Calcium Influx In Sensory Neurons Research Articles

(255) Effects of Cannabinoids on Responses of Human Dorsal Root Ganglia Neurons

Cannabinoids are suggested to be a potentially important alternative approach to analgesia. Rodent models have shown analgesic properties of cannabinoid receptor activation and suppressed nociceptive responses within sensory neurons. The goal of the present study was to determine whether activation of cannabinoid receptors in human sensory neurons could suppress nociceptive responses or hyperactivity. Lumbar level human dorsal root ganglia were recovered from cadaveric organ donors at the University of Cincinnati Medical Center, dissociated and cultured, or fixed and frozen for histology. Using Fura-2AM calcium imaging of viable human sensory neurons, responses to the peripherally active cannabinoid agonist CB-13, a potent CB1/CB2 agonist, in naive neurons were examined. Responses to capsaicin (250 nM) in presence and absence of the inflammatory mediator PGE2 (1 µM) were examined after pre-treatment with 1 µM CB-13, or with CB-13 bath applied in between capsaicin administrations. Our results show that CB-13 produced no calcium influx in sensory neurons on its own, and that responses to capsaicin in naive neurons as well as PGE2-exposed neurons were not altered by CB-13. Patch clamp recordings of human sensory neurons to test changes in membrane properties by cannabinoid receptor activation by CB-13 is reported. Our data do not presently support the concept of peripherally acting cannabinoid receptors as having the capacity to suppress sensory neuron nociceptive responses or sensitization in people.

Etomidate and propylene glycol activate nociceptive TRP ion channels.

BackgroundEtomidate 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.ResultsActivation 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.ConclusionsData 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.

A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors.

Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2-CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3-the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.

The inflammatory mediators serotonin, prostaglandin e 2 and bradykinin evoke calcium influx in rat sensory neurons

The inflammatory mediators bradykinin, prostaglandin E 2 and serotonin interact to excite and sensitize nociceptive neurons. All three mediators are coupled to signaling pathways that potentially induce rises in intracellular calcium concentration in other models. The aim of this study was therefore to investigate if the three mediators cause calcium rises in isolated rat sensory neurons that may explain their sensitizing action. Neurons exposed to serotonin, bradykinin, and prostaglandin E 2 exhibited reversible increases in intracellular calcium concentration, which were absent in calcium-free solution. The calcium increase induced by serotonin was preserved in the presence of extracellular cadmium suggesting calcium influx potentially through the serotonin receptor ion channel 5-HT 3. The bradykinin-induced calcium response was slower, showed pronounced tachyphylaxis and was absent in the presence of extracellular cadmium ions. Similar results were obtained for prostaglandin E 2 although the calcium rises were fast and not prone to tachyphylaxis. This suggests that prostaglandin E 2 as well as bradykinin via activation of G protein-coupled receptors seem to couple to calcium-permeant ion channels possibly the heat-transducing vanilloid receptor type 1 or related ion channels. The three mediators, however, did not cooperate to induce supra-additive calcium responses when applied simultaneously. In summary, our results suggest that the inflammatory mediators serotonin, prostaglandin E 2 and bradykinin induce calcium influx in sensory neurons. However, they do not utilize a calcium-dependent cooperative mechanism to facilitate proton-induced currents.