Cell culture models of human trophoblast II: trophoblast cell lines--a workshop report.
Explore the source record for details and available documents.
Adult↗
Biomedical subjects
G S Whitley
Publications and source records attributed to G S Whitley.
Endogenously produced nitric oxide inhibits endothelial cell growth as demonstrated using novel antisense cell lines.
Proliferation of endothelial cells is a vital component of vascular repair and angiogenesis. The endothelial cell mediator, nitric oxide (NO) has been reported both to inhibit and to promote endothelial cell proliferation. In this study we have generated cell lines which constitutively express antisense RNA to a region of inducible nitric oxide synthase (iNOS) from a murine endothelial cell line, sEnd-1. In response to stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) these antisense cells had no detectable RNA for endogenous iNOS, barely detectable iNOS protein and produced 82% less NO than did the control transfected line. Stimulation of the control transfected line caused significant NO production and inhibition of cell growth whereas for the antisense line, producing little NO in response to stimulation, proliferation remained the same as for unstimulated cells. No differences in cell death were observed between unstimulated and LPS/IFN-gamma stimulated cells. The data presented in this study directly demonstrate that NO derived endogenously from iNOS inhibits proliferation of endothelial cells. This approach overcomes problems in other studies where NO donors or non-isoform specific inhibitors of NO synthase have been used.
Animals↗
Thyrocyte release of asymmetric dimethylarginine does not account for human thyrocyte inhibition of endothelial cell cyclic GMP.
BACKGROUND: The thyroid gland produces and responds to the signalling molecule nitric oxide (NO). The activity of NO synthase (NOS) may be regulated by endogenous NOS inhibitors such as asymmetric dimethylarginine (ADMA). OBJECTIVE: To investigate whether human thyrocytes are capable of regulating NOS activity via the production of ADMA. DESIGN: Human thyrocytes were incubated with human umbilical vein endothelial cells (HUVEC) in order to determine the effect on HUVEC NOS activity. HUVEC cGMP production over a 3-h period was measured as an indicator of NOS activity in the absence and presence of thyrocytes. To determine thyrocyte production of ADMA, samples of conditioned media were analysed by HPLC. RESULTS: The presence of primary human thyrocytes or immortalized human thyrocyte SGHTL-189 cells caused a significant inhibition of both basal (approximately 57% inhibition) and thrombin-stimulated (approximately 42% inhibition) HUVEC cGMP production. Both primary human thyrocytes and SGHTL-189 cells released ADMA (approximately 0. 28 microg per 10(6) thyrocytes over a 3-day period). However, excess L-arginine, the natural substrate for NOS, was unable to overcome thyrocyte inhibition of HUVEC cGMP production. CONCLUSION: These data indicate that human thyrocytes potently reduce endothelial cell cGMP concentrations, and that thyrocytes produce the endogenous NOS inhibitor, ADMA. However, the inhibition of endothelial cGMP is not mediated via thyrocyte production of a competitive NOS inhibitor.
Arginine↗
Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases.
Methylarginines inhibit nitric oxide synthases (NOS). Cellular concentrations of methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3. 18). We have cloned human DDAH and identified and expressed a second novel DDAH isoform (DDAH I and II respectively). DDAH I predominates in tissues that express neuronal NOS. DDAH II predominates in tissues expressing endothelial NOS. These results strengthen the hypothesis that methylarginine concentration is actively regulated and identify molecular targets for the tissue and cell-specific regulation of methylarginine concentration.
Amidohydrolases↗
Hepatocyte growth factor regulates human trophoblast motility and invasion: a role for nitric oxide.
1. The expression of hepatocyte growth factor (HGF) is essential for normal placental development although its function is unknown. In this study we examined the effect of HGF on trophoblast cell motility and invasion of fibrin gels and investigated the possible role of nitric oxide (NO) in this process. 2. The human extravillous trophoblast cell line SGHPL-4 express both the constitutive and inducible isoforms of nitric oxide synthase (NOS). 3. HGF significantly stimulates cell motility in monolayer culture, the invasion of fibrin gels and the production of guanosine 3':5'-cyclic monophosphate (cyclic GMP). 4. Invasion, motility and cyclic GMP production were inhibited by Ng-monomethyl-L-arginine (L-NMMA). 5. Cell motility was also significantly inhibited by the inducible NOS specific inhibitor 1400 W. 6. Neither 8 Br-cyclic GMP nor the NO donor spermine-NO had any significant effect on basal trophoblast cell motility. 7. The data presented in this study demonstrate a direct effect of trophoblast-derived NO synthesis on trophoblast cell function and support the idea that HGF is involved in the regulation of trophoblast invasion through mechanisms that involve the production of NO. However neither exogenous NO nor activation of cyclic GMP-dependent pathways alone are sufficient to stimulate trophoblast cell motility.
