Peroxynitrite Stimulates l-Arginine Transport Systemy+ in Glial Cells

Abstract

We have reported previously that peroxynitrite stimulates L-arginine release from astrocytes, but the mechanism responsible for such an effect remains elusive. To explore this issue, we studied the regulation of Glial cells can be activated to endogenously produce ⅐ NO through the induction of the inducible NOS isoform (iNOS) (9 -13). Within the brain, free L-arginine is predominantly located in astrocytes (14), which produce large amounts of ⅐ NO upon iNOS (15) and L-arginine transporter (7) induction. However, unlike in astrocytes, L-arginine content in neurons is very low and is limiting for nNOS activity (16,17). Consistent with this, glial-derived L-arginine has been shown to be increased upon activation of ionotropic glutamate (non-N-methyl-Daspartate) receptors (18) and peroxynitrite (19), suggesting a neuronal-astrocytic signaling transduction pathway focused to provide NOS substrate for the neurons. However, direct demonstration of such a pathway and the elucidation of the precise transport system involved remain elusive.Plasma membrane L-arginine transport is brought about by two families of cationic amino acid transport proteins: Cat (cationic amino acid transporter) and Bat (broad scope amino acid transporter). The Cat family of transporters comprises three different genes encoding four isoform proteins (Cat1, Cat2, Cat2a, and Cat3) (20, 21), commonly referred to as the system y ϩ (22), which is mostly selective for cationic amino acids, although it does show a weak interaction with neutral amino acids in the presence of Na ϩ (23). Glial cells mainly, but not exclusively, express the high affinity (k t for L-arginine ϭ 40 -250 M) (24) 67-kDa Cat1 (constitutive) (25) and the 71.8-kDa Cat2 (inducible) (26) system y ϩ proteins (7, 27). The Bat family of constitutive transporter proteins is found in systems b o,ϩ , B o,ϩ , and y ϩ L (y ϩ Lat1 and y ϩ Lat2), which are mainly expressed in kidney and intestine, except for y ϩ Lat2, which is expressed in astrocytes (28).We have reported previously that the neurotoxic ⅐ NO derivative, peroxynitrite anion (ONOO Ϫ ), specifically stimulates Larginine release from astrocytes (19). Although the ONOO Ϫ -mediated stimulatory effect was inhibited by L-lysine, hence suggesting the involvement of system y ϩ (19), an in-depth study focused on elucidating the precise mechanism responsible for L-arginine transport activation has not yet been carried out. In view of the potential critical role of glial cells as neuronal L-arginine suppliers for ⅐ NO biosynthesis (8,18,29), we were prompted to investigate the mechanism through which ONOO Ϫ modulates L-arginine transport across the glial cell plasma membrane as well as the potential relevance of such modulation for neuronal L-arginine uptake.* This work was funded by grants from the Ministerio de Ciencia y Tecnología (Grant SAF2001-1961), the Junta de Castilla y León (Grant SA065/01) and Fundación Ramón Areces (to J. P. B.), and Grant RO1 DK53307-01 (to M. H.).§ The recipient of a fellowship from the Fundación Miguel Casado...

Peroxynitrite Protects Neurons against Nitric Oxide-mediated Apoptosis

“…Evidence for Activation of the G6PD-catalyzed Reaction by Endogenous Peroxynitrite in Astrocytes-To assess whether endogenous peroxynitrite promoted G6PD activation, astrocytes were incubated with LPS (1 g/ml) for 18 h as previously reported (43). Endogenous peroxynitrite production was evidenced by the ability of LPS-treated cells to oxidize dihydrorhodamine 123 to rhodamine 123 (45).…”

“…To estimate the endogenous formation of peroxynitrite from astrocytes (43), cells were treated with LPS (1 g/ml) for 18 h in either the absence or presence of the peroxynitrite scavenger methionine (10 mM) (44). Peroxynitrite generation was shown by the ability of these cells to oxidize dihydrorhodamine 123 as previously described (45).…”