The role of charged amphipathic helices in the structure and function of surfactant protein B.

Surfactant protein B (SP-B) is essential for normal lung surfactant function. Theoretical models predict that the disulfide cross-linked, N- and C-terminal domains of SP-B fold as charged amphipathic helices, and suggest that these adjacent helices participate in critical surfactant activities. This hypothesis is tested using a disulfide-linked construct (Mini-B) based on the primary sequences of the N- and C-terminal domains. Consistent with theoretical predictions of the full-length protein, both isotope-enhanced Fourier transform infrared (FTIR) spectroscopy and molecular modeling confirm the presence of charged amphipathic alpha-helices in Mini-B. Similar to that observed with native SP-B, Mini-B in model surfactant lipid mixtures exhibits marked in vitro activity, with spread films showing near-zero minimum surface tensions during cycling using captive bubble surfactometry. In vivo, Mini-B shows oxygenation and dynamic compliance that compare favorably with that of full-length SP-B. Mini-B variants (i.e. reduced disulfides or cationic residues replaced by uncharged residues) or Mini-B fragments (i.e. unlinked N- and C-terminal domains) produced greatly attenuated in vivo and in vitro surfactant properties. Hence, the combination of structure and charge for the amphipathic alpha-helical N- and C-terminal domains are key to SP-B function.

Function and inhibition sensitivity of the N-terminal segment of surfactant protein B (SP-B1-25) in preterm rabbits.

BACKGROUND: Surfactant protein B (SP-B) is an essential component of pulmonary surfactant, but shorter SP-B sequences may exert equivalent surface activity. METHODS: We synthesised a peptide based on the amino-terminal domain of SP-B (SP-B1-25), a full length SP-B1-78, and a full length palmitoylated SP-C peptide (SP-C1-35) and compared the in vivo function and sensitivity to plasma inhibition of preparations consisting of mixtures of phospholipids with SP-B1-25 or SP-B1-78 and/or SP-C1-35 to Survanta. Preterm rabbits born at 27 days of gestation were treated at birth with surfactant and ventilated for 60 minutes. At 15 minutes half of them received plasma intratracheally. Dynamic compliance was monitored every 15 minutes and postmortem pressure-volume curves were measured to define lung mechanics. RESULTS: Dynamic compliance and postmortem lung volumes were highest after treatment with a surfactant consisting of an SP-B peptide and SP-C1-35 or Survanta. Plasma instillation decreased dynamic compliance and lung volumes sharply, but the most effective activity was by prior instillation of surfactants containing SP-B1-25. CONCLUSION: These experiments suggest that the N-terminal domain of SP-B (SP-B1-25) exhibits in vitro and in vivo surface activity and is relatively insensitive to plasma inhibition.

Paradoxical effects of exogenous proteins on lung function in surfactant-deficient rats.

Leakage of plasma proteins into the alveolar space can inhibit pulmonary surfactant function and worsen respiratory failure in ventilated preterm infants. We tested the effect of intratracheal instillation of fetal calf serum (FCS) and fresh frozen plasma (FFP) on lung function in ventilated rats who were made surfactant-deficient by saline lavage. Post lavage, the rats were treated with air placebo, Survanta, FCS or FFP, air placebo + FCS or FFP 1 hour post lavage, or Survanta + FCS or FFP 1 hour post lavage. After 2 hours of ventilation, pressure volume curves were performed and the lungs relavaged. FCS instillation rapidly improved oxygenation when given immediately post lavage or 1 hour after placebo or Survanta instillation, whereas FFP instillation never improved oxygenation. FCS instillation increased post-treatment lavage phospholipid values, but FFP did not. Both FCS and FFP decreased lung volume, but the negative effect of FFP exceeded that of FCS. Surfactant aggregate sizing of the final lung lavages by dynamic light scattering showed a definite shift towards smaller aggregates after FFP, but not after FCS, instillation. These data suggest that intratracheal instillation of FCS improves oxygenation and preserves the alveolar presence of phospholipids and large surfactant aggregates, whereas FFP decreases oxygenation and surfactant aggregate size in surfactant-deficient lavaged rats.

Comparison of functional efficacy of surfactant protein B analogues in lavaged rats.

Leakage of plasma proteins into the alveoli inhibits pulmonary surfactant function and worsens respiratory failure. Surfactant protein B (SP-B), is essential for surfactant function, but the N-terminal domain of human SP-B (residues 1.25, SP-B1-25) can mimic the biophysical properties of full length SP-B1-78 in vitro. The authors compared the function and inhibition resistance of synthetic surfactant preparations containing SP-B analogues to a natural bovine surfactant preparation "Survanta". Eight groups of eight rats were lavaged to induce surfactant deficiency, fibrinogen was instilled as a surfactant inhibitor, and then they were rescued with exogenous surfactant. Five experimental surfactants were formulated by mixing 3% SP-B1-78, or an equimolar amount of SP-B1-25 and/or 1% palmitoylated surfactant protein C (SP-C)1-35, into a standard phospholipid (PL) mixture: B1-78, B1-25, C1-35, B1-78+C1-35, and B1-25+C1-35 surfactant preparations. Survanta was used as a positive control and PL and no treatment as a negative control. Lung function was assessed during a 2-h period using arterial blood gas and lung compliance measurements. Rats treated with B1-25+C1-35 surfactant and Survanta maintained the highest oxygenation and lung compliance values throughout the experiments. The surfactants could be ranked as B1-25+C1-35 surfactant and Survanta >B1-25 and B1-78+C1-35 surfactants >others. Because the N-terminal domain of surfactant protein B1-25 can improve inhibition resistance, it may be able to substitute for surfactant protein B in exogenous surfactant preparations.

