Polyamine transport regulation by calcium and calmodulin: Role of Ca2+-ATPase

The study was conducted on human leukemia (K 562) cells to characterize the mechanisms implicated in the regulation of the polyamine spermicine (Spd) transport process. The antagonists of calmodulin, trifluoperazine (TFP), W-7 (N-6-aminohexyl]-5-chloro-1-naphthelenesulfonamide), or mellitin inhiblted significantly polyamine Spd uptake in these cells. The translocation of calmodulin towards plasma membrane and a concomitant decrease in its contents in cytosol were directly correlated with the time course increases similar to that of Spd uptake, indicating that calmodulin is recruited towards plasma membrane during the Spd transport process. Diminution of free intracellular calcium, (Ca²⁺)i, by preincubating the cells in BAPTA (bis2-amino-5-methylphenoxyl]-ethane-N,N′,N′,-tetraacetate) buffer inhibited Spd transport significantly. Addition of lanthanum (LAN), a molecule known to inhibit Ca² efflux via Ca²⁺-ATPase, curtailed Spd uptake by these cells. LAN inhibited Vmax, but not the Km, of Spd uptake, indicating that the former does not directly interact with the polyamine transporter; rather it regulates the transport process, probably via its action on Ca²⁺-ATPase. Calmodulin-stimulated uptake of ⁴⁵Ca²⁺ by inside-out vesicles of K 562 cells, a measure of Ca²⁺-ATPase activity. Furthermore, addition of LAN inhibited both basal and calmodulin-stimulated activity of Ca²⁺-ATPase. Thapsigargin (THAP), a molecule known to elevate (Ca²⁺) i due to its action on the endoplasmic reticulum, increased Spd transport whereas addition of LAN inhibited THAP-stimulated Spd transport activity. THAP increased free (Ca²⁺)i in these cells, and a pre-addition of LAN to these cells curtailed the THAP-stimulated increases of (Ca²⁺)i concentrations. Addition of Spd brought about elevations in (Ca²⁺)i contents. Caffeine also increased (Ca²⁺)i in these cells; however, it failed to stimulate significantly the Spd uptake process, indicating that (Ca²⁺)i which is involved in the regulation of polyamine transport pathways does not belong to the calcium-induced calcium-release (CICR) pool. Replacement of Ca²⁺ from the incubation medium (i.e., 0% Ca²⁺) resulted in higher uptake activity as compared to that in 100% Ca²⁺ medium, demonstrating that in 100% Ca²⁺ medium the calcium efflux process is quickly compensated by calcium refilling/influx from the extracellular medium, while in 0% Ca²⁺ medium there is perpetual efflux of (Ca²⁺)i which contributes to higher Spd uptake process. The results of this study suggest that an increase in free (Ca²⁺)i and its release from the cells via Ca²⁺ ATPase, and concomitant activation of calmodulin, which controls Ca²⁺-pump activity, are involved in the regulation of the Spd uptake process in human leukemia cells. © 1993 Wiley-Liss, Inc.