apyrase and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

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.

Topics: Adenosine Triphosphate; Age Factors; Aniline Compounds; Animals; Animals, Newborn; Apyrase; Calcium; Calcium Channels; Coculture Techniques; Gadolinium; Ganglia, Spinal; Mechanotransduction, Cellular; Mice; Mice, Inbred BALB C; Neurites; Osteoblasts; Osteogenesis; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X; Satellite Cells, Perineuronal; Sensory Receptor Cells; Suramin; Xanthenes

This study investigates the role of extracellular nucleotides and apyrase enzymes in regulating stomatal aperture. Prior data indicate that the expression of two apyrases in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, is strongly correlated with cell growth and secretory activity. Both are expressed strongly in guard cell protoplasts, as determined by reverse transcription-polymerase chain reaction and immunoblot analyses. Promoter activity assays for APY1 and APY2 show that expression of both apyrases correlates with conditions that favor stomatal opening. Correspondingly, immunoblot data indicate that APY expression in guard cell protoplasts rises quickly when these cells are moved from darkness into light. Both short-term inhibition of ectoapyrase activity by polyclonal antibodies and long-term suppression of APY1 and APY2 transcript levels significantly disrupt normal stomatal behavior in light. Stomatal aperture shows a biphasic response to applied adenosine 5'-[γ-thio]triphosphate (ATPγS) or adenosine 5'-[β-thio] diphosphate, with lower concentrations inducing stomatal opening and higher concentrations inducing closure. Equivalent concentrations of adenosine 5'-O-thiomonophosphate have no effect on aperture. Two mammalian purinoceptor inhibitors block ATPγS- and adenosine 5'-[β-thio] diphosphate-induced opening and closing and also partially block the ability of abscisic acid to induce stomatal closure and of light to induce stomatal opening. Treatment of epidermal peels with ATPγS induces increased levels of nitric oxide and reactive oxygen species, and genetically suppressing the synthesis of these agents blocks the effects of nucleotides on stomatal aperture. A luciferase assay indicates that treatments that induce either the closing or opening of stomates also induce the release of ATP from guard cells. These data favor the novel conclusion that ectoapyrases and extracellular nucleotides play key roles in regulating stomatal functions.

Topics: Abscisic Acid; Adenosine Diphosphate; Adenosine Triphosphate; Apyrase; Arabidopsis; Arabidopsis Proteins; Enzyme Inhibitors; Extracellular Space; Gene Expression Regulation, Plant; Hydrogen Peroxide; Light; Models, Biological; Nitric Oxide; Nucleotides; Plant Stomata; Promoter Regions, Genetic; Pyridoxal Phosphate; RNA Interference; Thionucleotides; Triazines

In this work, we show that P2 nucleotide receptors control lipopolysaccharide (LPS)-induced neutrophil migration in the mouse air pouch model. Neutrophil infiltration in LPS-treated air pouches was reduced by the intravenous (iv) administration of the non-selective P2 receptor antagonist PPADS but not by suramin and RB-2. In addition, the iv administration of a P2 receptor ligand, UTP, enhanced LPS-induced neutrophil migration. In contrast, the iv injection of UDP had no effect on neutrophil migration. These data suggest that LPS-induced neutrophil migration in the air pouch could involve P2Y(4) receptor which is antagonized by PPADS, activated by UTP, but not UDP, and insensitive to suramin. The inhibition of neutrophil migration by PPADS correlated with a diminished secretion of chemokines macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC) in the air pouch exudates. As determined in vitro, PPADS did not affect MIP-2 and KC release from air pouch resident cells nor from accumulated neutrophils. MIP-2 and KC production in the LPS-treated air pouches correlated with an early neutrophil migration (1h after LPS injection), and both of these effects were significantly reduced in mice administered with PPADS. Altogether, these data suggest that P2Y(4) receptor expressed in circulating leukocytes and/or endothelium controls LPS-induced acute neutrophil recruitment in mouse air pouch.

Topics: Animals; Apyrase; Cell Movement; Chemokine CXCL2; Chemokines; Disease Models, Animal; Exudates and Transudates; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Neutrophils; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Synovial Membrane; Toll-Like Receptors; Uridine Triphosphate

Capsaicin-sensitive lung vagal afferents (CSLVAs) are important in detecting pulmonary reactive oxygen species (ROS). We investigated the mechanisms underlying the stimulation of CSLVAs by inhaled cigarette smoke (CS) in 216 anesthetized rats. In spontaneously breathing rats, CS evoked a CSLVA-mediated reflex bradypnea that was prevented by N-acetyl-L-cysteine (NAC; an antioxidant), HC-030031 [a transient receptor potential ankyrin 1 (TRPA1) receptor antagonist], and iso-pyridoxalphosphate-6-azophenyl-2',5'-disulfonate (iso-PPADS; a P2X receptor antagonist). In paralyzed, artificially ventilated rats, CS evoked an increase in CSLVA fiber activity (DeltaFA) that was abolished by NAC and was attenuated by HC-030031, iso-PPADS, indomethacin (Indo; a cyclooxygenase inhibitor), and a combination of apyrase and adenosine deaminase (ADA) (ATP scavengers); the response to CS was reduced to 11.7+/-4.0%, 39.5+/-10.0%, 52.9+/-14.4%, 68.7+/-10.1%, and 47.2+/-12.9% of control, respectively. The suppressive effect on this afferent response was not improved by a combination of HC-030031 and Indo (DeltaFA=39.5+/-10.1% of control) compared with that induced by HC-030031 alone. In contrast, the suppressive effect was enhanced by a combination of HC-030031 and apyrase+ADA (DeltaFA=5.3+/-4.9% of control) or a combination of iso-PPADS and Indo (DeltaFA=23.3+/-7.7% of control) compared with that induced by HC-030031 alone or iso-PPADS alone. This afferent response was not altered by the vehicles for these drugs. These results suggest that activations of TRPA1 receptors by cyclooxygenase metabolites and P2X receptors by ATP are both necessary for the ROS-mediated stimulation of CSLVA fibers by CS in rats.

Topics: Acetylcysteine; Adenosine Deaminase; Adenosine Triphosphate; Animals; Ankyrins; Antioxidants; Apyrase; Blood Pressure; Calcium Channels; Capsaicin; Cyclooxygenase Inhibitors; Heart Rate; Indomethacin; Inhalation Exposure; Lung; Male; Neurons, Afferent; Purinergic P2 Receptor Agonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Purinergic P2; Receptors, Purinergic P2X; Reflex; Respiration, Artificial; Respiratory Mechanics; Sensory System Agents; Signal Transduction; Smoke; Smoking; Time Factors; TRPA1 Cation Channel; TRPC Cation Channels; Vagus Nerve

The application of a polysaccharide elicitor from yeast extract, YE, to Salvia miltiorrhiza hairy root cultures induced transient release of ATP from the roots to the medium, leading to a dose-dependent increase in the extracellular ATP (eATP) level. The eATP level rose to a peak (about 6.5 nM with 100 mg l(-1) YE) at about 10 h after YE treatment, but dropped to the control level 6 h later. The elicitor-induced ATP release was dependent on membrane Ca2+ influx, and abolished by the Ca2+ chelator EGTA or the channel blocker La3+. The YE-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors. On the other hand, the application of exogenous ATP at 10-100 microM to the cultures also induced rapid and dose-dependent increases in H2O2 production and medium pH, both of which were effectively blocked by RB and EGTA. The non-hydrolyzable ATP analog ATPgammaS was as effective as ATP, but the hydrolyzed derivatives ADP or AMP were not so effective in inducing the pH and H2O2 increases. Our results suggest that ATP release is an early event and that eATP plays a signaling role in the elicitation of plant cell responses; Ca2+ is required for activation of the elicitor-induced ATP release and the eATP signal transduction. This is the first report on ATP release induced by a fungal elicitor and its involvement in the elicitor-induced responses in plant cells.

Topics: Adenosine Triphosphate; Apyrase; Calcium Signaling; Cell Membrane; Cell Survival; Egtazic Acid; Evans Blue; Extracellular Space; Hydrogen Peroxide; Hydrogen-Ion Concentration; Plant Roots; Pyridoxal Phosphate; Salvia miltiorrhiza; Signal Transduction

Brain astrocytes signal to each other and neurons. They use changes in their intracellular calcium levels to trigger release of transmitters into the extracellular space. These can then activate receptors on other nearby astrocytes and trigger a propagated calcium wave that can travel several hundred micrometers over a timescale of seconds. A role for endogenous ATP in calcium wave propagation in hippocampal astrocytes has been suggested, but the mechanisms remain incompletely understood. Here we explored how calcium waves arise and directly tested whether endogenously released ATP contributes to astrocyte calcium wave propagation in hippocampal astrocytes. We find that vesicular ATP is the major, if not the sole, determinant of astrocyte calcium wave propagation over distances between approximately 100 and 250 microm, and approximately 15 s from the point of wave initiation. These actions of ATP are mediated by P2Y1 receptors. In contrast, metabotropic glutamate receptors and gap junctions do not contribute significantly to calcium wave propagation. Our data suggest that endogenous extracellular astrocytic ATP can signal over broad spatiotemporal scales.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aniline Compounds; Animals; Apyrase; Astrocytes; Brefeldin A; Calcium; Calcium Signaling; Cells, Cultured; Cytoplasmic Vesicles; Diffusion; Enzyme Inhibitors; Fluorescent Dyes; Hippocampus; Macrolides; Neurons; Paracrine Communication; Proton-Translocating ATPases; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Suramin; Time Factors; Xanthenes

Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously, we showed that hypotonic swelling of alveolar A549 cells induces Ca(2+)-dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. In this study, we examined sources of Ca(2+) for the elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by acute 50% hypotonic stress and the role of autocrine purinergic signalling in Ca(2+)-dependent ATP release. We found that ATP release does not directly involve Ca(2+) influx from extracellular spaces, but depends entirely on Ca(2+) mobilization from intracellular stores. The [Ca(2+)](i) response consisted of slowly rising elevation, representing mobilization from thapsigargin (TG)-insensitive stores and a superimposed rapid spike due to Ca(2+) release from TG-sensitive endoplasmic reticulum (ER) Ca(2+) stores. The latter could be abolished by hydrolysis of extracellular triphospho- and diphosphonucleotides with apyrase; blocking P2Y(2)/P2Y(6) receptors of A549 cells with suramin; blocking UDP receptors (P2Y(6)) with pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS); emptying TG-sensitive stores downstream with TG or caffeine in Ca(2+)-free extracellular solution; or blocking the Ca(2+)-release inositol 1,4,5-triphosphate receptor channel of the ER with 2-aminoethyldiphenylborinate. These data demonstrate that the rapid [Ca(2+)](i) spike results from the autocrine stimulation of IP(3)/Ca(2+)-coupled P2Y, predominantly P2Y(6), receptors, accounting for approximately 70% of total Ca(2+)-dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress-induced ATP release from alveolar cells is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nucleotides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.

Topics: Adenosine Triphosphate; Apyrase; Autocrine Communication; Caffeine; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Inhibitors; Epithelial Cells; Humans; Hydrolysis; Hypotonic Solutions; Inositol 1,4,5-Trisphosphate Receptors; Lung Neoplasms; Osmotic Pressure; Paracrine Communication; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Suramin; Thapsigargin; Time Factors; Uracil Nucleotides

Inhalation of H2O2 is known to evoke bradypnea followed by tachypnea, which are reflexes resulting from stimulation by reactive oxygen species of vagal lung capsaicin-sensitive and myelinated afferents, respectively. This study investigated the pharmacological receptors and chemical mediators involved in triggering these responses. The ventilatory responses to 0.2% aerosolized H2O2 were studied before and after various pharmacological pretreatments in anesthetized rats. The initial bradypneic response was reduced by a transient receptor potential vanilloid 1 (TRPV1) receptor antagonist [capsazepine; change (Delta) = -53%] or a P2X purinoceptor antagonist [iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (PPADS); Delta = -47%] and was further reduced by capsazepine and iso-PPADS in combination (Delta = -78%). The initial bradypneic response was reduced by a cyclooxygenase inhibitor (indomethacin; Delta = -48%), ATP scavengers (apyrase and adenosine deaminase in combination; Delta = -50%), or capsazepine and indomethacin in combination (Delta = -47%), was further reduced by iso-PPADS and indomethacin in combination (Delta = -75%) or capsazepine and ATP scavengers in combination (Delta = -83%), but was not affected by a lipoxygenase inhibitor (nordihydroguaiaretic acid) or by any of the various vehicles. No pretreatment influenced delayed tachypnea. We concluded that 1) the initial bradypneic response to H2O2 results from activation of both TRPV1 and P2X receptors, possibly located at terminals of vagal lung capsaicin-sensitive afferent fibers; 2) the functioning of the TRPV1 and P2X receptors in triggering the initial bradypnea is, in part, mediated through the actions of cyclooxygenase metabolites and ATP, respectively; and 3) these mechanisms do not contribute to the H2O2-evoked delayed tachypnea.

Topics: Adenosine Deaminase; Adenosine Triphosphate; Animals; Apyrase; Arachidonic Acid; Capsaicin; Cyclooxygenase Inhibitors; Hydrogen Peroxide; Hyperventilation; Hypoventilation; Indomethacin; Lipoxygenase Inhibitors; Male; Oxidants; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Purinergic P2; Receptors, Purinergic P2X; Reflex; Respiratory Mechanics; TRPV Cation Channels

Using a guinea pig heart synaptosomal preparation, we previously observed that norepinephrine (NE) exocytosis was attenuated by a blockade of P2X purinoceptors, potentiated by inhibition of ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1)/CD39, and reduced by soluble CD39, a recombinant form of human E-NTPDase1/CD39. This suggests that norepinephrine and ATP are coreleased upon depolarization of cardiac sympathetic nerve endings and that ATP enhances norepinephrine exocytosis by an action modulated by E-NTPDase1/CD39 activity. Whether E-NTPDase1/CD39 is localized to cardiac neurons and modulates norepinephrine exocytosis in intact heart tissue remained untested. We report that E-NTPDase1/CD39 is selectively localized in human and porcine cardiac neurons and that depolarization of porcine heart tissue elicits omega-conotoxin-inhibitable release of both norepinephrine and ATP. Inhibition of E-NTPDase1/CD39 with ARL67156 markedly potentiated ATP release, demonstrating that E-NTPDase1/CD39 is a major determinant of ATP availability at sympathetic nerve terminals. Notably, inhibition of E-NTPDase1/CD39 enhanced both ATP and NE exocytosis, whereas administration of soluble CD39 reduced both ATP and NE exocytosis. The strong correlation between ATP and norepinephrine release was abolished in the presence of the purinergic P2X receptor (P2XR) antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). We conclude that released ATP governs norepinephrine exocytosis by activating presynaptic P2XR and that this action is controlled by neuronal E-NTPDase1/CD39. Clinically, excessive norepinephrine release is a major cause of arrhythmic and coronary vascular dysfunction during myocardial ischemia. By curtailing NE release, in addition to its effects as an antithrombotic agent, soluble CD39 may constitute a novel therapeutic approach to ischemic complications in the myocardium.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Exocytosis; Female; Humans; Male; Middle Aged; Myocardium; Neurons; Norepinephrine; Pyridoxal Phosphate; Swine

Central sensitization represents a sustained hypersensitive state of dorsal horn nociceptive neurons that can be evoked by peripheral inflammation or injury to nerves and tissues. It reflects neuroplastic changes such as increases in neuronal spontaneous activity, receptive field size, and responses to suprathreshold stimuli and a decrease in activation threshold. We recently demonstrated that purinergic receptor mechanisms in trigeminal subnucleus caudalis (Vc; medullary dorsal horn) are also involved in the initiation and maintenance of central sensitization in brain stem nociceptive neurons of trigeminal subnucleus oralis. The aim of the present study was to investigate whether endogenous ATP is involved in the development of central sensitization in Vc itself. The experiments were carried out on urethan/alpha-chloralose anesthetized and immobilized rats. Single neurons were recorded and identified as nociceptive-specific (NS) in the deep laminae of Vc. During continuous saline superfusion (0.6 ml/h it) over the caudal medulla, Vc neuronal central sensitization was readily induced by mustard oil application to the tooth pulp. However, this mustard-oil-induced central sensitization could be completely blocked by continuous intrathecal superfusion of the wide-spectrum P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2, 4-disulphonic acid tetra-sodium (33-100 microM) and by apyrase (an ectonucleotidase enzyme, 30 units/ml). Superfusion of the selective P2X1, P2X3 and P2X(2/3) receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (6-638 microM) partially blocked the Vc central sensitization. The two P2X receptor antagonists did not significantly affect the baseline nociceptive properties of the Vc neurons. These findings implicate endogenous ATP as an important mediator contributing to the development of central sensitization in nociceptive neurons of the deep laminae of the dorsal horn.

Topics: Action Potentials; Adenosine Triphosphate; Analysis of Variance; Animals; Apyrase; Brain Mapping; Drug Interactions; Male; Mustard Plant; Neurons; Nociceptors; Physical Stimulation; Plant Extracts; Plant Oils; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Sensory Thresholds; Stimulation, Chemical; Trigeminal Caudal Nucleus

The umbrella cells that line the bladder are mechanosensitive, and bladder filling increases the apical surface area of these cells; however, the upstream signals that regulate this process are unknown. Increased pressure stimulated ATP release from the isolated uroepithelium of rabbit bladders, which was blocked by inhibitors of vesicular transport, connexin hemichannels, ABC protein family members, and nucleoside transporters. Pressure-induced increases in membrane capacitance (a measure of apical plasma membrane surface area where 1 microF approximately equals 1 cm2) were inhibited by the serosal, but not mucosal, addition of apyrase or the purinergic receptor antagonist PPADS. Upon addition of purinergic receptor agonists, increased capacitance was observed even in the absence of pressure. Moreover, knockout mice lacking expression of P2X2 and/or P2X3 receptors failed to show increases in apical surface area when exposed to hydrostatic pressure. Treatments that prevented release of Ca2+ from intracellular stores or activation of PKA blocked ATPgammaS-stimulated changes in capacitance. These results indicate that increased hydrostatic pressure stimulates release of ATP from the uroepithelium and that upon binding to P2X and possibly P2Y receptors on the umbrella cell, downstream Ca2+ and PKA second messenger cascades may act to stimulate membrane insertion at the apical pole of these cells.

Topics: Adenosine Triphosphate; Animals; Apyrase; Calcium; Cell Membrane; Cyclic AMP-Dependent Protein Kinases; Electric Capacitance; Endocytosis; Exocytosis; Female; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Purinergic P2 Receptor Agonists; Pyridoxal Phosphate; Rabbits; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Signal Transduction; Urinary Bladder; Urothelium

We have investigated expression and function of the P2X7 receptor in fibroblasts from healthy subjects and patients with type 2 diabetes.. Fibroblasts were isolated from skin biopsies. P2X7 receptor expression in both cell populations was measured by functional assays, RT-PCR, fluorescence-activated cell sorter, and immunoblotting. We found that fibroblasts from diabetic subjects are characterized by enhanced P2X7-mediated responses as indicated by increased shape changes, microvesiculation, enhanced fibronectin and interleukin 6 secretion, and accelerated apoptosis. These responses were blocked by preincubation with the P2X blockers KN-62, oxidized ATP, or pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid). Furthermore, we also found a higher level of spontaneous fibronectin secretion and of apoptosis in fibroblasts from diabetic compared with healthy subjects. Both higher basal level of fibronectin secretion and spontaneous rate of apoptosis were likely attributable to the increased pericellular concentration of ATP because fibroblasts from diabetic subjects released 3x as much ATP into the supernatants compared with fibroblasts from healthy subjects.. We conclude that fibroblasts from type 2 diabetes patients are characterized by a hyperactive purinergic loop based either on a higher level of ATP release or on increased P2X7 reactivity.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Diphosphate; Adenosine Triphosphate; Apoptosis; Apyrase; Autocrine Communication; Cell Shape; Cytidine Triphosphate; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Interleukin-6; Membrane Potentials; Paracrine Communication; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Uridine Diphosphate; Uridine Triphosphate

Calcium signals can be communicated between cells by the diffusion of a second messenger through gap junction channels or by the release of an extracellular purinergic messenger. We investigated the contribution of these two pathways in endothelial cell lines by photoliberating InsP(3) or calcium from intracellular caged precursors, and recording either the resulting intercellular calcium wave or else the released ATP with a luciferin/luciferase assay. Photoliberating InsP(3) in a single cell within a confluent culture triggered an intercellular calcium wave, which was inhibited by the gap junction blocker alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptide gap 26, the purinergic inhibitors suramin, PPADS and apyrase and by purinergic receptor desensitisation. InsP(3)-triggered calcium waves were able to cross 20 microm wide cell-free zones. Photoliberating InsP(3) triggered ATP release that was blocked by buffering intracellular calcium with BAPTA and by applying gap 26. Gap 26, however, did not inhibit the gap junctional coupling between the cells as measured by fluorescence recovery after photobleaching. Photoliberating calcium did not trigger intercellular calcium waves or ATP release. We conclude that InsP(3)-triggered ATP release through connexin hemichannels contributes to the intercellular propagation of calcium signals.

Topics: Adenosine Triphosphate; Animals; Apyrase; Calcium; Calcium Signaling; Cell Communication; Cells, Cultured; Chelating Agents; Connexins; Endothelium; Fluorescence Recovery After Photobleaching; Gap Junctions; Glycyrrhetinic Acid; Inositol 1,4,5-Trisphosphate; Peptides; Photolysis; Pyridoxal Phosphate; Rats; Second Messenger Systems; Signal Transduction

ATP release from astrocytes contributes to calcium ([Ca(2+)]) wave propagation and may modulate neuronal excitability. In epithelial cells and hepatocytes, cell swelling causes ATP release, which leads to the activation of a volume-sensitive Cl(-) current (I(Cl,swell)) through an autocrine pathway involving purinergic receptors. Astrocyte swelling is counterbalanced by a regulatory volume decrease, involving efflux of metabolites and activation of I(Cl,swell) and K(+) currents. We used whole cell patch-clamp recordings in cultured astrocytes to investigate the autocrine role of ATP in the activation of I(Cl,swell) by hypo-osmotic solution (HOS). Apyrase, an ATP/ADP nucleotidase, inhibited HOS-activated I(Cl,swell), whereas ATP and the P2Y agonists, ADPbetaS and ADP, induced Cl(-) currents similar to I(Cl,swell). Neither the P2U agonist, UTP nor the P2X agonist, alpha,beta-methylene ATP, were effective. BzATP was less effective than ATP, suggesting that P2X7 receptors were not involved. P2 purinergic antagonists, suramin, RB2, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) reversibly inhibited activation of I(Cl,swell), suggesting that ATP-activated P2Y1 receptors. Thus ATP release mediates I(Cl,swell) in astrocytes through the activation of P2Y1-like receptors. The multidrug resistance protein (MRP) transport inhibitors probenicid, indomethacin, and MK-571 all potently inhibited I(Cl.swell). ATP release from astrocytes in HOS was observed directly using luciferin-luciferase and MK-571 reversibly depressed this HOS-induced ATP efflux. We conclude that ATP release via MRP and subsequent autocrine activation of purinergic receptors contributes to the activation of I(Cl,swell) in astrocytes by HOS-induced swelling.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Apyrase; Astrocytes; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cells, Cultured; Chloride Channels; Chlorides; Hypotonic Solutions; Membrane Potentials; Platelet Aggregation Inhibitors; Probenecid; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Suramin; Uricosuric Agents; Water-Electrolyte Balance

Intercellular spread of Ca2+ waves is the primary manifestation of cell-to-cell communication among astrocytes. Ca2+ waves propagate via the release of a diffusible extracellular messenger that has been identified as ATP. In dorsal spinal astrocytes, Ca2+ waves are mediated by activation of two functionally distinct subtypes of metabotropic purinoceptor: the P2Y1 receptor and a receptor previously classified as P2U. Here, we show that the P2U receptor is molecularly and pharmacologically identical to the cloned P2Y2 receptor. Both P2Y1 and P2Y2 receptors are necessary for full Ca2+ wave propagation in spinal astrocytes. Conversely, heterologous expression of either P2Y1 or P2Y2 receptors is sufficient for Ca2+ waves, and expressing these receptor subtypes together recapitulates the characteristics of Ca2+ waves in spinal astrocytes. Thus, P2Y1 and P2Y2 receptors are both necessary and sufficient for propagation of Ca2+ waves. Furthermore, we demonstrate that there are dramatic differences in the characteristics of Ca2+ waves propagating through each receptor subtype: Ca2+ waves propagating via P2Y2 receptors travel faster and further than those propagating via P2Y1 receptors. We find that the nucleotidase apyrase selectively blocks Ca2+ wave propagation through P2Y2 receptors but accelerates Ca2+ waves propagating through P2Y1 receptors. Taking our results together with those from the literature, we suggest that mediation of Ca2+ waves by ATP leading to activation of two subtypes of receptor, P2Y1 and P2Y2, may be a general principle for gliotransmission in the CNS. Thus, processes that alter expression or function of these receptors may control the rate and extent of astrocyte Ca2+ waves.

Topics: Adenosine Triphosphate; Animals; Apyrase; Astrocytes; Astrocytoma; Calcium; Calcium Signaling; Cells, Cultured; Enzyme Inhibitors; Humans; Physical Stimulation; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Suramin; Transfection; Uridine Triphosphate

The possibility was tested that endogenous ATP released upon alpha(1)-adrenoceptor activation causes relaxation of the rat vas deferens smooth muscle. ATP, 2-methylthio ATP and adenosine relaxed the vas deferens precontracted with 80 mM K(+). The metabolically stable P2 receptor agonists alpha,beta-methylene ATP (alpha,beta-MeATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) had little or no effect. The adenosine P1 receptor antagonist 8-(para-sulfophenyl)theophylline did not significantly affect the response to ATP. The P2 receptor antagonist reactive blue 2 markedly reduced the relaxation (by up to 73%); suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and acid blue 129 caused no change. ATP, but not alpha,beta-MeATP, also attenuated contractions elicited by noradrenaline at resting tension; reactive blue 2 blocked the inhibitory effect of ATP. Reactive blue 2, by itself, enhanced the response to noradrenaline (by up to 36%); suramin, PPADS and acid blue 129 caused no change. In the presence of the ATP-degrading enzymes apyrase and nucleotide pyrophosphatase, the facilitatory effect of reactive blue 2 was lost. Apyrase, by itself, enhanced the response to noradrenaline (by 13%). The results indicate that endogenous ATP, released from rat vas deferens smooth muscle upon alpha(1)-adrenoceptor stimulation, causes relaxation. The site of action of ATP is not a typical smooth muscle P2Y receptor.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Apyrase; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Muscle Relaxation; Norepinephrine; Nucleotides; Potassium; Purinergic P1 Receptor Antagonists; Pyridoxal Phosphate; Pyrophosphatases; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1; Suramin; Theophylline; Thionucleotides; Triazines; Vas Deferens; Vasoconstrictor Agents

Somatic sensation requires the conversion of physical stimuli into the depolarization of distal nerve endings. A single cRNA derived from sensory neurons renders Xenopus laevis oocytes mechanosensitive and is found to encode a P2Y1 purinergic receptor. P2Y1 mRNA is concentrated in large-fiber dorsal root ganglion neurons. In contrast, P2X3 mRNA is localized to small-fiber sensory neurons and produces less mechanosensitivity in oocytes. The frequency of touch-induced action potentials from frog sensory nerve fibers is increased by the presence of P2 receptor agonists at the peripheral nerve ending and is decreased by the presence of P2 antagonists. P2X-selective agents do not have these effects. The release of ATP into the extracellular space and the activation of peripheral P2Y1 receptors appear to participate in the generation of sensory action potentials by light touch.

Topics: Adenosine Triphosphate; Animals; Apyrase; Chickens; Female; Ganglia, Spinal; Membrane Potentials; Molecular Sequence Data; Nerve Fibers; Neurons, Afferent; Oocytes; Physical Stimulation; Purinergic P2 Receptor Agonists; Pyridoxal Phosphate; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Receptors, Purinergic P2Y1; RNA, Messenger; Sciatic Nerve; Skin; Suramin; Time Factors; Transcription, Genetic; Xenopus laevis