Protease-activated receptors: mechanisms by which proteases sensitize TRPV channels to induce neurogenic inflammation and pain

Proteolytic enzymes comprise approximately 2 percent of the human genome [1]. Given their abundance, it is not surprising that proteases have diverse biological functions, ranging from the degradation of proteins in lysosomes to the control of physiological processes such as the coagulation cascade. However, a subset of serine proteases (possessing serine residues within their catalytic sites), which may be soluble in the extracellular fluid or tethered to the plasma membrane, are signaling molecules that can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors (GPCRs). These serine proteases include members of the coagulation cascade (e.g., thrombin, factor VIIa, and factor Xa), proteases from inflammatory cells (e.g., mast cell tryptase, neutrophil cathepsin G), and proteases from epithelial tissues and neurons (e.g., trypsins). They are often generated or released during injury and inflammation, and they cleave PARs on multiple cell types, including platelets, endothelial and epithelial cells, myocytes, fibroblasts, and cells of the nervous system. Activated PARs regulate many essential physiological processes, such as hemostasis, inflammation, pain, and healing. These proteases and their receptors have been implicated in human disease and are potentially important targets for therapy. Proteases and PARs participate in regulating most organ systems and are the subject of several comprehensive reviews [2, 3]. Within the central and peripheral nervous systems, proteases and PARs can control neuronal and astrocyte survival, proliferation and morphology, release of neurotransmitters, and the function and activity of ion channels, topics that have also been comprehensively reviewed [4, 5]. This chapter specifically concerns the ability of PARs to regulate TRPV channels of sensory neurons and thereby affect neurogenic inflammation and pain transmission [6, 7].

Cigarette smoke-induced neurogenic inflammation is mediated by α,β-unsaturated aldehydes and the TRPA1 receptor in rodents

Cigarette smoke (CS) inhalation causes an early inflammatory response in rodent airways by stimulating capsaicin-sensitive sensory neurons that express transient receptor potential cation channel, subfamily V, member 1 (TRPV1) through an unknown mechanism that does not involve TRPV1. We hypothesized that 2 alpha,beta-unsaturated aldehydes present in CS, crotonaldehyde and acrolein, induce neurogenic inflammation by stimulating TRPA1, an excitatory ion channel coexpressed with TRPV1 on capsaicin-sensitive nociceptors. We found that CS aqueous extract (CSE), crotonaldehyde, and acrolein mobilized Ca2+ in cultured guinea pig jugular ganglia neurons and promoted contraction of isolated guinea pig bronchi. These responses were abolished by a TRPA1-selective antagonist and by the aldehyde scavenger glutathione but not by the TRPV1 antagonist capsazepine or by ROS scavengers. Treatment with CSE or aldehydes increased Ca2+ influx in TRPA1-transfected cells, but not in control HEK293 cells, and promoted neuropeptide release from isolated guinea pig airway tissue. Furthermore, the effect of CSE and aldehydes on Ca2+ influx in dorsal root ganglion neurons was abolished in TRPA1-deficient mice. These data identify alpha,beta-unsaturated aldehydes as the main causative agents in CS that via TRPA1 stimulation mediate airway neurogenic inflammation and suggest a role for TRPA1 in the pathogenesis of CS-induced diseases.

Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from approximately 42 degrees C to approximately 34 degrees C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds.

Diverse mechanisms underlying the regulation of ion channels by carbon monoxide

Carbon monoxide is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, nitric oxide and cGMP levels, as well as regulate MAP kinase signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca2+ -sensitive K+ channels. More recent studies have revealed the ability of CO to inhibit T-type Ca2+ channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

A flow reactor process for the synthesis of peptides utilizing immobilized reagents, scavengers and catch and release protocols

A general flow process for the multi-step assembly of peptides has been developed and this procedure has been used to successfully construct a series of Boc, Cbz and Fmoc N-protected dipeptides in excellent yields and purities, including an extension of the method to enable the preparation of a tripeptide derivative.

Simulated climate change impact on summer dissolved organic carbon release from peat and surface vegetation: implications for drinking water treatment

Uncertainty regarding changes in dissolved organic carbon (DOC) quantity and quality has created interest in managing peatlands for their ecosystem services such as drinking water provision. The evidence base for such interventions is, however, sometimes contradictory. We performed a laboratory climate manipulation using a factorial design on two dominant peatland vegetation types (Calluna vulgaris and Sphagnum Spp.) and a peat soil collected from a drinking water catchment in Exmoor National Park, UK. Temperature and rainfall were set to represent baseline and future conditions under the UKCP09 2080s high emissions scenario for July and August. DOC leachate then underwent standard water treatment of coagulation/flocculation before chlorination. C. vulgaris leached more DOC than Sphagnum Spp. (7.17 versus 3.00 mg g−1) with higher specific ultraviolet (SUVA) values and a greater sensitivity to climate, leaching more DOC under simulated future conditions. The peat soil leached less DOC (0.37 mg g−1) than the vegetation and was less sensitive to climate. Differences in coagulation removal efficiency between the DOC sources appears to be driven by relative solubilisation of protein-like DOC, observed through the fluorescence peak C/T. Post-coagulation only differences between vegetation types were detected for the regulated disinfection by-products (DBPs), suggesting climate change influence at this scale can be removed via coagulation. Our results suggest current biodiversity restoration programmes to encourage Sphagnum Spp. will result in lower DOC concentrations and SUVA values, particularly with warmer and drier summers.

Diverse mechanisms underlying the regulation of ion channels by carbon monoxide

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca2+ -sensitive K+ channels. More recent studies have revealed the ability of CO to inhibit T-type Ca2+ channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Impact of game bird release on the Adonis blue butterfly Polyommatus bellargus (Lepidoptera Lycaenidae) on chalk grassland

Polyommatus bellargus is a priority species of butterfly in the UK as a result of its scarcity and the rate of population decline over the last few years. In the UK, the species is associated with chalk grassland on hot, south-facing slopes suitable for the growth of the food plant Hippocrepis comosa. Shooting game birds is a popular pastime in the UK. Over 40 million game birds, principally Phasianus colchicus and Alectoris rufa, are bred and released into the countryside each year for shooting interests. There is a concern that the release of such a large number of non-native birds has an adverse effect on native wildlife. A study was carried out over a period of 3 years out to examine whether there was any evidence that A. rufa released into chalk grassland habitat negatively affects populations of P. bellargus. A comparison was made between sites where large numbers of A. rufa were released versus sites where no, or few, birds were released. The study involved the construction of exclosures in these sites to allow an examination of the number of butterflies emerging from H. comosa when the birds were excluded versus when the birds had free range across the area. Where birds were present the on-site vegetation was shorter than where they were absent indicating that the birds were definitely influencing habitat structure. However, the evidence that A. rufa was negatively influencing the number of adult butterflies emerging was not strong, although there was a largely non-significant tendency for higher butterfly emergence when the birds were excluded or absent.

Oxygen-dependent hydroxylation by Factor Inhibiting HIF (FIH) regulates the TRPV3 ion channel

Factor Inhibiting HIF (FIH) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ankyrin repeat domain. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygendependent asparaginyl hydroxylation is likely to extend to other ion channels.

A comparison between ion characteristics observed by the POLAR and DMSP spacecraft in the high-latitude magnetosphere

We study here the injection and transport of ions in the convection-dominated region of the Earth’s magnetosphere. The total ion counts from the CAMMICE MICS instrument aboard the POLAR spacecraft are used to generate occurrence probability distributions of magnetospheric ion populations. MICS ion spectra are characterised by both the peak in the differential energy flux, and the average energy of ions striking the detector. The former permits a comparison with the Stubbs et al. (2001) survey of He2+ ions of solar wind origin within the magnetosphere. The latter can address the occurrences of various classifications of precipitating particle fluxes observed in the topside ionosphere by DMSP satellites (Newell and Meng, 1992). The peak energy occurrences are consistent with our earlier work, including the dawn-dusk asymmetry with enhanced occurrences on the dawn flank at low energies, switching to the dusk flank at higher energies. The differences in the ion energies observed in these two studies can be explained by drift orbit effects and acceleration processes at the magnetopause, and in the tail current sheet. Near noon at average ion energies of _1 keV, the cusp and open LLBL occur further poleward here than in the Newell and Meng survey, probably due to convectionrelated time-of-flight effects. An important new result is that the pre-noon bias previously observed in the LLBL is most likely due to the component of this population on closed field lines, formed largely by low energy ions drifting earthward from the tail. There is no evidence here of mass and momentum transfer from the solar wind to the LLBL by nonreconnection coupling. At higher energies (_2–20 keV), we observe ions mapping to the auroral oval and can distinguish between the boundary and central plasma sheets. We show that ions at these energies relate to a transition from dawnward to duskward dominated flow, this is evidence of how ion drift orbits in the tail influence the location and behaviour of the plasma populations in the magnetosphere.

The dependence of cusp ion signatures on the reconnection rate

The interpretation of structure in cusp ion dispersions is important for helping to understand the temporal and spatial structure of magnetopause reconnection. “Stepped” and “sawtooth” signatures have been shown to be caused by temporal variations in the reconnection rate under the same physical conditions for different satellite trajectories. The present paper shows that even for a single satellite path, a change in the amplitude of any reconnection pulses can alter the observed signature and even turn sawtooth into stepped forms and vice versa. On 20 August 1998, the Defense Meteorological Satellite Program (DMSP) craft F-14 crossed the cusp just to the south of Longyearbyen, returning on the following orbit. The two passes by the DMSP F-14 satellites have very similar trajectories and the open-closed field line boundary (OCB) crossings, as estimated from the SSJ/4 precipitating particle data and Polar UVI images, imply a similarly-shaped polar cap, yet the cusp ion dispersion signatures differ substantially. The cusp crossing at 08:54 UT displays a stepped ion dispersion previously considered to be typical of a meridional pass, whereas the crossing at 10:38 UT is a sawtooth form ion dispersion, previously considered typical of a satellite travelling longitudinally with respect to the OCB. It is shown that this change in dispersed ion signature is likely to be due to a change in the amplitude of the pulses in the reconnection rate, causing the stepped signature. Modelling of the low-energy ion cutoff under different conditions has reproduced the forms of signature observed.

Cusp ion steps, field-aligned currents and poleward moving auroral forms

We predict the field-aligned currents around cusp ion steps produced by pulsed reconnection between the geomagnetic field and an interplanetary magnetic field (IMF) with a B-Y component that is large in magnitude. For B-Y > 0, patches of newly opened flux move westward and eastward in the Northern and Southern Hemispheres, respectively, under the influence of the magnetic curvature force. These flow directions are reversed for B-Y < 0. The speed of this longitudinal motion initially grows with elapsed time since reconnection, but then decays as the newly opened field lines straighten. We predict sheets of field-aligned current on the boundaries between the patches produced by successive reconnection pulses, associated with the difference in the speeds of their longitudinal motion. For low elapsed times since reconnection, near the equatorward edge of the cusp region where the field lines are accelerating, the field-aligned current sheets will be downward or upward in both hemispheres for positive or negative IMF B-Y, respectively. At larger elapsed times since reconnection, as events slow and evolve from the cusp into the mantle region, these field-aligned current directions will be reversed. Observations by the Polar spacecraft on August 26,1998, show the predicted upward current sheets at steps seen in the mantle region for IMF B-Y > 0. Mapped into the ionosphere, the steps coincide with poleward moving events seen by the CUTLASS HF radar. The mapped location of the largest step also coincides with a poleward moving arc seen by the UVI imager on Polar. We show that the arc is consistent with a region of upward field-aligned current that has become unstable, such that a potential drop of about 1 kV formed below the spacecraft. The importance of these observations is that they confirm that the poleward moving events, as seen by the HF radar and the UV imager, are due to pulsed magnetopause reconnection. Milan et al. [2000] noted that the great longitudinal extent of these events means that the required reconnection pulses would have contributed almost all the voltage placed across the magnetosphere at this time. The observations also show that auroral arcs can form on open field lines in response to the pulsed application of voltage at the magnetopause.

Ionospheric ion and electron heating at the poleward boundary of a poleward expanding substorm-disturbed region

We present observations of a poleward propagating substorm-disturbed region which was observed by the European Incoherent SCATter (EISCAT) radar and the Svalbard International Monitor for Auroral Geomagnetic Effects (IMAGE) magnetometers in the postmidnight sector. The expansion of the disturbance was launched by a substorm intensification which started similar to 25 min after the initial onset, and similar to 10 min before the disturbance arrived over Svalbard. In association with the magnetic disturbance, a poleward expanding enduring enhancement in the F region electron temperature was observed, indicative of soft electron precipitation, with a narrow band of enhanced ion temperature straddling its poleward edge, indicative of fast ion flows and ion-neutral collisional heating. This electron temperature boundary was coincident with the poleward propagating electrojet current system detected by the high-latitude IMAGE magnetometer stations and is taken to be a proxy for the observation of a substorm auroral bulge. The electron temperature boundary is inferred to have a width comparable or less than one radar range gate (similar to 60 km transverse to the magnetic field), while the region of high ion temperature was found to be approximately three gates wide, extending approximately two gates (similar to 120 km) poleward of the electron temperature boundary, and approximately one gate (similar to 60 km) equatorward. The two-beam radar line-of-sight velocity data are found to be consistent with the existence of a layer of high-speed flow in the boundary, peaking at values similar to1.5-3 km s(-1), roughly consistent with the ion temperature data. The flow is directed either east or west along the boundary depending on the direction of the flow in the poleward region. We infer that the flow is deflected along and around the boundary of the substorm-disturbed region due to the high conductivity of the latter. Variations in the flow poleward of the boundary produced no discernible magnetic effects on the ground, confirming the low conductivity of the preboundary ionosphere.

Modelling signatures of pulsed magnetopause reconnection in cusp ion dispersion signatures seen at middle altitudes

It is shown that the open magnetosphere model can reproduce both the down-going and the up-going magnetosheath ions seen in the cusp and mantle regions by the Polar satellite at middle altitudes. ?he pass studied shows a series of discontinuities in the ion dispersion, most of which are shown to arise from pulses of magnetopause reconnection rate. A total of 9 pulses are detected in an interval estimated to be about 30 min long, giving a mean repetition period of about 3 min: they vary in length between 0.5 min and 3.5 min and are separated by periods of much slower reconnection of duration 1-3 min. One step is not as predicted for reconnection rate pulses but is explained in terms of compressive motions caused by a pulse of solar wind dynamic pressure. The reconnection site is found to be 16 +/- 3 R-E from the ionosphere along the separatrix field line, placing it at low latitudes on the dayside magnetopause.

Ion populations on open field lines within the dayside low-latitude boundary layer: theory and observations during a transient event

We analyze of ion populations observed by the NOAA-12 satellite within dayside auroral transients. The data are matched with an open magnetopause model which allows for the transmission of magnetosheath ions across one or both of the two Alfvén waves which emanate from the magnetopause reconnection site. It also allows for reflection and acceleration of ions of magnetospheric origin by these waves. From the good agreement found between the model and the observations, we propose that the events and the low-latitude boundary precipitation are both on open field lines.