Anwar R. Baydoun

Anti-thrombotic effects of a nitric oxide-releasing, gastric-sparing aspirin derivative.

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.

Substrate-dependent regulation of intracellular amino acid concentrations in cultured bovine aortic endothelial cells

Amino acid deprivation induces adaptive changes in amino acid transport and the intracellular amino acid pool in cultured cells. In this study intracellular amino acid levels were determined in cultured bovine aortic endothelial cells (EC) deprived of L-arginine or total amino acids for 1, 3, 6 and 24 h. Amino acid concentrations were analyzed by reverse phase HPLC after precolumn derivatisation. Under normal culture conditions levels of L-arginine L-citrulline, total essential and non-essential amino acids were 840 +/- 90 microM, 150 +/- 40 microM, 11.4 +/- 0.9 mM and 53.3 +/- 3.4 mM (n = 9), respectively. In EC deprived of L-arginine or all amino acids for 24 h L-arginine and L-citrulline levels were 200 microM and 50 microM, and 670 microM and 100 microM Deprivation of L-arginine or total amino acids induced rapid (1 h) decreases (30 - 50%) in the levels of other cationic (lysine, ornithine) and essential branched-chain (valine, isoleucine, leucine) and aromatic (phenylalanine, tryptophan) amino acids. L-glutamine was reduced markedly in EC deprived of total amino acids for 1 h - 6 h but actually increased 3-fold in EC deprived of L-arginine for 6 h or 24 h. Arginine deprivation resulted in a rapid decrease in the total intracellular amino acid pool, however concentrations were restored after 24 h. Increased amino acid transport and/or reduced protein synthesis may account for the restoration of amino acid levels in EC deprived of L-arginine. The sustained reduction in the free amino acid pool of EC deprived of all amino acids may reflect utilization of intracellular amino acids for protein synthesis.

Lysophosphatidic acid protects mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis.

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.

Regulation of L-arginine transport and nitric oxide release in superfused porcine aortic endothelial cells.

1. We have investigated whether changes in extracellular ion composition and substrate deprivation modulate basal and/or bradykinin‐stimulated L‐arginine transport and release of nitric oxide (NO) and prostacyclin (PGI2) in porcine aortic endothelial cells cultured and superfused on microcarriers. 2. Saturable L‐arginine transport (Km = 0.14 +/‐ 0.03 mM; Vmax = 2.08 +/‐ 0.54 nmol min‐1 (5 x 10(6) cells)‐1) was pH insensitive and unaffected following removal of extracellular Na+ or Ca2+. 3. Cationic arginine analogues, including L‐lysine and L‐ornithine, inhibited L‐arginine transport, whilst 2‐methylaminoisobutyric acid, beta‐2‐amino‐bicyclo[2,2.1]‐heptane‐2‐carboxylic acid, L‐phenylalanine, 6‐diazo‐5‐oxo‐norleucine, L‐glutamine, L‐cysteine and L‐glutamate were poor inhibitors. 4. Deprivation of L‐arginine (30 min to 24 h) reduced intracellular free L‐arginine levels from 0.87 +/‐ 0.07 to 0.40 +/‐ 0.05 mM (P < 0.05) and resulted in a 40% stimulation of L‐arginine, L‐lysine and L‐ornithine transport. 5. L‐arginine and NG‐monomethyl‐L‐arginine (L‐NMMA), but not N omega‐nitro‐L‐arginine methyl ester (L‐NAME), trans‐stimulated efflux of L‐[3H]arginine. 6. Depolarization of endothelial cells with 70 mM K+ reduced L‐arginine influx and prevented the stimulation of transport by 100 nM bradykinin, but agonist‐induced release of NO and PGI2 was still detectable. 7. Basal rates of L‐arginine transport and NO release were unaffected during superfusion of cells with a nominally Ca(2+)‐free solution. Bradykinin‐stimulated L‐arginine transport was insensitive to removal of Ca2+, whereas agonist‐induced NO release was abolished. 8. Although bradykinin‐stimulated NO release does not appear to be coupled directly to the transient increase in L‐arginine transport, elevated rates of L‐arginine influx via system y+ in response to agonist‐induced membrane hyperpolarization or substrate deprivation provide a mechanism for enhanced L‐arginine supply to sustain NO generation.

