Role of the hydrophobic face of amphipathic alpha-helical peptides in synthetic pulmonary surfactants.

The sequence requirements for the peptide component of a totally synthetic lung surfactant mixture were examined. A series of model amphipathic alpha-helical peptides (MAP) with six to 18 residues were synthesized by solid phase techniques, mixed with dipalmitoylphosphatidylcholine (DPPC) and tested for efficacy in an in vitro adult rat lung model. The most effective peptide in these mixtures contained 10 residues. Peptides containing eight and 14 residues were also highly active when mixed with DPPC in buffer, but a six-residue peptide was inactive. Longer peptides were active only when mixed with DPPC in trifluoroethanol before swelling in buffer; no other lipids were required to elicit high activity. Biologically effective peptides, when combined with DPPC, formed translucent mixtures that were significantly less turbid than ineffective mixtures, dramatically decreased the enthalpy of the main phase transition of DPPC and reduced the gamma min in the pulsating bubble surfactometer. Turbidity and minimal surface tensions were significantly correlated with activity in this series of peptides. These data show that effective synthetic lung surfactants may be prepared with mixtures of DPPC and idealized amphipathic alpha-helical peptides containing as few as eight to 10 residues. They lend further support to the hypothesis that amphipathic alpha-helical peptides with hydrophobic surface areas greater than approximately 6.5 nm2 in simple mixtures with DPPC are biologically active.

An amphipathic alpha-helical decapeptide in phosphatidylcholine is an effective synthetic lung surfactant.

An idealized model amphipathic alpha-helical decapeptide was synthesized and tested for efficacy as a totally synthetic lung surfactant in simple mixtures with dipalmitoylphosphatidylcholine (DPPC). Quasi-static lung compliance was restored to 92 +/- 3% of the unlavaged value at a pressure of 5 cm H2O in an in vitro lavaged rat lung model. A sustained improvement in gas exchange was also observed when guinea pigs were treated with the synthetic lung surfactant in an in vivo lavaged lung model. DPPC/peptide mixtures rapidly formed low surface tension films in the pulsating bubble surfactometer consistent with a mechanism in which the lipid and peptide mixture spreads rapidly in the lavaged lung to minimize the surface tension at the air/tissue interface. This decapeptide sequence is active in mixtures with DPPC whether the residues are in the all L or all D conformation. However, a peptide with identical sequence, but with alternating D and L amino acid residues, is relatively inactive. Positive charge interactions are not important since a peptide with formylated lysine residues is active. The activity of these decapeptides, with sequences unrelated to any of those in natural lung surfactants, shows that the classic amphipathic alpha-helical hypothesis may be useful in designing peptides that will be effective synthetic lung surfactants in binary mixtures with DPPC. The data demonstrate that a small water-soluble synthetic peptide containing an amphipathic alpha-helical structure combined solely with the major lipid of natural lung surfactant is effective in restoring lung compliance and gas exchange in surfactant-deficient lungs and may be useful in treatment of the respiratory distress syndromes.

Glucocorticoid effects on lipid metabolism in HeLa cells: inhibition of cholesterol synthesis and increased sphingomyelin synthesis.

Glucocorticoid treatment had a variety of effects on lipid composition of HeLa S3G (HeLa 65) cells. Total cellular cholesterol levels were lower in treated cultures than in controls within 6 h of the addition of glucocorticoids and remained lower for up to 48 h. Total cell protein and total lipid phosphorus levels were also decreased, but only after more prolonged exposure. The major phospholipid components, viz. phosphatidylcholine and phosphatidylethanolamine, representing, respectively, 48% and 29% of the total phospholipids, were not affected by glucocorticoid treatment. Sphingomyelin, however, increased from 4.8 to 8.3% of the total phospholipid content after 48 h of exposure, whereas lysolecithin increased from 0.1 to 1.9%. The incorporation of labeled precursors was also measured after glucocorticoid addition. As related to cellular protein content, only slight increases were found for all major phospholipids. In contrast, sphingomyelin synthesis increased up to 200% after treatment with glucocorticoids. The fatty acid and plasmalogen compositions were unchanged by glucocorticoids.

Corticosteroids and phosphatidylcholine biosynthesis in microsomal fractions from L5178Y lymphoma.

Microsomal fractions from mouse lymphoma L5178Y and from rat thymocytes were used to follow incorporation of radiolabel from cytidine diphosphate-[methyl-14C]choline into microsomal lipids. Dexamethasone, at concentrations ranging from 2.8 X 10(-6)M to 2.8 X 10(-5)M, partially inhibited this transfer reaction. Microsomes prepared from freshly isolated thymocytes were more sensitive to the effects of dexamethasone showing inhibition at concentrations of steroid as low as 2.8 X 10(-8)M. The inhibitory effect did not depend on the amount of the available endogenous diglycerides and was not related to a possible stimulation of cytidine diphosphate choline transferase back reaction by the steroid. The survey of a broad selection of different steroids revealed a lack of correlation between the known lymphocytolytic properties of steroids and their effects on cytidine diphosphate choline transferase. Dexamethasone was the only steroid of the glucocorticoid group that inhibited this reaction in microsomal fractions of L5178Y lymphoma. The structural requirement for the inhibitory effect was related to the absence of oxygen functions in positions 11 and 17 of the steroid and, possibly, to the presence of both C-20 and C-21 on the side chain.