Purpose To determine the source(s) of vitamin D in tear fluid
Purpose To determine the source(s) of vitamin D in tear fluid and examine the expression of the endocytic proteins and putative vitamin D transporters megalin and cubilin in lacrimal and Harderian glands. showed apical duct cell megalin staining and weaker megalin staining in VDR knockout mice compared with controls. Vitamin D2 was more prevalent in rabbit lacrimal and accessory gland fluid than vitamin D3 and greater amounts of Vitamin D2 were found in in tear fluid obtained directly from lacrimal and accessory glands as compared with plasma concentrations. Conclusions This is the first study to demonstrate the presence of megalin and cubilin in lacrimal and accessory glands responsible for producing tear Proscillaridin A fluid. The results strengthen the hypothesis that megalin and cubilin are likely involved in the secretory pathway of vitamin D into tear fluid by the duct cells. = 5 per group). Three New Zealand white rabbits were used to collect serum along with lacrimal and accessory gland fluid. All animal studies were approved by the University institutional animal care and use committee and animals Proscillaridin A were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Lacrimal Cannulation Three white New Zealand rabbits (2.2 kg) were used to collect lacrimal and accessory gland fluid. Before any manipulation the rabbits were weighed to adjust anesthetic dosage with ketamine and xylazine. Vascular access was secured at the marginal ear vein and blood samples were taken for vitamin D metabolite analysis. Lacrimal cannulation was performed as described previously. 20 21 Briefly microcapillary tubes were fire polished and used to cannulate the lacrimal duct. After successful access to the lacrimal duct 0.2 mL of 2 mg/mL pilocarpine was injected every 20 Proscillaridin A minutes for up to 3 hours to stimulate lacrimal fluid production. Pilocarpine injection results in an immediate increase in lacrimal secretion. CCHL1A1 A total of 100 to 200 μL was collected directly from the microcapillary tube with a micropipette. At the same time fluid was collected from Proscillaridin A the combined accessory glands (Harderian and Wolfing) by collecting fluid from the fornix that pooled while the lacrimal gland was cannulated. Rabbits were euthanized with pentobarbital after the cannulation procedure. Mass Spectroscopy Vitamin D metabolites were analyzed by mass spectroscopy. The samples were extracted derivatized and analyzed as described previously22 with modification. In brief approximately 500 μL of samples spiked with internal standards (d6 vitamin D3 d6 25(OH) vitamin D3 and d6 1 25 (OH)2 vitamin D3) were extracted with 500 μL of methyl (4°C) for 10 minutes followed by supernatant recollection. Sample protein concentration was measured using the bicinchoninic acid protein assay reagent (Pierce Rockford IL USA). Equal amounts of protein (20 μg) were loaded onto an Proscillaridin A 8% gel and separated by SDS-PAGE. Rabbit anti-megalin (Santa Cruz Biotechnology Santa Cruz CA USA) and goat anti-cubilin (Santa Cruz Biotechnology) were used as primary antibodies. Horseradish peroxidase conjugated goat and rabbit anti-goat and anti-rabbit secondary antibodies were used to enhance detection. For loading controls membranes were stripped and Proscillaridin A reprobed with β-actin antibody (CP01; Calbiochem San Diego CA USA). Western blots were digitally photographed and blot density was determined by Fiji (Wayne Rasband National Institutes of Health Bethesda MD USA). For megalin immunohistochemistry paraffin sections (5 μm) were cut from 10-week-old mice on a Leica microtome (Wetzlar Germany). Sections were dried at 60°C in an oven for 1 hour placed in xylene overnight rehydrated in graded alcohols and blocked for endogenous peroxidase. Demasking was achieved by heating the sections in TEG buffer (Tris-EGTA buffer pH 9) at approximately 100°C for 10 minutes after which the sections were cooled at room temperature for 30 minutes and incubated for 30 minutes in 50 nM NH4Cl in 0.01 M PBS. Permeabilization was obtained with 0.05% saponin (1% BSA 0.2% gelatine 0.05% saponin in 0.01 M PBS) and sections were incubated with 1:500 of Protein A purified sheep anti-megalin (kindly provided by Pierre Verroust MD) or Protein A purified sheep Ig (DAKO Glostrup Denmark) in 0.01 M PBS 0.1% BSA and 0.3% Triton X-100 followed by washing and incubation with HRP-conjugated secondary antibody..