scholarly journals Extracellular l-arginine Enhances Relaxations Induced by Opening of Calcium-Activated SKCa Channels in Porcine Retinal Arteriole

2019 ◽  
Vol 20 (8) ◽  
pp. 2032
Author(s):  
Ulf Simonsen ◽  
Anna K. Winther ◽  
Aida Oliván-Viguera ◽  
Simon Comerma-Steffensen ◽  
Ralf Köhler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Potassium Channels ◽  
No Production ◽  
Response Curves ◽  
Retinal Arteriole ◽  
Cationic Amino Acid ◽  
Low Sodium ◽  
Arginine Uptake ◽  
Retinal Arterioles ◽  
System L

We investigated whether the substrate for nitric oxide (NO) production, extracellular l-arginine, contributes to relaxations induced by activating small (SKCa) conductance Ca2+-activated potassium channels. In endothelial cells, acetylcholine increased 3H-l-arginine uptake, while blocking the SKCa and the intermediate (IKCa) conductance Ca2+-activated potassium channels reduced l-arginine uptake. A blocker of the y+ transporter system, l-lysine also blocked 3H-l-arginine uptake. Immunostaining showed co-localization of endothelial NO synthase (eNOS), SKCa3, and the cationic amino acid transporter (CAT-1) protein of the y+ transporter system in the endothelium. An opener of SKCa channels, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) induced large currents in endothelial cells, and concentration-dependently relaxed porcine retinal arterioles. In the presence of l-arginine, concentration-response curves for CyPPA were leftward shifted, an effect unaltered in the presence of low sodium, but blocked by l-lysine in the retinal arterioles. Our findings suggest that SKCa channel activity regulates l-arginine uptake through the y+ transporter system, and we propose that in vasculature affected by endothelial dysfunction, l-arginine administration requires the targeting of additional mechanisms such as SKCa channels to restore endothelium-dependent vasodilatation.

Cytoskeletal regulation of the l-arginine/NO pathway in pulmonary artery endothelial cells

2001 ◽  
Vol 280 (3) ◽  
pp. L465-L473 ◽  
Author(s):  
Sergey I. Zharikov ◽  
Alla A. Sigova ◽  
Sifeng Chen ◽  
Michael R. Bubb ◽  
Edward R. Block
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
The State ◽  
No Production ◽  
Actin Microfilaments ◽  
Cationic Amino Acid ◽  
Arginine Transport ◽  
Arginine Uptake

We investigated possible involvement of the actin cytoskeleton in the regulation of thel-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton,l-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca2+/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50–100 nM, 2 h treatment) induced a reversible activation ofl-[3H]arginine uptake by PAEC, whereas Swinh (10–50 nM) decreased l-[3H]arginine uptake. The two drugs could abrogate the effect of each other onl-[3H]arginine uptake. The effects of both drugs on l-[3H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular l-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates l-arginine transport and that this regulation can affect NO production by PAEC.

Pertussis toxin activates l-arginine uptake in pulmonary endothelial cells through downregulation of PKC-α activity

2004 ◽  
Vol 286 (5) ◽  
pp. L974-L983 ◽  
Author(s):  
Sergey I. Zharikov ◽  
Karina Y. Krotova ◽  
Leonid Belayev ◽  
Edward R. Block
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Pertussis Toxin ◽  
Term Treatment ◽  
No Production ◽  
Arginine Transport ◽  
Arginine Uptake ◽  
Long Term Treatment

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 β-oligomer-binding subunit of PTX did not affect l-arginine transport in PAEC, suggesting that Gαi 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)-α but did not affect the activities of PKC-ϵ and PKC-ζ in PAEC. An activator of PKC-α, 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-α (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-α. 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-[3H]citrulline production from extracellular l-[3H]arginine without affecting endothelial NO synthase activity. These results demonstrate that PTX increased NO production through activation of l-arginine transport in PAEC.

Amino acids as modulators of endothelium-derived nitric oxide

2006 ◽  
Vol 291 (2) ◽  
pp. F297-F304 ◽  
Author(s):  
Masao Kakoki ◽  
Hyung-Suk Kim ◽  
Cora-Jean S. Edgell ◽  
Nobuyo Maeda ◽  
Oliver Smithies ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Amino Acids ◽  
Endothelial Cells ◽  
Cultured Cells ◽  
Rat Kidney ◽  
No Production ◽  
Renal Vasculature ◽  
Cationic Amino Acid ◽  
Neutral Amino Acids ◽  
Cationic Amino Acids

