Protein kinase C mediated inhibition of endothelial L-arginine transport is mediated by MARCKS protein.
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<0.05). This resulted from a 53% reduction in the Vmax (p<0.05), with no change in the 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<0.05). Moreover antisense inhibition of MARCKS abolished the PMA effect on L-arginine transport. PKC dependent mechanisms regulate the transport of L-arginine, mediated via process involving MARCKS.
Venardos
Kylie
K
Heart Failure Research Group, Baker Heart Research Institute, Melbourne VIC, Australia.
Enriquez
Carla
C
Marshall
Tanneale
T
Chin-Dusting
Jaye P F
JP
Ahlers
Belinda
B
Kaye
David M
DM
D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D027201:Cationic Amino Acid Transporter 1 / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*
D027201:Cationic Amino Acid Transporter 1; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D001120:Arginine; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C