Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 95, Num. 3, 2000, pp. 415-428

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 95(3) May/Jun. 2000, pp: 415-428

Procedures to Characterize and Study P_2Z/P2X₇ Purinoceptor: Flow Cytometry as a Promising Practical, Reliable Tool

Oscar Kenji Nihei, Wilson Savino, Luiz Anastacio Alves⁺

Laboratório de Pesquisas sobre o Timo, Departamento de Imunologia, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil

⁺Corresponding author. Fax: +55-21-280.1589. E-mail: alveslaa@gene.dbbm.fiocruz.br.

Received 9 August 1999
Accepted 27 October 1999

Code Number: oc00067

The expression of P_2Z/P2X₇ purinoceptor in different cell types is well established. This receptor is a member of the ionotropic P2X receptor family, which is composed by seven cloned receptor subtypes (P2X₁ - P2X₇). Interestingly, the P_2Z/P2X₇ has a unique feature of being linked to a non-selective pore which allows the passage of molecules up to 900 Da depending on the cell type. Early studies of P_2Z/P2X₇ purinoceptor were exclusively based on classical pharmacological studies but the recent tools of molecular biology have enriched the analysis of the receptor expression. The majority of assays and techniques chosen so far to study the expression of P_2Z/P2X₇ receptor explore directly or indirectly the effects of the opening of P_2Z/P2X₇ linked pore. In this review we describe the main techniques used to study the expression and functionality of P_2Z/P2X₇ receptor. Additionally, the increasing need and importance of a multifunctional analysis of P_2Z/P2X₇ expression based on flow cytometry technology is discussed, as well as the adoption of a more complete analysis of P_2Z/P2X₇ expression involving different techniques.

Key words: flow cytometry - purinergic receptors - P2X₇ receptor - ethidium bromide - extracellular ATP

NUCLEOTIDES AND NUCLEOSIDES AS EXTRACELLULAR SIGNALING MOLECULES

Nucleotides and nucleosides comprise a recently established new family of extracellular messengers (see Ralevic & Burnstock 1998). The first evidence that such molecules can play a physiological role when applied extracellularly came from experiments of Drury and Szent-Györgyi (1929), where perfusion of adenosine and adenosine 5'-monophosphate promoted hipotension and bradicardia in the guinea pig cardiovascular system. Based on systematic investigations, Burnstock (1971, 1996) proposed the existence of a purinergic component in the vegetative nervous system, where the ATP is released by synaptic terminals as a neurotransmitter or co-transmitter in both sympathetic and parasympathetic systems.

In fact, the discovery of such purinergic component in the autonomic nervous system was a landmark that established the importance of nucleotides and their derivatives as extracellular messengers. The proposed new role of nucleotides and nucleosides as extracellular messengers was initially viewed with skepticism because of its importance in cell maintenance and survival. Yet, further studies, showing the direct physiological effect of such molecules in every system (respiratory, muscular, vascular, haemopoietic, immune and nervous system), stand for their relevance (Dubyak & El-Moatassim 1993, Alves et al. 1999). Additionally, ATP and derivatives have been found stored within vesicles of platelets, basophils and mast cells, being released with other known compounds when the appropriate stimulus is applied (Dubyak & El-Moatassim 1993).

Extracellular nucleotides and nucleosides, released from neural and non-neural sources, interact with a specific family of membrane associated-molecules named purinergic receptors (reviewed by Fredholm et al. 1994). The purinergic receptors have been classified into two types, P1 and P2, as originally proposed by Burnstock (1978); both types being ubiquitous (Ralevic & Burnstock 1998). P1 purinoceptors are specific for adenosine, being sub-classified into four subtypes, namely A1, A2a, A2b and A3, according to pharmacological, functional and molecular criteria. Specific agonists as well as molecular biology techniques are available to distinguish each P1 receptor subtype (Olah & Stiles 1995, Ralevic & Burnstock 1998). P2 purinoceptors are specific for nucleotides and were classified in six subtypes: P_2D, P_2T, P_2U, P_2X, P_2Y and P_2Z. This classification was later sanctioned by the International Union of Pharmacology (IUPHAR) Purinoceptor Classification Subcommittee. It was based on differences in terms of agonist potency rank order and selectivity, sensitivity to antagonist, functional response, signal transduction mechanism and desensitization features of the receptor after continuous agonist application (Fredholm et al. 1994). P_2D, P_2T, P_2U and P_2Y are G-protein coupled while P_2Z and P_2X are ligand-gated intrinsic ion channels. Interestingly, despite the differences in signal transduction all the receptor subtypes tested are able to induce an increase in the intracellular calcium concentration. Molecular cloning of P2 receptors led to the discovery and addition of new purinergic receptor subtypes that established the existence of two major families, P2Y and P2X (Abbracchio & Burnstock 1994). P2Y family is composed by metabotropic receptors, and is structurally related to G protein-coupled receptors, with seven putative alpha-helical transmembrane segments, extracellular amino-terminal, and intracellular carboxyl-terminal. The P2Y family comprises at least 5 functional cloned mammalian receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ ) that have been described in several different cell types (Ralevic & Burnstock 1998, King et al. 1998). By contrast, the P2X family is composed by ionotropic receptors, i.e. ligand-gated ion channels, and so far seven receptor subtypes have been identified (P2X₁ _ P2X₇) (Table I). The P2X receptors bear two putative transmembrane domains connected by a large extracellular ligand-binding loop and intracellular amino- and carboxyl- termini (Valera et al. 1994, Hansen et al. 1997). P2X receptors have been found expressed preferentially in neurons and muscle cells, except for P2X₇, as we shall discuss here. Thus, P2 receptors are similar to other known classical neurotransmitter receptors such as those that recognize acethylcholine, gamma-amino butyric acid (GABA), glutamate and serotonine, which present functionally and structurally distinct families of ionotropic and metabotropic receptors. These families mediate fast and slow responses, respectively, via different signaling mechanisms (Burnstock 1997). Probably during the evolution, each type of signaling pathway has accomplished a different advantage to the organism survival. In this context, ATP and other energetic nucleotides might have been chemotaxic molecules, indicating position of damage or dead cells. Thus, cells which had receptors for such molecules could have been positively selected, determining survival and reproductive success. In agreement with this hypothesis some unicellular organisms and invertebrate species express ATP and AMP nucleotide receptors, indicating that use of nucleotides as signaling molecules may be very ancient (Carr et al. 1986, Devreotes & Zigmond 1988).

THE P_2Z/P2X₇ RECEPTOR

The wide expression of P1 and P2 receptors in different systems has fostered the recent search for its physiological importance as well as its pharmacological applications. One P2 receptor subtype, the P_2Z, has interested mostly immunologists, hematologists and biophysicists. The P_2Z is the endogenous native counterpart of the cloned P2X₇ purinoceptor and for this reason it is also called P_2Z/P2X₇ by some authors. The existence of a distinct purinergic receptor named P_2Z was proposed by Gordon (1986) based on pharmacological analysis. Subsequent investigation established more precisely other P_2Z receptor properties. It has been proposed that P_2Z receptor is restrictively activated by the fully anionic ATP^-4, requiring higher concentrations of ATP to be activated (EC₅₀: 0.1 to 1 mM) when compared with other P2X purinoceptors (~ 1 - 10 µM). The P_2Z receptor is sensitive to few other agonists, particularly to BzATP, usually 10 to 100 times more potent than ATP (Dubyak & El-Moatassim 1993, Di Virgilio 1995). The agonist potency rank order of P_2Z was BzATP>ATP= ATPgS>>>ADP=AMP. Agonists such as adenosine and UTP, potent agonists for other P1 and P2 receptor subtypes, were ineffective for P_2Z (Steinberg et al. 1987, Dubyak & El-Moatassim 1993, Nuttle & Dubyak 1994). Additionally the P_2Z was specifically antagonized by oxidized ATP (in all cell types tested so far) and KN-62 (particularly in lymphocytes) (Murgia et al. 1993, Wiley et al. 1993, Gargett & Wiley 1997). Functionally, the hallmark for this receptor is the opening of a low selective pore permeable to molecules up to 900 Da (Steinberg et al. 1987). Interestingly, permeability differences have been found depending on cell type analyzed: thymocytes and peripheral lymphocytes present lower permeability, limiting the passage of molecules until 200-414 Da (Pizzo et al. 1991, Wiley et al. 1993, Nagy et al. 1995, Chused et al. 1996). This raises the possibility existing other P_2Z receptor subtypes or different pores, or the existence of pore subconductances.

