Dataset Information

Arginase II activity regulates cytosolic Ca²⁺ level in a p32-dependent manner that contributes to Ca²⁺-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells.

ABSTRACT: Although arginase II (ArgII) is abundant in mitochondria, Ca²⁺-accumulating organelles, the relationship between ArgII activity and Ca²⁺ translocation into mitochondria and the regulation of cytosolic Ca²⁺ signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca²⁺ uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprotein stimulation induced an increase in [Ca²⁺]m as measured by CoCl₂-quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII^-/- mice. Conversely, [Ca²⁺]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca²⁺]c resulted in CaMKII and MLC 20 phosphorylation, which was associated with enhanced vasoconstriction activity to phenylephrine (PE) in the vascular tension assay. Cy5-tagged siRNA against mitochondrial p32 mRNA (sip32m) abolished mitochondrial Ca²⁺ uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca²⁺ uptake and dephosphorylation of CaMKII and was completely inhibited by sip32m incubation. In mAoSMCs from ApoE-null mice fed a high-cholesterol diet (ApoE^-/- +HCD), Arg activity was increased, and spermine concentration was higher than that of wild-type mice. Furthermore, [Ca²⁺]m and p32m levels were elevated, and CaMKII phosphorylation was reduced in mAoSMCs from ApoE^-/- +HCD. In vascular tension experiments, an attenuated response to vasoconstrictors in de-endothelialized aorta from ApoE^-/- +HCD was recovered by incubation of sip32m. ArgII activity-dependent production of spermine augments Ca²⁺ transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.

SUBMITTER: Koo BH

PROVIDER: S-EPMC6545325 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Arginase II activity regulates cytosolic Ca2+ level in a p32-dependent manner that contributes to Ca2+-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells.

Koo Bon-Hyeock BH Hong Dongeui D Hong Hyeon Don HD Lim Hyun Kyo HK Hoe Kwang Lae KL Won Moo-Ho MH Kim Young Myeong YM Berkowitz Dan E DE Ryoo Sungwoo S

Experimental & molecular medicine 20190603 6

Although arginase II (ArgII) is abundant in mitochondria, Ca2+-accumulating organelles, the relationship between ArgII activity and Ca2+ translocation into mitochondria and the regulation of cytosolic Ca2+ signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca2+ uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprote ...[more]

PMID: 31155612

Similar Datasets

Project description:Background 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.

Project description:AimsArginase II (ArgII) plays a key role in the regulation of Ca2+ between the cytosol and mitochondria in a p32-dependent manner. p32 contributes to endothelial nitric oxide synthase (eNOS) activation through the Ca2+/CaMKII/AMPK/p38MAPK/Akt signalling cascade. Therefore, we investigated a novel function of ArgII in the regulation of p32 stability.Methods and resultsmRNA levels were measured by quantitative reverse transcription-PCR, and protein levels and activation were confirmed by western blot analysis. Ca2+ concentrations were measured by FACS analysis and a vascular tension assay was performed. ArgII bound to p32, and ArgII protein knockdown using siArgII facilitated the ubiquitin-dependent proteasomal degradation of p32. β-lactone, a proteasome inhibitor, inhibited the p32 degradation associated with endothelial dysfunction in a Ca2+-dependent manner. The amino acids Lys154, Lys 180, and Lys220 of the p32 protein were identified as putative ubiquitination sites. When these sites were mutated, p32 was resistant to degradation in the presence of siArgII, and endothelial function was impaired. Knockdown of Pink/Parkin as an E3-ubiquitin ligase with siRNAs resulted in increased p32, decreased [Ca2+]c, and attenuated CaMKII-dependent eNOS activation by siArgII. siArgII-dependent Parkin activation was attenuated by KN93, a CaMKII inhibitor. Knockdown of ArgII mRNA and its gene, but not inhibition of its activity, accelerated the interaction between p32 and Parkin and reduced p32 levels. In aortas of ArgII-/- mice, p32 levels were reduced by activated Parkin and inhibition of CaMKII attenuated Parkin-dependent p32 lysis. siParkin blunted the phosphorylation of the activated CaMKII/AMPK/p38MAPK/Akt/eNOS signalling cascade. However, ApoE-/- mice fed a high-cholesterol diet had greater ArgII activity, significantly attenuated phosphorylation of Parkin, and increased p32 levels. Incubation with siArgII augmented p32 ubiquitination through Parkin activation, and induced signalling cascade activation.ConclusionThe results suggest a novel function for ArgII protein in Parkin-dependent ubiquitination of p32 that is associated with Ca2+-mediated eNOS activation in endothelial cells.

Project description:BackgroundHypoxic pulmonary vasoconstriction (HPV) optimizes the match between ventilation and perfusion in the lung by reducing blood flow to poorly ventilated regions. Sepsis and endotoxemia impair HPV. We previously showed that nitric oxide synthase 2 (NOS2) is required, but not sufficient, for the effect of endotoxin on HPV. The aim of the current study was to identify additional factors that might contribute to the impairment of HPV during endotoxemia.MethodsGene expression profiling was determined using pulmonary tissues from NOS2-deficient (NOS2-/-) and wild-type mice subjected to endotoxin or saline challenge (control). HPV was accessed as the percentage increase in left pulmonary vascular resistance (LPVR) in response to left main bronchus occlusion (LMBO) in wild-type mice.ResultsAmong the 22,690 genes analyzed, endotoxin induced a greater than three-fold increase in 59 and 154 genes in the lungs of wild-type and NOS2-/- mice, respectively. Of all the genes induced by endotoxin in wild-type mice, arginase 1 (Arg1) showed the greatest increase (16.3-fold compared to saline treated wild-type mice). In contrast, endotoxin did not increase expression of Arg1 in NOS2-/- mice. There was no difference in the endotoxin-induced expression of Arg2 between wild-type and NOS2-deficient mice. We investigated the role of arginase in HPV by treating the mice with normal saline or the arginase inhibitor Nω-hydroxy-nor-L-arginine (norNOHA). In control mice (in the absence of endotoxin) treated with normal saline, HPV was intact as determined by profound LMBO-induced increase in LPVR (121 ± 22% from baseline). During endotoxemia and treatment with normal saline, HPV was impaired compared to normal saline treated control mice (33 ± 9% vs. 121 ± 22%, P < 0.05). HPV was restored in endotoxin-exposed mice after treatment with the arginase inhibitor norNOHA as shown by the comparison to endotoxemic mice treated with normal saline (113 ± 29% vs, 33 ± 9%, P < 0.05) and to control mice treated with normal saline (113 ± 29% vs, 121 ± 22%, P = 0.97).ConclusionsThe results of this study suggest that endotoxemia induces Arg1 and that arginase contributes to the endotoxin-induced impairment of HPV in mice.

Dataset Information

Arginase II activity regulates cytosolic Ca²⁺ level in a p32-dependent manner that contributes to Ca²⁺-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells.

Publications

Arginase II activity regulates cytosolic Ca<sup>2+</sup> level in a p32-dependent manner that contributes to Ca<sup>2+</sup>-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets