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Nitric oxide enhances PGI2production by human pulmonary artery smooth muscle cells
Prostaglandins, Leukotrienes and Essential FattyAcids (2000) 62(6), 369^378 & 2000 Harcourt Publishers Ltd doi:10.1054/plef.2000.0168, available online at http://www.idealibrary.com on
Nitric oxide enhances PGI2 production by human pulmonary artery smooth muscle cells F. -Q.Wen, K.Watanabe, M.Yoshida Second Department of Internal Medicine, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan
Summary To evaluate the effect of exogenous nitric oxide (NO) and endogenous NO on the production of prostacyclin (PGI2) by cultured human pulmonary artery smooth muscle cells (HPASMC) treated with lipopolysaccharide (LPS), interleukin-1b (IL-1b), tumor necrosis factor a (TNFa) or interferon g (IFNg), HPASMC were treated with LPS and cytokines together with or without sodium nitroprusside (SNP), NO donor, NG-monomethyl-L-arginine (L-NMMA), NO synthetase inhibitor, and methylene blue (MeB), an inhibitor of the soluble guanylate cyclase. After incubation for 24 h, the postculture media were collected for the assay of nitrite by chemiluminescence method and the assay of PGI2 by radioimmunoassay. The incubation of HPASMC with various concentrations of LPS,IL-1b orTNFa for 24 h caused a significant increase in nitrite release and PGI2 production. However,IFNg slightly increased the release of nitrite and had little effect on PGI2 production. Although the incubation of these cells for 24 h with SNP did not cause a significant increase in PGI2 production, the incubation of HPASMC with SNP and10 mg/ml LPS, or with SNPand100 U/ml IL-1b further increase PGI2 production and this enhancement was closely related to the concentration of SNP. However, stimulatory effect of SNP on PGI2 production was not found inTNFa- and IFNg- treated HPASMC. Addition of L-NMMA to a medium containing LPS or IL-1b reduced nitrite release and attenuated the stimulatory effect of those agents on PGI2 production. MeB significantly suppressed the production of PGI2 by HPASMC treated with or without LPS or IL-1b .The addition of SNP partly reversed the inhibitory effect of MeB on PGI2 production by HPASMC.These experimental results suggest that NO might stimulate PGI2 production by HPASMC. Exogenous NO together with endogenous NO induced by LPS or cytokines from smooth muscle cells might synergetically enhance PGI2 production by these cells, possibly in clinical disorders such as sepsis and acute respiratory distress syndrome. & 2000 Harcourt Publishers Ltd
INTRODUCTION Nitric oxide (NO) is formed from L-arginine by NO synthase (NOS) in a wide variety of cells.1 In the cardiovascular system, NO, generated by endothelial NOS (eNOS) in vascular endothelial cells, plays a fundamental role in maintaining non thrombogenic properties of the endothelial surface.2 Bacterial lipopolysaccharide (LPS) and various cytokines induce the expression of inducible NOS (iNOS) by macrophage, vascular smooth
Received 29 February 2000 Accepted 28 March 2000 Correspondence to: Kentaro Watanabe, MD. Present address: 4th Department of Internal Medicine, School of Medicine, Fukuoka University, 45-1-7-Chome Nanakuma, Jonan-ku, Fukuoka, Japan.Tel.: 81 92 8011011 (3376); Fax: 8192 873 8008; E-mail: [email protected]
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muscle cells and lead to the formation of large amounts of NO,1 which plays an important role in the circulatory failure associated with septic and haemorrhagic shock.3,4 Prostacyclin (PGI2) exerts vasodilator and antithrombotic effects and is formed during the bioconversion of arachidonic acid by either constitutive cyclooxygenase (COX-1) or cytokine/LPS-inducible cyclooxygenase (COX2).5,6 The cardiovascular effects exerted by endogenously produced NO are often mediated in conjunction with PGI2. Endothelial cells contain eNOS and COX-17,8 and hence release both NO and PGI2 when stimulated with agonists such as bradykinin or ADP.9 Likewise, iNOS and COX-2 may be also induced in some cells including vascular smooth muscle cells after exposure to LPS or cytokines.3,8,10,11 Previous experiments showed that sodium nitroprusside (SNP) enhances prostaglandin E2 release by cultured rat vascular smooth muscle cells,12
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and that inhibition of NO formation reduces the release of prostanoids in LPS-treated mouse macrophages RAW 264.7, and other cell types.13 However, Keen et al. found that the inhibition of NO release potentiated the coupled release of PGI2, whereas exogenously added NO or SNP inhibits bradykinin-stimulated release of PGI2 by bovine aortic endothelial cells.14 Varying with the species of cell lines, the role of NO on the production of prostaglandins is still controversial. For years, many studies on circulatory hemodynamics have been focused on the role of vascular endothelial cells, a monolayer lining of vessels, which predominantly form PGI2 and NO in blood vessels.8,9,15 During inflammatory conditions such as ARDS and septic shock, endothelial cells could be injured and the underlying vascular smooth muscle cells, the multiple layer structure of vessel wall, acquire the ability to release NO and prostaglandins.15±18 Both the endothelial and smooth muscle cells are crucial for modulating pulmonary vascular resistance and affecting many aspects of pulmonary pathophysiology. However, few data could be found about human pulmonary artery smooth muscle cells. How NO regulates the prostaglandin production in human vascular smooth muscle cells remains unclear. In the present study, we asked whether cultured human pulmonary artery smooth muscle cells (HPASMC) could produce enough NO to affect PGI2 production by HPASMC when exposed to LPS or cytokine and, if so, what are the roles of endogenous and exogenous NO on regulating PGI2 production by HPASMC? Furthermore, we asked whether the action of NO depends on cGMP pathway. To address these questions, we have investigated the effect of NO donor, NOS inhibitor, and also guanylate cyclase inhibitor on PGI2 production by HPASMC treated with or without LPS and cytokines. MATERIALS AND METHODS
Materials Dulbecco's modified Eagle's medium (DMEM) was purchased from GIBCO (Grand Island, NY). Fetal calf serum (FCS) was obtained from Whittaker Bioproducts (Walkersville, MD). Penicillin, streptomycin, amphotericin B, trypsin, SNP, NG-monomethyl-L-arginine (L-NMMA) and LPS from E. coli (O55:B5) were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human interleukin-1b (IL-1b) and interferong (IFNg) were generously provided by Otsuka Pharmaceutical Co. (Tokyo, Japan). Recombinant human tumor necrosis factor a (TNFa) was obtained from Genzyme (Cambridge, MA). Monoclonal mouse anti-human a-smooth muscle actin antibody was obtained from Dako A/S (Glostrup, Denmark). [3H]-6-keto-prostaglandin F1a (6-keto-PGF1a) was
obtained from New England Nuclear (Boston, MA). 6keto-PGF1a and rabbit anti-6-keto-PGF1a antiserum were generously provided by Ono Pharmaceutical Co. (Osaka, Japan). Methylene blue trihydrate (MeB), sodium nitrite, acetic acid and potassium iodide were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Culture of HPASMC HPASMC were cultured as previously described.19,20 Briefly, specimens of human pulmonary artery were obtained at autopsy and placed in phosphate-buffered saline. Endothelial cells and adventitia were carefully removed. The layers of smooth muscle were then cut into pieces measuring 363 mm or less, placed in 35 mm petri dishes, and cultured in DMEM that contained 1.6 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml amphotericin B, and 10% FCS at 378C under 5% CO2 in air. The culture medium was changed at intervals of 72±96 h. When the cultured cells began to grow, the explants were removed. After confluence was achieved, cells were passaged by harvesting with 0.02% trypsin and 0.01% EDTA. HPASMC at passages 3Ð5 were used in these experiments. Cultured cells were identified as smooth muscle cells by staining with mouse antihuman a-smooth muscle actin monoclonal antibody.17 All treatments described below were carried out in the presence of 10% fetal calf serum.
