Expression of the transient receptor potential channels TRPV1, TRPA1 and TRPM8 in mouse trigeminal primary afferent neurons innervating the dura

ABSTRACT: BACKGROUND: Migraine and other headache disorders affect a large percentage of the population and cause debilitating pain. Activation and sensitization of the trigeminal primary afferent neurons innervating the dura and cerebral vessels is a crucial step in the "headache circuit". Many dural afferent neurons respond to algesic and inflammatory agents. Given the clear role of the transient receptor potential (TRP) family of channels in both sensing chemical stimulants and mediating inflammatory pain, we investigated the expression of TRP channels in dural afferent neurons. METHODS: We used two fluorescent tracers to retrogradely label dural afferent neurons in adult mice and quantified the abundance of peptidergic and non-peptidergic neuron populations using calcitonin gene-related peptide immunoreactivity (CGRP-ir) and isolectin B4 (IB4) binding as markers, respectively. Using immunohistochemistry, we compared the expression of TRPV1 and TRPA1 channels in dural afferent neurons with the expression in total trigeminal ganglion (TG) neurons. To examine the distribution of TRPM8 channels, we labeled dural afferent neurons in mice expressing farnesylated enhanced green fluorescent protein (EGFPf) from a TRPM8 locus. We used nearest-neighbor measurement to predict the spatial association between dural afferent neurons and neurons expressing TRPA1 or TRPM8 channels in the TG.Results and conclusionsWe report that the size of dural afferent neurons is significantly larger than that of total TG neurons and facial skin afferents. Approximately 40% of dural afferent neurons exhibit IB4 binding. Surprisingly, the percentage of dural afferent neurons containing CGRP-ir is significantly lower than those of total TG neurons and facial skin afferents. Both TRPV1 and TRPA1 channels are expressed in dural afferent neurons. Furthermore, nearest-neighbor measurement indicates that TRPA1-expressing neurons are clustered around a subset of dural afferent neurons. Interestingly, TRPM8-expressing neurons are virtually absent in the dural afferent population, nor do these neurons cluster around dural afferent neurons. Taken together, our results suggest that TRPV1 and TRPA1 but not TRPM8 channels likely contribute to the excitation of dural afferent neurons and the subsequent activation of the headache circuit. These results provide an anatomical basis for understanding further the functional significance of TRP channels in headache pathophysiology.     

The transient receptor potential family of ion channels

The transient receptor potential (TRP) multigene superfamily encodes integral membrane proteins that function as ion channels. Members of this family are conserved in yeast, invertebrates and vertebrates. The TRP family is subdivided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like); the latter is found only in invertebrates and fish. TRP ion channels are widely expressed in many different tissues and cell types, where they are involved in diverse physiological processes, such as sensation of different stimuli or ion homeostasis. Most TRPs are non-selective cation channels, only few are highly Ca²⁺ selective, some are even permeable for highly hydrated Mg²⁺ ions. This channel family shows a variety of gating mechanisms, with modes of activation ranging from ligand binding, voltage and changes in temperature to covalent modifications of nucleophilic residues. Activated TRP channels cause depolarization of the cellular membrane, which in turn activates voltage-dependent ion channels, resulting in a change of intracellular Ca²⁺ concentration; they serve as gatekeeper for transcellular transport of several cations (such as Ca²⁺ and Mg²⁺), and are required for the function of intracellular organelles (such as endosomes and lysosomes). Because of their function as intracellular Ca²⁺ release channels, they have an important regulatory role in cellular organelles. Mutations in several TRP genes have been implicated in diverse pathological states, including neurodegenerative disorders, skeletal dysplasia, kidney disorders and pain, and ongoing research may help find new therapies for treatments of related diseases.     

Phosphoinositide regulation of non-canonical transient receptor potential channels

Transient receptor potential (TRP) channels are involved in a wide range of physiological processes, and characterized by diverse activation mechanisms. Phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PIP₂, or PtdIns(4,5)P₂] recently emerged as regulators of many TRP channels. Several TRP channels require PIP₂ for activity, and depletion of the lipid inhibits them. For some TRP channels, however, phosphoinositide regulation seems more complex, both activating and inhibitory effects have been reported. This review will discuss phosphoinositide regulation of members of the TRPM (Melastatin), TRPV (Vanilloid), TRPA (Ankyrin) and TRPP (Polycystin) families. Lipid regulation of TRPC (Canonical) channels is discussed elsewhere in this volume.     

