Activity and inhibition resistance of a phospholipase-resistant synthetic surfactant in rat lungs

Abstract

This study investigates the activity and inhibition resistance in excised rat lungs of a novel synthetic surfactant containing the phospholipase-resistant diether phosphonolipid DEPN-8 plus 1.5% bovine surfactant protein (SP)-B/C compared to calf lung surfactant extract (CLSE). DEPN-8 + 1.5% SP-B/C surpassed CLSE in normalizing surfactant-deficient pressure-volume (P-V) deflation mechanics in lavaged excised lungs in the presence of phospholipase A(2) (PLA(2)) or C18:1 lyso-phosphatidylcholine (LPC). DEPN-8 + 1.5% SP-B/C had activity equal to CLSE in normalizing P-V mechanics in the absence of inhibitors or in the presence of serum albumin. These physiologic activity findings were directly consistent with surface activity measurements on the pulsating bubble surfactometer. In the absence of inhibitors, DEPN-8 + 1.5% SP-B/C and CLSE rapidly reached minimum surface tensions < 1 mN/m (0.5 and 2.5 mg surfactant phospholipid/ml). DEPN-8 + 1.5% SP-B/C maintained its high surface activity in the presence of PLA(2), while the surface activity of CLSE was significantly inhibited by exposure to this enzyme. DEPN-8 + 1.5% SP-B/C also had greater surface activity than CLSE in the presence of LPC, and the two surfactants had equivalent surface activity in the presence of albumin. DEPN-8 + 1.5% SP-B/C also had slightly greater surface activity than CLSE when exposed to peroxynitrite in pulsating bubble studies. These results support the potential of developing highly active and inhibition-resistant synthetic exogenous surfactants containing DEPN-8 + apoprotein/peptide constituents for use in treating direct pulmonary forms of clinical acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS).

Figure 1.

Effects of DEPN-8 + 1.5% surfactant protein (SP)-B/C in improving pressure–volume (P–V) deflation mechanics compared with calf lung surfactant extract (CLSE) in surfactant-deficient rat lungs. Lung volume is graphed as a fraction of total lung capacity at 30 cm H₂O. Lungs were excised from adult rats, and mechanics were measured during quasistatic deflation in the normal freshly excised state (surfactant-sufficient), after depletion of endogenous surfactant by lavage (surfactant-deficient), and after tracheal instillation of surfactant at a dose of 100 mg phospholipid or phosphonolipid per kg rat bodyweight (see Materials and Methods). Calculated % volume recoveries at selected transpulmonary pressures are given in Table 2. Data are mean ± SEM for n = 6. There are no statistically significant differences between P–V curves for DEPN-8 + 1.5% SP-B/C and CLSE by ANOVA.

Figure 2.

Effects of DEPN-8 + 1.5% SP-B/C and CLSE on P–V deflation mechanics in surfactant-deficient rat lungs in the presence of PLA₂. Surfactants were incubated in vitro with 0.1 U/ml of PLA₂ for 30 min at 37°C before instillation into excised lavaged rat lungs. Other model details are as in the legend to Figure 1. Calculated % volume recoveries at selected transpulmonary pressures are given in Table 2. Data are mean ± SEM for n = 6. Differences between curves for DEPN-8 + 1.5% SP-B/C + PLA₂ and CLSE + PLA₂ are statistically significant by ANOVA (P < 0.05).

Figure 3.

Effects of DEPN-8 + 1.5% SP-B/C and CLSE on P–V deflation mechanics in surfactant-deficient rat lungs in the presence of C18:1 lysophosphatidylcholine (LPC). Surfactants were incubated in vitro with 15% by weight of C18:1 LPC for 15 to 30 minutes at room temperature before instillation into excised lavaged rat lungs. Other model details are as in the legend to Figure 1. Calculated % volume recoveries at selected transpulmonary pressures are given in Table 2. Data are mean ± SEM for n = 4. Differences between curves for DEPN-8 + 1.5% SP-B/C + 15% LPC and CLSE + 15% LPC are statistically significant by ANOVA (P < 0.05).

Figure 4.

Effects of DEPN-8 + 1.5% SP-B/C and CLSE on P–V deflation mechanics in surfactant-deficient rat lungs in the presence of bovine serum albumin (BSA). Surfactants were incubated in vitro with 3 mg/ml of BSA for 15 to 30 minutes at room temperature before instillation into excised lavaged rat lungs. Other model details are as in the legend to Figure 1. Calculated % volume recoveries at selected transpulmonary pressures are given in Table 2. Data are mean ± SEM for n = 3–4. There are no differences between P–V curves for DEPN-8 + 1.5% SP-B/C + BSA and CLSE + BSA by ANOVA.