Mu Opioid Receptor Regulation in Neonatal Brainstem

描述（由申请人提供）：本研究的长期目标是了解子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）的调节和功能的机制。呼吸抑制是由内源性阿片肽和外源性阿片肽激活MOR引起的。这些影响的一个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孕产妇滥用阿片类药物的一个有充分证据的后果。这些新生儿表现出戒断性过度通气和婴儿猝死综合征（SIDS）的发生率增加。这些研究对于了解正常的呼吸发育、药物引起的变化以及对怀孕海洛因依赖者和美沙酮维持患者的有效治疗至关重要。NTS MOR在新生儿先天性麻醉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好模型。虽然已经克隆了豚鼠kappa阿片受体，但目前只获得了部分MOR和delta阿片受体的序列。然而，我们的实验室最近确定了完整的豚鼠MOR cDNA序列。该序列的可用性将使我们能够首次确定豚鼠MOR的药理学，并系统地将其与人类和小鼠MOR进行比较。研究基于以下四个假设：1)豚鼠MOR在mu激动剂功效、结合动力学和g蛋白活化方面与人类MOR功能相似，但与小鼠MOR不同；2)美沙酮诱导新生豚鼠呼吸抑制，与吗啡有同等作用，但持续时间较长；3)子宫内慢性吗啡和美沙酮暴露导致细胞表面功能性MOR升高，但降低了MOR与NTS中g蛋白的偶联效率；4)子宫内慢性吗啡和美沙酮暴露上调NTS中MOR mRNA。假设通过四个具体目的进行探讨：1)比较mu激动剂的选择性和效力，以及对稳定转染CHO细胞中表达的豚鼠、人和小鼠MOR的细胞耐受性的发展；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豚鼠新生儿脑干NTS区MOR的影响；4)在子宫内暴露于吗啡和美沙酮的豚鼠新生儿NTS中MOR mRNA的定量表达。这些研究将提供豚鼠NTS MOR在子宫内慢性阿片类药物暴露后的发育信息。

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.