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

Although arginase II (ArgII) is abundant in mitochondria, Ca 2+ -accumulating organelles, the relationship between ArgII activity and Ca 2+ translocation into mitochondria and the regulation of cytosolic Ca 2+ signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca 2+ 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 [Ca 2+ ]m as measured by CoCl 2 -quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII −/− mice. Conversely, [Ca 2+ ]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca 2+ ]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 Ca 2+ uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca 2+ 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, [Ca 2+ ]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 Ca 2+ transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.