Physiological and morphological effects of alendronate on
rabbit esophageal epithelium
A.
Dobrucali1,
N.
A.
Tobey1,
M. S.
Awayda2,
C.
Argote1,
S.
Abdulnour-Nakhoul1,
W.
Shao2, and
R. C.
Orlando1
1 Department of Medicine, Section of
Gastroenterology and Hepatology and 2 Department of
Physiology, Tulane University School of Medicine and Veterans
Affairs Medical Center, New Orleans, Louisiana 70112
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ABSTRACT |
Alendronate, an aminobisphosphonate,
produces as a side effect a topical (pill induced) esophagitis. To gain
insight into this phenomenon, we assessed the effects of luminal
alendronate on both esophageal epithelial structure and function.
Sections of rabbit esophageal epithelium were exposed to luminal
alendronate at neutral or acidic pH while mounted in Ussing chambers to
monitor transmural electrical potential difference (PD), short-circuit current (Isc), and resistance (R).
Morphological changes were sought by light microscopy in hematoxylin
and eosin-stained sections. Impedance analysis was used for
localization of alendronate-induced effects on ion transport. Luminal,
but not serosal, alendronate (pH 6.9-7.2), increased PD and
Isc in a dose- and time-dependent manner, with
little change in R and mild edema of surface cell layers.
The changes in Isc (and PD) were reversible with
drug washout and could be prevented either by inhibition of Na,K-ATPase activity with serosal ouabain or by inhibition of apical Na channels with luminal acidification to pH 2.0 with HCl. An effect on apical Na
channel activity was also supported by impedance analysis. Luminal
alendronate at acidic pH was more damaging than either alendronate at
neutral pH or acidic pH alone. These data suggest that alendronate
stimulates net ion (Na) transport in esophageal epithelium by
increasing apical membrane sodium channel activity and that this occurs
with limited morphological change and no alteration in barrier
function. Also alendronate is far more damaging at acidic than at
neutral pH, suggesting its association with esophagitis requires
gastric acid for expression. This expression may occur either by
potentiation between the damaging effects of (refluxed) gastric acid
and drug or by acid-induced conversion of the drug to a more toxic form.
biphosphonates; esophagitis; transepithelial ion transport; Ussing
chamber; sodium channels
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INTRODUCTION |
ALENDRONATE, AN
AMINOBISPHOSPHONATE, is a potent inhibitor of osteoclast-mediated
bone resorption, which makes this an effective agent for the treatment
of osteoporosis (3, 7, 10, 13). Although generally well
tolerated, an early important side effect of taking alendronate 10 mg
once a day (alendronate is now also available in 70-mg tablets for
treatment once weekly) was the development of erosive esophagitis
(1, 2, 9, 12, 14, 19). Indeed, in some cases, the
esophagitis was severe enough to require hospitalization due to
epithelial ulceration and healing with stricture formation (5, 9,
11, 12). On the basis of clinical history (patients ingesting
medication with little liquid or reclining shortly after taking the
medication) and endoscopic observations of the pattern of damage,
initial reports concluded that alendronate damaged the esophagus by
direct (luminal) contact with the esophageal epithelium, resulting in a
form of "pill-induced" esophagitis. To gain insight into this
phenomenon, therefore, we assessed the effects of luminal alendronate
on both esophageal epithelial structure and function and did this at
both neutral and acidic pH, the latter to take into account the
potential in humans for refluxed gastric acid to alter the potential
for alendronate toxicity. The stratified squamous epithelium from the
rabbit esophagus was chosen for these studies because it is
1) structurally and functionally similar to that of the
human esophageal epithelium, 2) well characterized in terms
of its transport and barrier physiology, and 3) devoid of
submucosal glands and is ideally suited for investigation of squamous
epithelial structure/function alone when mounted in Ussing chambers
(18, 20, 22). Also noteworthy is that the concentration
range of alendronate selected for study, 1-10 mg/ml, was by
necessity empiric because pill-induced esophagitis principally occurs
because of adherence of ingested tablets to the epithelial surface, an
occurrence that would create, as the tablets dissolve, high
concentrations of chemical in a localized area of epithelium.
