ATP Increases Ciliary Beat Frequency and Ciliary Bend Angle through Distinct Purinergic Receptors in Bronchial Ciliary Cells Isolated from Mice.

Airway ciliary cells are components of the mucociliary transport system and play an important role in sweeping out small particles, such as bacteria and viruses, towards the oropharynx by the action of beating cilia. Several lines of evidence have shown that the ciliary beat is under the regulation of the purinergic system; however, the subtype of receptor and the intracellular signaling pathways involved in the activation of ciliary movement remain to be elucidated. In addition, although the activity of ciliary movement comprises two parameters, the ciliary beat frequency (CBF) and ciliary bend angle (CBA), few reports have analyzed CBA. In this study, we examined the effects of ATP and other purinergic ligands on both CBF and CBA and demonstrated that the purinergic signaling requirements for CBF and CBA are different, with CBF mediated by P2Y1 receptor activation and CBA mediated by the P2X4 receptor.

Temporal Dynamics of Purinergic Receptor Expression in the Lungs of Marek's Disease (MD) Virus-Infected Chickens Resistant or Susceptible to MD.

Marek's disease virus (MDV) is an economic concern for the poultry industry due to its poorly understood pathophysiology. Purinergic receptors (PRs) are potential therapeutic targets for viral infections, including herpesviruses, prompting our investigation into their role in MDV pathogenesis. The current study is part of an experimental series analyzing the expression of PRs during MDV infection. To address the early or short-acting P2 PR responses during natural MDV infection, we performed an "exposure" experiment where age-matched chickens were exposed to experimentally infected shedders to initiate natural infection. In addition, select non-PR regulatory gene responses were measured. Two groups of naïve contact chickens (n = 5/breed/time point) from MD-resistant (White Leghorns: WL) and -susceptible (Pure Columbian) chicken lines were housed separately with experimentally infected PC (×PC) and WL (×WL) chickens for 6 or 24 h. Whole lung lavage cells (WLLC) were collected, RNA was extracted, and RT-qPCR assays were used to measure specific PR responses. In addition, other potentially important markers in pathophysiology were measured. Our study revealed that WL chickens exhibited higher P1 PR expression during natural infection. WL chickens also showed higher expression of P1A3 and P2X3 at 6 and 24 h when exposed to PC-infected chickens. P2X5 and P2Y1 showed higher expression at 6 h, while P2Y5 showed higher expression at 6 and 24 h; regardless of the chicken line, PC chickens exhibited higher expression of P2X2, P2Y8, P2Y10, P2Y13, and P2Y14 when exposed to either group of infected chickens. In addition, MDV infection altered the expression of DDX5 in both WL and PC groups exposed to PC-infected birds only. However, irrespective of the source of exposure, BCL2 and ANGPTL4 showed higher expression in both WL and PC. The expression of STAT1A and STAT5A was influenced by time and breed, with major changes observed in STAT5A. CAT and SOD1 expression significantly increased in both WL and PC birds, regardless of the source of infection. GPX1 and GPX2 expression also increased in both WL and PC, although overall lower expression was observed in PC chickens at 24 h compared to 6 h. Our data suggest systemic changes in the host during early infection, indicated by the altered expression of PRs, DDX5, BCL2, ANGPTL4, and other regulatory genes during early MDV infection. The relative expression of these responses in PC and WL chickens suggests they may play a key role in their response to natural MDV infection in the lungs and long-term pathogenesis and survival.

The role of pannexin/purinergic signaling in intervascular communication from capillaries during skeletal muscle contraction in male Golden hamsters.

