Walker S W, Strachan M W, Lightly E R, Williams B C, Bird I M
Department of Clinical Chemistry, University of Edinburgh, Royal Infirmary, U.K.
Mol Cell Endocrinol. 1990 Sep 10;72(3):227-38. doi: 10.1016/0303-7207(90)90147-z.
Zona fasciculata/reticularis (ZFR) cells, isolated from the bovine adrenal cortex, secreted cortisol in response to acetylcholine (AcCh). The response was present in freshly isolated cells and in cells maintained in primary culture, reaching a maximum after 48-72 h and thereafter declining. Cells maintained in primary culture for 72 h secreted cortisol with an ED50 at 1.2 x 10(-6) M. The potent inhibition of AcCh-stimulated secretion by atropine, and the relative ineffectiveness of nicotine or nicotinic antagonists, were consistent with a predominantly muscarinic response to AcCh in these cells. A selective M1-receptor agonist, McN-A-343, had no effect on cortisol secretion whereas the M3 antagonist, hexahydro-sila-difenidol, produced a dose-dependent inhibition of AcCh-stimulated cortisol secretion. These findings are consistent with AcCh mediating its effects on cortisol secretion through an M3 receptor. While AcCh had no effect on cAMP formation, a dose-dependent increase in [3H]phosphoinositols (identified using high-performance liquid chromatography (HPLC)) occurred in a manner that was not dependent on an influx of extracellular Ca2+. Detailed HPLC analysis of the formation of 3H-labelled phosphoinositols and glycerophosphoinositols from pre-labelled cells over the period 0-15 min showed that the earliest significant rise was in Ins(1,4,5)P3 at 5 s, followed by later rises in InsP1, InsP2 and Ins(1,3,4)P3. Additional studies using cells loaded with fura-2 indicator revealed a 1.6-fold increase in [Ca2+]i from a mean resting value of 75 nM in response to 10(-4) M AcCh. Furthermore, the rise in Ca2+ was not abolished by lowering extracellular Ca2+ to resting cytosolic levels, suggesting the mobilisation of an intracellular pool. These observations indicate that AcCh promotes rapid activation of a Ca2(+)-independent and polyphosphoinositide-specific phospholipase C, and that the Ins(1,4,5)P3 formed releases Ca2+ from an intracellular pool. The stimulation by AcCh of this signal transduction mechanism is consistent with our conclusion, based on the effects of the selective muscarinic agonist and antagonist on cortisol secretion, that the AcCh receptor is of the M3 subtype. We conclude that AcCh, acting through an M3 receptor coupled to phospholipase C, regulates cortisol secretion at the cellular level in bovine adrenal ZFR cells.
从牛肾上腺皮质分离出的束状带/网状带（ZFR）细胞，对乙酰胆碱（AcCh）有反应并分泌皮质醇。新鲜分离的细胞以及原代培养的细胞均有此反应，在48 - 72小时后达到最大值，此后下降。原代培养72小时的细胞分泌皮质醇，其半数有效浓度（ED50）为1.2×10⁻⁶ M。阿托品对AcCh刺激的分泌有强烈抑制作用，而尼古丁或烟碱拮抗剂的作用相对较弱，这与这些细胞对AcCh主要产生毒蕈碱样反应一致。选择性M1受体激动剂McN - A - 343对皮质醇分泌无影响，而M3拮抗剂六氢硅二苯哌啶产生剂量依赖性抑制AcCh刺激的皮质醇分泌。这些发现与AcCh通过M3受体介导其对皮质醇分泌的作用一致。虽然AcCh对环磷酸腺苷（cAMP）形成无影响，但[³H]磷酸肌醇（使用高效液相色谱法（HPLC）鉴定）呈剂量依赖性增加，其方式不依赖于细胞外Ca²⁺的内流。对预标记细胞在0 - 15分钟内³H标记的磷酸肌醇和甘油磷酸肌醇形成的详细HPLC分析表明，最早在5秒时显著升高的是肌醇-1,4,5-三磷酸（Ins(1,4,5)P3），随后肌醇一磷酸（InsP1）、肌醇二磷酸（InsP2）和肌醇-1,3,4-三磷酸（Ins(1,3,4)P3）也相继升高。使用负载fura-2指示剂的细胞进行的额外研究显示，对10⁻⁴ M AcCh的反应使细胞内钙离子浓度（[Ca²⁺]i）从平均静息值75 nM增加了1.6倍。此外，将细胞外Ca²⁺降低至静息胞质水平并不能消除Ca²⁺的升高，这表明动员了细胞内钙库。这些观察结果表明，AcCh促进了一种不依赖Ca²⁺且具有多磷酸肌醇特异性的磷脂酶C的快速激活，并且形成的Ins(1,4,5)P3从细胞内钙库释放Ca²⁺。基于选择性毒蕈碱激动剂和拮抗剂对皮质醇分泌的影响，我们得出结论：AcCh对这种信号转导机制的刺激与我们的结论一致，即AcCh受体是M3亚型。我们得出结论，AcCh通过与磷脂酶C偶联的M3受体起作用，在细胞水平上调节牛肾上腺ZFR细胞中的皮质醇分泌。
