Inhibitory effects of oxyradicals on surfactant function: utilizing in vitro Fenton reaction.

The inhibitory effects of reactive oxygen species (ROS) on the surface tension-lowering abilities of three surfactants were compared: natural lung surfactant (NLS), KL4 surfactant containing synthetic peptide resembling the hydrophobic/hydrophilic domains of SP-B in an aqueous dispersion of phospholipids, and Survanta (SUR) containing SP-B and SP-C. The inhibitory concentrations of Fenton reactants (i.e. 0.65 mM FeCl2, 0.65 mM EDTA, 30 mM H2O2), deduced from dose-response plots of FeCl2 on minimum surface tension (MST) of SUR, were used to assess the Fenton effect on biophysical properties of various surfactants. Neither H2O2 (30 mM) nor FeCl2 with EDTA (both 0.65 mM) alone affected surfactant function, but when mixed together significantly increased (p < 0.01) the MST of SUR compared with KL4 (p < 0.05) in a FeCl2 concentration-dependent manner. This effect on NLS was not significant (p = 0.05) at similar phospholipid concentrations. Also, the range of increases in surface adsorption in mN/m at equilibrium surface tension (EST) was 27-40 for SUR, 36-44 for KL4, and 24-25 for NLS. We speculate that the presence of SP-A and the catalase content in NLS may have protective effects on inactivation of NLS by ROS. We conclude that the in vitro Fenton effect could be a valuable test system for comparing the inactivation range of surfactants by oxyradicals.

Characterization of surfactant subtypes of beractant and a synthetic peptide containing surfactant KL4 following surface area cycling and addition of fibrinogen.

Surfactant is not a homogeneous material and can be separated into subtypes. Subtype conversion is clinically important because it is thought to occur naturally and because surface activity varies depending on the subtype. Fibrinogen, a naturally occurring serum protein, is known to affect this conversion. In this study we studied two surfactants, beractant and KL4, to examine their subtype characteristics. Surface area cycling, an in vitro method, was used in conjunction with sucrose gradient ultracentrifugation to separate subtypes in both surfactants. Activity, expressed as minimum surface tension of these subtypes, was measured using a pulsating bubble surfactometer. The effect of fibrinogen on subtype conversion and subsequent change in activity was elucidated. Our results indicate that following surface area cycling, beractant and KL4 have different subtypes and different responses to fibrinogen. Cycling of beractant resulted in two bands, representing a heavy and a light subtype. In the presence of fibrinogen, cycling resulted in two separate heavy subtypes. Cycling of KL4 surfactant also yielded light and heavy subtypes. However, in the presence of fibrinogen, cycling of KL4 resulted in ultraheavy subtypes. These ultraheavy subtypes retained minimum surface tension comparable to that of native KL4 surfactant. We conclude that these two surfactant preparations have different subtype conversions when subjected to surface area cycling and in the presence of fibrinogen. These conversions result in different activities toward lowering surface tension. We speculate that endogenous fibrinogen will also affect these two surfactants differently in vivo and thus affect their clinical effectiveness.

Combined effects of nitric oxide and hyperoxia on surfactant function and pulmonary inflammation.

NO and its derivative ONOO- are potent free radicals that can cause cell damage, especially in the presence of O2. To determine the potential pulmonary toxicities of nitric oxide (NO) and peroxynitrite (ONOO-) in vitro, Survanta (2.5 mg/ml) was exposed to ONOO- (0.3-8 mM) in the presence of two different buffering systems (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and phosphate buffer) and minimum surface tension (MST) was determined with an oscillating bubble surfactometer. Significant increases in MST were seen only with exposure to 8 mM ONOO-, indicating that in vitro, high concentrations of ONOO- can inhibit natural surfactant function. The in vivo effects of NO and hyperoxia were then studied in four groups of newborn piglets ventilated for 48 h with 21% O2, 100% O2, 21% O2 and 100 ppm NO, or with 90% O2 and 100 ppm NO. Five animals served as an untreated control group. Bronchoalveolar lavage fluid (BAL) obtained at 48 h was subjected to centrifugation and the surfactant pellet was reconstituted to 5 mg phospholipid/ml. Significant increases in MST were seen in surfactant from piglets ventilated with NO and 90% O2, compared with either untreated controls or piglets ventilated with 21% O2 for 48 h (P < 0.05, analysis of variance). Significant increases in neutrophil chemotactic activity (NCA) of BAL were also found in the NO and O2 group (P < 0.05), with significant positive interaction between NO and O2 found (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of pH on surface properties of lung surfactants.

Protein-lipid interactions at air-liquid interfaces are dependent on electrostatic charges, cations, anions, distribution of protons, and surface potential, which are influenced by pH changes. All of these factors may affect lung surfactant function. To verify the pH dependence of surface activity of pulmonary surfactant, we studied the in vitro effects of pH and Ca2+ on the surface tension-lowering abilities of various surfactants in an oscillating bubble surfactometer. Surface tension measurements were made of mixtures at known pHs or after the replacement of the subphase fluid of surfactant films with buffered saline at various pH values. At the pH range 4.0-7.0, the average equilibrium surface tension (EST)/minimum surface tension (MST) for natural surfactants human amniotic fluid and natural lung surfactant from rabbit lung lavage was 24/2 mN/m. At the same pH range, Exosurf and phospholipids alone had an EST/MST of 44/25 and 44/12 mN/m, respectively. Survanta (SUR) containing SP-B and SP-C and a phospholipid surfactant (KL4) containing a leucine/lysine peptide had an EST/MST of 29/5 and 36/3, respectively. Alkalinization of the subphase (pH > 7.4) significantly decreased the surface tension-lowering ability of SUR (P < 0.01) and to a lesser extent that of KL4 surfactant (P < 0.05), but natural lung surfactants were not significantly affected over a pH range of 3-7.5. These data demonstrate that natural surfactants maintain their optimal surface activities over a broader pH range than do the commercial products because of a lack of SP-A. Careful monitoring of the pH for optimal surface activity is recommended when evaluating surfactant function and the effects of specific inhibitors on this function.

Reduction of the surface-tension-lowering ability of surfactant after exposure to hypochlorous acid.

The reactive species hypochlorous acid (HOCl/OCl-) is a major product of the respiratory burst in activated neutrophils. We studied the effects of HOCl/OCl- on human surfactant and upon surfactants Survanta, KL4 and Exosurf, utilizing a pulsating surfactometer for measuring surface tension. HOCl/OCl- induced a marked dose-dependent decrease in the surface-tension-lowering activity of human surfactant. The surfactant containing surfactant proteins B and C (Survanta) was less sensitive; however, synthetic surfactants with or without peptides were not affected by HOCl/OCl- (KL4, Exosurf). Ascorbic acid and GSH protected human surfactant against inactivation by HOCl/OC1-. We suggest that HOCl/OCl- produced by activated phagocytes in the alveolar compartment of the lung could damage endogenous surfactant and affect the function of exogenously administered natural or other surfactants, especially if ascorbic acid and GSH levels in the lung lining fluids are subnormal, as is known to be the case in some inflammatory lung diseases.

Full length synthetic surfactant proteins, SP-B and SP-C, reduce surfactant inactivation by serum.

