Anti-pruritic effect of L-carnitine against chloroquine-induced pruritus mediated via nitric oxide pathway.

BACKGROUND: Pruritus, or itching, is a distressing symptom associated with various dermatological and systemic diseases. L-carnitine (ßeta hydroxy-γ-tri methyl amino-butyric acid), is a naturally occurring substance, it controls numerous physiological processes. The present research aims to identify L-carnitine for its anti-pruritic effect via nitric oxide-dependent mechanism. METHODS: Chloroquine-induced pruritus serves as an experimental model to investigate possible therapeutic interventions. In this study, we evaluated the efficacy of L-carnitine in combating oxidative stress, nitric oxide, and inflammatory cytokines in a chloroquine-induced pruritus model. RESULTS: L-carnitine treatment significantly reduced scratching behavior compared to the disease group (***P < 0.001 vs. chloroquine group), indicating its antipruritic potential. The markers of oxidative stress, GST, GSH, Catalase, and LPO were dysregulated in the disease model, but administration of L-carnitine restored GST, GSH, and Catalase levels and decreased LPO levels (***P < 0.001 vs. chloroquine group), thereby alleviating oxidative stress. L-carnitine also reduced nitric oxide synthase (NOS) activity, suggesting that it modulates nitric oxide signaling pathways involved in pruritus. In addition, L-carnitine lowered levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), inflammatory marker nuclear factor kappa B (p-NFκB) and also reduces an inflammatory enzyme, cyclooxygenase-2 (COX-2), determined by ELISA (Enzyme-Linked Immunosorbent Assay) (***P < 0.001 vs. chloroquine group). It downregulates nNOS mRNA expression confirmed by real-time polymerase chain reaction (RT-PCR). CONCLUSION: These findings highlight the therapeutic effects of L-carnitine in alleviating chloroquine-induced pruritus.

Curcumin inhibits the pruritus in mice through mast cell MrgprB2 receptor.

BACKGROUND: Curcumin is a diketone compound extracted from the rhizomes of some plants in the Zingiberaceae and Araceae family. It possesses a variety of biological activities, including antioxidant, anti-inflammatory and anti-cancer properties. However, the cellular and molecular antipruritic mechanisms of curcumin remain to be explored. OBJECTIVE: Our objective was to study the role of curcumin in pruritus and determine whether its antipruritic effect is related to MrgprB2 receptor. METHODS: The effect of curcumin on pruritus in mice was examined by scratching behavior test. The antipruritic mechanism of curcumin was explored by using transgenic mice (MrgprB2-/- mice, MrgprB2CreTd/tomato mice), histological analysis, western blot and immunofluorescence. In addition, the relationship between curcumin and MrgprB2/X2 receptor was studied in vitro by using calcium imaging, plasmid transfection and molecular docking RESULTS: In the current study, we found that curcumin had obvious antipruritic effect. Its antipruritic effect was related to the regulation of MrgprB2 receptor activation and mast cells tryptase release. In vitro, mouse peritoneal mast cells activated by compound 48/80 could be inhibited by curcumin. In addition, curcumin was also found to suppress the calcium flux in MrgprX2 or MrgprB2-overexpression HEK cells induced by compound 48/80, substance P, and PAMP 9-20, displaying the specific relation with the MrgprB2/X2 receptor. Moreover, molecular docking results showed that curcumin had affinity to MrgprX2 protein. CONCLUSIONS: Overall, these results indicated that curcumin has the potential to treat pruritus induced by mast cell MrgprB2 receptor.

Antipruritic Effects of Kappa Opioid Receptor Agonists: Evidence from Rodents to Humans.

Centrally administered bombesin induces scratching and grooming in rats. These behaviors were blocked by early benzomorphan kappa opioid receptor (KOR) agonists as reported by Gmerek and Cowan in 1984. This was the first evidence that KORs may be involved in the sensation of itch-like behaviors. Subsequent development of additional animal models for acute and chronic itch has led to important discoveries since then. For example, it was found that (a) gastrin-releasing peptide (GRP), natriuretic polypeptide b and their cognate receptors are keys for the transmission of itch sensation at the spinal cord level, (b) dynorphins (Dyns), the endogenous KOR agonists, work as inhibitory neuromodulators of itch at the spinal cord level, (c) in a mouse model for acute itch, certain KOR antagonists elicit scratching, (d) in mouse models of acute or chronic itch, KOR agonists (e.g., U50,488, nalfurafine, CR 845, nalbuphine) suppress scratching induced by different pruritogens, and (e) nalfurafine, CR 845, and nalbuphine are in the clinic or in clinical trials for pruritus associated with chronic kidney disease and chronic liver disease, as well as pruritus in chronic skin diseases.

Capsaicin and Gut Microbiota in Health and Disease.

Capsaicin is a widespread spice known for its analgesic qualities. Although a comprehensive body of evidence suggests pleiotropic benefits of capsaicin, including anti-inflammatory, antioxidant, anti-proliferative, metabolic, or cardioprotective effects, it is frequently avoided due to reported digestive side-effects. As the gut bacterial profile is strongly linked to diet and capsaicin displays modulatory effects on gut microbiota, a new hypothesis has recently emerged about its possible applicability against widespread pathologies, such as metabolic and inflammatory diseases. The present review explores the capsaicin-microbiota crosstalk and capsaicin effect on dysbiosis, and illustrates the intimate mechanisms that underlie its action in preventing the onset or development of pathologies like obesity, diabetes, or inflammatory bowel diseases. A possible antimicrobial property of capsaicin, mediated by the beneficial alteration of microbiota, is also discussed. However, as data are coming mostly from experimental models, caution is needed in translating these findings to humans.

The influence of menthol dose on human temperature regulation and perception.

INTRODUCTION: This study assessed the influence of High (H, 4.13%), Medium (M, 2.0%) and Low (L, 0.1%) doses of menthol on temperature perception and regulation, compared to a Placebo Condition (P). METHOD: Sixteen participants underwent the aforementioned conditions on four separate days. During each test participants rested supine (Environmental conditions: 30 °C, 50% rh) for 30-min before 40 mL of L, M, H or P gel was applied to the anterior upper body, then rested 30-min thereafter. Primary measures included thermal sensation (TS), thermal comfort (TC), irritation (IRR), rectal temperature (Tre), and skin temperature (chest, forearm, thigh, calf), and EMG (trapezius, pectoralis major, sternocleidomastoid). The area under the curve (AUC) from minute 30 to 60 was compared between conditions using relevant non/parametric tests (alpha level = 0.05). RESULTS: A cooling trend in Tre was observed following Placebo gel application, but this significantly (p < 0.05) reversed into a heat storage response in M and H. Both TS and TC significantly differed by condition (p < 0.001) in a dose-dependent manner, with L, M, and H doses eliciting significantly cooler sensations and more discomfort than P (p < 0.05). Irritation significantly differed by condition (p < 0.01) in a dose-dependent manner, with L and M eliciting significantly greater irritation than P (p < 0.01). No other differences were observed. CONCLUSIONS: Menthol exerts perceptual and thermoregulatory effects independent of skin temperature. A menthol dose-dependent perceptual cooling effect was evident with possible saturation at the moderate dose. A dose-dependent alteration in deep body temperature was also evident.

Menthol can be safely applied to improve thermal perception during physical exercise: a meta-analysis of randomized controlled trials.

Menthol is often used as a cold-mimicking substance to allegedly enhance performance during physical activity, however menthol-induced activation of cold-defence responses during exercise can intensify heat accumulation in the body. This meta-analysis aimed at studying the effects of menthol on thermal perception and thermophysiological homeostasis during exercise. PubMed, EMBASE, Cochrane Library, and Google Scholar databases were searched until May 2020. Menthol caused cooler thermal sensation by weighted mean difference (WMD) of - 1.65 (95% CI, - 2.96 to - 0.33) and tended to improve thermal comfort (WMD = 1.42; 95% CI, - 0.13 to 2.96) during physical exercise. However, there was no meaningful difference in sweat production (WMD = - 24.10 ml; 95% CI, - 139.59 to 91.39 ml), deep body temperature (WMD = 0.02 °C; 95% CI, - 0.11 to 0.15 °C), and heart rate (WMD = 2.67 bpm; 95% CI - 0.74 to 6.09 bpm) between the treatment groups. Menthol improved the performance time in certain subgroups, which are discussed. Our findings suggest that different factors, viz., external application, warmer environment, and higher body mass index can improve menthol's effects on endurance performance, however menthol does not compromise warmth-defence responses during exercise, thus it can be safely applied by athletes from the thermoregulation point of view.

Non-electrophilic TRPA1 agonists, menthol, carvacrol and clotrimazole, open epithelial tight junctions via TRPA1 activation.

Activation of the transient receptor potential A1 channel (TRPA1) by electrophilic agonists was reported to induce the opening of tight junctions (TJs). Because compounds that increase TJ permeability can be paracellular permeability enhancers, we investigated the effect of non-electrophilic TRPA1 activators, including food ingredients (menthol and carvacrol) and medication (clotrimazole), on epithelial permeability. We show that all three compounds induced increase of the permeability of fluorescein isothiocyanate-conjugated dextran (4 kDa) and decrease of transepithelial electrical resistance, accompanied by Ca2+ influx and cofilin activation in epithelial MDCK II monolayers. These phenotypes were attenuated by pretreatment of a TRPA1 antagonist, suggesting TRPA1-mediated opening of TJs. These results suggest that non-electrophilic TRPA1 activators with established safety can be utilized to regulate epithelial barriers.

PAR2 Mediates Itch via TRPV3 Signaling in Keratinocytes.

Animal studies have suggested that transient receptor potential ion channels and G-protein coupled receptors play important roles in itch transmission. TRPV3 gain-of-function mutations have been identified in patients with Olmsted syndrome, which is associated with severe pruritus. However, the mechanisms causing itch remain poorly understood. Here, we show that keratinocytes lacking TRPV3 impair the function of protease-activated receptor 2 (PAR2), resulting in reduced neuronal activation and scratching behavior in response to PAR2 agonists. Moreover, we show that TRPV3 and PAR2 were upregulated in skin biopsies from patients and mice with atopic dermatitis, whereas their inhibition attenuated scratching and inflammatory responses in mouse atopic dermatitis models. These results reveal a previously unrecognized link between TRPV3 and PAR2 in keratinocytes to convey itch information and suggest that a blockade of PAR2 or TRPV3 individually or both may serve as a potential approach for antipruritic therapy in atopic dermatitis.

Capsaicinoids enhance chemosensitivity to chemotherapeutic drugs.

Cytotoxic chemotherapy is the mainstay of cancer treatment. Conventional chemotherapeutic agents do not distinguish between normal and neoplastic cells. This leads to severe toxic side effects, which may necessitate the discontinuation of treatment in some patients. Recent research has identified key molecular events in the initiation and progression of cancer, promoting the design of targeted therapies to selectively kill tumor cells while sparing normal cells. Although, the side effects of such drugs are typically milder than conventional chemotherapies, some off-target effects still occur. Another serious challenge with all chemotherapies is the acquisition of chemoresistance upon prolonged exposure to the drug. Therefore, identifying supplementary agents that sensitize tumor cells to chemotherapy-induced apoptosis and help minimize drug resistance would be valuable for improving patient tolerance and response to chemotherapy. The use of effective supplementary agents provides a twofold advantage in combination with standard chemotherapy. First, by augmenting the activity of the chemotherapeutic drug it can lower the dose needed to kill tumor cells and decrease the incidence and severity of treatment-limiting side effects. Second, adjuvant therapies that lower the effective dose of chemotherapy may delay/prevent the development of chemoresistance in tumors. Capsaicinoids, a major class of phytochemical compounds isolated from chili peppers, have been shown to improve the efficacy of several anti-cancer drugs in cell culture and animal models. The present chapter summarizes the current knowledge about the chemosensitizing activity of capsaicinoids with conventional and targeted chemotherapeutic drugs, highlighting the potential use of capsaicinoids in novel combination therapies to improve the therapeutic indices of conventional and targeted chemotherapeutic drugs in human cancers.

Package of menthol measures for thirst relief: a randomized clinical study.

OBJECTIVE: To evaluate the effectiveness of the menthol package (lip hydration and ice popsicles) compared to a package of non-menthol measures (lip hydration and ice popsicles) as a way to relieve thirst in patients in the Anesthetic Recovery Room. METHOD: Randomized and parallel trial study, with 120 patients randomized patients in an experimental group - menthol measurements (n=59) and control group - measures without menthol (n = 61). RESULTS: There was a significant (p<0.05) decrease in intensity, hydration, dryness and taste in the oral cavity between the three moments of assessment/intervention in the two groups. The difference was significant in the experimental group for thirst intensity at the second assessment/intervention point (p<0.05) after a single administration of the menthol package. CONCLUSION: There was a reduction in thirst intensity in both groups. Patients who received menthol packages showed a significant decrease in intensity after a single evaluation/intervention time. NCT: 02869139.

Package of menthol measures for thirst relief: a randomized clinical study / Paquete de medidas mentoladas para el alivio de lased: estudio clínico aleatorizado / Pacote de medidas mentoladas para o alívio da sede: estudo clínico randomizado

ABSTRACT Objective: To evaluate the effectiveness of the menthol package (lip hydration and ice popsicles) compared to a package of non-menthol measures (lip hydration and ice popsicles) as a way to relieve thirst in patients in the Anesthetic Recovery Room. Method: Randomized and parallel trial study, with 120 patients randomized patients in an experimental group - menthol measurements (n=59) and control group - measures without menthol (n = 61). Results: There was a significant (p<0.05) decrease in intensity, hydration, dryness and taste in the oral cavity between the three moments of assessment/intervention in the two groups. The difference was significant in the experimental group for thirst intensity at the second assessment/intervention point (p<0.05) after a single administration of the menthol package. Conclusion: There was a reduction in thirst intensity in both groups. Patients who received menthol packages showed a significant decrease in intensity after a single evaluation/intervention time. NCT: 02869139.

RESUMEN Objetivo: Evaluar la efectividad del paquete de medidas mentoladas (hidratación labial y picolé de hielo) comparado a un paquete de medidas no mentoladas (hidratación labial y picolé de hielo) como un método de alivio de la sed en pacientes en la Sala de Recuperación Post-Anestésica. Método: En un ensayo clínico aleatorizado, paralelo, 120 pacientes aleatorizados en un grupo experimental - medidas mentoladas (n=59) y un grupo control - medidas sin el mentol (n=61). Resultados: Hubo una disminución significativa (p<0,05) sobre la intensidad, hidratación, sequedad y gusto en la cavidad oral entre los tres momentos de evaluación/intervención en los dos grupos, indistintamente. La diferencia fue significativa en el grupo experimental para la intensidad de la sed en el segundo momento de la evaluación/intervención (p<0,05) después de una única administración del paquete de medidas mentoladas. Conclusión: Se encontró una reducción de la intensidad de la sed en los dos grupos. Los pacientes que recibieron las medidas mentoladas presentaron una disminución significativa de la intensidad después de un único momento de evaluación/intervención. NCT: 02869139.

RESUMO Objetivo: Avaliar a efetividade do pacote de medidas mentoladas (hidratação labial e picolé de gelo) comparado a um pacote de medidas não mentoladas (hidratação labial e picolé de gelo) como método de alívio da sede em pacientes na Sala de Recuperação Anestésica. Método: Ensaio clínico randomizado, paralelo, 120 pacientes aleatorizados em grupo experimental - medidas mentoladas (n=59) e grupo controle - medidas sem o mentol (n=61). Resultados: Houve diminuição significativa (p<0,05) sobre a intensidade, hidratação, secura e gosto na cavidade oral entre os três momentos de avaliação/intervenção nos dois grupos indistintamente. A diferença foi significativa no grupo experimental para intensidade da sede no segundo momento de avaliação/intervenção (p<0,05) após uma única administração do pacote de medidas mentoladas. Conclusão: Houve redução da intensidade da sede nos dois grupos. Os pacientes que receberam as medidas mentoladas apresentaram diminuição significativa da intensidade após um único momento de avaliação/intervenção. A NCT: 02869139.

Dual Anti-cancer and Anti-Itch Activity of PD176252 Analogues: Design, Synthesis and Biological Evaluation.

BACKGROUND: Cancer patients treated with targeted anti-cancer drug suffer from itch or pruritus. Itch or pruritus is an unpleasant sensation that brings about a negative impact on quality of life, and serious itch may lead to dose reduction and even discontinuation. Gastrin releasing peptide receptor (GRPR) plays a critical role in itch, inflammation and cancer, and GRPR antagonist has obvious effect on cancer, inflammation and itch. The aim of this paper is to develop a new agent with anti-cancer and anti-itch activity. METHODS: A series of GRPR antagonist PD176252 analogues (3a-3l) were designed and synthesized. Both anticancer and anti-itch activities were evaluated. Anti-cancer activity was evaluated in three human cancer cell lines in vitro, the anti-itch activity in evaluated with Kunming mice by intrathecal injection of chloroquine phosphate as a modeling medium. And the cytotoxicity on normal cells was evaluated. RESULTS: Of the tested compounds, compound 3i showed potently anti-cancer activity to all cancer cell lines tested with IC50 values of 10.5µM (lung), 11.6µM (breast) and 12.8µM (liver) respectively and it also showed significant inhibition of the scratching behavior. Comparing with PD17625, compound 3i and 3g gave better inhibition activities against all cancer cell lines, compound 3b, 3c and 3i showed better anti-itch activity. The compound 3i is safe for normal breast and liver normal cells, but it has high cytotoxicity on normal lung cell. CONCLUSION: The synthesized compounds have dual anti-cancer and anti-itch activity, so the development of drug with dual anti-tumor and anti-itch property is possible.

Itching, chloroquine, and malaria: a review of recent molecular and neuroscience advances and their contribution to mechanistic understanding and therapeutics of chronic non-histaminergic pruritus.

Chloroquine (CQ) is an antimalarial drug that elicits severe pruritus in black Africans with malaria fever. This acute itching (2-7 days duration) exhibits age dependency and a racial and genetic predilection. CQ itch is non-histaminergic, which makes it both a good model and a tool to probe the mechanisms of chronic itch. This review focuses on recently discovered mechanisms, neuroscience, mediators, and receptors that are implicated in molecular studies of CQ pruritus. CQ pruritus mechanisms are also compared to that of itching following other systemic diseases, such as chronic kidney disease, chronic liver disease, skin disorders, and burns. There are striking similarities between CQ itching pathways and other chronic itch secondary to systemic disease with or without skin lesions, which have not been previously highlighted. Prominent among these are the shared roles of skin, neural and spinal µ opiate receptors, kappa opiate receptor, nitric oxide, serotonin via 5HT1B/D receptors, cytokines, especially interleukins, and tumor necrosis factor. There is elaborate "cross talk" among the diverse mediators and receptors involved in CQ-induced pruritus. CQ also binds to the mas-related G protein coupled receptors MrgprA3/MrgprX1 present in a small proportion (4-5%) of dorsal root ganglion neurons and skin. The mrgprA3 CQ receptors are coupled to PLC-ß3 and a chloride channel to initiate skin itch action potentials in C nerve fibers. Mrgpra3/X1 couples to TRPA1 for calcium influx into neuronal cells at noncutaneous sites. Central CQ itch occurs via gastrin-related peptide (GRP) and its receptor (GRPR) in the dorsal spinothalamic tracts, as well as glutamic mediated GRP projection to parabrachial nucleus. The possibility of chronic itch therapy based on personalized medicine, genetics, and transcriptomics or the use of itch "polypill/polycream" are discussed.

Monoacylglycerol Lipase Inhibition in Human and Rodent Systems Supports Clinical Evaluation of Endocannabinoid Modulators.

Monoacylglycerol lipase (MGLL) is the primary degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). The first MGLL inhibitors have recently entered clinical development for the treatment of neurologic disorders. To support this clinical path, we report the pharmacological characterization of the highly potent and selective MGLL inhibitor ABD-1970 [1,1,1,3,3,3-hexafluoropropan-2-yl 4-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-4-chlorobenzyl)piperazine-1-carboxylate]. We used ABD-1970 to confirm the role of MGLL in human systems and to define the relationship between MGLL target engagement, brain 2-AG concentrations, and efficacy. Because MGLL contributes to arachidonic acid metabolism in a subset of rodent tissues, we further used ABD-1970 to evaluate whether selective MGLL inhibition would affect prostanoid production in several human assays known to be sensitive to cyclooxygenase inhibitors. ABD-1970 robustly elevated brain 2-AG content and displayed antinociceptive and antipruritic activity in a battery of rodent models (ED50 values of 1-2 mg/kg). The antinociceptive effects of ABD-1970 were potentiated when combined with analgesic standards of care and occurred without overt cannabimimetic effects. ABD-1970 also blocked 2-AG hydrolysis in human brain tissue and elevated 2-AG content in human blood without affecting stimulated prostanoid production. These findings support the clinical development of MGLL inhibitors as a differentiated mechanism to treat pain and other neurologic disorders.

Capsaicin protects against testicular torsion injury through mTOR-dependent mechanism.

SCOPE: Testicular torsion and subsequent release of reactive oxygen species (ROS) can cause infertility in adults. Oxidative stress following testicular torsion plays an important role in the ýonset and development of apoptotic cell death through dysregulation of the cellular signaling pathways. Anti-inflammatory and antioxidant properties of capsaicin, a bioactive composition present in red peppers, has already been exploited for treatment of the cancer and pain relief. In present work, we evaluated the role of the mammalian target of rapamycin (mTOR) in antioxidant effect of capsaicin against reperfusion injury following testicular torsion. METHODS: Male Wistar rats weighing 200-220 g were randomly assigned into four major groups: (i) a sham operated group, (ii) a testicular torsion (TT) group, (iii) three groups treated with different doses of capsaicin (TT + 100, 500 and 1000 µg/ml Cap), and (iv) three groups of healthy rats treated with different doses of capsaicin (100, 500 and 1000 µg/ml). Western blotting assay was used to examine the anti-apoptotic effects of capsaicin in testicular cells following torsion. H&E and TUNEL methods were used to evaluate testicular morphology and apoptosis activity. RESULTS: Compared to control group, phosphorylation of mTOR was significantly increased in the TT groups. Capsaicin administration remarkably decreased the phosphorylation of mTOR at the highest dose (P < 0.05). Capsaicin decreased apoptosis and preserved tubular morphology in testes. CONCLUSION: Our results showed that antioxidant properties of capsaicin minimizes cell death and reperfusion injury following testicular torsion.

TrpM8-mediated somatosensation in mouse neocortex.

Somatosensation is a complex sense mediated by more than a dozen distinct neural subtypes in the periphery. Although pressure and touch sensation have been mapped to primary somatosensory cortex in rodents, it has been controversial whether pain and temperature inputs are also directed to this area. Here we use a well-defined somatosensory modality, cool sensation mediated by peripheral TrpM8-receptors, to investigate the neural substrate for cool perception in the mouse neocortex. Using activation of cutaneous TrpM8 receptor-expressing neurons, we identify candidate neocortical areas responsive for cool sensation. Initially, we optimized TrpM8 stimulation and determined that menthol, a selective TrpM8 agonist, was more effective than cool stimulation at inducing expression of the immediate-early gene c-fos in the spinal cord. We developed a broad-scale brain survey method for identification of activated brain areas, using automated methods to quantify c-fos immunoreactivity (fos-IR) across animals. Brain areas corresponding to the posterior insular cortex and secondary somatosensory (S2) show elevated fos-IR after menthol stimulation, in contrast to weaker activation in primary somatosensory cortex (S1). In addition, menthol exposure triggered fos-IR in piriform cortex, the amygdala, and the hypothalamus. Menthol-mediated activation was absent in TrpM8-knock-out animals. Our results indicate that cool somatosensory input broadly drives neural activity across the mouse brain, with neocortical signal most elevated in the posterior insula, as well as S2 and S1. These findings are consistent with data from humans indicating that the posterior insula is specialized for somatosensory information encoding temperature, pain, and gentle touch.

