British Journal of Pharmacology

TRPA1 is a non-selective cation channel that plays a crucial role in several pain and inflammatory conditions. Agents reducing membrane cholesterol decrease TRPA1 activation, but it remains unclear how cholesterol-lowering medications affect TRPA1 function. Given that TRPA1 is activated by a wide variety of chemicals, we explored whether statins have acute effects on this channel. We found that five commonly used statins activate human and mouse TRPA1 in a reversible and concentration-dependent manner. The effective concentrations were above the micromolar range, in the order: simvastatin {approx} lovastatin < fluvastatin < atorvastatin < pravastatin. Statin-induced activation was not correlated to changes in membrane order, nor mediated by N-terminal cysteine residues contributing to electrophilic compound agonism. Molecular docking calculations and the functional characterization of single-point mutants revealed two separate putative binding sites, one situated close to the kink of transmembrane segment 5 (TM5) and the other at the interface between TM4 and TM5. The mTRPA1 inhibitor A-967079 largely abrogated the response to the electrophilic agonist allyl isothiocyanate, but had weaker and varied effects across different statins and menthol. Mutation T877L strongly altered the effect of A-967079, also in an agonist-dependent manner, suggesting competitive binding between this antagonist and the non-electrophilic agonists. The identification of two distinct agonist binding sites may help explaining how TRPA1 is able to respond to a large variety of non-electrophilic compounds, while the finding of competitive interactions at one of these sites may help guide the development of agonist-specific antagonists of therapeutic relevance.

Opioid withdrawal is associated with heightened pain sensitivity, including allodynia. Although opioid-induced allodynia is well-documented in humans and animal models, the relationship between the severity of opioid withdrawal-induced allodynia and individual addiction-like behaviors remains poorly understood. To address this gap, Heterogeneous Stock rats underwent long access (12 hours/day) intravenous oxycodone self-administration, followed by measurement of mechanical sensitivity at six timepoints across three weeks of abstinence. Rats were stratified by an Addiction Index derived from individual differences in the escalation of oxycodone intake, motivation to consume oxycodone, tolerance to oxycodones analgesic effects, and acute withdrawal-induced mechanical pain sensitivity. Here, we show that oxycodone withdrawal induces significant and prolonged allodynia for up to three weeks, with High Addiction Index rats exhibiting greater intensity and longer duration of pain sensitivity than Low Addiction Index rats. Results remained consistent even when excluding allodynia from the Addiction Index, highlighting the robustness of the association between addiction-like severity and protracted allodynia. Linear regression associations revealed that self-administration behaviors, particularly oxycodone intake escalation and motivation to seek oxycodone, predicted subsequent withdrawal-induced allodynia severity. These findings demonstrate that greater addiction-like severity is associated with more intense and prolonged withdrawal-induced pain, supporting mechanical allodynia as a marker of addiction severity. These results motivate future work to define the mechanisms linking addiction severity to protracted opioid withdrawal-induced pain, with the goal of informing targeted clinical interventions for individuals most susceptible to severe abstinence-related allodynia.

Chronic pain and nicotine use frequently co-occur, and individuals with chronic pain often experience greater difficulty quitting. Therefore, we examined nicotine withdrawal behaviors and analgesic-like effects in pain-naive and chronic pain conditions. Adult male and female rats underwent chronic constriction injury or sham surgery. After pain establishment, rats received twice-daily subcutaneous nicotine (0.3 or 0.7 mg/kg) or saline for 14 days. 24 h after the final injection, withdrawal was assessed, including physical signs and anxiety-like behavior. Depressive-like responses were evaluated at 72 h. Pain sensitivity and nicotines analgesic-like effects were assessed throughout. Chronic pain increased physical signs of withdrawal in both sexes, with greater effects in females. It also induced anxiety-like behavior in controls of both sexes. In rats with comorbid chronic pain and withdrawal, anxiety-like behavior was further enhanced in males, whereas females showed variable responses across assays, with increases or decreases depending on the test. Chronic pain induced depressive-like behavior in males but not in females. During withdrawal, depressive-like responses in males with chronic pain were not greater than those in the chronic pain alone group, while chronic nicotine exposure reduced depressive-like behavior in females. Nicotine produced acute analgesic-like effects that diminished over time in both pain-naive and chronic pain conditions, indicating tolerance. In pain-naive rats, repeated nicotine exposure induced mechanical hypersensitivity. Chronic pain intensified nicotine withdrawal severity in a nicotine concentration- and sex-dependent manner. These findings highlight the importance of considering pain status and sex when developing effective cessation strategies, particularly for individuals with comorbid chronic pain. SummaryChronic pain exacerbates nicotine withdrawal severity. Chronic nicotine exposure induces pain hypersensitivity and tolerance to analgesic effects. These effects vary by nicotine concentration and sex.