Vanilloid receptor (VR1) expression in vagal afferent neurons innervating the gastrointestinal tract

The vanilloid receptor VR1 is a nonselective cation channel activated by capsaicin as well as increases in temperature and acidity, and can be viewed as molecular integrator of chemical and physical stimuli that elicit pain. The distribution of VR1 receptors in peripheral and central processes of rat primary vagal afferent neurons innervating the gastrointestinal tract was investigated by immunohistochemistry. Forty-two percent of neurons in the nodose ganglia retrogradely labeled from the stomach wall expressed low to moderate VR1 immunoreactivity (VR1-IR). VR1-IR was considerably lower in the nodose ganglia as compared to the jugular and dorsal root ganglia. In the vagus nerve, strongly VR1-IR fibers ran in separate fascicles that supplied mainly cervical and thoracic targets, leaving only weakly VR1-IR fibers in the subdiaphragmatic portion. Vagal afferent intraganglionic laminar endings (IGLEs) in the gastric and duodenal myenteric plexus did not express VR1-IR. Similarly, VR1-IR was contained in fibers running in perfect register with vagal afferents, but was not colocalized with horseradish peroxidase in the same varicosities of intramuscular arrays (IMAs) and vagal afferent fibers in the duodenal submucosa anterogradely labeled from the nodose ganglia. Only in the gastric mucosa did we find evidence for colocalization of VR1-IR in vagal afferent terminals. In contrast, many nerve fibers coursing through the myenteric and submucosal plexuses contained detectable VR1-IR, the majority of which colocalized calcitonin gene-related peptide immunoreactivity. In the dorsal medulla there was a dense plexus of VR1-IR varicose fibers in the commissural, dorsomedial and gelatinosus subnuclei of the medial NTS and the lateral aspects of the area postrema, which was substantially reduced, but not eliminated on the ipsilateral side after supranodose vagotomy. It is concluded that about half of the vagal afferents innervating the gastrointestinal tract express low levels of VR1-IR, but that presence in most of the peripheral terminal structures is below the immunohistochemical detection threshold.     

Non-selective cation channels, transient receptor potential channels and ischemic stroke

Several pathways to neural cell death are involved in ischemic stroke, and all require monovalent or divalent cation influx, implicating non-selective cation (NC) channels. NC channels are also likely to be involved in the dysfunction of vascular endothelial cells that leads to formation of edema following cerebral ischemia. Two newly described NC channels have emerged as potential participants in ischemic stroke, the acid sensing ion channel (ASIC), and the sulfonylurea receptor-1 (SUR1)-regulated NC(Ca-ATP) channel. Non-specific blockers of NC channels, including pinokalant (LOE 908 MS) and rimonabant (SR141716A), have beneficial effects in rodent models of ischemic stroke. Evidence is accumulating that NC channels formed by members of the transient receptor potential (TRP) family are also up-regulated in ischemic stroke and may play a direct role in calcium-mediated neuronal death. The nascent field of NC channels, including TRP channels, in ischemic stroke is poised to provide novel mechanistic insights and therapeutic strategies for this often devastating human condition.     

Cholecystokinin activates both A- and C-type vagal afferent neurons

Patch-clamp electrophysiological methods were used on dissociated rat nodose neurons maintained in culture to determine whether responses to cholecystokinin (CCK) were associated with capsaicin-resistant (A type) or capsaicin-sensitive (C type) neurons. Nodose neurons were classified as A or C type on the basis of the characteristics of the Na+ current, a hyperpolarization-activated current, and sensitivity to a low concentration of capsaicin to ascertain the presence of vanilloid receptor 1 that has been associated with C-type neurons in sensory ganglia. It was expected that only capsaicin-sensitive C-type neurons would respond to CCK, because most vagally mediated actions of CCK are blocked by capsaicin treatment. However, we found that subpopulations of both A- and C-type neurons responded to CCK (24 and 38%, respectively). Thus some vagally mediated actions of CCK may be mediated by capsaicin insensitive A-type neurons.     

