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Originally published online as doi:10.1189/jlb.0105048 on June 8, 2005

Published online before print June 8, 2005
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(Journal of Leukocyte Biology. 2005;78:696-704.)
© 2005 by Society for Leukocyte Biology

Effect of boldine, secoboldine, and boldine methine on angiotensin II-induced neurtrophil recruitment in vivo

Rossana Estellés*, Lara Milian*, Yafa Naim Abu Nabah*, Teresa Mateo*, Miguel Cerdá-Nicolás{dagger}, Mercedes Losada*, María Dolores Ivorra*, Andrew C. Issekutz{ddagger}, Julio Cortijo*,§, Esteban J. Morcillo*, María Amparo Blázquez* and María-Jesús Sanz*,1

* Departments of Pharmacology and
{dagger} Pathology, Faculty of Medicine, Universidad de Valencia, Spain;
{ddagger} Department of Pediatrics, Division of Immunology, Dalhousie University, Halifax, Nova Scotia, Canada; and
§ Valencia General Hospital Foundation, Spain

1Correspondence: Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Av. Blasco Ibañez, 15, 46010 Valencia, Spain. E-mail: Maria.J.Sanz{at}uv.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiotensin-II (Ang-II) has inflammatory activity and is involved in different diseases associated with the cardiovascular system. This study has evaluated the effect of boldine (B), and two phenanthrene alkaloids semisynthesized by us, secoboldine (SB) and boldine methine (BM), on Ang-II-induced neutrophil recruitment. Intraperitoneal administration of 1 nM Ang-II induced significant neutrophil accumulation, which was maximal at 4–8 h. BM inhibited neutrophil infiltration into the peritoneal cavity at 4 h and 8 h by 73% and 77%, respectively, SB at 8 h by 55%, and B had no effect on this response. Although BM inhibited the release of cytokine-inducible neutrophil chemoattractant/keratinocyte-derived chemokine, macrophage inflammatory protein-2 (MIP-2), and platelet-activating factor (PAF) elicited by Ang-II, SB only reduced the release of MIP-2 after 4 h of its administration. Sixty-minute superfusion of the rat mesentery with 1 nM Ang-II induced a significant increase in the leukocyte-endothelial cell interactions and P-selectin up-regulation, which were inhibited by 1 µM BM and SB. The generation of reactive oxygen species (ROS) in endothelial cells stimulated with Ang-II was inhibited significantly by the three alkaloids tested. BM also diminished Ang-II-induced interleukin-8 release from endothelial cells and blocked the PAF receptor on human neutrophils (concentration of the compound needed to produce 50% inhibition value: 28.2 µM). Therefore, BM is a potent inhibitor of Ang-II-induced neutrophil accumulation in vivo. This effect appears to be mediated through inhibition of CXC chemokine and PAF release, ROS scavenging activity, and blockade of the PAF receptor. Thus, it may have potential therapeutic interest for the control of neutrophil recruitment that occurs in inflammation associated with elevated levels of Ang-II.

Key Words: aporphine alkaloids • phenanthrene alkaloids • leukocyte • endothelium • intravital microscopy • chemokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nowadays, it is well accepted that there is a direct relation between blood pressure and the incidence of different cardiovascular events, such as stroke and myocardial infarction. In this regard, activation of the renin-angiotensin (Ang) system has been demonstrated in myocardial ischemia, acute myocardial infarction, coronary occlusion, and reperfusion models, as well as in chronic left ventricular dysfunction post-myocardial infarction [1 2 3 ]. Ang-II is the main effector peptide of the renin-Ang system, and in addition to its role as a potent vasoconstrictor and blood pressure and fluid homeostasis regulator, it has been shown to exert proinflammatory activity. Ang-II receptors have been demonstrated on human neutrophils [4 ]. In vitro, it has been shown that Ang-II releases a neutrophil chemoattractant factor from cultures of human and bovine arterial endothelial cells, which appears to be a partially characterized arachidonic acid metabolite generated via the lypoxygenase pathway [5 ]. In addition, it has been observed that Ang-converting enzyme inhibition can attenuate postischemic adhesion of neutrophils in isolated, perfused hearts [6 ]. More recently, we have revealed that acute Ang-II superfusion of the rat mesenteric microcirculation provokes neutrophil accumulation in vivo at subvasoconstrictor doses through rapid endothelial P-selectin up-regulation and CXC chemokine release, which is primarily mediated via Ang-II type 1 receptor interaction [7 , 8 ].

