Microfluorimetric analysis of a purinergic receptor (P2X7) in GH4C1 rat pituitary cells: effects of a bioactive substance produced by Pfiesteria piscicida.

Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH4C1 rat pituitary cells. Extracellular adenosine 5'-triphosphate (ATP) acting on P2X7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH4C1 rat pituitary cells express functional P2X7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2'-3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH4C1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH4C1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH4C1 cells express a purinoceptor with characteristics consistent with the P2X7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP.

Extracellular adenosine 5´-triphosphate (ATP) acting on purinergic (P2) receptors regulates a variety of functions, using transduction pathways that are dependent on the type of the P2-receptor and on the type of cells where the action occurs (1,2). For example, in the nervous system, ATP is co-released with neurotransmitters by exocytosis to modulate the synaptic transmission via the activation of P2X-receptor cation channels (3). Macrophages and mast cells express a different functional type of P2X-receptor that changes its ionic selectivity when exposed to extracellular ATP (4). In this type of receptor, first designated P2Z and later recognized as P2X 7 , the formation of a nonselective cation channel is followed by a progressive permeabilization of the cell membrane to ions of larger molecular weight and finally leading to cell lysis (5). This distinctive second mode of action involving an apparent increase in pore diameter has been the focus of many studies (5)(6)(7)(8)(9)(10). P2 receptors have been classified in two different classes: the ionotropic P2X, which forms a nonselective cation channel when activated by an agonist, and the metabotropic G-protein-coupled P2Y (11). Structurally, P2X receptors are constituted by the assemblage of subunits, each one with two transmembrane domains (12,13), whereas P2Y has the typical structure of a Gprotein-coupled receptor with seven membrane-spanning domains (13). Seven types of the P2X class of P2 receptors have been cloned (P2X 1 -P2X 7 ) and are distinguished pharmacologically in two groups on the basis of sensitivity to the ATP congener αβ-ATP. A third group, the P2X 7 receptors, is distinguished functionally by ionic conductance (14). The P2X 7 activity was first identified in mast cells and macrophages and was suggested to have a role in cytolysis and apoptotic death in cells of the immune system (15,16). P2X 7 was then cloned from the rat brain (6), where it is found in the microglia (17). Its presence has been documented in other cell types such as human fibroblasts (18) and hepatocytes (19), and in the cell lines CHO-K1 (9) and GH 4 C 1 (28). Pharmacological studies identified a characteristic higher affinity of P2X 7 to the ATP congener 2´-3´-O-(benzoyl-4-benzoyl)-ATP (BzATP) than to ATP itself, and revealed a sensitivity to the ionic environment in the kinetics of cell permeabilization (21). The finding that the replacement of extracellular ionic sodium by the larger ion N-methyl-Dglucamine (NMDG) facilitates the permeabilization mode of the activated receptor, causing a delay or inhibition of cell lysis (5), has been widely used in the functional characterization of the P2X 7 (21).
It is well known that toxins synthesized by a variety of organisms can selectively bind to ion conduction channels, altering their functional properties and leading to toxicity. The heterotrophic estuarine dinoflagellate Pfiesteria piscicida Steidinger & Burkholder was discovered in 1991 by Burkholder and co-workers (22) and has been implicated as the causative agent of major fish kills and fish disease in the two largest U.S. mainland estuaries (the Albemarle-Pamlico of North Carolina and Chesapeake Bay in Maryland) (23). A second toxic Pfiesteria species has subsequently been identified (among a number of organisms morphologically similar to P. piscicida, termed "Pfiesteria-like" or "Pfiesteria look-alike" organisms) from fish-kill/fish-disease events in aquaculture and coastal areas from Delaware to the Gulf of Mexico (23). The health hazards attributed to this organism (24)(25)(26) led to the need to characterize bioactive substances produced by P. piscicida capable of causing adverse effects. Research to fully characterize the chemical structure and biological effects of this putative toxin is a current challenge. P. piscicida produces a bioactive substance that induces cytotoxicity in GH 4 C 1 rat pituitary cells and at nontoxic Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH 4 C 1 rat pituitary cells. Extracellular adenosine 5'-triphosphate (ATP) acting on P2X 7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH 4 C 1 rat pituitary cells express functional P2X 7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2´-3´-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6azophenyl-2´-4´-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH 4 C 1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH 4 C 1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH 4 C 1 cells express a purinoceptor with characteristics consistent with the P2X 7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP. concentrations induces a c-fos luciferase reporter gene (27). The expression of c-fos in neural cells is often associated with elevated cytosolic free calcium [Ca 2+ ] i (28).
