BLOCKADE OF NITRIC-OXIDE SYNTHESIS BY TYROSINE KINASE INHIBITORS IN NEURONS

In striatal neurones in culture, N-methyl-D-aspartate-(NMDA), kainate-(Kai) and K+-dependent cGMP production is entirely mediated via nitric oxide (NO). Low concentrations of lavendustin-A (less than or equal to 0.3 mu M), a highly specific tyrosine kinase inhibitor, reduced irreversibly and in a time-dependent manner NMDA-stimulated cGMP production. After a preincubation period of 20 min with lavendustin-A (0.3 mu M), the inhibition of NMDA-induced cGMP production was equal to 56 +/- 8% (n = 6). After the same preincubation period, the IC50 of the lavendustin-A blockade was 30 +/- 15 nM. Genistein, another tyrosine kinase inhibitor also inhibited NMDA-dependent cGMP production with high potencies (less than or equal to 3 mu M). Whatever the tyrosine kinase inhibitor tested, the basal cGMP production remained unaffected. Kai-, K+-, and ionomycin-induced cGMP production was also inhibited by lavendustin-A, and genistein. In contrast, tyrosine kinase inhibitors were unable to block NO donor-induced cGMP production. Using patch clamp experiments, we have also found that lavendustin-A (0.3-1 mu M), the most potent tyrosine kinase inhibitor used, (a) did not reduce the NMDA receptor-mediated current, (b) only slighly affected Kai receptor-mediated current (16.4 +/- 3.4% inhibition) and (c) had a marked effect on voltage-sensitive Ca2+ channel- (VSCC) mediated currents (44.4 +/- 4.9% inhibition). A reduction in VSCC activity certainly explains the inhibition of K+-, Kai- and possibly part of the NMDA-induced cGMP production. However, two observations (inhibition of ionomycin-induced cGMP production and absence of inhibition of NO donor-induced cGMP production) indicated that tyrosine kinase inhibitors also inhibit another step localized between Ca2+ entry into neurones and NO production. We have no information regarding the step involved because the cascade of kinases and phosphatases leading to NO-S regulation could be very complex. In any case, it can be concluded that a tyrosine phosphorylation is needed to obtain a full brain NO-synthase (NO-S) activity upon glutamate receptor stimulation.