Effect of cytokines and LPS on the production of nitrite and PGI2 by HPASMC HPASMC were incubated with various concentrations of LPS (0±10 mg/ml) and cytokines, i.e., IL-1b (0±2000 U/ml), TNFa (0±5000 U/ml) and IFNg (0±1000 U/ml) as the previous experiments showed that LPS (0±10 mg/ml), IL-1b (0±3000 picomolar), TNFa (0±5000 U/ml) and IFNg (0±1000 U/ml) alone or the combination of these agents promoted NO production in systemic and pulmonary artery smooth muscle cells;11,12,21±23 and the very high serum concentrations of cytokines and endotoxin were found in early stages of ARDS and multiple organ dysfunction syndrome.24±26 When HPASMC grew confluent in 35 mm petri dishes, the culture medium was replaced and cultured with fresh culture medium containing various concentrations of LPS, IL-1b, TNFa or IFNg for 24 h. After 24 h, the culture medium was collected for the assays of nitrite and PGI2.
Effect of SNP and L-NMMA on the production of PGI2 by HPASMC To examine the effect of exogenous and endogenous NO on LPS-, IL-1b-, TNFa- or IFNg- induced production of PGI2
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Nitric oxide enhances PGI2 production
by HPASMC, cells were incubated with LPS (10 mg/ml), IL-1b (100 or 200 U/ml), TNFa (500 U/ml) and IFNg (200 U/ ml) together with various concentrations of SNP or L-NMMA for 24 h. After 24 h, culture medium was collected for the assay of PGI2.
Effect of MeB on the production of PGI2 by HPASMC To determine the effects of MeB, a soluble inhibitor of guanylate cyclase, on PGI2 production by HPASMC, confluent cells were incubated with the medium containing MeB with or without 10 mg/ml LPS or 100 U/ml IL-1b for 24 h. Cells were also incubated with medium containing MeB in the presence of SNP and LPS or IL-1b for 24 h. The postculture medium was then collected for PGI2 assay.
Measurement of PGI2 The levels of 6-keto-PGF1a, the stable end product of PGI2, were determined by radioimmunoassay as previously described.19,20 The cross reactivity of rabbit antiserum to 6-keto-PGF1a was 100% with 6-keto-PGF1a, 5.0% with PGE1, 0.76% with PGE2, 0.82% with PGF2a and 0.01% with thromboxane B2.
Nitrite assay Nitrite in the samples was converted to NO under acidic conditions, and the NO was measured with a sensitive chemiluminescence analyzer.27,28 Briefly, samples collected were diluted three times with deionized water and incubated at room temperature for 1 h. Then samples were injected into the purge vessel of NO analyzer, NOATM 280 (Sievers, Boulder, CO) containing 1% potassium iodide in glacial acetic acid which converts nitrite to NO. NO released from the reaction vessel was measured by a chemiluminescence. Standard curves were drawn by known amounts of nitrite in each experiment.
Measurement of lactate dehydrogenase (LDH) release After incubating HPASMC with various concentrations of cytokines, LPS and SNP or the combination of SNP and LPS or cytokine for 24 h, LDH in the postculture medium was measured using a LDH commercial kit (Sigma, St. Louis, MO). The LDH values were corrected by subtracting the LDH activity measured in the medium before culture. The maximal release of LDH was determined by scraping and sonicating the control cells, and measuring LDH in the medium. & 2000 Harcourt Publishers Ltd
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Statistical analyses Data were expressed as mean+SEM. Data were evaluated by one-way ANOVA in the experiments examining dose-dependent effects of LPS, cytokines, SNP or LNMMA on the production of PGI2, nitrite and on the release of LDH. If the overall F statistic was significant at the 0.05 level, subsequent intergroup significance testing was performed by the Scheffe's F-test. A two-tailed unpaired Student's t-test was also performed to compare the levels of nitrite, 6-keto-PGF1a, and LDH in cells treated with or without LPS, cytokines or those reagents examined. A level of p50.05 was accepted as statistically significant.
RESULTS Phase-contrast microscopy revealed that confluent HPASMC were aligned in parallel and exhibited the typical `hill and valley' pattern of intertwined, overlapping cells similar to that of cultures of vascular smooth muscle cells. These cultured cells were positively stained by mouse anti-human a-smooth muscle actin monoclonal antibody. No positive staining was observed in control cells. Fig. 1 shows nitrite release from HPASMC measured by chemiluminescence method. HPASMC were treated with various concentrations of LPS, IL-1b, TNFa or IFNg for 24 h. LPS, IL-1b and TNFa induced significant increase in nitrite release from HPASMC ( p50.01, respectively, one way ANOVA). Nitrite level was slightly higher in HPASMC treated with 1000 U/ml of IFNg than in control HPASMC, but without dose-dependency. When HPASMC were incubated with 1 mg/ml of LPS, 20 U/ml of IL-1b or 50 U/ml of TNFa for 24 h, PGI2 release by HPASMC was enhanced ( p50.01, 50.01 and 50.05, respectively, unpaired Student's t-test) (Fig. 2). However, incubation of HPASMC with 1000 U/ml of IFNg or higher concentration up to 1000 U/ml for 24 h did not augment PGI2 production. Fig. 3 shows PGI2 production by HPASMC that were treated with various concentrations of SNP and 10 mg/ml of LPS (Fig. 3a), 100 U/ml of IL-1b (Fig. 3b), 500 U/ml of TNFa (Fig. 3c) and 200 U/ml of IFNg (Fig. 3d) for 24 h. Incubation with 10, 20 and 50 mM of SNP without LPS or cytokines did not induce significant increase in PGI2 production when compared with control cells (data not shown). Similar to the results in Fig. 2, the levels of 6-ketoPGF1a released from HPASMC treated with 10 mg/ml of LPS, 100 U/ml of IL-1b and 500 U/ml of TNFa significantly exceeded those in the absence of LPS, IL-1b or TNFa. Addition of SNP to the medium containing LPS or IL-1b further enhanced the stimulated production of PGI2 by HPASMC treated with LPS or IL-1b, and these enhancements were closely related to the dosages of
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Fig. 1 Effects of cytokines and LPS on nitrite release by HPASMC. Confluent HPASMC were incubated with the medium containing increasing concentrations of LPS (a), IL-1b (b),TNFa (c) and IFNg (d) for 24 h. Nitrite released in culture medium was then measured by chemiluminescence method. Data are shown as means+SEM of 8^28 replicate samples in 2^5 experiments. Numbers in parentheses indicate those of samples tested. One way ANOVA showed the significantly increased levels of nitrite in LPS-, IL-1b- and TNFa- treated HPASMC (p50.01).*:p50.05 shows significance compared with control levels in post hoc Scheffe's F-test.
SNP used ( p50.01, respectively, one-way ANOVA). In spite of the stimulatory effect of TNFa on PGI2 production, addition of SNP did not augment PGI2 production in HPASMC incubated with TNFa. Fig. 4 shows PGI2 production by HPASMC that were treated with various concentrations of L-NMMA with the addition of 10 mg/ml of LPS (Fig. 4a) or 200 U/ml of IL-1b (Fig. 4b) for 24 h. Incubation with various concentrations of L-NMMA alone did not affect production of PGI2 by HPASMC (data not shown). Enhanced production of PGI2
by LPS- and IL-1b-treated HPASMC was significantly attenuated, when L-NMMA was added to the incubation medium containing LPS or IL-1b ( p50.01, one way ANOVA) (Fig. 4a, b). The addition of 0.5 mM L-NMMA caused 48.7 and 37% decrease in PGI2 production by LPS- and IL-1b-treated cells, respectively; and incubation with 5 mM L-NMMA caused further decrease in PGI2 production. The effect of MeB, SNP, LPS or combination of those agents on PGI2 production by HPASMC is shown in
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3. L-NMMA suppressed PGI2 production by HPASMC treated with LPS or cytokines 4. MeB inhibited LPS- and IL-1b-induced increase in PGI2 production. Addition of SNP partly reversed the suppresive effect of MeB on PGI2 production 5. Less concentrations of LPS or cytokines were needed for the efficient production of PGI2 than for the nitrite release by HPASMC.
