1 Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555; and 2 Department of Immunology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Eosinophil
peroxidase (EPO), a cationic protein found in eosinophils, has been
reported to be cytotoxic independent of its peroxidase activity. This
study investigated with electrophysiological methods whether EPO is
toxic to mammalian urinary bladder epithelium. Results indicate that
EPO, when added to the mucosal solution, increases apical membrane
conductance of urinary bladder epithelium only when the apical membrane
potential is cell interior negative. The EPO-induced conductance was
concentration dependent, with a maximum
conductance of 411 µS/cm2 and a
Michaelis-Menten constant of 113 nM. The EPO-induced conductance was
nonselective for K+ and
Cl. The conductance was
partially reversed using voltage but not by removal of EPO from the
bulk solution. Mucosal Ca2+
reversed the EPO-induced conductance by a mechanism involving reversible block of the conductance. Prolonged exposure (up to 1 h) to
EPO was toxic to the urinary bladder epithelium, as indicated by an
irreversible increase in transepithelial conductance. These results
suggest that EPO is indeed toxic to urinary bladder epithelium via a
mechanism that involves an increase in membrane permeability.
cationic proteins; tight epithelium; cytotoxic protein; ion conductance; calcium
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INTRODUCTION |
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EOSINOPHILS KILL PATHOGENIC organisms using cationic proteins, including eosinophil peroxidase (EPO), which are contained within the eosinophil in specific granules and are released after appropriate stimulation. EPO is a 65.7-kDa protein that is ~15% positively charged. The amino acid sequence of EPO is similar to myeloperoxidase and thyroid peroxidase (6, 22). EPO is toxic in the presence and in the absence of H2O2 and halide, suggesting that there are two mechanisms by which EPO is cytotoxic (18). Acting as a peroxidase, EPO is a potent toxin, causing ciliostasis, bleb formation, and exfoliation of guinea pig tracheal epithelium after 12 h of exposure at 52 nM. Acting as a cationic protein in the absence of H2O2 and halide, 10 times as much EPO (520 nM) is required for the same effect. EPO has also been demonstrated to be toxic to parasites independent of its peroxidase activity (9).
Eosinophil degranulation has been associated with a number of pathological conditions in the urinary bladder. Patients with urinary schistosomiasis (20), bladder cancer (16), and interstitial cystitis, a noninfectious, inflammatory disease that affects the bladder epithelium (4, 15, 26) have been demonstrated to have eosinophils and/or free eosinophil granular proteins in their urine. The effects of eosinophil granular proteins on urinary bladder epithelium are unknown.
To determine the effects of EPO on urinary bladder epithelium, we tested nanomolar concentrations of this protein on rabbit urinary bladder epithelium using electrophysiological techniques. The data presented in this study indicate that EPO is toxic to the bladder epithelium. The mechanism of toxicity involves a voltage-dependent increase in the apical membrane conductance. In addition, these experiments describe several factors that affect the extent of toxicity and thus may suggest ways to limit tissue damage in disease states.
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MATERIALS AND METHODS |
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Tissue Preparation
Urinary bladders were excised from 3-kg male New Zealand White rabbits and washed in NaCl Ringer (see Solutions below). After the smooth muscle was dissected away, the epithelium was mounted on a ring of 2-cm2 exposed area and transferred to a temperature-controlled modified Ussing chamber (13). Both sides of the epithelium were initially bathed in NaCl Ringer. The serosal side of the epithelium was held against a nylon mesh by a slightly greater hydrostatic pressure in the mucosal chamber than in the serosal chamber. The volumes of the chambers were 4.5 ml for the mucosal chamber and 15 ml for the serosal chamber. The solutions in both the mucosal and serosal chambers was aerated with 95% O2-5% CO2. The solutions were stirred by a magnetic spin bar at the bottom of the serosal chamber and by injecting 95% O2-5% CO2 into the bottom of the mucosal chamber and allowing it to bubble upwards. Integral water jackets maintained the temperature of the bathing solution at 37°C.Protein Purification
Purification of EPO from eosinophils has previously been described (7, 9, 21, 22). Briefly, eosinophils were obtained from patients with marked blood eosinophilia by cytapheresis. After thorough washing to remove plasma proteins, erythrocytes were lysed, and the cells were washed again. Eosinophil granules were obtained by lysing the cells and centrifuging to remove unbroken cells and cellular debris. Isolated granules were lysed by dissolving them in 10 mM HCl and briefly sonicating them and then centrifuged at 40,000 g for 5 min. EPO was isolated by fractionating the supernatant on a Sephadex G-50 column. Fractions containing EPO were dialyzed against 0.5 M Na2PO4-0.5 M KH2PO4 and applied to a CM Sepharose column. EPO was eluted with a linear NaCl gradient, dialyzed against PBS, and concentrated. The concentration of EPO was determined spectrophotometrically (17). EPO was added to the mucosal solution in microliter quantities from a stock solution.Solutions
The NaCl Ringer contained (in mM) 111.2 NaCl, 25 NaHCO3, 10 glucose, 5.8 KCl, 2.0 CaCl2, 1.2 KH2PO4, and 1.2 MgSO4. In KCl Ringer, all Na+ salts were replaced with the appropriate K+ salts. In nominally Ca2+- and Mg2+-free (CMF) KCl Ringer, Ca2+ and Mg2+ salts were omitted. The effects of increasing mucosal Ca2+ and Mg2+ on the EPO-induced membrane conductance were determined by adding CaCl2 or MgCl2, which were dissolved in distilled deionized water to make concentrated stock solutions.Transepithelial Electrophysiological Methods
Electrical measurements. All electrical measurements were made under voltage-clamp conditions unless otherwise noted. The transepithelial voltage (Vt) was measured with Ag-AgCl wires placed close to and on opposite sides of the epithelium (serosal solution ground), and current was passed from Ag-AgCl electrodes placed in the rear of each hemichamber. Current-passing and voltage-measuring electrodes were connected to an automatic voltage clamp (Warner Instruments). The transepithelial resistance and its inverse, the transepithelial conductance (Gt), were calculated from Ohm's law and the measured current required to clamp the epithelium 10 mV from the holding voltage.
Data acquisition. Voltage and current outputs of the voltage clamp were connected to an analog-to-digital converter (Warner Instruments) interfaced to a computer that calculated values for resistance and short-circuit current (Isc). Vt and current were continuously monitored on an oscilloscope. All data were printed out with the time of data acquisition and also stored on the hard disk.
Equivalent Circuit Analysis
The site of the EPO-induced conductance (cell membrane and/or tight junction) was determined using the method of Yonath and Civan (27). Gt was plotted as a function of Isc and fitted by the equation
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(1) |
Current-Voltage Relationship
The current-voltage (I-V) relationship of the EPO-induced conductance was calculated as the difference of the transepithelial I-V relationships in the presence and absence of added EPO. First, the tissue was voltage clamped to Vt = 0 mV, and then the transepithelial current responses to computer-generated voltage pulses 30 ms in duration and of increasing magnitude and alternating polarity were measured. Then, Vt was voltage clamped toData Analysis and Statistics
Curve fitting was performed using NFIT (Island Products, Galveston, TX) on a small laboratory computer. Data are shown as means ± SE. Statistics were calculated using INSTAT (GraphPAD Software, San Diego, CA). ![]() |
RESULTS |
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In this section, the effect of EPO on the permeability properties of the rabbit urinary bladder epithelium is described. Several characteristics of the effects of EPO were determined, including the dose-response relationship, voltage sensitivity, ion selectivity, divalent cation sensitivity, and reversibility. Unless otherwise noted, all experiments were done under voltage-clamp conditions with divalent cation-free mucosal solution (CMF KCl Ringer; see Solutions).
Effect of EPO on Membrane Conductance
The effect of EPO on the permeability properties of rabbit urinary bladder epithelium was tested as follows. EPO was added to the mucosal solution at a Vt of
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The EPO-induced conductance increase followed 2 different time courses; of 32 time courses on 13 tissues, 18 (57%) saturated and 14 (43%) did not; typical examples are given in Fig. 2. The saturating time courses were fitted by the single-exponential equation
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(2) |
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Dose-Response Relationships
The effect of the concentration of EPO on the magnitude and rate constant of the saturating conductance change was examined. EPO was added in various concentrations to the mucosal solution while the epithelium was voltage clamped at
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For the linear time course conductance change, the slope of the time
course
(Gt/
t)
was plotted as a function of EPO concentration. Over the range of
concentrations tested, the rate of change was independent of the EPO
concentration (data not shown).
Site of Action
The EPO-induced increase in Gt may be the result of an increase in the cellular conductance and/or Gj. The site of action was determined using the following protocol. EPO was added to the mucosal solution at Vt =
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The magnitude of the conductance change suggests not only that this is
a cellular membrane effect but also that EPO primarily affects the
apical membrane. Basolateral membrane resistance (the inverse of
conductance) is much smaller [1,500
· cm2,
determined previously using microelectrodes (13)] than apical membrane resistance (which is ~24,000
· cm2 in the
example for the linear time course). The magnitude of the conductance
increase for the linear time course corresponds to a resistance
decrease of 19,200
· cm2.
Consequently, the effect must be primarily on the apical membrane; however, a concurrent effect on the basolateral membrane cannot be
ruled out.
Voltage Sensitivity
As previously indicated, the ability of EPO to induce an increase in Gt is a voltage-sensitive phenomenon. This section more fully characterizes the relationship between the applied voltage and the EPO-induced conductance. Three minutes after EPO was added to the mucosal solution at Vt =
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(3) |
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Because EPO acts on the apical membrane, the effect of the changes in
Vt on the voltage
gradient across the apical membrane is of interest.
Vt is the sum of
the voltages across both the apical and the basolateral membranes
(serosal solution is ground). The voltage across the apical membrane
relative to the mucosal solution
(Va) can be
calculated as the difference between the basolateral membrane voltage,
which is 55 mV [as previously determined using
microelectrodes (13)] and the
Vt. Therefore,
during the equilibration, when
Vt is
70
mV, Va is 15 mV
(mucosal solution ground). When
Vt is clamped to
0 mV, then Va is
55 mV. Thus, as shown in Fig. 5, EPO induces a conductance
change only when Va is cell
interior negative. When
Va is cell
interior positive, there is no change in the
Gt.
I-V Relationship
The ionic permeability of the EPO-induced conductance was determined from the difference between the steady-state I-V relationships before and after the addition of EPO using the constant field equation (see Current-Voltage Relationship, Fig. 6). The I-V relationships for the EPO-induced conductance were generated after the induced conductance had reached a steady state at 0 mV; for linear time courses, a steady state was achieved by washing the mucosa with EPO-free solution at Vt = 0 mV. The steady-state difference I-V was then generated by subtracting the I-V relationship generated in the absence of EPO. The result is the I-V relationship for the EPO-induced conductance, which was fitted by the constant field equation to determine the K+ and Cl
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Reversibility of EPO-Induced Conductance
Once EPO induced an increase in the Gt, the reversibility of the induced conductance was tested as a function of voltage and removal of bath EPO. First, because the conductance increase was voltage sensitive, the ability of voltage to reverse the conductance was tested by changing Vt back toVoltage-induced reversal.
When Vt was
returned from 0 to 70 mV, the EPO-induced conductance partially
reversed (Fig. 7). The time course for the
reversal was biphasic, with an initial small rapid phase followed by a larger slow phase, and can be modeled as two conductive states leaving
the membrane in either a parallel or a series arrangement. Data are
shown fitted by the following equation based on the parallel model
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(4) |
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Removal of mucosal EPO.
Reversibility was also determined by reducing the concentration of free
EPO in the mucosal solution. This was accomplished by replacing the
mucosal solution with EPO-free solution at 0 mV after a significant
conductance change had been induced. Removal of EPO (Fig.
8) resulted in a seemingly paradoxical
increase in Gt
(n = 5). This increase in conductance
is more fully addressed below (see Comparison of EPO
With Other Cytotoxic Proteins).
