ATP and Pain

Abstract: Many decades have passed since the pain‐producing properties of ATP were demonstrated in both animals and humans. ^{1 , 2} However, the more recent discovery of a family of ion channels for which ATP is a ligand and which are expressed by nociceptive neurons, has led to a resurgence of interest into the physiological and pathophysiological actions of ATP. This article considers the extent to which available evidence supports the notion that ATP receptors might be important novel analgesic targets. The hypothesis that ATP is a pain mediator is considered in terms of: the distribution of ATP receptors (specifically the P2X ion channel family); whether ATP release occurs under appropriate conditions; the evidence that ATP is capable of initiating pain in humans and pain‐related behaviour in animals; and, lastly, the analgesic effects of pharmacological or molecular block of ATP receptors.     

Ethanol upregulates the P2X7 purinergic receptor in human macrophages

Alcohol consumption is considered to be the third leading cause of death in the United States. In addition to its direct toxicity, ethanol has two contrasting effects on the immune system: the nucleotide oligomerization domain‐like receptor pyrin domain‐containing‐3 (NLRP3) inflammasome is inhibited by acute ethanol exposure but activated by chronic ethanol exposure. Purinergic receptors (especially the P2X7 receptor) are able to activate the NLRP3 inflammasome and are involved in many ethanol‐related diseases (such as gout, pulmonary fibrosis, alcoholic steatohepatitis, and certain cancers). We hypothesized that ethanol regulates purinergic receptors and thus modulates the NLRP3 inflammasome's activity. In experiments with monocyte‐derived macrophages, we found that interleukin (IL)‐1β secretion was inhibited after 7 h of exposure (but not 48 h of exposure) to ethanol. The disappearance of ethanol's inhibitory effect on IL‐1β secretion after 48 h was not mediated by the upregulated production of IL‐1β, IL‐1α, IL‐6 or the inflammasome components NLRP3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain, and caspase 1. P2X7R expression was upregulated by ethanol, whereas expression of the P2X4 and P2X1 receptors was not. Taken as a whole, our results suggest that ethanol induces NLRP3 inflammasome activation by upregulating the P2X7 receptor. This observation might have revealed a new mechanism for inflammation in ethanol‐related diseases.     

P2X7R/NLRP3 signaling pathway-mediated pyroptosis and neuroinflammation contributed to cognitive impairment in a mouse model of migraine

Migraine is the second most common form of headache disorder and the second leading cause of disability worldwide. Cognitive symptoms ranked second resulting in migraine-related disability, after pain. P2X7 receptor (P2X7R) was recently shown to be involved in hyperalgesia in migraine. However, the role of P2X7R in migraine-related cognitive impairment is still ill-defined. The aim of this study was to explore the molecular mechanisms underlying migraine-related cognitive impairment and the role of P2X7R in it. Here we used a well-established mouse model of migraine that triggered migraine attacks by application of inflammatory soup (IS) to the dura. Our results showed that repeated dural IS stimulation triggered upregulation of P2X7R, activation of NLRP3 inflammasome, release of proinflammatory cytokines (IL-1β and IL-18) and activation of pyroptotic cell death pathway. Gliosis (microgliosis and astrogliosis), neuronal loss and cognitive impairment also occurred in the IS-induced migraine model. No significant apoptosis or whiter matter damage was observed following IS-induced migraine attacks. These pathological changes occurred mainly in the cerebral cortex and to a less extent in the hippocampus, all of which can be prevented by pretreatment with a specific P2X7R antagonist Brilliant Blue G (BBG). Moreover, BBG can alleviate cognitive impairment following dural IS stimulation. These results identified P2X7R as a key contributor to migraine-related cognitive impairment and may represent a potential therapeutic target for mitigating cognitive impairment in migraine. Supplementary InformationThe online version contains supplementary material available at 10.1186/s10194-022-01442-8.     

Adenosine receptors: promising targets for the development of novel therapeutics and diagnostics for asthma

Interest in the role of adenosine in asthma has escalated considerably since the early observation of its powerful bronchoconstrictor effects in asthmatic but not normal airways. A growing body of evidence has emerged in support of a proinflammatory and immunomodulatory role for the purine nucleoside adenosine in the pathogenic mechanisms of chronic inflammatory disorders of the airways such as asthma. The fact that adenosine enhances mast cell allergen-dependent activation, that elevated levels of adenosine are present in chronically inflamed airways, and that adenosine given by inhalation cause dose-dependent bronchoconstriction in subjects with asthma emphasizes the importance of adenosine in the initiation, persistence and progression of these common inflammatory disorders of the airways. These distinctive features of adenosine have been recently exploited in the clinical and research setting to identify innovative diagnostic applications for asthma. In addition, because adenosine exerts its multiple biological activities by interacting with four adenosine receptor subtypes, selective activation or blockade of these receptors may lead to the development of novel therapies for asthma.     

Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain.

Evidence is accumulating which supports a role for ATP in the initiation of pain by acting on P2X receptors expressed on nociceptive afferent nerve terminals. To investigate whether these receptors play a role in temporomandibular (TMJ) pain, we studied the presence of functional P2X receptors in rat TMJ by examining the nociceptive behavioral response to the application of the selective P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-meATP) into the TMJ region of rat. The involvement of endogenous ATP in the development of TMJ inflammatory hyperalgesia was also determined by evaluating the effect of the general P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on carrageenan-induced TMJ inflammatory hyperalgesia. Application of alpha,beta-meATP into the TMJ region of rats produced significant nociceptive responses that were significantly reduced by the co-application of lidocaine N-ethyl bromide quaternary salt, QX-314, (2%) or of the P2 receptor antagonist PPADS. Co-application of PPADS with carrageenan into the TMJ significantly reduced inflammatory hyperalgesia. The results indicate that functional P2X receptors are present in the TMJ and suggest that endogenous ATP may play a role in TMJ inflammatory pain mechanisms possibly by acting primarily in these receptors.     

P2X4受体对活化的小胶质细胞的调控作用

小胶质细胞是中枢神经系统内的免疫细胞,当脑组织出现损伤时,小胶质细胞被激活,对脑组织起到保护作用。在自身免疫性脑脊髓炎模型大鼠和视神经脊髓炎患者体内,均观察到在脊髓炎性病灶及小胶质细胞内,P2X4受体表达上调。本课题组进行在体和离体实验,用LPS活化小胶质细胞,观察P2X4受体在小胶质细胞炎性反应中的作用。膜片钳检测显示,在LPS激活的小胶质细胞内,P2X4受体活性增加。P2X4受体阻断剂可显著降低小胶质细胞的膜皱缩、TNFα的分泌、细胞形态的改变及LPS导致的小胶质细胞死亡。在体研究显示,LPS髓内注射后会诱发炎性反应,迅速导致小胶质细胞丢失;给予P2X4受体阻断剂可显著减少小胶质细胞的丢失,而P2X4受体激活剂则可显著增加小胶质细胞的丢失。海马齿状回的小胶质细胞特别容易被LPS诱导的炎症反应激活。注射LPS后2 h,位于海马齿状回的小胶质细胞即被激活,大约24 h后死亡,P2X4受体阻断剂可减少LPS诱导的小胶质细胞活化和死亡。上述数据提示,P2X4受体对于小胶质细胞的激活和存活有重要的调控作用。     

Involvement of α2‐adrenoceptors,imidazoline, and endothelin‐A receptors in the effect of agmatine on morphine and oxycodone‐induced hypothermia in mice

Potentiation of opioid analgesia by endothelin‐A (ET_A) receptor antagonist, BMS182874, and imidazoline receptor/α₂‐adrenoceptor agonists such as clonidine and agmatine are well known. It is also known that agmatine blocks morphine hyperthermia in rats. However, the effect of agmatine on morphine or oxycodone hypothermia in mice is unknown. The present study was carried out to study the role of α₂‐adrenoceptors, imidazoline, and ET_A receptors in morphine and oxycodone hypothermia in mice. Body temperature was determined over 6 h in male Swiss Webster mice treated with morphine, oxycodone, agmatine, and combination of agmatine with morphine or oxycodone. Yohimbine, idazoxan, and BMS182874 were used to determine involvement of α₂‐adrenoceptors, imidazoline, and ET_A receptors, respectively. Morphine and oxycodone produced significant hypothermia that was not affected by α₂‐adrenoceptor antagonist yohimbine, imidazoline receptor/α₂ adrenoceptor antagonist idazoxan, or ET_A receptor antagonist, BMS182874. Agmatine did not produce hypothermia; however, it blocked oxycodone but not morphine‐induced hypothermia. Agmatine‐induced blockade of oxycodone hypothermia was inhibited by idazoxan and yohimbine. The blockade by idazoxan was more pronounced compared with yohimbine. Combined administration of BMS182874 and agmatine did not produce changes in body temperature in mice. However, when BMS182874 was administered along with agmatine and oxycodone, it blocked agmatine‐induced reversal of oxycodone hypothermia. This is the first report demonstrating that agmatine does not affect morphine hypothermia in mice, but reverses oxycodone hypothermia. Imidazoline receptors and α₂‐adrenoceptors are involved in agmatine‐induced reversal of oxycodone hypothermia. Our findings also suggest that ET_A receptors may be involved in blockade of oxycodone hypothermia by agmatine.     

