Carboxyl-terminal Domain of Transient Receptor Potential Vanilloid 1 Contains Distinct Segments Differentially Involved in Capsaicin- and Heat-induced Desensitization

Multiple Ca²⁺-dependent processes are involved in capsaicin-induced desensitization of transient receptor potential vanilloid 1 (TRPV1), but desensitization of TRPV1 by heat occurs even in the absence of extracellular Ca²⁺, although the mechanisms are unknown. In this study, we tested the hypothesis that capsaicin and heat desensitize TRPV1 through distinct mechanisms involving distinct structural segments of TRPV1. In HEK293 cells that heterologously express TRPV1, we found that heat-induced desensitization was not affected by the inclusion of intracellular ATP or alanine mutation of Lys¹⁵⁵, both of which attenuate capsaicin-induced desensitization, suggesting that heat-induced desensitization occurs through mechanisms distinct from capsaicin-induced desensitization. To determine protein domains involved in heat-induced desensitization, we generated chimeric proteins between TRPV1 and TRPV3, a heat-gated channel lacking heat-induced desensitization. We found that TRPV1 with the carboxyl-terminal domain (CTD) of TRPV3 retained heat activation but was impaired in heat-induced desensitization. Further experiments using chimeric or deletion mutants within TRPV1 CTD indicated that the distal half of CTD regulates the activation and desensitization of TRPV1 in modality-specific manners. Within the distal CTD, we identified two segments that distinctly regulated capsaicin- and heat-induced desensitization. The results suggest that the activation and desensitization of TRPV1 by capsaicin and heat can be modulated differentially and disproportionally through different regions of TRPV1 CTD. Identifying the domains involved in thermal regulation of TRPV1 may facilitate the development of novel anti-hyperalgesic approaches aimed at attenuating activation and enhancing desensitization of TRPV1 by thermal stimuli.     

Desensitization of capsaicin-activated currents in the vanilloid receptor TRPV1 is decreased by the cyclic AMP-dependent protein kinase pathway

Proinflammatory prostaglandin E2 is known to sensitize sensory neurons to noxious stimuli. This sensitization is mediated by the cAMP-dependent protein kinase (PKA) signal pathway. The capsaicin receptor TRPV1, a non-selective cation channel of sensory neurons involved in the sensation of inflammatory pain, is a target of PKA-mediated phosphorylation. Our goal was to investigate the influence of PKA on Ca(2+)-dependent desensitization of capsaicin-activated currents. By using site-directed mutagenesis, we created point mutations at PKA consensus sites and studied wild-type and mutant channels transiently expressed in HEK293t cells under whole-cell voltage clamp. We found that forskolin, a stimulator of adenylate cyclase, decreased desensitization of TRPV1. The selective PKA inhibitor H89 inhibited this effect. Mimicking phosphorylation at PKA consensus sites by replacing Ser-6, Ser-116, Thr-144, Thr-370, Ser-502, Ser-774, or Ser-820 with aspartate resulted in five mutations (S116D, T144D, T370D, S774D, and S820D) that exhibited decreased desensitization as well. However, disrupting phosphorylation by replacing respective sites with alanine resulted in four mutations (S6A, T144A, T370A, and S820A) with desensitization properties resembling those of the aspartate mutations. Significant changes in relative permeabilities for Ca2+ over Na+ or in capsaicin sensitivity could not explain changes in desensitization properties of mutant channels. In mutations S116A, S116D, T370A, and T370D, pretreatment of cells with forskolin did not reduce desensitization as compared with wild-type and other mutant channels. We conclude that Ser-116 and possibly Thr-370 are the most important residues involved in the mechanism of PKA-dependent reduction of desensitization of capsaicin-activated currents.     

Dendritic cells do not transduce inflammatory stimuli via the capsaicin receptor TRPV1

Inflammatory stimuli provide critical activation signals for dendritic cells (DC). Signaling through the capsaicin receptor TRPV1 is reported to initiate DC maturation and migration. We attempted to characterize TRPV1 channels in DC. Capsaicin or extracellular protons failed to elicit a change in intracellular [Ca(2+)] or membrane current in DC. In contrast, capsaicin evoked a sustained increase in [Ca(2+)] and large inwards currents in sensory neurons and TRPV1-expressing HEK293 cells. TRPV1 expression was confirmed by RT-PCR in sensory neurons, but was undetectable in DC. Interestingly, and in contrast to capsaicin, the inflammatory neuropeptide substance P evoked Ca(2+) transients in DC. Thus, our data do not support the hypothesis that DC express TRPV1 channels. Rather, signaling through TRPV1 in sensory nerves may modulate DC via neurogenic actions.     

Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya alata

Coumarin and its derivatives are fragrant natural compounds isolated from the genus Murraya that are flowering plants widely distributed in East Asia, Australia, and the Pacific Islands. Murraya plants have been widely used as medicinal herbs for relief of pain, such as headache, rheumatic pain, toothache, and snake bites. However, little is known about their analgesic components and the molecular mechanism underlying pain relief. Here, we report the bioassay-guided fractionation and identification of a novel coumarin derivative, named muralatin L, that can specifically activate the nociceptor transient receptor potential vanilloid 1 (TRPV1) channel and reverse the inflammatory pain in mice through channel desensitization. Muralatin L was identified from the active extract of Murraya alata against TRPV1 transiently expressed in HEK-293T cells in fluorescent calcium FlexStation assay. Activation of TRPV1 current by muralatin L and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV1-expressing HEK-293T cells and dorsal root ganglion neurons isolated from mice. Furthermore, muralatin L could reverse inflammatory pain induced by formalin and acetic acid in mice but not in TRPV1 knock-out mice. Taken together, our findings show that muralatin L specifically activates TRPV1 and reverses inflammatory pain, thus highlighting the potential of coumarin derivatives from Murraya plants for pharmaceutical and medicinal applications such as pain therapy.     

Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line.

The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma x rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N-glycosylation sites, the predicted transient receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly. We used site-directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross-linking with dimethyladipimidate yielded almost exclusively dimers. Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti-VR1 immunoreactivity in neurite-like structures of F-11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry.     

Novel gating and sensitizing mechanism of capsaicin receptor (TRPV1): tonic inhibitory regulation of extracellular sodium through the external protonation sites on TRPV1

Transient receptor potential V1 (TRPV1) is a nonselective cation channel expressed in nociceptors and activated by capsaicin. TRPV1 detects diverse stimuli, including acid, heat, and endogenous vanilloids, and functions as a molecular integrator of pain perception. Herein we demonstrate a novel regulatory role of extracellular Na(+) ([Na(+)](o)) on TRPV1 function. In human embryonic kidney 293 cells expressing porcine TRPV1, low [Na(+)](o) evoked increases of [Ca(2+)](i) that were suppressed by TRPV1 antagonists and facilitated responses to capsaicin, protons, heat, and an endovanilloid. [Na(+)](o) removal simultaneously elicited a [Ca(2+)](i) increase and outward-rectified current with a reversal potential similar to those of capsaicin. Neutralization of the two acidic residues which confer the proton sensitivity to TRPV1 resulted in a reduction of low [Na(+)](o)-induced responses. In primary culture of porcine sensory neurons, the removal of [Na(+)](o) produced a [Ca(2+)](i) increase and current responses only in the cells responding to capsaicin. Low [Na(+)](o) evoked a [Ca(2+)](i) increase in sensory neurons of wild type mice, but not TRPV1-null mice, and in human embryonic kidney 293 cells expressing human TRPV1. The present results suggest that [Na(+)](o) negatively regulates the gating and polymodal sensitization of the TRPV1 channel. [Na(+)](o) surrounding several proton-sensitive sites on the extracellular side of the pore-forming loop of the TRPV1 channel may play an important role as a brake to suppress the excessive activity of this channel under physiological conditions.     

