Association of l-arginine transporters with fodrin: implications for hypoxic inhibition of arginine uptake

2000 ◽  
Vol 278 (1) ◽  
pp. L111-L117 ◽  
Author(s):  
S. I. Zharikov ◽  
E. R. Block
Keyword(s):  
Plasma Membranes ◽  
Calpain Inhibitor ◽  
Cationic Amino Acid Transporter ◽  
Transport Activity ◽  
Cationic Amino Acid ◽  
Arginine Transport ◽  
Arginine Uptake ◽  
Hydrolysis Of ◽  
Expression And Activity

In this study, we investigated the possible interaction between the cationic amino acid transporter (CAT)-1 arginine transporter and ankyrin or fodrin. Because ankyrin and fodrin are substrates for calpain and because hypoxia increases calpain expression and activity in pulmonary artery endothelial cells (PAEC), we also studied the effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC. Exposure to long-term hypoxia (24 h) inhibited l-arginine uptake by PAEC, and this inhibition was prevented by calpain inhibitor 1. The effects of hypoxia and calpain inhibitor 1 were not associated with changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in PAEC, and this could be prevented by calpain inhibitor 1. Incubation of solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60% decrease in l-arginine transport activity in reconstituted proteoliposomes (3,291 ± 117 vs. 8,101 ± 481 pmol ⋅ mg protein−1 ⋅ 3 min−1 in control). Incubation with anti-ankyrin antibodies had no effect on CAT-1 content or l-arginine transport in reconstituted proteoliposomes. These results demonstrate that CAT-1 arginine transporters in PAEC are associated with fodrin, but not with ankyrin, and that long-term hypoxia decreases l-arginine transport by a calpain-mediated mechanism that may involve fodrin proteolysis.

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.

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 l-Arginine and Cationic Amino Acid Transporter 2B Regulate Growth and Survival of Leishmania amazonensis Amastigotes in Macrophages

2007 ◽  
Vol 75 (6) ◽  
pp. 2802-2810 ◽  
Author(s):  
Nanchaya Wanasen ◽  
Carol L. MacLeod ◽  
Lesley G. Ellies ◽  
Lynn Soong
Keyword(s):  
Amino Acid ◽  
Amino Acid Transporter ◽  
Parasite Growth ◽  
Leishmania Amazonensis ◽  
Growth Enhancement ◽  
Cationic Amino Acid Transporter ◽  
Cationic Amino Acid ◽  
Arginine Transport ◽  
Ifn Γ ◽  
Acid Transporter

ABSTRACT Leishmania spp. are obligate intracellular parasites, requiring a suitable microenvironment for their growth within host cells. We previously reported that the growth of Leishmania amazonensis amastigotes in murine macrophages (Mφs) was enhanced in the presence of gamma interferon (IFN-γ), a Th1 cytokine normally associated with classical Mφ activation and killing of intracellular pathogens. In this study, we provided several lines of evidence suggesting that IFN-γ-mediated parasite growth enhancement was associated with l-arginine transport via mouse cationic amino acid transporter 2B (mCAT-2B). (i) mRNA expression of Slc7A2, the gene encoding for mCAT-2B, as well as l-arginine transport was increased in IFN-γ-treated Mφs. (ii) Supplementation of l-arginine in Mφ cultures increased parasite growth. (iii) Parasite growth enhancement in wild-type Mφs was inhibited in the presence of nonmetabolized l-arginine analogues. (iv) IFN-γ-mediated parasite growth was absent in Mφs derived from mCAT-2B-deficient mice. Although we detected a clear upregulation of mCAT-2B and l-arginine transport, no measurable iNOS or arginase activities were observed in IFN-γ-treated, infected Mφs. Together, these data suggest an involvement of a novel l-arginine usage independent of iNOS and arginase activities during IFN-γ-mediated parasite growth enhancement. A possible role of mCAT-2B in supplying l-arginine directly to the parasites for their proliferation is discussed.

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.

scholarly journals Arginine uptake is attenuated through modulation of cationic amino-acid transporter-1, in uremic rats

2006 ◽  
Vol 69 (2) ◽  
pp. 298-303 ◽  
Author(s):  
I.F. Schwartz ◽  
R. Ayalon ◽  
T. Chernichovski ◽  
R. Reshef ◽  
G. Chernin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Transporter ◽  
Cationic Amino Acid Transporter ◽  
Cationic Amino Acid ◽  
Arginine Uptake ◽  
Acid Transporter

Differential regulation of glomerular arginine transporters (CAT-1 and CAT-2) in lipopolysaccharide-treated rats

2003 ◽  
Vol 284 (4) ◽  
pp. F788-F795 ◽  
Author(s):  
Doron Schwartz ◽  
Idit F. Schwartz ◽  
Ehud Gnessin ◽  
Yoram Wollman ◽  
Tamara Chernichovsky ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mesangial Cells ◽  
Primary Cultures ◽  
Northern Blotting ◽  
Cationic Amino Acid ◽  
Arginine Transport ◽  
Arginine Uptake ◽  
Endothelial Nitric Oxide ◽  
In Vivo Administration

The decrease in glomerular filtration rate (GFR) that is characteristic of sepsis has been shown to result from inhibition of glomerular endothelial nitric oxide synthase (eNOS) by nitric oxide (NO) generated from the inducible isoform of NOS (iNOS). Although l-arginine is the sole precursor for NO biosynthesis, its intracellular availability in glomeruli from septic animals has never been investigated. Arginine uptake was measured in freshly harvested glomeruli from the following experimental groups: 1) untreated rats; 2) rats pretreated with LPS (4 mg/kg body wt, 4 h before experiments); 3) rats treated with LPS as above with eitherl- N 6-(1-iminoethyl)lysine hydrochloride (l-NIL), a selective iNOS antagonist, or 7-nitroindazole, a selective neuronal NOS antagonist; and 4) rats treated with l-NIL only. Both glomeular and mesangial arginine transport characteristics were found compatible with a y+ system. Arginine uptake was augmented in glomeruli from LPS-treated rats. Treatment with l-NIL completely abolished this effect whereas l-NIL alone had no effect. Similar results were obtained when primary cultures of rat mesangial cells were preincubated with LPS (10 μg/ml for 24 h) with or withoutl-NIL. Using RT-PCR, we found that in vivo administration of LPS resulted in a significant increase in glomerular cationic amino acid transporter-2 (CAT-2) mRNA expression whereas CAT-1 mRNA was undetected. Northern blotting further confirmed a significant increase in glomerular CAT-2 by LPS. In mesangial cells, the expression of both CAT-1 and CAT-2 mRNA was augmented after incubation with LPS. In conclusion, in vivo administration of LPS augments glomerular arginine transport through upregulation of steady-state CAT-2 mRNA while downregulating CAT-1 mRNA. These results may correspond to the changes in glomerular iNOS and eNOS activity in sepsis.

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