Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC112
β-NAD mediates purinergic post-junctional enteric inhibitory responses via P2Y1 receptors in the colon
S. M. Ward1
1. Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States.
Inhibitory motor neurons regulate complex motor patterns in the GI tract such as peristalsis, receptive relaxation, and sphincter opening. The neurotransmitters responsible for inhibitory responses have been investigated for decades, and numerous substances proposed. β-nicotinamide adenine dinucleotide (β-NAD) has recently been suggested to mediate inhibitory neurotransmission in mouse and human colons via P2Y1 receptors (P2Y1R; Hwang et al.,2011). However, evidence supporting a role for P2Y1 receptors in enteric responses is based on pharmacological agents, MRS2179 and MRS2500, thought to be highly specific P2Y1R antagonists. In order to confirm (i) the role of P2Y1R in inhibitory responses, and (ii) the importance of P2Y1R in colonic transit, we compared post-junctional responses of wildtype (WT) and P2Y1-/- mice using intracellular and isometric force recordings in vitro. Video imaging with spatio-temporal analysis (STMap) was used to examine how loss of P2Y1R affects colonic transit. Electrical field stimulation (EFS; 0.1-0.5 ms;150V) of enteric inhibitory nerves evoked bi-phasic inhibitory junction potentials (IJPs) in mouse and cynomolgus monkey colons. IJPs consisted of an initial fast hyperpolarization, followed by a slower secondary hyperpolarization. Both phases of the IJPs were blocked by tetrodotoxin (TTX; 1µM), and the secondary hyperpolarization blocked by N(ω)-nitro-L-arginine (L-NNA;100 μM), whereas the initial IJP was inhibited by MRS2500 (1 μM). In colons of P2Y1R-/- mice EFS produced large atropine (AT, 1 μM) sensitive excitatory junction potentials (EJPs), followed by slow, L-NNA sensitive IJPs. In AT and L-NNA, post-junctional responses were abolished in P2Y1R-/- mice. Force measurements revealed abnormal activity in colons of P2Y1R-/- mice. EFS (5 Hz;0.3 ms,30 s) of P2Y1R-/- colons caused relaxation that was potentiated by AT and inhibited by L-NNA. In AT and L-NNA, relaxation was abolished in P2Y1R-/- mice compared to the relaxations of WT muscles. Picosprizing ATP or ADP onto WT or P2Y1R-/- muscles caused hyperpolarization that was not affected by TTX or MRS2500, however picospritzing β-NAD caused hyperpolarization in WT muscles, but these agents did not cause hyperpolarization in P2Y1-/- muscles. Video imaging and STMaps revealed fecal transit of 0.53±0.06 mm s-1. Transit was disrupted in colons of P2Y1-/- mice; pellets traveled to a point mid way along the colons at 0.25±0.07 mm s-1 (P<0.05,t-test), where transit terminated. These data reveal the importance of P2Y1R in colonic motor responses. P2Y1R mediate the fast component of IJPs. Responses mediated by P2Y1R were mimicked by β-NAD, but not to ATP or ADP. P2Y1 receptors represent an important transduction mechanism for enteric inhibitory responses and are critical for normal colonic transit.
Where applicable, experiments conform with Society ethical requirements