Development of Conjunctival Goblet Cells and Their Neuroreceptor Subtype Expression

PURPOSE. To investigate expression of muscarinic, cholinergic, and adrenergic receptors on developing conjunctival goblet cells. METHODS. Eyes were removed from rats 9 to 60 days old, fixed, and used for microscopy. For glycoconjugate expression, sections were stained with Alcian blue/periodic acid-Schiff's reagent (AB/PAS) and with the lectins Ulex europeus agglutinin I (UEA-I) and Helix pomatia agglutinin (HPA). Goblet cell bodies were identified using anti-cytokeratin 7 (CK7). Nerve fibers were localized using anti-protein gene product 9.5. Location of muscarinic and adrenergic receptors was investigated using anti-muscarinic and ␤-adrenergic receptors. RESULTS. At days 9 and 13, single apical cells in conjunctival epithelium stained with AB/PAS, UEA-I, and CK7. At days 17 and 60, increasing numbers of goblet cells were identified by AB/PAS, UEA-I, HPA, and CK7. Nerve fibers were localized around stratified squamous cells and at the epithelial base at days 9 and 13, and around goblet cells and at the epithelial base at days 17 and 60. At days 9 and 13, M 2 -and M 3 -muscarinic and ␤ 2 -adrenergic receptors were found in stratified squamous cells, but M 1 -muscarinic and ␤ 1 -adrenergic receptors were not detected. At days 17 and 60, M 2 -and M 3 -muscarinic receptors were found in goblet cells, whereas M 1 -muscarinic receptors were in stratified squamous cells. ␤ 1 -and ␤ 2 -Adrenergic receptors were found on both cell types. ␤ 3 -Adrenergic receptors were not detected. CONCLUSIONS. In conjunctiva, nerves, M 2 -and M 3 -muscarinic, and ␤ 1 -and ␤ 2 -adrenergic receptors are present on developing goblet cells and could regulate secretion as eyelids open. (Invest Ophthalmol Vis Sci. 2000;41:2127-2137 T he tear film mucus layer consists of high molecular weight glycoconjugates including mucins, which are secreted mainly by conjunctival goblet cells. This layer plays an important role in protecting the ocular surface from exogenous agents (bacterial or chemical) and provides lubrication during all types of eye movements. 1 Goblet cells can release their secretory granules in a reflex response mediated by the activation of either parasympathetic or sympathetic nerves that surround them. 2,3 Previous reports from this laboratory showed the localization of nerve fibers adjacent to goblet cells in rat conjunctiva. 5 Use of immunofluorescence techniques demonstrated that M 2 -and M 3 -, but not M 1 -muscarinic acetylcholine receptors (MAchRs), are present on goblet cells and are located on membranes subjacent to secretory granules. VIP type 2 receptors (VIPR2s) are located in the basolateral membranes of goblet cells. 3 Although the role of the sympathetic agonists in stimulating goblet cell secretion is unknown, ␤ 1 -and ␤ 2 -adrenergic receptor (␤AR) subtypes appear to be present in goblet cells as well as in stratified squamous cells. Morphologic studies in developing conjunctiva suggest that based on changes in the acidity of glycoproteins in the secretory granules, goblet cells may differentiate from basal epithelial cells in the forniceal zone. 7 Watanabe et al