Use of synthetic CaV2.2 Ca2+ channel peptides to study presynaptic function

Small, synthetic peptides based on specific regions of voltage-gated Ca2+ channels (VGCCs) have been widely used to study Ca2+ channel function and have been instrumental in confirming the contribution of specific amino acid sequences to interactions with putative binding partners. In particular, peptides based on the Ca2+ channel Alpha Interaction Domain (AID) on the intracellular region connecting domains I and II (the I-II loop) and the SYNaptic PRotein INTerction (synprint) site on the II-III loop have been widely used. Emerging evidence suggests that such peptides may themselves possess inherent functionality, a property that may be exploitable for future drug design. Here, we review our recent work using synthetic Ca2+ channel peptides based on sequences within the CaV2.2 amino terminal and I-II loop, originally identified as molecular determinates for G protein modulation, and their effects on VGCC function. These CaV2.2 peptides act as inhibitory modules to decrease Ca2+ influx with direct effects on VGCC gating, ultimately leading to a reduction of synaptic transmission. CaV2.2 peptides also attenuate G protein modulation of VGCCs. Amino acid substitutions generate CaV2.2 peptides with increased or decreased inhibitory effects suggesting that synthetic peptides can be used to further probe VGCC function and, potentially, form the basis for novel therapeutic development.

Statistical uncertainty of changes in winter storms over the North Atlantic and Europe in an ensemble of transient climate simulations

Winter storms are among the most important natural hazards affecting Europe. We quantify changes in storm frequency and intensity over the North Atlantic and Europe under future climate scenarios in terms of return periods (RPs) considering uncertainties due to both sampling and methodology. RPs of North Atlantic storms' minimum central pressure (CP) and maximum vorticity (VOR) remain unchanged by 2100 for both the A1B and A2 scenarios compared to the present climate. Whereas shortened RPs for VOR of all intensities are detected for the area between British Isles/North-Sea/western Europe as early as 2040. However, the changes in storm VOR RP may be unrealistically large: a present day 50 (20) year event becomes approximately a 9 (5.5) year event in both A1B and A2 scenarios by 2100. The detected shortened RPs of storms implies a higher risk of occurrence of damaging wind events over Europe.

Assessment of winter cyclone activity in a transient ECHAM4-OPYC3 GHG experiment

Winter cyclone activity over the Northern Hemisphere is investigated in an ECHAM4/OPYC3 greenhouse gas scenario simulation. The goal of this investigation is to identify changes in cyclone activity associated with increasing concentrations. To this aim, two 50-year time periods are analysed, one representing present day climate conditions and the other a perturbed climate when CO2 concentrations exceed twice the present concentrations. Cyclone activity is assessed using an automatic algorithm, which identifies and tracks cyclones based on sea level pressure fields. The algorithm detects not only large and long living cyclones over the main ocean basins, but also their smaller counterparts in secondary storm track regions like the Mediterranean Basin. For the present climate, results show a good agreement with NCEP-reanalysis, provided that the spectral and time resolutions of the reanalysis are reduced to those available for the model. Several prominent changes in cyclone activity are observed for the scenario period in comparison to the present day climate, especially over the main ocean basins. A significant decrease of overall cyclone track density is found between 35 and 55 degrees North, together with a small increase polewards. These changes result from two different signals for deep and medium cyclones: for deep cyclones (core pressure below 990 hPa) there is a poleward shift in the greenhouse gas scenario, while for medium cyclones (core pressure between 990 and 1010 hPa) a general decrease in cyclone counts is found. The same kind of changes (a shift for intense cyclones and an overall decrease for the weaker ones) are detected when distinguishing cyclones from their intensity, quantified in terms of ���2p. Thus, the simulated changes can not solely be attributed to alterations in mean sea level pressure. Instead, corresponding increases in upper-tropospheric baroclinicity suggest more favourable conditions for the development of stronger systems at higher latitudes, especially at the delta regions of the North Atlantic and the North Pacific storm tracks.

