Interactions of cholecystokinin and mu opioid receptors

在本研究中，我们共注射编码CCK-B受体的cDNA到非洲爪蟾卵母细胞作为模型系统，以检查受体拮抗作用在单细胞水平。 我们早期的工作已经表明，激活CCK-B受体会导致其自身的显著脱敏，也会导致μ阿片受体的交叉脱敏。 假设CCK-B受体脱敏剂是一种蛋白激酶，因此我们测试了7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。 共检测了95个卵母细胞。 抑制剂及其靶点为staurosporine（非特异性PKC抑制剂）、氨基普伐他汀（CDK 1和CDK 2）、SB 203580（SAPK 2a/p38 MAP激酶）、SB 202190（p38 MAP激酶）、PD 98059（MEK）、SP 600125（JNK）和U 0126（MAPK级联抑制剂）。 其中，U 0126和SB 203580显然没有效果，但其他药物似乎阻断了脱敏作用（统计分析仍在进行中）。 检查μ阿片样物质和CCK- B受体之间相互作用的第二种方法使用在从大鼠幼仔获得的分离的锥体神经元中表达的天然受体。 用振动玻璃探针将大鼠海马区的活体脑片分离成带有突起的神经元细胞体。 因为在解离过程中没有使用酶，所以突触前元件的片段仍然附着在一起，通过我们可以在全细胞膜片钳条件下记录的自发突触电流来宣布自己。 我们已经广泛地研究了这些急性分离的神经元，并证明突触前片段表达CCK-B和μ阿片受体。 初步实验表明，CCK-B激动剂CCK 8-S减慢抑制性突触后电流（IPSC）的速率并降低其幅度。μ阿片受体，由激动剂去甲吗啡（20微摩尔）激活，也减慢了速率，但似乎对振幅没有影响。 这些实验表明，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.