Activation of TRPA1 by membrane permeable local anesthetics

<p>Abstract</p> <p>Background</p> <p>Low concentrations of local anesthetics (LAs) suppress cellular excitability by inhibiting voltage-gated Na⁺channels. In contrast, LAs at high concentrations can be excitatory and neurotoxic. We recently demonstrated that LA-evoked activation of sensory neurons is mediated by the capsaicin receptor TRPV1, and, to a lesser extent by the irritant receptor TRPA1. LA-induced activation and sensitization of TRPV1 involves a domain that is similar, but not identical to the vanilloid-binding domain. Additionally, activation of TRPV1 by LAs involves PLC and PI(4,5)P₂-signalling. In the present study we aimed to characterize essential structural determinants for LA-evoked activation of TRPA1.</p> <p>Results</p> <p>Recombinant rodent and human TRPA1 were expressed in HEK293t cells and investigated by means of whole-cell patch clamp recordings. The LA lidocaine activates TRPA1 in a concentration-dependent manner. The membrane impermeable lidocaine-derivative QX-314 is inactive when applied extracellularly. Lidocaine-activated TRPA1-currents are blocked by the TRPA1-antagonist HC-030031. Lidocaine is also an inhibitor of TRPA1, an effect that is more obvious in rodent than in human TRPA1. This species-specific difference is linked to the pore region (transmembrane domain 5 and 6) as described for activation of TRPA1 by menthol. Unlike menthol-sensitivity however, lidocaine-sensitivity is not similarly determined by serine- and threonine-residues within TM5. Instead, intracellular cysteine residues known to be covalently bound by reactive TRPA1-agonists seem to mediate activation of TRPA1 by LAs.</p> <p>Conclusions</p> <p>The structural determinants involved in activation of TRPA1 by LAs are disparate from those involved in activation by menthol or those involved in activation of TRPV1 by LAs.</p

The intravenous anesthetic propofol excites nociceptve neurons by activation of TRPA1-, TRPV1- and GABAA-receptors

Hintergrund: Klinisch-gebräuchliche Anästhetika können bei intravasaler Injektion Schmerzen verursachen und sind daher in der Lage, nozizeptive Neurone zu aktivieren und möglicherweise auch zu sensibilisieren. In dieser Studie identifizierten wir die molekularen Mechanismen, die dem Injektionsschmerz des Hypnotikums 2,6-Diisopropylphenol (Propofol) unterliegen. Methoden: Mit der Whole-cell-Konfiguration der Patch-clamp- Technik und der Ca2+-Imaging-Technik wurden Human embryonic kidney (HEK)-Zellen, die transient unterschiedliche nozizeptive Proteine exprimierten sowie nozizeptive Spinalganglienzellen von Wildtyp- und Knockout-Mäusen untersucht. Mit Hilfe eines ELISA (Enzyme-linked Immunosorbent Assay) wurde die Freisetzung des Neuropeptids Calcitonin gene-related peptide (CGRP) aus isolierten peripheren Nerven von Wildtyp- und Knockout-Mäusen bestimmt. In Humanexperimenten wurde die Schmerzhaftigkeit der intrakutanen Injektion mehrerer Substanzen getestet. Ergebnisse: Klinisch-relevante Dosen von Propofol aktivierten die Rezeptoren TRPA1 und TRPV1 aus der „Transient receptor potential“ (TRP)-Familie in Spinalganglienzellen sowie in HEK-Zellen. In Spinalganglienzellen korrelierte die Aktivierung besser mit der Expression von TRPA1 als mit der Expression von TRPV1. Nach Vorbehandlung der Spinalganglien- oder HEK-Zellen mit dem PKC-Aktivator 4-Phorbol12-Myristat13-acetat (PMA) zeigte sich jedoch eine signifikante Sensibilisierung der durch Propofol induzierten und durch TRPV1 vermittelten Ströme in Spinalganglienzellen und HEK-Zellen. Die Blockade der Rezeptoren in Spinalganglienzellen von Wildtyp-Mäusen mit bekannten Rezeptor-Antagonisten sowie der Knock-out der Gene für TRPV1 und/ oder TRPA1 in Mäusen führt zu einer deutlichen Verkleinerung der Propofol-induzierten Aktivierung. Die verbleibende Restaktivierung konnte durch den selektiven GABAA-Rezeptor-Antagonisten Picrotoxin vollständig geblockt werden. Propofol verursachte an isolierten peripheren Nervenpräparaten von Wildtyp-Mäusen eine Freisetzung von CGRP, die Applikation von GABA an isolierten peripheren Nerven führte zu keiner CGRP-Freisetzung. Nach Applikation von Propofol an isolierten Nerven von TRPV1-/-/ TRPA1-/-- Mäusen zeigte sich ebenfalls keine CGRP-Freisetzung. Im Humanversuch verursachte nur Propofol, nicht aber GABA einen intensiven, brennenden Schmerz nach intrakutaner Injektion. Schlussfolgerungen: Da sowohl die Freisetzung von CGRP aus peripheren Nerven als auch der Injektionsschmerz nicht durch GABAA-Rezeptoren vermittelt zu sein scheinen, belegen unsere Daten, dass TRPV1 und TRPA1 die Schlüsselelemente bei der Propofol-induzierten Aktivierung von nozizeptiven Neuronen sind. Unsere Studie eröffnet neue Aspekte zum Thema der Nozizeptor-Sensibilisierung durch Anästhetika sowie einer dadurch induzierten, möglicherweise verstärkten, postoperativen Schmerzwahrnehmung.Background: Anesthetic agents can induce a paradox activation and sensitization of nociceptive sensory neurons and, thus, potentially facilitate pain processing. Here we identify distinct molecular mechanisms that mediate an activation of sensory neurons by 2,6-diisopropylphenol (propofol), a commonly used intravenous anesthetic known to elicit intense pain upon injection. Methods: By means of the whole cell configuration of the patch clamp technique and the Ca2+ -imaging technique human embryonic kidney (HEK) cells transiently expressing different nociceptive proteins and dorsal root ganglia (DRG) cells from wild-type and knock-out mice were studied. The release of calcitonin gene-related peptide (CGRP) from isolated peripheral nerves from wild-type and knock-out mice was explored by an enzyme-linked immunosorbent assay. In human experiments the painfulness of intracutaneous injection of different substances was tested. Results: Clinically relevant concentrations of propofol activated the transient receptor potential (TRP) receptors TRPA1 and TRPV1 in HEK and DRG cells. In DRG neurons, propofol-induced activation correlated better to expression of TRPA1 than of TRPV1. However, pretreatment with the protein kinase C-activator 4-phorbol 12-myristate 13-acetate (PMA) resulted in a significantly sensitized propofol-induced activation of TRPV1 in DRG neurons as well as in HEK cells. Pharmacological and genetic silencing of both TRPA1 and TRPV1 only partially abrogated propofol-induced responses in DRG neurons. The remaining propofol-induced activation was abolished by the selective GABAA receptor antagonist picrotoxin. Propofol but not GABA evoked a release of CGRP, a key component of neurogenic inflammation, from isolated peripheral nerves of wild-type but not TRPV1 and TRPA1-deficient mice. Moreover, propofol but not GABA induced an intense pain upon intracutaneous injection. Conclusions: As both the release of calcitonin gene-related peptide and injection pain by propofol seem to be independent of GABAA receptors, our data identify TRPV1 and TRPA1 as key molecules for propofol-induced excitation of sensory neurons. This study warrants further investigations into the role of anesthetics to induce nociceptor sensitization and to foster postoperative pain

Etomidate and propylene glycol activate nociceptive TRP ion channels

Background Etomidate is a preferred drug for the induction of general anesthesia in cardiovascular risk patients. As with propofol and other perioperatively used anesthetics, the application of aqueous etomidate formulations causes an intensive burning pain upon injection. Such algogenic properties of etomidate have been attributed to the solubilizer propylene glycol which represents 35% of the solution administered clinically. The aim of this study was to investigate the underlying molecular mechanisms which lead to injection pain of aqueous etomidate formulations. Results Activation of the nociceptive transient receptor potential (TRP) ion channels TRPA1 and TRPV1 was studied in a transfected HEK293t cell line by whole-cell voltage clamp recordings of induced inward ion currents. Calcium influx in sensory neurons of wild-type and trp knockout mice was ratiometrically measured by Fura2-AM staining. Stimulated calcitonin gene-related peptide release from mouse sciatic nerves was detected by enzyme immunoassay. Painfulness of different etomidate formulations was tested in a translational human pain model. Etomidate as well as propylene glycol proved to be effective agonists of TRPA1 and TRPV1 ion channels at clinically relevant concentrations. Etomidate consistently activated TRPA1, but there was also evidence for a contribution of TRPV1 in dependence of drug concentration ranges and species specificities. Distinct N-terminal cysteine and lysine residues seemed to mediate gating of TRPA1, although the electrophile scavenger N-acetyl-L-cysteine did not prevent its activation by etomidate. Propylene glycol-induced activation of TRPA1 and TRPV1 appeared independent of the concomitant high osmolarity. Intradermal injections of etomidate as well as propylene glycol evoked severe burning pain in the human pain model that was absent with emulsification of etomidate. Conclusions Data in our study provided evidence that pain upon injection of clinical aqueous etomidate formulations is not an unspecific effect of hyperosmolarity but rather due to a specific action mediated by activated nociceptive TRPA1 and TRPV1 ion channels in sensory neurons

Local Anesthetic-like Inhibition of Voltage-gated Na Channels by the Partial -opioid Receptor Agonist Buprenorphine

ABSTRACT Background: Opioids induce analgesia mainly by inhibiting synaptic transmission via G protein-coupled opioid receptors. In addition to analgesia, buprenorphine induces a pronounced antihyperalgesia and is an effective adjuvant to local anesthetics. These properties only partially apply to other opioids, and thus targets other than opioid receptors are likely to be employed. Here we asked if buprenorphine inhibits voltage-gated Na ϩ channels. Methods: Na ϩ currents were examined by whole cell patch clamp recordings on different recombinant Na ϩ channel ␣-subunits. The effect of buprenorphine on unmyelinated mouse C-fibers was examined with the skin-nerve preparation. Data are presented as mean Ϯ SEM. Results: Buprenorphine induced a concentration-dependent tonic (IC 50 33 Ϯ 2 M) and use-dependent block of endogenous Na ϩ channels in ND7/23 cells. This block was state-dependent and displayed slow on and off characteristics. The effect of buprenorphine was reduced on local anesthetic insensitive Nav1.4-mutant constructs and was more pronounced on the inactivation-deficient Nav1.4-WCW mutant

The General Anesthetic Propofol Excites Nociceptors by Activating TRPV1 and TRPA1 Rather than GABAA Receptors*

Anesthetic agents can induce a paradox activation and sensitization of nociceptive sensory neurons and, thus, potentially facilitate pain processing. Here we identify distinct molecular mechanisms that mediate an activation of sensory neurons by 2,6-diisopropylphenol (propofol), a commonly used intravenous anesthetic known to elicit intense pain upon injection. Clinically relevant concentrations of propofol activated the recombinant transient receptor potential (TRP) receptors TRPA1 and TRPV1 heterologously expressed in HEK293t cells. In dorsal root ganglion (DRG) neurons, propofol-induced activation correlated better to expression of TRPA1 than of TRPV1. However, pretreatment with the protein kinase C activator 4β-phorbol 12-myristate 13-acetate (PMA) resulted in a significantly sensitized propofol-induced activation of TRPV1 in DRG neurons as well as in HEK293t cells. Pharmacological and genetic silencing of both TRPA1 and TRPV1 only partially abrogated propofol-induced responses in DRG neurons. The remaining propofol-induced activation was abolished by the selective γ-aminobutyric acid, type A (GABAA) receptor antagonist picrotoxin. Propofol but not GABA evokes a release of calcitonin gene-related peptide, a key component of neurogenic inflammation, from isolated peripheral nerves of wild-type but not TRPV1 and TRPA1-deficient mice. Moreover, propofol but not GABA induced an intense pain upon intracutaneous injection. As both the release of calcitonin gene-related peptide and injection pain by propofol seem to be independent of GABAA receptors, our data identify TRPV1 and TRPA1 as key molecules for propofol-induced excitation of sensory neurons. This study warrants further investigations into the role of anesthetics to induce nociceptor sensitization and to foster postoperative pain