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(Journal of Leukocyte Biology. 2001;70:199-206.)
© 2001 by Society for Leukocyte Biology

Reactive oxygen species mediate angiotensin II-induced leukocyte-endothelial cell interactions in vivo

Department of Pharmacology, Faculty of Medicine, University of Valencia, Spain

Correspondence: Dr. Maria-Jesus Sanz, Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Av. Blasco Ibañez, 17, 46010 Valencia, Spain. E-mail: maria.j.sanz{at}uv.es

	ABSTRACT

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Chronically elevated angiotensin II (Ang-II)-induced hypertension is partly mediated by superoxide production. In this study, we have investigated whether the leukocyte-endothelial cell interactions elicited by Ang-II involve reactive oxygen species (ROS) generation. Intravital microscopy within the rat mesenteric microvessels was used. Superfusion (60 min) with Ang-II (1 nM) induced significant increases in leukocyte rolling flux, adhesion, and emigration, which were inhibited by pretreatment with superoxide dismutase or catalase. Dihydrorhodamine-123 oxidation indicated that ROS are primarily produced by the vessel wall. Administration of dimethylthiourea, desferrioxamine, or N-acetylcisteine provoked significant reductions in Ang-II-induced leukocyte-endothelial cell interactions. In addition, a blockade of platelet-activating factor or leukotrienes also attenuated such responses significantly. The results presented indicate that in vivo Ang-II-induced leukocyte recruitment is dependent on the generation of intra- and extracellular ROS. Therefore, the use of anti-oxidants might constitute an alternative therapy for the control of the subendothelial leukocyte infiltration associated with hypertension and atherosclerosis.

Key Words: superoxide • hydrogen peroxide • endothelium • intravital microscopy

	INTRODUCTION

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The release of endothelial and leukocyte-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H₂O₂) plays an active role in host-mediated destruction of foreign pathogens [¹ ]. However, they have also been implicated as a cause of the vascular and tissue damage associated with acute inflammatory reactions [¹ , ² ]. Indeed, there is evidence that implicates ROS in initiating and/or amplifying the inflammatory response through activation of leukocyte recruitment mechanisms. In this context, superoxide and H₂O₂ promote increases in endothelial-associated P-selectin expression, the production of platelet activating factor (PAF), and CD11/CD18-dependent leukocyte adhesion [^{3 4 5} ]. In addition, ROS production is involved in the pathogenesis of several cardiovascular disease states such as atherosclerosis, hypertension, and ischemia-reperfusion injury [^{6 7 8} ], and local leukocyte accumulation in the vessel wall constitutes a key stage in the onset and progression of such pathological conditions [^{8 9 10 11} ].

Clinical observations suggest a link between augmented renin-angiotensin system activity and the development of cardiac ischemic events [¹² , ¹³ ]. Angiotensin II (Ang-II), the main effector peptide in this system, has been shown to exert pro-inflammatory activity. With regard to this, angiotensin converting enzyme (ACE) inhibition has effectively been shown to reduce the number of infiltrating cells in different inflammatory conditions such as in glomerular diseases of the kidney, hypertension, and atherosclerosis [¹⁰ , ¹⁴ , ¹⁵ ]. Furthermore, we have revealed recently that Ang-II shows pro-inflammatory activity in vivo at sub-vasoconstrictor doses. In particular, it induces leukocyte trafficking into the rat mesenteric microvasculature through endothelial P-selectin up-regulation in the vessel wall, and this effect is mediated primarily via an Ang-II AT₁ receptor interaction [¹⁶ ].

Chronically elevated Ang-II-induced hypertension is mediated in part by superoxide production [⁷ ]. Moreover, in a rabbit model of early atherosclerosis, pretreatment with an AT₁ receptor antagonist normalized superoxide and endothelial function and reduced atherosclerotic lesion formation [¹⁷ ]. Xanthine oxidase is a prime source of endothelium-dependent superoxide production, and membrane-associated reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase is a major source of this oxygen radical in vascular smooth muscle cells (VSMCs) and myocytes [⁶ , ¹⁸ ]. It has been demonstrated that vascular oxidase activity is increased by Ang-II, which can indeed augment NADH- and NADPH-driven superoxide production in cultured VSMCs and in aortic adventitial fibroblasts [⁶ ]. Furthermore, Ang-II-induced cellular hypertrophy in VSMCs is mediated by intracellular production of H₂O₂ through interaction with its subtype AT₁ receptor [⁶ ].

