Calcium channel blockade enhances nitric oxide synthase expression by cultured endothelial cells

In a recent study, we found marked increases in nitric oxide (NO) production and endothelial and inducible NO synthase (eNOS and iNOS) expressions with calcium channel blockade in rats with chronic renal failure. This study was undertaken to determine whether enhanced NO production with calcium channel blockade is a direct effect of this therapy or a consequence of the associated hemodynamic and humoral changes. We tested the effects of a calcium channel blocker, felodipine (10(-5), 10(-6), and 10(-7) mol/L), on nitrate and nitrite (NOx) generation, Ca2+-dependent and -independent NOS activity, and eNOS and iNOS protein masses in proliferating and quiescent rat aortic endothelial cells in culture. Compared with vehicle alone, felodipine significantly increased NOx generation, Ca2+-dependent NOS activity, and eNOS protein mass in proliferating and quiescent endothelial cells. Felodipine did not modify the stimulatory action of 10% fetal calf serum on DNA synthesis (thymidine incorporation) and cell proliferation. Ca2+-independent NOS activity and iNOS protein expression were negligible and unaffected by calcium channel blockade. NOx production and NOS expression were greater in proliferating cells than in quiescent cells. Thus, calcium channel blockade upregulates endothelial NO production in vitro, confirming our previous in vivo study. This observation indicates that the reductions in cytosolic [Ca2+] and vasodilation with calcium channel blockade are not only due to inhibition of Ca2+ entry but also to an NO-cGMP mediated mechanism.     

Eicosapentaenoic acid potentiates the production of nitric oxide evoked by interleukin-1 beta in cultured vascular smooth muscle cells

Experiments were designed to determine whether the omega 3-unsaturated fatty acid eicosapentaenoic acid affects the production of nitric oxide evoked by interleukin-1 beta in cultured vascular smooth muscle cells. Incubation of cultured rat or human aortic smooth muscle cells with interleukin-1 beta evoked a time- and concentration-dependent release of nitrite, an oxidation product of nitric oxide. The exposure of cells to interleukin-1 beta in combination with eicosapentaenoic acid caused a significantly larger production of nitrite than that evoked by the cytokine alone. The potentiation by eicosapentaenoic acid was concentration-dependent. The production of nitrite evoked by equieffective concentrations of interleukin-1 beta in the presence and absence of eicosapentaenoic acid were inhibited to a similar extent by nitro L-arginine (an inhibitor of nitric oxide synthase), transforming growth factor beta 1, platelet-derived growth factorAB and thrombin. The addition of interleukin-1 beta-activated smooth muscle cells to suspensions of washed and indomethacin-treated platelets inhibited the aggregation caused by thrombin. The inhibitory effect was enhanced when the smooth muscle cells were exposed to the cytokine in the presence of eicosapentaenoic acid prior to the experiment. Smooth muscle cells exposed to interleukin-1 beta and eicosapentaenoic acid did not affect platelet aggregation in the presence of oxyhemoglobin or methylene blue. Untreated cells or cells exposed to the fatty acid alone did not have such effects. These observations suggest that eicosapentaenoic acid potentiates the production of nitric oxide evoked by interleukin-1 beta in vascular smooth muscle.     

Quantitative measurement for endothelial constitutive nitric oxide synthase in cultured human endothelial cells

The development and validation of family-based alternatives to out-of-home placements for children is an important goal in the mental health services field. The rigorous evaluation of such alternatives, however, can be difficult to accomplish. The purpose of this article is to describe initial barriers experienced during the pilot study of a randomized trial, funded by the National Institute of Mental Health, conducted in a field setting, and the strategies that were used to overcome these barriers. The randomized trial is examining home-based multisystemic therapy as an alternative to the psychiatric hospitalization of youths presenting psychiatric emergencies. The pilot study illuminated the interface of treatment and services research issues, prompting significant changes in the project's clinical procedures, organization, and supervisory processes, as well as in the project's interface with existing community resources for serving youths with serious emotional disturbances.     