We sought to determine the physiological relevance of pannexin/purinergic-dependent signaling in mediating conducted vasodilation elicited by capillary stimulation through skeletal muscle contraction. Using hamster cremaster muscle and intravital microscopy we stimulated capillaries through local muscle contraction while observing the associated upstream arteriole. Capillaries were stimulated with muscle contraction at low and high contraction (6 and 60CPM) and stimulus frequencies (4 and 40 Hz) in the absence and presence of pannexin blocker mefloquine (MEF; 10-5 M), purinergic receptor antagonist suramin (SUR 10-5 M) and gap-junction uncoupler halothane (HALO, 0.07%) applied between the capillary stimulation site and the upstream arteriolar observation site. Conducted vasodilations elicited at 6CPM were inhibited by HALO while vasodilations at 60CPM were inhibited by MEF and SUR. The conducted response elicited at 4 Hz was inhibited by MEF while the vasodilation at 40 Hz was unaffected by any blocker. Therefore, upstream vasodilations resulting from capillary stimulation via muscle contraction are dependent upon a pannexin/purinergic-dependent pathway that appears to be stimulation parameter-dependent. Our data highlight a physiological importance of the pannexin/purinergic pathway in facilitating communication between capillaries and upstream arteriolar microvasculature and, consequently, indicating that this pathway may play a crucial role in regulating blood flow in response to skeletal muscle contraction.

Drugs Targeting Cough Receptors: New Therapeutic Options in Refractory or Unexplained Chronic Cough.

Refractory chronic cough is a disabling disease with very limited therapeutic options. A better understanding of cough pathophysiology has led to the development of emerging drugs targeting cough receptors. Recent strides have illuminated novel therapeutic avenues, notably centred on modulating transient receptor potential (TRP) channels, purinergic receptors, and neurokinin receptors. By modulating these receptors, the goal is to intervene in the sensory pathways that trigger cough reflexes, thereby providing relief without compromising vital protective mechanisms. These innovative pharmacotherapies hold promise for improvement of refractory chronic cough by offering improved efficacy and potentially mitigating adverse effects associated with current recommended treatments. A deeper comprehension of their precise mechanisms of action and clinical viability is imperative for optimising therapeutic interventions and elevating patient care standards in respiratory health. This review delineates the evolving landscape of drug development in this domain, emphasising the significance of these advancements in reshaping the paradigm of cough management.

Influence of extracellular ATP on mammalian sperm physiology.

In addition to its central role in cellular metabolism, adenosine 5'-triphosphate (ATP) is an important extracellular signalling molecule involved in various physiological processes. In reproduction, extracellular ATP participates in both autocrine and paracrine paths regulating gametogenesis, gamete maturation and fertilisation. This review focusses on how extracellular ATP modulates sperm physiology with emphasis on the mammalian acrosome reaction. The presence of extracellular ATP in the reproductive tract is primarily determined by the ion channels and transporters that influence its movement within the cells comprising the tract. The main targets of extracellular ATP in spermatozoa are its own transporters, particularly species-specific sperm purinergic receptors. We also discuss notable phenotypes from knock-out mouse models and human Mendelian inheritance related to ATP release mechanisms, along with immunological, proteomic, and functional observations regarding sperm purinergic receptors and their involvement in sperm signalling.

The impacts of obesity in rheumatoid arthritis and insights into therapeutic purinergic modulation.

Rheumatoid Arthritis (RA) is an autoimmune condition responsible for the impairment of synovia and joints, endangering the functionality of individuals and contributing to mortality. Currently, obesity is increasing worldwide, and recent studies have suggested an association between such condition and RA. In this sense, obese individuals present a lower capacity for achieving remission and present more intense symptoms of the disease, demonstrating a link between both disorders. Different studies aim to understand the possible connection between the conditions; however, few is known in this sense. Therefore, knowing that obesity can alter the activity of multiple body systems, this work's objective is to evaluate the main modifications caused by obesity, which can be linked to the pathophysiology of RA, highlighting as relevant topics obesity's negative impact triggering systemic inflammation, intestinal dysbiosis, endocrine disbalances. Furthermore, the relationship between oxidative stress and obesity also deserves to be highlighted, considering the influence of reactive oxygen species (ROS) accumulation in RA exacerbation. Additionally, many of those characteristics influenced by obesity, along with the classic peculiarities of RA pathophysiology, can also be associated with purinergic signaling. Hence, this work suggests possible connections between the purinergic system and RA, proposing potential therapeutic targets against RA to be studied.

Murine and Human-Purified very Small Embryonic-like Stem Cells (VSELs) Express Purinergic Receptors and Migrate to Extracellular ATP Gradient.

Purinergic signaling is an ancient primordial signaling system regulating tissue development and specification of various types of stem cells. Thus, functional purinergic receptors are present in several types of cells in the body, including multiple populations of stem cells. However, one stem cell type that has not been evaluated for expression of purinergic receptors is very small embryonic stem cells (VSELs) isolated from postnatal tissues. Herein, we report that human umbilical cord blood (UCB) and murine bone marrow (BM) purified VSELs express mRNA for P1 and P2 purinergic receptors and CD39 and CD73 ectonucleotidases converting extracellular ATP (eATP) into its signaling metabolite extracellular adenosine (eAdo), that antagonizes eATP effects. More importantly, we demonstrate that human and murine VSELs respond by chemotaxis to eATP, and eAdo inhibits this migration. These responses to eATP are mediated by activation of Nlrp3 inflammasome, and exposure of VSELs to its specific inhibitor MCC950 abolished the chemotactic response to ATP. We conclude that purinergic signaling plays an essential, underappreciated role in the biology of these cells and their potential role in response to tissue/organ injuries.

Crosstalk between purinergic receptor P2Y11 and chemokine receptor CXCR7 is regulated by CXCR4 in human macrophages.

P2Y11 is a G protein-coupled ATP receptor that activates IL-1 receptor (IL-1R) in a cyclic AMP dependent manner. In human macrophages, P2Y11/IL-1R crosstalk with CCL20 as a prime target is controlled by phosphodiesterase 4 (PDE4), which mediates breakdown of cyclic AMP. Here, we used gene expression analysis to identify activation of CXCR4 and CXCR7 as a hallmark of P2Y11 signaling. We found that PDE4 inhibition with rolipram boosts P2Y11/IL-1R-induced upregulation of CXCR7 expression and CCL20 production in an epidermal growth factor receptor dependent manner. Using an astrocytoma cell line, naturally expressing CXCR7 but lacking CXCR4, P2Y11/IL-1R activation effectively induced and CXCR7 agonist TC14012 enhanced CCL20 production even in the absence of PDE4 inhibition. Moreover, CXCR7 depletion by RNA interference suppressed CCL20 production. In macrophages, the simultaneous activation of P2Y11 and CXCR7 by their respective agonists was sufficient to induce CCL20 production with no need of PDE4 inhibition, as CXCR7 activation increased its own and eliminated CXCR4 expression. Finally, analysis of multiple CCL chemokines in the macrophage secretome revealed that CXCR4 inactivation and CXCR7 activation selectively enhanced P2Y11/IL-1R-mediated secretion of CCL20. Altogether, our data establish CXCR7 as an integral component of the P2Y11/IL-1R-initiated signaling cascade and CXCR4-associated PDE4 as a regulatory checkpoint.

Recent research advances in pain mechanisms in McCune-Albright syndrome thinking about the pain mechanism of FD/MAS.

BACKGROUND: The lack of effective understanding of the pain mechanism of McCune-Albright syndrome (MAS) has made the treatment of pain in this disease a difficult clinical challenge, and new therapeutic targets are urgently needed to address this dilemma. OBJECTIVE: This paper summarizes the novel mechanisms, targets, and treatments that may produce pain in MAS and fibrous dysplasia (polyfibrous dysplasia, or FD). METHODS: We conducted a systematic search in the PubMed database, Web of Science, China Knowledge Network (CNKI) with the following keywords: "McCune-Albright syndrome (MAS); polyfibrous dysplasia (FD); bone pain; bone remodeling; G protein coupled receptors; GDNF family receptors; purinergic receptors and glycogen synthase kinase", as well as other keywords were systematically searched. Papers published between January 2018 and May 2023 were selected for finding. Initial screening was performed by reading the titles and abstracts, and available literature was screened against the inclusion and exclusion criteria. RESULTS: In this review, we systematically analyzed the cutting-edge advances in this disease, synthesized the findings, and discussed the differences. With regard to the complete mechanistic understanding of the pain condition in FD/MAS, in particular, we collated new findings on new pathways, neurotrophic factor receptors, purinergic receptors, interferon-stimulating factors, potassium channels, protein kinases, and corresponding hormonal modulation and their respective strengths and weaknesses. CONCLUSION: This paper focuses on basic research to explore FD/MAS pain mechanisms. New nonneuronal and molecular mechanisms, mechanically loaded responsive neurons, and new targets for potential clinical interventions are future research directions, and a large number of animal experiments, tissue engineering techniques, and clinical trials are still needed to verify the effectiveness of the targets in the future.

