Transient receptor potential A1 mediates an osmotically activated ion channel

Transient receptor potential (TRP)A1 channel has been implicated in various physiological processes, including thermosensation and pain. A recent study of TRPA1 knockout mice demonstrated deficits in sensing mechanical stimuli, suggesting a role for TRPA1 also in somatic mechanosensation. However, direct evidence of TRPA1 activation by mechanical forces has thus far been lacking. Here we show, using an intracellular calcium assay, that hypertonic solution (HTS) activates TRPA1 channels in human embryonic kidney 293 cells transiently expressing rat TRPA1. In contrast, hypotonic solution has no effect. Single-channel recordings reveal that HTS opens an ion channel that displays similar single-channel conductance to that evoked by the TRPA1 agonist allyl isothiocyanate (AITC) in both recombinant rat TRPA1 cell lines and rat dorsal root ganglia neurons. Ruthenium red reduces the open probability of the single-channel currents and blocks the whole-cell currents evoked by HTS. Camphor also blocks the whole-cell currents evoked by HTS. HTS-activated channel openings are only observed in patches that are also sensitive to AITC. Finally, like AITC, HTS depolarizes the membrane potential of dorsal root ganglia neurons leading to the generation of action potentials. Taken together, these findings indicate that TRPA1 mediates an osmotically-activated ion channel and support a role for TRPA1 in mechanosensation.     

Transient receptor potential channel A1 involved in calcitonin gene-related peptide release in neurons

Transient receptor potential channel A1 is one of the important transducers of noxious stimuli in the primary afferents, which may contribute to generation of neurogenic inflammation and hyperalgesia. The present study was designed to investigate if activation of transient receptor potential channel A1 may induce calcitonin gene-related peptide release from the primary afferent neurons. We found that application of allyl isothiocyanate, a transient receptor potential channel A1 activator, caused calcitonin gene-related peptide release from the cultured rat dorsal root ganglion neurons. Knock- down of transient receptor potential channel A1 with an antisense oligodeoxynucleotide prevented calcitonin gene-related peptide release by allyl isothiocyanate application in cultured dorsal root ganglion neurons. Thus, we concluded that transient receptor potential channel A1 activation caused calcitonin gene-related peptide release in sensory neurons.     

Transient receptor potential vanilloid 1 and xenobiotics

Over the last couple of years, transient receptor potential vanilloid 1(TRPV1) channels have been a hot topic in ion channel research. Since this research field is still rather new, there is not much known about the working mechanism of TRPV1 and its ligands. Nevertheless, the important physiological role and therapeutic potential are promising. Therefore, extensive research is going on and a lot of natural as well as synthetic compounds are already described. In this review, we briefly give an overview of capsaicin's history and the current knowledge of its working mechanism and physiological role. We discuss the best known plant molecules acting on TRPV1 and highlight the latest discovery in TRPV1 research: animal venoms and toxins acting on TRPV1 channels. In an effort to give the complete image of TRPV1 ligands known today, the most promising synthetic compounds are presented. Finally, we present a novel pharmacophore model describing putative ligand binding domains.     

Subtype-dependent postnatal development of taste receptor cells in mouse fungiform taste buds

Taste buds contain two types of taste receptor cells, inositol 1,4,5-triphosphate receptor type 3-immunoreactive cells (type II cells) and synaptosomal-associating protein-25-immunoreactive cells (type III cells). We investigated their postnatal development in mouse fungiform taste buds immunohistochemically and electrophysiologically. The cell density, i.e. the number of cells per taste bud divided by the maximal area of the horizontal cross-section of the taste bud, of type II cells increased by postnatal day (PD)49, where as that of type III cells was unchanged throughout the postnatal observation period and was equal to that of the adult cells at PD1. The immunoreactivity of taste bud cell subtypes was the same as that of their respective subtypes in adult mice throughout the postnatal observation period. Almost all type II cells were immunoreactive to gustducin at PD1, and then the ratio of gustducin-immunoreactive type II cells to all type II cells decreased to a saturation level, ～60% of all type II cells, by PD15. Type II and III cells generated voltage-gated currents similar to their respective adult cells even at PD3. These results show that infant taste receptor cells are as excitable as those of adults and propagate in a subtype-dependent manner. The relationship between the ratio of each taste receptor cell subtype to all cells and taste nerve responses are discussed.     

