Richards M, van Giersbergen P, Zimmermann A, Lesur B, Hoflack J
Marion Merrell Research Institute, Strasbourg, France.
Biochem Pharmacol. 1997 Oct 1;54(7):825-32. doi: 10.1016/s0006-2952(97)00258-x.
Activation of endogenous neurotensin (NT) receptors and P2-purinoceptors expressed by human colonic adenocarcinoma HT-29 cells increased extracellular acidification rates that were detected in the microphysiometer. NT (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu), NT[8-13] (Arg-Arg-Pro-Tyr-Ile-Leu), NT[9-13] (Arg-Pro-Tyr-Ile-Leu), and NT1 (N alpha methyl-Arg-Lys-Pro-Trp-Tle-Leu [Tle = tert-leucine]) were full agonists, whereas XL 775 (N-[N-[2-[3-[[6-amino-1-oxo-2-[[(phenylmethoxy)carbonyl]-amino]hex yl]amino]phenyl]-3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-L-isoleucyl]-L-le ucine) was a partial agonist for activating NT receptors expressed by HT-29 cells. Desensitization induced by NT was rapid and monophasic with 85% of the initial response lost by a 30-s exposure. Once initiated, the rate and extent of desensitization were similar for different concentrations of a given agonist, for agonists of different potencies, and for agonists of different efficacies, which suggests that desensitization may be independent of receptor occupancy or agonist efficacy. Resensitization was a much slower process, requiring 60 min before the full agonist response to NT was recovered. ATP, via P2-purinoceptors, also activated cellular acidification rates in a concentration-dependent manner. ATP induced a biphasic desensitization of purinoceptors with a loss of ca. 50% of the initial stimulation detectable between 30 and 90 s of exposure to the agonist. Desensitization of NT receptors did not influence the activation of P2-purinoceptors by ATP, suggesting there was no heterologous desensitization between the two types of receptors. Superfusion with NT receptor agonists for 15 min at concentrations that did not elicit changes in extracellular acidification rates blocked, in a concentration-dependent manner, the agonist response induced by 100 nM NT. This may reflect sequestration of the receptor. These results suggest that the high agonist affinity state of NT receptors may modulate receptor sequestration, whereas activation of the low agonist affinity state may be linked to cellular metabolism. Comparison of our results with published data found differences as well as similarities of NT responses among three lines of HT-29 cells.
人结肠腺癌HT - 29细胞表达的内源性神经降压素（NT）受体和P2 - 嘌呤受体的激活增加了在微生理仪中检测到的细胞外酸化速率。NT（焦谷氨酸 - 亮氨酸 - 酪氨酸 - 谷氨酸 - 天冬酰胺 - 赖氨酸 - 脯氨酸 - 精氨酸 - 精氨酸 - 脯氨酸 - 酪氨酸 - 异亮氨酸 - 亮氨酸）、NT[8 - 13]（精氨酸 - 精氨酸 - 脯氨酸 - 酪氨酸 - 异亮氨酸 - 亮氨酸）、NT[9 - 13]（精氨酸 - 脯氨酸 - 酪氨酸 - 异亮氨酸 - 亮氨酸）和NT1（Nα - 甲基 - 精氨酸 - 赖氨酸 - 脯氨酸 - 色氨酸 - 叔亮氨酸 - 亮氨酸）是完全激动剂，而XL 775（N - [N - [2 - [3 - [[6 - 氨基 - 1 - 氧代 - 2 - [[(苄氧基)羰基] - 氨基]己基]氨基]苯基] - 3 - (4 - 羟基苯基) - 1 - 氧代 - 2 - 丙烯基] - L - 异亮氨酰] - L - 亮氨酸）是激活HT - 29细胞表达的NT受体的部分激动剂。NT诱导的脱敏迅速且呈单相，30秒暴露后初始反应丧失85%。一旦开始，对于给定激动剂的不同浓度、不同效力的激动剂以及不同效能的激动剂，脱敏的速率和程度相似，这表明脱敏可能与受体占有率或激动剂效能无关。再敏化是一个慢得多的过程，在对NT的完全激动剂反应恢复之前需要60分钟。ATP通过P2 - 嘌呤受体也以浓度依赖的方式激活细胞酸化速率。ATP诱导嘌呤受体的双相脱敏，在暴露于激动剂30至90秒之间可检测到初始刺激丧失约50%。NT受体的脱敏不影响ATP对P2 - 嘌呤受体的激活，表明两种类型的受体之间不存在异源脱敏。以不引起细胞外酸化速率变化的浓度用NT受体激动剂灌注15分钟，以浓度依赖的方式阻断了100 nM NT诱导的激动剂反应。这可能反映了受体的隔离。这些结果表明，NT受体的高激动剂亲和力状态可能调节受体隔离，而低激动剂亲和力状态的激活可能与细胞代谢有关。将我们的结果与已发表的数据进行比较，发现三株HT - 29细胞系之间NT反应既有差异也有相似之处。
