Effects of nerve growth factor on dihydrotetrabenazine binding to PC12 cells

Tetrabenazine and dihydrotetrabenzaine (TBZOH) are potent inhibitors of substrate transport by the predominant forms of the vesicular monoamine transporter (VMAT) present in bovine brain synaptic vesicles and bovine adrenal medullary chromaffin vesicles. Radiolabeled TBZOH binds to these preparations with apparent dissociation constants in the low nanomolar range. However, tetrabenazine is a much less potent inhibitor of transport by rVMAT1, a form of the transporter cloned from a rat pheochromocytoma (PC12) cDNA library and expressed in CHO cells. Reported attempts to observe binding of [3H]TBZOH to rVMAT1 have not been successful. We examined binding of [3H]TBZOH to a crude membrane fraction from PC12 cells. Computerized nonlinear least squares curve fitting revealed two classes of binding sites (Kd1 = 1.5 nM, R1 = 0.2 pmol/mg protein, Kd2 = 340 nM, R2 = 15.2 pmol/mg protein). While the identity of the higher affinity sites is not certain, their high affinity for TBZOH suggests that they may be associated with rVMAT2. The lower affinity sites are likely to be associated with rVMAT1 on the basis of their affinity for TBZOH and sensitivity to inhibition of TBZOH binding by transporter substrates and inhibitors. NGF-treated PC12 cells also exhibited two classes of sites (Kd1 = 1.9 nM, R1 = 0.18 pmol/mg protein; Kd2 = 370 nM, R2 = 23.7 pmol/mg protein). While there were no significant differences between control and NGF-treated cells in binding capacity of the higher affinity sites, nor in apparent dissociation constants for either class of sites, there was a highly significant increase in number of lower affinity binding sites in the NGF-treated cells (p = 0.001). These results provide direct evidence that the differential sensitivity of rat brain and adrenal catecholamine stores to depletion by tetrabenazine and its derivatives is due to a much lower affinity of rVMAT1 for these compounds, and that NGF treatment may increase levels of rVMAT1 expression in PC12 cells.