Thermosensitive ion channel TRPV1 is endogenously expressed in the sperm of a fresh water teleost fish (Labeo rohita) and regulates sperm motility

Sperm cells exhibit extremely high sensitivity in response to slight changes in temperature, osmotic pressure and/or presence of various chemical stimuli. In most cases throughout the evolution, these physico-chemical stimuli trigger Ca²⁺-signaling and subsequently alter structure, cellular function, motility and survival of the sperm cells. Few reports have recently demonstrated the presence of Transient Receptor Potential (TRP) channels in the sperm cells from higher eukaryotes, mainly from higher mammals. In this work, we have explored if the sperm cells from lower vertebrates can also have thermo-sensitive TRP channels. In this paper, we demonstrate the endogenous presence of one specific thermo-sensitive ion channel, namely Transient Receptor Potential Vanilloid family member sub type 1 (TRPV1) in the sperm cells collected from fresh water teleost fish, Labeo rohita. By using western blot analysis, fluorescence assisted cell sorting (FACS) and confocal microscopy; we confirm the presence of this non-selective cation channel. Activation of TRPV1 by an endogenous activator NADA significantly increases the quality as well as the duration of fish sperm movement. The sperm cell specific expression of TRPV1 matches well with our in silico sequence analysis. The results demonstrate that TRPV1 gene is conserved in various fishes, ranging from 1–3 in copy number, and it originated by fish-specific duplication events within the last 320 million years (MY). To the best of our knowledge, this is the first report demonstrating the presence of any thermo-sensitive TRP channels in the sperm cells of early vertebrates as well as of aquatic animals, which undergo external fertilization in fresh water. This observation may have implications in the aquaculture, breeding of several fresh water and marine fish species and cryopreservation of fish sperms.     

Contraction of human colonic circular smooth muscle cells is inhibited by the calcium channel blocker pinaverium bromide

The effects of L-type calcium channel blockers (CCBs) selective for the gastrointestinal tract (pinaverium) or non-selective (nicardipine and diltiazem), were investigated on CCK-, CCh- or KCl-induced contraction of smooth muscle cells (SMC) isolated from the circular muscle layer of normal or of inflamed human colons. In the normal tissue colon, whatever the contractile agent used, CCK-8 (1nM), CCh (1nM) or KCl (20mM), a micromolar concentration of pinaverium significantly inhibited contraction (88.36%, 93.10%, 93.92% inhibition respectively); this effect was concentration-dependent for CCh (IC50 = 0.73 +/- 0.08nM) and for CCK (IC50 = 0.92 +/- 0.12nM). In parallel, both nicardipine and diltiazem inhibit significantly contraction of isolated SMC. In inflamed colons, pinaverium (1 microM) display a significant higher efficacy than diltiazem or nicardipine to reduce cell contraction induced by CCK-8 or by KCl. In addition, RT-PCR experiments were performed to evidence tissue specificity of the L-type calcium channel. They revealed the expression of the messenger of the a-1 subunit L-type calcium channel (binding site of such CCBs), consistent with the expression of the rbC-2 splice variant of the alpha1-C gene.In conclusion: (i) the inhibition by calcium channel blockers of agonist-induced contractile activity suggest a modulation of SMC contraction upon extracellular calcium via 'L-type' voltage-dependent calcium channel; (ii) this study provides a rationale for the clinical use of pinaverium in colonic motor disoders affecting the contractility of SMC, since it appeared to decrease the contraction even in pathological situation; and (iii) RT-PCR experiments confirms the presence in human colon SMC of the alpha-1 subunit mRNA of calcium channel.     

SLC26A9 is expressed in gastric surface epithelial cells, mediates Cl-/HCO3- exchange, and is inhibited by NH4+

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylori) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9.     

TRPV3, a thermosensitive channel is expressed in mouse distal colon epithelium

The thermo-transient receptor potential (thermoTRP) subfamily is composed of channels that are important in nociception and thermo-sensing. Here, we show a selective expression of TRPV3 channel in the distal colon throughout the gastrointestinal tract. Expression analyses clearly revealed that TRPV3 mRNA and proteins were expressed in the superficial epithelial cells of the distal colon, but not in those of the stomach, duodenum or proximal colon. In a subset of primary epithelial cells cultured from the distal colon, carvacrol, an agonist for TRPV3, elevated cytosolic Ca²⁺concentration in a concentration-dependent manner. This response was inhibited by ruthenium red, a TRPV channel antagonist. Organotypic culture supported that the carvacrol-responsive cells were present in superficial epithelial cells. Moreover, application of carvacrol evoked ATP release in primary colonic epithelial cells. We conclude that TRPV3 is present in absorptive cells in the distal colon and may be involved in a variety of cellular functions.     

Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.     

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na⁺ channel, we examined the effect of Nedd4-2 on the apical Ca²⁺ channel TRPV6, which is involved in transcellular Ca²⁺ transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When co-expressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca²⁺ influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.     

Basal cells express functional TRPV4 channels in the mouse nasal epithelium

Basal cells in the nasal epithelium (olfactory and airway epithelia) are stem/progenitor cells that are capable of dividing, renewing and differentiating into specialized cells. These stem cells can sense their biophysical microenvironment, but the underlying mechanism of this process remains unknown. Here, we demonstrate the prominent expression of the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca²⁺-permeable channel that is known to act as a sensor for hypo-osmotic and mechanical stresses, in the basal cells of the mouse nasal epithelium. TRPV4 mRNA was expressed in the basal portions of the prenatal mouse nasal epithelium, and this expression continued into adult mice. The TRPV4 protein was also detected in the basal layers of the nasal epithelium in wild-type but not in TRPV4-knockout (TRPV4-KO) mice. The TRPV4-positive immunoreactions largely overlapped with those of keratin 14 (K14), a marker of basal cells, in the airway epithelium, and they partially overlapped with those of K14 in the olfactory epithelium. Ca²⁺ imaging analysis revealed that hypo-osmotic stimulation and 4α-phorbol 12,13 didecanoate (4α-PDD), both of which are TRPV4 agonists, caused an increase in the cytosolic Ca²⁺ concentration in a subset of primary epithelial cells cultured from the upper parts of the nasal epithelium of the wild-type mice. This response was barely noticeable in cells from similar parts of the epithelium in TRPV4-KO mice. Finally, there was no significant difference in BrdU-labeled proliferation between the olfactory epithelia of wild-type and TRPV4-KO mice under normal conditions. Thus, TRPV4 channels are functionally expressed in basal cells throughout the nasal epithelium and may act as sensors for the development and injury-induced regeneration of basal stem cells.     

Regulation of the epithelial calcium channel TRPV6 by the serum and glucocorticoid-inducible kinase isoforms SGK1 and SGK3

Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ-domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.     

Molecular determinants for the chemical activation of the warmth-sensitive TRPV3 channel by the natural monoterpenoid carvacrol

Transient receptor potential vanilloid 3 (TRPV3), robustly expressed in the skin, is a nonselective calcium-permeable cation channel activated by warm temperature, voltage, and certain chemicals. Natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel. However, how carvacrol activates TRPV3 mechanistically remains to be understood. Here, we describe the molecular determinants for chemical activation of TRPV3 by the agonist carvacrol. Patch clamp recordings reveal that carvacrol activates TRPV3 in a concentration-dependent manner, with an EC₅₀ of 0.2 mM, by increasing the probability of single-channel open conformation. Molecular docking of carvacrol into cryo-EM structure of TRPV3 combined with site-directed mutagenesis further identified a unique binding pocket formed by the channel S2-S3 linker important for mediating this interaction. Within the binding pocket consisting of four residues (Ile505, Leu508, Arg509, and Asp512), we report that Leu508 is the most critical residue for the activation of TRPV3 by carvacrol, but not 2-APB, a widely used nonspecific agonist and TRP channel modulator. Our findings demonstrate a direct binding of carvacrol to TRPV3 by targeting the channel S2-S3 linker that serves as a critical domain for chemical-mediated activation of TRPV3. We also propose that carvacrol can function as a molecular tool in the design of novel specific TRPV3 modulators for the further understanding of TRPV3 channel pharmacology.     

Galectin-1 is expressed by thymic epithelial cells in myasthenia gravis

Galectin-1, a member of a family of carbohydrate binding proteins, is synthesized by thymic epithelial cells in normal juvenile thymus, and mediates adhesion of immature T cells to thymic epithelium. Because cell adhesion molecules are proposed to play a role in the thymic hyperplasia and neoplasia seen in the autoimmune disease myasthenia gravis, we examined the expression of galectin-1 in myasthenic thymi. We detected abundant galectin-1 expression in thymic epithelial cells in 27 hyperplastic and neoplastic thymi from patients with myasthenia gravis. Primary cultures of neoplastic epithelial cells from a thymoma continued to express galectin-1, and bound immature T cells; T cell binding was inhibited by the addition of the -galactosides lactose and thiodigalactoside, suggesting that galectin-1 on the thymoma cells and a saccharide ligand on the T cells participated in cell-cell adhesion. Expression of galectin-1 by thymic epithelial cells may play a role in the thymic pathology seen in myasthenia gravis.     

