that were exposed to [3H]choline contained [3H]CDP-choline as the principal water-soluble choline derivative. As the extracellular concentration of choline was increase, however, the amount of 3H in phosphocholine greatly exceeded that in all other water-soluble derivatives. Choline-depletion of cells resulted in an increase in the specific activity of CTP:phosphocholine cytidylyltransferase in cell homogenates (from 0.40 +/- 0.15 to 1.31 +/- 0.20 nmol X min-1 X mg of protein-1). These data are indicative that the biosynthesis of phosphatidylcholine is integrated with that of phosphatidylglycerol and are consistent with the proposed involvement of CMP in this integration. The choline-depleted type II pneumonocyte provides a new model for investigating the regulation of CTP:phosphocholine cytidylyltransferase activity.     

Botulinum toxin a inhibits acetylcholine release from cultured neurons in vitro

Summary Clostridium botulinum type toxin A (BoTx) blocks stimulus-induced acetylcholine (ACh) release from presynaptic nerve terminals at peripheral neuromuscular junctions. However, the detailed mechanism of this effect remains elusive. One obstacle in solving this problem is the lack of a suitable in vitro homogenous cholinergic neuronal model system. We studied the clonal pheochromocytoma PC12 cell line to establish such a model. PC12 cells were differentiated in culture by treatment with 50 ng/ml nerve growth factor (NGF) for 4 days to enhance cellular ACh synthesis and release properties. Stimulation of these cells with high K⁺ (80 mM) in the perfusion medium markedly increased calcium-dependent [³H]ACh release compared to undifferentiated cells. Stimulated [³H]ACh release was totally inhibited by pretreatment of cells with 2 nM BoTx for 2 h. BoTx inhibition of [³H]ACh release was time- and concentration-dependent. A 50% inhibition was obtained after 2 h incubation with a low (0.02 nM) toxin concentration. The time required for 2 nM BoTx to cause a measurable inhibition (18%) of stimulated [³H]ACh release was 30 min. Botulinum toxin inhibition of stimulated ACh release was prevented by toxin antiserum and heat treatment, suggesting the specificity of the toxin effect. Our results show that by differentiation with NGF, PC12 cells can be shifted from an insensitive to a sensitive state with respect to BoTx inhibition of stimulated ACh release. This cell line, therefore, may serve as a valuable in vitro cholinergic model system to study the mechanism of action of BoTx.     

Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells

To determine whether small hydrophobic surfactant peptides (SP-B and SP-C) participate in recycling of pulmonary surfactant phospholipid, we determined the effect of these peptides on transfer of 3H- or 14C-labelled phosphatidylcholine from liposomes to isolated rat alveolar Type II cells and Chinese hamster lung fibroblasts. Both natural and synthetic SP-B and SP-C markedly stimulated phosphatidylcholine transfer to alveolar Type II cells and Chinese hamster lung fibroblasts in a dose- and time-dependent fashion. Effects of the peptides on phospholipid uptake were dose-dependent, but not saturable and occurred at both 4 and 37 degrees C. Uptake of labelled phospholipid into a lamellar body fraction prepared from Type II cells was augmented in the presence of SP-B. Neither SP-B nor SP-C augmented exchange of labelled plasma membrane phosphatidylcholine from isolated Type II cells or enhanced the release of surfactant phospholipid when compared to liposomes without SP-B or SP-C. Addition of native bovine SP-B and SP-C to the phospholipid vesicles perturbed the size and structure of the vesicles as determined by electron microscopy. To determine the structural elements responsible for the effect of the peptides on phospholipid uptake, fragments of SP-B were synthesized by solid-phase protein synthesis and their effects on phospholipid uptake assessed in Type II epithelial cells. SP-B (1-60) stimulated phospholipid uptake 7-fold. A smaller fragment of SP-B (15-60) was less active and the SP-B peptide (40-60) failed to augment phospholipid uptake significantly. Like SP-B and SP-C, surfactant-associated protein (SP-A) enhanced phospholipid uptake by Type II cells. However, SP-A failed to significantly stimulate phosphatidylcholine uptake by Chinese hamster lung fibroblasts. These studies demonstrate the independent activity of surfactant proteins SP-B and SP-C on the uptake of phospholipid by Type II epithelial cells and Chinese hamster lung fibroblasts in vitro.     

