Browning Kirsteen N, Kalyuzhny Alexander E, Travagli R Alberto
Department of Internal Medicine-Gastroenterology, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
J Neurosci. 2004 Aug 18;24(33):7344-52. doi: 10.1523/JNEUROSCI.1676-04.2004.
Whole-cell recordings were made from identified gastric-projecting rat dorsal motor nucleus of the vagus (DMV) neurons. The amplitude of evoked IPSCs (eIPSCs) was unaffected by perfusion with met-enkephalin (ME) or by mu-, delta-, or kappa-opioid receptor selective agonists, namely D-Ala2-N-Me-Phe4-Glycol5-enkephalin (DAMGO), cyclic [D-Pen2-D-Pen5]-enkephalin, or trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolytinil)-cyclohexyl]-benzeneacetamide methane sulfonate (U50,488), respectively. Brief incubation with the adenylate cyclase activator forskolin or the nonhydrolysable cAMP analog 8-bromo-cAMP, thyrotropin releasing hormone, or cholecystokinin revealed the ability of ME and DAMGO to inhibit IPSC amplitude; this inhibition was prevented by pretreatment with the mu-opioid receptor (MOR1) selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2. Conversely, incubation with the adenylate cyclase inhibitor dideoxyadenosine, with the protein kinase A (PKA) inhibitor N-[2-(p-Bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), or with the Golgi-disturbing agent brefeldin A, blocked the ability of forskolin to facilitate the inhibitory actions of ME. Immunocytochemical experiments revealed that under control conditions, MOR1 immunoreactivity (MOR1-IR) was colocalized with glutamic acid decarboxylase (GAD)-IR in profiles apposing DMV neurons only after stimulation of the cAMP-PKA pathway. Pretreatment with H89 or brefeldin A or incubation at 4 degrees C prevented the forskolin-mediated insertion of MOR1 on GAD-IR-positive profiles. These results suggest that the cAMP-PKA pathway regulates trafficking of mu-opioid receptors into the cell surface of GABAergic nerve terminals. By consequence, the inhibitory actions of opioid peptides in the dorsal vagal complex may depend on the state of activation of brainstem vagal circuits.
采用全细胞膜片钳记录法，对已鉴定的大鼠迷走神经背核（DMV）中投射至胃的神经元进行记录。脑啡肽（ME）灌流或μ-、δ-或κ-阿片受体选择性激动剂（即D-Ala2-N-Me-Phe4-Glycol5-脑啡肽（DAMGO）、环[D-Pen2-D-Pen5]-脑啡肽或反式-3,4-二氯-N-甲基-N-[2-(1-吡咯烷基)-环己基]-苯乙酰胺甲磺酸盐（U50,488））均未影响诱发的抑制性突触后电流（eIPSCs）的幅度。用腺苷酸环化酶激活剂毛喉素、不可水解的环磷酸腺苷（cAMP）类似物8-溴-cAMP、促甲状腺激素释放激素或胆囊收缩素短暂孵育后，发现ME和DAMGO具有抑制IPSC幅度的能力；μ-阿片受体（MOR1）选择性拮抗剂D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2预处理可阻止这种抑制作用。相反，用腺苷酸环化酶抑制剂双脱氧腺苷、蛋白激酶A（PKA）抑制剂N-[2-(对溴肉桂酰胺基)乙基]-5-异喹啉磺酰胺二盐酸盐（H89）或高尔基体干扰剂布雷菲德菌素A孵育，可阻断毛喉素促进ME抑制作用的能力。免疫细胞化学实验显示，在对照条件下，仅在cAMP-PKA途径激活后，MOR1免疫反应性（MOR1-IR）才与谷氨酸脱羧酶（GAD）-IR在与DMV神经元相对的结构中共定位。H89或布雷菲德菌素A预处理或在4℃孵育可阻止毛喉素介导的MOR1插入GAD-IR阳性结构中。这些结果表明，cAMP-PKA途径调节μ-阿片受体向GABA能神经末梢细胞表面的转运。因此，阿片肽在迷走神经背复合体中的抑制作用可能取决于脑干迷走神经回路的激活状态。
