Stable tetrahydrobiopterin analogue has vasodilator activity in experimental pulmonary hypertension

Suma R Kunuthur1, Colin J Suckling2, Colin L Gibson2 & Roger M Wadsworth1 . 1Department of Physiology and Pharmacology, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR. 2Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL.

Pulmonary hypertensive patients have diminished NO formation (Demoncheaux et al., 2005), and thus restoring pulmonary endothelial NO output may be beneficial. Deficiency of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS), causes pulmonary hypertension in experimental models (Nandi et al., 2003; Khoo et al., 2004). BH4 is a highly polar molecule that is subject to autooxidation with the production of superoxide (Vásquez-Vivar et al., 2002). The analogue of BH4, 6-acetyl-7-dimethyldihydropterin (ADDP), was selected because it has a more stable oxidation state and is more cell permeable than BH4.

Male Sprague-Dawley rats (150-350 g) were used in all experiments. For in vitro studies, pulmonary arteries were incubated for 6 hrs at 37°C in a minimum essential medium with 2,4-diamino-6-hydroxypyrimidine (DAHP; 10-2 M), an inhibitor of BH4 synthesis, and then preconstricted with phenylephrine (3.6×10-8 M). In such BH4-depleted pulmonary arteries, ADDP (10-9-3×10-7 M) produced a relaxation (maximum relaxation 28.1±4.3 %; n=5; P=0.001), but was without effect in arteries that had not been depleted of BH4. In contrast to ADDP, BH4 produced a contraction (n=5). Inhibition of NOS by L-NAME (Nω-Nitro-L-arginine methyl ester, 300 µM) inhibited ADDP-induced pulmonary vasodilation (n=5,6; P<0.001).

Pulmonary hypertension (PH) was induced in rats by placing them in a hypobaric chamber (580 millibar atmospheric pressure) for two weeks. The rats were injected with ADDP subcutaneously (14.1 mg/kg/day) throughout the period that the rats were placed in the hypobaric chamber. Rats were removed from the chamber after 2 weeks, and their lungs were perfused with physiological salt solution. Vasodilation to calcium ionophore-A23187 (endothelium-dependent) was increased in ADDP-treated PH rats compared to untreated PH rats (IC50=1.09±0.1 µM and IC50=1.72±0.06 µM respectively; n=6,20; P=0.0002). Vasodilation produced by the NO donor, SNAP (S-Nitroso-N-acetyl penicillamine), was maintained in ADDP-treated PH rats: IC50=3.8±1.2 µM; and ADDP-treated normotensive rats: IC50=2.9±1.0 µM (n=7,8). Acute hypoxic challenge resulted in elevated perfusion pressure in both the ADDP-treated and untreated PH rats (n=8). Right ventricular hypertrophy (RVH) was present in ADDP-treated PH rats (0.26±0.01, normotensive; 0.39±0.03, PH; n=9; P=0.0018), which is similar to RVH present in the absence of ADDP. The present results indicate that the novel analogue of BH4, ADDP, has improved pulmonary vasodilator properties compared to BH4 and is effective in experimental pulmonary hypertension.

Demoncheaux E. A. G. et al. (2005) J. Vasc. Res., 42:133-136.
Khoo J. P. et al. (2004) Heart, 90:150.
Nandi M. et al. (2003) Br. J. Pharmacol., 138:65.
Vásquez-Vivar J. et al. (2002) Biochem. J., 362:733-739.