The role of calcium in mucin packaging within goblet cells Helline B. Paz, Ann S. Tisdale, Yukitaka Danjo, Sandra J. Spurr-Michaud, Pablo Argu¨eso, Ilene K. Gipson * Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114–2500, USA Received 8 November 2002; received in revised form 21 February 2003; accepted 5 March 2003 Abstract Recent reports hypothesize that calcium plays an important role in providing cationic shielding to keep negatively charged mucins condensed and tightly packed within mucus granules of goblet cells. Vitamin D controls mineral ion homeostasis and intestinal calcium absorption, which is mediated by the nuclear vitamin D receptor (VDR). Hypocalcemia is observed in mice in which the VDR has been ablated. The purpose of this study was to test the hypothesis that normal levels of calcium are required for the physiological packaging of mucins, by comparing the morphology and mucin extractability of conjunctival goblet cells of VDR-ablated to wild-type control mice. Whole eyes from C57/129/sv hybrid wild-type, VDR-ablated, and VDR-ablated mice fed a diet high in calcium to normalize serum ionized calcium levels were fixed in situ and processed for light and transmission electron microscopy (TEM). Mucin extractability from sections of mouse eyes was assessed by lectin-blot, using helix pomatia agglutinin (HPA), and mucin content within goblet cells was assessed by immunohistochemistry, using an antibody specific to the goblet cell mucin Muc5AC. Altered mucin packaging in the goblet cells of VDR-ablated mice as compared to control mice was observed by both light and electron microscopy. In the VDR-ablated mice, the mucin packets varied in size and staining. In contrast, in the controls, the secretory granules appeared regular and uniform. By TEM, mucin packets in the VDR-ablated mice showed dispersed fibrillar and less electron-dense material compared to the homogeneous and more electron-dense packets in wild type. The appearance of mucin packets in the VDR-ablated mice with restored calcium levels was comparable to those of the wild-type control mice. HPA binding to mucin extracted from sections of VDR mouse eyes was reduced when compared to that from wild type. By immunohistochemistry, there was markedly less binding of the antibody to the mucin Muc5AC to goblet cells of VDR-ablated mice compared to controls. VDR-ablated mice presented altered conjunctival mucin packaging. There were lower levels of extractable and immunohistochemically localizable mucin in VDR-ablated mouse conjunctivas than in the wild-type controls. Restoration of ionized calcium levels in the VDR- ablated mice prevented altered mucin packaging, supporting the hypothesis that calcium is required for the physiological packaging of mucins in goblet cells. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: mucins; goblet cells; conjunctiva; tear film; ocular surface 1. Introduction Mucus in the tear film on the surface of the eye provides a protective covering that prevents pathogen penetrance and desiccation of the underlying epithelium (Gipson and Inatomi, 1997). High molecular weight glycoproteins known as mucins are the major component of mucus, giving it its protective, hydrophilic and rheological proper- ties (Verdugo et al., 1983). The primary source of the mucins on the ocular surface is conjunctival goblet cells. These cells express and secrete a very large gel-forming mucin, MUC5AC. MUC5AC mucin multimers are tightly packed in intracellular secretory granules of the conjunctival goblet cell and are negatively charged due to their associated sialic acid and sulfate residues (Argu¨eso and Gipson, 2001; Kuver et al., 2000). Upon stimulation, secretory granules within goblet cells fuse to one another and to the apical cell membrane, thus releasing mucins onto the epithelial surface (Forstner, 1995). Recent reports suggest that increased calcium concen- tration plays an important role in providing cationic 0014-4835/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. DOI:10.1016/S0014-4835(03)00084-8 Experimental Eye Research 77 (2003) 69–75 www.elsevier.com/locate/yexer * Corresponding author. Dr I. K. Gipson, Schepens Eye Research Institute, 20 Staniford St, Boston, MA 02114–2500, USA. E-mail address: gipson@vision.eri.harvard.edu (I.K. Gipson).