Cyclosporin inhibits nitric oxide production in medullary ascending limb cultured cells

BACKGROUND: Nitric oxide (NO) has been shown to play a role in cyclosporin (CsA) nephrotoxicity, but its mechanism of action is still unclear. As inducible NO synthase (iNOS) mRNA has been found to be expressed in rat medullary thick ascending limb (mTAL) cells, we investigated the effects of CsA on NO production in a model of mouse cultured mTAL cells. MATERIALS AND METHODS: The experiments were carried out on sub-cultured cells derived from isolated mTAL microdissected from the kidney of C57BL/6 mice. The identification of the iNOS mRNA in mTAL microdissected segment and cultured cell was confirmed by RT PCR and RsaI digestion. Nitrite (NO2) released by mTAL cells was determined using the modified Griess reagent method and taken as an index of nitric oxide production. The cultured cells were treated with various concentrations of CsA and different signal transduction regulators to assess the effect and possible pathway(s) of action of CsA on NO production in mTAL cells. RESULTS: The basal production of NO by mTAL cells increased by 1.8-fold following incubation with bacterial lipopolysaccaride (LPS). Both aminoguanidine and L-NAME inhibited NO production. CsA (10-300 ng/ml) also inhibited NO production in a dose-dependent manner and prevented its increase induced by LPS. Phorbol 12-myristate 13-acetate (PMA), a PKC stimulator, enhanced slightly the production of NO under basal conditions and prevented the inhibitory action of CsA on NO production. These results suggest that the NO secreted by mouse cultured mTAL cells is dependent on the PKC pathway. CONCLUSION: These results show that CsA may down-regulate the production of NO by cultured mTAL cells expressing iNOS mRNA and that the PKC pathway is involved in this process.     

High glucose inhibits nitric oxide production in cultured rat mesangial cells

Hyperglycemia directly contributes to the development of diabetic nephropathy. A high-serum glucose concentration alters intraglomerular hemodynamics and promotes deposition of extracellular matrix in the kidney. Nitric oxide (NO) is a short-lived messenger molecule that participates in the regulation of renal blood flow, GFR, and mesangial matrix accumulation. Therefore, in this study it was tested whether high glucose directly modulates NO synthesis by rat mesangial cells in vitro by measuring the accumulation of nitrite, the stable metabolite of NO, in the incubation media. Raising the external glucose concentration to 33.3 mM for 24 to 72 h reduced nitrite levels in cell supernatants in a time-dependent manner to a nadir of 14 +/- 3% of the amount in normal glucose media (5.6 mM) (P < 0.01). The decline in NO synthesis in high glucose media was paralleled by decreased cyclic guanosine monophosphate generation; however, there was no alteration in rat mesangial cell expression of inducible NO synthase protein. The suppressive effect of high glucose on NO production by mesangial cells was not modified by inhibition of protein kinase C (H-7), the addition of antioxidants (vitamin E or superoxide dismutase), or a pan-specific anti-transforming growth factor-beta antibody. An elevated ambient glucose caused a time-dependent reduction in mesangial cell L-arginine content. Addition of L-arginine (10 to 20 mM) to external media partially reversed the inhibitory effect of high glucose on mesangial cell NO production in a dose-dependent manner. The highest dose of L-arginine (20 mM) increased mesangial cell L-arginine content to comparable levels in normal and high glucose media. These results indicate that high glucose causes depletion of L-arginine in mesangial cells and compromises NO synthesis. Limitation in the metabolic precursor and other, as yet unidentified, factors act to reduce NO production by mesangial cells in the presence of an elevated ambient glucose level, a change that may play a role in the development of diabetic glomerulosclerosis.     

Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis

BACKGROUND: NO regulates vascular tone and structure, platelets, and monocytes. NO is synthesized by endothelial NO synthase (eNOS). Endothelial dysfunction occurs in atherosclerosis. METHODS AND RESULTS: With a porphyrinic microsensor, NO release was measured in atherosclerotic human carotid arteries and normal mammary arteries obtained during surgery. eNOS protein expression was analyzed by immunohistochemistry. In normal arteries, the initial rate of NO release after stimulation with calcium ionophore A23187 (10 micromol/L) was 0.42+/-0.05 (micromol/L)/s (n=10). In contrast, the initial rate of NO release was markedly reduced in atherosclerotic segments, to 0.08+/-0.04 (micromol/L)/s (n=10, P<0.0001). NO peak concentration in normal arteries was 0.9+/-0.09 micromol/L (n=10) and in atherosclerotic segments, 0.1+/-0.03 micromol/L (n=10, P<0.0001). Reduced NO release in atherosclerotic segments was accompanied by marked reduction of immunoreactive eNOS in luminal endothelial cells, although specific endothelial cell markers (CD31) were present (n=13). Endothelial cells of vasa vasorum of atherosclerotic segments, however, remained positive for eNOS, as was the endothelium of normal arteries. CONCLUSIONS: In clinically relevant human atherosclerosis, eNOS protein expression and NO release are markedly reduced. This may be involved in the progression of atherosclerosis.     

Monocyte-vascular smooth muscle cell interaction enhances nitric oxide production

OBJECTIVE: The adhesive interaction of monocytes and vascular smooth muscle cells (VSMCs) has been suggested to be a regulatory signal in the cellular activation that is involved in the pathogenesis of atherosclerosis. We investigated the effects of monocyte-VSMC interaction on inducible nitric oxide (NO) synthase expression. METHODS: NO production by the cultured cells was determined by measuring the nitrite content of the culture media using the Griess reagent. The expression of inducible NO synthase protein was assayed by Western blotting. RESULTS: Interleukin-1 beta (IL-1 beta) induced nitrite production by VSMCs in a time-dependent manner. The addition of the mouse monocyte cell line J774 to IL-1 beta-stimulated VSMCs further increased nitrite production in a monocyte number-dependent manner. Enhanced nitrite production by coculture was accompanied by increased inducible NO synthase protein accumulation. Addition of tumor necrosis factor-alpha (TNF-alpha) also enhanced IL-1 beta-induced nitrite production by VSMCs, but TNF-alpha showed no effect in the presence of monocytes. Coculture of monocytes and VSMCs in the presence of IL-1 beta secreted substantial amounts of TNF-alpha. The production of nitrite by coculture was markedly inhibited by an anti-TNF-alpha antibody. CONCLUSIONS: The present study revealed that direct cell-to-cell interaction between monocytes and VSMCs enhances NO production, suggesting an important role for their interaction in the pathogenesis of atherosclerosis.     

Dietary fish oil enhances macrophage production of nitric oxide

Melanotic neuroectodermal tumor of infancy (MNTI) is a rare but well-documented lesion of neuroectodermal derivation. Maturation of the neural elements has been reported only occasionally. We report a case of MNTI of the maxilla showing maturation of neural elements to ganglionic cells.     

Estrogen upregulates endothelial constitutive nitric oxide synthase expression in human osteoblast-like cells

The protective effects of estrogen on the cardiovascular system are thought to be mediated, in part, by nitric oxide (NO). Estrogen also has protective effects on bone although the mechanisms of action have not been fully established. Since nitric oxide synthase (NOS) inhibitors have been found to abrogate the protective effect of estrogen on bone in ovariectomised rats, we studied the effects of 17beta-estradiol on NOS activity and NOS mRNA levels in cultured human osteoblast-like cells. 17beta-Estradiol stimulated NOS activity by approximately 2.0 fold and this effect was reversed by the calcium chelator, EGTA, and the NOS inhibitor, L-NMMA, implicating activation of a constitutive, calcium-dependent isoform. Further studies using RT/PCR indicated that only the endothelial nitric oxide synthase (ecNOS) isoform was expressed and RNase protection assays showed that 17beta-Estradiol treatment resulted in a 2.2 fold increase in ecNOS mRNA levels. These findings suggest that estrogen stimulates NOS activity in osteoblastic cells by activation of the ecNOS pathway, and taken together with previous data, is consistent with the possibility that NO may act as a mediator of estrogen actions on bone.     