Fig. 2 Effects of LPS, IL-1b,TNFaand IFNg on PGI2 production by HPASMC. HPASMC were treated with medium containing1 mg/ml of LPS, 20 U/ml of IL-1b, 50 U/ml of TNFaand100 U/ml of IFNg or culture medium alone for 24 h. After 24 h, the postculture media were collected and assayed for 6-keto-PGF1aas described in`Materials and Methods'. Data are expressed as mean+SEM of 8^14 samples in two experiments. Numbers in parentheses indicate those of samples tested.**p50.01and *p50.05, compared with control HPASMC, unpaired Student's t -test.
Fig. 5a. Similar experiments were done after the substitution of IL-1b for LPS (Fig. 5b). MeB inhibited PGI2 production by HPASMC and also markedly suppressed LPS- or IL-1b-induced enhancement of PGI2 production when HPASMC were treated with MeB and LPS or IL-1b; the addition of 20 mM SNP partly reversed the inhibitory effect of MeB on the production of PGI2 by HPASMC ( p50.05±0.01, unpaired Student's t -test). During incubation with various concentrations of cytokines, LPS and SNP or combination of those agents there was no morphological change in HPASMC by phase contrast microscopy. A 24 h incubation with any cytokine, LPS and SNP or the combination of these compounds in the present study did not induce a significant increase in LDH release from HPASMC when compared with the control (Tables 1 and 2).
DISCUSSION Our findings in the present experiments are: 1. Both SNP and L-NMMA had no effect on PGI2 production by control HPASMC 2. SNP markedly augmented PGI2 production by HPASMC treated with LPS or cytokines & 2000 Harcourt Publishers Ltd
These findings suggest that enhanced production of PGI2 by HPASMC treated with LPS and IL-1b may be caused not only by exogenous NO from NO donor but also by endogenous NO produced from HPASMC. Previous experiments show controversial data on the effect of NO in the regulation of prostaglandin production. NO has been reported to activate,10,12,13,29±31 inhibit32±34 or have no effect35 on the activity of COX, a key rate-limiting enzyme in the synthesis of eicosanoids from arachidonic acid, in different kinds of cell lines. In our experiments, no apparent potentiation of SNP on basal PGI2 production by HPASMC was noted. Only in the presence of LPS or IL-1b together with exogenous NO from SNP, PGI2 production by HPASMC was significantly enhanced. Moreover, LPS- and cytokine-induced production of PGI2 by HPASMC was inhibited by L-NMMA. These results show that either exogenous or endogenous NO plays an important role in the enhancement of PGI2 production by HPASMC in spite that the increased release of nitrite by LPS- or IL-1b-treated HPASMC was not as marked as those noted in vascular smooth muscle cells originated from animal species.11,12 The enhanced production of PGI2 by NO might reflect the increased activity of COX, possibly COX-2, for the basal PGI2 production by HPASMC was not affected by SNP in the present study. Vascular smooth muscle cells constitutively produce prostaglandins by COX-1 but do not release NO for lack of eNOS.6 Although both NO and prostaglandins were produced by HPASMC treated with LPS or cytokines, less concentrations of LPS or cytokines were needed for the efficient production of PGI2 than for the nitrite production. Several possibilities are raised for its mechanism: 1) COX-2 is more sensitively induced than iNOS by LPS or cytokines; 2) the COX metabolites have been conversely reported to influence iNOS induction. PGE2 and iloprost, a stable PGI2 analog, at micromolar concentrations suppressed the iNOS induction in a murine macrophage cell line J774.36 PGD2 dose-dependently inhibited IL-1binduced NO production and also the expression of iNOS mRNA and protein in cultured rat vascular smooth muscle cells.37 NO acts by cGMP-independent and -dependent mechanisms. As the relaxation of vascular smooth muscle and inhibition of platelet aggregation are resulted from an elevation of cGMP,38,39 we tested whether the effect of
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Fig. 3 Effect of SNPon the production of PGI2 by LPS- and cytokine-treated HPASMC.HPASMC were incubated with medium containing10 mg/ml of LPS (a),100 U/ml of IL-1b (b), 500 U/ml of TNFa (c) and 200 U/ml of IFNg (d) with increasing concentrations of SNP (0,10, 20, and 50 mM) for 24 h. After 24 h, postculture media were collected for the assay of 6-keto-PGF1a. Data are expressed as mean+SEM of 8^15 samples in 2^3 experiments. Numbers in parentheses indicate those of samples tested. One way ANOVA showed the significantly increased levels of PGI2 in LPS- and IL-1b-treated HPASMC (p50.01).*p50.05, compared with no SNP, Scheffe's F-test.
NO on PGI2 production is cGMP-dependent in HPASMC. Salvemini et al. showed that MeB blocked the increase in cGMP levels in human fetal fibroblasts exposed to SNP or glyceryl trinitrite (GTN) but it did not affect the ability of SNP or GTN to augment PGE2 release by arachidonic acid.13 MeB diminished A23187-stimulated PGI2 production by bovine coronary endothelial cells, but another guanylate cyclase inhibitor, LY83583, had no effect on PGI2 production.31 Besides, 8-bromo-cGMP, a cGMP
analogue, did not increase eicosanoid production from bovine coronary microvascular endothelial cells and rat aortic smooth muscle cells.12,31 These findings suggest that NO may induce prostaglandin synthesis via a cGMPindependent pathway. However, our experimental results showed that the production of PGI2 by both control cells and cells treated with LPS or IL-1b was markedly inhibited by MeB. Moreover, the inhibitory effect of MeB on PGI2 production was partly reversed by SNP. Okamura et al.
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Fig. 4 Effect of L-NMMA on PGI2 production by LPS- and IL-1b-treated HPASMC. HPASMC were incubated with medium containing10 mg/ml of LPS (a) and 200 U/ml of IL-1b (b) with increasing concentrations of L-NMMA (0, 0.05, 0.5 and 5 mM) for 24 h. After 24 h, postculture media were collected and then assayed for 6-keto-PGF1a. Data are expressed as mean+SEM of13^14 samples in 3 experiments. Numbers in parenthesis indicate those of samples tested. One way ANOVA showed the significantly suppressed levels of PGI2 in LPS- and IL-1b-treated HPASMC (p50.01).*p50.05, compared with no L-NMMA, Scheffe's F-test.
Fig. 5 Effect of MeB on PGI2 production by LPS- and IL-1b-treated HPASMC. HPASMC were treated with10 mM MeB,10 mg/ml of LPS shown in panel a (IL-1b: panel b), or the same concentrations of LPS (panel a) (IL-1b: panel b) and MeB together with or without 20 mM SNP for 24 h. After the incubation, postculture media were then assayed for 6-keto-PGF1a. Data are expressed as mean+SEM of 8^13 samples in 2^3 experiments.Numbersin parenthesis indicate those of samples tested.*p50.05, **p50.01vs LPSMeB or IL-1bMeB; # P50.05, vs control or MeB SNP, unpaired Student's t-test.
