Localization of TRP Channels in Healthy Oral Mucosa from Human Donors.

The oral cavity is exposed to a remarkable range of noxious and innocuous conditions, including temperature fluctuations, mechanical forces, inflammation, and environmental and endogenous chemicals. How such changes in the oral environment are sensed is not completely understood. Transient receptor potential (TRP) ion channels are a diverse family of molecular receptors that are activated by chemicals, temperature changes, and tissue damage. In non-neuronal cells, TRP channels play roles in inflammation, tissue development, and maintenance. In somatosensory neurons, TRP channels mediate nociception, thermosensation, and chemosensation. To assess whether TRP channels might be involved in environmental sensing in the human oral cavity, we investigated their distribution in human tongue and hard palate biopsies. TRPV3 and TRPV4 were expressed in epithelial cells with inverse expression patterns where they likely contribute to epithelial development and integrity. TRPA1 immunoreactivity was present in fibroblasts, immune cells, and neuronal afferents, consistent with known roles of TRPA1 in sensory transduction and response to damage and inflammation. TRPM8 immunoreactivity was found in lamina propria and neuronal subpopulations including within the end bulbs of Krause, consistent with a role in thermal sensation. TRPV1 immunoreactivity was identified in intraepithelial nerve fibers and end bulbs of Krause, consistent with roles in nociception and thermosensation. TRPM8 and TRPV1 immunoreactivity in end bulbs of Krause suggest that these structures contain a variety of neuronal afferents, including those that mediate nociception, thermosensation, and mechanotransduction. Collectively, these studies support the role of TRP channels in oral environmental surveillance and response.

Somatosensory innervation of healthy human oral tissues.

The oral somatosensory system relays essential information about mechanical stimuli to enable oral functions such as feeding and speech. The neurochemical and anatomical diversity of sensory neurons across oral cavity sites have not been systematically compared. To address this gap, we analyzed healthy human tongue and hard-palate innervation. Biopsies were collected from 12 volunteers and underwent fluorescent immunohistochemistry (≥2 specimens per marker/structure). Afferents were analyzed for markers of neurons (ßIII tubulin), myelinated afferents (neurofilament heavy, NFH), and Merkel cells and taste cells (keratin 20, K20). Hard-palate innervation included Meissner corpuscles, glomerular endings, Merkel cell-neurite complexes, and free nerve endings. The organization of these somatosensory endings is reminiscent of fingertips, suggesting that the hard palate is equipped with a rich repertoire of sensory neurons for pressure sensing and spatial localization of mechanical inputs, which are essential for speech production and feeding. Likewise, the tongue is innervated by afferents that impart it with exquisite acuity and detection of moving stimuli that support flavor construction and speech. Filiform papillae contained end bulbs of Krause, as well as endings that have not been previously reported, including subepithelial neuronal densities, and NFH+ neurons innervating basal epithelia. Fungiform papillae had Meissner corpuscles and densities of NFH+ intraepithelial neurons surrounding taste buds. The differing compositions of sensory endings within filiform and fungiform papillae suggest that these structures have distinct roles in mechanosensation. Collectively, this study has identified previously undescribed neuronal endings in human oral tissues and provides an anatomical framework for understanding oral mechanosensory functions.

New natural agonists of the transient receptor potential Ankyrin 1 (TRPA1) channel.

The transient receptor potential (TRP) channels family are cationic channels involved in various physiological processes as pain, inflammation, metabolism, swallowing function, gut motility, thermoregulation or adipogenesis. In the oral cavity, TRP channels are involved in chemesthesis, the sensory chemical transduction of spicy ingredients. Among them, TRPA1 is activated by natural molecules producing pungent, tingling or irritating sensations during their consumption. TRPA1 can be activated by different chemicals found in plants or spices such as the electrophiles isothiocyanates, thiosulfinates or unsaturated aldehydes. TRPA1 has been as well associated to various physiological mechanisms like gut motility, inflammation or pain. Cinnamaldehyde, its well known potent agonist from cinnamon, is reported to impact metabolism and exert anti-obesity and anti-hyperglycemic effects. Recently, a structurally similar molecule to cinnamaldehyde, cuminaldehyde was shown to possess anti-obesity and anti-hyperglycemic effect as well. We hypothesized that both cinnamaldehyde and cuminaldehyde might exert this metabolic effects through TRPA1 activation and evaluated the impact of cuminaldehyde on TRPA1. The results presented here show that cuminaldehyde activates TRPA1 as well. Additionally, a new natural agonist of TRPA1, tiglic aldehyde, was identified and p-anisaldehyde confirmed.

