title: Regulation of the functional activity of the human dopamine transporter by the arachidonic acid pathway.
abstract: 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 microM) stimulated [3H]dopamine uptake when pre-incubated for short times (15-30 min); 160 microM 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 [3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([3H]WIN 35,428). The reduction in dopamine transporter activity by arachidonic acid (at 160 microM for 60 min) was caused by a decrease in the Vmax (from 202 to 44 pmol/mg/min) opposed by a smaller reduction in K(m) (from 1.2 to 0.8 microM), whereas the effect of arachidonic acid (at 160 microM for 15 min) on [3H]WIN 35,428 binding was caused by a reduction in the Bmax (from 1.8 to 1.3 pmol/mg) without a change in Kd (7.2 nM). Upon 15-min exposure, melittin, an activator of phospholipase A2, and nordihydroguaiaretic acid, a lipooxygenase inhibitor, both expected to cause enhanced endogenous arachidonic acid, inhibited [3H]dopamine uptake and [3H]WIN 35,428 binding with an IC50 value close to 1 microM, whereas thimerosal, which raises arachidonic acid by inhibiting lipid reacylation, caused similar reductions at the sub-millimolar level. Co-presence of stauroporine (0.3-2 microM), an inhibitor of protein kinase C, had little or no effect on the melittin- or arachidonic acid-induced inhibition of [3H]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.
authors.affiliation: Department of Biomedical and Therapeutic Sciences (formerly Basic Sciences), University of Illinois, College of Medicine, Peoria 61656, USA.
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: M E
authors.initials: ME
authors.lastname: Reith
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D001932:Brain Neoplasms / Q000378:metabolism*; D002352:Carrier Proteins / Q000502:physiology*; D002417:Cattle; D003042:Cocaine / Q000031:analogs & derivatives / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D004305:Dose-Response Relationship, Drug; D005910:Glioma / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins
chemical_list: D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D016718:Arachidonic Acid; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D003042:Cocaine; D004298:Dopamine