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 o xide biosynthesis occurs in brain cells only if extracellular cerebros pinal 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 enc oding high affinity L-arginine transporter System y(+) in cultured rat astrocytes, Inhibition profiles of radiolabeled L-arginine and L-leuc ine uptake identified the dominance of Na+-independent transport Syste m y(+) serving cationic amino acids, with insignificant activities of Systems y(+)L, b(o,+), or B-o,B-+. A reverse transcription-polymerase chain reaction/sequencing/cloning strategy was used to identify a sing le 123-base nucleotide sequence coding the high affinity domain of alt ernatively spliced CAT-2 (not CAT-2a) in astrocytes activated by lipop olysaccharide/interferon-gamma. Using this sequence as a cDNA probe, i t was determined that CAT-2 mRNA, iNOS mRNA, and System y(+) activity were concomitantly and strongly induced in astrocytes. Constitutive CA T-1 mRNA was weakly present in neurons and astrocytes, was not inducib le in either cell type, and contributed <3% to total System y(+) activ ity. Although astroglial iNOS K-m similar to 10 mu M L-arginine for in tracellular substrate, hyperbolic kinetics of inducible iNOS activity measured as a function of extracellular L-arginine concentration gave K-m similar to 50 mu M L-arginine with intact cells. The same K-m simi lar to 50 mu M was obtained for induced membrane transport System y(+) activity, iNOS activity was reduced to zero in the absence of extrace llular L-arginine uptake via System y(+). These findings expand the cu rrent understanding of NO biosynthesis modulation and implicate a coor dinated regulation of intracellular iNOS enzyme activity with membrane L-arginine transport in brain.