Asymmetric dimethylarginine (ADMA) is an endogenously produced inhibitor of nitric oxide synthases (NOSs) that is synthesised by the action of Type I protein arginine N-methyltransferases: PRMTs (PRMT 1, 3, 4 and 6) (Tang et al, 1998) and metabolised by dimethylarginine dimethylaminohydrolases (DDAH 1 and 2) (Leiper et al, 1999). While the process of NO mediated signalling from endothelium to smooth muscle cells (SMC) is well established, it is not clear whether SMC also signal to endothelium. If it were generated by SMC, ADMA might act as a signal between SMC and endothelium.

Levels of PRMT and DDAH mRNA were determined by quantitative-polymerase chain reaction (Q-PCR) and differences compared using a two-tailed t-test, n=3. DDAH activity in whole cells was measured by assessing conversion of L-NMMA to citrulline. Cells were treated with the DDAH inhibitor 4124W (10^-3 M, MacAllister et al, 1996) or the NO-donor (DEA NONOate 10^-3M). Differences were analysed using a 2-tailed t-test. ADMA and SDMA levels in cell culture medium were measured by HPLC. Data are presented as mean ± s.e.m.

Type I PRMT and DDAH 1 and 2 gene expression was determined in Human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs). All of the genes were expressed, with the level of mRNA being similar in both cell types for each gene. HCAECs had a 3.59±1.027 fold increase in mRNA levels of PRMT6 compared to HCASMCs (n=3, P<0.01). Treatment with 4124W reduced DDAH activity by 40.0±3.541 % (n=3, P<0.01) and treatment with DEA NONOate reduced it by 27.60±4.697 % (n=5, p<0.01). HCASMCs cultured under control conditions released ADMA and SDMA into the culture medium. Inhibition of DDAH activity with 4124W increased ADMA by 7.90±1.943 fold (n=3, P<0.01). We have previously demonstrated that DDAH activity can be inhibited by S-nitrosation of the enzyme. Consistent with these observations, incubation of HCASMCs with DEA NONOate increased ADMA (but not SDMA) release from the HCASMC by 8.26±1.738 fold (n=3, P<0.05).

Human SMC are a potentially major source of ADMA and both synthesise and metabolise this endogenous NOS inhibitor. Inhibition of DDAH caused a major increase in ADMA release. Since ADMA is stable and diffusible this would be expected to inhibit endothelial NO synthesis and provide a mechanism for SMC to influence endothelial cells. The observation that an NO donor inhibits DDAH in SMC and leads to an increase in ADMA release, is consistent with our previous finding that nitrosation of DDAH inhibits enzyme activity. It may also explain why exposure to nitrates appears to inhibit endothelium-dependent relaxation but that addition of arginine reverses the defect (Abou-Mohamed et al, 2000).

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