ATP-dependent intercellular Ca2+ signaling in the developing cochlea: Facts,fantasies and perspectives

Hearing relies on a sensitive mechanoelectrical transduction process in the cochlea of the inner ear. The cochlea contains sensory, secretory, neural, supporting and epithelial cells which are all essential to the sound transduction process. It is well known that a complex extracellular purinergic signaling system contributes to cochlear homeostasis, altering cochlear sensitivity and neural output via ATP-gated ion channels (P2X receptors) and G protein-coupled P2Y receptors. This review focuses on the emerging roles of ATP that are currently under investigation in the developing sensory epithelium, with particular emphasis on the link between ATP release, Ca²⁺ signaling, the expression and function of gap junction proteins connexin26 and connexin30, and the acquisition of hearing.     

ATP-mediated potassium recycling in the cochlear supporting cells

Gap junction-mediated K⁺ recycling in the cochlear supporting cell has been proposed to play a critical role in hearing. However, how potassium ions enter into the supporting cells to recycle K⁺ remains undetermined. In this paper, we report that ATP can mediate K⁺ sinking to recycle K⁺ in the cochlear supporting cells. We found that micromolar or submicromolar levels of ATP could evoke a K⁺-dependent inward current in the cochlear supporting cells. At negative membrane potentials and the resting membrane potential of −80 mV, the amplitude of the ATP-evoked inward current demonstrated a linear relationship to the extracellular concentration of K⁺, increasing as the extracellular concentration of K⁺ increased. The inward current also increased as the concentration of ATP was increased. In the absence of ATP, there was no evoked inward current for extracellular K⁺ challenge in the cochlear supporting cells. The ATP-evoked inward current could be inhibited by ionotropic purinergic (P2X) receptor antagonists. Application of pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50 μM) or pre-incubation with an irreversible P2X7 antagonist oxidized ATP (oATP, 0.1 mM) completely abolished the ATP-evoked inward current at the negative membrane potential. ATP also evoked an inward current at cell depolarization, which could be inhibited by intracellular Cs⁺ and eliminated by positive holding potentials. Our data indicate that ATP can activate P2X receptors to recycle K⁺ in the cochlear supporting cells at the resting membrane potential under normal physiological and pathological conditions. This ATP-mediated K⁺ recycling may play an important role in the maintenance of cochlear ionic homeostasis.     

Differential expression of P2Y receptors in the rat cochlea during development

Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂, during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the driving force for sound transduction. In addition, early postnatal P2Y₂ and P2Y₄ protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca²⁺-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance of extracellular nucleotide and nucleoside signaling to hearing.     

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5′-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5′-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca²⁺]_i) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca²⁺]_i transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca²⁺]_i transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca²⁺]_i response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca²⁺]_i transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9468-1) contains supplementary material, which is available to authorized users.     

Activation of Protein Kinase C Blocks Astroglial Gap Junction Communication and Inhibits the Spread of Calcium Waves

The following two processes related to astrocytes are thought to depend on intercellular coupling through gap junctions: the spatial buffering of K+o and the spread of calcium waves in the astrocytic syncytium. We have used the following two independent methods to measure the open state of gap junctions: injection of lucifer yellow, and optical calcium imaging of calcium waves in response to probing the cells with a micropipette. The spread of lucifer yellow and calcium waves was inhibited if the cells were treated with either phorbol 12-myristate 13-acetate (PMA) or a synthetic diacylglycerol that activates protein kinase C. Down-regulation of protein kinase C by a 24-h treatment with PMA inhibited the uncoupling effect of PMA, supporting a direct involvement of protein kinase C in the regulation of astroglial gap junctions. Purinergic P2Y receptors, which are coupled to the inositol phospholipid pathway, are expressed by most astroglia in culture. Activation of the P2Y purinergic receptor with the selective agonist 2-methylthio-ATP uncoupled astroglia in a manner similar to the effect of treatment with PMA. Modulation of gap junctional conductance could isolate specific pathways within the astrocytic syncytium to form an extraneuronal information transfer network in brain.     