The sensitivity to BzATP and the unique pore-forming capacity of the P_2Z/P2X₇ receptor make its characterization less uncertain than for the other purinoceptors.

The P2X₇ receptor was cloned from rat brain and expressed in HEK293 cells by Surprenant et al. (1996). This receptor presents 595 amino acids (a.a.) where the first 395 a.a. share 40% structural homology with other P2X receptors with the same putative structure: two transmembrane domains and a large extracellular loop. Conversely, the P2X₇ receptor presents a longer COOH-terminal compared to other P2X purinoceptors. It has been proposed that such extra intra-domain is responsible for, or participates in the formation of the pore. This hypothesis was based on an experiment where the expression of the P2X₇ protein truncated at 418 a.a. position in HEK293 cells did not induced pore formation as ascertained by dye uptake assay (Surprenant et al. 1996). This cloned rat P2X₇ receptor presented the following agonist potency rank order: BzATP>>ATP>2MeATP>ATPgS>> ADP. Additionally, it required high ATP concentrations to be activated (EC₅₀: 115 ± 9 µM), was antagonized by oxidized ATP (oATP), and promoted the non-selective pore formation. More recently, the human and mouse P2X₇ receptor was cloned and presented 80 to 85% of homology with the rat orthologue receptor (Rassendren et al. 1997, Chessell et al. 1998a).

Since these initial studies the native P_2Z purinoceptor and the cloned P2X₇ receptor matched many features, becoming possible the establishment of a consistent correlation between them. That was in opposition to some P2 receptors. The P_2Z/P2X₇ receptor has been expressed mostly in cells of haemopoietic origin, although it also may be found expressed in other cell types as well as in different cell lines (Table II). Such particularities distinguish the P_2Z/P2X₇ purinoceptor, making it quite different from all other known P2X receptors.

P_2Z/P2X₇ PURINOCEPTOR CHARACTERIZATION APPROACHES

Methodologically different techniques have been used to characterize P_2Z/P2X₇ receptor (Table II). Most of them directly or indirectly investigate pores, ion channels and membrane alterations, comprising: (1) analysis of membrane biophysics (electrophysiological techniques); (2) analysis of transductional signaling that follows the P_2Z/P2X₇ activation (calcium microfluorometry); (3) indirect analysis of the receptor activation (dye uptake assay, membrane depolarization, different ion influx analysis); (4) analysis of physiological alterations that follow the P_2Z/P2X₇ receptor activation (for ex. cytotoxicity, cytokine secretion). More recently, with the availability of the P2X₇ receptor cDNA (Surprenant et al. 1996), molecular biology techniques have been gradually adopted to study the expression of the P2X₇ receptor at mRNA and protein level, making receptor study more accurate. Some of the most frequently used techniques to characterize and study P_2Z/P2X₇ receptor are presented below including the discoveries associated with each one.

Electrophysiology - One of the most common electrophysiological technique to study P_2Z/P2X₇ receptor is the patch clamp which was developed by Neher and Sakmann (1976) and has revolutionized the study of membrane biophysics. The patch clamp method isolates a tiny portion of the cell membrane and makes it possible to study single ion channels and pores individually or collectively (Sakmann & Neher 1995). Such electrophysiological technique has been widely used to study P_2Z/P2X₇ receptor and have been proved to be appropriate to elucidate important receptor properties such as its kinetics of activation, pore permeability, selectivity and conductance, current reversal potential and rectification, as well as the analysis of agonist and antagonist selectivity (Albuquerque et al. 1993, Surprenant et al. 1996, Coutinho-Silva & Persechini 1997, Rassendren et al. 1997, Virginio et al. 1997, Chessell et al. 1997, 1998a,b). Patch clamp studies in whole cell configuration have revealed that the receptor activation induces a fast inward current of Na⁺ and Ca⁺⁺ ions that appear in the first milliseconds. This current generally lacked inward rectification (or had it low) and showed a reversal potential near 0 mV, which is consistent with the non-selective ion channel. In addition, the current presented no or low desensitization under continuous agonist exposure. In murine macrophages and phagocytic cells of thymic reticulum, a secundary outward current due to the activation of Ca²⁺-dependent K⁺ channels was also recorded (Albuquerque et al. 1993, Coutinho Silva et al. 1996a). Nuttle and Dubyak (1994) showed a biphasic inward current response during the kinetics of activation of the P_2Z/P2X₇ receptor, consisting of an initial fast current due to the opening of a poorly selective cation channel, followed by a delayed large current due to the opening of an non-selective pore. This led to the notion that P_2Z/P2X₇ receptor has two transient forms (ion channel/pore). Patch clamp studies in cell attached and out-side out configurations have attributed low conductances and subconductances (2 to 17 pS) to P_2Z/P2X₇ receptor triggered single channels (Tatham & Lindau 1990, Naumov et al. 1995, Coutinho-Silva et al. 1996b, Markwardt et al. 1997, Persechini et al. 1998), what is consistent with the fast activating ion channel activity, but not with the low selectivity pore formation. More recently, the conductance compatible with the non-selective pore was reported by Coutinho-Silva and Persechini (1997). In this work two pores were described, showing conductances of 280 pS and 409 pS. Nevertheless, it is not yet clear if such findings actually represent two types of pores or two subconductance states of the pore.

In addition, there is an unsolved controversy whether the pore linked to P_2Z/P2X₇ receptor corresponds to the receptor itself or represents a distinct chemical entity. When the rat P2X₇ receptor was expressed in HEK293 cells, ATP application induced cell permeabilization to YO PRO-1 (Surprenant et al. 1996), but the same did not happen when the rat P2X₇ was expressed in Xenopus oocyte system (Petrou et al. 1997). Furthermore, studies of P2X₇ expression on chinese hamster ovarian variant cell line (CHO-K1) cells have demonstrated that at 22^oC the permeabilization to YO-PRO-1 delayed up to 8 min when compared to that at 37^oC. In contrast, calcium influx delayed just 10 seconds, suggesting two transient forms of the receptor (ion channel/pore) or two distinct entities (Michel et al. 1998). In keeping with this, electrophysiological studies in cell attached configuration conducted by Coutinho-Silva and Persechini (1997) reinforce the receptor/pore dissociation hypothesis. In such a configuration the P2X₇ receptor/pore structure is confined within the recording pipette tip isolated from extracellular bulk by a gigaseal. However, the authors observed that the external ATP application induced a 409 pS inward current, showing that the pore may be dissociated from the receptor, possibly been gated by an intracellular messenger. Thus, electrophysiological techniques have been critical to elucidate novel aspects of P_2Z/P2X₇ receptor activation that otherwise would be impossible.

Analysis of signal transduction: Calcium microfluorometry - The signal most commonly associated with the activation of all P2 receptors is the increase of intracellular calcium (Dubyak & El-Moatassim 1993). In regard to the receptors of the P2Y family, the intracellular calcium increase is dependent on triphosphate inositol pathway, what induces the calcium release from intracellular stores. On the other hand in the P2X receptor family, calcium chiefly comes from the extracellular milieu and enters the cell through ligand-gated ion channels according to its electrochemical gradient (Harden et al. 1995). This response in particular has been explored to complement the characterization of P_2Z/P2X₇ receptor in different cell types. In keeping with this, calcium microfluorometry has been directed to pharmacological and functional studies since the calcium response amplitude is correlated with the agonist potency and concentration, when it is non-saturating. The calcium response induced by P_2Z/P2X₇ receptor activation has been described in the majority of cells analyzed (Table II). In general, the rise of intracellular calcium due to activation of P_2Z/P2X₇ receptor initiates milliseconds after the agonist application and presents a fast elevation, although its amplitude depends on the cell type. In thymocytes, intracellular calcium increases four times the baseline (0.1-1 µM) whereas in macrophages, it reaches at least ten times the baseline values under the same P_2Z/P2X₇ stimulation condition (Greenberg et al. 1988, Pizzo et al. 1991). Additionally, after a single agonist application, the P_2Z/P2X₇ receptor induces a sustained increase of intracellular calcium that is maintained for many seconds or even minutes (Greenberg et al. 1988, Ross et al. 1997). In this way, calcium microfluo-rometric evaluation allowed the distinction of the P_2Z/P2X₇ response from that of the other P2 receptors, as ascertained by distinct variables such as the required ATP concentration to trigger calcium response (high ATP concentrations), calcium response amplitude (high amplitude), duration of the response (sustained calcium response), agonist and antagonist selectivity (responsive to ATP and BzATP) as well as the calcium source (extracellular) (Greenberg et al. 1988, Macmillian et al. 1993).

