Mayur J. Patil

Inhibition of M Current in Sensory Neurons by Exogenous Proteases: A Signaling Pathway Mediating Inflammatory Nociception

Inflammatory pain is thought to be mediated in part through the action of inflammatory mediators on membrane receptors of peripheral nerve terminals, however, the downstream signaling events which lead to pain are poorly understood. In this study we investigated the nociceptive pathways induced by activation of protease-activated receptor 2 (PAR-2) in damage-sensing (nociceptive) neurons from rat dorsal root ganglion (DRG). We found that activation of PAR-2 in these cells strongly inhibited M-type potassium currents (conducted by Kv7 potassium channels). Such inhibition caused depolarization of the neuronal resting membrane potential leading, ultimately, to nociception. Consistent with this mechanism, injection of the specific M channel blocker XE991 into rat paw induced nociception in a concentration-dependent manner. Injection of a PAR-2 agonist peptide also induced nociception but coinjection of XE991 and the PAR-2 agonist did not result in summation of nociception, suggesting that the action of both agents may share a similar mechanism. We also studied the signaling pathway of M current inhibition by PAR-2 using patch-clamp and fluorescence imaging of DRG neurons. These experiments revealed that the PAR-2 effect was mediated by phospholipase C (PLC). Further experiments demonstrated that M current inhibition required concurrent rises in cytosolic Ca2+ concentration and depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP2). We propose that PLC- and Ca2+/PIP2-mediated inhibition of M current in sensory neurons may represent one of the general mechanisms underlying pain produced by inflammatory mediators, and may therefore open up a new therapeutic window for treatment of this major clinical problem.

Transient receptor potential V1 regulates activation and modulation of transient receptor potential A1 by Ca2

The transient receptor potential A1 (TRPA1) channel contributes to nociceptive signaling in certain pain models. It has been suggested that Ca(2+), which activates and modulates TRPA1, could play a critical regulatory role in this process. Since TRPA1 and transient receptor potential V1 (TRPV1) channels are co-expressed and interact in neurons, we investigated whether activation and modulation of TRPA1 by Ca(2+) is regulated by TRPV1. Cell-attached recordings showed that TRPA1 is activated by extracellular Ca(2+) ([Ca(2+)](e)) in concentration-response fashion. This activation, especially by 2 mM [Ca(2+)](e) was substantially suppressed by co-expression with TRPV1. Inside-out recordings demonstrated that intracellular Ca(2+) ([Ca(2+)](i))-triggered activation of TRPA1 was attenuated by the presence of TRPV1 only at 2 mM [Ca(2+)](e), but not in Ca(2+)-free conditions. Further, depletion of internal Ca(2+) stores by thapsigargin generated TRPA1-mediated currents, which is affected by TRPV1 in both Chinese hamster ovary cells and sensory neurons. Since mustard oil current (I(MO)) is modulated by [Ca(2+)](e), we next examined whether alterations in the Ca(2+)-permeability of TRPV1 by mutating Y671 effect I(MO) properties. First it was demonstrated that the mutations in TRPV1 did not affect association of the TRPA1 and TRPV1 channels. However, these TRPV1 mutations, particularly Y671K, altered the following characteristics of TRPA1: magnitude of I(MO) in presence and absence of [Ca(2+)](e); the influence of [Ca(2+)](e) on the voltage-dependency of I(MO), and open probability of single-channel I(MO). In summary, activation of TRPA1 by [Ca(2+)](e) and [Ca(2+)](i) is controlled by the TRPV1 channel, and characteristics of I(MO) depend on Ca(2+) permeability of the TRPV1 channel.

Cannabinoid receptor antagonists AM251 and AM630 activate TRPA1 in sensory neurons.

Cannabinoid receptor antagonists have been utilized extensively in vivo as well as in vitro, but their selectivity has not been fully examined. We investigated activation of sensory neurons by two cannabinoid antagonists - AM251 and AM630. AM251 and AM630 activated trigeminal (TG) sensory neurons in a concentration-dependent fashion (threshold 1 μM). AM251 and AM630 responses are mediated by the TRPA1 channel in a majority (90-95%) of small-to-medium TG sensory neurons. AM630 (1-100 μM), but not AM251, was a significantly more potent agonist in cells co-expressing both TRPA1 and TRPV1 channels. We next evaluated AM630 and AM251 effects on TRPV1- and TRPA1-mediated responses in TG neurons. Capsaicin (CAP) effects were inhibited by pre-treatment with AM630, but not AM251. Mustard oil (MO) and WIN55,212-2 (WIN) TRPA1 mediated responses were also inhibited by pre-treatment with AM630, but not AM251 (25 uM each). Co-treatment of neurons with WIN and either AM630 or AM251 had opposite effects: AM630 sensitized WIN responses, whereas AM251 inhibited WIN responses. WIN-induced inhibition of CAP responses in sensory neurons was reversed by AM630 pre-treatment and AM251 co-treatment (25 μM each), as these conditions inhibit WIN responses. Hindpaw injections of AM630 and AM251 did not produce nocifensive behaviors. However, both compounds modulated CAP-induced thermal hyperalgesia in wild-type mice and rats, but not TRPA1 null-mutant mice. AMs also partially regulate WIN inhibition of CAP-induced thermal hyperalgesia in a TRPA1-dependent fashion. In summary, these findings demonstrate alternative targets for the cannabinoid antagonists, AM251 and AM630, in peripheral antihyperalgesia which involve certain TRP channels.

Prolactin receptor in regulation of neuronal excitability and channels

Prolactin (PRL) activates PRL receptor isoforms to exert regulation of specific neuronal circuitries, and to control numerous physiological and clinically-relevant functions including; maternal behavior, energy balance and food intake, stress and trauma responses, anxiety, neurogenesis, migraine and pain. PRL controls these critical functions by regulating receptor potential thresholds, neuronal excitability and/or neurotransmission efficiency. PRL also influences neuronal functions via activation of certain neurons, resulting in Ca2+ influx and/or electrical firing with subsequent release of neurotransmitters. Although PRL was identified almost a century ago, very little specific information is known about how PRL regulates neuronal functions. Nevertheless, important initial steps have recently been made including the identification of PRL-induced transient signaling pathways in neurons and the modulation of neuronal transient receptor potential (TRP) and Ca2+-dependent K+ channels by PRL. In this review, we summarize current knowledge and recent progress in understanding the regulation of neuronal excitability and channels by PRL.

Prolactin regulates TRPV1, TRPA1, and TRPM8 in sensory neurons in a sex-dependent manner: Contribution of prolactin receptor to inflammatory pain.

Prolactin (PRL) is a hormone produced in the anterior pituitary but also synthesized extrapituitary where it can influence diverse cellular processes, including inflammatory responses. Females experience greater pain in certain inflammatory conditions, but the contribution of the PRL system to sex-dependent inflammatory pain is unknown. We found that PRL regulates transient receptor potential (TRP) channels in a sex-dependent manner in sensory neurons. At >20 ng/ml, PRL sensitizes TRPV1 in female, but not male, neurons. This effect is mediated by PRL receptor (PRL-R). Likewise, TRPA1 and TRPM8 were sensitized by 100 ng/ml PRL only in female neurons. We showed that complete Freund adjuvant (CFA) upregulated PRL levels in the inflamed paw of both male and female rats, but levels were higher in females. In contrast, CFA did not change mRNA levels of long and short PRL-R in the dorsal root ganglion or spinal cord. Analysis of PRL and PRL-R knockout (KO) mice demonstrated that basal responses to cold stimuli were only altered in females, and with no significant effects on heat and mechanical responses in both sexes. CFA-induced heat and cold hyperalgesia were not changed in PRL and PRL-R KO compared with wild-type (WT) males, whereas significant reduction of heat and cold post-CFA hyperalgesia was detected in PRL and PRL-R KO females. Attenuation of CFA-induced mechanical allodynia was observed in both PRL and PRL-R KO females and males. Thermal hyperalgesia in PRL KO females was restored by administration of PRL into hindpaws. Overall, we demonstrate a sex-dependent regulation of peripheral inflammatory hyperalgesia by the PRL system.

