yo-pro-1 and lucifer-yellow

P2X7 receptor has gained an increasing importance as a drug target. One important response to P2X7 receptor stimulation is the uptake of large molecular weight tracers into cells. However, mechanism for this response is not understood clearly, but it is generally believed that a nonselective large pore protein forms this P2X7 receptor-activated permeability pathway. We examined human embryonic kidney (HEK) 293 cells transfected with rat P2X7 receptors (HEK-rP2X7) and a macrophage derived cell line, RAW 264.7, that expresses an endogenous P2X7 receptor. We used confocal microscopy to investigate uptake of different types of dyes into these cells after ATP application. Stimulation of P2X7 receptors in HEK-rP2X7 cells activated two different dye uptake pathways. The first was permeable to the cationic fluorescent dyes YO-PRO-1 and TO-TO-1 but not to the anionic dyes lucifer yellow and calcein and did not require intracellular Ca2+ concentration ([Ca2+](i)) increase to be activated. The second pathway permeated only lucifer yellow and was completely dependent on [Ca2+](i) for activation. In RAW 264.7 cells, P2X7 receptor stimulation activated uptake of ethidium, YO-PRO-1, TO-TO-1, lucifer yellow, and calcein. Again, two different permeation pathways were discerned in RAW 264.7 cells: one permeated only ethidium and the other one, only lucifer yellow. We did observed no clear [Ca2+](i) dependence for these permeation pathways. Our results demonstrate that instead of a single nonselective pore, P2X7 receptor seems to activate at least two permeation pathways, one for cationic and one for anionic dyes with different activation properties.

Topics: Adenosine Triphosphate; Animals; Benzoxazoles; Calcium; Cell Line; Cell Membrane Permeability; Fluorescent Dyes; Humans; Isoquinolines; Kidney; Macrophages; Quinolinium Compounds; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Transcriptional Activation; Transfection

The P2X(7) receptor, which induces cation channel opening imparting significant permeability to Ca2+ and pore formation with changes in the plasma membrane potential, has been known to be rather restrictedly expressed in cells of the macrophage lineage including dendrites, mature macrophages, and microglial cells. However, we show here that the P2X(7) receptor is also expressed in cells of granulocytic lineage such as HL-60 promyelocytes, granulocytic differentiated cells, and neutrophils. Exposure of these cells to 2',3'-O-(4-benzoyl)benzoyl-ATP (BzATP) triggered intracellular Ca2+ rise through the mediation of phospholipase C-independent and suramin-sensitive pathways. BzATP also induced depolarization of the plasma membrane in the absence of extracellular Ca2+, whereas it hyperpolarized the cells in the presence of external Ca2+, probably in part through the activation of Ca2+-activated K(+) channels. However, the hyperpolarization phenomenon was markedly attenuated in differentiated HL-60 cells and neutrophils. RT-PCR and Northern blot analysis revealed the presence of P2X(7) receptors on both HL-60 and neutrophil-like cells. This was further confirmed by pore formation through which the uptake of Lucifer yellow and YO-PRO1 occurred on BzATP treatment. BzATP stimulated in a concentration-dependent manner the production of superoxide in differentiated HL-60 cells via a pathway partially dependent on extracellular Ca2+. Moreover, in human neutrophils, BzATP was a more effective inducer of superoxide generation than PMA. Taken together, this is a first demonstration of the expression of P2X(7) receptors on neutrophils, which shows that the receptor is functionally involved in the defense mechanism by activation of the respiratory burst pathway.

Topics: Adenosine Triphosphate; Benzoxazoles; Calcium; Calcium Channels; Cations, Divalent; Cell Membrane Permeability; Cell Separation; Extracellular Space; Fluorescent Dyes; HL-60 Cells; Humans; Isoquinolines; Membrane Potentials; Myeloid Cells; Neutrophils; Photoaffinity Labels; Quinolinium Compounds; Reactive Oxygen Species; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Superoxides; Type C Phospholipases

The present study shows that human mononuclear phagocytes express a P2Z-like purinergic membrane receptor activity. Extracellular adenosine triphosphate (ATP) induces the formation of nonselective membrane pores in human mononuclear phagocytes that allow the entry of otherwise membrane impermeant fluorescent dyes (YO-PRO-1 or Lucifer yellow) into the cytoplasm of these cells. The percentage of mononuclear phagocytes that was permeabilized by ATP increased as monocytes matured into macrophages. Their response to ATP was inhibited by Mg2+ and oxidized ATP. Benzoylbenzoic-ATP (BzBzATP) was approximately 60% as effective as ATP and adenosine-5 -O-(thiophosphate) (ATP gamma S) was less than 20% as effective as ATP in permeabilizing human macrophages to YO-PRO-1 or Lucifer Yellow. Thus, the human P2Z-like receptor differs from its murine counterpart because BzBzATP, ATP, and ATP gamma S are equally efficacious in permeabilizing murine macrophage-like J774 cells to these dyes. UTP, GTP, and CTP were ineffective in permeabilizing human or murine macrophages to YO-PRO-1. Taken together, these data indicate that human monocyte-derived macrophages express a P2Z-like activity that is pharmacologically distinct from that expressed by their murine counterparts and that expression of these receptors is developmentally regulated in human mononuclear phagocytes.

Topics: Adenosine Triphosphate; Benzoxazoles; Cell Membrane Permeability; Cells, Cultured; Fluorescent Dyes; Humans; Isoquinolines; Macrophages; Magnesium; Monocytes; Oxidation-Reduction; Quinolinium Compounds; Receptors, Purinergic P2; Time Factors