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

Tetrahydrobiopterin (BH4) has been shown to be required for dimerization and acquisition of nitric oxide (NO) generating capacity by nitric oxide synthase (NOS). In the present study we have investigated the hypothesis that BH4 may affect NOS activity through a novel mechanism-namely, modulating arginine transport in rat cardiac myocytes. Cardiac myocytes have been previously 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 augments arginine transport in 1 mmol/L arginine media (no BH4, 558 +/- 42 fmol arginine/microg protein/min; 0.1 mmol/L BH4, 580 +/- 11 fmol arginine/microg protein/min; 0.5 mmol/L BH4, 944 +/- 71* fmol arginine/microg protein/min; 1.0 mmol/L BH4, 983+/-84* fmol arginine/microg protein/min, n = 4; *: P <.05 vs no BH4). Treating the cells with lipopolysaccharide (LPS) (10 microg/mL) significantly augmented arginine transport only in the presence of BH4 (no BH4, 600 +/- 33 fmol arginine/microg protein/min; 0.1 mmol/L BH4, 691 +/- 29*dagger fmol arginine/microg protein/min; 0.5 mmol/L BH4, 1123 +/- 32*dagger fmol arginine/microg protein/min; 1.0 mmol/L BH4, 1296 +/- 42*dagger fmol arginine/microg protein/min, n = 4; *: P <.01 vs no BH4, dagger: P <.05 vs no LPS). The administration of biopterin, sodium nitroprusside (NO donor), 2,4-diamino-6-hydroxy-pyrimidine (inhibitor of BH4 synthesis), and sepiapterin (the precursor of de novo synthesis of BH4) to unstimulated cells had no effect on arginine uptake values. Using reverse trancriptase-polymerase chain reaction, we 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 blotting analysis further confirmed this observation. We also found that in the presence of BH4 in these concentrations, CAT-1 mRNA expression was abolished. We suggest that BH4 augments intracellular arginine availability by modulating CAT-2 mRNA expression and suggest that its presence is required for the LPS effect on trans-membrane arginine traffic.