The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells

Articles in this issue by A. C. Casiglia, A. LoCoco, and C. Zappulla; D. S. Crystal, H. Watanabe, K. Weinfurt, and C. Wu; M. Keller, N. Edelstein, C. Schmid, F. Fang, and G. Fang; and J. J. Han, M. D. Leichtman, and Q. Wang are discussed according to (a) the extent to which cultural variability can be reconciled with developmental theory and (b) the dimensions of cultural variability that matter most for development. It is argued that (a) cross-cultural research needs to be predicted on a model of how culture interacts with the forces that underlie and guide development and (b) the interpretation of cross-cultural research is severely limited without the direct measurement of the specific culture-related variables and processes that are hypothesized to account for diversity in development. Finally, within-culture variability needs to be studied in conjunction with between-culture variability so that a full model of diversity and development can be constructed.     

Aspirin and salicylate enhances the induction of inducible nitric oxide synthase in cultured rat smooth muscle cells

Aspirin and sodium salicylate enhance to a similar extent the production of nitric oxide (NO) in cultured smooth muscle cells following stimulation by interleukin-1beta (IL-1beta). The similar potencies of aspirin and sodium salicylate indicate that acetylation of cellular macromolecules is not essential for the enhancement of NO production. The failure of added prostaglandin E2 (PGE2) or Thromboxane A2 (TXA2) to overcome the effects of aspirin or sodium salicylate indicates that these effects are not simply the result of inhibition of prostaglandin synthesis. The enhancement of NO production occurs dependent of the effects of these agents on induction of inducible nitric oxide synthase (iNOS) expression by IL-1beta. Aspirin and sodium salicylate enhance the induction of iNOS expression by IL-1beta. We previously reported that pretreatment of vascular smooth muscle cells (VSMCs) with high glucose decreased the response of the cells by IL-1beta, that is, the induction of iNOS expression and NO production. We investigated the effect of aspirin and sodium salicylate on the response by IL-1beta of VSMCs pretreated with high glucose (25 mM). Aspirin and sodium salicylate ameliorate the down-regulation of iNOS expression and the decrease of NO production caused by pretreatment with high glucose (25 mM). These results suggest a possible therapeutic role in atherosclerotic disease and diabetes mellitus for aspirin and sodium salicylate by enhancing the level of iNOS expression and NO production.     

Acetate intolerance is mediated by enhanced synthesis of nitric oxide by endothelial cells

The clinical picture of acetate intolerance strictly mimics the nitric oxide (NO) effect, including smooth muscle relaxation and extreme vasodilation. Because acetate induces production of cAMP, which is a powerful stimulus of NO synthase (NOS), we evaluated the effect of different dialysate solutions with and without acetate on NOS activity in endothelial cells (EC). NOS activity of EC, evaluated as H3-citrulline produced from H3-arginine, was modulated by the dialysate composition (e.g., 38 mmol/L acetate produced an increase of 3.2 +/- 0.39-fold compared with basal values (P < 0.0005), and the small amount of acetate (4 mmol/L) in 35 mmol/L bicarbonate solution increased the NOS activity by 2 +/- 0.49-fold (P < 0.05). Conversely, the acetate-free solution produced no effect on NOS activity. The mRNA encoding for inducible NOS was highly expressed in EC incubated with acetate buffer and also with acetate in bicarbonate dialysis buffer. The EC proliferative index was depressed by acetate (P < 0.0005), and tumor necrosis factor synthesis was increased (P < 0.0005) compared with acetate-free buffer. This study suggests that dialytic "acetate intolerance" can be induced by the activation, through cAMP and tumor necrosis factor release, of NOS. The small amount of acetate in bicarbonate dialysate, although capable of inducing in vitro NOS activation, is likely to be rapidly metabolized, whereas the large amounts of this anion in acetate fluids overwhelm metabolism by the liver. Acetate-free dialysate is the only solution that provides an acceptable level of biocompatibility both in vivo and in vitro.     

