Protein kinase C mediated inhibition of endothelial L-arginine transport is mediated by MARCKS protein.

Abstract The endothelium plays a vital role in the maintenance of vascular tone and structural vascular integrity, principally mediated via the actions of nitric oxide (NO). l -arginine is the immediate substrate for NO synthesis, and the availability of extracellular l -arginine is critical for the production of NO. Activation of protein kinase C (PKC) dependent signalling pathways are a feature of a number of cardiovascular disease states, and in this study we aimed to systematically evaluate the mechanism by which PKC regulates l -arginine transport in endothelial cells. In response to PKC activation (PMA 100 nM, 30 min), [ 3 H] l -arginine uptake by bovine aortic endothelial cells (BAEC) was reduced to 45 + 4% of control ( p V max ( p K m for l -arginine. Western blot analysis and confocal microscopy revealed no change in the expression or membrane distribution of CAT-1, the principal BAEC l -arginine transporter. Moreover in 32 P-labeling studies, PMA exposure did not result in CAT-1 phosphorylation. We therefore explored the possibility that PKC altered and interaction with MARCKS protein, a candidate membrane associated protein. By co-immunoprecipitation we show that CAT-1 interacts with, a membrane associated protein, that was significantly inhibited by PKC activation ( p l -arginine transport. PKC dependent mechanisms regulate the transport of l -arginine, mediated via process involving MARCKS.