Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X₇channel

Abstract

Extracellular ATP is known to trigger apoptosis of thymocytes and lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of 3′- O-(4-benzoyl)benzoyl-adenosine 5′-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel are Ca2+ (and Ba2+) selective but also pass molecules up to the size of ethidium cation (314 Da). RT-PCR showed identity between lymphocyte P2Z and the hP2X7 gene recently cloned from human monocytes. When human leukemic B lymphocytes were incubated with ATP and133Ba2+, an immediate influx of isotope occurred. It was augmented by 45% when ATP was added 10 min before isotope. Time-resolved flow cytometry was used to examine kinetics of ethidium uptake in cells incubated with BzATP or the partial agonists ATP, 2-methylthioadenosine 5′-triphosphate, or adenosine 5′- O-(3-thiotriphosphate). Maximally effective concentrations of BzATP (50 μM) induced immediate uptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition of maximally effective ATP concentrations (500 μM) at 37°C, and the delay was longer at 24°C. ATP addition 2–10 min before ethidium abolished the delay. The delay was longer with other partial agonists and inversely related to maximal flux produced by agonist. A delay was also observed for submaximal BzATP concentrations (10–20 μM). P2Z/P2X7 inhibitors, KN-62 and 5-( N, N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened the delay. The results support a model in which agonists for P2Z/P2X7 receptor mediate an immediate channel opening allowing passage of small inorganic cations, followed by a slow further permeability increase allowing passage of larger permeant cations like ethidium. The rate of the second step depends on time and temperature and the efficacy and concentration of agonist and is slowed by antagonists, suggesting it depends on the fraction of P2Z/P2X7 channels held in the initial open state.

Antileukemia activity of perillyl alcohol (POH): uncoupling apoptosis from G0/G1 arrest suggests that the primary effect of POH on Bcr/Abl-transformed cells is to induce growth arrest

“…At 10.0 M, EIPA was toxic as seen by the appearance of apoptotic cells with subdiploid DNA content (Figure 6b). 75 As shown before, POH treatment reduced the proportion of cells in S phase and caused apoptosis (Figure 6e), while TPA reversed the POH-mediated apoptosis (Figure 6f). The ability of TPA to protect the cells from POH-mediated apoptosis was abrogated by EIPA.…”

Extracellular Nucleotides Differentially Regulate Interleukin-1β Signaling in Primary Human Astrocytes: Implications for Inflammatory Gene Expression

“…As a control, RNA samples were also subjected to the first strand cDNA preparation protocol without reverse transcriptase. Primers were based on the reported sequences of the human P2Y 1 , P2Y 2 , P2Y 4 , and P2X 7 receptors (Parr et al, 1994;Communi et al, 1995;Ay yanathan et al, 1996;Rassendren et al, 1997), and the primers for P2Y 2 , P2Y 4 , and P2X 7 have been published previously (Gorodeski et al, 1998;Merten et al, 1998;Wiley et al, 1998): P2Y 1 forward, 5Ј-GAC TTC TTG TAC GTG C TG AC T C T-3Ј; and reverse, 5Ј-GAC C TC TTG TCA CC T GAT ACG TG-3Ј; P2Y 2 forward, 5Ј-C TC TAC TTT GTC ACC ACC AGC GCG-3Ј; and reverse, 5Ј-TTC TGC TCC TAC AGC CGA ATG TCC -3Ј; P2Y 4 forward, 5Ј-ATC C TG CCA CCC TCA C TT C TC C -3Ј; and reverse, 5Ј-AGG CGA GAA GAC GAC TGT GC -3Ј; and P2X 7 forward, 5Ј-AC T CC T AGA TCC AGG GAT AGC C -3Ј; and reverse, 5Ј-TCA C TC TTC GGA AAC TC T TTC C-3Ј. Conditions applied for PCR were as follows: 95°for 5 min, followed by 31 cycles of 1 min 95°C, 1 min 64.4°C (P2Y 1 , P2Y 2 , P2X 7 ) or 61.8°C (P2Y 4 ), and 1 min 72°C, followed by 72°C for 7 min.…”

P2X7 polymorphism and chronic lymphocytic leukaemia: lack of correlation with incidence, survival and abnormalities of chromosome 12

“…Whereas short stimulation of the receptor by ATP is believed to facilitate the passage of cations and promote proliferation, strong or prolonged stimulation enables the passage of larger molecules that may act as a trigger for apoptosis. [3][4][5] A common singlenucleotide polymorphism (SNP) within the P2X7 gene has been described in exon 13 (nucleotide 1513A to C), which results in the substitution of glutamic acid at amino-acid position 496 by alanine (E496A). This polymorphism occurs in the region of the gene that encodes the carboxy-terminal tail of the protein, a structure that controls the permeability properties of the P2X7 receptor, 4 and biochemical studies have demonstrated that homozygosity for the C allele (C / C) leads to almost complete loss of P2X7 function, whereas heterozygosity (A / C) give a function that is half that of cells with the germline (A / A) P2X7 sequence.…”