P2 Receptor Antagonist Research Articles

Uridine adenosine tetraphosphate is a novel vasodilator in the coronary microcirculation which acts through purinergic P1 but not P2 receptors

Uridine adenosine tetraphosphate (Up4A) has been identified as an endothelium-derived contracting factor, which acts through purinergic P2X and P2Y receptors. Since the coronary vascular actions of Up4A are unknown, we investigated the vasoactive profile of Up4A in coronary microvessels, and studied the involvement of purinergic receptor subtypes. Studies were performed in isolated porcine coronary small arteries (diameter∼250μm), with and without endothelial denudation, mounted on a Mulvany wire myograph. Purinergic receptor expression was assessed by real-time PCR. Up4A (10−9–10−5M) failed to induce contraction at basal tone, but produced concentration-dependent vasorelaxation in precontracted microvessels. Up4A was slightly less potent than adenosine, ATP, and ADP in producing vasorelaxation, but significantly more potent than UTP and UDP. mRNA expression of P2X4, P2Y1, P2Y2, P2Y4, P2Y6 and A2A, but not P2X1, receptors was observed. Up4A-induced vasodilation was unaffected by non-selective P2 receptor antagonist PPADS, P2X1 antagonist MRS2159, P2Y1 antagonist MRS2179 and P2Y6 antagonist MRS2578, but was markedly attenuated by non-selective P1 receptor antagonist 8PT and A2A antagonist SCH58261. Up4A-induced vasodilation was not affected by ectonucleotidase inhibitor ARL67156, suggesting that A2A stimulation was not the result of Up4A breakdown to adenosine. Up4A-induced vasodilation was blunted in denuded vessels; additional A2A receptor blockade further attenuated Up4A-induced vasodilation, suggesting that A2A receptor-mediated vasodilation is only partly endothelium-dependent. In conclusion, Up4A exerts a vasodilator rather than a vasoconstrictor influence in coronary microvessels, which is mediated via A2A receptors and is partly endothelium-dependent.

Leukotoxin fromAggregatibacter actinomycetemcomitanscauses shrinkage and P2X receptor-dependent lysis of human erythrocytes

Leukotoxin (LtxA) is a virulence factor secreted by the bacterium Aggregatibacter actinomycetemcomitans, which can cause localized aggressive periodontitis and endocarditis. LtxA belongs to the repeat-in-toxin (RTX) family of exotoxins of which other members inflict lysis by formation of membrane pores. Recently, we documented that the haemolytic process induced by another RTX toxin [α-haemolysin (HlyA) from Escherichia coli] requires P2X receptor activation and consists of sequential cell shrinkage and swelling. In contrast, the cellular and molecular mechanisms of LtxA-mediated haemolysis are not fully understood. Here, we investigate the effect of LtxA on erythrocyte volume and whether P2 receptors also play a part in LtxA-mediated haemolysis. We observed that LtxA initially decreases the cell size, followed by a gradual rise in volume until the cell finally lyses. Moreover, LtxA triggers phosphatidylserine (PS) exposure in the erythrocyte membrane and both the shrinkage and the PS-exposure is preceded by increments in the intracellular Ca(2+) concentration ([Ca(2+)](i)). Interestingly, LtxA-mediated haemolysis is significantly potentiated by ATP release and P2X receptor activation in human erythrocytes. Furthermore, the LtxA-induced [Ca(2+)](i) increase and following volume changes partially depend on P2 receptor activation. Theseobservations imply that intervention against local P2-mediated auto- and paracrine signalling may prevent LtxA-mediated cell damage.

Trauma-induced expression of astrocytic thrombospondin-1 is regulated by P2 receptors coupled to protein kinase cascades

Thrombospondin-1 (TSP-1) is an extracellular matrix protein produced by astrocytes, which can promote synaptogenesis. The regulation of astrocytic TSP-1 involves extracellular ATP through the activation of P2Y receptors coupled to various protein kinase signaling pathways. However, not much is known about the mechanisms regulating TSP-1 expression in primary cortical astrocytes after a traumatic brain injury. Using an in-vitro model of central nervous system trauma that stimulates the release of ATP, we found that trauma-induced expression and release of TSP-1 involved purinergic signaling as both expression and release were significantly attenuated by pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid, a P2 receptor antagonist. Further antagonist studies with reactive blue 2 point to a role for P2Y4, as reactive blue 2 is a potent antagonist for rat P2Y4 receptors. In addition, the injury-induced expression of TSP-1 was significantly attenuated by the inhibition of extracellular signal-regulated kinase and p38/mitogen-activated protein kinase, whereas injury-induced release of TSP-1 was significantly blocked by the inhibition of extracellular signal-regulated kinase and Akt. Using an in-vivo model of a moderate parasagittal fluid-percussion brain injury, we found that TSP-1 levels were increased when compared with those in sham animals in the cortex, thalamus, and hippocampus. We conclude that TSP-1 expression after injury can be regulated by the activation of P2 receptors coupled with protein kinase signaling pathways and suggest that purinergic signaling, by regulating TSP expression, may play an important role in cell-matrix and cell-cell interactions such as those occurring during central nervous system repair.

Discovery of purinergic signalling, the initial resistance and current explosion of interest.

There has been a remarkable growth of papers published about purinergic signalling via ATP since 1972. I am most grateful to the wonderful PhD students and postdoctoral fellows who have worked with me over the years to pursue the purinergic hypothesis despite early opposition and to the many outstanding scientists around the world who are currently extending the story. Recently, therapeutic approaches to pathological disorders include the development of selective P1 and P2 receptor subtype agonists and antagonists, as well as of inhibitors of extracellular ATP breakdown and of ATP transport enhancers and inhibitors. Medicinal chemists are starting to develop small molecule purinergic drugs that are orally bioavailable and stable in vivo.

Purinergic modulation of hypoxic regulation via the rostral ventral lateral medulla in rats

Anatomical studies have demonstrated the existence of purinergic receptors in the rostral ventral lateral medulla (RVLM), a site containing some respiratory-related neurons. However, little is known about the functional role of these receptors in acute hypoxia. In the present study, we found that both the amplitude and frequency of phrenic nerve discharges were increased during hypoxia. Microinjection of adenosine 5′-triphosphate (ATP) (0.2M, 10–70nl) into the RVLM increased the hypoxic respiratory response and showed significant dose-dependency. An identical microinjection protocol of pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate (PPADS), a broad-spectrum P2 receptor antagonist, into the RVLM markedly attenuated the respiratory effects evoked by hypoxic ventilation. Immunohistochemical analysis showed that the P2X2 receptor was present in the postsynaptic membrane of the RVLM neuronal cell bodies and levels of this receptor were significantly increased after acute hypoxic challenge. These results suggest that RVLM purinergic P2 receptors may contribute to respiratory control by regulating the acute hypoxic ventilatory response.

Reactive oxygen species contribute to the promotion of the ATP-mediated proliferation of mouse skeletal myoblasts

Reactive oxygen species (ROS) and extracellular adenosine 5′-triphosphate (ATP) participate in autocrine and paracrine regulation in skeletal muscle. However, the link between these two signaling systems is not well established. Here, we studied cell proliferation as a possible consequence of the trophic effect of ATP in cultured skeletal mouse myoblasts and we tested the possibility that low concentrations of ROS represent the intermediate signaling molecule mediating this effect. Exposure to 10μM ATP increased proliferation of mouse myoblasts by ∼20%. ATP also induced intracellular Ca2+ oscillations, which were independent of extracellular Ca2+. Both effects of ATP were prevented by suramin, a broad-spectrum purinergic P2 receptor antagonist. In contrast, the adenosine receptor blocker CGS-15943 did not modify the ATP-mediated effects. Consistent with this, adenosine per se did not change myoblast growth, indicating the direct action of ATP via P2 receptor activation. The proliferative effect of ATP was prevented after depletion of hydrogen peroxide (H2O2) by the peroxidase enzyme catalase. Low-micromolar concentrations of exogenous H2O2 mimicked the stimulatory effect of ATP on myoblast growth. DCF imaging revealed ATP-induced catalase and DPI-sensitive ROS production in myoblasts. In conclusion, our results indicate that extracellular ATP controls mouse myoblast proliferation via induction of ROS generation.

Secretion of matrix metalloproteinase-9 from astrocytes by inhibition of tonic P2Y14-receptor-mediated signal(s).

Glial cells have various important roles in regulation of brain functions. For such events, extracellular nucleotides/P2 receptors have central roles. Although there have been huge amount of literature about activation of P2 receptors and glial functions, little is known about what happens in glia or the brain if glial P2 receptor is inhibited. Here we show that the inhibition of P2 receptors in astrocytes, the most abundant glial cells and cause a constitutive release of nucleotides, resulted in secretion of metalloproteinase-9 (MMP-9), a metal-dependent endopeptidase that degrades extracellular matrix molecules and is important in regulation of brain remodeling. When cultured astrocytes were treated with apyrase (ecto-nucleotidase), reactive blue 2 (P2 receptor antagonist), and pertussis toxin, they secreted MMP-9, suggesting that Gi-coupled P2Y receptor-mediated signals constitutively suppress the production of MMP-9. Among Gi-coupled P2Y receptors, we found that an inhibition of P2Y(14) receptor, a receptor for nucleotide-sugars such as UDP-glucose, is responsible for the production of MMP-9 by pharmacological and molecular biochemical analysis. As for the mechanisms, the inhibition of P2Y(14) receptors resulted in the release of tumor necrosis factor (TNF)-α which then acted on astrocytes to induce MMP-9. Taken together, our results suggest that the constitutive releases of nucleotide-sugars in astrocytes should play an important role in maintaining the normal status of the cell, through Gi-coupled P2Y(14) receptors, and when the signal is removed, the cells start to release TNF-α, which then acts on astrocytes in a feedback fashion to boost MMP-9 synthesis and secretion.

Cell type‐specific effects of Adenosine 5′‐triphosphate and pyrophosphate on the antitumor activity of doxorubicin

Extracellular ATP is an important signaling molecule mediating quite divergent specific biological effects. Even though recent studies suggest a potential role of ATP in cancer progress, its real impact in chemotherapeutic efficacy remains unclear. In the present study, we investigated the effect of ATP on the cytotoxicity of doxorubicin in various cancer cell types and found that ATP had no effect on doxorubicin cytotoxicity in colon, prostate, breast, and cervical cancers or in osteosarcoma. In contrast, ATP has divergent effects on lung cancer cells: it can protect against doxorubicin-induced cell death in non-metastatic lung cancer CL1.0 cells, but not in highly metastatic CL1.5 cells. Both apoptotic (characterized by sub-G(1) peak, caspase 3 activation, poly(ADP-ribose) polymerase-1 cleavage) and necrotic (characterized by propidium iodide uptake and ROS production) features induced by doxorubicin in CL1.0 cells were reduced by ATP. In addition, ATP attenuated p53 accumulation, DNA damage (assessed by poly(ADP-ribose) formation and the comet assay) and topoisomerase II inhibition after doxorubicin treatment, and doxorubicin cytotoxicity was diminished by the p53 inhibitor pifithrin-α. Moreover, UTP, UDP, ADP, and pyrophosphate sodium pyrophosphate tetrabasic decahydrate diminished the antitumor effect of doxorubicin in CL1.0 cells, whereas purinergic P2 receptors antagonists did not abrogate the action of ATP. In summary, ATP fails to alter the antitumor efficacy of doxorubicin in most cancer cell types, except in CL1.0 cells, in which pyrophosphate mediates the cell protection afforded by ATP via attenuation of reactive oxygen species production, DNA damage, p53 accumulation, and caspase activation.

Melittin Modulates Keratinocyte Function through P2 Receptor-dependent ADAM Activation

Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-α, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent.

Prospects for using agonists and antagonists of P2 receptors in clinical ophthalmology

This literature review focuses on the role of purinergic P2-receptors in the physiology and pathophysiology of the eye, as well as on the possibilities of pharmacologic stimulation of these receptors. The most important studies from the clinical point of view on the involvement of purinergic neuronal transmission in the physiological processes have been analyzed, ranging from normal embryogenesis to cell apoptosis in age-related degenerative diseases of the eye. Data on the effect of agonists and antagonists of P2 receptors on corneal wound healing, lacrimal fluid production, regulation of intraocular pressure, neurotransmission, proliferation of glial tissue in the retina has been presented along with data on the possibilities of modulating these processes by using potential drugs acting via P2 receptors.

Adenosine‐5′‐triphosphate up‐regulates proliferation of human cardiac fibroblasts

ATP is a potent signalling molecule that regulates biological activities including increasing or decreasing proliferation in different types of cells. The aim of the present study was to investigate how ATP regulates the proliferation of human cardiac fibroblasts. Reverse transcription (RT)-PCR, Western blot analysis, cell proliferation and migration assays were employed to investigate the effects of ATP on human adult ventricular fibroblasts. ATP increased cell proliferation in a concentration-dependent manner. Similarly, the P2X receptor agonist α,β-methylene ATP and P2Y receptor agonist ATP-γS also up-regulated cell proliferation. The P2 receptor antagonists suramin and reactive blue-2 prevented the ATP-induced increase in proliferation and RT-PCR and Western blot analysis revealed that mRNAs of P2X(4/7) and P2Y(2) are abundant in cardiac fibroblasts. ATP increased phosphorylated PKB (Akt) and ERK1/2 levels; an effect antagonized by suramin, reactive blue-2, the PI3-kinase inhibitor, wortmannin, PKB inhibitor, API-2, and MAPK inhibitor, PD98059. These kinase inhibitors also prevented the ATP-induced increase in proliferation. In addition, ATP enhanced the progression of cells from the G0/G1 phase to the S phase by increasing the expression of proteins for cyclin D1 and cyclin E. Silencing the P2X(4/7) and P2Y(2) receptors with siRNA targeting the corresponding receptor diminished ATP-stimulated proliferation and migration of the cardiac fibroblasts. ATP activates P2X(4/7) and P2Y(2) receptors and up-regulates the proliferation of human cardiac fibroblasts by promoting cell cycling progression. It also increases the migration of these cells. These effects of ATP may be involved in cardiac remodelling of injured hearts.

Regulation of ventral surface CO2/H+‐sensitive neurons by purinergic signalling

Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO(2)H+. A brainstem region called the retrotrapezoid nucleus (RTN) contains a population of CO2/H+-sensitive neurons that appears to function as an important chemoreceptor. Evidence also indicates that CO2-evoked ATP release from RTN astrocytes modulates activity of CO2/H+-sensitive neurons; however, the extent to which purinergic signalling contributes to chemoreception by RTN neurons is not clear and the mechanism(s) underlying CO2/H+-evoked ATP release is not fully elucidated. The goals of this study are to determine the extent to which ATP contributes to RTN chemoreception both in vivo and in vitro, and whether purinergic drive to chemoreceptors relies on extracellular Ca(2+) or gap junction hemichannels. We also examine the possible contribution of P2Y1 receptors expressed in the RTN to the purinergic drive to breathe.We show that purinergic signalling contributes, in part, to the CO(2)/H+ sensitivity of RTN neurons. In vivo, phrenic nerve recordings of respiratory activity in adult rats show that bilateral injections of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS, a P2 receptor blocker) decreased the ventilatory response to CO2 by 30%. In vitro, loose-patch recordings from RTN neurons show that P2 receptor blockers decreased responsiveness to both 10% and 15% CO2 also by 30%. In the slice, the contribution of purinergic signalling to RTN chemoreception did not increase with temperature (22–35◦C) and was retained in low extracellular Ca2+ medium. Conversely, the gap junction blockers carbenoxolone and cobalt decreased neuronal CO2/H+ sensitivity by an amount similar to P2 receptor antagonists. Inhibition of the P2Y1 receptor in the RTN had no effect on CO2 responsivness in vitro or in vivo; thus, the identity of P2 receptors underlying the purinergic component of RTN chemoreception remains unknown. These results support the possibility that CO2/H+-evoked ATP release is mediated by a mechanism involving gap junction hemichannels.

ATP mediates flow-induced NO production in thick ascending limbs

Mechanical stimulation caused by increasing flow induces nucleotide release from many cells. Luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in thick ascending limbs. However, the factors that mediate flow-induced NO production are unknown. We hypothesized that luminal flow stimulates thick ascending limb NO production via ATP. We measured NO in isolated, perfused rat thick ascending limbs using the fluorescent dye DAF FM. The rate of increase in dye fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nl/min stimulated NO production from 17 ± 16 to 130 ± 37 arbitrary units (AU)/min (P < 0.02). Increasing flow from 0 to 20 nl/min raised ATP release from 4 ± 1 to 21 ± 6 AU/min (P < 0.04). Hexokinase (10 U/ml) plus glucose, which consumes ATP, completely prevented the measured increase in ATP. Luminal flow did not increase NO production in the presence of luminal and basolateral hexokinase (10 U/ml). When flow was increased with the ATPase apyrase in both luminal and basolateral solutions (5 U/ml), NO levels did not change significantly. The P2 receptor antagonist suramin (300 μmol/l) reduced flow-induced NO production by 83 ± 25% (P < 0.03) when added to both and basolateral sides. Luminal hexokinase decreased flow-induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min (P < 0.02). Basolateral hexokinase also reduced flow-induced NO production. The P2X receptor-selective antagonist NF023 (200 μmol/l) prevented flow-induced NO production when added to the basolateral side but not the luminal side. We conclude that ATP mediates flow-induced NO production in the thick ascending limb likely via activation of P2Y receptors in the luminal and P2X receptors in the basolateral membrane.

Pannexin1-Mediated ATP Release Provides Signal Transmission Between Neuro2A Cells

Pannexin1 (Panx1), a protein related to the gap junction proteins of invertebrates, forms nonjunctional channels that open upon depolarization and in response to mechanical stretch and purinergic receptor stimulation. Importantly, ATP can be released through Panx1 channels, providing a possible role for these channels in non-vesicular signal transmission. In this study we expressed exogenous human and mouse Panx1 in the gap junction deficient Neuro2A neuroblastoma cell line and explored the contribution of Panx1 channels to cell-cell communication as sites of ATP release. Electrophysiological (patch clamp) recordings from Panx1 transfected Neuro2A cells revealed membrane conductance that increased beyond 0 mV when applying voltage ramps from -60 to +100 mV; threshold was correlated with extracellular K+, so that at 10 mM K+, channels began to open at -30 mV. Evaluation of cell-cell communication using dual whole cell recordings from cell pairs revealed that activation of Panx1 current in one cell of the pair induced an inward current in the second cell after a latency of 10-20 s. This paracrine response was amplified by an ATPase inhibitor (ARL67156, 100 μM) and was blocked by the ATP-degrading enzyme apyrase (6.7 U/ml), by the P2 receptor antagonist suramin (50 μM) and by the Panx1 channel blocker carbenoxolone. These results provide additional evidence that ATP release through Panx1 channels can mediate nonsynaptic bidirectional intercellular communication. Furthermore, current potentiation by elevated K+ provides a mechanism for enhancement of ATP release under pathological conditions.

Autocrine regulation of TGF-β1-induced cell migration by exocytosis of ATP and activation of P2 receptors in human lung cancer cells

TGF-β1 plays a key role in cancer progression through induction of various biological effects, including cell migration. Extracellular nucleotides, such as ATP, released from cells play a role in signaling through activation of P2 receptors. We show here that exocytosis of ATP followed by activation of P2 receptors play a key role in TGF-β1-induced actin remodeling associated with cell migration. Treatment with TGF-β1 facilitated migration of human lung cancer A549 cells, which was blocked by pretreatment with ecto-nucleotidase and P2 receptor antagonists. ATP and P2 agonists facilitated cell migration. TGF-β1-induced actin remodeling, which contributes to cell migration, was also suppressed by pretreatment with ecto-nucleotidase and P2 receptor antagonists. Knockdown of P2X7 receptor suppressed TGF-β1-induced migration and actin remodeling. These results indicate the involvement of TGF-β1-induced ATP release in cell migration, at least in part, through activation of P2X7 receptors. TGF-β1 caused release of ATP from A549 cells within 10 minutes. Both ATP-enriched vesicles and expression of a vesicular nucleotide transporter (VNUT) SLC17A9, which is responsible for exocytosis of ATP, were found in cytosol of A549 cells. TGF-β1 failed to induce release of ATP from SLC17A9-knockdown cells. TGF-β1-induced cell migration and actin remodeling were also decreased in SLC17A9-knockdown cells. These results suggest the importance of exocytosis of ATP in cell migration. We conclude that autocrine signaling through exocytosis of ATP and activation of P2 receptors is required for the amplification of TGF-β1-induced migration of lung cancer cells.

Purinergic modulation of the excitatory synaptic input onto rat striatal neurons

There is no in situ evidence hitherto for a modulation by ATP of the glutamatergic excitatory transmission onto medium spiny neurons (MSNs) in the rat striatum. In order to resolve this question, we used the patch-clamp technique in brain slice preparations to record excitatory postsynaptic currents (EPSCs) evoked by intrastriatal electrical stimulation and applied N-methyl-d-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to activate transmembrane currents of MSNs. In the absence of external Mg2+, ATP caused a higher maximum inhibition of the EPSCs than adenosine. Only P1 (A1), but not P2 receptor antagonists interfered with the effects of both ATP and adenosine. Moreover, A1 receptor antagonists were less potent in blocking the inhibition by ATP than that by adenosine. Eventually, adenosine deaminase (ADA) almost abolished the adenosine-induced inhibition, but only moderately decreased the ATP-induced inhibition. Antagonists of A1 receptors (but not of P2 receptors) counteracted the depression by ATP of the current responses to exogenous NMDA, without altering those to AMPA. It is suggested that ATP indirectly, via its degradation product adenosine, stimulates presynaptic inhibitory A1 receptors situated at glutamatergic nerve terminals of striatal afferents; these nerve terminals are devoid of P2 receptors. However, ATP, in contrast to adenosine, also activates postsynaptic A1 receptors at the MSN neurons themselves. The resulting negative interaction with NMDA receptors requires localized extracellular catabolism of ATP by ectonucleotidases.

An ATP signalling pathway in plant cells: extracellular ATP triggers programmed cell death in Populus euphratica

We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose- and time-dependent reduction in viability, and the agonist-treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca(2+) levels, resulting in Ca(2+) uptake by the mitochondria and subsequent H(2) O(2) accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP-induced increase of intracellular ATP, essential for the activation of caspase-like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca(2+) concentration but plays a negligible role in eATP-stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.

Signaling Mechanisms Mediating Uridine Adenosine Tetraphosphate–induced Proliferation of Human Vascular Smooth Muscle Cells

Proliferation of vascular smooth muscle cells (SMCs) plays an important role in the development of atherosclerosis and restenosis. Extracellular mononucleotides, such as adenosine triphosphate and uridine-5'-triphosphate stimulate SMC proliferation. However, the effects of dinucleotides on SMC proliferation and their underlying signaling mechanisms are less well defined. Recently, increasing evidence suggests that the dinucleotide, uridine adenosine tetraphosphate (Up4A) plays a role in the regulation of cardiovascular function. We have previously demonstrated that Up4A stimulates DNA synthesis and proliferation of human SMCs. This study investigated the signaling mechanisms underlying the proliferative effect of Up4A. Up4A-induced increase in bromodeoxyuridine incorporation was blocked by the mammalian target of rapamycin inhibitor, rapamycin, and the MEK inhibitor, PD98059. Up4A-stimulated phosphorylation and kinase activity of S6 kinase (S6K) and Erk1/2 were inhibited by PD98059, whereas phosphorylation and kinase activity of S6K, but not Erk1/2, were inhibited by rapamycin. Up4A also increased the phosphorylation of Akt, which was blocked by the PI3-kinase inhibitor, LY294002. Up4A-stimulated activation of S6K, but not Erk1/2, was also prevented by LY294002. Furthermore, Up4A-stimulated phosphorylation and kinase activity of S6K and Erk1/2 were inhibited by the P2 receptor antagonist, suramin, but not by the P2X receptor antagonist, Ip5I. Up4A also stimulated an increase in the protein expression of cycle-dependent kinase 2, which was prevented by rapamycin, PD98059, and suramin. These results suggest that the signaling mechanisms underlying the Up4A-stimulated proliferation of SMCs are mediated by P2Y receptors and involve the PI3-K/Akt and mitogen-activated protein kinase pathways, leading to the independent activation of S6K and an increase in cycle-dependent kinase 2 expression. This work stresses the concept that dinucleotides, like mononucleotides, play potentially important roles in the regulation of vascular function.

Basolateral P2X receptors mediate inhibition of NaCl transport in mouse medullary thick ascending limb (mTAL)

Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors, which mediate significant inhibition of solute absorption. Recently, we identified several P2 receptors in the medullary thick ascending limb (mTAL) including luminal and basolateral P2Y(2) receptors (Jensen ME, Odgaard E, Christensen MH, Praetorius HA, Leipziger J. J Am Soc Nephrol 18: 2062-2070, 2007). In addition, we found evidence for a basolateral P2X receptor. Here, we investigate the effect of basolateral ATP on NaCl absorption in isolated, perfused mouse mTALs using the electrical measurement of equivalent short-circuit current (I'(sc)). Nonstimulated mTALs transported at a rate of 1,197 ± 104 μA/cm(2) (n = 10), which was completely blockable with luminal furosemide (100 μM). Basolateral ATP (100 μM) acutely (1 min) and reversibly reduced the absorptive I'(sc). After 2 min, the reduction amounted to 24.4 ± 4.0% (n = 10). The nonselective P2 receptor antagonist suramin blocked the effect. P2Y receptors were found not to be involved in this effect. The P2X receptor agonist 2-methylthio ATP mimicked the ATP effect, and the P2X receptor antagonist periodate-oxidized ATP blocked it. In P2X(7)(-/-) mice, the ATP effect remained unaltered. In contrast, in P2X(4)(-/-) mice the ATP-induced inhibition of transport was reduced. A comprehensive molecular search identified P2X(4), P2X(5), and P2X(1) receptor subunit mRNA in isolated mouse mTALs. These data define that basolateral ATP exerts a significant inhibition of Na(+) absorption in mouse mTAL. Pharmacological, molecular, and knockout mouse data identify a role for the P2X(4) receptor. We suggest that other P2X subunits like P2X(5) are part of the P2X receptor complex. These data provide the novel perspective that an ionotropic receptor and thus a nonselective cation channel causes transport inhibition in an intact renal epithelium.

Age-related changes in afferent responses in sensory neurons to mechanical stimulation of osteoblasts in coculture system

Bone homeostasis is regulated by mechanical stimulation (MS). The sensory mechanism of bone tissue for MS remains unknown in the maintenance of bone homeostasis. We aimed to investigate the sensory mechanism from osteoblasts to sensory neurons in a coculture system by MS of osteoblasts. Primary sensory neurons isolated from dorsal root ganglia (DRG) of neonatal, juvenile, and adult mice and osteoblasts isolated from calvaria of neonatal mice were cocultured for 24 h. The responses in DRG neurons elicited by MS of osteoblasts with a glass micropipette were detected by increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) with fluo 3-AM. In all developmental stages mice, [Ca(2+)](i)-increasing responses in osteoblasts were promptly elicited by MS. After a short delay, [Ca(2+)](i)-increasing responses were observed in neurites of DRG neurons. The osteoblastic response to second MS was largely attenuated by a stretch-activated Ca(2+) channel blocker, gadolinium. The increases of [Ca(2+)](i) in DRG neurons were abolished by a P2 receptor antagonist; suramin, a P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate; and an ATP-hydrolyzing enzyme, apyrase. Satellite cells were found around DRG neurons in cocultured cells of only neonatal and juvenile mice. After satellite cells were removed, excessive abnormal responses to MS of osteoblasts were observed in neonatal neurites with unchanged osteoblast responses. The present study indicated that MS of bone tissue elicited afferent P2X receptor-mediated purinergic transmission to sensory neurons in all stages mice. This transmission is modulated by satellite cells, which may have protective actions on sensory neurons.