Conversely, our group isolated two phenanthrene alkaloids, uvariopsine and stephenanthrine, from the fresh root of Dennettia tripetala [9 ] and proved that they could inhibit Ang-II-induced leukocyte-endothelial cell interactions in vivo [10 ]. This effect was partly mediated through inhibition of the generation of reactive oxygen species (ROS), down-regulation of P-selectin expression on the endothelial cell, and blockade of platelet-activating factor (PAF)-induced responses by interacting with its receptor [10 ]. Based on their structural features, we semisynthesized four phenanthrene and one aporphine alkaloid from natural boldine (B) and evaluated their inhibitory effect on ROS generation [11 ]. The reasons for this semisynthetic process were two: first, to increase the number of phenolic groups in the structure of these alkaloids to enhance their ROS scavenging properties and second, to obtain enough quantities of the compounds to test their pharmacological activity in different in vivo and in vitro models. Indeed, in that study, B, secoboldine (SB), and boldine methine (BM) displayed more powerful ROS scavenger activity than other semisynthesized alkaloids or even uvariopsine and stephenanthrine. These alkaloids may present broad anti-inflammatory activity, which can make them interesting candidates to be used in the treatment of several inflammatory diseases not necessarily related to the cardiovascular system. However, as Ang-II is a proinflammatory molecule, which is associated with the development of different cardiovascular disorders, the search for new compounds that ameliorate its activity may constitute an alternative therapy for the control of inflammatory disease states in which this peptide hormone is involved. Therefore, in the present study, we have evaluated the effect of B, SB, and BM (Fig. 1 ) on Ang-II-induced neutrophil accumulation in vivo. For this purpose, we have investigated their effect on the neutrophil recruitment elicited by the intraperitoneal (i.p.) injection of Ang-II. Furthermore, we have used intravital microscopy within the rat mesenteric microcirculation and tested its effect on the leukocyte-endothelial cell interactions elicited by this peptide hormone. Finally, we have also characterized the mechanisms that may contribute to their anti-inflammatory activity.



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  		Figure 1. Chemical structure of B, SB, and BM. Aporphine skeleton is showed by dotted lines.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ethical approval
All of the experimental protocols were approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine, University of Valencia (Spain), and were conducted humanely.

Neutrophil leukocyte migration into the peritoneal cavity
Male Sprague-Dawley rats (200–250 g) were sedated with ether and i.p.-injected with 5 ml phosphate-buffered saline (PBS) or 1 nM Ang-II. After 1, 4, 8, and 24 h of the Ang-II administration, animals were killed by an anesthetic overdose, and the peritoneal cavity was first lavaged with 5 ml PBS and then with 30 ml heparinized PBS (10 U ml–1). The exudates were then centrifuged separately to obtain the cell pellets and the supernatant fluids. The cell pellets were then combined for total leukocyte counts in a hemocytometer and differential cell analysis of 500 cells/slide on cytospins stained with May-Grünwald and Giemsa stains. The results were expressed as the number of neutrophils recovered from each cavity.

The supernatant from the first (5 ml) lavage, after addition of carrier protein [0.5% bovine serum albumin (BSA)] and storage at –20°C, was used for determination of cytokine-inducible neutrophil chemoattractant/keratinocyte-derived chemokine (CINC/KC), macrophage inflammatory protein-2 (MIP-2), and PAF concentrations. Rat CINC/KC and MIP-2 levels were determined by conventional sandwich enzyme-linked immunosorbent assays (ELISAs). Results are expressed as pM chemokine in the supernatant from the 5 ml lavage. No cross-reactions were detected in the CINC/KC and MIP-2 assays with any rat chemokines tested: regulated on activation normal T expressed and secreted, monocyte chemoattractant protein-1, and MIP-2 or KC, respectively (<0.01%).

PAF was extracted from 1 ml sample of each peritoneal exudate using chloroform/methanol (2:1, v/v). The organic phases were dried under N2 atmosphere. The quantitave measurement of PAF from dried samples was checked using a PAF-3H scintillation proximity assay system (TRK-990). Before the assay, the dried PAF samples were reconstituted with 100 µl working assay buffer supplied with the kit. All determinations were made in duplicate, and PAF levels were measured as recommended by the manufacturer. Results are expressed as pg/ml peritoneal exudate.

The effect of the different alkaloids investigated in Ang-II-induced responses after its i.p. administration was determined by pretreatment of the animals i.p. with 20 nmol/kg B, SB, or BM, 15 min prior to Ang-II injection. The dose selected for testing these alkaloids was that which caused the total inhibition of Ang-II-induced, leukocyte-endothelial cell interactions in vivo by two related alkaloids, uvariopsine and stephenanthrine, previously studied by us [10 ]. A concentration response on this assay was not carried out as a result of the amount of animals to be used.

Finally, blood samples (2 ml) from rats exposed to saline or 1 nM Ang-II for 1 h were taken to determine the effect of the alkaloids on circulating CINC/KC and MIP-2 levels. Heparine was added to have a final concentration of 100 units/ml to promote the release of the chemokines bound to erythrocytes. Samples were centrifuged, and the plasma was stored at –20°C for ELISA.

