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-gamma. 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 Vmax. of 15 +/- 2 nmol/h per 10(6) cells. LPS (1 microgram/ml) induced a time-dependent stimulation of L-arginine transport, and after 24 h the Vmax. increased to 34 +/- 2 nmol/h per 10(6) 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 microM 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.

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 Giproteins, 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 LPA1 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 LPA1 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.Disclosure of potential conflicts of interest is found at the end of this article.

The aim of this study was to investigate the role of nitric oxide (NO) in a cellular model of early preconditioning (PC) in cultured neonatal rat ventricular myocytes. Cardiomyocytes “preconditioned” with 90 min of stimulated ischemia (SI) followed by 30 min reoxygenation in normal culture conditions were protected against subsequent 6 h of SI. PC was blocked by N^G-monomethyl-l-arginine monoacetate but not by dexamethasone pretreatment. Inducible nitric oxide synthase (NOS) protein expression was not detected during PC ischemia. Pretreatment (90 min) with the NO donor S-nitroso- N-acetyl-l,l-penicillamine (SNAP) mimicked PC, resulting in significant protection. SNAP-triggered protection was completely abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but was unaffected by chelerythrine or the presence of glibenclamide and 5-hydroxydecanoate. With the use of RIA, SNAP treatment increased cGMP levels, which were blocked by ODQ. Hence, NO is implicated as a trigger in this model of early PC via activation of a constitutive NOS isoform. After exposure to SNAP, the mechanism of cardioprotection is cGMP dependent but independent of protein kinase C or ATP-sensitive K⁺channels. This differs from the proposed mechanism of NO-induced cardioprotection in late PC.

The kinetics, specificity, pH‐ and Na⁺‐dependency of l‐citrulline transport were examined in unstimulated and lipopolysaccharide (LPS)‐activated murine macrophage J774 cells. The dependency of nitric oxide production on extracellular arginine or citrulline was investigated in cells activated with LPS (1 μg ml⁻¹) for 24 h. In unstimulated J774 cells, transport of citrulline was saturable (K_t = 0.16 mm and V_max = 32 pmol μg⁻¹ protein min⁻¹), pH‐insensitive and partially Na⁺‐dependent. In contrast to arginine, transport of citrulline was unchanged in LPS‐activated (1 μg ml⁻¹, 24 h) cells. Kinetic inhibition experiments revealed that arginine was a relatively poor inhibitor of citrulline transport, whilst citrulline was a more potent inhibitor (K_i = 3.4 mm) of arginine transport but only in the presence of extracellular Na⁺. Neutral amino acids inhibited citrulline transport (K_i = 0.2–0.3 mm), but were poor inhibitors of arginine transport. Activated J774 cells did not release nitrite in the absence of exogenous arginine. Addition of citrulline (0.01 − 10 mm), in the absence of exogenous arginine, could only partially restore the ability of cells to synthesize nitrite, which was abolished by 100 μm N^G‐nitro‐l‐arginine methyl ester or N^G‐iminoethyl‐l‐ornithine. Intracellular metabolism of l‐[¹⁴C]‐citrulline to l‐[¹⁴C]‐arginine was detected in unstimulated J774 cells and was increased further in cells activated with LPS and interferon‐γ. We conclude that J774 macrophage cells transport citrulline via a saturable but nonselective neutral carrier which is insensitive to induction by LPS. In contrast, transport of arginine via the cationic amino acid system y⁺ is induced in J774 cells activated with LPS. Our findings also confirm that citrulline can be recycled to arginine in activated J774 macrophage cells. Although this pathway provides a mechanism for enhanced arginine generation required for NO production under conditions of limited arginine availability, it cannot sustain maximal rates of NO synthesis.