Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Lipopolysaccharide (LPS) and/or cytokines induce the expression of the inducible nitric oxide synthase (iNOS) isoform (Forstermann et al., 1998) in many cell types, including macrophages (Stuehr et al., 1989) and vascular smooth muscle (Busse and Mulsch, 1990; Gross and Levi, 1992). The increased expression of this protein and the production of large quantities of nitric oxide (NO) are associated with hypotension and hyporesponsiveness to vasoconstrictor stimuli in endotoxemia or sepsis (Julou-Schaeffer et al., 1990; Thiemermann and Vane, 1990; Hollenberg et al., 1993). Moreover, 6 null mutant iNOS mice were reported to be resistant to the hypotension and death caused by LPS (Wei et al., 1995; MacMicking et al., 1995). Thus, it suggests that iNOS plays a crucial role in LPS-induced death. Although questions remain about potentially negative impacts of iNOS inhibitors on cardiac function (Klabunde and Ritger, 1991) and vascular integrity (Hutcheson et al., 1990), the selective inhibitors of iNOS (activity and/or iNOS protein expression) were suggested to be beneficial to endotoxic or septic shock; i.e., via the restoration of blood pressure (Kilbourn et al., 1990). Therefore, attempts have been made to find new chemicals for the treatment of endotoxemia. Isoquinoline compounds attract special interests on their pharmacological actions in relation to inflammation and related disorders (see below). For example, tetrandrine, an anti-inflammatory agent, was observed to inhibit the activation of NF-B and NF-B-dependent reporter gene expression induced by LPS in rat alveolar macrophages (Chen et al., 1997). Other isoquinoline series such as cepharanthine, isotetrandrine, and cycleanine were shown to suppress LPS-induced hepatitis and tumor necrosis factor (TNF) production in mice, and NO production in macrophages (Kondo et al., 1993). We also reported that higenamine, a benzylisoquinoline alkaloid (Kang et al., 1999a), and YS 49, a synthetic 1-naphthylmethyl analog of higenamine (Kang et al., 1999b), inhibited iNOS mRNA and protein expression in rat aorta and RAW 264.7 cells induced by LPS and cytokine via inhibition of NF-B activation. Thus, isoquinoline and related alkaloids provide us ample reason to investigate the mechanism of action for the suppression of iNOS gene expression. In general, positional isomers display quite different pharmacological characteristics since any or some conformational perturbations by positional isomers may result in completely new biochemical environments. Thus, YS 51 (Fig. 1), a positional isomer of YS 49, was expected to display quite different effects on LPS-stimulated iNOS induction from those of YS 49 reported by Kang et al. (1999b). So far, known actions of YS 51 are increasing contractile force of isolated guinea pig papillary muscle by increasing intracellular Ca²⁺ (Chang et al., 1998), and inhibiting TNF- mRNA expression in mouse peritoneal macrophages activated with LPS (Jung et al., 2000) and antithrombotic and antiplatelet aggregating action (Yun-Choi et al., 2001). The present studies were carried out to clarify 1) whether YS 51 inhibits iNOS expression and NO production, 2) if so, what is the mechanism of action for the inhibition of iNOS expression, and finally 3) whether it is beneficial against LPS-induced mortality.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Chemical structure of YS 51.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Cell Culture. RAW 264.7 cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were grown in RPMI-1640 medium supplemented with 25 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal calf serum.

Cell Stimulation. RAW 264.7 cells were plated at a density of 1 × 10⁷ cells per 100-mm dish. The cells were rinsed with fresh medium and stimulated with LPS (10 ng/ml) plus IFN- (10 U/ml) in the presence or absence of different concentrations of YS 51 (1-100 µM). YS 51 was dissolved in sterile distilled water and was filtered through a 0.2-µm filter.

Assay for Nitrite Production. NO was measured as its stable oxidative metabolite, nitrite, as described previously (Green et al., 1981; Kang et al., 1999a). At the end of the incubation, 100 µl of the culture medium were mixed with an equal volume of Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). The absorbance at 550 nm was measured, and the nitrite concentration was determined using a curve calibrated on sodium nitrite standards.

Analysis of iNOS mRNA. Total RNA was extracted as described previously (Kang et al., 1999a). A sample 15 µg of total RNA per lane was subjected to electrophoresis on 1% agarose gels containing formaldehyde and transferred to nylon filters. The filter was then hybridized with a random-primed ³²P-labeled iNOS cDNA probe in rapid hybridization solution (Quikhyb; Stratagene, La Jolla, CA) at 68°C for 1 h. The sense and antisense sequence of partial cDNA probe for iNOS was 5'-TGGACCAGTATAAGGCAAGC-3' (sense) and 5'-GCTCTGGATGAGCCTATATTG-3' (antisense). The hybridized filter was subsequently washed twice for 15 min at room temperature [(sodium chloride/sodium citrate)/0.1% SDS] and then twice for 15 min at 42°C with 0.2× standard saline citrate/0.1% SDS. The filter was then exposed to X-ray film. The filter was subsequently stripped and rehybridized with ³²P-labeled glyceraldehyde-3-phosphate dehydrogenase.

Assay for iNOS Protein. iNOS protein was analyzed by immunoblotting with the anti-iNOS antibody as described previously (Kang et al., 1999a). Briefly, the lung tissues were homogenized in a buffer containing 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM leupeptin, 1 mM pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol and sonicated. The homogenates were then centrifuged at 7500g for 15 min four times, and the supernatants were subjected to SDS-polyacrylamide gel electrophoresis (7.5% gel). The separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes, and the membrane was incubated with anti-iNOS antibody for 2 h followed by peroxidase-labeled goat anti-rabbit IgG for 1 h. Antigen-antibody complexes were detected using Enhanced chemiluminescence Western blotting detection reagents (Amersham) according to the manufacturer's instruction.

iNOS Promoter Activity Assay. RAW 264.7 cells were transfected by a LipofectAMINE method with 1.6 kbp of murine iNOS promoter fragment linked upstream of luciferase. After 16 h recovery in fresh medium containing 5% serum, cells were treated with LPS and IFN- in the presence or absence of YS 51 or pyrrolidine dithiocarbamate (PDTC) for 14 h. Cells were harvested, washed with ice-cold PBS, and lysed with Reporter lysis buffer (Promega, Madison, WI) or buffer containing 1% Triton X-100, 5 mM dithiothreitol, 50% glycerol, 10 mM EDTA, and 125 mM Tris-phosphate (pH 7.8). Luciferase activity was assayed with 20 µl of lysate by luminometer.

Ex Vivo Vascular Reactivity. Sprague-Dawley rats (male, 250-300 g) were intraperitoneally injected with 1) LPS (10 mg/kg, n = 4), 2) YS 51 (10 and 20 mg/kg, n = 4) 30 min before LPS, 3) saline (n = 4), or 4) YS 51 (20 mg/kg, n = 4). At 8 h after injection, the thoracic aortas were taken under pentobarbital anesthesia. The aortas were cleared of adhering periadventitial fat and cut into rings of 3-4 mm width. Endothelium was removed by gently rubbing the intimal surface with a wooden stick as reported previously (Chang et al., 1993). The rings were mounted in an organ bath (5 ml) filled with Krebs' solution (pH 7.4) consisting of 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 25 mM NaHCO₃, 11 mM glucose, and 0.03 mM EDTA. Isometric force was measured with a force transducer (FT 03; Grass Instruments, Quincy, MA). A tension of 1 g was applied, and the rings were equilibrated for 60 min, changing the Krebs' solution every 20 min (Chang et al., 1993). Indomethacin (10 µM) was used to prevent the production of cyclooxygenase metabolites that are predominantly vasoconstrictors in this experimental setting. Concentration-response curves for PE (1 nM-10 µM) were obtained. For relaxation studies, rings were contracted with U46619 (20 nM), and after plateau contraction was reached, L-arginine (1-100 µM) was introduced cumulatively.