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Effects of Divalent Cations
In experiments described in the previous sections, the mucosal solution contained no added divalent cations. Divalent cations have previously been found to inhibit or reverse the protein-induced conductance for a number of cationic proteins (11, 25). Therefore, the effects of Ca2+ and Mg2+ on the EPO-induced conductance were determined.Ca2+ reverses
EPO-induced conductance.
Adding millimolar CaCl2 to the
mucosal solution decreased the EPO-induced conductance despite
Vt being held at
0 mV (data not shown). The dose-response relationship for the
Ca2+ reversal of EPO was generated
by allowing the EPO-induced conductance to approach a steady state and
then adding increasing amounts of
Ca2+ at 0 mV. The new steady-state
conductance was then normalized to the maximal steady-state conductance
and plotted as a function of Ca2+
concentration (Fig. 9). The data are shown
fitted by an inhibition equation based on Michaelis-Menten kinetics,
and the best fit value for the Michaelis-Menten inhibitor constant was
0.1 mM.
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Effect of
Ca2+ is
reversible.
After Ca2+ decreased the induced
conductance, removal of the mucosal
Ca2+ resulted in a partial
restoration of the EPO conductance. First, EPO was allowed to induce a
conductance change at
Vt = 0 mV, and then it was washed from the mucosal solution. The resulting conductance was then reversed by adding millimolar
CaCl2 (Fig.
10). When
Gt reached a
steady state, Ca2+ was washed away
by replacement of the mucosal solution with
Ca2+-free solution. When
Ca2+ was removed from the bath,
Gt increased,
although not to pre-Ca2+ levels.
This is due at least in part to time-dependent dissociation of EPO from
the membrane without the usual concurrent conductance increase from the
mucosal solution pool of EPO.
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Site of Ca2+
action.
The site of Ca2+ action is
indicated by the plot of
Gt vs.
Isc for the
EPO-induced conductance increase and the
Ca2+-induced reversal (Fig.
11). The fact that this relationship for the Ca2+-induced reversal follows
the same line as the EPO-induced conductance suggests that
Ca2+ acts directly on the
EPO-induced conductance rather than on the tight junctions or another
permeability pathway in the membrane. Best fit values to
Eq. 1 are given in Table
1.
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Time course in 2 mM mucosal
Ca2+.
In the previous time course experiments (see Figs. 1 and 2), the
mucosal solution contained no added divalent cations. When the
experimental protocol was repeated with a mucosal solution that
contained 2 mM Ca2+ and 2 mM
Mg2+ (KCl Ringer, see
Solutions), there was a marked delay
in the EPO-induced conductance increase when voltage was clamped from 70 to 0 mV (Fig. 12). In many
cases, there was a small initial decrease before the conductance
increased. This delay may be due to divalent cations competing with EPO
for a membrane binding site. The alteration in the time course due to
the different mucosal solutions is most likely due to the presence of
mucosal Ca2+, since addition of
Mg2+ to the mucosal solution
was found to have no effect on the EPO-induced conductance (data not
shown). Note also that a much higher concentration of EPO was required
to induce a smaller and slower increase in conductance in divalent
cation-containing solution (228 nM) than was required in divalent
cation-free mucosal solution (30 nM in Fig. 2).
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Effect of
Ca2+ on
I-V
relationship.
Mucosal Ca2+ had no effect on the
ionic selectivity for EPO. This was determined by comparing the
steady-state difference
I-V relationships for EPO generated with divalent cation-containing mucosal
solution (KCl Ringer) with the
I-V
relationships for divalent cation-free solution (CMF KCl Ringer). There
was no significant difference in the selectivity of the EPO-induced
conductance for K+ and
Cl in the presence or
absence of Ca2+ as indicated by
the best fit values for
PCl/PK
(Table 2).
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Mg2+ had no
effect on EPO.
To determine the effect of Mg2+ on
the EPO-induced conductance, EPO (30 nM) was added to the mucosal
solution and allowed to equilibrate for 3 min. At
time
0,
Vt was clamped
from 70 to 0 mV. After a significant
Gt change,
MgCl2 was added to the mucosal solution in microliter amounts from a concentrated stock. Up to 12 mM
MgCl2 was added with no apparent
effect on the EPO-induced increase in
Gt
(n = 7; data not shown).
