Gábor Pethő

Sensory and Signaling Mechanisms of Bradykinin, Eicosanoids, Platelet-Activating Factor, and Nitric Oxide in Peripheral Nociceptors

Peripheral mediators can contribute to the development and maintenance of inflammatory and neuropathic pain and its concomitants (hyperalgesia and allodynia) via two mechanisms. Activation or excitation by these substances of nociceptive nerve endings or fibers implicates generation of action potentials which then travel to the central nervous system and may induce pain sensation. Sensitization of nociceptors refers to their increased responsiveness to either thermal, mechanical, or chemical stimuli that may be translated to corresponding hyperalgesias. This review aims to give an account of the excitatory and sensitizing actions of inflammatory mediators including bradykinin, prostaglandins, thromboxanes, leukotrienes, platelet-activating factor, and nitric oxide on nociceptive primary afferent neurons. Manifestations, receptor molecules, and intracellular signaling mechanisms of the effects of these mediators are discussed in detail. With regard to signaling, most data reported have been obtained from transfected nonneuronal cells and somata of cultured sensory neurons as these structures are more accessible to direct study of sensory and signal transduction. The peripheral processes of sensory neurons, where painful stimuli actually affect the nociceptors in vivo, show marked differences with respect to biophysics, ultrastructure, and equipment with receptors and ion channels compared with cellular models. Therefore, an effort was made to highlight signaling mechanisms for which supporting data from molecular, cellular, and behavioral models are consistent with findings that reflect properties of peripheral nociceptive nerve endings. Identified molecular elements of these signaling pathways may serve as validated targets for development of novel types of analgesic drugs.

Actions of 3-methyl-N-oleoyldopamine, 4-methyl-N-oleoyldopamine and N-oleoylethanolamide on the rat TRPV1 receptor in vitro and in vivo.

N-oleoyldopamine (OLDA) has been identified as an agonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor. A related fatty acid amide, N-oleoylethanolamide (OEA), was found to excite sensory neurons and produce visceral hyperalgesia via activation of the TRPV1 receptor, however, a recent study described this agent as an antinociceptive one. The aim of the present paper was to characterize two newly synthesized derivatives of N-oleoyldopamine, 3-methyl-N-oleoyldopamine (3-MOLDA) and 4-methyl-N-oleoyldopamine (4-MOLDA) as well as OEA with regard to their effects on the TRPV1 receptor. Radioactive 45Ca2+ uptake was measured in HT5-1 cells transfected with the rat TRPV1 receptor and intracellular Ca2+ concentration was monitored by fura-2 microfluorimetry in cultured trigeminal sensory neurons. Thermonociception was assessed by determining the behavioral noxious heat threshold in rats. 3-MOLDA induced 45Ca2+ uptake in a concentration-dependent manner, whereas 4-MOLDA and OEA were without effect. 4-MOLDA and OEA, however, concentration-dependently reduced the 45Ca2+ uptake-inducing effect of capsaicin. In trigeminal sensory neurons, 3-MOLDA caused an increase in intracellular Ca2+ concentration and this effect exhibited tachyphylaxis upon repeated application. Again, 4-MOLDA and OEA failed to alter intracellular Ca2+ levels. Upon intraplantar injection, 3-MOLDA caused an 8-10 degrees C drop of the noxious heat threshold in rats which was inhibited by the TRPV1 receptor antagonist iodo-resiniferatoxin. 4-MOLDA and OEA failed to alter the heat threshold but inhibited the threshold drop induced by the TRPV1 receptor agonist resiniferatoxin. These data show that 3-MOLDA behaves as an agonist, whereas 4-MOLDA and OEA appear to be antagonists, at the rat TRPV1 receptor.

Divergent peripheral effects of pituitary adenylate cyclase-activating polypeptide-38 on nociception in rats and mice

Abstract Pituitary adenylate cyclase‐activating polypeptide‐38 (PACAP‐38) and its receptors have been shown in the spinal dorsal horn, on capsaicin‐sensitive sensory neurons and inflammatory cells. The role of PACAP in central pain transmission is controversial, and no data are available on its function in peripheral nociception. Therefore, the aim of the present study was to analyze the effects of locally or systemically administered PACAP‐38 on nocifensive behaviors, inflammatory/neuropathic hyperalgesia and afferent firing. Intraplantar PACAP‐38 (0.2 nmol) injection inhibited carrageenan‐evoked inflammatory mechanical allodynia, mild heat injury‐induced thermal hyperalgesia, as well as nocifensive behaviors in the early and late phases of the formalin test in rats. However, the above dose did not alter basal mechanical or heat thresholds. In mice, PACAP‐38 (0.2 nmol/kg s.c.) significantly diminished acetic acid‐induced abdominal contractions, but exerted no effect on sciatic nerve ligation‐induced neuropathic mechanical hyperalgesia. In contrast, local PACAP‐38 injection markedly increased rotation‐induced afferent firing in the inflamed rat knee joint clearly demonstrating a peripheral sensitization in this organ. These actions were blocked by VPAC1/VPAC2 receptor antagonist pretreatment, but were not altered by PAC1 receptor antagonism. This paper presents the first data for the peripheral actions of PACAP‐38 on nociceptive transmission mediated by VPAC receptors. These effects seem to be divergent depending on the mechanisms of nociceptor activation and the targets of PACAP actions. In acute somatic and visceral inflammatory pain models, PACAP exerts anti‐nociceptive, anti‐hyperalgesic and anti‐allodynic effects. It has no significant peripheral role in traumatic mononeuropathy, but induces mechanical sensitization of knee joint primary afferents.

Antinociceptive desensitizing actions of TRPV1 receptor agonists capsaicin, resiniferatoxin and N-oleoyldopamine as measured by determination of the noxious heat and cold thresholds in the rat.

Agonists of the TRPV1 receptor excite TRPV1‐expressing polymodal nociceptors that is followed after higher doses by a state of diminished responsiveness called desensitization which ensues at two levels: (i) diminished responsiveness of the ion channel (TRPV1 receptor desensitization); (ii) diminished responsiveness of the nerve endings to all stimuli including noxious heat. The aim was to compare these desensitizing actions of TRPV1 agonists in the rat by measuring with an incremental hot/cold plate the noxious heat and cold thresholds, i.e. the lowest hot and highest cold plate temperature, respectively, that evokes nocifensive behaviour. Capsaicin (3.3–1000 nmol) or resiniferatoxin (0.016–0.5 nmol) applied intraplantarly evoked a sustained dose‐dependent elevation of the noxious heat threshold lasting for 2–11 days. N‐oleoyldopamine failed to elevate the heat threshold. The noxious cold threshold was decreased by capsaicin or resiniferatoxin with a recovery within 2–4 days. The diminished acute nocifensive and heat threshold‐lowering effects of resiniferatoxin or N‐oleoyldopamine by pretreatment with doses that failed to elevate the heat threshold and to alter the nocifensive action of the TRPA1 activator formaldehyde, were taken as indication of TRPV1 receptor desensitization. In conclusion, using measurement of threshold temperatures eliciting nocifensive reactions in rats both in the hot and cold range revealed that capsaicin and RTX impair thermosensation in both noxious ranges due to a functional desensitization of peripheral terminals of TRPV1‐expressing sensory neurons responsible for noxious heat and cold responsiveness. This could be differentiated from desensitization of TRPV1 receptor evoked by lower doses of resiniferatoxin or N‐oleoyldopamine.

Effect of transient receptor potential vanilloid 1 (TRPV1) receptor antagonist compounds SB705498, BCTC and AMG9810 in rat models of thermal hyperalgesia measured with an increasing-temperature water bath

The transient receptor potential vanilloid 1 (TRPV1) receptor is activated by noxious heat, various endogenous mediators and exogenous irritants. The aim of the present study was to compare three TRPV1 receptor antagonists (SB705498, BCTC and AMG9810) in rat models of heat hyperalgesia. The behavioural noxious heat threshold, defined as the lowest temperature evoking nocifensive reaction, was measured with an increasing-temperature water bath. The effects of TRPV1 receptor antagonists were assessed in thermal hyperalgesia induced by the TRPV1 agonist resiniferatoxin (RTX), mild heat injury (51 degrees C, 20s) or plantar incision in rats. The control heat threshold was 43.2+/-0.4 degrees C. RTX induced an 8-10 degrees C decrease in heat threshold which was dose-dependently inhibited by oral pre-treatment with any of the TRPV1 receptor antagonists with a minimum effective dose of 1mg/kg. The mild heat injury-evoked 7-8 degrees C heat threshold drop was significantly reversed by all three antagonists injected i.p. as post-treatment. The minimum effective doses were as follows: SB705498 10, BCTC 3 and AMG9810 1mg/kg. Plantar incision-induced heat threshold drop (7-8 degrees C) was dose-dependently diminished by an oral post-treatment with any of the antagonists with minimum effective doses of 10, 3 and 3mg/kg, respectively. Assessment of RTX hyperalgesia by measurement of the paw withdrawal latency with a plantar test apparatus yielded 30 mg/kg minimum effective dose for each antagonist. In conclusion, measurement of the noxious heat threshold with the increasing-temperature water bath is suitable to sensitively detect the effects of TRPV1 receptor antagonists in thermal hyperalgesia models.

Noxious heat threshold measured with slowly increasing temperatures: novel rat thermal hyperalgesia models.

The conventional methods for the study of thermal pain in animals apply constant suprathreshold heat stimuli and measure the reflex latency of pain-avoiding reactions. The latency measured by these methods may greatly vary upon repeated measurements which is a major disadvantage concerning reliability. The presently introduced novel approach involves applying a slowly increasing thermal stimulus which allows determination of the noxious heat threshold i.e. the lowest temperature evoking pain-avoiding behaviour. An increasing-temperature hot plate and an increasing-temperature water bath are presented which are both suitable to determine the noxious heat threshold with high reproducibility. Acute thermal hyperalgesia models based on the drop of the heat threshold are also described for each equipment which proved to be highly sensitive to standard analgesics.

Effects of Bradykinin on Nociceptors

Abstract Bradykinin is a potent mediator formed upon tissue damage and inflammation. It can both excite and sensitize nociceptors to heat, mechanical, and chemical stimuli. Two types of bradykinin receptors (B1 and B2) have been identified of which the constitutive B2 receptors mediate most of the acute effects of bradykinin in uninflamed tissues while the B1 receptors are induced and become activated during inflammation. Both receptor subtypes utilize similar signaling pathways including activation of protein kinase C (PKC), elevation of intracellular Ca2+ concentration, and release of arachidonic acid. PKC activation is the major mechanism underlying the neuronal excitatory and heat-sensitizing actions of bradykinin while Ca2+ accumulation induces formation of nitric oxide within sensory neurons which is involved – together with receptor downregulation – in the development of tachyphylaxis of B2 receptor-mediated effects of bradykinin. Nitric oxide, however, may also contribute to the excitatory and sensitizing actions of bradykinin. Cyclooxygenase metabolites of arachidonic acid (prostanoids) may also be involved in both the excitatory and the sensitizing effects of bradykinin. Recently, a new signaling mechanism has been revealed for bradykinin which involves activation of the capsaicin TRPV1 receptor through PKC activation and formation of 12-lipoxygenase products of arachidonic acid. According to a novel hypothesis, the neuronal excitatory action of bradykinin is not a separate effect but in fact a heat response as a result of a massive heat sensitization with a threshold drop below the ambient temperature. Prostanoids can also sensitize nociceptors to heat, mechanical, and chemical stimuli predominantly via the cyclic adenosine 3′,5′-monophosphate–protein kinase A pathway that modulates various membrane channels including Ca2+-dependent or voltage-gated K+ channels, tetrodotoxin-resistant Na+ channels as well as ligand-gated or noxious heat-gated ion channels.

Evidence for a novel, neurohumoral antinociceptive mechanism mediated by peripheral capsaicin-sensitive nociceptors in conscious rats

Stimulation of capsaicin-sensitive peripheral sensory nerve terminals induces remote anti-inflammatory effects throughout the body of anesthetized rats and guinea-pigs mediated by somatostatin. As somatostatin has also antinociceptive effects, the study aimed at investigating whether similar remote antinociceptive effects can be demonstrated in awake animals. In conscious rats, nociceptive nerve endings of the right hind paw decentralized by cutting the sciatic and saphenous nerves 18h before were chemically stimulated, and drop of the noxious heat threshold (heat hyperalgesia) induced by prior (18h before) plantar incision was measured on the contralateral, left hind paw using an increasing-temperature water bath. 18h after nerve transection, mustard oil-evoked plasma extravasation was not significantly reduced in the right hind paw as tested by in vivo fluorescence imaging. Applying agonist of either transient receptor potential vanilloid 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1) receptor (capsaicin or mustard oil, respectively) to the nerve-transected paw inhibited the plantar incision-induced drop of the noxious heat threshold on the contralateral paw. The onset of these remote antihyperalgesic effects was 10-20min. A similar contralateral inhibitory effect of capsaicin or mustard oil treatment was observed on neuropathic mechanical hyperalgesia evoked by partial sciatic nerve injury 2days before nerve transection and measured by a Randall-Selitto apparatus. The remote thermal antihyperalgesic effect was prevented by chronic (5days) denervation or local capsaicin desensitization of the stimulated paw; reduced by intraperitoneally applied antagonist of somatostatin (cyclosomatostatin) or opioid receptors (naloxone). The response was mimicked by intraperitoneally applied somatostatin and associated with a 72±27% increase in plasma somatostatin-like immunoreactivity that was absent after chronic (5days) denervation. In conclusion, chemical activation of decentralized peripheral capsaicin-sensitive nociceptors evokes remote antihyperalgesic responses initiated outside the central nervous system and mediated by somatostatin and endogenous opioids.

Noxious heat threshold temperature and pronociceptive effects of allyl isothiocyanate (mustard oil) in TRPV1 or TRPA1 gene-deleted mice

AIMS To investigate the roles of TRPV1 and TRPA1 channels in baseline and allyl isothiocyanate (AITC)-evoked nociceptive responses by comparing wild-type and gene-deficient mice. MAIN METHODS In contrast to conventional methods of thermonociception measuring reflex latencies, we used our novel methods to determine the noxious heat threshold. KEY FINDINGS It was revealed that the heat threshold of the tail measured by an increasing-temperature water bath is significantly higher in TRPV1(-/-), but not TRPA1(-/-), mice compared to respective wild-types. There was no difference between the noxious heat thresholds of the hind paw as measured by an increasing-temperature hot plate in TRPV1(-/-), TRPA1(-/-) and the corresponding wild-type mice. The withdrawal latency of the tail from 0°C water was prolonged in TRPA1(-/-), but not TRPV1(-/-), mice compared to respective wild-types. In wild-type animals, dipping the tail or paw into 1% AITC induced an 8-14°C drop of the noxious heat threshold (heat allodynia) of both the tail and paw, and 40-50% drop of the mechanonociceptive threshold (mechanical allodynia) of the paw measured by dynamic plantar esthesiometry. These AITC-evoked responses were diminished in TRPV1(-/-), but not TRPA1(-/-), mice. Tail withdrawal latency to 1% AITC was significantly prolonged in both gene-deleted strains. SIGNIFICANCE Different heat sensors determine the noxious heat threshold in distinct areas: a pivotal role for TRPV1 on the tail is contrasted with no involvement of either TRPV1 or TRPA1 on the hind paw. Noxious heat threshold measurement appears appropriate for preclinical screening of TRP channel ligands as novel analgesics.

Effects of Reference Analgesics and Psychoactive Drugs on the Noxious Heat Threshold of Mice Measured by an Increasing-Temperature Water Bath

The study aimed at validating an increasing‐temperature water bath suitable for determining the noxious heat threshold for use in mice. The noxious heat threshold was determined by immersing the tail of the gently held awake mouse into a water container whose temperature was near‐linearly increased at a rate of 24°C/min. until the animal withdrew its tail, that is, heating attained the noxious threshold. The effects of standard analgesic, neuroleptic and anxiolytic drugs were investigated in a parallel way on both the noxious heat threshold and the psychomotor activity assessed by the open field test. Morphine, diclofenac and metamizol (dipyrone) elevated the heat threshold of mice with minimum effective doses of 6, 30 and 1000 mg/kg i.p., respectively. These doses of morphine and diclofenac failed to induce any remarkable effect on psychomotor activity in the open field test while that of metamizol exerted a profound inhibition. The anxiolytic diazepam and the neuroleptic droperidol at doses evoking a mild and moderate, respectively, psychomotor inhibition failed to alter the heat threshold. Combination of a subliminal dose of morphine (regarding both antinociceptive and psychomotor inhibitory action) with diclofenac, metamizol, diazepam or droperidol at doses also subliminal regarding the thermal antinociceptive effect elevated the noxious heat threshold without major additional effects in the open field test. It is concluded that the increasing‐temperature water bath is suitable for studying the thermal antinociceptive effects of morphine and diclofenac as well as the morphine‐sparing action of diclofenac, metamizol, droperidol and diazepam. Behavioural testing is recommended when testing analgesics.