Induced Nitric Oxide Synthesis Is Dependent on Induced Alternatively Spliced CAT-2 Encoding L-Arginine Transport in Brain Astrocytes

Abstract

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 L-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 L-arginine transporter System y+ in cultured rat astrocytes. Inhibition profiles of radiolabeled L-arginine and L-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-γ. 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∼ 10 μML-arginine for intracellular substrate, hyperbolic kinetics of inducible iNOS activity measured as a function of extracellular L-arginine concentration gave Km∼50 μML-arginine with intact cells. The same Km∼ 50 μM 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. 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 L-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 L-arginine transporter System y+ in cultured rat astrocytes. Inhibition profiles of radiolabeled L-arginine and L-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-γ. 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∼ 10 μML-arginine for intracellular substrate, hyperbolic kinetics of inducible iNOS activity measured as a function of extracellular L-arginine concentration gave Km∼50 μML-arginine with intact cells. The same Km∼ 50 μM 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.