Cr. Adamson et al., EFFECTS OF NERVE GROWTH-FACTOR ON DIHYDROTETRABENAZINE BINDING TO PC12 CELLS, Neurochemistry international, 30(4-5), 1997, pp. 411-415
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.