Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X(7) receptor (previously termed P2Z). These responses include opening of a cation-selective channel/pore that allows entry of the fluorescent dye ethidium and activation of a membrane metalloprotease that sheds the adhesion molecule L-selectin. The surface expression of P2X(7) receptors was measured in normal leucocytes, platelets, and B-CLL lymphocytes and correlated with their functional responses. Monocytes showed four- to fivefold greater expression of P2X(7) than B, T, and NK lymphocytes, whereas P2X(7) expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed lymphocyte P2X(7) at about the same level as B lymphocytes from normal subjects. P2X(7) function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes (n = 47, r = 0.70; P < 0.0001). However, in three patients the ATP-induced uptake of ethidium into the malignant B lymphocytes was low or absent. The lack of P2X(7) function in these B lymphocytes was confirmed by the failure of ATP to induce Ba(2+) uptake into their lymphocytes. This lack of function of the P2X(7) receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule that directs the recirculation of lymphocytes from blood into the lymph node.

ATP receptors and giant cell formation.

We have investigated the role of the purinergic P2X7 receptor in the formation of multinucleated giant cells in human monocyte/macrophage cultures stimulated with either concanavalin A or phytohemagglutinin. Macrophage fusion can be blocked by a P2X7-selective pharmacological antagonist or by a mAb directed against the extracellular P2X7 domain. Furthermore, macrophage cell clones expressing high P2X7 levels spontaneously fuse in culture, whereas macrophage clones lacking P2X7 are unable to fuse. Our findings suggest that the newly identified purinergic P2X7 receptor plays a central role in the complex chain of events leading to generation of macrophage-derived giant cells.

Gene structure and chromosomal localization of the human P2X7 receptor.

The genomic organization for the human P2X7 receptor gene was determined to comprise 13 exons. Alignment of the exon-intron junctions with those for the rat P2X2 gene demonstrated a precise conservation of the boundaries for the first 10 introns. The human P2X7 receptor gene was localized by in situ hybridization to chromosome 12q24. Radiation hybrid mapping indicated that this is within 130 kb of the gene for the homologous P2X4 receptor.

Spontaneous cell fusion in macrophage cultures expressing high levels of the P2Z/P2X7 receptor.

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207- 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion.

Radiolabeling of the rat P2X4 purinoceptor: evidence for allosteric interactions of purinoceptor antagonists and monovalent cations with P2X purinoceptors.

The rat recombinant P2X4 purinoceptor was expressed in CHO-K1 cells, and binding studies were performed using the radioligand [35S]adenosine-5'-O-(3-thio)triphosphate ([35S]ATPgammaS). In 50 mM Tris/1 mM EDTA assay buffer, pH 7.4 at 4 degrees, [35S]ATPgammaS bound with high affinity to the P2X4 purinoceptor (KD = 0.13 nM, Bmax = 151 pmol/mg of protein). The purinoceptor agonists ATP and 2-methylthioadenosine triphosphate possessed nanomolar affinity for the P2X4 purinoceptor, whereas the antagonist suramin possessed much lower affinity (IC50 = 0.5 mM). Cibacron blue was more potent than suramin but produced a biphasic competition curve, whereas d-tubocurarine potentiated binding at concentrations in excess of 10 microM. The complex effects of cibacron blue and d-tubocurarine seemed to be due to an allosteric interaction with the P2X4 purinoceptor because these compounds affected radioligand dissociation, measured after isotopic dilution with unlabeled ATPgammaS. Cibacron blue (1-100 microM) and d-tubocurarine (0.1-1 mM) produced rapid (10 sec to 5 min) decreases or increases, respectively, in the level of [35S]ATPgammaS binding measured immediately after initiation of the dissociation reaction. However, the subsequent rates of radioligand dissociation were not markedly different from those measured in their absence. Monovalent cations produced similar affects on the P2X4 purinoceptor and, like d-tubocurarine, increased [35S]ATPgammaS binding. The actions of d-tubocurarine and sodium were not additive. The findings from this study indicate that [35S]ATPgammaS can be used to label the P2X4 purinoceptor and suggest that this binding can be enhanced by monovalent cations and d-tubocurarine and may be subject to negative allosteric modulation to varying degrees by different purinoceptor antagonists.

The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA.

A cDNA was isolated from a human monocyte library that encodes the P2X7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2', 3'-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.

Domains of P2X receptors involved in desensitization.

ATP-gated ion channels (P2X receptors) are abundantly expressed in both neuronal and nonneuronal tissues, where they can serve as postsynaptic receptors. The response to ATP shows marked desensitization in some tissues but not others. Currents induced by ATP in Xenopus oocytes expressing cloned P2X1 (or P2X3) receptor had strong desensitization, whereas currents in cells expressing P2X2 receptors desensitized relatively little (90% vs. 14% decline of current in a 10-s application). In chimeric receptors, substitution into the P2X1 receptor of either one of two 34-residue segments from the P2X2 receptor removed the desensitization; these segments included the first or the second hydrophobic domain. In contrast, desensitization was introduced into the P2X2 receptor only by providing both these segments of the P2X1 (or P2X3) receptor. This suggests that desensitization requires interaction between the two hydrophobic domains of the receptor, and supports the view that these are membrane-spanning segments.

A comparison of the binding characteristics of recombinant P2X1 and P2X2 purinoceptors.

1. We have recently provided evidence that [35S]-adenosine 5'-O-[3-thiotriphosphate] ([35S]-ATP gamma S) can label the human bladder recombinant P2X1 purinoceptor (human P2X1 purinoceptor). In this study we have characterized the binding of [35S]-ATP gamma S to a second P2X purinoceptor subtype, the rat PC12 phaeochromocytoma cell recombinant P2X2 purinoceptor (rat P2X2 purinoceptor), and compared its binding properties with those of both endogenous and recombinant P2X1 purinoceptors. 2. Infection of CHO-K1 cells with the rat P2X2 purinoceptor using Semliki forest virus (SFV) resulted in the expression of high affinity (pKd = 9.3; Bmax = 18.1 pmol mg-1 protein) binding sites for [35S]-ATP gamma S but not for [3H]-alpha, beta-methylene ATP ([3H]-alpha beta meATP). Since functional P2X purinoceptors could be detected electrophysiologically in these cells, but not in non-infected or CHO-K1 cells infected with SFV containing the LacZ gene, these results suggest that the rat P2X2 purinoceptor can be labelled using [35S]-ATP gamma S. 3. The binding characteristics of the rat P2X2 purinoceptor were compared with those of the human P2X1 purinoceptor, which was also expressed in the CHO-K1 cells using SFV. A major difference between the two recombinant P2X purinoceptor types was in the binding characteristics of alpha, beta-methylene ATP (alpha beta meATP). Thus, in the absence of divalent cations, alpha beta meATP possessed low affinity for both the human P2X1 purinoceptor (pIC50 = 7.2) and rat P2X2 purinoceptor (pIC50 = 7.1) labelled using [35S]-ATP gamma S. However, when the recombinant P2X purinoceptors were labelled with [3H]-alpha beta meATP in the presence of 4 mM CaCl2, the affinity of alpha beta meATP for the human P2X1 purinoceptor increased (pIC50 for alpha beta meATP = 8.2), while the affinity of the rat P2X2 purinoceptor for alpha beta meATP did not change (pIC50 for alpha beta meATP = 6.8). 4. Affinity estimates of 15 other nucleotide analogues for the [35S]-ATP gamma S binding sites on the two recombinant P2X purinoceptor subtypes were surprisingly similar (less than 5 fold difference), the only exception being 2'-deoxy ATP which possessed 8 fold higher affinity for rat P2X2 than for human P2X1 purinoceptors. In contrast dextran sulphate and the P2 purinoceptor antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid and 4,4'-diisothiocyanatostilbene-2,2' disulphonic acid, possessed 7 to 33 fold higher affinity for the human P2X1 than for the rat P2X2 purinoceptor. These data provide a correlation coefficient (r) of 0.894. 5. There was some evidence for species differences in the P2X1 purinoceptor. Thus, most nucleotides possessed slightly greater (up to 9-10 fold), while the P2 purinoceptor antagonists possessed slightly lower (up to 7-16 fold), affinity for the endogenous rat vas deferens and rat bladder P2X1 purinoceptors than for the human recombinant P2X1 purinoceptor. These differences were reflected in a slightly lower correlation coefficient, when comparing across species between the human recombinant P2X1 purinoceptor and the endogenous P2X1 purinoceptors labelled in either the rat deferens (r = 0.915) or the rat bladder (r = 0.932), than when comparing within species between the endogenous rat vas deferens and rat bladder P2X1 purinoceptors (r = 0.995). 6. In summary, [35S]-ATP gamma S can be used to label the recombinant P2X1 and P2X2 purinoceptors. Despite the marked differences reported between these two forms of P2X purinoceptor in functional studies, the differences in binding studies were more limited. However, a number of antagonists could discriminate between the P2X purinoceptor subtypes in the binding studies raising expectations that selective antagonists for these receptors can be developed.

The binding characteristics of a human bladder recombinant P2X purinoceptor, labelled with [3H]-alpha beta meATP, [35S]-ATP gamma S or [33P]-ATP.

1. The binding of [3H]-alpha beta meATP, [35s]-ATP gamma S and [alpha 33P]-ATP to a human bladder P2X purinoceptor, transiently expressed in CHO-K1 cells using the Semliki Forest Virus (SFV) expression system, was examined. The characteristics of the binding sites were compared with results obtained in rat vas deferens, a tissue in which the radioligands are thought to label P2X purinoceptors and in which the endogenous P2X purinoceptor displays high homology with the human bladder P2X purinoceptor. 2. In non-infected CHO-K1 cells, 100 microM ATP evoked only small inward currents (40 pA) in approximately 30% of the cells when studied by the whole-cell voltage clamp technique. In membranes prepared from either these non-infected cells or cells infected with SFV containing the LacZ gene (SFV-LacZ), [3H]-alpha beta meATP bound with low affinity (pKd = 7.04; Bmax = 8.88 pmol ml-1 protein) and there was only a low density of [35S]-ATP gamma S binding sites (pKd = 8.74; Bmax = 358 fmol ml-1 protein). These binding sites differed from those present in rat vas deferens. Thus, pIC50 values for alpha beta meATP (6.5) and L-beta gamma meATP (4.0) at the [3H]-alpha beta meATP binding sites in non-infected CHO-K1 cells were much lower than the respective pIC50 values of 8.3 and 7.7, determined in rat vas deferens. Similarly, affinity estimates (pIC50 values) for ATP (6.82), 2-meS-ATP (5.43), ATP gamma S (7.06) and alpha beta meATP (4.84) at the [35S]-ATP gamma S binding sites in non-infected CHO-K1 cells were up to 2291 fold lower than the respective values of 9.01, 8.79, 8.73 and 7.57, determined in rat vas deferens. 3. In CHO-K1 cells infected using SFV containing the cDNA for the human bladder P2X purinoceptor (SFV-h.P2X), ATP, 2-meS-ATP and alpha beta meATP evoked large inward currents (2-7 nA) in whole cell voltage clamp studies. In membranes prepared from these SFV-h.P2X infected cells, [3H]-alpha beta meATP binding was increased, compared to that measured in the non infected or SFV-LacZ infected cells, with only high affinity [3H]-alpha beta meATP binding sites being detected (pKd = 9.21; Bmax = 3.54 pmol mg-1 protein). The pIC50 values for alpha beta meATP (8.2) and L-beta gamma meATP (7.2) in competing for these sites were the same or similar to the values determined in rat vas deferens. 4. A high density of [35H]-ATP gamma S binding sites (pKd = 9.09; Bmax = 6.82 pmol mg-1 protein) was also present in the membranes from CHO-K1 cells infected with SFV-h.P2X and affinity estimates (pIC50 values) for ATP (8.93), 2-meS-ATP (8.23), ATP gamma S (8.08), and alpha beta meATP (7.17) at competing for these sites were as much as 631 fold higher than the respective values determined in non-infected CHO-K1 cells but were close to the values determined in rat vas deferens. Similar data were obtained with [alpha 33P]-ATP as radioligand. 5. These data suggest that [3H]-alpha beta meATP, [35S]-ATP gamma S and [33P]-ATP label the human bladder recombinant P2X purinoceptor expressed in CHO-K1 cells following infection with SFV-h.P2X and provide further corroborative evidence to support the contention that the high affinity binding sites for these radioligands in rat vas deferens are P2X purinoceptors.

Characterization and chromosomal localization of a human P2X receptor from the urinary bladder.

A cDNA encoding an ion channel (hP2X), gated by extracellular ATP, was isolated from the human urinary bladder. It encodes a 399 amino acid protein, composed of a cysteine-rich central domain, flanked by two hydrophobic regions. A comparison of the sequence with those of the corresponding rat and mouse proteins shows predominantly conservative substitutions of hydrophilic residues. Northern blot analysis demonstrated the presence of the mRNA in several human tissues and established that the distal untranslated portion of the mRNA includes an 'expressed sequence tag' for the differentiation of the hemopoetic cell line, HL60. By fluorescent in situ hybridization the hP2X gene was mapped to the short arm of human chromosome 17. Expressed in Xenopus oocytes, the receptor was sensitive to the purinergic agonists ATP and alpha,beta-methylene ATP.

Vasoactive intestinal peptide: expression of the prohormone in bacterial cells.

A complementary DNA (cDNA) to the messenger RNA for the preprohormone of human vasoactive intestinal peptide (VIP) has been isolated and characterized. This cDNA extends from 65 bases 5' of the AUG translation start codon through the entire 3' untranslated region. Using this cDNA we have constructed expression plasmids which allow the synthesis of 120 out of the 150 amino acids of the prohormone in E. coli. This portion of the prohormone gene was either fused to a segment of a bacteriophage structural gene or expressed alone. When expression was induced the fusion protein constituted 15% of the total bacterial cell protein while the prohormone alone was 5%. Both proteins are recognized by antiserum raised against porcine VIP. They provide protein to study the precursor-product relationship of the hormone plus the possibility of identifying cryptic regulatory peptides contained within the prohormone.

Cloning overlapping DNA fragments from the B95-8 strain of Epstein-Barr virus reveals a site of homology to the internal repetition.

Overlapping, sheared DNA fragments from the B95-8 strain of Epstein-Barr virus were cloned in Charon 4A. Eleven recombinant phages plus one recombinant plasmid contained all of the sequences found in B95-8 virion DNA. Analysis of recombinant DNA molecules revealed a previously undetected site of homology to the internal repetition found in Epstein-Barr virus DNA. This site was adjacent to or at a site which was unstable when the recombinant DNA was propagated as phage DNA in procaryotic hosts.

Isolation of recombinant plasmids bearing cDNA to hen ovomucoid and lysozyme mRNAs.

A large library of hen oviduct cDNA-pCR1 recombinant plasmids has been established in Escherichia coli X1776. From this library, ovomucoid cDNA and lysozyme cDNA-bearing plasmids have been identified. One of these plasmids, pMu7, yielded the sequence of the 3'-untranslated region of ovomucoid mRNA.

Restriction map of the region surrounding the EcoRI site in the pCR1 plasmid and analysis of an inserted ovalbumin gene.

We have determined a restriction map of a 1650 base pair region surrounding the EcoRI site of the bacterial plasmid, pCR1. We have used pCR1 as a vector in cloning synthetic ovalbumin double-stranded cDNA. Using the pCR1 restriction map, we have characterized the ovalbumin sequences inserted in one recombinant plasmid, pOvE12. POvE12 appears to contain all, or nearly all, of the sequences found in full length, double-stranded cDNA synthesized in vitro.

Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. Optimization for full length second strand synthesis by Escherichia coli DNA polymerase I.

Sequential reverse transcriptase, DNA polymerase, and S1 nuclease reactions can be employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, monovalent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing RNA.

Synthesis of full length cDNAs from four partially purified oviduct mRNAs.

Total poly(A)-containing RNA prepared from hen oviduct and centrifuged on an isokinetic sucrose gradient displays four peaks of optical absorbance. These have been identified by translation in vitro as lysozyme, ovomucoid, ovalbumin, and conalbumin mRNAs. Isolation and recentrifugation of the peaks results in partial purification of each mRNA. Molecular weights have been determined for the mRNAs on agarose gels containing 20 mM methylmercury hydroxide. Each mRNA possesses a number of apparently untranslated nucleotides ranging from approximately 900 bases for ovalbumin and conalbumin mRNAs to 200 bases for ovomucoid and lysozyme mRNAs. The mRNAs have been copied with avian myeloblastosis virus reverse transcriptase. Each mRNA with the exception of conalbumin gives rise to a high proportion of full length cDNA. Several parameters previously reported to influence the size distribution of cDNA had no effect on the length of cDNA made from any mRNA fraction. The proportion of full length copy does depend on the reverse transcriptase lot.