Toxicity
Although the EPO-induced conductance was partially reversible when Vt was clamped from 0 to
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DISCUSSION |
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EPO has been demonstrated to be cytotoxic to both respiratory epithelial cells and parasites in the absence of peroxidase substrates and the presence of catalase [which rapidly converts H2O2 into water (9, 18)]. This toxic effect was attributed to an increase in membrane permeability. However, detailed analysis of the ability of EPO to increase membrane permeability has not been previously described. In this report, the effects of EPO on membrane permeability properties are characterized on rabbit urinary bladder epithelium.
Properties of EPO-Induced Conductance
EPO, when added to the mucosal solution in nanomolar concentrations, increased the Gt of rabbit urinary bladder epithelium. The data suggest that this effect acted predominantly on the apical membrane. This increase was dependent on EPO concentration and was sensitive to Va. When Va was cell interior positive, EPO did not alter the Gt. When Va was cell interior negative, the apical membrane conductance increased. As the voltage was made more negative, both the rate constant and the magnitude of the induced conductance change increased. The ability of EPO to increase the membrane conductance at negative cell membrane potentials suggests that EPO might increase the membrane conductance under physiological conditions. Because most cell membrane potentials are negative (1), a prediction is that EPO might increase the membrane conductance of most cells. The EPO-induced conductance could be partially reduced by reversing the voltage gradient so that the cell interior was again cell interior positive. A possible mechanism to explain these observations has been previously described for other cytotoxic, cationic proteins (11). The cationic protein forms a loose association with the membrane, possibly with anionic sugars or lipids, when Va is positive. When Va is negative, the positively charged amino acids sense the voltage gradient and are attracted toward the cell interior. This drives the protein into the membrane, forming the conductive unit. When Va is then made positive, the protein is driven out of the membrane by the voltage field, reducing the protein-induced conductance. The conductance does not fully reverse, suggesting that some of the EPO may be irreversibly associated with the membrane and is insensitive to the membrane voltage.Comparison of EPO With Other Cytotoxic Proteins
The EPO-induced conductance shares a number of properties with the conductances induced by other cationic proteins, particularly histones and protamine sulfate (11, 23, 25). Some common characteristics are that the protein-induced conductance is not ion selective, is dependent on the Va gradient, and is reversible by the addition of mucosal Ca2+.An unusual observation was that removal of EPO from the mucosal solution resulted in a rapid increase in Gt. This increase in conductance when a protein is removed from the bath has been observed for protamine sulfate and other cationic peptides (23, 24). It was proposed that the cationic protein in the bulk solution blocks the protein-induced conductance, a process called self-inhibition. The fact that the conductance increased when EPO was removed from the bath suggests that EPO can partially inhibit the EPO-induced conductance.
Divalent cations have been demonstrated to inhibit cationic proteins via a number of mechanisms (25). Several lines of evidence suggest that Ca2+ reversal of EPO is at least partially due to a direct block of the EPO-induced conductance. 1) The Ca2+-induced decrease in the EPO-induced conductance was reversible. 2) The plot of Gt vs. Isc for the EPO-induced conductance increase and the Ca2+-induced reversal suggests that Ca2+ acts directly on the EPO-induced conductance rather than on the tight junctions or another permeability pathway in the membrane. 3) Ca2+ had no effect on the ionic selectivity for the EPO-induced conductance. These observations support a mechanism of conductive block by mucosal Ca2+. Other potential mechanisms for the Ca2+ reversal, such as competition for a membrane binding site or an increased rate of EPO dissociation from the membrane, may also be important.
