EFFECTS OF NERVE GROWTH-FACTOR ON DIHYDROTETRABENAZINE BINDING TO PC12 CELLS

Tetrabenazine and dihydrotetrabenazine (TBZOH) are potent inhibitors o f substrate transport by the predominant forms of the vesicular monoam ine transporter (VMAT) present in bovine brain synaptic vesicles and b ovine 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 inhibito r of transport by rVMAT1, a form of the transporter cloned from a rat pheochromocytoma (PC12) cDNA library and expressed in CHO cells. Repor ted attempts to observe binding of [H-3]TBZOH to rVMAT1 have not been successful. We examined binding of [H-3]TBZOH to a crude membrane frac tion from PC12 cells. Computerized nonlinear least squares curve fitti ng revealed two classes of binding sites (K(d)1 = 1.5 nM, R(1) = 0.2 p mol/mg protein, K(d)2 = 340 nM, R(2) = 15.2 pmol/mg protein). While th e identity of the higher affinity sites is not certain, their high aff inity for TBZOH suggests that they may be associated with rVMAT2. The lower affinity sites are likely to be associated with rVMAT1 on the ba sis of their affinity for TBZOH and sensitivity to inhibition of TBZOH binding by transporter substrates and inhibitors. NGF-treated PC12 ce lls also exhibited two classes of sites (K(d)1 = 1.9 nM, R(1) = 0.18 p mol/mg protein; K(d)2 = 370 nM, R(2) = 23.7 pmol/mg protein). While th ere were no significant differences between control and NGF-treated ce lls in binding capacity of the higher affinity sites, nor in apparent dissociation constants for either class of sites, there was a highly s ignificant increase in number of lower affinity binding sites in the N GF-treated cells (p = 0.001). These results provide direct evidence th at 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 treatm ent may increase levels of rVMAT1 expression in PC12 cells. (C) 1997 E lsevier Science Ltd.