Superoxide anion production and phagocytosis of crystals by cultured endothelial cells

OBJECTIVE: To show that cultured human umbilical vein endothelial cells (HUVEC) are capable of phagocytizing inflammation-causing crystals and of generating superoxide anion (SOA) during phagocytosis. METHODS: The superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT) dye was used as a measure of SOA production. Phagocytosis was quantified by light microscopy and confirmed by transmission electron microscopy. Cytochrome C was also studied but was found to undergo spontaneous reduction by monosodium urate (MSU) without cells. RESULTS: Crystals of MSU, calcium oxalate, hydroxyapatite, and calcium pyrophosphate dihydrate (CPPD) were phagocytized and, except for the CPPD crystals, induced NBT reduction. Cholesterol and cholesterol monohydrate were neither phagocytized nor did they induce NBT reduction. CONCLUSIONS: Endothelial cells may be a significant source of oxygen radicals in crystal-associated and other arthritides.     

Angiotensin II induces apoptosis of human endothelial cells. Protective effect of nitric oxide

Angiotensin II (Ang II) importantly contributes to the pathobiology of atherosclerosis. Since endothelial injury is a key event early in the pathogenesis of atherosclerosis, we tested the hypothesis that Ang II may injure endothelial cells by activation of cellular suicide pathways leading to apoptosis. Human umbilical venous endothelial cells (HUVECs) were incubated with increasing doses of Ang II for 18 hours. Apoptosis of HUVECs was measured by ELISA specific for histone-associated DNA fragments and confirmed by DNA laddering and nuclear staining. Ang II dose-dependently induced apoptosis of HUVECs. Simultaneous blockade of both the AT1 and AT2 receptor prevented Ang II-induced apoptosis, whereas each individual receptor blocker alone was not effective. Selective agonistic stimulation of the AT2 receptor also dose-dependently induced apoptosis. Ang II-mediated as well as selective AT2 receptor stimulation-mediated apoptosis was associated with the activation of caspase-3, a central downstream effector of the caspase cascade executing the cell death program. Specific inhibition of caspase-3 activity abrogated Ang II-induced apoptosis. In addition, the NO donors sodium nitroprusside and S-nitrosopenicillamine completely inhibited Ang II-induced apoptosis and eliminated caspase-3 activity. Thus, Ang II induces apoptosis of HUVECs via activation of the caspase cascade, the central downstream effector arm executing the cell death program. NO completely abrogated Ang II-induced apoptosis by interfering with the activation of the caspase cascade.     

Up-regulation of inducible nitric oxide synthase and production of nitric oxide by the Swarm rat and human chondrosarcoma

Phospholipids are the major constituents of cell membranes, and have numerous structural and functional roles in the nervous system. Although the metabolic pathways responsible for the syntheses of the phosphatides phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and phosphatidylserine (PtdSer) are well understood, the mechanisms controlling these pathways in neural tissue have not been fully characterized. Recent studies have suggested that the main factors controlling PtdCho and PtdEtn synthesis by the Kennedy cycle tend to be the intracellular levels of key substrates for the biosynthetic enzymes, or changes in the activities of the rate-limiting enzymes. Moreover, different control mechanisms may operate, depending upon the functional state of the tissue.     

Plasma from women with preeclampsia increases endothelial cell nitric oxide production

In preeclampsia, a factor in the maternal circulation alters endothelial function via a reduction in nitric oxide synthesis. We measured the in vitro effects of 2% plasma from women with preeclampsia, compared with 2% plasma from normotensive pregnant women, on cultured endothelial cell nitrite production and nitric oxide synthase activity. On finding differential effects, we measured the effects on cellular viability (assessed by lactate dehydrogenase levels) and performed a time course study. Endothelial cell nitrite production was found to be higher after exposure to plasma from the preeclamptic group than the normotensive pregnant group. The effects of long-term exposure (120 hours) were similar to those of short-term exposure (24 hours). In addition, nitric oxide synthase activity was significantly greater after exposure to preeclamptic plasma than after exposure to normotensive pregnant plasma. No differential effect on cellular viability was found. Contrary to our hypothesis, exposure of endothelial cells to preeclamptic plasma resulted in increased nitric oxide production and nitric oxide synthase activity.