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P2X2 receptors, with other P2X receptor subtypes, have an important role mediating synaptic transmission in regulating the functions of the gastrointestinal tract. Our recent work has found a new regulator of P2X receptor function, called phosphoinositide-interacting regulator of transient receptor potential channels (Pirt). In the present work, we have shown that Pirt immunoreactivity was localized in nerve cell bodies and nerve fibers in the myenteric plexus of the stomach, ileum, proximal, and distal colon and in the submucosal plexus of the jejunum, ileum, proximal, and distal colon. Almost all the Pirt-immunoreactive (ir) neurons were also P2X2-ir, and co-immunoprecipitation experiments have shown that Pirt co-precipitated with the anti-P2X2 antibody. This work provides detailed information about the expression of Pirt in the gut and its co-localization with P2X2, indicating its potential role in influencing P2X2 receptor function.

Single- and double-immunostaining techniques were used systematically to study the distribution pattern and neurochemical density of oxytocin-immunoreactive (-ir) neurons in the digestive tract of the guinea pig. Oxytocin immunoreactivity was distributed widely in the guinea pig gastrointestinal tract; 3%, 13%, 17%, 15%, and 10% of ganglion neurons were immunoreactive for oxytocin in the myenteric plexuses of the gastric corpus, jejunum, ileum, proximal colon, and distal colon, respectively, and 36%, 40%, 52%, and 56% of ganglion neurons were immunoreactive for oxytocin in the submucosal plexuses of the jejunum, ileum, proximal colon, and distal colon, respectively. In the myenteric plexus, oxytocin was expressed exclusively in the intrinsic enteric afferent neurons, as identified by calbindin 28 K. In the submucosal plexuses, oxytocin was expressed in non-cholinergic secretomotor neurons, as identified by vasoactive intestinal polypeptide. Oxytocin-ir nerve fibers in the inner circular muscle layer possibly arose from the myenteric oxytocin-ir neurons, and oxytocin-ir nerve fibers in the mucosa possibly arose from both the myenteric and submucosal oxytocin-ir neurons. Thus, oxytocin in the digestive tract might be involved in gastrointestinal tract motility mainly via the regulation of the inner circular muscle and the balance of the absorption and secretion of water and electrolytes.

Expression of P2X(4) and P2X(6) receptor subunits in the gastrointestinal tract of the rat was studied with double-labeling fluorescence immunohistochemistry. The results showed that P2X(6) receptors were expressed widely in the submucosal and myenteric plexuses. In the myenteric plexus, P2X(6) receptors were expressed mainly in large size neurons which resembled Dogiel type II neurons. These P2X(6) receptor-immunoreactive (ir) neurons also expressed calbindin 28K, calretinin and neuronal nuclei (NeuN), proteins that are markers of intrinsic sensory neurons. In the submucosal plexus, all the calbindin 28K, calretinin and NeuN-ir cells were immunoreactive for P2X(6) receptors. P2X(6) receptors do not form homomultimers, but rather heteromultimers with either P2X(2) or P2X(4) receptors. P2X(4) receptors were not expressed in neurons, but were expressed in macrophages of the rat gastrointestinal tract. These data indicate that P2X(6) receptors are mainly expressed on intrinsic sensory neurons and that ATP, via P2X(6) receptors probably in heteromeric combination with P2X(2) receptors, may be involved in regulating the physiological functions of these neurons.

The distribution of P2Y(6) and P2Y(12) receptor-immunoreactive (ir) neurons and fibers and their coexistence with calbindin, calretinin and nitric oxide synthase (NOS) has been investigated with single and double labeling immunostaining methods. The results showed that 30-36% of the ganglion cells in the myenteric plexus are strongly P2Y(6) receptor-ir neurons; they are distributed widely in the myenteric plexus of stomach, jejunum, ileum and colon, but not in the submucosal plexus, with a typical morphology of multipolar neurons with a long axon-like process. About 42-46% of ganglion cells in both the myenteric and submucosal plexuses show P2Y(12) receptor-ir. About 28-35% of P2Y(6) receptor-ir neurons were found to coexist with NOS and 41-47% of them coexist with calretinin, but there was no coexistence of P2Y(6) receptor-ir with calbindin. In contrast, all P2Y(12) receptor-ir neurons were immunopositive for calbindin, although occasionally P2Y(12) receptor-ir neurons without calbindin immunoreactivity were found, while none of the P2Y(12) receptor-ir neurons were found to coexist with calretinin or NOS in the gastrointestinal system of guinea pig. The P2Y(12) receptor-ir neurons coexpressing calbindin-ir in the small intestine are Dogiel type II/AH, intrinsic primary afferent neurons.

Adenosine 5'-triphosphate receptors are known to be involved in fast excitatory postsynaptic currents in myenteric neurons of the digestive tract. In the present study, the distribution of P2X2 and P2X3 receptor mRNA was examined by in situ hybridisation while P2X2 and P2X3 receptor protein was localised by immunohistochemical methods. In addition, P2X2 and P2X3 receptors were colocalised with calbindin and calretinin in the myenteric and submucosal plexus. P2X2- and P2X3- immunoreactive neurons were found in the myenteric and submucosal plexuses throughout the entire length of the rat digestive tract from the stomach to the colon. Approximately 60%, 70% and 50% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 56% and 45% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X2 receptor. Approximately 10%, 2% and 15% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 62% and 40% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X3 receptor. Double-labelling studies showed that about 10-25% of the neurons with P2X2 immunoreactivity in myenteric plexus and 30-50% in the submucosal plexus were found to express calbindin or calretinin. About 80% of the neurons with P2X3 receptor immunoreactivity in the myenteric plexus and about 40% in the submucosal plexus expressed calretinin. Approximately 30-75% of the neurons with P2X3 receptor immunoreactivity in the submucosal plexus expressed calbindin, while none of them were found to express calbindin in the myenteric plexus., (Copyright 2004 Springer-Verlag)