Introduction

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Bacterial lipopolysaccharide (LPS; endotoxin) and a number of cytokines, including interferon (IFN)-, induce an isoform of nitric oxide (NO) synthase in macrophages, resulting in NO formation that destroys bacterial pathogens and tumor cells (Marletta et al., 1990). The presence of NO in inflammatory cells modulates local cytotoxicity, edema formation, and leukocyte traffic and is thought to be involved in the pathophysiology of inflammatory disorders (Middleton et al., 1993). However, there is evidence that an enhanced formation of NO by inducible NO synthase (iNOS) also contributes to circulatory failure (hypotension and vascular hyporeactivity to vasopressor agents), multiple organ dysfunction, and death caused by endotoxin in rodents (Thiemermann and Vane, 1990). Inhibitors of NO synthase (NOS) can reverse or prevent the hypotension induced in animals by LPS, hemorrhage, and anaphylatic shock. Moreover, Wei et al. (1995) and MacMicking et al. (1995) showed that null mutant iNOS mice are resistant to the hypotension and death caused by LPS. Thus, it is suggested that iNOS plays a crucial role in LPS-induced death. The selective inhibitors of iNOS activity and/or iNOS protein expression may be beneficial for the treatment of systemic inflammatory diseases. Some isoquinoline alkaloids, including higenamine, have been used as folk remedies for the treatment of inflammation in oriental countries (Deng, 1990). Although the anti-inflammatory mechanism of action of these isoquinoline alkaloids is unclear, some of them significantly suppressed NO production in murine peritoneal macrophages by LPS, and they attenuated the LPS-induced hepatitis by suppression of tumor necrosis factor (TNF) production in mice (Kondo et al., 1993a,b). Specifically, tetrandrine, another isoquinoline analog, inhibited nuclear factor B (NF-B ) activation in rat alveolar macrophages by LPS (Chen et al., 1997). Furthermore, the synthesized isoquinoline analog HMN-1180 was shown to inhibit glutamate-stimulated NO production generated by neuronal NOS in the human neuroblastomoa cell line SK-N-MC (Nishio et al., 1998). It seems likely that isoquinoline moiety may have some modulatory role in the inhibition of iNOS expression and/or of iNOS activity. Although higenamine was reported to scavenge free radicals and to inhibit superoxidization of lipid in synovial fluid (Zhang and Chen, 1985), its effect on production of NO or iNOS mRNA expression, which is an important causative factor for inflammation and sepsis, has not been investigated. Thus, the purpose of this study was to determine whether higenamine has inhibitory action on NO production and iNOS mRNA expression in RAW 264.7 cells activated by LPS and IFN-. If so, we wanted to determine the inhibitory mechanism of action of NO production by higenamine. Finally, we wanted to determine whether higenamine shows beneficial effects in LPS-treated endotoxemia in rodents.

	Materials and Methods

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Materials. RPMI 1640, fetal calf serum, penicillin, streptomycin, and glutamine were supplied by Gibco Laboratories (Gaithersburg, MD). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), HEPES, LPS (Escherichia coli; serotype 0128:B12), indomethacin, phenylephrine (PE) HCl, sulfanilaminde, N-[1-naphthyl]ethylenediamine, sodium chloride, leupeptin, pepstatin A, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), pyrrolidine dithiocarbamate, and sodium citrate were obtained from Sigma Chemical Co. (St. Louis, MO). [-³²P]ATP was purchased from NEN-DuPont (Boston, MA). 9,11-Dideoxy-11,9-epoxymethanoprostaglandin F₂ (U46619) was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Higenamine was synthesized and purified as described in Chang et al. (1986).

Cell Culture and Stimulation. RAW 264.7 cells were obtained from the American Type Culture Collection (Rockville, MD). RAW 264.7 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. RAW 264.7 cells were stimulated with LPS (10 ng/ml) + IFN- (10 U/ml) in the presence or absence of different concentrations of higenamine (1, 10, or 100 µM). Higenamine was administered simultaneously or 1 h before stimulation with LPS + IFN-. Higenamine was dissolved in sterile, distilled water.

