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BACKGROUND: The characterization of neuropathic pain is maladaptive plasticity within the nociceptive system. Multiple alterations contribute to complex pain phenotypes. Adrenomedullin (AM) has been documented to be a pain mediator. However, its involvement in pathological pain is poorly understood. We studied the contribution of AM to chronic neuropathic pain in the spinal nerve ligation (SNL) model. METHODS: Daily injection of the AM receptor antagonist AM22-52 (10 nmol) via an intrathecal (i.t.) route after SNL inhibited mechanical allodynia starting on day 6. Single administration of AM22-52 produced an immediate attenuation on pain hypersensitivity on day 2 or 10 post-SNL. Protein and mRNA levels were assayed by immunofluorescent staining and qRT-PCR, respectively, on days 1, 3, 7 and 15 post-SNL. RESULTS: The results showed that AM at both protein and mRNA levels was increased in both injured (L5) and adjacent uninjured (L4) nerves starting on day 3 post-SNL. In dorsal root ganglion (DRG) at L5, AM was increase on days 1-7 at mRNA level but only on day 7 at protein level. However, AM was increase at mRNA level on days 1-7 and at protein level on days 3-15 in L4 DRG. AM mRNA expression was upregulated on days 1-7 in the spinal cord. Expression of receptor activity-modifying protein 2 (RAMP2), an essential AM1 receptor component, was upregulated in small and medium-diameter neurons on days 1-15 in both L5 and L4 DRG. Furthermore, single administration of AM22-52 suppressed the increase of nNOS in DRG induced by SNL and daily injection of AM22-52 for 7 days inhibited SNL-induced increase of CGRP mRNA in the spinal dorsal horn. CONCLUSIONS: This study indicates that the increased AM bioactivity in injured and uninjured peripheral nerves, uninjured adjacent DRG and the spinal dorsal horn play a critical role mainly in the late-phase development of neuropathic pain. The mechanism may involve the recruitment of nNOS and CGRP. ispartof: FRONTIERS IN BIOSCIENCE-LANDMARK vol:27 issue:7 ispartof: location:Singapore status: published

Mechanosensation - by which mechanical stimuli are converted into a neuronal signal - is the basis for the sensory systems of hearing, balance, and touch. Mechanosensation is unmatched in speed and its diverse range of sensitivities, reaching its highest temporal limits with the sense of hearing; however, hair cells (HCs) and the auditory nerve (AN) serve as obligatory bottlenecks for sounds to engage the brain. Like other sensory neurons, auditory neurons use the canonical pathway for neurotransmission and millisecond-duration action potentials (APs). How the auditory system utilizes the relatively slow transmission mechanisms to achieve ultrafast speed, and high audio-frequency hearing remains an enigma. Here, we address this paradox and report that the mouse, and chinchilla, AN are mechanically sensitive, and minute mechanical displacement profoundly affects its response properties. Sound-mimicking sinusoidal mechanical and electrical current stimuli affect phase-locked responses. In a phase-dependent manner, the two stimuli can also evoke suppressive responses. We propose that mechanical sensitivity interacts with synaptic responses to shape responses in the AN, including frequency tuning and temporal phase locking. Combining neurotransmission and mechanical sensation to control spike patterns gives the mammalian AN a secondary receptor role, an emerging theme in primary neuronal functions. ispartof: ELIFE vol:11 ispartof: location:England status: published

The transient receptor potential vanilloid type-2 (TRPV2) protein is a nonselective Ca2+ permeable channel member of the TRPV subfamily, still considered an orphan TRP channel due to the scarcity of available selective and potent pharmacological tools and endogenous modulators. Here we describe the discovery of novel synthetic long-chain capsaicin derivatives as potent TRPV2 antagonists in comparison to the totally inactive capsaicin, the role of their hydrophobic chain, and how the structure-activity relationships of such derivatives led, through a ligand-based approach, to the identification of endogenous long-chain fatty acid ethanolamides or primary amides acting as TRPV2 antagonists. Both synthetic and endogenous antagonists exhibited differential inhibition against known TRPV2 agonists characterized by distinct kinetic profiles. These findings represent the first example of both synthetic and naturally occurring TRPV2 modulators with efficacy in the submicromolar/low-micromolar range, which will be useful for clarifying the physiopathological roles of this receptor, its regulation, and its targeting in pathological conditions. We gratefully acknowledge financial support from Universitat de Lleida, Ministerio de Educación, Cultura y Deporte and Banco Santander (Programa UdL-Impuls). The authors are grateful to the Serveis Cientifictècnics (SCT) of the Universitat de Lleida for providing us with spectroscopic and chromatographic facilities. We acknowledge Dr. Alberto Minassi, Dipartimento di Scienze del Farmaco, Universitàdel Piemonte Orientale, Novara, Italy, for the kind gift of olvanil.

Objectives Recent observations demonstrated the expression of the insulin receptor (InsR) and its functional interaction with the transient receptor potential vanilloid type 1 receptor (TRPV1) in sensory ganglion neurons. Because sensory nerves are implicated in pancreatic inflammatory processes, we studied the colocalization of the InsR with TRPV1 and proinflammatory neuropeptides in spinal and vagal pancreatic afferent neurons. Methods Immunohistochemistry and quantitative morphometry were used to analyze the expression of TRPV1, InsR, substance P (SP), and calcitonin gene-related peptide (CGRP) in retrogradely labeled pancreatic dorsal root ganglion (DRG) and nodose ganglion (NG) neurons. Results The proportions of retrogradely labeled pancreatic TRPV1-, InsR-, SP-, and CGRP-immunoreactive neurons amounted to 68%, 48%, 33%, and 54% in DRGs and 64%, 49%, 40%, and 25% in the NGs. Of the labeled DRG and NG neurons, 23% and 35% showed both TRPV1 and InsR immunoreactivity. Colocalization of the InsR with SP or CGRP was demonstrated in 14% and 28% of pancreatic DRG and 24% and 8% of pancreatic NG neurons. Conclusions The present findings provide morphological basis for possible functional interactions among the nociceptive ion channel TRPV1, the InsR, and the proinflammatory neuropeptides SP and CGRP expressed by pancreatic DRG and NG neurons.

Negative affective state has a significant impact on pain, and genetic background is an important moderating influence on this interaction. The Wistar-Kyoto (WKY) inbred rat strain exhibits a stress-hyperresponsive, anxiety/depressive-like phenotype and also displays a hyperalgesic response to noxious stimuli. Transient receptor potential subfamily V member 1 (TRPV1) within the midbrain periaqueductal grey (PAG) plays a key role in regulating both aversive and nociceptive behaviour. In the present study, we investigated the role of TRPV1 in the sub-columns of the PAG in formalin-evoked nociceptive behaviour in WKY versus Sprague-Dawley (SD) rats. TRPV1 mRNA expression was significantly lower in the dorsolateral (DL) PAG and higher in the lateral (L) PAG of WKY rats, compared with SD counterparts. There were no significant differences in TRPV1 mRNA expression in the ventrolateral (VL) PAG between the two strains. TRPV1 mRNA expression significantly decreased in the DLPAG and increased in the VLPAG of SD, but not WKY rats upon intra-plantar formalin administration. Intra-DLPAG administration of either the TRPV1 agonist capsaicin, or the TRPV1 antagonist 5'-lodoresiniferatoxin (5'-IRTX), significantly increased formalin-evoked nociceptive behaviour in SD rats, but not in WKY rats. The effects of capsaicin were likely due to TRPV1 desensitisation, given their similarity to the effects of 5'-IRTX. Intra-VLPAG administration of capsaicin or 5'-IRTX reduced nociceptive behaviour in a moderate and transient manner in SD rats, and similar effects were seen with 5'-IRTX in WKY rats. Intra-LPAG administration of 5'-IRTX reduced nociceptive behaviour in a moderate and transient manner in SD rats, but not in WKY rats. These results indicate that modulation of inflammatory pain by TRPV1 in the PAG occurs in a sub-column-specific manner. The data also provide evidence for differences in the expression of TRPV1, and differences in the effects of pharmacological modulation of TRPV1 in specific PAG sub-columns, between WKY and SD rats, suggesting that TRPV1 expression and/or functionality in the PAG plays a role in hyper-responsivity to noxious stimuli in a genetic background prone to negative affect. (C) 2016 Elsevier Ltd. All rights reserved. This work was funded by grants from Science Foundation Ireland (10/IN.1/B2976), the Irish Research Council, and a PhD scholarship from the College of Science, National University of Ireland Galway. peer-reviewed

Neural crest stem cells (NCPCs) have been shown to differentiate into various cell types and tissues during embryonic development, including sensory neurons. The few studies addressing the generation of NCPCs and peripheral sensory neurons (PSNs) from human induced pluripotent stem cells (hiPSCs), generated sensory cells without displaying robust activity. Here, we describe an efficient strategy for hiPSCs differentiation into NCPCs and functional PSNs using chemically defined media and factors to achieve efficient differentiation, confirmed by the expression of specific markers. After 10 days hiPSCs differentiated into NCPCs, cells were then maintained in neural induction medium containing defined growth factors for PSNs differentiation, followed by 10 days in neonatal human epidermal keratinocytes- (HEKn-) conditioned medium (CM). We observed a further increase in PSN markers expression and neurites length after CM treatment. The resulting neurons elicited action potentials after current injection and released substance P (SP) in response to nociceptive agents such as anandamide and resiniferatoxin. Anandamide induced substance P release via activation of TRPV1 and not CB1. Transcriptomic analysis of the PSNs revealed the main dorsal root ganglia neuronal markers and a transcriptional profile compatible with C fiber-low threshold mechanoreceptors. TRPV1 was detected by immunofluorescence and RNA-Seq in multiple experiments. In conclusion, the developed strategy generated PSNs useful for drug screening that could be applied to patient-derived hiPSCs, consisting in a powerful tool to model human diseases in vitro. Made available in DSpace on 2019-02-07T23:37:27Z (GMT). No. of bitstreams: 1 APGenerationiPSCFelipe.pdf: 7505321 bytes, checksum: 135c87796cec61c67776b2a6e5db7f0f (MD5) Previous issue date: 2019-02-07 Article 277.

The Nociceptin/Orphanin FQ peptide receptor (NOP), activated by its endogenous peptide ligand Nociceptin/Orphanin FQ (N/OFQ), exerts several effects including modulation of pain signalling. We have examined, for the first time, the tissue distribution of the NOP receptor in clinical visceral and somatic pain disorders by immunohistochemistry, and assessed functional effects of NOP and [micro] opioid receptor activation in cultured human and rat dorsal root ganglion (DRG) neurons. Quantification of NOP-positive nerve fibres within the bladder sub-urothelium revealed a remarkable several-fold increase in Detrusor Overactivity (p

Summary Topical capsaicin formulations are used for pain management. Safety and modest efficacy of low-concentration capsaicin formulations, which require repeated daily self-administration, are supported by meta-analyses of numerous studies. A high-concentration capsaicin 8% patch (Qutenza™) was recently approved in the EU and USA. A single 60-min application in patients with neuropathic pain produced effective pain relief for up to 12 weeks. Advantages of the high-concentration capsaicin patch include longer duration of effect, patient compliance, and low risk for systemic effects or drug–drug interactions. The mechanism of action of topical capsaicin has been ascribed to depletion of substance P. However, experimental and clinical studies show that depletion of substance P from nociceptors is only a correlate of capsaicin treatment and has little, if any, causative role in pain relief. Rather, topical capsaicin acts in the skin to attenuate cutaneous hypersensitivity and reduce pain by a process best described as ‘defunctionalization’ of nociceptor fibres. Defunctionalization is due to a number of effects that include temporary loss of membrane potential, inability to transport neurotrophic factors leading to altered phenotype, and reversible retraction of epidermal and dermal nerve fibre terminals. Peripheral neuropathic hypersensitivity is mediated by diverse mechanisms, including altered expression of the capsaicin receptor TRPV1 or other key ion channels in affected or intact adjacent peripheral nociceptive nerve fibres, aberrant re-innervation, and collateral sprouting, all of which are defunctionalized by topical capsaicin. Evidence suggests that the utility of topical capsaicin may extend beyond painful peripheral neuropathies.

Bladder small DRG neurons, which are putative nociceptors pivotal to urinary bladder function, express more than a dozen different ionic membrane mechanisms: ion channels, pumps and exchangers. Small-conductance Ca2+-activated K+ (SKCa) channels which were earlier thought to be gated solely by intracellular Ca2+ concentration ([Ca]i) have recently been shown to exhibit inward rectification with respect to membrane potential. The effect of SKCa inward rectification on the excitability of these neurons is unknown. Furthermore, studies on the role of KCa channels in repetitive firing and their contributions to different types of afterhyperpolarization (AHP) in these neurons are lacking. In order to study these phenomena, we first constructed and validated a biophysically detailed single compartment model of bladder small DRG neuron soma constrained by physiological data. The model includes twenty-two major known membrane mechanisms along with intracellular Ca2+ dynamics comprising Ca2+ diffusion, cytoplasmic buffering, and endoplasmic reticulum (ER) and mitochondrial mechanisms. Using modelling studies, we show that inward rectification of SKCa is an important parameter regulating neuronal repetitive firing and that its absence reduces action potential (AP) firing frequency. We also show that SKCa is more potent in reducing AP spiking than the large-conductance KCa channel (BKCa) in these neurons. Moreover, BKCa was found to contribute to the fast AHP (fAHP) and SKCa to the medium-duration (mAHP) and slow AHP (sAHP). We also report that the slow inactivating A-type K+ channel (slow KA) current in these neurons is composed of 2 components: an initial fast inactivating (time constant ∼ 25-100 ms) and a slow inactivating (time constant ∼ 200-800 ms) current. We discuss the implications of our findings, and how our detailed model can help further our understanding of the role of C-fibre afferents in the physiology of urinary bladder as well as in certain disorders., Author summary The small dorsal root ganglion (DRG) neurons of the bladder carry information related to pain from the bladder to the spinal cord. The level of electrical activity of these neurons is generally low but rises in abnormal conditions such as bladder inflammation and spinal cord injury. In order to investigate the reasons for this hyperactivity, we constructed a model of the bladder small DRG neuron soma including its membrane ion channels and intracellular Ca2+ dynamics. One of the channels, the small-conductance Ca2+-activated K+ (SKCa) channel present in these neurons which was earlier thought to be activated solely by intracellular Ca2+ concentration was recently shown to exhibit inward rectification with respect to the neuron’s membrane potential. It was found that SKCa inward rectification can alter the repetitive firing of the bladder small DRG neurons. We also delineated the role SKCa and BKCa (large-conductance Ca2+-activated K+) channels to the complex afterhyperpolarization (AHP) of the neuron’s action potentials, shedding light on the mechanisms that govern repetitive firing in these sensory neurons. Our findings have implications for the normal biophysics and dysfunctional states of urinary bladder arising from changes in sensory axon function, and these are discussed.

Background: Oxaliplatin chemotherapy induced neuropathy is a dose related cumulative toxicity that manifests as tingling, numbness, and chronic pain, compromising the quality of life and leading to discontinued chemotherapy. Patients report marked hypersensitivity to cold stimuli at early stages of treatment, when sensory testing reveals cold and heat hyperalgesia. This study examined the morphological and functional effects of oxaliplatin treatment in cultured adult rat DRG neurons. Results: 48 hour exposure to oxaliplatin resulted in dose related reduction in neurite length, density, and number of neurons compared to vehicle treated controls, using Gap43 immunostaining. Neurons treated acutely with 20 μg/ml oxaliplatin showed significantly higher signal intensity for cyclic AMP immunofluorescence (160.5 ± 13 a.u., n = 3, P < 0.05), compared to controls (120.3 ± 4 a.u.). Calcium imaging showed significantly enhanced capsaicin (TRPV1 agonist), responses after acute 20 μg/ml oxaliplatin treatment where the second of paired capsaicin responses increased from 80.7 ± 0.6% without oxaliplatin, to 171.26 ± 29% with oxaliplatin, (n = 6 paired t test, P < 0.05); this was reduced to 81.42 ± 8.1% (P < 0.05), by pretretreatment with the cannabinoid CB2 receptor agonist GW 833972. Chronic oxaliplatin treatment also resulted in dose related increases in capsaicin responses. Similarly, second responses to icilin (TRPA1/TRPM8 agonist), were enhanced after acute (143.85 ± 7%, P = 0.004, unpaired t test, n = 3), and chronic (119.7 ± 11.8%, P < 0.05, n = 3) oxaliplatin treatment, compared to control (85.3 ± 1.7%). Responses to the selective TRPM8 agonist WS-12 were not affected. Conclusions: Oxaliplatin treatment induces TRP sensitization mediated by increased intracellular cAMP, which may cause neuronal damage. These effects may be mitigated by co-treatment with adenylyl cyclase inhibitors, like CB2 agonists, to alleviate the neurotoxic effects of oxaliplatin.

Important pain transducers of noxious stimuli are small- and medium-diameter sensory neurons that express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors whose activity is upregulated by endogenous neuropeptides in acute and chronic pain models. Little is known about the role of endogenous modulators in restraining the expression and function of TRPV1 and P2X3 receptors. In dorsal root ganglia, evidence supports the involvement of the natriuretic peptide system in the modulation of nociceptive transmission especially via the B-type natriuretic peptide (BNP) that activates the natriuretic peptide receptor-A (NPR-A) to downregulate sensory neuron excitability. Since the role of BNP in trigeminal ganglia (TG) is unclear, we investigated the expression of BNP in mouse TG in situ or in primary cultures and its effect on P2X3 and TRPV1 receptors of patch-clamped cultured neurons. Against scant expression of BNP, almost all neurons expressed NPR-A at membrane level. While BNP rapidly increased cGMP production and Akt kinase phosphorylation, there was no early change in passive neuronal properties or responses to capsaicin, α,β-meATP or GABA. Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,β-meATP or GABA. Anantin alone decreased basal cGMP production and enhanced control α,β-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP. These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.

1 We have investigated the mechanism of regulation of 5-HT3 receptor channel sensitivity in voltage-clamped (-80 mV) NG108-15 neuroblastoma cells. 2 The 5-HT-induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, tau(1) (1.1+/-0.21s) and tau(2) (8.9+/-1.6s), respectively. Brief applications of 5-HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5-HT3 receptor-mediated peak inward currents. 3 Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5-HT-induced loss of responsiveness of 5-HT3 receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness. 4 Inclusion of the protein kinase inhibitor, staurosporine (1 mu M) or of okadaic acid (1 mu M), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5-HT3 receptor sensitivity. 5 Injection of cyclosporin A-cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5-HT response tau(1) (1.4+/-0.28s) and tau(2) (11.3+/-1.7s). The complex, however, prevented the loss of 5-HT3 receptor responsiveness upon repeated application of 5-HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 mu M). 6 The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 mu M) showed a half-life time (tau 1/2 of 2.6+/-0.12 min in control cells which was reduced to 1.6+/-0.09 min in cells treated with cyclosporin A-cyclophilin A (20 nM) complex. 7 We conclude that calcineurin does not affect the fast decay of the 5-HT3 receptor response but may be involved in a slower process which regulates channel activity.

The bioelectrical signalling within neural networks has to be monitored in real-time and localized in space in order to unravel the mechanisms behind pathologies and diseases of the nervous systems. Organic materials have significant potential for bio-functional neural interfacing given that their "soft" nature offers better mechanical compatibility with the nerve tissues than conventional semiconductors, and their flexibility allows realization of the non-planar forms typically required for biomedical implants. The integration of living cells into organic semiconductors is an important step towards the development of bio-organic electronic transducers of cellular activity from neurons. Here, we report on the use and characterization of n-type perylene derivatives as a suitable interface platform for organic neuro-electronic devices. We demonstrate that primary neurons can adhere, grow and differentiate on a suitably engineered perylene-based field-effect transistor platform, while maintaining their firing properties even after a prolonged time of cell-culturing. It is noteworthy that the field-effect transistors preserve their electrical characteristics even after 10 days of incubation in cell culture media. These results validate n-type perylene derivatives as a suitable long-term interface platform for organic neuro-electronic devices, which is particularly relevant in view of the recently reported perylene-based field-effect transistor structure capable of providing bidirectional stimulation and recording of primary neurons.

Depending on their primary structure, the 28 mammalian transient receptor potential (TRP) cation channels identified so far can be sorted into 6 subfamilies: TRPC ("Canonical"), TRPV ("Vanilloid"), TRPM ("Melastatin"), TRPP ("Polycystin"), TRPML ("Mucolipin"), and TRPA ("Ankyrin"). The TRPV subfamily (vanilloid receptors) comprises channels critically involved in nociception and thermosensing (TRPV1, TRPV2, TRPV3, and TRPV4), whereas TRPV5 and TRPV6 are involved in renal Ca2+ absorption/reabsorption. Apart from TRPV1, the pharmacology of these channels is still insufficiently known. Furthermore, only few small-molecule ligands for non-TRPV1 vanilloid receptors have been identified, and little is known of their endogenous ligands, resulting in a substantial "orphan" state for these channels. In this review, we summarize the pharmacological properties of members of the TRPV subfamily, highlighting the critical issues and challenges facing their "deorphanization" and clinical exploitation. ispartof: Molecular Pharmacology vol:75 issue:6 pages:1262-1279 ispartof: location:United States status: published

Background Reports of Ca(2+) current I(Ca) loss after injury to peripheral sensory neurons do not discriminate between axotomized and spared neurons. The spinal nerve ligation model separates axotomized from spared neurons innervating the same site. The authors hypothesized that I(Ca) loss is a result of neuronal injury, so they compared axotomized L5 dorsal root ganglion neurons to spared L4 neurons, as well as neurons from rats undergoing skin incision alone. Methods After behavioral testing, dissociated neurons from L4 and L5 dorsal root ganglia were studied in both current and voltage patch clamp modes. The biophysical consequence of I(Ca) loss on the action potential was confirmed using selective I(Ca) antagonists. Data were grouped into small, medium, and large cells for comparison. Results Reduced I(Ca) was predominantly a consequence of axotomy (L5 after spinal nerve ligation) and was most evident in small and medium neurons. ICa losses were associated with action potential prolongation in small and medium cells, whereas the amplitude and duration of after hyperpolarization was reduced in medium and large neurons. Blockade with Ca(2+) channel antagonists showed that action potential prolongation and after hyperpolarization diminution were alike, attributable to the loss of I(Ca). Conclusion Axotomy is required for I(Ca) loss. I(Ca) loss correlated with changes in the biophysical properties of sensory neuron membranes during action potential generation, which were due to I(Ca) loss leading to decreased outward Ca(2+)-sensitive K currents. Taken together, these results suggest that neuropathic pain may be mediated, in part, by loss of I(Ca) and the cellular processes dependent on Ca(2+).

The function of ATP-activated P2X3 receptors involved in pain sensation is modulated by desensitization, a phenomenon poorly understood. The present study used patch-clamp recording from cultured rat or mouse sensory neurons and kinetic modeling to clarify the properties of P2X3 receptor desensitization. Two types of desensitization were observed, a fast process (t1/2 = 50 ms; 10 microM ATP) following the inward current evoked by micromolar agonist concentrations, and a slow process (t1/2 = 35 s; 10 nM ATP) that inhibited receptors without activating them. We termed the latter high-affinity desensitization (HAD). Recovery from fast desensitization or HAD was slow and agonist-dependent. When comparing several agonists, there was analogous ranking order for agonist potency, rate of desensitization and HAD effectiveness, with 2-methylthioadenosine triphosphate the strongest and beta,gamma-methylene-ATP the weakest. HAD was less developed with recombinant (ATP IC50 = 390 nM) than native P2X3 receptors (IC50 = 2.3 nM). HAD could also be induced by nanomolar ATP when receptors seemed to be nondesensitized, indicating that resting receptors could express high-affinity binding sites. Desensitization properties were well accounted for by a cyclic model in which receptors could be desensitized from either open or closed states. Recovery was assumed to be a multistate process with distinct kinetics dependent on the agonist-dependent dissociation rate from desensitized receptors. Thus, the combination of agonist-specific mechanisms such as desensitization onset, HAD, and resensitization could shape responsiveness of sensory neurons to P2X3 receptor agonists. By using subthreshold concentrations of an HAD-potent agonist, it might be possible to generate sustained inhibition of P2X3 receptors for controlling chronic pain.

The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. Removing external Ca2+ enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium-dependent process. Heat stimuli of above about 43 °C elicit a sensation of pain in humans, and initiate action potentials in nociceptive nerve terminals in humans and animals (Belmonte & Giraldez, 1981; Robinson et al. 1983). In experiments on isolated nociceptive neurones, heat was found to activate an inward current that has the properties expected from psychophysical and whole-animal experiments for the detector of painful levels of heat (Cesare & McNaughton, 1996; Reichling & Levine, 1997; Kirschstein et al. 1997; Nagy & Rang, 1999). Inflammation causes the threshold for the initiation both of a sensation of pain and of action potentials in nociceptive nerve fibres to fall to a lower temperature, a process known as sensitization (Treede et al. 1992). The heat-gated current also shows sensitization, in that the temperature threshold for activation of the current is lowered on exposure of the nociceptor to bradykinin, a nonapeptide released during inflammation (Cesare & McNaughton, 1996). Bradykinin sensitizes the heat-gated current by activating the ɛ isoform of protein kinase C (PKCɛ) (Cesare et al. 1999a). An important molecular sensor for heat is the receptor for capsaicin, which was recently cloned and named vanilloid receptor 1, or VR1 (Caterina et al. 1997). Capsaicin, the active ingredient of chilli peppers, excites nociceptive neurones (Baumann et al. 1991; LaMotte et al. 1992) resulting in a burning pain sensation (Buck & Burks, 1986). VR1 is a non-selective cation channel that is activated by a broad spectrum of stimuli, including capsaicin, heat, low pH and the endocannabinoid anandamide (Caterina et al. 1997; Tominaga et al. 1998; Zygmunt et al. 1999; Smart et al. 2000). Targeted disruption of the VR1 gene in mice produces animals that do not respond adversely to even high doses of capsaicin (Caterina et al. 2000), showing that VR1 is the only receptor for capsaicin linked to pain perception. VR1 knockout animals still respond to noxious heat, showing that other heat-sensitive nociceptor mechanisms exist in addition to VR1. A major difference between wild-type and VR1 knockout mice, however, was that the heat hyperalgesia observed in inflamed tissue was greatly reduced in the knockout, suggesting that VR1 is the principal mechanism responsible for heat hyperalgesia (Davis et al. 2000; Caterina et al. 2000). The experiments in the present study explore the role of PKC in the sensitization of VR1, using both nociceptive neurones and heterologously expressed VR1. In nociceptive neurones we found that the response to capsaicin is sensitized by PKC activation, in a similar manner to the response to heat, adding to the evidence that the same molecular mechanism is responsible for the response to the two stimuli. In HEK293 cells expressing VR1 (hVR1-HEK293 cells) the responses to heat, capsaicin, low pH and anandamide were found to be enhanced by PKC activation. The enhancement of current in response to capsaicin, in both nociceptive neurones and hVR1-HEK293 cells, was abolished in the absence of external Ca2+, consistent with the demonstration by other authors that desensitization of VR1 occurs by a calcium-sensitive process (Cholewinski et al. 1993; Docherty et al. 1996; Liu & Simon, 1996; Koplas et al. 1997). Finally, while heat, capsaicin, low pH and anandamide activate current through VR1, activation of PKC potentiates the effect of these stimuli but does not itself activate current, showing that PKC activation does not directly gate the VR1 channel. Preliminary reports of the data reported here have appeared elsewhere (Vellani et al. 1999).

We demonstrate increased intra-ensemble energy transfer (ET) in monodispersed semiconducting quantum dots (QDs), mediated by localized plasmons on metallic thin films with nano-scale wrinkles. The increased ET results in a net spectral red-shift, up to three-fold increase in emission intensity, and a faster radiative recombination rate of the ensemble. The extent of the red-shift is dependent on QD size, and is largest for the QDs where the absorption spectrum overlaps the plasmonic resonance of the film. This effect has a uniform, macroscopic manifestation and may provide an inexpensive option of improving performance of QD based photovoltaic devices.

1. We have investigated the mechanism of regulation of 5-HT3 receptor channel sensitivity in voltage-clamped (-80 mV) NG108-15 neuroblastoma cells. 2. The 5-HT-induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, tau 1 (1.1 +/- 0.21s) and tau 2 (8.9 +/- 1.6s), respectively. Brief applications of 5-HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5-HT3 receptor-mediated peak inward currents. 3. Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5-HT-induced loss of responsiveness of 5-HT3 receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness. 4. Inclusion of the protein kinase inhibitor, staurosporine (1 microM) or of okadaic acid (1 microM), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5-HT3 receptor sensitivity. 5. Injection of cyclosporin A-cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5-HT response tau 1 (1.4 +/- 0.28s) and tau 2 (11.3 +/- 1.7s). The complex, however, prevented the loss of 5-HT3, receptor responsiveness upon repeated application of 5-HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 microM). 6. The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 microM) showed a half-life time (tau 1/2) of 2.6 +/- 0.12 min in control cells which was reduced to 1.6 +/- 0.09 min in cells treated with cyclosporin A-cyclophilin A (20 nM) complex. 7. We conclude that calcineurin does not affect the fast decay of the 5-HT3 receptor response but may be involved in a slower process which regulates channel activity.

The endocannabinoid anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca2+ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca(2+)-ATPase inhibitor thapsigargin leads to formation of anandamide, and subsequent TRPV1-dependent Ca2+ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1-mediated Ca2+ entry. In DRG neurons, this mechanism was found to lead to TRPV1-mediated currents that were enhanced by selective blockade of anandamide cellular efflux. Thus, endogenous anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5-triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release.