Acetylcholine content of normal and denervated cat carotid bodies measured by pyrolysis gas chromatography/mass fragmentometry
Acetylcholine content of normal and denervated cat carotid bodies measured by pyrolysis gas chromatography/mass fragmentometry
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通过热解气相色谱/质量碎片测定法测量正常和去神经猫颈动脉体的乙酰胆碱含量
DOI：
10.1111/j.1471-4159.1976.tb06492.x
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发表时间：
1976
影响因子：
4.7
通讯作者：
W. B. Stavinoha
中科院分区：
文献类型：
--
作者：
S. Fidone;S. Weintraub;W. B. Stavinoha
德·卡斯特罗的先驱研究（1928，1951）。与HEYMANS等（1930）的结果一起，提出颈动脉体的血管球（I型）细胞是对血液pOz、pCOz和pH敏感的化学感受器元件，并且终止于这些细胞的颈动脉神经纤维是感觉神经元，其将化学感受器信息从血管球细胞传递到CNS。SCHWEITZER & WRIGHT（1938）首先指出ACh可能在颈动脉体化学感受中起作用，但EYZAGUIRRE和他的合作者（见EYZAG U I R t & ZAPATA，1968）系统地研究了这一问题，并提出ACh可能是从血管球细胞释放的感觉递质，以兴奋邻近的传入神经末梢。然而，在颈动脉体组织中从未化学鉴定出ACh，尽管生物测定表明在颈动脉体提取物中存在ACh样物质（EYZAGUIRRE等人，1965; JONES，1975）。此外，BI-COE（1971）; OSBORNE & BUTLER（1975）的相反观点认为颈动脉体突触的极性和ACh的作用方式与EYZAGUIRRE等人提出的相反，1965，即ACh不存在于血管球细胞中，而是存在于颈动脉神经纤维中，并且从神经末梢释放以作用于细胞，并且因此颈动脉体突触在功能上是传出的而不是传入的。为了更好地了解ACh在颈动脉体中的作用，我们首先（1）使用热解气相色谱法和质谱法重新评估组织中内源性ACh的水平，（2）通过颈动脉神经切片确定组织中的ACh是否在慢性去神经支配后丢失，如果ACh包含在颈动脉神经末梢中，则可能会出现这种情况。数据收集自11只成年猫的两种性别。在4只猫中，在切除颈动脉体进行ACh分析前14天，通过切除颈动脉体至与舌咽神经交界处的颈动脉神经的长度来使一侧的颈动脉体失神经支配。在大多数动物中，将颈动脉体从动物中快速取出，并立即置于充满用100%02平衡的含有30 pM-艾司氯胺酮（Sigma）的冰冷洛克溶液的室中。将颈动脉体快速清除周围的结缔组织，在Cahn电子天平上称重（平均颈动脉体重= 719 pg），并在液氮中冷冻直至分析。为了确定在样品制备过程中是否发生了ACh的显著降解，如已经报道的大鼠脑ACh的降解（STAVINOHA等人，1973; STAVINOHA & WEINTRAUBB，1974~）。在切除之前，通过将整个手术区域浸泡在含有艾司氯胺酮的冰冷洛克溶液中，原位冷却六个颈动脉体。此外，这些颈动脉体中的两个在切除后立即在液氮中快速冷冻，没有时间清洁组织或称重。由于结果显示，原位冷却颈动脉体，或原位冷却并在切除后立即速冻，得到的ACh值与未经这些程序获得的值没有显著差异（P > 0.2，非配对，双尾r检验），因此我们得出结论，组织ACh降解在本研究中不是问题，并且我们的测量准确地反映了组织ACh水平。对于ACh分析，将每个冷冻组织样品置于冷微均化管中的100 μ l 15%（v/v）甲酸的丙酮溶液中，该丙酮溶液含有5 μ l 3 μ m-碘化丁酰胆碱，并快速均化。均质化后，将样品在冰浴中静置30 min，然后转移至Beckman Microfuge管中，并在Beckman Microfuge中离心8 min。将上清液转移至1 ml锥形玻璃管中，加入100 μ l 5 M氯化钠。用200 μ l水饱和的乙醚进行两次萃取，用干燥氮气流将水层干燥。将所得残余物溶解于10 μ l蒸馏水中，并将溶液转移至微量离心管中。用10 μ l高碘酸盐溶液（2.0 g KI、1.8 g碘溶于10 ml蒸馏水中）处理各样品，并在Vortex-Genie混合器上充分混合。在Microfuge中离心4分钟后，通过抽吸除去上清液，并将沉淀物溶于20 μ l色谱级乙腈（Analabs）中。将约Img份的阴离子交换树脂MP-AGlX 8-CL（BioRad）加入到每个管中以除去过量的碘，并且将5-10 - 1的溶液用于每次分析。通过裂解气相色谱-质谱联用技术对乙酰胆碱进行定量分析。热解使胆碱衍生物去甲基化，从而允许二甲基氨基乙醇的酯的方便的气相色谱分离。对（CH，），NCH：离子（m/c 58）的耦合质量碎片分析允许其定量（STAVINOHA & WEINTRAUR，1974 h）。为此目的，Finnigan 1015 C型四极质谱仪与Varian 1400型气相色谱仪和Varian 425型热解器一起使用。气相色谱条件为：6 ft x 2 mm i.d.在100/120目Gas Chrom Q（Applied Science）上填充有5%OV 101、5%十二烷基二甲基三胺琥珀酰胺的玻璃柱;氦气流速，25 ml/min;柱温，110°C;注射器温度，170°C。质谱仪条件为：电子能量，70 eV;源温度，125°C;压力，约5x Torr;单离子扫描质谱仪的质量。
THE PIONEERING studies of DE CASTRO (1928, 1951). ‘together with those of HEYMANS et al. (1930), suggested that the glomus (Type I) cells of the carotid body are chemoreccptor elements which are sensitive to blood pOz, pCOz and pH, and that the fibers of the carotid nerve which terminate upon these cells are sensory neurons which convey chemoreceptor information from the glomus cells to the CNS. SCHWEITZER & WRIGHT (1938) first noted that ACh may play a role in carotid body chemoreception, but it was EYZAGUIRRE and his collaborators (see EYZAG U I R R t & ZAPATA, 1968) who systematically studied this problem and suggested that ACh might be a sensory transmitter released from the glomus cells to excite neighboring afferent nerve terminals. However, ACh has nevcr been chemically identified in carotid body tissue, although bioassays have suggested the presence of an ACh-like substance in carotid body extracts (EYZAGUIRRE et al., 1965; JONES, 1975). Furthermore, opposing views by BI~COE (1971); OSBORNE & BUTLER (1975) regard the polarity of carotid body synapses and the action of ACh in a manner which is the reverse of that proposed by EYZAGUIRRE et nl., 1965, i.e. that ACh is present, not in the glomus cells, but in the carotid ncrve fibers, and is released from the nerve terminals to act upon the cells, and that consequently carotid body synapses are efferent rather than afferent in function. As a first step toward a better understanding of the role of ACh in the carotid body, we endeavored to (1) reassess the levels of endogenous ACh in the tissue, using pyrolysis gas