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

ATP increases intracellular calcium concentration ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca(2+)-sensitive dye, fura 2-AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin release from hypothalamo-neurohypophyseal system explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca(2+)](i) in dissociated SON neurons. However, the ATP-induced increase in [Ca(2+)](i) persisted after removal of Ca(2+) from the perifusate ([Ca(2+)](o)). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca(2+) release from intracellular stores, whereas P2XRs are Ca(2+)-permeable ion channels. Depletion of [Ca(2+)](i) stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca(2+)](i) in zero, but not in 2 mM [Ca(2+)](o), indicating that both Ca(2+) influx and release of intracellular Ca(2+) contribute to the ATP response. Ca(2+) influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca(2+) channels. PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), and iso-PPADS, P2XR antagonists, attenuated, but did not abolish, the ATP-induced increase in [Ca(2+)](i). Combined treatment with PPADS or iso-PPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-methylthio-ADP increased [Ca(2+)](i) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca(2+)](i) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero [Ca(2+)](o). Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca(2+)](i), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cadmium; Calcium; Diagnostic Imaging; Drug Synergism; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; In Vitro Techniques; Male; Neurons; Neurotransmitter Agents; Osmolar Concentration; Purinergic Agonists; Purinergic Antagonists; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2X; Supraoptic Nucleus; Tetrodotoxin; Thapsigargin

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

The mechanisms by which uridine triphosphate (UTP) stimulates ATP release from Schwann cells cultured from the sciatic nerve were investigated using online bioluminescence techniques. UTP, a P2Y(2) and P2Y(4) receptor agonist, stimulated ATP release from Schwann cells in a dose-dependent manner with an ED(50) of 0.24 microm. UTP-stimulated ATP release occurs through P2Y(2) receptors as it was blocked by suramin which inhibits P2Y(2) but not P2Y(4) receptors. Furthermore, positive immunostaining of P2Y(2) receptors on Schwann cells was revealed and GTP, an equipotent agonist with UTP at rat P2Y(4) receptors, did not significantly stimulate ATP release. UTP-stimulated ATP release involved second messenger pathways as it was attenuated by the phospholipase C inhibitor U73122, the protein kinase C inhibitor chelerytherine chloride, the IP(3) formation inhibitor lithium chloride, the cell membrane-permeable Ca(2+) chelator BAPTA-AM and the endoplasmic reticulum Ca(2+)-dependent ATPase inhibitor thapsigargin. Evidence that ATP may be stored in vesicles that must be transported to the cell membrane for exocytosis was found as release was significantly reduced by the Golgi-complex inhibitor brefeldin A, microtubule disruption with nocodazole, F-actin disruption with cytochalasin D and the specific exocytosis inhibitor botulinum toxin A. ATP release from Schwann cells also involves anion transport as it was significantly reduced by cystic fibrosis transmembrane conductance regulator inhibitor glibencamide and anion transporter inhibitor furosemide. We suggest that UTP-stimulated ATP release is mediated by activation of P2Y(2) receptors that initiate an IP(3)-Ca(2+) cascade and protein kinase C which promote exocytosis of ATP from vesicles as well as anion transport of ATP across the cell membrane.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Animals, Newborn; Benzophenanthridines; Botulinum Toxins; Botulinum Toxins, Type A; Brefeldin A; Calcium; Cyclic AMP-Dependent Protein Kinases; Cytochalasin D; Diagnostic Imaging; Dose-Response Relationship, Drug; Drug Interactions; Estrenes; Furosemide; Glyburide; Glycyrrhetinic Acid; Guanosine Triphosphate; Immunohistochemistry; Isoquinolines; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Phenanthridines; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Synthesis Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Schwann Cells; Sciatic Nerve; Sulfonamides; Suramin; Thapsigargin; Time Factors; Type C Phospholipases; Uridine Triphosphate

We investigated the effects of P2-receptor agonists on cell size, intracellular calcium levels ([Ca(2+)](i)), and permeation of FITC-labeled dextran (FD-4) as well as the relationship between these effects in human umbilical vein endothelial cells (HUVEC). FD-4 concentration, cell size, and [Ca(2+)](i) were analyzed by HPLC with fluorescence, phase contrast microscopic imaging, and fluorescent confocal microscopic imaging, respectively. The P2Y(1)-receptor agonists 2-methylthio ATP (2meS-ATP) and ADP decreased cell size and increased [Ca(2+)](i) in HUVEC. The P2Y(2)-receptor agonist UTP increased [Ca(2+)](i), but did not influence cell size. The P2X-receptor agonist alpha,beta-methylene ATP did not induce either response. The decrease in size and increase in [Ca(2+)](i) by 2meS-ATP were blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, P2Y(1)-antagonist), thapsigargin (Ca(2+)-pump inhibitor), and U73122 (phospholipase C inhibitor). Furthermore, 2meS-ATP (P2Y(1)-receptor agonist) enhanced permeation of FD-4 through the endothelial cell monolayer. The 2meS-ATP-induced enhancement of the permeation was also prevented by PPADS, thapsigargin, and U73122. These results indicate that activation of P2Y receptors induces a decrease in cell size, an increase in [Ca(2+)](i), and may participate in facilitating macromolecular permeability in HUVEC.

Topics: Adenosine Triphosphate; Biological Transport; Calcium; Calcium Signaling; Cell Membrane Permeability; Cell Size; Chelating Agents; Endothelial Cells; Estrenes; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Muscle, Smooth, Vascular; Purinergic P2 Receptor Agonists; Pyridoxal Phosphate; Pyrrolidinones; Receptors, Purinergic P2; Signal Transduction; Thapsigargin; Thionucleotides

The effects of P2 receptor agonists on cell size and intracellular calcium levels, [Ca(2+)](i), was investigated using cultured endothelial cells isolated from the caudal artery of male Wistar rats. Cell size and [Ca(2+)](i) were measured using a phase-contrast and fluorescent confocal microscopic image analyzer and a Calcium Green fluorescence probe. P2Y receptor agonists, 2-methylthio ATP (2meS-ATP), ADP, UTP and ATP decreased the cell size and increased [Ca(2+)](i) in endothelial cells from rat caudal artery. However, alpha,beta-methylene ATP, a P2X receptor agonist, did not induce these responses. The decrease in size and the increase in [Ca(2+)](i), by 2meS-ATP were blocked by PPADS (P2-antagonist), suramin (P2-antagonist), thapsigargin (Ca(2+) pump inhibitor) and U-73122 (phospholipase C inhibitor). The present results show that activation of P2Y receptors, not P2X receptors, induces a decrease in cell size and an increase in [Ca(2+)](i), and the pharmacological properties of these two responses are the same. We concluded that the size of endothelial cells is regulated by P2Y receptors via intracelluar Ca(2+) derived from Ca(2+) stores.

Topics: Adenosine Triphosphate; Animals; Calcium; Cell Size; Endothelium, Vascular; Enzyme Inhibitors; Estrenes; Male; Osmotic Pressure; Phosphodiesterase Inhibitors; Purinergic P2 Receptor Agonists; Pyridoxal Phosphate; Pyrrolidinones; Rats; Rats, Wistar; Receptors, Purinergic P2; Suramin; Thapsigargin; Thionucleotides

1. Mechanisms underlying spontaneous rhythmical contractions have been studied in irideal arterioles of the rat using video microscopy and electrophysiology. 2. Rhythmical contractions (4 min-1) were more common during the second and third postnatal weeks and were always preceded by large, slow depolarizations (5-40 mV). 3. Spontaneous contractions were unaffected by tetrodotoxin (1 microM), neurotransmitter receptor antagonists, the sympathetic neurone blocker, guanethidine (5 microM) or sensory neurotoxin, capsaicin (1 microM). 4. Stimulation of sensory nerves inhibited spontaneous activity and this was not prevented by L-NAME (10 microm). 5. L-NAME (10 microm) caused an increase in frequency of spontaneous contractions, while forskolin (30 nM), in the presence of L-NAME, abolished spontaneous, but not nerve-mediated, contractions. 6. Spontaneous activity was not affected by felodipine (1 nM) or nifedipine (1 microM), but was abolished by cadmium chloride (1 microM) or superfusion with calcium-free solution. 7. Caffeine (1 mM), thapsigargin (2 microM) and cyclopiazonic acid (3 microM), but not ryanodine (3 microM), abolished spontaneous and nerve-mediated contractions. After preincubation in L-NAME (10 microM), cyclopiazonic acid abolished spontaneous contractions only. 8. Spontaneous depolarizations and contractions were abolished by 18alpha-glycyrrhetinic acid (20 microM). 9. Results suggest that spontaneous rhythmical contractions are myogenic and result from the cyclical release of calcium from intracellular stores, without a contribution from voltage-dependent calcium channels. Intercellular coupling through gap junctions appears to be essential for co-ordination of these events which could be modulated by nitric oxide and increases in cAMP. The possibility that different intracellular stores underly spontaneous and nerve-mediated contractions is discussed.

Topics: Adenosine Triphosphate; Age Factors; Animals; Arterioles; Cadmium Chloride; Caffeine; Calcium; Capsaicin; Central Nervous System Stimulants; Colforsin; Cyclic AMP; Dinucleoside Phosphates; Enzyme Inhibitors; Felodipine; Female; Gap Junctions; Iris; Male; Membrane Potentials; Muscle, Smooth, Vascular; Neuropeptide Y; NG-Nitroarginine Methyl Ester; Peptides, Cyclic; Periodicity; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Wistar; Ryanodine; Thapsigargin; Vasoconstriction; Vasodilator Agents

1. The relationship between the stimulation of ATP receptors, the increase in intracellular free calcium concentration ([Ca2+]i; measured using the fluorescent indicator fura-2), contraction and the subtypes of purinoceptors involved were investigated in the small mesenteric artery of the rat. 2. In normal physiological solution, ATP (0.001-3 mM) caused concentration-dependent increases in both [Ca2+]i and contraction. Both responses produced by ATP (1 mM) were inhibited by 50% in the presence of nitrendipine (1 microM) and were abolished in the presence of nitrendipine plus SK&F 96365 (30 microM). 3. In Ca(2+)-free medium, ATP (3 mM) elicited a transient increase in both [Ca2+]i and tension which were abolished by caffeine and decreased by 65% by thapsigargin (1 microM). Moreover, ATP (1 and 3 mM) produced increases in the [3H]D-myo-inositol 1,4,5-trisphosphate ([3H]IP3) content of vessels in a concentration-dependent manner. 4. Treatment of the vessels with Bordetella pertussis toxin (PTX) inhibited contractions to ATP linked to the influx of calcium through nitrendipine-sensitive mechanisms, but not those linked to the release of Ca2+ from intracellular stores nor the capacity of ATP in increasing IP3 content of the vessels. 5. The order of potency of ATP and its analogues in eliciting contraction was alpha, beta-methylene-ATP (alpha, beta-MeATP) > 2-methylthio-ATP (2-MeSATP) > ATP = ADP. The response to ATP was inhibited by suramin. Reactive Blue 2 (up to 100 microM) did not affect the contractile response to ATP. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) and alpha, beta-MeATP abolished the response to low concentrations of ATP and reduced contractions elicited by high concentrations of ATP. 6. After blockade of P2X-purinoceptors with PPADS, the order of potency of ATP and its analogues was 2-MeSATP > ATP = ADP. UTP produced concentration-dependent contractions which were not affected by suramin, Reactive Blue 2, PPADS or alpha, beta-MeATP, suggesting the presence of P2U-purinoceptors. 7. The results suggest that low concentrations of ATP activate P2X-purinoceptors and produce an influx of calcium through both voltage-dependent calcium channels sensitive to nitrendipine and through receptor-operated calcium channels sensitive to SK&F 96365. High concentrations of ATP activate P2Y-purinoceptors which promote firstly a nitrendipine-sensitive calcium influx via a PTX-sensitive G protein and secondly a release of Ca2+ from an

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcium; Calcium Channel Blockers; Imidazoles; Male; Mesenteric Arteries; Muscle Contraction; Nitrendipine; Pertussis Toxin; Platelet Aggregation Inhibitors; Purinergic Agonists; Purinergic Antagonists; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic; Terpenes; Thapsigargin; Vascular Resistance; Virulence Factors, Bordetella