Tetrahydrobiopterin augments arginine transport in rat cardiac myocytes through modulation of CAT-2 mRNA

Tetrahydrobiopterin (BH4) has been shown to be required for dimerization an d acquisition of nitric oxide (NO) generating capacity by nitric oxide synt hase (NOS). in the present study we have investigated the hypothesis that B H4 may affect NOS activity through a novel mechanism-namely modulating argi nine transport in rat cardiac myocytes, Cardiac myocytes have been previous ly shown to express cationic amino acid transport proteins (y+ system) CAT- 1 and CAT-2. Increasing extracellular BH4 concentrations up to 0.5 mmol/L a ugments arginine transport in 1 mmol/L arginine media no (BH4, 558 +/- 42 f mol arginine/mug protein/min; 0.1 mmol/L BH4, 580 +/- 11 fmol arginine/mug protein/min; 0.5 mmol/L BH4 944 +/- 71* fmol arginine/mug protein/min; 1.0 mmol/L BH4, 983+/-84* fmol arginine/mug protein/min, n = 4: *P < .05 vs no BH4). Treating the cells with lipopolysaccharide (LPS) (10 <mu>g/mL) signif icantly augmented arginine transport only in the presence of BH4 (no BH4, 6 00 +/- 33 fmol arginine/mug protein/min; 0.1 mmol/L BH4, 691 +/- 29*dagger fmol arginine/mug protein/min; 0.5 mmol/L BH4, 1123 +/- 32*dagger fmol argi nine/mug protein/min; 1.0 mmol/L BH4, 1296 +/- 42*dagger fmol arginine/mug protein/min, n = 4; *P < .01 vs no BH4, <dagger>P < .05 vs no LPS). The adm inistration of biopterin, sodium nitroprusside (NO donor), 2,4-diamino-6-hy droxy-pyrimidine (inhibitor of BH4 synthesis), and sepiapterin (the precurs or of de novo synthesis of BH4) to unstimulated cells had no effect on argi nine uptake values. Using reverse trancriptase-polymerase chain reaction, w e next studied the steady state levels for CAT-1 and CAT-2 mRNA. Incubation with BH4 significantly increased CAT-2 mRNA expression in a concentration- dependent manner in 0.1, 0.5, and 1 mmol/L BH4, respectively, Northern blot ting analysis further confirmed this observation. We also found that in the presence of BH4 in these concentrations, CAT-1 mRNA expression was abolish ed. We suggest that BH4 augments intracellular arginine availability by mod ulating CAT-2 mRNA expression and suggest that its presence is required for the LPS effect on trans-membrane arginine traffic.