Effects of degranulation of mast cells on proliferation of mesenchymal cells in the mesentery of mice

Summary A mast-cell activator, compound 48/80, causes proliferation of mesenchymal cells in the mesentery of rats. Its effect on W/W ^vmice deficient in mast cells was tested to determine whether the proliferation is mediated in the degranulation of mast cells. Incorporation of [³H]thymidine into mesenchymal cells in the mesentery of these mice with or without compound 48/80 was very small compared to their normal litter mates. However, bone marrow transplantation markedly enhanced the effect of compound 48/80, and resulted in an incorporation of [³H]thymidine almost comparable to that observed in normal mice. Our results provide evidence that mesenchymal cell proliferation is caused by a product secreted by mast cells when stimulated by compound 48/80.Supported by a Grant-in-Aid for Scientific Research, No. 366, from the Japanese Ministry of Health and WelfareThe authors are indebted to Drs. Motomu Minamiyama and Yukio Hirata for valuable advices, and to Miss Mitsuko Inoue for technical assistance     

Interruption of TPA-induced signals by an antiviral and antitumoral xanthate compound: inhibition of a phospholipase C-type reaction

The effect of tricyclodecan-9-yl-xanthogenate on the phorbolester TPA induced changes in phosphatidylcholine metabolism was investigated. In the simultaneous presence of the xanthate TPA failed to stimulate the metabolic [32P] turnover of the major phospholipids. The precursor molecule [3H] choline was incorporated into phosphatidylcholine after pulse labeling in TPA/D609-treated cells. Thus, the reduction of the [32P] phosphatidylcholine turnover did not appear to result from an inhibition of the TPA-stimulated phosphatidylcholine biosynthesis. However, the xanthate exerted an inhibitory effect on the TPA-stimulated liberation of [3H] phosphorylcholine from [3H] phosphatidylcholine in cells prelabeled with [3H] choline. Furthermore, the TPA-induced rise in the diacylglycerol level was reduced in the presence of the compound. Thus, these results provide evidence that the xanthate inhibits a TPA-induced phospholipase C activity in the intact cell.     

Stimulation of choline release from NG108-15 cells by 12-O-tetradecanoylphorbol 13-acetate.

The effects of the potent tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on phosphatidylcholine (PtdCho) metabolism were investigated in the neuroblastoma X glioma hybrid cell line NG108-15. TPA (100 nM) stimulated by 150-200% the release into the medium of 3H radioactivity from cells that had been pre-labelled with [3H]choline. H.p.l.c. analysis of the medium revealed that TPA stimulated the release of only free [3H]choline (212 +/- 11% of control), without affecting such other labelled metabolites as [3H]phosphocholine and [3H]glycerophosphocholine. This effect was concentration-dependent, with a half-maximal effect obtained at 27.5 +/- 6.8 nM, and was observable as early as 5-10 min after exposure to TPA. The TPA-induced release of [3H]choline into the medium was accompanied by a small and variable decrease in cellular [3H]PtdCho (to 93 +/- 4% of control). However, the radioactivity associated with water-soluble cellular choline metabolites (mainly [3H]phosphocholine and [3H]glycerophosphocholine) remained unchanged. TPA also stimulated the release of [3H]choline derived from [3H]PtdCho that had been produced via the methylation pathway from [3H]methionine. These data suggest that phosphatidylcholine may serve as the source of free choline released from the cells in response to TPA. The possible enzymic mechanisms underlying this response are discussed.     