Arginine↗
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.
Animals↗
The effects of angiogenic growth factors on extravillous trophoblast invasion and motility.
There is accumulating evidence that deficient trophoblast invasion of the placental bed spiral arteries is crucial to the pathogenesis of pre-eclampsia and intrauterine growth restriction. However, the factors which regulate the process of trophoblast invasion remain unclear. We have investigated whether extravillous trophoblast invasion and motility are mediated by the angiogenic growth factors, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). The SGHPL-4 extravillous trophoblast cell line was utilized. Expression of mRNA for the receptors of VEGF and PlGF (KDR and flt-1) was determined using the reverse transcriptase polymerase chain reaction. An in vitro model of invasion assessed the number and length of trophoblast processes invading into an extracellular matrix. The motility of cells under standard culture conditions was also quantified. The effect of the addition of VEGF and PlGF (+/-heparin) on trophoblast invasion and motility was determined. The effect of VEGF and PlGF (+/-heparin) on SGHPL-4 cell proliferation was assessed by cell counts at 24, 48 and 72 h post-addition of growth factor. The SGHPL-4 cells expressed mRNA for the flt-1 but not the KDR receptor. The addition of VEGF resulted in a significant decrease in the number of trophoblast processes formed (P< 0.02); this effect was not influenced by the addition of heparin. However, there was no effect on the length of processes formed in response to VEGF (+/-heparin). The addition of PlGF had no effect on either the number or the length of processes formed. The addition of VEGF increased the motility of the SGHPL-4 cells (P< 0.002); the addition of heparin prevented this VEGF-induced increase in motility. The addition of PlGF had no effect on SGHPL-4 motility (+/-heparin). Neither growth factor had any effect on the proliferative ability of SGHPL-4 cells. Contrary to our hypothesis, we did not find that the angiogenic growth factors, VEGF and PlGF, mediated the in vitro invasion of trophoblast cells into an extracellular matrix. However, VEGF did increase trophoblast motility. Our findings of an effect of VEGF on trophoblast motility (and possibly invasion) suggests the presence of functional receptors, which can mediate the actions of VEGF. Caution must be exercised before any extrapolation to the in vivo situation, however, it could be speculated that the increased motility in response to VEGF may be an initial response to attract trophoblast cells to the decidua, and that VEGF might then limit the degree to which trophoblast cells invade.
Cell Movement↗
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.
Adult↗
Nitric oxide enhances thyroid peroxidase activity in primary human thyrocytes.
Recent studies have demonstrated the production of the multi-functional messenger molecule nitric oxide (NO) by the thyroid gland. To examine a possible role for NO in thyroid function, we studied the acute and chronic effect of NO donors on thyroid peroxidase (TPO) activity in monolayer cultures of primary human thyrocytes, using a colorimetric assay technique. The presence of either S-nitrosoglutathione (GS-NO) or sodium nitroprusside (SNP) (10(-6)-10(-4) M) at the time of the assay caused a significant increase in TPO activity. Pre-incubation of thyrocytes with 10(-5) M GS-NO for 3 days had no effect on the level of TPO activity when the assay was performed in the absence of NO donors. However, GS-NO pre-incubation significantly enhanced the acute stimulatory effect of GS-NO and SNP on TPO activity. These results suggest a possible role for NO in the regulation of TPO activity and thus thyroid hormone synthesis.
Cells, Cultured↗
Endothelial cell adhesion molecule expression and lymphocyte adhesion to endothelial cells: effect of nitric oxide.
Interactions between circulating leukocytes and vascular endothelial cells are of fundamental importance in controlling normal recirculation and migration of cells into sites of inflammation. Nitric oxide (NO), which is synthesized by vascular endothelial cells, has been reported to decrease the binding of platelets, monocytes, macrophages, and neutrophils to endothelial cells. Using NO donors and inhibitors of the enzyme NO synthase, we found no evidence that physiologically relevant levels of NO alter adhesion of purified lymphocytes to an endothelial cell line derived from human umbilical vein endothelial cells (SGHEC-7). In addition, NO donors did not alter the cell surface expression of VCAM-1, ICAM-1, or E-selectin on SGHEC-7 cells.
