Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.

The TGR5 receptor mediates bile acid-induced itch and analgesia

Patients with cholestatic disease exhibit pruritus and analgesia, but the mechanisms underlying these symptoms are unknown. We report that bile acids, which are elevated in the circulation and tissues during cholestasis, cause itch and analgesia by activating the GPCR TGR5. TGR5 was detected in peptidergic neurons of mouse dorsal root ganglia and spinal cord that transmit itch and pain, and in dermal macrophages that contain opioids. Bile acids and a TGR5-selective agonist induced hyperexcitability of dorsal root ganglia neurons and stimulated the release of the itch and analgesia transmitters gastrin-releasing peptide and leucine-enkephalin. Intradermal injection of bile acids and a TGR5-selective agonist stimulated scratching behavior by gastrin-releasing peptide- and opioid-dependent mechanisms in mice. Scratching was attenuated in Tgr5-KO mice but exacerbated in Tgr5-Tg mice (overexpressing mouse TGR5), which exhibited spontaneous pruritus. Intraplantar and intrathecal injection of bile acids caused analgesia to mechanical stimulation of the paw by an opioid-dependent mechanism. Both peripheral and central mechanisms of analgesia were absent from Tgr5-KO mice. Thus, bile acids activate TGR5 on sensory nerves, stimulating the release of neuropeptides in the spinal cord that transmit itch and analgesia. These mechanisms could contribute to pruritus and painless jaundice that occur during cholestatic liver diseases.

The du2J mouse model of ataxia and absence epilepsy has deficient cannabinoid CB1 receptor-mediated signalling

Key point summary • Cerebellar ataxias are progressive debilitating diseases with no known treatment and are associated with defective motor function and, in particular, abnormalities to Purkinje cells. • Mutant mice with deficits in Ca2+ channel auxiliary α2δ-2 subunits are used as models of cerebellar ataxia. • Our data in the du2J mouse model shows an association between the ataxic phenotype exhibited by homozygous du2J/du2J mice and increased irregularity of Purkinje cell firing. • We show that both heterozygous +/du2J and homozygous du2J/du2J mice completely lack the strong presynaptic modulation of neuronal firing by cannabinoid CB1 receptors which is exhibited by litter-matched control mice. • These results show that the du2J ataxia model is associated with deficits in CB1 receptor signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity due to reduced α2δ-2 subunit expression. Knowledge of such deficits may help design therapeutic agents to combat ataxias. Abstract Cerebellar ataxias are a group of progressive, debilitating diseases often associated with abnormal Purkinje cell (PC) firing and/or degeneration. Many animal models of cerebellar ataxia display abnormalities in Ca2+ channel function. The ‘ducky’ du2J mouse model of ataxia and absence epilepsy represents a clean knock-out of the auxiliary Ca2+ channel subunit, α2δ-2, and has been associated with deficient Ca2+ channel function in the cerebellar cortex. Here, we investigate effects of du2J mutation on PC layer (PCL) and granule cell (GC) layer (GCL) neuronal spiking activity and, also, inhibitory neurotransmission at interneurone-Purkinje cell(IN-PC) synapses. Increased neuronal firing irregularity was seen in the PCL and, to a less marked extent, in the GCL in du2J/du2J, but not +/du2J, mice; these data suggest that the ataxic phenotype is associated with lack of precision of PC firing, that may also impinge on GC activity and requires expression of two du2J alleles to manifest fully. du2J mutation had no clear effect on spontaneous inhibitory postsynaptic current (sIPSC) frequency at IN-PC synapses, but was associated with increased sIPSC amplitudes. du2J mutation ablated cannabinoid CB1 receptor (CB1R)-mediated modulation of spontaneous neuronal spike firing and CB1Rmediated presynaptic inhibition of synaptic transmission at IN-PC synapses in both +/du2J and du2J/du2J mutants; effects that occurred in the absence of changes in CB1R expression. These results demonstrate that the du2J ataxia model is associated with deficient CB1R signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity and the ataxic phenotype.

Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1.

Prostaglandins (PG) are known to induce pain perception indirectly by sensitizing nociceptors. Accordingly, the analgesic action of nonsteroidal anti-inflammatory drugs (NSAIDs) results from inhibition of cyclooxygenases and blockade of PG biosynthesis. Cyclopentenone PGs, 15-d-PGJ(2), PGA(2), and PGA(1), formed by dehydration of their respective parent PGs, PGD(2), PGE(2), and PGE(1), possess a highly reactive alpha,beta-unsaturated carbonyl group that has been proposed to gate the irritant transient receptor potential A1 (TRPA1) channel. Here, by using TRPA1 wild-type (TRPA1(+/+)) or deficient (TRPA1(-/-)) mice, we show that cyclopentenone PGs produce pain by direct stimulation of nociceptors via TRPA1 activation. Cyclopentenone PGs caused a robust calcium response in dorsal root ganglion (DRG) neurons of TRPA1(+/+), but not of TRPA1(-/-) mice, and a calcium-dependent release of sensory neuropeptides from the rat dorsal spinal cord. Intraplantar injection of cyclopentenone PGs stimulated c-fos expression in spinal neurons of the dorsal horn and evoked an instantaneous, robust, and transient nociceptive response in TRPA1(+/+) but not in TRPA1(-/-) mice. The classical proalgesic PG, PGE(2), caused a slight calcium response in DRG neurons, increased c-fos expression in spinal neurons, and induced a delayed and sustained nociceptive response in both TRPA1(+/+) and TRPA1(-/-) mice. These results expand the mechanism of NSAID analgesia from blockade of indirect nociceptor sensitization by classical PGs to inhibition of direct TRPA1-dependent nociceptor activation by cyclopentenone PGs. Thus, TRPA1 antagonism may contribute to suppress pain evoked by PG metabolites without the adverse effects of inhibiting cyclooxygenases.

Nonpsychotropic plant cannabinoids, cannabidivarin (CBDV) and cannabidiol (CBD), activate and desensitize transient receptor potential vanilloid 1 (TRPV1) channels in vitro: potential for the treatment of neuronal hyperexcitability

Epilepsy is the most common neurological disorder, with over 50 million people worldwide affected. Recent evidence suggests that the transient receptor potential cation channel subfamily V member 1 (TRPV1) may contribute to the onset and progression of some forms of epilepsy. Since the two nonpsychotropic cannabinoids cannabidivarin (CBDV) and cannabidiol (CBD) exert anticonvulsant activity in vivo and produce TRPV1-mediated intracellular calcium elevation in vitro, we evaluated the effects of these two compounds on TRPV1 channel activation and desensitization and in an in vitro model of epileptiform activity. Patch clamp analysis in transfected HEK293 cells demonstrated that CBD and CBDV dose-dependently activate and rapidly desensitize TRPV1, as well as TRP channels of subfamily V type 2 (TRPV2) and subfamily A type 1 (TRPA1). TRPV1 and TRPV2 transcripts were shown to be expressed in rat hippocampal tissue. When tested on epileptiform neuronal spike activity in hippocampal brain slices exposed to a Mg2+-free solution using multielectrode arrays (MEAs), CBDV reduced both epileptiform burst amplitude and duration. The prototypical TRPV1 agonist, capsaicin, produced similar, although not identical effects. Capsaicin, but not CBDV, effects on burst amplitude were reversed by IRTX, a selective TRPV1 antagonist. These data suggest that CBDV antiepileptiform effects in the Mg2+-free model are not uniquely mediated via activation of TRPV1. However, TRPV1 was strongly phosphorylated (and hence likely sensitized) in Mg2+-free solution-treated hippocampal tissue, and both capsaicin and CBDV caused TRPV1 dephosphorylation, consistent with TRPV1 desensitization. We propose that CBDV effects on TRP channels should be studied further in different in vitro and in vivo models of epilepsy.

Controversial role of toll-like receptor 4 in adult stem cells

Adult or somatic stem cells are tissue-resident cells with the ability to proliferate, exhibit self-maintenance as well as to generate new cells with the principal phenotypes of the tissue in response to injury or disease. Due to their easy accessibility and their potential use in regenerative medicine, adult stem cells raise the hope for future personalisable therapies. After infection or during injury, they are exposed to broad range of pathogen or damage-associated molecules leading to changes in their proliferation, migration and differentiation. The sensing of such damage and infection signals is mostly achieved by Toll-Like Receptors (TLRs) with Toll-like receptor 4 being responsible for recognition of bacterial lipopolysaccharides (LPS) and endogenous danger-associated molecular patterns (DAMPs). In this review, we examine the current state of knowledge on the TLR4-mediated signalling in different adult stem cell populations. Specifically, we elaborate on the role of TLR4 and its ligands on proliferation, differentiation and migration of mesenchymal stem cells, hematopoietic stem cells as well as neural stem cells. Finally, we discuss conceptual and technical pitfalls in investigation of TLR4 signalling in stem cells.

Exhaustive Exercise Increases Inflammatory Response via Toll Like Receptor-4 and NF-kappa Bp65 Pathway in Rat Adipose Tissue

Cytokines (IL-6, IL-10, and TNF-alpha) are increased after exhaustive exercise in the retroperitoneal adipose tissue (RPAT) and mesenteric adipose tissue (MEAT). An exhaustive acute exercise protocol induces inflammation in adipose tissue that lasts 6 h after the exercise has ended. It is well-established that this protocol increases circulating plasma levels of non-esterified fatty acids (NEFAs) and lipopolysaccharides (LPS), compounds that are important in stimulating signaling via toll like receptor-4 (TLR-4) in different type cells. In the present study, we investigated the regulation of TLR-4 and DNA-binding of nuclear factor-kappa Bp65 (NF-kappa Bp65) in different depots of adipose tissue in rats after exhaustive exercise. Rats were killed by decapitation immediately (E0 group, n = 6), 2 (E2 group, n = 6), and 6 h (E6 group, n = 6) after the exhaustive exercise, which consisted of running on a treadmill (approximately 70% V(O2max)) for 50 min and then running at an elevated rate that increased at 1 m/min, until exhaustion. The control group (C group, n = 6) was not subjected to exercise. In RPAT, TLR-4, MYD-88, and IkB alpha increased in the E2 group after exercise. MYD-88 and TRAF6 remained increased in the E6 group in comparison with the control group. DNA-binding of NF-kappa Bp65 was not altered. In MEAT, TLR-4, MYD-88, TRAF6, and DNA-binding of NF-kappa Bp65 were increased only in the E6 group. In conclusion, we have shown that increases in pro-inflammatory cytokines in adipose tissue pads after exhaustive exercise may be mediated via TLR-4 signaling, leading to increases in NF-kappa Bp65 binding to DNA in MEAT. J. Cell. Physiol. 226: 1604-1607, 2011. (C) 2010 Wiley-Liss, Inc.

Novel endogenous peptide agonists of cannabinoid receptors

Hemopressin (Hp), a 9-residue alpha-hemoglobin-derived peptide, was previously reported to function as a CB(1) cannabinoid receptor antagonist (1). In this study, we report that mass spectrometry (MS) data from peptidomics analyses of mouse brain extracts identified N-terminally extended forms of Hp containing either three (RVD-Hp alpha) or two (VD-Hp alpha) additional amino acids, as well as a beta-hemoglobinderived peptide with sequence similarity to that of hemopressin (VD-Hp beta). Characterization of the alpha-hemoglobin-derived peptides using binding and functional assays shows that in contrast to Hp, which functions as a CB(1) cannabinoid receptor antagonist, both RVD-Hp alpha and VD-Hp alpha function as agonists. Studies examining the increase in the phosphorylation of ERK1/2 levels or release of intracellular Ca(2+) indicate that these peptides activate a signal transduction pathway distinct from that activated by the endo-cannabinoid, 2-arachidonoylglycerol, or the classic CB(1) agonist, Hu-210. This finding suggests an additional mode of regulation of endogenous cannabinoid receptor activity. Taken together, these results suggest that the CB(1) receptor is involved in the integration of signals from both lipid-and peptide-derived signaling molecules.-Gomes, I., Grushko, J. S., Golebiewska, U., Hoogendoorn, S., Gupta, A., Heimann, A. S., Ferro, E. S., Scarlata, S., Fricker, L. D., Devi, L. A. Novel endogenous peptide agonists of cannabinoid receptors. FASEB J. 23, 3020-3029 (2009). www.fasebj.org

Toll-Like Receptor 4 Signaling Leads to Severe Fungal Infection Associated with Enhanced Proinflammatory Immunity and Impaired Expansion of Regulatory T Cells

Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecular patterns and influence immunity to control the host-parasite interaction. The objective of this study was to characterize the involvement of TLR4 in the innate and adaptive immunity to Paracoccidioides brasiliensis, the most important primary fungal pathogen of Latin America. We compared the responses of C3H/HeJ mice, which are naturally defective in TLR4 signaling, with those of C3H/HePas mice, which express functional receptors, after in vitro and in vivo infection with P. brasiliensis. Unexpectedly, we verified that TLR4-defective macrophages infected in vitro with P. brasiliensis presented decreased fungal loads associated with impaired synthesis of nitric oxide, interleukin-12 (IL-12), and macrophage chemotactic protein 1 (MCP-1). After intratracheal infection with 1 million yeasts, TLR4-defective mice developed reduced fungal burdens and decreased levels of pulmonary nitric oxide, proinflammatory cytokines, and antibodies. TLR4-competent mice produced elevated levels of IL-12 and tumor necrosis factor alpha (TNF-alpha), besides cytokines of the Th17 pattern, indicating a proinflammatory role for TLR4 signaling. The more severe infection of TLR4-normal mice resulted in increased influx of activated macrophages and T cells to the lungs and progressive control of fungal burdens but impaired expansion of regulatory T cells (Treg cells). In contrast, TLR4-defective mice were not able to clear their diminished fungal burdens totally, a defect associated with deficient activation of T-cell immunity and enhanced development of Treg cells. These divergent patterns of immunity, however, resulted in equivalent mortality rates, indicating that control of elevated fungal growth mediated by vigorous inflammatory reactions is as deleterious to the hosts as low fungal loads inefficiently controlled by limited inflammatory reactions.

Toll-like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin-specific allergic airway disease: role of MyD88 adaptor molecule and interleukin-12/interferon-gamma axis

Background Epidemiological and experimental data suggest that bacteria] lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll-like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro-Type 1 T helper cells (Th 1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways, we used TLR4-, MyD88-, TRIF-, or IL-12/IFN-gamma-deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co-adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co-adsorbed onto alum impaired in dose-dependent manner OVA-induced Th2-mediated allergic responses such as airway eosinophilia, type-2 cytokines secretion, airway hyper-reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1 -affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL-12/IFN-gamma axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll-like receptor 4 agonists co-adsorbed with allergen onto alum down-modulate allergic lung disease and prevent the development of polarized T cell-mediated airway inflammation.

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Alteration of purinergic P2X(4) and P2X(7) receptor expression in rats with temporal-lobe epilepsy induced by pilocarpine

Although ATP and P2X receptor activity have been lately associated with epilepsy, little is known regarding their exact roles in epileptogenesis. Temporal-lobe epilepsy (TLE) in rat was induced by pilocarpine in order to study changes of hippocampal P2X(2), P2X(4) and P2X(7) receptor expression during acute, latent or chronic phases of epilepsy. During acute and chronic phases increased P2X(7) receptor expression was principally observed in glial cells and glutamatergic nerve terminals, suggesting participation of this receptor in the activation of inflammatory and excitotoxic processes during epileptogenesis. No significant alterations of hippocampal P2X(2) and P2X(4) receptor expression was noted during the acute or latent phase when compared to the control group, indicating that these receptors are not directly involved with the initiation of epilepsy. However, the reduction of hippocampal P2X(4) receptor immunostaining in the chronic phase could reflect neuronal toss or decreased GABAergic signaling. (C) 2008 Elsevier B.V. All rights reserved.

Inhibition Mechanism of Rat alpha(3)beta(4) Nicotinic Acetylcholine Receptor by the Alzheimer Therapeutic Tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer`s disease, on currents elicited by activation of rat alpha(3)beta(4) nAChR heterologously expressed in KX alpha(3)beta(4)R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 +/- 0.2 mu M and 4.3 +/- 1.3 for the channel opening equilibrium constant, Phi(-1). Experiments were performed to investigate whether tacrine is able to activate the alpha(3)beta(4) nAChR. Tacrine did not activate whole-cell currents in KX alpha(3)beta(4)R2 cells but inhibited receptor activity at submicromolar concentration. Dose response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 mu M. The increase of Phi(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.

Response of macrophage Toll-like receptor 4 to a Sporothrix schenckii lipid extract during experimental sporotrichosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)