Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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Effect of Various Pathological Conditions on Nitric Oxide Level and L-Citrulline Uptake in Motor Neuron-Like (NSC-34) Cell Lines

  • Shashi Gautam (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Sookmyung Women's University) ;
  • Sana Latif (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Sookmyung Women's University) ;
  • Young-Sook Kang (Research Institute of Pharmaceutical Sciences, College of Pharmacy, Sookmyung Women's University)
  • Received : 2023.06.07
  • Accepted : 2023.10.21
  • Published : 2024.01.01
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Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a nonessential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [14C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [14C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [14C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [14C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

Keywords

Acknowledgement

The authors thank Professor Hoon Ryu from the Korea Institute of Science and Technology for providing the cell lines. This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2019R1F1A1044048) (YSK).

References

  1. Ahmed, M. and Phillips, M. (2011) Neurodegenerative disorders. In Problem Solving in Neuroradiology, pp. 333-360.
  2. Allerton, T. D., Proctor, D. N., Stephens, J. M., Dugas, T. R., Spielmann, G. and Irving, B. A. (2018) L-citrulline supplementation: Impact on cardiometabolic health. Nutrients 10, 921. https://doi.org/10.3390/nu10070921
  3. Almer, G., Vukosavic, S., Romero, N. and Przedborski, S. (1999) Inducible nitric oxide synthase up-regulation in a transgenic mouse model of familial amyotrophic lateral sclerosis. J. Neurochem. 72, 2415-2425. https://doi.org/10.1046/j.1471-4159.1999.0722415.x
  4. Bahri, S., Zerrouk, N., Aussel, C., Moinard, C., Crenn, P., Curis, E., Chaumeil, J. C., Cynober, L. and Sfar, S. (2013) Citrulline: from metabolism to therapeutic use. Nutrition 29, 479-484. https://doi.org/10.1016/j.nut.2012.07.002
  5. da Silva, M. P., Cedraz-Mercez, P. L. and Varanda, W. A. (2014) Effects of nitric oxide on magnocellular neurons of the supraoptic nucleus involve multiple mechanisms. Braz. J. Med. Biol. Res. 47, 90-100. https://doi.org/10.1590/1414-431X20133326
  6. Dawson, V. L., Dawson, T. M., Bartley, D. A., Uhl, G. R. and Snyder, S. H. (1993) Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures. J. Neurosci. 13, 2651-2661. https://doi.org/10.1523/JNEUROSCI.13-06-02651.1993
  7. Fonseca, S. G., Romao, P. R. T., Figueiredo, F., Morais, R. H., Lima, H. C., Ferreira, S. H. and Cunha, F. Q. (2003) TNF-α mediates the induction of nitric oxide synthase in macrophages but not in neutrophils in experimental cutaneous leishmaniasis. Eur. J. Immunol. 33, 2297-2306. https://doi.org/10.1002/eji.200320335
  8. Forstermann, U. and Sessa, W. C. (2012) Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829-837. https://doi.org/10.1093/eurheartj/ehr304
  9. Gubert, F., Bonacossa-Pereira, I., Decotelli, A. B., Furtado, M., Vasconcelos-dos-Santos, A., Mendez-Otero, R. and Santiago, M. F. (2019) Bone-marrow mononuclear cell therapy in a mouse model of amyotrophic lateral sclerosis: functional outcomes from different administration routes. Brain Res. 1712, 73-81. https://doi.org/10.1016/j.brainres.2019.02.003
  10. Gyawali, A., Gautam, S., Hyeon, S. J., Ryu, H. and Kang, Y. S. (2021a) L-citrulline level and transporter activity are altered in experimental models of amyotrophic lateral sclerosis. Mol. Neurobiol. 58, 647-657. https://doi.org/10.1007/s12035-020-02143-6
  11. Gyawali, A. and Kang, Y. S. (2019) Blood-to-retina transport of imperatorin involves the carrier-mediated transporter system at the inner blood-retinal barrier. J. Pharm. Sci. 108, 1619-1626. https://doi.org/10.1016/j.xphs.2018.11.040
