2-Aryl substituted pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as highly potent TRPV1 antagonists

A series of 2-aryl pyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Multiple compounds showed highly potent TRPV1 antagonism toward capsaicin comparable to previous lead 7. Among them, compound 9 demonstrated anti-allodynia in a mouse neuropathic pain model and blocked capsaicin-induced hypothermia in a dose-dependent manner. Docking analysis of 9 with our hTRPV1 homology model provided insight into its specific binding mode.     

Expression and characteristics of vanilloid receptor 1 in the rabbit submandibular gland

Vanilloid receptor 1 (VR1) is a polymodal receptor originally found in sensory neurons of the central nervous system. Recent evidence indicates that VR1 is also expressed in non-neuronal tissues. We report here endogenous expression of VR1 in rabbit submandibular gland (SMG) and its possible role in regulating saliva secretion based on: (i) the expression of VR1 mRNA and protein detected in SMG; (ii) VR1 was mainly localized in the basolateral membrane of duct cells and the cytoplasm of acinar cells and also in cytoplasm of primary cultured neonatal rabbit SMG cells; (iii) stimulation of neonatal rabbit SMG cells with capsaicin induced a significant increase in intracellular calcium, and capsazepine, a VR1 antagonist, abolished this increase; (iv) infusion of capsaicin via the external carotid artery to isolated SMG increased saliva secretion of the gland. These findings indicated that VR1 was expressed in SMG and appeared to play an important role in regulating saliva secretion.     

Overexpression and purification of the vanilloid receptor in yeast (Saccharomyces cerevisiae)

The vanilloid receptor type 1 (VR1) is a novel membrane receptor activated by heat or chemical ligands conveying information about chemosensitive and thermosensitive pain. We have overexpressed and purified wild type VR1 (wtVR1) as well as several mutant forms using the yeast strain Saccharomyces cerevisiae with the goal of obtaining sufficient protein for structural studies. To facilitate the rapid assaying of protein production and purification we used PCR to construct mutant VR1-green fluorescent protein fusion genes. All recombinant inserts were engineered with 12 HIS tags on the C-terminus for metal affinity column purification. The yield of purified protein from 16L fermentation was about 1mg following a single-step purification procedure. By taking advantage of the calcium permeability of VR1 we measured changes in [Ca(2+)](i) in capsaicin-stimulated fura-2 loaded yeast cells expressing VR1.     

4-Aminophenyl acetamides and propanamides as potent transient receptor potential vanilloid 1 (TRPV1) ligands

A series of 2-(3,5-substituted 4-aminophenyl)acetamide and propanamide derivatives were investigated as human TRPV1 antagonists. The analysis of the structure-activity relationship indicated that 2-(3,5-dihalo 4-aminophenyl)acetamide analogues displayed excellent antagonism of hTRPV1 activation by capsaicin and showed improved potency compared to the corresponding propanamides. The most potent antagonist (36) exhibited potent and selective antagonism for hTRPV1 not only to capsaicin but also to NADA and elevated temperature; however, it only displayed weak antagonism to low pH. Further studies indicated that oral administration of antagonist 36 blocked the hypothermic effect of capsaicin in vivo but demonstrated hyperthermia at that dose. A docking study of 36 was performed in our established hTRPV1 homology model to understand its binding interactions with the receptor and to compare with that of previous antagonist 1.     

Distribution of the vanilloid (capsaicin) receptor type 1 in the human stomach

Vanilloid receptor type 1 (TRPV1) is expressed in a capsaicin-sensitive and peptide-containing sub-population of primary sensory nerves that in the rat stomach seems involved in regulation of chlorhydropeptic secretion and gastroprotection. Our aim was to identify which cell types express TRPV1 in the human stomach in order to gain a better insight in the role of this receptor in the regulation of HCl secretion. Immunohistochemistry, by using three different commercially available anti-capsaicin antibodies, in situ hybridisation and Western blot analysis were performed on fragments surgically obtained from the gastric body on the large curvature. TRPV1 labelling was found in the parietal cells at the level of intra-cytoplasmatic granules matching mitochondrial features and distribution. Immunolabelled neurons and nerve fibres were also seen, the latter numerous in the submucosa and mucosa and often ending close to the parietal cells. TRPV1 presence was confirmed by Western blot analysis and in situ hybridisation. TRPV1 presence in nerve structures and parietal cells suggests the possibility of a combined effect of both neuronal and epithelial TRPV1 on chlorhydropeptic secretion. The presumed TRPV1 mitochondrial location inside parietal cells is in favour of the existence of a local pathway of auto-regulation of HCl secretion. Therefore, TRPV1 might modulate chlorhydropeptic secretion in the human stomach through more complex pathways than previously thought.     

Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo

RNA interference (RNAi) has proven to be a powerful technique to study the function of genes by producing knock-down phenotypes. Here, we report that intrathecal injection of an siRNA against the transient receptor potential vanilloid receptor 1 (TRPV1) reduced cold allodynia of mononeuropathic rats by more than 50% over a time period of approximately 5 days. A second siRNA targeted to a different region of the TRPV1 gene was employed and confirmed the analgesic action of a TRPV1 knock-down. Furthermore, siRNA treatment diminished spontaneous visceral pain behavior induced by capsaicin application to the rectum of mice. The analgesic effect of siRNA-mediated knockdown of TRPV1 in the visceral pain model was comparable to that of the low-molecular weight receptor antagonist BCTC. Our data demonstrate that TRPV1 antagonists, including TRPV1 siRNAs, have potential in the treatment of both, neuropathic and visceral pain.     

Transient receptor potential vanilloid 1-mediated expression and secretion of endothelial cell-derived calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP), the principal transmitter in sensory nerves, could also be expressed in vascular endothelium. Transient receptor potential vanilloid 1(TRPV1), which modulates the synthesis and release of CGRP in sensory nerves, is also present in endothelial cells. The present study tested whether TRPV1 modulates the release and synthesis of CGRP in endothelial cells, and evaluated the protective effect of endothelial cell-derived CGRP. Human umbilical vein endothelial cells (HUVECs) were treated with capsaicin or hyperthermia. The level of CGRP mRNA was detected by RT-PCR, and protein level was measured by radioimmunoassay. Endothelial cell injury was induced by lysophosphatidylcholine, and evaluated by cell viability and lactate dehydrogenase activity. HUVECs expressed CGRP, both alpha- and beta-subtype. Capsaicin increased the level of CGRP in the culture medium, and up-regulated the expression of CGRP in endothelial cells. Hyperthermia also increased the level of CGRP mRNA. These effects were abolished by capsazepine, a competitive antagonist of TRPV1. Capsaicin significantly attenuated the endothelial cell damage induced by LPC, which was prevented and aggravated by capsazepine or CGRP(8-37,) antagonist of CGRP receptor. These results indicate that TRPV1 also regulates the expression and secretion of endothelial cell-derived CGRP, which affords protective effects on endothelial cells.     

Discovery of potent transient receptor potential vanilloid 1 antagonists: Design and synthesis of phenoxyacetamide derivatives

We aimed to discover a novel type of transient receptor potential vanilloid 1 (TRPV1) antagonist because such antagonists are possible drug candidates for treating various disorders. We modified the structure of hit compound 7 (human TRPV1 IC₅₀ = 411 nM) and converted its pyrrolidino group to a (hydroxyethyl)methylamino group, which substantially improved inhibitory activity (15d; human TRPV1 IC₅₀ = 33 nM). In addition, 15d ameliorated bladder overactivity in rats in vivo.     

[11C]BCTC: Radiosynthesis and in vivo binding to transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor in the mouse trigeminal nerve

The purpose of this study was to synthesize a new positron emission tomography radiotracer, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-[¹¹C]carboxamide ([¹¹C]BCTC, [¹¹C]1), and assess its in vivo binding to the transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor in mice. [¹¹C]BCTC was synthesized by reacting the hydrochloride of 4-tertiarybutylaniline (2·HCl) with [¹¹C]phosgene ([¹¹C]COCl₂) to give isocyanate [¹¹C]4, followed by reaction with 4-(3-chloropyridin-2-yl)tetrahydropyrazine (3). [¹¹C]BCTC was obtained at a 16–20% radiochemical yield, based on the cyclotron-produced [¹¹C]CO₂ (decay-corrected to the end of bombardment), with >98% radiochemical purity and 65–110 GBq/μmol specific activity at the end of synthesis. An ex vivo biodistribution study in mice confirmed the specific binding of [¹¹C]BCTC to TRPV1 in the trigeminal nerve, which is a tissue with high TRPV1 expression.     

