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

Kylie Venardos, Carla Enriquez, Tanneale Marshall, J. P F Chin-Dusting, Belinda Ahlers, David M. Kaye

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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), [3H]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 Km 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 32P-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.

Original languageEnglish
Pages (from-to)86-92
Number of pages7
JournalJournal of Molecular and Cellular Cardiology
Volume46
Issue number1
DOIs
Publication statusPublished - Jan 2009
Externally publishedYes

Keywords

  • Amino acid transport
  • Cationic Amino Acid Transporter-1
  • Endothelium
  • L-arginine
  • Nitric oxide
  • Protein kinase C
  • Protein-protein interaction

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