Serum albumin enhances the uptake of [3H]cholesterol from phosphatidylcholine vesicles by cultured human fibroblasts

Cultured human lung fibroblasts, incubated with cholesterol/phosphatidylcholine vesicles (cholesterol: phosphatidylcholine molar ratio 1:1) incorporated vesicle [3H]-cholesterol linearly for at least 48 h by an exchange process without gaining sterol mass. The incorporation of [3H]cholesterol by the cells was markedly enhanced in the presence of purified bovine serum albumin. A fraction of the incorporated vesicle [3H]cholesterol was esterified by the cells.     

Inhibitory effect of fulvic acid extracted from Canadian sphagnum peat on chemical mediator release by RBL-2H3 and KU812 cells

Fulvic acid (FA) was extracted and purified from Canadian Sphagnum peat (CP-FA) and characterized by using an element analysis meter, Fourier transform infrared (FT-IR) spectroscopy, electron spin resonance (ESR) spectroscopy, and (13)C-nuclear magnetic resonance ((13)C-NMR) spectroscopy. To investigate the antiallergic effect of CP-FA, we incubated rat basophilic leukemia (RBL-2H3) cells with 0.001-10.0 microg/ml of CP-FA and determined the beta-hexosaminidase release inhibition at different response stages. The intracellular calcium [Ca(2+)](i) level was also determined by using Fluo 3-AM, a calcium-specific fluorescent probe, and the cytotoxicity of CP-FA was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The results revealed that RBL-2H3 cells incubated for 48 h with 0.001-10.0 microg/ml of CP-FA did not show any decreased viability. CP-FA inhibited the beta-hexosaminidase release by IgE-sensitized, antigen-stimulated RBL-2H3 cells at the antigen-antibody binding stage and the antibody-receptor binding stage. CP-FA also inhibited histamine release from A23187 plus PMA- or compound 48/80-stimulated KU812 cells. Furthermore, there was a decrease in the intracellular [Ca(2+)](i) level in IgE-sensitized cells incubated with CP-FA and stimulated with antigen. Our results show that CP-FA may be useful for the treatment or prevention of allergic diseases.     

Altered lipid synthesis in type II pneumonocytes exposed to lung surfactant.

When type II pneumonocytes were exposed to purified lung surfactant that contained 1-palmitoyl-2-[3H]palmitoyl-glycero-3-phosphocholine, radiolabelled surfactant was apparently taken up by the cells since it could not be removed by either repeated washing or exchange with non-radiolabelled surfactant, but was released when the cells were lysed. After 4 h of exposure to [3H]surfactant, more than half of the 3H within cells remained in disaturated phosphatidylcholine. Incorporation of [3H]choline, [14C]palmitate and [14C]acetate into glycerophospholipids was decreased in type II cells exposed to surfactant and this inhibition, like surfactant uptake, was half-maximal when the extracellular concentration of surfactant was approx. 0.1 mumol of lipid P/ml. Inhibition of incorporation of radiolabelled precursors by surfactant occurred rapidly and reversibly and was not due solely to dilution of the specific radioactivity of intracellular precursors. Activity of dihydroxyacetone-phosphate acyltransferase, but not glycerol-3-phosphate acyltransferase, was decreased in type II cells exposed to surfactant and this was reflected by a decrease in the 14C/3H ratio of total lipids synthesized when cells incubated with [U-14C]glycerol and [2-3H]glycerol were exposed to surfactant. Phosphatidylcholine, phosphatidylglycerol and cholesterol, either individually or mixed in the molar ratio found in surfactant, did not mimic purified surfactant in the inhibition of glycerophospholipid synthesis. In contrast, an apoprotein fraction isolated from surfactant inhibited greatly the incorporation of [3H]choline into lipids and this inhibitory activity was labile to heat and to trypsin. It is concluded that the apparent uptake of surfactant by type II cells in vitro is accompanied by an inhibition of glycerophospholipid synthesis via a mechanism that involves a surfactant apoprotein.