  12. Gyawali, A., Kim, M. H. and Kang, Y. S. (2021b) A novel organic cation transporter involved in paeonol transport across the inner blood-retinal barrier and changes in uptake in high glucose conditions. Exp. Eye Res. 202, 108387. https://doi.org/10.1016/j.exer.2020.108387
  13. Hensley, K., Abdel-Moaty, H., Hunter, J., Mhatre, M., Mhou, S., Nguyen, K., Potapova, T., Pye, Q. N., Qi, M., Rice, H., Stewart, C. and West, M. (2006) Primary glia expressing the G93A-SOD1 mutation present a neuroinflammatory phenotype and provide a cellular system for studies of glial inflammation. J. Neuroinflammation 3, 2. https://doi.org/10.1186/1742-2094-3-2
  14. Hsu, H. Y. and Wen, M. H. (2002) Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression. J. Biol. Chem. 277, 22131-22139. https://doi.org/10.1074/jbc.M111883200
  15. Jung, M.-K., Kim, K. Y., Lee, N.-Y., Kang, Y.-S., Hwang, Y. J., Kim, Y., Sung, J.-J., McKee, A., Kowall, N., Lee, J. and Ryu, H. (2013) Expression of taurine transporter (TauT) is modulated by heat shock factor 1 (HSF1) in motor neurons of ALS. Mol. Neurobiol. 47, 699-710. https://doi.org/10.1007/s12035-012-8371-9
  16. Kang, Y. S., Ohtsuki, S., Takanaga, H., Tomi, M., Hosoya, K. and Terasaki, T. (2002) Regulation of taurine transport at the blood-brain barrier by tumor necrosis factor-α, taurine and hypertonicity. J. Neurochem. 83, 1188-1195. https://doi.org/10.1046/j.1471-4159.2002.01223.x
  17. Kuhlmann, C. R. W., Gerigk, M., Bender, B., Closhen, D., Lessmann, V. and Luhmann, H. J. (2008) Fluvastatin prevents glutamate-induced blood-brain-barrier disruption in vitro. Life Sci. 82, 1281-1287. https://doi.org/10.1016/j.lfs.2008.04.017
  18. Latif, S. and Kang, Y.-S. (2022) Protective effects of choline against inflammatory cytokines and characterization of transport in motor neuron-like cell lines (NSC-34). Pharmaceutics 14, 2374. https://doi.org/10.3390/pharmaceutics14112374
  19. Latif, S. and Kang, Y.-S. (2021) Change in cationic amino acid transport system and effect of lysine pretreatment on inflammatory state in amyotrophic lateral sclerosis cell model. Biomol. Ther. (Seoul) 29, 498-505. https://doi.org/10.4062/biomolther.2021.037
  20. Lee, J., Hyeon, S. J., Im, H., Ryu, H., Kim, Y. and Ryu, H. (2016) Astrocytes and microglia as non-cell autonomous players in the pathogenesis of ALS. Exp. Neurobiol. 25, 233-240. https://doi.org/10.5607/en.2016.25.5.233
  21. Lee, J., Ryu, H. and Kowall, N. W. (2009a) Differential regulation of neuronal and inducible nitric oxide synthase (NOS) in the spinal cord of mutant SOD1 (G93A) ALS mice. Biochem. Biophys. Res. Commun. 387, 202-206. https://doi.org/10.1016/j.bbrc.2009.07.007
  22. Lee, J., Ryu, H. and Kowall, N. W. (2009b) Motor neuronal protection by L-arginine prolongs survival of mutant SOD1 (G93A) ALS mice. Biochem. Biophys. Res. Commun. 384, 524-529. https://doi.org/10.1016/j.bbrc.2009.05.015
  23. Lee, K.-E. and Kang, Y.-S. (2017) Characteristics of L-citrulline transport through blood-brain barrier in the brain capillary endothelial cell line (TR-BBB cells). J. Biomed. Sci. 24, 28. https://doi.org/10.1186/s12929-017-0336-x
  24. Lee, N. Y. and Kang, Y. S. (2015) The changes by hypoxia inducible factor- 1alpha (HIF-1α) on taurine uptake in brain capillary endothelial cells at high glucose conditions. Adv. Exp. Med. Biol. 803, 501-511. https://doi.org/10.1007/978-3-319-15126-7_40
  25. Lee, N. Y. and Kang, Y. S. (2016) In vivo and in vitro evidence for brain uptake of 4-phenylbutyrate by the monocarboxylate transporter 1 (MCT1). Pharm. Res. 33, 1711-1722. https://doi.org/10.1007/s11095-016-1912-6
