Regulation of the functional activity of the human dopamine transporter by the arachidonic acid pathway

The role of arachidonic acid was examined in the regulation of dopamine transport in C6 glioma cells stably expressing the human dopamine transporter. Exogenously added arachidonic acid (20-160 mu M) stimulated [H-3]dopamine uptake when pre-incubated for short times (15-30 min); 160 mu M arachidonic acid inhibited following longer pre-exposures (45-60 min). Under the same conditions, only decreases were observed in the binding of the cocaine analog [H-3]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane([H-3]WIN 35,428). The reduction in dopamine transporter activity by arachidonic acid (at 160 mu M for 60 min) was caused by a decrease in the V-max (from 202 to 44 pmol/mg/min) opposed by a smaller reduction in K-m (from 1.2 to 0.8 mu M), whereas the effect of arachidonic acid (at 160 mu M for 15 min) on [H-3]WIN 35,428 binding was caused by a reduction in the B-max (from 1.8 to 1.3 pmol/mg) without a change in K-d (7.2 nM). Upon 15-min exposure, melittin, an activator of phospholipase A(2), and nordihydroguaiaretic acid, a lipooxygenase inhibitor, both expected to cause enhanced endogenous arachidonic acid, inhibited [H-3]dopamine uptake and [H-3]WIN 35,428 binding with an IC50 value close to 1 mu M, whereas thimerosal, which raises arachidonic acid by inhibiting lipid reacylation, caused similar reductions at the sub-millimolar level. Co-presence of staurosporine (0.3-2 mu M), an inhibitor of protein kinase C, had little or no effect on the melittin- or arachidonic acid-induced inhibition of [H-3]dopamine uptake. Both the melittin- and arachidonic acid-, but not phorbol 12-myristate 13-acetate-induced inhibition of uptake were counteracted by bovine serum albumin (0.1 and 1 mg/ml) which binds arachidonic acid. The data taken together suggest that the inhibitory effects of arachidonic acid activators and those of protein kinase C activators on dopamine uptake are mediated by separate mechanisms.