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1.
Br J Pharmacol ; 148(5): 732-40, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16715118

ABSTRACT

1. Myristoylated pseudosubstrate of PKCzeta (mPS) - a synthetic myristoylated peptide with a sequence (13 amino acids) mimicking the endogenous PKCzeta pseudosubstrate region -- is considered a selective cell-permeable inhibitor of PKCzeta. We present strong evidence that in endothelial cells the action of mPS is not limited to inhibition of PKC activity and that myristoylation of certain peptides can activate eNOS (endothelial nitric oxide synthase) through Akt phosphorylation. 2. mPS at micromolar concentrations (1-10 microM) induced profound phosphorylation of eNOS, Akt, ERK 1/2, and p38 MAPK in cultured pulmonary artery endothelial cells (PAEC). The same changes were observed after treatment of PAEC with a myristoylated scrambled version of mPS (mScr), whereas a cell-permeable version of PKCzeta pseudosubstrate fused to the HIV-TAT membrane-translocating peptide did not induce analogous changes, suggesting that myristoylation confers new properties on the peptides consisting of activation of different signaling pathways in endothelial cells. 3. In addition to mPS and mScr, a number of other myristoylated peptides induced phosphorylation of eNOS suggesting that myristoylation of peptides can activate eNOS by mechanisms unrelated to inhibition of PKC. All active myristoylated peptides contained basic amino acids motif and were longer than six amino acids. 4. Activation of eNOS by myristoylated peptides was dependent on the PI3K/Akt pathway and the rise of intracellular calcium and was associated with an elevation of cGMP levels in PAEC and with relaxation of precontracted isolated pulmonary artery segments. 5. Myristoylated peptides can be considered a new class of activators of NO production in endothelial cells and that using mPS as a specific inhibitor of PKC should be done with caution, especially in endothelial cells.


Subject(s)
Endothelial Cells/drug effects , Fatty Acids, Monounsaturated/chemistry , Nitric Oxide Synthase Type III/metabolism , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , 3T3-L1 Cells , Animals , Calcium/physiology , Cells, Cultured , Cyclic GMP/biosynthesis , Humans , Isoenzymes/chemistry , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Organ Specificity/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/physiology , Phosphorylation/drug effects , Placebos/pharmacology , Protein Kinase C/chemistry , Protein Kinase C/pharmacology , Proto-Oncogene Proteins c-akt/physiology , Pulmonary Artery/cytology , Pulmonary Artery/drug effects , Swine , Vasodilation/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism
2.
Am J Physiol Lung Cell Mol Physiol ; 286(5): L1066-74, 2004 May.
Article in English | MEDLINE | ID: mdl-14729513

ABSTRACT

Catalytic activity of eNOS is regulated by multiple posttranscriptional mechanisms, including a 40-amino acid (604-643) autoinhibitory domain (AID) located in the reductase domain of the eNOS protein. We examined whether an exogenous synthetic AID, an 11-amino acid (626-636) fragment of AID (AAF), or scrambled AAF (AAF-SR), enhanced eNOS activity and NO-cGMP-mediated vasorelaxation using pulmonary artery (PA) endothelial/smooth muscle cell (PAEC/PASM) coculture, isolated PA segment, and isolated lung perfusion models. Incubation of isolated total membrane fraction of PAEC with AID or AAF resulted in concentration-dependent loss of eNOS activity. In contrast, incubation of intact PAEC with AID or AAF but not AAF-SR caused concentration- and time-dependent activation of eNOS. Because AID and AAF had similar effects on activation of eNOS, AAF and AAF-SR were used for further evaluation. Although AAF stimulation increased catalytic activity of PKC-alpha in PAEC, AAF-mediated activation of eNOS was independent of phosphorylation of Ser1177 or Thr495 and/or expression of eNOS protein. AAF stimulation of PAEC increased NO and cGMP production, which were attenuated by pretreatment with the eNOS inhibitor l-NAME. AAF caused time-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PA segments, and this response was attenuated by l-NAME. AAF, but not AAF-SR, also caused vasorelaxation in an ex vivo isolated mouse lung perfusion model precontracted with U-46619. Incubation with fluorescence-labeled AAF demonstrated translocation of AAF in PAEC in culture, isolated PA, and isolated intact lungs. These results demonstrate that AAF-stimulated vasodilation is mediated via activation of eNOS and enhanced NO-cGMP production in PA and intact lung.


Subject(s)
Cyclic GMP/physiology , Nitric Oxide Synthase/pharmacology , Nitric Oxide/physiology , Pulmonary Artery/physiology , Vasodilation/drug effects , Amino Acid Sequence , Animals , Cells, Cultured , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Models, Animal , Molecular Sequence Data , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase Type III , Peptide Fragments/chemistry , Pulmonary Artery/drug effects , Swine
3.
Am J Physiol Lung Cell Mol Physiol ; 286(5): L974-83, 2004 May.
Article in English | MEDLINE | ID: mdl-14695118

ABSTRACT

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.


Subject(s)
Arginine/metabolism , Cyclic AMP/analogs & derivatives , Endothelium, Vascular/metabolism , Pertussis Toxin/pharmacology , Protein Kinase C/metabolism , Pulmonary Artery , Animals , Biological Transport/drug effects , Cationic Amino Acid Transporter 1/drug effects , Cationic Amino Acid Transporter 1/metabolism , Cells, Cultured , Citrulline/metabolism , Colforsin/pharmacology , Cyclic AMP/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Enzyme Inhibitors/pharmacology , Protein Kinase C/drug effects , Protein Kinase C-alpha , Swine , Thionucleotides/pharmacology
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