Analytical and clinical performance of a new,automated assay panel for the diagnosis of antiphospholipid syndrome

Summary. Background: Anticardiolipin (aCL) and anti‐β₂glycoprotein I (aβ₂GPI) antibodies are part of the criteria for antiphospholipid syndrome (APS). Therefore they are widely measured and about 30 commercial kits are available. Objectives: To investigate the analytical and clinical performances of four fully automated, chemiluminescent assays: HemosIL AcuStar aCL IgG, HemosIL AcuStar aCL IgM, HemosIL AcuStar aβ₂GPI IgG, and HemosIL AcuStar aβ₂GPI IgM. Methods: Cut‐off values were assessed by testing 250 blood donors. Total coefficients of variation (CV) were determined with six plasma pools and two controls ranging from 4.3 to 2694 U mL^?1 depending on the assay. Samples from 218 well‐characterized patients and 103 controls were measured in three laboratories to determine inter‐laboratory variation. The results of the 321 samples were compared with three commercial assays (REAADS, INOVA and VARELISA). Results: Cut‐off values were assigned to 20 arbitrary units for all the tests. Total CV ranged from 4.3 to 11.2%. No interference of hemoglobin, bilirubin, triglycerides, heparins and rheumatoid factor was observed. Inter‐laboratory variability was low and no sample changed status. Overall status agreement between HemosIL assays and the comparator kits ranged from 82 to 96%. Sensitivity, specificity, agreement when predicting APS and the odds ratios when predicting a thrombotic or obstetric event gave comparable results between HemosIL AcuStar and the three other assays. Conclusions: Our study demonstrates that the fully automated HemosIL AcuStar aPL assay panel showed similar performances to the three commercial ELISAs commonly used by various laboratories to detect antiphospholipid antibodies.     

Potential of telmisartan in the treatment of benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is a common health problem in ageing men. This study was carried out to investigate the protective effect of telmisartan on testosterone‐induced BPH in rats. Fifty‐four male Wistar rats (200–250 g) were randomly divided into nine groups (n = 6) and orally treated for 28 consecutive days: group 1 – vehicle normal, olive oil (10 mL/kg); group 2 – BPH model control (10 mL/kg); groups 3–5 – telmisartan (5, 10 or 20 mg/kg, respectively); group 6 – pioglitazone (20 mg/kg); group 7 – celecoxib (20 mg/kg); group 8 – combination of telmisartan (5 mg/kg) and pioglitazone (20 mg/kg); group 9 – combination of telmisartan (5 mg/kg) and celecoxib (20 mg/kg). Animals in groups 2–9 were given testosterone propionate in olive oil (3 mg/kg) subcutaneously 15 min after pretreatments. On day 29, blood was collected for the estimation of serum testosterone and prostate‐specific antigen (PSA). The prostates were excised, weighed and subjected to biochemical and histological studies. Testosterone injection induced significant increase in prostatic index, serum testosterone and PSA suggesting BPH as well as increased prostate oxidative stress which were ameliorated with the pretreatment of rats with telmisartan or co‐administration of celecoxib and pioglitazone. Histological examination showed that testosterone disrupted the morphology of the prostate epithelial cells evidenced in the involution of the epithelial lining of the acini into the lumen indicating BPH which was reversed by telmisartan. Findings from this study showed that telmisartan alone or in combination with pioglitazone prevented the development of testosterone‐induced prostatic hyperplasia.     

Therapeutic Potentials of Ecto‐Nucleoside Triphosphate Diphosphohydrolase,Ecto‐Nucleotide Pyrophosphatase/Phosphodiesterase,Ecto‐5′‐Nucleotidase,and Alkaline Phosphatase Inhibitors

The modulatory role of extracellular nucleotides and adenosine in relevance to purinergic cell signaling mechanisms has long been known and is an object of much research worldwide. These extracellular nucleotides are released by a variety of cell types either innately or as a response to patho‐physiological stress or injury. A variety of surface‐located ecto‐nucleotidases (of four major types; nucleoside triphosphate diphosphohydrolases or NTPDases, nucleotide pyrophosphatase/phosphodiesterases or NPPs, alkaline phosphatases APs or ALPs, and ecto‐5′‐nucleotidase or e5NT) are responsible for meticulously controlling the availability of these important signaling molecules (at their respective receptors) in extracellular environment and are therefore crucial for maintaining the integrity of normal cell functioning. Overexpression of many of these ubiquitous ecto‐enzymes has been implicated in a variety of disorders including cell adhesion, activation, proliferation, apoptosis, and degenerative neurological and immunological responses. Selective inhibition of these ecto‐enzymes is an area that is currently being explored with great interest and hopes remain high that development of selective ecto‐nucleotidase inhibitors will prove to have many beneficial therapeutic implications. The aim of this review is to emphasize and focus on recent developments made in the field of inhibitors of ecto‐nucleotidases and to highlight their structure activity relationships wherever possible. Most recent and significant advances in field of NTPDase, NPP, AP, and e5NT inhibitors is being discussed in detail in anticipation of providing prolific leads and relevant background for research groups interested in synthesis of selective ecto‐nucleotidase inhibitors.     

Transactivation of sphingosine-1-phosphate receptors by FcepsilonRI triggering is required for normal mast cell degranulation and chemotaxis

Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) in RBL-2H3 and bone marrow-derived mast cells activates sphingosine kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P). In turn, S1P activates its receptors S1P1 and S1P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcepsilonRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S1P1 and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcepsilonRI-triggered degranulation. However, S1P2, whose expression is up-regulated by FcepsilonRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S1PRs by FcepsilonRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.     

Nicotine and metabolites determination in human plasma by ultra performance liquid chromatography–tandem mass spectrometry: a simple approach for solving contamination problem and clinical application

A quantitative method using ultra performance liquid chromatography–tandem mass spectrometry is described for simultaneous determination of nicotine and its metabolites (cotinine and trans‐3′‐ hydroxycotinine) in human plasma. Aliquots of 0.25 mL of plasma specimens were used for analysis, and 3 analytes were extracted by liquid–liquid extraction. The main problem was blank plasma contamination with environmental nicotine. Activated charcoal was used to avoid this analytical interference. For optimized chromatographic performance, a basic mobile phase consisting of 0.2% ammonia in water (mobile phase A, pH10.6) and acetonitrile (mobile phase B) was selected. The analytes were separated on a 50 mm × 2.1 mm BEH C18 column, 1.7 μm particle size, and quantified by MS/MS using multiple‐reaction monitoring (MRM) in positive mode. The chromatographic separation was achieved in 3 min followed by 1.2 min of column equilibration. The calibration curves were linear in the concentration range of 10–1000 ng/mL with correlation coefficients exceeding 0.99. Within‐day precisions and between‐day precisions (CV, %) were <15 %. The accuracy expressed as bias was within ±15% for all analytes. The recovery values ranged from 50% to 97%. The ions used for quantification of nicotine, cotinine and 3‐OH‐cotinine were 166.9 > 129.7; 176.9 > 79.7; 192.9 > 79.7 m/z, respectively. The original blank sample preparation solved the problem of contamination in a cost‐effective and efficient way. The validated method has been routinely used for analysis of nicotine and metabolites and determination of hydroxycotinine/cotinine metabolic ratio. This biomarker seems to be interesting at predicting response of nicotine patch replacement therapies.     

Inhaled hypertonic saline for cystic fibrosis: Reviewing the potential evidence for modulation of neutrophil signalling and function

Cystic fibrosis(CF) is a multisystem disorder with significantly shortened life expectancy. The major cause of mortality and morbidity is lung disease with increasing pulmonary exacerbations and decline in lung function predicting significantly poorer outcomes. The pathogenesis of lung disease in CF is characterised in part by decreased airway surface liquid volume and subsequent failure of normal mucociliary clearance. This leads to accumulation of viscous mucus in the CF airway, providing an ideal environment for bacterial pathogens to grow and colonise, propagating airway inflammation in CF. The use of nebulised hypertonic saline(HTS) treatments has been shown to improve mucus clearance in CF and impact positively upon exacerbations, quality of life, and lung function. Several mechanisms of HTS likely improve outcome, resulting in clinically relevant enhancement in disease parameters related to increase in mucociliary clearance. There is increasing evidence to suggest that HTS is also beneficial through its anti-inflammatory properties and its ability to reduce bacterial activity and biofilm formation. This review will first describe the use of HTS in treatment of CF focusing on its efficacy and tolerability. The emphasis will then change to the potential benefits of aerosolized HTS for the attenuation of receptor mediated neutrophil functions, including down-regulation of oxidative burst activity, adhesion molecule expression, and the suppression of neutrophil degranulation of proteolytic enzymes.     