Other transductional signaling pathways have been associated to P_2Z/P2X₇ receptor such as phospholipase A2 (Alzola et al. 1998) and phospholipase D (PLD) activation (El-Moatassim & Dubyak 1992,1993, Gargett et al. 1996, Humphrey & Dubyak 1996). Interestingly, in THP-1 monocytic cell line the PLD activation was explored as a marker of P_2Z/P2X₇ activation in the study of its modulation by pro-inflammatory factors such as interferon-g (INFg) and lipopolysaccharide (LPS) (Humphreys & Dubyak 1996).

Dye uptake assay - The P_2Z/P2X₇ purinoceptor differs from other known ligand-gated receptors due its link to a non-selective pore. The P_2Z/P2X₇ pore opening induces the exchange of ions and molecules up to 900 Da according to an electrochemical gradient. Electrophysiological, fluorometric and radiometric studies have revealed that the P_2Z/P2X₇ channel/pore is permeable to several ions such as Na⁺, K⁺, Li⁺, Rb⁺, Cl^-, Mn²⁺, Ca²⁺, Sr²⁺ and Ba²⁺, as well as larger molecules such as tris(hidroxymethyl)aminomethane (TRIS) (121.1 Da) and N-methyl-D-glucamine (NMDG+) (195.2 Da) (Steinberg et al. 1987, Naumov et al. 1992, Albuquerque et al. 1993, Wiley et al. 1993, Nuttle & Dubyak 1994).

Several studies have also demonstrated that the P_2Z/P2X₇ pore is permeable to different fluorescent markers such as 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) (290 Da), ethidium bromide (314 Da), YO PRO-1 (375.5 Da), 6-carboxyfluorescein (376 Da), propidium iodide (414 Da), TO-PRO-3 (417 Da), lucifer yellow (443 Da), eosine yellowish (646 Da) and FURA-2 (831 Da), but is not permeable to trypan blue (961 Da), evans blue (961 Da) and high molecular weight dextran conjugates (Steinberg et al. 1987, El-Moatassim et al. 1990, Picello et al. 1990, Wiley et al. 1993, Nuttle & Dubyak 1994, Nagy et al. 1995, Surprenant et al. 1996, Persechini et al. 1998). This permeability is a functional hallmark and has been explored in the majority of studies that characterize the P_2Z/P2X₇ receptor.

Additionally, this assay has been used to conduct pharmacological and functional studies. In macrophages, it was demonstrated that the pore opening is temperature and pH dependent; being inactive below 18^oC and at pH 6.5 and optimally active at 37^oC and pH 8.0-8.5 (Steinberg et al. 1987). Such phenomenon is also inhibited by Mg²⁺, indicating that the active P_2Z/P2X₇ receptor ligand is the non-complexed ATP^-4 (Steinberg et al. 1987). The dye uptake assay has also been useful to verify the functional expression of P_2Z/P2X₇ purinoceptor in studies that involve the generation of cell lines with altered expression of P_2Z/P2X₇ receptor (Chiozzi et al. 1996, 1997). P_2Z/P2X₇ receptor hiper (ATP-sensitive) and hipo (ATP-resistant) expressed in J774 cell lines can be clearly differentiated by the degree of perme-abilization to fluorescent dyes (Chiozzi et al. 1996, 1997). Dye uptake assay may also demonstrate modulation of the P_2Z/P2X₇ receptor expression. Human THP-1 monocyte cell line presents increased expression of P_2Z/P2X₇ receptor when treated concomitantly with different pro-inflammatory and inflammatory factors such as INF-g and LPS or INF-g and tumor necrosis factor-a (TNFa), as ascertained by permeabilization assay (Humphreys & Dubyak 1996, 1998). The primary characterization of the P_2Z/P2X₇ receptor by dye uptake assay can thus be used as a fast and practical method to ascertain its functionality.

However, additional techniques are required to ascertain the uncertain expression of P_2Z/P2X₇ purinoceptor when the cell analyzed is resistant to ATP-induced permeabilization. It has been the case with neutrophils and, in a lesser extent, monocytes and B lymphocytes (Walker et al. 1991, Hickman et al. 1994, Chused et al. 1996).

FLOW CYTOMETRIC ANALYSIS OF P_2Z/P2X₇ PURINOCEPTOR

The flow cytometry was developed by a collective effort that began in the 1950s. This invention allowed automatic counting and quantification of cell size for the first time, what significantly increased the reliability of such analysis (Melamed et al. 1991). Later a fluorescence detection system was coupled and evolved making it possible cell multiparametric studies. Additionally, the development of monoclonal antibody technology by Koehler and Milstein in the 70s increased the availability of reagents directed to research and clinical studies, and, as a consequence, the availability of fluorescent coupled ones (Koehler & Milstein 1975). Such technological evolvements made it real the use of flow cytometry as a powerful tool to improve research in immunobiology. Nowadays the flow cytometry usage is widespread in clinical and research laboratories, being applied to analyze multiple cell parameters such as cell cycle, cell membrane alterations, alterations of intracellular calcium and cell phenotype.

The P_2Z/P2X₇ receptor pore-formation capacity has been explored by several groups to permeabilize different cell types in order to introduce cell membrane impermeant molecules that could have clinical or research interest (Picello et al. 1990, Jaffar & Pearce 1993, Munerati et al. 1994, Gan et al. 1998). Additionally, using different techniques, these studies have analyzed the important parameters that could be affected by such ATP-induced membrane permeabilization such as the cell viability, morphology, intracellular calcium and pH, and apoptosis. Using flow cytometry all these analyses can be performed with accuracy.

Several studies have used the flow cytometry to study the P_2Z/P2X₇ receptor (Wiley et al. 1993, 1998, Hickman et al. 1994, Nagy et al. 1995, Chused et al. 1996, Persecchini et al. 1998). The main focus of such reports is the indirect P_2Z/P2X₇ receptor detection by means of dye uptake assays. A pioneer work that used this technology was that of Wiley et al. (1993), which demonstrated that lymphocytes obtained from B-cell chronic lymphocytic leukemia patients became permeable to ethidium bromide but not to propidium iodide after ATP-treatment. Later on, Hickman et al. (1994), demonstrated a possible augmented expression of P_2Z/P2X ₇ receptor in monocytes, which varied with cultured time using the flow cytometry as an complementary technique and the YO-PRO-1 dye. In these studies, DNA binding dyes such as ethidium bromide, propidium iodide and YO-PRO-1 have preferentially been chosen to analyze dye uptake by flow cytometry. These fluorescent dyes presented two major advantages over other dyes such as lucifer yellow, that do not bind DNA: (1) these dyes are almost unaffected by diffusion; (2) they do not suffer the subtraction by organic transporters that could decrease its concentration in cytoplasm and, consequently, diminish the associated fluorescent signal.

Another important point before performing flow cytometry analyses of dye uptake assays is to ascertain by fluorescence microscopy if the phenomenon is simply due to P_2Z/P2X₇ activation, i.e., only pore opening, rather than endocytosis. In this regard, our group has identified the P_2Z/P2X₇ purinoceptor in primary cultured murine dendritic cells. In this study, permeabilization analyses were performed by flow cytometry using ethidium bromide as the standard dye. In this case the dye uptake analysis was also viewed by fluorescence microscopy in order to avoid any unwanted artifact. Dendritic cells treated with ATP concentrations compatible with that necessary to activate P_2Z/P2X₇ became permeabilized to ethidium bromide as shown in Fig. 1. Additionally, dendritic cells were sensitive to the agonist BzATP and the ATP-induced permeabilization was antagonized by oxidized ATP (oATP), thus showing that dendritic cells permeabilization is due to specific P_2Z/P2X₇ receptor activation (Fig. 1). In this regard, other P1 and P2 agonists such as adenosine, AMPc, ADP and UTP were ineffective (Fig. 2).

The time-resolved flow cytometry has also been explored to study the P_2Z/P2X₇ receptor properties. In this case, the mean fluorescence intensity of a pre-determined number of cells that pass in different time intervals is collected, what provides a continuous observation of the analyzed phenomenon. Wiley et al. (1998), using this method, confirmed that BzATP was a full agonist of the P_2Z/P2X₇ receptor-dependent permeabilization of human leukemic lymphocytes.