A CHO cell line engineered to express XBP1 and ERO1‐Lα has increased levels of transient protein expression

Transient gene expression (TGE) systems currently provide rapid and scalable (up to 100 L) methods for generating multigram quantities of recombinant heterologous proteins. Product titers of up to 1 g/L have been demonstrated in HEK293 cells 1 but reported yields from Chinese hamster ovary (CHO) cells are lower at ～300 mg/L. 2 We report on the establishment of an engineered CHOS cell line, which has been developed for TGE. This cell line has been engineered to express both X‐box binding protein (XBP‐1S) and endoplasmic reticulum oxidoreductase (ERO1‐Lα) and has been named CHOS‐XE. CHOS‐XE cells produced increased antibody (MAb) yields (5.3– 6.2 fold) in comparison to CHOS cells. Product quality was unchanged as assessed by size, charge, propensity to aggregate, major glycosylation species, and thermal stability. To further develop and test this TGE system, five commercial media were assessed, and one was shown to offer the greatest increase in antibody yields. With the addition of a commercial feed, MAb titers reached 875 mg/L. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:697–706, 2013     

Dissection of protease-activated receptor-1-dependent and -independent responses to thrombin in skeletal myoblasts

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.     

Pigment epithelium-derived factor (PEDF) binds to caveolin-1 and inhibits the pro-inflammatory effects of caveolin-1 in endothelial cells

Pigment epithelium-derived factor (PEDF) exerts atheroprotective effects both in cell culture and animal models through its anti-oxidative and anti-inflammatory properties. Caveolin-1 (Cav), a major protein component of caveolae in endothelial cells (ECs), plays a role in the progression of atherosclerosis. However, effects of PEDF on Cav-exposed ECs remain unknown. In this study, we examined whether and how PEDF could inhibit the Cav-induced inflammatory and thrombogenic reactions in human umbilical vein ECs (HUVECs). Surface plasmon resonance revealed that PEDF bound to Cav at the dissociation constant of 7.36 × 10⁻⁷ M. Further, one of the major Cav-interacting proteins in human serum was identified as PEDF by peptide mass fingerprinting analysis using BIAcore 1000 combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Exogenously added Cav was taken up into the membrane fraction of HUVECs and dose-dependently increased monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels, all of which were blocked by the simultaneous treatment with 10 nM PEDF. Small interfering RNAs directed against Cav decreased endogenous Cav levels and suppressed gene expression of MCP-1, VCAM-1 and PAI-1 in HUVECs. This study indicates that PEDF binds to Cav and could block the inflammatory and thrombogenic reactions in Cav-exposed HUVECs. Our present study suggests that atheroprotective effects of PEDF might be partly ascribed to its Cav-interacting properties.     

Germanium oxide inhibits the transition from G2 to M phase of CHO cells

We report here for the first time that germanium oxide (GeO(2)) blocks cell progression. GeO(2) is not genotoxic to Chinese hamster ovary (CHO) cells and has limited cytotoxicity. However, GeO(2) arrests cells at G2/M phase. The proportion of cells stopped at G2/M phase increased dose-dependently up to 5 mM GeO(2) when treated for 12 h, but decreased at GeO(2) concentration was greater than 5 mM. Analysis of 5-bromodeoxyuridine-labeled cells indicated that GeO(2) delayed S phase progression in a dose-dependent manner, and blocked cells at G2/M phase. Microscopic examination confirmed that GeO(2) treatment arrested cells at G2 phase. Similar to several other events that cause G2 block, the GeO(2)-induced G2 block can also be ameliorated by caffeine in a dose- and time-dependent manner. To explore the mechanism of G2 arrest by GeO(2), cyclin content and cyclin-dependent kinase activity were examined. Cyclin B1 level was not affected after GeO(2) treatment in CHO cells. However, GeO(2) decreased p34(cdc2) kinase (Cdk1) activity. The kinase activity recovered within 9 h after GeO(2) removal and correlated with the transition of G2/M-G1 phase of the cells. This result suggests that GeO(2) treatment reduces Cdk1 activity and causing the G2 arrest in CHO cells.     

Mercuric chloride inhibits the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

Glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1). Mercuric chloride (HgCl₂) is a highly toxic compound that inhibits glutamate uptake in astrocytes, resulting in excessive extracellular glutamate accumulation, leading to excitotoxicity and neuronal cell death. The mechanisms associated with the inhibitory effects of HgCl₂ on glutamate uptake are unknown. This study examines the effects of HgCl₂ on the transport of ³H-d-aspartate, a nonmetabolizable glutamate analog, using Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2), as a model system. Additionally, studies were undertaken to determine the effects of HgCl₂ on mRNA and protein levels of these transporters. The results indicate that (1) HgCl₂ leads to significant (p<0.001) inhibition of glutamate uptake via both transporters, but is a more potent inhibitor of glutamate transport via GLAST and (2) the effect of HgCl₂ on inhibition of glutamate uptake in transfected CHO cells is not associated with changes in transporter protein levels despite a significant decrease in mRNA expression; thus, (3) HgCl₂ inhibition is most likely related to its direct binding to the functional thiol groups of the transporters and interference with their uptake function.     

