Association of L-arginine transporters with fodrin: implications for hypoxic inhibition of arginine uptake

In this study, we investigated the possible interaction between the cationi c amino acid transporter (CAT)-1 arginine transporter and ankyrin or fodrin . Because ankyrin and fodrin are substrates for calpain and because hypoxia increases calpain expression and activity in pulmonary artery endothelial cells (PAEC), we also studied the effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC. Exposure to long-term hypoxia (24 h) inhibited L-a rginine uptake by PAEC, and this inhibition was prevented by calpain inhibi tor 1. The effects of hypoxia and calpain inhibitor 1 were not associated w ith changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in PAEC, and this could be prevented by calpain inhibitor 1. Incubation of solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60% decrease in L-arginine transport act ivity in reconstituted proteoliposomes (3,291 +/- 117 vs. 8,101 +/- 481 pmo l.mg protein(-1).3 min(-1) in control). Incubation with anti-ankyrin antibo dies had no effect on CAT-1 content or L-arginine transport in reconstitute d proteoliposomes. These results demonstrate that CAT-1 arginine transporte rs in PAEC are associated with fodrin, but not with ankyrin, and that long- term hypoxia decreases L-arginine transport by a calpain-mediated mechanism that may involve fodrin proteolysis.