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.
Highlights
Arginase hydrolyzes L-arginine to urea and L-ornithine, a precursor for polyamines, including spermine, spermidine, and putrescine
We previously demonstrated that arginase II (ArgII) played an important role in the regulation of mitochondrial Ca2+ concentration in native low-density lipoprotein-treated Human aortic smooth muscle cells (hAoSMCs), we did not define the target protein[7]
Stimulated hAoSMCs We first wished to determine the effect of ArgII inhibition on mitochondrial Ca2+ uptake in cultured hAoSMCs (Supplementary Fig. 1A). native low-density lipoprotein (nLDL) stimulation for 30 min resulted in increased [Ca2+]m (Fig. 1a, * vs. untreated, 147 ± 6.6 vs. 100 ± 7.9%, P < 0.01), which was abolished by pretreatment with the ArgII inhibitor ABH and small interfering RNA against ArgII
Summary
Arginase hydrolyzes L-arginine to urea and L-ornithine, a precursor for polyamines, including spermine, spermidine, and putrescine. Arginase I (ArgI) is found in Official journal of the Korean Society for Biochemistry and Molecular Biology. Koo et al Experimental & Molecular Medicine (2019) 51:60 the cytosol, and arginase II (ArgII) is targeted to the mitochondria. ArgI can stimulate proliferation through a polyamine-dependent mechanism in aortic smooth muscle cells[6], the mechanism underlying the role of ArgII in mitochondrial function is yet unknown. We previously demonstrated that ArgII played an important role in the regulation of mitochondrial Ca2+ concentration in native low-density lipoprotein (nLDL)-treated hAoSMCs, we did not define the target protein[7]. Together with the endoplasmic reticulum, the mitochondria play a critical role as a Ca2+-handling organelle in cells, and mitochondrial Ca2+ influx from the cytosol affects cellular physiology and pathophysiology through contributions to the spatial and temporal profiles of intracellular Ca2+ signaling
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