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.
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