title: Analysis of uptake and release of newly synthesized acetylcholine in PC12 cells overexpressing the rat vesicular acetylcholine transporter (VAChT).
abstract: 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.
authors: Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA. AliRoghani@ttmc.ttuhsc.edu
authors: Ali
authors: A
authors: Roghani
authors: Paul T
authors: PT
authors: Carroll
mesh_terms: D000085:Acetates / Q000378:metabolism; D000109:Acetylcholine / Q000096:biosynthesis* / Q000378:metabolism*; D000818:Animals; D001923:Brain Chemistry / Q000235:genetics; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002490:Central Nervous System / Q000378:metabolism*; D002794:Choline / Q000378:metabolism; D002795:Choline O-Acetyltransferase / Q000378:metabolism*; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents / Q000494:pharmacology; D016716:PC12 Cells; D010880:Piperidines / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism; D009435:Synaptic Transmission / Q000235:genetics*; D013572:Synaptic Vesicles / Q000378:metabolism*; D014162:Transfection; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
chemical_list: D000085:Acetates; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D009467:Neuromuscular Depolarizing Agents; D010880:Piperidines; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; C006189:vesamicol; D002795:Choline O-Acetyltransferase; D002794:Choline; D000109:Acetylcholine; D011188:Potassium