Recently we identified the presence of P2X 7 receptor expression in GH 4 C 1 rat pituitary cells and provided evidence for a role of this receptor type to mediate the action of the putative Pfiesteria toxin (pPfTx) in the induction of c-fos luciferase (20). Here we investigated the ionic properties of the P2X 7 receptor in GH 4 C 1 cells, and the activation of these characteristic ionic conductances by the pPfTx. We found that GH 4 C 1 cells have ionotropic purinergic receptors with pharmacologic and functional properties consistent with the P2X 7 subtype and that pPfTx mimics the kinetics of cell permeabilization by ATP.

Cell Culture
Clonal GH 4 C 1 cells were maintained in monolayer cultures in Ham's F10 medium supplemented with 15% horse serum and 2.5% fetal bovine serum (F10 + ) in a water-saturated atmosphere of 5% CO 2 and 95% air at 37 ± 1°C. Before experiments, cells were harvested from one donor culture dish with 0.02% EDTA and reseeded on glass coverslips at a density of approximately 200,000 cells/cm 2 . The cells were grown for 2-5 days, with change of medium each second day. Only cultures with less than 10 passages were used.

Calcium Analysis
GH 4 C 1 cells plated in monolayer on glass coverslips were loaded with Fura-2 just before the experiment. Cells were washed twice with Hepes-buffered saline Hanks' balanced salt solution (HBSS)-II (Hepes 20, Dglucose 10, NaCl 118, KCl 4.6, CaCl 2 0.4 mM, pH 7.2) and incubated with 2 µM Fura-2/AM in the same buffered saline in a water-saturated atmosphere of 5% CO 2 at 37 ± 1°C for 30 min. Cells were then washed twice, incubated at room temperature for 20 min, then mounted in a flow-through chamber (FCSII; Bioptechs Inc., Butler, PA, USA). Experiments were performed with an inverted fluorescence microscope [Zeiss 100, oil immersion Zeiss F-Fluor 40× lens (Carl Zeiss, Inc., Thornwood, NY, USA)], using the Attofluor ratio imaging analysis system (Atto Instruments, Rockville, MD, USA). Temperature in the chamber was controlled at 37 ± 1°C and HBSS-II was pumped in a laminar flow over the cells at a constant rate of 100 µL/min. Calcium concentration was calculated by the method of Grynkiewicz (29). Excitation wavelengths were 340 and 380 nm, and emission intensity was measured at 510 nm. The cytosolic free calcium concentration in single cells was monitored in real time. At least 30 cells were monitored simultaneously in each experiment. The values of cytosolic free calcium from each cell were averaged after the experiment. We used the in vitro method of calibration modified from the Attofluor manual. This method uses two calibration points, a maximum and a minimum correspondent respectively to a saturated calcium solution (10 mM CaCl 2 ) and a solution calcium free (with 10 µM EGTA). One µM Fura-2 pentapotassium was added to each solution, which was used to calibrate the system before each experimental session.

Cell-Permeabilization Analysis
The dye YO-PRO-1, a fluorescent analog of propidium iodide of 629 Da molecular weight, was used to detect cell permeabilization (5). GH 4 C 1 cells plated in monolayer on glass coverslips were washed twice with HBSS-II and mounted in the flow-through chamber. The experiments were performed using an inverted microscope and the Attofluor imaging system (described for the calcium analysis experiments) but in a single wavelength mode of analysis. A modified Hepes-buffered saline solution, made with NMDG substituting for NaCl (Hepes 20, D-glucose 10, NMDG 118, KCl 4.6, CaCl 2 0.4 mM, pH 7.2) and henceforth designated HBSS-YO, was used in the permeabilization assays (5). HBSS-YO was pumped in a laminar flow at the rate of 100 µL/min. Test reagents were diluted in 200 µL HBSS-YO and injected in one of the inflows (see flow-through description below). The flow of the buffered saline was constant throughout the experiments. The excitation wavelength was 460 nm, and emission was measured at 510 nm. At least 30 cells were individually monitored simultaneously in real time during each experiment. Experiments with BzATP were conducted five times, ATP four times, and pPfTx five times. For the experiments where the inhibition by oxATP was tested, cells were preincubated with 300 µM of oxATP in F10 + for 40 min in a watersaturated atmosphere of 5% CO 2 at 37 ± 1°C. Cells were then washed twice and mounted in the flow-through chamber, and the experiments were performed as above.

Flow-Through System
All of our experiments were completed in a closed chamber (FCSII; Bioptechs Inc.) through which a constant laminar flow of the Hepes-buffered saline was maintained over the cells. The inflow was fed by one of two  alternate peristaltic pumps that were digitally controlled. A check valve just before the chamber inlet separated the inflows driven from each pump. The test solution was injected into a loop inserted into one of the inflows, and was pumped through the chamber when the respective pump was digitally switched on. Both pumps were set to the flow rate of 100 µL/min. The temperature in the chamber was controlled at 37 ± 1°C.

Data Analysis
Values of light emission from individual cells were averaged and standard errors calculated.
One-way analysis of variance was applied to the emission data, corresponding to time points at and just before stimulation by the test substance.

Results
The action of the P2 receptor agonist BzATP on [Ca 2+ ] i was investigated in GH 4 C 1 cells loaded with the fluorescent calcium chelator Fura-2. The baseline [Ca 2+ ] i was approximately 200 nM and was elevated to a peak of 390 nM within 3-5 sec after exposure to 300 µM BzATP (Figure 1). [Ca 2+ ] i was maintained above 350 nM in the continuous presence of BzATP and decreased to a plateau of 280 nM when BzATP was washed out.
The permeabilization of the cell membrane to larger ions, a functional characteristic of P2X 7 subtype receptors, was next investigated. GH 4 C 1 cells were mounted in an inverted microscope under the same conditions of temperature (37 ± 1°C) and flowthrough (100 µL/min) used for the first experiment with Fura-2. The dye YO-PRO-1 was present in the flowing solution only at the times indicated in the chart (Figure 2). The background emission was an average of 20 relative fluorescence units [(RFU), used to measure the intensity of light emission] in the absence of the dye and was elevated to less than 25 RFU when the dye was present. The mean emission (n = 30 cells) was nearly 25 RFU in the absence of dye. The mean emission was elevated to less then 40 RFU in the presence of 5 µM YO-PRO-1 and returned to 25 RFU after the dye was washed out. Return of the emission to the initial levels revealed that the fluorescent dye was not trapped in the cells and indicated that the cells were not permeable to the dye. When the dye was delivered simultaneously with BzATP, a gradual but steady increase of emission intensity from the cells reached a level 4-fold higher (96 vs 23 RFU) than the initial emission. The high emission level was maintained after the dye and BzATP were washed out, indicating that the dye had permeated the cells.