found that arachidonic acid-stimulated production of PGI2 released from dog renal arterial strips was significantly reduced by the treatment with MeB.40 In addition to suppressive effect on soluble guanylate cyclase, MeB is & 2000 Harcourt Publishers Ltd
known to generate oxy-radicals, which inactivate NO, and also act as a direct inhibitor of NOS.41,42 The inhibitory effect of MeB on PGI2 production by HPASMC might be not only due to the possible inhibition of the endogenous
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NO production but also due to the clearance of exogenous NO, since SNP, a NO donor, partly reversed the inhibitory effect of MeB on PGI2 production. However, the inhibitory effect of MeB on PGI2 production by control cells in which NO is not involved suggests that NO might regulate the production of PGI2 in HPASMC via a cGMP-dependent pathway. Interestingly, TNFa increased nitrite release and PGI2 production from HPASMC, but PGI2 production in TNFatreated HPASMC was not augmented by the coincubation with SNP. TNFa enhances NO production in vascular smooth muscle cells by inducing iNOS. However, it downregulates eNOS,43 and decreases pulmonary artery endothelial nitrovasodilator activity by the generation of oxy-radicals which react with NO to form the peroxynitrite radical (ONOO7). ONOO7 has the capability to cause cell dysfunction and cell death, and reduce the vasodilator activity of NO.44 Whether such effects would also impair the synthesis of PGI2 and the contractility of the smooth muscle cells needs investigating. Table 1 Effect of cytokines and LPS on the release of LDH from HPASMC Treatment
LDH (%)
Control
3+1 (18)
LPS (mg/ml) 0.1 1.0 10.0
6+2 (5) 5+3 (5) 4+2 (10)
IL-1b (U/ml) 20 200 2000
6+3 (5) 6+2 (5) 8+3 (5)
TNFa (U/ml) 50 500 5000
3+3 (5) 7+2 (10) 5+2 (5)
IFNg (U/ml) 10 100 1000
5+3 (5) 5+2 (5) 5+3 (5)
All data are expressed as percentage to maximum release (%) (mean+SEM). Numbers tested are indicated in parentheses. Table 2
Despite a lot of in vitro and in vivo studies on the interaction between NO and prostaglandins, most of these data came from animal experiments and few were directly related to human pulmonary circulation.3,4,15,21,29,31,45 Meyrick et al.46 reported that cultured human pulmonary artery endothelial cells (HPAEC) responded to LPS (0.001±10 mg/ml) to produce PGI2 with modest cellular cytotoxicity. The level of 6-keto-PGF1a produced by HPAEC during 24 h incubation with normal medium was about 250 pg/ml and HPAEC treated with 1 mg/ml of LPS for 24 h showed three-fold increase in PGI2 production. In contrast, the level of 6-keto-PGF1a by control HPASMC during 24 h incubation was about 750 pg/ml, which was a three-fold increase compared with that of HPAEC.20 HPASMC incubated with the same serotype of LPS (O55:B5) as Meyrick et al. used for 24 h also showed a three-fold increase in PGI2 production.20 Based on these experimental results, pulmonary artery smooth muscle cells as a major component of the vessel wall may play important roles in either physiological or pathological conditions such as hypoxic vasoconstriction, pulmonary hypertension and ARDS.45 The present observations show the importance in smooth muscle cells as well as endothelial cells in regulating the pulmonary vascular tonus. In conclusion, our results suggest that both exogenous and endogenous NO from smooth muscles cells enhance PGI2 production from HPASMC treated with LPS or IL-1b, and PGI2 production by HPASMC was suppressed by guanylate cyclase inhibitor. In pathophysiological states involving endotoxin and cytokines, pulmonary vascular tension and blood distributions in the lung are probably affected by the increased production of prostaglandins which might be further augmented by NO. Further study is still needed to elucidate the mechanism of the effect of NO on PGI2 production by pulmonary artery smooth muscle cells.
ACKNOWLEDGEMENT This work was supported by a Grant-in-Aid for Scientific Research (C), No. 09670636 from the Ministry of Education, Science, and Culture of Japan.
Effect of SNP on the release of LDH from HPASMC SNP (mM)
Control LPS10 mg/ml IL-1b100 U/ml TNFa 500 U/ml IFNg 200 U/ml
0
10
20
2+1 5+3 3+3 7+3 5+3
4+2 5+4 3+2 3+2 4+3
5+2 3+2 5+4 4+2 5+3
50 2+1 4+3 7+4 5+3 5+3
All data are expressed as percentage to maximum release of LDH(%) (mean+SEM); n=10 in control cells and cells treated with SNPalone; n=5 in the rest groups. Prostaglandins, Leukotrienes and Essential FattyAcids (2000) 62(6), 369^378
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REFERENCES 1. Forstermann U., Schmidt H. W., Pollack J. S. et al. Isoforms of nitric oxide synthase. Characterization and purification from different cell types. Biochem Pharmacol 1991; 42: 1849±1857. 2. Moncada S., Palmer R. M. I., Higgs E. A. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Res 1991; 43: 109±142. 3. Szabo C., Mitchell J. A., Thiemermann C. et al. Nitric oxide mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock. Br J Pharmacol 1993; 108: 786±792. 4. Thiemermann C., Szabo C., Mitchell J. A. et al. Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide. Proc Natl Acad Sci USA 1993; 90: 267±271. 5. Wu K. K. Cyclooxygenase 2 induction: molecular mechanism and pathophysiologic roles. J Lab Clin Med 1996; 128: 242±245. 6. Maclouf J., Folco G., Patrono C. Eicosanoids and iso-eicosanoids: constitutive, inducible and transcellular biosynthesis in vascular disease. Thromb Haemost 1998; 79: 691±705. 7. Sessa W. C., Harrison J. K., Barber C. M. et al. Molecular cloning and expression of cDNA encoding endothelial cell nitric oxide synthase. J Biol Chem 1992; 267: 15274±15276. 8. Mitchell J. A., Akarasereenont P., Thiemermann C. et al. Selectivity of nonsteroid anti-inflammatory drugs as inhibitors of constitutive and inducible cyclo-oxygenase. Proc Natl Acad Sci USA 1993; 90: 11693±11697. 9. Mitchell J. A., De Nucci G., Warner T. D. et al. Different patterns of release of endothelium derived relaxing factor and prostacyclin. Br J Pharmacol 1992; 105: 485±489. 10. Corbett J. A., Kwon G., Turk J. et al. IL-1b induces the coexpression of both nitric oxide synthase and cyclooxygenase by islets of Langerhans: activation of cyclooxygenase by nitric oxide. Biochemistry 1993; 32: 13767±13770. 11. Koide M., Kawahara Y., Tsuda T. et al. Cytokine-induced expression of an inducible type of nitric oxide synthase gene in cultured vascular smooth muscle cells. FEBS 1993; 318: 213±217. 12. Inoue R., Shigeto M., Koh E. et al. Nitric oxide mediates interleukin-1 prostaglandin E2 production by vascular smooth muscle cells. Biochem Biophys Res Commun 1993; 194: 420±424. 13. Salvemini D., Misko T. P., Masferrer J. et al. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci USA 1993; 90: 7240±7244. 14. Keen M., Pickering S. A. W., Hunt J. A. Modulation of bradykininstimulated release of prostacyclin from endothelial cells. Br J Pharmacol 1990; 101: 524. 15. Bishop-Bailey D., Larkin S. W., Warner T. D. et al. Characterization of the induction of nitric oxide synthase and cyclo-oxygenase in rat aorta in organ culture. Br J Pharmacol 1997; 121: 125±133. 16. Royall J. A., Berkow R. L., Beckman J. S. et al. Tumor necrosis factor and interleukin 1 a increase vascular endothelial permeability. Am J Physiol 1989; 257: L399±L410. 17. Fullerton D. A., Mclntyre R. C. Jr., Hahn A. R. et al. Dysfunction of cGMP-mediated pulmonary vasorelaxation in endotoxininduced acute lung injury. Am J Physiol 1995; 268: L1029±L1035. 18. Sheridan B. C., Mclntyre R. C., Agrafojo J. et al. Neutrophil depletion attenuates endotoxin-induced dysfunction of cGMPmediated pulmonary vasorelaxation. Am J Physiol 1996; 271: L820±L828. 19. Watanabe K., Ishida T., Yoshitomi F. et al. Fibrinogen degradation products influence PGI2 synthesis by cultured porcine aortic endothelial and smooth muscle cells. Atherosclerosis 1984; 51: 151±161.
