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M A Meneray, D J Bennett; Identification of G proteins in lacrimal gland.. Invest. Ophthalmol. Vis. Sci. 1995;36(6):1173-1180.
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PURPOSE: The intent of this study was to identify and characterize guanine nucleotide binding proteins (G proteins) that are a component of cell signaling of stimulus-secretion coupling in lacrimal gland. METHODS: Membranes were isolated from the lacrimal glands of male Sprague-Dawley rats and New Zealand rabbits and were used to identify G proteins in lacrimal gland by Western blot analysis. Solubilized membrane proteins from lacrimal glands and from a positive tissue control (SH-SY5Y cells) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The alpha subunits of the G proteins were identified by immunoreactivity, with specific peptide directed antisera against Gs alpha, Gi alpha 1, Gi alpha 1/2, and Go alpha. The initial characterization of coupling of specific G proteins to receptors was accomplished by preincubation of membranes with nonimmune serum, anti-Gs alpha, or anti-Go alpha antiserum and assay of adenylyl cyclase activity in the presence of the neuropeptide, vasoactive intestinal peptide (VIP), or forskolin. RESULTS: Gs alpha, Gi alpha 3, and Go alpha were present in all three membrane preparations. Gs alpha subunits were detected as two bands at 45 to 48 kd and 48 to 54 kd. Gi alpha 3 was detected as a single protein at 40 to 41 kd, and at least one form of Go alpha with a molecular weight of 40 kd was detected in all three preparations. Gi alpha 1 was detected in immunoblots of rat lacrimal and SH-SY5Y membranes at 41 kd, and the density of the band was enhanced in blots probed with anti-Gi alpha 1/2 antiserum. Immunoreactivity to anti-Gi alpha 1 or anti-Gi alpha 1/2 was faint or not detectable in rabbit lacrimal membranes. A prominent band was detected in rabbit and rat lacrimal but not in SH-SY5Y membranes at 31 kd with anti-Gi alpha 1/2 antiserum, which may represent a G protein involved in exocytosis. Coupling of VIP receptor activation to adenylyl cyclase by Gs alpha was evidenced by the reduction of VIP stimulation of the enzyme by preincubation of rabbit membranes with anti-Gs alpha antiserum. In contrast, preincubation of membranes with anti-Go alpha antiserum resulted in an increase in activity of adenylyl cyclase in the presence of VIP. CONCLUSIONS: Detection of specific alpha subunits in lacrimal gland indicates that Gs, Gi, and Go are present in rabbit and rat lacrimal gland. Both Gs and Go influence VIP stimulation of lacrimal adenylyl cyclase and thus are presumed to be involved in signal transduction, leading to second-messenger accumulation and subsequent regulation of lacriminal function, including secretion. In addition, an unidentified protein is present in lacrimal gland that may represent one of the guanine nucleotide binding proteins involved in exocytosis.
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