The impact of commercially available purinergic ligands on purinergic signalling research

Pannexin1 is a prime candidate to represent an ATP release channel. The pannexin1 channel can be activated by extracellular ATP through purinergic receptors P2X7 or P2Y. Recent studies have shown that the Pannexin1 channel is inhibited by its own permeant ion, ATP, and also by P2X7 receptor agonists and antagonists. However, the dose dependence of this inhibition indicated that significant inhibition was prominent at ATP concentrations higher than required for activation of purinergic receptors, including P2X7 and P2Y2. The inhibitory effect of ATP is largely decreased when R75 in the first extracellular loop of Pannexin1 is mutated to alanine, indicating that ATP regulates this channel presumably through binding. To further investigate the structural property of the putative ATP binding site, we performed alanine-scanning mutagenesis of the extracellular loops of pannexin1. Mutations on W74, S237, S240, I247 and L266 in the extracellular loops 1 and 2 severely impaired the inhibitory effect of BzATP, indicating that they might be the essential amino acids in the putative binding site. Mutations on R75, S82, S93, L94, D241, S249, P259 and I267 moderately (≥50%) decreased BzATP sensitivity, suggesting their supporting roles in the binding. Mutations of other residues did not change the BzATP potency compared to wild-type except for some nonfunctional mutants. These data demonstrate that several amino acid residues on the extracellular loops of Pannexin1 mediate ATP sensitivity. Cells expressing mutant Panx1W74A exhibited an enhanced release of ATP, consistent with the removal of a negative feedback loop controlling ATP release.     

P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium⁺ uptake into MEL cells in a concentration-dependent fashion and with an EC₅₀ of ∼ 154 μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ and YO-PRO-1²⁺ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.     

High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca²⁺ responses to ATP, including a transition of Ca²⁺ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10⁻⁴ and 10⁻² M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP₃) mediated Ca²⁺ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca²⁺ responses due to the biphasic nature of IP₃Rs and the interaction of SERCA pumps with IP₃Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP₃Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca²⁺ response signatures, which are important in mediating bone mechanoadaptation.     

Purinergic receptor-induced Ca2+ signaling in the neuroepithelium of the vomeronasal organ of larval Xenopus laevis

Purinergic signaling has considerable impact on the functioning of the nervous system, including the special senses. Purinergic receptors are expressed in various cell types in the retina, cochlea, taste buds, and the olfactory epithelium. The activation of these receptors by nucleotides, particularly adenosine-5′-triphosphate (ATP) and its breakdown products, has been shown to tune sensory information coding to control the homeostasis and to regulate the cell turnover in these organs. While the purinergic system of the retina, cochlea, and taste buds has been investigated in numerous studies, the available information about purinergic signaling in the olfactory system is rather limited. Using functional calcium imaging, we identified and characterized the purinergic receptors expressed in the vomeronasal organ of larval Xenopus laevis. ATP-evoked activity in supporting and basal cells was not dependent on extracellular Ca²⁺. Depletion of intracellular Ca²⁺ stores disrupted the responses in both cell types. In addition to ATP, supporting cells responded also to uridine-5′-triphosphate (UTP) and adenosine-5′-O-(3-thiotriphosphate) (ATPγS). The response profile of basal cells was considerably broader. In addition to ATP, they were activated by ADP, 2-MeSATP, 2-MeSADP, ATPγS, UTP, and UDP. Together, our findings suggest that supporting cells express P2Y₂/P2Y₄-like purinergic receptors and that basal cells express multiple P2Y receptors. In contrast, vomeronasal receptor neurons were not sensitive to nucleotides, suggesting that they do not express purinergic receptors. Our data provide the basis for further investigations of the physiological role of purinergic signaling in the vomeronasal organ and the olfactory system in general.     

