The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation. The transient receptor potential (TRP)1 superfamily of cation channels consists of a large number of recently identified molecules that share sequence homology with the Drosophila protein named after a phototransduction mutant called trp. According to sequence similarities, the TRP channels are further divided into subfamilies, such as TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) (see reviews in Refs. 1 and 2). These channels are involved in diverse cellular functions including receptor and store-operated Ca 2ϩ entry (3), Ca 2ϩ transport (4, 5), trace metal detection (6), and temperature (7-9) and osmolality (10, 11) sensations. The activation mechanisms for most of the TRP channels remain to be elucidated. Specific ligands have been found for TRPC3, TRPC6, TRPC7, TRPV1, TRPV4, TRPM2, TRPM4, TRPM5, TRPM7, and TRPM8. These include endogenous substances, such as lipids (diacylglycerol (12), anandamide (13, 14), and phosphatidylinositol 4,5-bisphosphate (15)), nucleotides (ADP-ribose (16) (23,24), 2APB was soon found to directly block native store-operated channels (25-27), sarco/ endoplasmic reticulum Ca 2ϩ -ATPase pumps (28), mitochondrial permeability transition pore (29), and a few other ion channels (30). The mechanism of action for 2APB is likely to be complex. In addition to inhibition, low concentrations of 2APB enhanced the activity of store-operated channels (26). At greater than 50 M, 2APB activated a Ca 2ϩ -permeable nonselective cation channel with a 50-picosiemens single channel conductance and very low open probability in rat basophilic leukemia cells (31).2APB has been perceived as a general inhibitor of TRP channels (1). However, except for TRPC3 (24,32), the effects of this drug...

Peripheral inhibitory nerves are physiological regulators of the contractile behavior of visceral smooth muscles. One of the transmitters responsible for inhibitory neurotransmission has been reputed to be a purine, possibly ATP. However, the exact identity of this substance has never been verified. Here we show that ␤-nicotinamide adenine dinucleotide (␤-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of ␤-NAD mimics the endogenous neurotransmitter better than ATP. Levels of ␤-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of ␤-NAD depends on the frequency of nerve stimulation. ␤-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or -conotoxin GVIA. ␤-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous ␤-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to ␤-NAD and inhibitory junction potentials are blocked by the P2Y1-selective antagonist, MRS2179, and the nonselective P2 receptor antagonists, pyridoxal phosphate 6-azophenyl-2,4-disulfonic acid and suramin. Responses to ATP are not blocked by these P2Y receptor inhibitors. The expression of CD38 in gastrointestinal muscles, and specifically in interstitial cells of Cajal, provides a means of transmitter disposal after stimulation. ␤-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.enteric nervous system ͉ gastrointestinal motility ͉ P2Y receptor ͉ purinergic neurotransmission ͉ interstitial cells of Cajal

Alterations in gastrointestinal motility and secretion underlie the constipating action of therapeutically administered opiates. The prototype opiate is morphine, which acts to delay gastric emptying and intestinal transit, to suppress intestinal secretion of water and electrolytes and to suppress transport of bile into the duodenum. The effects of opiates, synthetic opioids and endogenously released opioid peptides on these organ-level gastrointestinal functions reflect actions on electrical and synaptic behaviour of neurones in the enteric nervous system. Adverse effects and positive therapeutic effects of administration of opioid-receptor-blocking drugs on the digestive tract must be understood in the context of the neurophysiology of the enteric nervous system and mechanisms of neural control of gastrointestinal smooth muscle, secretory glands and blood-lymphatic vasculature. We review here the integrated systems of physiology and cellular neurobiology that are basic to understanding the actions of opioid agonists and antagonists in the digestive tract.

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Slow synaptic excitation (slow EPSP) in enteric neurones is recorded as a slowly activating depolarization of the membrane potential in specific populations of enteric neurones when neurotransmitters are released experimentally by focal electrical stimulation of presynaptic axons in the myenteric and submucosal plexuses (reviewed by Surprenant, 1989;Wood, 1994;Galligan, 1998;Gershon, 1998). Mediators released to the enteric nervous system in paracrine fashion from non-neuronal cell types (e.g. histamine and cytokines from enteric mast cells) can evoke responses that mimic slow synaptic excitation (Wood, 1992;Liu et al. 2003). Two kinds of slow EPSPs are recorded in enteric neurones. An increase in input resistance is associated with the depolarization and augmented excitability for one kind of slow EPSP. The input resistance decreases or remains unchanged during the depolarization and augmented excitability of the second kind. Slow EPSPs with increased input resistance are found generally in AH-type neurones with multipolar Dogiel Type II morphology. Most evidence suggests that the principal ionic mechanism for this type of slow EPSP is suppression of resting Ca 2+ -dependent K + conductance that accounts for the membrane depolarization, increased input resistance, and suppression of the Ca 2+ component of the rising phase of the action potential (e.g. Grafe et al. 1980). Signal transduction for the slow EPSP with increased input resistance involves coupling of metabotropic receptors through heterotrimeric G proteins to adenylate cyclase, and elevation of intraneuronal cyclic adenosine monophosphate (Palmer et al. 1986(Palmer et al. , 1987.Whereas slow EPSPs characterized by increased input resistance during the depolarizing response predominate in AH-type neurones in the myenteric plexus, slow EPSPs characterized by decreased input resistance are routinely found in S-type uniaxonal neurones in the small and large intestinal submucosal plexus. Likewise, application of putative neurotransmitters and paracrine mediators (e.g. serotonin, ATP and substance P) evoke slowly activating depolarizing responses associated with decreased input resistance in S-type neurones in the submucosal plexus.This report presents evidence that synaptically released ATP acts at P2Y 1 purinergic receptors to evoke slow EPSPs that are characterized by decreased input resistance in the submucosal plexus. The evidence suggests that the signal transduction cascade for the submucosal P2Y 1 receptor includes activation of phospholipase C, release of inositol 1,4,5-trisphosphate and elevation of cytosolic free Ca