Therefore, in the present study, we aimed to investigate whether the leukocyte-endothelial cell interactions elicited by Ang-II are also mediated through ROS production. To test this hypothesis, we used intravital microscopy within the rat mesenteric microcirculation and examined the effect of superoxide dismutase (SOD) and catalase on Ang-II-mediated leukocyte responses. In addition, because anti-oxidants have the potential to limit ROS-induced cell recruitment in vivo and thereby regulate amplification of the inflammatory response, we have also explored the modulatory effect of different anti-oxidants and free-radical scavengers, which act at intracellular and extracellular levels on leukocyte responses induced by Ang-II. Finally, because ROS can activate phospholipase A₂ (PLA₂), we have studied the role of PAF and leukotrienes on leukocyte infiltration caused by Ang-II.

	MATERIALS AND METHODS

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Animal preparation
Male Sprague-Dawley rats (200–250 g) were fasted for 20–24 h prior to experiments with free access to water. The animals were anesthetized with sodium pentobarbital [Sigma Química, Madrid, Spain; 65 mg/kg, intraperitoneally (i.p.)]. A tracheotomy was performed to facilitate breathing, and the right jugular vein was cannulated for intravenous administration of drugs or additional anesthetic as required. The right carotid artery was cannulated to monitor systemic arterial blood pressure through a pressure transducer (Spectramed Stathan P-23XL) connected to a recorder (GRASS RPS7C8B, Quincy, MA).

Intravital microscopy
A mid-line, abdominal incision was made, and a segment of the mid-jejunal mesentery was exteriorized and carefully placed on an optically clear-viewing pedestal to allow transillumination of a 3-cm² segment of the mesenteric microvasculature. The temperature of the pedestal was maintained at 37°C. Animal temperature was monitored using a rectal electrothermometer and was maintained at the same temperature with an infrared heat lamp. The exposed intestine was superfused continuously with a bicarbonate buffer saline (BBS; pH 7.4, 2 ml/min, 37°C) and covered with a BBS-soaked gauze to prevent evaporation. Mesenteric microcirculation was observed through an orthostatic microscope (Nikon Optiphot-2, SMZ1, Badhoevedorp, The Netherlands) with a 20x objective lens (Nikon SLDW) and a 10x eyepiece as described previously [¹⁶ ]. 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 captured on videotape (Sony SVT-S3000P) with superimposed time and date for subsequent playback analysis. The final magnification of the image on the monitor was 1300x.

Single, unbranched mesenteric venules with diameters ranging between 25 and 40 µm were studied. Venular diameter was measured on-line using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station, TX). Center-line red blood cell velocity (V_rbc) was also measured on-line with an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University). Venular blood flow was calculated from the product of mean V_rbc (V_mean=V_rbc/1.6) and microvascular cross-sectional area, assuming cylindrical geometry. Venular wall shear rate () was calculated based on the Newtonian definition: = 8 x (V_mean/D_v)s^-1, in which D_v is venular diameter [¹⁹ ].

The number of rolling, adherent, and emigrated leukocytes was determined off-line during playback analysis of videotaped images. Rolling leukocyte flux was determined by counting the number of rolling leukocytes passing a fixed reference point in the microvessel per min. The same reference point was used throughout the experiment, because leukocytes may roll for only a section of the vessel before rejoining the blood flow or becoming firmly adherent. Leukocyte rolling velocity (V_wbc) was determined by measuring the time required for a leukocyte to traverse a distance of 100 µm along the length of the venule and was expressed as µm/s. A leukocyte was considered to be adherent to venular endothelium if it remained stationary for a period equal to or exceeding 30 s. Adherent cells were expressed as the number per 100 µm length of venule. Leukocyte emigration was expressed as the number of white blood cells per microscopic field. The rate of emigration was determined from the difference between the number of any interstitial leukocytes present at the beginning of the experiment and the number of cells present in the interstitium at the end of the experiment.

