Mechanisms of action of Pseudomonas aeruginosa pyocyanin on human ciliary beat in vitro

Abstract

Pyocyanin is a blue redox active pigment produced by Pseudomonas aeruginosa. It is present at concentrations of up to 10(-4) M in sputa from patients with cystic fibrosis and bronchiectasis who are heavily colonized with this organism. Pyocyanin, at physiologically relevant concentrations, slows human nasal ciliary beat frequency (CBF) in vitro and leads to disruption of the epithelium. Pyocyanin-induced slowing of CBF after 2 h was associated with a significant fall in intracellular cyclic AMP (cAMP) (90%) and ATP (66%) and was reversible after the pyocyanin was removed by washing. These effects were not mediated through interaction with neutrophils. The pyocyanin-induced fall in CBF was not affected by EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], pyrazinamide, 8-phenyltheophylline, indomethacin, or antioxidants, including catalase (500 U/ml), superoxide dismutase, and N-acetylcysteine. Ciliary slowing was, however, prevented (> 70%) by isobutylmethylxanthine and forskolin, both of which increase intracellular cAMP, and also by the cAMP analog, dibutyryl cAMP. There was also a concomitant protection against the fall in both cAMP and ATP. These agents also delayed the onset of epithelial disruption associated with pyocyanin treatment. In contrast, treatment with the iron chelator desferrioxamine prevented epithelial disruption, although it had no effect on pyocyanin-induced slowing of CBF. It appears that ciliary slowing can be dissociated from epithelial disruption and that the effects of pyocyanin on CBF are associated with a fall in both intracellular cAMP and ATP.