Rose C, Camus A, Schwartz J C
Unité de Neurobiologie et Pharmacologie (U. 109) de l'INSERM, Centre Paul Broca, Paris, France.
Neuroscience. 1989;29(3):583-94. doi: 10.1016/0306-4522(89)90132-2.
Endogenous cholecystokinin immunoreactivity released by depolarization of slices of rat cerebral cortex undergoes extensive degradation (85% of released immunoreactivity) before reaching the incubation medium. In order to identify the responsible peptidases, a large number of inhibitors of the four catalytic classes were tested for their protective effects. Inhibitors of metallopeptidases (bestatin, amastatin, puromycin, Thiorphan, captopril, o-phenantroline), thiol-peptidases, (leupeptin, antipain, p-hydroxymercuribenzoate) or carboxyl-peptidases (pepstatin) had generally low if any protective effect. By contrast, several serine peptidase inhibitors, i.e. diisopropyl-fluorophosphate, phenylmethylsulphonylfluoride or the chloromethylketone Ala-Ala-Pro-Val-CH2Cl, doubled the recovery of cholecystokinin immunoreactivity and the effect was amplified in the co-presence of bestatin, an aminopeptidase inhibitor and/or Thiorphan, an enkephalinase inhibitor. High-performance liquid chromatographic analysis of the cholecystokinin immunoreactivity recovered in medium in the absence of any inhibitor showed cholecystokinin-8 to be the major peak, representing 8% of the released immunoreactive material. Non-sulphated cholecystokinin-8 represented less than 1%, indicating that desulphation does not constitute a major inactivation pathway for the endogenous octapeptide. Cholecystokinin-5 was the major clearly identifiable immunoreactive fragment, representing 9% of released immunoreactivity in the absence of inhibitors. Its formation was decreased by about 50% in the presence of either diisopropyl-fluorophosphate or bestatin and Thiorphan and abolished when they were associated, suggesting that it resulted from the actions of a serine peptidase(s) and an aminopeptidase(s). Cholecystokinin-6 (or cholecystokinin-7) was less abundant, representing 4% of the released immunoreactivity, and its level was augmented in the presence of diisopropyl-fluorophosphate. Hence a serine endopeptidase cleaving the Met3-Gly4 bond of cholecystokinin-8 may represent a major inactivating peptidase for the endogenous neuropeptide. Additional metabolic pathways not blocked by serine peptidase inhibitors and resulting in the formation of cholecystokinin-6 (or cholecystokinin-7) and, possibly, cholecystokinin-4, are also suggested by the present approach.
大鼠大脑皮层切片去极化释放的内源性胆囊收缩素免疫反应性在到达孵育培养基之前会经历广泛降解（释放的免疫反应性的85%）。为了鉴定起作用的肽酶，测试了四大催化类别的大量抑制剂的保护作用。金属肽酶抑制剂（苯丁抑制素、氨肽酶抑制剂、嘌呤霉素、硫醇苯丙氨酸、卡托普利、邻菲罗啉）、巯基肽酶抑制剂（亮抑酶肽、抑肽酶、对羟基汞苯甲酸）或羧基肽酶抑制剂（胃蛋白酶抑制剂）通常即使有保护作用也很弱。相比之下，几种丝氨酸肽酶抑制剂，即二异丙基氟磷酸酯、苯甲基磺酰氟或氯甲基酮丙氨酸-丙氨酸-脯氨酸-缬氨酸-CH2Cl，使胆囊收缩素免疫反应性的回收率提高了一倍，并且在氨基肽酶抑制剂苯丁抑制素和/或脑啡肽酶抑制剂硫醇苯丙氨酸共同存在时效果增强。在没有任何抑制剂的情况下，对培养基中回收的胆囊收缩素免疫反应性进行高效液相色谱分析表明，胆囊收缩素-8是主要峰，占释放的免疫反应性物质的8%。非硫酸化的胆囊收缩素-8占比不到1%，这表明脱硫不是内源性八肽的主要失活途径。胆囊收缩素-5是主要的可明确识别的免疫反应性片段，在没有抑制剂的情况下占释放的免疫反应性的9%。在二异丙基氟磷酸酯或苯丁抑制素和硫醇苯丙氨酸存在时，其形成减少约50%，当它们联合使用时则被消除，这表明它是由一种丝氨酸肽酶和一种氨基肽酶的作用产生的。胆囊收缩素-6（或胆囊收缩素-7）含量较少，占释放的免疫反应性的4%，并且在二异丙基氟磷酸酯存在时其水平升高。因此，切割胆囊收缩素-8的Met3-Gly4键的丝氨酸内肽酶可能是内源性神经肽的主要失活肽酶。本研究方法还提示了未被丝氨酸肽酶抑制剂阻断且导致胆囊收缩素-6（或胆囊收缩素-7）以及可能的胆囊收缩素-4形成的其他代谢途径。
