PURINERGIC REGULATION OF ENTERIC NEURAL REFLEXES

修订后的补助金的总体目标是确定P1和P2嘌呤受体在神经反射中的作用。四个一般假设进行测试：1。内源性腺苷作用于神经A1 R以抑制蠕动反射。(i)在体内的运动性是记录与应变仪响应于电刺激，扩张或药物。 将空肠血液供应分为肛门、感觉和口腔，以测试腺苷（i.a.输注）对上行收缩和下行舒张的影响。（ii）对推进力的影响通过人造弹丸的传播速度来量化。(iii)CM运动神经元上的神经A1 R和A2 bR是否有助于腺苷的作用通过以下方式进行测试：（a）从S/Dil标记的CM运动神经元记录;（B）使用2室系统用于通过CM扩张的VIP释放;（c）绘制A1/A2 bR免疫反应性- 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A1受体抑制初级传入AH神经元的慢突触传递。(i)5-HT在肌间AH细胞的外周部位可诱发慢EPSP。 这用于测试腺苷/ATP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A1 R是否通过降低cAMP水平抑制AH细胞中的慢突触传递-研究了对慢EPSP模拟物或慢EPSP的反应。3. ATP激活P2 Y2（或P2 Y 4）嘌呤受体，升高[Ca 2 +]i，导致AH神经元缓慢超极化。AH细胞中的Ca 2+激光共聚焦成像用于研究（i）P2 Y受体药理学或（ii）通过G蛋白、磷脂酶C、CICR或cADPR途径起作用的药物的信号传导。4.内源性腺苷通过作用于粘膜下传入AH神经元上的A1 Rs抑制肾上腺素能反射。（i）在具有附着粘膜岛的分离的粘膜下层中测试药物对AH细胞响应于N2抽吸或5-HT的反射激活的作用。（ii）通过SP、CGRP或Dil鉴定充满神经生物素的神经元。(iii)通量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新的见解嘌呤能受体的传入和运动神经元在肠神经反射和运动的作用。

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.