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Ca2+ Imaging (ca2+ + imaging)
Selected AbstractsThe patterns of spontaneous Ca2+ signals generated by ventral spinal neurons in vitro show time-dependent refinementEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2009Sara Sibilla Abstract Embryonic spinal neurons maintained in organotypic slice culture are known to mimic certain maturation-dependent signalling changes. With such a model we investigated, in embryonic mouse spinal segments, the age-dependent spatio-temporal control of intracellular Ca2+ signalling generated by neuronal populations in ventral circuits and its relation with electrical activity. We used Ca2+ imaging to monitor areas located within the ventral spinal horn at 1 and 2 weeks of in vitro growth. Primitive patterns of spontaneous neuronal Ca2+ transients (detected at 1 week) were typically synchronous. Remarkably, such transients originated from widespread propagating waves that became organized into large-scale rhythmic bursts. These activities were associated with the generation of synaptically mediated inward currents under whole-cell patch-clamp. Such patterns disappeared during longer culture of spinal segments: at 2 weeks in culture, only a subset of ventral neurons displayed spontaneous, asynchronous and repetitive Ca2+ oscillations dissociated from background synaptic activity. We observed that the emergence of oscillations was a restricted phenomenon arising together with the transformation of ventral network electrophysiological bursting into asynchronous synaptic discharges. This change was accompanied by the appearance of discrete calbindin immunoreactivity against an unchanged background of calretinin-positive cells. It is attractive to assume that periodic oscillations of Ca2+ confer a summative ability to these cells to shape the plasticity of local circuits through different changes (phasic or tonic) in intracellular Ca2+. [source] Multiple functions of GABAA and GABAB receptors during pattern processing in the zebrafish olfactory bulbEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Rico Tabor Abstract ,-Aminobutyric acid (GABA)ergic synapses are thought to play pivotal roles in the processing of activity patterns in the olfactory bulb (OB), but their functions have been difficult to study during odor responses in the intact system. We pharmacologically manipulated GABAA and GABAB receptors in the OB of zebrafish and analysed the effects on odor responses of the output neurons, the mitral cells (MCs), by electrophysiological recordings and temporally deconvolved two-photon Ca2+ imaging. The blockade of GABAB receptors enhanced presynaptic Ca2+ influx into afferent axon terminals, and changed the amplitude and time course of a subset of MC responses, indicating that GABAB receptors have a modulatory influence on OB output activity. The blockade of GABAA receptors induced epileptiform firing, enhanced excitatory responses and abolished fast oscillations in the local field potential. Moreover, the topological reorganization and decorrelation of MC activity patterns during the initial phase of the response was perturbed. These results indicate that GABAA receptor-containing circuits participate in the balance of excitation and inhibition, the regulation of total OB output activity, the synchronization of odor-dependent neuronal ensembles, and the reorganization of odor-encoding activity patterns. GABAA and GABAB receptors are therefore differentially involved in multiple functions of neuronal circuits in the OB. [source] Differential responses to NMDA receptor activation in rat hippocampal interneurons and pyramidal cells may underlie enhanced pyramidal cell vulnerabilityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2005E. Avignone Abstract Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults. Because NMDA receptors play a crucial role in neurodegeneration, we have compared the response to exogenous NMDA in CA1 pyramidal cells and interneurons of the stratum oriens using combined whole-cell patch-clamp recording and ratiometric Ca2+ imaging. In voltage-clamp, current-clamp or in nominally Mg2+ -free medium, NMDA (10 µm; 3,5 min exposure in the presence of tetrodotoxin) induced a markedly larger inward current and Ca2+ rise in pyramidal cells than in interneurons. Pyramidal cells also showed a more pronounced voltage dependence in their response to NMDA. We hypothesized that this enhanced response to NMDA receptor activation in pyramidal cells could underlie their increased vulnerability to excitotoxicity. Using loss of dye as an indicator of degenerative membrane disruption, interneurons tolerated continuous exposure to a high concentration of NMDA (30 µm) for longer periods than pyramidal cells. This acute neurodegeneration in pyramidal cells was independent of intracellular Ca2+, because high intracellular BAPTA (20 mm) did not prolong survival time. Thus, a plausible explanation for the enhanced sensitivity of pyramidal neurons to excitotoxic insults associated with cerebral ischemia is their greater response to NMDA receptor activation, which may reflect differences in NMDA receptor expression and/or subunit composition. [source] Long-range oscillatory Ca2+ waves in rat spinal dorsal hornEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2005Ruth Ruscheweyh Abstract Synchronous activity of large populations of neurons shapes neuronal networks during development. However, re-emergence of such activity at later stages of development could severely disrupt the orderly processing of sensory information, e.g. in the spinal dorsal horn. We used Ca2+ imaging in spinal cord slices of neonatal and young rats to assess under which conditions synchronous activity occurs in dorsal horn. No spontaneous synchronous Ca2+ transients were detected. However, increasing neuronal excitability by application of 4-aminopyridine after pretreatment of the slice with blockers of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, ,-aminobutyric acid (GABA)A and glycine receptors evoked repetitive Ca2+ waves in dorsal horn. These waves spread mediolaterally with a speed of 1.0 ± 0.1 mm/s and affected virtually every dorsal horn neuron. The Ca2+ waves were associated with large depolarizing shifts of the membrane potential of participating neurons and were most likely synaptically mediated because they were abolished by blockade of action potentials or N -methyl- d -aspartate (NMDA) receptors. They were most pronounced in the superficial dorsal horn and absent from the ventral horn. A significant proportion of the Ca2+ waves spread to the contralateral dorsal horn. This seemed to be enabled by disinhibition as primary afferent-induced dorsal horn excitation crossed the midline only when GABAA and glycine receptors were blocked. Interestingly, the Ca2+ waves occurred under conditions where AMPA/kainate receptors were blocked. Thus, superficial dorsal horn NMDA receptors are able to sustain synchronous neuronal excitation in the absence of functional AMPA/kainate receptors. [source] Calcium dynamics are altered in cortical neurons lacking the calmodulin-binding protein RC3EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2003Jacqueline J. W. Van Dalen Abstract RC3 is a neuronal calmodulin-binding protein and protein kinase C substrate that is thought to play an important regulatory role in synaptic transmission and neuronal plasticity. Two molecules known to regulate synaptic transmission and neuronal plasticity are Ca2+ and calmodulin, and proposed mechanisms of RC3 action involve both molecules. However, physiological evidence for a role of RC3 in neuronal Ca2+ dynamics is limited. In the current study we utilized cultured cortical neurons obtained from RC3 knockout (RC3,/,) and wildtype mice (RC3+/+) and fura-2-based microscopic Ca2+ imaging to investigate a role for RC3 in neuronal Ca2+ dynamics. Immunocytochemical characterization showed that the RC3,/, cultures lack RC3 immunoreactivity, whereas cultures prepared from wildtype mice showed RC3 immunoreactivity at all ages studied. RC3+/+ and RC3,/, cultures were indistinguishable with respect to neuron density, neuronal morphology, the formation of extensive neuritic networks and the presence of glial fibrillary acidic protein (GFAP)-positive astrocytes and ,-aminobutyric acid (GABA)ergic neurons. However, the absence of RC3 in the RC3,/, neurons was found to alter neuronal Ca2+ dynamics including baseline Ca2+ levels measured under normal physiological conditions or after blockade of synaptic transmission, spontaneous intracellular Ca2+ oscillations generated by network synaptic activity, and Ca2+ responses elicited by exogenous application of N-methyl- d -aspartate (NMDA) or class I metabotropic glutamate receptor agonists. Thus, significant changes in Ca2+ dynamics occur in cortical neurons when RC3 is absent and these changes do not involve changes in gross neuronal morphology or neuronal maturation. These data provide direct physiological evidence for a regulatory role of RC3 in neuronal Ca2+ dynamics. [source] Nucleotide-induced Ca2+ signaling in sustentacular supporting cells of the olfactory epitheliumGLIA, Issue 15 2008Thomas Hassenklöver Abstract Extracellular purines and pyrimidines are important signaling molecules acting via purinergic cell-surface receptors in neurons, glia, and glia-like cells such as sustentacular supporting cells (SCs) of the olfactory epithelium (OE). Here, we thoroughly characterize ATP-induced responses in SCs of the OE using functional Ca2+ imaging. The initial ATP-induced increase of the intracellular Ca2+ concentration [Ca2+]i always occurred in the apical part of SCs and subsequently propagated toward the basal lamina, indicating the occurrence of purinergic receptors in the apical part of SCs. The mean propagation velocity of the Ca2+ signal within SCs was 17.10 ± 1.02 ,m/s. ATP evoked increases in [Ca2+]i in both the presence and absence of extracellular Ca2+. Depletion of the intracellular Ca2+ stores abolished the responses. This shows that the ATP-induced [Ca2+]i increases were in large part, if not entirely, due to the activation of G protein-coupled receptors followed by Ca2+ mobilization from intracellular stores, suggesting an involvement of P2Y receptors. The order of potency of the applied purinergic agonists was UTP > ATP > ATP,S (with all others being only weakly active or inactive). The ATP-induced [Ca2+]i increases could be reduced by the purinergic antagonists PPADS and RB2, but not by suramin. Our findings suggest that extracellular nucleotides in the OE activate SCs via P2Y2/P2Y4 -like receptors and initiate a characteristic intraepithelial Ca2+ wave. © 2008 Wiley-Liss, Inc. [source] Axon-glia communication evokes calcium signaling in olfactory ensheathing cells of the developing olfactory bulbGLIA, Issue 4 2007Anne Rieger Abstract Olfactory ensheathing cells (OECs) accompany receptor axons in the olfactory nerve and promote axonal growth into the central nervous system. The mechanisms underlying the communication between axons and OECs, however, have not been studied in detail yet. We investigated the effect of activity-dependent neuronal transmitter release on Ca2+ signaling of OECs in acute mouse olfactory bulb slices using confocal Ca2+ imaging. TTX-sensitive axonal activity upon electrical nerve stimulation triggers a rise in cytosolic Ca2+ in OECs, which can be mimicked by application of DHPG, an agonist of metabotropic glutamate receptors (mGluRs). Both stimulation- and DHPG-induced Ca2+ transients in OECs were abolished by depletion of intracellular Ca2+ stores with cyclopiazonic acid (CPA). The mGluR1 -specific antagonist CPCCOEt completely inhibited DHPG-evoked Ca2+ transients, but reduced stimulation-induced Ca2+ transients only partly, suggesting the involvement of another neurotransmitter. Application of ATP evoked CPA-sensitive Ca2+ transients in OECs, which were inhibited by the P2Y1 -specific antagonist MRS2179. Co-application of CPCCOEt and MRS2179 almost completely blocked the stimulation-induced Ca2+ transients, indicating that they were mediated by mGluR1 and P2Y1 receptors. Our results show that OECs are able to respond to olfactory nerve activity with an increase in cytosolic Ca2+ due to glutamate and ATP release. © 2006 Wiley-Liss, Inc. [source] Calcium control of gene regulation in rat hippocampal neuronal culturesJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Giulietta Pinato Blockage of GABA-A receptors in hippocampal neuronal cultures triggers synchronous bursts of spikes initiating neuronal plasticity, partly mediated by changes of gene expression. By using specific pharmacological blockers, we have investigated which sources of Ca2+ entry primarily control changes of gene expression induced by 20,µM gabazine applied for 30,min (GabT). Intracellular Ca2+ transients were monitored with Ca2+ imaging while recording electrical activity with patch clamp microelectrodes. Concomitant transcription profiles were obtained using Affymetrix oligonucleotide microarrays and confirmed with quantitative RT-PCR. Blockage of NMDA receptors with 2-amino-5-phosphonovaleric acid (APV) did not reduce significantly somatic Ca2+ transients, which, on the contrary, were reduced by selective blockage of L, N, and P/Q types voltage gated calcium channels (VGCCs). Therefore, we investigated changes of gene expression in the presence of blockers of NMDA receptors and L, N, and P/Q VGCCs. Our results show that: (i) among genes upregulated by GabT, there are genes selectively dependent on NMDA activation, genes selectively dependent on L-type VGCCs and genes dependent on the activation of both channels; (ii) the majority of genes requires the concomitant activation of NMDA receptors and Ca2+ entry through VGCCs; (iii) blockage of N and P/Q VGCCs has an effect similar but not identical to blockage of L-type VGCCs. J. Cell. Physiol. 220: 727,747, 2009. © 2009 Wiley-Liss, Inc. [source] Glutamate-induced internalization of Cav1.3 L-type Ca2+ channels protects retinal neurons against excitotoxicityTHE JOURNAL OF PHYSIOLOGY, Issue 6 2010Fengxia Mizuno Glutamate-induced rise in the intracellular Ca2+ level is thought to be a major cause of excitotoxic cell death, but the mechanisms that control the Ca2+ overload are poorly understood. Using immunocytochemistry, electrophysiology and Ca2+ imaging, we show that activation of ionotropic glutamate receptors induces a selective internalization of Cav1.3 L-type Ca2+ channels in salamander retinal neurons. The effect of glutamate on Cav1.3 internalization was blocked in Ca2+ -free external solution, or by strong buffering of internal Ca2+ with BAPTA. Downregulation of L-type Ca2+ channel activity in retinal ganglion cells by glutamate was suppressed by inhibitors of dynamin-dependent endocytosis. Stabilization of F-actin by jasplakinolide significantly reduced the ability of glutamate to induce internalization suggesting it is mediated by Ca2+ -dependent reorganization of actin cytoskeleton. We showed that the Cav1.3 is the primary L-type Ca2+ channel contributing to kainate-induced excitotoxic death of amacrine and ganglion cells. Block of Cav1.3 internalization by either dynamin inhibition or F-actin stabilization increased vulnerability of retinal amacrine and ganglion cells to kainate-induced excitotoxicity. Our data show for the first time that Cav1.3 L-type Ca2+ channels are subject to rapid glutamate-induced internalization, which may serve as a negative feedback mechanism protecting retinal neurons against glutamate-induced excitotoxicity. [source] Glutamate-mediated astrocyte-to-neuron signalling in the rat dorsal hornTHE JOURNAL OF PHYSIOLOGY, Issue 5 2010Rita Bardoni By releasing neuroactive agents, including proinflammatory cytokines, prostaglandins and neurotrophins, microglia and astrocytes are proposed to be involved in nociceptive transmission, especially in conditions of persistent, pathological pain. The specific action on dorsal horn neurons of agents released from astrocytes, such as glutamate, has been, however, poorly investigated. By using patch-clamp and confocal microscope calcium imaging techniques in rat spinal cord slices, we monitored the activity of dorsal horn lamina II neurons following astrocyte activation. Results obtained revealed that stimuli that triggered Ca2+ elevations in astrocytes, such as the purinergic receptor agonist BzATP and low extracellular Ca2+, induce in lamina II neurons slow inward currents (SICs). Similarly to SICs triggered by astrocytic glutamate in neurons from other central nervous system regions, these currents (i) are insensitive to tetrodotoxin (TTX), (ii) are blocked by the NMDA receptor (NMDAR) antagonist d -AP5, (iii) lack an AMPA component, and (iv) have slow rise and decay times. Ca2+ imaging also revealed that astrocytic glutamate evokes NMDAR-mediated episodes of synchronous activity in groups of substantia gelatinosa neurons. Importantly, in a model of peripheral inflammation, the development of thermal hyperalgesia and mechanical allodynia was accompanied by a significant increase of spontaneous SICs in dorsal horn neurons. The NMDAR-mediated astrocyte-to-neuron signalling thus represents a novel pathway that may contribute to the control of central sensitization in pathological pain. [source] Efficient Ca2+ buffering in fast-spiking basket cells of rat hippocampusTHE JOURNAL OF PHYSIOLOGY, Issue 8 2008Yexica Aponte Fast-spiking parvalbumin-expressing basket cells (BCs) represent a major type of inhibitory interneuron in the hippocampus. These cells inhibit principal cells in a temporally precise manner and are involved in the generation of network oscillations. Although BCs show a unique expression profile of Ca2+ -permeable receptors, Ca2+ -binding proteins and Ca2+ -dependent signalling molecules, physiological Ca2+ signalling in these interneurons has not been investigated. To study action potential (AP)-induced dendritic Ca2+ influx and buffering, we combined whole-cell patch-clamp recordings with ratiometric Ca2+ imaging from the proximal apical dendrites of rigorously identified BCs in acute slices, using the high-affinity Ca2+ indicator fura-2 or the low-affinity dye fura-FF. Single APs evoked dendritic Ca2+ transients with small amplitude. Bursts of APs evoked Ca2+ transients with amplitudes that increased linearly with AP number. Analysis of Ca2+ transients under steady-state conditions with different fura-2 concentrations and during loading with 200 ,m fura-2 indicated that the endogenous Ca2+ -binding ratio was ,200 (,S= 202 ± 26 for the loading experiments). The peak amplitude of the Ca2+ transients measured directly with 100 ,m fura-FF was 39 nm AP,1. At ,23°C, the decay time constant of the Ca2+ transients was 390 ms, corresponding to an extrusion rate of ,600 s,1. At 34°C, the decay time constant was 203 ms and the corresponding extrusion rate was ,1100 s,1. At both temperatures, continuous theta-burst activity with three to five APs per theta cycle, as occurs in vivo during exploration, led to a moderate increase in the global Ca2+ concentration that was proportional to AP number, whereas more intense stimulation was required to reach micromolar Ca2+ concentrations and to shift Ca2+ signalling into a non-linear regime. In conclusion, dentate gyrus BCs show a high endogenous Ca2+ -binding ratio, a small AP-induced dendritic Ca2+ influx, and a relatively slow Ca2+ extrusion. These specific buffering properties of BCs will sharpen the time course of local Ca2+ signals, while prolonging the decay of global Ca2+ signals. [source] Sustained granule cell activity disinhibits juvenile mouse cerebellar stellate cells through presynaptic mechanismsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2008Simone Astori GABA release from cerebellar molecular layer interneurons can be modulated by presynaptic glutamate and/or GABAB receptors upon perfusing the respective agonists. However, it is unclear how release and potential spillover of endogenous transmitter lead to activation of presynaptic receptors. High frequency firing of granule cells, as observed in vivo upon sensory stimulation, could lead to glutamate and/or GABA spillover. Here, we established sustained glutamatergic activity in the granule cell layer of acute mouse cerebellar slices and performed 190 paired recordings from connected stellate cells. Train stimulation at 50 Hz reduced by about 30% the peak amplitude of IPSCs evoked by brief depolarization of the presynaptic cell in 2-week-old mice. A presynaptic mechanism was indicated by changes in failure rate, paired-pulse ratio and coefficient of variation of evoked IPSCs. Furthermore, two-photon Ca2+ imaging in identified Ca2+ hot spots of stellate cell axons confirmed reduced presynaptic Ca2+ influx after train stimulation within the granular layer. Pharmacological experiments indicated that glutamate released from parallel fibres activated AMPARs in stellate cells, evoking GABA release from surrounding cells. Consequential GABA spillover activated presynaptic GABABRs, which reduced the amplitude of eIPSCs. Two-thirds of the total disinhibitory effect were mediated by GABABRs, one-third being attributable to presynaptic AMPARs. This estimation was confirmed by the observation that bath applied baclofen induced a more pronounced reduction of evoked IPSCs than kainate. Granule cell-mediated disinhibition persisted at near-physiological temperature but was strongly diminished in 3-week-old mice. At this age, GABA release probability was not reduced and presynaptic GABABRs were still detectable, but GABA uptake appeared to be advanced, attenuating GABA spillover. Thus, sustained granule cell activity modulates stellate cell-to-stellate cell synapses, involving transmitter spillover during a developmentally restricted period. [source] Modulation of Ca2+ signalling in rat atrial myocytes: possible role of the ,1c carboxyl terminalTHE JOURNAL OF PHYSIOLOGY, Issue 2 2003Sun-Hee Woo Ca2+ influx through L-type Cav1.2 (,1c) Ca2+ channels is a critical step in the activation of cardiac ryanodine receptors (RyRs) and release of Ca2+ via Ca2+ -induced Ca2+ release(CICR). The released Ca2+, in turn, is the dominant determinant of inactivation of the Ca2+ current (ICa) and termination of release. Although Ca2+ cross-signalling is mediated by high Ca2+ fluxes in the microdomains of ,1c -RyR complexes, ICa -gated Ca2+ cross-signalling is surprisingly resistant to intracellular Ca2+ buffering and has steeply voltage-dependent gain, inconsistent with a strict CICR mechanism, suggesting the existence of additional regulatory step(s). To explore the possible regulatory role of the carboxyl (C)-terminal tail of ,1c in modulating Ca2+ signalling, we tested the effects of introducing two ,1c C-terminal peptides, LA (1571,1599) and K (1617,1636) on the central ,1c -unassociated Ca2+ -release sites of atrial myocytes, using rapid (240 Hz) two-dimensional confocal Ca2+ imaging. The frequency of spontaneously activating central sparks increased by approximately fourfold on dialysing LA- but not K-peptide into myocytes voltage-clamped at -80 mV. The rate but not the magnitude of caffeine (10 mM)-triggered central Ca2+ release was significantly accelerated by LA- but not K-peptide. Individual Ca2+ spark size and flux were larger in LA- but not in K-peptide-dialysed myocytes. Although LA-peptide did not change the amplitude or inactivation kinetics of ICa, LA-peptide did strongly enhance the central Ca2+ transients triggered by ICa at -30 mV (small ICa) but not at +20 mV (large ICa). In contrast, K-peptide had no effect on either ICa or the local Ca2+ transients. LA-peptide with a deleted calmodulin-binding region (LM1-peptide) had no significant effects on the central spark frequency but suppressed spontaneous spark frequency in the periphery. Our results indicate that the calmodulin-binding LA motif of the ,1c C-terminal tail may sensitize the RyRs, thereby increasing their open probability and providing for both the voltage-dependence of CICR and the higher frequency of spark occurrence in the periphery of atrial myocytes where the native ,1c -RyR complexes are intact. [source] Antimuscarinic antibodies in primary Sjögren's syndrome reversibly inhibit the mechanism of fluid secretion by human submandibular salivary acinar cellsARTHRITIS & RHEUMATISM, Issue 4 2006L. J. Dawson Objective Sjögren's syndrome (SS) is an autoimmune condition affecting salivary glands, for which a clearly defined pathogenic autoantibody has yet to be identified. Autoantibodies that bind to the muscarinic M3 receptors (M3R), which regulate fluid secretion in salivary glands, have been proposed in this context. However, there are no previous data that directly show antisecretory activity. This study was undertaken to investigate and characterize the antisecretory activity of anti-M3R. Methods Microfluorimetric Ca2+ imaging and patch clamp electrophysiologic techniques were used to measure the secretagogue-evoked increase in [Ca2+]i and consequent activation of Ca2+ -dependent ion channels in individual mouse and human submandibular acinar cells. Together, these techniques form a sensitive bioassay that was used to determine whether IgG isolated from patients with primary SS and from control subjects has antisecretory activity. Results IgG (2 mg/ml) from patients with primary SS reduced the carbachol-evoked increase in [Ca2+]i in both mouse and human acinar cells by ,50%. IgG from control subjects had no effect on the Ca2+ signal. Furthermore, the inhibitory action of primary SS patient IgG on the Ca2+ signal was acutely reversible. We repeated our observations using rabbit serum containing antibodies raised against the second extracellular loop of M3R and found an identical pattern of acutely reversible inhibition. Anti-M3R,positive serum had no effect on Ca2+ -dependent ion channel activation evoked by the direct intracellular infusion of inositol 1,4,5-triphosphate. Conclusion These observations show for the first time that IgG from patients with primary SS contains autoantibodies capable of damaging saliva production and contributing to xerostomia. The unusual but not unprecedented acute reversibility of the effects of anti-M3 autoantibodies is the subject of further research. [source] Resolvin D1 attenuates activation of sensory transient receptor potential channels leading to multiple anti-nociceptionBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2010S Bang BACKGROUND AND PURPOSE Temperature-sensitive transient receptor potential ion channels (thermoTRPs) expressed in primary sensory neurons and skin keratinocytes play a crucial role as peripheral pain detectors. Many natural and synthetic ligands have been found to act on thermoTRPs, but little is known about endogenous compounds that inhibit these TRPs. Here, we asked whether resolvin D1 (RvD1), a naturally occurring anti-inflammatory and pro-resolving lipid molecule is able to affect the TRP channel activation. EXPERIMENTAL APPROACH We examined the effect of RvD1 on the six thermoTRPs using Ca2+ imaging and whole cell electrophysiology experiments using the HEK cell heterologous expression system, cultured sensory neurons and HaCaT keratinocytes. We also checked changes in agonist-specific acute licking/flicking or flinching behaviours and TRP-related mechanical and thermal pain behaviours using Hargreaves, Randall-Selitto and von Frey assay systems with or without inflammation. KEY RESULTS RvD1 inhibited the activities of TRPA1, TRPV3 and TRPV4 at nanomolar and micromolar levels. Consistent attenuations in agonist-specific acute pain behaviours by immediate peripheral administration with RvD1 were also observed. Furthermore, local pretreatment with RvD1 significantly reversed mechanical and thermal hypersensitivity in inflamed tissues. CONCLUSIONS AND IMPLICATIONS RvD1 was a novel endogenous inhibitor for several sensory TRPs. The results of our behavioural studies suggest that RvD1 has an analgesic potential via these TRP-related mechanisms. [source] Genistein potentiates activity of the cation channel TRPC5 independently of tyrosine kinasesBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2010Ching-On Wong Background and purpose:, TRPC5 is a Ca2+ -permeable channel with multiple modes of activation. We have explored the effects of genistein, a plant-derived isoflavone, on TRPC5 activity, and the mechanism(s) involved. Experimental approach:, Effects of genistein on TRPC5 channels were investigated in TRPC5-over-expressing human embryonic kidney 293 (HEK) cells and bovine aortic endothelial cells (BAECs) using fluorescent Ca2+ imaging and electrophysiological techniques. Key results:, In TRPC5-over-expressing HEK cells, genistein stimulated TRPC5-mediated Ca2+ influx, concentration dependently (EC50= 93 µM). Genistein and lanthanum activated TRPC5 channels synergistically. Effects of genistein on TRPC5 channels were mimicked by daidzein (100 µM), a genistein analogue inactive as a tyrosine kinase inhibitor, but not by known tyrosine kinase inhibitors herbimycin (2 µM), PP2 (20 µM) and lavendustin A (10 µM). Action of genistein on TRPC5 channels was not affected by an oestrogen receptor inhibitor ICI-182780 (50 µM) or a phospholipase C inhibitor U73122 (10 µM), suggesting genistein did not act through oestrogen receptors or phospholipase C. In BAECs, genistein (100 µM) stimulated TRPC5-mediated Ca2+ influx. In patch clamp studies, both genistein (50 µM) and daidzein (50 µM) augmented TRPC5-mediated whole-cell cation current in TRPC5 over-expressing HEK cells. Genistein stimulated TRPC5 channel activity in excised inside-out membrane patch, suggesting that its action was relatively direct and did not require cytosolic factors. Conclusions and implications:, The present study is the first to demonstrate stimulation of a TRP channel by isoflavones. Genistein is a lipophilic compound able to stimulate TRPC5 activity in TRPC5-over-expressing HEK cells and in native vascular endothelial cells. [source] Modulation of sensory neuron potassium conductances by anandamide indicates roles for metabolitesBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2008R M Evans Background and purpose: The endogenous cannabinoid anandamide (AEA) acts at cannabinoid (CB1) and vanilloid (TRPV1) receptors. AEA also shows antinociceptive properties; although the underlying mechanism for this is not fully understood, both CB1 and TRPV1 may be involved. Voltage-activated Ca2+ channels in rat-cultured dorsal root ganglion (DRG) neurons are modulated by AEA. However, AEA in different populations of neurons enhanced or attenuated KCl-evoked Ca2+ influx; these effects were linked with soma size. The aim of this study was to determine how AEA or its metabolites might produce these variable responses. Experimental approach: The whole cell patch-clamp technique and fura-2 Ca2+ imaging were used to characterize the actions of AEA on action potential firing and voltage-activated K+ currents and to determine whether AEA metabolism plays any role in its effects on cultured DRG neurons. Key results: AEA attenuated multiple action potential firing evoked by 300 ms depolarizing current commands in a subpopulation of DRG neurons. Application of 1 ,M AEA attenuated voltage-activated K+ currents and the recovery of KCl-evoked Ca2+ transients. The insensitivity of these responses to the CB1 receptor antagonist rimonabant (100 nM) and preincubation of DRG neurons with pertussis toxin suggested that these actions are not CB1 receptor-mediated. Preincubating DRG neurons with the fatty acid amide hydrolase (FAAH) inhibitor phenylmethylsulphonyl fluoride (PMSF) attenuated the inhibitory actions of AEA on K+ currents and Ca2+ influx. Conclusion and implications: These data suggest that the products of AEA metabolism by FAAH contribute to the attenuation of K+ conductances and altered excitability of cultured sensory neurons. British Journal of Pharmacology (2008) 154, 480,492; doi:10.1038/bjp.2008.93; published online 31 March 2008 [source] | |||||||||||||