Intravital microscopy
The experimental preparation used in this study was similar to that described previously [10 ]. Male Sprague-Dawley rats (200–250 g) were fasted for 24 h and anesthetized with sodium pentobarbital (50 mg/kg, i.p.). A tracheostomy was performed to maintain a patent airway throughout the experiment. A polyethylene catheter was inserted in the right carotid artery to monitor mean arterial blood pressure (MABP) through a pressure transducer (Spectramed Statham P-23XL, Hato Rey, P. R.) connected to a recorder (GRASS Instrument Co. RPS7C8B, Quincy, MA), and a second catheter was placed in the contralateral jugular vein to permit the intravenous administration of anesthetic. A midline abdominal incision was made, and the rats were then placed in a supine position on an adjustable Plexiglass microscope stage. A segment of the midjejunum was exteriorized and draped over an optically clear viewing pedestal, which allowed transillumination of a 2-cm2 segment of the tissue. The temperature of the pedestal was maintained at 37°C, and the exposed tissue was covered with saline-soaked gauze to minimize tissue dehydration. The exposed mesentery was suffused continuously at a rate of 1 ml/min with a warmed bicarbonate-buffered salt solution (pH 7.4).

The mesenteric preparation was then observed using an intravital orthostatic microscope (Nikon Optiphot-2, SMZ1, Badhoevedorp, The Netherlands), equipped with a 20x objective lens (Nikon SLDW) and a 10x eyepiece. A video camera (Sony SSC-C350P, Koeln, Germany) mounted on the microscope projected the image onto a color monitor (Sony Trinitron PVM-14N2E), and the images were video-recorded (Sony SVT-S3000P) for playback analysis. The final magnification of the video screen was 1300x. Animal temperature, monitored using a rectal electrothermometer, was maintained at 37°C with an infrared heat lamp.

Single, unbranched mesenteric venules (20–40 µm in diameter) were selected for study, and their diameters were measured on-line using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station). Centerline red blood cell velocity (Vrbc) was also measured on-line using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University). Venular blood flow was calculated from the product of mean red blood cell velocity (Vmean=Vrbc/1.6) and cross-sectional area, assuming cylindrical geometry. Venular wall shear rate ({gamma}) was calculated based on the Newtonian definition: {gamma} = 8 x (Vmean/Dv)s–1, in which Dv is venular diameter [12 ].

The number of rolling, adherent, and emigrated leukocytes was determined off-line during playback of videotaped images. Rolling leukocytes were defined as white blood cells moving at a slower velocity than erythrocytes. Rolling leukocyte flux was determined by counting the number of rolling leukocytes/min, passing a reference point in the microvessel. The same reference point was used throughout the experiment, as leukocytes may roll for only a section of the vessel before rejoining the blood flow or becoming firmly adherent. Leukocyte rolling white blood cell velocity was determined by measuring the time required for a leukocyte to move along 100 µm length of the microvessel and is expressed as µm/s. A leukocyte was defined as adherent to venular endothelium if it were stationary for at least 30 s. Leukocyte adhesion was expressed as the number of cells adhered per 100 µm length of venule. Leukocyte emigration was expressed as the number of white blood cells per microscopic field surrounding the venule. The rate of emigration was determined from the difference between the number of any interstitial leukocytes present at the beginning of the experiment and those present at the end of the experiment.

Experimental protocol
After a 30-min stabilization period, baseline measurements (time 0) of MABP, Vrbc, Dv, shear rate, leukocyte rolling flux, and velocity and leukocyte adhesion and emigration were taken. The buffered-saline superfusion was continued or supplemented with Ang-II (1 nM), and recordings were performed for 5 min at 15-min intervals over a 60-min period during which the aforementioned leukocyte and hemodynamic parameters were measured.

In the first group of experiments, B (1 µM) was applied topically 10 min prior to Ang-II suffusion, then it was cosuperfused with Ang-II, and responses were evaluated for 5 min at 15-min intervals over a 60-min period. In the second group of experiments, SB (1 µM) was similarly applied prior to Ang-II suffusion, and in a third group of experiments, the same protocol was followed to test the activity of BM. Alkaloids were dissolved in dimethyl sulfoxide (DMSO) and diluted further in bicarbonate-buffered salt solution. The same percentage of DMSO (0.01% at 1 µM) was used in the control groups (buffer or Ang-II 1 nM).

Immunohistochemistry
Immunohistochemistry was used to examine the expression of P-selectin. Once the experiment using intravital microscopy was completed, the portion exposed to buffer or Ang-II, with or without the alkaloids (1 µM concentration) for 1 h, was then isolated and further fixed in 4% paraformaldehyde for 90 min at 4°C as described previously [13 ]. After fixation, the tissue was dehydrated using graded acetone washes at 4°C and embedded in paraffin wax, and 4 µm-thick sections were cut.

Immunohistochemical localization of P-selectin was accomplished using a modified avidin and biotin immunoperoxidase technique, as described previously by Sanz et al. [13 ]. Slides were immersed in antigen unmasking fluid, placed in a plastic coplin jar, and heated to boil in a microwave oven for 3–5 min. This was refilled if necessary with deionized water and microwaved for 3–5 min. The jar was placed at room temperature for 15 min and rinsed in deionized water. Tissue sections were incubated with the anti-rat P-selectin (RP-2) monoclonal antibody (mAb) for 24 h at 200 µg/ml. Control preparations consisted in the incubation with the isotype-matched murine antibody MOPC 21 (immunoglobulin G1) as primary antibody for the same period of time at 200 µg/ml. Positive staining was defined as a venule displaying brown reaction product.

Measurement of ROS generation in human umbilical vein endothelial cells (HUVECs)
HUVECs were isolated by collagenase treatment [14 ] and maintained in human endothelial cells’ specific medium EBM-2, supplemented with EGM-2 and 10% fetal calf serum (FCS). HUVECs were grown to confluence and used up to Passage 2 for the experiment. HUVECs were placed on 24-well culture plates, and prior to every experiment, they were incubated for 16 h in medium containing 1% FCS. Cells were washed twice with Hanks’ balanced salt solution. Then, 250 µl phenol red-free medium 199 with 0.25% BSA containing 140 µM ferricytochrome C, with or without Ang-II 0.1 µM, was added to each well, according to the protocol described by Zhang et al. [15 ]. In some wells, the cells were pretreated with B, SB, or BM (0.1–100 µM) for 30 min before Ang-II was added. The plate was kept in the cell culture incubator for 1 h. The supernatant from each reaction was pipetted out and analyzed by using a Wallac 1420 Victor2 multilabel counter (EG&G, Turku, Finland) at a wavelength of 550 nm. Superoxide dismutase (SOD) was used as positive control (1000 U/ml). The amount of superoxide released was calculated by dividing the difference in absorbance of the samples with or without SOD by the extinction coefficient for reduction of ferricytochrome C to ferrocytochrome C ({varepsilon}=21.1 cm–1 M).

Measurement of interleukin (IL)-8 release in HUVECs
HUVECs were isolated as described previously and preincubated with IL-1ß (1000 U/ml) for 24 h to induce synthesis and storage of IL-8 in Weibel-Palade bodies [16 ]. Then, they were washed twice and incubated for 1 h in fresh medium, with or without or 100 nM Ang-II. At the end of the experiment, cell-free supernatants were stored at –20°C for IL-8 quantification. Alkaloids (0.1–100 µM) were added to some wells 30 min prior to Ang-II (100 nM). IL-8 was measured by conventional sandwich ELISA.

In vitro [3H]PAF-binding studies
[3HPAF] (C18; 1-O-[3H] octadecyl-2-acetyl-sn-3-phosphocholine) receptor binding was studied in human polymorphonuclear leukocytes (PMNs; 2.5x106 cells/ml) by use of a modified method described previously by Vasänge et al. [17 ]. Incubations were performed for 120 min at 20°C in a total volume of 1 ml incubation buffer (0.6 mM NaH2PO4, 25 mM Tris HCl, 130 mM NaCl, 5.5 mM KCl, BSA 0.5%, pH 7.4) with [3H]PAF (0.4 nM) and different concentrations of inhibitors (B, SB, BM, final concentration: 1–1000 µM). The incubation was initiated by the addition of the cells. Nonspecific binding was determined with unlabeled PAF (2.0 µM). The binding reactions were terminated by rapid vacuum filtration using a Brandel cell harvester (M24R, Gaithersburg, MD) with glass fiber filters (Schleicher and Schuell, Keene, NH, nº. 30, presoaked in 0.3% polyethylenimine). The filters were then washed with 3 x 2 ml incubation buffer containing 0.1% BSA, and the filter-bound radioactivity was determined by liquid scintillation counting. Assays were conducted in triplicate. The concentration of the compound needed to produce 50% inhibition of [3H]PAF binding (IC50) value for BM was calculated from a nonlinear regression plot (Graph Pad Software, San Diego, CA).