Because BzATP caused both a pharmacological and functional action characteristic of P2X 7 receptors, we next investigated ATP, the physiologic agonist of the P2 receptors. The mean activity of 60 cells that were monitored simultaneously was determined after normalization by reducing the background level, which also was measured simultaneously ( Figure 3). During the periods indicated (Figure 3), the cells were exposed to 5 µM YO-PRO-1 only, or to 5 µM YO-PRO-1 with 200, 400, or 600 µM ATP. There was a concentration-dependent elevation of light emission from the cells, demonstrating that ATP had permeabilized GH 4 C 1 cell membranes to the YO-PRO-1 dye A small response (an increase of 2-fold the initial level) was obtained with 200 µM ATP. When the cells were stimulated with 400 or 600 µM ATP, there was a 10-fold increase in activity Environmental Health Perspectives • VOLUME 109 | SUPPLEMENT 5 | October 2001  To reexamine whether P2 receptors mediate the ATP permeabilization of GH 4 C 1 , we co-administered PPADS, an antagonist of P2 receptors, and ATP (13). GH 4 C 1 cells were first treated with 300 mM ATP, leading to accumulation of YO-PRO-1. PPADS given at 300 µM caused a small increase emission, and the addition of 300 µM ATP led to a small increase in dye accumulation. However, this partial inhibitory effect was reversible, and after the antagonist was washed out, led to further accumulation of YO-PRO-1 in the cells (Figure 4). We next investigated the action of oxATP, an irreversible and more selective P2X antagonist (30,31) in GH 4 C 1 cells. Cells were preincubated with 300 µM oxATP for 30 min in HBSS-II buffered saline, washed twice, and mounted in the flowthrough chamber using the Hepes buffer with NMDG substituting NaCl (HBSS-YO), as in the previous experiments. Pretreatment with oxATP fully inhibited the accumulation of YO-PRO-1 in GH 4 C 1 cells exposed to 300 and 600 µM ATP ( Figure 5).
To evaluate the role of P2X 7 as a potential target in mediating the toxic effects of Pfiesteria, we used a partially purified fraction from a toxic culture of the dinoflagellate. The action of pPfTx on cytosolic free calcium was investigated in GH 4 C 1 cells loaded with the fluorescent calcium chelator Fura-2. Cells were mounted on an open coverslip in an inverted fluorescence microscope, and individual cells were monitored under static incubation conditions. With each incremental addition of the pPfTx caused a gradual sustained elevation of [Ca 2+ ] i that reached near plateau level in about 90 sec ( Figure 6). Enhanced effects were observed with further additions, which eventually reached very high values consistent with cytotoxicity. We next examined the effect of the pPfTx on GH 4 C 1 cell permeability to YO-PRO-1. Cells were mounted in the flow-through chamber and were exposed to 200 µL of 1 µL/100 µL, 2 µL/100 µL, or 4 µL/100 µL pPfTx. The pPfTx fraction induced the accumulation of YO-PRO-1 in GH 4 C 1 cells in a concentration-dependent manner. The intensity of emission was monitored simultaneously in 60 cells (Figure 7). Exposure of the cells to 1 µL/100 µL caused a small elevation of emission from 1 to 5 RFU. Exposure to 2 µL/100 µL was repeated, and in both exposures the average cell emission was elevated with a reproducible effect that doubled the effect produced by exposure to 1 µL/100 µL. Exposure of GH 4 C 1 cells to 4 µL/100 µL led to an increase of average cell emission to nearly 58 RFU, an effect 5-fold higher than that observed from exposure to 2 µL/100 µL (Figure 7). When we used GH 4 C 1 cells that previously had been incubated with 300 mM oxATP, the accumulation of YO-PRO-1 in GH 4 C 1 cells exposed to 4 µL/100 µL pPfTx was inhibited (Figure 8). Further exposure to 400 µM ATP also failed to induce the accumulation of dye in these cells.