The failure of some infants with respiratory distress syndrome to respond to therapy with surfactant may be explained in part by inactivation of surfactant caused by leakage of plasma constituents into air spaces. Surfactant-associated proteins (SP-A, SP-B and SP-C) reduce the susceptibility of surfactants to inactivation in vitro. To study this phenomenon further, we used full length synthetic proteins, SP-B [1-78] and SP-C [1-31], mixed with surfactant lipids in different ratios and different concentrations. Equilibrium and minimum surface tensions of these mixtures, with or without serum and calcium, were measured using a pulsating surfactometer. Mixtures containing both SP-B and SP-C had optimal minimum and equilibrium surface tensions of < 5 and < 28 mN/m, respectively. Mixtures with SP-B had optimal minimum surface tensions, but equilibrium surface tensions averaged 35 mN/m. Mixtures with SP-C had high minimal (19 mN/m) and high equilibrium surface tensions (35 mN/m). When serum was added to these mixtures, the least inactivation was found with mixtures containing 3% protein at 1:1 ratio of SP-B/SP-C with 2 mM calcium chloride. These data indicate that SP-B and SP-C, particularly in the presence of calcium, reduce surfactant inactivation that may be caused by plasma constituents. The results lead to the hypothesis that charge interactions among ions, lipids, surfactant proteins, and serum inactivators are a major element in pathophysiological surfactant inactivation.

Reversible and irreversible inactivation of preformed pulmonary surfactant surface films by changes in subphase constituents.

Several mechanisms for surfactant inactivation have been reported. In this study, we have measured the reversibility of surfactant inactivation caused by various lipid or protein constituents of plasma or by pH changes. A surfactant of bovine origin was studied in a pulsating surfactometer either when surfactants were premixed with different serum constituents (inactivators) or when inactivators were introduced into subphase fluid surrounding surfactant films formed at an air-liquid interface. Subphase exchanges with sodium bicarbonate or sodium borate raised pH and raised minimal surface tensions either when premixed with surfactant or when introduced with saline subphase beneath a preformed surfactant surface film. The pH effects on surfactant film function were reversible for sodium bicarbonate but not for sodium borate when the subphase with bicarbonate or borate was replaced with saline. Lipids (platelet-activating factor or lysophosphatidylcholine) had non-reversible effects on preformed films. Proteins (fibrinogen or C reactive protein) had reversible effects at low concentrations, but reversibility was less evident at high concentrations. Effects with whole serum were non-reversible at low protein concentrations (0.5 mg/ml). These results add evidence that surfactant inactivation can be caused by multiple mechanisms, both reversible and irreversible.

Inhibition of mixtures of surfactant lipids and synthetic sequences of surfactant proteins SP-B and SP-C.

The respiratory distress syndrome of premature infants is caused by both surfactant deficiency and surfactant inhibition by capillary-alveolar leakage of serum factors. Dispersions of a standard surfactant lipid mixture, with and without various synthetic peptides, modeled on human surfactant proteins SP-B (residues 1-25, 49-66, 1-78) and SP-C (residues 1-10), were evaluated for inhibition by serum and by plasma constituents using a pulsating bubble surfactometer. Inhibition was derived from the changes in surface properties of these mixtures after addition of human serum or plasma constituents. Modified bovine surfactant (TA) containing native SP-B and SP-C was used as a control. In the absence of serum inhibitors, mixtures with synthetic peptides gave results similar to surfactant TA. However, inhibition was more evident in the dispersions with synthetic peptides when compared with surfactant TA. The peptide/phospholipid mixture with the entire sequence of SP-B and the first 10 residues of SP-C were more resistant to inhibition than mixtures with synthetic peptides containing fewer domains. Addition of calcium reduced the inhibitory effects of serum both in mixtures containing synthetic peptides and in surfactant TA. Therefore, synthetic SP-B and SP-C peptides in surfactant lipids, in cooperation with calcium, permit resistance to inhibition by several plasma constituents that probably inactivate surfactant by a variety of different mechanisms.

Genetic characterization of the new morphological and UV-sensitive mutants in Coprinus cinereus. I. A UV-sensitive mutation rad 1 associated with elevated frequencies of mitotic and meiotic recombination.

Studies on the effect of an UV-sensitive mutation, rad 1, in meiotic and mitotic recombination in Coprinus indicated that, in homozygous condition, rad 1 increased the spontaneous meiotic recombination by 50% and UV-induced mitotic intergenic recombination by about 5-fold. The homozygous rad 1 diploid was shown to be much more sensitive to the recombinogenic effects of polyfunctional than than of mono- or non-functional alkylating agents.