Extracellular ATP Receptors Research Articles

Differences in the neurotoxicity profile induced by ATP and ATPγS in cultured cerebellar granule neurons

Extracellular ATP and P2 receptors may play a crucial role in the neurodegeneration of the CNS. Here, we investigated in neuronal cerebellar granule cultures the biological effect of the quite stable P2 receptor agonist ATPγS and compare it to the cytotoxic action of ATP. Time-course experiments showed that 500 μM ATPγS causes 50–100% cell death in 15–24 h. As proved by pharmacological means, ATPγS toxicity apparently involves neither indirect activation of NMDA receptors, nor ectonucleotidase activities, nor nucleoside transport and intracellular purine metabolism. Moreover, ATPγS induces detrimental effects without modifying the expression of several P2X and P2Y receptor proteins. Cell death instead occurs after extracellular release of the cytosolic enzyme lactic dehydrogenase and inhibition of the overall activity of the intracellular dehydrogenases. Moreover, ATPγS causes transient outflow of cytochrome c from mitochondria (maximal 2.5-fold stimulation in 4 h), it raises the intracellular reactive oxygen species (about four-fold in 1 h) and cAMP levels (about 40% in 15 min–4 h). Among several P2 receptor antagonists, only pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid 4-sodium promotes 80–100% neuroprotection.

Basal Activation of the P2X7 ATP Receptor Elevates Mitochondrial Calcium and Potential, Increases Cellular ATP Levels, and Promotes Serum-independent Growth

P2X7 is a bifunctional receptor (P2X7R) for extracellular ATP that, depending on the level of activation, forms a cation-selective channel or a large conductance nonselective pore. The P2X7R has a strong proapoptotic activity but can also support growth. Here, we describe the mechanism involved in growth stimulation. Transfection of P2X7R increases resting mitochondrial potential (delta psi(mt)), basal mitochondrial Ca2+ ([Ca2+]mt), intracellular ATP content, and confers ability to grow in the absence of serum. These changes require a full pore-forming function, because they are abolished in cells transfected with a mutated P2X7R that retains channel activity but cannot form the nonselective pore, and depend on an autocrine/paracrine tonic stimulation by secreted ATP. On the other hand, sustained stimulation of P2X7R causes a delta psi(mt) drop, a large increase in [Ca2+]mt, mitochondrial fragmentation, and cell death. These findings reveal a hitherto undescribed mechanism for growth stimulation by a plasma membrane pore.

Traumatic Injury Activates Protein Kinase B/Akt in Cultured Astrocytes: Role of Extracellular ATP and P2 Purinergic Receptors

Protein kinase B/Akt is a key signaling molecule that regulates cell survival, growth, and metabolism, and inhibits apoptosis. Traumatic brain injury (TBI) activates Akt, and Akt has been implicated in neuronal survival after TBI, but little is known about injury-induced Akt activation in astrocytes, cells that exhibit hypertrophic and hyperplastic responses to CNS injury. Here we have investigated the effect of mechanical strain on Akt activation in primary cultures of rat cortical astrocytes growing on deformable Silastic membranes. When astrocytes were subjected to mechanical strain (50 msec; 5-7.5 mm displacement), we observed an increase in phosphorylation of serine 473, a key indicator of Akt activation. Akt phosphorylation was increased at 3 min postinjury, was maximal from 5 to 10 min, and declined gradually thereafter. Akt activation was also dependent on the severity of the injury. Stretch-induced Akt phosphorylation was attenuated by blocking calcium influx and phosphoinositide 3-kinase (PI3K), an upstream activator of Akt. In addition, we found that ATP is rapidly released after mechanical strain and that the P2 purinergic receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',5'disulfonate (PPADS) attenuated trauma-induced Akt activation. We conclude that mechanical strain causes activation of Akt in astrocytes via stimulation of P2 receptors. This suggests that P2 receptor/Akt signaling promotes astrocyte survival and growth, and this process may play a role in the generation of reactive gliosis after TBI.

