Ca2+/calmodulin mediated pathways regulate the uptake of L-DOPA in mouse neuroblastoma neuro 2A cells.
The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca2+/calmodulin mediated pathways on the uptake of L-DOPA through the L-type amino acid transporter in Neuro 2A cells, an in vitro model of neuronal cells. Non-linear analysis of the saturation curve for L-DOPA revealed a Km value (in microM) of 54+/-2 and a Vmax value (in nmol mg protein/6 min) of 34+/-1. L-DOPA uptake was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 mM), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC50=82 microM). The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC50's of 33 and 105 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutirate, phorbol 12-myristate 13-acetate and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in Neuro 2A cells is promoted through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin mediated pathways.
Sampaio-Maia
B
B
Institute of Pharmacology & Therapeutics, Faculty of Medicine, Porto, Portugal.
Soares-da-Silva
P
P
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism*; D051379:Mice; D009447:Neuroblastoma; D009474:Neurons / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D007980:Levodopa; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium