Meneray M A, Fields T Y, Bennett D J
Department of Physiology, Louisiana State University Medical Center, New Orleans 70119, USA.
Invest Ophthalmol Vis Sci. 1997 May;38(6):1261-70.
The intent of this study was to determine the physiological role of selected G proteins in receptor-mediated protein release by lacrimal acini.
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.
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.
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.
本研究旨在确定特定G蛋白在泪腺腺泡受体介导的蛋白质释放中的生理作用。
采用成年雄性新西兰白兔泪腺组织，研究G蛋白在泪液分泌中的作用。通过用百日咳毒素处理原代腺泡培养物，用链球菌溶血素 - O使培养的腺泡通透化，并插入GDPβS或针对Gs或Gq/11α亚基的抗体，来确定G蛋白在血管活性肠肽（VIP）和卡巴胆碱刺激的泪液分泌中的作用。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和免疫印迹法，在从新鲜分离的泪腺片段、新鲜分离的腺泡和培养的腺泡中获得的泪膜中鉴定Gs和Gq/11。
链球菌溶血素 - O通透化并向培养的腺泡中引入硫代二磷酸鸟苷，可使100 nM VIP或100 μM卡巴胆碱刺激的蛋白质释放减少约50%。将培养的腺泡暴露于100 ng/ml百日咳毒素36至48小时，对两种激动剂刺激的释放均无影响，表明VIP和卡巴胆碱的三磷酸鸟苷依赖性作用是通过百日咳毒素不敏感的G蛋白介导的。用针对Gs和Gq/11α亚基的多克隆抗体鉴定从新鲜分离的腺体、新鲜分离的腺泡和培养的腺泡中获得的泪膜中的百日咳毒素不敏感G蛋白。用抗Gsα抗血清对泪膜进行免疫印迹显示在44和47 kDa处有两条免疫反应带。抗Gq/11α抗血清在类似的膜制剂中检测到一条46 kDa的条带。抗Gsα抗血清使对VIP的分泌反应降低64%，对卡巴胆碱的反应降低37%。引入抗Gq/11α抗血清使对卡巴胆碱的反应降低70%；然而，对VIP的反应未改变。同时引入两种抗血清对VIP刺激释放的抑制作用并不比单独使用抗Gsα抗血清更强。然而，同时引入抗Gsα和抗Gq/11α抗血清可完全抑制卡巴胆碱对培养腺泡蛋白质释放的作用。
这些结果表明，泪腺蛋白质释放的VIP受体激活是通过Gs介导的，而胆碱能刺激涉及Gs和Gq/11。根据作者的结果，作者得出结论，Gs将VIP受体激活与腺苷酸环化酶和环磷酸腺苷生成以及腺泡细胞最终的蛋白质释放联系起来，并且Gq/11将毒蕈碱受体激活与磷脂酶C以及肌醇三磷酸和二酰基甘油积累联系起来，这也导致蛋白质释放。此外，推测Gs在调节囊泡运输和胞吐作用中具有额外作用。
