Eosinophil peroxidase increases membrane permeability in mammalian urinary bladder epithelium

Teri J. Kleine1, Gerald J. Gleich2, and Simon A. Lewis1

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


    ABSTRACT
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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


    INTRODUCTION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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.


    MATERIALS AND METHODS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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
<IT>G</IT><SUB>t</SUB> = (<IT>I</IT><SUB>sc</SUB>/<IT>E</IT><SUB>c</SUB>) + <IT>G</IT><SUB>j</SUB> (1)
If EPO alters only the cell resistance when Vt is clamped to 0 mV, the plot of Eq. 1 will be linear, with a y-intercept equal to the junctional conductance (Gj). The slope is equal to the inverse of the cellular electromotive force (Ec), which is the sum of the apical and basolateral membrane equivalent batteries.

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 to -70 mV, followed by the addition of EPO to the mucosal solution. After a 5-min incubation with EPO at -70 mV, Vt was clamped to 0 mV. When the conductance reached a steady state, the I-V relationship was again measured. The difference between the two I-V relationships is the voltage dependence of the current flowing through the EPO-induced conductance. The difference I-V relationship was fitted by the constant field equation to determine the relative ionic permeabilities of the EPO-induced conductance (23).

Data 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
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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 -70 mV (serosal solution ground) and equilibrated for a period of at least 3 min. To determine whether there were any voltage-independent effects of EPO, the equilibration period was extended to as long as 20 min in three experiments. Over the equilibration period, permeability properties of the bladder epithelium did not change (Fig. 1). When Vt was clamped to 0 mV, there was a large, rapid increase in Gt. In the absence of EPO, voltage clamping from Vt = -70 to 0 mV induced a small increase in Gt due to an intrinsic voltage sensitivity of the urinary bladder epithelium in divalent cation-free solution. The fact that the conductance increase in the absence of EPO was much smaller than the increase in the presence of EPO demonstrates that EPO can induce a voltage-sensitive increase in Gt.


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Fig. 1.   Effect of eosinophil peroxidase (EPO) on transepithelial conductance (Gt) is voltage dependent (black-triangle). EPO (300 nM), when added to mucosal solution at transepithelial voltage (Vt) = -70 mV, had no effect on Gt for periods as long as 20 min. When Vt was clamped to 0 mV (dotted line), Gt rapidly increased. Data are shown fitted by Eq. 2. Delta Gt, change in Gt. Best fit values are maximal protein-induced conductance (Gp) = 795 µS/cm2 and rate constant of conductance change (ko) = 0.28 s-1. triangle , Effect of voltage on Gt in absence of EPO. Mucosal solution was a divalent cation-free [Ca2+- and Mg2+-free (CMF)] KCl Ringer. Serosal solution was a divalent cation-containing NaCl Ringer.

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
&Dgr;<IT>G</IT><SUB>t</SUB>(<IT>t</IT>) = <IT>G</IT><SUB>p</SUB> · (1 − <IT>e</IT><SUP>−<IT>k</IT><SUB>o</SUB><IT>t</IT></SUP>) (2)
where Delta Gt(t) is the time-dependent change in Gt, Gp is the maximal protein (EPO)-induced conductance, t is time, and ko is the rate constant of the conductance change. The best fit value for the magnitude (Gp, normalized for EPO concentration) is 2,400 ± 330 µS · cm-2 · µM-1 (n = 18), and the best fit value for ko is 0.022 ± 0.004 s-1 (n = 15). The nonsaturating data were fitted by a line to determine the rate of the conductance change (Delta Gt/Delta t). For 30 nM EPO, Delta Gt/Delta t = 0.76 ± 0.27 µS · cm-2 · s-1 (n = 8). At present, it is not known why there were two different shapes for the time courses of the conductance increase.