A randomized clinical trial on the acute therapeutic effect of TRPA1 and TRPM8 agonists in patients with oropharyngeal dysphagia.

BACKGROUND: Oropharyngeal dysphagia (OD) treatment is moving away from compensatory strategies toward active treatments that improve swallowing function. The aim of this study was to assess the acute therapeutic effect of TRPA1/M8 agonists in improving swallowing function in OD patients. METHODS: Fifty-eight patients with OD caused by aging, stroke, or neurodegenerative disease were included in a three-arm, quadruple-blind, randomized clinical trial (NCT02193438). Swallowing safety and efficacy and the kinematics of the swallow response were assessed by videofluoroscopy (VFS) during the swallow of 182 ± 2 mPa·s viscosity (nectar) boluses of a xanthan gum thickener supplemented with (a) 756.6 µmol/L cinnamaldehyde and 70 µmol/L zinc (CIN-Zn) (TRPA1 agonists), (b) 1.6 mmol/L citral (CIT) (TRPA1 agonist), or (c) 1.6 mmol/L citral and 1.3 mmol/L isopulegol (CIT-ISO) (TRPA1 and TRPM8 agonists). The effects on pharyngeal event-related potentials (ERP) were assessed by electroencephalography. KEY RESULTS: TRPA1 stimulation with either CIN-Zn or CIT reduced time to laryngeal vestibule closure (CIN-Zn P = .002, CIT P = .023) and upper esophageal sphincter opening (CIN-Zn P = .007, CIT P = .035). In addition, CIN-Zn reduced the penetration-aspiration scale score (P = .009), increased the prevalence of safe swallows (P = .041), and reduced the latency of the P2 peak of the ERP. CIT-ISO had no positive effect on biomechanics or neurophysiology. No significant adverse events were observed. CONCLUSIONS AND INFERENCES: TRPA1 stimulation with CIN-Zn or CIT improves the swallow response which, in the case of CIN-Zn, is associated with a significant improvement in cortical activation and safety of swallow. These results provide the basis for the development of new active treatments for OD using TRPA1 agonists.

Claudin-based permeability barriers in taste buds.

Tight junctions operate as semipermeable barriers in epithelial tissue, separating the apical from the basolateral sides of the cells. Membrane proteins of the claudin family represent the major tight junction constituents, and some reinforce permeability barriers, whereas others create pores based on solute size and ion selectivity. To outline paracellular permeability pathways in gustatory tissue, all claudins expressed in mouse taste buds and in human fungiform papillae have been characterized. Twelve claudins are expressed in murine taste-papillae-enriched tissue, and five of those are expressed in human fungiform papillae. A subset of the claudins expressed in mouse papillae is uniquely found in taste buds. By immunohistochemistry, claudin 4 has been found in mouse taste epithelium, with high abundance around the taste pore. Claudin 6 is explicitly detected inside the pore, claudin 7 was found at the basolateral side of taste cells, and claudin 8 was found around the pore. With the ion permeability features of the different claudins, a highly specific permeability pattern for paracellular diffusion is apparent, which indicates a peripheral mechanism for taste coding.

Effects of TRP channel agonist ingestion on metabolism and autonomic nervous system in a randomized clinical trial of healthy subjects.

Various lines of published evidence have already demonstrated the impact of TRPV1 agonists on energetic metabolism through the stimulation of the sympathetic nervous system (SNS). This study presents a trial investigating if stimulation of the two related sensory receptors TRPA1 and TRPM8 could also stimulate the SNS and impact the energetic metabolism of healthy subjects. The trial was designed to be double-blinded, randomized, cross-over, placebo-controlled with healthy subjects and the impact on the energetic metabolism and the autonomic nervous system (ANS) of cinnamaldehyde, capsaicin and a cooling flavor was measured during the 90 min after ingestion. Energy expenditure and substrate oxidation were measured by indirect calorimetry. An exploratory method to measure ANS activity was by facial thermography and power spectral analysis of heart rate variability using ECG was also used. Following cinnamaldehyde ingestion, energy expenditure was increased as compared to placebo. Furthermore, postprandial fat oxidation was maintained higher compared to placebo after cinnamaldehyde and capsaicin ingestion. Similar peripheral thermoregulation was observed after capsaicin and cinnamaldehyde ingestion. Unlike capsaicin, the dose of cinnamaldehyde was not judged to be sensorially 'too intense' by participants suggesting that Cinnamaldehyde would be a more tolerable solution to improve thermogenesis via spicy ingredients as compared to capsaicin.