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20. Wen F. Q., Watanabe K., Yoshida M. Inhibitory effects of interleukin-6 on release of PGI2 by cultured human pulmonary artery smooth muscle cells. Prostaglandin 1996; 52: 93±102. 21. Nakayama D. K., Geller D. A., Lowenstein J. et al. Cytokines and lipopolysaccharide induce nitric oxide synthase in cultured rat pulmonary artery smooth muscle. Am J Respir Cell Mol Biol 1992; 7: 471±476. 22. Durante W., Liao L., Iftikhar I. et al. Differential regulation of Larginine transport and nitric oxide production by vascular smooth muscle and endothelium. Circ Res 1996; 78: 1075±1082. 23. Li S., Fan S. X., McKenna T. M. Vascular smooth muscle cells on matrigel as a model for LPS-induced hypocontractility and NO formation. Am J Physiol 1997; 272: H576±H584. 24. Meduri G. U., Headley S., Kohler G. et al. Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Chest 1995; 107: 1062±1073. 25. Roumen R. M., Hendriks T., van der Ven-Jongekrijg J. et al. Cytokine patterns in patients after major vascular surgery, hemorrhagic shock, and severe blunt trauma. Relation with subsequent adult respiratory distress syndrome and multiple organ failure. Ann Surg 1993; 218: 769±776. 26. Casey L. C., Balk R. A., Bone R. C. Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome. Ann Intern Med 1993; 119: 771±778. 27. Archir S. Measurement of nitric oxide in biological models. FASEB J 1993; 7: 349±360. 28. Robbins R. A., Jinkins P. A., Bryan T. W. et al. Methotrexate inhibition of inducible nitric oxide synthase in murine lung epithelial cells in vitro. Am J Respir Cell Mol Biol 1998; 18: 853±859. 29. Akarasereenont P., Bakhle Y. S., Thiemermann C. et al. Cytokinemediated induction of cycloxygenase-2 by activation of tyrosine kinase in bovine endothelial cells stimulated by bacterial lipopolysaccharide. Br J Pharmacol 1995; 115: 401±408. 30. Salvemini D., Currie M., Mollace V. Nitric oxide-mediated cyclooxygenae activation. J Clin Invest 1996; 97: 2562±2568. 31. Davidge S. T., Baker P. P., McLaughlin M. K. et al. Nitric oxide produced by endothelial cells increases production of eicosanoids through activation of prostaglandin H synthase. Cir Res 1995; 77: 274±283. 32. Matthews J. S., McWilliams P. J., Key B. J. et al. Inhibition of prostacyclin release from cultured endothelial cells by nitrovasodilator drugs. Biochim Biophys Acta 1995; 1269: 237±242. 33. Stadler J., Harbrecht B. G., Di Silvio M. et al. Endogenous nitric oxide inhibits the synthesis of cyclooxygenase products and interleukin-6 by rat Kupffer cells. J Leukot Biol 1993; 53: 165±172. 34. Swierkosz T., Mitchell J. A., Warner T. D. et al. Co-induction of nitric oxide synthase and cyclo-oxygenase: interaction between nitric oxide and prostanoids. Br J Pharmacol 1995; 114: 1335±1342. 35. Tsai A. L., Wei C., Kulmacz R. Interaction between nitric oxide and prostaglandin H synthase. Arch Biochem Biophys 1994; 313: 367±372. 36. Morotta P., Sautebin L., DiRosa M. Modulation of the induction of NO synthase by eicosanoids in the murine macrophage cell line J774. Br J Pharmacol 1992; 107: 640±641. 37. Nagoshi H., Uehara Y., Kanai F. et al. Prostaglandin D2 inhibits inducible nitric oxide synthase expression in rat vascular smooth muscle cells. Cir Res 1998; 82: 204±209. 38. Lowenstein C. J., Dinerman J. L., Synder S. H. Nitric oxide: a physiologic messenger. Am Intern Med 1994; 120: 227±237.
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39. Kibourn R. Nitric oxide and shock. In: Bone R, ed. Disease-aMonth. St. Louis, MO: Mosby-Year Book Inc., 1997; 298±305. 40. Okamura T., Yoshida K., Toda N. Suppression by methylene blue of prostaglandin I2 synthesis in isolated dog renal arteries. J Pharmacol Exp Ther 1990; 254: 198±203. 41. Mayer B., Brunner F., Schmidt K. Inhibition of nitric oxide synthesis by methylene blue. Biochem Pharmacol 1993; 45: 367±374. 42. Marczin N., Tekeres M., Salzman A. et al. Methylene blue infusion in septic shock. Crit Care Med 1996; 23: 1936±1938. 43. Yoshizumi M., Perrella M. A., Burnett J. C. Jr. et al. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Cir Res 1993; 73: 205±209.
44. Johnson A., Phelps D. T., Ferro T. J. Tumor necrosis factor-a decreases pulmonary artery endothelial nitrovasodilator via protein kinase C. Am J Physiol 1994; 267: L318±L325. 45. Johnson B. A., Lowenstein C. J., Schwartz M. A. et al. Culture of pulmonary microvascular smooth muscle cells from intraacinar arteries of the rat: Characterization and inducible production of nitric oxide. Am J Respir Cell Mol Biol 1994; 10: 604±612. 46. Meyrick B., Berry Jr. L. C. Christman B. W. Response of cultured human pulmonary artery endothelial cells to endotoxin. Am J Physiol 1995; 268: L239±L244.
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Nitric oxide enhances PGI2 production by human pulmonary artery smooth muscle cells F. -Q.Wen, K.Watanabe, M.Yoshida Second Department of Internal Medicine, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan
Summary To evaluate the effect of exogenous nitric oxide (NO) and endogenous NO on the production of prostacyclin (PGI2) by cultured human pulmonary artery smooth muscle cells (HPASMC) treated with lipopolysaccharide (LPS), interleukin-1b (IL-1b), tumor necrosis factor a (TNFa) or interferon g (IFNg), HPASMC were treated with LPS and cytokines together with or without sodium nitroprusside (SNP), NO donor, NG-monomethyl-L-arginine (L-NMMA), NO synthetase inhibitor, and methylene blue (MeB), an inhibitor of the soluble guanylate cyclase. After incubation for 24 h, the postculture media were collected for the assay of nitrite by chemiluminescence method and the assay of PGI2 by radioimmunoassay. The incubation of HPASMC with various concentrations of LPS,IL-1b orTNFa for 24 h caused a significant increase in nitrite release and PGI2 production. However,IFNg slightly increased the release of nitrite and had little effect on PGI2 production. Although the incubation of these cells for 24 h with SNP did not cause a significant increase in PGI2 production, the incubation of HPASMC with SNP and10 mg/ml LPS, or with SNPand100 U/ml IL-1b further increase PGI2 production and this enhancement was closely related to the concentration of SNP. However, stimulatory effect of SNP on PGI2 production was not found inTNFa- and IFNg- treated HPASMC. Addition of L-NMMA to a medium containing LPS or IL-1b reduced nitrite release and attenuated the stimulatory effect of those agents on PGI2 production. MeB significantly suppressed the production of PGI2 by HPASMC treated with or without LPS or IL-1b .The addition of SNP partly reversed the inhibitory effect of MeB on PGI2 production by HPASMC.These experimental results suggest that NO might stimulate PGI2 production by HPASMC. Exogenous NO together with endogenous NO induced by LPS or cytokines from smooth muscle cells might synergetically enhance PGI2 production by these cells, possibly in clinical disorders such as sepsis and acute respiratory distress syndrome. & 2000 Harcourt Publishers Ltd
INTRODUCTION Nitric oxide (NO) is formed from L-arginine by NO synthase (NOS) in a wide variety of cells.1 In the cardiovascular system, NO, generated by endothelial NOS (eNOS) in vascular endothelial cells, plays a fundamental role in maintaining non thrombogenic properties of the endothelial surface.2 Bacterial lipopolysaccharide (LPS) and various cytokines induce the expression of inducible NOS (iNOS) by macrophage, vascular smooth
Received 29 February 2000 Accepted 28 March 2000 Correspondence to: Kentaro Watanabe, MD. Present address: 4th Department of Internal Medicine, School of Medicine, Fukuoka University, 45-1-7-Chome Nanakuma, Jonan-ku, Fukuoka, Japan.Tel.: 81 92 8011011 (3376); Fax: 8192 873 8008; E-mail: [email protected]
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muscle cells and lead to the formation of large amounts of NO,1 which plays an important role in the circulatory failure associated with septic and haemorrhagic shock.3,4 Prostacyclin (PGI2) exerts vasodilator and antithrombotic effects and is formed during the bioconversion of arachidonic acid by either constitutive cyclooxygenase (COX-1) or cytokine/LPS-inducible cyclooxygenase (COX2).5,6 The cardiovascular effects exerted by endogenously produced NO are often mediated in conjunction with PGI2. Endothelial cells contain eNOS and COX-17,8 and hence release both NO and PGI2 when stimulated with agonists such as bradykinin or ADP.9 Likewise, iNOS and COX-2 may be also induced in some cells including vascular smooth muscle cells after exposure to LPS or cytokines.3,8,10,11 Previous experiments showed that sodium nitroprusside (SNP) enhances prostaglandin E2 release by cultured rat vascular smooth muscle cells,12
