Maitotoxin activates a nonselective cation channel and a P2Z/P2X7-like cytolytic pore in human skin fibroblasts

William P. Schilling, William G. Sinkins, and Mark Estacion

Rammelkamp Center for Education and Research and Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44109


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Maitotoxin (MTX), a potent cytolytic agent, activates Ca2+ entry via nonselective cation channels in virtually all types of cells. The identity of the channels involved and the biochemical events leading to cell lysis remain unknown. In the present study, the effect of MTX on plasmalemmal permeability of human skin fibroblasts was examined. MTX produced a time- and concentration-dependent increase in cytosolic free Ca2+ concentration that depended on extracellular Ca2+ and was relatively insensitive to blockade by extracellular lanthanides. MTX also produced a time- and concentration-dependent increase in plasmalemma permeability to larger molecules as indicated by 1) uptake of ethidium (314 Da), 2) uptake of YO-PRO-1 (375 Da), 3) release of intracellular fura 2 (636 Da), 4) uptake of POPO-3 (715 Da), and, ultimately, 5) release of lactate dehydrogenase (relative molecular weight of 140,000). At the single cell level, uptake of YO-PRO-1 correlated in time with the appearance of large MTX-induced membrane currents carried by the organic cation, N-methyl-D-glucamine (167 Da). Thus MTX initially activates Ca2+-permeable cation channels and later induces the formation of large pores. These effects of MTX on plasmalemmal permeability are similar to those seen on activation of P2Z/P2X7 receptors in a variety of cell types, raising the intriguing possibility that MTX and P2Z/P2X7 receptor stimulation activate a common cytolytic pore.

calcium; fura 2; whole cell currents; ethidium uptake; cytolysis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NECROTIC CELL DEATH occurs as a consequence of cellular damage, often resulting from an oxidative insult. Significant oxidative stress occurs during ischemia-reperfusion injury in the myocardium following infarction and in the brain following stroke. Reactive metabolites generated during drug metabolism by hepatocytes can also produce changes in the redox status of the cell, ultimately leading to necrotic cell death (for recent reviews, see Refs. 43 and 44). This type of cell death is associated with cell and organelle swelling and lysis and has been termed oncosis. In contrast, apoptosis, or programmed cell death, is associated with cell shrinkage and nuclear condensations. Although it was originally thought that these two types of cell death were the result of separate biochemical pathways, recent evidence indicates that the strength and duration of the insult and the energy status of the cell play important roles in defining the path taken on the way to ultimate cell demise (10, 21, 30, 31).

A rise in cytosolic free Ca2+ concentration ([Ca2+]i) appears to be one of the earliest detectable events associated with both apoptotic and necrotic cell death (22, 23, 29, 43, 44). The increase in [Ca2+]i is associated with expression of immediate-early genes (c-fos, c-jun, and c-myc) and the activation of a variety of enzymes, including phospholipases, endonucleases, and proteases. The mechanisms responsible for the alteration in Ca2+ homeostasis and the subsequent biochemical events leading to cell lysis remain unknown. Originally, cell lysis was thought to occur by a rather nonspecific, ill-defined mechanism ending in "membrane perturbation" or "membrane breakdown" and osmotic lysis. However, recent studies on the P2Z purinergic receptor have demonstrated that necrotic cell death can reflect an ordered sequence of permeability changes (9, 13, 28, 48). The P2Z receptor is a member of the P2X ionotropic purinergic receptor family recently cloned and identified as P2X7 (27, 39). Functional P2Z/P2X7 receptors are found in mononuclear phagocytes, macrophages, and microglial cells, suggesting that they play an important role in the inflammatory reaction (5, 11, 19). P2Z/P2X7 receptors are also found in adult rat lung, spleen, salivary gland, testes, bone marrow, brain ependyma (5, 45), retina (2), parotid acinar cells (42), human and mouse fibroblasts (15, 34, 37), and Chinese hamster ovary cells (24). Thus the expression of P2Z/P2X7 receptors is widespread. Activation of P2Z/P2X7 is thought to be responsible for ATP-induced cytolysis. High concentrations of ATP (3-5 mM) in the presence of physiological concentrations of Mg2+ cause a rapid increase in [Ca2+]i followed in time by a progressive increase in permeability of the membrane to molecules with molecular mass up to ~900 Da. The initial change in [Ca2+]i is associated with Ca2+ influx via ATP-activated ion "channels" and the subsequent increase in permeability to larger molecules is equated to "pore" formation. The ultimate consequence of pore formation is cell death by oncosis; however, recent studies have shown that activation of the P2Z/P2X7 channel and pore gives rise to both apoptosis and necrosis in mesangial cells (36). The molecular mechanisms associated with channel activation by ATP and subsequent steps involved in pore formation remain unknown. Likewise, the biochemical link between P2Z/P2X7 channel/pore formation and apoptosis/necrosis has not been defined.

