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Matthias Grueb MD Department of Ophthalmology University of Tuebingen Schleichstrasse 12 D-72076 Tuebingen Germany Tel: + 49 7071 298 4761 Fax: + 49 7071 294762 Email: email@example.com
Purpose: Monoamine receptors are found throughout the body. Reports about the presence of monoamine receptors in the human cornea are inconsistent.
Methods: Immunohistochemistry, immunofluorescence and immunoblotting were used to localize monoamine receptor sites on human corneal epithelium and endothelium.
Results: Antibodies to alpha-1, beta-1 and beta-2 adrenergic receptors and to D1-like and 5HT-7 receptors were bound in corneal epithelium. Antibodies to alpha-1, alpha-2A, beta-1 and beta-2 adrenergic receptors and to 5HT-7 receptors were bound in corneal endothelium.
Conclusions: Our data demonstrate the presence of several monoamine receptors in the human cornea. These receptors may play a role in the regulation of fluid transport or corneal homeostasis.
Monoamine receptors are membrane-bound receptors located throughout the body in neuronal and non-neuronal tissues where they mediate a diverse range of responses to the endogenous catecholamines epinephrine, norepinephrine, dopamine and serotonin.
Adrenergic receptors are members of the 7-transmembrane domain G-protein-coupled receptor superfamily that bind epinephrine and norepinephrine. Pharmacological, structural and molecular cloning data indicate significant heterogeneity within the receptor family. Several receptor subtypes have been identified thus far, including alpha-1, alpha-2 and beta adrenergic receptor subtypes (Lands 1967; Langer 1974). Dopaminergic receptors are members of the same superfamily. There are at least five dopamine receptors (D1−D5) which can be divided into two subfamilies whose properties are defined pharmacologically and biochemically. The two subfamilies are termed D1-like receptors (D1, D5) and D2-like receptors (D2, D3, D4) (Strange 1992). Serotonin (5-hydroxytryptamine, 5-HT) produces its effects through a variety of membrane-bound receptors. Except for the 5-HT3 receptor, which is a ligand-gated ion channel, 5-HT receptors belong to the G-protein-coupled receptor superfamily and, with at least 14 distinct members, collectively represent one of the most complex families of neurotransmitter receptors (Pauwels 2000).
Reports about the presence of monoamine receptors on corneal epithelial and endothelial cells are contradictory and differ considerably according to specimen and technique (Neufeld et al. 1978, 1982; Walkenbach et al. 1985, 1991, 1992; Cavallotti et al. 1999; Crider et al. 2003). Using freshly fixed human corneal tissue for immunohistochemistry, immunofluorescence and immunoblotting, our studies are the first to show a complete profile of monoamine receptors on the human cornea with identical tissue preparation and assay protocols.
Material and Methods
Corneal tissue was provided by the Department of Ophthalmology, University of Tuebingen and by the University Cornea Bank. Informed consent was obtained from all patients or their relatives according to the Declaration of Helsinki. The specimens used for immunohistochemistry and immunofluorescence were:
1a corneal explant from a 30-year-old man with a small central scar after penetrating trauma; time to preparation < 1 hour;
2a corneal explant from a 46-year-old man with few disseminated superficial lesions after explosion trauma; time to preparation < 1 hour;
3a donor cornea from a 58-year-old man; time to preparation 12 hours post-mortem, not in culture, endothelial cell density 2000 cells/mm2;
4a donor cornea from a 39-year-old woman; time to preparation 10 hours post-mortem, not in culture, endothelial cell density 1600 cells/mm2;
5a corneoscleral ring from a 76-year-old man; time to preparation 9 hours post-mortem, in culture for 10 days, endothelial cell density 2200 cells/mm2, and
6a corneoscleral ring from a 39-year-old woman; time to preparation 10 hours post-mortem, in culture for 14 days, endothelial cell density 2000 cells/mm2.
Immunoblotting was performed using:
1a corneal explant from a 30-year-old woman with few peripheral scars after keratitis; time to preparation < 1 hour, and
2a corneal explant from a 43-year-old man with a central scar after penetrating trauma; time to preparation < 1 hour.
For immunohistochemistry we used the following primary antibodies:
All primary antibodies were diluted in ChemMate antibody diluent (Dako, Glostrup, Denmark) to prevent non-specific staining. They were applied either to cryostat or paraffin sections of human corneal tissue for 24 hours at 4 °. Prior to sectioning the tissues were fixed in 2% paraformaldehyde (PFA) for 10 mins (cryosections) or in 4.5% formaline overnight (paraffin sections). After being rinsed in phosphate- buffered saline (PBS) the sections were incubated with the secondary antibody using either goat antirabbit antibody conjugated to Alexa-488 (Molecular Probes, Eugene, Oregon, USA) for immunofluorescence or the ChemMate detection kit, alkaline phosphatase/RED, rabbit/mouse by (Dako) for immunocytochemistry. Negative controls were performed for each series.