Cell Adhesion↗
Inhibition of nitric oxide synthase by antisense techniques: investigations of the roles of NO produced by murine macrophages.
1. An antisense approach to block nitric oxide (NO) synthesis was developed, complementing the widely used chemical inhibitors and overcoming problems associated with their use in studying the roles of NO. 2. Murine macrophage cell lines (J774.2) were generated expressing a 500 bp sequence from inducible NO synthase (iNOS) in either the antisense or sense orientation, driven by the SV40 promoter/enhancer region. 3. Messenger RNA derived from the transfected sequences was detected by a specific cDNA probe. Cells expressing sense and antisense iNOS RNA were characterized further. 4. The antisense lines produced 22-97% less NO than the sense lines on stimulation with lipopolysaccharide (LPS) in the range 1 ng ml-1 - 10 micrograms ml-1, as determined by nitrite production. One antisense line in particular, A10, expressed substantially less iNOS protein on LPS stimulation as determined by western blot analysis. 5. Adhesion of the antisense line, A10, to cytokine-stimulated murine endothelial cells (sEnd.1 line) was significantly higher than adhesion of the sense lines. There was a negative correlation between the amount of NO produced, as determined by nitrite accumulation, and the level of adhesion of the transfected lines. This indicates and anti-adhesive role of NO, produced by macrophages during the 15 min of the assay, in adhesion to endothelial cells. 6. This novel approach allowed the roles of NO in adhesion to be investigated with the substantial advantage that the contribution of NO produced rapidly by activated macrophages could be studied separately from that produced in a continuous manner by endothelial cells. 7. These lines, and the extension of this approach, will be of great use in dissecting the contributions of NO produced by different cell types to its many potential functions.
Animals↗
Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase.
1. Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors NG-monomethyl-arginine and NG,NG-dimethy-L-arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates. 2. S-2-amino-4(3-methylguanidino)butanoic acid (4124W) inhibited the metabolism of [14C]-NG-monomethyl-L-arginine to [14C]-citrulline by rat liver homogenates (IC50 416 +/- 66 microM; n = 9), human cultured endothelial cells (IC50 250 +/- 34 microM; n = 9) and isolated purified dimethylarginine dimethylaminohydrolase. 3. Addition of 4124W to culture medium increased the accumulation of endogenously-generated NG,NG-dimethy-L-arginine in the supernatant of human cultured endothelial cells from 3.1 +/- 0.3 to 5 +/- 0.7 microM (n = 15; P < 0.005). 4. 4124W (1 microM - 1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium-dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 +/- 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L-arginine (100 microM). 4124W reversed endothelium-dependent relaxation of human saphenous vein (19.2 +/- 6.7% reversal of bradykinin-induced relaxation at 1 mM 4124W). 5. These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular contraction of NG,NG-dimethyl-L-arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.
Amidohydrolases↗
The expression and release of adhesion molecules by human endothelial cell lines and their consequent binding of lymphocytes.
We report the characterization of a novel series of human endothelial cell lines (designated SGHEC) regarding the expression and release of adhesion molecules and their binding of lymphocytes. SGHEC expressed significant levels of intercellular adhesion molecule-1 (ICAM-1; CD54) which increased after stimulation with tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or interferon-gamma (IFN-gamma). Vascular cell adhesion molecule-1 (VCAM-1; CD106) and E-selectin (CD62E) were not detectable on unstimulated SGHEC but substantial levels were expressed after stimulation with either TNF alpha or IL-1 beta but not with IFN-gamma. The increased expression of ICAM-1 and VCAM-1 was evident after 4 h stimulation and was even higher after 24 h; E-selectin was maximal after 4 h and returned almost to basal levels by 24 h. Substantial quantities of immunoreactive ICAM-1 and VCAM-1 also accumulated as soluble material in the supernatants of TNF alpha-stimulated SGHEC (VCAM-1 was substantially higher than ICAM-1), but E-selectin remained below the limits of detection. Various quantitative data suggest that this is a controlled release regulated by cytokine and provide support for a physiological function for these soluble molecules. Primary human lymphocytes and lymphoblastoid cell lines expressing lymphocyte function-associated antigen-1 (LFA-1) bound to SGHEC; this binding increased substantially after activation of either cell type. The binding was inhibited by monoclonal antibodies against LFA-1 and, to a lesser extent, ICAM-1, thus demonstrating the importance of these molecules in the observed binding; neither anti-VCAM-1 nor anti-E-selectin antibodies affected the binding. From these various data, we conclude that LFA-1/ICAM-1 interactions are partially responsible for the binding of lymphocytes to endothelial cells. The SGHEC lines should prove useful in investigating leukocyte-endothelial interactions and the mechanism of release of soluble adhesion molecules.