Experimental protocol
After a 30-min stabilization period, baseline measurements (time 0) of mean arterial blood pressure (MABP), V_rbc, vessel diameter, shear rate, leukocyte rolling flux and V_wbc, and leukocyte adhesion and emigration were determined. The superfusion buffer was maintained or supplemented with Ang-II (Sigma Química, 1 nM) because previous studies by our laboratory have demonstrated that this dose causes the maximum and most consistent increase in leukocyte recruitment after 60-min superfusion [¹⁶ ]. Recordings were performed for 5 min at 15-min intervals over a 60-min period, and the aforementioned leukocyte and hemodynamic parameters were measured.

To determine the role of extracellular superoxide and H₂O₂ on Ang-II-induced leukocyte-endothelial cell interactions, animals were pretreated with SOD [Sigma Química, 8 mg/kg, intravenously (i.v.)] 5 min before the Ang-II suffusion or with catalase (Sigma Química, 5 mg/ kg, i.v.) 5 min before and 30 min after Ang-II 1-nM suffusion (to maintain adequate plasma levels) or with a combination of both enzymes. The doses of both anti-oxidants were based on those used in previous in vivo studies [⁴ , ²⁰ ].

To test the possible implication of intracellular ROS generation on Ang-II-induced leukocyte infiltration, one group of animals received the hydroxyl radical scavenger, dimethylthiourea (Sigma Química, 500 mg/kg, i.v.), 30 min before Ang-II superfusion. Another group of rats was pretreated with the iron-chelator, desferrioxamine mesylate (Sigma Química, 50 mg/kg, i.v.), 60 min prior to Ang-II suffusion. The doses used for the different treatments were those used by Mitchell et al. [²⁰ ] and Suematsu et al. [²¹ ] in similar studies.

To examine the effect of N-acetylcysteine (NAC; Sigma Química), an anti-oxidant and ROS scavenger, on Ang-II-induced leukocyte responses, animals were pretreated with NAC (150 mg/kg, i.v.) 15 min before Ang-II suffusion. Schmidt et al. [²² ] have proved that this dose of NAC attenuated endotoxin-induced leukocyte adherence in rat mesenteric, postcapillary venules.

Finally, to determine the involvement of chemotactic mediators such as PAF and leukotrienes on leukocyte infiltration elicited by Ang-II, a PAF receptor antagonist (WEB2086) and a 5-lipoxygenase inhibitor (ICI 230,487) were used. Animals were pretreated with WEB2086 (10 mg/kg, i.v.), or the exposed mesentery was superfused continuously with ICI 230,487 (100 µM) 15 min before Ang-II suffusion. WEB2086 was a generous gift from Boehringer-Ingelheim (Germany), and ICI 230,487 was kindly donated by AstraZeneca (Macclesfield, U.K.). Similarly, all the doses administered were used as stated in previous in vivo studies [⁴ , ²³ ].

In vivo assessment of free-radical generation
To quantify the generation of oxidants by cells in the area under study, the oxidant-sensitive fluorochrome dihydrorhodamine (DHR)-123 (Molecular Probes, Eugene, OR; 10 µM) was superfused onto the mesentery as previously described [²⁴ ].

During an initial, 30-min stabilization period, the mesenteric preparation was superfused with DHR-free BBS, and a background auto-fluorescence image was recorded. The preparation was then superfused with the working solution for 15 min and finally rinsed with DHR-123-free BBS to eliminate the precursor dye from the preparation. Fluorescence intensity (excitation wavelength, 500 nm; emission wavelength, 536 nm) was detected just before (baseline value) and following administration of BBS or Ang-II 1 nM using a charge-coupled device camera model XC-77 (Hamamatsu Photonics, Hamamatsu-City, Japan) with a C2400-68 intensifier head (Hamamatsu Photonics) and a C240-60 charge-coupled device camera control unit. Fluorescence intensity of the venule under investigation and background fluorescence were measured using an image analyzer program (analySIS 2.11, analysis DOCU). An index of free-radical generation within the venule was obtained after subtracting background fluorescence from the fluorescence intensity in the area of interest.

