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were reduced following co-incubation with Nrnitro-L-arginine methylester (L-NAME; 1 mM), NGnitro-Larginine (L-NOARG; mM) and L-canavanine (1 mM) to 47.1 ± 6.2, 24.7 ± 3.6 and 12.5 ± 2.8% of control levels (P< 0.001; n = 9). ADMA (1 mM; n = 3) reduced intracellular [14C]-citrulline levels to 4 ± 4% of control (P<0.01) but SDMA (1 mM; n = 3) had no effect. 5 The accumulation of endogenously synthesized ADMA in the culture supernatant of SGHEC-7 cells was increased by co-incubation with L-NMMA (1 mM) from 1.98 ± 0.08 to 2.74 ± 0.36 nmol mg-cell protein, an increase of 40%. 6 These results demonstrate that human vasculature possesses an enzyme which has similar properties to dimethylarginase; human endothelial cells and human saphenous vein metabolize L-NMMA to citrulline via a process inhibited by ADMA but not SDMA. The increase in endothelium-derived ADMA following co-incubation with L-NMMA is consistent with competition between ADMA and L-NMMA for dimethylarginase. Inhibition of this enzyme might increase the intracellular concentration of ADMA, an endogenously produced compound that inhibits nitric oxide synthesis.

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The periodate method was found to be most effective for preparing horseradish peroxidase-sheep anti-human and horseradish peroxidase-donkey anti-mouse immunoglobulin (IgG) conjugates. The conjugates were improved by carrying out the oxidation of the enzyme at low pH. Anti-human and anti-mouse IgG-peroxidase conjugates (0·5 mg/mL IgG and 0.7 mg/mL 19G, respectively) were used at 1:15 000 and 1:8000 dilutions, respectively, in a sandwich ELISA to detect human and mouse IgG in buffer or in a growth medium containing 20% foetal calf serum. Using the peroxidase conjugates, it was possible to detect human and mouse IgG at concentrations as low as I ng/mL. The glutaraldehyde method was found to be much more effective than the periodate method for conjugating alkaline phosphatase to the antibodies. The optimum dilutions for anti/human and anti-mouse IgG-alkaline phosphatase conjugates (0.18 mg/mL IgG and 0.3 mg/mL IgG, respectively) in ELISA were 1:500 and 1:1000, respectively. The detection limit with alkaline phosphatase conjugates was 7 ng/ml for human IgG and 4 ng/ml for mouse IgG.

NG,NG-dimethylarginine is an endogenous inhibitor of nitric oxide synthesis produced by endothelial cells and found in the plasma and urine of normal adults. We have examined the ability of NG, NG-dimethylarginine, produced by endothelial cells (SGHEC-7), to regulate the production of nitric oxide by lipopolysaccharide-stimulated mouse macrophage cells (J774.2). Stimulation of SGHEC-7 or J774.2 cells with lipopolysaccharide had no effect on their release of NG,NG-dimethylarginine into the culture supernatant. Stimulation of J774.2 cells with lipopolysaccharide for 24 h significantly stimulated nitric oxide production by J774.2 but not SGHEC-7 cells. When lipopolysaccharide-stimulated J774.2 cells were co-cultured with endothelial cells for 24 h, there was a significant inhibition of nitrite accumulation. The inhibition observed was dependent on the endothelial cell number (12 +/- 5% [mean +/- SEM] following incubation with 0.6 x 105 cells, up to 47 +/- 8% with 4.8 x 105 cells). The inhibitory effect of endothelial cells was prevented by incubation with increasing concentrations of L-arginine; the IC50 was 2.9 +/- 0.6 mM arginine. Western blot analysis indicated that the expression of inducible nitric oxide synthase was not inhibited by co-culture with SGHEC-7 cells. The results presented here demonstrate that NG,NG-dimethylarginine synthesized by endothelial cells may inhibit nitric oxide synthase in adjacent cells and play a role in the regulation of nitric oxide synthesis by macrophages.

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