RESUMEN
The potential induction of cationic and zwitterionic amino acid transport systems and mRNA transcripts was investigated in primary neuronal cultures from rat hypothalamus/brainstem. Cultures exposed to bacterial lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) were assessed with respect to northern blot analyses, L-leucine/L-arginine cross-inhibition uptake profiles in the presence and absence of Na+, and initial rate sodium-independent L-arginine transport kinetics. L-Arginine uptake activity was constitutively expressed along with uninduced steady-state levels of CAT1 and 4F2hc transcripts. However, neither the high-affinity nor the low-affinity alternatively spliced inducible isoforms of CAT2 or CAT2a transcripts (encoding system y+ in control astrocytes, lymphocytes, or liver) nor the rBAT transcripts (encoding system b(o,+) in control intestinal epithelial cells) were detected by northern analysis of neuronal mRNA. Cross-inhibition profiles were consistent with physiologic system y+ activity, but not system b(o,+) or system y+ L. Transport kinetics gave a single component with Vmax = 113 +/- 7 pmol/min/mg of protein and Km = 47 +/- 8 microM L-arginine; these kinetic parameters were not influenced by addition of LPS/IFNgamma at concentrations that up-regulated CAT2 mRNA and system y+ activity in control astroglia from the same area of the brain. The data are consistent with L-arginine membrane uptake occurring via only system y+ encoded by constitutive CAT1, with possible physiologic contribution by constitutive 4F2hc transcripts in primary neuronal cultures.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos , Antígenos CD/genética , Arginina/metabolismo , Proteínas Portadoras/genética , Catalasa/genética , Glicoproteínas de Membrana/genética , Óxido Nítrico Sintasa/metabolismo , Proteínas , Aminoácidos/metabolismo , Animales , Animales Recién Nacidos , Antígenos CD/metabolismo , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Arginina/farmacocinética , Transporte Biológico/fisiología , Proteínas Portadoras/metabolismo , Catalasa/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Proteína-1 Reguladora de Fusión , Regulación Enzimológica de la Expresión Génica , Leucina/farmacocinética , Glicoproteínas de Membrana/metabolismo , Neuronas/citología , Neuronas/enzimología , Óxido Nítrico Sintasa de Tipo I , Ratas , Ratas Sprague-Dawley , Rombencéfalo/citología , Canales Catiónicos TRPV , TritioRESUMEN
The inducible isoform II of nitric-oxide synthase (iNOS) was recently cloned from brain and identified in astroglial cells. Induced nitric oxide biosynthesis occurs in brain cells only if extracellular cerebrospinal fluid contains -arginine. This study demonstrates for the first time that induced iNOS activity is strictly dependent on concomitant induction of an alternatively spliced transcript of the cat-2 gene encoding high affinity -arginine transporter System y+ in cultured rat astrocytes. Inhibition profiles of radiolabeled -arginine and -leucine uptake identified the dominance of Na+-independent transport System y+ serving cationic amino acids, with insignificant activities of Systems y+L, bo,+, or Bo,+. A reverse transcription-polymerase chain reaction/sequencing/cloning strategy was used to identify a single 123-base nucleotide sequence coding the high affinity domain of alternatively spliced CAT-2 (not CAT-2a) in astrocytes activated by lipopolysaccharide/interferon-gamma. Using this sequence as a cDNA probe, it was determined that CAT-2 mRNA, iNOS mRNA, and System y+ activity were concomitantly and strongly induced in astrocytes. Constitutive CAT-1 mRNA was weakly present in neurons and astrocytes, was not inducible in either cell type, and contributed <3% to total System y+ activity. Although astroglial iNOS Km approximately 10 microM L-arginine for intracellular substrate, hyperbolic kinetics of inducible iNOS activity measured as a function of extracellular L-arginine concentration gave Km approximately 50 microM L-arginine with intact cells. The same Km approximately 50 microM was obtained for induced membrane transport System y+ activity. iNOS activity was reduced to zero in the absence of extracellular L-arginine uptake via System y+. These findings expand the current understanding of NO biosynthesis modulation and implicate a coordinated regulation of intracellular iNOS enzyme activity with membrane L-arginine transport in brain.