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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Arginase inhibition by rhaponticin increases L-arginine concentration that contributes to Ca2+-dependent eNOS activation

  • Koo, Bon-Hyeock (Department of Biological Sciences, Kangwon National University) ;
  • Lee, Jonghoon (Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine) ;
  • Jin, Younghyun (Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine) ;
  • Lim, Hyun Kyo (Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine) ;
  • Ryoo, Sungwoo (Department of Biological Sciences, Kangwon National University)
  • Received : 2021.04.12
  • Accepted : 2021.05.17
  • Published : 2021.10.31
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Abstract

Although arginase primarily participates in the last reaction of the urea cycle, we have previously demonstrated that arginase II is an important cytosolic calcium regulator through spermine production in a p32-dependent manner. Here, we demonstrated that rhaponticin (RPT) is a novel medicinal-plant arginase inhibitor and investigated its mechanism of action on Ca2+-dependent endothelial nitric oxide synthase (eNOS) activation. RPT was uncompetitively inhibited for both arginases I and II prepared from mouse liver and kidney. It also inhibited arginase activity in both aorta and human umbilical vein endothelial cells (HUVECs). Using both microscope and FACS analyses, RPT treatments induced increases in cytosolic Ca2+ levels using Fluo-4 AM as a calcium indicator. Increased cytosolic Ca2+ elicited the phosphorylations of both CaMKII and eNOS Ser1177 in a time-dependent manner. RPT incubations also increased intracellular L-arginine (L-Arg) levels and activated the CaMKII/AMPK/Akt/eNOS signaling cascade in HUVECs. Treatment of L-Arg and ABH, arginase inhibitor, increased intracellular Ca2+ concentrations and activated CaMKII-dependent eNOS activation in ECs of WT mice, but, the effects were not observed in ECs of inositol triphosphate receptor type 1 knockout (IP3R1-/-) mice. In the aortic endothelium of WT mice, RPT also augmented nitric oxide (NO) production and attenuated reactive oxygen species (ROS) generation. In a vascular tension assay using RPT-treated aortic tissue, cumulative vasorelaxant responses to acetylcholine (Ach) were enhanced, and phenylephrine (PE)-dependent vasoconstrictive responses were retarded, although sodium nitroprusside and KCl responses were not different. In this study, we present a novel mechanism for RPT, as an arginase inhibitor, to increase cytosolic Ca2+ concentration in a L-Arg-dependent manner and enhance endothelial function through eNOS activation.

Keywords

Acknowledgement

This work was supported by the Basic Science Research Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2018 R1D1A1B07047959 to SR, 2019R1A6A3A1309450512 to BK). We thank to the Central Laboratory of Kangwon National University for technical assistance with the instruments.

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