Anti-obesity and anti-hyperglycemic effects of cinnamaldehyde via altered ghrelin secretion and functional impact on food intake and gastric emptying.

Cinnamon extract is associated to different health benefits but the active ingredients or pathways are unknown. Cinnamaldehyde (CIN) imparts the characteristic flavor to cinnamon and is known to be the main agonist of transient receptor potential-ankyrin receptor 1 (TRPA1). Here, expression of TRPA1 in epithelial mouse stomach cells is described. After receiving a single-dose of CIN, mice significantly reduce cumulative food intake and gastric emptying rates. Co-localization of TRPA1 and ghrelin in enteroendocrine cells of the duodenum is observed both in vivo and in the MGN3-1 cell line, a ghrelin secreting cell model, where incubation with CIN up-regulates expression of TRPA1 and Insulin receptor genes. Ghrelin secreted in the culture medium was quantified following CIN stimulation and we observe that octanoyl and total ghrelin are significantly lower than in control conditions. Additionally, obese mice fed for five weeks with CIN-containing diet significantly reduce their cumulative body weight gain and improve glucose tolerance without detectable modification of insulin secretion. Finally, in adipose tissue up-regulation of genes related to fatty acid oxidation was observed. Taken together, the results confirm anti-hyperglycemic and anti-obesity effects of CIN opening a new approach to investigate how certain spice derived compounds regulate endogenous ghrelin release for therapeutic intervention.

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.

ERK1/2 controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell of the cortical collecting duct of the mouse kidney.

The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 pathway as shown in vivo by immunostaining of phosphorylated active ERK1/2 (pERK1/2). The MEK1/2-ERK1/2 pathway controls many different ion transports both in proximal and distal nephron, raising the question of whether this pathway is involved in the basal and/or hormone-dependent transepithelial sodium reabsorption in the principal cell of the cortical collecting duct (CCD), a process mediated by the apical epithelial sodium channel and the basolateral sodium pump (Na,K-ATPase). To answer this question we used ex vivo microdissected CCDs from normal mouse kidney or in vitro cultured mpkCCDcl4 principal cells. Significant basal levels of pERK1/2 were observed ex vivo and in vitro. Aldosterone and vasopressin, known to up-regulate sodium reabsorption in CCDs, did not change ERK1/2 activity either ex vivo or in vitro. Basal and aldosterone- or vasopressin-stimulated sodium transport was down-regulated by the MEK1/2 inhibitor PD98059, in parallel with a decrease in pERK1/2 in vitro. The activity of Na,K-ATPase but not that of epithelial sodium channel was inhibited by MEK1/2 inhibitors in both unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface biotinylation showed that intrinsic activity rather than cell surface expression of Na,K-ATPase was controlled by pERK1/2. PD98059 also significantly inhibited the activity of Na,K-ATPase ex vivo. Our data demonstrate that the ERK1/2 pathway controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell and indicate that basal constitutive activity of the ERK1/2 pathway is a critical component of this control.

A novel vasopressin-induced transcript promotes MAP kinase activation and ENaC downregulation.

In the principal cell of the renal collecting duct, vasopressin regulates the expression of a gene network responsible for sodium and water reabsorption through the regulation of the water channel and the epithelial sodium channel (ENaC). We have recently identified a novel vasopressin-induced transcript (VIT32) that encodes for a 142 amino acid vasopressin-induced protein (VIP32), which has no homology with any protein of known function. The Xenopus oocyte expression system revealed two functions: (i) when injected alone, VIT32 cRNA rapidly induces oocyte meiotic maturation through the activation of the maturation promoting factor, the amphibian homolog of the universal M phase trigger Cdc2/cyclin; and (ii) when co-injected with the ENaC, VIT32 cRNA selectively downregulates channel activity, but not channel cell surface expression. In the kidney principal cell, VIP32 may be involved in the downregulation of transepithelial sodium transport observed within a few hours after vasopressin treatment. VIP32 belongs to a novel gene family ubiquitously expressed in oocyte and somatic cells that may be involved in G to M transition and cell cycling.