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Robin R. Hodges, Darlene A. Dartt; Identification of Altered Signaling Pathways in Lacrimal Glands from a Mouse Model of Sjogren’s Syndrome. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3719.
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To determine if cholinergic, α1D-adrenergic, or purinergic signaling pathways that stimulate lacrimal gland protein secretion are altered in a mouse model of Sjogren’s Syndrome.
Intracellular Ca2+ Measurements: Acini from lacrimal glands from female 11 week old wild type (WT) and TSP-1 null mice were isolated by collagenase digestion. Acini were incubated with the Ca2+ indicator dye fura 2 and stimulated with the cholinergic agonist carbachol (Cch, 10-5 M), the α1D-adrenergic agonist phenylephrine (Ph, 10-5 M), the purinergic agonist ATP (10-6 M), and the P2X7 receptor agonist benzylATP (BzATP, 10-4 M). Fluorescent images of cells were analyzed with a digital fluorescence imaging system (InCyte Im2, Intracellular Imaging Inc) and intracellular Ca2+ concentration ([Ca2+]i) was determined. Measurement of Pore Formation: To measure P2X7 receptor pore formation, acini were incubated with or without 1 µM Yo-Pro, a molecule which enters cells through pores and fluoresces only when intercalated in the DNA, in the presence of BzATP at10-5 and 10-4 M. Fluorescence was measured every 30s for 10 min and the area under the curve calculated
In WT mice, Cch increased [Ca2+]i to 486.5 ± 109.5 nM, while the Cch response in TSP-1 null mice was 874.6 ± 45.0 nM. This was significantly increased from WT. In WT mice, Ph increased [Ca2+]i to 478.2 ± 32.1. This was not significantly different from TSP-1 null mice, which was 687.3 ± 96.3 nM. ATP increased [Ca2+]i to 471.9 ± 32.5 in WT mice and to 702.1 ± 48.2 nm in TSP-1 null mice. The TSP-1 response was significantly increased from WT. The BzATP response in WT mice was 541.8 ± 54.8 nM which was not significantly different from TSP-1 null mice. There was no difference in the amount of Yo-Pro fluorescence between acini from WT and TSP-1 null mice.
Cholinergic and purinergic, but not α1D-adrenergic or P2X7, receptor pathways are altered in TSP-1 null compared to WT mice.
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