Paul A. Stevens

Effect of hyperoxia on the composition of the alveolar surfactant and the turnover of surfactant phospholipids, cholesterol, plasmalogens and vitamin E.

Experimental and clinical studies have provided evidence for the involvement of oxygen free radicals in development of acute and chronic lung diseases. Hyperoxia is very often an indispensable therapeutic intervention which seems to impose oxidative stress on lung tissue. We measured the effect of hyperoxia (80% O2 for 20 h) (1) on the lipid composition of pulmonary surfactant treated in vitro, (2) on surfactant lipid synthesis and secretion of type II pneumocytes in primary culture, (3) on the lipid composition and on the SP-A content of rat lung lavages and (4) on the turnover of phospholipids, cholesterol, plasmalogens and vitamin E in type II pneumocytes, lamellar bodies and lavages of adult rat lungs. (1) Hyperoxia of lung lavages in vitro reduces the vitamin E content significantly but does not change the relative proportion of PUFA or the content of plasmalogens. (2) Hyperoxia does not affect the biosynthesis or secretion of surfactant lipids and plasmalogens by type pneumocytes in primary culture. (3) Hyperoxic treatment of rats increases the SP-A content and reduces the vitamin E content significantly but does not change the concentration of other lipid components of lung lavage. (4) The vitamin E turnover, measured in type II pneumocytes, lamellar bodies and lung lavages, is increased 2-fold in these fractions. In contrast, the turnover of surfactant cholesterol and surfactant lipids does not change. (5) Hyperoxia caused an increase of the vitamin E uptake by type II pneumocytes resulting in a vitamin E enrichment of lamellar bodies. From these results we conclude that type II pneumocytes are able to regulate the turnover of lipophilic constituents of the alveolar surfactant independently of each other. Hyperoxia caused type II pneumocytes to increase the vitamin E content of lamellar bodies. The lipid and SP-A content of alveolar fluid can be regulated independently each other.

Interaction of pulmonary surfactant protein A with phospholipid liposomes: a kinetic study on head group and fatty acid specificity

Recent work on surfactant protein A (SP-A) has shown that Ca(2+) induces an active conformation, SP-A, which binds rapidly to liposomes and mediates their aggregation. Employing sensitive real time assays, we have now studied the lipid binding characteristics of the SP-A liposome interaction. From the final equilibrium level of the resonant mirror binding signal, an apparent dissociation constant of ca. K(d)=5 microM is obtained for the complex between SP-A and dipalmitoylphosphatidylcholine (DPPC) liposomes. At nanomolar SP-A concentrations, this complex is formed with a subsecond (0.3 s) reaction time, as measured by light-scattering signals evoked by photolysis of caged Ca(2+). With palmitoyloleoylphosphatidylcholine (POPC), the complex formation proceeds at half the rate, compared to DPPC, leading to a lower final equilibrium level of SP-A lipid interaction. Distearoylphosphatidylcholine (DSPC) shows a stronger interaction than DPPC. Regarding the phospholipid headgroups, phosphatidylinositol (PI) and sphingomyelin (SM) interact comparable to DPPC, while less interaction is seen with phosphatidylethanolamine (PE) or with phosphatidylglycerol (PG). Thus both headgroup and fatty acid composition determine SP-A phospholipid interaction. However, the protein does not exhibit high specificity for either the polar or the apolar moiety of phospholipids.

Perfluorocarbons are taken up by isolated type II pneumocytes and influence its lipid synthesis and secretion.

ObjectiveBecause alveoli fill with perfluorocarbons during liquid ventilation, an uptake of perfluorocarbons by type II pneumocytes can be postulated that might affect synthesis and secretion of pulmonary surfactant. The study was performed to answer the following questions: Do isolated type II pneumocytes take up perfluorocarbons? Do perfluorocarbons affect lipid synthesis of type II cells? Do perfluorocarbons change surfactant secretion of type II pneumocytes? DesignControlled experiments that used isolated type II pneumocytes. SettingExperimental laboratory of a university hospital. SubjectsMale Wistar rats. InterventionsTo study perfluorocarbon uptake, isolated type II cells were incubated with fluorescence-labeled perfluorocarbons and examined with a laser scanning microscope. The effect of perfluorocarbons on biosynthesis of phospholipids and triglycerides was measured by incubating cells that were pulse-labeled with [H]-palmitic acid for 30 secs, with two different perfluorocarbons (PF 5080 or RM 101) for 10 mins. The effect of perfluorocarbon incubation on lipid secretion was studied by transmission electron microscopy. To quantify secretion, adherent type II pneumocytes (containing radioactively labeled phospholipids) were incubated with perfluorocarbons, and extra- and intracellular radioactivity was measured. Measurements and Main ResultsWe found a significant uptake of labeled perfluorocarbons into lamellar bodies within 10 mins. Both perfluorocarbon species significantly (p < .05) reduced the biosynthesis of phospholipids when compared with control. Perfluorocarbon incubation did not affect mitochondrial activity, tested by MitoTracker staining. Transmission electron microscopy revealed changes that suggest an increased secretion of surfactant by type II cells. Studies with radioactively labeled surfactant revealed a significantly (p < .01) higher amount of extracellular lipids after RM 101 and PF 5080 treatment (RM 101, 17 ± 7.9%; PF 5080, 9 ± 1.9%) compared with control (5.3 ± 1.9%). ConclusionsOur results suggest that perfluorocarbons are taken up by type II pneumocytes and cause an increased secretion of surfactant, despite a relative reduction in the synthesis of phospholipids.

Different Pathways of Internalization of the Somatostatin Receptor sst3 and the Possible Autoreceptor sst1

Different Pathways of Internalization of the Somatostatin Receptor sst3 and the Possible Autoreceptor sst1

THE ANTIOXIDATIVE POTENTIAL OF ALVEOLAR SURFACTANT

Alveolar surfactant is exposed to a variety of oxidants that can oxidize functionally important lipids and proteins. We examined the hypothesis that the type II pneumocyte equips surfactant with antioxidants to counteract its oxidation.Rat type II cells, cultured in the presence of 14C-palmitic acid and either 3H-vitamin E or 3H-vitamin D, responded to stimulation with isoproterenol with a time-dependent increase in secretion of 14C-labelled phospholipids and 3H-vitamin E, but not of 3H-vitamin D.Plasmatogens - a subclass of phospholipids - also act as antioxidants in animal cells. Type II cells, cultured in presence of 3H palmitic acid and 14C hexadecanol, synthesize and secrete 3H labeled phospholipids and 14C labeled plasmalogens spontaneously and in response to isoproterenol stimulation.In pharyngeal aspirates of healthy newborns vitamin Eand plasmalogen contents range from 2-10 nmol/μmol polyunsaturated fatty acids (PUFA) and from 8-20 nmol/μmol PUFA respectively.We conclude that alveolar surfactant is equipped with lipophilic antioxidants of its own during its formation in type II cells. These lipophilic components could be of use as clinical parameters to evaluate the antioxidantive potential of alveolar surfactant.Supported by BMFT Project “Risikoneugeborenes”.

219 MECHANISM OF SP-A-MEDIA TED SURFACTANT ENDOCYTOSIS BY TYPE II CELLS

AIM: Surfactant protein A (SP-A) enhances surfactant lipid uptake by type II pneumocytes. In the presence of SP-A internalized surfactant lipids are reported to bypass the degradative pathway and are recycled towards lamellar bodies. We wanted to further clarify the role of SP-A in surfactant lipid endocytosis.METHODS: A previously described antibody (2H5) against a type II cell membrane protein which stimulates surfactant lipid uptake by type II cells (Pediatr. Res. 1994, 35: 278) was used in parallel with SP-A to study uptake and intracellular fate of liposomes with surfactant-like composition in rat type II cells.RESULTS: In the presence of 2H5 or SP-A significantly more labeled lipid is internalized in a time- and concentration-dependent fashion by type II cells than in their absence (2H5 2-fold, SP-A 3-fold above control). In cells in solution no difference in the distribution of label in phospholipid classes between control cells and cells incubated with either 2H5 on SP-A was found. In adherent 24 hour-old cells after one hour of incubation 82% of the internalized 3H-label is still associated with PC in control cells vs. 87% in the presence of 2H5 and 94% with SP-A. Surprisingly, inhibition of coated pit formation (uptake pathway for SP-A) by K+-depletion enhanced lipid uptake by type II cells significantly. Also, inhibition of protein kinase C (PKC) (staurosporine 100 nM) enhanced lipid uptak^ by type II cells in the presence of SP-A.CONCLUSIONS: SP-A and 2H5 do quantitatively enhance lipid uptake in type II cells. The subsequent intracellular fate of the PC molecule may depend on the type of assay used. Uptake via coated pits and PKC activation are involved in these processes.

INFLUENCE OF TYP-II CELL-CONDITONED MEDIUM ON THE PROLIFERATION OF FIBROBLAST CELL CULTURES

Lung fibroblast proliferation is an important component of diseases such as bronchopulmonary dysplasia. A possible mechanism contributing to this phenomenon could be the loss of inhibitory influences by the adjacent epithelial layer, which is damaged early in the course of the disease.In the present study the influence of rat type-II cell-conditioned medium (CM) on the proliferation of human fetal fibroblasts was measured.Methods: CM from type II cells cultured in 1 or 10% fetal calf serum for 1 day (1%ld; 10%ld) or 3 days (1%3d; 10%3d) were added (1:10) to fibroblast cultures.Fibroblast proliferation was measured by incorporation of bromodeoxyuridine (BrdU), as estimated by ELISA.Results: CM 1%ld inhibited incorporation of BrdU by 35% (p<0.05), CM 10%ld by 24% (p<0.05) of control (no CM). CM 1%3d stimulated the incorporation of BrdU by 8% (n.s.), CM 10%3d by 13% (p<0.05) above control.Conclusions: Freshly prepared type-II cells in culture inhibit the proliferation of human fetal fibroblasts.After 3 days in culture this property is lost, and may be replaced by stimulatory influences.