ARGININOSUCCINATE SYNTHETASE OVEREXPRESSION IN VASCULAR SMOOTH-MUSCLECELLS POTENTIATES IMMUNOSTIMULANT-INDUCED NO PRODUCTION

Immunostimulants trigger vascular smooth muscle cells (VSMC) to expres s both the inducible isoform of NO synthase (iNOS) and argininosuccina te synthetase (AS). With constitutively expressed argininosuccinate ly ase (AL), AS confers cells with an Arg/Cit cycle that can sustain NO p roduction via continuous regeneration of the NOS substrate, L-arginine (Arg), from the NOS coproduct, L-citrulline (Cit). To assess whether NO synthesis can be rate-limited by Arg recycling, we tested whether A S-overexpressing cells have an enhanced capacity for immununostimulant -induced NO synthesis. Rat VSMC were stably transfected with human AS cDNA in a eukaryotic cell expression vector, driven by a strong viral promoter. AS activity in transfected VSMC exceeded that induced in unt ransfected cells treated for 24 h with a combination of bacterial lipo polysaccharide and interferon-gamma (LPS/IFN). AS activity was predomi nantly associated with membranes but was also found in cytosol. Recomb inant AS was purified from cytosol and possessed a specific activity e xceeding that reported for native AS. Western blotting verified the ba sal expression of AS antigen in membranes from untreated AS-transfecte d VSMC and from untransfected VSMC after 24 h exposure to LPS/IFN. Epi fluorescence histochemistry revealed a punctate distribution of AS ant igen in transfected cells, consistent with a predominant membrane loca lization. Remarkably, on a per cell basis, LPS/IFN-induced NO producti on was 3-4-fold greater in AS-transfected cells than untransfected VSM C. In untransfected VSMC, maximal NO production during 48 h required m illimolar Arg; notably, Cit was needed at approximate to 3-fold higher concentrations than Arg for a comparable NO synthesis rate. In contra st, AS-transfected VSMC utilized Arg and Cit equi-effectively and at m uch lower concentrations; 100 mu M of either precursor supported a max imal rate of NO synthesis for 48 h. The enhanced ability of AS-transfe cted cells to produce NO, compared with untransfected cells, could not be ascribed to differences in iNOS protein content or LPS/IFN potency for immunoactivation. We conclude that transfection with AS provides a continuous flux of Arg which drives NO synthesis in immunoactivated VSMC. Arg regeneration by AS is rate-limiting to NO synthesis and appa rently provides iNOS with a preferred cellular source of Arg. In accor d with the reported ''channeling'' of substrates by urea cycle enzymes , we hypothesize that the Arg/Cit cycle sequesters a discrete pool of recyclable substrate that sustains high-output NO synthesis.