Transcriptome analysis of cultured human vascular endothelial cells after γ-ray irradiation and correlation analysis with ATP, ADP, and adenosine as secondary soluble factors.

Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing radiation is a common physical stimulant that induces a bystander effect whereby irradiated cells influence neighboring cells through signalings, including purinergic receptor signaling, activated by adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine as secondary soluble factors. Human vascular endothelial EA.hy926 cells were cultured and irradiated with γ-rays or treated with ATP, ADP, or adenosine under non-toxic conditions. RNA-seq, gene ontology, and hierarchical clustering analyses were performed. The transcriptome analysis of differentially expressed genes in vascular endothelial cells after γ-ray irradiations suggests that the change of gene expression by γ-irradiation is mediated by ATP and ADP. In addition, the expression and activity of the proteins related to blood coagulation and fibrinolysis systems appear to be secondarily regulated by ATP and ADP in vascular endothelial cells after exposure to γ-irradiation. Although it is unclear whether the changes of the gene expression related to blood coagulation and fibrinolysis systems by γ-irradiation affected the increased hemorrhagic tendency through the exposure to γ-irradiation or the negative feedback to the activated blood coagulation system, the present data indicate that toxicity associated with γ-irradiation involves the dysfunction of vascular endothelial cells related to the blood coagulation-fibrinolytic system, which is mediated by the signalings, including purinergic receptor signaling, activated by ATP and ADP.

The purinergic receptor P2X3 promotes facial pain by activating neurons and cytokines in the trigeminal ganglion.

The mechanism underlying allodynia/hyperalgesia caused by dental pulpitis has remained enigmatic. This investigation endeavored to characterize the influence of the purinergic receptor P2X3 on pain caused by experimental pulpitis and the mechanism involved. An experimental model of irreversible pulpitis was produced by the drilling and exposure of the dental pulp of the left upper first and second molars in rats, followed by measuring nociceptive responses in the oral and maxillofacial regions. Subsequently, neuronal activity and the expression of P2X3 and pertinent cytokines in the trigeminal ganglion (TG) were meticulously examined and analyzed. Histological evidence corroborated that significant pulpitis was produced in this model, which led to a distinct escalation in nociceptive responses in rats. The activation of neurons, coupled with the upregulated expression of c-fos, P2X3, p-p38, TNF-α and IL-1ß, was identified subsequent to the pulpitis surgery within the TG. The selective inhibition of P2X3 with A-317491 effectively restrained the abnormal allodynia/hyperalgesia following the pulpitis surgery and concurrently inhibited the upregulation of p-p38, TNF-α and IL-1ß within the TG. These findings suggest that the P2X3 signaling pathway plays a pivotal role in instigating and perpetuating pain subsequent to the induction of pulpitis in rats, implicating its association with the p38 MAPK signaling pathway and inflammatory factors.

Mathematical Modeling of PI3K/Akt Pathway in Microglia.

The motility of microglia involves intracellular signaling pathways that are predominantly controlled by changes in cytosolic Ca2+ and activation of PI3K/Akt (phosphoinositide-3-kinase/protein kinase B). In this letter, we develop a novel biophysical model for cytosolic Ca2+ activation of the PI3K/Akt pathway in microglia where Ca2+ influx is mediated by both P2Y purinergic receptors (P2YR) and P2X purinergic receptors (P2XR). The model parameters are estimated by employing optimization techniques to fit the model to phosphorylated Akt (pAkt) experimental modeling/in vitro data. The integrated model supports the hypothesis that Ca2+ influx via P2YR and P2XR can explain the experimentally reported biphasic transient responses in measuring pAkt levels. Our predictions reveal new quantitative insights into P2Rs on how they regulate Ca2+ and Akt in terms of physiological interactions and transient responses. It is shown that the upregulation of P2X receptors through a repetitive application of agonist results in a continual increase in the baseline [Ca2+], which causes the biphasic response to become a monophasic response which prolongs elevated levels of pAkt.

Renal ischemia and reperfusion impact the purinergic signaling in a vascular bed distant from the injured site.

AIMS: Acute kidney injury (AKI) is a public health problem and represents a risk factor for cardiovascular diseases (CVD) and vascular damage. This study aimed to investigate the impact of AKI on purinergic components in mice aorta. MAIN METHODS: The kidney ischemia was achieved by the occlusion of the left kidney pedicle for 60 min, followed by reperfusion for 8 (IR8) and 15 (IR15) days. Renal function was assessed through biochemical assays, while gene expression levels were evaluated by RT-qPCR. KEY FINDINGS: Analyses of renal parameters showed renal remodeling through mass loss in the left kidney and hypertrophy of the right kidney in the IR15 group. Furthermore, after 15 days, local inflammation was evidenced in the aorta. Moreover, the aorta purinergic components were significantly impacted by the renal ischemia and reperfusion model, with increases in gene expression of the pro-inflammatory purinoceptors P2Y1, P2Y2, P2Y6, and P2X4, potentially contributing to the vessel inflammation. The expression of NTPDase2 and ecto-5'-nucleotidase were also significantly increased in the aorta of the same group. In addition, both ATP and AMP hydrolysis were significantly increased in the aorta from IR15 animals, driving the entire purinergic cascade to the production of the anti-inflammatory adenosine. SIGNIFICANCE: In short, this is the first time that inflammation of the aorta due to AKI was shown to have an impact on purinergic signaling components, with emphasis on the adenosinergic pathway. This seems to be closely implicated in the establishment of vascular inflammation in this model of AKI and deserves to be further investigated.

ATP-Induced Contractile Response of Esophageal Smooth Muscle in Mice.

The tunica muscularis of mammalian esophagi is composed of striated muscle and smooth muscle. Contraction of the esophageal striated muscle portion is mainly controlled by cholinergic neurons. On the other hand, smooth muscle contraction and relaxation are controlled not only by cholinergic components but also by non-cholinergic components in the esophagus. Adenosine triphosphate (ATP) is known to regulate smooth muscle contraction and relaxation in the gastrointestinal tract via purinergic receptors. However, the precise mechanism of purinergic regulation in the esophagus is still unclear. Therefore, the aim of the present study was to clarify the effects of ATP on the mechanical responses of the esophageal muscle in mice. An isolated segment of the mouse esophagus was placed in a Magnus's tube and longitudinal mechanical responses were recorded. Exogenous application of ATP induced contractile responses in the esophageal preparations. Tetrodotoxin, a blocker of voltage-dependent sodium channels in neurons and striated muscle, did not affect the ATP-induced contraction. The ATP-evoked contraction was blocked by pretreatment with suramin, a purinergic receptor antagonist. RT-PCR revealed the expression of mRNA of purinergic receptor genes in the mouse esophageal tissue. The findings suggest that purinergic signaling might regulate the motor activity of mouse esophageal smooth muscle.

Lipopolysaccharide-mediated ATP signaling regulates interleukin-6 mRNA expression via the P2-purinoceptor in human dental pulp cells.

Dental pulp cells play a crucial role in maintaining the balance of the pulp tissue. They actively respond to bacterial inflammation by producing proinflammatory cytokines, particularly interleukin-6 (IL-6). While many cell types release adenosine triphosphate (ATP) in response to various stimuli, the mechanisms and significance of ATP release in dental pulp cells under inflammatory conditions are not well understood. This study aimed to investigate ATP release and its relationship with IL-6 during the inflammatory response in immortalized human dental pulp stem cells (hDPSC-K4DT) following lipopolysaccharide (LPS) stimulation. We found that hDPSC-K4DT cells released ATP extracellularly when exposed to LPS concentrations above 10 µg/mL. ATP release was exclusively attenuated by N-ethylmaleimide, whereas other inhibitors, including clodronic acid (a vesicular nucleotide transporter inhibitor), probenecid (a selective pannexin-1 channel inhibitor), meclofenamic acid (a selective connexin 43 inhibitor), suramin (a nonspecific P2 receptor inhibitor), and KN-62 (a specific P2X7 antagonist), did not exhibit any effect. Additionally, LPS increased IL-6 mRNA expression, which was mitigated by the ATPase apyrase enzyme, N-ethylmaleimide, and suramin, but not by KN-62. Moreover, exogenous ATP induced IL-6 mRNA expression, whereas ATPase apyrase, N-ethylmaleimide, and suramin, but not KN-62, diminished ATP-induced IL-6 mRNA expression. Overall, our findings suggest that LPS-induced ATP release stimulates the IL-6 pathway through P2-purinoceptor, indicating that ATP may function as an anti-inflammatory signal, contributing to the maintenance of dental pulp homeostasis.

María Teresa Miras Portugal: a pioneer in the study of purinoceptors in chromaffin cells.

María Teresa Miras Portugal devoted most of her scientific life to the study of purinergic signalling. In an important part of her work, she used a model system: the chromaffin cells of the adrenal medulla. It was in these cells that she identified diadenosine polyphosphates, from which she proceeded to the study of adrenomedullary purinome: nucleotide synthesis and degradation, adenosine transport, nucleotide uptake into chromaffin granules, exocytotic release of nucleotides and autocrine regulation of chromaffin cell function via purinoceptors. This short review will focus on the current state of knowledge of the purinoceptors of adrenal chromaffin cells, a subject to which María Teresa made seminal contributions and which she continued to study until the end of her scientific life.

Effects of Noise Damage on the Purinergic Signal of Cochlear Spiral Ganglion Cells in Guinea Pigs.

To observe the expression changes of P2 protein in cochlear spiral ganglion cells before and after noise injury, and to explore the relationship between the changes of purinergic receptors in spiral ganglion cells and noise-induced hearing loss, so that the signal transduction of purinergic receptors can be used to treat SNHL The target point provides a theoretical basis. The experimental animals were randomly divided into normal and experimental groups. The experimental group was given 120 dB white noise continuous exposure for 10 days and 3 h a day. The auditory brainstem response was measured before and after the noise exposure. After the noise exposure, the two groups of animals were collected. Do immunofluorescence staining, western blot, fluorescence real-time quantitative PCR to observe the expression of P2 protein. The average hearing threshold of the animals in the experimental group increased to 38.75 ± 6.44 dB SPL after 7 days of noise exposure, and the high-frequency hearing loss was lower and severe; the average hearing threshold increased to 54.38 ± 6.80 dB SPL after 10 days of noise exposure, and the hearing loss at 4 k Hz was relatively high. Light; Frozen sections of cochlear spiral ganglion cells and staining of isolated spiral ganglion cells found that P2X2, P2X3, P2X4, P2X7, P2Y2, and P2Y4 proteins were all expressed in cochlear spiral ganglion cells before noise exposure. Among them, P2X3 expression increased and P2X4, the down-regulation of P2Y2 expression was statistically significant (P < 0.05); Western blot and real-time quantitative PCR detection results showed that the expression of P2X3 was significantly increased after noise exposure than before noise exposure (P < 0.05), and P2X4 and P2Y2 were expressed after noise exposure The amount was significantly lower than before noise exposure (P < 0.05). (Figure. 4). After noise exposure, the expression of P2 protein is upregulated or downregulated. By affecting the Ca2+ cycle, the transmission of sound signals to the auditory center is blocked, which provides a theoretical basis for the signal transduction of purinergic receptors to become a target for the treatment of SNHL.