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and that inhibition of NO formation reduces the release of prostanoids in LPS-treated mouse macrophages RAW 264.7, and other cell types.13 However, Keen et al. found that the inhibition of NO release potentiated the coupled release of PGI2, whereas exogenously added NO or SNP inhibits bradykinin-stimulated release of PGI2 by bovine aortic endothelial cells.14 Varying with the species of cell lines, the role of NO on the production of prostaglandins is still controversial. For years, many studies on circulatory hemodynamics have been focused on the role of vascular endothelial cells, a monolayer lining of vessels, which predominantly form PGI2 and NO in blood vessels.8,9,15 During inflammatory conditions such as ARDS and septic shock, endothelial cells could be injured and the underlying vascular smooth muscle cells, the multiple layer structure of vessel wall, acquire the ability to release NO and prostaglandins.15±18 Both the endothelial and smooth muscle cells are crucial for modulating pulmonary vascular resistance and affecting many aspects of pulmonary pathophysiology. However, few data could be found about human pulmonary artery smooth muscle cells. How NO regulates the prostaglandin production in human vascular smooth muscle cells remains unclear. In the present study, we asked whether cultured human pulmonary artery smooth muscle cells (HPASMC) could produce enough NO to affect PGI2 production by HPASMC when exposed to LPS or cytokine and, if so, what are the roles of endogenous and exogenous NO on regulating PGI2 production by HPASMC? Furthermore, we asked whether the action of NO depends on cGMP pathway. To address these questions, we have investigated the effect of NO donor, NOS inhibitor, and also guanylate cyclase inhibitor on PGI2 production by HPASMC treated with or without LPS and cytokines. MATERIALS AND METHODS
Materials Dulbecco's modified Eagle's medium (DMEM) was purchased from GIBCO (Grand Island, NY). Fetal calf serum (FCS) was obtained from Whittaker Bioproducts (Walkersville, MD). Penicillin, streptomycin, amphotericin B, trypsin, SNP, NG-monomethyl-L-arginine (L-NMMA) and LPS from E. coli (O55:B5) were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human interleukin-1b (IL-1b) and interferong (IFNg) were generously provided by Otsuka Pharmaceutical Co. (Tokyo, Japan). Recombinant human tumor necrosis factor a (TNFa) was obtained from Genzyme (Cambridge, MA). Monoclonal mouse anti-human a-smooth muscle actin antibody was obtained from Dako A/S (Glostrup, Denmark). [3H]-6-keto-prostaglandin F1a (6-keto-PGF1a) was
obtained from New England Nuclear (Boston, MA). 6keto-PGF1a and rabbit anti-6-keto-PGF1a antiserum were generously provided by Ono Pharmaceutical Co. (Osaka, Japan). Methylene blue trihydrate (MeB), sodium nitrite, acetic acid and potassium iodide were purchased from Wako Pure Chemical Industries (Osaka, Japan).
Culture of HPASMC HPASMC were cultured as previously described.19,20 Briefly, specimens of human pulmonary artery were obtained at autopsy and placed in phosphate-buffered saline. Endothelial cells and adventitia were carefully removed. The layers of smooth muscle were then cut into pieces measuring 363 mm or less, placed in 35 mm petri dishes, and cultured in DMEM that contained 1.6 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml amphotericin B, and 10% FCS at 378C under 5% CO2 in air. The culture medium was changed at intervals of 72±96 h. When the cultured cells began to grow, the explants were removed. After confluence was achieved, cells were passaged by harvesting with 0.02% trypsin and 0.01% EDTA. HPASMC at passages 3Ð5 were used in these experiments. Cultured cells were identified as smooth muscle cells by staining with mouse antihuman a-smooth muscle actin monoclonal antibody.17 All treatments described below were carried out in the presence of 10% fetal calf serum.
Effect of cytokines and LPS on the production of nitrite and PGI2 by HPASMC HPASMC were incubated with various concentrations of LPS (0±10 mg/ml) and cytokines, i.e., IL-1b (0±2000 U/ml), TNFa (0±5000 U/ml) and IFNg (0±1000 U/ml) as the previous experiments showed that LPS (0±10 mg/ml), IL-1b (0±3000 picomolar), TNFa (0±5000 U/ml) and IFNg (0±1000 U/ml) alone or the combination of these agents promoted NO production in systemic and pulmonary artery smooth muscle cells;11,12,21±23 and the very high serum concentrations of cytokines and endotoxin were found in early stages of ARDS and multiple organ dysfunction syndrome.24±26 When HPASMC grew confluent in 35 mm petri dishes, the culture medium was replaced and cultured with fresh culture medium containing various concentrations of LPS, IL-1b, TNFa or IFNg for 24 h. After 24 h, the culture medium was collected for the assays of nitrite and PGI2.
Effect of SNP and L-NMMA on the production of PGI2 by HPASMC To examine the effect of exogenous and endogenous NO on LPS-, IL-1b-, TNFa- or IFNg- induced production of PGI2
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by HPASMC, cells were incubated with LPS (10 mg/ml), IL-1b (100 or 200 U/ml), TNFa (500 U/ml) and IFNg (200 U/ ml) together with various concentrations of SNP or L-NMMA for 24 h. After 24 h, culture medium was collected for the assay of PGI2.
Effect of MeB on the production of PGI2 by HPASMC To determine the effects of MeB, a soluble inhibitor of guanylate cyclase, on PGI2 production by HPASMC, confluent cells were incubated with the medium containing MeB with or without 10 mg/ml LPS or 100 U/ml IL-1b for 24 h. Cells were also incubated with medium containing MeB in the presence of SNP and LPS or IL-1b for 24 h. The postculture medium was then collected for PGI2 assay.
Measurement of PGI2 The levels of 6-keto-PGF1a, the stable end product of PGI2, were determined by radioimmunoassay as previously described.19,20 The cross reactivity of rabbit antiserum to 6-keto-PGF1a was 100% with 6-keto-PGF1a, 5.0% with PGE1, 0.76% with PGE2, 0.82% with PGF2a and 0.01% with thromboxane B2.
Nitrite assay Nitrite in the samples was converted to NO under acidic conditions, and the NO was measured with a sensitive chemiluminescence analyzer.27,28 Briefly, samples collected were diluted three times with deionized water and incubated at room temperature for 1 h. Then samples were injected into the purge vessel of NO analyzer, NOATM 280 (Sievers, Boulder, CO) containing 1% potassium iodide in glacial acetic acid which converts nitrite to NO. NO released from the reaction vessel was measured by a chemiluminescence. Standard curves were drawn by known amounts of nitrite in each experiment.
Measurement of lactate dehydrogenase (LDH) release After incubating HPASMC with various concentrations of cytokines, LPS and SNP or the combination of SNP and LPS or cytokine for 24 h, LDH in the postculture medium was measured using a LDH commercial kit (Sigma, St. Louis, MO). The LDH values were corrected by subtracting the LDH activity measured in the medium before culture. The maximal release of LDH was determined by scraping and sonicating the control cells, and measuring LDH in the medium. & 2000 Harcourt Publishers Ltd
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Statistical analyses Data were expressed as mean+SEM. Data were evaluated by one-way ANOVA in the experiments examining dose-dependent effects of LPS, cytokines, SNP or LNMMA on the production of PGI2, nitrite and on the release of LDH. If the overall F statistic was significant at the 0.05 level, subsequent intergroup significance testing was performed by the Scheffe's F-test. A two-tailed unpaired Student's t-test was also performed to compare the levels of nitrite, 6-keto-PGF1a, and LDH in cells treated with or without LPS, cytokines or those reagents examined. A level of p50.05 was accepted as statistically significant.