In Vitro Vascular Contractility. As described above, endothelium-denuded thoracic aortic ring preparations (3- to 4-mm width) were prepared from untreated rats under pentobarbital anesthesia. To know the effects of YS 51 on iNOS induction in vitro, the rings were divided into groups: 1) an LPS (300 ng/ml, n = 4)-treated group, 2) a YS 51 (30 µM) with LPS-treated group (n = 4), 3) a control (Krebs' solution) group (n = 3), and 4) a YS 51 (30 µM) group (n = 3). The tissues were incubated at 37°C, 95% O₂/5% CO₂ for 8 h. After completion of incubation, isometric tension experiments were determined as described above.

Plasma Nitrite/Nitrate Measurement. Rats were divided into four groups: 1) LPS (10 mg/kg, i.p., n = 4), 2) LPS plus YS 51 (10 and 20 mg/kg, i.p, n = 4), 3) saline (i.p., n = 3), and 4) YS 51 (i.p., n = 3). When used, YS 51 was given 30 min before LPS. After an 8-h LPS treatment, a whole blood sample was withdrawn by cardiac puncture. The plasma nitrate concentration was determined by reducing the nitrate enzymatically, using nitrate reductase from Aspergillus species. Briefly, plasma samples were diluted 1:10 with distilled water and incubated with assay buffer: 50 mM KH₂PO₄, 0.6 mM NADPH, 5 mM FAD, and 10 U/ml nitrate reductase, pH 7.5, for 30 min at 37°C. Subsequently, culture medium was mixed with an equal volume of the Griess reagent (mixture of 1 part 1% sulfanilamide in 5% phosphoric acid and 1 part 0.1% naphthylethylenediamine dihydrochloride in water) and incubated at room temperature for 10 min. A standard curve for nitrate was constructed by incubation of nitrate (1-100 µM) with assay buffer. The resultant nitrite concentrations were determined with Griess reagent with sodium nitrite as standard (model 550; Bio-Rad Laboratories, Hercules, CA).

Survival Studies. ICR mice (22-25 g, male) were divided into four groups with 20 mice in each group, and the drug was injected intraperitoneally 30 min before LPS: 1) the LPS (20 mg/kg) group, 2) the LPS plus YS 51 (10 mg and 20 mg/kg) group, 3) the saline group, and 4) the YS 51 (20 mg/kg) group. The survival was monitored every 6 h until 48 h.

Electrophoretic Mobility Shift Assay. Macrophages were treated with LPS or IFN- in the presence or absence of YS 51 for 2 h. Nuclear extracts were prepared by the method of Staal et al. (1990). Protein concentrations of nuclear extracts were determined by the BCA method (Pierce Chemical, Rockford, IL). A double-stranded NF-B-specific oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') was end-labeled with [-³²P]ATP using T4 polynucleotide kinase and purified on a G-50 Sephadex column. The nuclear extracts (10 µg of protein) were incubated with ~40,000 cpm (~0.5 ng) of ³²P-labeled oligonucleotide (labeled probe) in the presence or absence of cold probe (100 folds over labeled probe) or NF-B subunit p65 antibody in the incubation buffer [4.2 mM HEPES (pH 7.4), 4.2 mM KCl, 0.02 mM EDTA, 1 mM MgCl₂, 2.5% glycerol, 2% Ficoll, 21 mM dithiothreitol, and 2 µg of poly(dI-dC)] for 30 min at room temperature as described previously (Kim et al., 1997). DNA-protein complexes were electrophoretically separated on a native 5% polyacrylamide gel in TBE running buffer (450 mM Tris-borate and 1 µM EDTA, pH 8.0). The gel was then dried and subjected to autoradiography.