Carbon monoxide inhibition of Cav3.2 T-type Ca2+ channels reveals tonic modulation by thioredoxin

T-type Ca2+ channels play diverse roles in tissues such as sensory neurons, vascular smooth muscle, and cancers, where increased expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1) is often found. Here, we report regulation of T-type Ca2+ channels by carbon monoxide (CO) a HO-1 by-product. CO (applied as CORM-2) caused a concentration-dependent, poorly reversible inhibition of all T-type channel isoforms (Cav3.1-3.3, IC50 ���3 ��M) expressed in HEK293 cells, and native T-type channels in NG108-15 cells and primary rat sensory neurons. No recognized CO-sensitive signaling pathway could account for the CO inhibition of Cav3.2. Instead, CO sensitivity was mediated by an extracellular redox-sensitive site, which was also highly sensitive to thioredoxin (Trx). Trx depletion (using auranofin, 2-5 ��M) reduced Cav3.2 currents and their CO sensitivity by >50% but increased sensitivity to dithiothreitol ���3-fold. By contrast, Cav3.1 and Cav3.3 channels, and their sensitivity to CO, were unaffected in identical experiments. Our data propose a novel signaling pathway in which Trx acts as a tonic, endogenous regulator of Cav3.2 channels, while HO-1-derived CO disrupts this regulation, causing channel inhibition. CO modulation of T-type channels has widespread implications for diverse physiological and pathophysiological mechanisms, such as excitability, contractility, and proliferation

A spectral view of nonlinear fluxes and stationary-transient interaction in the atmosphere

Nonlinear spectral transfers of kinetic energy and enstrophy, and stationary-transient interaction, are studied using global FGGE data for January 1979. It is found that the spectral transfers arise primarily from a combination, in roughly equal measure, of pure transient and mixed stationary-transient interactions. The pure transient interactions are associated with a transient eddy field which is approximately locally homogeneous and isotropic, and they appear to be consistently understood within the context of two-dimensional homogeneous turbulence. Theory based on spatial wale separation concepts suggests that the mixed interactions may be understood physically, to a first approximation, as a process of shear-induced spectral transfer of transient enstrophy along lines of constant zonal wavenumber. This essentially conservative enstrophy transfer generally involves highly nonlocal stationary-transient energy conversions. The observational analysis demonstrates that the shear-induced transient enstrophy transfer is mainly associated with intermediate-scale (zonal wavenumber m > 3) transients and is primarily to smaller (meridional) scales, so that the transient flow acts as a source of stationary energy. In quantitative terms, this transient-eddy rectification corresponds to a forcing timescale in the stationary energy budget which is of the same order of magnitude as most estimates of the damping timescale in simple stationary-wave models (5 to 15 days). Moreover, the nonlinear interactions involved are highly nonlocal and cover a wide range of transient scales of motion.

Transient heparin-induced platelet activation linked to generation of platelet 12-lipoxygenase: findings from a randomised controlled trial

Previously we demonstrated that heparin administration during carotid endarterectomy (CEA) caused a marked, but transient increase in platelet aggregation to arachidonic acid (AA) and adenosine diphosphate (ADP), despite effective platelet cyclo-oxygenase-1 (COX-1) inhibition with aspirin. Here we investigated the metabolism of AA via platelet 12-lipoxygenase (12-LOX) as a possible mediator of the observed transient aspirin resistance, and compared the effects of unfractionated (UFH) and low-molecular-weight (LMWH) heparin. A total of 43 aspirinated patients undergoing CEA were randomised in the trial to 5,000 IU UFH (n=22) or 2,500 IU LMWH (dalteparin, n=21). Platelet aggregation to AA (4x10�����) and ADP (3x10������) was determined, and the products of the COX-1 and 12-LOX pathways; thromboxane B��� (TXB���) and 12-hydroxyeicosatretraenoic acid (12-HETE) were measured in plasma, and in material released from aggregating platelets.Aggregation to AA increased significantly (~10-fold) following heparinisation (p<0.0001), irrespective of heparin type (p=0.33). Significant, but smaller (~2-fold) increases in aggregation to ADP were also seen, which were significantly lower in the platelets of patients randomised to LMWH (p<0.0001). Plasma levels of TxB2 did not rise following heparinisation (p=0.93), but 12-HETE increased significantly in the patients' plasma, and released from platelets stimulated in vitro withADP, with both heparin types (p<0.0001). The magnitude of aggregation to ADP correlated with 12-HETE generation (p=0.03). Heparin administration during CEA generates AA that is metabolised to 12-HETE via the 12-LOX pathway, possibly explaining the phenomenon of transient heparin-induced platelet activation. LMWH has less effect on aggregation and 12-HETE generation than UFH when the platelets are stimulated with ADP.

The transient atmospheric response to midlatitude SST anomalies

To study the transient atmospheric response to midlatitude SST anomalies, a three-layer quasigeostrophic (QG) model coupled to a slab oceanic mixed layer in the North Atlantic is used. As diagnosed from a coupled run in perpetual winter conditions, the first two modes of SST variability are linked to the model North Atlantic Oscillation (NAO) and eastern Atlantic pattern (EAP), respectively, the dominant atmospheric modes in the Atlantic sector. The two SST anomaly patterns are then prescribed as fixed anomalous boundary conditions for the model atmosphere, and its transient responses are established from a large ensemble of simulations. In both cases, the tendency of the air���sea heat fluxes to damp the SST anomalies results in an anomalous diabatic heating of the atmosphere that, in turn, forces a baroclinic response, as predicted by linear theory. This initial response rapidly modifies the transient eddy activity and thus the convergence of eddy momentum and heat fluxes. The latter transforms the baroclinic response into a growing barotropic one that resembles the atmospheric mode that had created the SST anomaly in the coupled run and is thus associated with a positive feedback. The total adjustment time is as long as 3���4 months for the NAO-like response and 1���2 months for the EAP-like one. The positive feedback, in both cases, is dependent on the polarity of the SST anomaly, but is stronger in the NAO case, thereby contributing to its predominance at low frequency in the coupled system. However, the feedback is too weak to lead to an instability of the atmospheric modes and primarily results in an increase of their amplitude and persistence and a weakening of the heat flux damping of the SST anomaly.

A self-similar expansion model for use in solar wind transient propogation studies

Since the advent of wide-angle imaging of the inner heliosphere, a plethora of techniques have been developed to investigate the three-dimensional structure and kinematics of solar wind transients, such as coronal mass ejections, from their signatures in single- and multi-spacecraft imaging observations. These techniques, which range from the highly complex and computationally intensive to methods based on simple curve ���tting, all have their inherent advantages and limitations. In the analysis of single-spacecraft imaging observations, much use has been made of the ���xed �� ���tting (FPF) and harmonic mean ���tting (HMF) techniques, in which the solar wind transient is considered to be a radially propagating point source (���xed ��, FP, model) and a radially expanding circle anchored at Sun centre (harmonic mean, HM, model), respectively. Initially, we compare the radial speeds and propagation directions derived from application of the FPF and HMF techniques to a large set of STEREO/Heliospheric Imager (HI) observations. As the geometries on which these two techniques are founded constitute extreme descriptions of solar wind transients in terms of their extent along the line of sight, we describe a single-spacecraft ���tting technique based on a more generalized model for which the FP and HM geometries form the limiting cases. In addition to providing estimates of a transient���s speed and propagation direction, the self-similar expansion ���tting (SSEF) technique provides, in theory, the capability to estimate the transient���s angular extent in the plane orthogonal to the ���eld of view. Using the HI observations, and also by performing a Monte Carlo simulation, we assess the potential of the SSEF technique.

Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).

Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice.

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

Cigarette smoke-induced neurogenic inflammation is mediated by ��,��-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.

The transient versus the equilibrium response of sea ice to global warming

To examine the long-term stability of Arctic and Antarctic sea ice, idealized simulations are carried out with the climate model ECHAM5/MPIOM. Atmospheric CO2 concentration is increased over 2000 years from pre-industrial levels to quadrupling, is then kept constant for 5940 years, is afterwards decreased over 2000 years to pre-industrial levels, and finally kept constant for 3940 years.Despite these very slow changes, the sea-ice response significantly lags behind the CO2 concentration change. This lag, which is caused by the ocean���s thermal inertia, implies that the sea-ice equilibrium response to increasing CO2 concentration is substantially underestimated by transient simulations. The sea-ice response to CO2 concentration change is not truly hysteretic and in principle reversible.We find no lag in the evolution of Arctic sea ice relative to changes in annual-mean northern-hemisphere surface temperature. The summer sea-ice cover changes linearly with respect to both CO2 concentration and temper...

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

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.

Transient atmospheric response to interactive SST anomalies

The transient atmospheric response to interactive SST anomalies in the midlatitudes is investigated using a three-layer QG model coupled in perpetual winter conditions to a slab oceanic mixed layer in the North Atlantic. The SST anomalies are diagnosed from a coupled run and prescribed as initial conditions, but are free to evolve. The initial evolution of the atmospheric response is similar to that obtained with a prescribed SST anomaly, starting as a quasi-linear baroclinic and then quickly evolving into a growing equivalent barotropic one. Because of the heat flux damping, the SST anomaly amplitude slowly decreases, albeit with little change in pattern. Correspondingly, the atmospheric response only increases until it reaches a maximum amplitude after about 1���3.5 months, depending on the SST anomaly considered. The response is similar to that at equilibrium in the fixed SST case, but it is 1.5���2 times smaller, and then slowly decays away.