Jackie D. Wood

2-Aminoethoxydiphenyl Borate Is a Common Activator of TRPV1, TRPV2, and TRPV3

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.

Fundamentals of neurogastroenterology

Current concepts and basic principles of neurogastroenterology in relation to functional gastrointestinal disorders are reviewed. Neurogastroenterology is emphasized as a new and advancing subspecialty of clinical gastroenterology and digestive science. As such, it embraces the investigative sciences dealing with functions, malfunctions, and malformations in the brain and spinal cord, and the sympathetic, parasympathetic and enteric divisions of the autonomic innervation of the digestive tract. Somatomotor systems are included insofar as pharyngeal phases of swallowing and pelvic floor involvement in defecation, continence, and pelvic pain are concerned. Inclusion of basic physiology of smooth muscle, mucosal epithelium, and the enteric immune system in the neurogastroenterologic domain relates to requirements for compatibility with neural control mechanisms. Psychologic and psychiatric relations to functional gastrointestinal disorders are included because they are significant components of neurogastroenterology, especially in relation to projections of discomfort and pain to the digestive tract.

Function of opioids in the enteric nervous system

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.

Fundamentals of Neurogastroenterology: Basic Science

This review examines the fundamentals of neurogastroenterology that may underlie the pathophysiology of functional GI disorders (FGIDs). It was prepared by an invited committee of international experts and represents an abbreviated version of their consensus document that will be published in its entirety in the forthcoming book and online version entitled ROME IV. It emphasizes recent advances in our understanding of the enteric nervous system, sensory physiology underlying pain, and stress signaling pathways. There is also a focus on neuroimmmune signaling and intestinal barrier function, given the recent evidence implicating the microbiome, diet, and mucosal immune activation in FGIDs. Together, these advances provide a host of exciting new targets to identify and treat FGIDs and new areas for future research into their pathophysiology.

Synaptic modulation of calcium‐dependent potassium conductance in myenteric neurones in the guinea‐pig.

1. Ganglion cells of the myentric plexus of the guinea‐pig small intestine were studied with intracellular recording methods. 2. Electrical stimulation of the interganglionic connectives elicited slow synaptic excitation (slow e.p.s.p.) that was associated with an increase in the input resistance of the cell. The slow e.p.s.p.s continued for several seconds after termination of stimulation, and they occurred only in neurones in which prolonged hyperpolarizing after‐potentials followed an action potential. 3. Superfusion of the neurones with solutions containing either 1‐5 mM‐Mn2+ or 16 mM‐Mg2+ and 1 mM‐Ca2+ mimicked the slow e.p.s.p. The common characteristics of Mn2+, Mg2+ and the slow e.p.s.p. were: (a) depolarization of the membrane potential, (b) increased input resistance of the cell, (c) augmented excitability, (d) blockade of post‐pike hyperpolarizing potentials and (e) reversal potential between ‐70 and ‐75 mV. 4. Analyses based on the ‘constant field equation’ indicated that the permeability ratios of K+ to other permeant ionic species were reduced when Ca2+ influx was blocked by Mn2+ or Mg2+. 5. The organic Ca antagonist D‐600 did not affect the neurones. 6. The results suggest that slow synaptic modulation of excitability within the myenteric plexus involves a reduction of both resting GK and post‐spike GK which is secondary to suppression of Ca2+ influx by the neurotransmitter for the slow e.p.s.p.

β-Nicotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle

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 β-nicotinamide adenine dinucleotide (β-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of β-NAD mimics the endogenous neurotransmitter better than ATP. Levels of β-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of β-NAD depends on the frequency of nerve stimulation. β-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or ω-conotoxin GVIA. β-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous β-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to β-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. β-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.

Potentiation of TRPV3 channel function by unsaturated fatty acids.

Transient receptor potential vanilloid (TRPV) channels are polymodal detectors of multiple environmental factors, including temperature, pH, and pressure. Inflammatory mediators enhance TRPV function through multiple signaling pathways. The lipoxygenase and epoxygenase products of arachidonic acid (AA) metabolism have been shown to directly activate TRPV1 and TRPV4, respectively. TRPV3 is a thermosensitive channel with an intermediate temperature threshold of 31–39°C. We have previously shown that TRPV3 is activated by 2‐aminoethoxydiphenyl borate (2APB). Here we show that AA and other unsaturated fatty acids directly potentiate 2APB‐induced responses of TRPV3 expressed in HEK293 cells, Xenopus oocytes, and mouse keratinocytes. The AA‐induced potentiation is observed in intracellular Ca2+ measurement, whole‐cell and two‐electrode voltage clamp studies, as well as single channel recordings of excised inside‐out and outside‐out patches. The fatty acid‐induced potentiation is not blocked by inhibitors of protein kinase C and thus differs from that induced by the kinase. The potentiation does not require AA metabolism but is rather mimicked by non‐metabolizable analogs of AA. These results suggest a novel mechanism regulating the TRPV3 response to inflammation, which differs from TRPV1 and TRPV4, and involves a direct action of free fatty acids on the channel. J. Cell. Physiol.