P2Y 2 nucleotide receptor up-regulation in submandibular gland cells from the NOD.B10 mouse model of Sjögren's syndrome

Sjögren's syndrome (SS) is an autoimmune disease that specifically targets exocrine glands, including salivary glands, and results in an impairment of secretory function. P2Y(2) nucleotide receptors for extracellular ATP and UTP are up-regulated in response to stress or injury in a variety of tissues including submandibular glands (SMGs) [Ahn JS, Camden JM, Schrader AM, Redman RS, Turner JT. Reversible regulation of P2Y(2) nucleotide receptor expression in the duct-ligated rat submandibular gland. Am J Physiol Cell Physiol 2000;279:C286-94; Hou M, Malmsjö M, Möller S, Pantev E, Bergdahl A, Zhai X-H, et al. Increase in cardiac P2X(1)- and P2Y(2)-receptor mRNA levels in congestive heart failure. Life Sci 1999;65:1195-206; Kishore BK, Wang Z, Rab H, Haq M, Soleimani M. Upregulation of P2Y(2) purinoceptor during ischemic reperfusion injury (IRI): possible relevance to diuresis of IRI. J Am Soc Nephrol 1998;9:581 (abstract); Koshiba M, Apasov S, Sverdlov V, Chen P, Erb L, Turner JT, et al. Transient up-regulation of P2Y(2) nucleotide receptor mRNA expression is an immediate early gene response in activated thymocytes. Proc Natl Acad Sci U S A 1997;94:831-6; Turner JT, Landon LA, Gibbons SJ, Talamo BR. Salivary gland P2 nucleotide receptors. Crit Rev Oral Biol Med 1999;10:210-24; Seye CI, Gadeau AP, Daret D, Dupuch F, Alzieu P, Capron L, et al. Overexpression of the P2Y(2) purinoceptor in intimal lesions of the rat aorta. Arterioscler Thromb Vasc Biol 1997;17:3602-10; Seye C, Kong Q, Erb L, Garrad RC, Krugh B, Wang M, et al. Functional P2Y(2) nucleotide receptors mediate uridine 5'-triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries. Circulation 2002;106:2720-6]. To assess whether P2Y(2) receptor expression is up-regulated in SMGs of the NOD.B10 mouse model of primary SS as compared to SMGs of normal C57BL/6 mice. SMG cells were isolated from normal C57BL/6 and diseased NOD.B10 mice. P2Y(2) receptor mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization, whereas functional P2Y(2) receptor activity was analyzed by measuring UTP-induced increases in [Ca(2+)](i). In contrast to SMG cells from C57BL/6 mice, SMG cells from 4- to 19-week-old NOD.B10 mice exhibited increased P2Y(2) receptor mRNA localized to both ductal and acinar cell types. The levels of mRNA for other uridine nucleotide receptors, i.e., P2Y(4) and P2Y(6) receptors, showed no significant differences between SMG cells of C57BL/6 and NOD.B10 mice, suggesting that only the P2Y(2) receptor was up-regulated in NOD.B10 mice. Moreover, P2Y(2) receptor activity in SMG cells from NOD.B10 mice increased with age (i.e., disease progression). P2Y(2) receptor up-regulation in SMGs is associated with the SS phenotype in NOD.B10 mice, which encourages further attempts to determine the role of this pathway in the development of SS.

Ca2+ waves in keratinocytes are transmitted to sensory neurons: the involvement of extracellular ATP and P2Y2 receptor activation.

ATP acts as an intercellular messenger in a variety of cells. In the present study, we have characterized the propagation of Ca2+ waves mediated by extracellular ATP in cultured NHEKs (normal human epidermal keratinocytes) that were co-cultured with mouse DRG (dorsal root ganglion) neurons. Pharmacological characterization showed that NHEKs express functional metabotropic P2Y2 receptors. When a cell was gently stimulated with a glass pipette, an increase in [Ca2+]i (intracellular Ca2+ concentration) was observed, followed by the induction of propagating Ca2+ waves in neighbouring cells in an extracellular ATP-dependent manner. Using an ATP-imaging technique, the release and diffusion of ATP in NHEKs were confirmed. DRG neurons are known to terminate in the basal layer of keratinocytes. In a co-culture of NHEKs and DRG neurons, mechanical-stimulation-evoked Ca2+ waves in NHEKs caused an increase in [Ca2+]i in the adjacent DRG neurons, which was also dependent on extracellular ATP and the activation of P2Y2 receptors. Taken together, extracellular ATP is a dominant messenger that forms intercellular Ca2+ waves in NHEKs. In addition, Ca2+ waves in NHEKs could cause an increase in [Ca2+]i in DRG neurons, suggesting a dynamic cross-talk between skin and sensory neurons mediated by extracellular ATP.

Synaptic P2X7 and oxygen/glucose deprivation in organotypic hippocampal cultures.