Cytosolic calcium regulation in rat afferent vagal neurons during anoxia

Sensory neurons are able to detect tissue ischaemia and both transmit information to the brainstem as well as release local vasoactive mediators. Their ability to sense tissue ischaemia is assumed to be primarily mediated through proton sensing ion channels, lack of oxygen however may also affect sensory neuron function. In this study we investigated the effects of anoxia on isolated capsaicin sensitive neurons from rat nodose ganglion. Acute anoxia triggered a reversible increase in [Ca²⁺]_i that was mainly due to Ca²⁺-efflux from FCCP sensitive stores and from caffeine and CPA sensitive ER stores. Prolonged anoxia resulted in complete depletion of ER Ca²⁺-stores. Mitochondria were partially depolarised by acute anoxia but mitochondrial Ca²⁺-uptake/buffering during voltage-gated Ca²⁺-influx was unaffected. The process of Ca²⁺-release from mitochondria and cytosolic Ca²⁺-clearance following Ca²⁺ influx was however significantly slowed. Anoxia was also found to inhibit SERCA activity and, to a lesser extent, PMCA activity. Hence, anoxia has multiple influences on [Ca²⁺]_i homeostasis in vagal afferent neurons, including depression of ATP-driven Ca²⁺-pumps, modulation of the kinetics of mitochondrial Ca²⁺ buffering/release and Ca²⁺-release from, and depletion of, internal Ca²⁺-stores. These effects are likely to influence sensory neuronal function during ischaemia.     

NMDA channels control meal size via central vagal afferent terminals

The N-methyl-D-aspartate (NMDA) ion channel blocker MK-801 administered systemically or as a nanoliter injection into the nucleus of the solitary tract (NTS), increases meal size. Furthermore, we have observed that ablation of the NTS abolishes increased meal size following systemic injection of dizocilpine (MK-801) and that MK-801-induced increases in intake are attenuated in rats pretreated with capsaicin to destroy small, unmyelinated, primary afferent neurons. These findings led us to hypothesize that NMDA receptors on central vagal afferent terminals or on higher-order NTS neurons innervated by these vagal afferents might mediate increased food intake. To evaluate this hypothesis, we examined 15% sucrose intake after 50-nl MK-801 injections ipsilateral or contralateral to unilateral nodose ganglion removal (ganglionectomy). On the side contralateral to ganglionectomy, vagal afferent terminals would be intact and functional, whereas ipsilateral to ganglionectomy vagal afferent terminals would be absent. Three additional control preparations also were included: 1) sham ganglionectomy and 2) subnodose vagotomy either contralateral or ipsilateral to NTS cannula placement. We found that rats with subnodose vagotomies increased their sucrose intake after injections of MK-801 compared with saline, regardless of whether injections were made contralateral (12.6 +/- 0.2 vs. 9.6 +/- 0.3 ml) or ipsilateral (14.2 +/- 0.6 vs. 9.7 +/- 0.4 ml) to vagotomy. Rats with NTS cannula placements contralateral to nodose ganglionectomy also increased their intake after MK-801 (12.2 +/- 0.9 and 9.2 +/- 1.1 ml for MK-801 and saline, respectively). However, rats with placements ipsilateral to ganglionectomy did not respond to MK-801 (8.0 +/- 0.5 ml) compared with saline (8.3 +/- 0.4 ml). We conclude that central vagal afferent terminals are necessary for increased food intake in response to NMDA ion channel blockade. The function of central vagal afferent processes or the activity of higher-order NTS neurons driven by vagal afferents may be modulated by NMDA receptors to control meal size.     

The role of canonical transient receptor potential 7 in B-cell receptor-activated channels

Phospholipase C signaling stimulates Ca2+ entry across the plasma membrane through multiple mechanisms. Ca2+ store depletion stimulates store-operated Ca2+-selective channels, or alternatively, other phospholipase C-dependent events activate Ca2+-permeable non-selective cation channels. Transient receptor potential 7 (TRPC7) is a non-selective cation channel that can be activated by both mechanisms when ectopically expressed, but the regulation of native TRPC7 channels is not known. We knocked out TRPC7 in DT40 B-cells, which expresses both forms of Ca2+ entry. No difference in the store-operated current I(crac) was detected between TRPC7-/- and wild-type cells. Wild-type cells demonstrated nonstore-operated cation entry and currents in response to activation of the B-cell receptor or protease-activated receptor 2, intracellular dialysis with GTPgammaS, or application of the synthetic diacylglycerol oleyl-acetyl-glycerol. These responses were absent in TRPC7-/- cells but could be restored by transfection with human TRPC7. In conclusion, in B-lymphocytes, TRPC7 appeared to participate in the formation of ion channels that could be activated by phospholipase C-linked receptors. This represents the first demonstration of a physiological function for endogenous TRPC7 channels.     

Diet-induced obesity leads to the development of leptin resistance in vagal afferent neurons

Ingestion of high-fat, high-calorie diets is associated with hyperphagia, increased body fat, and obesity. The mechanisms responsible are currently unclear; however, altered leptin signaling may be an important factor. Vagal afferent neurons (VAN) integrate signals from the gut in response to ingestion of nutrients and express leptin receptors. Therefore, we tested the hypothesis that leptin resistance occurs in VAN in response to a high-fat diet. Sprague-Dawley rats, which exhibit a bimodal distribution of body weight gain, were used after ingestion of a high-fat diet for 8 wk. Body weight, food intake, and plasma leptin levels were measured. Leptin signaling was determined by immunohistochemical localization of phosphorylated STAT3 (pSTAT3) in cultured VAN and by quantifaction of pSTAT3 protein levels by Western blot analysis in nodose ganglia and arcuate nucleus in vivo. To determine the mechanism of leptin resistance in nodose ganglia, cultured VAN were stimulated with leptin alone or with lipopolysaccharide (LPS) and SOCS-3 expression measured. SOCS-3 protein levels in VAN were measured by Western blot following leptin administration in vivo. Leptin resulted in appearance of pSTAT3 in VAN of low-fat-fed rats and rats resistant to diet-induced obesity but not diet-induced obese (DIO) rats. However, leptin signaling was normal in arcuate neurons. SOCS-3 expression was increased in VAN of DIO rats. In cultured VAN, LPS increased SOCS-3 expression and inhibited leptin-induced pSTAT3 in vivo. We conclude that VAN of diet-induced obese rats become leptin resistant; LPS and SOCS-3 may play a role in the development of leptin resistance.     

Zinc inactivates melastatin transient receptor potential 2 channels via the outer pore

Zinc ion (Zn(2+)) is an endogenous allosteric modulator that regulates the activity of a wide variety of ion channels in a reversible and concentration-dependent fashion. Here we used patch clamp recording to study the effects of Zn(2+) on the melastatin transient receptor potential 2 (TRPM2) channel. Zn(2+) inhibited the human (h) TRPM2 channel currents, and the steady-state inhibition was largely not reversed upon washout and concentration-independent in the range of 30-1000 μM, suggesting that Zn(2+) induces channel inactivation. Zn(2+) inactivated the channels fully when they conducted inward currents, but only by half when they passed outward currents, indicating profound influence of the permeant ion on Zn(2+) inactivation. Alanine substitution scanning mutagenesis of 20 Zn(2+)-interacting candidate residues in the outer pore region of the hTRPM2 channel showed that mutation of Lys(952) in the extracellular end of the fifth transmembrane segment and Asp(1002) in the large turret strongly attenuated or abolished Zn(2+) inactivation, and mutation of several other residues dramatically changed the inactivation kinetics. The mouse (m) TRPM2 channels were also inactivated by Zn(2+), but the kinetics were remarkably slower. Reciprocal mutation of His(995) in the hTRPM2 channel and the equivalent Gln(992) in the mTRPM2 channel completely swapped the kinetics, but no such opposing effects resulted from exchanging another pair of species-specific residues, Arg(961)/Ser(958). We conclude from these results that Zn(2+) inactivates the TRPM2 channels and that residues in the outer pore are critical determinants of the inactivation.     

