Thymopoietin interacts at the α-bungarotoxin site of and induces process formation in PC12 pheochromocytoma cells

Thymopoietin, a polypeptide isolated from thymus and involved in immune regulation, potently inhibited [¹²⁵I]α-bungarotoxin binding in both pheochromocytoma (PC12) cells in culture (ic₅₀ of 3.9 nM) and in PC12 cell membranes (ic₅₀ of 2.2 nM). The degree of inhibition produced by thymopoietin was similar to that observed with α-bungarotoxin; in contrast, nicotinic receptor ligands affected α-bungarotoxin binding only at micromolar concentrations, in agreement with previous work. Binding of thymopoietin was reversible. Studies with PC12 cell membranes suggested that the interaction between α-bungarotoxin and thymopoietin at the receptor was competitive. The effect of thymopoietin was subsequently assessed on various morphological characteristics of PC12 cells in culture. Exposure of the cells to the polypeptide resulted in neurite extension, which was evident as early as 1-2 days in culture and was maximal after 4-6 days; this response was observed with concentrations of thymopoietin as low as 10^-8M. Nerve growth factor also induced neurite extension in PC12 cells; however, the effects of nerve growth factor were qualitatively and quantitatively distinct from those which occurred with thymopoietin. Moreover, a monoclonal antibody to nerve growth factor completely prevented the nerve growth factor-induced process formation without affecting the thymopoietin-induced response. On the other hand, α-bungarotoxin resulted in the formation of processes which appeared morphologically similar to those induced by thymopoietin, although α-bungarotoxin appeared less potent than the thymic polypeptide. The effect of thymopoietin appeared to be specific; thysplenin, a polypeptide with approximately 80% homology with thymopoietin, did not elicit process formation. The thymopoietin-induced effect was reversed upon removal of the polypeptide from the culture medium. These results show that thymopoietin, a polypeptide endogenous to mammalian systems, potently interacted at the α-bungarotoxin site in a neuronal cell line. Furthermore, thymopoietin could elicit process formation in PC12 cells, suggesting that it may be a neuronotrophic factor.