Alterations in lung arginine metabolism in lambs with pulmonary hypertension associated with increased pulmonary blood flow

Abstract

Previous studies demonstrate impaired nitric oxide (NO) signaling in children and animal models with congenital heart defects and increased pulmonary blood flow. However, the molecular mechanisms underlying these alterations remain incompletely understood. The purpose of this study was to determine if early changes in arginine metabolic pathways could play a role in the reduced NO signaling demonstrated in our lamb model of congenital heart disease with increased pulmonary blood flow (Shunt lambs). The activities of the arginine recycling enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) were both decreased in lung tissues of Shunt lambs while arginase activity was increased. Associated with these alterations, lung L-arginine levels were decreased. These changes correlated with an increase in NO synthase-derived reactive oxygen species (ROS) generation. This study provides further insights into the molecular mechanisms leading to decreased NO signaling in Shunt lambs and suggests that altered arginine metabolism may play a role in the development of the endothelial dysfunction associated with pulmonary hypertension secondary to increased pulmonary blood flow.

Figures

Fig. 1

The arginine–nitric oxide pathway showing the role of enzymes involved in the arginine metabolism and NO signaling. ASS: argininosuccinate synthetase; ASL: argininosuccinate lyase; CAT-1: cationic amino acid transporter; ARG: arginase; ROS: reactive oxygen species; OTC: ornithine transcarbamylase; eNOS: endothelial nitric oxide synthase; NO: nitric oxide.

Fig. 2

Activity and expression of the arginine biosynthetic enzymes. The specificity of the ASS and ASL antibodies were confirmed by transfecting COS-7 cells with expression plasmids containing coding sequences for ASS (A) or ASL (D). ASS (B) or ASL (E) protein levels were measured using Western blot analysis. Protein loading was normalized using β-actin. There was a significant increase in normalized densitometric values for ASS (B) but a significant decrease in ASL expression (E) in Shunt lambs. There is a significant decrease in both ASS (C) and ASL (F) activities in Shunt lambs. Values are mean±SEM. N=6 for each group. *p<0.05 vs. control lambs.

Fig. 3

Arginase expression and activity. Arginase 1 and 2 protein levels were measured using Western blot analysis with liver (A) and kidney (B) extracts as positive controls, respectively. In addition, arginase 1 and arginase 2 mRNA levels were quantified by real-time RT-PCR analysis. There were no significant changes in the protein or mRNA levels of either arginase 1 or 2 (A–C). However, a significant increase in arginase enzyme activity is observed in Shunt lambs (D). CAT-1 expression in peripheral lung tissue of control and Shunt lambs was also determined using Western blot analysis (E). There was a significant increase in normalized (using β-actin) densitometric values for CAT-1 in Shunt lambs. Values are mean±SEM, N=6 for each group. *p<0.05 vs. control lambs.

Fig. 4

Altered NO signaling in Shunt lambs. eNOS protein levels were measured using Western blot analysis and protein loading was normalized using β-actin. There was no significant change in eNOS protein levels (A). However, relative NOS activity (calculated from tissue NOx levels as a fraction of calcium-dependent [3H]–L-arginine to [3H]–L-citrulline conversion) is significantly decreased in Shunt lambs (B) while NOS-derived superoxide levels are increased (C). Protein loading was normalized using the mitochondrial specific marker, VDAC. Values are mean±SEM. N=6 for each group. *p<0.05 compared to control lambs, †p<0.05 compared to untreated (no ETU).