Fibulin-1 binds the amino-terminal head of beta-amyloid precursor protein and modulates its physiological function

Genetic studies have implicated amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease. While accumulating lines of evidence indicate that APP has various functions in cells, little is known about the proteins that modulate its biological activity. Toward this end, we employed a two-hybrid system to identify potential interacting factors. We now report that fibulin-1, which contains repetitive Ca(2+)-binding EGF-like elements, binds to APP at its amino-terminal growth factor-like domain, the region that is responsible for its neurotrophic activities. Fibulin-1 expression in the brain is confined to neurons, and is not expressed significantly by astrocytes or microglia. Direct binding of fibulin-1 to the secreted form of APP (sAPP) was demonstrated with a pull-down assay using fragments of both fibulin-1 fused with glutathione-S transferase and sAPP, produced in bacteria and yeast, respectively. The fibulin-1/sAPP heteromer was shown to form in the conditioned medium of transfected COS-7 cells. Furthermore, fibulin-1 blocks sAPP-mediated proliferation of primary cultured rat neural stem cells. These results suggest that fibulin-1 may play a significant role in modulating the neurotrophic activities of APP.     

Combination of insulinomimetic agents H2O2 and vanadate enhances insulin receptor mediated tyrosine phosphorylation of IRS-1 leading to IRS-1 association with the phosphatidylinositol 3-kinase

To analyze the mechanism of action of the insulinomimetic agents H₂O₂, vanadate, and pervanadate (H₂O₂ and vanadate), CHO cells or CHO cells that overexpress wild-type or mutant insulin receptor and/or the insulin receptor substrate (IRS-1) were used. H₂O₂ or vanadate treatment alone had little or no effect on tyrosine phosphorylation of cellular proteins; however, pevanadate treatment dramatically enhanced tyrosine phosphorylation of a number of proteins including the insulin receptor and IRS-1. However, the insulin receptor and IRS-1 coimmunoprecipitate from insulin-treated but not from pervanadate-treated cells. Pervanadate-induced tyrosine phosphorylation of the insulin receptor led to an increase in insulin receptor tyrosine kinase activity toward IRS-1 in vivo and IRS-1 peptides in vitro equal to that induced by insulin treatment. Pervanadate-enhanced phosphorylation of IRS-1 led to a fifteenfold increase in IRS-1–associated phosphatidylinositol (Ptdlns) 3-kinase activity. However, insulin receptor–associated Ptdlns 3-kinase activity from pervanadate-treated cells was not detectable, while insulin receptor–associated Ptdlns 3-kinase activity from insulin-treated cells was 20% of the IRS-1-associated activity. Thus, pervanadate but not H₂O₂ or vanadate alone under these conditions mimics many of insulin actions, but pervanadate treatment does not induce insulin receptor/IRS-1 association.     

Role of the Outer Pore Domain in Transient Receptor Potential Vanilloid 1 Dynamic Permeability to Large Cations

Transient receptor potential vanilloid 1 (TRPV1) has been shown to alter its ionic selectivity profile in a time- and agonist-dependent manner. One hallmark of this dynamic process is an increased permeability to large cations such as N-methyl-d-glucamine (NMDG). In this study, we mutated residues throughout the TRPV1 pore domain to identify loci that contribute to dynamic large cation permeability. Using resiniferatoxin (RTX) as the agonist, we identified multiple gain-of-function substitutions within the TRPV1 pore turret (N628P and S629A), pore helix (F638A), and selectivity filter (M644A) domains. In all of these mutants, maximum NMDG permeability was substantially greater than that recorded in wild type TRPV1, despite similar or even reduced sodium current density. Two additional mutants, located in the pore turret (G618W) and selectivity filter (M644I), resulted in significantly reduced maximum NMDG permeability. M644A and M644I also showed increased and decreased minimum NMDG permeability, respectively. The phenotypes of this panel of mutants were confirmed by imaging the RTX-evoked uptake of the large cationic fluorescent dye YO-PRO1. Whereas none of the mutations selectively altered capsaicin-induced changes in NMDG permeability, the loss-of-function phenotypes seen with RTX stimulation of G618W and M644I were recapitulated in the capsaicin-evoked YO-PRO1 uptake assay. Curiously, the M644A substitution resulted in a loss, rather than a gain, in capsaicin-evoked YO-PRO1 uptake. Modeling of our mutations onto the recently determined TRPV1 structure revealed several plausible mechanisms for the phenotypes observed. We conclude that side chain interactions at a few specific loci within the TRPV1 pore contribute to the dynamic process of ionic selectivity.     

Lactobacillus plantarum AS1 binds to cultured human intestinal cell line HT-29 and inhibits cell attachment by enterovirulent bacterium Vibrio parahaemolyticus

Aim: Lactobacillus plantarum AS1 was incubated with HT‐29 adenocarcinoma cell line to assess its adhesion potency and examined for its inhibitory effect on the cell attachment by an enterovirulent bacterium Vibrio parahaemolyticus. Methods and Results: Lactobacillus plantarum AS1 attached efficiently to HT‐29 cells as revealed by scanning electron microscopy and bacterial adhesion assay. Lactobacillus plantarum AS1 significantly reduced V. parahaemolyticus attached to HT‐29 cells by competition, exclusion and displacement mode. Lactobacillus plantarum AS1 seems to adhere to human intestinal cells via mechanisms that involve different combinations of carbohydrate and protein factors on the bacteria and eukaryotic cell surface. Conclusion: Strain Lact. plantarum AS1 inhibits the cell attachment of a pathogen V. parahaemolyticus by steric hindrance mechanism. Also, antibacterial factors such as bacteriocins, lactic acid and exopolysaccharides could be involved. Significance and Impact of the Study: The ability to inhibit the adhesion of V. parahaemolyticus to intestinal cell line warrants further investigation to explore the use of probiotic strain Lact. plantarum AS1 in the management of gastroenteritis caused by V. parahaemolyticus.     

Identification and characterization of a Ca2+ -sensitive interaction of the vanilloid receptor TRPV1 with tubulin

The vanilloid receptor TRPV1 plays a well-established functional role in the detection of a range of chemical and thermal noxious stimuli, such as those associated with tissue inflammation and the resulting pain. TRPV1 activation results in membrane depolarization, but may also trigger intracellular Ca2+ -signalling events. In a proteomic screen for proteins associated with the C-terminal sequence of TRPV1, we identified beta-tubulin as a specific TRPV1-interacting protein. We demonstrate that the TRPV1 C-terminal tail is capable of binding tubulin dimers, as well as of binding polymerized microtubules. The interaction is Ca2+ -sensitive, and affects microtubule properties, such as microtubule sensitivity towards low temperatures and nocodazole. Our data thus provide compelling evidence for the interaction of TRPV1 with the cytoskeleton. The Ca2+ -sensitivity of this interaction suggests that the microtubule cytoskeleton at the cell membrane may be a downstream effector of TRPV1 activation.     

Augmented sensitivity to methotrexate by curcumin induced overexpression of folate receptor in KG-1 cells

Folate receptors are targets of various strategies aimed at efficient delivery of anti-cancer drugs. Folate receptors also play a role in the uptake of antifolate drugs which are used for therapeutic intervention in leukemia. Therefore, it is important to identify compounds which regulate expression of folate receptors in leukemic cells. The present study examined if curcumin could modulate the uptake and cytotoxicity of the antifolate drug methotrexate, in KG-1 leukemic cells. This is the first report to show that curcumin (10–50 μM) causes a significant, dose-dependent, 2–3 fold increase in uptake of radiolabelled folic acid and methotrexate into KG-1 cells both at 24 h and 48 h of treatment. Interestingly, pre-treatment of KG-1 leukemic cells with curcumin (10 μM and 25 μM) also caused a statistically significant enhancement in the cytotoxicity of methotrexate. We performed Real Time Quantitative RT-PCR to confirm the upregulation of FRβ mRNA in curcumin treated cells. Immunocytochemistry and Western blotting showed that curcumin caused increased expression of folate receptor βin KG-1 cells. Our data show that the mechanism of curcumin action involves up-regulation of folate receptor β mRNA and protein in KG-1 cells. Therefore, combination of non-toxic concentrations of curcumin and methotrexate, may be a viable strategy for therapeutic intervention for leukemias using a folate receptor-targeted drug delivery system.