PKCalpha regulates both CAT1 and CAT2A/B transporters in Human Umbilical Vein Endothelial cells

PKC is involved in the modulation of arginine transport in various mammalian cell types. Contrasting results have been obtained on the effects of PKC activators, such as phorbols, on system y+-mediated arginine transport in endothelial models. Moreover, those studies mostly concern CAT1 transporter, while little is known about the role of the inducible isoforms CAT2B and CAT2A. In the present contribution we have investigated the effect of PKC activation on CAT transporters in human umbilical vein endothelial cells (HUVECs). When HUVECs were incubated with PDBu (100 nM), an activator of classical and novel PKCs, a 2-fold stimulation of system y+ transport activity was observed with a maximal effect after 8h of treatment. A marked, 10-fold induction of the mRNAs for the high affinity CAT2B and the low affinity CAT2A transporters was also observed after a 2h-incubation with PDBu, while CAT1 mRNA doubled. PKC-dependent induction of CAT2A/B was also confirmed at protein level through Western blot analysis. The effects of PDBu were associated to the specific translocation of PKCalpha from the cytosol to the membrane fraction. Consistently, a selective activator of classical PKCs, thymeleatoxin (100 nM), induced comparable changes in L-arginine uptake, CAT2 mRNAs and protein expression, as well as in PKCalpha intracellular distribution. Conversely, phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl (PIP, 5 µM), which activates novel PKCs, did not affect system y+ transport activity. The effects of both PDBu and thymeleatoxin were abolished by a specific inhibitor of classical PKCs, Gö6976 (1 µM). These data demonstrate that PKCalpha activation stimulates endothelial arginine transport through the induction of both CAT2A/B and CAT1. Preliminary results point to the involvement of the core IkBalpha-NF-kB cascade in PKC-dependent system y+ stimulation.