Ca2+/calmodulin modulates TRPV1 activation by capsaicin

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159-212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487-517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816-824). When activated, they produce depolarization through the influx of Na+, but their high Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience. 55:1015-1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525-3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of approximately 30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin.     

Localization of the PIP2 sensor of TRPV1 ion channels

Although a large number of ion channels are now believed to be regulated by phosphoinositides, particularly phosphoinositide 4,5-bisphosphate (PIP2), the mechanisms involved in phosphoinositide regulation are unclear. For the TRP superfamily of ion channels, the role and mechanism of PIP2 modulation has been especially difficult to resolve. Outstanding questions include: is PIP2 the endogenous regulatory lipid; does PIP2 potentiate all TRPs or are some TRPs inhibited by PIP2; where does PIP2 interact with TRP channels; and is the mechanism of modulation conserved among disparate subfamilies? We first addressed whether the PIP2 sensor resides within the primary sequence of the channel itself, or, as recently proposed, within an accessory integral membrane protein called Pirt. Here we show that Pirt does not alter the phosphoinositide sensitivity of TRPV1 in HEK-293 cells, that there is no FRET between TRPV1 and Pirt, and that dissociated dorsal root ganglion neurons from Pirt knock-out mice have an apparent affinity for PIP2 indistinguishable from that of their wild-type littermates. We followed by focusing on the role of the C terminus of TRPV1 in sensing PIP2. Here, we show that the distal C-terminal region is not required for PIP2 regulation, as PIP2 activation remains intact in channels in which the distal C-terminal has been truncated. Furthermore, we used a novel in vitro binding assay to demonstrate that the proximal C-terminal region of TRPV1 is sufficient for PIP2 binding. Together, our data suggest that the proximal C-terminal region of TRPV1 can interact directly with PIP2 and may play a key role in PIP2 regulation of the channel.     

Agonist- and Ca2+-dependent desensitization of TRPV1 channel targets the receptor to lysosomes for degradation

TRPV1 receptor agonists such as the vanilloid capsaicin and the potent analog resiniferatoxin are well known potent analgesics. Depending on the vanilloid, dose, and administration site, nociceptor refractoriness may last from minutes up to months, suggesting the contribution of different cellular mechanisms ranging from channel receptor desensitization to Ca(2+) cytotoxicity of TRPV1-expressing neurons. The molecular mechanisms underlying agonist-induced TRPV1 desensitization and/or tachyphylaxis are still incompletely understood. Here, we report that prolonged exposure of TRPV1 to agonists induces rapid receptor endocytosis and lysosomal degradation in both sensory neurons and recombinant systems. Agonist-induced receptor internalization followed a clathrin- and dynamin-independent endocytic route, triggered by TRPV1 channel activation and Ca(2+) influx through the receptor. This process appears strongly modulated by PKA-dependent phosphorylation. Taken together, these findings indicate that TRPV1 agonists induce long-term receptor down-regulation by modulating the expression level of the channel through a mechanism that promotes receptor endocytosis and degradation and lend support to the notion that cAMP signaling sensitizes nociceptors through several mechanisms.     

Activation of protein kinase C reverses capsaicin-induced calcium-dependent desensitization of TRPV1 ion channels

Ca2+ selective ion channels of vanilloid receptor subtype-1 (TRPV1) in capsaicin-sensitive dorsal root ganglion (DRG) neurons and TRPV1 transfected Chinese hamster ovarian (CHO) cells are desensitized following calcium-dependent tachyphylaxis induced by successive applications of 100 nM capsaicin. Tachyphylaxis of TRPV1 to 100 nM capsaicin stimuli was not observed in the absence of extracellular calcium. Capsaicin sensitivity of desensitized TRPV1 ion channels recovered on application of phorbol-12-myristate-13-acetate (PMA). PMA-induced recovery of desensitized TRPV1 was primarily due to influx of extracellular calcium observed during re-application of capsaicin following desensitization. Capsazepine blocked the re-sensitization to capsaicin by PMA. Protein kinase C (PKC) inhibitory peptide PKC fragment 19-36 also inhibited re-sensitization to capsaicin by PMA. Reversal of capsaicin-induced desensitization by PMA was prevented by a mutation of TRPV1 where phosphorylation sites serine502 and serine800 were replaced with alanine. This study provides evidence for a role of PKC in reversing capsaicin-induced calcium-dependent desensitization of TRPV1 ion channels.     

The Endoplasmic Reticulum of Dorsal Root Ganglion Neurons Contains Functional TRPV1 Channels

Transient receptor potential vanilloid type 1 (TRPV1) is a plasma membrane Ca²⁺ channel involved in transduction of painful stimuli. Dorsal root ganglion (DRG) neurons express ectopic but functional TRPV1 channels in the endoplasmic reticulum (ER) (TRPV1_ER). We have studied the properties of TRPV1_ER in DRG neurons and HEK293T cells expressing TRPV1. Activation of TRPV1_ER with capsaicin or other vanilloids produced an increase of cytosolic Ca²⁺ due to Ca²⁺ release from the ER. The decrease of [Ca²⁺]_ER was directly revealed by an ER-targeted aequorin Ca²⁺ probe, expressed in DRG neurons using a herpes amplicon virus. The sensitivity of TRPV1_ER to capsaicin was smaller than the sensitivity of the plasma membrane TRPV1 channels. The low affinity of TRPV1_ER was not related to protein kinase A- or C-mediated phosphorylations, but it was due to inactivation by cytosolic Ca²⁺ because the sensitivity to capsaicin was increased by loading the cells with the Ca²⁺ chelator BAPTA. Decreasing [Ca²⁺]_ER did not affect the sensitivity of TRPV1_ER to capsaicin. Disruption of the TRPV1 calmodulin-binding domains at either the C terminus (Δ35AA) or the N terminus (K155A) increased 10-fold the affinity of TRPV1_ER for capsaicin, suggesting that calmodulin is involved in the inactivation. The lack of TRPV1 sensitizers, such as phosphatylinositol 4,5-bisphosphate, in the ER could contribute to decrease the affinity for capsaicin. The low sensitivity of TRPV1_ER to agonists may be critical for neuron health, because otherwise Ca²⁺ depletion of ER could lead to ER stress, unfolding protein response, and cell death.     

Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase

The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A.cyclophilin A complex (CsA.CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA.CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA.CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA.CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.     

Marked structural and functional heterogeneity in CXCR4: separation of HIV-1 and SDF-1alpha responses

CXCR4, the chemotactic cell receptor for SDF-1alpha, is essential for immune trafficking and HIV infection. CXCR4 is remarkably heterogeneous and the purpose of this study was to better identify the isoforms expressed by cells and compare their structure and function. We found that cells express either a predominant isoform or multiple isoforms. These were best resolved on SDS-PAGE using sucrose-gradient-fractionated, triton-insoluble, membrane extracts. We hypothesized that glycosyl modification may underpin some of this heterogeneity and that cell isoform(s) differences may underscore CXCR4's multiple cell functions. A comparison of wild-type (WT) and dual N-linked glycosylation site, N11A/N176A, mutant CXCR4 expressed in 3T3 and HEK-293 cells served to implicate variabilities in glycosylation and oligomerization in almost half of the isoforms. Immunoprecipitation of CXCR4 revealed monomer and dimer non-glycosylated forms of 34 kDa and 68 kDa from the N11A/N176A mutant, compared with glycosylated 40 kDa and 47 kDa and 73 kDa and 80 kDa forms from WT. The functional specificity of isoform action was also implicated because, despite CEMT4 cells expressing high levels of CXCR4 and 11 different isoforms, a single 83 kDa form was found to bind gp120 for HIV-1 IIIB infection. Furthermore, comparative studies found that in contrast to SDF-1alpha-responsive Nalm-6 cells that expressed similar levels of a single isoform, CEMT4 cells did not show a Ca(++) flux or a chemotactic response to SDF-1alpha. Thus, CXCR4 can differ both structurally and functionally between cells, with HIV-1 infection and chemotaxis apparently mediated by different isoforms. This separation of structure and function has implications for understanding HIV-1 entry and SDF-1alpha responses and may indicate therapeutic possibilities.     

Renal cells activate the platelet receptor CLEC-2 through podoplanin

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.     

Modulation of TRPV1 by nonreceptor tyrosine kinase, c-Src kinase

The capsaicin receptor TRPV1 is a nonselective cation channel that is expressed in sensory neurons. In this study, we examined the role of the nonreceptor cellular tyrosine kinase c-Src kinase in the modulation of the rat TRPV1. Capsaicin-induced currents in identified colonic dorsal root ganglion neurons were blocked by the c-Src kinase inhibitor PP2 and enhanced by the tyrosine phosphatase inhibitor sodium orthovandate. PP2 also abolished currents in human embryonic kidney-293 cells transfected with rat TRPV1, whereas cotransfection of TRPV1 with v-Src resulted in fivefold increase in capsaicin-induced currents. In cells transfected with dominant-negative c-Src and TRPV1, capsaicin-induced currents were decreased by approximately fourfold. TRPV1 co-immunoprecipitated with Src kinase and was tyrosine phosphorylated. These studies demonstrate that TRPV1 is a potential target for cellular tyrosine kinase-dependent phosphorylation.     

Transient receptor potential vanilloid 1, calcitonin gene-related peptide, and substance P mediate nociception in acute pancreatitis

The mechanism of pancreatitis-induced pain is unknown. In other tissues, inflammation activates transient receptor potential vanilloid 1 (TRPV1) on sensory nerves to liberate CGRP and substance P (SP) in peripheral tissues and the dorsal horn to cause neurogenic inflammation and pain, respectively. We evaluated the contribution of TRPV1, CGRP, and SP to pancreatic pain in rats. TRPV1, CGRP, and SP were coexpressed in nerve fibers of the pancreas. Injection of the TRPV1 agonist capsaicin into the pancreatic duct induced endocytosis of the neurokinin 1 receptor in spinal neurons in the dorsal horn (T10), indicative of SP release upon stimulation of pancreatic sensory nerves. Induction of necrotizing pancreatitis by treatment with L-arginine caused a 12-fold increase in the number of spinal neurons expressing the proto-oncogene c-fos in laminae I and II of L1, suggesting activation of nociceptive pathways. L-arginine also caused a threefold increase in spontaneous abdominal contractions detected by electromyography, suggestive of referred pain. Systemic administration of the TRPV1 antagonist capsazepine inhibited c-fos expression by 2.5-fold and abdominal contractions by 4-fold. Intrathecal, but not systemic, administration of antagonists of CGRP (CGRP(8-37)) and SP (SR140333) receptors attenuated c-fos expression in spinal neurons by twofold. Thus necrotizing pancreatitis activates TRPV1 on pancreatic sensory nerves to release SP and CGRP in the dorsal horn, resulting in nociception. Antagonism of TRPV1, SP, and CGRP receptors may suppress pancreatitis pain.     

β-Arrestin mediates oxytocin receptor signaling, which regulates uterine contractility and cellular migration

Desensitization of the oxytocin receptor (OXTR) in the setting of prolonged oxytocin exposure may lead to dysfunctional labor, which increases the risk for cesarean delivery, and uterine atony, which may result in postpartum hemorrhage. The molecular mechanism for OXTR desensitization is through the agonist-mediated recruitment of the multifunctional protein β-arrestin. In addition to its desensitizing function, β-arrestins have recently been shown to simultaneously activate downstream signaling. We tested whether oxytocin stimulation promotes β-arrestin-mediated OXTR desensitization in vivo and activates β-arrestin-mediated mitogen-activated protein kinase (MAPK) growth signaling. Uterine muscle strips isolated from wild-type mice exhibited diminished uterine contractility following repeated exposure to oxytocin, whereas uterine muscle strips from β-arrestin-1 and β-arrestin-2 knockout mice showed no desensitization. Utilizing siRNA knockdown of β-arrestin-1 and β-arrestin-2 in HEK-293 cells expressing the OXTR, we demonstrated oxytocin-mediated MAPK signaling that was dependent on β-arrestin-1 and β-arrestin-2. Wild-type and β-arrestin-1 and β-arrestin-2 knockout mice receiving intravenous oxytocin also demonstrated oxytocin-mediated MAPK signaling that was dependent on β-arrestin-1 and β-arrestin-2. Finally, to test the significance of β-arrestin-mediated signaling from the OXTR, HEK-293 cells expressing the OXTR showed β-arrestin-dependent proliferation in a cell migration assay following oxytocin treatment. In conclusion, β-arrestin is a multifunctional scaffold protein that mediates both desensitization of the OXTR, leading to decreases in uterine contractility, and MAPK growth signaling following stimulation by oxytocin. The development of unique OXTR ligands that prevent receptor desensitization may be a novel approach in the treatment of adverse clinical events secondary to prolonged oxytocin therapy.     

Structure-activity relationships of the ultrapotent vanilloid resiniferatoxin (RTX): the homovanillyl moiety

Starting from ROPA (2), analogues of RTX (1a) modified on the acyl side chain were prepared and evaluated for vanilloid activity in HEK-293 cells over-expressing the human recombinant TRPV1. The ROPA motif provided an enhancement of potency sufficient to expand the range of vanillyl surrogates to structural elements (e.g., an unsubstituted phenyl ring) that afford inactive analogues in compounds from the capsaicin series.     

Differential cytotoxicity and intracellular calcium-signalling following activation of the calcium-permeable ion channels TRPV1 and TRPA1

Several members of the transient receptor channel (TRP) family can mediate a calcium-dependent cytotoxicity. In sensory neurons, vanilloids like capsaicin induce neurotoxicity by activating TRPV1. The closely related ion channel TRPA1 is also activated by irritants, but it is unclear if and how TRPA1 mediates cell death. In the present study we explored cytotoxicity and intracellular calcium signalling resulting from activation of TRPV1 and TRPA1, either heterologously expressed in HEK 293 cells or in native mouse dorsal root ganglion (DRG) neurons. While activation of TRPV1 by the vanilloids capsaicin, resiniferatoxin and anandamide results in calcium-dependent cell death, activation by protons and the oxidant chloramine-T failed to reduce cell viability. The TRPA1-agonists acrolein, carvacrol and capsazepine all induced cytotoxicity, but this effect is independent of TRPA1. Activation of both TRPA1 and TRPV1 triggers a strong influx of external calcium, but also a strong calcium-release from intracellular stores most likely including the endoplasmic reticulum (ER). Activation of TRPV1, but not TRPA1 also results in a strong increase of mitochondrial calcium both in HEK 293 cells and mouse DRG neurons. Our data demonstrate that activation of TRPV1, but not TRPA1 mediates a calcium-dependent cell death. While both receptors mediate a release of calcium from intracellular stores, only activation of TRPV1 seems to mediate a robust and probably lethal increase in mitochondrial calcium.     

Glycosylation of beta(1)-adrenergic receptors regulates receptor surface expression and dimerization

The beta(1)-adrenergic receptor (beta(1)AR) has one predicted site of N-linked glycosylation on its extracellular amino-terminus, but the glycosylation and potential functional importance of this site have not yet been examined. We show here that the beta(1)AR is glycosylated in various cell types and that mutation of the single predicted site of N-linked glycosylation (N15A) results in the formation of receptors that are not N-glycosylated. The beta(1)AR N15A mutant exhibited significantly decreased basal surface expression relative to the wild-type receptor but had no detectable deficits in ligand binding or agonist-promoted internalization. Co-immunoprecipitation experiments using Flag-tagged and HA-tagged receptors demonstrated that the beta(1)AR-N15A mutant receptor exhibits a markedly reduced capacity for dimerization relative to wild-type beta(1)AR. These data reveal that the beta(1)AR is glycosylated on Asn15 and that this glycosylation plays a role in regulating beta(1)AR surface expression and dimerization.