IL-1β and IL-6 excite neurons and suppress nicotinic and noradrenergic neurotransmission in guinea pig enteric nervous system

Conventional intracellular microelectrodes and injection of biocytin were used to study the actions of IL-1β and IL-6 on electrical and synaptic behavior in morphologically identified guinea pig small intestinal submucous neurons. Exposure to nanomolar concentrations of either IL-1β or IL-6 stimulated neuronal excitability. The excitatory action consisted of depolarization of the membrane potential, decreased membrane conductance, and increased discharge of action potentials. Excitatory action of IL-1β was suppressed by the natural IL-1β human receptor antagonist. Electrical stimulation of sympathetic postganglionic axons evoked inhibitory postsynaptic potentials (IPSPs), and stimulation of cholinergic axons evoked nicotinic fast excitatory postsynaptic potentials (EPSPs). Both kinds of synaptic potentials occurred in neurons with uniaxonal morphology believed to be secretomotor neurons. Either IL-1β or IL-6 suppressed the noradrenergic IPSPs and the fast EPSPs, and the two acted synergistically when applied in combination. Suppression of the IPSP resulted from presynaptic inhibition of the release of norepinephrine from sympathetic nerves. The results suggest that the presence of either or both inflammatory cytokines will release the sympathetic brake from secretomotor neurons to the intestinal crypts and from nicotinic synapses in the integrative microcircuits, where norepinephrine is known to have a presynaptic inhibitory action. This, in concert with excitation of secretomotor neurons, may lead to neurogenic secretory diarrhea. J. Clin. Invest. 103:1309–1316 (1999).

Slow excitatory synaptic transmission mediated by P2Y1 receptors in the guinea-pig enteric nervous system.

Electrophysiological recording was used to study a type of slow excitatory postsynaptic potential (slow EPSP) that was mediated by release of ATP and its action at P2Y1 receptors on morphologically identified neurones in the submucosal plexus of guinea‐pig small intestine. MRS2179, a selective P2Y1 purinergic receptor antagonist, blocked both the slow EPSP and mimicry of the EPSP by exogenously applied ATP. Increased conductance accounted for the depolarization phase of the EPSP, which occurred exclusively in neurones with S‐type electrophysiological behaviour and uniaxonal morphology. The purinergic excitatory input to the submucosal neurones came from neighbouring neurones in the same plexus, from neurones in the myenteric plexus and from sympathetic postganglionic neurones. ATP‐mediated EPSPs occurred coincident with fast nicotinic synaptic potentials evoked by the myenteric projections and with noradrenergic IPSPs evoked by sympathetic fibres that innervated the same neurones. The P2Y1 receptor on the neurones was identified as a metabotropic receptor linked to activation of phospholipase C, synthesis of inositol 1,4,5‐trisphosphate and mobilization of Ca2+ from intracellular stores.

Immunoreactivity of Hu proteins facilitates identification of myenteric neurones in guinea-pig small intestine.

Hu proteins, together with neurone‐specific enolase (NSE), protein gene product 9.5 (PGP‐9.5), microtubule‐associated protein‐2 (MAP‐2) and tubulin beta III isoform, were evaluated immunohistochemically as neuronal markers in whole‐mount preparations and cultures obtained from the myenteric plexus of guinea‐pig small intestine. Anti‐Hu immunostaining marked the ganglion cell somas and nuclei without staining of the neuronal processes in the whole‐mounts and cultures. The ganglion cell bodies were not obscured by staining of multiple neuronal fibres and this facilitated accurate counting of the neurones. MAP2 immunostaining also provided clear images of individual neurones in both whole mounts and cultures. Immunoreactivity for NSE, PGP‐9.5 and tubulin beta III isoform provided sharp images of the ganglion cells in culture, but not in whole‐mount preparations. Strong staining of the neuronal processes in the whole‐mount preparations obscured the profiles of the ganglion cell bodies to such an extent that accurate counting of the total neuronal population was compromised. Anti‐Hu immunostaining was judged to be an acceptable method for obtaining reliable estimates of total numbers of myenteric neurones in relation to other specific histochemical properties such as histamine binding.