-
Cell Biology and Toxicology May 2024Purinergic receptor P2Y11, a G protein-coupled receptor that is stimulated by extracellular ATP, has been demonstrated to be related to the chemotaxis of granulocytes,...
Purinergic receptor P2Y11, a G protein-coupled receptor that is stimulated by extracellular ATP, has been demonstrated to be related to the chemotaxis of granulocytes, apoptosis of neutrophils, and secretion of cytokines in vitro. P2Y11 mutations were associated with narcolepsy. However, little is known about the roles of P2RY11 in the occurrence of narcolepsy and inflammatory response in vivo. In this study, we generated a zebrafish P2Y11 mutant using CRISPR/Cas9 genome editing and demonstrated that the P2Y11 mutant replicated the narcolepsy-like features including reduced HCRT expression and excessive daytime sleepiness, suggesting that P2Y11 is essential for HCRT expression. Furthermore, we accessed the cytokine expression in the mutant and revealed that the P2RY11 mutation disrupted the systemic inflammatory balance by reducing il4, il10 and tgfb, and increasing il6, tnfa, and il1b. In addition, the P2RY11-deficient larvae with caudal fin injuries exhibited significantly slower migration and less recruitment of neutrophils and macrophages at damaged site, and lower expression of anti-inflammatory cytokines during tissue damage. All these findings highlight the vital roles of P2RY11 in maintaining HCRT production and secreting anti-inflammatory cytokines in the native environment, and suggested that P2RY11-deficient zebrafish can serve as a reliable and unique model to further explore narcolepsy and inflammatory-related diseases with impaired neutrophil and macrophage responses.
Topics: Animals; Zebrafish; Neutrophils; Macrophages; Inflammation; Zebrafish Proteins; Cytokines; Mutation; Receptors, Purinergic P2
PubMed: 38771396
DOI: 10.1007/s10565-024-09882-5 -
BioRxiv : the Preprint Server For... May 2024Rotavirus causes life-threatening diarrhea in children, resulting in ∼200,000 deaths/year. The current treatment during infection is Oral Rehydration Solution which...
Rotavirus causes life-threatening diarrhea in children, resulting in ∼200,000 deaths/year. The current treatment during infection is Oral Rehydration Solution which successfully replenishes fluids but does not alleviate diarrhea volume or severity. As a result, there is an urgent need to better understand rotavirus pathophysiology and develop more effective pediatric therapeutics. Rotavirus primarily infects the tips of small intestinal villi, yet has far-reaching effects on cell types distant from infected cells. We recently identified that rotavirus infected cells release the purinergic signaling molecule ADP, which activates P2Y1 receptors on nearby uninfected cells . To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection , we used the mouse-like rotavirus strain D6/2 which generates a severe infection in mice. C57BL/6J mouse pups were given an oral gavage of D6/2 rotavirus and assessed over the course of 5-7 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4 mg/kg MRS2500, a selective P2Y1 antagonist. Mice were monitored for diarrhea severity, diarrhea incidence, and viral shedding. Neonatal mice were euthanized at days 3 and 5 post-infection and small intestine was collected to observe infection. MRS2500 treatment decreased the severity, prevalence, and incidence of rotavirus diarrhea. Viral stool shedding, assessed by qPCR for rotavirus gene levels, revealed that MRS2500 treated pups had significantly lower viral shedding starting at day 4 post infection compared to saline treated pups, which suggests P2Y1 signaling may enhance rotavirus replication. Finally, we found that inhibition of P2Y1 with MRS2500 limited transmitted rotavirus diarrhea to uninfected pups within a litter. Together, these results suggest that P2Y1 signaling is involved in the pathogenesis of a homologous murine rotavirus strain, making P2Y1 receptors a promising anti-diarrheal, anti-viral therapeutic target to reduce rotavirus disease burden.
PubMed: 38765995
DOI: 10.1101/2024.05.07.592953 -
JBMR Plus Jun 2024Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in...
Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in patients with CKD. Extracellular nucleotides (ie, ATP or UTP) regulate the arterial calcification process by interacting with (1) purinergic receptors and (2) breakdown via ecto-nucleotidases, such as ectonucleotide pyrophosphatase/phosphodiesterase NPP1 or NPP3, affecting the local levels of calcification inhibitor, pyrophosphate, and stimulator inorganic phosphate (PP/P ratio). Also, it has been shown that ATP analogs (ie, β,γ-methylene-ATP [β,γ-meATP]) inhibit vascular smooth muscle cell calcification in vitro. In the first experiment, daily dosing of β,γ-meATP (2 mg/kg) was investigated in rats fed a warfarin diet to trigger the development of non-CKD-related arterial medial calcifications. This study showed that β,γ-meATP significantly lowered the calcium scores in the aorta and peripheral vessels in warfarin-exposed rats. In a second experiment, daily dosing of 4 mg/kg β,γ-meATP and its metabolite medronic acid (MDP) was analyzed in rats fed an adenine diet to promote the development of CKD-related arterial medial calcification. Administration of β,γ-meATP and MDP did not significantly decrease aortic calcification scores in this model. Moreover, both compounds induced deleterious effects on physiological bone mineralization, causing an imminent risk for worsening the already compromised bone status in CKD. Due to this, it was not possible to raise the dosage of both compounds to tackle CKD-related arterial calcification. Again, this points out the difficult task of targeting solely ectopic calcifications without negatively affecting physiological bone mineralization. On the other hand, aortic mRNA expression of and was significantly and positively associated with aortic calcification scores, suggesting that normalizing the aortic NPP1/3 activity to control values might be a possible target to treat (CKD-induced) arterial media calcifications.