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METHODS |
Male New Zealand White rabbits weighing 8-9 lb were killed
by administration of an intravenous overdose of pentobarbital sodium (60 mg/ml). The esophagus was dissected free, opened lengthwise, and
pinned mucosal surface down in a paraffin tray containing ice-cold
oxygenated normal Ringer solution. The submucosa and muscularis propria
were grasped with hemostats, lifted up, and dissected free of the
underlying mucosa with a scalpel. This process left attached to the
paraffin tray a sheet of tissue consisting of stratified squamous
epithelium and a small amount of underlying connective tissue. From
this tissue, four sections were cut and mounted as flat sheets between
Lucite half-chambers with an aperture of 1.13 cm2, which
permitted contact with separate bathing solutions for the luminal and
serosal sides of the tissue.
Tissues mounted in chambers were bathed initially with normal Ringer
solution composed of the following (in mM): 140 Na+, 119.8 Cl
, 5.2 K+, 25 HCO3
, 1.2 Ca2+, 1.2 Mg2+, 2.4 HPO4
2, 0.4 H2PO4
2, with osmolality 268 mosmol/kgH2O and pH 7.4 when gassed with 95% O2-5%
CO2 at 37°C. Luminal and serosal solutions
were connected to calomel and Ag-AgCl electrodes by Ringer agar bridges
for measurements of potential difference (PD) and short-circuit current
(Isc) with a voltage clamp (World Precision
Instruments, Sarasota, FL). Tissues were continuously short circuited,
except for 5-10 s when the open circuit PD was read. Tissue
electrical resistance (R), a marker of epithelial
permeability, was calculated from the PD and Isc
using Ohm's law (R = PD/Isc).
Tissues were equilibrated in Ringer for 45 min before examination to
allow stabilization of electrical parameters. After equilibration,
tissues having R > 1,000
/cm2 were
paired by R (R within 25%) for comparative
studies between those treated with alendronate and untreated controls.
Alendronate solutions (1-10 mg/ml) were prepared by pulverizing to
a powder with mortar and pestle commercially available 10-mg tablets of
alendronate (Fosamax; Merck, West Point, PA). The powder was dissolved
in 10 ml of normal Ringer solution, yielding solutions whose pH ranged
from pH 6.9 to 7.2. For alendronate exposure, after equilibration in 10 ml normal Ringer solution, the luminal bathing solution was drained and
substituted for 10 ml normal Ringer containing the desired
concentration of alendronate. The control was handled in a similar
manner, except that the luminal solution was replaced with normal
Ringer solution, which was titrated with 0.1 N HCl, when necessary, to
match the pH of the alendronate solution. After initial titration with
HCl, pH of all solutions remained constant. In some experiments, the
effect of pretreatment with alendronate on the ability of the
esophageal epithelium to resist injury by acid was tested by
acidification of the luminal bath to pH 2.0 with 3 N HCl. Injury was
assessed by monitoring R and by fixing tissues with 10%
formaldehyde for histology. Tissue sections were cut and subsequently
stained with hematoxylin and eosin for evaluation by light microscopy.
Morphological injury, which was evaluated by an observer with no
knowledge of treatment groups was scored as follows: 0, normal; 1, inter/intracellular edema; 2, patchy necrosis; 3, diffuse necrosis; 4, transmural necrosis (ulceration).
Impedance analysis.
This was carried out as previously described by Van Driessche et al.
(23). Briefly, tissues were mounted in modified Ussing chambers and clamped to 0 mV using a low noise four-electrode clamp
with agar bridges connected by low-resistance Ag/AgCl electrodes. Impedance was continuously measured at five frequencies: 128 Hz, 343 Hz, 1.0 kHz, 4.1 kHz, and 16.4 kHz. The direct current (DC) conductance
(gm) was measured using a low-frequency (0.25 Hz) signal, and this value was used to calculate the capacitance
(Cm) at each of the individual frequencies.
Every 10 min an impedance spectrum was measured at 99 frequencies
ranging from 1 Hz to 16 kHz.
Junction potentials were measured for all solutions according to a
method modified following that of Read and Fordtran (15). Inasmuch as these values were found to be equal or less than +1.1 mV,
no correction was made for them in the presentation of results.
All chemicals were obtained from Sigma (St. Louis, MO), except where
otherwise indicated. Ouabain, 10
4 M, was prepared by
dissolving in a small volume of DMSO.
Statistics.
Statistical significance was determined using Student's
t-test for parametric data (electrical parameters), the
Mann-Whitney U test for nonparametric data (morphological
injury scores), and the regression coefficient analysis for
dose-response studies. All data were reported as the means ± SE,
and P < 0.05 was accepted as indicating a
statistically significant difference among groups.
All studies were approved by the Animal Welfare Committee.
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RESULTS |
To determine if alendronate had any effect on the barrier or ion
transport properties of esophageal epithelium, rabbit esophageal epithelium was initially exposed to a high concentration (10 mg/ml) of
alendronate in normal Ringer solution while mounted in Ussing chambers
for 1 h. The results, as shown in Fig.
1, illustrate that acute exposure to
alendronate increases PD and Isc with time while
having little effect on R. The PD increased by 54 ± 6% and Isc increased by 64 ± 7% at
1 h vs. the control, which PD decreased by 10 ± 3% and
Isc decreased by 15 ± 2% at 1 h
(n = 4/group, P < 0.01). This increase
translates into an increase of 5 ± 0.2 mV and 5.5 ± 2 µAmps/cm2 in alendronate-treated tissues. Moreover, this
effect was reversible because removal of alendronate by replacement of
the luminal bath with normal Ringer solution results in complete
reversibility by 45 min. At the end of the washout period (1 h
post-alendronate removal), tissues were fixed and examined
morphologically. The histology of alendronate-exposed tissues showed
minimal changes with some membrane "wrinkling" and mild edema of
the surface cell layers when compared with normal Ringer-exposed
controls (Fig. 2, A and
B), and this was reflected in
scores for alendronate-treated tissues of 1.2 ± 0.5 vs.
Ringer-exposed controls of 0.3 ± 0.2 (n = 4/group, P < 0.01).

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Fig. 1.
Effect of luminal alendronate, 10 mg/ml, on the potential
difference (PD; A), short-circuit current
(Isc; B), and electrical resistance
(R; C) of rabbit esophageal epithelium mounted in
Ussing chamber in normal Ringer solution, pH 6.9, for 1 h. Control
tissues are exposed to normal Ringer at pH 6.9. At 60 min after
exposure to alendronate, the luminal bathing solutions were removed for
both alendronate-treated and control tissues and replaced with normal
Ringer solution (washout). Data are expressed as percentage of initial
values before alendronate exposure. Values are means ± SE;
n = 4 for each group; *P < 0.05 compared with controls. Note: absolute values for PD,
Isc, and R for each group were
similar before treatment and were 12.8 ± 1.4 mV and 17.4 ± 1.4 mV, 10.5 ± 2.6 µAmp/cm2 and 11 ± 1.2 µAmp/cm2, and 1,649 ± 227 /cm2 and
1,869 ± 234 /cm2 for alendronate and controls,
respectively.
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Fig. 2.
Representative light micrographs of rabbit esophageal epithelium
exposed to normal Ringer solution, pH 6.9 (A), or
alendronate, 10 mg/ml, in normal Ringer solution, pH 6.9 (B), for 1 h. Note that the alendronate-exposed tissues
exhibit minor changes in the surface layers of epithelium with
some "wrinkling" of the membranes and mild edema.
Hematoxylin and eosin, ×150.
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Notably, alendronate exists commercially as a sodium salt, and when
dissolved in normal Ringer solution, this salt liberates Na ions such
that it increased the luminal Na concentration by 20 meq/l. To
determine whether the effect of alendronate observed above was the
result of the higher Na concentration, tissues were exposed to normal
Ringer solution or to normal Ringer solution to which 20 meq/l of Na
was added in the form of Na isothionate. The results of this experiment
establish that the additional Na in the luminal bath has no effect on
the electrical parameters (PD, Isc,
R) of the tissue as illustrated for the
Isc in Fig. 3.
This suggests that the stimulation of ion transport in
alendronate-exposed tissues was a direct effect of the agent on the
epithelium. Moreover, and suggesting that the effect of alendronate is
a drug class effect, luminal exposure to risedronate, 10 mg/ml, also
resulted in stimulation of the Isc; however, the
magnitude of the current rise, 17 ± 4.6% (n = 4), above basal levels was significantly lower than that for
alendronate, 64 ± 7% (n = 4, P < 0.05).

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Fig. 3.
Effect on the Isc of rabbit
esophageal epithelium mounted in Ussing chambers of luminal addition of
20 meq/l of Na in the form of Na isothionate to normal Ringer solution,
pH 6.9. Controls contain normal Ringer solution, pH 6.9, alone. Values
are means ± SE; n = 4 for each group. Note that
the Isc of esophageal epithelium was unaffected
by the additional Na in the luminal bath. (Note: absolute values
for Isc before treatment were 12.6 ± 0.8 and 12 ± 1.5 µAmp/cm2 for Na isothionate-added
Ringer and regular Ringer, respectively.)
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The dose at which alendronate exerted its effects on PD and
Isc was subsequently evaluated by exposing
rabbit esophageal epithelium in Ussing chambers to varying
concentrations of alendronate for 1 h. In Fig.
4, the results of these experiments are
depicted as a plot of the concentration of alendronate vs. percent
change in PD, Isc, and R at 1 h.
Note the strong linear relationship for the increase in PD and
Isc over the entire dose range up to 10 mg/ml
with R2 > 0.93 for each parameter.

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Fig. 4.
A strong linear relationship is shown between the dose of
luminal alendronate and its effects on the PD (A) and
Isc (B) of rabbit esophageal
epithelium mounted in Ussing chambers. There is no effect of varying
dose on electrical R (C). The values plotted
represent those taken after 1 h of exposure to the agent.
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To address the possible mechanism by which alendronate stimulates
Isc (and so PD) across the esophageal
epithelium, tissues were pretreated with serosal ouabain,
10
4 M, for 30 min to block active transport before
exposure to luminal alendronate, 10 mg/ml. As shown in Fig.
5, ouabain progressively reduced PD and
Isc with time such that by 30 min both
parameters were significantly lower than untreated normal
Ringer-exposed controls. When alendronate was then added to the luminal
bath, PD and Isc increased in the untreated
tissues, whereas treatment of the tissues with ouabain prevented the
increase in both Isc and PD. This suggests that
alendronate's ability to increase PD and Isc
was a direct effect on net (transcellular) ion transport rather than an
effect on the paracellular pathway.

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Fig. 5.
Effect of pretreatment with serosal ouabain,
10 4 M, on the ability of luminal alendronate, 10 mg/ml,
to increase the PD (A) and Isc
(B) of rabbit esophageal epithelium mounted in Ussing
chambers in normal Ringer solution, pH 6.9, for 1 h. Alendronate
treatment was initiated at 30 min after ouabain treatment. Control
tissues were exposed to luminal alendronate only at 30 min. Note that
pretreatment with ouabain abolished the increase in PD and
Isc associated with the addition of alendronate
to the luminal bath. Data are expressed as percentage of initial
values. Values are means ± SE; n = 4 for each
group; *P < 0.05 compared with controls. (Note:
absolute PD and Isc values before treatment were
15.9 ± 4.4 and 19.3 ± 4.7 mV and 13.6 ± 2.8 and
17.6 ± 4.3 µAmp/cm2 for tissue pretreated with
ouabain and control, respectively.)
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To better understand the site of action of alendronate, tissues were
also exposed to alendronate serosally in doses of 10, 5, and 3 mg/ml.
Unlike luminal alendronate, however, the results revealed a prompt
dose-dependent decline in Isc (and PD). For 10 mg/ml, Isc declined by 38%; for 5 mg/ml, by
11%; and for 3 mg/ml, by 13% within 15 min, and these reductions were
completely reversible inasmuch as replacement of the alendronate
solution with normal Ringer resulted in return of the
Isc (and PD) to baseline values within 15 min
(data not shown). (Note: serosal pH 6.9 in the absence of alendronate
has no effect on current.) Because these results suggest that
alendronate's luminal action was not related to absorption and action
on the basolateral membrane, attention was focused on alendronate's
effect on apical membrane sodium channels in this actively
sodium-transporting tissue. This was done by first exposing the
esophageal epithelium to alendronate, 10 mg/ml, to produce an increase
in Isc and PD, and then titrating the luminal
bath with 3 N HCl to reach a pH of 2.0. The luminal bath was acidified
to pH 2.0 as a means of inhibiting Na transport through apical membrane
Na channels (see DISCUSSION and Refs. 18 and
21). As illustrated in Fig. 6, luminal
acidification almost completely abolished the alendronate-induced
increase in PD and Isc. [Note: in the absence
of acidification, alendronate-treated tissues exhibit continued
elevations in Isc and PD over baseline and these
elevations continue to slowly increase over the hour of these
experiments. Also luminal acidification to pH 2.0 alone has no
significant effect on PD, Isc, or R
or on tissue histology in esophageal epithelium. This is evident by
comparison of luminal acidification to pH 2.0 for 1 h (60- to
120-min time period in Ussing chamber) in Fig. 6 with that of
nonacidified controls exposed to normal Ringer solution for 1 h
(over the same 60- to 120-min time period) in Fig. 1]. Further luminal
acidification to pH 2.0 produced a similar inhibitory effect on
Isc (and PD; data not shown) over the entire
dose range up to 10 mg/ml (Fig. 7).
Additional evidence to support the increase in PD and
Isc by alendronate was due to stimulation of
active Na transport; tissues were exposed to alendronate, 10 mg/ml, for
1 h in Cl-free bathing solution (both luminal and serosal). (Note:
Na-free experiments cannot be done technically because alendronate
releases Na from its salt.) Similar to alendronate in Cl-containing
Ringer solution, alendronate in Cl-free solution produced similar
increases in Isc (61 ± 10% over controls)
and PD (46 ± 1.6% over controls) (n = 3).

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Fig. 6.
Effect of luminal acidification with HCl, pH 2.0, on the
increase in PD (A) and Isc
(B) produced by luminal alendronate, 10 mg/ml, in rabbit
esophageal epithelium mounted in Ussing chambers in normal Ringer
solution, pH 6.9, for 1 h. Luminal acidification was initiated at
60 min after alendronate treatment. Control tissues were only exposed
to normal Ringer solution before luminal acidification at 60 min. Note
that luminal acidification at pH 2.0 has little if any effect on PD or
Isc in controls (see Fig. 1 for comparison to
normal Ringer alone from 60 to 120 min) but substantially reduces the
increase in PD and Isc produced by exposure to
alendronate. Data are expressed as percentage of initial values. Values
are means ± SE; n = 7 for each group;
*P < 0.05 compared with controls. (Note: absolute PD
and Isc values for alendronate and control group
before treatment were 12.1 ± 0.9 and 10.1 ± 0.6 mV and
13.5 ± 1.8 and 12.4 ± 0.6 µAmp/cm2,
respectively.)
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Fig. 7.
Effect of luminal acidification with HCl, pH 2.0, on the
increase in PD and Isc produced by varying doses
of luminal alendronate, 3-10 mg/ml, in rabbit esophageal
epithelium mounted in Ussing chambers in normal Ringer solution for
1 h. Luminal acidification was initiated at 60 min after
alendronate treatment. Control tissues were only exposed to normal
Ringer solution before luminal acidification at 60 min. Note that
luminal acidification at pH 2.0 has little if any effect on PD or
Isc in controls (see Fig. 1 for comparison to normal
Ringer alone from 60 to 120 min) but reduces the increase in PD and
Isc produced by exposure to all doses of
alendronate. Data are expressed as percentage of initial values. Values
are means ± SE; n = 5-7 for each group;
*P < 0.05 compared with controls. (Note: absolute
values for Isc before treatment and at 60 min
were 10.8 ± 1.4. and 10 ± 1.6 µAmp/cm2 for
control; 10.6 ± 1 and 12.4 ± 1.3 µAmp/cm2 for
alendronate, 3 mg/ml; 9 ± 1.1 and 12.2 ± 1.4 µAmp/cm2 for alendronate, 5 mg/ml; 8.2 ± 0.8 and
10.8 ± 0.8 µAmp/cm2 for alendronate, 7 mg/ml; and
13.5 ± 1.8 and 22 ± 2.8 µAmp/cm2 for
alendronate, 10 mg/ml.)
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To determine the effects of alendronate on transepithelial properties
we used impedance analysis. As shown in Fig.
8, alendronate, 0.67 mg/ml, stimulated
both conductance and the Isc. This stimulation was reversible, and both parameters returned to control after washout
of alendronate. The increase of conductance was in the range of 10%
and was smaller than the ~45% increase of
Isc. As the transepithelial conductance of
rabbit esophageal epithelia is dominated by the paracellular pathway
(20), the smaller change of conductance vs. current rules
out appreciable changes of the paracellular pathway and indicates that
the likely target of alendronate is the cellular apical and basolateral
membrane resistance. This is also consistent with the lack of
detectable changes of the high-frequency capacitance (see Ref.
23 and Fig. 8). To demonstrate the effects of low-dose
alendronate on the apical membrane, impedance spectra were also
collected (Fig. 9). Under these
conditions, the time constant of the apical and basolateral membranes
were sufficiently different from each other to permit resolution of the
relative contributions of each membrane to the transepithelial impedance. In these experiments, a small decrease of transepithelial resistance with alendronate (leftward shift of the impedance spectrum) was noted. Although the absolute value of the apical and basolateral resistances cannot be determined without a priori knowledge of the
resistance of the paracellular pathway, the relative contribution of
each of the membrane resistances could be assessed. Therefore, the
decrease of impedance was found to be predominantly due to a decrease
of apical membrane impedance and more specifically its DC resistance
(Fig. 9, left). These data support the concept that the
changes of Isc are mediated via effects on the
apical membrane.