  26. Lee, N. Y., Kim, Y., Ryu, H. and Kang, Y. S. (2017) The alteration of serine transporter activity in a cell line model of amyotrophic lateral sclerosis (ALS). Biochem. Biophys. Res. Commun. 483, 135-141. https://doi.org/10.1016/j.bbrc.2016.12.178
  27. Li, C., He, A., Guo, Y., Yang, X., Luo, M., Cheng, Z., Huang, L., Xia, Y. and Luo, S. (2021) Hypertonic stress modulates eNOS function through O-GlcNAc modification at Thr-866. Sci. Rep. 11, 11272. https://doi.org/10.1038/s41598-021-90321-4
  28. Lively, S. and Schlichter, L. C. (2018) Microglia responses to pro-inflammatory stimuli (LPS, IFNγ+TNFα) and reprogramming by resolving cytokines (IL-4, IL-10). Front. Cell. Neurosc. 12, 215. https://doi.org/10.3389/fncel.2018.00215
  29. Luiking, Y. C., Engelen, M. P. K. J. and Deutz, N. E. P. (2010) Regulation of nitric oxide production in health and disease. Curr. Opin. Clin. Nutr. Metab. Care 13, 97-104. https://doi.org/10.1097/MCO.0b013e328332f99d
  30. McManus, M. L., Churchwell, K. B. and Strange, K. (1995) Regulation of cell volume in health and disease. N. Engl. J. Med. 333, 1260-1267. https://doi.org/10.1056/NEJM199511093331906
  31. Moinard, C., MacCario, J., Walrand, S., Lasserre, V., Marc, J., Boirie, Y. and Cynober, L. (2016) Arginine behaviour after arginine or citrulline administration in older subjects. Br. J. Nutr. 115, 399-404. https://doi.org/10.1017/S0007114515004638
  32. Nomura, E., Ohta, Y., Tadokoro, K., Shang, J., Feng, T., Liu, X., Shi, X., Matsumoto, N., Sasaki, R., Tsunoda, K., Sato, K., Takemoto, M., Hishikawa, N., Yamashita, T., Kuchimaru, T., Kizaka-Kondoh, S. and Abe, K. (2019) Imaging hypoxic stress and the treatment of amyotrophic lateral sclerosis with dimethyloxalylglycine in a mice model. Neuroscience 415, 31-43. https://doi.org/10.1016/j.neuroscience.2019.06.025
  33. Pollari, E., Goldsteins, G., Bart, G., Koistinaho, J. and Giniatullin, R. (2014) The role of oxidative stress in degeneration of the neuromuscular junction in amyotrophic lateral sclerosis. Front. Cell. Neurosci. 8, 131.
  34. Renton, A. E., Chio, A. and Traynor, B. J. (2014) State of play in amyotrophic lateral sclerosis genetics. Nat. Neurosci. 17, 17-23. https://doi.org/10.1038/nn.3584
  35. Ryu, H., Lee, J., Hagerty, S. W., Byoung, Y. S., McAlpin, S. E., Cormier, K. A., Smith, K. M. and Ferrante, R. J. (2006) ESET/SETDB1 gene expression and histone H3 (K9) trimethylation in Huntington's disease. Proc. Natl. Acad. Sci. U. S. A. 103, 19176-19181. https://doi.org/10.1073/pnas.0606373103
  36. Sasabe, J., Chiba, T., Yamada, M., Okamoto, K., Nishimoto, I., Matsuoka, M. and Aiso, S. (2007) D-serine is a key determinant of glutamate toxicity in amyotrophic lateral sclerosis. EMBO J. 26, 4149-4159. https://doi.org/10.1038/sj.emboj.7601840
  37. Satsu, H., Miyamoto, Y. and Shimizu, M. (1999) Hypertonicity stimulates taurine uptake and transporter gene expression in Caco-2 cells. Biochim. Biophys. Acta Biomembr. 1419, 89-96. https://doi.org/10.1016/S0005-2736(99)00058-9
  38. Sharp, C. D., Hines, I., Houghton, J., Warren, A., Jackson, T. H., IV, Jawahar, A., Nanda, A., Elrod, J. W., Long, A., Chi, A., Minagar, A. and Alexander, J. S. (2003) Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor. Am. J. Physiol. Heart Circ. Physiol. 285, H2592-H2598. https://doi.org/10.1152/ajpheart.00520.2003
  39. Yabuki, Y., Shioda, N., Yamamoto, Y., Shigano, M., Kumagai, K., Morita, M. and Fukunaga, K. (2013) Oral L-citrulline administration improves memory deficits following transient brain ischemia through cerebrovascular protection. Brain Res. 1520, 157-167. https://doi.org/10.1016/j.brainres.2013.05.011
  40. Zhang, X., Chen, S., Li, L., Wang, Q. and Le, W. (2008) Folic acid protects motor neurons against the increased homocysteine, inflammation and apoptosis in SOD1G93A transgenic mice. Neuropharmacology 54, 1112-1119. https://doi.org/10.1016/j.neuropharm.2008.02.020