To examine the mechanisms whereby amino acids modulate nitric oxide (NO) production and blood flow in the renal vasculature, chemiluminescence techniques were used to quantify NO in the renal venous effluent of the isolated, perfused rat kidney as different amino acids were added to the perfusate. The addition of 10−4 or 10−3 M cationic amino acids (l-ornithine, l-lysine, or l-homoarginine) or neutral amino acids (l-glutamine, l-leucine, or l-serine) to the perfusate decreased NO and increased renal vascular resistance. Perfusion with anionic amino acids (l-glutamate or l-aspartate) had no effect on either parameter. The effects of the cationic and neutral amino acids were reversed with 10−3 M l-arginine and prevented by deendothelialization or NO synthase inhibition. The effects of the neutral amino acids but not the cationic amino acids were dependent on extracellular sodium. Cationic and neutral amino acids also decreased calcimycin-induced NO, as assessed by DAF-FM-T fluorescence, in cultured EA.hy926 endothelial cells. Inhibition of system y+ or y+L by siRNA for the cationic amino acid transporter 1 or the CD98/4F2 heavy chain diminished the NO-depleting effects of these amino acids. Finally, transport studies in cultured cells demonstrated that cationic or neutral amino acids in the extracellular space stimulate efflux of l-arginine out of the cell. Thus the present experiments demonstrate that cationic and neutral amino acids can modulate NO production in endothelial cells by altering cellular l-arginine transport through y+ and y+L transport mechanisms.

Estradiol augments while progesterone inhibits arginine transport in human endothelial cells through modulation of cationic amino acid transporter-1

2015 ◽  
Vol 309 (4) ◽  
pp. R421-R427 ◽  
Author(s):  
Ohad S. Bentur ◽  
Doron Schwartz ◽  
Tamara Chernichovski ◽  
Merav Ingbir ◽  
Talia Weinstein ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Amino Acid ◽  
Protein Content ◽  
Amino Acid Transporter ◽  
Female Rats ◽  
Cationic Amino Acid Transporter ◽  
Cationic Amino Acid ◽  
Arginine Transport ◽  
Arginine Uptake ◽  
Acid Transporter

Decreased generation of nitric oxide (NO) by endothelial NO synthase (eNOS) characterizes endothelial dysfunction (ECD). Delivery of arginine to eNOS by cationic amino acid transporter-1 (CAT-1) was shown to modulate eNOS activity. We found in female rats, but not in males, that CAT-1 activity is preserved with age and in chronic renal failure, two experimental models of ECD. In contrast, during pregnancy CAT-1 is inhibited. We hypothesize that female sex hormones regulate arginine transport. Arginine uptake in human umbilical vein endothelial cells (HUVEC) was determined following incubation with either 17β-estradiol (E2) or progesterone. Exposure to E2 (50 and 100 nM) for 30 min resulted in a significant increase in arginine transport and reduction in phosphorylated CAT-1 (the inactive form) protein content. This was coupled with a decrease in phosphorylated MAPK/extracellular signal-regulated kinase (ERK) 1/2. Progesterone (1 and 100 pM for 30 min) attenuated arginine uptake and increased phosphorylated CAT-1, phosphorylated protein kinase Cα (PKCα), and phosphorylated ERK1/2 protein content. GO-6976 (PKCα inhibitor) prevented the progesterone-induced decrease in arginine transport. Coincubation with both progesterone and estrogen for 30 min resulted in attenuated arginine transport. While estradiol increases arginine transport and CAT-1 activity through modulation of constitutive signaling transduction pathways involving ERK, progesterone inhibits arginine transport and CAT-1 via both PKCα and ERK1/2 phosphorylation, an effect that predominates over estradiol.

Modulation of the l-arginine/nitric oxide signalling pathway in vascular endothelial cells

2004 ◽  
Vol 71 ◽  
pp. 143-156 ◽  
Author(s):  
Amanda W. Wyatt ◽  
Joern R. Steinert ◽  
Giovanni E. Mann
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Amino Acid ◽  
Protein Kinase ◽  
Signalling Pathway ◽  
No Production ◽  
Dependent Manner ◽  
Amino Acid Transport System ◽  
Membrane Hyperpolarization ◽  
Cationic Amino Acid

Nitric oxide (NO) is synthesized from l-arginine, and in endothelial cells influx of l-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes l-arginine to NO and l-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of l-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of l-arginine despite having sufficient intracellular l-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17ϐ-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on l-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the l-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.

scholarly journals CAT2-mediated l-arginine transport and nitric oxide production in activated macrophages

1999 ◽  
Vol 340 (2) ◽  
pp. 549-553 ◽  
Author(s):  
Donald K. KAKUDA ◽  
Matthew J. SWEET ◽  
Carol L. MACLEOD ◽  
David A. HUME ◽  
Daniel MARKOVICH
Keyword(s):  
Amino Acid ◽  
Transport System ◽  
Transport Systems ◽  
No Production ◽  
Activated Macrophages ◽  
Amino Acid Transport System ◽  
Cationic Amino Acid ◽  
Arginine Transport ◽  
Arginine Uptake ◽  
Expression Studies

Activated macrophages require L-arginine uptake to sustain NO synthesis. Several transport systems could mediate this L-arginine influx. Using competition analysis and gene-expression studies, amino acid transport system y+ was identified as the major carrier responsible for this activity. To identify which of the four known y+ transport-system genes is involved in macrophage-induced L-arginine uptake, we used a hybrid-depletion study in Xenopus oocytes. Cationic amino acid transporter (CAT) 2 antisense oligodeoxyribonucleotides abolished the activated-macrophage-mRNA-induced L-arginine transport. Together with expression studies documenting that CAT2 mRNA and protein levels are elevated with increased L-arginine uptake, our data demonstrate that CAT2 mediates the L-arginine transport that is required for the raised NO production in activated J774 macrophages.

