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This study evaluated the effects of varying tidal volumes (VT) and positive end-expiratory pressure (PEEP) levels on surfactant aggregate conversion and lung function in an animal model of lung injury induced by N-nitroso-N-methylurethane. Lung-injured adult rabbits were initially ventilated using a VT of 10 ml/kg (VT10), a respiratory rate of 30 breaths/min (RR30), and a PEEP of 3.5 cm H2O. A trace dose of radiolabeled rabbit large surfactant aggregates was instilled after the onset of ventilation, and animals were then ventilated at different ventilator settings for 1 h. Ventilation strategies involving a lower VT (VT5, RR60) resulted in significantly superior oxygenation and lower surfactant aggregate conversion rates than strategies involving a higher VT ([VT10, RR30], [VT15, RR20], p < 0.05). Increasing the PEEP level to 8.0 cm H2O improved oxygenation, but it was sustained only with a low VT (VT5, RR60), and deteriorated with a high VT (VT10, RR30). Varying VT but not PEEP levels resulted in significant changes in surfactant aggregate conversion. We conclude that increased surfactant aggregate conversion resulting from suboptimal ventilation of injured lungs may play an important role in the pathophysiology of ventilation-induced lung dysfunction in acute lung injury.

The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that PDE4 exists in two conformations that bind the inhibitor rolipram with affinities differing by more than 100-fold. Here we report that these two conformations are the consequence of PDE4 binding to its metal cofactor such as Mg(2+). Using a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay, we identified that L-791,760, a fluorescent inhibitor, binds to the apoenzyme (free enzyme) and the holoenzyme (enzyme bound to Mg(2+)) with comparable affinities (K(d) approximately 30 nM). By measuring the displacement of the bound L-791,760, we have also identified that other inhibitors bind differentially with the apoenzyme and the holoenzyme depending upon their structure. CDP-840, SB-207499, and RP-73401 bind preferentially to the holoenzyme. The conformational-sensitive inhibitor (R)-rolipram binds to the holoenzyme and apoenzyme with affinities (K(d)) of 5 and 300 nM, respectively. In contrast to its high affinity (K(d) approximately 2 microM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K(d) approximately 170 microM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.

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