Jayakar Selwyn S, Pugh Phyllis C, Dale Zack, Starr Eric R, Cole Samantha, Margiotta Joseph F
Department of Neurosciences, University of Toledo, College of Medicine and Life Sciences, United States.
Mol Cell Neurosci. 2014 Nov;63:1-12. doi: 10.1016/j.mcn.2014.08.007. Epub 2014 Aug 25.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide found at synapses throughout the central and autonomic nervous system. We previously found that PACAP engages a selective G-protein coupled receptor (PAC1R) on ciliary ganglion neurons to rapidly enhance quantal acetylcholine (ACh) release from presynaptic terminals via neuronal nitric oxide synthase (NOS1) and cyclic AMP/protein kinase A (PKA) dependent processes. Here, we examined how PACAP stimulates NO production and targets resultant outcomes to synapses. Scavenging extracellular NO blocked PACAP-induced plasticity supporting a retrograde (post- to presynaptic) NO action on ACh release. Live-cell imaging revealed that PACAP stimulates NO production by mechanisms requiring NOS1, PKA and Ca(2+) influx. Ca(2+)-permeable nicotinic ACh receptors composed of α7 subunits (α7-nAChRs) are potentiated by PKA-dependent PACAP/PAC1R signaling and were required for PACAP-induced NO production and synaptic plasticity since both outcomes were drastically reduced following their selective inhibition. Co-precipitation experiments showed that NOS1 associates with α7-nAChRs, many of which are perisynaptic, as well as with heteromeric α3*-nAChRs that generate the bulk of synaptic activity. NOS1-nAChR physical association could facilitate NO production at perisynaptic and adjacent postsynaptic sites to enhance focal ACh release from juxtaposed presynaptic terminals. The synaptic outcomes of PACAP/PAC1R signaling are localized by PKA anchoring proteins (AKAPs). PKA regulatory-subunit overlay assays identified five AKAPs in ganglion lysates, including a prominent neuronal subtype. Moreover, PACAP-induced synaptic plasticity was selectively blocked when PKA regulatory-subunit binding to AKAPs was inhibited. Taken together, our findings indicate that PACAP/PAC1R signaling coordinates nAChR, NOS1 and AKAP activities to induce targeted, retrograde plasticity at autonomic synapses. Such coordination has broad relevance for understanding the control of autonomic synapses and consequent visceral functions.
垂体腺苷酸环化酶激活多肽（PACAP）是一种多效性神经肽，存在于整个中枢神经系统和自主神经系统的突触中。我们之前发现，PACAP与睫状神经节神经元上的一种选择性G蛋白偶联受体（PAC1R）结合，通过神经元型一氧化氮合酶（NOS1）以及环磷酸腺苷/蛋白激酶A（PKA）依赖性过程，快速增强突触前终末的量子化乙酰胆碱（ACh）释放。在此，我们研究了PACAP如何刺激一氧化氮生成并将产生的结果靶向至突触。清除细胞外一氧化氮可阻断PACAP诱导的可塑性，这支持了一氧化氮对ACh释放的逆行（突触后至突触前）作用。活细胞成像显示，PACAP通过需要NOS1、PKA和Ca²⁺内流的机制刺激一氧化氮生成。由α7亚基组成的Ca²⁺通透型烟碱型ACh受体（α7-nAChRs）通过PKA依赖性的PACAP/PAC1R信号传导而增强，并且是PACAP诱导的一氧化氮生成和突触可塑性所必需的，因为在它们被选择性抑制后，这两种结果都大幅降低。共沉淀实验表明，NOS1与许多位于突触周围的α7-nAChRs以及产生大部分突触活动的异源α3* - nAChRs相关联。NOS1与nAChR的物理结合可促进突触周围和相邻突触后位点的一氧化氮生成，以增强并列突触前终末的局部ACh释放。PACAP/PAC1R信号传导的突触结果由PKA锚定蛋白（AKAPs）定位。PKA调节亚基覆盖分析在神经节裂解物中鉴定出五种AKAPs，包括一种突出的神经元亚型。此外，当PKA调节亚基与AKAPs的结合被抑制时，PACAP诱导的突触可塑性被选择性阻断。综上所述，我们的研究结果表明，PACAP/PAC1R信号传导协调nAChR、NOS1和AKAP的活性，以在自主突触处诱导靶向性的逆行可塑性。这种协调对于理解自主突触的控制以及随之而来的内脏功能具有广泛的相关性。
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