Statistical analysis
All data are expressed as mean ± SE. The data within groups were compared using an analysis of variance (one-way ANOVA) with a Newman-Keuls post hoc correction for multiple comparisons. Statistical significance was set at *P < 0.05.

	RESULTS

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Ang-II promoted significant increases in leukocyte rolling flux (80.0±8.5 vs. 18.4±2.6 cells/min), adhesion (10.2±1.5 vs. 1.0±0.3 cells/100 µm), and emigration (2.8±0.5 vs. 0.6±0.4 cells/field) after 60-min superfusion compared with basal values (Fig. 1 ). Buffer superfusion did not cause significant changes in the leukocyte or the hemodynamic parameters measured during the whole experimental protocol (Fig. 1 and Table 1 ). These results are consistent with previous studies from our laboratory [¹⁶ ]. In addition, circulating leukocyte counts were unaltered after 60-min mesenteric superfusion with Ang-II (82.5±7.8x10⁵ cells/ml at time 0 vs. 86.5±9.8x10⁵ cells/ml after 60-min superfusion). SOD pretreatment significantly reduced Ang-II induced leukocyte rolling flux, adhesion, and emigration by 79.6%, 56.5%, and 45.5%, respectively, after 60-min Ang-II suffusion (Fig. 1) . When catalase was administered, leukocyte rolling flux and adhesion caused by 60-min Ang-II suffusion were attenuated significantly by 50.3% and 37.1%, respectively, but this pretreatment did not alter Ang-II-induced emigration significantly (18.1%). The co-administration of both anti-oxidants almost completely inhibited leukocyte-endothelial cell interactions elicited by Ang-II (Fig. 1) . In addition, although the decrease in V_wbc induced by Ang-II was not reversed by pretreatment with either of these enzymes when administered separately, co-administration of the two returned V_wbc to basal levels (Table 2 ).

View larger version (33K): [in this window] [in a new window]   Figure 1. Effect of the extracellular anti-oxidants superoxide dismutase and catalase on Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B), and leukocyte emigration (C) in the rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30, and 60 min after superfusion with buffer (n=5), with Ang-II (1 nM) in animals untreated (n=5) or pretreated with SOD (8 mg/kg, i.v., n=5), with catalase (5 mg/kg, i.v., n=5), or with a combination of both. Results are represented as mean ± SE. **P < 0.01 relative to buffer group. +P < 0.05 or ++P < 0.01 relative to the Ang-II-untreated group.

View larger version (17K): [in this window] [in a new window]   Figure 2. Free-radical generation in response to BBS or Ang-II superfusion within the rat mesenteric postcapillary venules. Fifteen min after superfusion of DHR-123 alone, baseline DHR oxidation (0 min) was determined. Animals were divided into two groups: The BBS-superfusion was continued (n=4) or supplemented with Ang-II 1 nM (n=4), and the DHR oxidation was measured as total vessel rhodamine fluorescence intensity at 15, 30, and 60 min. Results are represented as mean ± SE. *P < 0.05 or **P < 0.01 relative to the control value (0 min) in the Ang-II-superfused group. ++P < 0.01 relative to the BBS-superfused group.

View larger version (22K): [in this window] [in a new window]   Figure 3. Effect of the intracellular anti-oxidant, dimethylthiourea, and the iron-chelator, desferrioxamine, on Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B), and leukocyte emigration (C) in the rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30, and 60 min after Ang-II (1 nM) superfusion in animals untreated (n=5) or pretreated with dimethylthiourea (500 mg/kg, i.v., n=7) or with desferrioxamine (50 mg/kg, i.v., n=5). Results are represented as mean ± SE. *P < 0.05 or **P < 0.01 relative to the control value (0 min) in the untreated group. +P < 0.05 or ++P < 0.01 relative to the untreated group.

