Pertussis toxin activates L-arginine uptake in pulmonary endothelial cells through downregulation of PKC-alpha activity.
Pertussis toxin (PTX) induces activation of l-arginine transport in pulmonary artery endothelial cells (PAEC). The effects of PTX on l-arginine transport appeared after 6 h of treatment and reached maximal values after treatment for 12 h. PTX-induced changes in l-arginine transport were not accompanied by changes in expression of cationic amino acid transporter (CAT)-1 protein, the main l-arginine transporter in PAEC. Unlike holotoxin, the beta-oligomer-binding subunit of PTX did not affect l-arginine transport in PAEC, suggesting that Galpha(i) ribosylation is an important step in the activation of l-arginine transport by PTX. An activator of adenylate cyclase, forskolin, and an activator of protein kinase A (PKA), Sp-cAMPS, did not affect l-arginine transport in PAEC. In addition, inhibitors of PKA or adenylate cyclase did not change the activating effect of PTX on l-arginine uptake. Long-term treatment with PTX (18 h) induced a 40% decrease in protein kinase C (PKC)-alpha but did not affect the activities of PKC-epsilon and PKC-zeta in PAEC. An activator of PKC-alpha, phorbol 12-myristate 13-acetate, abrogated the activation of l-arginine transport in PAEC treated with PTX. Incubation of PTX-treated PAEC with phorbol 12-myristate 13-acetate in combination with an inhibitor of PKC-alpha (Go 6976) restored the activating effects of PTX on l-arginine uptake, suggesting PTX-induced activation of l-arginine transport is mediated through downregulation of PKC-alpha. Measurements of nitric oxide (NO) production by PAEC revealed that long-term treatment with PTX induced twofold increases in the amount of NO in PAEC. PTX also increased l-[(3)H]citrulline production from extracellular l-[(3)H]arginine without affecting endothelial NO synthase activity. These results demonstrate that PTX increased NO production through activation of l-arginine transport in PAEC.
Zharikov
Sergey I
SI
Div. of Pulmonary Medicine, UF College of Medicine, P. O. Box 100225, Gainesville, FL 32610-0225, USA. zharikov@ufl.edu
Krotova
Karina Y
KY
Belayev
Leonid
L
Block
Edward R
ER
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D027201:Cationic Amino Acid Transporter 1 / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology*; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011651:Pulmonary Artery; D013552:Swine; D013873:Thionucleotides / Q000494:pharmacology
D027201:Cationic Amino Acid Transporter 1; D004791:Enzyme Inhibitors; D013873:Thionucleotides; D005576:Colforsin; C016957:adenosine-3',5'-cyclic phosphorothioate; D002956:Citrulline; D001120:Arginine; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha