1 Children's Hospital Oakland Research Institute, Oakland 94609; and 2 Cardiovascular Research Institute, University of California, San Francisco, California 94143
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ABSTRACT |
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This study was designed to test
the in vivo efficacy of the chemical chaperone trimethylamine oxide
(TMAO) in correcting the Cl transport defect in a mouse
model of cystic fibrosis (CF). Rectal potential difference (RPD)
measurements were done in matched wild-type and
F508 CF
mice. Mice were treated by subcutaneous injections of TMAO. Wild-type
mice demonstrated a forskolin-stimulated, Cl
-dependent
hyperpolarization of
6.4 ± 0.8 mV (n = 11),
which was significantly increased to
13.1 ± 1.4 mV after
treatment with TMAO.
F508 CF mice showed no significant responses to
forskolin. Treatment with TMAO recovered a forskolin-activated RPD in
F508 CF mice (
1.1 ± 0.2 mV; n = 17) but not
in CFTR null mice. The effects of TMAO were dose dependent, resulting
in a slope of
0.4 ± 0.1 mV · g
1 · kg
1 in
F508 CF
mice. The forskolin-stimulated RPD in TMAO-treated
F508 CF mice was
partially blocked by glibenclamide and further stimulated by apigenin.
The total response to forskolin plus apigenin was
2.5 ± 0.45 mV
(n = 6 mice), corresponding to 39% of the response evoked by forskolin only in wild-type mice.
apigenin; F508 cystic fibrosis transmembrane conductance
regulator; cystic fibrosis transmembrane conductance regulator
knockout; glibenclamide; rectum; epithelia
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INTRODUCTION |
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CYSTIC
FIBROSIS (CF) is an autosomal recessively inherited disease
caused by a mutation of the CF transmembrane conductance regulator
(CFTR) gene, which encodes for the cAMP-regulated Cl
channel in the apical membranes of epithelial cells. Numerous CF-causing mutations of CFTR have been described. The most common mutation is a deletion of phenylalanine at position 508 of CFTR (
F508 CFTR) that is present on 66% of CF chromosomes; 88% of CF
patients carry at least one
F508 allele (32). This
mutation leads to a misfolded protein that is retained in the
endoplasmic reticulum and is targeted for rapid degradation (5,
14, 37). Epithelial cells homozygous for the
F508 mutation
have an extremely reduced plasma membrane Cl
conductance
(25, 29, 35). When
F508 CFTR was overexpressed so that
it could translocate to the cell membrane,
F508 CFTR formed a
functional Cl
channel with a reduced open probability
(9, 11, 19) and a reduced half-life in the membrane
(28).
Currently, there is no treatment for CF that targets the molecular
defect of CFTR. Possible targets for pharmacological interventions are
1) the CFTR channel in the membrane, 2) signaling
cascades that regulate CFTR, and 3) the maturation and
trafficking pathway of CFTR. A number of small, hydrophilic molecules
have been recently introduced as chemical chaperones (3,
34), which promoted protein folding and maturation. When 3T3
cells expressing F508 CFTR were treated with the chemical chaperone
trimethylamine oxide (TMAO) at concentrations of 50-100 mM, fully
glycosylated mature
F508 CFTR was found and the Cl
permeability of the cell membrane was partially restored
(3). This result suggested that treatment with TMAO was
able to correct the processing defect of
F508 CFTR. To test the
effects of TMAO in vivo, Bai et al. (1)
developed a treatment protocol for mice that resulted in serum
concentrations of TMAO (50-100 mM) that were effective in cell
culture models. The current study was designed to determine the in vivo
efficacy of TMAO treatment for the correction of the cAMP-stimulated,
Cl
-dependent rectal potential difference (RPD) in CF
mice. The measurement of the rectal difference was chosen because,
unlike humans, CF mice develop CF-related intestinal but not airway
pathology (31). Furthermore, Cl
transport in
CF mouse intestine and colon is impaired (8, 16-18).
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METHODS |
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Study protocol.
Transgenic F508 CF mice were bred at the University of California,
San Francisco Animal Facility and genotyped with standard protocols
(36). Transgenic CFTR knockout mice (31) were
bred and genotyped in the laboratory of Dr. B. H. Koller
(University of North Carolina, Chapel Hill, NC) and were kindly made
available for this study. The mice were fed a standard diet. The mice
were subcutaneously injected with TMAO from a 2 M stock solution in water (or with water as a negative control) every 8 h for 48 h. The mice were treated with 1-7 g/kg body wt. The highest dose used (7 g/kg body wt) resulted in ~50% deaths in wild-type mice, i.e., approximates the half-maximal lethal dose for TMAO as shown in a
previous study by Bai et al. (1). In CF mice, the
highest dose used was 4 g/kg body wt. RPD was measured before treatment and 24 and 48 h after the start of treatment.
Measurement of RPD.
RPD was measured with a protocol similar to that for nasal potential
difference measurements in humans (21). Mice were
anesthetized with 1 µl/g body wt of 0.4 mg/ml of acepromazine and 11 mg/ml of ketamine. RPD was sensed with a 1 M NaCl-agar bridge inserted ~2 cm into the rectum and connected through a Ag-AgCl electrode to a
high-impedance (>1-G) digital voltmeter. Readings were amplified and interfaced to a computer for continuous recording. Potentials were
measured with respect to a subcutaneous, 1 M NaCl-filled needle. A
second rectal tube was used for continuous perfusion of and drug
administration into the rectum. Solutions were perfused by gravity flow
at ~1 ml/min at room temperature. The Cl
-containing
solution had the following composition (in mM): 145 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 0.1 amiloride, pH 7.4. The Cl
-free solution contained (in mM) 145 sodium
gluconate, 4 potassium gluconate, 4 calcium gluconate, 1 magnesium
gluconate, 10 HEPES, and 0.1 amiloride, pH 7.4.
Drugs and chemicals. Forskolin (Calbiochem, La Jolla, CA) was used at 20 µM from a 100 mM stock solution in dimethyl sulfoxide (DMSO). Amiloride was used at 100 µM from a 10 mM stock solution in water. Glibenclamide was used at 300 µM from a 100 mM stock solution in DMSO. Apigenin (4',5,7-trihydroxyflavone) was used at 30 µM from a 100 mM stock solution in DMSO. If not mentioned otherwise, chemicals were from Sigma (St. Louis, MO).
Statistics. The effects of treatment were analyzed with factorial ANOVAs with Fisher's corrections for multiple t-comparisons. In addition, all responses were tested for significance (i.e., difference from zero) with one-group t-tests. The effects of dose and length of treatment were tested with a multiple regression analysis. P < 0.05 was considered significant. All statistical calculations were done with StatView version 4.5 (Abacus Concepts, Berkeley, CA).
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RESULTS |
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RPD was measured in F508 CF mice and their wild-type
littermates before and 24 and 48 h after the start of treatment
with TMAO. To amplify the CFTR-dependent RPD, all measurements were done in the presence of amiloride (to block the
Na+-dependent RPD) and with Cl
-free
solutions. The change of perfusion solution from a
Cl
-containing to a Cl
-free solution (in the
presence of amiloride) did not result in significant changes in RPD in
any of the treatment groups (average for all groups,
0.11 ± 0.51 mV; n = 55 mice; P = 0.84 by
one-sample t-test). Figure
1A shows representative
recordings of the responses to forskolin, and Fig. 1B shows
the average responses measured in control and TMAO-treated wild-type
mice. Perfusion with forskolin induced a hyperpolarization of RPD,
which indicated activation of a Cl
conductance in the
rectal epithelium. In control mice, perfusion with forskolin
hyperpolarized RPD by
6.4 ± 0.79 mV (n = 11). Treatment with TMAO significantly increased the responses to forskolin in normal mice to
12.6 ± 2.0 mV (n = 7) at
24 h and to
13.5 ± 2.0 mV (n = 10) at
48 h, which were not different from one another (Fig.