Natural toxins and poisons (e.g., cholera toxin, pertussis toxin, TTX, conotoxin, digitalis, ryanodine, thapsigargin) have proved useful in the identification and functional characterization of specific proteins in biochemical pathways critical for cell homeostasis and signaling. In general, these compounds are potent, specific, and highly selective agents that are derived from plant, bacterial, or animal sources. Maitotoxin (MTX), isolated from the dinoflagellate Gambierdiscus toxicus, is one of the most potent toxins known. MTX causes a profound increase in [Ca2+]i in all cells tested (17, 25). Originally, it was thought that MTX was a specific activator of voltage-gated Ca2+ channels, since the rise in [Ca2+]i depended on the presence of extracellular Ca2+ and could be attenuated by organic and inorganic Ca2+ channel antagonists (41). However, it was later discovered that MTX activates nonselective cation channels in all cells examined, including both excitable and nonexcitable cells (1, 7, 8, 12, 25, 49). In nonexcitable cells, activation of these channels by MTX allows the influx of Ca2+ and the subsequent secondary effects such as activation of phospholipase C and release of arachidonic acid. In excitable cells, MTX-induced activation of nonselective cation channels causes membrane depolarization, activation of voltage-gated channels, and secondary effects such as contraction of cardiac and smooth muscle and the release of neurotransmitters from nerve terminals. After prolonged incubation, MTX has been shown to induce lactate dehydrogenase (LDH) release from rat hepatocytes (20) and from neonatal rat cardiac myocytes (33), consistent with osmotic cell lysis. The identity of the MTX-activated channels and the intervening biochemical events leading to oncosis remain unknown. In the present study, the effect of MTX on plasmalemma permeability was examined in human skin fibroblasts. With the use of fluorescence techniques to measure changes in [Ca2+]i and uptake of large organic vital dyes and electrophysiological techniques to measure whole cell membrane currents, the results show that MTX initially activates Ca2+-permeable cation channels and subsequently induces formation of a P2Z/P2X7-like cytolytic pore.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Solutions and reagents. Unless otherwise indicated, HEPES-buffered saline (HBS) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM D-glucose, 1.8 mM CaCl2, 15 mM HEPES, and 0.1% BSA, pH adjusted to 7.40 at 37°C with NaOH. Ca2+-free HBS contained 0.3 mM EGTA and the same salts as HBS without added CaCl2. Mg2+-free HBS contained the same salts as HBS without added MgCl2. Fura 2-AM, ethidium bromide, YO-PRO-1, and POPO-3 were obtained from Molecular Probes (Eugene, OR). MTX was obtained from CalBiochem (San Diego, CA) or from LC Laboratories (Woburn, MA). In preliminary experiments, we found that the apparent MTX potency varied substantially from week to week. This variability occurred as a result of instability of the MTX stock solutions. Consistent results were obtained when MTX was stored in amber-colored glass or plastic vials at -20°C as an aqueous stock solution (>1 µM) with 0.1% BSA. All other salts and chemicals were of reagent grade.

Cell culture. Human skin fibroblasts (cell line SK45), obtained from American Type Culture Collection (ATCC), were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin-neomycin (PSN) antibiotic mixture, and 2 mM glutamine. For passage, cells were dispersed by trypsin treatment and seeded at a density of ~3 × 103 cells/cm3. The medium was changed every 2-3 days after seeding. Experiments were performed with cells in passages 6-18 and at 7 days postseeding. The fibroblasts were allowed to proliferate in serum-containing medium until the day before experimentation, at which time the medium was changed to serum-free DMEM and the cells were incubated for an additional 18-24 h. For experimentation, fibroblasts were harvested by trypsinization, washed, and resuspended in HBS at 37°C. The THP-1 monocytic cell line was obtained from ATCC and grown in RPMI 1640 containing 10% heat-inactivated FBS, 1% PSN antibiotic mixture, and 2 mM L-glutamine in spinner flasks as previously described (19). In some experiments, the P2Z/P2X7 receptor was upregulated by pretreatment of THP-1 cells for 48 h in complete RPMI 1640 medium containing 1 µg/ml bacterial lipopolysaccharide (LPS) and 1,000 U/ml interferon-gamma .

Measurement of the apparent [Ca2+]i. [Ca2+]i was measured using the fluorescent indicator fura 2 as previously described (35). Briefly, dispersed cells were suspended in HBS containing 20 µM fura 2-AM. After a 30-min incubation at 37°C, the cell suspension was diluted ~10-fold with HBS, incubated for an additional 30 min, washed, and resuspended in fresh HBS. Aliquots from this final suspension were centrifuged and washed twice immediately before fluorescence measurement. Fluorescence was recorded using an SLM 8100 spectrophotofluorometer; excitation wavelength alternated between 340 and 380 nm, and fluorescence intensity was monitored at an emission wavelength of 510 nm. All measurements were performed at 37°C. Calibration of the fura 2 associated with the cells was accomplished using Triton X-100 lysis in the presence of saturating divalent cation concentration followed by addition of EGTA (pH 8.5). [Ca2+]i was calculated by the equation of Grynkiewicz et al. (16) using a dissociation constant value for Ca2+ binding to fura 2 of 224 nM. As shown in RESULTS, MTX causes fura 2 efflux from the cells. Therefore, [Ca2+]i values are reported as "apparent" changes in [Ca2+]i. Estimates of Ba2+ and Sr2+ influx using fura 2 were obtained from the ratio of fluorescence at excitation wavelengths of 350 and 390 nm as previously described (35). Figures 1, 2, 4, 5, 6, and 9 show representative traces from experiments performed at least three times. Where indicated, n equals the number of independent experiments.