Targeting the kappa opioid receptor (KOR) system has emerged as a potential alternative to current analgesics, however, advancing the therapeutic development of KOR requires further elucidation of its intracellular signaling events and modulators. Among these intracellular modulators, Regulators of G protein signaling (RGS) proteins act as key modulators of GPCR signaling to shape nociceptive circuits and influence pain processing. Despite this, the molecular diversity of RGS proteins that shape KOR signaling and its behavioral consequences remains largely unexplored. Here we report that RGS6, a member of the R7 RGS family, is highly expressed in nociceptive areas and modulates multiple modalities of KOR-dependent anti-nociception and nocifensive behaviors. Using global single and double knockout mouse models we show that this anti-nociceptive phenotype was highly specific to RGS6 within the R7 RGS family. Further we demonstrate that the R7 RGS family displays a lack of functional redundancy in regulation of KOR signaling and behaviors. Using peripherally restricted KOR agonists, we found that KOR-RGS6 anti-nociceptive signaling displays sex differences in a site-specific manner, as females but not males displayed enhanced anti-nociceptive and blunted nocifensive behaviors. Our findings suggest that RGS6 is a highly specific modulator of KOR-dependent anti-nociceptive signaling and plays an essential role in modulating nociceptive circuits, potentially aiding in the development of novel analgesic drugs and therapeutics.

BackgroundNeuropathic pain is a major source of disability and distress with few pharmacological options for treatment. Opioid drugs can be effective, but high doses are needed, leading to unwanted effects. BMS-986122 is a positive allosteric modulator of the mu opioid receptor that potentiates acute opioid antinociception without increasing opioid-induced constipation, reward, or respiratory depression. Therefore, we asked if BMS-986122 could increase the effects of low-dose opioid analgesics in chronic neuropathic pain. MethodsWe employed the spared nerve injury and tibial neuroma models in rats and assessed the tactile hypersensitivity of the hind paw and site of neuroma, respectively. ResultsAdministration of low doses of (R)-methadone, morphine, or buprenorphine slightly reduced the tactile hypersensitivity of the hind paw the in spared nerve injury model. Pretreatment with BMS-986122 significantly enhanced the reversal of hypersensitivity, reaching the effect of high-dose gabapentin, a standard of care in neuropathic pain. Pretreatment with BMS-986122 similarly increased the anti-allodynic effects of low dose (R)-methadone on neuroma pain. A similar effect of (R)-methadone in the absence of BMS-986122 was only observed at a dose where respiratory distress was seen. ConclusionsThese findings show that allosteric modulators of the mu opioid receptor such as BMS-986122 can enhance opioid activity that could translate to a safe and effective treatment for chronic neuropathic pain.

Crush syndrome (CS) is characterised by ischaemia/reperfusion-induced rhabdomyolysis, leading to systemic inflammation and high mortality. Building on our previous findings that intravenous nitric oxide (NO) donors improve survival in this condition, we investigated the therapeutic efficacy of inhaled NO delivered via a portable, controlled-release device in an experimental rat model of CS. Anaesthetised rats underwent bilateral hindlimb compression using rubber tourniquets for 5 h, followed by reperfusion. Among the various inhalation conditions tested, administration of NO (160 parts per million) for 2 h after reperfusion significantly increased survival rate from 20 to 90%. Improvements in haemodynamic parameters, biochemical markers, and histopathological findings correlated with enhanced survival outcomes. These results suggest that on-site NO inhalation therapy may serve as an effective first-line, emergency intervention for CS, particularly in disaster settings. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/710439v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1de2a5forg.highwire.dtl.DTLVardef@b0048eorg.highwire.dtl.DTLVardef@1fb310borg.highwire.dtl.DTLVardef@50da9a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Previous studies have linked opioid use to altered metabolic profiles, but findings have been inconsistent and mechanisms remain unclear. One potential mechanism involves increased adiposity, leading to chronic low-grade inflammation that elevates metabolic risk. Here, we examined metabolic profiles in individuals with opioid use disorder (OUD) and matched non-OUD controls, focusing on the sequential mediating roles of BMI and inflammation. Data from individuals with OUD (n=281) and non-OUD (n=246) were drawn from a natural history screening protocol from the National Institute on Alcohol Abuse and Alcoholism intramural program. Groups were matched on age, sex, race, ethnicity, socioeconomic status, and education via propensity score matching. Metabolic measures included BMI, blood glucose, hemoglobin A1c (HbA1c), and lipid profiles, with lipid imbalance indexed by the atherogenic index of plasma (AIP). Inflammatory markers included C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Individuals with OUD had significantly higher BMI (F1,481=12.9, p<0.001), HbA1c (F1,481=10.5, p=0.001), lower high-density lipoprotein cholesterol (HDL-C; F1,481= 46.2, p< 0.001), higher low-density lipoprotein cholesterol (LDL-C; F1, 481=11.9, p< 0.001), and higher AIP (F1,481=20.7, p< 0.001) compared to non-OUD. Inflammatory markers were also elevated in individuals with OUD, including CRP (F1,481=9.4, p=0.002) and ESR (F1,481=7.4, p= 0.007), and statistically mediated group differences in AIP and HbA1c, respectively. Our results are consistent with prior evidence of metabolic dysfunctions in individuals with OUD and suggest inflammation as a contributing mechanism. Targeting metabolic health and inflammation may offer new avenues for improving long-term health outcomes in OUD.

Preclinical evidence indicates that cocaine exerts acute and chronic effects on astrocyte functioning, which in turn modulate cocaine-related impacts on neural integrity and brain function. However, human evidence for astrocytic involvement in cocaine users (CU) remains limited. Glial fibrillary acidic protein (GFAP) is a marker of astrocyte activation with promising clinical utility in neurological conditions, yet its relevance in the addiction field is unclear. Hence, we investigated plasma GFAP levels in chronic CU (n=41) and cocaine-naive controls (HC; n=34) at baseline and after a 4-month follow-up. GFAP was assessed alongside plasma neurofilament light chain (NfL) levels, a marker of neuroaxonal injury previously associated with cocaine use in the same sample. Contrary to our hypothesis, we found no group differences in plasma GFAP concentrations between CU and HC. Neither cross-sectional nor longitudinal associations between GFAP levels and objective indices of cocaine use (derived from hair testing) were detected. However, exploratory analyses revealed higher plasma GFAP levels among CU with recent cocaine consumption (within the last 7 days), suggesting transient astrocytic responses following acute exposure. Additionally, GFAP and NfL were positively correlated across participants, supporting their functional association. Overall, these findings suggest that while GFAP might not be chronically elevated in CU, it may exhibit transient increases related to recent cocaine use. Further research is warranted to characterize the temporal dynamics and biological significance of these glial responses.