Identification of two domains involved in the assembly of transient receptor potential canonical channels

Transient receptor potential canonical (TRPC) channels are associated with calcium entry activity in nonexcitable cells. TRPCs can form homo- or heterotetrameric channels, in which case they can assemble together within a subfamily groups. TRPC1, 4, and 5 represent one group, and TRPC3, 6, and 7 represent the other. The molecular determinants involved in promoting subunit tetramerization are not known. To identify them, we generated chimeras by swapping the different domains of TRPC4 with the same regions in TRPC6. We showed that TRPC4 coimmunoprecipitated with the chimeras containing the ankyrin repeats and coiled-coil domains of TRPC4 into TRPC6. However, chimeras containing only the ankyrin repeats or only the coiled-coil domain of TRPC4 did not coimmunoprecipitate with TRPC4. We also showed that a second domain of interaction composed of the pore region and the C-terminal tail is involved in the oligomerization of TRPC4. However, chimeras containing only the pore region or only the C-terminal tail of TRPC4 did not coimmunoprecipitate with TRPC4. Furthermore, we showed that the N terminus of TRPC6 coimmunoprecipitated with the C terminus of TRPC6. Overexpression in HEK293T cells of chimeras that contained an N terminus and a C terminus from different subfamily groups increased intracellular calcium entry subsequent to stimulation of G(q) protein-coupled receptors. These results suggest that two types of interactions are involved in the assembly of the four subunits of the TRPC channel. The first interaction occurs between the N termini and involves two regions. The second interaction occurs between the N terminus and the C terminus and does not appear to be necessary for the activity of TRPCs.     

Cholecystokinin increases cytosolic calcium in a subpopulation of cultured vagal afferent neurons

Imaging fluorescent measurements with fura 2 were used to examine cytosolic calcium signals induced by sulfated CCK octapeptide (CCK-8) in dissociated vagal afferent neurons from adult rat nodose ganglia. We found that 40% (184/465) of the neurons responded to CCK-8 with a transient increase in cytosolic calcium. The threshold concentration of CCK-8 for inducing the response varied from 0.01 to 100 nM. In most neurons (13/16) the response was eliminated by removing extracellular calcium. Depleting intracellular calcium stores with thapsigargin slightly augmented the response. Most neurons were unresponsive to nonsulfated CCK-8. The response was eliminated by the CCK-A receptor antagonist lorglumide. Low concentrations of JMV-180 had no effect; however, high concentrations of JMV-180 reduced responses to CCK-8. These results demonstrate that CCK acts at the low-affinity site of the CCK-A receptor to trigger the entry of extracellular calcium into vagal afferent neurons. Increased cytosolic calcium may participate in acute activation of vagal afferent neurons, or it may initiate long-term changes, which modulate future neuronal responses to sensory stimuli.     

Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas

Abstract.  Objectives : Glial-derived primary brain tumours, gliomas, are among the fastest growing malignancies and present a huge clinical challenge. Research suggests an important, yet poorly understood, role of ion channels in growth control of normal and malignant cells. In this study, we sought to functionally characterize Transient Receptor Potential Canoncial (TRPC) channels in glioma cell proliferation. TRPC channels form non-selective cation channels that have been suggested to represent a Ca²⁺ influx pathway impacting cellular growth. Materials and Methods : Employing a combination of molecular, biochemical and biophysical techniques, we characterized TRPC channels in glioma cells. Results : We showed consistent expression of four channel family members (TRPC-1, -3, -5, -6) in glioma cell lines and acute patient-derived tissues. These channels gave rise to small, non-voltage-dependent cation currents that were blocked by the TRPC inhibitors GdCl₃, 2-APB, or SKF96365. Importantly, TRPC channels contributed to the resting conductance of glioma cells and their acute pharmacological inhibition caused an ~10 mV hyperpolarization of the cells' resting potential. Additionally, chronic application of the TRPC inhibitor SKF96365 caused near complete growth arrest. A detailed analysis, by fluorescence-activated cell sorting and time-lapse microscopy, showed that growth inhibition occurred at the G₂+ M phase of the cell cycle with cytokinesis defects. Cells underwent incomplete cell divisions and became multinucleate, enlarged cells. Conclusions : Nuclear atypia and enlarged cells are histopathological hallmarks for glioblastoma multiforme , the highest grade glioma, suggesting that a defect in TRPC channel function may contribute to cellular abnormalities in these tumours.     