Measurement of fura 2 efflux. Cells were loaded with fura 2 as described above. Aliquots of the cell suspension (2 ml) were incubated at 37°C in the presence and absence of MTX. At various times, the cells were pelleted by centrifugation for 2 min in a tabletop centrifuge, and the supernatants were removed for measurement of fura 2 released from the cells. In some experiments, the efflux of fura 2 was examined in the absence of extracellular Ca2+. In these experiments, the supernatant was supplemented with 1.8 mM CaCl2 before fluorescence measurement. Values are expressed as the percentage of fura 2 remaining associated with the cells relative to the value obtained following permeabilization of the cells with 0.03% saponin.

Measurement of ethidium bromide uptake. An aliquot (2 ml) of dispersed cells suspended in HBS at 37°C was placed in a cuvette. After addition of ethidium bromide (final concentrations of 5 µM), fluorescence was recorded as a function of time with excitation and emission wavelengths of 302 and 560 nm, respectively. All ethidium bromide fluorescence values were corrected for background (extracellular) dye fluorescence and expressed as a percentage relative to the value obtained following complete permeabilization of the cells with 0.0075% saponin or 0.003% digitonin. Where indicated, ethidium fluorescence was corrected for a small concentration-dependent quenching of fluorescence by 2' and 3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP). Uptake of POPO-3 and YO-PRO-1 was determined as described for ethidium with excitation/emission wavelengths of 530/565 and 468/510 nm, respectively.

Measurement of LDH release. Aliquots of dispersed cells (2 ml) were incubated at 37°C for various lengths of time in the presence and absence of MTX. The cells were pelleted by centrifugation for 2 min in a tabletop centrifuge, and the supernatants were removed and frozen for subsequent measurement of LDH activity. Enzyme activity in aliquots (20-50 µl) of the supernatants was determined using the LD-L kit from Sigma. All values are expressed as percent LDH released relative to the value obtained following permeabilization of the cells with 0.03% saponin.

Total RNA extraction, first-strand cDNA synthesis, and PCR amplification. Total RNA was extracted by the method of Chomcyznski and Sacchi (4). First-strand cDNA was generated using Moloney murine leukemia virus-RT and oligo(dT) and random primers as previously described (14). PCR was performed with Taq polymerase for 35 cycles using Perkin-Elmer DNA thermal cycler 480. Primers used for amplification of P2Z/P2X7 were based on the human sequence (sense, 5'-GGCAATTCAGGGCGGAATAATGG-3'; antisense, 5'-GAAGCGCCAGGTGGCGTAGCAC-3') and generated the predicted 940-bp product. The thermocycler was programmed to give an initial cycle consisting of 94°C denaturation for 5 min, 50°C annealing for 1 min, and 72°C extension for 1 min followed by 35 cycles of 94, 50, and 72°C for 1 min each with a final extension at 72°C for 10 min. A "no-template control" was performed alongside all experimental samples. PCR products were separated on 1% agarose, stained with SYBR green and visualized using a STORM 860 imager (Molecular Dynamics) in blue fluorescence mode.

Simultaneous measurement of membrane current and YO-PRO-1 uptake. The patch-clamp technique for current recording was utilized in the whole cell mode (18, 46). All experiments were performed on human skin fibroblasts attached to glass coverslip chambers. The extracellular bath was a modified Ringer solution containing (in mM) 160 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.4). The pipette solution contained (in mM) 145 potassium aspartate, 2 MgCl2, 0.1 CaCl2, 1.1 EGTA, and 10 HEPES (pH 7.2 and pCa 8). In some experiments, the Na+ in the bath solution was isosmotically replaced with N-methyl-d-glucamine (NMDG-Ringer). Data were obtained using an Axopatch 1-D amplifier (Pacer Scientific, Los Angeles, CA) and sampled on-line using pCLAMP 5.5 software. The ground electrode was an Ag-AgCl wire connected to the bath via an agar bridge containing 150 mM NaCl. All recordings were made at room temperature (~22°C). To generate current-voltage (I-V) relations, voltage ramps were applied from -120 to +60 mV every 10-20 s. Unless otherwise indicated, the holding potential between ramps was 0 mV. Figures 7 and 8 show representative traces not corrected for leak currents but corrected for liquid junction potential. For single cell measurement of vital dye uptake, fibroblasts on glass coverslips were challenged with MTX in the presence of 1 µM YO-PRO-1 in the extracellular solution. The cells were illuminated with light from a 75-watt xenon lamp using an O-5717 filter cube obtained from Molecular Probes. Epifluorescence was recorded using a Hamamatsu intensified charge-coupled device camera (model XC-77), and images were acquired and analyzed using Image 1 software (Universal Imaging, West Chester, PA). Regions were defined over single cells, and the average fluorescence intensity of the region was quantified as a function of time.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of MTX on [Ca2+]i of human skin fibroblasts. MTX produced a concentration-dependent increase in the apparent [Ca2+]i of human skin fibroblasts with an ED50 of ~0.5 nM (Fig. 1A). The change in [Ca2+]i occurred with a slight lag following addition of MTX to the cuvette, and the major effect of MTX was on the rate of increase in [Ca2+]i. At 1 nM MTX, [Ca2+]i increased with a lag time of ~10 s and subsequently increased in an exponential fashion with a half time of ~2 min. At the highest MTX concentration tested (5 nM), fura 2 fluorescence increased to a value that was close to saturation with Ca2+; apparent [Ca2+]i was 1.5-2 µM 6 min after MTX addition.


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Fig. 1.   Effect of maitotoxin (MTX) on fura 2 fluorescence in human skin fibroblasts. Fura 2-loaded fibroblasts were suspended in HEPES-buffered saline (HBS), and fluorescence ratio was recorded as described in MATERIALS AND METHODS as a function of time. A: at arrow, MTX was added during the individual recordings at the final concentrations indicated (right). Seven traces are shown superimposed. B: effect of extracellular divalent cations on MTX-induced changes in fura 2 fluorescence. Three traces are shown superimposed. Fura 2-loaded fibroblasts were suspended in Ca2+-free HBS. At the time indicated by first arrow, MTX (final concentration = 2 nM) was added to the cuvette followed by either Ca2+, Sr2+, or Ba2+ (final free concentration of each divalent was 1.7 mM) at the second arrow. [Ca2+]i, cytosolic free Ca2+ concentration.

MTX had no effect on the apparent [Ca2+]i when added to fibroblasts in the absence of extracellular Ca2+ (Fig. 1B). Subsequent addition of Ca2+ produced a rapid increase in fluorescence ratio indicative of Ca2+ influx, consistent with previous reports in which the effect of MTX depended on extracellular Ca2+. Two commonly used Ca2+ surrogates, Ba2+ and Sr2+, substitute for Ca2+ in supporting activation of cation influx by MTX; addition of these divalent cations to the extracellular solution after MTX produced a rapid increase in fluorescence ratio. Both Sr2+ and Ba2+ are permeable through most known Ca2+ channels and bind to and produce the same spectral changes in fura 2 as those seen with Ca2+ (35). Thus the increase in fluorescence ratio seen in Fig. 1B when Sr2+ and Ba2+ were added suggest that the MTX-activated channels are permeable to these divalent cations.

As seen in Fig. 2A, the effect of MTX on apparent [Ca2+]i was unaffected when 100 µM Gd3+ was added after MTX (trace b). When Gd3+ was added before MTX (trace a), the increase in fluorescence ratio was actually increased over that seen in the absence of Gd3+. The response to MTX was partially blocked when the concentration of Gd3+ was increased to 1 mM (Fig. 2B). This inhibition was seen when 1 mM Gd3+ was added either before or after MTX. Interestingly, addition of 1 mM Gd3+ after MTX only partially reduced the fluorescence ratio, i.e., there appears to be a component of the response that is resistant to blockade. Previous studies have also noted that blockade of MTX-induced changes in [Ca2+]i by lanthanides is partial and transient (26). Lanthanides will bind to fura 2 with high affinity and produce a greater increase in fluorescence at the 340-nm excitation wavelength compared with Ca2+. Thus there are two possible explanations for the effects seen in Fig. 2: 1) Gd3+ may be permeable through the MTX-activated pathway, and/or 2) MTX may produce a time-dependent release of fura 2 from the cell.


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Fig. 2.   Effect of Gd3+ on MTX-induced changes in fura 2 fluorescence. In A and B, 2 traces are shown superimposed. A: MTX (0.5 nM) was added at times indicated in absence (trace b) or presence (trace a) of 100 µM Gd3+. Gd3+ was subsequently added to trace b at 8 min. B: same procedure as in A, with 1 mM Gd3+ added before and after MTX.

Effect of MTX on fura 2 efflux. To investigate the possibility that MTX channels are permeable to fura 2, the accumulation of fura 2 in the extracellular space was examined (Fig. 3A). In the absence of MTX, fura 2 leakage from the fibroblasts occurred at a slow rate of ~0.3%/min at 37°C. MTX produced a concentration-dependent increase in the fura 2 efflux rate; 50% of cell-associated fura 2 was released within 10 min in the presence of 1 nM MTX, and 50% was released within 20 min in the presence of 0.2 nM MTX. Efflux of fura 2 from the cells on addition of MTX also occurred, with a delay of 4 and 5 min for 1 and 0.2 nM MTX, respectively. The increase in fura 2 leakage was dependent on extracellular Ca2+, and both Sr2+ and Ba2+ could substitute for Ca2+ (Fig. 3B). The concentration of Ca2+ needed to support this effect of MTX was in the millimolar range; very little effect was observed at concentrations below 0.5 mM (Fig. 3C). These results demonstrate that the MTX-induced change in apparent [Ca2+]i, as seen in Fig. 1, reflects Ca2+ binding to fura 2 in both the intracellular and extracellular space. Furthermore, these results suggest that the membrane channels activated by MTX are of sufficient size to allow fura 2, a polyanion with a molecular mass of 636 Da, to exit the cell.