Cell Respiration. Cell respiration, an indicator of cell viability, was assessed by mitochondrial-dependent reduction of MTT to formazan (Gross and Levi, 1992). Cells in 96-well plates were incubated (37°C) with MTT (0.2 mg/ml for 60 min). Culture medium was removed by aspiration and cells were solubilized in dimethyl sulfoxide (100 µl). The extent of reduction of MTT to formazan within cells was quantified by measurement of the absorbance at 570 nm, A₅₇₀.

Analysis of iNOS mRNA. Total RNA was extracted from the cellular lysate. A sample 20 µg of total RNA per lane was subjected to electrophoresis on 1% agarose gels containing formaldehyde (Formalin) and transferred to nylon filters. The filter was then hybridized with a random primed ³²P-labeled murine macrophage iNOS cDNA probe (from 85 to 169 bp) in rapid hybridization solution (Quikhyb, Stratagene, CA) at 68°C for 1 h. The hybridized filter was subsequently washed twice for 15 min at room temperature with 2 × SSC/0.1% SDS and then twice for 15 min at 42°C with 0.2 × SSC/0.1% SDS. The filter was then exposed to an X-ray film. The filter was subsequently stripped and rehybridized with a [³²P]glyceraldehyde-3-phosphate dehydrogenase cDNA probe.

Assay for Nitrite Production. The RAW 264.7 cells were washed once with the RPMI 1640, and 5 × 10⁵ cells in the RPMI 1640 were then added to each well of 6-well plates. The cells were incubated overnight at 37°C in a humidified 5% CO₂ incubator. Various concentrations of higenamine were added to the cells 1 h before treatment of LPS (10 ng/ml) and IFN- (10 U/ml). The cells were then incubated at 37°C in a humidified 5% CO₂ incubator for 24 h. NO was measured as its stable oxidative metabolite, nitrite. At the end of the incubation, 100 µl of the culture medium was mixed with an equal volume of Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). The absorbance at A₅₅₀ was measured, and the nitrite concentration was determined with a curve calibrated on sodium nitrite standards.

NF-B Activation. Raw 264.7 cells were treated with LPS and IFN- in the presence (10 µM, 100 µM) or absence of higenamine for 60 min. Cells were harvested and lysed by hypotonic buffer (10 mM HEPES, 1.5 mM MgCl₂, pH 7.5), and the nuclei were pelleted by centrifugation at 3000 g for 5 min. Nuclear lysis was performed with a hypertonic buffer (30 mM HEPES, 10% glycerol, 1.5 mM MgCl₂, 450 mM KCl, 1 mM DTT, 0.3 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) and incubated on ice for 30 min. The suspension was centrifuged at 14,500 rpm at 4°C for 20 min, and the supernatant was retained for use in the DNA-binding assay. NF-B consensus oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') was end-labeled with [-³²P]ATP. Nuclear extracts (10 µg) were incubated with 2 µg of poly(dI-dC) and the ³²P-labeled DNA probe in the binding buffer (100 mM KCl, 30 mM HEPES, 1.5 mM MgCl₂, 0.3 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1 µg/ml of leupeptin, and 1 µg/ml of aprotinin) for 20 min at room temperature. DNA-binding activity was separated from free probe with a 4.8% polyacrylamide gel in 0.5 × TBE (44.5 mM Tris, 44.5 mM boric acid, and 1 mM EDTA). After electrophoresis, the gel was dried and subjected to autoradiography.