Upregulation of P2X3 receptors by neuronal calcium sensor protein VILIP-1 in dorsal root ganglions contributes to the bone cancer pain in rats

Primary and metastatic cancers that affect bone are frequently associated with severe and intractable pain. The mechanisms underlying the development of bone cancer pain are largely unknown. In this study, we first demonstrated that a functional upregulation of P2X3 receptors in dorsal root ganglion (DRG) neurons is closely associated with the neuronal hyperexcitability and the cancer-induced bone pain in MRMT-1 tumor cell–inoculated rats. Second, we revealed that visinin-like protein 1 (VILIP-1), a member of visinin-like proteins that belong to the family of neuronal calcium sensor proteins is responsible for the observed upregulation of P2X3 receptors in DRG neurons. The interaction between the amino terminus of VLIP-1 and the carboxyl terminus of the P2X3 receptor is critical for the surface expression and functional enhancement of the receptor. Finally, overexpression of VILIP-1 increases the expression of functional P2X3 receptors and enhances the neuronal excitability in naive rat DRG neurons. In contrast, knockdown of VILIP-1 inhibits the development of bone cancer pain via downregulation of P2X3 receptors and repression of DRG excitability in MRMT-1 rats. Taken together, these results suggest that functional upregulation of P2X3 receptors by VILIP-1 in DRG neurons contributes to the development of cancer-induced bone pain in MRMT-1 rats. Hence, P2X3 receptors and VILIP-1 could serve as potential targets for therapeutic interventions in cancer patients for pain management. Pharmacological blockade of P2X3 receptors or knockdown of VILIP-1 in DRGs would be used as innovative strategies for the treatment of bone cancer pain.     

Sumatriptan reduces severity of status epilepticus induced by lithium–pilocarpine through nitrergic transmission and 5‐HT1B/D receptors in rats: A pharmacological‐based evidence

Status epilepticus (SE) is a life‐threatening neurologic disorder that can be as both cause and consequence of neuroinflammation. In addition to previous reports on anti‐inflammatory property of the anti‐migraine medication sumatriptan, we have recently shown its anticonvulsive effects on pentylenetetrazole‐induced seizure in mice. In the present study, we investigated further (i) the effects of sumatriptan in the lithium–pilocarpine SE model in rats, and (ii) the possible involvement of nitric oxide (NO), 5‐hydroxytryptamin 1B/1D (5‐HT_1B/1D) receptor, and inflammatory pathways in such effects of sumatriptan. Status epilepticus was induced by lithium chloride (127 mg/kg, i.p) and pilocarpine (60 mg/kg, i.p.) in Wistar rats. While SE induction increased SE scores and mortality rate, sumatriptan (0.001‐1 mg/kg, i.p.) improved it (P < 0.001). Administration of the selective 5‐HT_1B/1D antagonist GR‐127935 (0.01 mg/kg, i.p.) reversed the anticonvulsive effects of sumatriptan (0.01 mg/kg, i.p.). Although both tumor necrosis factor‐α (TNF‐α) and NO levels were markedly elevated in the rats' brain tissues post‐SE induction, pre‐treatment with sumatriptan significantly reduced both TNF‐α (P < 0.05) and NO (P < 0.001) levels. Combined GR‐127935 and sumatriptan treatment inhibited these anti‐inflammatory effects of sumatriptan, whereas combined non‐specific NOS (L‐NAME) or selective neuronal NOS (7‐nitroindazole) inhibitors and sumatriptan further reduced NO levels. In conclusion, sumatriptan exerted a protective effect against the clinical manifestations and mortality rate of SE in rats which is possibly through targeting 5‐HT_1B/1D receptors, neuroinflammation, and nitrergic transmission.     

β‐Cell subcellular localization of glucose‐stimulated Mn uptake by X‐ray fluorescence microscopy: implications for pancreatic MRI

Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β‐cells and showed that, in the presence of MnCl₂, glucose‐activated pancreatic islets yield significant signal enhancement in T₁‐weigheted MR images. In this study, we exploited for the first time the unique capabilities of X‐ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β‐cells at cellular and subcellular levels. MIN‐6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 × 10^?11 µg/µm², homogenously distributed across the cell. Exposure to 2 m m glucose and 50 µ m MnCl₂ for 20 min resulted in nonglucose‐dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 × 10^?11 µg/µm². When cells were activated by incubation in 16 m m glucose in the presence of 50 µ m MnCl₂, a significant increase in cytoplasmic Mn was measured, reaching 2.57 ± 1.34 × 10^?10 µg/µm². A further rise in intracellular concentration was measured following KCl‐induced depolarization, with concentrations totaling 1.25 ± 0.33 × 10^?9 and 4.02 ± 0.71 × 10^?10 µg/µm² in the cytoplasm and nuclei, respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β‐cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast. Copyright © 2011 John Wiley & Sons, Ltd.