Sequential activation of constitutive and inducible nitric oxide synthase (NOS) in rat cerebellar granule neurons by pseudomonas fluorescens and invasive behaviour of the bacteria.

Previous studies have shown that Pseudomonas fluorescens and its lipopolysaccharide (LPS) exert dose-related cytotoxic effects on neurons and glial cells. In the present work, we investigated the time course effect of P. fluorescens MF37 and its LPS on cultured rat cerebellar granule neurons. The kinetics of binding of P. fluorescens to cerebellar granule neurons is rapid and reaches a mean of 3 bacteria/cell after 5 h. As demonstrated by measurement of the concentration of nitrite in the culture medium, P. fluorescens induces a rapid stimulation (3 h) of the nitric oxide synthase (NOS) activity of the cells. In contrast, LPS extracted from P. fluorescens requires a long lag phase (24 h) before observation of an activation of NOS. Measurement of the membrane resting potential of granule neurons showed that within 3 h of incubation there was no difference of effect between the action of P. fluorescens and that of its endotoxin. Two complementary approaches allowed to demonstrate that P. fluorescens MF37 presents a rapid invasive behaviour suggesting a mobilisation of calcium in its early steps of action. The present study reveals that P. fluorescens induces the sequential activation of a constitutive calcium-dependent NOS and that of an inducible NOS activated by LPS. Our results also suggest that in P. fluorescens cytotoxicity and invasion are not mutually exclusive events.