Statistical Evaluations. Data are expressed as mean ± S.E.M. of results obtained from number (n) of animals used. Differences between data sets were assessed by one-way analysis of variance followed by Dunnett's test. A level of P < 0.05 was accepted as statistically significant.

Materials. Lipopolysaccharide (E. coli; serotype 0128:B12), indomethacin, phenylephrine, HCl, sulfanilamide, N-[1-naphthyl]ethyleneamine, sodium chloride, leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, and dithiothreitol were from Sigma (St. Louis, MO). U46619 (9,11-dideoxy-11,9-epoxymethanoprostaglandin F₂). iNOS antibody was from Transduction Laboratories (Lexington, KY). The p65 antibody was from Santa Cruz Biotechnology, (Santa Cruz, CA). Horseradish peroxidase-labeled goat antirabbit IgG was purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Enhanced chemiluminescence Western blotting detection reagent was from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effects of YS 51 on NO Production. In cultured RAW cells, stimulation with LPS plus IFN- resulted in accumulation of nitrite in a time-dependent manner (data not shown). The accumulated nitrite was 9.8 ± 1.07 µM in control media, which was increased to 66.05 ± 1.54 µM by LPS plus IFN- for 18 h. Cotreatment of YS 51 decreased the nitrite production in a concentration-dependent manner (Fig. 2). The concentration of 50% inhibition of iNOS mRNA expression (IC₅₀) by YS 51 was 23.5 µM.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent inhibition of NO production by YS 51 in RAW 264.7 cells activated with LPS plus IFN-. RAW 264.7 cells were incubated for 18 h. Control (1), LPS (10 ng/ml) plus IFN- (10 units/ml), (2) LPS + INF- with different concentrations of YS 51 [10 µM (3), 30 µM (4), or 50 µM (5)]. Data represent the mean ± S.E.M. of triple determinations from a representative experiment performed at least three separate times with comparable results. *, significantly different from LPS plus IFN- group at P < 0.05.

Effects of YS 51 on iNOS mRNA Expression and iNOS Promoter Activity. To understand the reduced production of NO by YS 51 was ascribed to the inhibition of iNOS expression, Northern analysis was performed. YS 51 decreased the expression of iNOS mRNA in a concentration-dependent manner (Fig. 3). To confirm that YS 51 regulates iNOS gene expression at the transcriptional level, we measured iNOS promoter activity in RAW 264.7 cells, which were transfected by incorporating 1.6-kbp iNOS promoter fragments linked upstream of luciferase. As shown in Fig. 4, YS 51 inhibited the luciferase activity in a concentration-dependent manner. For example, LPS plus IFN- increased the luciferase activity about 7-fold compared with the control, which was decreased to 6 (10 µM)-, 4 (30 µM)-, and 2 (50 µM)-fold, depending on the concentration of YS 51, indicating that YS 51 inhibits iNOS gene expression transcriptionally. For comparison, PDTC was used, and the inhibitory potency of YS 51 was almost the same as that of PDTC.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent inhibition of the expression of iNOS mRNA by YS 51 in RAW 264.7 cells activated with LPS plus IFN-. RAW 264.7 cells (1) were incubated for 10 h. Control (1), LPS (10 ng/ml) plus IFN- (10 units/ml) alone (2), LPS + INF- with different concentrations of YS 51 [10 µM (3), 30 µM (4), or 50 µM (5)]. The total RNA was extracted and subjected to Northern blot. Upper panel, densitometric intensities of expressed iNOS mRNA. Lower panel, ratio of expressed iNOS mRNA and GADPH mRNA. Data are expressed as mean ± S.E.M. of determinations from a representative experiment performed at least three separate times with comparable results. *, significantly different from LPS plus IFN- group at P < 0.05.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effects of YS 51 on activity of iNOS promoter construct. Promoter activity was evaluated using iNOS luciferase constructs, which contained sequentially deleted 5'-flanking regions of the mouse iNOS gene and a luciferase reporter gene. The RAW cells transfected with the constructs were incubated with LPS plus IFN- in the presence or absence of YS 51. For comparison, PDTC was involved in separate experiments. Data are expressed as mean ± S.E.M. of determinations from a representative experiment performed at least three separate times with comparable results.

Effects of YS 51 on LPS-Induced Vascular Reactivity in Vitro. To understand the functional significance of iNOS expression in blood vessels, the vascular reactivity to PE in LPS-treated aorta was examined in vitro. Figure 5A shows concentration-response curves to PE. A typical physiological recording of vascular contractility to PE (10 nM-10 µM) in LPS (300 ng/ml)-treated aorta is shown in Fig. 5B. Coadministration of YS 51 (30 µM) with LPS prevented the LPS-induced hypocontractile response to PE; for example, maximum contractile force to PE (10 µM) was 0.53 ± 0.04 g (n = 4) in the LPS-treated group and 1.38 ± 0.25 g (n = 4) in YS51 + LPS-treated groups, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effects of YS 51 on vascular contractility by vasoconstrictor in LPS-induced rat aorta in vitro. A, a representative tracing of PE-induced contraction in rat thoracic aorta that had been incubated with either LPS (300 ng/ml) or LPS + YS 51 (30 µM) for 8 h in vitro. Numbers indicate log molar concentration. B, concentration-response curves of PE-induced concentration. , control; , LPS + YS 51; , LPS. Values are expressed as mean ± S.E.M. of contractile forces in grams. *, significantly different from all other groups at P < 0.05.

Effects of YS 51 on LPS-Induced Vascular Reactivity ex Vivo. To test vascular reactivity ex vivo, aortas were isolated after 8 h either from LPS (10 mg/kg, i.p.)-treated or YS 51 plus LPS-treated rats, and the vascular reactivity was examined as described under Experimental Procedures. Aortas from LPS-treated rats caused significant depression in the contractile ability to PE ex vivo, which was prevented by YS 51 (10 and 20 mg/kg, i.p.) pretreatment (Fig. 6, A and B). The maximum contractile force to 10 µM PE was 0.60 ± 0.01 g (n = 4) in the LPS-treated group, 1.1± 0.35 g in the YS 51 (10 mg/kg) plus LPS-treated group, and 1.9 ± 0.17 g (n = 4) in the YS 51 (20 mg/kg) plus LPS-treated group, respectively. In contrast, YS 51 (20 mg/kg) alone did not affect the contractile ability to PE as compared with saline-treated groups, in which the maximum contraction to 10 µM PE was 2.35 ± 0.53 g (n = 4) and 2.6 ± 0.47 g (n = 4) in saline and YS 51-treated rings, respectively. To confirm that the diminished contraction to PE in aortas from LPS-treated rats was due to the induction of iNOS in the vessel, aorta was contracted with U46619, and when maximum contraction was reached, L-arginine, a substrate of NOS, was added to the bath cumulatively. As shown in Fig. 6, aortas from LPS-injected rats treated with L-arginine relaxed in a concentration-dependent manner, whereas the relaxation response was significantly diminished in aortas from rats treated with YS 51 along with LPS.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effects of YS 51 on LPS-induced vascular hyporeactivity ex vivo. Rats were injected (i.p.) with either saline () or LPS (10 mg/kg; ), YS 51 (10 mg/kg; ) + LPS, YS 51 (20 mg/kg; ) + LPS, or YS 51 (20 mg/kg), and then sacrificed 8 h after injection. Thoracic aortas were removed and isometric tension was recorded. A, contractile force to PE (10 nM-10 µM) in aortas taken from LPS-treated rats was significantly (P < 0.01) lower than that in controls. Contractile force to PE in aortas taken from YS 51 + LPS-treated rats was significantly (P < 0.01) greater than that in LPS-treated ones. B, L-Arginine, a NOS substrate, 6 relaxed, in a concentration-dependent manner, in aortas taken from LPS-treated rats, but it was almost resistant to 6 relax in aortas taken from YS 51 + LPS-treated rats. Values are expressed as mean ± S.E.M. of contractile forces in grams. *, significantly different from all other groups at P < 0.05.

