Upregulation of Na(+)-dependent alanine transport in vascular endothelial cells by serum: role of intracellular Ca2+.
OBJECTIVE
Amino acid transport and its regulation in vascular endothelial cells remains a largely unexplored area. In this study, we evaluated alanine transport in bovine aortic endothelial cells to assess possible mechanisms of regulation.


METHODS
Alanine transport into confluent monolayers of endothelial cells was measured using 100 microM [3H]alanine in the presence and absence of external Na+, in cells deprived of serum for 24 hr (SD), and in SD cells exposed to 10% serum (S) for 3 hr (SD + S cells).


RESULTS
Our results indicate that although SD did not significantly affect the Na(+)-independent transport of alanine when compared to normal cells, serum addition to serum-deprived cells markedly stimulated the Na(+)-dependent uptake of this amino acid through system A. The stimulation of alanine transport pathway(s) by serum was totally abolished by pretreatment of endothelial cells with 10 microM cycloheximide, suggesting a role of protein synthesis. Serum also induced a marked increase in calcium recycling at the cell membrane, suggesting that calcium is a key element of the serum signaling pathway. Indeed, both BAPTA (20 microM), a cellular calcium chelator, and thapsigargin (1 microM), an agent that depletes intracellular calcium stores, prevented the stimulation of alanine uptake by serum. Finally, pertussis toxin (400 ng/ml), an agent known to inactivate certain G-protein-dependent pathways, significantly reduced the serum-dependent 45Ca uptake and [3H]alanine entry. However, the protein kinase C activator PMA (100 nM), significantly reduced the stimulation of alanine uptake by serum but did not affect the stimulation of calcium uptake.


CONCLUSIONS
Altogether these findings suggest that cell calcium is involved in the regulation of system A by serum in vascular endothelial cells.
Escobales
N
N
Department of Physiology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico 00936-5067.
Martinez
J
J
Gonzalez
O
O
D000409:Alanine / Q000378:metabolism*; D026905:Amino Acid Transport Systems; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D001769:Blood; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D037342:Pertussis Toxin; D011188:Potassium / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin; D015854:Up-Regulation; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D026905:Amino Acid Transport Systems; D002352:Carrier Proteins; D013729:Terpenes; D010566:Virulence Factors, Bordetella; D004533:Egtazic Acid; D010042:Ouabain; D019284:Thapsigargin; D003513:Cycloheximide; D037342:Pertussis Toxin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine; D011188:Potassium; D002118:Calcium