Cell Adhesion↗
Detection of NG,NG-dimethylarginine dimethylaminohydrolase in human tissues using a monoclonal antibody.
Two endogenous inhibitors of nitric oxide synthesis, NG-monomethylarginine (MMA) and NG,NG-dimethylarginine (DMA), are broken down by NG,NG-dimethylarginine dimethylaminohydrolase (DDAH) [EC 3.5.3.18]. This enzymatic activity is widely distributed in rat tissues, and is correlated with the distribution of free MMA and DMA, and the production of nitric oxide. In this study, immunoblotting analyses with a monoclonal antibody directed towards rat kidney DDAH showed that immunoreactive proteins exist in human pancreas, kidney, and liver. Enzyme activity was definitely detected in these tissues, but the specific activity was lower than that in the corresponding rat tissue. These results are interesting in connection with the idea that the enzyme might play a role as a regulator of nitric oxide generation in human tissues.
Amidohydrolases↗
The cell adhesion molecule, VCAM-1, is selectively elevated in serum in pre-eclampsia: does this indicate the mechanism of leucocyte activation?
Explore the source record for details and available documents.
Cell Adhesion Molecules↗
Induction of NG-monomethyl-L-arginine uptake: a mechanism for differential inhibition of NO synthases?
The properties, selectivity, and regulation of NG-monomethyl-L-arginine (L-NMMA) uptake were examined in a human cultured vascular endothelial cell line SGHEC-7 and murine macrophage J774 cells. In both cell types the uptake of L-[14C]NMMA was time and temperature dependent. In endothelial cells L-[14C]NMMA uptake occurred via a single saturable carrier-mediated system with an apparent Kt of 77 +/- 2 microM. In murine macrophage cells a saturable component with an apparent Kt of 51 +/- 6 microM and a nonsaturable component of L-NMMA uptake were identified. In both cell types uptake of L-[14C]NMMA (10 microM) was significantly inhibited in the presence of 100-fold excess of L-NMMA, asymmetric NG,NG-dimethyl-L-arginine (ADMA), symmetric NG,NG-dimethyl-L-arginine (SDMA), L-canavanine, L-arginine, and to a lesser extent D-arginine. Uptake of L-[14C]NMMA was inhibited weakly (approximately 30%) by NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, aminoguanidine, and L-citrulline. Incubation of macrophage J774 cells with lipopolysaccharide (LPS; 1 or 10 micrograms/ml) resulted in the induction of nitric oxide (NO) synthase activity determined by the accumulation of nitrite in the culture medium. In these cells an enhanced uptake of L-NMMA uptake was observed which was prevented by pretreatment with cycloheximide (1 microM) but not dexamethasone (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effects of guanidino and uremic compounds on nitric oxide pathways.
Aminoguanidine, NG-monomethyl-L-arginine (L-NMMA), NGNGdimethyl-L-arginine (asymmetric dimethylarginine; ADMA), creatinine, guanidinosuccinic acid, guanidinoproprionic acid and methylguanidine were added to cultures of activated murine macrophages. Only aminoguanidine, ADMA, L-NMMA and methylguanidine inhibited nitrite production in a dose-dependent manner. In the presence of 100 microM arginine, nitrite production was inhibited by 31.8 +/- 7.1% by ADMA (100 microM; P < 0.01) but the same dose of methylguanidine was without effect. A higher dose of methylguanidine (1000 microM) inhibited nitrite production by 47.6 +/- 5.6% (P < 0.001). The effects of these compounds were also tested on relaxation of human saphenous veins. L-NMMA and ADMA inhibited endothelium-dependent relaxations (EC50 = 4.7 +/- 1.1 microM and 17.9 +/- 4.9 microM, respectively); methylguanidine caused endothelium-independent contractions and reversed the relaxations to bradykinin and sodium nitroprusside (EC50 > 100 microM); aminoguanidine was without effect. The results of this study suggest that of the guanidino compounds which accumulate in renal failure, only ADMA is a potent inhibitor of nitric oxide (NO) synthesis. Methylguanidine is a weak inhibitor of nitric oxide synthesis, whereas the closely related compound aminoguanidine appears to inhibit selectively the inducible isoform of nitric oxide synthase and has no effect on constitutive NO synthase in human veins.
Amino Acid Oxidoreductases↗
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.
Amino Acid Oxidoreductases↗