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Fig. 8.
Time course of the effects of alendronate on transepithelial
impedance. After a 90-min control period, alendronate was added to the
apical side at 0.67 mg/ml. This caused a stimulation of both membrane
conductance (gm) and short-circuit current
(Im). The values of the capacitance
(Cm) calculated at low frequencies is highly
dependent on the direct current resistance. Therefore changes at 128 Hz
and other low frequencies represent changes of resistance rather than
changes of capacitance and therefore membrane area. At high frequency,
the equivalent capacitance is the sum of both membrane capacitances and
is highly affected by the resistance of the lateral intercellular
spaces (23). As the capacitance at 4.1 and 16.4 kHz is
essentially unaffected by alendronate, we interpret this finding in
support of appreciable effects on the paracellular pathway
(n = 5).
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Fig. 9.
Nyquist plot of the transepithelial impedance in rabbit esophageal
epithelia and the effects of alendronate. The real and imaginary
components of the impedance (ZT) were calculated and
plotted in the Nyquist format. In this representation, a biological
membrane consisting of a resistor parallel with a capacitor yields a
semi-circle and an epithelium consisting of 2 series membranes results
in 2 semi-circles. Owing to lower capacitance, the apical membrane
impedance is dominant at high frequency (left), whereas the
basolateral membrane is dominant at low frequencies. As seen in this
figure, the decrease of resistance (0 frequency extrapolation) is
predominantly due to a decrease of the apical (high frequency)
semi-circle, providing evidence for effects of alendronate on the
apical membrane and presumably the apical Na conductive pathway
(n = 5).
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To determine the effects of a high dose of alendronate on tissue
impedance, tissues were exposed to 10 mg/ml of alendronate. As shown in
Fig. 10, luminal alendronate caused a
marked stimulation of the Isc. This stimulation
was similar in magnitude to that observed with 0.67 mg/ml, indicating
potential saturation of the response of Na transport. The changes of
conductance were also similar with the high dose of alendronate.
Moreover, addition of basolateral alendronate, as shown in Fig. 10,
reversed the effects on transport but further potentiated the
stimulation of conductance. These effects indicate the potential for a
complex response at the higher doses, which may involve separate
effects on both apical and basolateral membranes.

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Fig. 10.
Time course of a high dose of alendronate (10 mg/ml). Apical (Ap)
addition of alendronate increases both gm and
Im. The changes of current were, however, larger
than those of conductance. Addition of alendronate to the basolateral
solution (BL) caused a small further increase of conductance. However,
the stimulation observed with apical addition was reversed and the
Im returned to control prealendronate levels.
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To address the effects of high dose alendronate on both membranes,
impedance spectral analysis was also carried out. As shown in Fig.
11, alendronate caused a large increase
of apical resistance (Ra) and a compensatory
decrease of basolateral resistance (Rbl), such
that the total resistance was decreased as observed in Fig. 10. The
increase of Ra was initially (within the first
10 min) preceded by a small decrease as observed with the lower doses of alendronate (data not shown). Furthermore, addition of sequential intermediate doses of alendronate (2, 4, 6 mg/ml) indicated that the
sustained decrease of Ra with 0.67 mg/ml was
also observed at 2 mg/ml but not at the higher doses (data not shown).
These data are consistent with the interpretation that low doses of alendronate, up to 2 mg/ml, stimulate the apical conductance, whereas
higher doses block it. This is, however, not reflected as inhibition of
transport with high dose luminal alendronate, as these changes are
accompanied by stimulation of basolateral conductance. This decrease in
Rbl is likely due to increased basolateral K
conductance, which in turn increases the electrical driving force for
apical Na entry into the cells. Notably, addition of basolateral
alendronate inhibits the Isc despite large
stimulation of the K channels. This is due, as shown in Fig. 11, to
severe inhibition of apical (Na channel) conductance.

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Fig. 11.
