ARTICLE |
Correspondence to: Agnes Kittel, Dept. of Cell Biology, Inst. of Experimental Medicine, Hungarian Academy of Sciences, PO Box 67, 1450 Budapest, Hungary.
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Summary |
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The aim of this study was to investigate the distribution pattern of Ca2+- and Mg2+-dependent ecto-ATPases on the surface of rat brain capillary endothelial cells (ECs) in control and lipopolysaccharide (LPS)-treated animals. Ecto-ATPases in the membrane of vascular endothelial cells are suggested to play a crucial role in thromboregulation. Loss of this enzyme activity after oxidative stress and upregulation of the enzyme chain hydrolyzing extracellular ATP after transient forebrain ischemia have also been reported. We used histochemistry to localize the activities of this enzyme on ECs and found pH- and cation-dependent changes in the localization of enzyme activity both in control and in LPS-treated animals. These findings suggest the presence of more than one ecto-ATPase enzyme on the surface of rat capillary ECs. The different behavior of ECs after LPS treatment is the target of further investigations. The increased ecto-nucleotidase activity might play a role in nucleotide-mediated cellular responses after bacterial infections. (J Histochem Cytochem 47:393399, 1999)
Key Words: ecto-ATPase, CD39, capillary endothelial cell, LPS treatment
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Introduction |
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Extracellular nucleotides such as ATP, ADP, and UTP are signaling molecules in many extracellular activities, i.e., from neurotransmission to immune responses (
The source of nucleotides and their release mechanisms have been studied mostly in the vascular and nervous systems. Nucleotides are released into the bloodstream by damaged cells in the vessel wall (
Ecto-ATPases are related to several different proteins (
In this study, we used an enzyme histochemical method at the electron microscopic level, which is appropriate for localizing putative ecto-ATPase enzyme activity. We investigated the effect of lipopolysaccharide (LPS) on capillary endothelial cells (ECs). This bacterial endotoxin has potent proinflammatory properties toward many cell types, including ECs (
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Materials and Methods |
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All of the general reagents were purchased from Sigma Chemical, (St Louis, MO) unless otherwise specified. This study was carried out in accordance with the guidelines of the Institutional Ethical Committee for Experimental Animals.
LPS Treatment
Male Wistar rats (about 200250 g) were injected with LPS (2 mg/kg body weight) IP 2 hr before sacrifice (
Electron microscopic demonstration of Ca2+- or Mg2+-dependent ecto-ATPases at pH 7.5 was as described by ,ß-methylene-ADP (5'-nucleotidase inhibitor), as well as CaCl2 (1.5 mM) and EDTA (0.1 mM) or MgCl2 (1.5 mM) and EGTA (0.1 mM) for 45 min at 37C. Incubation was followed by three rinses in Tris-maleate buffer. The samples were postfixed in 1% OsO4 (Taab) dissolved in cacodylate buffer for 30 min. After rinses in distilled water, tissue slices were dehydrated in graded ethanol (en bloc-stained with 2% uranyl acetate in 70% ethanol for 30 min), processed through propylene oxide, and embedded in Epon (Fluka; Buchs, Switzerland). Ultrathin sections were cut and examined in a Hitachi 2001 transmission electron microscope (Hitachi; Tokyo, Japan). To demonstrate the specificity of the reaction product, in some experiments either the ATP or the ions were omitted from the incubation medium. Control experiments were carried out in the presence of both cations at pH 7.5 and pH 9.5.
Demonstration of Ca2+- or Mg2+-dependent Ecto-ATPases After Fixation at pH 9.5
The procedure was the same as above except for the washing and the fixative solutions. We used a 0.9% NaCl solution for rinsing the vessels. The fixative solution contained 4% paraformaldehyde, 0.1 M NaOH, and 0.1 M Na2B4O7·10 H2O at pH 9.5. After fixation and washing, the sectioning was carried out in Tris-maleate buffer.
