Analysis of uptake and release of newly synthesized acetylcholine in PC12 cells overexpressing the rat vesicular acetylcholine transporter (VAChT).

Rat VAChT cDNA was stably transfected into PC12 cells to generate cell clones overexpressing different quantities of VAChT protein. Membrane fractions prepared from one cell clone (#3) in which VAChT was highly expressed (as determined by Western blot and [(3)H]vesamicol binding analyses) accumulated approximately two and half times as much [(3)H]ACh during incubation as did membrane fractions prepared from control cells. Vesamicol inhibited this ATP-dependent uptake. Membrane fractions isolated from a second cell clone (#6), which contained considerably less VAChT protein than did clone #3, accumulated no more [(3)H]ACh than did control cells. We compared the accumulation of newly synthesized [(14)C]ACh by a particulate fraction prepared from clones #3 and #6 with untransfected cells when these cells were incubated with either labeled acetate or choline. The results indicated that particulate fractions of clones #3 and #6 did not accumulate any more newly synthesized [(14)C]ACh than did the particulate fraction of untransfected cells. Furthermore, vesamicol reduced the filling of a particulate fraction of untransfected cells with newly synthesized ACh better than it reduced the refilling of a particulate fraction of VAChT-transfected cells. High K(+) depolarization did not release any more newly synthesized [(14)C]ACh from VAChT-transfected than it did from untransfected cells. In related studies, overexpression of VAChT in clone #3 induced a slight but significant increase in ChAT activity. Overall, our results indicate that an increase in the amount of VAChT protein associated with a particulate fraction of PC12 cells does not augment the amount of newly synthesized [(14)C]ACh acquired by that particulate fraction. However, it does reduce the effectiveness of vesamicol in blocking the filling of the particulate fraction with newly synthesized [(14)C]ACh. In summary, our results suggest that the vesicular release of ACh from PC12 cells is not regulated at the level of its uptake into synaptic vesicles.