Effects of YS 51 on iNOS Protein Expression in LPS-Treated Rat. To determine whether YS 51 decreases iNOS protein expression, Western blot analysis was performed, in which lung tissues were isolated from the same rats that were used for vascular reactivity experiments ex vivo as described above. Lung tissue was deliberately chosen because it is known to express iNOS protein abundantly when LPS is injected to rats. As shown in Fig. 7, significant induction of iNOS protein was noticed after LPS challenge, but treatment with YS 51 (10 mg/kg) reduced the increased iNOS protein expression. Moreover, higher concentrations of YS 51 (20 mg/kg) almost completely abolished the iNOS protein expression (Fig. 7).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effects of YS 51 on iNOS protein expression in lung tissues and on plasma nitrite/nitrate levels by LPS-injected rat. A, lane 1, control; lane 2, LPS 10 mg/kg alone; lane 3, LPS plus 10 mg/kg YS 51; lane 4, LPS plus 20 mg/kg YS 51; lane 5, 20 mg/kg YS 51 alone. Pretreatment of YS 51 significantly reduced LPS-induced iNOS protein expression in lung tissues. YS 51 alone had no effect. B, blood samples were collected from cardiac puncture before sacrifice for the iNOS protein expression experiment; the samples were centrifuged and serum fraction was subjected to analysis to measure nitrite/nitrate concentrations. Values are expressed as mean ± S.E.M. of four separate experiments. *, significantly different from all other groups at P < 0.05.

Effects of YS 51 on Plasma Nitrite/Nitrate Levels in LPS-Treated Rat. The plasma concentration of NO was expected to decrease by treatment with YS 51 in LPS-injected rats since YS 51 reduced expression of iNOS protein in lung tissues stimulated by LPS. As shown in Fig. 7, the concentration of nitrite/nitrate (NOx) in plasma after saline and YS 51 (20 mg/kg, i.p) was 7.72 ± 1.07 µM and 7.8 ± 1.04 µM, respectively (n = 4). After 8 h of LPS administration (10 mg/kg, i.p), the plasma NOx was elevated to 52 ± 6 µM, which was significantly (P < 0.05) decreased to 39.8 ± 5.1 µM (10 mg/kg) and 19.7 ± 2.8 µM (20 mg/kg) by the treatment with YS 51, respectively (n = 4).

Survival Rates. The bolus injection of LPS (20 mg/kg, i.p.) to ICR mice was associated with a 48-h survival rate of only 20% (i.e., 4 of 20 of animals). In contrast, pretreatment with YS 51 increased the survival rate to more than 90% (10 mg/kg, i.p.) and 95% (20 mg/kg, i.p.) at 48 h after LPS injection (Fig. 8). Thus, YS 51 significantly increased the survival rate of animals injected with LPS. Saline- and YS 51-injected groups had a 100% survival rate over the 48 h (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Effects of YS 51 on the survival rate of LPS-treated mice. Each group consisted of 20 animals. Saline was injected in control groups. LPS (20 mg/kg) was injected (i.p.) in LPS-treated groups. Two different doses of YS 51 (10 and 20 mg/kg) were injected 30 min before the LPS injection in each group. Thereafter, survival was monitored every 6 h up to 48 h. , significantly different at P < 0.01.

Inhibition of NF-B-DNA Complex by YS 51. To understand the mechanism of the inhibition of iNOS mRNA expression by YS 51, we compared the binding capacity of NF-B-DNA in nuclear extract of RAW cells stimulated with LPS plus IFN- for 2 h. As shown in Fig. 9, LPS plus IFN- caused a significant increase in the level of the NF-B-DNA complex, which was decreased by treatment with YS 51 in a concentration-dependent manner. Excess amounts of unlabeled NF-B abolished the binding, and the p65 subunit of NF-B was examined by immunoblot analysis using a p65 antibody.