P2Y purinoceptors induce changes in intracellular calcium in acinar cells of rat lacrimal glands

Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca²⁺]_i in acinar cells. The removal of extracellular Ca²⁺ or the use of Ca²⁺ channel blockers partially inhibited the ATP-induced [Ca²⁺]_i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP₃ receptors) did not completely inhibit the ATP-induced [Ca²⁺]_i increase. P1 purinoceptor agonists did not induce any changes in [Ca²⁺]_i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca²⁺]_i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca²⁺]_i, although UTP (a P2Y_2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y₁ played a significant role in the cellular response to ATP. BzATP (a P2X₇ receptor agonist) induced a small increase in [Ca²⁺]_i, but α,β-meATP (a P2X_1,3 receptor agonist) had no effect. RT-PCR indicated that P2X_2,3,4,5,6,7 and P2Y_1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y₁) as well as by P2X purinoceptors.     

The effects of extracellular nucleotides on [Ca2+]i signalling in a human‐derived renal proximal tubular cell line (HKC‐8)

HKC‐8 cells are a human‐derived renal proximal tubular cell line and provide a useful model system for the study of human renal cell function. In this study, we aimed to determine [Ca²⁺]_i signalling mediated by P2 receptor in HKC‐8. Fura‐2 and a ratio imaging method were employed to measure [Ca²⁺]_i in HKC‐8 cells. Our results showed that activation of P2Y receptors by ATP induced a rise in [Ca²⁺]_i that was dependent on an intracellular source of Ca²⁺, while prolonged activation of P2Y receptors induced a rise in [Ca²⁺]_i that was dependent on intra‐ and extracellular sources of Ca²⁺. Pharmacological and molecular data in this study suggests that TRPC4 channels mediate Ca²⁺ entry in coupling to activation of P2Y in HKC‐8 cells. U73221, an inhibitor of PI‐PLC, did not inhibit the initial ATP‐induced response; whereas D609, an inhibitor of PC‐PLC, caused a significant decrease in the initial ATP‐induced response, suggesting that P2Y receptors are coupled to PC‐PLC. Although P2X were present in HKC‐8, The P2X agonist, α,β me‐ATP, failed to cause a rise in [Ca²⁺]_i. However, PPADS at a concentration of 100 µM inhibits the ATP‐induced rise in [Ca²⁺]_i. Our results indicate the presence of functional P2Y receptors in HKC‐8 cells. ATP‐induced [Ca²⁺]_i elevation via P2Y is tightly associated with PC‐PLC and TRP channel. J. Cell. Biochem. 109: 132–139, 2010. © 2009 Wiley‐Liss, Inc.     

Damage-induced cell–cell communication in different cochlear cell types via two distinct ATP-dependent Ca<Superscript>2+</Superscript> waves

Intercellular Ca²⁺ waves can coordinate the action of large numbers of cells over significant distances. Recent work in many different systems has indicated that the release of ATP is fundamental for the propagation of most Ca²⁺ waves. In the organ of hearing, the cochlea, ATP release is involved in critical signalling events during tissue maturation. ATP-dependent signalling is also implicated in the normal hearing process and in sensing cochlear damage. Here, we show that two distinct Ca²⁺ waves are triggered during damage to cochlear explants. Both Ca²⁺ waves are elicited by extracellular ATP acting on P2 receptors, but they differ in their source of Ca²⁺, their velocity, their extent of spread and the cell type through which they propagate. A slower Ca²⁺ wave (14 μm/s) communicates between Deiters’ cells and is mediated by P2Y receptors and Ca²⁺ release from IP₃-sensitive stores. In contrast, a faster Ca²⁺ wave (41 μm/s) propagates through sensory hair cells and is mediated by Ca²⁺ influx from the external environment. Using inhibitors and selective agonists of P2 receptors, we suggest that the faster Ca²⁺ wave is mediated by P2X₄ receptors. Thus, in complex tissues, the expression of different receptors determines the propagation of distinct intercellular communication signals.     

Differential sensitivity to nickel and SK&F96365 of second messengerm operated and receptormoperated calcium channels in rat submandibular ductal cells

The intracellular concentration of calcium ([Ca²⁺]_i) of rat submandibular ductal cells was measured with the intracellular fluorescent dye Fura-2. Carbachol (100 μM) and ATP (1 mM) both increased the [Ca²⁺]_j. The late response to ATP was blocked by 0.5 mM Ni²⁺. This concentration of Ni²⁺ also blocked the increase of the [Ca²⁺]_i and the uptake of manganese and calcium in response to 2′- and 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP, 100 μM), a specific agonist of P2X receptors from salivary glands. The increase of the [Ca²⁺]_i in response to 2-methylthioadenosine 5′-triphosphate (2-McSATP, 100 μM) a specific P2Y agonist in salivary glands or to a muscarinic agonist (carbachol) was not affected by 0.5 mM Ni²⁺. Only higher concentrations of Ni²⁺ (in the millimolar range) inhibited the uptake of extracellular calcium in response to carbachol. SK&F 96365, a blocker of store-operated calcium channels, inhibited the uptake of extracellular calcium in response to carbachol without affecting the response to BzATP. It is concluded that at low concentrations (below 0.5 mM), Ni²⁺ inhibits the non-specific cation channel coupled to P2X receptors. The uptake of extracellular calcium by store-operated calcium channels is inhibited by higher concentrations of Ni²⁺ and by SK&F96365.     

Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells

In blood vessels, stimulation of the vascular endothelium by the Ca(2+)-mobilizing agonist ATP initiates a number of cellular events that cause relaxation of the adjacent smooth muscle layer. Although vascular endothelial cells are reported to express several subtypes of purinergic P2Y and P2X receptors, the major isoform(s) responsible for the ATP-induced generation of vasorelaxant signals in human endothelium has not been well characterized. To address this issue, ATP-evoked changes in cytosolic Ca(2+), membrane potential and acute nitric oxide production were measured in isolated human umbilical vein endothelial cells (HUVECs) and profiled using established P2X and P2Y receptor probes. Whereas selective P2X agonist (i.e. α,β-methyl ATP) and antagonists (i.e. TNP-ATP and PPADS) could neither mimic nor block the observed ATP-evoked cellular responses, the specific P2Y receptor agonist UTP functionally reproduced all the ATP-stimulated effects. Furthermore, both ATP and UTP induced intracellular Ca(2+) mobilization with comparable EC(50) values (i.e. 1-3μM). Collectively, these functional and pharmacological profiles strongly suggest that ATP acts primarily via a P2Y2 receptor sub-type in human endothelial cells. In support, P2Y2 receptor mRNA and protein were readily detected in isolated HUVECs, and siRNA-mediated knockdown of endogenous P2Y2 receptor protein significantly blunted the cytosolic Ca(2+) elevations in response to ATP and UTP, but did not affect the histamine-evoked response. In summary, these results identify the P2Y2 isoform as the major purinergic receptor in human vascular endothelial cells that mediates the cellular actions of ATP linked to vasorelaxation.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.     

ATP enhances spontaneous calcium activity in cultured suburothelial myofibroblasts of the human bladder

Background

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves. They express purinergic receptors and show calcium transients in response to ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Since ATP concentration is likely to be very low during the initial filling phase, we hypothesized that sMF Ca²⁺ activity is affected even at very low ATP concentrations. We investigated ATP induced modulation of spontaneous activity, intracellular calcium response and purinergic signaling in cultured sMF.

Methodology/Principal Findings

Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10⁻¹⁶ to 10⁻⁴ mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18%±1.65 of the sMF (N = 48 experiments). ATP significantly increased calcium activity even at 10⁻¹⁶ mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1 µM; A-317491, 1 µM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence.

Conclusions/Significance

Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca²⁺ response in cultured sMF at very low concentrations, most likely involving P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.     

Differential Regulation of Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Extracellular ATP triggers changes in intracellular Ca²⁺, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca²⁺ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca²⁺ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca²⁺ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y₂, P2Y₆, and P2Y₁₂ metabotropic receptors and P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, and P2X₇ ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca²⁺, while 100 μM suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca²⁺ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 μM PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.     

