Cationic amino acid transporters and their modulation by nitric oxide in cardiac muscle cells

Nitric oxide (NO) plays a central role as a cellular signaling molecule in health and disease. In the heart, NO decreases the rate of spontaneous beating and the velocity and extent of shortening and accelerates the velocity of relengthening. Since the cationic amino acid l-arginine (l-Arg) is the substrate for NO production by NO synthases (NOS), we tested whether the transporters that mediate l-Arg import in cardiac muscle cells represent an intervention point in the regulation of NO synthesis. Electrical currents activated by l-Arg with low apparent affinity in whole cell voltage-clamped rat cardiomyocytes were found to be rapidly and reversibly inhibited by NO donors. Radiotracer uptake studies performed on cardiac sarcolemmal vesicles revealed the presence of high-affinity/low-capacity and low-affinity/high-capacity components of cationic amino acid transport that were inhibited by the NO donor S-nitroso- N-acetyl-dl-penicillamine. NO inhibited uptake in a noncompetitive manner with Ki values of 275 and 827 nM for the high- and low-affinity component, respectively. Fluorescence spectroscopy experiments showed that millimolar concentrations of l-Arg initially promoted and then inhibited the release of endogenous NO in cardiomyocytes. Likewise, l-Arg currents measured in cardiac myocytes voltage clamped in the presence of 460 nM free intracellular Ca2+, a condition in which a Ca-CaM complex should activate endogenous NO production, showed fast activation followed by inhibition of l-Arg transport. The NOS inhibitor N-nitro-l-arginine methyl ester, but not blockers of downstream reactions, specifically removed this inhibitory component. These results demonstrate that NO acutely regulates its own biosynthesis by modulating the availability of l-Arg via cationic amino acid transporters.

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L-Lysine uptake in giant vesicles from cardiac ventricular sarcolemma: two components of cationic amino acid transport

Bioscience Reports ◽

10.1042/bsr20080159 ◽

2009 ◽

Vol 29 (4) ◽

pp. 271-281 ◽

Cited By ~ 7

Author(s):

Xiaodong Lu ◽

Ruifang Zheng ◽

Jorge Gonzalez ◽

Lawrence Gaspers ◽

Eldo Kuzhikandathil ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cardiac Muscle ◽

Muscle Cells ◽

Significant Activity ◽

Cardiac Muscle Cells ◽

Membrane Hyperpolarization ◽

Apparent Affinity ◽

Cationic Amino Acid ◽

Vesicle Protein

Cationic L-amino acids enter cardiac-muscle cells through carrier-mediated transport. To study this process in detail, L-[14C]lysine uptake experiments were conducted within a 103-fold range of L-lysine concentrations in giant sarcolemmal vesicles prepared from rat cardiac ventricles. Vesicles had a surface-to-volume ratio comparable with that of an epithelial cell, thus representing a suitable system for initial uptake rate studies. Two Na+-independent, N-ethylmaleimide-sensitive uptake components were found, one with high apparent affinity (Km=222±71 μM) and low transport capacity (Vmax=121±36 pmol/min per mg of vesicle protein) and the other with low apparent affinity (Km=16±4 mM) and high capacity (Vmax=4.0±0.4 nmol/min per mg of vesicle protein). L-Lysine uptake mediated by both components was stimulated by the presence of intravesicular L-lysine as well as by valinomycin-induced membrane hyperpolarization. Altogether, this behaviour is consistent with the functional properties of the CAT-1 and CAT-2A members of the system y+ family of cationic amino acid transporters. Furthermore, mRNA transcripts for these two carrier proteins were identified in freshly isolated rat cardiac myocytes, the amount of CAT-1 mRNA, relative to β-actin, being 33-fold larger than that of CAT-2A. These two transporters appear to function simultaneously as a homoeostatic device that supplies cardiac-muscle cells with cationic amino acids under a variety of metabolic conditions. Analysis of two carriers acting in parallel with such an array of kinetic parameters shows significant activity of the low-affinity component even at amino acid plasma levels far below its Km.

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Characterization of cationic amino acid transporters and expression of endothelial nitric oxide synthase in human placental microvascular endothelial cells

The FASEB Journal ◽

10.1096/fj.02-0916fje ◽

2003 ◽

Vol 18 (1) ◽

pp. 125-127 ◽

Cited By ~ 41

Author(s):

Julian F. Dye ◽

Sarah Vause ◽

Tracey Johnston ◽

Peter Clark ◽

J. Anthony Firth ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Amino Acid ◽

Endothelial Nitric Oxide Synthase ◽

Microvascular Endothelial Cells ◽

Amino Acid Transporters ◽

Cationic Amino Acid ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

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Role of cationic amino acid transporters in the regulation of nitric oxide synthesis in vascular cells

10.36866/pn.52.12 ◽

2003 ◽

pp. 12-15 ◽

Cited By ~ 1

Author(s):

Anwar Baydoun ◽

Giovanni Mann

Keyword(s):

Nitric Oxide ◽

Amino Acid ◽

Amino Acid Transporters ◽

Vascular Cells ◽

Nitric Oxide Synthesis ◽

Cationic Amino Acid

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Expression Profile of Cationic Amino Acid Transporters in Rats with Endotoxin-Induced Uveitis

Mediators of Inflammation ◽

10.1155/2016/6586857 ◽

2016 ◽

Vol 2016 ◽

pp. 1-14 ◽

Cited By ~ 2

Author(s):

Yung-Ray Hsu ◽

Shu-Wen Chang ◽

Chang-Hao Yang ◽

Yi-An Lee ◽

Tzu-Yun Kao

Keyword(s):

Nitric Oxide ◽

Amino Acid ◽

Histopathological Examination ◽

Raw 264.7 Cells ◽

Amino Acid Transporters ◽

Raw 264.7 ◽

Mobility Shift ◽

Cationic Amino Acid ◽

Cat Isoforms

Purpose. The transcellular arginine transportation via cationic amino acid transporter (CAT) is the rate-limiting step in nitric oxide (NO) synthesis, which is crucial in intraocular inflammation. In this study, CAT isoforms and inducible nitric oxide synthase (iNOS) expression was investigated in endotoxin-induced uveitis (EIU).Methods.EIU was induced in Lewis rats by lipopolysaccharide (LPS) injection. In the treatment group, the rats were injected intraperitoneally with the proteasome inhibitor bortezomib before EIU induction. After 24 hours, leukocyte quantification, NO measurement of the aqueous humor, and histopathological examination were evaluated. The expression of CAT isoforms and iNOS was determined by reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence staining. Nuclear factor-kappa B (NF-κB) binding activity was evaluated by electrophoretic mobility shift assay. The mouse macrophage cell line RAW 264.7 was used to validate thein vivofindings.Results. LPS significantly stimulated iNOS, CAT-2A, and CAT-2B mRNA and protein expression but did not affect CAT-1 in EIU rats and RAW 264.7 cells. Bortezomib attenuated inflammation and inhibited iNOS, CAT-2A, and CAT-2B expression through NF-κB inhibition.Conclusions.CAT-2 and iNOS, but not CAT-1, are specifically involved in EIU. NF-κB is essential in the induction of CAT-2 and iNOS in EIU.

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Transmembrane signalling mechanisms regulating expression of cationic amino acid transporters and inducible nitric oxide synthase in rat vascular smooth muscle cells

Biochemical Journal ◽

10.1042/bj3440265 ◽

1999 ◽

Vol 344 (1) ◽

pp. 265-272 ◽

Cited By ~ 19

Author(s):

Anwar R. BAYDOUN ◽

Samantha M. WILEMAN ◽

Caroline P. D. WHEELER-JONES ◽

Michael S. MARBER ◽

Giovanni E. MANN ◽

...

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Amino Acid ◽

Nitric Oxide Synthase ◽

P38 Mapk ◽

Amino Acid Transporters ◽

Cationic Amino Acid ◽

Arginine Transport ◽

Ifn Γ ◽

Oxide Synthase

The signalling mechanisms involved in the induction of nitric oxide synthase and L-arginine transport were investigated in bacterial lipopolysaccharide (LPS)- and interferon-γ (IFN-γ)-stimulated rat cultured aortic smooth muscle cells (RASMCs). The expression profile of transcripts for cationic amino acid transporters (CATs) and their regulation by LPS and IFN-γ were also examined. Control RASMCs expressed mRNA for CAT-1, CAT-2A and CAT-2B. Levels of all three transcripts were significantly elevated in activated cells. Stimulated CAT mRNA expression and L-arginine transport occurred independently of protein kinase C (PKC), protein tyrosine kinase (PTK) and p44/42 mitogen-activated kinases (MAPKs), but were inhibited by the p38 MAPK inhibitor SB203580, which at 3 μM caused maximum inhibition of both responses. Induction of NO synthesis was independent of p44/42 MAPK activation and only marginally dependent on PKC, but was attenuated markedly by the PTK inhibitors genistein and herbimycin A. SB203580 differentially regulated inducible NO synthase expression and NO production, potentiating both processes at low micromolar concentrations and inhibiting at concentrations of ⩾ 1 μM. In conclusion, our results suggest that RASMCs constitutively express transcripts for CAT-1, CAT-2A and CAT-2B, and that expression of these transcripts is significantly enhanced by LPS and IFN-γ. Moreover, stimulation of L-arginine transport and induction of NO synthesis by LPS and IFN-γ appear to be under critical regulation by the p38 MAPK, since both processes were significantly modified by SB203580 at concentrations so far shown to have no effect on other signalling pathways. Thus, in RASMCs, the p38 MAPK cascade represents an important signalling mechanism, regulating both enhanced L-arginine transport and induced NO synthesis.

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