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Keywords:

  • ecto-5′-nucleotidase;
  • immunohistochemistry;
  • ciliary body;
  • choroid

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Adenosine is known to exert multiple functions within the eye. The aim of this report was to find out if adenosine can be produced locally in the choroid and ciliary body. Therefore, I investigated the distribution of ecto-5′-nucleotidase (5′-NT), the key enzyme for the production of extracellular adenosine. This report provides evidence that 5′-NT is expressed in the choroid and in the ciliary body (and its processes) of the rat eye, predominantly in endothelial cells. These locations of 5′-NT indicate strategically important production sites of adenosine regulating choroid and ciliary body functions (e.g., blood flow, aqueous fluid production, and immune response). Anat Rec, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

The cell membrane enzyme ecto-5′-nucleotidase (5′-NT) is expressed in many mammalian organs and cell types. 5′-NT is expressed on various endothelial cells (e.g., heart capillaries, umbilical veins, or aortic endothelial cells) with considerable species-specific differences (e.g., almost 10 times lower in pig endothelium than in human endothelial cells) (Smolenski et al., 2006). 5′-NT activities were four to five times higher in human and mice hearts (highest in rat) compared with pigs and baboon hearts (Khalpey et al., 2007). In the eye of four rodent species, 5′-NT is localized in the external portion of the retina (photoreceptor, external nuclear, and external plexiform layers), in retinal Müller cells, and in the retinal pigment epithelium (Kreutzberg and Hussain, 1982, 1984). It is unknown, if 5′-NT is expressed in the choroid and ciliary body of the eye.

5′-NT is the key enzyme for the production of extracellular adenosine by regulating the hydrolytic cleavage of adenosine monophosphate (Centelles et al., 1992). Another pathway of adenosine formation is the hydrolysis of S-adenosylhomocysteine (SAH) by SAH hydrolase (Achterberg et al., 1985). Adenosine acts on A1, A2, or A3 receptors that regulate (A1 and A3 inhibit, A2 stimulates) adenylate cyclase activity and mediate multiple effects (Yaar et al., 2005).

5′-NT is one of the main regulators of endothelial function (Ledoux et al., 2003). Its product adenosine has potent vascular functions. In the kidney, it constricts afferent arterioles and reduces glomerular filtration rate, whereas in deep renal cortex and medulla it causes vasodilation (Vallon et al., 2006). In the heart, adenosine is a potent vasodilator of coronary arteries of many species (e.g., rabbits, rats, and dogs) (Tabrizchi and Bedi, 2001). In the pulmonary circulation, adenosine can cause contraction or relaxation depending on the basal vessel tone and on the activation of A1 (vasoconstriction) or A2 (vasodilation) receptors (Cheng et al., 1996). In the rabbit eye, adenosine causes vasodilation and increase of blood flow in iris, retina, choroid, and ciliary body (Braunagel et al., 1988; Campochiaro and Sen, 1989). These results have been confirmed later in many other species including rats, pigs, and humans (Gidday and Park, 1993; Polska et al., 2003; Nakazawa et al., 2008). Adenosine also regulates and modulates many other functions, such as neurotransmission, and immune responses, for example, in rats (Marak et al., 1988).

Considering these multiple effects of adenosine in the eye, both in health and disease, and because adenosine acts locally as an autacoid at the site where it is produced (Vallon et al., 2006; Jacobson, 2008), it is important to know the exact localization of its main producing enzyme, 5′-NT. Furthermore, 5′-NT is known to be expressed in the choroid plexus (Braun et al., 1994). This raises the question if 5′-NT is also expressed in the ciliary body which is structurally and functionally related to the choroid plexus. Their functional relationship relates to aqueous humor production by the ciliary processes and cerebrospinal fluid production by the choroid plexus. The present immunohistochemical study detected new locations of adenosine-producing 5′-NT in the choroid and ciliary body of the rat eye.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Five healthy male albino rats (Wistar strain) with body weights of ∼250 g were anesthetized with thiopental, 100 mg/kg body weight, intraperitoneally. Thereafter, rats were perfused with 2.5% paraformaldehyde, 0.1% glutaraldehyde, and 3 mM magnesium chloride dissolved in a 6:4 mixture of 0.1 M cacodylate buffer (pH 7.4 adjusted to 300 mOsmol with sucrose) and of 10% hydroxyethyl starch. After 5 min, fixation was stopped by perfusion rinsing with 0.1 M cacodylate buffer, pH 7.4, adjusted to 300 mOsmol with sucrose, for 10 min. Subsequently, eyes were stored in 0.1 M cacodylate buffer (pH 7.4, adjusted to 300 mOsmol with sucrose) containing 0.04% NaN3 at 4°C overnight and then frozen.