The involvement of transient receptor potential channels in mast cell activation by microbubbles

This study was to explore the activation of mast cells by microbubbles, with the focus on transient receptor potential (TRP) channels mediated degranulation and calcium influx. Bone marrow-derived mast cells (BMMCs) were primarily obtained from femurs in mice and induced differentiation for 4 weeks. After the purity identification, BMMCs were contacted by homogeneous microbubbles with the diameter of 1 mm for 1 h. β-hexosaminidase and histamine levels in supernatants were assessed by enzyme-linked immunosorbent assay (ELISA) and the CD63 expression was tested by flow cytometry. The intracellular calcium binding with Fluo-4 AM dyes in BMMCs was observed under the fluorescence microscope and the mean fluorescence intensity was quantitatively measured by flow cytometry. β-hexosaminidase release, histamine concentration, CD63 expression and calcium influx were significantly increased in BMMCs group upon microbubble stimulation compared to the control groups. After preconditioning with the available inhibitors and microbubble contact, only transient receptor potential vanilloid 1 (TRPV1) and TRPV4 inhibitors robustly suppressed the microbubble-induced degranulation. Likewise, the elevated fluorescence intensity of cytosolic calcium level was also significantly weaken. The results demonstrated microbubble stimulus effectively promoted BMMCs degranulation, which could be substantially restrained by inhibitors targeted for blocking TRPV1 or TRPV4 channel. The alternation of intracellular calcium level in BMMCs was consistent with the changes of degranulation capacity. It's suggested that the activation of BMMCs by microbubbles may involve specific TRP calcium dependent channels.     

Microscale grooves regulate maturation development of hPSC-CMs by the transient receptor potential channels (TRP channels)

The use of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited in drug discovery and cardiac disease mechanism studies due to cell immaturity. Micro-scaled grooves can promote the maturation of cardiomyocytes by aligning them in order, but the mechanism of cardiomyocytes alignment has not been studied. From the level of calcium activity, gene expression and cell morphology, we verified that the W20H5 grooves can effectively promote the maturation of cardiomyocytes. The transient receptor potential channels (TRP channels) also play an important role in the maturation and development of cardiomyocytes. These findings support the engineered hPSC-CMs as a powerful model to study cardiac disease mechanism and partly mimic the myocardial morphological development. The important role of the TRP channels in the maturation and development of myocardium is first revealed.     

Afidopyropen: New and potent modulator of insect transient receptor potential channels

The commercial insecticides pymetrozine and pyrifluquinazon control plant-sucking pests by disturbing their coordination and ability to feed. We have previously shown that these compounds act by overstimulating and eventually silencing vanilloid-type transient receptor potential (TRPV) channels, which consist of two proteins, Nanchung and Inactive, that are co-expressed exclusively in insect chordotonal stretch receptor neurons. Here we show that a new insecticidal compound, afidopyropen, modulates chordotonal organs of American grasshoppers (Schistocerca americana) in the same fashion. Afidopyropen stimulated heterologously expressed TRPV channels from two different insect species - fruit fly (Drosophila melanogaster) and pea aphid (Acyrthosiphon pisum) - but did not affect function of the mammalian TRPV channel TRPV4. Activation of the insect TRPVs required simultaneous expression of both Nanchung and Inactive proteins. Tritium-labeled afidopyropen bound fruit fly TRPVs with higher affinity than pymetrozine and competed with pymetrozine for binding. Nanchung protein formed the main binding interface for afidopyropen, whereas co-expression of Inactive dramatically increased binding affinity. Another modulator of chordotonal organs, flonicamid, did not activate insect TRPV channels, nor did it compete with afidopyropen for binding, indicating that it has a different target site. These results define afidopyropen as a new, potent and specific modulator of insect TRPV channels, and provide insight into the unique binding mode of these compounds.