Pertussis toxin (PTX) induces concentration- and time-dependent 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 (about a 2-fold increase) after treatment for 12 h. PTX-induced changes in L-arginine transport were not accompanied by changes in the expression of CAT-1 protein - the main L-arginine transporter in PAEC. We studied possible mechanisms by which long-term treatment of PAEC with PTX activates CAT-1-mediated L-arginine transport. Unlike the holotoxin, the -oligomer-binding subunit of PTX did not affect L-arginine transport in PAEC suggesting that G_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 Larginine uptake. Long-term treatment with PTX (500 ng/ml, 18 h) induced a 40% decrease in PKC but did not affect the activities of PKC and PKC/ in PAEC. An activator of PKC, phorbol-12-myristate-13-acetate (100 nM, 60 min), 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 (Go6976) restored the activating effects of PTX on L-arginine uptake suggesting that PTX-induced activation of L-arginine transport is mediated through down-regulation of PKC. Measurements of NO production by PAEC using a specific probe for NO (DAF-FM) revealed that long-term treatment with PTX induced 2-fold increases in the amount of NO in PAEC. PTX also increased [³H]-L-citrulline production from extracellular [³H]-L-arginine without affecting eNOS activity. These results demonstrate that PTX increased NO production through the activation of L-arginine transport into PAEC.