Endothelin-converting enzyme-1 regulates trafficking and signalling of the neurokinin 1 receptor in endosomes of myenteric neurones

Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by beta-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by beta-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and beta-arrestin at the plasma membrane, and the SP-NK(1)R-beta-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-beta-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating beta-arrestin-mediated endosomal signalling.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with beta-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of beta-arrestin1 and PP2A with noninternalized NK(1)R. beta-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that beta-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping beta-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires beta-arrestin1. ECE-1 promotes this process by releasing beta-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

Pungent general anesthetics activate transient receptor potential-A1 to produce hyperalgesia and neurogenic bronchoconstriction

BACKGROUND: Volatile anesthetics such as isoflurane and halothane have been in clinical use for many years and represent the group of drugs most commonly used to maintain general anesthesia. However, despite their widespread use, the molecular mechanisms by which these drugs exert their effects are not completely understood. Recently, a seemingly paradoxical effect of general anesthetics has been identified: the activation of peripheral nociceptors by irritant anesthetics. This mechanism may explain the hyperalgesic actions of inhaled anesthetics and their adverse effects in the airways. METHODS: To test the hypothesis that irritant inhaled anesthetics activate the excitatory ion-channel transient receptor potential (TRP)-A1 and thereby contribute to hyperalgesia and irritant airway effects, we used the measurement of intracellular calcium concentration in isolated cells in culture. For our functional experiments, we used models of isolated guinea pig bronchi to measure bronchoconstriction and withdrawal threshold to mechanical stimulation with von Frey filaments in mice. RESULTS: Irritant inhaled anesthetics activate TRPA1 expressed in human embryonic kidney cells and in nociceptive neurons. Isoflurane induces mechanical hyperalgesia in mice by a TRPA1-dependent mechanism. Isoflurane also induces TRPA1-dependent constriction of isolated bronchi. Nonirritant anesthetics do not activate TRPA1 and fail to produce hyperalgesia and bronchial constriction. CONCLUSIONS: General anesthetics induce a reversible loss of consciousness and render the patient unresponsive to painful stimuli. However, they also produce excitatory effects such as airway irritation and they contribute to postoperative pain. Activation of TRPA1 may contribute to these adverse effects, a hypothesis that remains to be tested in the clinical setting.

Trafficking and signaling of G protein-coupled receptors in the nervous system: implications for disease and therapy

G protein-coupled receptors (GPCRs) are expressed throughout the nervous system where they regulate multiple physiological processes, participate in neurological diseases, and are major targets for therapy. Given that many GPCRs respond to neurotransmitters and hormones that are present in the extracellular fluid and which do not readily cross the plasma membrane, receptor trafficking to and from the plasma membrane is a critically important determinant of cellular responsiveness. Moreover, trafficking of GPCRs throughout the endosomal system can initiate signaling events that are mechanistically and functionally distinct from those operating at the plasma membrane. This review discusses recent advances in the relationship between signaling and trafficking of GPCRs in the nervous system. It summarizes how receptor modifications influence trafficking, discusses mechanisms that regulate GPCR trafficking to and from the plasma membrane, reviews the relationship between trafficking and signaling, and considers the implications of GPCR trafficking to drug development.

Endosomes: a legitimate platform for the signaling train

Although long regarded as a conduit for the degradation or recycling of cell surface receptors, the endosomal system is also an essential site of signal transduction. Activated receptors accumulate in endosomes, and certain signaling components are exclusively localized to endosomes. Receptors can continue to transmit signals from endosomes that are different from those that arise from the plasma membrane, resulting in distinct physiological responses. Endosomal signaling is widespread in metazoans and plants, where it transmits signals for diverse receptor families that regulate essential processes including growth, differentiation and survival. Receptor signaling at endosomal membranes is tightly regulated by mechanisms that control agonist availability, receptor coupling to signaling machinery, and the subcellular localization of signaling components. Drugs that target mechanisms that initiate and terminate receptor signaling at the plasma membrane are widespread and effective treatments for disease. Selective disruption of receptor signaling in endosomes, which can be accomplished by targeting endosomal-specific signaling pathways or by selective delivery of drugs to the endosomal network, may provide novel therapies for disease.

Endosomal deubiquitinating enzymes control ubiquitination and down-regulation of protease-activated receptor 2

The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR(2)), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR(2) deubiquitination and its importance in trafficking and signaling of endocytosed PAR(2) are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR(2) ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR(2). Trapping PAR(2) in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR(2) with beta-arrestin2 or the duration of PAR(2)-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR(2) but not for regulating PAR(2) dissociation from beta-arrestin2 or PAR(2)-mediated ERK2 activation.

Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, beta-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls beta-arrestin-dependent signaling of endocytosed GPCRs.

Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease-activated receptor 2

Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR(2)) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR(2) activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR(2) stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCepsilon. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR(2), and neuropeptides. PAR(2) agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR(2) agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR(2) agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies.