Tension Experiment for In Vitro Study. Male Sprague-Dawley rats (250-300 g) were sacrificed by decapitation (total of 20 animals). Thoracic aortae from rats were cleared of adhering periadventitial fat and cut into rings of 3 to 4 mm in width. Endothelium was removed by gently rubbing the intimal surface with a wooden stick as reported previously (Chang et al., 1993a). The rings were divided into five groups: LPS (300 ng/ml)-treated group (n = 5), different concentrations of higenamine (10, 30, and 100 µM) with LPS (300 ng/ml)-treated groups (n = 4, each group), and a control (Krebs' solution) group (n = 3). The tissues were incubated at 37°C and 95% O₂/5% CO₂ for 8 h. After completion of incubation, isometric force was measured with a force transducer (FT 03; Grass Instrument Co., Quincy, MA) as described in Chang et al. (1993a). In brief, the rings were mounted in 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. A tension of 1g was applied, and the rings were equilibrated for 60 min, changing the Krebs' solution every 20 min. Indomethacin (10 µM) was used to prevent the production of cyclooxygenase metabolites that are predominantly vasoconstrictors in this experimental setting. Concentration-response curves to PE (1 nM to10 µM) were obtained. For relaxation studies, rings were contracted with 20 nM U46619 and L-arginine, D-arginine (1, 3, 10, 30, 100 µM), or sodium nitroprusside (SNP; 1, 3, 10, 30, 100 nM) were introduced cumulatively after plateau contraction had been reached.

In Vivo Hemodynamic Experiments. Male Sprague-Dawley rats (270-300 g) were anesthetized with sodium pentobarbital (50 mg/kg i.p.). The trachea was cannulated to facilitate respiration and allowed to ventilate at room temperature. The right carotid artery was cannulated and connected to a pressure transducer for the measurement of phasic and mean arterial pressure (MAP) and heart rate (HR), which were digitized by a Maclab A/D converter (AD Instruments, Milford, MA), and stored and displayed on a Macintosh personal computer. The left femoral vein was cannulated for the administration of drugs. Upon completion of the surgical procedure, cardiovascular parameters were allowed to stabilize from 20 to 30 min. After recording baseline hemodynamic parameters, animals [higenamine (10 mg/kg i.p.) pretreated group (n = 3) and control group (n = 3)] were given norepinephrine (NE; 1 µg/kg bolus i.v.), and 10 min later animals received LPS (5 mg/kg i.v.) as a slow injection over 5 min. The pressor responses to NE were reassessed at 60, 120, and 180 min after LPS injection.

Serum Nitrite/Nitrite (NOx) Measurement. Twelve rats were divided into four groups. LPS (5 mg/kg i.p.)-treated group (n = 4), LPS + higenamine (10 mg/kg i.p.)-treated group (n = 5), saline-treated group (n = 2), and higenamine (n = 3)-treated group. Higenamine was administered 90 min before LPS. After an 8-h LPS treatment, a whole-blood sample was withdrawn by cardiac puncture. The plasma nitrite concentration was determined by reducing the nitrate enzymatically with 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 flavin adenine dinucleotide, and 10 U/ml nitrate reductase, pH 7.5) for 30 min at 37°C. 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.

Survival Experiment. Mice (ICR) were injected i.p. with 20 mg/kg of LPS (n = 20) in a volume of 200 µl. Higenamine was administered i.p. 30 min before injection of LPS in a concentration of 10 mg/kg (n = 20) or 20 mg/kg (n = 20). After LPS injections, mice were observed every 6 h for 48 h.

Statistical Evaluations. Data are expressed as mean ± S.E. of results obtained from the number (n) of animals used. Differences between data sets were assessed by one-way ANOVA followed by Dunnett's t test. A level of P < .05 was accepted as statistically significant.

	Results

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Higenamine Inhibits NO Production and iNOS mRNA Expression in RAW 264.7 Cells. In control media, nitrite was accumulated 5 ± 0.8 µM, which was increased 60 ± 2.6 µM by LPS + IFN- (Fig. 1A). Pre- or cotreatment of higenamine concentration-dependently decreased the nitrite, which correlated well with the decreased expression of iNOS mRNA (Fig. 1B). The concentration of 50% inhibition of iNOS mRNA expression (IC₅₀) by higenamine was 53 ± 2.6 µM. MTT tests indicated that the inhibitory expression of iNOS mRNA by higenamine in RAW 264.7 cells was not due to cell damage (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effects of higenamine on NO production and iNOS mRNA expression in RAW 264.7 cells activated by LPS (10 ng/ml) + INF- (10 U/ml). A, higenamine was added 1 h before cytokines. Higenamine inhibited production of NO concentration-dependently. Nitrite was measured by the Griess reagent in 0.5 ml of the conditioned medium from cells treated for 24 h. B, densitometric analysis of the gel photograph of messages for the iNOS:NADPH ratio. Higenamine (1, 0 µM; 3, 1 µM; 4, 10 µM; 5, 100 µM) effectively inhibited the expression of iNOS mRNA in RAW 264.7 cells activated by cytokines (lane 2). One representative experiment of at least three is presented. Values represent means ± S.E. of triplicate determinations from a representative experiment performed three separate times with comparable results. *, significantly different from all other groups at P < .05.

Higenamine Protects Vascular Hyporeactivity against LPS In Vitro. Fig. 2A shows a typical physiological recordings of vascular contractility to PE (10 nM to 10 µM) in LPS-treated aorta and higenamine (100 µM)- + LPS-treated aorta. Contractile response to PE was significantly diminished in LPS-treated aorta compared with that in higenamine-cotreated aorta. Concentration-response curves for PE are depicted in Fig. 2B. To investigate whether iNOS expression in vascular smooth muscles is responsible for the diminished contractions in this experimental setting, L-arginine, NO substrate, was cumulatively administered after reaching a plateau contraction with U46619 in both LPS- and LPS- + higenamine-treated aortae. As shown in Fig. 3A and B, LPS-treated aorta was relaxed by L-arginine in a concentration-dependent manner, but the relaxation response was significantly diminished in aorta that was coincubated with LPS + higenamine. However, D-arginine was without effect in both aortae (data not shown). To confirm that this relaxation response was specific to L-arginine, SNP was added. As shown in Fig. 3C, SNP relaxed both aortae concentration-dependently.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects of higenamine on vascular contractility by vasoconstrictor in LPS-treated 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 + higenamine (Hig, 100 µM) for 8 h in vitro. Arabic numbers indicate log molar concentration. B, concentration-response curves of PE-induced contraction. Values are expressed as means ± S.E. of contractile forces in grams. *, significantly different from all other groups at P < .05.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effects of higenamine on L-arginine- and SNP-induced relaxation in rat aorta that had been incubated with LPS. A, a representative tracing of L-arginine (L-Arg)-induced relaxation in rat thoracic aorta that had been incubated with either LPS (300 ng/ml) or LPS + higenamine (Hig, 100 µM) for 8 h in vitro. Contraction was induced by U46619 (20 nM), and arabic numbers indicate log molar concentration. B, concentration-response curves of L-arginine (B)- and SNP (C)-induced relaxation. Values are expressed as means ± S.E. of at least three experiments. **, significantly different from all other groups at P < .01.

Higenamine Protects Hypotension in Endotoxin-Treated Rats In Vivo. At the end of the 20- to 30-min stabilization period, values for MAP ranged from 125 ± 7 to 130 ± 5 mm Hg and were not significantly different among any of the animal groups studied. In sham-operated animals treated with vehicle, MAP and HR were stable throughout the experimental period, whereas in sham-operated animals treated with higenamine i.p., the hypotension lasted >90 min, and thereafter it slowly returned to basal level (initial MAP, 129 ± 3 mm Hg; 10 min after higenamine, 80 ± 5 mm Hg; 30 min after higenamine, 93 ± 8 mm Hg, 90 min after higenamine, 120 ± 7 mm Hg; n = 4). Therefore, higenamine was administered i.p. at least 90 to 100 min before LPS. Administration of LPS caused a rapid but transient fall in MAP from 130 mm Hg to 66 mm Hg (n = 3) within 5 min, which had partly recovered by 120 min (n = 3). After 120 min, there was a second fall in MAP from 92 ± 8 mm Hg to 63 ± 7 mm Hg (n = 3) at 180 min. This delayed and the early (within 60 min) fall in MAP was abolished by higenamine pretreatment (Fig. 4). Baseline values for HR were significantly (P < .05) different between sham-operated vehicle- and higenamine-treated groups because of the cardiac  adrenoceptor-stimulating action of higenamine (Park et al., 1984) [i.e., 406 ± 8 beats/min in sham-operated vehicle-treated groups (n = 4) and 477 ± 11 beats/min in sham-operated higenamine-treated groups (n = 5)]. LPS injection reduced HR immediately but followed by an increase in HR from 30 to 180 min. However, LPS did not change the HR in higenamine-pretreated rats (Fig. 4).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Time-course effects of higenamine (Hig) on heart rate and mean arterial blood pressure after bolus injection of LPS in anesthetized rat. *, significant difference between LPS groups and higenamine + LPS groups at P < .05. Values are expressed as means ± S.E. of at least three experiments.

