Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions

Extracellular ATP is suspected to contribute to migraine pain but regulatory mechanisms controlling pro-nociceptive purinergic mechanisms in the meninges remain unknown. We studied the peculiarities of metabolic and signaling pathways of ATP and its downstream metabolites in rat meninges and in cultured trigeminal cells exposed to the migraine mediator calcitonin gene-related peptide (CGRP). Under resting conditions, meningeal ATP and ADP remained at low nanomolar levels, whereas extracellular AMP and adenosine concentrations were one-two orders higher. CGRP increased ATP and ADP levels in meninges and trigeminal cultures and reduced adenosine concentration in trigeminal cells. Degradation rates for exogenous nucleotides remained similar in control and CGRP-treated meninges, indicating that CGRP triggers nucleotide release without affecting nucleotide-inactivating pathways. Lead nitrate-based enzyme histochemistry of whole mount meninges revealed the presence of high ATPase, ADPase, and AMPase activities, primarily localized in the medial meningeal artery. ATP and ADP induced large intracellular Ca²⁺ transients both in neurons and in glial cells whereas AMP and adenosine were ineffective. In trigeminal glia, ATP partially operated via P2X7 receptors. ATP, but not other nucleotides, activated nociceptive spikes in meningeal trigeminal nerve fibers providing a rationale for high degradation rate of pro-nociceptive ATP. Pro-nociceptive effect of ATP in meningeal nerves was reproduced by α,β-meATP operating via P2X3 receptors. Collectively, extracellular ATP, which level is controlled by CGRP, can persistently activate trigeminal nerves in meninges which considered as the origin site of migraine headache. These data are consistent with the purinergic hypothesis of migraine pain and suggest new targets against trigeminal pain.     

Transmission of Mechanical Information by Purinergic Signaling

The skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically relevant mechanical forces lead to small repairable membrane injuries in bone-forming osteoblasts, resulting in release of ATP and stimulation of purinergic (P2) calcium responses in neighboring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, their extracellular diffusion and degradation, and purinergic responses in neighboring cells. After validation using experimental data for intracellular free calcium elevations, ATP, and vesicular release after mechanical stimulation of a single osteoblast, the model was scaled to a tissue-level injury to investigate how purinergic signaling communicates information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration, and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed spatial factors, such as the maximal distance from the injury at which purinergic responses were stimulated. The peak ATP concentration reflected the severity of an individual cell injury, allowing to discriminate between minor and severe injuries that released similar amounts of ATP because of differences in injury repair, and determined temporal aspects of the response, such as signal propagation velocity. ADP-mediated signaling became relevant only in larger tissue-level injuries, conveying information about the distance to the injury site and its geometry. Thus, we identified specific features of extracellular ATP and ADP spatiotemporal signals that depend on tissue mechanoresilience and encode the severity, scope, and proximity of the mechanical stimulus.     

Real-time monitoring of circadian clock oscillations in primary cultures of mammalian cells using Tol2 transposon-mediated gene transfer strategy

Background  

The circadian rhythm in mammals is orchestrated by a central pacemaker in the brain, but most peripheral tissues contain their own intrinsic circadian oscillators. The circadian rhythm is a fundamental biological system in mammals involved in the regulation of various physiological functions such as behavior, cardiovascular functions and energy metabolism. Thus, it is important to understand the correlation between circadian oscillator and physiological functions in peripheral tissues. However, it is still difficult to investigate the molecular oscillator in primary culture cells.     

Cystic fibrosis transmembrane conductance regulator facilitates ATP release by stimulating a separate ATP release channel for autocrine control of cell volume regulation

These studies provide evidence that cystic fibrosis transmembrane conductance regulator (CFTR) potentiates and accelerates regulatory volume decrease (RVD) following hypotonic challenge by an autocrine mechanism involving ATP release and signaling. In wild-type CFTR-expressing cells, CFTR augments constitutive ATP release and enhances ATP release stimulated by hypotonic challenge. CFTR itself does not appear to conduct ATP. Instead, ATP is released by a separate channel, whose activity is potentiated by CFTR. Blockade of ATP release by ion channel blocking drugs, gadolinium chloride (Gd(3+)) and 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid (DIDS), attenuated the effects of CFTR on acceleration and potentiation of RVD. These results support a key role for extracellular ATP and autocrine and paracrine purinergic signaling in the regulation of membrane ion permeability and suggest that CFTR potentiates ATP release by stimulating a separate ATP channel to strengthen autocrine control of cell volume regulation.     

The role of pannexin1 in the induction and resolution of inflammation

Extracellular ATP is an important signaling molecule throughout the inflammatory cascade, serving as a danger signal that causes activation of the inflammasome, enhancement of immune cell infiltration, and fine-tuning of several signaling cascades including those important for the resolution of inflammation. Recent studies demonstrated that ATP can be released from cells in a controlled manner through pannexin (Panx) channels. Panx1-mediated ATP release is involved in inflammasome activation and neutrophil/macrophage chemotaxis, activation of T cells, and a role for Panx1 in inducing and propagating inflammation has been demonstrated in various organs, including lung and the central and peripheral nervous system. The recognition and clearance of dying cells and debris from focal points of inflammation is critical in the resolution of inflammation, and Panx1-mediated ATP release from dying cells has been shown to recruit phagocytes. Moreover, extracellular ATP can be broken down by ectonucleotidases into ADP, AMP, and adenosine, which is critical in the resolution of inflammation. Together, Panx1, ATP, purinergic receptors, and ectonucleotidases contribute to important feedback loops during the inflammatory response, and thus represent promising candidates for new therapies.     

Activation of 5'-AMP-activated kinase with diabetes drug metformin induces casein kinase Iepsilon (CKIepsilon)-dependent degradation of clock protein mPer2

Metformin is one of the most commonly used first line drugs for type II diabetes. Metformin lowers serum glucose levels by activating 5'-AMP-activated kinase (AMPK), which maintains energy homeostasis by directly sensing the AMP/ATP ratio. AMPK plays a central role in food intake and energy metabolism through its activities in central nervous system and peripheral tissues. Since food intake and energy metabolism is synchronized to the light-dark (LD) cycle of the environment, we investigated the possibility that AMPK may affect circadian rhythm. We discovered that the circadian period of Rat-1 fibroblasts treated with metformin was shortened by 1 h. One of the regulators of the period length is casein kinase Iepsilon (CKIepsilon), which by phosphorylating and inducing the degradation of the circadian clock component, mPer2, shortens the period length. AMPK phosphorylates Ser-389 of CKIepsilon, resulting in increased CKIepsilon activity and degradation of mPer2. In peripheral tissues, injection of metformin leads to mPer2 degradation and a phase advance in the circadian expression pattern of clock genes in wild-type mice but not in AMPK alpha2 knock-out mice. We conclude that metformin and AMPK have a previously unrecognized role in regulating the circadian rhythm.     

Extracellular ATP as a signaling molecule for epithelial cells

The charge of this invited review is to present a convincing case for the fact that cells release their ATP for physiological reasons. Many of our "purinergic" colleagues as well as ourselves have experienced resistance to this concept, because it is teleologically counter-intuitive. This review serves to integrate the three main tenets of extracellular ATP signaling: ATP release from cells, ATP receptors on cells, and ATP receptor-driven signaling within cells to affect cell or tissue physiology. First principles will be discussed in the Introduction concerning extracellular ATP signaling. All possible cellular mechanisms of ATP release will then be presented. Use of nucleotide and nucleoside scavengers as well as broad-specificity purinergic receptor antagonists will be presented as a method of detecting endogenous ATP release affecting a biological endpoint. Innovative methods of detecting released ATP by adapting luciferase detection reagents or by using "biosensors" will be presented.Because our laboratory has been primarily interested in epithelial cell physiology and pathophysiology for several years, the role of extracellular ATP in regulation of epithelial cell function will be the focus of this review. For ATP release to be physiologically relevant, receptors for ATP are required at the cell surface. The families of P2Y G protein-coupled receptors and ATP-gated P2X receptor channels will be introduced. Particular attention will be paid to P2X receptor channels that mediate the fast actions of extracellular ATP signaling, much like neurotransmitter-gated channels versus metabotropic heptahelical neurotransmitter receptors that couple to G proteins. Finally, fascinating biological paradigms in which extracellular ATP signaling has been implicated will be highlighted. It is the goal of this review to convert and attract new scientists into the exploding field of extracellular nucleotide signaling and to convince the reader that extracellular ATP is indeed a signaling molecule.     

