Abstract on Research Topic - Guido Wollmann

Neurotensin in the Bovine Ciliary Epithelium - Distribution, Modulation and Release Wollmann G, Coca-Prados, M; Department of Ophthalmology and Visual Science, Yale University School of Medicine

The ocular ciliary epithelium is composed of a bilayer of neuroepithelial cells responsible for the secretion of aqueous humor and the regulation of intraocular pressure. In former studies its neuroendocrine properties have been revealed. Several neuropeptides, such as neurotensin (NT), ANP, galanin, neuropeptide Y and angiotensinogen, and specific prohormone convertases (e.g. PC1, PC2, CPE and 7B2) have been detected by RT-PCR, Northern analysis, radioimmunoassay and indirect immunofluorescence. In this study we compare the expression, modulation, distribution and releasing mechanism of NT and related convertases in two different bovine cell lines derived from the nonpigmented (NPE) and pigmented (PE) ciliary epithelium. Both cell lines express Neurotensin whereas the expression of the convertases shows a cell specific pattern. PC1 expression seems to be restricted to PE cells; PC2 and CPE expression was found only in the nonpigmented cell line. Unexpectedly, both cell lines express 7B2, a protein usually involved in activation of PC2. The level of NT expression could be up-regulated in both the NPE and the PE cells by dexamethasone (1mM) 2 fold, by forskolin (1mM) 2 fold and by a combination of both 12 fold, indicating a synergistic effect. PC1 expression in PE cells responded to the same inducers, whereas PC2 expression was detectable only after forskolin stimulation. 7B2 was up-regulated in PE and down-regulated in NPE cells by phorbol ester (PMA; 50nM). Previous radioimmunoassay results (Ortego et al.) indicate different releasing mechanisms of NT. In order to address the question of a calcium dependent secretion pathway, we plan to apply a radioimmunoassay to measure the NT concentration in the culture medium and in the cell extract under different calcium conditions after treatment with thapsigargin (1mM), BAPTA AM (10mM), EGTA (10mM) or KCl (20mM). Collectively, our data may support the view that the two cell types display different neuroendocrine features. It is well known that PC1 produces a specific NT/NN pattern in the gut that is distinct from that produced by PC2 in the brain. Therefore, it is reasonable to speculate that the neuroendocrine properties of both the NPE and PE cells are cell specific.

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