CA2+ CALMODULIN AND PROTEIN-KINASE-C REGULATION OF SEROTONIN TRANSPORT IN HUMAN K562 LYMPHOCYTES/

This study was conducted on human K562 lymphocytes to investigate-the mechanisms implicated in the regulation of the serotonin transport pro cess. The uptake of serotonin in these cells was saturable (K-m, 3.37 mu M; V-max, 2.03 nmol/10(6) cells) and Na+ dependent; isoosmotic repl acement of Na+ with choline chloride in the assay medium resulted in t he decreased uptake process, Augmentation of intracellular free calciu m, [Ca2+](i), by thapsigargin decreased the uptake of serotonin in the se cells. Similarly, addition of calcium ionophores (A23187) and ionom ycin also inhibited serotonin transport. In Fura-2-loaded cells, these agents increased the [Ca2+](i) contents. These results suggest that a n increase in [Ca2+](i) is implicated with a decrease in serotonin tra nsport; Since an increase in [Ca2+](i) is known to activate calmodulin (CaM), we employed CaM antagonists, Calmodulin antagonists W-7 [6-ami nohexyl]-5-chloro-1-naphthalene-sulfonamide) and mellitin inhibited se rotonin uptake in K562 cells, suggesting that CaM is involved in serot onin transport regulation. Furthermore, acute exposure of K562 cells t o known protein kinase C (PKC) activators, phorbol-12-myristate-13-ace tate (PMA) and sn-1,2-dioctanoylglycerol (DiC8), curtailed serotonin u ptake by these cells. However, staurosporine (a PKC inhibitor) failed to abolish the inhibitory effects of PMA on serotonin transport in the se cells, indicating that the target of PMA is not PKC. Nonetheless, t he PMA-induced inhibitory effects are specific as 4 alpha-phorbol-12,1 3-didecanoate (a phorbol ester known not to activate PKC) failed to mi mic PMA-like actions on serotonin transport in K562 cells. DiC8 not on ly exerted higher inhibitory effects than PMA but also had additive ef fects in the presence of the latter on serotonin transport. These resu lts suggest that in addition to PKC, there are other cellular targets (of PMA) implicated in serotonin transport regulation. (C) 1996 Academ ic Press, Inc.