Endothelin-converting enzyme-1 degrades internalized somatostatin-14

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase

Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

Suppression of a pro-apoptotic K+ channel as a mechanism for hepatitis C virus persistence

An estimated 3% of the global population are infected with hepatitis C virus (HCV), and the majority of these individuals will develop chronic liver disease. As with other chronic viruses, establishment of persistent infection requires that HCV-infected cells must be refractory to a range of pro-apoptotic stimuli. In response to oxidative stress, amplification of an outward K(+) current mediated by the Kv2.1 channel, precedes the onset of apoptosis. We show here that in human hepatoma cells either infected with HCV or harboring an HCV subgenomic replicon, oxidative stress failed to initiate apoptosis via Kv2.1. The HCV NS5A protein mediated this effect by inhibiting oxidative stress-induced p38 MAPK phosphorylation of Kv2.1. The inhibition of a host cell K(+) channel by a viral protein is a hitherto undescribed viral anti-apoptotic mechanism and represents a potential target for antiviral therapy.

Mesolithic to Neolithic and medieval coastal environmental change: intertidal survey at Woolaston, Gloucestershire

At Woolaston on the western shores of the middle Severn Estuary c. 7 km upstream of Chepstow intertidal Holocene sediment exposures have been surveyed and the stratigraphic sequence established by coring and limited excavation. There are two main peats each with a submerged forest. An existing dendrochronological sequence for the Upper Submerged Forest has been extended and the preliminary results of pollen analysis from the peat sequence are summarised. A few flint flakes were found but were not stratified in the mid-Holocene sequence. There is evidence for late Mesolithic / early Neolithic burning episodes which may relate to human activity. Evidence is reported for Medieval activity and the extensive modification of drainage in this period is suggested.

A global assessment of the effects of climate policy on the impacts of climate change

This study presents the first global-scale multi-sectoral regional assessment of the magnitude and uncertainty in the impacts of climate change avoided by emissions policies. The analysis suggests that the most stringent emissions policy considered here – which gives a 50% chance of remaining below a 2oC temperature rise target - reduces impacts by 20-65% by 2100 relative to a ‘business-as-usual’ pathway (A1B) which reaches 4oC, and can delay impacts by several decades. Effects vary between sector and region, and there are few noticeable effects of mitigation policy by 2030. The impacts avoided by 2100 are more strongly influenced by the date and level at which emissions peak than the rate of decline of emissions, with an earlier and lower emissions peak avoiding more impacts. The estimated proportion of impacts avoided at the global scale is relatively robust despite uncertainty in the spatial pattern of climate change, but the absolute amount of avoided impacts is considerably more variable and therefore uncertain.

A method for incorporating climate variability in climate change impact assessments: sensitivity of river flows in the Eden catchment to precipitation scenarios

Interest in the impacts of climate change is ever increasing. This is particularly true of the water sector where understanding potential changes in the occurrence of both floods and droughts is important for strategic planning. Climate variability has been shown to have a significant impact on UK climate and accounting for this in future climate cahgne projections is essential to fully anticipate potential future impacts. In this paper a new resampling methodology is developed which includes the variability of both baseline and future precipitation. The resampling methodology is applied to 13 CMIP3 climate models for the 2080s, resulting in an ensemble of monthly precipitation change factors. The change factors are applied to the Eden catchment in eastern Scotland with analysis undertaken for the sensitivity of future river flows to the changes in precipitation. Climate variability is shown to influence the magnitude and direction of change of both precipitation and in turn river flow, which are not apparent without the use of the resampling methodology. The transformation of precipitation changes to river flow changes display a degree of non-linearity due to the catchment's role in buffering the response. The resampling methodology developed in this paper provides a new technique for creating climate change scenarios which incorporate the important issue of climate variability.

How will organic carbon stocks in mineral soils evolve under future climate? Global projections using RothC for a range of climate change scenarios

We use a soil carbon (C) model (RothC), driven by a range of climate models for a range of climate scenarios to examine the impacts of future climate on global soil organic carbon (SOC) stocks. The results suggest an overall global increase in SOC stocks by 2100 under all scenarios, but with a different extent of increase among the climate model and emissions scenarios. The impacts of projected land use changes are also simulated, but have relatively minor impacts at the global scale. Whether soils gain or lose SOC depends upon the balance between C inputs and decomposition. Changes in net primary production (NPP) change C inputs to the soil, whilst decomposition usually increases under warmer temperatures, but can also be slowed by decreased soil moisture. Underlying the global trend of increasing SOC under future climate is a complex pattern of regional SOC change. SOC losses are projected to occur in northern latitudes where higher SOC decomposition rates due to higher temperatures are not balanced by increased NPP, whereas in tropical regions, NPP increases override losses due to higher SOC decomposition. The spatial heterogeneity in the response of SOC to changing climate shows how delicately balanced the competing gain and loss processes are, with subtle changes in temperature, moisture, soil type and land use, interacting to determine whether SOC increases or decreases in the future. Our results suggest that we should stop looking for a single answer regarding whether SOC stocks will increase or decrease under future climate, since there is no single answer. Instead, we should focus on improving our prediction of the factors that determine the size and direction of change, and the land management practices that can be implemented to protect and enhance SOC stocks.