Characterization of 5-HT1A receptor-mediated [35S]GTPgammaS binding in rat hippocampal membranes.
Characterization of 5-HT1A receptor-mediated [35S]GTPgammaS binding in rat hippocampal membranes.
复制标题
大鼠海马膜中 5-HT1A 受体介导的 [35S]GTPgammaS 结合的表征。
DOI：
10.1016/s0014-2999(97)01547-1
复制
发表时间：
1998
影响因子：
5
通讯作者：
Nelson,DL
中科院分区：
文献类型：
--
作者：
Alper,RH;Nelson,DL
在大鼠海马膜中表征了血清素（5-羟色胺，5-HT）受体配体对[35 S]GTPγS结合的刺激。优化的试验含有30-50 μg蛋白、300 μM GDP和0.1 nM [35 S]GTPγS，在37°C下孵育20 min。在10 μM时，5-HT 1A受体激动剂R（+）-8-羟基-2-（二正丙基氨基）四氢萘[R（+）-8-OH-DPAT]刺激GTPγS结合，从27.1±2.5 fmol/mg蛋白增加到45.7±4.2 fmol/mg蛋白。增加蛋白浓度不影响基础和最大GTPγS结合之间的绝对差异，也不影响EC 50，但降低刺激百分比。与R（+）-8-OH-DPAT相比，非选择性激动剂5-羟色胺和5-羧酰胺色胺的有效性高30-35%，而部分激动剂丁螺环酮和S（−）-8-羟基-2-（二正丙基氨基）四氢萘分别刺激GTPγS结合19± 1%和43± 3%。5-HT 2受体激动剂[（±）1-（2，5-二甲氧基-4-碘苯基）-2-氨基丙烷盐酸盐]（DOI）和5-HT 1A受体拮抗剂WAY 100，635（n-[2-[4-（2-甲氧基苯基）-1-哌嗪基]乙基]-n-（2-吡啶基）环己烷甲酰胺三盐酸盐）和螺哌隆均未改变基础GTPγS结合。WAY 100，635消除了R（+）-8-OH-DPAT的作用，但仅使5-羟色胺的作用降低了88± 3%。最后，甲硫替平拮抗R（+）-8-OH-DPAT刺激的GTPγS结合，并降低其自身的基础GTPγS结合。减少不受100，635号公路的影响。本研究通过测定激动剂诱导的[35 S]GTPγS结合来评价大鼠海马膜5-HT 1A受体的功能活性。
Stimulation of [35S ]GTPγS binding by serotonin (5-hydroxytryptamine, 5-HT) receptor ligands was characterized in rat hippocampal membranes. The optimized assay contained 30–50 μg protein, 300 μM GDP and 0.1 nM [35S ]GTPγS, incubated at 37°C for 20 min. At 10 μM, the 5-HT1Areceptor agonist R(+)-8-hydroxy-2-(di-n-propylamino)tetralin [R(+)-8-OH-DPAT] stimulated GTPγS binding from 27.1±2.5 to 45.7±4.2 fmol/mg protein. Increasing the protein concentration did not affect the absolute difference between basal and maximal GTPγS binding nor the EC50, but decreased the percent stimulation. The non-selective agonists serotonin and 5-carboxamidotryptamine were 30–35% more efficacious, whereas the partial agonists buspirone and S(−)-8-hydroxy-2-(di-n-propylamino)tetralin stimulated GTPγS binding by 19±1 and 43±3%, respectively, compared to R(+)-8-OH-DPAT. Neither the 5-HT2receptor agonist [(±)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl] (DOI) nor the 5-HT1Areceptor antagonists WAY 100,635 (n-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-n-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride) and spiperone altered basal GTPγS binding. WAY 100,635 abolished the effect of R(+)-8-OH-DPAT, but only reduced the effect of serotonin by 88±3%. Finally, methiothepin antagonized R(+)-8-OH-DPAT-stimulated GTPγS binding and reduced basal GTPγS binding by itself. The reduction was not affected by WAY 100,635. We have characterized a method to assess functional activity at 5-HT1Areceptors in rat hippocampal membranes by measuring agonist-induced [35S ]GTPγS binding.