For Western blotting epithelial cells were collected by trypsinization and spun at approximately 10 000 g in an Eppendorf microfuge for 10 mins at 4 °. Supernatant was transferred and protein concentration was determined. Proteins were separated using SDS-polyacrylamid gel electrophoresis and transferred to nitrocellulose membrane for 1 hour at 1 amp at 4 °. The nitrocellulose membrane was incubated with the primary antibodies diluted in blocking buffer for 60 mins at room temperature, washed with PBS, incubated with the secondary antibody in blocking buffer for 45 mins at room temperature, washed again with PBS and detected with the Amersham enhanced chemiluminescence (ECL) kit. Due to the small quantity of endothelial tissue, Western blot analysis was only performed on corneal epithelium tissue.
Using freshly fixed human corneal tissue for either immunofluorescence or immunocytochemistry of monoamine receptors, we were able to show the presence of alpha-1, beta-1 and beta-2 adrenergic receptors and of D1-like dopaminergic and 5HT-7 serotonergic receptors in corneal epithelium, and of alpha-1, alpha-2A, beta-1 and beta-2 adrenergic receptors and 5HT-7 serotonergic receptors in corneal endothelium. In contrast, antibodies to alpha-2A and alpha-2C adrenergic receptors and to D2-like dopaminergic receptors did not bind in corneal epithelium and antibodies to alpha-2C adrenergic receptors and to D1-like and D2-like dopaminergic receptors did not bind in corneal endothelium (Figures 1-4). In controls, where the primary antibody was omitted and only the secondary fluorescent or enzyme coupled antibody was used, a light staining of the epithelium, endothelium and stroma could be seen. This was taken as background staining.
Western blot analysis confirmed the presence of alpha-1, beta-1 and beta-2 adrenergic receptors and of D1-like dopaminergic and 5HT-7 serotonergic receptors in corneal epithelium and the absence of alpha-2A and alpha-2C adrenergic receptors and D2-like dopaminergic receptors in epithelial cells.
There was no difference in receptor staining between the corneal explants, the donor corneas and the corneoscleral rings. Although, due to organ culture, the epithelial quality in corneoscleral rings was poorer than in the other specimens, there was a clear staining in the basal epithelial layer. No variations in receptor staining for age, gender, post-mortem time or time to preparation were found.
Adrenergic receptors mediate a diverse range of responses to the endogenous catecholamines. The adrenoceptor family includes three alpha-1 adrenergic receptor subtypes (alpha-1A/D, alpha-1B and alpha-1C), three alpha-2 adrenergic receptor subtypes (alpha-2A, alpha-2B and alpha-2C), and three beta adrenergic receptor subtypes (beta-1, beta-2 and beta-3). The alpha-1 adrenergic receptors mediate their response via G-protein-coupled receptors through a Gp/Gq mechanism. All the subtypes are coupled to phospholipase C and activation of the receptor results in the production of inositol phosphate-3 (IP3) and diacylglycerol (DAG). The production of these second messengers results in an activation of both voltage-dependent and independent Ca2 + channels as well as stimulation of protein kinase C, phospholipase A and D, arachidonic acid release and cyclic adenosine monophosphate (AMP) formation (Berridge & Irvine 1989). The alpha-2 adrenoceptors mediate their functions through a variety of G-proteins including Gi/G0. All the sub- types have been shown to be negatively coupled to adenylyl cyclase and mediate an inhibitory effect through the inhibition of cyclic AMP production (Bylund 1995). The beta adrenoceptors are also coupled to G-proteins and subsequent intracellular second messenger systems. The beta adrenoceptor is positively coupled to adenylyl cyclase via activation of Gs G-proteins. There is also evidence to suggest beta adrenoceptors are linked via a stimulatory G-protein to voltage-gated Ca2 + channels (Bylund et al. 1994). Our data demonstrate the presence of alpha-1, beta-1 and beta-2 adrenergic receptors in freshly fixed human corneal epithelium and endothelium and of alpha-2A adrenergic receptors in corneal endothelium. The existence of beta adrenergic receptors on corneal epithelial and endothelial cells has been established (Neufeld et al. 1978; Walkenbach et al. 1985). Corneal epithelial beta adrenergic receptors have been associated with stimulation of adenylyl cyclase and cyclic AMP-dependent protein kinase, as well as chloride secretion (Walkenbach et al. 1991). Corneal endothelial beta adrenoceptors also appear to be linked to adenylyl cyclase and cyclic AMP synthesis (Walkenbach et al. 1985). The presence of alpha adrenergic receptors in corneal epithelium and endothelium is still under discussion. Several previous studies using broken cell preparations were unable to detect alpha adrenergic receptors (Walkenbach et al. 1991), whereas direct radioligand binding studies indicate that intact corneal epithelial and endothelial cells exhibit alpha-1 adrenergic receptors (Walkenbach et al. 1983). Alpha-1 adrenergic receptors appear to be associated with regulation of inositol-1,4,5-tris-phosphate formation (Walkenbach et al. 1991, 1992). The physiological role of alpha-1 and beta adrenergic receptors and their second messengers phosphatidyl inositol and cyclic AMP in corneal epithelium and endothelium is unclear. They may be involved in the regulation of corneal homeostasis and fluid transport. Walkenbach et al. (1991, 1992) did not detect an effect of alpha-1 adrenergic agents on rabbit corneal thickness in vitro; however, Nielsen & Nielsen (1985) found timolol increased and isoprenaline decreased corneal thickness in human corneas. These data suggest that corneal beta adrenergic receptors may be involved in the regulation of corneal thickness. Although there is functional evidence for the presence of alpha-2 adrenergic receptors in corneal tissue (Chu & Candia 1988), the presence of alpha-2 receptors has not yet been determined on human corneal epithelial or endothelial cells. Our data demonstrate the presence of alpha-2A adrenergic receptors in corneal endothelium for the first time. Endothelial alpha-2A adrenergic receptors may be negatively coupled to adenylyl cyclase and mediate an inhibitory effect through the inhibition of cyclic AMP production in corneal endothelium. This may contribute to the regulation of corneal fluid transport.
Dopaminergic receptors are members of the G-protein-coupled receptor superfamily. Two subfamilies can be distinguished: D1-like and D2-like receptors. D1-like receptors are able to stimulate adenylyl cyclase. Interaction between D1-like receptors and GABA-A receptors has been described. The D2-like receptor subtypes have each been shown to inhibite adenylyl cyclase. The D2-like receptors will also stimulate mitogenesis and extracellular acidification. Effects have been shown on arachidonic acid release and mitogen-activated protein kinase as well as on Ca2 + channels (Strange 1992). Previous reports failed to show D1-like or D2-like dopamine receptors in structures other than the retina of the rabbit or rat eye (Elena et al. 1989). Cavallotti et al. 1999 showed D1-like and D2-like dopamine receptors in sections of the rabbit cornea using autoradiographic techniques, suggesting a possible role of the dopaminergic system in the control of corneal functions. Using freshly fixed human corneal tissue we could only show the presence of D1-like dopaminergic receptors in corneal epithelium. Cavallotti et al. 1999 used [3H]spiroperidol as a ligand of dopamine D2-like receptors, but [3H]spiroperidol is not selective to D2-like dopamine receptors and also binds to 5-HT2 serotonin receptors (McGonigle 1988). However, the presence of 5-HT2 serotonin receptors in corneal tissue has not yet been investigated. We used a specific antihuman dopamin D2 receptor antibody and were unable to find any receptor staining in any of our specimens. The difference might be explained by the different specimens and techniques.
Serotonin (5-hydroxytryptamine, 5-HT) produces its effects through a variety of membrane-bound receptors. Most of the 5-HT receptors belong to the G-protein-coupled receptor superfamily. The receptors are divided into seven distinct classes (5-HT1-7), largely on the basis of their structural and operational characteristics. The 5-HT7 receptor was shown to positively modulate cyclic AMP formation via Gs. The receptor also activates the mitogen-activated protein kinase ERK in primary neuronal cultures. Alternate splicing has been reported to generate four 5-HT7 receptor isoforms, which differ in their C-termini. However, these isoforms, to date, have not been shown to differ in their respective pharmacology, signal transduction or tissue distribution (Jasper et al. 1997). 5-HT receptors have been found in rabbit corneal tissues and in human corneal epithelial cell lines. 5-HT has been shown to increase cyclic AMP levels in incubated rabbit corneas (Neufeld et al. 1982; Crider et al. 2003). The only 5-HT receptors known to stimulate cyclic AMP production by activating adenylyl cyclase are the 5-HT4, 5-HT6 or 5-HT7 receptors. For the first time our studies were able to show the presence of 5-HT7 receptors in human corneal epithelium and endothelium, suggesting a possible role for serotonin in corneal fluid transport, as a physiological or pathological role for 5-HT has been hypothesized by the detection of 5-HT in human tears (Martin & Brennan 1994).
Taken together, our studies show the presence of several monoamine receptors on human corneal epithelial and endothelial cells. Future studies are required to show whether these receptors play a role in the regulation of corneal fluid transport and homeostasis.