chromatography and mass fragmentometry, and (2), to determine whether the ACh in the tissue was lost following chronic denervation by section of the carotid nerve, as might be expected if the ACh were contained in the carotid nerve terminals. Data were collected from eleven adult cats of both sexes. In four cats, the carotid body on one side was denervated by removal of a length of carotid nerve, from the carotid body to the junction with the glossopharyngeal nerve, 14 days prior to excision of the carotid bodies for ACh analysis. In most animals, carotid bodies were quickly removed from the animals and immediately placed in a chamber filled with ice-cold Locke’s solution containing 30 pM-eserine (Sigma) equilibrated with 100% 02. The carotid bodies were rapidly cleaned of surrounding connective tissue, weighcd on a Cahn Electrobalance (mean carotid body weight = 719 pg), and frozen in liquid nitrogen until analysis. ln order to determine whether significant degradation of ACh occurred during sample preparation, as has been reported for rat brain ACh (STAVINOHA e f al., 1973; STAVINOHA & WEINTRAUBB, 1974~). six carotid bodies were cooled in situ prior to excision by bathing the entire surgical field in ice-cold Locke’s solution containing eserine. Also, two of these carotid bodies were quickly frozen in liquid nitrogen immediately upon excision, without allowing time for cleaning of the tissue or weighing. Since the results showed that cooling the carotid body in situ, or cooling in situ and quick-freezing immediately upon excision, gave ACh values which were not significantly different from those obtained without these procedures (P > 0.2, non-paired, double-tailed r-test), we concluded that degradation of tissue ACh was not a problem in this study, and that our measurements accurately reflected tissue ACh levels. For ACh analysis, each frozen tissue sample was placed into 100 pl of 15% (v/v) formic acid in acetone containing 5 pl of 3 pM-butyrylcholine iodide in a cold micro-homogenization tube and was quickly homogenized. The samples were allowed to stand in an ice bath for 30 min after homogenization and were then transferred to Beckman Microfuge tubes and were centrifuged for 8 min in a Beckman Microfuge. The supernatants were transferred to I ml conical glass tubes, and 100 p1 of 5 M-sodium chloride was added. Two extractions with 200 p1 portions of water-saturated diethyl ether were performed and the aqueous layer was taken to dryness by a stream of dry nitrogen. The resultant residue was dissolved in 1 0 pl of distilled water and the solution was transferred to a Microfuge tube. Each sample was treated with 10 pl of periodide solution (2.0 g KI, 1.8 g iodine in 10 ml of distilled water) and mixed thoroughly on a Vortex-Genie mixer. After centrifugation in the Microfuge for 4 min, the supernatants were removed by aspiration and the precipitate dissolved in 20 pl of chromatographic grade acetonitrile (Analabs). Approximately 1 mg portions of anion exchange resin MP-AGlX8-CL (BioRad) was added to each tube to remove excess iodine and 5-10~1 of the solution was used for each analysis. Quantitation of ACh was obtained through combination pyrolysis gas chromatography-mass fragmentometry. Pyrolysis demethylates the choline derivatives, thereby allowing the convenient gas chromatographic separation of the esters of dimethylaminoethanol. Coupled mass fragmentometric analysis of the (CH,), NCH: ion (m/c 58) permits their quantitation (STAVINOHA & WEINTRAUR, 1974h). For this purpose, a Finnigan model 1015C quadrupole mass spectrometer was used in conjunction with a Varian model 1400 gas chromatograph and a Varian model 425 pyrolyzer. Gas chromatographic conditions were: 6 ft x 2 mm i.d. glass column packed with 5% OV 101, 5% dodecyldimethylenctriamine succinamide on 100/120 mesh Gas Chrom Q (Applied Science); helium flow rate, 25 ml/min; column temperature, 110°C; injector temperature, 170°C. Mass spectrometer conditions were: electron energy, 70 eV; source temperature, 125°C; pressure, approx 5 x Torr; single ion scan for mass of