Alterations in the distribution of glycoproteins in epithelial cells of murine colon after injection of tunicamycin

Summary Glycoproteins are associated with several structures of colonic absorptive cells of the mouse. These include the cell coat, Golgi apparatus and vesicles that transport the glycoproteins from the apparatus to the cell surface (Michaels and Leblond 1976). In many in vitro systems, the antibiotic tunicamycin inhibits the glycosylation of asparagine residues yielding carbohydrate-poor glycoproteins. In the present in vivo study, tunicamycin was injected into mice. The murine colonic epithelial cells were prepared routinely for electron microscopy and cytochemistry. Cells from the experimental and control animals were similar morphologically. However, staining by the periodic acid-chromic acid-silver methenamine technique, revealed differences in the distribution of glycoproteins. In animals that received the higher dosages of tunicamycin there was a substantial reduction in silver staining in both the Golgi apparatus and the vesicles of colonic epithelial cells compared to these structures in cells of identically treated control tissues, whereas the staining over the cell coat was not significantly altered. Possible explanations for the staining of the cell coat in the treated animals were provided in the text. This report demonstrates the feasibility of using tunicamycin in vivo and detection of the changes obtained by the silver methenamine method.     

Ion permeation properties of a cloned human 5-HT3 receptor transiently expressed in HEK 293 cells

Summary Human 5-HT₃ receptors expressed in HEK 293 cells were studied using patch-clamp techniques. The permeability ratios of cations to Na⁺ were Li⁺, 1.16; K⁺, 1.04; Rb⁺, 1.11; Cs⁺ 1.11; NMDG⁺, 0.04; Ca²⁺, 0.49, and Mg²⁺, 0.37. The permeability sequence of the alkali metal cations was Lⁱ⁺ > Rb⁺ = Cs⁺ > K⁺ > Na⁺. Increased external concentrations of Ca²⁺ or Mg²⁺ decreased 5-HT-induced currents at all potentials tested in a voltage-independent manner. The single-channel conductance of human 5-HT₃ receptors measured by fluctuation analysis of whole-cell currents was 790 ± 100fS. Differences in the basic properties of 5-HT₃ receptors between species may explain interspecies differences in pharmacological properties.     

Enzastaurin-induced apoptosis in glioma cells is caspase-dependent and inhibited by BCL-XL

The novel protein kinase C-beta inhibitor enzastaurin (ENZA) induced apoptosis in LNT-229 and T98G cells whereas A172 cells were resistant. Further, ENZA reduced proliferation in glioblastoma-initiating cells T 269 and T 323 but did not induce apoptosis. ENZA-induced apoptosis involved cleavage of caspases 3, 8, and 9 and led to mitochondrial cytochrome c release and was strongly suppressed by the broad spectrum caspase inhibitor zVAD-fmk but only slightly by the expression of the viral caspase 1/8 inhibitor cytokine response modifier-A. ENZA did not reduce the phosphorylation of protein kinase B (Akt), but of p70 S6 kinase and of its substrate S6 protein in T98G cells. Inhibition of the phosphatidylinositol 3 kinase signaling pathway did not restore sensitivity of A172 cells towards ENZA, and constitutively active Akt did not protect LNT-229 and T98G cells from ENZA-induced apoptosis. Dephosphorylation of glycogen synthase kinase 3beta, a biomarker of ENZA action, and cell death induction by ENZA were separately regulated. Inhibition or activation of Akt only weakly modulated ENZA-induced dephosphorylation of glycogen synthase kinase 3beta. In ENZA-resistant A172 cells, apoptosis ligand 2 (Apo2L.0)-induced cleavage of caspases 3, 8, and 9 was increased by ENZA, resulting in synergistic activity of ENZA and Apo2L.0.     

Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells

Airway epithelial inflammation associated with emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and asthma is regulated in part by alveolar type II cell chemokine signaling. Data suggest that resident lung cells use CCR3, CCR5 and CCR2 chemokine receptor/ligand systems to regulate the profile of leukocytes recruited in disease-associated inflammatory conditions. Thus studies were designed to test whether alveolar type II cells possess a Th1-activated CCR5-ligand system that modulates the Th2-activated CCR3/eotaxin-2 (CCL24), eotaxin-3 (CCL26) chemokine systems. The A549 alveolar type II epithelial-like cell culture model was used to demonstrate that alveolar type II cells constitutively express CCR5 which may be upregulated by MIP-1alpha (CCL3) whose expression was induced by the Th1 cytokines IL-1beta and IFN-gamma. Selective down-regulation of CCL26, but not CCL24, was observed in CCL3 and IL-4/CCL3 stimulated cells. Down-regulation was reversed by anti-CCR5 neutralizing antibody treatment. Thus, one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Results suggest that the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to abrogate the chronic inflammation associated with airway diseases.