View larger version (63K): [in this window] [in a new window]   Fig. 9.   Effects of YS 51 on DNA-binding activity of NF-B in activated RAW 264.7 cells. RAW cells were stimulated with LPS plus IFN- for 2 h in the absence or presence of YS 51. Nuclear extracts from these cells were obtained and combined with a labeled NF-B probe nucleotide.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Previously, we reported that isoquinoline alkaloids such as higenamine and YS 49 inhibited iNOS induction in vascular smooth muscle cells and RAW 264.7 cells (Kang et al., 1999a,b). To obtain more information about the beneficial effects of isoquinoline alkaloids in endotoxemia, the effects of YS 51, a positional isomer of YS 49, were investigated on NO production and iNOS expression in macrophage cells that was activated with LPS plus IFN-, on reactivity of LPS-stimulated vascular muscle, and on survival rate in LPS-treated rodents. NO, which is massively generated from iNOS in various cells several hours after a challenge with LPS and/or cytokines, has been known as an important pathologic mediator of endotoxin shock (Kilbourn et al., 1990). In the present study, it was demonstrated that YS 51 reduces, in a concentration-dependent manner, the production of NO and the expression of iNOS mRNA in LPS plus IFN-- treated RAW cells, as well as restores the vascular contractile response to vasoconstrictor, PE, in rat aortas treated with LPS in vitro and ex vivo. These results signify the importance of YS 51 as a benefit in endotoxemia, since the major problem of endotoxemia in human and experimental animal is a rapid lowering of blood pressure (shock) and impaired responsiveness to vasoconstrictor agents (vasophlegia). In fact, LPS plays a pivotal role in triggering the development of both clinical and laboratory manifestations of Gram-negative septicemia, such as impaired responsiveness to vasoconstrictor agents (Julou-Schaeffer et al., 1990). Indeed, blood vessels isolated from animals given endotoxin in vivo (Pomerantz et al., 1982) and in vitro have been shown to express iNOS isoform (Deakin et al., 1995; Kang et al., 1999a), which is believed to be responsible for the diminished vasoconstrictor responsiveness of the vascular smooth muscle. The incubation of vasculature for 8 h with 300 ng/ml was reported to be sufficient for induction of iNOS activity in vitro (Kang et al., 1999a). Although the expression of iNOS protein in rat aorta was not confirmed in in vitro experiments, YS 51 might reduce the iNOS protein expression in aorta as it did in RAW cells, which accounts for the restoration of vascular hyporeactivity to PE. This explanation was further verified with the in vivo experimental results that the expression of iNOS protein in lung tissues isolated from rats pretreated with LPS was significantly inhibited by YS 51. In addition, induction of iNOS in vascular tissues was examined indirectly by a relaxation study in which U46619, a thromboxane A₂ mimetic, was used as a vasoconstrictor (Vallance et al., 1989; Kang et al., 199a). L-Arginine relaxed the U46619-contracted aortas isolated from LPS-injected rats in a concentration-dependent fashion. This response was almost entirely absent in aortas taken from LPS plus YS 51-treated rats. D-Arginine did not relax the U46619-induced contraction of the aortas taken from either LPS- or LPS plus YS 51-treated rats (data not shown). The concentration of YS 51 necessary for inhibition of the LPS-induced vascular hyporeactivity ex vivo was parallel with that necessary for inhibition for iNOS protein expression in vivo. To further elucidate the mechanism of action for YS 51 on inhibition of iNOS expression, we measured iNOS promoter activity in RAW 264.7 cells. We found that YS 51 caused a concentration-dependent reduction in luciferase activity, indicating that YS 51 regulates iNOS gene at the transcriptional level. The effect of YS 51 is, thus, to prevent de novo synthesis of iNOS protein. The inducibility of iNOS by LPS has been already shown to be dependent on transcription NF-B (Xie et al., 1994). Pretreatment of RAW cells with YS 51 interfered with the LPS plus IFN--induced NF-B-DNA binding