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Protease-activated receptor ([PAR.sub.2]) is expressed by nociceptive neurons and activated during inflammation by proteases from mast cells, the intestinal lumen, and the circulation. Agonists of [PAR.sub.2] cause hyperexcitability of intestinal sensory neurons and hyperalgesia to distensive stimuli by unknown mechanisms. We evaluated the role of the transient receptor potential vanilloid 4 (TRPV4) in [PAR.sub.2]-induced mechanical hyperalgesia of the mouse colon. Colonic sensory neurons, identified by retrograde tracing, expressed immunoreactive TRPV4, [PAR.sub.2], and calcitonin generelated peptide and are thus implicated in nociception. To assess nociception, visceromotor responses (VMR) to colorectal distension (CRD) were measured by electromyography of abdominal muscles. In [TRPV4.sup.+/+] mice, intraluminal [PAR.sub.2] activating peptide ([PAR.sub.2]-AP) exacerbated VMR to graded CRD from 6-24 h, indicative of mechanical hyperalgesia. [PAR.sub.2]-induced hyperalgesia was not observed in [TRPV4.sup.-/-] mice. [PAR.sub.2]-AP evoked discharge of action potentials from colonic afferent neurons in [TRPV4.sup.+/+] mice, but not from [TRPV4.sup.-/-] mice. The TRPV4 agonists 5',6'-epoxyeicosatrienoic acid and 4[alpha]-phorbol 12,13-didecanoate stimulated discharge of action potentials in colonic afferent fibers and enhanced current responses recorded from retrogradely labeled colonic dorsal root ganglia neurons, confirming expression of functional TRPV4. [PAR.sub.2]-AP enhanced these responses, indicating sensitization of TRPV4. Thus TRPV4 is expressed by primary spinal afferent neurons innervating the colon. Activation of [PAR.sub.2] increases currents in these neurons, evokes discharge of action potentials from colonic afferent fibers, and induces mechanical hyperalgesia. These responses require the presence of functional TRPV4. Therefore, TRPV4 is required for [PAR.sub.2]-induced mechanical hyperalgesia and excitation of colonic afferent neurons. visceral pain; proteases; protease-activated receptors; transient receptor potential channels

Ghrelin is a peptide released from gastric endocrine cells that has an orexigenic effect via a vagal pathway. Here we determine the effect of ghrelin on mechanosensitivity of upper-intestinal vagal afferent fibers in ferret and mouse. The responses of gastroesophageal vagal afferents to graded mechanical stimulation were determined in vitro before and during application of ghrelin to their peripheral endings. Three types of vagal afferent were tested: tension receptors responding to circumferential tension, mucosal receptors responding only to mucosal stroking, and tension/mucosal (TM) receptors in ferret esophagus that responded to both stimuli. In the mouse, ghrelin did not significantly affect the response of mucosal receptors to mucosal stroking with calibrated von Frey hairs. However, it significantly reduced responses of tension receptors to circumferential tension (P < 0.005; two-way ANOVA) by up to 40%. This inhibition was reversed by the ghrelin receptor antagonist [D-Lys-3]-growth hormone-releasing peptide (GHRP)-6. In the ferret, ghrelin significantly reduced the response of mucosal and TM receptors to mucosal stroking with calibrated von Frey hairs. Surprisingly, ghrelin did not significantly alter the response to circumferential tension in either tension or TM receptors. RT-PCR analysis indicated that both ghrelin and its receptor are expressed in vagal afferent cell bodies in mouse nodose ganglia. In conclusion, ghrelin selectively inhibits subpopulations of mechanically sensitive gastroesophageal vagal afferents; there is also potential for ghrelin release from vagal afferents. However, the subpopulation of afferents inhibited differs between species. These data have broad implications for ghrelin's role in food intake regulation and reflex control of gastrointestinal function. in vitro gastroesophageal preparation; neuromodulation; mechanoreceptor doi:10.1152/ajpgi.00536.2006.

