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. 1998 Jul;39(8):1339-45.
The intent of this study was to identify the pertussis toxin-sensitive G proteins that couple met-enkephalin to the inhibition of cholinergically stimulated secretion in rabbit lacrimal gland acini.
The authors detected G proteins in membranes from freshly isolated glands, freshly isolated acini, and cultured lacrimal acini from rabbits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Antibodies against the alpha subunits of Gi1, Gi1 and Gi2, or Gi3 were used in cultured acini permeabilized by streptolysin-O to determine the role of the G proteins in met-enkephalin inhibition of cholinergic stimulation of lacrimal acinar protein release.
Western blot analysis showed the presence of the alpha subunits of Gi2 and Gi3, but not Gi1, in all three membrane preparations. The met-enkephalin analog D-Ala2-methionine enkephalinamide (DALA) inhibited cholinergic stimulation of secretion by cultured rabbit acinar cells to near basal levels. Inhibition of secretion by DALA was blocked by insertion of antibody to a peptide sequence common to Gialpha1 and Gialpha2, but was not blocked by antibody against a specific Gialpha1 sequence. The inhibitory effect of DALA also was blocked by antibody to a Gialpha3 sequence. At low doses of anti-Gialpha1/2 and anti-Gialpha3 in combination, the effect on reversal of inhibition was additive. However, at higher doses, the effect of the combination was no greater than the effect of either antibody alone.
These results demonstrate that met-enkephalin inhibition of cholinergic secretion is mediated by way of the pertussis toxin-sensitive G proteins Gi2 and Gi3 in cultured rabbit lacrimal acini. Because the effects of the G proteins are not additive, the intracellular events distal to G protein activation most likely converge at some point before exocytosis.
本研究旨在确定在兔泪腺腺泡中，将甲硫氨酸脑啡肽与胆碱能刺激分泌的抑制相偶联的百日咳毒素敏感型G蛋白。
作者通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹法，检测了来自新鲜分离的腺体、新鲜分离的腺泡以及培养的兔泪腺腺泡的膜中的G蛋白。使用针对Gi1、Gi1和Gi2或Gi3的α亚基的抗体，在经链球菌溶血素-O通透处理的培养腺泡中，确定G蛋白在甲硫氨酸脑啡肽抑制泪腺腺泡蛋白释放的胆碱能刺激中的作用。
蛋白质印迹分析显示，在所有三种膜制剂中均存在Gi2和Gi3的α亚基，但不存在Gi1的α亚基。甲硫氨酸脑啡肽类似物D-丙氨酸2-甲硫氨酸脑啡肽酰胺（DALA）将培养的兔腺泡细胞的胆碱能刺激分泌抑制至接近基础水平。DALA对分泌的抑制作用被针对Gialpha1和Gialpha2共有的肽序列的抗体所阻断，但未被针对特定Gialpha1序列的抗体所阻断。DALA的抑制作用也被针对Gialpha3序列的抗体所阻断。在低剂量的抗Gialpha1/2和抗Gialpha3联合使用时，对抑制逆转的作用是相加的。然而，在较高剂量时，联合使用的效果并不比单独使用任何一种抗体的效果更大。
这些结果表明，在培养的兔泪腺腺泡中，甲硫氨酸脑啡肽对胆碱能分泌的抑制是通过百日咳毒素敏感型G蛋白Gi2和Gi3介导的。由于G蛋白的作用不是相加的，G蛋白激活后的细胞内事件很可能在胞吐作用之前的某个点汇聚。
