Gerard P. Ahern

Induction of vanilloid receptor channel activity by protein kinase C

Capsaicin or vanilloid receptors (VRs) participate in the sensation of thermal and inflammatory pain. The cloned (VR1) and native VRs are non-selective cation channels directly activated by harmful heat, extracellular protons and vanilloid compounds. However, considerable attention has been focused on identifying other signalling pathways in VR activation; it is known that VR1 is also expressed in non-sensory tissue and may mediate inflammatory rather than acute thermal pain. Here we show that activation of protein kinase C (PKC) induces VR1 channel activity at room temperature in the absence of any other agonist. We also observed this effect in native VRs from sensory neurons, and phorbol esters induced a vanilloid-sensitive Ca2+ rise in these cells. Moreover, the pro-inflammatory peptide, bradykinin, and the putative endogenous ligand, anandamide, respectively induced and enhanced VR activity, in a PKC-dependent manner. These results suggest that PKC may link a range of stimuli to the activation of VRs.

cGMP and S-nitrosylation: two routes for modulation of neuronal excitability by NO

The nitric oxide (NO)-cGMP signaling cascade has been implicated in synaptic plasticity and, more broadly, in the control of many forms of electrical activity. This raises the issue of how these second messengers regulate ion channels. The field of ion-channel modulation is dominated by G proteins; NO and cGMP are often treated as poor cousins. However, recent advances surveyed here could change this perception. A surprising new dimension to NO signaling is the direct cGMP-independent action of NO on channel proteins through S-nitrosylation. The existence of two effector pathways has important functional implications, expanding and enriching the possibilities for modulating neuronal excitability.

Capsaicin Receptor: TRPV1 A Promiscuous TRP Channel

TRPV1, the archetypal member of the vanilloid TRP family, was initially identified as the receptor for capsaicin, the pungent ingredient in hot chili peppers. The receptor has a diverse tissue distribution, with high expression in sensory neurons. TRPV1 is a nonselective cation channel with significant permeability to calcium, protons, and large polyvalent cations. It is the most polymodal TRP channel, being activated by numerous stimuli, including heat, voltage, vanilloids, lipids, and protons/cations. TRPV1 acts as a molecular integrator of physical and chemical stimuli in peripheral nociceptor terminals and plays a critical role in thermal inflammatory hyperalgesia. In addition, TRPV1 may regulate a variety of physiological functions in different organ systems. Various second messenger systems regulate TRPV1 activity, predominantly by serine-threonine phosphorylation. In this review, we provide a concise summary of the information currently available about this channel.

General anesthetics activate a nociceptive ion channel to enhance pain and inflammation

General anesthetics (GAs) have transformed surgery through their actions to depress the central nervous system and blunt the perception of surgical insults. Counterintuitively, many of these agents activate peripheral nociceptive neurons. However, the underlying mechanisms and significance of these effects have not been explored. Here, we show that clinical concentrations of noxious i.v. and inhalation GAs excite sensory neurons by selectively activating TRPA1, a key ion channel in the pain pathway. Further, these GAs induce pain-related responses in mice that are abolished in TRPA1-null animals. Significantly, TRPA1-dependent neurogenic inflammation is greater in mice anesthetized with pungent compared with nonpungent anesthetics. Thus, our results show that TRPA1 is essential for sensing noxious GAs. The pronociceptive effects of GAs combined with surgical tissue damage could lead to a paradoxical increase in postoperative pain and inflammation.

Extracellular Cations Sensitize and Gate Capsaicin Receptor TRPV1 Modulating Pain Signaling

Transient receptor potential (TRP) channels detect diverse sensory stimuli, including alterations in osmolarity. However, a molecular detector of noxious hypertonic stimuli has not yet been identified. We show here that acute pain-related behavior evoked by elevated ionic strength is abolished in TRP vanilloid subtype 1 (TRPV1)-null mice and inhibited by iodoresiniferatoxin, a potent TRPV1 antagonist. Electrophysiological recordings demonstrate a novel form of ion channel modulation by which extracellular Na+, Mg2+, and Ca2+ ions sensitize and activate the capsaicin receptor, TRPV1. At room temperature, increasing extracellular Mg2+ (from 1 to 5 mm) or Na+ (+50 mm) increased ligand-activated currents up to fourfold, and 10 mm Mg2+ reduced the EC50 for activation by capsaicin from 890 to 450 nm. Moreover, concentrations of divalent cations >10 mm directly gate the receptor. These effects occur via electrostatic interactions with two glutamates (E600 and E648) formerly identified as proton-binding residues. Furthermore, phospholipase C-mediated signaling enhances the effects of cations, and physiological concentrations of cations contribute to the bradykinin-evoked activation of TRPV1 and the sensitization of the receptor to heat. Thus, the modulation of TRPV1 by cationic strength may contribute to inflammatory pain signaling.