Discussion
This research provides evidence for a P2 receptor with the functional characteristics of the P2X 7 type in GH 4 C 1 cells. We used two signal transduction measurements to identify this receptor. One was the ability of the selective agonist BzATP to elevate the cytosolic free calcium. The other was the subsequent accumulation of the fluorescent dye YO-PRO-1 due to the permeabilization of the cells to larger ions. In GH 4 C 1 cells, BzATP  272  432  499  564  632  699  764  831  897  963  1033  1103  1170  1237  1303  1370  1437  1503  1570  1636 induced a rapid calcium response that elevated cytosolic free calcium 2-fold. After the agonist was washed out, the effect persisted for at least 3 min. When GH 4 C 1 cells were exposed to BzATP in the presence of the fluorescent dye YO-PRO-1, a gradual accumulation of the dye in the cells was observed. The comparison between the effect of BzATP on the concentration of cytosolic calcium, and on the accumulation of YO-PRO-1 in the cells, corresponds to the dynamics of pore formation characteristic of the purinoceptor P2X 7 (8,12,18). Once activated by an agonist, P2X 7 forms a nonselective cation channel, a characteristic of the ionotropic P2X class of P2 receptors. The influx of calcium ions causes the elevation of the cytosolic free calcium. P2X 7 differs from the other members of P2X receptors in that its activation leads to the cell membrane permeability to larger ions (6). This characteristic has been studied using fluorescent dyes, particularly YO-PRO-1 (a derivative of propidium iodide), which are ions of large molecular weight with affinity to nucleic acids (5). The sensitivity to the selective agonist BzATP and the accumulation of YO-PRO-1 in GH 4 C 1 cells provides evidence for the presence in these cells of an ionotropic purinoceptor with the functional characteristics of P2X 7 .
To further characterize the induction of cell permeability in GH 4 C 1 cells by the activation of P2 receptors, we exposed cells to increasing concentrations of ATP. We observed that ATP induces the permeability of GH 4 C 1 cells to YO-PRO-1 in a concentration-dependent manner. The reversibility of the ATP-induced permeability of the cells was also shown in this experiment, as the cells did not accumulate YO-PRO-1 when they were exposed to the dye both in the absence of ATP and after observed induction by 400 µM of the agonist. We next used two classes of antagonists of P2 receptors to inhibit the accumulation of YO-PRO-1 in GH 4 C 1 cells in response to ATP: PPADS, a reversible antagonist of P2 receptors, and oxATP, a selective and irreversible inhibitor of P2X 7 (6). PPADS partially inhibited cell permeability to YO-PRO-1 by ATP, and this partial inhibition was reversed after the antagonist was washed out. To investigate the effect of the P2X 7 selective antagonist oxATP, we pretreated cells with the antagonist (30,31). Pretreatment of GH 4 C 1 cells with oxATP fully inhibited the accumulation of YO-PRO-1 during exposure to 300 and 600 µM ATP. The ability of a selective antagonist to prevent the accumulation of YO-PRO-1 provided a second line of evidence for functional P2X 7 receptors in GH 4 C 1 cells.
We next investigated the role of the receptor functionally characterized in this work as a potential target for the toxicity of the dinoflagellate Pfiesteria. A bioactive substance has been identified in toxic Pfiesteria cultures with cytotoxic selectivity to GH 4 C 1 cells based upon a panel of different cell types (27). We report here that the pPfTx elevates cytosolic free calcium in Fura2-loaded GH 4 C 1 cells. The elevation of calcium is sustained and is consistent with activation of a membrane channel that gates calcium. The time course of elevated cytosolic free calcium is consistent with activation of P2X 7 receptors (32). Calcium is a second messenger, and one of its downstream signaling events is among those factors that induce the immediate response gene c-fos. For that reason, the sustained elevation of cytosolic free calcium by pPfTx is consistent with the induction of the c-fos Environmental Health Perspectives • VOLUME 109 | SUPPLEMENT 5 | October 2001 Figure 6. Elevation of the concentration of cytosolic free calcium in the presence of putative Pfiesteria toxin pPfTx. Cytosolic free calcium concentrations were calculated following the method of Grynkiewicz (29), using a K d of 224 nM Fura-2 for calcium ions. GH 4 C 1 cells loaded with Fura-2 were treated with increasing amounts of pPfTx, and the concentration of free cytosolic calcium was monitored individually in 7 cells in real time. Data are given as the activities (RFU) from individual cells measured simultaneously in an experiment repeated 4 times with similar results. The pPfTx is expressed as microliters of an assay-guided fraction of a methanol/water extract of toxic Pfiesteria culture medium. Exposure of the cells to 15 µL pPfTx led to an increase of cytosolic free calcium, which was enhanced in a dose-dependent manner by further additions of 5 µL each.

Figure 7.
Concentration-dependent permeabilization of GH 4 C 1 cells to the fluorescent dye YO-PRO-1 in the presence of pPfTx. Data are given as the mean RFU ± 1 SD from 60 cells that were individually measured simultaneously in a single real-time experiment after subtracting the background light intensity, which was also monitored simultaneously from five selected areas free of cells in the field of view. The pPfTx was always delivered simultaneously with 5 µM YO-PRO-1 in 200 µL buffer at the same flow rate. Where indicated, only the dye (without pPfTx) was delivered in the same manner.  (20,27). Activation of P2X 7 receptors leads to strong induction of additional transcription factors as well, including nuclear factor (NF)-kappaB and the nuclear factor of activated T cells (NFAT), both of which are associated with cytokine production (33). NFAT is induced rapidly and requires the entry of extracellular calcium. NFAT has a unique DNA binding region, yet binds cooperatively with the Fos/Jun heterodimer to the regulation region of the interleukin-2 gene (34). By contrast, NF-kappaB is induced more slowly and may require more downstream signaling events (35). Subsequent to elevating cytosolic free calcium, pPfTx induced the permeabilization of GH 4 C 1 cells to YO-PRO-1. This action of pPfTx is consistent with its cytotoxic effect (27) and the decreased c-fos luciferase activity (20). The permeabilizing effect of pPfTx in GH 4 C 1 cells was completely inhibited by oxATP. These results provide an additional line of evidence that the previously described cytotoxic effect of pPfTx (27) likely is mediated by P2X 7 receptors (20). Defining the role for P2 receptors has been hampered partly because few, if any, natural specific agonists have been identified for purinergic receptors. One toxin that also shows a strong cytotoxic response in GH 4 C 1 and other cell types is maitotoxin (36,37). Although the primary target for maitotoxin has not been identified, it does elevate cytosolic calcium in GH 4 C 1 cells by activating a calcium-dependent membrane depolarization (38,39), one that appears to involve chloride conductances (40). Maitotoxin induces c-fos luciferase and at higher concentrations cytotoxicity in GH 4 C 1 cells expressing this construct (41). Maitotoxin has been found to activate a cytolytic pore in several other cell types; however, overexpressing P2X 7 receptors in HEK-292 cells, which leads to substantial BzATP responses, fails to enhance the response to maitotoxin (42,43). These results indicate that P2X 7 is not a primary target for maitotoxin, although activation of a cytolytic pore is a likely downstream response. Thus pPfTx may prove to be a unique pharmacological probe for P2 receptors. More definitive characterization of pPfTx will require larger-scale production of toxic Pfiesteria for structural resolution of the toxin.
A role of P2X 7 receptors in the action of Pfiestera toxin is of interest, considering that this toxic dinoflagellate has been reported to cause a range of health impacts in both finfish and humans (22)(23)(24)(25)(26). The effects linked to Pfiesteria toxicity may be related to an inflammatory response, either in macrophages in the periphery or microglia in brain tissue. Implication of P2X 7 receptors as a potential target for the bioactive substance produced by toxic P. piscicida provides a common basis for the investigation of symptoms that previously have been regarded as unrelated, such as ulcers in menhaden and cognitive dysfunction in humans. Figure 8. Permeabilization of GH 4 C 1 cells by the pPfTx to the fluorescent dye YO-PRO-1 and its inhibition by oxATP, which is a specific antagonist of the P2X 7 purinergic receptor. Data are given as the mean RFU ± 1 SD of the activity from 60 cells individually measured simultaneously in a single real-time experiment, after subtracting the background light intensity which was also monitored simultaneously from five selected areas free of cells in the field of view. We used 400 µM ATP as a positive control. Test chemicals were always delivered simultaneously with 5 µM YO-PRO-1 in 200 µL buffer at the same flow rate.