Bradykinin-Induced Collapse of Rat Pheochromocytoma (PC12) Cell Growth Cones: A Role for Tyrosine Kinase Activity

Abstract

Pathfinding of growing nerve processes is guided by extracellular guidance cues. Here we report growth cone collapse of NGF-differentiated PC12 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 together 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 confocal microscopy. (2) Bradykinin-induced Ca 2ϩ release and retraction of the neurite occurred at nanomolar concentrations. Ca 2ϩ responses at single growth cones were measured using a 1:1 mixture of fura-red and fluo-3 Ca 2ϩ -sensitive dyes. The [Ca 2ϩ ] i rise is not a prerequisite for the observed effects, because F-actin loss and retraction occurred during inhibition of Ca 2ϩ responses. In contrast, inhibition by genistein pointed to a tyrosine kinase activity in the bradykinin-evoked cellular events. Subsequent analysis of phosphotyrosine proteins revealed that bradykinin stimulated tyrosine phosphorylation of the cytoskeleton-associated protein paxillin and the nonreceptor protein tyrosine kinase pp60 c-src . Paxillin and pp60c-src co-precipitated after bradykinin 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 Gprotein-and tyrosine kinase-dependent pathways in these cellular events.

Complex formation between EphB2 and Src requires phosphorylation of tyrosine 611 in the EphB2 juxtamembrane region

“…These are similar to the modi®cations of Src occurring upon activation of the PDGF receptor (Ralston and Bishop, 1985), which reportedly re¯ect phosphorylation of residues in the amino terminus of Src and increased Src kinase activity (Gould and Hunter, 1988;Stover et al, 1996). Interestingly, Src activity has been associated with changes in cytoskeletal organization (Thomas et al, 1995;Parsons and Parsons, 1997;Brunton et al, 1997), such as those that might occur during the migration of cells (Hall et al, 1996;Boyer et al, 1997) and the retraction of neurites (Schindelholz et al, 1997).…”

Characterization of the Tyrosine Kinases RAFTK/Pyk2 and FAK in Nerve Growth Factor-induced Neuronal Differentiation

“…8, B and C). Bradykinin, a neuropeptide activating the G proteincoupled receptor and producing intracellular Ca 2ϩ increase, induces neurite retraction (43). We found that bradykinin also induces the tyrosine phosphorylation of RAFTK and paxillin in a Ca 2ϩ -dependent manner in differentiated PC12 cells.…”

Bradykinin decreases K+ and increases Cl− conductances in vagal afferent neurones of the guinea pig

“…It is well documented that BK receptors signal IP 3 Ca 2+ pools, and thus, BK could serve as a stimulus for neuronal growth. In support of this possibility are the observations that BK can cause neurite extension via IP 3 ‐sensitive Ca 2+ signalling upon activation of B 2 receptors in pheochromocytoma (PC12) cells (Kozlowski et al 1989; Reber & Schindelholz, 1996; Schindelholz & Reber, 1997).…”