activity and significantly reduced iNOS mRNA expression. Although we did not measure NF-B-DNA binding activity in vivo in the present study, significant reduction of the expression of iNOS protein in rat lung by YS 51 in LPS-injected rats may reflect the ability of YS 51 to inhibit the activation of NF-B. Indeed, in the rat model, LPS was shown to activate NF-B in vivo, and its activation led to transcription of the iNOS gene and expression of the iNOS protein (Liu et al., 1997). The precise mechanism of action of YS 51 on inhibition of NF-B activation is not elucidated in the present study; however, it is likely that prevention of IB-kinase activation can result in interference of the translocation of NF-B from cytosol into nucleus. We are now investigating this possibility. These detailed studies will give us information about how isoquinoline structures share the ability to inhibit the activation of NF-B. For example, isoquinoline alkaloids, tetrandrine and higenamine, have been reported to inhibit the activation of NF-B and NF-B-dependent reporter gene expression in rat alveolar macrophages and RAW 264.7 cells, respectively, by LPS (Chen et al., 1997; Kang et al., 1999a). Therefore, more detailed study on the mechanism of inhibition of NF-B by YS 51 remains to be established. Our data show a direct effect of YS 51 on LPS-simulated generation of iNOS protein. However, inhibition of other mediators by YS 51 may additionally have contributed to the inhibitory effect of YS 51 on iNOS expression and NOx production. Because activation of NF-B and, in turn, induction of iNOS are regarded as pivotal events for the development of a variety of proinflammatory mediators such as TNF-, IL-1, and NO, the effects of YS 51 on these cytokines are of much interest for additional study. In fact, YS 51 decreased the TNF- mRNA expression in mouse peritoneal macrophages activated with LPS (Jung et al., 2000). Most importantly, YS 51 significantly suppressed the LPS-induced lethality in the mouse model. When observed up to 48 h, more than 90% survived with the given dose of YS 51 in LPS-treated mice compared with a 20% survival rate in LPS-alone-treated mice. The combination of LPS and a NOS inhibitor significantly prevented an LPS-induced elevation in plasma NOx in animals (Cobb et al., 1992; Minnard et al., 1994); however, NOS inhibitor increased the mortality rates in LPS-challenged mice (Cobb et al., 1992). Furthermore, NOS inhibitors were observed to decrease the survival of LPS-treated animals in a dose-dependent manner (Tracey et al., 1995). The increased mortality by combined use of both LPS and a NOS inhibitor appears to be related specifically to NOS inhibition (Tracey et al., 1995). The authors concluded that the decrease in survival may be associated with the inhibition of eNOS or nNOS isoform rather than iNOS isoforms (Tracey et al., 1995). Other possible mechanisms for the decreased survival suggested are deleterious effects on vascular integrity (Hutcheson et al., 1990; Laszlo et al., 1994) or cardiac function (Klabunde and Ritger, 1991; Cobb et al., 1992) of iNOS inhibitors. In this sense, it seems important to know whether YS 51 inhibits eNOS. When we measured cGMP in human umbilical vein endothelial cells that were treated with different concentrations of YS 51 for 10 h, YS 51 partially increased cGMP production, but that result was not statistically significant (data not shown). This suggested that YS 51 may not influence the NO production by eNOS in vascular endothelial cells. However, other actions of YS 51, such as inhibition of platelet aggregation, antithrombotic effects (Yun-Choi et al., 2001), and strong positive inotropic action on the heart (Kilbourn et al., 1994; Chang et al., 1998) may contribute to improving the survival.

In conclusion, the ability of YS 51 to suppress iNOS gene expression is suggested to be responsible for the restoration of LPS-induced vascular hyporeactivity, the reduction of serum NOx, and the increase in survival in LPS-injected mice. Therefore, the present data demonstrate that YS 51, a positional isomer of YS 49, might be beneficial against LPS-induced circulatory failure and mortality.