Mu Opioid Receptor Regulation in Neonatal Brainstem

描述(申请人提供)：拟议研究的长期目标是了解宫内慢性吗啡和美沙酮暴露影响新生儿脑干呼吸控制区MU阿片受体(MOR)调节和功能的机制。呼吸抑制是由激活MOR的内源性阿片肽和外源性阿片肽诱导的。这些效应的关键脑干部位是孤束核(NTS)，它整合感觉信号并驱动呼吸肌。新生儿呼吸严重障碍是母亲滥用阿片类药物的一个有据可查的后果。这些新生儿表现出停药、过度换气和婴儿猝死综合征(SID)发生率增加。拟议的研究对于了解正常的呼吸道发育、药物诱导的变化以及对怀孕的海洛因成瘾者和美沙酮维持患者的有效治疗至关重要。NTS MOR在新生儿先天性毒品依赖和这些呼吸障碍中的确切作用尚不清楚。由于解剖学、生理学和药理学的原因，豚鼠是研究母体阿片类药物滥用的极佳模型。豚鼠Kappa阿片受体已被克隆，但目前只有MOR和Delta阿片受体的部分序列可用。然而，我们的实验室最近已经确定了豚鼠MOR的完整cDNA序列。该序列的可获得性将使我们能够首次定义豚鼠的MOR药理，并系统地将其与人和小鼠的MOR进行比较。这项研究以四个假设为指导：1)豚鼠MOR在功能上与人MOR相似，但在MU激动剂效力、结合动力学和G蛋白激活方面与小鼠MOR不同；2)美沙酮在新生豚鼠引起呼吸抑制，与吗啡相同，但持续时间长于吗啡；3)宫内长期暴露吗啡和美沙酮可导致细胞表面功能性MOR增加，但降低了MOR与NTS中G蛋白的偶联效率；4)宫内长期暴露吗啡和美沙酮可上调NTS中MOR mRNA的表达。通过四个特定的目的探索假说：1)比较MU激动剂的选择性和效力，以及在稳定表达的CHO细胞中表达的豚鼠、人和小鼠的细胞耐受性；2)证明美沙酮对新生豚鼠的呼吸作用与吗啡相似；3)研究吗啡和美沙酮对宫内暴露的豚鼠新生鼠脑干NTS中MOR的影响；以及4)对宫内暴露于吗啡和美沙酮的豚鼠新生NTS中MOR的mRNA进行定量。这些研究将提供慢性宫内阿片类药物暴露后豚鼠NTS MOR的发育信息。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand mechanisms by which chronic in utero morphine and methadone exposure affect regulation and function of mu opioid receptors (MOR) in respiratory control areas of newborn brainstem. Respiratory depression is induced by endogenous opioid peptides and exogenous opioids that activate MOR. A critical brainstem site for these effects is the nucleus tractus solitarius (NTS), which integrates sensory signals and drives respiratory muscles. Profound disturbance of neonatal breathing is a well-documented consequence of maternal opioid abuse. These neonates exhibit withdrawal hyperventilation and an increased incidence of sudden infant death syndrome (SIDS). The proposed studies are essential for understanding normal respiratory development, drug-induced changes, and effective treatment of pregnant heroin addicts and methadone maintained patients. The exact role of NTS MOR in neonatal congenital narcotic dependence and these respiratory disturbances is unknown. For anatomical, physiological, and pharmacological reasons, the guinea pig is a superb model for study of maternal opioid abuse. Guinea pig kappa opioid receptor has been cloned, but only partial sequences of MOR and delta opioid receptors have been available. However, our laboratory has recently determined the complete guinea pig MOR cDNA sequence. Availability of this sequence will enable us to define for the first time guinea pig MOR pharmacology, and systematically compare it to human and mouse MOR. Research is guided by four hypotheses: 1) guinea pig MOR is functionally similar to human MOR with respect to mu agonist efficacy, binding kinetics, and activation of G-protein, but different from murine MOR; 2) methadone induces respiratory depression and is equipotent to, but of longer duration than morphine in the neonatal guinea pig; 3) chronic in utero morphine and methadone exposure results in increased functional MOR on the cell surface, but decreases the coupling efficiency of MOR with G-proteins in the NTS; 4) chronic in utero morphine and methadone exposure up-regulates MOR mRNA in the NTS. Hypotheses are explored through four specific aims: 1) to compare mu agonist selectivity and potency, and development of cellular tolerance for guinea pig, human and murine MOR each expressed in stably transfected CHO cells; 2) to prove that the respiratory effects of methadone are similar to morphine in the neonatal guinea pig; 3) to study the effects of morphine and methadone on guinea pig MOR in NTS of brainstem sections from guinea pig neonates exposed in utero; and 4) to quantitate MOR mRNA in NTS from guinea pig neonates exposed to morphine and methadone in utero. These studies will provide developmental information on guinea pig NTS MOR following chronic in utero opioid exposure.