PURINERGIC REGULATION OF ENTERIC NEURAL REFLEXES

修订拨款的总体目标是确定P1和P2嘌呤受体在神经反射中的作用。四个基本假设被检验：1.内源性腺苷作用于神经A1Rs以抑制蠕动反射。(I)体内运动是用应变计记录对电刺激、扩张或药物的反应。空肠血供分为肛门室、感觉室和口腔室，以测试腺苷对上行收缩和下行松弛的影响。(Ii)对推进的影响是通过人造弹丸的繁殖速度来量化的。(3)神经元A1R和A2bR在CM运动神经元上是否参与腺苷的作用：(A)记录S/Dil标记的CM运动神经元；(B)使用双室系统通过CM扩张释放血管活性肠肽；(C)定位A1/A2bR免疫反应-Dil标记的CM运动神经元，共同标记上行/下行CM运动神经元的递质。2.内源性腺苷通过作用于与cAMP通路相关的A1Rs，抑制初级传入AH神经元的慢突触传递。(1)粘膜应用5-羟色胺可引起肌间AH细胞在周缘部位出现慢的EPSP。这是用来测试腺苷/三磷酸腺苷对反射诱发的慢EPSP的影响。(Ii)FICRhR/cAMP激光共聚焦成像将评估内源性腺苷激活A1R是否通过降低cAMP水平来抑制AH细胞中缓慢的突触传递--对慢EPSP模拟物或慢EPSP的反应进行了研究。3.三磷酸腺苷可激活P2Y2(或P2Y4)嘌呤受体，使[Ca~(2+)]i升高，从而引起AH神经元的缓慢超极化。AH细胞中的钙激光共聚焦成像用于研究(I)P2Y受体药理或(Ii)通过G蛋白、磷脂酶C、CICR或cADPR途径作用的药物的信号转导。4.内源性腺苷通过作用于粘膜下传入AH神经元的A1Rs抑制5-羟色胺能反射。(I)在带有粘膜岛的分离的粘膜下层，测试药物对对N_2喷雾或5-羟色胺反应的AH细胞反射激活的影响。(2)SP、CGRP或DIL标记神经生物素阳性神经元。(Iii)通透室研究将评估反射诱发的ISC反应是否涉及ATP。整合这些信息提供了对嘌呤能受体在肠神经反射和运动中传入和运动神经元的作用的新的见解。

The overall aim of the revised grant is to determine the role of P1 and P2 purinoceptors in neural reflexes. Four general hypotheses are tested: 1. Endogenous adenosine acts at neural A1Rs to suppress the peristaltic reflex. (i) In vivo motility is recorded with strain-gauges in response to electrical stimulation, distension or drugs. Jejunal blood supply is separated into anal, sensory and oral chambers for testing effects of adenosine (i.a.infusion) on ascending contraction and descending relaxation. (ii)Effects on propulsion are quantitated by velocity of propagation of artificial pellets. (iii) Whether neural A1R and A2bRs on CM motor neurons contribute to effects of adenosine is tested by:(a)recording from S/Dil labeled CM motor neurons; (b)using a 2-chamber system for VIP release by CM distension; (c)mapping of A1/A2bR immunoreactive - Dil labeled CM motor neurons, co-labeled for transmitters of ascending/descending CM motor neurons. 2. Endogenous adenosine inhibits slow synaptic transmission in primary afferent AH neurons by acting at A1Rs linked to a cAMP pathway. (i) Mucosal application of 5-HT elicits a slow EPSP in myenteric AH cells at circumferential sites. This is used to test effects of adenosine/ATP on reflex-evoked slow EPSPs. (ii) FICRhR/cAMP-Laser confocal imaging would assess if A1R activation by endogenous adenosine inhibits slow synaptic transmission in AH cells by reducing cAMP levels-Responses to slow EPSP-mimetics or slow EPSPs are studied. 3. ATP activates P2Y2 (or P2Y4) purinoceptors to elevate [Ca2+]i leading to slow hyperpolarization in AH neurons. Ca2+ -Laser confocal imaging in AH cells is used to study (i) P2Y receptor pharmacology or (ii)Signalling with agents acting through G-proteins, phospholipase C, CICR, or cADPR pathways. 4. Endogenous adenosine inhibits the serotonergic reflex by acting at A1Rs on submucous afferent AH neurons. (i)Effects of drugs are tested on reflex-activation of AH cells in response to N2 puffs or 5-HT in isolated submucosa with an island of attached-mucosa. (ii)Neurobiotin-filled neurons are identified by SP,CGRP or Dil. (iii) flux-chamber studies will assess if the reflex-evoked Isc response involves ATP. Integrating such information provides new insights on the role of purinergic receptors on afferent and motor neurons in enteric neural reflexes and motility.