Regulation of macrophage nitric oxide synthesis by endothelial cells: a role for NG,NG-dimethylarginine.

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.

Plasma concentrations of asymmetric dimethylarginine, a natural inhibitor of nitric oxide synthase, in normal pregnancy and preeclampsia.

OBJECTIVE: We investigated the change in the plasma concentration of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, in early-, mid-, and late-gestation normotensive pregnancies and in gestational age-matched preeclamptic pregnancies and compared the observed changes with changes in blood pressure. STUDY DESIGN: Blood pressure and peripheral plasma asymmetric dimethylarginine concentrations were measured in 20 nonpregnant and 145 pregnant women (33 first-trimester, 50 second-trimester, and 44 third-trimester normotensive pregnancies and 18 third-trimester pregnancies complicated by preeclampsia). In 23 normotensive pregnancies serial plasma asymmetric dimethylarginine concentrations were measured. Statistical analysis was by analysis of variance and linear regression. RESULTS: The blood pressures recorded throughout normal pregnancy were significantly lower than in nonpregnant subjects (p < 0.0001). The mean systolic, diastolic, and average blood pressures were significantly higher in the second-trimester groups than in the first-trimester groups, whereas in the third trimester average and diastolic blood pressures were significantly higher than in the second trimester. The mean (+/-SD) systolic and diastolic blood pressures in third-trimester preeclamptic patients was 157.7 +/- 11.2 and 110.9 +/- 8.5 mm Hg. The mean plasma asymmetric dimethylarginine concentration in nonpregnant women was 0.82 +/- 0.31 micromol/L (significantly higher than in normotensive pregnancy, p < 0.0001). The plasma asymmetric dimethylarginine concentration was also significantly higher in second-trimester than in first-trimester normotensive groups (respectively, 0.52 +/- 0.20 micromol/L and 0.40 +/- 0.15 micromol/L, p = 0.001) and was higher in third-trimester normotensive pregnancy 0.56 +/- 0.23 micromol/L than it was in the second trimester. The asymmetric dimethylarginine concentration in third-trimester preeclamptic patients was 1.17 +/- 0.42 micromol/L (p < 0.0001 vs normotensive third-trimester subjects). CONCLUSIONS: It is well recognized that blood pressure falls in early normal pregnancy and rises again toward term. These studies show that the early fall in blood pressure is accompanied by a significant fall in the plasma asymmetric dimethylarginine concentration. Later in pregnancy circulating concentrations increase and, when pregnancy is complicated by preeclampsia, concentrations are higher than in the nonpregnant state. Our data support a role for both asymmetric dimethylarginine and nitric oxide in the changes in blood pressure seen in both normal and preeclamptic pregnancy.

Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis.

1. The metabolism of methylarginines by human cultured endothelial cells and human saphenous vein was studied in vitro. The human endothelial cell line (SGHEC-7), primary cultures of human umbilical vein endothelial cells (HUVEC) and human saphenous vein were incubated with [14C]-monomethyl-L-arginine ([14C]-L-NMMA) and the cytosolic extract analysed by high performance liquid chromatography (h.p.l.c.) with on-line radioisotope detection. 2. SGHEC-7, HUVEC and human saphenous vein metabolized [14C]-L-NMMA to a compound which co-eluted with [14C]-citrulline. A second metabolite which co-eluted with [14C]-arginine was evident on the radiochromatograms of HUVEC cytosol and saphenous vein extracts. 3. The intracellular levels of [14C]-L-NMMA and [14C]-citrulline in SGHEC-7 cells incubated with [14C]-L-NMMA (0.5 microCi ml-1: 8.9 microM) for 1 h were 113 +/- 22 and 67.6 +/- 6.2 pmol mg-1 cell protein respectively (n = 7). Co-incubation with NGNGdimethyl-L-arginine (ADMA; 100 microM) but not NGNGdimethyl-L-arginine (SDMA; 100 microM) reduced the intracellular level of [14C]-citrulline to 26.3 +/- 3.7 pmol mg-1 cell protein (P < 0.01; n = 3) without reducing the intracellular level of [14C]-L-NMMA. 4. The intracellular levels of [14C]-citrulline in SGHEC-7 cells incubated with [14C]-L-NMMA for 1 h were reduced following co-incubation with NGnitro-L-arginine methylester (L-NAME; 1 mM), NGnitro-L-arginine (L-NOARG; 1 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 to4 +/- 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-derivedADMA 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.

Characterization of human umbilical vein endothelial cell lines produced by transfection with the early region of SV40.

Human umbilical vein endothelial cells were transfected by electroporation with the plasmid pSV3neo, containing the early region of simian virus 40. The resultant "cell lines" divide rapidly (population doubling time of 33 h) for up to 24 passages in medium supplemented with 5% (v/v) serum and 2.5 micrograms/ml endothelial cell growth supplement. Several of these lines express basal levels of ICAM-1 and MHC class I but not MHC class II. One cell line, designated SGHEC-7, retained a number of differentiated endothelial cell functions throughout its lifespan. These functions include increased production of tissue plasminogen activator in response to histamine, thrombin, and PMA. Stability of function and rapid growth over 24 passages endow these cells with a number of advantages over primary cultures. The homogeneous cell population and consistency of response make them ideal for biochemical and immunological studies hereto impractical with primary human endothelial cells. The success of this approach may allow the production of functional cell lines from other vascular beds.

Development of an enzyme-linked immunosorbent assay for caffeine.

A competitive enzyme-linked immunosorbent assay suitable for the measurement of caffeine in plasma and serum has been developed. Sheep immunised with an immunogen prepared by coupling 7-(5-carboxypentyl)1,3-dimethylxanthine to egg albumin produced antibodies with little crossreactivity with the metabolites of caffeine. The enzyme label was prepared by coupling 7-(5-carboxypentyl)-1,3-dimethylxanthine to peroxidase using the mixed anhydride method. The assay, which has a sensitivity of 0.01 mumol/l, permits direct measurement of caffeine in plasma and serum samples. 50 plasma samples measured by ELISA and by an established radioimmunoassay showed a correlation of r = 0.97 (P less than 0.001).