Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Bacterial lipopolysaccharide (LPS) induced a dose-and time-dependent stimulation of nitrite production, which was further increased in the presence of interferon-y. Nitrite synthesis was absolutely dependent on extracellular L-arginine and inhibited in the presence of L-lysine or L-ornithine. In unactivated J774 cells L-arginine transport was saturable, with an apparent Km of 0. 14 + 0.04 mm and Vma. of 15 + 2 nmol/h per 106 cells. LPS (1 ,ug/ml) induced a time-dependent stimulation of L-arginine transport, and after 24 h the Vm.. increased to 34 + 2 nmol/h per 106 cells. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of NO.

Effects of a nitroxybutylester derivative of aspirin (NCX 4215) on platelet aggregation and prostanoid synthesis were compared to the effects of aspirin. NCX 4215 was approximately seven times more potent than aspirin as an inhibitor of thrombin-induced human platelet aggregation in vitro, but did not inhibit platelet thromboxane synthesis or gastric prostaglandin synthesis. NCX 4215 released nitric oxide when incubated in the presence of platelets and increased platelet levels of cGMP within 10 min of exposure, while aspirin did not. The anti-aggregatory effects of NCX 4215 in vitro were significantly attenuated by 10 juM hemoglobin.In ex vivo studies of ADP-or collagen-or thrombin-induced rat platelet aggregation, aspirin and NCX 4215 had comparable inhibitory effects 3 h after administration. Aspirin (10-120 mg/kg) caused extensive hemorrhagic erosion formation in the stomach of the rat within 3 h of oral administration, while NCX 4215 did not produce significant damage at doses of up to 300 mg/kg, nor when given daily for two weeks at 166 mg/kg. NCX 4215 did not alter systemic arterial blood pressure when administered intravenously to the rat. These studies demonstrate that NCX 4215 has comparable or enhanced anti-thrombotic activity to that of aspirin, but does not cause gastric damage or alter systemic blood pressure. The anti-thrombotic actions of NCX 4215 are, at least in part, due to generation of nitric oxide. (J. Clin. Invest. 1995. 96:2711-2718

Bone marrow-derived mesenchymal stem cells (MSCs) have shown great promise for cardiac repair. However, poor viability of transplanted MSCs within the ischemic heart has limited their therapeutic potential. Our previous studies have documented that hypoxia and serum deprivation (hypoxia/SD), induced MSCs apoptosis through the mitochondrial apoptotic pathway. Since serum lysophosphatidic acid (LPA) levels are known to be significantly elevated after acute myocardial infarction and that LPA enhanced survival of other cell systems, we embarked on determining whether LPA protects MSCs against hypoxia/SD-induced apoptosis. We have also investigated the potential mechanism(s) that may mediate such actions of LPA. All experiments were carried out on rat bone marrow MSCs. Apoptosis was induced by exposure of cells to hypoxia/SD in a sealed GENbox hypoxic chamber. Effects of LPA were investigated in the absence and presence of inhibitors that target either G i proteins, the mitogen activated protein kinases ERK1/2, or phosphoinositide 3-kinase (PI3K). The data obtained showed that hypoxia/SD-induced apoptosis was significantly attenuated by LPA through Gi-coupled LPA 1 receptors linked to the downstream ERK1/2 and PI3K/Akt signaling pathways that function in parallel. Additional studies have demonstrated that hypoxia/SD-induced activation of mitochondrial dysfunction was virtually abolished by LPA treatment and that inhibition of the LPA 1 receptor, Gi proteins, the PI3K/Akt pathway, or ERKs effectively reversed this protective action of LPA. Taken together, our findings indicate that LPA is a novel, potent survival factor for MSCs and this may prove to be of considerable therapeutic significance in terms of exploiting MSC-based therapy in the infracted myocardium. STEM CELLS 2008; 26:135-145 Disclosure of potential conflicts of interest is found at the end of this article.

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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.