Materials
Ang-II, BSA, DMSO, PAF, MOPC 21, ferricytochrome C, and SOD were purchased from Sigma Chemical Co. (St. Louis, MO). EBM-2 medium supplemented with EGM-2 was from Clonetics (Barcelona, Spain). Rat CINC/KC and MIP-2, antibodies to rat CINC/KC and MIP-2, and human IL-8 and IL-1ß were from PeproTech (London, UK). The antibody pair for human IL-8 ELISA was from R&D Systems (Madrid, Spain). Neutravidin-horseradish peroxidase was from Perbio Science (Cheshire, UK). K-Blue substrate was from Neogen (Lexington, KY); [3H]PAF (specific activity 159 Ci/mmol) and PAF-3H scintillation proximity assay system (TRK-990) were from Amersham Pharmacia Biotech (Uppsala, Sweden). Phenol red-free medium 199 was from Life Technologies (Paisley, UK). mAb against RP-2 was acquired as previously stated [18 ].

Statistical analysis
All data are expressed as mean ± SE. The data within groups were compared using a one-way ANOVA with a Newman-Keuls post hoc correction for multiple comparisons. A P value <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
As previously found by Nabah et al. [8 ], i.p. administration of 5 ml 1 nM Ang-II in rats induced a significant neutrophil recruitment, which was maximal at 4–8 h (Fig. 2 ). Pretreatment of the animals with B did not affect Ang-II-induced neutrophil accumulation into the peritoneal cavity (Fig. 2) . In contrast, although SB significantly inhibited the neutrophil recruitment at 8 h post-Ang-II administration by 55% (Fig. 2) , pretreatment with BM resulted in a significant reduction of neutrophil numbers 4 h and 8 h after Ang-II i.p. injection by 73% and 77%, respectively (Fig. 2) .



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  		Figure 2. Effect of B, SB, and BM on Ang-II-induced neutrophil recruitment in rat peritoneal cavity. Neutrophil counts in the peritoneal exudate in the following experimental groups: untreated rats i.p.-injected with 5 ml PBS; untreated rats exposed to 1 nM Ang-II i.p.; and Ang-II-exposed rats pretreated with 20 nmol/kg B, SB, or BM i.p. Results are mean ± SE for n = 5–8 animals per group: *, P < 0.05, or **, P < 0.01, relative to values in the PBS-injected group. +, P < 0.05, or ++, P < 0.01, relative to the Ang-II-untreated group.

 
To investigate whether the reduction in Ang-II-induced neutrophil accumulation by these alkaloids is a result of inhibition of the release and generation of CXC chemokines, CINC/KC and MIP-2 levels were measured in the peritoneal exudate of untreated and treated animals after 1 h and 4 h exposure to Ang-II. CINC/KC and MIP-2 levels were elevated following Ang-II injection, peaking at 1 h (Fig. 3A and 3C ), and significant CXC chemokine levels were still evident at 4 h (Fig. 3B and 3D) . B failed to decrease the Ang-II-induced augmentation of CINC/KC and MIP-2 content in the peritoneal exudate (Fig. 3) . Although SB had no effect on the release of CINC/KC elicited by Ang-II, it significantly reduced the levels of MIP-2 at 4 h after Ang-II exposure (Fig. 3) . Conversely, BM significantly decreased the levels of both CXC chemokines at 1 h and 4 h after Ang-II i.p. administration (Fig. 3) . Although clearly, most of MIP-2 is gone by 4 h, the inhibitory effects of SB and BM remain apparent but on a much smaller scale.



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  		Figure 3. Effect of B, SB, and BM on Ang-II-induced release of CINC/KC (A and B), MIP-2 (C and D), and PAF (E) in rat peritoneal exudates. CINC/KC, MIP-2, and PAF levels in the peritoneal exudate in the following experimental groups: untreated rats i.p.-injected with 5 ml PBS; untreated rats exposed to 1 nM Ang-II i.p.; and Ang-II-exposed rats pretreated with 20 nmol/kg B, SB, or BM i.p. Results are mean ± SE for n = 5–8 animals per group: *, P < 0.05, or **, P < 0.01, relative to values in the PBS-injected group. +, P < 0.05, relative to the Ang-II-untreated group.

 
As these alkaloids may provoke a reduction in the circulating levels of CINC/KC and MIP-2, plasma concentration of these two chemokines was measured. CINC/KC and MIP-2 levels were not significantly increased after 1 h Ang-II i.p. injection compared with PBS-injected animals (Table 1 ). In addition, administration of the alkaloids neither affected the plasma levels of CINC/KC nor those of MIP-2 after 1 h exposure to 1 nM Ang-II (Table 1) .


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  		Table 1. Circulating Levels of CINC/KC and MIP-2 in Animals Untreated and Treated with B, SB, and BM after 1 h Ang-II (1 nM) i.p. Injection

 
Ang-II can cause rapid PAF release, which may contribute to the neutrophil infiltration elicited by this peptide hormone [19 ]. As it is illustrated in Figure 3E , Ang-II induces a significant increase in PAF levels 1 h after its i.p. injection. Although pretreatment of the animals with B or SB did not affect the augmentation of this inflammatory mediator elicited by Ang-II, BM significantly reduced this effect by 80% (Fig. 3E) .

