Enhancement of Ca 2+-induced noradrenaline release by vanadate in PC12 cells: possible involvement of tyrosine phosphorylation

Abstract

Tyrosine phosphorylation has been shown to participate in the signal cascade after receptor stimulation with neurotransmitters and neurotrophins. However, the role of tyrosine phosphorylation in the process(es) of neurotransmitter release has not been well established. The effects of orthovanadate (Na 3VO 4), an inhibitor of protein-tyrosine phosphatases, on cytosolic free Ca 2+ concentrations ([Ca 2+] i ), phosphotyrosine accumulation and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. Addition of Na 3VO 4 enhanced ionomycin-stimulated [ 3 H ]NA release in a concentration-dependent manner, although Na 3VO 4 alone had no effect. Na 3VO 4 also enhanced [ 3 H ]NA release induced by P2 receptor stimulation with adenosine 5′- O-(3-thiotriphosphate) (ATPγS) or by depolarization with 50 mM KCl, which stimulated a [Ca 2+] i increase. A cell permeable inhibitor of protein-tyrosine phosphatases, l- p-bromotetramisole oxalate, at 0.3 mM enhanced ionomycin-stimulated [ 3 H ]NA release, although pervanadate had no effect. Addition of 5 mM Na 3VO 4 stimulated phosphotyrosine accumulation in several protein bands such as p130 cas, but did not increase [Ca 2+] i in PC12 cells. These findings suggest that the tyrosine phosphorylation pathway regulates Ca 2+-stimulated NA release without changes of [Ca 2+] i in PC12 cells.