Results were obtained from five different experiments with parallel sampling for both fixation procedures.
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Results |
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To demonstrate the Ca2+- and Mg2+-dependence of ecto-ATPDase activity in the capillary ECs in rat brain cortex, two fixative solutions were used at pH 7.5 and 9.5. The ecto-enzyme activity was represented as a Ce-phosphate deposit at the site of liberation of phosphate during the hydrolysis of extracellular ATP or, as the next step of the enzyme activity, from ADP. As the control experiments demonstrate, when Ca2+ and Mg2+ were used together, the ecto-ATPase activity was very strong on both (luminal and basal) surfaces of the ECs. This activity was independent of the pH of the fixative solution. There was also some activity in the extracellular matrix (ECM) (Figure 1a and Figure 1b).
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Control experiments without substrate ATP or Ca2+/Mg2+ did not show any deposit around the cells or on the cell membrane (Figure 1c).
After fixation at pH 7.5 and when the incubation medium contained only Ca2+ as cation, a fine cerium deposit was found both on the luminal and basal sides of the cell and in the ECM, as in the case when both cations were used (Figure 2a). However, if Mg2+ replaced Ca2+ in the incubation medium at pH 7.5 fixation, the activity on the luminal side was absent or reduced (Figure 2b). Enzyme activity did not change in the ECM.
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At pH 7.5 fixation, LPS treatment did not change the pattern in the presence of Ca2+ (Figure 2c). However, in the presence of Mg2+ some ecto-ATPase activity appeared on the luminal side, whereas the activity on the basal side remained at the same level (Figure 2d).
After fixation at pH 9.5, we observed only a slight difference in the appearance of ecto-ATPase activity, using either Ca2+ or Mg2+ in control animals. Some enzyme activity was present on the luminal and basal sides of the ECs (Figure 2e and Figure 2f). This slight difference concerns the deposit on the luminal side of ECs using Mg2+ only (Figure 2f), which was not visible with fixation at pH 7.5.
LPS treatment after fixation at pH 9.5 changed the pattern of the enzyme activity with both cations used individually. With Ca2+ only, the enzyme activity of the basal side of the cell increased, especially in the pinocytotic vesicles. The presence of these caveola-like membrane structures in considerable numbers was observable after LPS treatment and in the presence of Ca2+ only (Figure 2g). If Mg2+ was used in the incubation solution, an abundant Ce deposit showed the increased ecto-enzyme activity on the luminal surface of the ECs. Staining on the basal side was observable only in trace amounts (Figure 2h).
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Discussion |
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The distribution of the Ca- and Mg-dependent ecto-ATPases on rat brain capillary ECs was studied in control and LPS-treated animals. Cation preferences of ATPases showed region-dependence as previously suggested (
Effects of LPS have been published on several enzymes such as Na+-K+-ATPase, Ca2+-ATPase, GTPase and nitric oxide synthase (
Significant alterations occurred in the localization of the ecto-ATPase after LPS administration and fixation at pH 9.5. (Alternatively, when fixation was done at pH 7.5, the treatment with LPS did not induce visible changes.) With only Ca2+ in the incubation medium, many caveola-like membrane structures full of deposit indicating strong ecto-ATPase activity appeared on the basal side, whereas the activity on the luminal surface did not change. What might be the role of these membrane invaginations in the capillary ECs during inflammation? Caveolae, dynamic pieces of membrane, were first identified in ECs. They are suggested to take part in signal transduction and to play a major role in coordinating the interaction of the cell with its environment (
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Acknowledgments |
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Supported by a grant (OTKA) 019860 from the Hungarian Scientific Research Fund.
I wish to thank Drs E. Bacsy, S.C. Robson, and K.J. Kovacs for critical review of the manuscript and Ms A. Foldes, K. Lengyel, and O. Szalay for excellent assistance.
Received for publication June 23, 1998; accepted October 27, 1998.
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