Amol M. Patwardhan

Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia

Transient receptor potential vanilloid 1 (TRPV1) plays a major role in hyperalgesia and allodynia and is expressed both in the peripheral and central nervous systems (CNS). However, few studies have evaluated mechanisms by which CNS TRPV1 mediates hyperalgesia and allodynia after injury. We hypothesized that activation of spinal cord systems releases endogenous TRPV1 agonists that evoke the development of mechanical allodynia by this receptor. Using in vitro superfusion, the depolarization of spinal cord triggered the release of oxidized linoleic acid metabolites, such as 9-hydroxyoctadecadienoic acid (9-HODE) that potently activated spinal TRPV1, leading to the development of mechanical allodynia. Subsequent calcium imaging and electrophysiology studies demonstrated that synthetic oxidized linoleic acid metabolites, including 9-HODE, 13-HODE, and 9 and 13-oxoODE, comprise a family of endogenous TRPV1 agonists. In vivo studies demonstrated that intrathecal application of these oxidized linoleic acid metabolites rapidly evokes mechanical allodynia. Finally, intrathecal neutralization of 9- and 13-HODE by antibodies blocks CFA-evoked mechanical allodynia. These data collectively reveal a mechanism by which an endogenous family of lipids activates TRPV1 in the spinal cord, leading to the development of inflammatory hyperalgesia. These findings may integrate many pain disorders and provide an approach for developing analgesic drugs.

Heat generates oxidized linoleic acid metabolites that activate TRPV1 and produce pain in rodents

The transient receptor potential vanilloid 1 (TRPV1) channel is the principal detector of noxious heat in the peripheral nervous system. TRPV1 is expressed in many nociceptors and is involved in heat-induced hyperalgesia and thermoregulation. The precise mechanism or mechanisms mediating the thermal sensitivity of TRPV1 are unknown. Here, we have shown that the oxidized linoleic acid metabolites 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE) are formed in mouse and rat skin biopsies by exposure to noxious heat. 9- and 13-HODE and their metabolites, 9- and 13-oxoODE, activated TRPV1 and therefore constitute a family of endogenous TRPV1 agonists. Moreover, blocking these substances substantially decreased the heat sensitivity of TRPV1 in rats and mice and reduced nociception. Collectively, our results indicate that HODEs contribute to the heat sensitivity of TRPV1 in rodents. Because oxidized linoleic acid metabolites are released during cell injury, these findings suggest a mechanism for integrating the hyperalgesic and proinflammatory roles of TRPV1 and linoleic acid metabolites and may provide the foundation for investigating new classes of analgesic drugs.

Modulation of trigeminal sensory neuron activity by the dual cannabinoid–vanilloid agonists anandamide, N‐arachidonoyl‐dopamine and arachidonyl‐2‐chloroethylamide

Peripheral cannabinoids have been shown to suppress nociceptive neurotransmission in a number of behavioral and neurophysiological studies. It is not known, however, whether cannabinoids exert this action through direct interactions with nociceptors in the periphery and/or if other processes are involved. To gain a better understanding of the direct actions of cannabinoid‐vanilloid agonists on sensory neurons, we examined the effects of these compounds on trigeminal ganglion (TG) neurons in vitro. AEA (EC50=11.0 μM), NADA (EC50=857 nM) and arachidonyl‐2‐chloroethylamide ACEA (EC50=14.0 μM) each evoked calcitonin gene‐related peptide (CGRP) release from TG neurons. The TRPV1 antagonists iodo‐resiniferatoxin (I‐RTX) and capsazepine (CPZ) each obtunded AEA‐, NADA‐, ACEA‐ and capsaicin (CAP)‐evoked CGRP release with individually equivalent IC50s for each of the compounds (I‐RTX IC50 range=2.6–4.0 nM; CPZ IC50 range=523–1140 μM). The pro‐inflammatory mediator prostaglandin E2 significantly increased the maximal effect of AEA‐evoked CGRP release without altering the EC50. AEA, ACEA and CAP stimulated cAMP accumulation in TG neurons in a calcium‐ and TRPV1‐dependent fashion. Moreover, the protein kinase inhibitor staurosporine significantly inhibited AEA‐ and CAP‐evoked CGRP release. The pungency of AEA, NADA, ACEA and CAP in the rat eye‐wipe assay was also assessed. Interestingly, when applied intraocularly, NADA or CAP each produced nocifensive responses, while AEA or ACEA did not. Finally, the potential inhibitory effects of these cannabinoids on TG nociceptors were evaluated. Neither AEA nor ACEA decreased CAP‐evoked CGRP release. Furthermore, neither of the cannabinoid receptor type 1 antagonists SR141716A nor AM251 had any impact on either basal or CAP‐evoked CGRP release. AEA also did not inhibit 50 mM K+‐evoked CGRP release and did not influence bradykinin‐stimulated inositol phosphate accumulation. We conclude that the major action of AEA, NADA and ACEA on TG neurons is excitatory, while, of these, only NADA is pungent. These findings are discussed in relation to our current understanding of interactions between the cannabinoid and vanilloid systems and nociceptive processing in the periphery.

Bradykinin-Induced Functional Competence and Trafficking of the δ-Opioid Receptor in Trigeminal Nociceptors

Peripheral opioid analgesia is increased substantially after inflammation. We evaluated the hypothesis that an inflammatory mediator, bradykinin (BK), evokes functional competence of the δ-opioid receptor (DOR) for inhibiting trigeminal ganglia (TG) sensory neurons. We also evaluated whether BK evokes trafficking of the DOR to the plasma membrane. Rat TG cultures were pretreated with BK (10 μm) or vehicle, and the effects of DOR agonists ([d-Pen2,5]-enkephalin or [d-Ala2, d-Leu5]-enkephalin) on BK (10μm)/prostagladin E2 (PGE2; 1 μm)-stimulated immunoreactive calcitonin gene-related peptide (iCGRP) release or PGE2 (1 μm)-stimulated cAMP accumulation were measured. The effect of BK treatment on opioid receptor trafficking was evaluated by DOR immunohistochemistry, cell-surface DOR biotinylation, and live imaging of neurons transfected with mDOR–green fluorescent protein. BK pretreatment rapidly and significantly increased DOR agonist inhibition of evoked iCGRP release and cAMP accumulation. These effects of BK pretreatment were blocked by a B2 receptor antagonist (HOE-140; 10μm) or a protein kinase C (PKC) inhibitor [bisindolymaleimide (BIS); 1μm]. Moreover, BK treatment rapidly and significantly increased the accumulation of DOR in the plasma membrane. However, BK-induced trafficking of DOR was not reversed by pretreatment with BIS, nor was trafficking evoked by application of a PKC activator PMA (200 nm). These data suggest that BK, in a PKC-dependent manner, induces rapid functional competence of DOR for inhibiting TG nociceptors and in a PKC-independent manner rapidly induces trafficking of DOR to the plasma membrane. These findings indicate that exposure to certain inflammatory mediators rapidly alters the signaling properties and neuronal localization of DOR, possibly contributing to peripheral opioid analgesia.

Cannabinoid WIN 55,212-2 Regulates TRPV1 Phosphorylation in Sensory Neurons

Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr144 and Thr370 were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.

Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion

BackgroundNerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) all play important roles in the development of the peripheral sensory nervous system. Additionally, these growth factors are proposed to modulate the properties of the sensory system in the adult under pathological conditions brought about by nerve injury or inflammation. We have examined the effects of NGF, GDNF and BDNF on adult rat trigeminal ganglion (TG) neurons in culture to gain a better understanding of how these growth factors alter the cytochemical and functional phenotype of these neurons, with special attention to properties associated with nociception.ResultsCompared with no growth factor controls, GDNF, at 1 and 100 ng/ml, significantly increased by nearly 100% the number of neurons in culture at 5 days post-plating. A significant, positive, linear trend of increasing neuron number as a function of BDNF concentration was observed, also peaking at nearly 100%. NGF treatment was without effect. Chronic treatment with NGF and GDNF significantly and concentration-dependently increased 100 nM capsaicin (CAP)-evoked calcitonin gene-related peptide (CGRP) release, reaching approximately 300% at the highest concentration tested (100 ng/ml). Also, NGF and GDNF each augmented anandamide (AEA)- and arachidonyl-2-chloroethylamide (ACEA)-evoked CGRP release, while BDNF was without effect. Utilizing immunohistochemistry to account for the proportions of TRPV1- or CGRP-positive neurons under each growth factor treatment condition and then standardizing evoked CGRP release to these proportions, we observed that NGF was much more effective in enhancing CAP- and 50 mM K+-evoked CGRP release than was GDNF. Furthermore, NGF and GDNF each altered the concentration-response function for CAP- and AEA-evoked CGRP release, increasing the Emax without altering the EC50 for either compound.ConclusionsTaken together, our results illustrate that NGF, GDNF and BDNF differentially alter TG sensory neuron survival, neurochemical properties and TRPV1-mediated neuropeptide release in culture. In particular, our findings suggest that GDNF and NGF differentially modulate TRPV1-mediated neuropeptide secretion sensitivity, with NGF having a much greater effect on a per neuron basis than GDNF. These findings are discussed in relation to possible therapeutic roles for growth factors or their modulators in pathological pain states, especially as these relate to the trigeminal system.

Cannabinoids Desensitize Capsaicin and Mustard Oil Responses in Sensory Neurons via TRPA1 Activation

Although the cannabinoid agonists R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)-(1-naphthalenyl) methanone mesylate [WIN 55,212-2 (WIN)] and (R,S)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) exert peripheral antihyperalgesia in inflammatory pain models, the mechanism for cannabinoid-induced inhibition of nociceptive sensory neurons has not been fully studied. Because TRPV1 and TRPA1 channels play important roles in controlling hyperalgesia in inflammatory pain models, we investigated their modulation by WIN and AM1241. The applications of WIN (>5 μm) and AM1241 (>30 μm) inhibit responses of sensory neurons to capsaicin and mustard oil. To determine potential mechanisms for the inhibition, we evaluated cannabinoid effects on nociceptors. WIN and AM1241 excite sensory neurons in a concentration-dependent manner via a nonselective Ca2+-permeable channel. The expression of TRP channels in CHO cells demonstrates that both WIN and AM1241 activate TRPA1 and, by doing so, attenuate capsaicin and mustard oil responses. Using TRPA1-specific small interfering RNA or TRPA1-deficient mice, we show that the TRPA1 channel is a sole target through which WIN and mustard oil activate sensory neurons. In contrast, AM1241 activation of sensory neurons is mediated by TRPA1 and an unknown channel. The knockdown of TRPA1 activity in neurons completely eliminates the desensitizing effects of WIN and AM1241 on capsaicin-activated currents. Furthermore, the WIN- or AM1241-induced inhibition of capsaicin-evoked nocifensive behavior via peripheral actions is reversed in TRPA1 null-mutant mice. Together, this study demonstrates that certain cannabinoids exert their peripheral antinocifensive actions via activation of the TRPA1 channel on sensory neurons.

Prolactin modulates TRPV1 in female rat trigeminal sensory neurons.

Sex dependency in pain perception is well documented and is thought to be attributable to the effect of reproductive hormones on nociceptive processing. In the present study, we evaluated whether estradiol alters gene transcription in the trigeminal ganglia (TG) of ovariectomized rats (OVX). These experiments demonstrated a dramatic (40-fold) upregulation of prolactin (PRL) expression in TG by 17-β-estradiol (E2). PRL expression was restricted to TG neurons and was highly overlapped with transient potential receptor vanilloid type 1 (TRPV1) (∼90%) in TG. Additionally, PRL is released from neurons during stimulation. Both forms of PRL receptors (PRLRs), short and long, were also present in TG neurons. Moreover, expression of the long PRLRs was under control of estradiol. We next evaluated the novel hypothesis that PRL acts as a neuromodulator of sensory neurons. PRL pretreatment significantly enhanced capsaicin-evoked inward currents, calcium influx, and immunoreactive calcitonin gene-related peptide release from cultured TG neurons. This PRL modulation of capsaicin responses was abolished by withdrawal of E2 from TG cultures. Biochemical analysis demonstrated that PRL increased (>50%) phosphorylation levels of TRPV1 in TG. In a behavioral test, PRL pretreatment significantly potentiated capsaicin-evoked nocifensive behavior in female rats at proestrous and in OVX rats after E2 treatment. The in vivo potentiating effect of PRL on capsaicin responses was also dependent on E2. Collectively, these data demonstrate that PRL is a novel modulator of sensory neurons tightly regulated by E2. These findings are consistent with the hypothesis that PRL could contribute to the development of certain pain disorders, possibly including those modulated by estrogen.

Role of ionotropic cannabinoid receptors in peripheral antinociception and antihyperalgesia

Despite the wealth of information on cannabinoid-induced peripheral antihyperalgesic and antinociceptive effects in many pain models, the molecular mechanism(s) for these actions remains unknown. Although metabotropic cannabinoid receptors have important roles in many pharmacological actions of cannabinoids, recent studies have led to the recognition of a family of at least five ionotropic cannabinoid receptors (ICRs). The known ICRs are members of the family of transient receptor potential (TRP) channels and include TRPV1, TRPV2, TRPV4, TRPM8 and TRPA1. Cannabinoid activation of ICRs can result in desensitization of the TRPA1 and TRPV1 channel activities, inhibition of nociceptors and antihyperalgesia and antinociception in certain pain models. Thus, cannabinoids activate both metabotropic and ionotropic mechanisms to produce peripheral analgesic effects. Here, we provide an overview of the pharmacology of TRP channels as ICRs.

PAR-2 agonists activate trigeminal nociceptors and induce functional competence in the delta opioid receptor

&NA; The role of protease activated receptor‐2 (PAR‐2) activation in trigeminal nociception and in induction of functional competence in the delta opioid receptor (DOR) is not known. In this study, we evaluated whether agonists of PAR‐2 activate the capsaicin‐sensitive subclass of trigeminal nociceptors in a PLC–PKC‐dependent manner and induce functional competence in the DOR. Adult male rat trigeminal ganglion (TG) cultured neurons were treated with the PAR‐2 agonist (SL‐NH2) or an enzyme activator of PAR (trypsin) and the activation of TG nociceptors was assessed using three independent methods: neuropeptide release, calcium influx, and whole cell patch‐clamp. The specificity of SL‐NH2 and trypsin responses was evaluated using TG cultures transfected with siRNA against PAR‐2. The in vivo role of PAR‐2 activation was determined measuring SL‐NH2 and trypsin‐evoked nocifensive behavior and increase in blood flow. Trigeminal neurons were treated with SL‐NH2/vehicle and then the DOR agonist to determine DOR inhibition of evoked neuropeptide release and cAMP accumulation. The results showed that SL‐NH2 (100 &mgr;M) and trypsin (1–600 nM) activate TG nociceptors, which is partly reversible by the PKC inhibitor bisindolylmaleimide (500 nM) and by ruthenium red (10 &mgr;M). In cultures treated with siRNA against PAR‐2, both SL‐NH2 and trypsin responses were significantly diminished. Both SL‐NH2 and trypsin evoke nocifensive behavior and increases in blood flow in an orofacial pain model. Application of SL‐NH2 rapidly produced functional competence of DOR for inhibiting nociceptor function. In inflamed tissue, endogenous proteases may activate TG nociceptors and generate pain. Moreover, activation of PAR‐2 can also induce functional competence in DOR.