Nyquist plot of high dose alendronate, 10 mg/ml, on both apical
and basolateral membranes. Impedance (ZT) spectra showed
that the addition of luminal alendronate caused a small initial
decrease of apical resistance similar to that observed with 0.67 mg/ml
alendronate (Figs. 8 and 9). However, this was followed by an increase
of apical resistance (see data at 30 and 60 min). The overall
resistance was decreased as shown in Fig. 10, owing to the additional
effects on the basolateral membrane resulting in a large decrease of
its resistance. These data indicate that at higher doses alendronate
affects both the apical and basolateral membranes.
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In another set of experiments, the morphological effects of luminal
alendronate, 10 mg/ml, for 1 h were compared at acidic (HCl, pH 2)
and neutral pH (Ringer, pH 6.9) in Ussing chamber-mounted tissue
sections from the same rabbit. Alendronate at neutral pH again, as
noted in Fig. 2, produced only minor changes in histology. Similarly,
luminal acidity, pH 2, had little effect on esophageal morphology. In
contrast, luminal alendronate at pH 2 resulted in significant injury as
evidenced by the presence of diffuse edema and cell necrosis (Figs.
12 and
13, A and B).
In addition, pretreatment with ouabain for 30 min to inhibit active Na
transport before exposure to alendronate, 10 mg/ml, plus HCl, pH 2.0, for 1 h failed to alter the ability of this combination to produce morphological injury (morphological injury score was 2.16 ± 0.3, n = 3).

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Fig. 12.
Difference was significant between the morphological
scores of the tissues treated for 1 h with luminal acidification,
HCl pH 2.0, alone (n = 7), or luminal acidification
plus alendronate, 10 mg/ml (n = 7) (morphological
scores were 0.6 ± 0.3 and 2.3 ± 0.5, respectively,
P < 0.05). The addition of alendronate to the acidic
bathing solution produces significantly greater damage than
acidification alone. Also we detected that the tissues exposed to
alendronate at pH 6.9 (n = 4) were significantly more
damaged than tissues exposed to Ringer alone at pH 2 (morphological
scores were 1.2 ± 0.5 and 0.6 ± 0.3, respectively,
P < 0.05). There was no significant difference between
morphological scores of tissues exposed luminally to Ringer at pH 6.9 (n = 4) and pH 2 (morphological scores were 0.4 ± 0.2 and 0.6 ± 0.3, respectively, P > 0.05).
*P < 0.05; §P > 0.05.
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Fig. 13.
Representative light micrographs of rabbit esophageal epithelium
exposed for 1 h to HCl-acidified Ringer solution, pH 2.0 (A), or to HCl-acidified Ringer solution containing
alendronate, 10 mg/ml (B). Tissues exposed to HCl, pH 2.0, show no evidence of significant morphological damage (see Fig. 2 for
comparison to normal Ringer exposure), whereas tissues exposed to acid
plus alendronate show both diffuse edema and cell necrosis. Hematoxylin
and eosin, ×150.
|
|
 |
DISCUSSION |
In the present study we observed that luminal alendronate at
neutral pH has little effect on the morphology or barrier function of
the esophageal epithelium. However, interestingly, it produced a
dose-dependent stimulatory effect on active ion transport (increase in
Isc), active ion transport in rabbit esophageal
epithelium being principally the result of active Na absorption (Na
absorption is ~85% of Isc; Ref.
16). Support for an effect of alendronate on Na absorption
is provided in our studies by inhibition of this stimulatory effect on
Isc by ouabain, an Na,K-ATPase inhibitor, or by
luminal acidification, the latter a maneuver previously shown by our
lab to inhibit apical membrane Na channels in this tissue (16,
21) and by the fact that alendronate produced similar increases
in Isc and PD in Cl-free Ringer solution.
Furthermore, because serosal alendronate did not increase
Isc (actually decreased Isc), the action of luminal alendronate was
unlikely to be mediated by its absorption and action on the squamous
cell basolateral membranes. Additional support for the conclusion that
alendronate stimulates active transport in esophageal epithelium by an
action on the apical membrane was obtained using impedance analysis. In
these experiments, luminal alendronate, up to 2 mg/ml, reversibly stimulated both Isc and conductance, with the
effect on Isc being much greater than that on
conductance. Because conductance is dominated by the paracellular
pathway in rabbit epithelium (20), this difference coupled
with a lack of change in capacitance at high frequency (Fig. 8)
mitigates against an appreciable change in paracellular permeability
and in favor of a change in membrane permeability (23).
Furthermore, based on time constants, the impedance spectra indicated
that the primary effect of alendronate was on the apical membrane.