Development of voltage-dependent currents in taste receptor cells

Taste buds, the specialized end organs of gustation, comprise a renewing sensory epithelium. Undifferentiated basal cells become taste receptor cells by elongating and extending processes apically toward the taste pore. Mature taste cells are electrically excitable and express voltage-dependent Na⁺ Ca²⁺, and K⁺ currents, whereas basal stem cells exhibit only slowly activating K⁺ currents. The question we have addressed in the present study is whether contact with the taste pore is required for expression of voltage-dependent inward currents in Necturus taste cells. Mature taste cells were distinguished from developing cells by labeling the apical surface of the cells with fluorescein-isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), while the tissue was still intact. Elongate cells lacking FITC-WGA staining were interpreted as developing taste cells that had not yet reached the taste pore. Giga-seal whole-cell recording revealed that most developing taste cells lacked inward currents. Although some developing cells expressed inward currents, they were much smaller than those of mature cells, and the activation kinetics of the K⁺ currents were slower than in mature cells. Electron microscopy confirmed the identity of labeled and unlabeled cells. All FITC-WGA-labeled cells exhibited the ultrastructural characteristics of mature taste receptor cells, whereas most unlabeled taste cells had a characteristic morphology that was markedly different from mature taste receptor cells or basal stem cells. These data suggest that contact with the taste pore is required for the development of inward currents in taste cells. © 1996 Wiley-Liss, Inc.     

Transient receptor potential A1 mediates acetaldehyde-evoked pain sensation

Six transient receptor potential (TRP) ion channels expressed in the sensory afferents play an important role as body thermosensors and also as peripheral pain detectors. It is known that a number of natural compounds specifically activate those sensory neuronal TRP channels, and a well-known example is cinnamaldehyde for TRPA1. Here we show that human and mouse TRPA1 are activated by acetaldehyde, an intermediate substance of ethanol metabolism, in the HEK293T cell heterologous expression system and in cultured mouse trigeminal neurons. Acetaldehyde failed to activate other temperature-sensitive TRP channels expressed in sensory neurons. TRPA1 antagonists camphor and gadolinium, and a general TRP blocker ruthenium red inhibited TRPA1 activation by acetaldehyde. Camphor, gadolinium and ruthenium red also suppressed the acute nociceptive behaviors induced by the intradermal administration of acetaldehyde into the mouse footpads. Intradermal co-application of prostaglandin E2 and acetaldehyde greatly potentiated the acetaldehyde-induced nociceptive responses, and this effect was reversed by treatment with the TRPA1 antagonist camphor. These results suggest that acetaldehyde causes nociception via TRPA1 activation. Our data may also help elucidate the mechanisms underlying acetaldehyde-related pathological symptoms such as hangover pain.     

Transient receptor potential protein subunit assembly and membrane distribution in human platelets

We have previously suggested that the human homologue of the Drosophila transient receptor potential protein, TRPC1, is involved in conducting store-operated Ca2+ entry (SOCE) in human platelets since an antibody raised against the pore-forming region of TRPC1 inhibited SOCE. Here we have investigated plasma membrane expression of TRPC1 in human platelets and have probed for the presence of other TRPC proteins in these cells. Biotinylation revealed the presence of TRPC1 in the plasma membrane of resting platelets. Surface expression was not detectibly changed following Ca2+ store depletion or stimulation with thrombin. Western blotting demonstrated the presence of TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 in platelet lysates. TRPC1, TRPC4 and TRPC5 coimmunoprecipitated, as did TRPC3 and TRPC6. TRPC1, TRPC4 and TRPC5 were associated with detergent-resistant platelet membranes, from which they were partially released when the cells were cholesterol-depleted using methyl-beta-cyclodextrin. The distributions of TRPC3 and TRPC6 between soluble and membrane fractions were not affected by methyl-beta-cyclodextrin treatment. These results suggest that TRPC1, TRPC4 and TRPC5 form a heteromultimer associated with platelet lipid raft domains, whereas TRPC3 and TRPC6 associate independently of lipid rafts.     