View larger version (17K): [in this window] [in a new window]   Figure 4. Effect of the anti-oxidant and free-radical scavenger NAC on Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B), and leukocyte emigration (C) in the rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30, and 60 min after superfusion with Ang-II (1 nM) in animals untreated (n=5) or pretreated with NAC (150 mg/kg, i.v., n=5). Results are represented as mean ± SE. *P < 0.05 or **P < 0.01 relative to the control value (0 min) in the untreated group. ++P < 0.01 relative to the untreated group.

View larger version (25K): [in this window] [in a new window]   Figure 5. Effect of the PAF receptor antagonist WEB2086 and the specific 5-lipoxygenase inhibitor ICI 230,487 on Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B), and leukocyte emigration (C) in the rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30, and 60 min after superfusion with Ang-II (1 nM) in animals untreated (n=5) or pretreated with WEB2086 (10 mg/kg, i.v., n=5) or in animals co-superfused with ICI 230,487 (100 µM, n=5). Results are represented as mean ± SE. *P < 0.05 or **P < 0.01 relative to the control value (0 min) in the untreated group. +P < 0.05 or ++P < 0.01 relative to the untreated group.

	DISCUSSION

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To further investigate direct, free-radical formation on Ang-II stimulation in our system, we used the oxidant-sensitive fluorescent probe DHR-123, which specifically converts to a fluorescent (rhodamine) form following reaction with ROS [²⁵ ] and has been successfully used in monitoring in vivo free-radical generation in the rat mesentery [²⁶ , ²⁷ ]. In our study, the temporary responses of DHR oxidation in mesenteric tissue exposed to Ang-II suggest that there is an early rise in DHR oxidation that occurs within the first 30 min of Ang-II superfusion and is localized within the vessel. This is in agreement with previous in vitro findings that encountered superoxide production by NADH and NADPH from cultured VSMCs after Ang-II stimulation [⁶ ]. In addition, it has been shown that lipoxygenase metabolites of arachidonic acid mediate Ang-II stimulation of the NAD(P)H oxidase in VSMCs [²⁸ ], and we have demonstrated in this study that inhibition of lipoxygenase provokes a significant reduction in the leukocyte-endothelial cell interactions caused by Ang-II. Another possible source of superoxide-anion production is the endothelial cell, which can produce H₂O₂ via the mitochondrial electron transport chain, xanthine oxidase, and/or the biosynthesis of prostaglandins [²¹ , ²⁹ ]. However, because of the methodology of this study, it is difficult to see whether Ang-II acts on the endothelium, on the vascular smooth muscle, or on both, because receptors for this peptide hormone can be found in both cell types. Furthermore, the significant increase in DHR oxidation in the venule was detected at the same stage where the exacerbated leukocyte rolling and adhesion were encountered. Consequently, it seems that the vascular wall is the primary source of the Ang-II-induced oxygen free-radical generation, which leads to leukocyte recruitment through increased adhesion-molecule expression.

Superoxide can be dismutated spontaneously or by SOD to yield H₂O₂, H₂O₂ can be metabolized by catalase to form oxygen and water, or a Fenton reaction may occur in the presence of iron, resulting in the formation of hydroxyl radical. Thus, it is also relevant to this study that administration of the intracellular anti-oxidant and hydroxyl radical scavenger, dimethylthiourea, and inhibition of iron-catalyzed oxyradical formation by desferrioxamine pretreatment provoked significant reductions in leukocyte-endothelial cell interactions elicited by Ang-II. These results implicate intracellular secondarily derived oxygen radicals other than superoxide as important chemical mediators of the adhesive interactions observed in postcapillary venules exposed to Ang-II. Indeed, in vitro observations suggest that oxyradical propagation may involve ferritin-binding iron in endothelial cells, because iron-chelating reagents or free-radical scavengers can prevent the expression of adhesion molecules such as P-selectin [³⁰ ]. It is conceivable that dimethylthiourea or desferrioxamine act on the endothelial cell. Consequently, the effects observed with these compounds suggest that they prevent the intracellular generation of oxygen radicals induced by Ang-II and cause the down-regulation of the increased endothelial P-selectin expression.

