Investigative Ophthalmology & Visual Science, Vol 38, 1261-1270, Copyright © 1997 by Association for Research in Vision and Ophthalmology

ARTICLES AND REPORTS

Gs and Gq/11 couple vasoactive intestinal peptide and cholinergic stimulation to lacrimal secretion

MA Meneray, TY Fields and DJ Bennett
Department of Physiology, Louisiana State University Medical Center, New Orleans 70119, USA.

PURPOSE: The intent of this study was to determine the physiological role of selected G proteins in receptor-mediated protein release by lacrimal acini. METHODS: The role of G proteins in lacrimal secretion was determined in tissues obtained from the lacrimal glands of adult male New Zealand White rabbits. Pertussis toxin treatment of primary acinar cultures and permeabilization of cultured acini with streptolysin-O and insertion of GDP beta S or antibodies against the alpha subunit of Gs or Gq/11 were used to determine the role of G proteins in vasoactive intestinal peptide (VIP) and carbachol- stimulated lacrimal secretion. Gs and Gq/11 were identified in lacrimal membranes obtained from freshly isolated lacrimal gland fragments, freshly isolated acini, and cultured acini by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting. RESULTS: Permeabilization by streptolysin-O and introduction of guanosine thiodiphosphate into cultured acini blocked stimulation of protein released by either 100 nM VIP or 100 microM carbachol by approximately 50%. Exposure of cultured acini to 100 ng/ml pertussis toxin for 36 to 48 hours did not affect stimulated release by either agonist, indicating that the guanosine triphosphate-dependent actions of VIP and carbachol are mediated through pertussis toxin-insensitive G proteins. Pertussis toxin-insensitive G proteins in lacrimal membranes obtained from freshly isolated glands, freshly isolated acini, and cultured acini were identified with polyclonal antibodies to the alpha subunits of Gs and Gq/11. Immunoblotting of lacrimal membranes with anti-Gs alpha antiserum showed two immunoreactive bands at 44 and 47 kDa. Anti- Gq/11 alpha antiserum detected a single band at 46 kDa in similar membrane preparations. Anti-Gs alpha antiserum reduced the secretory response to VIP by 64% and to carbachol by 37%. Introduction of anti- Gq/11 alpha antiserum reduced the response to carbachol by 70%; however, the response to VIP was unchanged. Simultaneous introduction of both antisera caused no further reduction of VIP-stimulated release than did anti-Gs alpha antiserum alone. However, simultaneous introduction of both anti-Gs alpha and anti-Gq/11 alpha antisera resulted in complete inhibition of the effects of carbachol on protein release by cultured acini. CONCLUSIONS: These results show that VIP receptor activation of lacrimal protein release is mediated through Gs, whereas cholinergic stimulation involves both Gs and Gq/11. From the authors' results, the authors conclude that Gs links VIP receptor activation to adenylyl cyclase and cyclic adenosine 3'-5' monophosphate production and the ultimate release of protein by acinar cells and that Gq/11 links muscarinic receptor activation to phospholipase C and IP3 and diacylglycerol accumulation, which also leads to protein release. Furthermore, it is hypothesized that Gs has an additional role in the regulation of vesicular traffic and exocytosis.

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