Extracellular guanosine 5′ triphosphate enhances nerve growth factor-induced neurite outgrowth via increases in intracellular calcium

Extracellular guanosine 5' triphosphate (GTP) enhances nerve growth factor-dependent neurite outgrowth from rat pheochromocytoma (PC12) cells; cultures of PC12 cells exposed to GTP and nerve growth factor together contain significantly more neurite-bearing cells than do those exposed to either nerve growth factor or GTP alone [Gysbers J. W. and Rathbone M. P. (1996) Int. J. devl Neurosci. 14, 19-34]. PC12 cells contain specific cell surface binding sites for extracellular GTP, which do not bind ATP or uridine 5' triphosphate. Exposure of PC12 cells to extracellular GTP (300microM) produced a robust and sustained increase in intracellular Ca(2+) ([Ca(2+)](i)), different from the transient response to the addition of ATP. The GTP-induced [Ca(2+)](i) increase was blocked by the L-type calcium channel inhibitor, nifedipine. The L-type Ca(2+) channel inhibitors, nifedipine or verapamil, also inhibited the enhancement of neurite outgrowth by GTP, but did not affect neurite outgrowth stimulated by nerve growth factor alone. Pre-treatment of PC12 cells with ryanodine (0.5-50microM) depleted calcium from internal stores and prevented the further release of calcium by GTP. Similarly, pre-treatment of PC12 cells with thapsigargin (an inhibitor of internal store Ca(2+)/ATPase) or dantrolene (which blocks Ca(2+) release from some of these stores) also reduced the enhancement of neurite outgrowth by GTP. Therefore, Ca(2+)-induced Ca(2+) release from specific stores, present in PC12 cells, is involved in the enhancement of nerve growth factor-induced neurite outgrowth by GTP, possibly acting at specific binding sites on the cell surface. GTP is proving to be an important extracellular trophic modulator in the central nervous system. These studies show that the neuritogenic actions of GTP involve moderate but sustained increases in intracellular Ca(2+) which are likely due to activation of L-type Ca(2+) channels and Ca(2+)-induced Ca(2+) release from intracellular stores. These effects of extracellular GTP are likely mediated at the cell surface and may be related to specific GTP binding sites which are distinct from G-proteins and from hitherto described purine nucleotide (P2) receptors. These data indicate a mechanism whereby the neuritogenic effects of GTP are mediated and emphasize the importance of considering GTP as a neurotrophic mediator.