For immunohistochemistry, sections of 5-μm thickness were prepared in a cryomicrotome. The sections were thawed onto chrom-alaun-gelatine-coated slides and kept in a moist chamber. Free aldehyde groups in the tissue were quenched by rinsing the slides for 30 min in PBS with 50 mM NH4Cl. Unspecific binding of the antibodies was further decreased by incubating the sections for 30 min in PBS with 10% normal goat serum and 1% bovine serum albumin. The sections were incubated 90 min with a purified monoclonal mouse antibody against rat CD73 (5′-NT) (BD Biosciences, Franklin Lakes, NJ), diluted 1:2,000 in PBS. The binding sites of the primary antibodies were visualized by biotinylated goat anti-mouse IgG (Dakopatts, Glostrup, Denmark) diluted 1:100 in PBS and streptavidin-Texas red (Amersham, Buckingshamshire, England). The nuclei were stained with 2 mg/mL DAPI (Boehringer, Mannheim, Germany). Controls were prepared by omitting the anti-CD73 antibody or by replacing it with preimmune serum. The specificity of the anti-CD73 antibody has been proven in many previous publications (Gandhi et al., 1990). Images were created with conventional fluorescence microscopy.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Immunohistochemistry demonstrated dense expression of 5′-NT in the retina. 5′-NT was detected mainly in the external plexiform and nuclear layers, in the receptor and pigment cell layers of the retina (Fig. 1a,b). These results confirm previous data of Kreutzberg and Hussain (1982, 1984), and Irons (1989).

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Figure 1. Ecto-5′-nucleotidase (5′-NT)-positive cells in the rat eye are visualized in 5-μm cryostat sections by immunohistochemistry. 5′-NT is known to be located in the retinal pigment epithelium (RPE), photoreceptors (PR), outer nuclear layer (ONL), and outer plexiform layer (OPL). In addition, 5′-NT is strongly expressed in the ciliary body and choroid. In the optic nerve head (ONH) (a) and choroid (c), 5′-NT-positive cells (Texas red) are located in the endothelium of vessels (with the exception of a few arterial walls marked with n = negative). Also retinal vessels (RV) express 5′-NT (b). Replacement of the immune serum by preimmune serum resulted in absence of staining in all structures, for example, ciliary body (d). In the ciliary body processes, 5′-NT-positive cells are predominantly expressed in the vessel walls but also in interstitial cells (e, f). 5′-NT is expressed by endothelial cells and pericytes (g) but only weakly by epithelial cells (f). Nuclei are blue (DAPI). Bars = 20 μm. Arrows (d–f) indicate ciliary body epithelium, arrowheads (c, e, f) indicate interstitial cells.

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Moreover, 5′-NT was also found in the ciliary body and choroid. In the choroid and optic nerve head connective tissue, 5′-NT was prominent in the endothelium of vessels and in the connective tissue cells and their processes, which surround the vessels (Fig. 1a,c). Not all vessel walls were 5′-NT-positive. Vessels with a thin vessel wall (resembling arterioles, capillaries, and venules) were mostly 5′-NT-positive, whereas a few vessels with a multilayered vessel wall (resembling arteries) were 5′-NT-negative both in the choroid and in the optic nerve head connective tissue (Fig. 1a,c). Also retinal vessels express 5′-NT (Fig. 1b).

There was no staining of the ciliary body stroma or epithelium in control sections where the immune serum was replaced by preimmune serum (Fig. 1d).