Intravital microscopy was used to examine leukocyte trafficking in the mesentery, as leukocyte-endothelial cell interactions would be expected to precede the tissue accumulation of neutrophils. Figure 4 illustrates acute Ang-II-induced leukocyte responses. Leukocyte rolling flux, adhesion, and emigration were significantly increased within 30 min of 1 nM Ang-II superfusion. After 60-min superfusion with Ang-II, increases in leukocyte rolling flux and concomitant significant decreases in the leukocyte rolling velocity were observed versus buffer (Fig. 4A and 4B) . Similarly, at the same time-point, Ang-II induced increases in leukocyte adhesion and emigration (Fig. 4C and 4D) . Pretreatment with B only significantly affected an Ang-II-induced increase in leukocyte rolling flux, which was diminished by 44% (Fig. 4) . However, cosuperfusion of Ang-II with 1 µM SB or 1 µM BM markedly diminished Ang-II-induced increase in the leukocyte rolling flux, adhesion, and emigration, which were nearly reduced to basal levels (Fig. 4) . In addition, the decrease in leukocyte rolling velocity induced by Ang-II at 60 min was reversed by the administration of either of these two phenanthrene alkaloids (Fig. 4) . Ang-II superfusion for 60 min neither affected MABP nor venular shear rate (Table 2 ). Similarly, alkaloid cosuperfusion with Ang-II had no effect on these responses. None of these treatments affected circulating leukocyte counts (data not shown).



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  		Figure 4. Effect of B, SB, and BM on Ang-II-induced leukocyte rolling flux (A), leukocyte rolling velocity (B), leukocyte adhesion (C), and leukocyte emigration (D) in rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30, and 60 min after topical superfusion with buffer (n=5) or with Ang-II (1 nM) in animals untreated (n=6) or cosuperfused with B 1 µM (n=6), SB 1 µM (n=5), or BM 1 µM (n=5). Results are represented as mean ± SE. *, P < 0.05, or **, P< 0.01, relative to values in the buffer group. +, P < 0.05, or ++, P < 0.01, relative to the Ang-II-untreated group.

 

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  		Table 2. Hemodynamic Parameters in Animals Untreated and Treated with B, SB, and BM before (0 min) and after (60 min) Ang-II Superfusion (1 nM)

 
Immunohistochemical experiments revealed that when the mesenteric tissue was subjected to 60 min buffer superfusion, endothelial P-selectin expression was not detectable (Fig. 5A ). Ang-II superfusion caused a significant up-regulation of P-selectin (Fig. 5B) , which was partially inhibited by B (Fig. 5C) and reduced to basal levels by cosuperfusion with SB or BM at 1 µM (Fig. 5D yand 5E) .



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  		Figure 5. Effect of B, SB, and BM on Ang-II-induced endothelial P-selectin up-regulation. P-selectin expression after buffer (A), Ang-II (B), 1 nM Ang-II + 1 µM B (C), 1 nM Ang-II + 1 µM SB (D), and 1 nM Ang-II + 1 µM BM (E) 60-min topical superfusion. Brown reaction product indicates positive immunoperoxidase localization for P-selectin on the vascular endothelium. All five panels are lightly counterstained with hematoxylin and have the same original magnification (x400). Results are representative of n = 4–5 experiments with each treatment.

 
Ang-II-induced leukocyte endothelial cell interactions are partly mediated through the production of ROS [19 ]. In addition, we have recently proved that these three alkaloids display ROS scavenging activity in human PMNs stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or by the hypoxanthine-xanthine oxidase system [12 ]. Therefore, they could inhibit leukocyte responses elicited by Ang-II through inhibition of superoxide anion generation by endothelial cells. HUVECs were stimulated with 100 nM Ang-II for 1 h in the absence or presence of B, SB, and BM. All alkaloids at the doses assayed inhibited in a concentration-dependent manner the release of superoxide anion induced by 100 nM Ang-II (Fig. 6 ).



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  		Figure 6. Effect of B, SB, and BM on superoxide anion release in HUVECs stimulated with Ang-II. HUVECs were grown to confluence in a 24-well culture plate, then incubated in the absence or presence of B, SB, or BM (0.1–100 µM) for 30 min, and stimulated with 100 nM Ang-II for 1 h, as described in Materials and Methods. The assay of superoxide release was performed by measurement of SOD-inhibitable reduction of ferricytochrome C. Results are expressed as percentage inhibition of the control from duplicate determinations. Results are represented as mean ± SE of n = 5–7 preparations.