RESULTS Phase-contrast microscopy revealed that confluent HPASMC were aligned in parallel and exhibited the typical `hill and valley' pattern of intertwined, overlapping cells similar to that of cultures of vascular smooth muscle cells. These cultured cells were positively stained by mouse anti-human a-smooth muscle actin monoclonal antibody. No positive staining was observed in control cells. Fig. 1 shows nitrite release from HPASMC measured by chemiluminescence method. HPASMC were treated with various concentrations of LPS, IL-1b, TNFa or IFNg for 24 h. LPS, IL-1b and TNFa induced significant increase in nitrite release from HPASMC ( p50.01, respectively, one way ANOVA). Nitrite level was slightly higher in HPASMC treated with 1000 U/ml of IFNg than in control HPASMC, but without dose-dependency. When HPASMC were incubated with 1 mg/ml of LPS, 20 U/ml of IL-1b or 50 U/ml of TNFa for 24 h, PGI2 release by HPASMC was enhanced ( p50.01, 50.01 and 50.05, respectively, unpaired Student's t-test) (Fig. 2). However, incubation of HPASMC with 1000 U/ml of IFNg or higher concentration up to 1000 U/ml for 24 h did not augment PGI2 production. Fig. 3 shows PGI2 production by HPASMC that were treated with various concentrations of SNP and 10 mg/ml of LPS (Fig. 3a), 100 U/ml of IL-1b (Fig. 3b), 500 U/ml of TNFa (Fig. 3c) and 200 U/ml of IFNg (Fig. 3d) for 24 h. Incubation with 10, 20 and 50 mM of SNP without LPS or cytokines did not induce significant increase in PGI2 production when compared with control cells (data not shown). Similar to the results in Fig. 2, the levels of 6-ketoPGF1a released from HPASMC treated with 10 mg/ml of LPS, 100 U/ml of IL-1b and 500 U/ml of TNFa significantly exceeded those in the absence of LPS, IL-1b or TNFa. Addition of SNP to the medium containing LPS or IL-1b further enhanced the stimulated production of PGI2 by HPASMC treated with LPS or IL-1b, and these enhancements were closely related to the dosages of
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Fig. 1 Effects of cytokines and LPS on nitrite release by HPASMC. Confluent HPASMC were incubated with the medium containing increasing concentrations of LPS (a), IL-1b (b),TNFa (c) and IFNg (d) for 24 h. Nitrite released in culture medium was then measured by chemiluminescence method. Data are shown as means+SEM of 8^28 replicate samples in 2^5 experiments. Numbers in parentheses indicate those of samples tested. One way ANOVA showed the significantly increased levels of nitrite in LPS-, IL-1b- and TNFa- treated HPASMC (p50.01).*:p50.05 shows significance compared with control levels in post hoc Scheffe's F-test.
SNP used ( p50.01, respectively, one-way ANOVA). In spite of the stimulatory effect of TNFa on PGI2 production, addition of SNP did not augment PGI2 production in HPASMC incubated with TNFa. Fig. 4 shows PGI2 production by HPASMC that were treated with various concentrations of L-NMMA with the addition of 10 mg/ml of LPS (Fig. 4a) or 200 U/ml of IL-1b (Fig. 4b) for 24 h. Incubation with various concentrations of L-NMMA alone did not affect production of PGI2 by HPASMC (data not shown). Enhanced production of PGI2
by LPS- and IL-1b-treated HPASMC was significantly attenuated, when L-NMMA was added to the incubation medium containing LPS or IL-1b ( p50.01, one way ANOVA) (Fig. 4a, b). The addition of 0.5 mM L-NMMA caused 48.7 and 37% decrease in PGI2 production by LPS- and IL-1b-treated cells, respectively; and incubation with 5 mM L-NMMA caused further decrease in PGI2 production. The effect of MeB, SNP, LPS or combination of those agents on PGI2 production by HPASMC is shown in
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3. L-NMMA suppressed PGI2 production by HPASMC treated with LPS or cytokines 4. MeB inhibited LPS- and IL-1b-induced increase in PGI2 production. Addition of SNP partly reversed the suppresive effect of MeB on PGI2 production 5. Less concentrations of LPS or cytokines were needed for the efficient production of PGI2 than for the nitrite release by HPASMC.
Fig. 2 Effects of LPS, IL-1b,TNFaand IFNg on PGI2 production by HPASMC. HPASMC were treated with medium containing1 mg/ml of LPS, 20 U/ml of IL-1b, 50 U/ml of TNFaand100 U/ml of IFNg or culture medium alone for 24 h. After 24 h, the postculture media were collected and assayed for 6-keto-PGF1aas described in`Materials and Methods'. Data are expressed as mean+SEM of 8^14 samples in two experiments. Numbers in parentheses indicate those of samples tested.**p50.01and *p50.05, compared with control HPASMC, unpaired Student's t -test.
Fig. 5a. Similar experiments were done after the substitution of IL-1b for LPS (Fig. 5b). MeB inhibited PGI2 production by HPASMC and also markedly suppressed LPS- or IL-1b-induced enhancement of PGI2 production when HPASMC were treated with MeB and LPS or IL-1b; the addition of 20 mM SNP partly reversed the inhibitory effect of MeB on the production of PGI2 by HPASMC ( p50.05±0.01, unpaired Student's t -test). During incubation with various concentrations of cytokines, LPS and SNP or combination of those agents there was no morphological change in HPASMC by phase contrast microscopy. A 24 h incubation with any cytokine, LPS and SNP or the combination of these compounds in the present study did not induce a significant increase in LDH release from HPASMC when compared with the control (Tables 1 and 2).
DISCUSSION Our findings in the present experiments are: 1. Both SNP and L-NMMA had no effect on PGI2 production by control HPASMC 2. SNP markedly augmented PGI2 production by HPASMC treated with LPS or cytokines & 2000 Harcourt Publishers Ltd
These findings suggest that enhanced production of PGI2 by HPASMC treated with LPS and IL-1b may be caused not only by exogenous NO from NO donor but also by endogenous NO produced from HPASMC. Previous experiments show controversial data on the effect of NO in the regulation of prostaglandin production. NO has been reported to activate,10,12,13,29±31 inhibit32±34 or have no effect35 on the activity of COX, a key rate-limiting enzyme in the synthesis of eicosanoids from arachidonic acid, in different kinds of cell lines. In our experiments, no apparent potentiation of SNP on basal PGI2 production by HPASMC was noted. Only in the presence of LPS or IL-1b together with exogenous NO from SNP, PGI2 production by HPASMC was significantly enhanced. Moreover, LPS- and cytokine-induced production of PGI2 by HPASMC was inhibited by L-NMMA. These results show that either exogenous or endogenous NO plays an important role in the enhancement of PGI2 production by HPASMC in spite that the increased release of nitrite by LPS- or IL-1b-treated HPASMC was not as marked as those noted in vascular smooth muscle cells originated from animal species.11,12 The enhanced production of PGI2 by NO might reflect the increased activity of COX, possibly COX-2, for the basal PGI2 production by HPASMC was not affected by SNP in the present study. Vascular smooth muscle cells constitutively produce prostaglandins by COX-1 but do not release NO for lack of eNOS.6 Although both NO and prostaglandins were produced by HPASMC treated with LPS or cytokines, less concentrations of LPS or cytokines were needed for the efficient production of PGI2 than for the nitrite production. Several possibilities are raised for its mechanism: 1) COX-2 is more sensitively induced than iNOS by LPS or cytokines; 2) the COX metabolites have been conversely reported to influence iNOS induction. PGE2 and iloprost, a stable PGI2 analog, at micromolar concentrations suppressed the iNOS induction in a murine macrophage cell line J774.36 PGD2 dose-dependently inhibited IL-1binduced NO production and also the expression of iNOS mRNA and protein in cultured rat vascular smooth muscle cells.37 NO acts by cGMP-independent and -dependent mechanisms. As the relaxation of vascular smooth muscle and inhibition of platelet aggregation are resulted from an elevation of cGMP,38,39 we tested whether the effect of
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Fig. 3 Effect of SNPon the production of PGI2 by LPS- and cytokine-treated HPASMC.HPASMC were incubated with medium containing10 mg/ml of LPS (a),100 U/ml of IL-1b (b), 500 U/ml of TNFa (c) and 200 U/ml of IFNg (d) with increasing concentrations of SNP (0,10, 20, and 50 mM) for 24 h. After 24 h, postculture media were collected for the assay of 6-keto-PGF1a. Data are expressed as mean+SEM of 8^15 samples in 2^3 experiments. Numbers in parentheses indicate those of samples tested. One way ANOVA showed the significantly increased levels of PGI2 in LPS- and IL-1b-treated HPASMC (p50.01).*p50.05, compared with no SNP, Scheffe's F-test.