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Fig. 2.   Two different time courses were observed for EPO-induced conductance change. When Vt was clamped from -70 to 0 mV, time course for resulting conductance increase for EPO (30 nM) was either saturating or linear. Saturating time course (black-triangle) was fitted by Eq. 2. Best fit values are Gp = 93 µS/cm2 or 3,100 µS · cm-2 · µM-1 and ko = 0.012 s-1. Nonsaturating data (triangle ) were fitted by a line to determine rate of conductance change (Delta Gt/Delta t). Best fit value for Delta Gt/Delta t = 0.72 µS · cm-2 · s-1.

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 -70 mV. After equilibration of the EPO in solution, Vt was voltage clamped to 0 mV and the magnitude and the rate constant for the conductance increase were recorded and plotted as a function of EPO concentration. The results indicate that the magnitude of the EPO-induced conductance change was dose dependent (Fig. 3) whereas the rate constant was independent of EPO concentration (data not shown). When fitted by the Michaelis-Menten equation, the dose-response relationship for the magnitude of the conductance change gave a maximum conductance of 411 µS/cm2 and a Michaelis-Menten constant of 113 nM (data from 9 tissues).


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Fig. 3.   EPO dose-response relationship for saturating time courses. Various amounts of EPO were added to mucosal solution, allowed to equilibrate, and then clamped to a Vt of 0 mV. Data from 9 tissues were fitted by Eq. 2 to determine magnitude and rate constant of conductance change. Magnitude of EPO-induced conductance was a saturating function of EPO concentration and was fitted by Michaelis-Menten equation. Best fit values are maximum conductance = 411 µS/cm2 and Michaelis-Menten constant = 113 nM.

For the linear time course conductance change, the slope of the time course (Delta Gt/Delta 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 = -70 mV and equilibrated for 3 min before Vt was clamped to 0 mV. The resultant conductance change was plotted as a function of Isc and fitted by Eq. 1 (Fig. 4). Best fit values are Ec = -59 ± 2 mV and Gj = 23 ± 2 µS/cm2 (n = 14). These two examples correspond to the two time courses shown in Fig. 2. For both the saturating and the nonsaturating time courses, the conductance increased as a linear function of the Isc. Because Eq. 1 is an equation for a line if only Isc (and not Gj or membrane selectivity) varies with increase in conductance, these data suggest that the added EPO affects only the cellular conductance and not the tight junctions or the electrochemical driving force across the apical and basolateral membranes (Ec). Because the slope of this plot is 1/Ec, the slight change in slope at the beginning of the saturating time course data suggests that, in this case, the EPO-induced conductance may be slightly anion selective at first and then become nonselective as the conductance increases further.


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Fig. 4.   Site of EPO action was determined from a plot of Gt vs. short-circuit current (Isc). Data correspond to time courses shown in Fig. 2 and are shown fitted by Eq. 1. For linear time course (triangle ), EPO-induced conductance change at 0 mV was a linear function of cellular current, suggesting that EPO increased cellular conductance rather than tight junctional conductance (Gj). Best fit values are Gj = 25 µS/cm2 and cellular electromotive force (Ec) = -64 mV. For saturating time course (black-triangle), relationship between conductance and Isc was biphasic, suggesting an early anion-selective stage that becomes nonselective as conductance increases. Best fit values are Gj = 22 µS/cm2 and Ec = -45 mV. For both examples, value of Gj is an underestimation, since EPO causes a small change in membrane selectivity.

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 Omega  · cm2, determined previously using microelectrodes (13)] than apical membrane resistance (which is ~24,000 Omega  · 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 Omega  · 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 = -70 mV, the Vt was clamped to more positive potentials. The saturating time courses for the conductance changes were fitted by Eq. 2 to determine the magnitude and the rate constant at each voltage. These values were normalized to the magnitude and the rate constant at 0 mV to correct for tissue variability. Figure 5, A and B, demonstrates the relationship between Vt and both the magnitude and the rate of the EPO-induced conductance change; both parameters increased as exponential functions of voltage. The smooth curve through the data is the best fit by the equation
<IT>G</IT><SUB>t</SUB>(<IT>V</IT>) = <IT>G</IT><SUB>t</SUB>(0) · <IT>e</IT><SUP>ec<IT>NV</IT><SUB>t</SUB>/<IT>kT</IT></SUP> (3)
where Gt(0) is the conductance change at 0 mV, Gt(V) is the total conductance at a particular voltage, N is an empirical constant, ec is the electron charge (1.602 × 10-19 C), k is the Boltzmann constant (1.38 × 10-23 J/K), and T is absolute temperature (310 K). Best fit values for N are 1.5 for the magnitude and 0.97 for the rate constant of the induced conductance increase (n = 6).


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Fig. 5.   EPO voltage sensitivity. At Vt = -70 mV, EPO was added to mucosal solution and equilibrated, and then Vt was clamped to more positive voltages. Time course was fitted by Eq. 2, and Gp was normalized to EPO concentration. Best fit values for Gp and ko were normalized to values for Gp and ko at Vt = 0 mV and then plotted as a function of both Vt and apical membrane voltage. Data from 6 experiments were then fitted by Eq. 3. A: magnitude of conductance change was an exponential function of voltage. Best fit value for empirical constant (N) was 0.98, and mean magnitude of conductance change at 0 mV was 2,661 ± 706 µS · cm-2 · µM-1. B: rate constant is also an exponential function of voltage. Best fit value for N was 1.5, and mean rate constant at 0 mV was 0.02 ± 0.004 s-1.

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- permeabilities (PK and PCl). The intracellular ion activities used for the constant field equation were 70 mM K+ and 15 mM Cl- (14). The best fit value for PK is 2 ± 0.8 × 10-7 cm/s. The best fit for the ratio of PCl to PK is 0.7 ± 0.2 (n = 8), suggesting that the EPO-induced conductance was equally permeable to K+ and Cl-.


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Fig. 6.   Current-voltage (I-V) relationship for EPO-induced conductance. Steady-state difference I-V relationship was generated from difference between I-V relationship for steady-state EPO-induced conductance and control I-V relationship at a holding voltage of 0 mV. Data are shown fitted by constant field equation, which was used to determine ion permeabilities.

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 to -70 mV. Second, the reversal of EPO at Vt = 0 mV was measured by replacing the mucosal solution with EPO-free solution.

Voltage-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
&Dgr;<IT>G</IT><SUB>t</SUB>(<IT>t</IT>) = <IT>G</IT><SUB>r</SUB> · e<SUP>−<IT>k</IT><SUB>r</SUB><IT>t</IT></SUP> + <IT>G</IT><SUB>s</SUB> · <IT>e</IT><SUP>−<IT>k</IT><SUB>s</SUB><IT>t</IT></SUP> (4)
where Gr is the rapid component of the conductance reversal, Gs is the slow component, and kr and ks are the rate constants for leaving the rapid and slow conductance states, respectively. Best fit values of the reversals for 30 nM EPO are Gr = 28 ± 5 µS/cm2, kr = 0.24 ± 0.08 s-1, Gs = 70 ± 28 µS/cm2, and ks = 0.02 ± 0.006 s-1 (n = 6). However, as shown in Fig. 7, the Gt does not return to the initial baseline value. This irreversible increase in conductance will be described below (see Toxicity in both RESULTS and DISCUSSION).


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Fig. 7.   Voltage-dependent reversal of induced conductance. When Vt was clamped from 0 to -70 mV, conductance induced by EPO (46 nM) partially reversed. Reversal is shown fitted by Eq. 4. Best fit values are rapid and slow components of conductance reversal (Gr and Gs) = 32 and 63 µS/cm2, respectively, and rate constants of return from rapid and slow states (kr and ks) = 0.35 and 0.018 s-1, respectively.

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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Fig. 8.   Removal of EPO resulted in an increase in Gt. EPO was added to mucosal solution and equilibrated, and, at time 0, Vt was clamped from -70 to 0 mV. After a significant increase in Gt at Vt = 0 mV, mucosal solution was replaced with EPO-free solution (dotted line at left), which caused Gt to increase. On completion of wash (dotted line at right), voltage was clamped back to -70 mV, and induced conductance partially reversed.

To determine the proportion of the total EPO-induced conductance that was inhibited by the EPO in the mucosal solution, the magnitude of the increase in conductance with removal of EPO was divided by the total EPO-induced conductance (which was the sum of the increase in conductance with clamping to Vt = 0 mV and the increase in the conductance with the removal of EPO from the mucosal solution). The proportion of the EPO-induced conductance that was inhibited was 0.39 ± 0.04 (n = 5). On completion of the wash, Vt was clamped back to -70 mV and the conductance slowly reversed. Therefore, voltage was more effective than removal of EPO in reversing the induced conductance.

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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Fig. 9.   Dose-response relationship for Ca2+ reversal of EPO was determined by varying Ca2+ concentration ([Ca2+]) at Vt = 0 mV following a conductance change induced by 30 nM EPO. Gt was then allowed to reach a steady state that was normalized to total EPO-induced conductance. Initial conductance at -70 mV is defined as 0. Data are shown fitted by Michaelis-Menten inhibition curve. Best fit value for Michaelis-Menten inhibitor constant is 0.1 mM (6 tissues).

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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Fig. 10.   Effect of Ca2+ on EPO-induced conductance was reversible. At time 0, with 152 nM EPO in mucosal solution, Vt was clamped to 0 mV, and Gt increased. Next, EPO was removed by replacing mucosal solution with EPO-free CMF KCl Ringer. Then Ca2+ was added to mucosal solution, resulting in complete reversal of EPO-induced conductance. Next, Ca2+ was removed by replacing mucosal solution with CMF KCl Ringer, and Gt increased. Dotted lines, solution changes.

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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Fig. 11.   Site of Ca2+ action. Ca2+-induced reversal of conductance change was a linear function of Isc. Fact that relationship for Ca2+-induced reversal (black-triangle) followed same relationship as EPO-induced conductance (triangle ) suggests that Ca2+ acted directly on EPO-induced conductance rather than on tight junctions or other permeability pathways in membrane.

                              
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Table 1.   Ec and Gj estimated by Ca2+ and voltage-dependent reversal

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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Fig. 12.   Time course for EPO-induced conductance change in presence of 2 mM mucosal Ca2+. When mucosal solution contained 2 mM Ca2+, EPO (228 nM) was added at Vt = -70 mV and allowed to equilibrate for 3 min, and then Vt was clamped to 0 mV (at time 0). Mucosal solution was KCl Ringer solution that contained 2 mM Ca2+ in form of CaCl2. Serosal solution was NaCl Ringer.

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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Table 2.   Ion-selective permeability of EPO-induced conductance

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 -70 mV (see Voltage-induced reversal above), some of the induced increase in the Gt was irreversible. This irreversible increase in tissue conductance indicates tissue damage, or toxicity. The degree of toxicity increased as a function of time, as shown in Fig. 13. Time 0 was the last point of the incubation period at Vt = -70 mV before clamping to Vt = 0 mV. The ability of the tissue to recover from a protein-induced increase in conductance was determined by allowing a voltage-dependent reversal (clamp from 0 to -70 mV) to reach a steady-state value. This new baseline conductance was then normalized to the baseline conductance at time 0.


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Fig. 13.   Toxic effect of EPO. Ability of tissue to recover from an EPO-induced increase in conductance was determined by allowing a voltage-dependent reversal to reach a steady-state value. This new baseline conductance was then normalized to baseline conductance at time 0. Time 0 was last point of incubation period at Vt = -70 mV before clamping to Vt = 0 mV. Conductance increased as a function of exposure time, indicating irreversible damage to tissue (7 tissues). Smooth curve through points was fitted by hand.


    DISCUSSION
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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.


    ACKNOWLEDGEMENTS

We thank James Checkel and David Loegering for preparing the EPO used in these studies.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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Am J Physiol Cell Physiol 276(3):C638-C647
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