Characterization of umami receptor and coupling G protein in mouse taste cells

Taste receptor cells (TRCs) express multiple umami receptors. We performed physiological investigations to determine whether umami-responding cells in taste buds possess G protein-coupled receptors and to determine what type of G proteins exist if any. To clarify the components that participate in intracellular umami signal transduction in mouse, we recorded the activation of TRCs. TRCs treated with the G protein inhibitor GDP-beta-S lost umami-induced inward currents. Treatment with the Galphai inhibitor, pertussis toxin, did not increase the intracellular Ca2+ level in many TRCs. Immunohistochemical analysis revealed that a subset of TRCs responding to umami stimuli expressed alpha-gustducin. Thus, we demonstrated that umami stimuli were received by G protein-coupled receptors that function together with some of the Galphai family members.     

A method for isolating and patch-clamping single mammalian taste receptor cells

Individual taste receptor cells were isolated from the tongue of the mouse by enzymatic treatment followed by mechanical dissociation. The cells were morphologically identical with taste cells from amphibians. Whole-cell voltage-clamp recordings indicated that the murine taste cells possess a variety of voltage-dependent inward and outward currents. Delayed rectifier currents were blocked by denatonium benzoate, one of the most bitter compounds known. This preparation should permit a detailed electrophysiologcal investigation of taste transduction in mammals at the level of taste receptor cells.     

Epithelial Na+ channel subunits in rat taste cells: localization and regulation by aldosterone

Amiloride-sensitive Na+ channels play an important role in transducing Na+ salt taste. Previous studies revealed that in rodent taste cells, the channel shares electrophysiological and pharmacological properties with the epithelial Na+ channel, ENaC. Using subunit-specific antibodies directed against alpha, beta, and gamma subunits of rat ENaC (rENaC), we observed cytoplasmic immunoreactivity for all three subunits in nearly all taste cells of fungiform papillae, and in about half of the taste cells in foliate and vallate papillae. The intensity of labeling in cells of vallate papillae was significantly lower than that of fungiform papillae, especially for beta and gamma subunits. Dual localization experiments showed that immunoreactivity for the taste cell-specific G protein, gustducin, occurs in a subset ofrENaC positive taste cells. Aldosterone is known to increase the amiloride sensitivity of the NaCl taste response. In our study, increases in blood aldosterone levels enhanced the intensity of apical immunoreactivity for beta and gamma rENaC in taste cells of all papillae. In addition, whole cell recordings from isolated taste cells showed that in fungiform papillae, aldosterone increased the number of amiloride-sensitive taste cells and enhanced the current amplitude. In vallate taste cells, which are normally unresponsive to amiloride, aldosterone treatment induced an amiloride sensitive current in about half of the cells. Immunoreactivity for rENaC subunits also was present in nonsensory epithelial cells, especially in the anterior portion of the tongue. In addition, immunoreactivity for all subunits, but especially beta and gamma, was associated with some nerve fibers innervating taste papillae. These extragustatory sites of rENaC expression may indicate a role for this channel in paracellular transduction of sodium ions.     

Gustatory innervation and bax-dependent caspase-2: participants in the life and death pathways of mouse taste receptor cells

In the adult mouse tongue, an average of 11% of the gustatory receptor cells are replaced each day. In investigating homeostatic cell death mechanisms in gustatory renewing epithelium, we observed that taste receptor cells were selectively immunopositive for the bcl-2 family death factor, Bax, and for the protease Caspase-2 (Nedd2/Ich1). We determined that 8-10% of the taste receptor cells of the vallate papilla were Bax positive and that 11% were Caspase-2 positive. Some of these immunopositive taste cells had apoptotic morphological defects. Within the subset of vallate taste cells immunopositive for either Caspase-2 or Bax, up to 79% coexpressed both death factors. Bax and Caspase-2 first appeared in occasional vallate taste receptor cells on the same postnatal day-the day after birth. bax null mutation markedly reduced gustatory Caspase-2 immunoexpression. These observations suggest that taste cell death pathways utilize p53, Bax, and Caspase-2 to dispose of aged receptor cells. Apart from reducing Caspase-2 expression, Bax deficiency also altered taste organ development. bax(-/-) mice had a more profusely innervated vallate papilla, which grew to be 25% longer and taller, with the mean taste bud containing more than twice the normal number of taste cells. This augmentation of taste organ development with increased innervation is complementary to the well-documented reduction in taste organ development with sparse innervation. We propose that additional taste neurons survived programmed cell death in Bax-deficient mice, thereby providing an inductive boost to vallate gustatory development.     

Ionic mechanism of generation of receptor potential in response to quinine in frog taste cell

The ionic mechanism of generation of the receptor potential in a frog taste cell elicited by quinine-HCl (Q-HCl) was studied with an intracellular recording technique by replacing the superficial and interstitial fluids of the tongue with various saline solutions. The taste cells whose receptor membranes were adapted to normal saline and deionized water generated depolarizing receptor potentials at Q-HCl concentrations higher than 2 and 0.01 mM, respectively. The input resistance of taste cell during Q-HCl stimulation scarcely changed. The receptor potential did not change even when the membrane potential level was broadly changed. The magnitude of the receptor potential was increased by reducing the concentration of superficial Cl⁻ on the taste receptor membrane, but was independent to the concentration of superficial Na⁺. Injection of Cl⁻ into a taste cell increased the receptor potential to 170%. The magnitude of receptor potential was decreased to 20–30% by removing interstitial Na⁺ or Cl⁻ or both surrounding the basolateral membrane of taste cell. Furosemide (1 mM) added to the interstitial fluid decreased the receptor potential to 15%, while interstitial ouabain (0.1 mM) and superficial SITS (0.1 mM) did not influence it. From these results, we conclude: (1) an electroneutral Na⁺/Cl⁻ cotransport occurs through the basolateral membrane of a taste cell in the resting state, so that Cl⁻ accumulates inside the cell. (2) Q-HCl stimulation induces the active secretion of Cl⁻ across the taste receptor membrane, resulting in a depolarizing receptor potential.     

Effects of metabotropic glutamate receptor 5 on latent inhibition in conditioned taste aversion

Latent inhibition (LI) is a phenomenon by which pre-exposure of a conditioned stimulus (CS) prior to the CS-unconditioned stimulus (US) pairings retards conditioned responding (CR). LI has been demonstrated in a variety of learning tasks including conditioned taste aversion (CTA). Earlier work has shown that systemic administration of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective metabotropic glutamate receptor 5 (mGlu5) antagonist, is able to disrupt classical conditioning in CTA. The present study investigated the involvement of mGlu5 receptors in LI using a CTA procedure. In the first experiment, rats received either water (non-pre-exposed, NPE) or a saccharin solution (pre-exposed, PE) on 2 consecutive days. The animals then received conditioning in which a fixed amount of saccharin was paired with lithium chloride and then the CR to the taste was tested. Either MPEP (3, 6, 12 mg/kg) or vehicle was injected intraperitoneally prior to taste pre-exposure or testing. Animals in the vehicle control groups displayed LI. MPEP injections before pre-exposure trials attenuated LI but also reduced consumption during pre-exposure, which obscured interpretation of the LI effect. The second experiment used four pre-exposure trials and controlled access to fixed amount of the solutions during the pre-exposure as well as the conditioning trials. Rats were injected before pre-exposure trials but not before the test trial. The results found that MPEP attenuates latent inhibition suggesting that the mGlu5 receptor exerts an influence on the processes that underlie the effects of taste pre-exposure on conditioning.     

Tacrine interacts with different sites on nicotinic receptor subtypes in SH-SY5Y neuroblastoma and M10 cells

The effect of chronic treatment with the cholinesterase inhibitor tacrine on nicotinic receptor subtypes was investigated in human SH-SY5Y neuroblastoma cells and in a fibroblast cell line (M10 cells) stably transfected with alpha4beta2 nicotinic receptors. Tacrine significantly increased the number of nicotinic receptors in SH-SY5Y cells, in a concentration dependent manner (10(-9) to 10(-4) M), when using [3H]epibatidine as labelled ligand. Chronic tacrine treatment of M10 cells significantly increased and decreased the number of alpha4beta2 nicotinic receptors in a concentration dependent manner (10(-9) to 5 x 10(-6) M and 2 x 10(-5) to 10(-4) M, respectively). The tacrine induced increase of nicotinic receptors in SH-SY5Y cells, was not blocked in the presence of the nicotinic antagonists tubocurarine or mecamylamine. A further increase in the number of nicotinic receptors was, however, observed in the presence of mecamylamine. This study demonstrates that the effect of tacrine on the number of nicotinic receptor subtypes is different in human SH-SY5Y neuroblastoma and M10 cells. The up-regulation of different nicotinic receptor subtypes obtained with tacrine might be achieved through interaction via different binding sites on the receptor, i.e. the acetylcholine binding site as well as an allosteric site.