Interactions of cholecystokinin and mu opioid receptors

在本研究中，我们将编码CCK-B受体的cDNA共同注射到非洲爪哇卵母细胞中作为模型系统，在单细胞水平上检测受体拮抗作用。我们早期的工作表明，激活CCK-B受体会导致自身显着的脱敏，也会导致Mu阿片受体的交叉脱敏。假设CCK-B受体的脱敏剂是一种蛋白激酶，因此我们测试了七种激酶抑制剂。卵母细胞在这些抑制剂中孵育不同的时间，以确定它们是否对CCK-B受体或其下游效应物的脱敏恢复有任何影响。总共检测了95个卵母细胞。这些抑制剂及其靶点是星形孢子素(非特异性蛋白激酶C抑制剂)、氨基丙戊醇(CDK1和CDK2)、SB 203580(SAPK2a/p38 MAP激酶)、SB202190(p38 MAP激酶)、PD 98059(MEK)、SP 600125(JNK)和U0126(MAPK级联抑制因子)。其中，U0126和SB203580显然没有效果，但其他药物似乎阻止了脱敏(统计分析仍在进行中)。 第二种研究Mu阿片类药物和CCK-B受体相互作用的方法使用了从大鼠幼鼠分离的锥体神经元中表达的天然受体。来自海马区的大鼠大脑活切片被一个小的振动玻璃探头分离成带有一些连接的突起的神经元胞体。由于没有酶用于解离，突触前元件的片段仍然附着在一起，通过自发的突触电流宣布自己，我们可以在全细胞膜片钳条件下记录到这种电流。我们已经对这些严重分离的神经元进行了广泛的研究，并证明了突触前片段同时表达CCK-B和MU阿片受体。初步实验表明，CCK-B激动剂CCK_8-S使抑制性突触后电流(IPSCs)的速度减慢，幅度降低。由激动剂去甲吗啡(20微摩尔)激活的Mu阿片受体也减慢了速度，但似乎对波幅没有影响。这些实验证明，这两种受体都可以存在于突触前片段中，下一步是确定它们是否或如何相互作用。

In the present study, we co-injected cDNA encoding the CCK-B receptor into Xenopus oocytes as a model system to examine receptor antagonism at the single cell level. Our earlier work has shown that activating the CCK-B receptor causes marked desensitization of itself and also a cross desensitization of the mu opioid receptor. Hypothesizing that the agent of desensitization of the CCK-B receptor was a protein kinase, we therefore tested seven kinase inhibitors. Oocytes were incubated in these inhibitors for different times to determine if they had any influence over the recovery from desensitization of the CCK-B receptor or its downstream effectors. A total of 95 oocytes was tested. The inhibitors and their targets were staurosporine (nonspecific PKC inhibitor), aminopurvalanol (CDK1 and CDK2), SB 203580 (SAPK2a/p38 MAP kinase), SB202190 (p38 MAP kinase), PD 98059 (MEK), SP 600125 (JNK), and U0126 (MAPK cascade suppressor). Of these, U0126 and SB203580 clearly had no effect, but the other drugs seemed to block the desensitization (statistical analysis is still in progress). The second approach to examining the interactions between the mu opioid and CCK- B receptors uses native receptors expressed in isolated pyramidal neurons obtained from rat pups. Living slices of rat brain from the hippocampus region are dissociated by a small vibrating glass probe into neuronal cell bodies with some attached processes. Because no enzymes are used in the dissociation, fragments of presynaptic elements remain attached that announce themselves by spontaneous synaptic currents that we can record under whole cell patch clamp conditions. We have explored these acutely dissociated neurons extensively and have demonstrated that pre-synaptic fragments express both CCK-B and mu opioid receptors. Preliminary experiments show that the CCK-B agonist CCK8-S slows the rate of inhibitory post synaptic currents (IPSCs) and diminishes their amplitudes. The mu opioid receptor, activated by the agonist normorphine (20 micro molar), also slows the rate but seems to have no effect on amplitudes. These experiments demonstrate that both receptors can be present in the presynaptic fragments, and the next step is to determine if or how they interact.