Three Orai (Orai1, Orai2, and Orai3) and two stromal interaction molecule (STIM1 and STIM2) mammalian protein homologues constitute major components of the store‐operated Ca2+ entry mechanism. When co‐expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca2+ channels with properties of Ca2+ release‐activated Ca2+ (CRAC) channels. Despite the high level of homology between Orai proteins, CRAC channels formed by different Orai isoforms have distinctive properties, particularly with regards to Ca2+‐dependent inactivation, inhibition/potentiation by 2‐aminoethyl diphenylborinate and sensitivity to reactive oxygen species. This study characterises and compares the regulation of Orai1, Orai2‐ and Orai3‐mediated CRAC current (ICRAC) by intracellular pH (pHi). Using whole‐cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1, we show that ICRAC formed by each Orai homologue has a unique sensitivity to changes in pHi. Orai1‐mediated ICRAC exhibits a strong dependence on pHi of both current amplitude and the kinetics of Ca2+‐dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pHi, whereas Orai3 shows no dependence on pHi at all. Investigation of different Orai1–Orai3 chimeras suggests that pHi dependence of Orai1 resides in both the N‐terminus and intracellular loop 2, and may also involve pH‐dependent interactions with STIM1. Key points: It has been shown previously that Orai1/stromal interaction molecule 1 (STIM1)‐mediated Ca2+ release‐activated Ca2+ current (ICRAC) is inhibited by intracellular acidification and potentiated by intracellular alkalinisation.The present study reveals that CRAC channels formed by each of the Orai homologues Orai1, Orai2 and Orai3 has a unique sensitivity to changes in intracellular pH (pHi). The amplitude of Orai2 current is affected by the changes in pHi similarly to the amplitude of Orai1. However, unlike Orai1, fast Ca2+‐dependent inactivation of Orai2 is unaffected by acidic pHi. In contrast to both Orai1 and Orai2, Orai3 is not sensitive to pHi changes.Domain swapping between Orai1 and Orai3 identified the N‐terminus and intracellular loop 2 as the molecular structures responsible for Orai1 regulation by pHi.Reduction of ICRAC dependence on pHi seen in a STIM1‐independent Orai1 mutant suggested that some parts of STIM1 are also involved in ICRAC modulation by pHi. [ABSTRACT FROM AUTHOR]

Background: Muscarinic receptor 1 positive allosteric modulators (M1PAMs) enhance colonic propulsive contractions and defecation through the facilitation of M1 receptor (M1R)‐mediated signaling. We examined M1R expression in the colons of 5 species and compared colonic propulsion and defecation caused by the M1PAM, T440, the 5‐HT4 agonist, prucalopride, and the cholinesterase inhibitor, neostigmine, in rats and dogs. Methods: M1R expression was profiled by immunostaining and in situ hybridization. In vivo studies utilized male SD rats and beagle dogs. Colonic propulsive contractions were recorded by manometry in anesthetized rats. Gut contractions in dogs were assessed using implanted force transducers in the ileum, proximal, mid, and distal colons. Key Results: M1R was localized to neurons of myenteric and submucosal plexuses and the epithelium of the human colon. A similar receptor localization was observed in rat, dog, mouse, and pig. T440 enhanced normal defecation in rats in a dose‐dependent manner. Prucalopride also enhanced defecation in rats, but the maximum effect was half that of T440. Neostigmine and T440 were similarly effective in enhancing defecation, but the effective dose of neostigmine was close to its lethal dose. In rats, all 3 compounds induced colonic contractions, but the associated propulsion was strongest with T440. In dogs, intestinal contractions elicited by T440 propagated from ileum to distal colon. Prucalopride and neostigmine also induced intestinal contractions, but these were less well coordinated. No loss of effectiveness of T440 on defecation occurred after 5 days of repeated dosing. Conclusion and Inferences: These results suggest that M1PAMs produce highly coordinated propagating contraction by actions on the enteric nervous system of the colon. The localization of M1R to enteric neurons in both animals and humans suggests that the M1PAM effects would be translatable to human. M1PAMs provide a potential novel therapeutic option for constipation disorders. [ABSTRACT FROM AUTHOR]