cytochalasin-d and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

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

P2X7 receptors (P2X7Rs) affect many epithelial cell functions including transcellular ion transport, secretion, and cell death. Here we used parotid acinar and duct cells to reveal the unique cell-specific assembly and gating of the P2X7R channels. Immunolocalization indicated expression of P2X7Rs in the luminal membrane of both cell types. Stimulation with 5 mm ATP raised [Ca2+]i levels in a cell-specific manner and activated multiple currents. The current mediated by P2X7R was isolated by infusing the cells with high [EGTA]. The initial activation of acinar cell P2X7Rs by ATP was slow requiring approximately 2.5 min. Subsequent removal and addition of ATP, however, resulted in rapid inhibition and activation (gating) of the P2X7Rs. By contrast, P2X7Rs in duct cells displayed only rapid gating by ATP. Activation of P2X7Rs in both cell types was verified by (a) low Km for ATP, (b) sensitivity to external divalent ions, (c) lack of desensitization/inactivation, (d) permeability to Na+, and (e) inhibition by Brilliant Blue G, Cu2+, and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium. The slow P2X7R activation in acinar cells was not affected by manipulation of exo-/endocytosis. Rather, disassembly or solidification of the actin cytoskeleton prior to incubation with ATP prevented channel assembly. Remarkably, after completion of the slow activation, manipulation of the actin cytoskeleton no longer affected gating by ATP. Accordingly, manipulation of the actin cytoskeleton had no effect on P2X7R gating by ATP in duct cells. We concluded that P2X7Rs are not active in resting acinar cells. On exposure to ATP, P2X7Rs are assembled into functional channels with the aid of the actin cytoskeleton. Once assembled, P2X7Rs are subject to rapid gating by ATP. Duct cell P2X7Rs are preassembled and therefore continually subject to rapid gating by ATP. This cell-specific behavior may reflect the specific function of P2X7Rs in the two cell types.

Topics: Actins; Adenosine Triphosphate; Animals; Antineoplastic Agents; Benzenesulfonates; Calcium; Cell Death; Cell Membrane; Coloring Agents; Copper; Cytochalasin D; Cytoskeleton; Depsipeptides; Egtazic Acid; Electrophysiology; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Immunohistochemistry; Indicators and Reagents; Ions; Kinetics; Mice; Nucleic Acid Synthesis Inhibitors; Parotid Gland; Peptides, Cyclic; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sodium; Thionucleotides; Time Factors