ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

Extracellular purines from cells of seminiferous tubules

It has been long postulated that extracellular purines can modulate the function of the male reproductive system by interacting with different purinergic receptors of Sertoli and germinative cells. Many authors have described the biological changes induced by extracellular ATP and/or adenosine in these cells, and some hypothetical models for paracrine communication mediated by purines were proposed; however, the cellular source(s) of these molecules in seminiferous tubules remains unknown. In this study, we demonstrated for the first time that Sertoli cells are able to release ATP (0.3 nmol/mg protein) and adenosine (0.1 nmol/mg protein) in the extracellular medium, while germinative and myoid peritubular cells are able to secrete adenosine (0.02 and 0.37 nmol/mg protein, respectively). Indeed, all the three types of cells were able to release inosine at significant concentrations (about 0.4 nmol/mg protein). This differential secretion depending on the cellular type suggests that these molecules may be involved in the paracrine regulation and/or control of the maturation processes of these cells.     

Differential crosstalk between P2X7 and arachidonic acid in activation of mitogen-activated protein kinases

Accumulating evidence indicates that astroglial syncytium plays key role in normal and pathological brain functions. Astrocytes both in vitro and in situ respond to extracellular adenine-based nucleotides via the activation of P2 receptors. Massive release of ATP from neurons and glial cells occurs as a result of pathological conditions of the brain leading to neuroinflammation and involving P2X7 receptors. In this study, we investigated whether P2X7 stimulation on cultured cortical astrocytes promoted a differential activation of mitogen-activated protein kinases (MAPKs), and whether the second messenger arachidonic acid (AA), which is also a key modulator of neuroinflammation, affected the P2X7-mediated MAPK phosphorylation. The results show that the synthetic P2X7 receptor agonist 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP), induced a concentration-dependent phosphorylation of MAPK ERK1/2, JNK and p38. Stimulation of ERK1/2, JNK and p38 phosphorylation was also obtained by pathophysiological levels of extracellularly applied AA. Interestingly, a robust potentiation of ERK1/2 phosphorylation was elicited by co-application of BzATP and AA, whereas no differences were observed in JNK or p38 phosphosignals. The kinases activation showed a differential dependence on the presence of extracellular Ca²⁺. The potentiation of BzATP-mediated ERK1/2 phosphorylation was also observed in human embryonic kidney cells (HEK293) stably transfected with rat P2X7, but not in HEK cells expressing truncated P2X7 receptor lacking the full cytoplasmic carboxy-terminal or in those carrying the structurally related rat P2X2. AA and BzATP synergism in ERK1/2 activation was abolished by cyclo-oxygenase and lipoxygenase pathway inhibitors.The result that ERK1/2-mediated transduction pathway is synergistically modulated by ATP and AA signalling depicts possible novel pharmacological targets for interfering with pathological activation of astroglial cells.     

Purinoceptor-mediated calcium signaling in primary neuron-glia trigeminal cultures

Receptors for extracellular nucleotides (the P2X-calcium channels and the phospholipase C-coupled P2Y receptors) play key roles in pain signaling, but little is known on their function in trigeminal ganglia, whose hyperactivation leads to the development of migraine pain. Here we characterize calcium signaling via P2X(3) and P2Y receptors in primary mouse neuron-glia trigeminal cultures. Comparison with intact ganglion showed that, in dissociated cultures, sensory neurons retain, at least in part, their physical relationships with satellite glia. RT-PCR indicated expression of P2X(2)/P2X(3) (confirmed by immunocytochemistry) and of all cloned P2Y receptors. Single-cell calcium imaging with subtype-selective P2-agonists/antagonists revealed presence of functional neuronal P2X(3), as well as of ADP-sensitive P2Y(1,12,13) and UTP-activated P2Y(2)/P2Y(4) receptors on both neurons and glia. Calcium responses were much higher in glia, that also responded to UDP, suggesting functional P2Y(6) receptors. To study whether trigeminal ganglia P2 receptors are modulated upon treatment with pro-inflammatory agents, cultures were acutely (up to 3 min) or chronically (24 h) exposed to bradykinin. This resulted in potentiation of algogenic P2X(3) receptor-mediated calcium responses followed by their down-regulation at 24 h. At this exposure time, P2Y receptors responses in satellite glia were instead upregulated, suggesting a complex modulation of P2 receptors in pain signaling.     

Purinergic Signalling: ATP Release

Adenosine triphosphate (ATP) has a fundamental intracellular role as the universal source of energy for all living cells. The demonstration of its release into the extracellular space and the identification and localisation of specific receptors on target cells have been essential in establishing, after considerable resistance, its extracellular physiological roles. It is now generally accepted that ATP is a genuine neurotransmitter both in the central and peripheral nervous systems. As such, there are numerous arguments which prove that the release of ATP by nerve terminals is by exocytosis. In some non-neuronal cells, however, recent evidence suggests that ATP release could also be carrier-mediated and would involve ATP-binding cassette proteins (ABC), an ubiquitous family of transport ATPases.     

ATP-Induced Oscillations of Cytosolic Ca2+ Activity in Cultured Astrocytes from Rat Brain are Modulated by Medium Osmolarity Indicating a Control of [Ca2+]i Oscillations by Cell Volume

Oscillations of cytosolic Ca²⁺ activity ([Ca²⁺]_i) induced by stimulation with ATP in rat astrocytes in primary cultures were analysed. Astrocytes, prepared from the brains of newborn rats, loaded with the fluorescent Ca²⁺ indicator fura-2/AM, were continuously stimulated with ATP (10 M). ATP caused a large initial [Ca²⁺ peak, followed by regular [Ca²⁺]_i oscillations (frequencies 1–5/min). Astrocytes were identified by glial fibrillary acidic protein staining of cells after [Ca²⁺]_i recording. The oscillations were reversibly blocked by the P₂ purinoceptor antagonist suramin (30 M). Influx of extracellular Ca²⁺ and mobilization of Ca²⁺ from intracellular stores both contributed to the oscillations. The effects of hypertonic and hypotonic superfusion medium on ATP-induced [Ca²⁺]_i oscillations were examined. Hypertonic medium (430 mOsm) reversibly suppressed the ATP-induced oscillations. Hypotonic medium (250 mOsm), in spite of having heterogeneous effects, most frequently induced a rise in [Ca²⁺]_i, or reversibly increased the frequency of the oscillations. Thus, a change in cell volume might be closely connected with [Ca²⁺]_i oscillations in astrocytes indicating that [Ca²⁺]_i oscillations in glial cells play an important role in regulatory volume regulation in the brain.     

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

Mechanisms involved in the adenosine-induced vasorelaxation to the pig prostatic small arteries

Benign prostatic hypertrophy has been related with glandular ischemia processes and adenosine is a potent vasodilator agent. This study investigates the mechanisms underlying the adenosine-induced vasorelaxation in pig prostatic small arteries. Adenosine receptors expression was determined by Western blot and immunohistochemistry, and rings were mounted in myographs for isometric force recording. A_2A and A₃ receptor expression was observed in the arterial wall and A_2A-immunoreactivity was identified in the adventitia–media junction and endothelium. A₁ and A_2B receptor expression was not obtained. On noradrenaline-precontracted rings, P1 receptor agonists produced concentration-dependent relaxations with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA) = {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 > 2-Cl-IB-MECA = 2-Cl-cyclopentyladenosine = adenosine. Adenosine reuptake inhibition potentiated both NECA and adenosine relaxations. Endothelium removal and ZM241385, an A_2A antagonist, reduced NECA relaxations that were not modified by A₁, A_2B, and A₃ receptor antagonists. Neuronal voltage-gated Ca²⁺ channels and nitric oxide (NO) synthase blockade, and adenylyl cyclase activation enhanced these responses, which were reduced by protein kinase A inhibition and by blockade of the intermediate (IK_Ca)- and small (SK_Ca)-conductance Ca²⁺-activated K⁺ channels. Inhibition of cyclooxygenase (COX), large-conductance Ca²⁺-activated-, ATP-dependent-, and voltage-gated-K⁺ channel failed to modify these responses. These results suggest that adenosine induces endothelium-dependent relaxations in the pig prostatic arteries via A_2A purinoceptors. The adenosine vasorelaxation, which is prejunctionally modulated, is produced via NO- and COX-independent mechanisms that involve activation of IK_Ca and SK_Ca channels and stimulation of adenylyl cyclase. Endothelium-derived NO playing a regulatory role under conditions in which EDHF is non-functional is also suggested. Adenosine-induced vasodilatation could be useful to prevent prostatic ischemia.     

P2X4 purinoceptor signaling in chronic pain

ATP, acting via P2 purinergic receptors, is a known mediator of inflammatory and neuropathic pain. There is increasing evidence that the ATP-gated P2X4 receptor (P2X4R) subtype is a locus through which activity of spinal microglia and peripheral macrophages instigate pain hypersensitivity caused by inflammation or by injury to a peripheral nerve. The present article highlights the recent advances in our understanding of microglia-neuron interactions in neuropathic pain by focusing on the signaling and regulation of the P2X4R. We will also develop a framework for understanding converging lines of evidence for involvement of P2X4Rs expressed on macrophages in peripheral inflammatory pain.