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Fig. 3.   Effect of MTX on fura 2 efflux from human skin fibroblasts. A: fura 2 efflux was measured as described in materials and methods as a function of time in absence (control) or presence of the indicated concentration of MTX added at time 0. Values are means ± SE; n = no. of determinations (given in parentheses). A control data set was collected and is shown for each concentration of MTX tested. Note that, after cells were loaded with fura 2, they were maintained at 37°C. There is a short period of setup time that elapses before addition of MTX following the last centrifugation step in the fura 2-loading procedure. During this time, fura 2 will leak from the cells (~10% in this set of experiments), accounting for the initial decline in fura 2 from time 0 to 2 min. B: effect of extracellular divalent cations on MTX-induced fura 2 efflux. Fura 2 efflux from fibroblasts was measured for 15 min at 37°C in the presence of 2 nM MTX in Ca2+-free HBS (0) or Ca2+-free HBS supplemented with Ca2+, Sr2+, or Ba2+ at a final free concentration of 1.7 mM. Values are means ± SE; n = 3. C: effect of extracellular Ca2+ concentration on MTX-induced fura 2 efflux. Fura 2 efflux from fibroblasts was measured for 15 min at 37°C in the presence of 0.2 nM MTX in nominally Ca2+-free HBS supplemented with 0, 0.1, 0.25, 0.5, 1.0, or 2.0 mM CaCl2. Values are means ± SE; n = 3.

Effect of MTX on ethidium bromide uptake. To further investigate the ability of MTX to open large pores in the cell membrane, uptake of ethidium was determined in the absence and presence of MTX. Ethidium, a cation with molecular mass of 314 Da, is normally excluded from the cytoplasm. However, if ethidium gains access to the cytosol, a large fluorescence signal will be generated on binding to cellular DNA and RNA. Thus ethidium fluorescence can be used as an index of permeabilization of the plasmalemma. MTX caused a time- and concentration-dependent increase in ethidium uptake in human skin fibroblasts (Fig. 4A). At all concentrations tested, ethidium uptake occurred with a slight delay; the delay was ~1 min with 0.2 nM MTX and ~0.5 min with 1 nM MTX. Ethidium fluorescence increased to >80% of the maximum within 7 min of MTX (2 nM) addition. The effect of MTX on ethidium uptake depended on extracellular Ca2+, and both Sr2+ and Ba2+ could substitute for Ca2+ in this effect (Fig. 4B). These results suggest that MTX rapidly produces pores with sufficient size to allow ethidium to enter the cell.


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Fig. 4.   Effect of MTX on ethidium bromide uptake in human skin fibroblasts. A: ethidium bromide uptake was determined as the increase in fluorescence as described in MATERIALS AND METHODS as a function of time. Four traces are shown superimposed. Ethidium bromide (5 µM) was added at 50 s. At time indicated by arrow, MTX was added at the final concentrations indicated (right). Values shown are normalized to the maximum fluorescence obtained by addition of saponin (final concentration = 0.03%) at the end of each trace. B: effect of divalent cations on MTX-induced ethidium bromide uptake in fibroblasts. Four traces are shown superimposed. Fibroblasts were suspended in Ca2+-free HBS or Ca2+-free HBS supplemented with the indicated divalent cation at a final free concentration of 1.7 mM. Ethidium bromide (5 µM) was added at 50 s, and MTX (2 nM) was added to each trace at the time indicated by arrow.

Previous studies have suggested that the downstream effect of MTX on cell function may be either [Ca2+]i dependent or independent (for review, see Ref. 17). To determine if a rise in [Ca2+]i is sufficient to alter membrane permeability, fibroblasts were challenged with the Ca2+ ionophore ionomycin (Fig. 5). Ionomycin produced a biphasic increase in [Ca2+]i that was concentration dependent. At 20 µM, ionomycin produced a large, transient increase followed by a long-lasting elevation of [Ca2+]i that increased slowly with time. In contrast, ionomycin had no effect on ethidium uptake even at the highest concentration examined. Thus, although extracellular Ca2+ is necessary for MTX-induced changes in plasmalemmal permeability, a rise in [Ca2+]i is not sufficient.


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Fig. 5.   Effect of ionomycin on [Ca2+]i and ethidium uptake in human skin fibroblasts. Fura 2-loaded fibroblasts were suspended in HBS, and fluorescence (F) ratio was recorded as a function of time. Four traces are shown superimposed. At time indicated by arrow, ionomycin (I) was added to the cuvette at the final concentrations indicated (right). Inset: ethidium uptake was determined as described in Fig. 4. Ionomycin (20 µM) was added at time indicated by arrow.

Effect of MTX on LDH release. Because MTX stimulates the movement of ethidium and fura 2 across the cell membrane, we considered the possibility that MTX was simply producing cell lysis. To test this hypothesis, the release of LDH (relative molecular weight = 140,000) was determined in the absence and presence of MTX. LDH release from human skin fibroblasts during a 10-min incubation at 37°C was undetectable in the absence of MTX. LDH release was 4.5 ± 0.2% and 27.7 ± 1% (means ± SE; n = 5) when the fibroblasts were incubated with 0.2 or 1.0 nM MTX for 10 min, respectively. Thus, although cell lysis is observed after MTX, it occurs much more slowly and to a lesser extent compared with the MTX-induced change in membrane permeability to Ca2+, ethidium, or fura 2.

To investigate more closely the timing of the permeability change and the size of the MTX-induced pores, the change in fura 2 fluorescence was correlated with the uptake of three vital dyes. Specifically, the uptake of ethidium (314 Da), YO-PRO-1 (375 Da), and POPO-3 (715 Da) in response to MTX was examined in parallel on the same cells (Fig. 6). At both concentrations of MTX tested, the permeability time course follows the sequence of Ca2+, followed by ethidium, YO-PRO, and POPO-3. These results are consistent with the hypothesis that MTX causes a progressive increase in the permeability of the plasma membrane to small organic molecules with molecular masses of <800 Da.


