Abstract 1632: Protein Kinase C Regulates Endothelial L-Arginine Transport via a Calpain Sensitive Pathway
Background: Endothelial dysfunction is a hallmark of many forms of cardiovascular disease. Previous studies from our group indicate that the transport of L-arginine (L-ARG), the substrate for nitric oxide synthesis, is impaired although the mechanism(s) remain uncertain. In conjunction, activation of protein kinase C (PKC) dependent signaling pathways are a feature of a number of cardiovascular disease states, and it has been previously suggested that PKC activation alters L-ARG transport. In this study we aimed to determine the responsible mechanism.
Methods and Results: In response to PKC activation (PMA 100nM, 30 min), [3H]L-Arg 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-Arg. Western blot analysis and 32P-labeling studies indicated no change in the expression or phosphorylation of CAT-1, the principal L-ARG transporter. To determine the responsible mechanism, we considered potentially relevant PKC dependent regulators. In particular, the PKC-mediated effect on L-ARG transport was significantly reversed by the inhibition of calpain either pharmacologically (100%, p<0.05) or via adenoviral overexpression of the endogenous regulator of calpain, calpastatin (40% p<0.05). On the basis that eNOS, caveolin and CAT1 are known to be co-localized, we examined the investigated the effect of PKC activation, and demonstrated that the expression of eNOS and caveolin was unchanged.
Conclusion: Protein kinase C regulates the transport of L-ARG, via a calpain-sensitive intermediate pathway.