Toxicity
EPO had a toxic effect on the rabbit urinary bladder epithelium. Toxicity was defined as an irreversible increase in Gt, which is indicative of a loss of tissue integrity. The irreversible effects induced by EPO increased as a function of the length of time that the epithelium was exposed to EPO.The above data describe the early cellular events induced by EPO that lead to toxicity of the epithelium. Little is known about these early events; previous studies have reported events further downstream (hours to days after exposure). For example, Motojima et al. (18) reported that 600-1,800 nM EPO caused ciliostasis, bleb formation, and exfoliation in guinea pig tracheal epithelium (in the absence of peroxidase substrates and the presence of catalase) over a time course of 3-6 h. In a different report (9), 50 µM purified EPO caused death of helminth larvae over a 48-h period. The data presented in this paper suggest a possible mechanism for the toxic effect of EPO. Because the EPO-induced conductance is nonselective, it allows the influx of both cations and anions, which is followed by an obligate influx of water. The cell then swells and eventually bursts. An alternative possibility is that the formation of a conductance creates an entrance into the pathogen for EPO and/or other molecules released by the eosinophil. These molecules might then have intracellular effects that are toxic to the pathogen. It is unlikely that the toxic effect of EPO on urinary bladder is due to its peroxidase activity, since peroxidase substrates were not added to the mucosal solution and there was insufficient time for the bladder to generate a significant concentration of H2O2.
When an eosinophil recognizes a pathogenic organism, it binds to that organism, forming a small pocket between itself and the membrane of the organism. The eosinophil then releases its granular contents into this pocket, where they are free to act on the pathogen. Electron micrographs indicate that the eosinophil forms an intimate association with the pathogen. The distance between the two cells ranges from ~60 to 250 nm (5). The EPO concentration in the pocket is unknown but may be quite high. Therefore, EPO may contribute to the cytotoxic activity of the eosinophil by inducing a conductance in the membrane of the pathogen. However, the eosinophil membrane is also exposed to EPO. It is not known whether EPO is toxic to the eosinophil. It is possible that the eosinophil may have a protective mechanism, such as T lymphocytes have to protect them from perforin (for a review, see Ref. 19). On the other hand, after degranulation, many of the eosinophils died in the study by Glauert et al. (5), so eosinophils are not necessarily immune to their own cytotoxic proteins. Also, analyses of eosinophil-rich lesions have shown the presence of eosinophil debris (3, 8, 12), suggesting that eosinophils themselves are destroyed after degranulation (2). In addition, little is known about the ionic composition of the pocket, such as the Ca2+ concentration. This information is of interest for determination of the environment in which EPO would be exposed to both the eosinophil and the pathogen.
Eosinophiluria and elevated eosinophil cationic proteins have been demonstrated in several bladder diseases (15). EPO has been measured in the sputum of patients with asthma and chronic obstructive pulmonary disease (10). The EPO concentration ranged from undetectable to ~10 nM, with a mean of ~4 nM in asthma. This suggests that the EPO concentrations in some patients were in the range of the concentrations that had a conductive effect in rabbit urinary bladder epithelium. Moreover, the tissue concentrations of EPO were likely considerably higher. Overall, these findings are in keeping with a pathophysiological role for EPO.
The results presented in this report also suggest factors that may modulate the extent of tissue damage by EPO. Removal of EPO may result in a short-term increase in the toxic effect of EPO (because of the loss of self-inhibition). It is not clear whether removal of EPO results in eventual recovery of the epithelium. Ca2+ may reduce the extent of EPO toxicity. These data may have important implications for understanding and treating conditions associated with extensive eosinophil degranulation both in the bladder (such as tumors and infections) and in other tissues.
In summary, the cationic eosinophil granular protein EPO is cytotoxic to rabbit urinary bladder epithelium via a voltage-dependent mechanism involving an alteration of apical membrane permeability. Also, the permeability induced by EPO is similar to those induced by other cationic proteins. This suggests that EPO may contribute to disease processes in which significant eosinophil degranulation occurs. Further studies are needed to determine to what extent these observations are important in vivo.
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ACKNOWLEDGEMENTS |
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We thank James Checkel and David Loegering for preparing the EPO used in these studies.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-51382 (to S. A. Lewis) and AI-09728 (to G. J. Gleich) and by a James W. McLaughlin Fellowship to T. J. Kleine.
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: S. A. Lewis, Dept. of Physiology and Biophysics, University of Texas-Medical Branch, Galveston, TX 77555-0641.
Received 8 May 1998; accepted in final form 1 December 1998.
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