1B). Figure 1, C and D, shows
representative recordings and the average responses from
F508 CF
mice. In untreated CF mice, RPD was not significantly affected by
forskolin (
0.10 ± 0.14 mV; n = 14 mice; not
different from zero by one-group t-test). Treatment with
TMAO significantly increased the responses to forskolin in
F508 mice
to
1.3 ± 0.38 mV (n = 9) at 24 h and to
1.0 ± 0.3 mV (n = 7) at 48 h, which were
not different from one another (Fig. 1D). These data show
that the treatment of mice with TMAO increased the forskolin-activated
RPD in both wild-type and
F508 CF mice.
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The effects of dose and length of treatment with TMAO were tested with
a multiple regression analysis. In F508 CF mice, the responses of
RPD to forskolin correlated significantly with the administered dose
(P = 0.019) but not with length of treatment (P
= 0.89). Figure 2 shows the
relationship between dosage and forskolin-induced RPD responses, which
yielded a slope of
0.40 ± 0.10 mV · g
1 · kg
1.
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To verify the CFTR dependence of the forskolin-induced responses of RPD
by TMAO treatment, we carried out experiments that probe for CFTR by
using 1) CFTR knockout mice, 2) the CFTR blocker glibenclamide, and 3) the CFTR activator apigenin.
Representative experiments with CFTR knockout mice are shown in Fig.
3. CFTR knockout [(/
)] mice and
their heterozygous littermates were treated with 4 g/kg body wt of TMAO
for 24 h. In heterozygotes (Fig. 3A) but not in
CFTR(
/
) mice (Fig. 3B), forskolin hyperpolarized the
RPD, indicating the lack of a Cl
conductance in
TMAO-treated CFTR(
/
) mice. In CFTR(
/
) mice, forskolin caused a
depolarization of 1.6 ± 1.3 mV (n = 5), which was
possibly caused by the activation of K secretion in these mice, similar
to that reported for the mouse colon epithelium (8).
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The effect of perfusion of the rectal mucosa with 300 µM
glibenclamide is shown in Fig. 4. In
wild-type mice, glibenclamide partially (39 ± 16%;
n = 3) inhibited the forskolin-stimulated RPD (Fig.
4A). In TMAO-treated (4 g/kg body wt, 24 h) F508 CF mice, forskolin hyperpolarized RPD by
1.9 ± 0.1 mV
(n = 3) in this set of experiments, of which
glibenclamide inhibited 53 ± 7.8% (i.e., 1.0 ± 0.04 mV;
n = 3; Fig. 4B). The partial block of CFTR
by glibenclamide is comparable to its effects in other CFTR-expressing
epithelia such as Calu-3 cells (29 ± 5%) in the presence of a
mucosal Cl
-free solution (23). Partial block
of CFTR by glibenclamide in presence of mucosal Cl
-free
solution is consistent with the voltage-dependent blocker affinity of
glibenclamide, which has been shown to be reduced when the membrane
potential was depolarized (30).
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We also tested the CFTR activator apigenin (21) in
TMAO-treated F508 CF mice (4 g/kg body wt, 24 h). The effect of
acute perfusion of apigenin is shown in Fig.
5. In this set of experiments, perfusion
with forskolin hyperpolarized RPD to, on average,
1.4 ± 0.3 mV
(n = 6 mice). The addition of apigenin caused a further hyperpolarization and a total response of
2.5 ± 0.5 mV (P
= 0.036 by paired t-test; Fig. 5B). For
comparison, in untreated CF mice, rectal perfusion with apigenin did
not significantly affect RPD (change in RPD = 0.12 ± 0.19 mV; n = 5 mice; not different from 0, P =
0.56; Fig. 5B).
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DISCUSSION |
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Using F508 CFTR heterologously expressed in cell cultures,
Brown et al. (3) reported that the chemical chaperones
glycerol (1.2 M) and TMAO (100 mM) corrected the maturation defect of
F508 CFTR. These investigators measured the TMAO effect by the
appearance of the mature, fully glycosylated band C in Western blots of
CFTR and by Cl
efflux in cellular studies. When we
initially tested these chemical chaperones in an in vivo mouse model,
our goal was to achieve concentrations that were similar to the
effective concentrations in the cell culture model used by Brown et
al.. We found that it was not possible to achieve adequate serum levels
using glycerol without severe toxicity. However, with TMAO given in
subcutaneous injections every 8 h over 48 h, it was possible
to achieve similar serum concentrations of ~50-100 mM with only
50% lethality (1). These high concentrations were
necessary to achieve measurable effects in this in vivo study and are
similar to the effective TMAO concentration in the cell culture studies
(3). Thus the experiments for this study were designed to
test the efficacy of this chaperone in correcting the Cl
defect in CF mice.
The data show that TMAO treatment partially corrected the
forskolin-activated Cl conductance in CF mouse rectum. In
single-channel recordings, the
F508 CFTR mutant expresses a reduced
open probability (25-30% of normal value; 9, 11, 19) and the
lifetime in the cell membrane is reduced by ~50% (28).
Thus the restoration of normal trafficking of
F508 CFTR can be
predicted to yield only a partial recovery (~15%) of the
Cl
conductance found in wild-type cells. We found that
treatment with TMAO recovered a response to forskolin of
1.1 mV in
F508 CF mice, which is ~17% of the response in wild-type mice.
Although variations in a transepithelial potential difference do not
have a direct linear relationship with changes in conductance, the data
suggest a significant correction of the trafficking defect of
F508
CFTR. Because some studies support the hypothesis that only a small
fraction (5-10%) of normal CFTR-mediated Cl
conductance is required to ameliorate clinical CF symptoms (10, 20, 27), it is conceivable that a restoration of
F508
trafficking in humans, similar to the effect observed here in
F508
mice, could be associated with positive clinical effects in patients. However, the main value of these studies is that they constitute a
proof of principle that a chemical chaperone can be effective in vivo.
Because TMAO itself was associated with severe toxicity (i.e., the
compound was used near its half-maximal lethal dose), it cannot be
seriously considered for clinical use. Thus the present study
demonstrates the feasibility of a pharmacological approach with a
chemical chaperone. It also emphasizes the need to develop better
compounds, characterized by higher efficiency and lower toxicity.
A second aspect of our work is that treatment with a chemical chaperone
combined with a CFTR activator might be beneficial. Several CFTR
activators have been previously described (2, 15, 21, 22).
We selected apigenin for this study because of its high affinity and
low toxicity. We found that in TMAO-treated F508 CF mice, the
effects on Cl
-selective potentials were almost doubled
after the addition of apigenin (Fig. 5). Previously, Illek and
Fischer (21) reported that apigenin is a potent
CFTR activator by increasing the open probability of wild-type CFTR,
and apigenin can potentiate in vitro the effect of 4-phenylbutyrate on
F508 CFTR (24). The present study shows that the
combination of a chemical chaperone with a channel activator also
exhibits a synergistic effect in vivo.
Clearly, the extraordinarily high concentrations that we employed in
this study are likely to have caused various other physiological and
cellular effects, such as upregulation of hormones responsible for
osmotic homeostasis and induction of cellular stress proteins, possibly
including the heat shock proteins (HSPs). Therefore, it may be possible
that the effect of TMAO treatment may be indirectly mediated by a
response to the osmotic stress. Using the chemical chaperone glycerol
(1 M), Brown et al. (3) have shown that treatment of
NIH/3T3 mouse fibroblasts with glycerol did not induce expression of
the heat-inducible HSP72; however, glycerol treatment supported
maturation of recombinant F508 CFTR. When these cells were further
heat stressed, expression of HSP72 was induced in control cells but not
in glycerol-treated cells (3). Similarly, in HeLa cells,
heat shock-induced expression of HSP73 was blocked by glycerol
(4), and no effects of glycerol on HSP70 mRNA expression were detected in Madin-Darby canine kidney cells (6).
