Li Z W, Bijl W A, van Nispen J W, Brendel K, Davis T P
Department of Pharmacology, University of Arizona, Tucson.
J Pharmacol Exp Ther. 1990 May;253(2):851-7.
The central enzymatic stability of des-enkephalin-gamma-endorphin and its synthetic analogs [cycloN alpha 6, C delta 11]beta-endorphin-[6-17] and [Pro7, Lys(Ac)9]-beta-endorphin[6-17] was studied in vitro using a newly developed, regionally dissected rat brain slice, time course incubation procedure. Tissue slice viability was estimated as the ability of the brain slice to take up or release gamma-[3H]aminobutyric acid after high K+ stimulation. Results demonstrated stability of uptake/release up to 5 hr of incubation, suggesting tissue viability over this period. The estimated half-life of peptides based on the results obtained in our incubation protocol suggest that the peptides studied are metabolized at different rates in the individual brain regions tested. A good correlation exists between the high enzyme activity of neutral endopeptidase (EC 3.4.24.11) and the rapid degradation of des-enkephalin-gamma-endorphin and [cycloN alpha 6, C delata 11]beta-endorphin-[6-17] in caudate putamen. Proline substitution combined with lysine acetylation appears to improve resistance to enzymatic metabolism in caudate putamen and hypothalamus. However, cyclization of des-enkephalin-gamma-endorphin forming an amide bond between the alpha-NH2 of the N-terminal threonine and the gamma-COOH of glutamic acid did not improve peptide stability in any brain region tested. The present study has shown that the brain slice technique is a valid and unique approach to study neuropeptide metabolism in small, discrete regions of rat brain where peptides, peptidases and receptors are colocalized and that specific structural modifications can improve peptide stability.
使用新开发的、区域解剖的大鼠脑片时间进程孵育程序，在体外研究了去脑啡肽 - γ - 内啡肽及其合成类似物[环Nα6，Cδ11]β - 内啡肽 - [6 - 17]和[Pro7，Lys(Ac)9] - β - 内啡肽 - [6 - 17]的核心酶稳定性。通过高钾刺激后脑片摄取或释放γ - [3H]氨基丁酸的能力来评估组织切片的活力。结果表明，在长达5小时的孵育过程中摄取/释放稳定，表明在此期间组织具有活力。根据我们的孵育方案获得的结果估计的肽半衰期表明，所研究的肽在测试的各个脑区以不同速率代谢。在尾状核壳核中，中性内肽酶（EC 3.4.24.11）的高酶活性与去脑啡肽 - γ - 内啡肽和[环Nα6，Cδ11]β - 内啡肽 - [6 - 17]的快速降解之间存在良好的相关性。脯氨酸取代与赖氨酸乙酰化相结合似乎提高了尾状核壳核和下丘脑对酶代谢的抗性。然而，去脑啡肽 - γ - 内啡肽的环化，即在N端苏氨酸的α - NH2和谷氨酸的γ - COOH之间形成酰胺键，在任何测试的脑区中都没有提高肽的稳定性。本研究表明，脑片技术是一种有效且独特的方法，可用于研究大鼠脑中小的离散区域的神经肽代谢，在这些区域中肽、肽酶和受体共定位，并且特定的结构修饰可以提高肽的稳定性。