Plasticity of cytochrome P450 isozyme expression in rat trigeminal ganglia neurons during inflammation

TOC summary CYP enzymes are coexpressed with TRPV1 in trigeminal neurons, are upregulated after inflammation, and catalyze the formation of endogenous TRPV1 agonists after application of linoleic acid to cultured neurons. ABSTRACT Recently, specific oxidized linoleic acid metabolites (OLAMs) have been identified as transient receptor potential vanilloid 1 (TRPV1) channel agonists that contribute to inflammatory and heat hyperalgesia mechanisms, yet the specific mechanism responsible for OLAM synthesis in sensory neurons is unknown. Here, we use molecular, anatomical, calcium imaging, and perforated patch electrophysiology methods to demonstrate the specific involvement of cytochrome P450 enzymes (CYPs) in the oxidation of linoleic acid leading to neuronal activation and show that this is enhanced under inflammatory conditions. Additional studies evaluated CYP expressions in the native rat trigeminal ganglia (TG) tissue and cultures as well as changes in their expression pattern following the induction of peripheral inflammation. Fourteen of 20 candidate transcripts were detected in native TG, and 7 of these displayed altered expression under cultured conditions. Moreover, complete Freund’s adjuvant‐induced inflammation of vibrissal pad selectively increased expression of CYP3A23/3A1 and CYP2J4 transcripts in TG. In situ hybridization studies demonstrated broad expression pattern of CYP3A23/3A1 and CYP2J4 within TG neurons. Anatomical studies characterized the expression of CYP3A1 and the CYP2J families within TG sensory neurons, including those with TRPV1, with about half of all TRPV1‐positive neurons showing more prominent CYP3A1 and CYP2J expression. Together, these findings show that CYP enzymes play a primary role in mediating linoleic acid‐evoked activation of sensory neurons and furthermore, implicate the involvement of specific CYPs as contributing to the formation of OLAMs that act as TRPV1 agonists within this subpopulation of nociceptors.

Chronic alteration in phosphatidylinositol 4,5-biphosphate levels regulates capsaicin and mustard oil responses

There is an agreement that acute (in minutes) hydrolysis and accumulation of phosphatidylinositol 4,5‐bisphosphate (PIP2) modulate TRPV1 and TRPA1 activities. Because inflammation results in PIP2 depletion, persisting for long periods (hours to days) in pain models and in the clinic, we examined whether chronic depletion and accumulation of PIP2 affect capsaicin (CAP) and mustard oil (MO) responses. In addition, we wanted to evaluate whether the effects of PIP2 depend on TRPV1 and TRPA1 coexpression and whether the PIP2 actions vary in expression cells vs. sensory neurons. Chronic PIP2 production was stimulated by overexpression of phosphatidylinositol‐4‐phosphate‐5‐kinase, and PIP2‐specific phospholipid 5′‐phosphatase was selected to reduce plasma membrane levels of PIP2. Our results demonstrate that CAP (100 nM) responses and receptor tachyphylaxis are not significantly influenced by chronic changes in PIP2 levels in wild‐type (WT) or TRPA1 null‐mutant sensory neurons as well as CHO cells expressing TRPV1 alone or with TRPA1. However, low concentrations of CAP (20 nM) produced a higher response after PIP2 depletion in cells containing TRPV1 alone but not TRPV1 together with TRPA1. MO (25 μM) responses were also not affected by PIP2 in WT sensory neurons and cells coexpressing TRPA1 and TRPV1. In contrast, PIP2 reduction leads to pronounced tachyphylaxis to MO in cells with both channels. Chronic effect of PIP2 on TRPA1 activity depends on presence of the TRPV1 channel and cell type (CHO vs. sensory neurons). In summary, chronic alterations in PIP2 levels regulate magnitude of CAP and MO responses as well as MO tachyphylaxis. This regulation depends on coexpression profile of TRPA1 and TRPV1 and cell type.

Endogenous Prolactin Generated During Peripheral Inflammation Contributes to Thermal Hyperalgesia

Prolactin (PRL) is a hormone and a neuromodulator. It sensitizes TRPV1 (transient receptor potential cation channel subfamily V member 1) responses in sensory neurons, but it is not clear whether peripheral inflammation results in the release of endogenous PRL, or whether endogenous PRL is capable of acting as an inflammatory mediator in a sex‐dependent manner. To address these questions, we examined inflammation‐induced release of endogenous PRL, and its regulation of thermal hyperalgesia in female and male rats. PRL is expressed in several types of peripheral neuronal and non‐neuronal cells, including TRPV1‐positive nerve fibers, preadipocytes and activated macrophages/monocytes localized in the vicinity of nerves. Evaluation of PRL levels in hindpaws and plasma indicated that complete Freund’s adjuvant (CFA) stimulates release of peripheral, but not systemic, PRL within 6–48 h in both ovariectomized females with estradiol replacement (OVX‐E) and intact male rats. The time course of release varies in OVX‐E and intact male rats. We next employed the prolactin receptor (PRL‐R) antagonist Δ1‐9‐G129R‐hPRL to assess the role of locally produced PRL in nociception. Applied at a ratio of 1 : 1 (PRL:Δ1‐9‐G129R‐hPRL; 40 nm each), this antagonist was able to nearly (≈80%) reverse PRL‐induced sensitization of capsaicin responses in rat sensory neurons. CFA‐induced inflammatory thermal hyperalgesia in OVX‐E rat hindpaws was significantly reduced in a dose‐dependent manner by the PRL‐R antagonist at 6 h but not at 24 h. In contrast, PRL contributed to inflammatory thermal hyperalgesia in intact male rats at 24, but not at 6 h. These findings indicate that inflammation leads to accumulation of endogenous PRL in female and male rats. Furthermore, PRL acts as an inflammatory mediator at different time points for female and intact male rats.

Sex-dependent roles of prolactin and prolactin receptor in postoperative pain and hyperalgesia in mice

Although surgical trauma activates the anterior pituitary gland and elicits an increase in prolactin (PRL) serum levels that can modulate nociceptive responses, the role of PRL and the PRL-receptor (PRL-R) in thermal and mechanical hyperalgesia in postoperative pain is unknown. Acute postoperative pain condition was generated with the use of the hindpaw plantar incision model. Results showed endogenous PRL levels were significantly increased in serum, operated hindpaw and spinal cords of male and female rats 24h after incision. These alterations were especially pronounced in females. We then examined the role of the PRL system in thermal and mechanical hyperalgesia in male and female mice 3-168 h after plantar incision with the use of knock-out (KO) mice with PRL or PRL-R gene ablations and in wild-type (WT) mice. WT mice showed postoperative cold hyperalgesia in a sex-dependent manner (only in females), but with no effect on heat hyperalgesia or mechanical allodynia in either sex. Studies in KO mice showed no effect of PRL and PRL-R gene ablation on heat and cold hyperalgesia in male mice, while heat hyperlgesia were reduced 3-72 h post-surgery in female PRL and PRL-R KO mice. In contrast, PRL and PRL-R ablations significantly attenuated mechanical allodynia 3-72 h post-surgery in both male and female mice. Overall, we found elevated PRL levels in serum, hindpaws and spinal cords after incision, and identify a contributory role for the PRL system in postoperative pain responses to thermal stimuli in females and to mechanical stimuli in both males and females.

Mechanisms of Transient Signaling via Short and Long Prolactin Receptor Isoforms in Female and Male Sensory Neurons

Background: Prolactin regulates the activity of nociceptors in pain conditions. Results: Prolactin regulation of sensory neurons is acute and mediated via PI3K and PKCϵ following activation of prolactin receptor short isoform. Prolactin receptor short isoform actions are inhibited by the long isoform. Conclusion: Prolactin receptor short isoform mediates transient sensitization of nociceptors. Significance: The proposed mechanism could underlie prolactin involvement in hyperalgesia/pain. Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ϵ (PKCϵ) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3–5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCϵ, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells.