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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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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The eosinophil
granule protein major basic protein (MBP) is toxic to a wide variety of
cell types, by a poorly understood mechanism. To determine whether the
action of MBP involves an alteration in membrane permeability, we
tested purified MBP on rabbit urinary bladder epithelium using
transepithelial voltage-clamp techniques. Addition of nanomolar
concentrations of MBP to the mucosal solution caused an increase in
apical membrane conductance only when the voltage across the apical
membrane was cell interior negative. The magnitude of the MBP-induced
conductance was a function of MBP concentration, and the rate of the
initial increase in conductance was a function of the transepithelial
voltage. The MBP-induced conductance was nonselective for
K+ and
Cl
. Mucosal
Ca2+ reversed the induced
conductance, whereas mucosal Mg2+
partially blocked the induced conductance and slowed the rate of the
increase in conductance. The induced conductance was partially reversed
by changing the voltage gradient across the apical membrane to cell
interior positive. Prolonged exposure resulted in an irreversible loss
of the barrier function of the urinary bladder epithelium. These
results suggest that an increase in cell membrane ion permeability is
an initial step in MBP-induced loss of barrier function.
cationic protein; tight epithelium; cytotoxic proteins; ionic conductances; calcium
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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MAJOR BASIC PROTEIN (MBP) is a cationic and cytotoxic protein that is the major component of eosinophil specific granules (18, 1). It is a 14-kDa protein that contains 17 arginines, only 1 acidic residue, and has a calculated isoelectric point of 10.9 (38). Purified MBP is toxic to a number of cell types, including parasites (6, 8, 21, 24, 39), tumor cells (6), a variety of mammalian cells such as splenic, intestinal, and endothelial cells (15), and airway epithelium (3, 12, 29, 32). The cytotoxic effect of MBP is believed to be important for immunity, by killing pathogens, and in disease processes associated with eosinophil infiltration and degranulation. For example, MBP has been measured in inflammatory lesions in tissues including cornea (34), liver (28), and intestine (9, 20, 33). Furthermore, elevated MBP levels have been measured in the sputum of patients with asthma (13), and a considerable body of evidence suggests that MBP mediates the tissue damage associated in asthma (for a review, see Ref. 16).
The mechanism of cytotoxicity for MBP is unclear. MBP has been shown to interact with synthetic liposomes made of anionic phospholipids, altering the fluorescence characteristics and circular dichroism spectra of both the lipids and MBP, suggesting that the first step in the effect of MBP is through a protein-lipid interaction (1). The MBP-induced toxic effect is a direct consequence of its cationic amino acids; acidic amino acids abolished the effect of MBP on guinea pig tracheal epithelium (4). A number of reports suggest that MBP alters membrane permeability. Purified MBP causes 51Cr release from parasites, tumor cells (6), and cutaneous epidermal cells (15). Microscopic changes to cells exposed to MBP, including blebbing and lysis, are indicative of disruption of the cellular membrane (3, 12, 29). However, it is not clear whether the increase in cell membrane permeability is a direct effect of MBP on the cellular membrane or is instead the result of extensive cell damage via other mechanisms.
Evidence of eosinophil degranulation has been found in association with some bladder disorders. Eosinophiluria and elevated levels of eosinophil cationic protein (a protein that is also released from eosinophil-specific granules and therefore is a marker of eosinophil degranulation) have been measured in the urine of patients with urinary parasitic infections (30) and bladder tumors (27) as well as in some patients with interstitial cystitis, a noninfectious inflammatory disease affecting the bladder epithelium (11, 31, 41). Thus eosinophils may serve a protective function in the case of infections or neoplastic states, yet may be pathogenic in the case of the inflammatory bladder disease interstitial cystitis. However, the effects of eosinophil granule proteins on urinary bladder epithelium are unknown.
To determine the direct effects of MBP on cell membrane permeability, we tested purified MBP on rabbit urinary bladder epithelium using electrophysiological techniques. The data presented in this study indicate that MBP induces a voltage-dependent increase in apical membrane conductance that is nonselective for cations and anions. This increase in conductance might contribute to the MBP-induced cytotoxic effect found in other tissues, by allowing an influx of ions and water leading to cell swelling and eventual lysis.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Tissue Preparation
Urinary bladders were excised from 3-kg male New Zealand White rabbits and washed in a NaCl Ringer (see Solutions). 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 (26). Both sides of the epithelium were initially bathed in NaCl Ringer solution. The serosal side of the epithelium was held against a nylon mesh by a slight excess of solution in the mucosal chamber. The solution in both the mucosal and serosal chambers was aerated with 95% O2-5% CO2 and stirred by magnetic spin bars at the bottom of the chambers. Integral water jackets maintained the temperature of the bathing solution at 37°C.MBP Purification
Purification of MBP from eosinophils has previously been described (19, 38). Briefly, leukocytes were obtained from patients with eosinophilia by cytapheresis and were thoroughly washed, and the erythrocytes were lysed. Eosinophil granules were isolated by lysing the cells and centrifuging to remove unbroken cells and cellular debris. Isolated granules were lysed by dissolving in 10 mM HCl, briefly sonicating, and then centrifuging at 40,000 g for 5 min. MBP was isolated by fractionating the supernatant on a Sephadex G-50 column and collecting fractions from the third peak. These fractions were rechromatographed on a Sephadex G-50 column. Fractions containing MBP were stored in 0.025 M acetate buffer with 150 mM NaCl (pH 4.3) and adjusted to a final concentration of 1.4 mg/ml (1 × 104 M). MBP was then added
to the mucosal solution in microliter quantities from this concentrated
stock.
Solutions
NaCl Ringer solution contains (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 solution, all Na+ salts were substituted with the appropriate K+ salts. In nominally Ca2+-free, Mg2+-free (CMF) KCl Ringer solution, Ca2+ and Mg2+ salts were omitted. The effect of increasing mucosal Ca2+ and Mg2+ on the MBP-induced membrane conductance was determined by adding CaCl2 or MgCl2, which were dissolved in distilled deionized water to make concentrated stock solutions.Transepithelial Electrophysiological Methods
Electrical measurements. Unless otherwise noted, all electrical measurements were made under voltage-clamp conditions. The transepithelial voltage (Vt) was measured with Ag-AgCl wires placed adjacent to both sides of the epithelium (serosal solution ground), whereas current was passed from Ag-AgCl electrodes placed in the rear of each hemichamber. Both current-passing and voltage-measuring electrodes were connected to an automatic voltage clamp (Warner Instruments). Ohm's law was used to calculate the transepithelial resistance (Rt) and its inverse, the transepithelial conductance (Gt), from the current required to clamp the epithelium 10 mV from the holding voltage.
Data acquisition. Current and voltage outputs of the voltage clamp were connected to an analog-to-digital converter (Axon Instruments) that interfaced with 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 method of Yonath and Civan (42) was used to determine whether the site of protein action was at the cell membrane and/or the tight junctions. Gt was plotted as a function of Isc and fit by the equation
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(1) |
Current-voltage relationship.
The current-voltage
(I-V)
relationship of the MBP-induced conductance was calculated from the
transepithelial
I-V
relationships in the absence and presence of added MBP. First, the
tissue was voltage-clamped to a
Vt of 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. Next, the
Vt was voltage
clamped to 
70 mV, and protein was added to the mucosal solution.
After a 5-min incubation, the
Vt was clamped to
0 mV, the conductance was allowed to reach a steady state, and 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 protein-induced conductance and was fit by the constant field
equation to determine the relative ionic permeabilities of the
protein-induced conductance (35).
Data Analysis and Statistics
Curve fitting was done using NFIT (Island Products, Galveston, TX) on a laboratory computer. Data are shown as means ± SE. Statistics were calculated using INSTAT (GraphPAD Software, San Diego, CA).| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Here we describe the effects of MBP on permeability properties of the rabbit urinary bladder epithelium under voltage-clamp conditions. These effects are characterized in regard to a number of parameters including the potency, voltage sensitivity, ion selectivity, site of action, and reversibility. Unless otherwise noted, all experiments were performed with divalent cation-free mucosal solution (CMF KCl Ringer solution, see Solutions).
Effect of MBP on Membrane Conductance
Figure 1 shows a typical example of the effect of MBP on the Gt of rabbit urinary bladder epithelium. MBP (360 nM) was added to the nominally divalent cation-free mucosal solution at a Vt of70
mV and allowed to equilibrate for 3 min. Over the equilibration period,
there was slight decrease in
Gt (data not
shown). Then Vt
was clamped to 0 mV, which resulted in an increase in
Gt. In the
absence of MBP, clamping
Vt from 70
to 0 mV resulted in a slight increase in
Gt that was
smaller than the change in the presence of MBP. This intrinsic membrane
voltage sensitivity was apparent only in the absence of mucosal
divalent cations; if the mucosal solution contained both 2 mM
Ca2+ and 2 mM
Mg2+,
Gt did not change
in response to the change in
Vt. The effect of
divalent cations on
Gt was also
apparent at Vt of
70 mV. When the mucosal solution was changed from divalent
cation-containing to divalent-free solution, the baseline conductance
increased 19 ± 3 µS/cm2
(n = 15). When
Ca2+ was added to the mucosal
solution, Gt
fully recovered within seconds. Similarly, when MBP was added to the
mucosal solution at
Vt of 70
mV, Gt recovered
62 ± 8% or 11 ± 2 µS/cm2
(n = 15). Thus MBP mimicked the
stabilizing effect of Ca2+ on the
epithelium, possibly due to the cationic nature of both molecules.
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The time course of the MBP-induced increase in conductance displayed two different shapes (Fig. 2). Both time courses displayed a small but fast exponential increase in Gt. In some cases, the fast exponential was followed by a slower exponential increase in Gt. In other tissues, the initial rapid exponential increase was followed by a sigmoidal increase in conductance, i.e., a slow increase (delay) followed by an exponential-like increase. This delay typically lasted for 250 ± 30 s (n = 16) after Vt had been clamped to 0 mV. The delay was tissue dependent; the presence or absence of a delay as well as the length of the delay varied widely among tissues but was relatively consistent for a given tissue.
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For 360 nM MBP, the magnitude of the residual intrinsic membrane voltage sensitivity was much smaller than the MBP-induced increase in Gt. The magnitude of the fast exponential increase is ~6 µS/cm2, whereas the magnitude of the conductance induced by 360 nM MBP was 250 ± 40 µS/cm2 (n = 8).
Dose-Response Relationship
The relationship between the concentration of MBP and the magnitude of the conductance increase was determined from the time course data. The magnitude of the plateau was determined by deleting the delay period and then fitting the MBP-induced exponential increase by a single exponential equation. The magnitude of the initial conductance increase was not included. The conductance-concentration curve was hyperbolic and was fit by Michaelis-Menten kinetics (Fig. 3). The best fit for the maximal MBP-induced conductance was 403 µS/cm2, and the Michaelis constant was 228 nM.
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Site of MBP Action
The conductance changes induced by MBP might occur at two possible locations in the tissue: at the cellular membrane and/or the tight junctions. To differentiate between these two possible sites of action (see MATERIALS AND METHODS), the following protocol was used. MBP was added to the mucosal solution at a Vt of70 mV and equilibrated for 3 min, and
Vt was clamped to
0 mV. The resultant conductance was plotted as a function of the
Isc and fit by
Eq. 1 (a typical example is shown in
Fig. 4). Best-fit values are as follows:
Ec = 51 ± 1 mV and Gj = 25 ± 2 µS/cm2
(n = 15). The relationship was linear
for all time courses (with or without the delay). The relationship
remained linear even when Vt was held at 0 mV for as long as 2 h. This suggests that the MBP affects only the
cellular conductance and not the tight
Gj or the
Ec across the
apical and basolateral membranes.
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The large magnitude of the conductance change indicates that the effect
was primarily on the apical (rather than basolateral) membrane. This is
because basolateral membrane resistance (the inverse of conductance) is
much smaller (1,500 
· cm2)
than apical membrane resistance (~16,000
· cm2 in
this example). Because the magnitude of the conductance change corresponds to a resistance change of 10,570 · cm2, the
effect must be primarily on the apical membrane. A concurrent, smaller
effect on the basolateral membrane cannot be ruled out.
Voltage Sensitivity
As indicated in the time courses, the ability of MBP to induce a conductance is a voltage-dependent phenomenon; Gt increased at Vt of 0 mV but not at70 mV. In this section, the voltage sensitivity of the
MBP-induced conductance is more fully characterized. First, MBP was
added to the mucosal solution at a
Vt of 70
mV, allowed to equilibrate, and then the
Vt was clamped to
more positive values. The slope of the initial rate of the MBP-induced
increase in conductance (or
Gi/t)
was determined by fitting the initial portion of the MBP-induced
conductance with a linear equation. The slope was then plotted as a
function of the applied voltage (Fig. 5).
The relationship between the initial rate of the MBP-induced conductance increase and
Vt was then fit
by an exponential equation
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(2) |
Gi/t(0)
is the conductance change at 0 mV,
Gi/t(v)
is the total conductance at a particular voltage,
N is an empirical constant,
e is the electron charge (1.602 × 1019 C),
k is the Boltzmann constant (1.38 × 1023 J/K), and
T is temperature (310 K). The best-fit
value for N, which is a measure of the
steepness of the voltage sensitivity, is 1.2 (n = 7).
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As shown in Fig. 5, MBP induced a conductance change in the apical
membrane only when the voltage gradient across the apical membrane
(Va) was cell
interior negative. The
Va was calculated as follows. The
Vt is the sum of
the voltages across both the apical and the basolateral membrane with
the serosal solution as ground. The basolateral membrane voltage has
previously been determined to be 55 mV (using microelectrodes,
Ref. 26). During equilibration, when
Vt is 70
mV, the voltage gradient across the apical membrane is 15 mV (cell
interior positive). When
Vt is clamped to
0 mV, then Va is
55 mV (cell interior negative). Thus the rate of the MBP-induced
conductance increases as the voltage gradient across the apical
membrane becomes more cell interior negative. When the apical membrane
voltage gradient is cell interior positive, there is no conductance
change. This suggests that the cationic MBP molecules can sense the
voltage gradient across the apical membrane and are either
electrostatically attracted or repelled accordingly. When the voltage
gradient across the apical membrane is cell interior positive, the MBP
is repelled and does not induce a conductance. In contrast, when the
voltage gradient across the apical membrane is cell interior negative, MBP is attracted toward the cell interior and is able to interact with
the apical membrane to induce a conductance.
I-V Relationship
The ionic permeability of the MBP-induced conductance was determined from the difference between the I-V relationships in the presence and the absence of MBP (see MATERIALS AND METHODS). This I-V relationship for the MBP-induced conductance was fit by the constant field equation to determine the K+ and Cl permeabilities (Fig.
6). The intracellular ion activities used for the constant field equation were 70 mM
K+ and 15 mM
Cl. The best-fit values
were
PCl/PK = 0.4 ± 0.1 and a PK = 30 ± 7 × 108 cm/s.
Thus the MBP-induced conductance was permeable to both K+ and
Cl, i.e., a nonselective
conductance.
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Reversibility of the MBP-Induced Conductance
The ability of the MBP-induced conductance to be reversed was tested using two protocols. First, Vt was clamped from 0 mV back to70 mV to see if reversing the driving force
across the apical membrane resulted in a decrease in conductance.
Second, the mucosal solution was replaced with MBP-free solution at a Vt of 0 mV.
Voltage reversal.
When Vt was
returned from 0 to 70 mV, the MBP-induced conductance partially
reversed; the Gt
decreased but did not return to the baseline value (Fig.
7). The reversal can be modeled as either a
series or a parallel arrangement of two conductive states leaving the
membrane. Data are shown fit by a double exponential equation based on
the parallel model
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(3) |
1,
Gs = 27 ± 6 µS/cm2, and
ks = 0.009 ± 0.004 s1
(n = 4).
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Removal of bath MBP.
The MBP-induced conductance also partially reversed at 0 mV when the
mucosal solution was replaced with MBP-free solution (Fig.
8). The time course of the reversal is
shown fit by a single exponential equation. For 360 nM MBP, the
best-fit value for the magnitude of the conductance change was 71 ± 24 µS/cm2 and for the rate
constant was 0.03 ± 0.02 s1
(n = 3). After the completion of the
wash, the voltage was returned to a
Vt value of
70 mV, and the conductance reversed further. The
voltage-dependent reversal after removal of MBP followed the form of a
double exponential equation. The fraction of the MBP-induced conductance that decreased with removal of bath MBP was not dependent on the amount of time that
Vt had been
clamped to 0 mV. For clamp times ranging from 5 to 130 min, the
fraction of the induced conductance that decreased (by removal of bath
MBP) was relatively constant at 0.36 ± 0.06 (n = 5).
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Effect of Divalent Cations on the Induced Conductance
Because divalent cations have been reported to inhibit or reverse the protein-induced conductance for other cationic proteins and peptides (37, 25), the effects of Ca2+ and Mg2+ on the MBP-induced conductance were examined. Both divalent cations had an inhibitory effect on the MBP-induced conductance, with Ca2+ exerting the more potent effect.Ca2+ reverses the MBP-induced
conductance. Adding Ca2+ to the
nominally Ca2+-free mucosal
solution decreased the MBP-induced conductance (Fig. 9). At a
Vt of 70
mV, 360 nM MBP was added to the mucosal solution and equilibrated, then
Vt was clamped to
0 mV. After a significant Gt change,
millimolar concentrations of CaCl2
were added to the mucosal solution, resulting in a decrease in the
MBP-induced conductance. The degree of reversal by
Ca2+ was dependent on the mucosal
Ca2+ concentration (Fig.
10). The decrease in the magnitude of the MBP-induced conductance was plotted as a function of the mucosal Ca2+ concentration. Data were fit
by the Hill equation. Best-fit values were as follows: the inhibition
constant was 0.4 mM and the Hill coefficient was 2.5 (3 tissues),
possibly suggesting that there are two or three binding sites for
Ca2+.
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To determine if Ca2+ decreased Gt by acting on the tight junctions or by decreasing a permeability pathway in the membrane other than the MBP-induced conductance, the relationships between the change in Gt and Isc for the MBP-induced conductance and the Ca2+-induced decrease in conductance were compared (Fig. 11). The plot of Gt vs. Isc for the MBP-induced conductance increase and the Ca2+-induced reversal followed along the same line, with no significant changes in either Gj or Ec, as shown in Eq. 1 (see MATERIALS AND METHODS). This suggests that Ca2+ is acting directly on the MBP-induced conductance rather than on the tight junctions or another permeability pathway in the membrane.
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Mg2+ slowed the conductance increase for MBP. In contrast to the effect of Ca2+ addition, when Mg2+ was added to the mucosal solution, there was an initial slight decrease in the MBP-induced conductance followed by a resumption of the MBP-induced increase in Gt (Fig. 12). The protocol for Mg2+ addition was the same as for Ca2+ addition described above; Mg2+ was added at a Vt value of 0 mV after MBP had been allowed to induce a significant conductance change.
