Arginase ii contributes to the Ca2+/CaMKII/eNOS axis by regulating Ca2+ concentration between the cytosol and mitochondria in a p32-dependent manner

Bon Hyeock Koo, Hye Mi Hwang, Bong Gu Yi, Hyun Kyo Lim, Byeong Hwa Jeon, Kwang Lae Hoe, Young-Guen Kwon, Moo Ho Won, Young Myeong Kim, Dan E. Berkowitz, Sungwoo Ryoo

Research output: Contribution to journalArticle

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Abstract

Arginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase (eNOS). We tested the hypotheses that arginase II activity participates in the regulation of Ca2+/Ca2+/calmodulin‐dependent kinase II/eNOS activation, and this process is dependent on mitochondrial p32. Methods and Results Downregulation of arginase II increased the concentration of cytosolic Ca2+ ([Ca2+]c) and decreased mitochondrial Ca2+ ([Ca2+]m) in microscopic and fluorescence‐activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca2+/Ca2+/calmodulin‐dependent kinase II. These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca2+ from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II‐null mice resulted in reduced p32 protein level. In the aortas of wild‐type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO‐dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca2+]m were increased in the aortas from apolipoprotein E (ApoE−/−) mice fed a high‐cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca2+/Ca2+/calmodulin‐dependent kinase II‐dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice. Conclusions These data demonstrate a novel function of arginase II in regulation of Ca2+‐dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.

Original languageEnglish
Article numbere009579
JournalJournal of the American Heart Association
Volume7
Issue number18
DOIs
Publication statusPublished - 2018 Sep 1

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Arginase
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Nitric Oxide Synthase Type III
Cytosol
Mitochondria
Small Interfering RNA
Phosphorylation
Phosphotransferases
Spermine
Aorta
Down-Regulation
Gene Knockdown Techniques
Human Umbilical Vein Endothelial Cells
Apolipoproteins E
Immunoassay
Knockout Mice
Vasodilation
Intravenous Administration
Endothelium
Chromatography

All Science Journal Classification (ASJC) codes

  • Cardiology and Cardiovascular Medicine

Cite this

Koo, Bon Hyeock ; Hwang, Hye Mi ; Yi, Bong Gu ; Lim, Hyun Kyo ; Jeon, Byeong Hwa ; Hoe, Kwang Lae ; Kwon, Young-Guen ; Won, Moo Ho ; Kim, Young Myeong ; Berkowitz, Dan E. ; Ryoo, Sungwoo. / Arginase ii contributes to the Ca2+/CaMKII/eNOS axis by regulating Ca2+ concentration between the cytosol and mitochondria in a p32-dependent manner. In: Journal of the American Heart Association. 2018 ; Vol. 7, No. 18.
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abstract = "Arginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase (eNOS). We tested the hypotheses that arginase II activity participates in the regulation of Ca2+/Ca2+/calmodulin‐dependent kinase II/eNOS activation, and this process is dependent on mitochondrial p32. Methods and Results Downregulation of arginase II increased the concentration of cytosolic Ca2+ ([Ca2+]c) and decreased mitochondrial Ca2+ ([Ca2+]m) in microscopic and fluorescence‐activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca2+/Ca2+/calmodulin‐dependent kinase II. These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca2+ from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II‐null mice resulted in reduced p32 protein level. In the aortas of wild‐type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO‐dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca2+]m were increased in the aortas from apolipoprotein E (ApoE−/−) mice fed a high‐cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca2+/Ca2+/calmodulin‐dependent kinase II‐dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice. Conclusions These data demonstrate a novel function of arginase II in regulation of Ca2+‐dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.",
author = "Koo, {Bon Hyeock} and Hwang, {Hye Mi} and Yi, {Bong Gu} and Lim, {Hyun Kyo} and Jeon, {Byeong Hwa} and Hoe, {Kwang Lae} and Young-Guen Kwon and Won, {Moo Ho} and Kim, {Young Myeong} and Berkowitz, {Dan E.} and Sungwoo Ryoo",
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Arginase ii contributes to the Ca2+/CaMKII/eNOS axis by regulating Ca2+ concentration between the cytosol and mitochondria in a p32-dependent manner. / Koo, Bon Hyeock; Hwang, Hye Mi; Yi, Bong Gu; Lim, Hyun Kyo; Jeon, Byeong Hwa; Hoe, Kwang Lae; Kwon, Young-Guen; Won, Moo Ho; Kim, Young Myeong; Berkowitz, Dan E.; Ryoo, Sungwoo.

In: Journal of the American Heart Association, Vol. 7, No. 18, e009579, 01.09.2018.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Arginase ii contributes to the Ca2+/CaMKII/eNOS axis by regulating Ca2+ concentration between the cytosol and mitochondria in a p32-dependent manner

AU - Koo, Bon Hyeock

AU - Hwang, Hye Mi

AU - Yi, Bong Gu

AU - Lim, Hyun Kyo

AU - Jeon, Byeong Hwa

AU - Hoe, Kwang Lae

AU - Kwon, Young-Guen

AU - Won, Moo Ho

AU - Kim, Young Myeong

AU - Berkowitz, Dan E.

AU - Ryoo, Sungwoo

PY - 2018/9/1

Y1 - 2018/9/1

N2 - Arginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase (eNOS). We tested the hypotheses that arginase II activity participates in the regulation of Ca2+/Ca2+/calmodulin‐dependent kinase II/eNOS activation, and this process is dependent on mitochondrial p32. Methods and Results Downregulation of arginase II increased the concentration of cytosolic Ca2+ ([Ca2+]c) and decreased mitochondrial Ca2+ ([Ca2+]m) in microscopic and fluorescence‐activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca2+/Ca2+/calmodulin‐dependent kinase II. These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca2+ from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II‐null mice resulted in reduced p32 protein level. In the aortas of wild‐type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO‐dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca2+]m were increased in the aortas from apolipoprotein E (ApoE−/−) mice fed a high‐cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca2+/Ca2+/calmodulin‐dependent kinase II‐dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice. Conclusions These data demonstrate a novel function of arginase II in regulation of Ca2+‐dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.

AB - Arginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase (eNOS). We tested the hypotheses that arginase II activity participates in the regulation of Ca2+/Ca2+/calmodulin‐dependent kinase II/eNOS activation, and this process is dependent on mitochondrial p32. Methods and Results Downregulation of arginase II increased the concentration of cytosolic Ca2+ ([Ca2+]c) and decreased mitochondrial Ca2+ ([Ca2+]m) in microscopic and fluorescence‐activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca2+/Ca2+/calmodulin‐dependent kinase II. These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca2+ from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II‐null mice resulted in reduced p32 protein level. In the aortas of wild‐type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO‐dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca2+]m were increased in the aortas from apolipoprotein E (ApoE−/−) mice fed a high‐cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca2+/Ca2+/calmodulin‐dependent kinase II‐dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice. Conclusions These data demonstrate a novel function of arginase II in regulation of Ca2+‐dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.

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