Classical isoforms of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells

2003 ◽  
Vol 284 (6) ◽  
pp. L1037-L1044 ◽  
Author(s):  
Karina Y. Krotova ◽  
Sergey I. Zharikov ◽  
Edward R. Block
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Inhibitory Effect ◽  
Term Treatment ◽  
Transport Activity ◽  
Cationic Amino Acid ◽  
Arginine Transport ◽  
Arginine Uptake ◽  
Pkc Isoforms ◽  
Long Term Treatment

We examined which isoforms of protein kinase C (PKC) may be involved in the regulation of cationic amino acid transporter-1 (CAT-1) transport activity in cultured pulmonary artery endothelial cells (PAEC). An activator of classical and novel isoforms of PKC, phorbol 12-myristate-13-acetate (PMA; 100 nM), inhibited CAT-1-mediatedl-arginine transport in PAEC after a 1-h treatment and activated l-arginine uptake after an 18-h treatment of cells. These changes in l-arginine transport were not related to the changes in the expression of the CAT-1 transporter. The inhibitory effect of PMA on l-arginine transport was accompanied by a translocation of PKCα (a classical PKC isoform) from the cytosol to the membrane fraction, whereas the activating effect of PMA on l-arginine transport was accompanied by full depletion of the expression of PKCα in PAEC. A selective activator of Ca2+-dependent classical isoforms of PKC, thymeleatoxin (Thy; 100 nM; 1-h and 18-h treatments), induced the same changes inl-arginine uptake and PKCα translocation and depletion as PMA. The effects of PMA and Thy on l-arginine transport in PAEC were attenuated by a selective inhibitor of classical PKC isoforms Go 6976 (1 μM). Phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl (PIP; 5 μM), which activates novel PKC isoforms, did not affectl-arginine transport in PAEC after 1-h and 18-h treatment of cells. PIP (5 μM; 1 h) induced the translocation of PKCε (a novel PKC isoform) from the cytosolic to the particulate fraction and did not affect the translocation of PKCα. These results demonstrate that classical isoforms of PKC are involved in the regulation of CAT-1 transport activity in PAEC. We suggest that translocation of PKCα to the plasma membrane induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKCα after long-term treatment with PMA or Thy promotes dephosphorylation of the CAT-1 transporter and activation of its activity.

scholarly journals Increased l-arginine uptake and inducible nitric oxide synthase activity in aortas of rats with heart failure

2001 ◽  
Vol 280 (2) ◽  
pp. H859-H867 ◽  
Author(s):  
Peter B. Stathopulos ◽  
Xiangru Lu ◽  
Ji Shen ◽  
Jeremy A. Scott ◽  
James R. Hammond ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Heart Failure ◽  
Amino Acid ◽  
Nitric Oxide Synthase ◽  
No Production ◽  
Cationic Amino Acid ◽  
Arginine Transport ◽  
Arginine Uptake ◽  
Oxide Synthase

l-Arginine crosses the cell membrane primarily through the system y+ transporter. The aim of this study was to investigate the role of l-arginine transport in nitric oxide (NO) production in aortas of rats with heart failure induced by myocardial infarction. Tumor necrosis factor-α levels in aortas of rats with heart failure were six times higher than in sham rats ( P < 0.01). l-Arginine uptake was increased in aortas of rats with heart failure compared with sham rats ( P < 0.01). Cationic amino acid transporter-2B and inducible (i) nitric oxide synthase (NOS) expression were increased in aortas of rats with heart failure compared with sham rats ( P < 0.05). Aortic strips from rats with heart failure treated with l-arginine but not d-arginine increased NO production ( P < 0.05). The effect ofl-arginine on NO production was blocked byl-lysine, a basic amino acid that shares the same system y+ transporter withl-arginine, and by the NOS inhibitor N G-nitro-l-arginine methyl ester (l-NAME). Treatment with l-lysine andl-NAME in vivo decreased plasma nitrate and nitrite levels in rats with heart failure ( P < 0.05). Our data demonstrate that NO production is dependent on iNOS activity andl-arginine uptake and suggest that l-arginine transport plays an important role in enhanced NO production in heart failure.

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