Ca(2+)-dependent enhancement of [3H]noradrenaline uptake in PC12 cells through calmodulin-dependent kinases.
Ca(2+)-dependent regulation of [3H]noradrenaline ([3H]NA) uptake through the NA transporter was studied using PC12 cells. Preincubation for 10 min in the presence of 0.3-10 mM ca2+ in Krebs-Ringer (KR) buffer induced marked enhancement of the uptake (at 1 mM Ca2+, 6.6 times greater than that observed in the absence of Ca2+), which reflected both an increase in Vmax and a decrease in K(m) of the uptake process. Preincubation with 1 mM Ca2+ also induced a significant increase in the Bmax and Kd of [3H]desipramine binding. The uptake was still enhanced after washing cells with Ca(2+)-free buffer following preincubation with 1 mM Ca2+. 1-[N, O-bis(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-c hlo rocinnamyl) -N-methylbenzylamine (KN-93) (inhibitors of Ca2+/calmodulin-dependent kinase II), N-(6-aminohexyl)-5-chloro-1-naphthalenesulonamide (W-7) (a calmodulin antagonist), wortmannin (a myosin light chain kinase inhibitor) significantly reduced Ca(2+)-dependent enhancement of the uptake. Mycalolide B (an inhibitor of actin-myosin interaction) also inhibited the enhancement. Although calphostin C (a protein kinase C (PKC) inhibitor) did not affect the enhancement, 12-o-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake. A synthetic peptide with a sequence (KKVIYKFFS579 IRGSLW) contained in the intracellular COOH-terminal domain of a rat NA transporter was phosphorylated by purified brain Ca2+/calmodulin-dependent protein kinase II. These results suggest that Ca(2+)-dependent enhancement of the [3H]NA uptake in PC12 cells are mediated by activation of calmodulin-dependent protein kinases, probably through stimulation of translocation of the NA transporter to the plasma membrane and/or direct phosphorylation of the transporter itself.
Uchida
J
J
Department of Pharmacology, School of Medicine, Showa University, Tokyo, Japan.
Kiuchi
Y
Y
Ohno
M
M
Yura
A
A
Oguchi
K
K
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002273:Carcinogens / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D003891:Desipramine / Q000378:metabolism / Q000494:pharmacology; D004298:Dopamine / Q000493:pharmacokinetics; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D008387:Marine Toxins; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000037:antagonists & inhibitors / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D009638:Norepinephrine / Q000493:pharmacokinetics*; D050484:Norepinephrine Plasma Membrane Transport Proteins; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D051381:Rats; D013566:Sympathomimetics / Q000493:pharmacokinetics*; D027981:Symporters; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D002147:Calmodulin; D002273:Carcinogens; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D018994:Myosin Light Chains; D009281:Naphthalenes; D050484:Norepinephrine Plasma Membrane Transport Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D010080:Oxazoles; D010446:Peptide Fragments; C493226:Slc6a2 protein, rat; D013566:Sympathomimetics; D027981:Symporters; D014316:Tritium; C080978:mycalolide B; D015638:Cytochalasin D; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D003891:Desipramine; D004298:Dopamine; D009638:Norepinephrine; D000077191:Wortmannin