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

1 The interactions between pro‐inflammatory cytokines and bacterial lipopolysaccharide (LPS) on L‐arginine 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 μg ml−1) 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‐γ (IFN‐γ, 100 u ml−1), tumour necrosis factor‐α (TNF‐a, 300 u ml−1) or interleukin‐1α (IL‐1α, 100 u ml−1) failed to stimulate L‐arginine transport or increase nitrite accumulation. 4 When applied in combination with LPS (100 μg ml−1) both IFN‐γ and TNF‐α, but not IL‐1α, enhanced the effects observed with LPS alone. Furthermore, activation of cells with LPS and IFN‐γ had no effect on uptake of the neutral amino acid L‐citrulline but selectively increased the Vmax for L‐arginine transport 2.8 fold and nitrite levels from 24 ± 7 to 188 ± 14 pmol μg−1 protein 24 h−1. 5 The substrate specificity, Na+ and pH‐independence of saturable L‐arginine transport in both unactivated (Km = 44 μm, Vmax = 3 pmol μ−1 protein min−1) and activated (Km = 75 μm, Vmax = 8.3 pmol μg‐1 protein min−1) smooth muscle cells were characteristic of the cationic amino acid transport system y+. 6 Cycloheximide (1 μm) abolished induction of L‐arginine transport and nitrite accumulation in response to LPS and IFN‐γ. In contrast, the glucocorticoid dexamethasone (10 μm, 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.

Selective inhibition by dexamethasone of induction of NO synthase, but not of induction of L-arginine transport, in activated murine macrophage J774 cells.

1 Effects of dexamethasone on induction of nitric oxide (NO) synathase and l‐arginine transport by lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line J774. Metabolism of l‐arginine to l‐citrulline and subsequent changes in intracellular amino acids pools were correlated with changes in nitrite production. 2 Despite a high intracellular concentration of arginine in activated J774 cells, LPS (1 μg ml−1, 8 h) induced a 2.4 fold increase in arginine transport. Treatment of cells with cycloheximide (1 μg ml−1) inhibited the time‐dependent (1–8 h) induction of NO synthase and arginine transport mediated by LPS. 3 Induction of NO synthase by LPS (1 μg ml−1, 24 h) alone was accompanied by a marked increase in arginine utilisation leading to decreased intracellular arginine levels and elevated intracellular and extracellular l‐citrulline levels. These changes were further enhanced in the presence of interferon‐γ (IFN‐γ, 100 units ml−1, 24 h). 4 Dexamethasone (1 μm) abolished the increases in both nitrite and citrulline production induced by LPS alone but only partially reversed the combined effects of LPS and IFN‐γ. In contrast, treatment of cells with dexamethasone (10 μm) had no effect on the LPS‐mediated induction of arginine transport or the decrease in intracellular arginine concentration. 5 We conclude that induction of arginine transporter activity in LPS‐stimulated J774 cells involves de novo synthesis of carrier proteins, which increases transport of exogenous arginine during enhanced NO production. Moreover, the intracellular signalling pathways mediating induction of arginine transport and of NO synthase by LPS in activated macrophages diverge, since only the latter is sensitive to dexamethasone.

Effects of Endothelin-1 on the Rat Isolated Heart

Summary This study shows that bolus injections of endothelin-1 (ET-1) (1–30 pmol) produce transient vasodilator and prolonged coronary vasoconstrictor actions. The initial effect on cardiac contractility was a positive inotropic action, but with repeated doses a negative inotropic action developed. Verapamil (0.1

Discrimination between citrulline and arginine transport in activated murine macrophages: inefficient synthesis of NO from recycling of citrulline to arginine

mUM) antagonized the vasoconstrictor action of Bay K 8644 but did not affect ET-1-induced vasoconstriction. In contrast, removal of extracellular calcium did block the vasoconstrictor action of ET-1. This suggests that vasoconstriction is due to activation of receptor-rather than potential-operated calcium channels. The ET-1-induced vasoconstriction was not due to the release of platelet-activating factor (PAF) or thromboxane A2 since it was not inhibited by WEB 2086 (0.5

Inhibition of inducible nitric oxide synthase expression by novel nonsteroidal anti‐inflammatory derivatives with gastrointestinalsparing properties

mUM), fluribiprofen (2

Aspirin induces apoptosis in mesenchymal stem cells requiring Wnt/beta-catenin pathway.

mUM), or BW755C (7