The ATP4− Receptor-Operated Channel (P2Z Class) of Human Lymphocytes Allows Ba2+ and Ethidium+ Uptake: Inhibition of Fluxes by Suramin

doi:10.1006/abbi.1993.1392

Archives of Biochemistry and Biophysics

Volume 305, Issue 1, 15 August 1993, Pages 54-60

https://doi.org/10.1006/abbi.1993.1392 Get rights and content

Abstract

It is now recognized that extracellular ATP can open a receptor-operated ion channel in a variety of cell types. In human lymphocytes this P₂Z purinergic channel conducts Na⁺, K⁺, Rb⁺, Li⁺, and Ca²⁺ but its permeability to larger cations is not known. Fluorometric measurements were used to show that ATP⁴⁻ induced the entry of Sr²⁺ (87 Da) and Ba²⁺ (137 Da) into human lymphocytes loaded with fura-2. Flow cytometry was used to show that ATP⁴⁻ induced the entry of ethidium⁺ (314 Da) but that the larger propidium²⁺ cation (414 Da) was excluded. ATP⁴⁻ induced entry of both Ba²⁺ and ethidium⁺ showed features previously demonstrated for smaller cation permeants: (i) inhibition by amiloride analogs, (ii) sigmoid dependence of flux on ATP concentrations, and (iii) inhibition by extracellular Na⁺ ions. Specific inhibitors of L-type voltage-gated Ca²⁺ channels (nisoldipine and diltiazem) had no effect on ATP⁴⁻-induced Ba²⁺ influx. Suramin and reactive blue 2, which are recognized antagonists of ATP-operated purinergic receptors in other tissues, inhibited ATP-induced uptake of ethidium⁺ in lymphocytes with K^1/2 of 61 and 69 μM, respectively. However, hexamethylene amiloride was a more potent inhibitor of ATP-induced ethidium⁺ uptake with a K^1/2 of 13 μM. These data show that the ATP⁴⁻ receptor-operated channel of human lymphocytes allows influx of cations as large as Ba²⁺ or ethidium⁺ and that this influx is inhibited by suramin and reactive blue 2.

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Rozengurt and Heppel also demonstrated that, provided exposure to eATP was reasonably brief (5–6 min), the plasma membrane resealed, and cells regained the ability to grow to approximately the same rate as control cells which had not been exposed to eATP. A large body of evidence has accumulated ever since to support the view that tumor cells express an eATP-activated “permeabilizing” receptor, initially named P2Z and then P2X7 [60,64–70]. The role played by the P2X7R in tumor growth is not easy to rationalize since this receptor is expected to make tumor cells susceptible to killing by the high eATP concentrations usually found in the TME [71].
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The problem with exploiting these active transport pathways to enhance anticancer drug penetration, though, is that they often have a wide tissue distribution and do not have activity easily regulated pharmacologically. The experiments described in this paper were conducted based on the long known ability for extracellular adenosine 5′-triphosphate (ATP) to permeabilize certain cell types, such as mast cells and macrophages, to relatively large polyatomic ions including 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), N-methyl-d-glucamine (NMDG+) [9], ethidium [10,11], the Ca2+-sensor Fura-2 and Lucifer Yellow [12,13]. A previous study had some success with using ATP-evoked permeabilization in order to load macrophages with doxorubicin and use them as a release vehicle for the drug in tumors [14].
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The steroid hormone 1α,25(OH)₂D3 produces biological responses via both genomic and nongenomic mechanisms. Stimulation of rapid, nongenomic responses by 1α,25(OH)₂D3 has been postulated to result from interaction of the ligand with cell membrane 1α,25(OH)₂D3 receptors and to involve membrane receptors. We examined the rapid effects of 1α,25(OH)₂D3 on calcium mobilization and calcium entry into resting human peripheral blood mononuclear cells isolated from healthy volunteers. We also investigated the possible involvement of purinergic receptors in this action. 1α,25(OH)₂D3 induced a time-dependent increase in intracellular calcium concentration ([Ca²⁺]_i). The initial 1α,25(OH)₂D3-stimulated calcium increment was sensitive to thapsigargin (Tg), indicating its origins in calcium release from intracellular stores. 2-Aminoethyldiphenyl borate (2APB), an inhibitor of capacitative calcium entry, caused a significant [Ca²⁺]_i decrease in human cells treated with 1α,25(OH)₂D3. Furthermore, in contrast to observations in osteoblasts and skeletal muscle cells, nifedipine had no effect on 1α,25(OH)₂D3-induced calcium entry, suggesting that L-type calcium channels were not implicated in this action. Besides, 1α,25(OH)₂D3 prevented the calcium entry induced by 3′-O-(4-benzoyl)benzoyl-adenosine 5′-triphosphate (BzATP), a specific agonist of purinergic P2X₇ receptors. This finding was further confirmed by 1α,25(OH)₂D3-induced reduction of BzATP- and 4-aminopyridine (4AP)-stimulated ethidium bromide fluorescence. The presented results demonstrate, for the first time in healthy, resting human peripheral blood mononuclear cells that 1α,25(OH)₂D3 is capable of exerting a rapid, nongenomic effect on [Ca²⁺]_i, while inhibiting of the P2X₇ channel permeability.
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Citation Excerpt :
Such an apparent increase of a binding site affinity due to an increased efficacy has been described in detail by Colquhoun (37). It is well known that substitution of extracellular Na+ by K+ or organic cations increases the influx of Ba2+, ethidium+(22), and Ca2+(38) into P2Z receptor-expressing B lymphocytes. Likewise, extracellular Na+ removal has been reported to activate secondary divalent-dependent currents in hP2X7 receptor-expressing Xenopus oocytes (15), to permeabilize hP2X7 receptor-expressing HEK293 cells for NMDG+(39), and to increase airway ciliary motility by activating a native P2X7-like receptor (36).
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Regular ArticleThe ATP4− Receptor-Operated Channel (P2Z Class) of Human Lymphocytes Allows Ba2+ and Ethidium+ Uptake: Inhibition of Fluxes by Suramin

Abstract

Regular Article
The ATP⁴⁻ Receptor-Operated Channel (P₂Z Class) of Human Lymphocytes Allows Ba²⁺ and Ethidium⁺ Uptake: Inhibition of Fluxes by Suramin