BRADYKININ-INDUCED COLLAPSE OF RAT PHEOCHROMOCYTOMA (PC12) CELL-GROWTH CONES - A ROLE FOR TYROSINE KINASE-ACTIVITY

Pathfinding of growing nerve processes is guided by extracellular guid ance cues. Here we report growth cone collapse of NGF-differentiated P C12 cells in culture evoked by the neuropeptide bradykinin. The growth cone response is mediated by B-2 bradykinin receptors. Two different effects were distinguished. (1) Disappearance of filopodia occurred to gether with a loss of fibrillar actin (F-actin) in the growth cones at picomolar concentrations of bradykinin. The relative F-actin content was measured by means of rhodamine-phalloidin fluorescence using confo cal microscopy. (2) Bradykinin-induced Ca2+ release and retraction of the neurite occurred at nanomolar concentrations. Ca2+ responses at si ngle growth cones were measured using a 1:1 mixture of fura-red and fl uo-3 Ca2+-sensitive dyes. The [Ca2+](i) rise is not a prerequisite for the observed effects, because F-actin loss and retraction occurred du ring inhibition of Ca2+ responses. In contrast, inhibition by genistei n pointed to a tyrosine kinase activity in the bradykinin-evoked cellu lar events. Subsequent analysis of phosphotyrosine proteins revealed t hat bradykinin stimulated tyrosine phosphorylation of the cytoskeleton -associated protein paxillin and the nonreceptor protein tyrosine kina se pp60(c-src). Paxillin and pp60(c-src) co-precipitated after bradyki nin treatment. Immunostaining experiments showed punctate distribution of paxillin along PC12 neurites and in growth cones. Taken together, our data suggest that pp60(c-src) and paxillin are putative components of the intracellular signaling pathway of bradykinin-mediated neurite retraction and provide evidence for a crosstalk between G-protein- an d tyrosine kinase-dependent pathways in these cellular events.