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

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, b o,+ , or B o,+ . 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