GTP AND GUANOSINE SYNERGISTICALLY ENHANCE NGF-INDUCED NEURITE OUTGROWTH FROM PC12 CELLS

Six per cent of rat pheochromocytoma (PC12) cells extended neurites (p rocesses greater than one cell diameter in length) in the presence of 300 mu M extracellular GTP or 300 mu M guanosine for 48 hr, compared t o only 2.5% of cells in control cultures. In the presence of 40 ng/ml of 2.5S NGF, about 20-35% of PC12 cells had neurites after 48 hr, and the addition of 300 mu M guanosine or GTP together with NGF synergisti cally increased the proportion of cells with neurites to 40-65%. GTP a nd guanosine also increased the average number of branches per neurite , from 0.6 in NGF-treated cultures to 1.2 (guanosine) or 1.5 (GTP). Ne urites formed after exposure to NGF alone had axonal characteristics a s determined by immunocytochemistry with antibody, SMI-31, against axo nal-specific polyphosphorylated neurofilament epitopes. Neurites gener ated with the addition of both guanosine or GTP had the same character istics. GTP probably did not exert its effects via the P-2X or P-2Y pu rinoccptors because the adenine nucleotides ATP, ATP gamma S, ADP beta S, and ADP, which are all agonists of these receptors, inhibited rath er than enhanced, NGF-induced neurite outgrowth. UTP also enhanced the proportion of cells with neurites, although not to the same degree as did GTP. This may indicate activity through a P-2U-like nucleotide re ceptor. However, the response profile obtained, GTP>UTP much less than ATP, does not fit the profile of any known P-2Y, P-2X or P-2U recepto r. The poorly hydrolyzable GTP analogues, GTP gamma S and GDP beta s w ere also unable to enhance the proportion of cells with neurites. This implied that GTP may produce its effects through a GTP-specific ectoe nzyme or kinase. This idea was supported by results showing that anoth er poorly hydrolyzable analogue, GMP-PCP, competitively inhibited the effects of GTP on neurite outgrowth. GTP did not exert its effects aft er hydrolysis to guanosine since the metabolic intermediates GDP and G MP were also ineffective in enhancing the proportion of cells with neu rites. Moreover, the effects of GTP and guanosine were mutually additi ve, implying that these two purines utilized different signal transduc tion mechanisms. The effects of guanosine were not affected by the nuc leoside uptake inhibitors nitrobenzylthioinosine (NBTI) and dipyridamo le, indicating that a transport mechanism was not involved. Guanosine also did not activate the purinergic P-1 receptors, because the A(2) r eceptor antagonists, 1, 3-dipropyl-7-methylxanthine (DPMX) or CGS15943 , and the A(1) receptor antagonist, 1, 3-dipropyl-8-(2-amino-4-chloro) xanthine (PACPX) did not inhibit its reaction. Therefore guanosine enh anced neurite outgrowth by a signal transduction mechanism that does n ot include the activation of the P-1 purinoceptors. The enhancement of the neuritogenic effects of NGF by GTP and guanosine may have physiol ogical implications in sprouting and functional recovery after neurona l injury in the CNS, due to the high levels of nucleosides and nucleot ides released from dead or injured cells.