Pharmacological properties of a pore induced by raising intracellular Ca2+.
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 microM) increased intracellular free Ca(2+) concentration and induced pore formation with conductance of 421 +/- 14 pS, half-time (t(1/2)) for ethidium bromide uptake of 118 +/- 17 s, and t(1/2) 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.
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Laboratory of Cellular Communication, Oswaldo Cruz Institute, FIOCRUZ, Oswaldo Cruz Foundation, Rio de Janeiro Av. Brasil, 4365 Manguinhos 21045-900, Rio de Janeiro, Brasil.
Reis
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Casabulho
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Alberto
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de Farias
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Henriques-Pons
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Alves
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