Menthol Stereoisomers Exhibit Different Effects on α4ß2 nAChR Upregulation and Dopamine Neuron Spontaneous Firing.

Menthol contributes to poor cessation rates among smokers, in part because menthol enhances nicotine reward and reinforcement. Mentholated tobacco products contain (-)-menthol and (+)-menthol, in varying proportions. We examined these two menthol stereoisomers for their ability to upregulate α4ß2 nAChRs and to alter dopamine neuron firing frequency using long-term, low-dose (≤500 nm) exposure that is pharmacologically relevant to smoking. We found that (-)-menthol upregulates α4ß2 nAChRs while (+)-menthol does not. We also found that (-)-menthol decreases dopamine neuron baseline firing and dopamine neuron excitability, while (+)-menthol exhibits no effect. We then examined both stereoisomers for their ability to inhibit α4ß2 nAChR function at higher concentrations (>10 µm) using the Xenopus oocyte expression system. To probe for the potential binding site of menthol, we conducted flooding simulations and site-directed mutagenesis. We found that menthol likely binds to the 9´ position on the TM2 (transmembrane M2) helix. We found that menthol inhibition is dependent on the end-to-end distance of the side chain at the 9´ residue. Additionally, we have found that (-)-menthol is only modestly (∼25%) more potent than (+)-menthol at inhibiting wild-type α4ß2 nAChRs and a series of L9´ mutant nAChRs. These data reveal that menthol exhibits a stereoselective effect on nAChRs and that the stereochemical effect is much greater for long-term, submicromolar exposure in mice than for acute, higher-level exposure. We hypothesize that of the two menthol stereoisomers, only (-)-menthol plays a role in enhancing nicotine reward through nAChRs on dopamine neurons.

A possible role for CD26/DPPIV enzyme activity in the regulation of psoriatic pruritus.

BACKGROUND: Psoriasis (PSO) is one of the most common chronic inflammatory skin diseases, and pruritus affects approximately 60-90% of patients with PSO. However, the pathogenesis of pruritus in PSO remains unclear. Dipeptidyl peptidase IV (DPPIV) enzyme activity is involved in the regulation of peptide hormones, chemokines and neurotransmitters. OBJECTIVES: Our aim is to evaluate for a potential association between DPPIV and an increased risk of pruritus, and to identify possible underlying treatment targets in affected patients. METHODS: Utilizing clinical serum samples of PSO patients and in vivo experimental pruritus models, we evaluated for a potential association between DPPIV and an increased risk for pruritus, and attempted to identify possible underlying treatment targets in pruritus of PSO. RESULTS: We first showed that levels of DPPIV enzyme activity in sera of patients with PSO were significantly increased compared to those of healthy controls. We next evaluated levels of substance-P (SP), which is a neurotransmitter for pruritus and a substrate for DPPIV enzyme. Truncated form SP cleaved by DPPIV was significantly increased in sera of PSO. In an in vivo pruritus model induced by SP, scratching was decreased by treatment with a DPPIV inhibitor. Moreover, DPPIV-knockout mice showed attenuation of scratching induced by SP. Finally, scratching was decreased following the administration of a DPPIV inhibitor in an imiquimod-induced PSO model. On the other hand, scratching induced by imiquimod was increased in DPPIV overexpressing-mice. CONCLUSIONS: These results suggest that inhibition of DPPIV enzyme activity regulates pruritus in PSO.

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.

Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition.

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 µM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 µM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon).