The importance of cationic amino acid transporter expression in human skin.
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Inducible nitric oxide synthase and arginase activities are acknowledged as important players in human skin epidermal function. For proper enzyme function the substrate availability of L-arginine for both enzymes and thus its transport across the cell membrane via the y+-system (also named cationic amino acid transporters) is critical. Here, we examine the expression of cationic amino acid transporters and their functional role in modulating inducible nitric oxide synthase and arginase activities in human skin and primary keratinocytes, fibroblasts and endothelial cells as well as their impact on keratinocyte proliferation. Skin biopsies were found to express constitutively both cationic amino acid transporter-1 and cationic amino acid transporter-2 mRNA, an expression pattern known to occur in hepatocytes and muscle cells only. To determine the cellular components expressing cationic amino acid transporter, we analyzed the expression patterns in the different human skin cell types in vitro, i.e., in fibroblasts, dermal endothelial cells, and keratinocytes as well as in the HaCaT cell line. An ubiquitous cationic amino acid transporter-1 mRNA expression was found in all cells, whereas constitutive cationic amino acid transporter-2 mRNA expression occurs in resident keratinocytes and dermal endothelial cells only. De novo induction of cationic amino acid transporter-2 and inducible nitric oxide synthase by proinflammatory cytokines was seen in fibroblasts and HaCaT. Competitive inhibition of the cationic amino acid transporter-mediated L-arginine transport by culturing primary human keratinocytes in the presence of increased L-lysine concentration led to decreased inducible nitric oxide synthase and arginase activities with a concomitant significant decrease in keratinocyte proliferation. In summary, our results demonstrate that human keratinocytes constitutively express cationic amino acid transporters 1 and 2 and that cationic amino acid transporter mediated L-arginine influx, is essential for both inducible nitric oxide synthase and arginase enzyme activities, which in turn modulate proliferation and differentiation of human epidermal skin cells. (+info)
Transcriptional control of the arginine/lysine transporter, cat-1, by physiological stress.
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Cells respond to physiological stress by phosphorylating the alpha subunit of the translation initiation factor eIF2. This adaptive response inhibits protein synthesis and up-regulates genes essential for cell survival. Cat-1, the transporter for the essential amino acids, arginine and lysine, is one of the up-regulated genes. We previously showed that stress increases cat-1 expression by coordinated stabilization of the mRNA and increased mRNA translation. This induction is triggered by amino acid depletion and the unfolded protein response (UPR), which is caused by unfolded proteins in the endoplasmic reticulum. We show here that cat-1 gene transcription is also increased by cellular stress. Our studies demonstrate that the cat-1 gene promoter/regulatory region is TATA-less and is located in a region that includes 94 bases of the first exon. Transcription from this promoter is stimulated 8-fold by cellular stress. An amino acid response element within the first exon is shown to be required for the response to amino acid depletion but not to the UPR. The stimulation of transcription by amino acid depletion requires activation of GCN2 kinase, which phosphorylates eIF2alpha. This phosphorylation also induces translation of the cat-1 mRNA, demonstrating that stress-induced transcriptional and translational control of cat-1 are downstream targets of a signaling pathway initiating with eIF2alpha phosphorylation. Our studies show that the increase in cat-1 gene expression by cellular stress involves at least three types of coordinate regulation: regulation of transcription, regulation of mRNA stability, and regulation of mRNA translation. (+info)
Pertussis toxin activates L-arginine uptake in pulmonary endothelial cells through downregulation of PKC-alpha activity.
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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. (+info)
Intracellular accumulation of L-Arg, kinetics of transport, and potassium leak conductance in oocytes from Xenopus laevis expressing hCAT-1, hCAT-2A, and hCAT-2B.
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Cationic amino acid transporters play an important role in the intracellular supply of L-Arg and the generation of nitric oxide. Since the transport of L-Arg is voltage-dependent, we aimed at determining the intracellular L-Arg concentration and describing the transport of L-Arg in terms of Michaelis-Menten kinetics, taking into account membrane voltage. The human isoforms of the cationic amino acid transporters, hCAT-1, hCAT-2A, and hCAT-2B, were expressed in oocytes from Xenopus laevis and studied with the voltage clamp technique and in tracer experiments. We found that L-Arg was concentrated intracellularly by all hCAT isoforms and that influx and efflux, in the steady state of exchange, were nearly mirror images. Conductance measurements at symmetric concentrations of L-Arg (inside/outside) allowed us to determine KM and Vmax. The empty transporter of hCAT-2B featured an unexpected potassium conductance, which was inhibited by L-Arg. (+info)
Role of adenosine transport in gestational diabetes-induced L-arginine transport and nitric oxide synthesis in human umbilical vein endothelium.
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Gestational diabetes is associated with increased L-arginine transport and nitric oxide (NO) synthesis, and reduced adenosine transport in human umbilical vein endothelial cells (HUVEC). Adenosine increases endothelial L-arginine/NO pathway via A(2) purinoceptors in HUVEC from normal pregnancies. It is unknown whether the effect of gestational diabetes is associated with activation of these purinoceptors or altered expression of human cationic amino acid transporter 1 (hCAT-1) or human equilibrative nucleoside transporter 1 (hENT1), or endothelial NO synthase (eNOS) in HUVEC. Cells were isolated from normal or gestational diabetic pregnancies and cultured up to passage 2. Gestational diabetes increased hCAT-1 mRNA expression (2.4-fold) and activity, eNOS mRNA (2.3-fold), protein level (2.1-fold), and phosphorylation (3.8-fold), but reduced hENT1 mRNA expression (32%) and activity. Gestational diabetes increased extracellular adenosine (2.7 microM), and intracellular L-arginine (1.9 mM) and L-citrulline (0.7 mM) levels compared with normal cells (0.05 microM, 0.89 mM, 0.35 mM, respectively). Incubation of HUVEC from normal pregnancies with 1 microM nitrobenzylthioinosine (NBMPR) mimicked the effect of gestational diabetes, but NBMPR was ineffective in diabetic cells. Gestational diabetes and NBMPR effects involved eNOS, PKC and p42/44(mapk) activation, and were blocked by the A(2a) purinoceptor antagonist ZM-241385. Thus, gestational diabetes increases the L-arginine/NO pathway involving activation of mitogen-activated protein (MAP) kinases, protein kinase C (PKC) and NO cell signalling cascades following activation of A(2a) purinoceptors by extracellular adenosine. A functional relationship is proposed between adenosine transport and modulation of L-arginine transport and NO synthesis in HUVEC, which could be determinant in regulating vascular reactivity in diabetes mellitus. (+info)
Protein kinase C activation promotes the internalization of the human cationic amino acid transporter hCAT-1. A new regulatory mechanism for hCAT-1 activity.
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The human cationic amino acid transporter hCAT-1 is almost ubiquitously expressed and probably the most important entity for supplying cells with extracellular arginine, lysine, and ornithine. We have previously shown that hCAT-1-mediated transport is decreased after protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) (Graf, P., Forstermann, U., and Closs, E. I. (2001) Br. J. Pharmacol. 132, 1193-1200). In the present study, we examined the mechanism of this down-regulation. In both Xenopus laevis oocytes and U373MG glioblastoma cells, PMA treatment promoted the internalization of hCAT-1 (fused to the enhanced green fluorescence protein (EGFP)) as visualized by fluorescence microscopy. Biotinylation of cell surface proteins and subsequent Western blot analyses confirmed that the cell surface expression of hCAT-1.EGFP was significantly reduced upon PMA treatment. Pretreatment with the PKC inhibitor bisindolylmaleimide I prevented the reduction by PMA of both hCAT-1.EGFP-induced arginine transport and the internalization of the transporter. Similar results were obtained with hCAT-1 expressed endogenously in DLD-1 colon carcinoma cells. Inhibition of protein synthesis did not augment the PMA effect. In addition, the PMA effect was reverted in washout experiments without changing the hCAT-1 protein expression, suggesting that the PMA effect is reversible in these cells. PKC did not phosphorylate hCAT-1 directly as evidenced by in vivo phosphorylation experiments and mutational analysis, indicating an indirect action of PKC on hCAT-1. (+info)
Impaired L-arginine transport and endothelial function in hypertensive and genetically predisposed normotensive subjects.
