EXPRESSION OF DOPAMINE TRANSPORTER AT THE TIPS OF GROWING NEURITES OFPC12 CELLS

The four kinds of oligopeptides specific in amino acid sequence to a r at dopamine transporter (DAT), peptide-1-peptide-4, were chemically sy nthethized, An attempt to produce antipeptide antibodies against these oligopeptides was made with an in vitro immunization method, Two mono clonal antibodies, MAbs H-1a and H-1b, were produced against one of th e oligopeptides, peptide-1, Western blot analysis confirmed that the t wo antibodies recognized a similar to 85,000 Da protein in a synaptoso mal fraction prepared from the rat striatum but none in the fraction f rom the cerebellum, The specificity of the antibody to DAT was also co nfirmed by an antibody absorption test using two synthetic oligopeptid es, one of which is specific only to DAT, These results have confirmed the specificity of the present antibody to DAT, The expression and su bcelllar localization of DAT were immunohistochemically examined with MAbs H-1a and H-1b in PC12 cells treated with nerve growth factor (NGF ). The antibody labeled the surface of PC12 cells, When the cells were treated with NGF, the expression of DAT was significantly emphasized, first in the area mainly including the Golgi apparatus and rough endo plasmic reticulum and then on the surface of growth cones from the beg inning of neurite outgrowth, DAT was detected by Western blot analysis in a microsomal fraction prepared from PC12 cells, The activity of DA T in the PC12 cells was pharmacologically confirmed by the uptake of [ H-3]-dopamine and blockade by uptake inhibitors, The NGF treatment dou bled the dopamine uptake activity, GBR12909, a specific inhibitor of D AT, blocked the [H-3]-dopamine at a concentration of 10(-7) M. The exp ression of DAT and norepinephrine transporter (NET) mRNA in the PC12 c ells was examined by reverse transcriptase-polymerase chain reaction ( RT-PCR), DAT mRNA significantly increased in the NGF-treated cells aft er 7 days of incubation, whereas NET mRNA markedly decreased.