Ca 2+-dependent enhancement of [ [formula omitted]]noradrenaline uptake in PC12 cells through calmodulin-dependent kinases

Abstract

Ca 2+-dependent regulation of [ 3 H ]noradrenaline ([ 3 H ]NA) uptake through the NA transporter was studied using PC12 cells. Preincubation for 10 min in the presence of 0.3–10 mM Ca 2+ in Krebs–Ringer (KR) buffer induced marked enhancement of the uptake (at 1 mM Ca 2+, 6.6 times greater than that observed in the absence of Ca 2+), which reflected both an increase in V max and a decrease in K m of the uptake process. Preincubation with 1 mM Ca 2+ also induced a significant increase in the B max and K d of [ 3 H ]desipramine binding. The uptake was still enhanced after washing cells with Ca 2+-free buffer following preincubation with 1 mM Ca 2+. 1-[ N, O-bis(5-Isoquinolinesulfonyl)- N-methyl- l-tyrosyl]-4-phenylpiperazine (KN-62), 2-[ N-(2-hydroxyethyl)- N-(4-methoxybenzenesulfonyl)]amino- N-(4-chlorocinnamyl)- N-methylbenzylamine (KN-93) (inhibitors of Ca 2+/calmodulin-dependent kinase II), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (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 (KKVIYKFFS 579IRGSLW) contained in the intracellular COOH-terminal domain of a rat NA transporter was phosphorylated by purified brain Ca 2+/calmodulin-dependent protein kinase II. These results suggest that Ca 2+-dependent enhancement of the [ 3 H ]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 phosphrylation of the transporter itself.