Inhibition of calcium-independent luminal uptake of L-dopa by calmodulin antagonists in immortalized rat capillary cerebral endothelial 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 Ca(2+)/calmodulin mediated pathways on the luminal uptake of L-DOPA through the L-type amino acid transporter in immortalized rat capillary cerebral endothelial (REB-4) cells. Non-linear analysis of the saturation curve for L-DOPA revealed a K(m)value (in microM) of 71+/-9 and a V(max)value of 17+/-1 (in nmol mg protein/6 min). L-DOPA uptake at the luminal cell border was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 m m), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC(50)=140 microM). The Ca(2+)/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC(50)s of 23 and 33 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-dibutyrate, staurosporine 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 RBE-4 cells is promoted through the L-type amino acid transporter and appears to be under the control of calmodulin mediated pathways.
Sampaio-Maia
B
B
Institute of Pharmacology & Therapeutics, Faculty of Medicine, Porto, 4200-319, Portugal.
Soares-Da-Silva
P
P
D000598:Amino Acids, Cyclic / Q000494:pharmacology; D000621:Aminoisobutyric Acids / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007980:Levodopa / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D012964:Sodium / Q000494:pharmacology; D013687:Telencephalon; D013997:Time Factors; D014268:Trifluoperazine / Q000494:pharmacology
D000598:Amino Acids, Cyclic; D000621:Aminoisobutyric Acids; D020746:Calcium Channels, L-Type; D002147:Calmodulin; D004791:Enzyme Inhibitors; D007093:Imidazoles; C006504:2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; D014268:Trifluoperazine; C017911:2-(methylamino)isobutyric acid; D007980:Levodopa; C031938:calmidazolium; D012964:Sodium; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C