Extracellular ATP in the lymphohematopoietic system: P2Z purinoceptors and membrane permeabilization

Abstract

The effects of extracellular nucleosides and nucleotides on many organs and systems have been recognized for almost 50 years. The effects of extracellular ATP (ATP o ), UTP o , ADP o , and other agonists are mediated by P2 purinoceptors. One of the most dramatic effects of ATP o is the permeabilization of plasma membranes to low molecular mass solutes of up to 900 Da. This effect is evident in several cells of the lymphohematopoietic system and is supposed to be mediated by P2Z, an ATP 4--activated purinoceptor. Here, we review some basic information concerning P2 purinoceptors and focus our attention on P2Z-associated phenomena displayed by macrophages. Using fluorescent dye uptake, measurement of free intracellular Ca 2+ concentration and electrophysiological recordings, we elucidate some of the events that follow the application of ATP to the extracellular surface of macrophages. We propose a regulatory mechanism for the P2Z-associated permeabilization pore. The presence of P2 purinoceptors in cells of the lymphohematopoietic system makes them potential candidates to mediate immunoregulatory events. Key words Effects of extracellular ATPThe effects of extracellular nucleosides and nucleotides on many organs and systems have been recognized for almost 50 years (1-3). Early investigators concentrated on the actions of extracellular ATP (ATP o ) and adenosine on the cardiovascular system, including their shock-inducing properties and their applications to geriatric patients with cardiovascular disorders.In non-lymphoid tissues, both norepinephrine and acetylcholine can be found colocalized with ATP o . Actually, in the nervous system, ATP is stored and released with norepinephrine and acetylcholine, and can act either as a neurotransmitter or as a co-transmitter (1-3). In the endocrine system, ATP o can act as a secretagogue for hormones, as demonstrated in the pancreas and adrenals.In the lymphohematopoietic system, one of the most dramatic effects of ATP o is the permeabilization of plasma membranes of several cell types to low molecular mass solutes of up to 900 Da. This phenomenon requires mM concentrations of ATP o and has already been described in macrophages, mast cells, phagocytic cells of the thymic reticulum, bone marrow cells, and thymic as

ATP-induced P2X7-associated uptake of large molecules involves distinct mechanisms for cations and anions in macrophages

“…These channels were called Z pores and could only be recorded under cell-attached patch-clamping conditions. Because they carry currents of large cations (Tris, 121 Da and N-methyl-Dglucamine (NMDG), 195 Da) and anions (glutamate, 146 Da) we have proposed that they could be a pathway for dye uptake (CoutinhoSilva and Persechini, 1997;Persechini et al, 1998). We therefore investigated whether a similar large conductance non-selective channel could be detected in HEK-P2X 7 cells under cell-attached patch-clamping conditions.…”

“…Whole-cell patch-clamp recordings performed on cells transfected with P2X 7 receptors indicate a permeability shift from low to high M r molecules in the first seconds after stimulation by ATP e , while keeping the selectivity for cations (Virginio et al, 1999). In addition, although ATP e induces the uptake of both cationic and anionic dyes in macrophages (Steinberg et al, 1987a), the uptake of anions has not been reported either in HEK-293 cells or astrocytomas transfected with P2X 7 , neither has it been reported in lymphocytes (Wiley et al, 1993;Surprenant et al, 1996;Rassendren et al, 1997;Chessell et al, 1998;Ferrari et al, 2000;Paukert et al, 2002;Duan et al, 2003;Suadicani et al, 2006), suggesting that in these experimental situations, ATP e might not induce membrane permeabilization to anions.Based on cell-attached patch-clamping experiments demonstrating the opening of large cation-and anion-permeant channels (Z pores) induced by ATP e in macrophages, we have hypothesized that the permeabilization pore of macrophages could be distinct from the receptor itself, but coupled to P2X 7 through an unidentified signaling mechanism (Coutinho-Silva and Persechini, 1997;Persechini et al, 1998). This possibility has recently been corroborated by data showing that pannexin-1, a protein that forms large non-selective transmembrane channels (Barbe et al, 2006), is involved in the phenomenon of P2X 7 -associated, ATP e -induced membrane permeabilization (Pelegrin and Surprenant, 2006;Locovei et al, 2007).…”

“…However, the uptake of ethidium was resistant to the chelation of divalent cations, even in cells loaded with BAPTA-AM, a condition that completely abrogates the intracellular Ca 2+ signaling (Monteiroda-Cruz et al, 2006). Taken together these results also imply that it is unlikely that Z pores are involved in the uptake of large cationic dyes.We therefore propose that, besides the small (8-9 pS) and nonselective cation channels directly associated with the P2X 7 receptors (Persechini et al, 1998;Riedel et al, 2006), at least two additional transport mechanisms are activated by ATP e in macrophages: the anion uptake mechanism, which is associated with Z pores and allows the free diffusional flow of anionic dyes through the plasma membrane, and the mechanism for the non diffusional uptake of cations, which is yet to be elucidated. It is interesting to notice that besides the differences, both transport mechanisms require similar high concentrations of ATP e in the mM range to be activated (Fig.…”

Pharmacological properties of a pore induced by raising intracellular Ca2+

“…In one view, the channel formed by P2X 7 receptor is gradually open upon activation, increasing its permeability from small molecules to large ones (up to 900 Da) (80,81). In the other, P2X 7 channel is formed upon activation, inducing the production or release of a second messenger that may autoactivate P2X 7 receptor or an independent pore-forming membrane protein (16,61). Recently, we have demonstrated that the action of second messengers is necessary to form the pore (28).…”

Intracellular Signaling Pathways Integrating the Pore Associated with P2X7R Receptor with Other Large Pores

“…The initial suggestions for dependency of cytoplasmic factors in this event were originated from electrophysiological data in outside or inside out configurations with no large conductance channel recordings (Coutinho-Silva & Persechini, 1997;Persechini et al, 1998;Petrou et al, 1997). Posteriorly, other groups have investigated the P2X7R pore formation induced by intracellular signaling and how mutated amino acids or truncated regions affect functional availability of the receptor.…”