Dimethylarginine dimethylaminohydrolase-1 is the critical enzyme for degrading the cardiovascular risk factor asymmetrical dimethylarginine

Abstract

Objective: The objective of this study was to identify the role of dimethylarginine dimethylaminohydrolase-1 (DDAH1) in degrading the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and N(g)-monomethyl-L-arginine (L-NMMA).

Methods and results: We generated a global-DDAH1 gene-deficient (DDAH1(-/-)) mouse strain to examine the role of DDAH1 in ADMA and l-NMMA degradation and the physiological consequences of loss of DDAH1. Plasma and tissue ADMA and L-NMMA levels in DDAH1(-/-) mice were several folds higher than in wild-type mice, but growth and development of these DDAH1(-/-) mice were similar to those of their wild-type littermates. Although the expression of DDAH2 was unaffected, DDAH activity was undetectable in all tissues tested. These findings indicate that DDAH1 is the critical enzyme for ADMA and L-NMMA degradation. Blood pressure was ≈ 20 mm Hg higher in the DDAH1(-/-) mice than in wild-type mice, but no other cardiovascular phenotype was found under unstressed conditions. Crossing DDAH1(+/-) male with DDAH1(+/-) female mice yielded DDAH1(+/+), DDAH1(+/-), and DDAH1(-/-) mice at the anticipated ratio of 1:2:1, indicating that DDAH1 is not required for embryonic development in this strain.

Conclusions: Our findings indicate that DDAH1 is required for metabolizing ADMA and L-NMMA in vivo, whereas DDAH2 had no detectable role for degrading ADMA and l-NMMA.

Figure 1

Generation of the DDAH1^−/− mouse strain was achieved by crossing DDAH1^flox/flox with Protamine-Cre mice. Exon4 of DDAH1 was deleted in the sperm of Protamine-Cre/DDAH1^flox/+ during spermatogenesis. Heterozygous global DDAH1 mice were generated by crossing male Prm-cre/DDAH1^flox/+ to wild type female mice (a). Genomic DNA PCR shows that exon4 of DDAH1 was deleted in the DDAH1^−/− mice (b).

Figure 2

Global-DDAH1^−/− mice reveal that DDAH1 is essential for degradation of ADMA and L-NMMA. DDAH1^−/− abolished DDAH1 protein expression in all tissues tested (a), but had no effect on DDAH2 protein expression (b). DDAH1^−/− abolished DDAH activity in kidney, brain and lung as tested using either stable isotope labeled d6-ADMA (c) or d6-L-NMMA as substrate (d). * p<0.05 compared with samples from wild type littermates.

Figure 3

DDAH1^−/− caused significant increases of ADMA and L-NMMA in kidney (a), brain (b) and lung (c), but had no effect on SDMA or L-arginine content. DDAH1^−/− decreased the ratios of L-arginine to ADMA or L-NMMA in these samples (a–c). * p<0.05 compared with controls.

Figure 4

NO signaling was impaired in the DDAH1^−/− mice. DDAH1^−/− increased ADMA (a) and L-NMMA (b) content in plasma, but had no effect on plasma L-arginine (c) and SDMA (d) levels. The ratios of L-arginine to ADMA (e) and L-NMMA (f) in the DDAH1^−/− were significantly decreased. Total NOx in the urine and plasma of DDAH1^−/− mice were also significantly decreased; L-NAME caused further decreases of NOx, but ~40% of both urinary and plasma NOx were resistant to NOS inhibition with L-NAME (g, h). DDAH1^−/− decreased acetylcholine induced NO generation in aortic rings (i) and increased blood pressure (j, k). *p<0.05 compared with corresponding wild type controls. #p<0.05 compared with saline treated controls.

Figure 4

NO signaling was impaired in the DDAH1^−/− mice. DDAH1^−/− increased ADMA (a) and L-NMMA (b) content in plasma, but had no effect on plasma L-arginine (c) and SDMA (d) levels. The ratios of L-arginine to ADMA (e) and L-NMMA (f) in the DDAH1^−/− were significantly decreased. Total NOx in the urine and plasma of DDAH1^−/− mice were also significantly decreased; L-NAME caused further decreases of NOx, but ~40% of both urinary and plasma NOx were resistant to NOS inhibition with L-NAME (g, h). DDAH1^−/− decreased acetylcholine induced NO generation in aortic rings (i) and increased blood pressure (j, k). *p<0.05 compared with corresponding wild type controls. #p<0.05 compared with saline treated controls.