 
Ang-II produces the rapid release of IL-8 from HUVECs pretreated with IL-1 [8 ]. Under these conditions, Ang-II 100 nM caused a significant increase of IL-8 content in the culture medium compared with that seen without stimulation with this peptide hormone (2599±134 vs. 1534±182 pM). B and SB had little effect on the release of this chemokine elicited by Ang-II (Fig. 7 ). In contrast, BM was able to inhibit in a concentration-dependent manner the release of IL-8 caused by 100 nM Ang-II (Fig. 7) .



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  		Figure 7. Effect of B, SB, and BM on IL-8 release in HUVECs stimulated with Ang-II and preincubated with IL-1. HUVECs were stimulated with IL-1 1000 U/ml for 24 h. Then the cells were washed and incubated in the absence or presence of B, SB, or BM (0.1–100 µM) for 30 min and stimulated with 100 nM Ang-II for 1 h as described in Materials and Methods. Results are expressed as percentage inhibition of the control from duplicate determinations. Results are represented as mean ± SE of n = 5 preparations.

 
Finally, as in a previous study, demonstrating that two natural phenanthrene alkaloids behaved as PAF receptor antagonists [11 ], in the present study, we have investigated whether B, SB, and BM display such capability. To test this possibility, a [3H]PAF-binding assay was carried out on human PMNs. As it is shown in Figure 8 , only BM inhibited in a concentration-dependent manner [3H]PAF binding to human PMNs. The calculated IC50 value was 28.2 ± 0.8 µM. B and SB only inhibited [3H]PAF binding to human PMNs at 100 µM by 47% and 51%, respectively.



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  		Figure 8. Effect of BM on [3H]PAF binding to human PMNs, which were incubated in the absence or presence of BM (1–1000 µM) and [3H]PAF (0.4 nM) for 120 min at 20°C, as described in Materials and Methods. Nonspecific binding was determined with unlabeled PAF (2.0 µM). Assays were conducted in triplicate. Results are represented as mean ± SE of n = 5–6 preparations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have demonstrated that a phenanthrene alkaloid semisynthesized from natural B, BM, clearly inhibited the neutrophil accumulation elicited by Ang-II. This effect was partly mediated by a diminished release of two CXC chemokines, MIP-2 and CINC/KC, the rat functional homologues of human IL-8 [20 , 21 ] and PAF. Conversely, circulating levels of CINC/KC and MIP-2 were not affected by alkaloid treatment. In addition, at 1 µM, it markedly reduced the increase in the leukocyte-endothelial cell interactions induced by 1 nM Ang-II superfusion in the rat mesenteric postcapillary venules without affecting the different hemodynamic parameters measured. The other phenanthrene alkaloid, SB, only inhibited the Ang-II-induced neutrophil accumulation at 8 h. This effect appears to be mediated through inhibition of MIP-2 synthesis but not its acute release. In contrast, leukocyte-endothelial cell interactions elicited by acute stimulation with Ang-II were totally abolished by SB cosuperfusion. The aporphine alkaloid, B, had no effect on the neutrophil recruitment elicited this peptide hormone and only significantly diminished the enhancement on leukocyte rolling flux induced by Ang-II. These results suggest that these phenanthrene alkaloids display a more powerful anti-inflammatory activity than their corresponding equivalents with aporphine skeleton. Furthermore, although SB and BM present a similar structure, the smaller inhibitory effect provoked by SB than that exerted by BM cannot be explained by differences in their hydrophobicity or basicity; however, SB lacks a methyl group in its structure. Therefore, the existence of a tertiary amine in BM could give more steric stability to the alkaloid interacting more specifically to its sites of action.

Although we have proved that the effects displayed by BM and SB on the neutrophil recruitment elicited by Ang-II are partly mediated by inhibition of CXC chemokine release, the effect of these alkaloids on Ang-II-induced, leukocyte-endothelial cell interactions suggests the existence of other mechanisms by which they can exert their anti-inflammatory activity. In this context, superoxide anion production is involved in chronically elevated Ang-II-induced hypertension [22 ]. In addition, we demonstrated that free radical generation was also implicated in acute Ang-II-induced leukocyte-endothelial cell interactions in vivo [19 ]. Furthermore, we have shown that these alkaloids showed free radical scavenging activity in a concentration-dependent manner in ROS generation by human PMN-stimulated fMLP and by the hypoxanthine-xanthine oxidase system [12 ]. As the vascular endothelium seems to play a primary role in the acute leukocyte recruitment induced by Ang-II, we next examined whether these alkaloids could also inhibit Ang-II-induced superoxide anion generation by endothelial cells. When HUVECs were stimulated with Ang-II, superoxide anion levels in supernantants were inhibited by preincubation with any of the three alkaloids in a concentration-dependent manner. In agreement with previous observations [12 ], the order of potency was BM > SB > B. These findings suggest that free phenolic groups in the structure of the alkaloids are a relevant feature for inhibition of ferricytochrome C reduction caused by superoxide anion. Nevertheless, other functional groups in the alkaloid structure may also account for the differences encountered in the order of potency exerted by the compounds tested.