Addition of alpha1-antitrypsin to surfactant improves oxygenation in surfactant-deficient rats.

During its life cycle, surfactant converts from highly surface active, large aggregates to less surface active, smaller aggregates. This process is probably regulated by a serine protease. We tested whether adding alpha1-antitrypsin (alpha1-AT), an antiprotease, to surfactant improves its in vivo function. alpha1-AT was added to Survanta, to a standard phospholipid (PL) mixture, and to a synthetic surfactant (BC mixture = PL mixture + synthetic surfactant proteins B and C) at a dose of 100 mg alpha1-AT per 75 mg PL. Adding alpha1-AT did not affect in vitro surface activity, except for that of the PL mixture. Adult rats were ventilated with 100% O2, at a tidal volume of 7.5 ml/kg and a ventilatory rate of 60 breaths/ min. The rats' lungs were lavaged with saline until the PaO2 dropped below 100 mm Hg, at which time 100 mg/kg of surfactant with or without alpha1-AT or alpha1-AT alone was instilled. After 1 h of ventilation the rats were killed, pressure-volume curves were generated, and the rats' lungs were relavaged. Surfactant treatment improved oxygenation in the order: BC mixture > Survanta > PL mixture. Addition of alpha1-AT equalized oxygenation in all three alpha1-AT groups, but decreased respiratory system compliance in the groups given Survanta and PL mixture. Particle sizing of the final lung lavages showed preservation of large surfactant aggregates after treatment with alpha1-AT. These data suggest that the addition of alpha1-AT to surfactant can exert a positive effect on oxygenation and surfactant metabolism in surfactant-deficient rats.

Sensitivity of synthetic surfactants to albumin inhibition in preterm rabbits.

Surfactant can be inhibited in vivo by plasma proteins invading the alveolar space during acute lung injury. The resistance to protein inhibition of surfactant preparations with various synthetic surfactant proteins B and C (B and C) was tested in preterm rabbits. Surfactants consisted of a palmitic acid containing phospholipid mixture (PL) with full-length SP-B peptide (B1-78), one of two SP-B mutants (Bserine and BR236C), the synthetic SP-B mimic KL4 (UCLA-KL4), a natural SP-B (Bbovine), synthetic palmitoylated SP-C peptide (C1-35), a combination of B1-78 + C1-35, a combination of BR236C + C1-35, and the clinical surfactant Survanta. Preterm rabbits born at 28 days of gestation were ventilated and received 100 mg/kg of albumin intratracheally at 30 min and 100 mg/kg of surfactant at 45 min after birth. Dynamic lung compliance (tidal volume/mean airway pressure) decreased from 0.82 to 0.57 mL/kg/cm H2O after albumin instillation and to 0.43 mL/kg/cm H2O over a 60-min period after saline placebo. Treatment with B1-78 + C1-35 and BR236C + C1-35 surfactant and Survanta returned dynamic compliance to prealbumin values, B1-78, BR236C, Bbovine, and C1-35 surfactant stabilized dynamic compliance, but PL, Bserine, and UCLA-KL4 surfactant were unable to prevent a further deterioration in dynamic compliance. These data suggest that a combination of synthetic surfactant peptides B1-78 and C1-35 and the clinical surfactant Survanta confer a high degree of resistance to surfactant inhibition by human albumin in ventilated preterm rabbits.

Synthetic mimics of surfactant proteins B and C: in vitro surface activity and effects on lung compliance in two animal models of surfactant deficiency.

Synthetic surfactant peptides SP-B1-78 and SP-C1-31 in a standard phospholipid mixture have been employed to examine the correlation between in vitro surface activity and in vivo function of synthetic surfactant preparations in the isolated rat lung and premature rabbit models of respiratory distress syndrome. Monolayer techniques showed that SP-B peptides have a high propensity for association with a phospholipid structure. By dynamic respreading, synthetic SP-B and SP-C showed rapid spreading and attained low surface tensions. Used as replacement surfactants in two animal models, these synthetic surfactant preparations partially restored lung compliance in lavaged rats and premature rabbits better than a pure phospholipid preparation and to a degree comparable to clinical surfactant, measured by pressure/volume curves. Our data confirm that in vitro functional determinations of synthetic surfactant peptides are instrumental in the preparation of replacement surfactants, and that dispersions thus selected represent viable therapeutic alternatives to current treatments for respiratory distress syndrome.