These data show that treatment with the chemical chaperone glycerol did
not affect the expression of several HSPs, and the heat-induced expression of HSP did not support
F508 maturation. Thus it appears that the effect of chemical chaperones on
F508 CFTR maturation is
not related to the expression of HSPs. Although this has not been
tested for TMAO, the proposed common mechanism of action (see below) of
both TMAO and glycerol supports the notion that HSPs are not involved
in the treatment-induced recovery of
F508 CFTR. To test for side
effects of TMAO in vivo in mice, a panel of serum parameters, including
electrolytes and liver enzymes, was measured (1). Only
minor, insignificant effects on the concentrations of glucose, NaCl,
bicarbonate, phosphate, and albumin were detected, and it was concluded
that treatment with TMAO did not impair renal, liver, pancreas, or
muscle function (1). Thus these previous measurements did
not show significant changes in blood chemistry and on cellular stress
protein expression. Although an effect of TMAO on other factors (which
may mediate
F508 CFTR expression) cannot be ruled out from our
study, the simplest explanation of our data is a direct recovery of
F508 trafficking by TMAO.
The in vivo measurement of RPD is determined by the ion selectivity of
both the transcellular and paracellular pathways, both of which may
have been affected by TMAO treatment, and a contribution of the
paracellular pathway cannot be excluded. However, the following observations suggest that the TMAO-recovered RPD is mediated in large
part by CFTR activity: 1) stimulation by forskolin in
F508 but not in knockout mice, 2) sensitivity to
glibenclamide, and 3) sensitivity to apigenin.
The mechanism of action of chemical chaperones is not totally
understood. These molecules may support the formation of a stable conformation of CFTR in the endoplasmic reticulum. They are thought to
act through a mechanism of stabilizing protein structures by affecting
the degree of hydration and stabilize intramolecular interactions
(7, 12, 13). Interestingly, in our study, TMAO increased
the responses to forskolin in both wild-type and F508 CF mice. This
observation is consistent with the hypothesis that only a fraction
(25%) of wild-type CFTR trafficks correctly to the cell membrane,
whereas the majority of wild-type CFTR and probably all of
F508 CFTR
are targeted for degradation by intracellular proteasomes (26,
33, 37). Thus the stabilization of the CFTR protein in the
endoplasmic reticulum by TMAO may increase forskolin-activated RPDs in
both normal and CF mice.
In summary, these studies provide evidence that in vivo treatment with
TMAO can partially correct the Cl conductance defect in
F508 CF mice. Although TMAO is unlikely to be suitable for clinical
use, these studies in CF mice provide support for the potential
efficacy of a less toxic chemical chaperone for treatment of human CF.
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ACKNOWLEDGEMENTS |
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We thank Alan Verkman for helpful suggestions and encouragement.
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FOOTNOTES |
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This work was supported by Cystic Fibrosis Foundation Grant FISCHE99G0; National Heart, Lung, and Blood Institute Grants 1-P50-HL-60288-01 and HL-51854; the Centre National de la Recherche Scientifique; and the Vaincre la Mucoviscidose.
Present address of P. Barbry: Institut de Pharmacologie Moleculaire et Cellulaire, CNRS UPR411, 06560 Sophia Antipolis, France.
Original submission in response to a special call for papers on "CFTR Trafficking and Signaling in Respiratory Epithelium."
Address for reprint requests and other correspondence: H. Fischer, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609 (E-mail: hfischer{at}chori.org).
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.
Received 20 September 2000; accepted in final form 27 November 2000.
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