Current treatments for opioid use disorder (OUD) have major barriers to access. As such, researching new potential therapies for OUD is important to public health. Previous research has implicated glucagon-like peptide-1 (GLP-1) receptor agonists in decreasing the use of addictive substances by animals. In this study, female Wistar rats (N=32) were surgically implanted with jugular catheters and trained to self-administer fentanyl at a fixed-ratio 1 (FR1) schedule of reinforcement for 21 sessions under short- (ShA; 1 hour) or long-access (LgA; 8 hours) conditions. Next, the animals received injections of semaglutide (0.1 mg/kg, s.c.) or saline (0.9% NaCl, s.c.) prior to another FR1 session. The animals underwent a progressive ratio (PR) schedule of reinforcement while receiving saline (i.v.) or fentanyl (0.625-10 {micro}g/kg/inf, i.v.) and semaglutide (0.1 mg/kg, s.c.) or saline (s.c.). Next, the animals underwent a semaglutide (0-0.1 mg/kg, s.c.) dose response procedure at FR1 and a single dose of fentanyl (2.5 {micro}g/kg/inf, i.v.). Following drug discontinuation, spontaneous locomotor activity and withdrawal-like symptoms were measured. Semaglutide dose-dependently decreased fentanyl rewards under ShA and LgA conditions (p<0.05). Under a PR, semaglutide significantly decreased breakpoint (p<0.05), suggesting semaglutide decreases motivation to self-administer fentanyl. Semaglutide-treated ShA animals displayed significantly less withdrawal-like behavior (p<0.05) but not LgA animals. Overall, these findings suggest semaglutide may modulate motivation to seek opioid reward and could be useful in the development of pharmacotherapies to address OUD.

Background and PurposeWhile inflammation has been generally considered to exacerbate symptoms of schizophrenia, some clinical observations suggest that acute inflammation may alleviate positive symptoms. However, animal models often use excessive inflammatory stimuli, and the effects of acute inflammation--comparable to levels observed in patients--remain unknown. Experimental ApproachTo address this, we examined whether acute inflammation induced under relatively mild, clinically relevant conditions suppresses behavioural sensitization in methamphetamine (METH)-sensitized mice, a model of psychostimulant-induced psychosis with relevance to certain aspects of positive symptoms of schizophrenia. We used a repeated METH (1 mg/kg) sensitized model to evaluate the effects of acute inflammation on behavioural sensitization. Acute inflammation was induced via two methods using either lipopolysaccharides (LPS; 1 g/kg) to mimic peripheral immune activation or restraint stress (RS; single 2-h exposure) to model the neuroinflammation induced by psychological stress. LPS doses were adjusted with reference to the magnitude of peripheral cytokine elevation reported in patients, and RS was applied in short single sessions to avoid excessive inflammation. Key ResultsBoth LPS and RS significantly suppressed behavioural sensitization, without inducing other behavioural abnormalities. This suppression was dependent on toll-like receptor-4 activation. LPS-mediated suppression involved cyclooxygenase-2, whereas RS-mediated suppression was linked to the microglia-derived tumour necrosis factor-. LPS did not alter, whereas RS significantly reduced the striatal extracellular dopamine levels. Conclusion and ImplicationsThese findings suggest that acute inflammation suppresses behavioural sensitization through distinct mechanisms depending on the inflammatory trigger, providing a framework for understanding how inflammation may influence psychosis-related processes, with potential relevance to schizophrenia.

Background and Aims: Synthetic opioids cause tens of thousands of deaths each year in North America, and there are indications that synthetic opioids are also becoming increasingly prevalent in the European drug market. This study aimed to examine high-risk substance use in the German drug-using community with a particular focus on the synthetic opioids fentanyl and nitazenes and related awareness, concerns, overdose experiences, and harm-reduction behavior. Design: Cross-sectional, observational online survey. Setting: Open drug-use scenes, addiction clinics, and substitution practices at numerous geographic locations throughout Germany, August to September 2025. Participants: 235 individuals aged 14+ from the drug using community (mean age 43.4 years; 57.9% male), 79.6% recruited by peers in open drug-use scenes. Measurements: The primary outcome was substances used within the past 12 months. In addition, sources, forms, routes of administration, and perceived changes in availability and price of (synthetic) opioids were assessed as well as risk perceptions, fears, harm-reduction behavior, and overdose-related experiences. Findings: 227 respondents reported substance use with an average of 6.2 substances, and 73.1% (95% confidence interval [CI] = 67.0-78.5%) had used at least one opioid in the past year. Synthetic opioids were consumed in many parts of Germany and across all age and gender groups. Among participants who experienced a shortage of their primary opioid in the past year, 25% (95% CI = 15.8-37.2%) reported having used fentanyl instead. 56.5% (95% CI = 36.8-74.3%) of individuals using synthetic opioids reported having experienced an overdose in the past twelve months. Most of the respondents perceived synthetic opioids as posing a high risk, and a substantial proportion expressed fear that they could be mixed into their own substances. However, only 9.9% (95% CI = 6.6-14.7%) use drug checking, although the vast majority stated they would use it if it were available to them. Conclusions: Synthetic opioids, including fentanyl and nitazenes, have entered the German drug scene, with users reporting high rates of overdose and limited access to harm reduction measures. Germany may be in an early phase of a synthetic opioid transition, warranting urgent expansion of surveillance, naloxone distribution, and drug checking services.

NLRP3 inflammasome is a cytosolic multi-protein complex that plays a crucial role in the immune system, responding to various exogenous and endogenous stimuli by triggering protective inflammatory responses. However, aberrant NLRP3 inflammasome activation is implicated in numerous inflammatory diseases. Therefore, the NLRP3 inflammasome is an important pharmacological target for the treatment of multiple diseases. In this context, we screened various US-FDA-approved drugs for NLRP3 inflammasome inhibition. We found that among various drugs, minoxidil hydrochloride (MXL) effectively inhibits NLRP3 inflammasome, evidenced by reduced secretion of IL-1{beta} and IL-18 in J774A.1 cells treated with MXL. The IC50 values of MXL for inhibition of IL-1{beta} and IL-18 were calculated to be 1.2 and 1.06 {micro}M, respectively. MXL was found to prevent ASC oligomerization, thereby inhibiting the NLRP3 inflammasome and leading to CASP1 cleavage. Further investigation revealed that MXL also utilizes AMPK-mediated autophagy to modulate NLRP3 inflammasome activity. Using siAMPK and bafilomycin A1, an end-stage autophagy inhibitor, we elucidated crosstalk between the NLRP3 inflammasome and autophagic pathways, which was modulated by MXL. Furthermore, we demonstrated the efficacy of MXL in two different mouse models of inflammation, involving the NLRP3 inflammasome. MXL at doses of 10 and 20 mg/kg effectively inhibited the activation of NLRP3 inflammasome by monosodium urate in the air pouch model and by ATP in the peritoneal inflammation model, as evidenced by reduced secretion of 1{beta} and IL-18 in the lavage. Our study identifies MXL as a potent NLRP3 inflammasome inhibitor, warranting further investigation as a potential therapeutic agent for inflammatory diseases.

Temozolomide is the gold standard chemotherapeutic agent used in the treatment of glioblastoma multiforme. Yet its pharmacological use has been linked to the emergence of depressive- and/or anxiety-like behaviors, probably through the inhibition of hippocampal neurogenesis. Since prior studies reporting these negative effects were based on prolonged treatment paradigms (i.e., from 2 weeks to up to 6 months), and given the few reports that have included female rodents in their studies, our approach aimed at further characterizing the behavioral effects induced by temozolomide (25 mg/kg, 1 or 2 cycles, 5 days/cycle) in a mixed-sex cohort of adult rats. To do so, rats were scored across time through specific behavioral tests that capture diverse manifestations of affective-like responses (forced-swim, open field, novelty-suppressed feeding and sucrose preference) or cognitive performance (Barnes maze). At the neurochemical level, we ascertained the effects of 2 cycles of temozolomide on hippocampal neurogenesis (neural progenitors with NeuroD) and other potential neuroplasticity targets (i.e., FADD, BDNF). The main results showed that temozolomide induced unexpected antidepressant-like responses in a treatment-duration manner while decreased hippocampal FADD, a neuroplastic marker previously associated with the acute and repeated actions of most antidepressants. These results break the prior dogma linking increased hippocampal neurogenesis with antidepressant-like efficacy, and suggest that other mechanisms of action, such as the one described through the neuroplastic molecule FADD, might be responsible for the antidepressant-like actions of temozolomide, even in the presence of impaired neurogenesis. Our results, in conjunction with the prior data, suggested cycle- and/or length-dependent treatment effects in terms of temozolomides antidepressant- vs. depressant-like profile, while proposing a novel biomarker of its treatment response.