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Both the initial decrease in the conductance with Mg2+ addition and the rate of the subsequent increase were dependent on the mucosal Mg2+ concentration (Fig. 13, A and B). The decrease in the total MBP-induced conductance by mucosal Mg2+ was determined as follows. To determine the total amount of the MBP-induced conductance, the magnitude of the initial MBP-induced increase in Gt (before the addition of Mg2+) was measured. The magnitude of each subsequent increase in Gt (after the addition of Mg2+) was added to this value. The total of all the increases in Gt was defined as the total MBP-induced increase. Next, the magnitude of the conductance that was decreased by the addition of Mg2+ was measured. The magnitude of the conductance that was reversed by Mg2+ was then subtracted from the total MBP-induced conductance to determine the residual MBP-induced conductance. This value was then normalized to the total MBP-induced conductance and plotted as a function of Mg2+ concentration (Fig. 13A). Data are shown fit by the Michaelis-Menten equation, which yielded an inhibition constant of 14 mM. This suggests that Mg2+ was much less potent than Ca2+ (which had an inhibition constant of 0.4 mM) in decreasing the MBP-induced conductance.
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The rate of the MBP-induced conductance increase, or
Gt/t,
was determined by fitting the increase in conductance by a linear equation and then plotted as a function of
Mg2+ concentration. As shown in
Fig. 13B,
Gt/t
diminished as the concentration of mucosal
Mg2+ increased. The data were fit
by the Hill equation; best-fit values were as follows: the
inhibition constant = 1.9 mM and the Hill coefficient = 1.8. The
Hill coefficient suggests the possibility of two binding sites for
Mg2+. It can be concluded from
these relationships that mucosal
Mg2+ both slowed and partially
decreased the MBP-induced conductance.
Loss of Epithelial Barrier Function
As described above, the MBP-induced conductance was only partially reversible after voltage clamping back to70 mV even after MBP
had been removed from the bath (see Fig. 8). In other words, some
Rt was
irreversibly lost. This irreversible loss of resistance (or increase in
tissue conductance) indicates a loss of barrier function of the
epithelium. The degree of increase in conductance was a function of
both the time the epithelium was exposed to MBP and the time that the
tissue was clamped to 0 mV (Fig. 14). Time 0 was the last point of the
incubation period at a
Vt value of
70 mV before clamping to a
Vt value of 0 mV.
Vt was clamped to
0 mV for at least 4 min to allow a significant MBP-induced conductance.
Next, the recovery of the tissue was measured by clamping the
epithelium back to 70 mV, removing MBP, and allowing the
Gt to reach a
steady state. This new baseline conductance was normalized to the
baseline conductance at time 0. To
determine if MBP displayed any voltage-independent toxic effects, MBP
was added to the mucosal solution,
Vt was held at
70 mV, and the Gt was monitored
over time. The time courses for the effect of MBP on
Gt at both 0 mV
(n = 5) and 70 mV
(n = 4) were fit by a single
exponential equation. For the voltage-dependent MBP-induced loss of
barrier function, the best-fit value for the rate constant was 0.009 min1, whereas the value for
the voltage-independent MBP effects was 0.001 min1. This indicates that
the loss of recovery from the voltage-dependent effects of MBP on the
Gt was nine times
faster than loss of
Gt due to any
possible voltage-independent effects of MBP. Therefore, the
voltage-dependent increase in conductance caused by MBP resulted in a
significant acceleration of the loss of tissue barrier function.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Properties of the MBP-Induced Conductance
The results suggest that MBP induces an increase in the conductance of the apical membrane in rabbit urinary bladder epithelium. The MBP-induced conductance was dependent on the MBP concentration in the mucosal solution and on the voltage gradient across the apical membrane, occurring only when the voltage gradient across the apical membrane was cell interior negative. The time course of the MBP-induced conductance suggests a multiple-step process that in some cases has a considerable lag period. The MBP-induced conductance was nonselective for cations and anions and was partially reversible using voltage, removal of MBP, or addition of divalent cations. MBP caused a voltage-dependent irreversible increase in the Gt, indicating a loss of barrier function of the epithelium.The reason for the delay in some time courses is unknown. The delay occurred in some specimens but not in others, suggesting that the delay was because of some unknown differences between individual bladders. One possibility is that individual differences in bladder composition may result in different responses. Although the membrane binding site for MBP has not been identified, an anionic membrane binding site, such as an anionic phospholipid (1), is likely. Perhaps MBP has a different affinity for different anionic phospholipids (or other binding sites). The presence or absence of a delay might be attributable to differences in the relative proportion of the different binding sites in individual bladders. Another possibility is that because MBP has been demonstrated to form aggregates (17), the composition of the bladder or the nature of the binding site in some bladders may accelerate or impede aggregation. For example, some phospholipids have been demonstrated to be more fluid than others, which might allow for more easy aggregation of MBP molecules within the membrane. If the membrane composition impeded aggregation of small conductive states into larger conductive states, this might explain the delay period in some tissues.
Significance
MBP disrupts cell membranes and is cytotoxic. Purified MBP induces cell blebbing and other changes associated with increased membrane permeability in fungi, parasites, and bacteria (for review, see Ref. 16). MBP has also been found to cause 51Cr release from labeled parasites and tumor cells (6). MBP is believed to cause tissue destruction in asthma because lung tissue in asthmatics contains degranulated eosinophils, measurable quantities of MBP have been detected in the sputa of asthmatics (13), and MBP is deposited on damaged tissues (10). MBP exerts a cytotoxic effect on respiratory epithelium (3, 12, 15, 26). The data presented here suggest that MBP causes an irreversible loss of barrier function in a mammalian urinary epithelium. Of interest is that the ability of MBP to disrupt barrier function of the urinary bladder is reduced by the presence of bath Ca2+ (this study). Because total Ca2+ concentration in rabbit urine can range from 0.8 to 3 mM (2), this suggests that MBP would cause a smaller increase in apical membrane permeability of the urinary bladder than reported in this study. Two factors make it difficult to determine the influence of Ca2+ on the effect of MBP on urinary bladder barrier function. First, because of the presence of organic anions such as oxalate (which sequesters Ca2+), the free urine Ca2+ concentration will be lower than the total Ca2+ concentrations. Second, eosinophils do not release MBP into the bath solution, but rather they make intimate contact with their target leading to irreversible adherence (5). Eosinophil granule proteins are released into the small pocket formed by the membrane of the eosinophil and the target cell (6, 14). Thus the concentration of Ca2+ in this restricted space might be lower than urine Ca2+, whereas the MBP concentration will be much higher. If MBP is toxic to urinary bladder epithelium, this raises the question of the impact of MBP in disease states of the urinary bladder that involve eosinophil degranulation. Such states include urinary parasitic infections (30), bladder tumors (27), and some cases of interstitial cystitis (11, 31, 41).Irreversible Effects of MBP
MBP induces an irreversible loss of barrier function in the rabbit urinary bladder epithelium as indicated by an irreversible increase in Gt. The degree of loss was related to the voltage-dependent effects of MBP and to the length of time that the tissue had been exposed. MBP also has numerous effects in addition to cytotoxicity. As examples, MBP has been demonstrated to increase Cl
secretion in dog tracheal epithelium (22) and to increase secretion of
prostaglandins in guinea pig tracheal epithelium (40). The results
presented in this paper do not exclude these effects as contributing to
the MBP-induced loss of epithelial barrier function. The conductive
effect of MBP may occur in tandem with the other effects or could
possibly explain them. For example, an influx of
Ca2+ through the MBP-induced
conductance could stimulate prostaglandin production. The cellular
effects of MBP might alter the membrane permeability and thus might
either potentiate or diminish the direct effects of MBP on membrane
permeability. An irreversible increase in membrane permeability can
disrupt a number of cellular processes. For example, the increased ion
permeability leads to an influx of ions and water and depolarization of
the cell membrane potential. If the rate of ion and water influx occurs
more quickly than the cell can regulate cell volume, cell swelling will
lead to lysis.