NO on PGI2 production is cGMP-dependent in HPASMC. Salvemini et al. showed that MeB blocked the increase in cGMP levels in human fetal fibroblasts exposed to SNP or glyceryl trinitrite (GTN) but it did not affect the ability of SNP or GTN to augment PGE2 release by arachidonic acid.13 MeB diminished A23187-stimulated PGI2 production by bovine coronary endothelial cells, but another guanylate cyclase inhibitor, LY83583, had no effect on PGI2 production.31 Besides, 8-bromo-cGMP, a cGMP
analogue, did not increase eicosanoid production from bovine coronary microvascular endothelial cells and rat aortic smooth muscle cells.12,31 These findings suggest that NO may induce prostaglandin synthesis via a cGMPindependent pathway. However, our experimental results showed that the production of PGI2 by both control cells and cells treated with LPS or IL-1b was markedly inhibited by MeB. Moreover, the inhibitory effect of MeB on PGI2 production was partly reversed by SNP. Okamura et al.
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Fig. 4 Effect of L-NMMA on PGI2 production by LPS- and IL-1b-treated HPASMC. HPASMC were incubated with medium containing10 mg/ml of LPS (a) and 200 U/ml of IL-1b (b) with increasing concentrations of L-NMMA (0, 0.05, 0.5 and 5 mM) for 24 h. After 24 h, postculture media were collected and then assayed for 6-keto-PGF1a. Data are expressed as mean+SEM of13^14 samples in 3 experiments. Numbers in parenthesis indicate those of samples tested. One way ANOVA showed the significantly suppressed levels of PGI2 in LPS- and IL-1b-treated HPASMC (p50.01).*p50.05, compared with no L-NMMA, Scheffe's F-test.
Fig. 5 Effect of MeB on PGI2 production by LPS- and IL-1b-treated HPASMC. HPASMC were treated with10 mM MeB,10 mg/ml of LPS shown in panel a (IL-1b: panel b), or the same concentrations of LPS (panel a) (IL-1b: panel b) and MeB together with or without 20 mM SNP for 24 h. After the incubation, postculture media were then assayed for 6-keto-PGF1a. Data are expressed as mean+SEM of 8^13 samples in 2^3 experiments.Numbersin parenthesis indicate those of samples tested.*p50.05, **p50.01vs LPSMeB or IL-1bMeB; # P50.05, vs control or MeB SNP, unpaired Student's t-test.
found that arachidonic acid-stimulated production of PGI2 released from dog renal arterial strips was significantly reduced by the treatment with MeB.40 In addition to suppressive effect on soluble guanylate cyclase, MeB is & 2000 Harcourt Publishers Ltd
known to generate oxy-radicals, which inactivate NO, and also act as a direct inhibitor of NOS.41,42 The inhibitory effect of MeB on PGI2 production by HPASMC might be not only due to the possible inhibition of the endogenous
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NO production but also due to the clearance of exogenous NO, since SNP, a NO donor, partly reversed the inhibitory effect of MeB on PGI2 production. However, the inhibitory effect of MeB on PGI2 production by control cells in which NO is not involved suggests that NO might regulate the production of PGI2 in HPASMC via a cGMP-dependent pathway. Interestingly, TNFa increased nitrite release and PGI2 production from HPASMC, but PGI2 production in TNFatreated HPASMC was not augmented by the coincubation with SNP. TNFa enhances NO production in vascular smooth muscle cells by inducing iNOS. However, it downregulates eNOS,43 and decreases pulmonary artery endothelial nitrovasodilator activity by the generation of oxy-radicals which react with NO to form the peroxynitrite radical (ONOO7). ONOO7 has the capability to cause cell dysfunction and cell death, and reduce the vasodilator activity of NO.44 Whether such effects would also impair the synthesis of PGI2 and the contractility of the smooth muscle cells needs investigating. Table 1 Effect of cytokines and LPS on the release of LDH from HPASMC Treatment
LDH (%)
Control
3+1 (18)
LPS (mg/ml) 0.1 1.0 10.0
6+2 (5) 5+3 (5) 4+2 (10)
IL-1b (U/ml) 20 200 2000
6+3 (5) 6+2 (5) 8+3 (5)
TNFa (U/ml) 50 500 5000
3+3 (5) 7+2 (10) 5+2 (5)
IFNg (U/ml) 10 100 1000
5+3 (5) 5+2 (5) 5+3 (5)
All data are expressed as percentage to maximum release (%) (mean+SEM). Numbers tested are indicated in parentheses. Table 2
Despite a lot of in vitro and in vivo studies on the interaction between NO and prostaglandins, most of these data came from animal experiments and few were directly related to human pulmonary circulation.3,4,15,21,29,31,45 Meyrick et al.46 reported that cultured human pulmonary artery endothelial cells (HPAEC) responded to LPS (0.001±10 mg/ml) to produce PGI2 with modest cellular cytotoxicity. The level of 6-keto-PGF1a produced by HPAEC during 24 h incubation with normal medium was about 250 pg/ml and HPAEC treated with 1 mg/ml of LPS for 24 h showed three-fold increase in PGI2 production. In contrast, the level of 6-keto-PGF1a by control HPASMC during 24 h incubation was about 750 pg/ml, which was a three-fold increase compared with that of HPAEC.20 HPASMC incubated with the same serotype of LPS (O55:B5) as Meyrick et al. used for 24 h also showed a three-fold increase in PGI2 production.20 Based on these experimental results, pulmonary artery smooth muscle cells as a major component of the vessel wall may play important roles in either physiological or pathological conditions such as hypoxic vasoconstriction, pulmonary hypertension and ARDS.45 The present observations show the importance in smooth muscle cells as well as endothelial cells in regulating the pulmonary vascular tonus. In conclusion, our results suggest that both exogenous and endogenous NO from smooth muscles cells enhance PGI2 production from HPASMC treated with LPS or IL-1b, and PGI2 production by HPASMC was suppressed by guanylate cyclase inhibitor. In pathophysiological states involving endotoxin and cytokines, pulmonary vascular tension and blood distributions in the lung are probably affected by the increased production of prostaglandins which might be further augmented by NO. Further study is still needed to elucidate the mechanism of the effect of NO on PGI2 production by pulmonary artery smooth muscle cells.
ACKNOWLEDGEMENT This work was supported by a Grant-in-Aid for Scientific Research (C), No. 09670636 from the Ministry of Education, Science, and Culture of Japan.
Effect of SNP on the release of LDH from HPASMC SNP (mM)
Control LPS10 mg/ml IL-1b100 U/ml TNFa 500 U/ml IFNg 200 U/ml
0
10
20
2+1 5+3 3+3 7+3 5+3
4+2 5+4 3+2 3+2 4+3
5+2 3+2 5+4 4+2 5+3
50 2+1 4+3 7+4 5+3 5+3
All data are expressed as percentage to maximum release of LDH(%) (mean+SEM); n=10 in control cells and cells treated with SNPalone; n=5 in the rest groups. Prostaglandins, Leukotrienes and Essential FattyAcids (2000) 62(6), 369^378
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