Higenamine Restores Vascular Reactivity in Endotoxin-Treated Rats In Vivo. In sham-operated animals, higenamine reduced the pressor effects to NE (1 µg/kg) compared with that of controls (26 ± 7 mm Hg in higenamine-treated animals versus 39 ± 4 mm Hg in control animals; n = 4, P < .05). Administration of LPS depressed the pressor effect to NE in both groups. When the pressor response to NE at zero time (before the LPS) was considered as 100% in each group, the percent response to NE after 30 min in LPS- and higenamine- + LPS-treated animals was 55.7 ± 6% and 53.3 ± 5% (n = 4), respectively. The pressor response to NE between the two groups was significantly (P < .05) different at 120 and 180 min, respectively, after LPS treatment. As shown in Fig. 5, for example, 180 min after LPS treatment, the response was 84.6 ± 5% of the original level (22 ± 5 mm Hg; n = 4, P < .05) in higenamine- + LPS-treated animals, whereas it was reduced to 36% (14 ± 2 mm Hg; n = 4) in LPS-treated animals.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Effects of higenamine (Hig) on pressor responses to NE in pentobarbital-anesthetized rats treated with LPS. Pressor responses to NE (1 µg/kg bolus i.v.) were assessed at 30, 60, 120, and 180 min after LPS injection. Pressor responses to NE at time 0 were normalized between LPS and LPS + higenamine groups. Values are expressed as means ± S.E. of at least three experiments. *, significantly different from all other groups at P < .05.

Effects of Higenamine on Plasma NOx Levels in LPS-Treated Rat. As shown in Fig. 6, the concentration of NOx in the plasma after saline and higenamine (10 mg/kg i.p.) treatment was 7.2 ± 3 and 6.5 ± 2 µM, respectively (n = 4). After 8 h of LPS administration, the plasma NOx elevated to 78 ± 7 µM, which was significantly (P < .05) decreased to 43 ± 5 µM by the treatment with higenamine 90 min before LPS (n = 3).

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Effects of higenamine (Hig) on NOx production in LPS-injected rat plasma. Blood samples were collected from cardiac puncture 8 h after LPS (10 mg/kg i.p.) injection. Samples were centrifuged, and serum fraction was subjected to analysis to measure nitrite/nitrate concentrations. For the positive controls, saline and higenamine (10 mg/kg) also were injected i.p. in separate animals. Values represent means ± S.E. of three separate experiments. *, significantly different at P < .05.

Survival Experiment. About 4 h after LPS injection, all mice appeared to be febrile and cling together. In the LPS-treated group, mice began to die 14 h after the injection, but mice pretreated with higenamine (10 mg/kg, 20 mg/kg) did not die until 30 h after LPS injection (Fig. 7). The protective effect of higenamine also was seen when the agent was administered simultaneously with LPS (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effects of higenamine (hig) on 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 higenamine (10 mg/kg, 20 mg/kg) were injected i.p. 30 min before the LPS injection, and survival was monitored every 6 h up to 48 h. **, significantly different at P < .01.

