Pharmacological properties of a pore induced by raising intracellular Ca2

• Published in: American Journal of Physiology: Cell Physiology 2009-07, Vol.297 (1), p.C28-C42
• Main Authors: Faria, R.X, Reis, R.A.M, Casabulho, C.M, Alberto, A.V.P, de Farias, F.P, Henriques-Pons, A, Alves, L.A
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Animals; Calcium Signaling - drug effects; Calmodulin - antagonists & inhibitors; Calmodulin - metabolism; Cell Line; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Survival; Connexins - drug effects; Connexins - metabolism; Cyclic AMP-Dependent Protein Kinases - metabolism; Cytoskeleton - drug effects; Cytoskeleton - metabolism; Dose-Response Relationship, Drug; Enzyme Activators - pharmacology; Enzyme Inhibitors - pharmacology; Flow Cytometry; Fluorescent Dyes - metabolism; Humans; Ion Channel Gating - drug effects; Ionomycin - pharmacology; Ionophores - pharmacology; Kinetics; Macrophages - drug effects; Macrophages - metabolism; Membrane Potentials; Membrane Transport Modulators - pharmacology; Membrane Transport Proteins - drug effects; Membrane Transport Proteins - metabolism; Mice; Mitogen-Activated Protein Kinases - antagonists & inhibitors; Mitogen-Activated Protein Kinases - metabolism; Patch-Clamp Techniques; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Rats; Receptors, Purinergic P2 - drug effects; Receptors, Purinergic P2 - metabolism; Receptors, Purinergic P2X7; Tubulin Modulators - pharmacology; Type C Phospholipases - antagonists & inhibitors; Type C Phospholipases - metabolism
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00476.2008

1 Laboratory of Cellular Communication and 2 Laboratory of Cell Biology, Oswaldo Cruz Institute, Fiocruz; and 3 Program in Neurobiology, Institute of Biophysics, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Submitted 19 September 2008 ; accepted in final form 19 March 2009 Recent studies on the P2X 7 receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca 2+ concentration induces a pore opening similar to P2X 7 receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca 2+ concentration is associated to P2X 7 receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X 7 receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5–10 µM) increased intracellular free Ca 2+ concentration and induced pore formation with conductance of 421 ± 14 pS, half-time ( t ) for ethidium bromide uptake of 118 ± 17 s, and t for Lucifer yellow of 122 ± 11 s. P2X 7 receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular Ca 2+ concentration. However, 5-( N , N -hexamethylene)-amiloride, a P2X 7 receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular Ca 2+ at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca +2 induces a novel membrane pore pharmacologically different from the P2X 7 associated pore and hemigap-junction pore. P2X 7 receptor; pore formation; second messenger Address for reprint requests and other correspondence: L. Anastacio Alves, Laboratory of Cellular Communication, Oswaldo Cruz Institute, FIOCRUZ, Oswaldo Cruz Foundation, Rio de Janeiro Av. Brasil, 4365 Manguinhos