Selena Harrison

Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1.

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burning sensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis when they consume alcoholic beverages or are administered alcohol by injection as a medical treatment. We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release or plasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dorsal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin, protons and heat and lowered the threshold for heat activation of VR1 from ∼42°C to ∼34°C. This provides a likely mechanistic explanation for the ethanol-induced sensory responses that occur at body temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found in esophagitis, neuralgia or wounds.

The vanilloid receptor (VR1)-mediated effects of anandamide are potently enhanced by the cAMP-dependent protein kinase.

The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. Here, we demonstrate that the potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over‐expressing human VR1, AEA induces a rise in cytosolic Ca2+ concentration that is mediated by this receptor. The EC50 for this effect was decreased five‐fold in the presence of forskolin (FRSK, 1–5 µm) or the cAMP analogue, 8‐Br‐cAMP (10–100 µm). The effects of 8‐Br‐cAMP and FRSK were blocked by a selective PKA inhibitor. The FRSK (10 nm) also potently enhanced the sensory neurone‐ and VR1‐mediated constriction by AEA of isolated guinea‐pig bronchi, and this effect was abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA‐induced release of substance P, an effect mediated by VR1 activation, was enhanced three‐fold by FRSK (10 nm). Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions.

Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1 receptor stimulation

&NA; Platinum‐based anticancer drugs cause neurotoxicity. In particular, oxaliplatin produces early‐developing, painful, and cold‐exacerbated paresthesias. However, the mechanism underlying these bothersome and dose‐limiting adverse effects is unknown. We hypothesized that the transient receptor potential ankyrin 1 (TRPA1), a cation channel activated by oxidative stress and cold temperature, contributes to mechanical and cold hypersensitivity caused by oxaliplatin and cisplatin. Oxaliplatin and cisplatin evoked glutathione‐sensitive relaxation, mediated by TRPA1 stimulation and the release of calcitonin gene‐related peptide from sensory nerve terminals in isolated guinea pig pulmonary arteries. No calcium response was observed in cultured mouse dorsal root ganglion neurons or in naïve Chinese hamster ovary (CHO) cells exposed to oxaliplatin or cisplatin. However, oxaliplatin, and with lower potency, cisplatin, evoked a glutathione‐sensitive calcium response in CHO cells expressing mouse TRPA1. One single administration of oxaliplatin produced mechanical and cold hyperalgesia in rats, an effect selectively abated by the TRPA1 antagonist HC‐030031. Oxaliplatin administration caused mechanical and cold allodynia in mice. Both responses were absent in TRPA1‐deficient mice. Administration of cisplatin evoked mechanical allodynia, an effect that was reduced in TRPA1‐deficient mice. TRPA1 is therefore required for oxaliplatin‐evoked mechanical and cold hypersensitivity, and contributes to cisplatin‐evoked mechanical allodynia. Channel activation is most likely caused by glutathione‐sensitive molecules, including reactive oxygen species and their byproducts, which are generated after tissue exposure to platinum‐based drugs from cells surrounding nociceptive nerve terminals. TRPA1 is necessary and sufficient for mechanical‐ and cold‐hypersensitivity evoked by oxaliplatin/cisplatin. TRPA1 activation occurs through reactive molecules, after tissue exposure to platinum‐based drugs.

Hydrogen sulfide causes vanilloid receptor 1-mediated neurogenic inflammation in the airways

1 Hydrogen sulfide (H2S) is described as a mediator of diverse biological effects, and is known to produce irritation and injury in the lung following inhalation. Recently, H2S has been found to cause contraction in the rat urinary bladder via a neurogenic mechanism. Here, we studied whether sodium hydrogen sulfide (NaHS), used as donor of H2S, produces responses mediated by sensory nerve activation in the guinea‐pig airways. 2 NaHS evoked an increase in neuropeptide release in the airways that was significantly attenuated by capsaicin desensitization and by the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine. In addition, NaHS caused an atropine‐resistant contraction of isolated airways, which was completely prevented by capsaicin desensitization. Furthermore, NaHS‐induced contraction was reduced by TRPV1 antagonism (ruthenium red, capsazepine and SB366791), and was abolished by pretreatment with the combination of tachykinin NK1 (SR140333) and NK2 (SR48968) receptor antagonists. 3 In anesthetized guinea‐pigs, intratracheal instillation of NaHS increased the total lung resistance and airway plasma protein extravasation. These two effects were reduced by TRPV1 antagonism (capsazepine) and tachykinin receptors (SR140333 and SR48968) blockade. 4 Our results provide the first pharmacological evidence that H2S provokes tachykinin‐mediated neurogenic inflammatory responses in guinea‐pig airways, and that this effect is mediated by stimulation of TRPV1 receptors on sensory nerves endings. This novel mechanism may contribute to the irritative action of H2S in the respiratory system.

Antitussive activity of iodo-resiniferatoxin in guinea pigs

Background: Iodo-resiniferatoxin (I-RTX) has recently been described as an ultra potent antagonist of the transient receptor potential vanilloid-1 (TRPV1). Methods: The ability of I-RTX to inhibit cough induced by inhalation of two putative TRPV1 stimulants (capsaicin and citric acid) was tested in non-anaesthetised guinea pigs. Results: Pretreatment with I-RTX either intraperitoneally (0.03–0.3 µmol/kg) or by aerosol (0.1–3 µM) reduced the number of coughs produced by inhalation of citric acid (0.25 M) and capsaicin (30 µM) in a dose dependent manner. Capsazepine (CPZ) also reduced citric acid and capsaicin induced cough, but the activity of I-RTX was 10–100 times more potent than CPZ in all the experimental conditions tested. Conclusions: I-RTX is a novel and potent antitussive drug which inhibits cough mediated by agents possibly acting via TRPV1 activation.

Neurogenic responses mediated by vanilloid receptor‐1 (TRPV1) are blocked by the high affinity antagonist, iodo‐resiniferatoxin

Stimulation of the vanilloid receptor‐1 (TRPV1) results in the activation of nociceptive and neurogenic inflammatory responses. Poor specificity and potency of TRPV1 antagonists has, however, limited the clarification of the physiological role of TRPV1. Recently, iodo‐resiniferatoxin (I‐RTX) has been reported to bind as a high affinity antagonist at the native and heterologously expressed rat TRPV1. Here we have studied the ability of I‐RTX to block a series of TRPV1 mediated nociceptive and neurogenic inflammatory responses in different species (including transfected human TRPV1). We have demonstrated that I‐RTX inhibited capsaicin‐induced mobilization of intracellular Ca2+ in rat trigeminal neurons (IC50 0.87 nM) and in HEK293 cells transfected with the human TRPV1 (IC50 0.071 nM). Furthermore, I‐RTX significantly inhibited both capsaicin‐induced CGRP release from slices of rat dorsal spinal cord (IC50 0.27 nM) and contraction of isolated guinea‐pig and rat urinary bladder (pKB of 10.68 and 9.63, respectively), whilst I‐RTX failed to alter the response to high KCl or SP. Finally, in vivo I‐RTX significantly inhibited acetic acid‐induced writhing in mice (ED50 0.42 μmol kg−1) and plasma extravasation in mouse urinary bladder (ED50 0.41 μmol kg−1). In in vitro and in vivo TRPV1 activated responses I‐RTX was ∼3 log units and ∼20 times more potent than capsazepine, respectively. This high affinity antagonist, I‐RTX, may be an important tool for future studies in pain and neurogenic inflammatory models.

Halogenation of a capsaicin analogue leads to novel vanilloid TRPV1 receptor antagonists

The C‐5 halogenation of the vanillyl moiety of resiniferatoxin, an ultrapotent agonist of vanilloid TRPV1 receptors, results in a potent antagonist for these receptors. Here, we have synthesized a series of halogenated derivatives of ‘synthetic capsaicin’ (nonanoyl vanillamide=nordihydrocapsaicin) differing for the nature (iodine, bromine–chlorine) and the regiochemistry (C‐5, C‐6) of the halogenation. The activity of these compounds was investigated on recombinant human TRPV1 receptors overexpressed in HEK‐293 cells. None of the six compounds exerted any significant agonist activity, as assessed by measuring their effect on TRPV1‐mediated calcium mobilization. Instead, all compounds antagonized, to various extents, the effect of capsaicin in this assay. All 6‐halo‐nordihydrocapsaicins behaved as competitive antagonists against human TRPV1 according to the corresponding Schilds plots, and were more potent than the corresponding 5‐halogenated analogues. The iodo‐derivatives were more potent than the bromo‐ and chloro‐derivatives. Using human recombinant TRPV1, 6‐iodo‐nordihydrocapsaicin (IC50=10 nM against 100 nM capsaicin) was about four times more potent than the prototypical TRPV1 antagonist, capsazepine, and was tested against capsaicin also on native TRPV1 in: (i) rat dorsal root ganglion neurons in culture; (ii) guinea‐pig urinary bladder; and (iii) guinea‐pig bronchi. In all cases, except for the guinea‐pig bronchi, the compound was significantly more potent than capsazepine as a TRPV1 antagonist. In conclusion, 6‐iodo‐nordihydrocapsaicin, a stable and easily prepared compound, is a potent TRPV1 antagonist and a convenient replacement for capsazepine in most of the in vitro preparations currently used to assess the activity of putative vanilloid receptor agonists.

Pharmacological assessment of the duration of action of glycopyrrolate vs tiotropium and ipratropium in guinea-pig and human airways

1 Our study was aimed at investigating the duration of the bronchodilator action of the antimuscarinc drug glycopyrrolate compared to tiotropium and ipratropium. 2 In the guinea‐pig isolated trachea, the time (t1/2) necessary for a contractile response to carbachol (0.3 μM) to return to 50% recovery after washout of the antagonist was studied. The offset of the antagonist effect of glycopyrrolate, tiotropium and ipratropium (10 nM each) was t1/2=4.0±0.5, >4.5 and 0.5±0.1 h, respectively. At 4.5 h from the washout of the antagonist, the recovery of the response to carbachol was 50±8, 10±4 and 70±7%, respectively. 3 In the human isolated bronchus, the offset of the bronchodilator effects of glycopyrrolate (3 nM), tiotropium (1 nM) and ipratropium (10 nM) was t1/2=3.7±0.2; >6 and 3.0±0.2 h, respectively. At 6.0 h from the washout of the antagonist, the recovery of the response to carbachol (1 μM) was 101±10, 27±3 and 110±10%, respectively. 4 In anaesthetized guinea‐pigs, acetylcholine‐induced bronchoconstriction was markedly reduced by intratracheal instillation of glycopyrrolate (3 nmol kg−1; 88.1±4% inhibition), tiotropium (1.3 nmol kg−1; 86.2±5% inhibition) or ipratropium (1.45 nmol kg−1; 88.1±10% inhibition). These inhibitory effects assessed 3 or 24 h after antagonist administration were reduced to 69.9±5 and 29.7±6%; 28.3±5 and 14.2±5% for glycopyrrolate and ipratropium, respectively, whereas they remained stable (83.5±4; 70.6±6) for tiotropium. The residual inhibitory effect of glycopyrrolate was also assessed at 16 h from administration, and proved to be as low as that found at 24 h (31.2±10 vs 29.7±6%, respectively). 5 In conclusion, glycopyrrolate‐induced bronchodilation has a longer duration than that of ipratropium, but less than that of tiotropium. The efficacy of a possible glycopyrrolate‐based therapy for asthma or chronic obstructive pulmonary disease given once‐a‐day is not guaranteed by the present investigation.

Capsaicin-like effects of N-arachidonoyl-dopamine in the isolated guinea pig bronchi and urinary bladder

A capsaicin-like endogenous ligand of vanilloid (VR1) receptors, N-arachidonoyl-dopamine, was recently identified in bovine and rat nervous tissue, and found to be almost as potent as capsaicin, and 5-10-fold more potent than anandamide, on these receptors, both in isolated cells and in vivo. Here we have investigated if N-arachidonoyl-dopamine also exerts other capsaicin-like effects at VR1 receptors in some isolated organ preparations. N-arachidonoyl-dopamine exerted a potent contractile response of guinea pig isolated bronchi (EC50=12.6 +/- 1.7 microM, Emax=69.2 +/- 2.4% of carbachol Emax), which was blocked by pre-treatment with capsaicin or with the VR1 antagonist capsazepine, as well as by a combination of tachykinin NK1 and NK2 receptor antagonists. In this assay, N-arachidonoyl-dopamine was less and more potent and/or efficacious than capsaicin (EC50=40.0 nM; Emax=93.5%) and anandamide (EC50=15.2 microM, Emax=38.0%), respectively. Unlike capsaicin and anandamide, forskolin or ethanol did not enhance N-arachidonoyl-dopamine effect in this preparation, whereas epithelial denudation resulted in a 2.5-fold increase in potency without affecting the efficacy. N-arachidonoyl-dopamine also contracted the isolated guinea pig urinary bladder, although in this preparation, as well as in the isolated rat urinary bladder, the potency (EC50=3.7 +/- 0.3 and 19.9 +/- 0.1 microM) and/or efficacy (Emax=12.0 +/- 0.1% and 20.7 +/- 0.7% of carbachol Emax) of the compound were significantly lower than those of both capsaicin and anandamide. These data suggest that the extent to which exogenous N-arachidonoyl-dopamine activates VR1 receptor in isolated organs is largely dependent on pharmacodynamics and bioavailability.

Tachykinins in the respiratory tract.

Tachykinins as substance P and neurokinin A belong to a family of peptides, which are released from airway nerves after noxious stimulation. They influence numerous respiratory functions under both normal and pathological conditions including the regulation of airway smooth muscle tone, vascular tone, mucus secretion and immune functions. For the most part the synthesis/release of tachykinins is associated with neuronal cells; nevertheless, inflammatory and immune cells can synthesize and release tachykinins under certain physiological conditions. Moreover, this second cellular source of tachykinins may play an important role in inflammatory airway diseases such as bronchial asthma or chronic obstructive pulmonary disease (COPD). Dual tachykinin (NK1 and NK2) receptor antagonists demonstrate a significant bronchoprotection and a possible future role in the development of novel therapeutic approaches. In addition, NK3 receptors could also possess a bronchoprotective action, however, their presence in the human respiratory tract still needs to be confirmed. The family of tachykinins has recently been extended by the discovery of a third tachykinin gene that encodes the previously unknown NK1 receptor selective tachykinins hemokinin 1, endokinin A and B. Together with other novel tachykinin peptides such as C14TKL-1 and virokinin further research is required to define their respiratory biological role in health and disease.