Higenamine Inhibits LPS-Induced NF-B Activation in RAW 264.7 Cells. To understand the mechanism of action of higenamine on the inhibition of iNOS mRNA expression, we compared the appearance of the NF-B-DNA complex in nuclear extracts of RAW cells challenged with LPS ± higenamine for 60 min. As shown in Fig. 8, LPS caused a significant increase in the level of the NF-B-DNA complex. Higenamine decreased the level of the NF-B-DNA complex.

View larger version (124K): [in this window] [in a new window]   Fig. 8.   Effects of higenamine on DNA-binding activity of NF-B in activated RAW 264.7 cells. RAW 264.7 cells were stimulated with LPS + IFN- for 60 min in the presence or absence of higenamine. Nuclear extracts from these cells were obtained and combined with a labeled NF-B probe nucleotide. Lane 1, nonstimulated; lane 2, stimulated; lane 3, stimulated in the presence of 100 µM higenamine; lane 4, stimulated in the presence of 10 µM higenamine; lane 5, stimulated in the presence of 10 µM pyrrolidine dithiocarbamate; lane 6, competitive binding with excess unlabeled oligonucleotides.

	Discussion

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Inflammatory mediators such as interleukin-1, LPS, TNF-, and IFN- stimulate expression of iNOS in rodent macrophages in vitro. The high level of NO produced by iNOS appears to mediate the cytotoxic actions of macrophages on target cells (Green et al., 1990). However, the excess production of NO has been implicated in pathogenesis and tissue damage in a growing number of immunological and inflammatory diseases, including arthritis (Farrell et al., 1992; Sakurai et al., 1995). Expression of iNOS mRNA, iNOS protein, and NO production were shown in ex vivo organ cultures in both inflammatory synoviocytes and chondrocytes isolated from rheumatoid arthritis (RA) patients, in which the NO production was suppressed by N^G-monomethyl-L-arginine, an inhibitor of NO synthase (Sakurai et al., 1995). It seems likely that the increased NO production may contribute to the pathological features in inflammatory arthritis; thus, inhibition of NO production in the vicinity of inflammated tissues may be beneficial in this condition. We have demonstrated that higenamine concentration-dependently reduced NO production and iNOS mRNA expression in RAW 264.7 cells activated by LPS + IFN-. Higenamine has been widely used as traditional remedy for the treatment of RA in oriental countries (Deng, 1990). We propose that the inhibitory action of NO production by higenamine is responsible for the treatment of RA along with the scavenging action of oxygen free radicals (Zhang and Chen, 1985).

