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H. C. Turner, O. A. Candia, J. M. Wolosin; Mobilization and Secretion of sPLA2 by Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5303.
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Group IIA secretory phospholipase A2 (sPLA2-IIA), a powerful gram A+ microbiocide in human tears, is a lipolytic enzyme known to play intricate and critical roles in inflammatory states in multiple tissues. Although it is generally assumed that tear sPLA2-IIA is derived from the lacrimal gland, where its presence has been confirmed, we have identified sPLA2-IIA as a major, differentiation dependent, conjunctival (CNJ) epithelial gene that is highly expressed in the mucin-secreting CNJ Goblet cells (Turner et al., IOVS, 2006). The distribution pattern of sPLA2-IIA granules within the Goblets cells in various stages of maturation suggest that the protein is an important secretory component in these cells. Accordingly, the present study aimed to directly confirm sPLA2-IIA enzyme secretion from conjunctival tissue.
Goblet cell rich (forniceal/bulbar) and Goblet cell free (mucocutaneous) rabbit conjunctival segments, each ~ 4 mm2 were placed apical-side up in a specifically-designed perspex chamber and incubated in M199 culture media. After a 30 min equilibration period, the medium was refreshed with control M199 or forskolin/IBMX complemented media. Media aliquots were then collected every 5 min and sPLA2 lipolytic activity was measured using a fluorimetric assay based on the rate of hydrolysis of 1 hexadecanoly-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt. Activities were normalized according to the amount of epithelia/per specimen.
The fluorimetric phospholipase A2 assay revealed continuous sPLA2 secretion with a gradual decrease of the in vitro secretion rate over the first hour. The cAMP cocktail mix increased the secretory rate. In addition, the Goblet cell-rich CNJ regions showed a substantially higher sPLA2 secreted activity than the mucocutaneous Goblet cell poor zone.
This study presents for the first time evidence for pharmacologically controlled sPLA2 secretion from the conjunctiva. Furthermore, the results suggest that the conjunctiva and Goblet cells in particular may play an important role in the secretion of this important pro-inflammatory protein into the tear film.
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