Oleoylethanolamide excites vagal sensory neurones, induces visceral pain and reduces short‐term food intake in mice via capsaicin receptor TRPV1

Oleoylethanolamide (OEA) is an endogenous lipid that regulates feeding and body weight. Although the effects of OEA are believed to depend on activation of vagal sensory afferent neurones, the mechanisms involved in exciting these neurones are unclear. Here we show that OEA directly excited nodose ganglion neurones, the cell bodies of vagal afferents. OEA depolarized these neurones and evoked inward currents that were restricted to capsaicin‐sensitive cells. These currents were fully blocked by the TRPV1 inhibitor, capsazepine, and no responses to OEA were observed in neurones cultured from TRPV1‐null mice. Similarly, OEA induced a rise in Ca+ concentration in wild‐type but not TRPV1‐deficient neurones, and responses to OEA were greater at 37°C compared to room temperature. Significantly, OEA administration in mice induced visceral pain‐related behaviours that were inhibited by capsazepine and absent in TRPV1‐null animals. Further, OEA reduced 30‐min food intake in wild‐type but not in TRPV1‐null mice. Thus, the acute behavioural effects of OEA may result from visceral malaise via the activation of TRPV1.

Voltage is a partial activator of rat thermosensitive TRP channels.

TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage‐sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo‐sensitive TRP channels. We show here using whole‐cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20–25°C) membrane depolarization evokes responses that saturate at ∼50–60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 μm), resiniferatoxin (5 μm) and menthol (6 mm) reveal voltage‐independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C‐dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage‐sensitive mechanism is unlikely to represent a final, gating mechanism for these channels.

Polyamines Are Potent Ligands for the Capsaicin Receptor TRPV1

Polyamines are important endogenous regulators of ion channels and are known to modulate inflammation and nociception. Here we investigated effects of polyamines on the capsaicin receptor TRPV1, a major ion channel expressed in nociceptive sensory afferents. Extracellular spermine, spermidine, and putrescine directly activated TRPV1 in a charge-dependent manner, both in heterologous expression systems and sensory neurons. The threshold for activation by spermine was ∼500 μm at room temperature. At lower concentrations, spermine enhanced capsaicin-evoked currents with an EC50 of ∼5 μm. Further, polyamines freely permeated TRPV1 (estimated relative permeabilities compared with Na+ were between 3 and 16), and spermine reduced the single channel conductance from 96 to 49 pS. Experiments with TRPV1 mutants identified extracellular acidic residues critical for polyamine regulation. Neutralization of aspartate 646 (D646N) abolished direct activation by spermine, whereas neutralization of this same aspartate (D646N) or glutamate 648 (E648A) inhibited spermine-induced sensitization. These data show that polyamines, by virtue of their cationic charge, can regulate the activity of TRPV1. Extracellular polyamines are present in considerable concentrations in the gastrointestinal tract and at synapses, and these levels increase during inflammation and cancer. Therefore, polyamine regulation of TRPV1 in these tissues may be relevant to a variety of physiological and pathophysiological states.

TRPV1-null mice are protected from diet-induced obesity

We explored a role for the capsaicin receptor, transient receptor potential channel vanilloid type 1 (TRPV1), in the regulation of feeding and body mass. On a 4.5% fat diet, wild‐type and TRPV1‐null mice gained equivalent body mass. On an 11% fat diet, however, TRPV1‐null mice gained significantly less mass and adiposity; at 44 weeks the mean body weights of wild‐type and TRPV1‐null mice were ∼51 and 34 g, respectively. Both groups of mice consumed equivalent energy and absorbed similar amounts of lipids. TRPV1‐null mice, however, exhibited a significantly greater thermogenic capacity. Interestingly, we found that 3T3‐L1 preadipocytes expressed functional calcitonin gene‐related peptide receptors. Thus, these data support a potential neurogenic mechanism by which TRPV1‐sensitive sensory nerves may regulate energy and fat metabolism.

TRPV1 is a novel target for omega‐3 polyunsaturated fatty acids

Omega‐3 (n‐3) fatty acids are essential for proper neuronal function, and they possess prominent analgesic properties, yet their underlying signalling mechanisms are unclear. Here we show that n‐3 fatty acids interact directly with TRPV1, an ion channel expressed in nociceptive neurones and brain. These fatty acids activate TRPV1 in a phosphorylation‐dependent manner, enhance responses to extracellular protons, and displace binding of the ultrapotent TRPV1 ligand [3H]resiniferatoxin. In contrast to their agonistic properties, n‐3 fatty acids competitively inhibit the responses of vanilloid agonists. These actions occur in mammalian cells in the physiological concentration range of 1–10 μm. Significantly, docosahexaenoic acid exhibits the greatest efficacy as an agonist, whereas eicosapentaenoic acid and linolenic acid are markedly more effective inhibitors. Similarly, eicosapentaenoic acid but not docosahexaenoic acid profoundly reduces capsaicin‐evoked pain‐related behaviour in mice. These effects are independent of alterations in membrane elasticity because the micelle‐forming detergent Triton X‐100 only minimally affects TRPV1 properties. Thus, n‐3 fatty acids differentially regulate TRPV1 and this form of signalling may contribute to their biological effects. Further, these results suggest that dietary supplementation with selective n‐3 fatty acids would be most beneficial for the treatment of pain.