However, increased production of NO from an iNOS contributes to the pathophysiology of endotoxic- and cytokine-induced shock. LPS plays a 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; Chang et al., 1993b). The results of the current study confirmed that contractile responses to PE in aortae incubated with LPS in vitro are impaired, which is associated with induction of iNOS enzyme in the vascular smooth muscle. However, the depressed contractile response to PE was prevented in aortae coincubated with higenamine. Furthermore, the relaxation response to L-arginine, NOS substrate, but not to D-arginine was significantly diminished in aortae coincubated with higenamine. These observations indirectly suggest that higenamine may inhibit iNOS activity or iNOS expression in the vascular smooth muscle. This idea comes from the results that SNP-induced relaxation in both LPS- and LPS- + higenamine-treated aortae in a concentration-dependent manner. So, it is not nonspecific for the diminished relaxation to L-arginine in higenamine-cotreated aortae. In fact, incubation of 300 ng/ml LPS in rat aorta for 8 h was reported to be sufficient to increase cGMP and to induce relaxation by L-arginine in vitro (Moritoki et al., 1996). Because suppression of vascular contractile function by LPS requires de novo synthesis of protein (McKenna, 1990), possible intervention of higenamine in the process of iNOS induction may be the main mechanism of action. In septic shock, the release of cytokines that activate iNOS in cells such as macrophages and vascular smooth muscle is associated with extreme hypotension that has been reversed by NOS inhibitors in animals (Wu et al., 1995) and in humans (Petros et al., 1991). Although we did not measure iNOS activity and iNOS mRNA expression in vascular smooth muscles in this study, results from the Northern blot assay with RAW 264.7 cells and functional studies in aortae indicate that higenamine may inhibit iNOS induction in rat aorta as well. Many reports suggest that iNOS mRNA was expressed in both macrophages and in smooth muscle cells by LPS and cytokines. Of particular interest is that the concentration to inhibit iNOS mRNA expression of higenamine (IC₅₀ = 53 µM in this study) is enough to elicit positive inotropic action. In isolated murine atria, higenamine showed positive inotropic action at a concentration range from 12.5 to 800 nM, where the EC₅₀ value was 97.0 nM (Kimura et al., 1994). Although the administration of inotropic agents alone did not reverse the endotoxin-mediated hypotension in humans (Vincent et al., 1990), the combination of N^G-monomethyl-L-arginine with dobutamine did reverse the endotoxin-mediated myocardial depression and vascular dilation in animals (Kilbourn et al., 1994). In human and experimental animal models of sepsis, cytokines are released in a sequential manner and are thought to contribute to the clinical manifestations of the sepsis syndrome and possibly to the end organ dysfunction. Cardiac contractile dysfunction has been documented in patients (Parker et al., 1984) and in experimental animal models of systemic sepsis complicated by hypotension (Natanson et al., 1989). The relative importance of systemic sepsis-related cardiac contractile failure is not well understood, but it appears to contribute, at least in some patients, to a fatal outcome (Vincent et al., 1992). Our data show that higenamine inhibits the development of the delayed vascular failure caused by LPS, which is associated with induction of iNOS enzyme (Wu et al., 1995). Our finding that higenamine prevents the expression of iNOS implies that an agent like higenamine having positive inotropic activity (Park et al., 1984), along with inhibiting iNOS expression, as in the present study, could be more effective in a condition where myocardial contractility is likely to depress, such as in septic shock and/or the endotoxin-induced inflammatory disorders. Furthermore, higenamine did not inhibit neuronal constitutive NOS activity when checked from the rat cerebellum (data not shown). Thus, it may be useful in diseases associated with an ongoing local or systemic inflammatory response in which an enhanced formation of NO by iNOS has been reported to contribute to pathogenesis or pathophysiology. What is the mechanism of action of higenamine to inhibit iNOS expression? The inducibility of iNOS by cytokines has been already shown to be dependent on two transcription factors: IFN regulatory factor 1 (for IFN inducibility) (Kamijo et al., 1994) and NF-B (for LPS inducibility) (Xie et al., 1994). In particular, NF-B induces many inflammatory genes that decode for proinflammatory cytokines, chemokines that selectively attract inflammatory cells, and inflammatory enzymes such as iNOS. NF-B is probably involved in the generation of both proximal and distal portions of the cytokine cascade and appears to be an ideal target for modifying acute inflammation. Tetrandrine, another isoquinoline alkaloid, has been reported to inhibit the activation of NF-B and NF-B-dependent reporter gene expression in rat alveolar macrophages by LPS (Chen at al., 1997). Similarly, higenamine inhibited the activation of NF-B in LPS- + IFN--stimulated RAW 264.7 cells. Higenamine also decreased the plasma NOx levels, which indicates that this drug is effective in vivo and thus may reinforce our idea that the reduction in nitrite accumulation could be due to a transcriptional inhibition of iNOS expression.

In conclusion, we provided evidence that higenamine inhibited NO production and iNOS mRNA expression in RAW 264.7 cells activated by LPS and IFN-. Thus, it is clear that the ability to suppress iNOS gene expression by higenamine may be responsible for anti-inflammatory use of this drug in RA patients in oriental countries. Furthermore, our data demonstrate that higenamine may be beneficial against LPS-induced vascular hyporeactivity and LPS-induced circulatory failure and mortality. Thus, higenamine may be useful in inflammatory diseases in which enhanced formation of NO is the main causative factor.