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Fig. 6.   Comparison of MTX-induced fura 2 fluorescence and vital dye uptake. Fura 2-loaded fibroblasts were suspended in HBS, and fluorescence at an excitation wavelength of 340 nm was recorded as a function of time (trace a) following addition of either 0.2 nM (A) or 2 nM (B) MTX. Fluorescence intensity of ethidium (trace b), YO-PRO-1 (trace c), or POPO-3 (trace d) following addition of MTX are shown superimposed.

Measurement of MTX-induced pore current. Although the above results are consistent with MTX-induced pore formation, the possibility should be considered that the toxin either directly or indirectly activates an electrically silent organic transporter, e.g., the multidrug resistant P-glycoprotein or the probenecid-inhibitable anion exchanger. Such an effect could give rise to fluorescent dye uptake and/or fura 2 efflux. If MTX activates a pore, we reasoned that increases in membrane conductance should be observed in the presence of large, organic cations and this conductance should correlate in time with the appearance of vital dye uptake. As seen in Fig. 7, application of MTX (1 nM) to a human skin fibroblast with Na+ as the predominant cation in the bath and K+ the predominant cation in the pipette solution caused a time-dependent increase in inward and outward currents that reversed at +7 mV. These results are consistent with previous studies indicating that MTX initially activates a cation channel that poorly discriminates between Na+ and K+ (1, 8, 12). Subsequent isosmotic replacement of bath Na+ with the large organic cation NMDG (167 Da) produced a shift in current reversal potential to -35 mV, demonstrating that the MTX-activated channels are much less permeable to NMDG compared with K+ and Na+.


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Fig. 7.   Effect of MTX on whole cell currents. Whole cell membrane current was recorded from a human skin fibroblast in response to 1 nM MTX. Currents elicited using voltage ramps occurring at 10-s intervals were recorded in a modified Ringer solution. MTX produced an increase in inward and outward currents as a function of time; currents reversed at +7 mV (right arrow). At 4 min after MTX addition, bath solution was replaced with N-methyl-d-glucamine (NMDG)-Ringer (left arrow), which produced a leftward shift in reversal potential to -35 mV (heavy trace). Similar results were obtained in 4 of 4 cells examined.

To determine the time course of pore formation, MTX-induced YO-PRO-1 uptake and whole cell currents were simultaneously recorded from single fibroblasts with NMDG as the major cation in the bath solution (Fig. 8). Under these ionic conditions, activation of nonselective channels that were equally permeable to Ca2+ and K+, but relatively impermeable to NMDG (and YO-PRO-1), should produce an increase in membrane current with a reversal potential of approximately -35 mV, as seen in Fig. 7. Subsequent formation of large pores, permeable to all ions including NMDG, should produce a shift in reversal potential toward zero. This second phase should be associated with the uptake of YO-PRO-1. As seen in Fig. 8A, current recorded at a holding potential of -80 mV increased rapidly for the first 4 min following addition of MTX to the bath solution. This rapid increase in current seen at -80 mV occurred with little change in current recorded at the reversal potential of -35 mV; and the complete I-V relationships (Fig. 8B, inset) show that both inward current and outward current are substantially increased during this initial phase of MTX action. During this time, no increase in cell-associated fluorescence was observed. Between 4 and 11 min after MTX, current slowly increased at both -35 and -80 mV, suggesting a small increase in permeability to NMDG. This was associated with a small increase in the rate of YO-PRO-1 uptake into the cell. After 15 min in the presence of MTX, a dramatic increase in inward current was seen that correlated in time with a rapid change in fluorescence reflecting uptake of YO-PRO-1 into the cell. This final phase was associated with a dramatic shift in reversal potential toward zero (Fig. 8B). The MTX-induced shift in reversal potential during simultaneous recordings correlated in time with YO-PRO-1 uptake in five of five cells examined. Thus MTX-induced pore formation is associated with an increase in the conductance of the membrane to NMDG.


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Fig. 8.   Simultaneous recording of MTX-induced whole cell currents and YO-PRO-1 uptake. Whole cell membrane currents during voltage ramps were recorded from a single fibroblast bathed in NMDG-Ringer containing 1 µM YO-PRO-1. A: current recorded at -80 mV () and -35 mV (open circle ) and cell-associated fluorescence (black-diamond ) as a function of time. MTX (1 µM) was added at the arrow. B: complete current-voltages recorded at times indicated in A (1, 2, 3, 4 in inset; A, B, C, D in main panel). Results shown are representative of simultaneous recordings of membrane current and YO-PRO-1 uptake in 5 cells.