There is growing interest in understanding how hormonal signaling pathways contribute to the pathophysiology of mood disorders, based on the premise that fluctuations in sex hormones influence mood, a relationship particularly evident in conditions such as premenstrual dysphoric disorder, prenatal depression, postpartum depression, and perimenopausal depression. Estrogen receptor alpha (ER) is predominantly localized in the nucleus, but can also be associated with the cell membrane, thus mediating a broad range of genomic and non-genomic effects through distinct intracellular pathways. By employing a combination of computational simulations and in vitro biochemical and cell-based assays, we systematically evaluated the potential binding and functional interactions of antidepressant compounds with ER. Our results provide compelling evidence that antidepressants may not only affect classical monoaminergic targets but also modulate hormone receptor activity, particularly that of ER. These findings are consistent with the hypothesis that ER plays an important role in mood regulation and highlight it as a potential therapeutic target. Moreover, this work raises the possibility that the clinical efficacy of certain antidepressants may, at least in part, derive from their capacity to influence estrogen receptor-mediated signaling. Significance statementClinical observations suggest a link between female sex hormones and mood, highlighted by the higher prevalence of depression in women and increased vulnerability to depression during hormonal fluctuations. Here, we report that structurally diverse conventional and rapid-acting antidepressants directly interact with estrogen receptor alpha (ER). This interaction is associated with rapid intracellular signaling in cellular models. These findings indicate that, alongside their conventional targets, antidepressants may also engage sex steroid receptor components and signaling. This work broadens our basic understanding of antidepressant pharmacology at the cellular level, offering an additional perspective that may inform future research into the biological mechanisms of mood disorders and suggest a framework for developing targeted therapies for hormone-associated depressive disorders.

Crush syndrome (CS) is a serious medical condition characterized by damage to the muscle cells due to pressure and is associated with high mortality, even in patients receiving fluid therapy. We focused on adrenaline (Adr), a standard medication administered by medical teams dispatched during disasters. Adr is readily available for use in disaster scenarios owing to its inclusion in standard emergency kits. The effectiveness of Adr in the treatment of CS remains a subject of ongoing debate. This study aimed to evaluate the impact of Adr on acute complications, such as heart failure, shock, and renal failure, and explore whether its influence on inflammatory pathways is correlated with improved survival in rats with CS. The CS model involved subjecting anesthetized rats to bilateral hindlimb compression using a rubber tourniquet for 5 h. Subsequently, the rats were randomly divided into eight groups. Under continuous monitoring and recording of the arterial blood pressure, blood and tissue samples were collected for biochemical analyses at designated time points before and after reperfusion. The survival rate, vital signs, and blood gas parameters were higher in the CS group than in the sham group. They were improved in the Adr-treated group (0.01 or 0.01 mg/kg), which was not significantly different from that in the CS group, despite the improvement in shock and kidney dysfunction. In conclusion, intramuscular Adr provides immediate hemodynamic stabilization and renal protection during the early stages of CS. However, its use requires careful dose titration; low doses may promote the systemic release of lethal toxins, whereas high doses may worsen metabolic acidosis. These findings highlight the importance of combining Adr with other therapies, such as fluid resuscitation, to manage systemic toxemia inherent in CS.

The use of neuromodulation techniques for the treatment of alcohol use disorder is receiving increasing attention, especially non-invasive approaches, such as repetitive transcranial magnetic stimulation or transcranial direct current stimulation, while the hypothetical use of electroconvulsive therapy remains unexplored. Given our experience inducing electroconvulsive seizures (ECS) for therapeutic purposes in psychopathology rodent models, we evaluated the role of ECS on reducing the increased voluntary ethanol consumption caused by adolescent ethanol exposure in our validated preclinical model. Rats were treated in adolescence with a binge paradigm of ethanol (2 g/kg, i.p.; 3 rounds of 2 days at 48-h intervals; post-natal day, PND 29-30, PND 33-34 and PND 37-38) or saline. Following persistent withdrawal until adulthood, rats were allowed to: voluntarily drink ethanol (20%) by a two-bottle choice test, for 3 days (PND 80-82); treated with ECS (95 mA for 0.6 s, 100 Hz, pulse width 0.6 ms; ear-clip electrodes) or SHAM for 5 days (PND 86-90); re-exposed to voluntarily ethanol exposure (PND 94-96). Brains were collected on PND 97 to evaluate hippocampal markers of ethanol toxicity and/or treatment response (e.g., NeuroD, NF-L, BDNF and NF-L/BDNF ratio). Our results reproduced the increased voluntary ethanol consumption in adult rats induced by adolescent ethanol exposure and demonstrated that ECS could improve this abuse-prone response. Moreover, we suggested a possible role for BDNF in the beneficial effects induced by ECS, especially reducing the neurotoxic ratio NF-L/BDNF. Overall, we provide preclinical evidence for the potential use of ECS as an efficacious treatment for alcohol use disorder.

Opioid use disorder (OUD) is a chronic, relapsing disease driven by the reinforcing properties of opioids and perpetuated by avoidance of the negative affective states associated with the absence of the drug. Most available OUD treatments directly engage the {micro}-opioid receptor and may induce side effects that can compromise their therapeutic efficacy, thus underscoring the need for novel therapeutic alternatives. Calcitonin gene-related peptide (CGRP) is produced by a small population of neurons in the parabrachial nucleus (PBN) that has been shown to modulate itch, pain, as well as appetitive behaviors. Using a cell-specific nuclear labeling approach coupled with RNA-sequencing, we generated a baseline transcriptome of CGRPPBN neurons and confirmed expression of multiple genes associated with behavioral responses to appetitive stimuli, as well as enrichment of the {micro}-opioid receptor, suggesting that CGRPPBN neuron function may be sensitive to the presence of opioids. Indeed, cFos immunostaining showed that CGRPPBN neuron activity increases during early morphine abstinence and reduces gradually over 48 hours. Given the inhibitory effects of opioids on CGRPPBN neuron activity, we next tested whether these neurons could regulate opioid reinforcement. Using a mouse model of morphine intravenous self-administration, we found that chemogenetic inhibition of CGRPPBN neurons significantly reduced the number of morphine rewards earned in both single-dose and dose-response tests but did not affect context-induced morphine seeking after 21 days of abstinence. These results suggest that CGRPPBN neurons are sensitive to opioid administration and can regulate appetitive behaviors such as morphine-taking. Considering that CGRP signaling is regulated by opioid administration, molecular targets that regulate CGRP neurotransmission without direct -opioid receptor engagement may therefore serve as novel therapeutic avenues for the treatment of OUD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/712659v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1fb9c9borg.highwire.dtl.DTLVardef@1e6ba79org.highwire.dtl.DTLVardef@dc60f5org.highwire.dtl.DTLVardef@61adaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and purposePIEZO1 mechanosensitive cation channels translate mechanical cues into intracellular Ca2+ and Na+ elevations, enabling cells to respond to physical alterations in their environment. PIEZO1 contributes to red blood cells (RBC) volume homeostasis and gain-of-function PIEZO1 mutations cause hereditary xerocytosis (HX), a rare mostly compensated hemolytic anemia, and aberrant channel activation exacerbates sickling and vascular dysfunction in sickle cell disease. Despite strong genetic and physiological evidence supporting PIEZO1 as a therapeutic target, potent and selective inhibitors are limited, and existing compounds show modest specificity or poorly explored mechanisms. Improved pharmacological tools are needed. Experimental approachWe conducted a high-throughput screen of FDA-approved drugs to identify PIEZO1 inhibitors. Compounds were tested at concentrations of 10 {micro}M in a monocytic cell line, using intracellular Ca2+ elevations evoked by the PIEZO1 agonist Yoda1 as read-out. The inhibitory activity of the best hit was validated and compared to existing PIEZO1 inhibitors using electrophysiological analysis, orthogonal PIEZO1-dependent assays across cell lines and human RBCs. As functional proof, we investigated the impact of three PIEZO1 inhibitors on RBC deformability by ektacytometry, after Yoda1 pre-stimulation. Key resultsThis screen identified Otenabant, a selective Cannabinoid Receptor Type 1 (CB1) antagonist, as a potent PIEZO1 inhibitor. Otenabant dose-dependently inhibited Ca2+ elevations mediated by endogenous or exogenously expressed human PIEZO1, but was ineffective against mouse Piezo1, revealing species-specific channel differences. Otenabant inhibited mechanosensitive currents elicited by shear stress in fibroblasts and by repeated poking in PIEZO1-expressing HEK-293 cells, altering the currents activation and inactivation kinetics, and prevented Yoda1-induced hyperpolarization in RBCs. Otenabant was able to reverse the negative impact of Yoda1 on RBC deformability. Conclusions and implicationsThese findings demonstrate the utility of Yoda-based screening for discovering PIEZO1 antagonists and identify Otenabant as a promising chemical scaffold for developing selective PIEZO1 inhibitors with therapeutic potential.

