Identification of neuronal nitric oxide synthase (nNOS) and cationic amino acid transporter-1 (CAT-1) in rodent brain mitochondria: influence of anxiety status?

Dilwar Muhammed Hussain1,2, Abdel Ennaceur2, Paul Chazot1. 1University of Durham, Durham, County Durham, UK, 2Sunderland Pharmacy School, Sunderland, Tyne & Wear, UK.

The activity of a putative mitochondrial nitric oxide synthase (mtNOS) has been demonstrated in various tissue preparations and may be involved in the regulation of mitochondrial respiration and apoptosis. L-arginine is the primary substrate of nitric oxide synthase (NOS) and is transported in cells via four transport proteins, CAT-1, -2A, -2B, -3 and -4. Expression of the Cat-1 gene is induced in proliferating cells and in response to a variety of stress conditions which induce the unfolded protein response (UPR) (Huang et al., 2010). The location of CAT-1 and NOS proteins to mitochondria has not been formally demonstrated. Here, we investigated whether nNOS and CAT-1 are expressed within rodent brain mitochondria, and whether expression of these proteins is modulated by in vivo behavioural anxiety. Forebrain mitochondria from rat and mice (3-4 month female Wistar and male CD-1, respectively) were purified by a process of differential centrifugation (Morin et al., 2003), and purity was confirmed using electron microscopy (EM). Immunoblotting was performed (Connelly et al., 2009) using 15% SDS PAGE gels, 30 µg of protein applied per well and blots probed with validated commercial rabbit anti-CAT-1 or anti-nNOS (1:1000, Protein Tech Group, USA; Santa Cruz Europe) antibodies overnight at 4°C. Proteins were visualised by means of enhanced chemiluminescence methods. Blots were re-probed with murine anti-β-actin and rabbit anti-SDHC (succinate dehydrogenase complex subunit C) antibodies to validate mitochondrial preparation purity. Immunoblot analysis of mice and rat brain and heart mitochondrial preparations has revealed expression of CAT-1 protein (Mr ∼67 kDa) (n > 4). A Citrulline assay (Bredt & Schmid, 1996) detected NO production by heart mitochondria of 275 ± 44 fmoles NO 30 min-1 mg protein-1 and 160 ± 40 fmoles NO 30 min-1 mg protein-1 (n = 2-3) with and without the cofactors, CaCl2 (1mM)/Calmodulin (10 µg/ml)/NADPH (1mM)/BH4 (100 µM)/FAD (10 µM)/FMN (10 µM), respectively. The nNOS (Mr ∼155 kDa) protein was also detected in purified brain mitochondria. The purity of the preparations was confirmed by the presence of SDHC, and lack of detectable β-actin signal. These results provide the first evidence for a CAT-1 protein and a putative mtNOS in brain mitochondria. This is in accordance with previous studies where rat cerebral mitochondrial L-arginine uptake occurred via a high affinity component (KM = 0.08mM) with transport properties resembling that of the classical plasma membrane system (Dolinska & Albrecht, 1998). In our preliminary studies, forebrain CAT-1 expression was shown to be compromised in high anxiety male Balb/c mice exposed to a novel anxiogenic open space environment. Studies are in progress to confirm these data with immunogold EM approaches, and to further quantify changes in expression following behavioural stress using our novel open space anxiety tests (eg. Ennaceur et al., 2010).

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