Applying the immune serum, the ciliary body was clearly 5′-NT-positive. The epithelium of the ciliary body processes was 5′-NT-positive, but did not stain homogeneously. Some parts were 5′-NT-negative (Fig. 1e,f, left side), whereas other parts of the epithelial layer stained weakly positive for 5′-NT (Fig. 1e,f, right side). Mostly peripheral parts of the ciliary body processes showed a weaker 5′-NT staining. In contrast to the epithelium of ciliary body processes, the stroma showed a strong and consistent expression of 5′-NT in the vessel walls. In contrast to the choroid, all vessel walls were 5′-NT-positive in the ciliary body stroma. Both the vascular endothelium and perivascular cells were strongly 5′-NT-positive (Fig. 1g). Some interstitial cells of the stroma were also 5′-NT-positive (Fig. 1e,f).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Adenosine modulates intraocular hemodynamics and pressure, neurotransmission, epithelial transport, and the immune system in many species (Burnstock, 2002; Polska et al., 2003). Considering the multiple functions of adenosine, I hypothesized a broad distribution of 5′-NT in the eye, the key enzyme of extracellular adenosine production. 5′-NT has been reported in outer retinal layers and pigment epithelium of four rodent species (Kreutzberg and Hussain, 1982, 1984; Irons, 1989) and also in the rabbit ciliary body nonpigmented epithelium (Farahbakhsh, 2003) but not in the ciliary body stroma and choroid.

In this study, 5′-NT was detected in the rat choroid and ciliary body processes. 5′-NT was strongly expressed in endothelial and interstitial cells but only weakly in epithelial cells. Not all choroidal endothelial and not all ciliary body epithelial cells were 5′-NT-positive. This is consistent with other organs (such as liver, kidney, or spleen) where 5′-NT is distributed asymmetrically with organotypic subsets of vessels or fibroblasts being 5′-NT-positive (Thomson et al., 1990; Zimmermann, 1992). Differences in vascular 5′-NT expression might have several functional implications, for example, for spatial regulation of adenosine-mediated signaling, for mediating adhesion, for lymphocyte-endothelial cell interactions, for cell activation, and for cytoprotection (Airas et al., 1997; Smolenski et al., 2006; Khalpey et al., 2007).

The localization of 5′-NT indicates the sites of extracellular adenosine production. Extracellular adenine nucleotides released from epithelial cells, muscle cells, nerves, and blood circulation can reach interstitial and endothelial cells of the choroid and ciliary body and be converted in situ to adenosine by 5′-NT. Adenosine acts locally as an autacoid in situ which may explain the ability of the eye's autoregulation. Adenosine can reach targets, such as vessel walls, nerve endings, epithelial cells, and macrophages, and modulate their function. Adenosine receptors have been reported in the retina (A1 and A2A), pigment epithelium, ciliary processes (A1, A2A, and A2B), and in the choroid (A2A) of the rat eye (Kvanta et al., 1997). Indeed, adenosine modulates intraocular blood flow by dilating vessels and increasing blood flow in the choroid, ciliary body, and optic nerve head of rabbits, rats, and humans (Braunagel et al., 1988; Polska et al., 2003; Nakazawa et al., 2008). In addition, activation of A1 receptors reduces intraocular pressure via decreasing aqueous flow and increasing outflow facility, whereas activation of A2 receptors increases intraocular pressure, aqueous humor flow, and protein concentrations in rabbits and cats (Crosson and Gray, 1996). Adenosine's effects on ocular blood flow and aqueous humor production might be explained (in part) by its ability to modulate neurotransmission (Fredholm et al., 2005).

5′-NT activity in the choroid and ciliary body processes may also be detrimental for recycling of nucleotides released from neighboring cells. Metabolically active cells release nucleotides. Extracellular hydrolysis of these nucleotides would allow reuptake of adenosine for nucleotide synthesis instead of energetically expensive de novo synthesis. Such recycling via ectoenzymes and adenosine transporter has been reported in the luminal membranes of rat hepatocytes (Che et al., 1992).

Immune cells express adenosine receptors and are present in the ciliary body and the choroid of the rat, mouse, and human eye (McMenamin et al., 1994; McMenamin, 1997). Adenosine regulates immune cell functions of mice and humans, it limits endothelial cell inflammatory responses and it suppresses macrophage activation, oxidative burst, and cytokine release (Haskó et al., 2009). In experimental rat allergic uveitis, adenosine produced a marked reduction in choroidal inflammation (Marak et al., 1988). Adenosine functions in an autocrine manner (Vallon et al., 2006) and its half time is only a few seconds. Therefore, the sites of 5′-NT expression indicate where adenosine is produced in situ by endothelial and interstitial cells and where it is expected to be most effective in local downregulation of immune responses.

In conclusion, 5′-NT is expressed in the ciliary body processes and choroid endothelial and interstitial cells. Its product adenosine may activate neighboring adenosine receptors in ciliary body and choroid and thus modulate diverse function such as intraocular blood flow and pressure, neurotransmission, and immune function.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED