P2X receptors are ATP‐gated cation channels composed of one or more of seven different subunits. ATP acts at P2X receptors to contribute to fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons but the subunit composition of enteric P2X receptors is unknown. These studies used tissues from P2X2 wild‐type (P2X2+/+) and P2X2 gene knockout (P2X2‐/‐) mice to investigate the role of this subunit in enteric neurotransmission. Intracellular electrophysiological methods were used to record synaptic and drug‐induced responses from ileal myenteric neurons in vitro. Drug‐induced longitudinal muscle contractions and peristaltic contractions of ileal segments were also studied in vitro. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. RT‐PCR analysis of mRNA from intestinal tissues and data from immunohistochemical studies verified P2X2 gene deletion. The fEPSPs recorded from S neurons in tissues from P2X2+/+ mice were reduced by mecamylamine (nicotinic cholinergic receptor antagonist) and PPADS (P2X receptor antagonist). The fEPSPs recorded from S neurons from P2X2−/− mice were unaffected by PPADS but were blocked by mecamylamine. ATP depolarized S and AH neurons from P2X2+/+ mice. ATP depolarized AH but not S neurons from P2X2‐/‐ mice. α,β‐Methylene ATP (α,β‐mATP)(an agonist at P2X3 subunit‐containing receptors) did not depolarize S neurons but it did depolarize AH neurons in P2X2+/+ and P2X2‐/‐ mice. Peristalsis was inhibited in ileal segments from P2X2‐/‐ mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X2+/+ and P2X2‐/‐ mice. Gastrointestinal transit was similar in P2X2+/+ and P2X2‐/‐ mice. It is concluded that P2X2 homomeric receptors contribute to fEPSPs in neural pathways underlying peristalsis studied in vitro.

Purinergic P2X receptors contribute to neurotransmission in the gut. P2X receptors are ligand-gated cation channels that mediate synaptic excitation in subsets of enteric neurons. The present study evaluated colonic motility in vitro and in vivo in wild type (WT) and P2X2 and P2X3 subunit knockout (KO) mice. The muscarinic receptor agonist, bethanechol (0.3–3 μM), caused similar contractions of the longitudinal muscle in colon segments from WT, P2X2 and P2X3 subunit KO mice. Nicotine (1–300 μM), acting at neuronal nicotinic receptors, caused similar longitudinal muscle relaxations in colonic segments from WT and P2X2 and P2X3 subunit KO mice. Nicotine-induced relaxations were inhibited by nitro-l-arginine (NLA, 100 μM) and apamin (0.1 μM) which block inhibitory neuromuscular transmission. ATP (1–1000 μM) caused contractions only in the presence of NLA and apamin. ATP-induced contractions were similar in colon segments from WT, P2X2 and P2X3 KO mice. The mouse colon generates spontaneous migrating motor complexes (MMCs) in vitro. The MMC frequency was higher in P2X2 KO compared to WT tissues; other parameters of the MMC were similar in colon segments from WT, P2X2 and P2X3 KO mice. 5-Hydroxytryptophan-induced fecal output was similar in WT, P2X2 and P2X3 KO mice. These data indicate that nicotinic receptors are located predominately on inhibitory motor neurons supplying the longitudinal muscle in the mouse colon. P2X2 or P2X3 subunit containing receptors are not localized to motor neurons supplying the longitudinal muscle. Synaptic transmission mediated by P2X2 or P2X3 subunit containing receptors is not required for propulsive motility in the mouse colon.

There is evidence that R-type Ca2+ channels contribute to synaptic transmission in the myenteric plexus. It is unknown if R-type Ca2+ channels contribute to neuromuscular transmission. We measured the effects of the nitric oxide synthase (NOS) inhibitor, nitro L-arginine (NLA), Ca2+ channel blockers and apamin (SK channel blocker) on neurogenic relaxations and contractions of the guinea pig ileum longitudinal muscle-myenteric plexus (LMMP) in vitro. We used intracellular recordings to measure inhibitory junction potentials (IJPs). Immunohistochemical and western blot techniques localized R-type Ca2+ channel protein in the LMMP and circular muscle. CdCl2 (pan Ca2+ channel blocker) blocked and NLA and NiCl2 (R-type Ca2+ channel blocker) reduced neurogenic relaxations in a non-additive manner. NiCl2 did not alter neurogenic cholinergic contractions but it potentiated neurogenic non-cholinergic contractions. Relaxations were inhibited by apamin, NiCl2 and NLA and were blocked by combined application of these drugs. Relaxations were reduced by NiCl2 or ω-conotoxin (ω-CTX, N-type Ca2+ channel blocker) and were blocked by combined application of these drugs. Longitudinal muscle IJPs were inhibited by NiCl2, but not MRS 2179 (P2Y1 receptor antagonist). Circular muscle IJPs were blocked by apamin, MRS 2179, ω-CTX and CdCl2 but not NiCl2. We conclude that neuronal R-type Ca2+ channels contribute to inhibitory neurotransmission to longitudinal muscle but less so or not all in the circular muscle of the guinea pig ileum.