Comparison of MBP With Other Cationic Proteins
The conductive activity of MBP is similar to that reported for other cationic proteins. Histone, protamine sulfate, and polylysine have been found to increase apical membrane permeability in rabbit urinary bladder epithelium (25, 35, 36). Like MBP, the activity of these proteins occurred with a cell interior-negative voltage gradient across the apical membrane. The induced conductances were not ion selective and could be reversed by mucosal Ca2+. Mg2+ was inhibitory to protamine sulfate but not to histone. These similarities suggest that these proteins may be increasing apical membrane conductance in urinary bladder epithelium by a similar mechanism.Other than having a net positive charge, these proteins vary in
structure. Protamine is a random coil and contains high amounts of
arginine. Polylysine is also random coil but obviously contains no
arginine. Histones have either a globular or 
-helical central domain
flanked at either end by random coil tails. They contain both lysine
and arginine and vary in molecular mass from 11 to 25 kDa. In solution,
histones aggregate to form variously sized polymers. The secondary and
tertiary structure of MBP has not been well characterized. MBP is
arginine rich and has a molecular mass of 13.9 kDa (for a review, see
Ref. 16). MBP has also been demonstrated to form aggregates (17). It is
not known how these differently structured proteins can exert similar
effects on membrane permeability.
Some evidence points to an anionic phospholipid as the membrane-binding site. MBP interacted with vesicles formed of negatively charged phospholipids as indicated by alterations in the optical qualities of the vesicles (1). Whether MBP then forms a channel or activates a native membrane channel (either directly or via a second messenger system) remains unclear. Neutrophil defensins (23) and the frog secretory glandular protein Magainin-2 (7) have been found to create voltage-dependent channels in lipid bilayers. This suggests that cationic proteins increase membrane permeability by forming channels directly in the cell membrane via a phospholipid binding site.
Of the eosinophil proteins, eosinophil cationic protein, but not eosinophil peroxidase or eosinophil-derived neurotoxin, was found to form voltage-insensitive, nonselective channels in lipid bilayers (43). This author also reports that MBP did not form channels in preliminary experiments. More experiments are necessary to determine if MBP increases membrane permeability by forming channels in cell membranes.
In summary, this study indicates that MBP is able to increase apical membrane conductance in urinary bladder epithelium in a manner similar to that reported for other cationic proteins. This suggests that the mechanism of the cytotoxic effect of MBP on other tissues involves an increase in membrane permeability. Further studies characterizing the effect of MBP on epithelia are necessary to understand the potential pathophysiological role of MBP in eosinophil-associated epithelial disease.
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ACKNOWLEDGEMENTS |
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We thank James Checkel and David Loegering for preparing the MBP 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) as well as by a James W. McLaughlin Fellowship (to T. J. Kleine).
Address for reprint requests: S. A. Lewis, Dept. of Physiology and Biophysics, Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0641.
Received 23 October 1997; accepted in final form 25 March 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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). First, there was a small, rapid
jump in conductance when
Vt was clamped to
0 mV that was due to intrinsic membrane voltage sensitivity. This was
followed in some tissues by a delay and then a large, sigmoidal
increase in Gt.
In absence of both mucosal divalent cations and MBP, there was a much
smaller increase in
Gt when
Vt was clamped
from 
). Mucosal solution was a
Ca2+- and
Mg2+-free KCl Ringer solution;
serosal solution was a NaCl Ringer solution.