Differentiating connexin hemichannels and pannexin channels in cellular ATP release

Adenosine triphosphate (ATP) plays a fundamental role in cellular communication, with its extracellular accumulation triggering purinergic signaling cascades in a diversity of cell types. While the roles for purinergic signaling in health and disease have been well established, identification and differentiation of the specific mechanisms controlling cellular ATP release is less well understood. Multiple mechanisms have been proposed to regulate ATP release with connexin (Cx) hemichannels and pannexin (Panx) channels receiving major focus. However, segregating the specific roles of Panxs and Cxs in ATP release in a plethora of physiological and pathological contexts has remained enigmatic. This multifaceted problem has arisen from the selectivity of pharmacological inhibitors for Panxs and Cxs, methodological differences in assessing Panx and Cx function and the potential compensation by other isoforms in gene silencing and genetic knockout models. Consequently, there remains a void in the current understanding of specific contributions of Panxs and Cxs in releasing ATP during homeostasis and disease. Differentiating the distinct signaling pathways that regulate these two channels will advance our current knowledge of cellular communication and aid in the development of novel rationally-designed drugs for modulation of Panx and Cx activity, respectively.     

Feed forward cycle of hypotonic stress-induced ATP release, purinergic receptor activation, and growth stimulation of prostate cancer cells

ATP is released in many cell types upon mechanical strain, the physiological function of extracellular ATP is largely unknown, however. Here we report that ATP released upon hypotonic stress stimulated prostate cancer cell proliferation, activated purinergic receptors, increased intracellular [Ca(2+)](i), and initiated downstream signaling cascades that involved MAPKs ERK1/2 and p38 as well as phosphatidylinositol 3-kinase (PI3K). MAPK activation, the calcium response as well as induction of cell proliferation upon hypotonic stress were inhibited by preincubation with the ATP scavenger apyrase, indicating that hypotonic stress-induced signaling pathways are elicited by released ATP. Hypotonic stress increased prostaglandin E(2) (PGE(2)) synthesis. Consequently, ATP release was inhibited by antagonists of PI3K (LY294002 and wortmannin), phospholipase A(2) (methyl arachidonyl fluorophosphonate (MAFP)), cyclooxygenase-2 (COX-2) (indomethacin, etodolac, NS398) and 5,8,11,14-eicosatetraynoic acid (ETYA), which are involved in arachidonic acid metabolism. Furthermore, ATP release was abolished in the presence of the adenylate cyclase (AC) inhibitor MDL-12,330A, indicating regulation of ATP-release by cAMP. The hypotonic stress-induced ATP release was significantly blunted when the ATP-mediated signal transduction cascade was inhibited on different levels, i.e. purinergic receptors were blocked by suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), the Ca(2+) response was inhibited upon chelation of intracellular Ca(2+) by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ERK1,2 as well as p38 were inhibited by UO126 and SB203580, respectively. In summary our data demonstrate that hypotonic stress initiates a feed forward cycle of ATP release and purinergic receptor signaling resulting in proliferation of prostate cancer cells.     

Extracellular ATP exerts opposite effects on activated and regulatory CD4+ T cells via purinergic P2 receptor activation

It has been reported that ATP inhibits or stimulates lymphoid cell proliferation depending on the cellular subset analyzed. In this study, we show that ATP exerts strikingly opposite effects on anti-CD3/CD28-activated and regulatory CD4(+) T cells (T(regs)), based on nucleotide concentration. We demonstrate that physiological concentrations of extracellular ATP (1-50 nM) do not affect activated CD4(+) T cells and T(regs). Conversely, higher ATP concentrations have a bimodal effect on activated CD4(+) T cells. Whereas 250 nM ATP stimulates proliferation, cytokine release, expression of adhesion molecules, and adhesion, 1 mM ATP induces apoptosis and inhibits activated CD4(+) T cell functions. The expression analysis and pharmacological profile of purinergic P2 receptors for extracellular nucleotides suggest that activated CD4(+) T cells are induced to apoptosis via the upregulation and engagement of P2X7R and P2X4R. On the contrary, 1 mM ATP enhances proliferation, adhesion, migration, via P2Y2R activation, and immunosuppressive ability of T(regs). Similar results were obtained when activated CD4(+) T cells and T(regs) were exposed to ATP released by necrotized leukemic cells. Taken together, our results show that different concentrations of extracellular ATP modulate CD4(+) T cells according to their activated/regulatory status. Because extracellular ATP concentration highly increases in fast-growing tumors or hyperinflamed tissues, the manipulation of purinergic signaling might represent a new therapeutic target to shift the balance between activated CD4(+) T cells and T(regs).     

Quantitative analyses of circadian gene expression in mammalian cell cultures

The central circadian pacemaker is located in the hypothalamus of mammals, but essentially the same oscillating system operates in peripheral tissues and even in immortalized cell lines. Using luciferase reporters that allow automated monitoring of circadian gene expression in mammalian fibroblasts, we report the collection and analysis of precise rhythmic data from these cells. We use these methods to analyze signaling pathways of peripheral tissues by studying the responses of Rat-1 fibroblasts to ten different compounds. To quantify these rhythms, which show significant variation and large non-stationarities (damping and baseline drifting), we developed a new fast Fourier transform–nonlinear least squares analysis procedure that specifically optimizes the quantification of amplitude for circadian rhythm data. This enhanced analysis method successfully distinguishes among the ten signaling compounds for their rhythm-inducing properties. We pursued detailed analyses of the responses to two of these compounds that induced the highest amplitude rhythms in fibroblasts, forskolin (an activator of adenylyl cyclase), and dexamethasone (an agonist of glucocorticoid receptors). Our quantitative analyses clearly indicate that the synchronization mechanisms by the cAMP and glucocorticoid pathways are different, implying that actions of different genes stimulated by these pathways lead to distinctive programs of circadian synchronization.     

Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.     

P2 receptor-mediated signaling in mast cell biology

Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of mast cells to the extracellular ATP release and to the maintenance of extracellular nucleotides pool. Recent publications highlight the importance of purinergic signaling for the pathogenesis of chronic airway inflammation. Therefore, the role of ATP and P2 receptors in allergic inflammation with focus on mast cells was analyzed. Finally, ATP functions as mast cell autocrine/paracrine factor and as messenger in intercellular communication between mast cells, nerves, and glia in the central nervous system.     

Effects of adenylates on the circadian interaction of KaiB with the KaiC complex in the reconstituted cyanobacterial Kai protein oscillator

Cyanobacteria are photosynthetic prokaryotes that possess circadian oscillators. Clock proteins, KaiA, KaiB, KaiC compose the central circadian oscillator, which can be reconstituted in vitro in the presence of ATP. KaiC has ATPase, autokinase, and autophosphatase enzymatic activities. These activities are modulated by protein–protein interactions among the Kai proteins. The interaction of KaiB with the KaiC complex shows a circadian rhythm in the reconstituted system. We previously developed a quantitative, real-time monitoring system for the dynamic behavior of the complex using fluorescence correlation spectroscopy. Here, we examined the effects of ATP and ADP on the rhythmic interaction of KaiB. We show that increased concentration of ATP or ADP shortened period length. Adding ADP to the Kai protein oscillation shifted its phase in a phase-dependent manner. These results provide insight into how circadian oscillation entrainment mechanism is linked to cellular metabolism.     