It is interesting that when NAC was administered, Ang-II-induced leukocyte rolling, adhesion, and emigration were nearly returned to basal levels. NAC acts as a free-radical scavenger and anti-oxidant [³¹ ]. In addition, the synthesis of cellular glutathione (GSH) can be sustained by NAC, serving as a precursor for GSH and thus replenishing the intracellular pool of cysteine [³¹ ]. Among all the compounds tested in this study, NAC exerted the most powerful inhibition of the leukocyte-endothelial cell interactions elicited by Ang-II, which is probably because of the vast array of different anti-inflammatory mechanisms that have been attributed to this molecule. In fact, its anti-adhesive properties in vivo have been clearly demonstrated in animal models of liver ischemia-reperfusion injury and endotoxemia [²² , ³² ].

Finally, Ang-II-induced leukocyte-endothelial cell interactions were also ameliorated significantly by pretreatment with the PAF receptor antagonist, WEB2086, and by the 5-lipoxygenase inhibitor, ICI 230,487, in particular at the level of leukocyte adhesion and emigration. The results of these experiments support the view that PAF and leukotrienes mediate the leukocyte responses elicited by Ang-II and are consistent with the findings of Mangat et al. [³³ ] that confirmed the role of Ang-II in cytosolic PLA₂ activation, which is critical to the synthesis and release of these potent chemotactic mediators. Conversely, oxidants can also peroxidize endothelial cell membranes to activate PLA₂. Indeed, superoxide and H₂O₂ induce the endothelium to synthesize PAF, and it has been proved that leukocyte-endothelial cell interactions induced by both reactive-oxygen metabolites can be inhibited by PAF receptor-antagonist pretreatment [⁴ , ³⁴ ]. Unexpectedly, in our study, pretreatment with WEB2086 was also capable of reducing Ang-II-induced leukocyte rolling flux. Although some authors could only find a role for PAF on leukocyte adhesion [³⁵ , ³⁶ ], others have found that PAF superfusion causes significant increases on leukocyte rolling flux within 30 min of superfusion or that increases on the flux of rolling leukocytes induced by different mediators can be diminished significantly by PAF receptor-antagonist pretreatment [^{37 38 39 40} ]. Therefore, we suggest that in addition to the effect of PAF increasing CD11/CD18 integrin expression on leukocyte cell surface, it can also act on the endothelial cell-inducing, rapid P-selectin expression as demonstrated in previous studies [³⁹ , ⁴⁰ ].

In conclusion, the present study indicates that in vivo leukocyte recruitment elicited by Ang-II is dependent on the generation of intra- and extracellular ROS. Our proposal is that upon Ang-II stimulation, superoxide and H₂O₂ are released quickly and cause endothelial P-selectin up-regulation, which leads to the observed increase in leukocyte rolling flux. In parallel, the ROS released can stimulate PLA₂, which in turn provokes the synthesis and release of PAF and leukotriene B₄ (LTB₄), explaining in part the subsequent leukocyte adhesion and emigration detected. Additionally, newly formed secondarily derived oxidants from lipoxygenase metabolism could be responsible for an amplification mechanism, which results in subsequent accumulation of adherent leukocytes and in turn contributes to the overall response to Ang-II. This is relevant, because in hypertensive states with elevated Ang-II profiles, the release of ROS by Ang-II can also participate in the oxidation of low-density lipoproteins (LDL), a key step in the development of the atherosclerotic lesion, which can also provoke leukocyte adhesion and emigration [⁴¹ ]. In this way, the use of anti-oxidants could constitute an alternative therapy for the control of the subendothelial leukocyte infiltration associated with the vascular damage detected in hypertension and atherosclerosis.

	ACKNOWLEDGEMENTS

 
The present study has been supported by grant PM 98 205 from CICYT, Spanish Ministerio de Educación y Ciencia.

Received December 20, 2000; revised April 1, 2001; accepted April 5, 2001.

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