Previous studies have demonstrated that superoxide anion produced via a hypoxanthine-xanthine oxidase-generating system and hydrogen peroxide have been shown to induce leukocyte influx through increased P-selectin expression [23 , 24 ]. In the present study, SB and BM were capable of reducing to basal levels Ang-II-induced endothelial P-selectin up-regulation in vivo, an effect likely mediated by their ROS scavenging properties. It is interesting that the significant decrease in Ang-II-induced leukocyte rolling flux elicited by B could be explained by the antioxidant properties of this aporphine alkaloid, which would result in a minor decrease in endothelial P-selectin. With regard to this, a recent study indicates that CXC chemokines play a fundamental role in the leukocyte recruitment detected in a colonic model of ischemia-reperfusion-induced tissue injury [25 ]. In that study, inhibition of xanthine-oxidase activity with allopurinol markedly reduced the colonic levels of CINC/KC and MIP-2 and the leukocyte-endothelial cell interactions. Based on these findings, it could be assumed that the effect of BM on CXC chemokine release may be a consequence of its ROS scavenging properties. However, only BM inhibited in a concentration-dependent manner the rapid release of IL-8 elicited by Ang-II, whereas B and SB only affected this response at the highest concentration assayed, despite both alkaloids present ROS scavenging activity. These results indicate that inhibition of Ang-II-induced neutrophil recruitment by BM is a consequence of its ROS scavenging activity and its capability of reducing the generation and release of CXC chemokines.

Conversely, Ang-II-induced leukocyte adhesion is also a result of the release of endogenous-generated chemotactic mediators such as PAF, as the administration of the PAF receptor antagonist WEB2086 inhibits this response [19 ]. In this context, BM was capable of inhibiting the release of this chemoattractant in vivo when animals were i.p.-injected with Ang-II. Additionally, some phenanthrene alkaloids bind to the PAF receptor and behave as antagonists of this inflammatory mediator, as it has been demonstrated by using binding assays in rabbit platelets and human neutrophils [10 , 26 ]. In the present study, we have encountered that only BM was capable of blocking the PAF receptor in a concentration-dependent manner (IC50 28.2 µM), and B and SB only inhibited [3H]PAF binding to PMNs when they were assayed at 100 µM. The effect displayed by BM is of a lesser magnitude than that observed with uvariposine and stephenanthrine, which were twice and six times more potent than the alkaloid tested [10 ]. Nevertheless, all of these alkaloids are less potent than the well-known PAF receptor antagonist WEB2086 (IC50 0.15 µM). As opposed to uvariopsine and stephenanthrine, which present a methylenedioxy group in their structure [10 ], BM lacks this functional group, which would result in a lower potency as a PAF receptor antagonist.

In conclusion, in the present study, we have demonstrated that two phenanthrene alkaloids semisynthesized from natural B, BM and SB, can inhibit Ang-II-induced, leukocyte-endothelial cell interactions in vivo. The effects displayed by BM are mediated partly through inhibition of the generation of ROS, the down-regulation of P-selectin expression on the endothelial cell, the inhibition of CXC chemokine and PAF release, and the blockade of PAF-induced responses by interacting with its receptor. The inhibition of ROS generation elicited by SB seems to be the key mechanism by which this alkaloid inhibits Ang-II-induced responses, as P-selectin up-regulation and inhibition of CXC chemokine synthesis are linked to this capability. By contrast, the aporphine alkaloid B, although endowed with antioxidant properties, did not markedly affect the inflammatory activity elicited by Ang-II. In this way, the use of a phenanthrene alkaloid, such as BM, could constitute an alternative therapy for the control of Ang-II-induced neutrophilic inflammation in cardiovascular disease states where Ang-II plays a critical role.


   ACKNOWLEDGEMENTS
 
The present study was supported by Grants SAF-2002-01482, SAF-2002-04667, and SAF-2003-07206-C02-01 from CICYT, Spanish Ministry of Education and Science, Research Group 03/166 of Generalitat Valenciana, and by Grant MT-7684 from the Canadian Institutes of Health Research (A. C. I.). Y. N. A. N. and L. M. were supported by a grant from the Spanish Ministry of Education and Science. T. M. is primarily supported by a grant from Generalitat Valenciana. M. L. is supported by Alban Program and CDCH.

Received January 25, 2005; revised April 15, 2005; accepted May 3, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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