The P2X7 receptor for extracellular ATP is the main candidate, among P2 receptors, inducing cell death in the immune system. Here, we demonstrate the direct participation of this receptor to cell damage induced by oxygen/glucose deprivation, in the ex vivo model of organotypic hippocampal cultures. By pharmacological and immunological approaches, we show that P2X7 is rapidly and transiently up regulated in hippocampal areas eliciting metabolism impairment. Moreover, the P2 antagonists 2',3',-dialdehyde ATP and reactive blue 2 prevent both up regulation of this receptor and hypoxic/hypoglycemic damage. By confocal laser microscopy, we show that P2X7 is present at the synaptic level of fibers extending from the CA1-2 pyramidal cell layer throughout the strata oriens and radiatum, but absent on oligodendrocytes, astrocytes or neuronal cell bodies. Colocalization of P2X7 is obtained with neurofilament-L protein and with synaptophysin, not with myelin basic protein, glial fibrillary acidic protein or a marker for neuronal nuclei. P2X7 up regulation and diffuse cellular damage are also induced by 3'-O-(4-benzoyl) benzoyl-ATP, an agonist selective but not exclusive for P2X7. In summary, our study demonstrates that P2X7 not only directly participates to the hypoxic/hypoglycemic process, but also owns specific phenotypic localization. We do not exclude that it might serve as a sensor of dysregulated neuronal activity and ATP release, both occurring during oxygen/glucose deprivation.

ATP- and Adenosine-Mediated Signaling in the Central Nervous System: Adenosine Receptor Activation by ATP Through Rapid and Localized Generation of Adenosine by Ecto-nucleotidases

Extracellular ATP is now recognized as a neurotransmitter or neuromodilator in the nervous system, producing diverse physiological effects by activating multiple P2 receptors. Although P2-receptor signaling is terminated by hydrolysis of ATP by the ecto-nucleotidase cascade, such a metabolic step leads to adenosine generation, thereby initiating adenosine (P1)-receptor activation. Because most cells and tissues co-express P1 and P2 receptors, ecto-nucleotidase on target tissues, especially enzymes catalyzing adenosine formation, are determinants of the cellular response to ATP. Ecto-5′-nucleotidase (E-5′-NT) has been considered to play a principal role in conversion of AMP to adenosine. In addition to E-5′-NT, we have recently demonstrated that ecto-alkaline phosphatase is also involved in ATP-induced P1-receptor activation through a rapid and localized adenosine production on the membrane surface. In this minireview, we describe the pharmacological profile of ecto-nucleotidase-dependent P1-receptor activation by ATP and molecular bases of preferential delivery of metabolically generated adenosine to P1 receptors. Several lines of evidence suggest that the close association between ecto-nucleotidases and P1 receptors may constitute a functional receptor for extracellular ATP, and some physiological responses to ATP would occur through this mechanism.

Up-regulation of p2x 2, p2x 4 receptor and ischemic cell death: prevention by p2 antagonists

In the present work we examined the involvement of selected P2X receptors for extracellular ATP in the onset of neuronal cell death caused by glucose/oxygen deprivation. The in vitro studies of organotypic cultures from hippocampus evidenced that P2X 2 and P2X 4 were up-regulated by glucose/oxygen deprivation. Moreover, we showed that ischemic conditions induced specific neuronal loss not only in hippocampal, but also in cortical and striatal organotypic cultures and the P2 receptor antagonists basilen blue and suramin prevented these detrimental effects. In the in vivo experiments we confirmed the induction of P2X receptors in the hippocampus of gerbils subjected to bilateral common carotid occlusion. In particular, P2X 2 and P2X 4 proteins became significantly up-regulated, although to different extent and in different cellular phenotypes. The induction was confined to the pyramidal cell layer of the CA1 subfield and to the transition zone of the CA2 subfield and it was coincident with the area of neuronal damage. P2X 2 was expressed in neuronal cell bodies and fibers in the CA1 pyramidal cell layer and in the strata oriens and radiatum. Intense P2X 4 immunofluorescence was localized to microglia cells. Our results indicate a direct involvement of P2X receptors in the mechanisms sustaining cell death evoked by metabolism impairment and suggest the use of selected P2 antagonists as effective neuroprotecting agents.

Activation of Extracellular Signal-Regulated Kinase by Stretch-Induced Injury in Astrocytes Involves Extracellular ATP and P2 Purinergic Receptors

Gliosis is characterized by hypertrophic and hyperplastic responses of astrocytes to brain injury. To determine whether injury of astrocytes produced by an in vitro model of brain trauma activates extracellular signal-regulated protein kinase (ERK), a key regulator of cellular proliferation and differentiation, astrocytes cultured on deformable SILASTIC membranes were subjected to rapid, reversible strain (stretch)-induced injury. Activation of ERK was observed 1 min after injury, was maximal from 10 to 30 min, and remained elevated for 3 hr. Activation of ERK was dependent on the rate and magnitude of injury; maximum ERK activation was observed after a 20-60 msec, 7.5 mm membrane displacement. ERK activation was blocked by inhibiting MEK, the upstream activator of ERK. Activation of ERK was reduced when calcium influx was diminished. When extracellular ATP was hydrolyzed by apyrase or ATP/P2 receptors were blocked, injury-induced ERK activation was significantly reduced. P2 receptor antagonist studies indicated a role for P2X2 and P2Y1, but not P2X1, P2X3, or P2X7, receptors in injury-induced ERK activation. These findings demonstrate for the first time that ATP released by mechanical injury is one of the signals that triggers ERK activation and suggest a role for extracellular ATP, P2 purinergic receptors, and calcium-dependent ERK signaling in the astrocytic response to brain trauma.

Relief of inflammatory pain in rats by local use of the selective P2X7 ATP receptor inhibitor, oxidized ATP.

Oxidized ATP (oATP) is a selective inhibitor of the P2Z/P2X7 ATP receptor for extracellular ATP, which contributes to the antinociceptive effect. This study sought to determine the mechanism by which local administration of oATP is able to relieve inflammatory pain in arthritic rat paws. Arthritis was induced in Wistar rats by injections of Freund's complete adjuvant into one hind paw. Nociceptive thresholds were measured before and after local injection of oATP into the inflamed paws. The influence on pain transmission due to the presence of recruited inflammatory cells at the site of inflammation was determined by inhibiting the initial phase of their migration (by intravenous treatment with fucoidin, which blocks the adhesion molecules of the selectin family). ATP intraplantar content was determined in the different experimental conditions. Histologic features of the hind paws were evaluated by using the anti-P2X7 receptor polyclonal antibody. Intraplantar administration of oATP into inflamed paws significantly relieved inflammatory pain. The antinociceptive effect of oATP was independent of the immune-cell recruitment. ATP levels in inflamed tissues were significantly reduced by oATP treatment. A variable presence of P2X7 receptors on cutaneous sensory nerves with respect to the different treatments was observed. Following oATP treatment, there was a reduction in P2X7 expression in the endings of peripheral nerves, as well as in endothelial cells. Oxidized ATP inhibits inflammatory pain in arthritic rats by inhibition of the P2X7 receptor for ATP, which is localized on nerve terminals.

Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary neurons: role of P2 and NGFRp75 receptors.

In this study, we monitored the direct expression of P2 receptors for extracellular ATP in cerebellar granule neurons undergoing metabolism impairment. Glucose deprivation for 30-60 min inhibited P2Y1 receptor protein, only weakly modulated P2X1, P2X2 and P2X3, and up-regulated by about two-fold P2X4, P2X7 and P2Y4. The P2X/Y antagonist basilen blue, protecting cerebellar neurons from hypoglycemic cell death, maintained within basal levels only the expression of P2X7 and P2Y4 proteins, but not P2X4 or P2Y1. Glucose starvation transiently increased (up to three-fold) the expression of NGFRp75 receptor protein and strongly stimulated the extracellular release of nerve growth factor (NGF; about 10-fold). Exogenously added NGF then augmented hypoglycemic neuronal death by about 60%, increasing the percentage of Höechst-positive nuclei (from approximately 62 to 95%), reducing lactate dehydrogenase (LDH) release (from about 50 to 14%) and significantly overstimulating the hypoglycemia-induced expression of P2X7 and P2Y4. Conversely, extracellular ATP augmented hypoglycemic neuronal death by about 80%, reducing the number of Höechst-positive nuclei (from approximately 62% to 14%), augmenting LDH outflow (by about 30%) and further increasing the hypoglycemia-induced expression of NGFRp75. Our results indicate that P2 and NGFRp75 receptors are modulated during glucose starvation and that extracellular ATP and NGF drive features of, respectively, necrotic and apoptotic hypoglycemic cell death, aggravating the consequences of metabolism impairment in cerebellar primary neurons.

ATP selectively suppresses the synthesis of the inflammatory protein microglial response factor (MRF)-1 through Ca 2+ influx via P2X 7 receptors in cultured microglia

Microglia are known to express purinergic receptors for extracellular ATP of both P2Y and P2X subtypes. In the CNS, ATP is released from neurons and acts as a signal between neurons and glia. The mrf-1 gene encodes a 17-kDa protein with a single calcium-binding (EF-hand) motif and is expressed specifically in microglia. The gene was isolated from activated microglia in response to apoptosis of cerebellar granule neurons in culture and is upregulated in response to neuronal death and degeneration in vivo. We have found that ATP suppresses the synthesis of the inflammation-related protein MRF-1 in cultured rat microglia. When microglia were treated with ATP (1 mM) for 6 h, mrf-1 mRNA levels decreased to approximately 50% compared to those in the control. This effect was dependent on both the treatment period and the dose of ATP. After ATP (1 mM) treatment for 16 h, levels of mrf-1 mRNA decreased to 37.3% and MRF-1 levels decreased to 55.0% compared to those in the control. A decrease in MRF-1 or its mRNA was also induced by benzoylbenzoyl-ATP (0.1 mM), a P2X 7 receptor-selective agonist, and by the Ca 2+ ionophore A23187 (2 μM), dependent on extracellular Ca 2+. Moreover, ATP modified neither the MRF-1 degradation rate nor total protein synthesis. These results indicate that ATP selectively suppresses MRF-1 synthesis at the transcription level via Ca 2+ influx through P2X 7 receptors.

ATP regulates the differentiation of mammalian skeletal muscle by activation of a P2X5 receptor on satellite cells.

ATP is well known for its role as an intracellular energy source. However, there is increasing awareness of its role as an extracellular messenger molecule (Burnstock, 1997). Although evidence for the presence of receptors for extracellular ATP on skeletal myoblasts was first published in 1983 (Kolb and Wakelam), their physiological function has remained unclear. In this paper we used primary cultures of rat skeletal muscle satellite cells to investigate the role of purinergic signaling in muscle formation. Using immunocytochemistry, RT-PCR, and electrophysiology, we demonstrate that the ionotropic P2X5 receptor is present on satellite cells and that activation of a P2X receptor inhibits proliferation, stimulates expression of markers of muscle cell differentiation, including myogenin, p21, and myosin heavy chain, and increases the rate of myotube formation. Furthermore, we demonstrate that ATP application results in a significant and rapid increase in the phosphorylation of MAPKs, particularly p38, and that inhibition of p38 activity can prevent the effect of ATP on cell number. These results not only demonstrate the existence of a novel regulator of skeletal muscle differentiation, namely ATP, but also a new role for ionotropic P2X receptors in the control of cell fate.

Extracellular ATP and P2X7 receptors in neurodegeneration

Neuronal injury and cell death in the central nervous system (CNS) are underlying features of neurodegenerative disorders. However, our understanding of the fundamental mechanisms involved is still limited. Inflammatory processes mediated by cytokines, and interleukin-1 (IL-1) in particular, play a significant role in neuronal death following pathological insults. Despite this growing area of research, very little is known about the factors regulating the expression, cleavage and release of interleukin-1 in the brain. Recent studies on immune cells demonstrate that extracellular ATP can act as a potent stimulus for the maturation and release of interleukin-1β, via activation of P2X7 receptors. Stimulation of P2X7 receptors with ATP has dramatic cytotoxic properties and a wider role in neurodegenerative processes is possible. This review discusses the potential involvement of extracellular ATP and P2X7 receptors as regulators of interleukin-1-mediated neuropathologies and thus as a mediator of cell death following pathological insults.

Internal thiols and reactive oxygen species in candidacidal activity exerted by an N-terminal peptide of human lactoferrin.

We previously showed that the energized mitochondrion and extracellular ATP are essential for the candidacidal activity of the N-terminal peptide of human lactoferrin, subsequently referred to as hLF(1-11). The present study focuses on the involvement of internal thiols and reactive oxygen species (ROS) in the candidacidal activity exerted by hLF(1-11). Our results reveal that hLF(1-11) reduced the internal thiol level of Candida albicans by 20%. In agreement, N-acetyl-L-cysteine (NAC), which is a precursor of glutathione and an ROS scavenger, inhibited the candidacidal activity of hLF(1-11). In addition, azodicarboxylic acid bis(N,N-dimethylamide) (diamide), which oxidizes internal thiols, was candidacidal. Furthermore, hLF(1-11) increased the level of ROS production by C. albicans in a dose-dependent manner, and a correlation between ROS production and candidacidal activity was found. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), which is an ROS scavenger, partially inhibited the hLF(1-11)-induced, but not the diamide-triggered, candidacidal activity. It is of interest that hLF(1-11) and diamide acted synergistically in killing C. albicans and in ROS production. In agreement, oxidized ATP, an irreversible inhibitor of extracellular ATP receptors, partially blocked the hLF(1-11)-induced, but not the diamide-triggered, candidacidal activity. Finally, the hLF(1-11)-induced activation of mitochondria was inhibited by NAC, indicating that internal thiols and ROS affect mitochondrial activity. Therefore, the candidacidal activity of hLF(1-11) involves both generation of ROS and reduction of internal thiols.

ATP can enhance the proton-induced CGRP release through P2Y receptors and secondary PGE 2 release in isolated rat dura mater

Trigeminal afferent neurons express ionotropic P2X receptors for extracellular ATP which are known to be sensitive to low interstitial pH. Both conditions – ATP release and tissue acidosis – may occur in the dura following the ischemia phase of migraine attacks. Aim of this study was to investigate whether and how ATP and protons may cooperate in exciting meningeal afferents. After removal of the cerebral hemispheres hemisected scull cavities of adult Wistar rats were used as organ bath of their own lining, the dura mater. The dura was chemically stimulated and the amounts of immunoreactive calcitonin gene-related peptide (iCGRP) and prostaglandin E 2 (PGE 2) released into incubation fluid were measured using enzyme immunoassays. Stimulation with ATP (10 −4 and 10 −3 M) augmented iPGE 2 release dose-dependently whereas iCGRP secretion was minimally enhanced only if the dura had previously been depleted of extracellular ATP using hexokinase. Acid buffer solutions (pH 5.9 and 5.4) resulted in pH-dependent increase of iCGRP release but reduced iPGE 2 release. Purines (ATP 10 −3>UTP 10 −4 M>ATP 10 −4 M) and PGE 2 (10 −5 M) were found to facilitate the proton-induced increase in iCGRP release. The proton-reduction of PGE 2 release was overcome by adding ATP (10 −3 M). S(+)-flurbiprofen (10 −6 M) suppressed both the basal and stimulated iPGE 2 release and prevented the ATP(10 −4 M)-induced facilitation of the proton response. The facilitating effect of ATP was also blocked under suramin, a non-selective P2 antagonist, and under reactive blue, an non-selective P2Y-antagonist, but not under pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid, a P2X-antagonist. The present results provide evidence that ATP has poor, if at all, direct excitatory effects on CGRP-containing trigeminal nerve endings in the isolated dura and its facilitatory action seems to depend on G-protein coupled P2Y receptors and secondary PGE 2 release. The UTP effect and the antagonist profile is indicative for the P2Y 2 receptor subtype.

The extracellular ATP receptor, cP2Y1, inhibits cartilage formation in micromass cultures of chick limb mesenchyme

We have investigated the function of the G protein-coupled receptor for extracellular ATP, chick P2Y(1) (cP2Y(1)) during development of the chick limb. cP2Y(1) is strongly expressed in undifferentiated limb mesenchyme cells but appears to be lost from cells as they differentiate, raising the possibility that the function of this receptor may be to inhibit cell differentiation. This pattern of expression was particularly striking surrounding areas of cartilage formation. We tested whether cP2Y(1) was able to regulate cartilage formation by using an in-vitro micromass model of chondrogenesis. Because limb cells in micromass culture lose expression of cP2Y(1), we have used a gain-of-function approach to demonstrate that cP2Y(1) expression can inhibit cartilage differentiation. We also demonstrate that early limb mesenchyme cells release ATP into the extracellular medium and have mechanisms to breakdown extracellular ATP. These results suggest that extracellular ATP, signaling through cP2Y(1), can modulate the differentiation of limb mesenchyme cells in vitro, and the expression pattern of cP2Y(1) suggests that this type of signaling could play a similar role in ovo.

Inflammatory Pain Reduction In Rats By Local Treatment With oATP, A Selective Inhibitor Of P2X7 ATP Receptor

Peptide neurotransmitters, as substance P or ATP, are released during inflammatiory processes by the nerve endings of sensory fibers. ATP is also released from the cytoplasm of damaged cells at the site of inflammation. It acts at the level of many P2X subtypes of purinoreceptors. The receptor for extracellular ATP named P2Z/P2X7 is selectively blocked by the periodate oxidized ATP (oATP). We have hypothesized that P2X subunits present on peripheral sensory nerve terminals, able to initiate a nociceptive signal, could be blocked by local treatment with oATP, so inducing pain relief. Male inbred Fisher rats weighing about 250 g were used. Unilateral inflammation into rat hind paw was induced by intraplantar injection of Freund's complete adjuvant (FCA). The following signs of inflammation, from 3 to 48 h after FCA injection, were detected: increased paw volume, increased paw temperature and hyperalgesia. The latter was evaluated using an algesiometric test wich measured the paw pressure threshold (PPT, expressed in g). We treated some rats, bearing paw inflammation by 12 h, with local injection of 56 μM oATP. We showed a significant reduction of hyperalgesia in treated rats (PPT = 190 ± 2.3 in inflamed paw of oATP treated vs. PPT = 60 ± 1.6 in inflamed paw of untreated rats, at 60 min following oATP innoculation). We showed also that treatment with oATP was more efficient than treatment with diclofenac in reducing local inflammatory pain (PPT expressed as percentage of the maximum possible effect = 60 ± 0.5, at 120 min following intraplantar administration of oATP, vs. 25 ± 1.9 at the same time following intraplantar administration of diclofenac). The use of polyclonal antibody anti P2X7 receptor to perform immunohistochemical analysis of inflamed tissue, showed a reduction of receptor expression at the level of nerve endings in sections obtained from rat paw treated with oATP with respect to sections obtained from untreated rats. Such an effect was independent on the recruitment of immunocytes in inflamed tissue. Our results demonstrate that ATP exerts a key role in the pathophysiology of peripheral inflammation and that oATP may be effective in treating inflammatory pain.

Recombinant P2Y receptors: the UCL experience

The beginning of the last decade heralded three important and sequential developments in our understanding of cell-to-cell signalling by extracellular ATP via its cell surface receptors, the P2 purinoceptors. One major development in ATP signalling culminated in a timely review in 1991, when it was established in the clearest of terms that ATP receptors exploited discrete signal transduction pathways (Dubyak, G.R., 1991. Signal transduction by P2-purinergic receptors for extracellular ATP. Am. J. Respir. Cell. Mol. Biol. 4, 295–300; and later in Dubyak, G.R., El-Moatassim, C., 1993. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am. J. Physiol. 265, C577–C606). Henceforth, it was universally acknowledged that some P2 purinoceptors interacted with heterotrimeric G-proteins to activate intracellular signalling cascades (metabotropic ATP receptors), whereas others contained intrinsic ion-channels (ionotropic ATP receptors). A second key development can be traced to 1992, from the discovery that ATP receptors were involved in excitatory neurotransmission in the CNS and PNS (Edwards, F.A., Gibb, A.J., Colquhoun, D., 1992. ATP receptor-mediated synaptic currents in the central nervous system. Nature 359, 144–147; Evans, R.J., Derkach, V., Surprenant, A., 1992. ATP mediates fast synaptic transmission in mammalian neurons. Nature 357, 503–505; Silinsky, E.M., Gerzanich, V., Vanner, S.M., 1992. ATP mediates excitatory synaptic transmission in mammalian neurones. Br. J. Pharmacol., 106, 762–763). Thereafter, it was accepted that ATP could play a neurotransmitter and/or modulatory role throughout the entire nervous system. The third key development stemmed from the isolation of a cDNA, from chick brain, encoding a metabotropic ATP receptor (Webb, T.E., Simon, J., Krishek, B.J., Bateson, A.N., Smart, T.G., King, B.F., Burnstock, G., Barnard, E.A., 1993. Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett. 324, 219–225). The cloning of a membrane protein serving as an ATP receptor ignited a widespread international interest in purinergic signalling. Investigators at University College London (UCL) — colleagues and associates of Geoffrey Burnstock — were at the forefront of this rapid phase of discovery. In this review, we highlight the UCL experience when the fields of molecular biology, physiology and cell biology converged to help advance our understanding of ATP as an extracellular signalling molecule.

Dynamic selectivity filters in ion channels.

This work was supported by a Wellcome Trust (UK) International Prize Travelling Fellowship and the NIH (NS-11756).