Vesicular exocytosis contributes to volume-sensitive ATP release in biliary cells

Extracellular ATP is a potent autocrine/paracrine signal that regulates a broad range of liver functions through activation of purinergic receptors. In biliary epithelium, increases in cell volume stimulate ATP release through a phosphoinositide 3-kinase (PI3-kinase)-dependent mechanism. Because PI3-kinase also regulates vesicular exocytosis, the purpose of these studies was to determine whether volume-stimulated vesicular exocytosis contributes to cellular ATP release. In a human cholangiocarcinoma cell line, exocytosis was measured by using the plasma membrane marker FM1-43, whereas ATP release was assessed by using a luciferase-luciferin assay. Under basal conditions, cholangiocytes exhibited constitutive exocytosis at a rate of 1.6%/min, and low levels of extracellular ATP were detected at 48.2 arbitrary light units. Increases in cholangiocyte cell volume induced by hypotonic exposure resulted in a 10-fold increase in the rate of exocytosis and a robust 35-fold increase in ATP release. Both vesicular exocytosis and ATP release were proportional to cell volume, and both exhibited similar regulatory properties including: 1) dependence on intact PI3-kinase, 2) attenuation by inhibition of PKC, and 3) potentiation by activation of PKC before hypotonic exposure. These findings demonstrate that increases in cholangiocyte cell volume stimulate ATP release and vesicular exocytosis through similar regulatory paradigms. Functional interactions among cell volume, PKC, and PI3-kinase modulate exocytosis, thereby regulating ATP release and purinergic signaling in cholangiocytes. It is hypothesized that PKC is involved in the recruitment of a volume-sensitive vesicular pool to a readily releasable state.     

P2X7 receptor-pannexin 1 interaction mediates extracellular alpha-synuclein-induced ATP release in neuroblastoma SH-SY5Y cells

Abnormalities of alpha-synuclein (ASN), the main component of protein deposits (Lewy bodies), were observed in Parkinson’s disease (PD), dementia with Lewy bodies, Alzheimer’s disease, and other neurodegenerative disorders. These alterations include increase in the levels of soluble ASN oligomers in the extracellular space. Numerous works have identified several mechanisms of their toxicity, including stimulation of the microglial P2X7 receptor leading to oxidative stress. While the significant role of purinergic signaling—particularly, P2 family receptors—in neurodegenerative disorders is well known, the interaction of extracellular soluble ASN with neuronal purinergic receptors is yet to be studied. Therefore, in this study, we have investigated the effect of ASN on P2 purinergic receptors and ATP-dependent signaling. We used neuroblastoma SH-SY5Y cell line and rat synaptoneurosomes treated with exogenous soluble ASN. The experiments were performed using spectrofluorometric, radiochemical, and immunochemical methods. We found the following: (i) ASN-induced intracellular free calcium mobilization in neuronal cells and nerve endings depends on the activation of purinergic P2X7 receptors; (ii) activation of P2X7 receptors leads to pannexin 1 recruitment to form an active complex responsible for ATP release; and (iii) ASN greatly decreases the activity of extracellular ecto-ATPase responsible for ATP degradation. Thus, it is concluded that purinergic receptors might be putative pharmacological targets in the molecular mechanism of extracellular ASN toxicity. Interference with P2X7 signaling seems to be a promising strategy for the prevention or therapy of PD and other neurodegenerative disorders.     

Domain-specific purinergic signaling in polarized rat cholangiocytes

In cholangiocytes, adenine nucleotides function as autocrine/paracrine signals that modulate ductular ion transport by activation of purinergic receptors. The purpose of these studies was to identify cellular signals that modulate ATP release and nucleotide processing in polarized normal rat cholangiocytes. In Ussing chamber studies, selective exposure of the apical and basolateral membranes to ATP or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) stimulated increases in short-circuit current. Apical purinergic receptor agonist preference was consistent with the P2Y(2) subtype. In contrast, basolateral ADP was more potent in stimulating transepithelial currents, consistent with the expression of different basolateral P2 receptor(s). Luminometric analysis revealed that both membranes exhibited constitutive ATP efflux. Hypotonic exposure enhanced ATP release in both compartments, whereas decreases in ATP efflux during hypertonicity were more prominent at the apical membrane. Increases in intracellular cAMP, cGMP, and Ca(2+) also increased ATP permeability, but selective effects on apical and basolateral ATP release differed. Finally, the kinetics of ATP degradation in apical and basolateral compartments were distinct. These findings suggest that there are domain-specific signaling pathways that contribute to purinergic responses in polarized cholangiocytes.