IntroductionInadequately controlled inflammation is a key driver of adverse cardiac remodelling after acute myocardial infarction (AMI). Central to this process is activation of the NLRP3 inflammasome-gasdermin D (GSDMD) pathway, which promotes pyroptosis and the release of the pro-inflammatory cytokine interleukin-1{beta} (IL-1{beta}), a mediator strongly associated with infarct severity and poor clinical outcomes. This study investigates whether repurposing the FDA-approved therapeutic Disulfiram, recently shown to inhibit GSDMD pore formation, could reduce inflammation and thus improve cardiac injury after AMI. Methods and ResultsCardiac ischemia-reperfusion (I/R) injury was induced in C57BL/6 mice by 60-minute ligation of the left coronary artery followed by reperfusion. Disulfiram (25 or 50 mg/kg) was administered at reperfusion and daily thereafter. Cardiac function was assessed by echocardiography, while fibrosis and inflammation were evaluated by histology, RT-PCR, immunohistochemistry and immunoblotting. Leukocyte populations in blood, spleen, bone marrow and heart were analysed by flow cytometry. In vitro, mouse bone marrow-derived macrophages (BMDMs) and PMA-differentiated THP-1 cells were treated with Disulfiram. Cytokine secretion, inflammatory gene expression and changes in cell viability (propidium iodide (PI) staining and lactate dehydrogenase (LDH) release) were measured. Disulfiram (50 mg/kg) significantly improved cardiac function 7 days post-I/R. This was accompanied by a significant reduction in cardiac fibrosis and inflammation, as reflected by a lower abundance of inflammatory cells in circulation and cardiac tissue. In LPS- and ATP/Nigericin-stimulated BMDMs and THP-1 cells, Disulfiram dose-dependently (0.1-50 {micro}M) reduced IL-1{beta} and IL-6 secretion and attenuated membrane permeability and cell lysis. ConclusionsThis study demonstrates that Disulfiram improves cardiac function post-AMI by ameliorating inflammation and fibrosis, which was associated with reductions in cytokine release from inflammatory cells in vitro. Therefore, targeting GSDMD by "repurposing" the FDA-approved drug, Disulfiram, may represent a novel way to provide cardio-protection post-AMI. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/710794v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@7fd82eorg.highwire.dtl.DTLVardef@149de52org.highwire.dtl.DTLVardef@a2fe0eorg.highwire.dtl.DTLVardef@d56b4f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Acute kidney injury (AKI) is a serious and common clinical syndrome that currently has no effective treatment. Emerging evidence links coagulation pathways to kidney injury, particularly through coagulation proteases. Protease-activated receptors (PARs) are a family of G-protein coupled receptors (GPCRs) that are activated by proteolytic cleavage of their N termini, exposing a tethered ligand that initiates receptor signaling. PARs have been shown to play a major role in inflammation, vascular regulation, and tissue injury. PARs play key roles in inflammation, vascular regulation, and tissue injury. Previous work from the Hamm laboratory demonstrated that PAR4 contributes to AKI progression, as PAR4 knockout mice were protected in both unilateral ureteral obstruction and ischemia-reperfusion-based models of kidney disease. In this study, we investigated the potential of a PAR4 antagonist, VU6073819, at mitigating AKI progression in an ischemia-reperfusion injury (IRI) mouse model. PAR4 antagonism not only alleviated kidney injury and inflammatory response, but it significantly improved the survival. These findings identify PAR4 as a promising therapeutic target for AKI.