Induction of L‐arginine transport and nitric oxide synthase in vascular smooth muscle cells: synergistic actions of pro‐inflammatory cytokines and bacterial lipopolysaccharide

Abstract

1 The interactions between pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) on Larginine transporter and inducible nitric oxide synthase (iNOS) activities were examined in rat cultured aortic smooth muscle cells. 2 LPS induced a concentration (0.01-100 4g ml-') and time (8-24 h)-dependent stimulation of nitrite production which was accompanied by a parallel increase in L-arginine transport.3 Unlike LPS, activation of smooth muscle cells with either interferon-y (IFN-y, 100 u ml-'), tumour necrosis factor-a (TNF-a, 300 u ml-') or interleukin-la (IL-la, 100 u ml-') failed to stimulate L-arginine transport or increase nitrite accumulation.4 When applied in combination with LPS (100 pg ml-') both IFN-y and TNF-a, but not IL-la, enhanced the effects observed with LPS alone. Furthermore, activation of cells with LPS and IFN-y had no effect on uptake of the neutral amino acid L-citrulline but selectively increased the Vm,,, for L-arginine transport 2.8 fold and nitrite levels from 24+7 to 188+14 pmol ug-' protein 24 h-'. 5 The substrate specificity, Na+ and pH-independence of saturable L-arginine transport in both unactivated (Km= 44 jM, Vma. = 3 pmol pg' protein min') and activated (Km = 75 gM, V,, = 8.3 pmol pg-' protein min'-) smooth muscle cells were characteristic of the cationic amino acid transport system y+.6 Cycloheximide (1 pM) abolished induction of L-arginine transport and nitrite accumulation in response to LPS and IFN-y. In contrast, the glucocorticoid dexamethasone (10 pM, 24 h) selectively inhibited nitrite production. 7 Our results demonstrate that pro-inflammatory mediators selectively enhance transport of L-arginine under conditions of sustained NO synthesis by vascular smooth muscle cells. In addition, the differential inhibition of iNOS and L-arginine transporter activity by dexamethasone suggests that distinct signalling pathways mediate induction of the cationic transport protein and iNOS. The close coupling between substrate supply and NO production may have important implications in the pathogenesis of several disease states including endotoxin shock.

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

“…-Arginine is predominantly transported across cell membranes via a specific sodium-independent transport system selective for cationic amino acids and sensitive to trans-stimulation, thus referred to as system y + [12]. The specificity and characteristics of -arginine transport in RASMCs used in our study strongly support the existence of carrier system(s) resembling system y + [1]. At present, three related carriers that mediate the transport of cationic amino acids similar to system y + (K m : 0.1-0.25 mM) have been identified and referred to as cationic amino acid transporter-1 (CAT-1), CAT-2B and CAT-3 [13][14][15][16][17][18][19].…”

“…Induction of both processes is blocked by cycloheximide and therefore dependent on de no o protein synthesis. Expression of iNOS is attenuated selectively by dexamethasone, suggesting that the signalling events associated with regulation of iNOS expression may, at least in part, be distinct from those associated with upregulation of -arginine transport [1,2].…”

“…We have previously demonstrated that activation of rat cultured aortic smooth muscle cells (RASMCs) with pro-inflammatory mediators results in expression of inducible nitric oxide synthase (iNOS) and upregulation in transport of -arginine [1]. Induction of both processes is blocked by cycloheximide and therefore dependent on de no o protein synthesis.…”

“…Initial studies suggested that activation of PKC may be the key signal transduction pathway in several cell types including rat hepatocytes Abbreviations used : CAT, cationic amino acid transporter ; COX, cyclo-oxygenase ; DMEM, Dulbecco's modified Eagle's medium ; IFN-γ, interferon-γ ; IL-1β, interleukin-1β ; iNOS, inducible nitric oxide synthase ; JNK, c-Jun N-terminal kinase ; LPS, bacterial lipopolysaccharide ; MAPK, mitogenactivated protein kinase ; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide ; NOS, NO synthase ; PKC, protein kinase C ; PTK, protein tyrosine kinase ; RASMC, rat aortic smooth muscle cell ; mCAT, mouse CAT ; rCAT, rat CAT ; RT, reverse transcriptase. 1 To whom correspondence should be addressed (e-mail a.baydoun!herts.ac.uk). 2 Present address : Health Services Research Unit, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, Scotland, U.K.…”

Nitric oxide regulates nucleoside transport in activated B lymphocytes

“…15 for review], such as macrophages [16,17], muscle and endothelial cells [18][19][20][21], or T lymphocytes [22], a variety of agents, including LPS and TNF-␣, trigger NO synthesis and y ϩ transport activity. This transport system is involved in arginine uptake, and thus supplies substrate to nitric oxide synthase (NOS) [15].…”