Role of nitric oxide on purinergic signalling in the cochlea

In the inner ear, there is considerable evidence that extracellular adenosine 5′-triphosphate (ATP) plays an important role in auditory neurotransmission as a neurotransmitter or a neuromodulator, although the potential role of adenosine signalling in the modulation of auditory neurotransmission has also been reported. The activation of ligand-gated ionotropic P2X receptors and G protein-coupled metabotropic P2Y receptors has been reported to induce an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons (SGNs), and supporting cells in the cochlea. ATP may participate in auditory neurotransmission by modulating [Ca²⁺]_i in the cochlear cells. Recent studies showed that extracellular ATP induced nitric oxide (NO) production in IHCs, OHCs, and SGNs, which affects the ATP-induced Ca²⁺ response via the NO-cGMP-PKG pathway in those cells by a feedback mechanism. A cross-talk between NO and ATP may therefore exist in the auditory signal transduction. In the present article, I review the role of NO on the ATP-induced Ca²⁺ signalling in IHCs and OHCs. I also consider the possible role of NO in the ATP-induced Ca²⁺ signalling in SGNs and supporting cells.     

Purinergic receptors activating rapid intracellular Ca increases in microglia

We provide both molecular and pharmacological evidence that the metabotropic, purinergic, P2Y(6), P2Y(12) and P2Y(13) receptors and the ionotropic P2X(4) receptor contribute strongly to the rapid calcium response caused by ATP and its analogues in mouse microglia. Real-time PCR demonstrates that the most prevalent P2 receptor in microglia is P2Y(6) followed, in order, by P2X(4), P2Y(12), and P2X(7) = P2Y(13). Only very small quantities of mRNA for P2Y(1), P2Y(2), P2Y(4), P2Y(14), P2X(3) and P2X(5) were found. Dose-response curves of the rapid calcium response gave a potency order of: 2MeSADP>ADP=UDP=IDP=UTP>ATP>BzATP, whereas A2P4 had little effect. Pertussis toxin partially blocked responses to 2MeSADP, ADP and UDP. The P2X(4) antagonist suramin, but not PPADS, significantly blocked responses to ATP. These data indicate that P2Y(6), P2Y(12), P2Y(13) and P2X receptors mediate much of the rapid calcium responses and shape changes in microglia to low concentrations of ATP, presumably at least partly because ATP is rapidly hydrolyzed to ADP. Expression of P2Y(6), P2Y(12) and P2Y(13) receptors appears to be largely glial in the brain, so that peripheral immune cells and CNS microglia share these receptors. Thus, purinergic, metabotropic, P2Y(6), P2Y(12), P2Y(13) and P2X(4) receptors might share a role in the activation and recruitment of microglia in the brain and spinal cord by widely varying stimuli that cause the release of ATP, including infection, injury and degeneration in the CNS, and peripheral tissue injury and inflammation which is signaled via nerve signaling to the spinal cord.     

Purinergic receptor-mediated Ca<Superscript>2+</Superscript> signaling in the olfactory bulb and the neurogenic area of the lateral ventricles

Like in other vertebrates, the anterior part of the telencephalon of amphibians mainly consists of the olfactory bulb (OB), but different from higher vertebrates, the lateral telencephalic ventricles of larval Xenopus laevis expand deep into the anterior telencephalon. The neurogenic periventricular zone (PVZ) of the lateral ventricles generates new OB neurons throughout the animal’s lifetime. We investigated the ultrastructural organization of the PVZ and found that within a time period of 24 h, 42.54 ± 6.65% of all PVZ cells were actively proliferating. Functional purinergic receptors are widespread in the central nervous system and their activation has been associated with many critical physiological processes, including the regulation of cell proliferation. In the present study we identified and characterized the purinergic system of the OB and the PVZ. ATP and 2MeSATP induced strong [Ca²⁺]_i increases in cells of both regions, which could be attenuated by purinergic antagonists. However, a more thorough pharmacological investigation revealed clear differences between the two brain regions. Cells of the OB almost exclusively express ionotropic P2X purinergic receptor subtypes, whereas PVZ cells express both ionotropic P2X and metabotropic P1 and P2Y receptor subtypes. The P2X receptors expressed in the OB are evidently not involved in the immediate processing of olfactory information.