Effect of Bz-ATP on [Ca2+]i of human skin fibroblasts. The responses of the fibroblasts to MTX are reminiscent of the permeability changes observed following stimulation of P2Z/P2X7 receptors in a variety of cell types (9, 13, 28, 48). To determine if the fibroblasts used in the present study express this receptor, the effect of Bz-ATP, a specific P2Z/P2X7 agonist, was examined. Bz-ATP, at maximum concentrations, produced a small but significant increase in the apparent [Ca2+]i, from a basal resting level of 85.9 ± 4.3 to 111 ± 3.1 nM (n = 6; P < 0.0002). The increase in [Ca2+]i was unaffected by La3+ when added either before (Fig. 9B) or after (Fig. 9A) Bz-ATP. The response to Bz-ATP was dependent on extracellular Ca2+ (Fig. 9A, inset), suggesting that it is not related to release of Ca2+ from internal stores. For comparison, the response of the fibroblasts to bradykinin was examined. Bradykinin increases [Ca2+]i in human fibroblasts via a G protein-coupled receptor of the B2 type. As seen in Fig. 9, and as previously shown (3), bradykinin produced a biphasic response; the initial increase in [Ca2+]i reflects release of Ca2+ from internal stores, and the sustained elevation reflects Ca2+ influx from the extracellular space via the capacitative Ca2+ entry mechanism (CCE). La3+ inhibits CCE in fibroblasts when added either before addition of bradykinin (Fig. 9B) or during the sustained phase (Fig. 9A) but has no effect on Ca2+ release from internal stores. Thus the magnitude of the Bz-ATP-induced Ca2+ influx is similar to CCE in this cell line. To further test for the presence of P2Z/P2X7 receptors, a 940-bp fragment was amplified by RT-PCR (Fig. 9B, inset). For comparison, the same product was amplified from RNA isolated from control THP-1 monocytes and from THP-1 cells pretreated with bacterial LPS and interferon-gamma , a maneuver known to upregulate P2Z/P2X7 message and function in this cell type (19). Interestingly, the magnitude of the small [Ca2+]i response to Bz-ATP in fibroblasts is similar to that reported by Humphreys and Dubyak (19) in control THP-1 cells. Together, these experiments suggest that the fibroblasts used in the present study express both P2Z/P2X7 transcripts and functional receptors.


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Fig. 9.   Receptor-mediated changes in [Ca2+]i in human skin fibroblasts. Fura 2-loaded fibroblasts were suspended in 0 Mg2+ HBS. In A and B, 2 traces superimposed are shown. A: at times indicated, either 2' and 3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP) (trace a; 200 µM) or bradykinin (trace b; BK, 50 nM) was added. La3+ (10 µM) was added to both traces at 5 min. A second aliquot of La3+ (100 µM) was added to trace a at ~7 min. Inset: cells were initially suspended in 0 Ca2+, 0 Mg2+ HBS. Bz-ATP (100 µM) and 1.8 mM Ca2+ were added at times indicated. B: same as in A, with La3+ (10 µM) added before bradykinin or Bz-ATP. Inset: amplification of a 940-bp fragment of the human P2Z/P2X7 receptor using RT-PCR on RNA isolated from human skin fibroblasts, control untreated THP-1 cells, and THP-1 cells pretreated with lipopolysacharide and interferon-gamma for 48 h. A no template control is also shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a variety of cell types, MTX causes a rapid change in [Ca2+]i and subsequent release of LDH; however, the molecular mechanisms linking channel activation to cell lysis remain poorly understood. In the present study, we confirm that MTX causes a time- and concentration-dependent increase in [Ca2+]i that is unrelated to release of Ca2+ from internal stores but reflects a dramatic increase in Ca2+ entry via a plasmalemmal pathway that is relatively insensitive to blockade by lanthanides. These results are consistent with previous reports demonstrating that MTX activates Ca2+-permeable, nonselective cation channels (7, 8, 12, 25). An important finding in the present study is that the apparent increase in [Ca2+]i reflects, at least in part, the opening of large pores in the plasmalemma that allow small organic molecules with molecular masses of <800 Da to enter or exit the cell. The time-dependent changes in plasmalemmal permeability induced by MTX do not simply reflect cell lysis. If this were the case, a similar time course for vital dye uptake, fura 2 efflux, and LDH release would be expected. However, there appears to be a progression of changes that begins with the lowest molecular mass molecules and proceeds in a time- and concentration-dependent manner to larger macromolecules. In this regard, the size of the pore seems to increase with time. As judged by the initial fura 2 response and vital dye uptake, the MTX-induced flux of ions follows this sequence: Ca2+, ethidium, YO-PRO, fura 2, and finally, POPO-3. However, the simultaneous measurement of membrane current and fluorescence indicates that an increase in conductance of the membrane to NMDG correlates closely in time with YO-PRO-1 uptake. Thus the pore size may be fixed, and the apparent differences in time course of vital dye uptake may simply reflect relative differences in pore conductance for the various dyes examined.

Irrespective of the exact structural features of the pore, it is clear that MTX-induced activation of a Ca2+-permeable nonselective cation channel precedes the increase in conductance to NMDG and the uptake of vital dyes. The lag between channel activation and pore formation appears to be greater in the whole cell current recordings compared with the lag seen in the population studies performed in the cuvette. This may reflect the fact that the cell is dialyzed by the pipette solution during whole cell recording, suggesting that some cytoplasmic factor may be involved in coupling between channel activation and pore formation. Alternatively, because the electrophysiological experiments were performed at room temperature, the coupling between channel and pore may be sensitive to temperature. Preliminary studies suggest that the latter is true, i.e., decreasing temperature has little effect on channel activation but greatly attenuates MTX-induced pore formation.

Both the MTX-induced channel activation and the ability of MTX to open large pores in the plasmalemma depend on the presence of extracellular Ca2+, and, in this respect, both Sr2+ and Ba2+ can substitute for Ca2+. Furthermore, both Sr2+ and Ba2+ are permeable through the MTX-activated channels as judged by the increase in fura 2 fluorescence seen when these divalent cations were readded to the bath solution after MTX. This does not reflect the binding of these cations to fura 2 in the extracellular space, since fura 2 efflux (i.e., the formation of pores) also requires the presence of extracellular divalents. The finding that both Ba2+ and Sr2+ pass through the MTX-activated channels provides an explanation for previous reports that Ba2+ can substitute for Ca2+ in MTX-induced platelet aggregation (47), that Sr2+ can substitute for Ca2+ in MTX-induced histamine release from human basophils (6), and that both Ba2+ and Sr2+ can substitute for Ca2+ in MTX-induced depolarization of rat brain synaptosomes (40). Although a rise in [Ca2+]i is not sufficient for induction of the large pore, it is not clear if a rise in [Ca2+]i is necessary. In preliminary studies, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid loading of the fibroblasts only partially inhibits MTX-induced pore formation (Schilling, unpublished observations). Whether this reflects a change in kinetics of pore formation or a change in the number of pores formed will require further investigation.

Although the molecular identities of the MTX-sensitive channel and pore are unknown, the profile of permeability changes observed for MTX is similar to those observed following activation of P2Z/P2X7 receptors in a variety of cell types. When cells are stimulated with ATP or with Bz-ATP, a selective agonist for the P2Z/P2X7 receptor, large increases in [Ca2+]i are followed by uptake of vital dyes and release of intracellular fura 2. With continued stimulation of the receptor, vital dye uptake is followed by cell swelling and release of LDH, which presumably signals oncotic cell death (13). The effect of Bz-ATP on plasmalemmal permeability is similar to that observed for MTX both in time course and magnitude. For example, Humphreys and Dubyak (19) have shown that THP-1 monocytic cells, pretreated with LPS and interferon-gamma to upregulate the P2Z/P2X7 receptor, exhibit large increases in [Ca2+]i and ethidium uptake in response to Bz-ATP and ATP, and these changes are followed within 15 min by release of ~65% of cell-associated LDH. The size of the Bz-ATP-induced pores and the MTX-induced pores are very similar; the P2Z/P2X7 pores allow molecules with molecular masses of <900 Da to enter/exit the cell (13, 28, 38). Furthermore, whole cell membrane current changes seen following application of ATP to responsive cells are similar to those reported in the present study for MTX with respect to ion selectivity and the subsequent pore formation. In this regard, stimulation of P2Z/P2X7 receptors initially reveals a nonselective cation channel that is permeable to Ca2+, Na+, Li+, and K+ but not to NMDG or Tris. With continued receptor stimulation, conductance of the membrane to NMDG and Tris increases (28), and this phase is associated with uptake of fluorescent dyes (32, 39). Thus, in several respects, the MTX- and Bz-ATP-induced changes in plasmalemmal permeability are very similar. Weisman and colleagues (15, 34) have shown that stimulation of transformed mouse 3T6 fibroblasts by ATP or Bz-ATP increases the permeability of the plasmalemma to small (<1 kDa) and ultimately large (4-20 kDa) macromolecules, demonstrating the presence of ATP-sensitive pores in this cell line. Furthermore, human fibroblasts have been shown to express P2Z/P2X7 transcripts (37). Likewise, the fibroblasts used in the present study express P2Z/P2X7 transcripts and respond to Bz-ATP with a small increase in [Ca2+]i that is insensitive to lanthanides. Thus there are at least two possible explanations for the results of the present study: 1) MTX is a high-affinity ligand for the P2Z/P2X7 receptor, or 2) MTX and P2Z/P2X7 receptor stimulation activate distinct channels but activate a common cytolytic pore. Although the effects of Bz-ATP on [Ca2+]i are much smaller in magnitude than those seen with MTX, the results do not eliminate either possibility, since MTX may simply be a much more effective agonist for the P2Z/P2X7 channel. Experiments designed to distinguish between these hypotheses will be presented in the accompanying study (35a). Irrespective of the exact molecular mechanism of MTX action, the results of the present study clearly demonstrate that MTX is a potent and highly effective initiator of pore formation in the plasmalemma of human skin fibroblasts. As such, this toxin may prove useful for understanding the molecular steps involved in channel activation, pore formation, and necrotic cell death.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Tanya Wasylyna and Erin McGowan and helpful discussions with Drs. George Dubyak and Ben Humphreys.


    FOOTNOTES

This work was supported in part by National Institute of General Medical Sciences Grant GM-52019, American Heart Association-Ohio Affiliate Grant 9806267, and by funds awarded to Case Western Reserve University by the Howard Hughes Medical Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: W. P. Schilling, Rammelkamp Center for Education and Research, MetroHealth Medical Center, Rm. R322, 2500 MetroHealth Dr., Cleveland, OH 44109-1998 (E-mail: wschilling{at}metrohealth.org).

Received 1 April 1999; accepted in final form 24 June 1999.


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