ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells.

Abstract

We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by voltage.clamp measurements using the patch-clamp technique, and (c) by measurements of exocytos~is in response to entry of Ca 2+ and GTP')'S into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 #M. In normal saline containing 1 mM MgCl~ and 2 mM CaCl~, dye uptake and electric conductance are detectable at 100 #M ATP corresponding to 4 #M ATP 4-. The permeabilization is half-maximal at an ATP 4-concentration of 5-20 #M with a Hill coefficient near 2. The ATP-induced whole.cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant <65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of ~200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of ~40 pS and the single-channel conductance obtained from noise analysis is ~2-10 pS. Entry of Ca 2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTP~S is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.

P2 receptor-mediated signaling in mast cell biology

“…RBL-2H3 and rat peritoneal mast cells indeed express functional P2Y, P2X 1 , and P2X 7 receptors as was shown indirectly by pharmacological studies [7,[60][61][62][63][64].…”

“…Membrane permeabilization and Ca 2+ influx First observations of extracellular ATP effects on mast cells reported an increase in intracellular Ca 2+ [3,61,65,66]. Such elevation in the intracellular Ca 2+ concentration is a result of Ca 2+ influx across the cellular membrane (feature of P2X receptors) or Ca 2+ release from the intracellular stores (linked to the P2Y receptors) [34,38].…”

“…Pannexin-1 co-associates with P2X 7 , and this complex is not only essential for P2X 7 -induced dye uptake without altering Ca 2+ influx but also for the caspase-1 processing and release of mature IL-1β [67,68]. Degranulation ATP has long been known to induce histamine release from mast cells [3][4][5][60][61][62][63]. In 1979, Cockcroft and Gomperts have shown that ATP caused Ca 2+ -dependent histamine release and striking morphological changes in rat mast cells [5,60].…”

“…There is a tight cross-communication between neurons and mast cells which will be discussed below. ATP not only directly induces degranulation of mast cells but also modulates IgE/ antigen-mediated degranulation [60][61][62]70]. Moreover, ATP and other nucleotides are potent chemoattractants [48].…”

The P2Z‐purinoceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidized ATP

“…Many features of this P2Y-receptor are functionally similar to the endothelial P2U-receptor except that UTP is the preferred agonist for the latter. ATP also induces the formation of ion-conducting channels (pores) in mouse and human lymphocytes (Wiley & Dubyak, 1989;Wiley et al, 1990;El-Moatassim et al, 1990;Pizzo et al, 1991), rat peritoneal mast cells (Tatham & Lindau, 1990), rat parotid acinar cells (Soltoff et al, 1992) and mouse macrophages (Greenberg et al, 1988). A subclass of purinoceptors termed P2z was first proposed for the receptor associated with this channel based on a specificity for the fully ionized ATP4-and need for relatively high (ca.…”