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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.

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.

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

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).

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.