Differences of Transport Activity of Arginine and Regulation on Neuronal Nitric Oxide Synthase and Oxidative Stress in Amyotrophic Lateral Sclerosis Model Cell Lines

L-Arginine, a semi-essential amino acid, was shown to delay dysfunction of motor neurons and to prolong the lifespan, upon analysis of transgenic mouse models of amyotrophic lateral sclerosis (ALS). We investigated the transport function of arginine and neuronal nitric oxide synthase (nNOS) expression after pretreatment with L-arginine in NSC-34 hSOD1WT (wild-type, WT) and hSOD1G93A (mutant-type, MT) cell lines. [3H]L-Arginine uptake was concentration-dependent, voltage-sensitive, and sodium-independent in both cell lines. Among the cationic amino acid transporters family, including system y+, b0,+, B0,+, and y+L, system y+ is mainly involved in [3H]L-arginine transport in ALS cell lines. System b0,+ accounted for 23% of the transport in both cell lines. System B0,+ was found only in MT, and whereas, system y+L was found only in WT. Lysine competitively inhibited [3H]L-arginine uptake in both cell lines. The nNOS mRNA expression was significantly lower in MT than in WT. Pretreatment with arginine elevated nNOS mRNA levels in MT. Oxidizing stressor, H2O2, significantly decreased their uptake; however, pretreatment with arginine restored the transport activity in both cell lines. In conclusion, arginine transport is associated with system y+, and neuroprotection by L-arginine may provide an edge as a possible therapeutic target in the treatment of ALS.

Pravastatin induces NO synthesis by enhancing microsomal arginine uptake in healthy and preeclamptic placentas

Background Pravastatin, a known inducer of endothelial nitric-oxide synthase (eNOS) was demonstrated in human placenta, however the exact mechanism of it’s action is not fully understood. Since placental NO (nitric oxide) synthesis is of primary importance in the regulation of placental blood flow, we aimed to clarify the effects of pravastatin on healthy (n = 6) and preeclamptic (n = 6) placentas (Caucasian participants). Methods The eNOS activity of human placental microsomes was determined by the conversion rate of C14 L-arginine into C14 L-citrulline with or without pravastatin and Geldanamycin. Phosphorylation of eNOS (Ser1177) was investigated by Western blot. Microsomal arginine uptake was measured by a rapid filtration method. Results Pravastatin significantly increased total eNOS activity in healthy (28%, p<0.05) and preeclamptic placentas (32%, p<0.05) using 1 mM Ca2+ promoting the dissociation of a eNOS from it’s inhibitor caveolin. Pravastatin and Geldanamycin (Hsp90 inhibitor) cotreatment increased microsomal eNOS activity. Pravastatin treatment had no significant effects on Ser1177 phosphorylation of eNOS in either healthy or preeclamptic placentas. Pravastatin induced arginine uptake of placental microsomes in both healthy (38%, p < 0.05) and preeclamptic pregnancies (34%, p < 0.05). Conclusions This study provides a novel mechanism of pravastatin action on placental NO metabolism. Pravastatin induces the placental microsomal arginine uptake leading to the rapid activation of eNOS independently of Ser1177 phosphorylation. These new findings may contribute to better understanding of preeclampsia and may also have a clinical relevance.

Modulation of CAT-2B-Mediated l-Arginine Uptake and Nitric Oxide Biosynthesis in HCT116 Cell Line Through Biological Activity of 4′-Geranyloxyferulic Acid Extract from Quinoa Seeds

Chenopodium quinoa Wild is a “pseudocereal” grain which attracts a lot of attention in the scientific community as it has a positive effect on health. Here, we investigate the presence of biologically active O-prenylated phenylpropanoids in the ethanol extract of commercially available quinoa seeds. We claim that 4′-Geranyloxyferulic acid (GOFA) was the only phytochemical product found that belongs to quinoa’s group secondary metabolites. We studied the changes in the oxidative and inflammatory status of the cellular environment in HCT 116 cell line processed with quinoa extract and its component GOFA; the implementation was done through the analysis of the antioxidant enzymes (SOD and CAT), the pro-inflammatory components (iNOS, IL-6 and TNF-α), and the products of intermediary metabolism (ONOO−, O2−). Moreover, the l-arginine uptake was proposed as a target of the tested compounds. We demonstrated that the GOFA, through a decrease of the CAT-2B expression, leads to a reduction of the l-arginine uptake, downregulating the harmful iNOS and restoring the altered redox state. These results propose a new molecular target involved in the reduction of the critical inflammatory process responsible for the cancer progression.

