Nerve growth factor enhances [3H]nicotine binding to a nicotinic cholinergic receptor on PC 12 cells.

Abstract

Although nicotinic cholinergic agonists have functional effects on PC 12 cells, radioligand-binding sites have not been detected. We, therefore, studied PC 12 cells incubated in the presence of nerve growth factor (NGF) and determined that specific [3H]nicotine-binding sites were induced approximately 2.5-fold in the presence of NGF (50 or 100 ng/ml). Specific binding was maximal between the first (100 ng/ml NGF) and seventh (50 ng/ml NGF) days of treatment and was stable for 2 weeks with addition of NGF every 3 days. Using intact cells, average association and dissociation rates for [3H]nicotine were 0.00021 min-1 nM-1 (n = 2) and 0.048 min-1 (n = 2), respectively, at 4 C, yielding an average apparent Kd of 229 nM. At 22 C, stable equilibrium was not attained during association studies. A similar Kd value for broken cell preparations was obtained by kinetic analysis (i.e. an average association rate of 0.00042 min-1 nM-1 and dissociation rate of 0.087 min-1), yielding an average Kd value of 207 nM (n = 2) at 4 C. By saturation binding analysis of intact cells, an average Kd of 292 nM (n = 2) and a binding capacity (Bmax) of 15,118 molecules/cell were obtained. [3H]Nicotine binding was inhibited on an equimolar basis by L-(-)nicotine and N-methylcarbamylcholine. D-(+) Nicotine was 7-fold less potent, whereas alpha-bungarotoxin, mecamylamine, and atropine showed no significant inhibition. [3H]Nicotine binding was also inhibited quantitatively by mono-specific polyclonal antibodies raised against the predicted alpha 3-subunit sequence (amino acids 130-139) of the rat neuronal nicotinic cholinergic receptor. This study represents the first biochemical characterization of NGF-stimulated nicotine-binding sites on PC 12 cells and confirms previous evidence of the presence of functional nicotinic cholinergic receptors on these cells.