Flow cytometry has also been used to detect intracellular calcium alterations due to P_2Z/P2X₇ activation (Nagy et al. 1995, Chused et al. 1996). In theses studies indo-1 and fluo-3 dyes have been used. Chused et al. (1996) monitored the ATP dependent permeabilization and intracellular calcium alterations of different murine thymocyte and peripheral lymphocyte populations in a multipara-metric analysis performed by flow cytometry. This study demonstrated indirectly the presence of P_2Z/P2X₇ receptor in different cell types in the following decreasing expression sequence: spCD8+> thCD8+> spCD4+>thCD4+ >thCD4+CD8+ (sp:spleen; th:thymus). Persechini et al. (1998) and Alves-Neto and Persechini (pers. commun.) have used flow cytometry to determine the expression of the P_2Z/P2X₇ receptor in different peripheral blood mononuclear cell (PBMC) populations. The three color analysis of PBMC showed that T lymphocytes (CD3+) and monocytes (CD14+) became permeable to the TO-PRO-1 dye after ATP treatment. The same occurred with natural killer cells (CD16⁺/CD56⁺), thus evidencing the P_2Z/P2X₇ expression. Interestingly, among these PBMC populations monocytes presented the highest degree of permeabilization.

Recently, a monoclonal antibody (mAb) directed to the P_2Z/P2X₇ receptor was developed and tested (Chiozzi et al. 1997, Collo et al. 1997, Buell et al. 1998). Once largely adopted, it will facilitate the P_2Z/P2X₇ receptor expression analysis. Thus, the flow cytometry will consist in a more useful tool to study this purinergic receptor. Concomitantly with specific mAb labeling, P_2Z/P2X₇ pore functionality could be ascertained in the same experiment by dye uptake assay. Furthermore, multifunctional analyses of different cell populations can be performed through the use of other mAbs available.

CONCLUSIONS

Flow cytometry allows: (1) fast analysis of a large number of cells; (2) the sensitivity of a reliable fluorescence detection system; and (3) the possibility of distinguishing different cell populations due to the usage and availability of a variety of mAbs bearing distinct specificities. Such points are advantages that make flow cytometry distinctive from the all other technologies commonly used to detect cell fluorescence, such as fluorescence microscopy and fluorometry. Particularly regarding the study of purinergic receptors, this strategy has become gradually more used by different groups. Yet flow cytometry-based studies involving the analysis of P_2Z/P2X₇ receptor expression, its modulation and functionality are still underexplored. With the availability of the specific anti-P_2Z/P2X₇ mAb, the generation of new fluorescent dyes and the accessibility to flow cytometry apparatus coupled to two or more laser systems, different protocols could be envisioned for the investigation of more complex systemic parameters, such as the involvement of P_2Z/P2X₇ receptor in bone marrow cell differentiation and on thymocyte differentiation, including the intrathymic selection of the T cell repertoire.

It must be emphasized that the P2 receptor characterization based on classical pharmacological studies with the exclusive analysis of agonists and antagonist effects is nowadays considered incomplete, due to the cloning of many different new P2 receptors and the lack of specific pharmacological tools. The investigation of P_2Z/P2X₇ is not a exception, despite its unique hallmark properties. Its analysis must involve different techniques such as those described here as well as molecular biology approaches. Only the adoption of such procedures will clarify the precise characterization of the P_2Z/P2X₇ receptor in cells of different systems and distinguish the possible existence of different P_2Z/P2X₇ subtypes. This is the case of the P2 receptor characterized or just suggested in erythrocytes, gastric smooth muscle cells of toad, hepatocytes, rat pancreatic ducts, Leydig cells, supraoptic neurones and schwann cells (Parker & Snow 1972, Foresta et al. 1996, Zoeteweij et al. 1996, Ugur et al. 1997, Christoffersen et al. 1998, Grafe et al. 1999, Shibuya et al. 1999). In these cells the characterized P2 receptor shared some, almost all, or even all P_2Z/P2X₇ pharmacological properties, but it was not able to induce the formation of the large non-specific pore. In some cases such point was not investigated. This point also involves the controversy if the P_2Z/P2X₇ receptor and the linked pore are really the same or distinct entities. Further investigation involving different techniques will be necessary to clarify all these unsolved questions.

ACKNOWLEDGEMENTS

To Drs Robson Coutinho-Silva and Fernando P de Farias for the critical review of the manuscript.

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This work was partially supported with grants from CNPq, Pronex/CNPq, PADCT/CNPq and Faperj (Brasil).

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