Effects of Arginine and Inflammation on Protein Metabolism in Human Skeletal Muscle Cells (P01-034-19)

Objectives Chronic inflammation is a hallmark of skeletal muscle (SkM) with advancing age and is thought to augment age-related SkM atrophy (sarcopenia). Arginine, a conditionally essential amino acid, is necessary for protein synthesis. Intriguingly, arginine has been identified as being important for attenuating nuclear factor kappa B (NF-κB) activity in non-muscle cells. Our objective was to determine the age-related effects of arginine and inflammation on markers of protein synthesis and breakdown in human myotubes. Methods Muscle progenitor cells (MPCs) were obtained from SkM biopsy tissue of young (YNG, n = 5) and old (OLD, n = 5) women. MPCs were differentiated for 5 days (myotubes) followed by treatment with a pro-inflammatory cytokine, TNFα, for 6 h, 24 h, 48 h, or 96 h. Inflammatory activity and arginine uptake were determined using immunoblotting and radioactive isotopes. Protein synthesis was assessed with puromycin incorporation. MURF1 and MAFBX1 gene expression were used as indicators of proteolysis. Results Prior to TNFα treatment, OLD (vs. YNG) myotubes had greater arginine uptake (P = 0.02), but there were no age-related differences in basal p65 NF-κB activity, puromycin incorporation, or MAFBX1 and MURF1 expression (P > 0.05). Treatment with TNFα induced NF-κB activity in both YNG and OLD myotubes at 6 and 24 h of treatment (P < 0.0001), with no additional increase in activity from 24–96 h. CAT2, an arginine transporter, was transiently induced at 6 h of TNFα treatment (P < 0.01), with no age-related difference in response (P > 0.05). 96 h of TNFα increased arginine uptake in OLD (P < 0.05), but not YNG myotubes (P > 0.05). 6 h TNFα had no effect (P > 0.05), but 96 h of TNFα reduced puromycin incorporation in both YNG and OLD myotubes (P < 0.05). Compared to no treatment control, 96 h TNFα unexpectedly decreased MAFBX1 expression in both YNG and OLD myotubes (P < 0.001). Intriguingly, in the presence of TNFα, arginine increased MURF1 expression in YNG and OLD myotubes compared to no treatment control (P < 0.05). Conclusions Unexpectedly, arginine amplified markers of proteolysis, and did not affect protein synthesis. Future studies will investigate regulatory transcription factors involved in protein breakdown and a crude marker of protein balance (e.g., myotube diameter). Funding Sources President's Council for Cornell Women.

Extracellular l-arginine Enhances Relaxations Induced by Opening of Calcium-Activated SKCa Channels in Porcine Retinal Arteriole

We investigated whether the substrate for nitric oxide (NO) production, extracellular l-arginine, contributes to relaxations induced by activating small (SKCa) conductance Ca2+-activated potassium channels. In endothelial cells, acetylcholine increased 3H-l-arginine uptake, while blocking the SKCa and the intermediate (IKCa) conductance Ca2+-activated potassium channels reduced l-arginine uptake. A blocker of the y+ transporter system, l-lysine also blocked 3H-l-arginine uptake. Immunostaining showed co-localization of endothelial NO synthase (eNOS), SKCa3, and the cationic amino acid transporter (CAT-1) protein of the y+ transporter system in the endothelium. An opener of SKCa channels, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) induced large currents in endothelial cells, and concentration-dependently relaxed porcine retinal arterioles. In the presence of l-arginine, concentration-response curves for CyPPA were leftward shifted, an effect unaltered in the presence of low sodium, but blocked by l-lysine in the retinal arterioles. Our findings suggest that SKCa channel activity regulates l-arginine uptake through the y+ transporter system, and we propose that in vasculature affected by endothelial dysfunction, l-arginine administration requires the targeting of additional mechanisms such as SKCa channels to restore endothelium-dependent vasodilatation.

Thyroid hormones stimulate L-arginine transport in human endothelial cells

Thyroid hormone activity is associated with L-arginine metabolism and nitric oxide (NO) production, which participate in the cardiovascular manifestations of thyroid disorders. L-arginine transporters play an important role in activating L-arginine uptake and NO production. However, the effects of thyroid hormones on L-arginine transporters in endothelial cells have not yet been evaluated. The following methods were used. We measured L-arginine uptake, mRNA expression of L-arginine transporters, endothelial nitric oxide synthase (eNOS) mRNA and NO generation after the administration of T3, T4 and the T3 analog, 3,3′,5-triiodothyroacetic acid TRIAC in human umbilical vein endothelial cells (HUVECs). We also analyzed the role of αvβ3 integrin and of phosphatidyl-inositol-3 kinase (PI3K), mitogen-activated protein kinases (MAPKs: ERK1/2, p38 and SAPK-JNK) and intracellular calcium signaling pathways as underlying mechanisms. To this end, αvβ3 integrin was pharmacologically inhibited by tetraiodothyroacetic acid (TETRAC) or genetically blocked by silencing αv mRNA and PI3K, MAPKs and intracellular calcium by selective inhibitors. The following results were obtained. Thyroid hormones and the T3 analog TRIAC increased L-arginine uptake in HUVECs, the sodium-independent y+/CAT isoforms, except CAT2b, sodium-dependent y+L system and sodium-independent system b0,+L-arginine transporters, eNOS mRNA and NO production. These effects were suppressed by αvβ3 integrin inhibition with TETRAC or αv integrin downregulation or by PI3K, MAPK or intracellular Ca2+ signaling inhibitors. In conclusion, we report for the first time that activation of L-arginine uptake by thyroid hormones is related to an upregulation of L-arginine transporters. This effect seems to be mediated by activation of αvβ3 integrin receptor and subsequent PI3K, MAPK and intracellular Ca2+ signaling pathways.