Conference: 2008 International PHA Conference and Scientific Sessions
Release Date: 06.20.2008
Presentation Type: Abstracts
Zharikov S., Krotova K., Hu H., Patel J.M., Baylis C., Johnson R.J., Block E.R.
Department of Medicine, University of Florida, Gainesville, FL, USA
BACKGROUND: Elevated levels of serum uric acid (UA) are commonly associated with pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC).
METHODS: We studied effects of UA on vasodilation of isolated porcine pulmonary artery rings (PAR) and on L-arginine-eNOS and L-arginine-arginase pathways in cultured PAEC.
RESULTS: In isolated porcine PAR, UA in concentrations corresponding to plasma concentrations observed in patients with pulmonary hypertension inhibited ACh-induced vasorelaxation. Using the fluorescent probe DAF-FM we demonstrated that UA caused a dose dependent decrease in the NO production in cultured PAEC stimulated by bradykinin. We also measured intracellular concentrations of the NO second messenger cGMP in cell treated with UA. UA significantly decreased accumulation of cGMP in PAEC stimulated by a calcium-ionophore. Accumulation of cGMP was completely blocked in stimulated PAEC after preliminary incubation of cells with L-NAME suggesting that cGMP accumulation is NO-dependent. Taken together, these observations indicate that UA decreases NO production by PAEC. To investigate the mechanisms of reduced NO production in PAEC treated with UA, we tested the effects of UA on L-arginine transport and on eNOS activity and expression. Incubation of PAEC with different concentrations of UA (2.5 – 15 mg/dl) for 24 h did not affect [3H]-L-arginine uptake or activity/expression of eNOS. We also tested the effects of UA on stimulated phosphorylation of eNOS. Ionophore A23187 induced phosphorylation of eNOS at Ser1177, and this response to A23187 was not changed in PAEC treated with UA (7.5 mg/dl). To test the concept that UA affects arginase activity which in turn might diminish L-arginine availability for NO production, we studied changes in arginase activity in cultured PAEC. We found that PAEC treated with UA produced significantly more urea than control cells in a dose-dependent manner. According to our previous studies, arginase II is the major isoform of arginase expressed in PAEC. RT-PCR and Western blot analyses demonstrated that UA did not alter gene expression of arginase I or II or arginase II protein expression in PAEC suggesting that UA (or its metabolites) are able to stimulate arginase directly without changing enzyme expression. Kinetic analysis of arginase II activity in cell lysates demonstrated that UA activated arginase by increasing its affinity for L-arginine. An inhibitor of arginase (S)-(2-boronoethyl)-L-cysteine prevented UA-induced reduction of NO production by PAEC.
CONCLUSIONS: UA inhibits vasodilation in the lung by reducing NO production in endothelial cells. The reduction of NO production in lung endothelial cells by UA is mediated via arginase activation. These studies might explain a contribution of hyperuricemia to a severity of pulmonary hypertension.