These data, together, suggest that alendronate at low dose stimulates
active (Na) transport in esophageal epithelium by an effect on the
apical membrane Na channels. It remains unclear, however, whether this
effect is a direct effect on the Na channel itself or mediated
indirectly by changing apical membrane fluidity (4, 6). At
higher doses, 4-10 mg/ml, similar to the lower dose, alendronate
stimulated Isc. However, this was only briefly
associated with a decrease in apical resistance because the higher
doses then rapidly increased apical resistance. Nonetheless, Isc increased at the higher dose because of
coincident increases in basolateral conductance, as shown by the
decrease in Rbl in Fig. 10. This is most likely
a reflection of stimulation of basolateral K conductance, which
hyperpolarizes the cells and increases the apical driving force for Na
entry. The only previous reports of an effect of a bisphosphonate on
ion transport have been in osteoclasts. In these cells it was shown
that alendronate and tiludronate can inhibit Na-independent
H+ extrusion that occurs via a vacuolar
H+-ATPase (8, 24).
Although luminal alendronate at neutral pH had a significant effect on
esophageal epithelial ion transport, it had minimal effects even at
high concentration on esophageal morphology. This argues against the
fact that a topical action by the drug as ingested (at neutral pH) was
alone responsible for pill-induced esophagitis. A similar conclusion
was reached by Peter and colleagues (17) based on
experiments perfusing alendronate (0.2 mg/ml) in vivo into the
esophagus of dogs for 5 days. Moreover, they observed that unlike
neutral perfusions with alendronate, esophageal perfusion with
alendronate at acidic pH (pH 2) resulted in significant esophageal pathology as characterized by marked ulcerative esophagitis. Our findings are consistent with these observations in that luminal alendronate for 1 h at an acidic pH (pH 2) that was itself
nondamaging resulted in significant edema and cell necrosis. This
indicates that alendronate's potential for esophageal injury is
expressed on exposure to gastric acid. Furthermore, this injury appears to be unrelated to the ability of alendronate to stimulate active Na
transport because acid pH 2.0 is known to inhibit Na absorption via the
apical membrane Na channel in esophageal epithelium and prior treatment
with ouabain to inhibit active Na transport failed to alter the extent
of tissue injury induced by alendronate plus acid. In humans in vivo
this might occur with adherence of a pill containing alendronate to the
esophageal epithelium as a result of patients ingesting medication with
little liquid or reclining shortly after taking the medication. Damage
then occurs when the pill-containing esophagus is subsequently bathed
by gastric acid through either physiological, or in some patients with
coincident gastroesophageal reflux disease, pathologic reflux.
Alternatively, alendronate tablets dissolving in acidic gastric juice
in the stomach could, through physiological or pathologic reflux,
reenter and damage the esophagus at acid pH. [Note: This latter
possibility seems less likely because renewed attention on the proper
means for taking the medication (follow medication with full glass of water and remain upright for at least one-half hour) appears to have
greatly diminished the frequency of this clinical problem (personal
observations).] The mechanism for the toxicity of alendronate under
these conditions is unclear but Peter and colleagues showed that it was
not specific to aminobisphosphonates such as alendronate or risedronate
because similar injury in their model could be produced by perfusion of
a non-aminobisphosphonate (tiludronate) at acid pH. Moreover, they
suggested that the damage by alendronate may be due to conversion of
the medication at acid pH to a more toxic form (17).
Indeed alendronate exists as a monosodium salt at pH >3.5, whereas at
pH <3.5 it is primarily in a free acid form that is known to be more
irritating to mucosa (1, 17). The development of
pill-induced esophagitis could also in some circumstances result from
the potentiation between alendronate and acid such that two mildly
injurious processes synergize to produce severe esophagitis.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Veterans Affairs merit grant and an
National Institute of Diabetes and Digestive and Kidney Diseases Grant
DK-36013. Dr. Dobrucali is supported by the Akdamar Fellowship Program
in Gastroenterology.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. Dobrucali, Istanbul Universitesi, Cerrahpasa Tip Fakultesi,
Gastroenteroloji Bilim Dali, 34300 Cerrahpasa/Istanbul, Turkey (E-mail:
adobrucali{at}yahoo.com).
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. Section 1734 solely to indicate this fact.
April 24, 2002;10.1152/ajpgi.00014.2002
Received 14 January 2002; accepted in final form 16 April 2002.
 |
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