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BACKGROUND: Impaired endothelium-dependent NO-mediated vasodilation is a key feature of essential hypertension and may precede the increase in blood pressure. We investigated whether transport of the NO precursor L-arginine is related to decreased endothelial function. METHODS AND RESULTS: Radiotracer kinetics ([3H]L-arginine) were used to measure forearm and peripheral blood mononuclear cell arginine uptake in hypertensive subjects (n=12) and in 2 groups of healthy volunteers with (n=15) and without (n=15) a family history of hypertension. In conjunction, forearm blood flow responses to acetylcholine and sodium nitroprusside were measured before and after a supplemental intra-arterial infusion of L-arginine. In vivo and in vitro measures of L-arginine transport were substantially reduced in the essential hypertension and positive family history groups compared with the negative family history group; however, no difference was detected in peripheral blood mononuclear cell mRNA or protein expression levels for the cationic amino acid transporter CAT-1. Plasma concentrations of L-arginine and N(G),N(G')-dimethylarginine (ADMA) did not differ between groups. L-arginine supplementation improved the response to acetylcholine only in subjects with essential hypertension and positive family history. CONCLUSIONS: Similar to their hypertensive counterparts, normotensive individuals at high risk for the development of hypertension are characterized by impaired L-arginine transport, which may represent the link between a defective L-arginine/NO pathway and the onset of essential hypertension. The observed transport defect is not due to apparent alterations in CAT-1 expression or elevated endogenous ADMA. (+info)
Dexamethasone suppresses eNOS and CAT-1 and induces oxidative stress in mouse resistance arterioles.
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Long-term treatment with glucocorticoids is associated with mild to moderate hypertension. We reported previously that downregulation of endothelial NO synthase (eNOS) expression and activity is likely to contribute to this increase in blood pressure. In the present study, we tested the effects of dexamethasone on the vasodilation of microvascular arterioles using implanted dorsal skin-fold chambers in anesthetized C57BL/6J mice. Experiments were performed on control mice or on mice treated with dexamethasone (0.1-3 mg/kg of body wt). Endothelium-dependent vasodilation in response to ACh (0.1-10 microM) was reduced by dexamethasone in a dose-dependent fashion. Comparable inhibition was seen in tissues superfused with 30 microM N(G)-nitro-L-arginine methyl ester. In contrast, endothelium-independent vasodilation in response to S-nitroso-N-acetyl-D,L-penicillamine (10 microM) was not influenced by either dexamethasone or N(G)-nitro-L-arginine methyl ester. Levels of eNOS mRNA in murine hearts and NO(2)(-)/NO(3)(-) in serum were suppressed by dexamethasone (down to 63 and 50% of control values, respectively, at 3 mg/kg of body wt) along with a reduction in eNOS protein to 85.6%. Dexamethasone also concentration dependently reduced the expression of the cationic amino acid transporter-1 in murine hearts and cultured endothelial cells. The suppression by dexamethasone of the ACh-induced vasodilation could be partially reversed by dietary L-arginine (50 mg/kg of body wt) and by dietary vitamin C (10 g/kg of diet). We conclude that suppression by dexamethasone of the endothelium-mediated microvascular vasodilation involves several mechanisms including 1) downregulation of eNOS, 2) downregulation of cationic amino acid transporter-1, and 3) generation of reactive oxygen species. The demonstration that L-arginine and vitamin C can partially offset the effects of dexamethasone on microvascular arterioles suggests the potential clinical usefulness of these agents for the reduction of glucocorticoid-induced hypertension. (+info)