PubMed: 38764790
DOI: 10.1093/jbmrpl/ziae057 -
Channels (Austin, Tex.) Dec 2024P2X7 is a member of the Ionotropic Purinergic Receptor (P2X) family. The P2X family of receptors is composed of seven (P2X1-7), ligand-gated, nonselective cation... (Review)
Review
P2X7 is a member of the Ionotropic Purinergic Receptor (P2X) family. The P2X family of receptors is composed of seven (P2X1-7), ligand-gated, nonselective cation channels. Changes in P2X expression have been reported in multiple disease models. P2Xs have large complex extracellular domains that function as receptors for a variety of ligands, including endogenous and synthetic agonists and antagonists. ATP is the canonical agonist. ATP affinity ranges from nanomolar to micromolar for most P2XRs, but P2X7 has uniquely poor ATP affinity. In many physiological settings, it may be difficult to achieve the millimolar extracellular ATP concentrations needed for P2X7 channel activation; however, channel function is implicated in pain sensation, immune cell function, cardiovascular disease, cancer, and osteoporosis. Multiple high-resolution P2X7 structures have been solved in apo-, ATP-, and antagonist-bound states. P2X7 structural data reveal distinct allosteric and orthosteric antagonist-binding sites. Both allosteric and orthosteric P2X7 antagonists are well documented to inhibit ATP-evoked channel current. However, a growing body of evidence supports P2X7 activation by non-nucleotide agonists, including extracellular histone proteins and human cathelicidin-derived peptides (LL-37). Interestingly, P2X7 non-nucleotide agonism is not inhibited by allosteric antagonists, but is inhibited by orthosteric antagonists. Herein, we review P2X7 function with a focus on the efficacy of available pharmacology on P2X7 channel current activation by non-nucleotide agonists in effort to understand agonist/antagonist efficacy, and consider the impact of these data on the current understanding of P2X7 in physiology and disease given these limitations of P2X7-selective antagonists and incomplete knockout mouse models.
Topics: Animals; Humans; Adenosine Triphosphate; Purinergic P2X Receptor Agonists; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X7
PubMed: 38762911
DOI: 10.1080/19336950.2024.2355150 -
Cell Communication and Signaling : CCS May 2024Extracellular ATP-AMP-adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is...
BACKGROUND
Extracellular ATP-AMP-adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell-derived ATPases in the development and progression of colon cancer.
METHODS
Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8 T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues.
RESULTS
We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8 T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8 T cells.
CONCLUSION
Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP-adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.
Topics: Humans; Exosomes; Adenosine; Adenosine Triphosphate; Colonic Neoplasms; CD8-Positive T-Lymphocytes; Apyrase; Animals; Mice; Cell Line, Tumor; Male; Female; Metabolic Reprogramming; Receptor, Adenosine A2A
PubMed: 38755598
DOI: 10.1186/s12964-024-01654-2 -
CNS Neuroscience & Therapeutics May 2024Depression is a common psychiatric disorder in diabetic patients. Depressive mood associated with obesity/metabolic disorders is related to the inflammatory response...
BACKGROUND
Depression is a common psychiatric disorder in diabetic patients. Depressive mood associated with obesity/metabolic disorders is related to the inflammatory response caused by long-term consumption of high-fat diets, but its molecular mechanism is unclear. In this study, we investigated whether the antidepressant effect of transcutaneous auricular vagus nerve stimulation (taVNS) in high-fat diet rats works through the P2X7R/NLRP3/IL-1β pathway.
METHODS
We first used 16S rRNA gene sequencing analysis and LC-MS metabolomics assays in Zucker diabetic fatty (ZDF) rats with long-term high-fat diet (Purina #5008) induced significant depression-like behaviors. Next, the forced swimming test (FST) and open field test (OFT) were measured to evaluate the antidepressive effect of taVNS. Immunofluorescence and western blotting (WB) were used to measure the microglia state and the expression of P2X7R, NLRP3, and IL-1β in PFC.
RESULTS
Purina#5008 diet induced significant depression-like behaviors in ZDF rats and was closely related to purine and inflammatory metabolites. Consecutive taVNS increased plasma insulin concentration, reduced glycated hemoglobin and glucagon content in ZDF rats, significantly improved the depressive-like phenotype in ZDF rats through reducing the microglia activity, and increased the expression of P2X7R, NLRP3, and IL-1β in the prefrontal cortex (PFC).
CONCLUSION
The P2X7R/NLRP3/IL-1β signaling pathway may play an important role in the antidepressant-like behavior of taVNS, which provides a promising mechanism for taVNS clinical treatment of diabetes combined with depression.
Topics: Animals; Prefrontal Cortex; Diet, High-Fat; NLR Family, Pyrin Domain-Containing 3 Protein; Depression; Male; Rats; Interleukin-1beta; Vagus Nerve Stimulation; Receptors, Purinergic P2X7; Rats, Zucker; Phenotype
PubMed: 38752512
DOI: 10.1111/cns.14755 -
Molecular Pain 2024Recent studies have demonstrated that activated microglia were involved in the pathogenesis of central sensitization characterized by cutaneous allodynia in migraine....
Recent studies have demonstrated that activated microglia were involved in the pathogenesis of central sensitization characterized by cutaneous allodynia in migraine. Activation of microglia is accompanied by increased expression of its receptors and release of inflammatory mediators. Acupuncture and its developed electroacupuncture (EA) have been recommended as an alternative therapy for migraine and are widely used for relieving migraine-associated pain. However, it remains rare studies that show whether EA exerts anti-migraine effects via inhibiting microglial activation related to a release of microglial receptors and the inflammatory pathway. Therefore, this study aimed to investigate EA' ability to ameliorate central sensitization via modulation of microglial activation, microglial receptor, and inflammatory response using a rat model of migraine induced by repeated epidural chemical stimulation. In the present study, a rat model of migraine was established by epidural repeated inflammatory soup (IS) stimulation and treated with EA at Fengchi (GB20) and Yanglingquan (GB34) and acupuncture at sham-acupoints. Pain hypersensitivity was further determined by measuring the mechanical withdrawal threshold using the von-Frey filament. The changes in c-Fos and ionized calcium binding adaptor molecule 1 (Ibal-1) labeled microglia in the trigeminal nucleus caudalis (TNC) were examined by immunflurescence to assess the central sensitization and whether accompanied with microglia activation. In addition, the expression of Ibal-1, microglial purinoceptor P2X4, and its associated inflammatory signaling pathway mediators, including interleukin (IL)-1β, NOD-like receptor protein 3 (NLRP3), and Caspase-1 in the TNC were investigated by western blot and real-time polymerase chain reaction analysis. Allodynia increased of c-Fos, and activated microglia were observed after repeated IS stimulation. EA alleviated the decrease in mechanical withdrawal thresholds, reduced the activation of c-Fos and microglia labeled with Ibal-1, downregulated the level of microglial purinoceptor P2X4, and limited the inflammatory response (NLRP3/Caspase-1/IL-1β signaling pathway) in the TNC of migraine rat model. Our results indicate that the anti-hyperalgesia effects of EA ameliorate central sensitization in IS-induced migraine by regulating microglial activation related to P2X4R and NLRP3/IL-1β inflammatory pathway.
Topics: Animals; Electroacupuncture; Receptors, Purinergic P2X4; Microglia; Hyperalgesia; Migraine Disorders; Rats, Sprague-Dawley; Disease Models, Animal; Male; Inflammation; Central Nervous System Sensitization; Rats; NLR Family, Pyrin Domain-Containing 3 Protein; Proto-Oncogene Proteins c-fos
PubMed: 38744426
DOI: 10.1177/17448069241258113 -
Scientific Reports May 2024Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease that can compromise the functioning of various organs, including the salivary glands (SG). The...
Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease that can compromise the functioning of various organs, including the salivary glands (SG). The purinergic system is one of the most important inflammatory pathways in T2DM condition, and P2X7R and P2X4R are the primary purinergic receptors in SG that regulate inflammatory homeostasis. This study aimed to evaluate P2X7R and P2X4R expression, and morphological changes in the submandibular gland (SMG) in T2DM. Twenty-four 5-week-old mice were randomly assigned to control (CON) and diabetes mellitus (DM) groups (n = 12 each). Body weight, diet, and blood glucose levels were monitored weekly. The histomorphology of the SMG and the expression of the P2X7R, and P2X7R was evaluated by immunohistochemistry (IHC) staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 11 and 13 weeks of age. Our findings indicate a significant increase in food consumption, body weight, and blood glucose levels in the DM group. Although a significant increase in P2X7R and P2X4R expression was observed in the DM groups, the receptor location remained unchanged. We also observed a significant increase in the acinar area in the DM13w group, and a significant decrease in the ductal area in the DM11w and DM13w groups. Targeting purinergic receptors may offer novel therapeutic methods for diabetic complications.
Topics: Animals; Submandibular Gland; Receptors, Purinergic P2X4; Receptors, Purinergic P2X7; Diet, High-Fat; Diabetes Mellitus, Type 2; Mice; Diabetes Mellitus, Experimental; Male; Blood Glucose; Body Weight; Streptozocin; Mice, Inbred C57BL
PubMed: 38740782
DOI: 10.1038/s41598-024-60519-3 -
Purinergic Signalling May 2024Vascular P2Y receptors mediate many effects, but the role of individual subtypes is often unclear. Here we discuss how subtype-selective antagonists and receptor... (Review)
Review
Vascular P2Y receptors mediate many effects, but the role of individual subtypes is often unclear. Here we discuss how subtype-selective antagonists and receptor knockout/knockdown have helped identify these roles in numerous species and vessels. P2Y receptor-mediated vasoconstriction and endothelium-dependent vasodilation have been characterised using the selective antagonists, MRS2179 and MRS2216, whilst AR-C118925XX, a P2Y receptor antagonist, reduced endothelium-dependent relaxation, and signalling evoked by UTP or fluid shear stress. P2Y receptor knockdown reduced endothelial signalling and endothelial P2Y receptor knockout produced hypertensive mice and abolished vasodilation elicited by an increase in flow. UTP-evoked vasoconstriction was also blocked by AR-C118925XX, but the effects of P2Y receptor knockout were complex. No P2Y receptor antagonists are available and P2Y knockout did not affect the vascular actions of UTP and UDP. The P2Y receptor antagonist, MRS2578, identified endothelial P2Y receptors mediating vasodilation, but receptor knockout had complex effects. MRS2578 also inhibited, and P2Y knockout abolished, contractions evoked by UDP. P2Y receptors contribute to the myogenic tone induced by a stepped increase in vascular perfusion pressure and possibly to the development of atherosclerosis. The P2Y receptor antagonists, NF157 and NF340, inhibited ATP-evoked signalling in human endothelial cells. Vasoconstriction mediated by P2Y/P2Y and P2Y receptors was characterised using the antagonists, cangrelor, ticagrelor, AR-C67085 and MRS2211 or PPTN respectively. This has yet to be backed up by receptor knockout experiments. Thus, subtype-selective antagonists and receptor knockout/knockdown have helped identify which P2Y subtypes are functionally expressed in vascular smooth muscle and endothelial cells and the effects that they mediate.
PubMed: 38740733
DOI: 10.1007/s11302-024-10016-z -
International Journal of Molecular... Apr 2024Adenosine A receptor (AR) antagonists are the leading nondopaminergic therapy to manage Parkinson's disease (PD) since they afford both motor benefits and...
Adenosine A receptor (AR) antagonists are the leading nondopaminergic therapy to manage Parkinson's disease (PD) since they afford both motor benefits and neuroprotection. PD begins with a synaptic dysfunction and damage in the striatum evolving to an overt neuronal damage of dopaminergic neurons in the substantia nigra. We tested if AR antagonists are equally effective in controlling these two degenerative processes. We used a slow intracerebroventricular infusion of the toxin MPP in male rats for 15 days, which caused an initial loss of synaptic markers in the striatum within 10 days, followed by a neuronal loss in the substantia nigra within 30 days. Interestingly, the initial loss of striatal nerve terminals involved a loss of both dopaminergic and glutamatergic synaptic markers, while GABAergic markers were preserved. The daily administration of the AR antagonist SCH58261 (0.1 mg/kg, i.p.) in the first 10 days after MPP infusion markedly attenuated both the initial loss of striatal synaptic markers and the subsequent loss of nigra dopaminergic neurons. Strikingly, the administration of SCH58261 (0.1 mg/kg, i.p. for 10 days) starting 20 days after MPP infusion was less efficacious to attenuate the loss of nigra dopaminergic neurons. This prominent AR-mediated control of synaptotoxicity was directly confirmed by showing that the MPTP-induced dysfunction (MTT assay) and damage (lactate dehydrogenase release assay) of striatal synaptosomes were prevented by 50 nM SCH58261. This suggests that AR antagonists may be more effective to counteract the onset rather than the evolution of PD pathology.
Topics: Animals; Adenosine A2 Receptor Antagonists; Rats; Male; Disease Models, Animal; Parkinson Disease; Receptor, Adenosine A2A; Corpus Striatum; Dopaminergic Neurons; Pyrimidines; Triazoles; Substantia Nigra; Neuroprotective Agents; Rats, Sprague-Dawley
PubMed: 38732120
DOI: 10.3390/ijms25094903