1 Ion Transport Unit, National Heart and Lung Institute, London SW3 6LR, United Kingdom; 2 Department for Molecular and Cellular Biology, University of Queensland, Brisbane 4072, Australia; and 3 Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Some cystic
fibrosis transmembrane conductance regulator (CFTR) mutations, such as
G551D, result in a correctly localized Cl channel at the cell
apical membrane, albeit with markedly reduced function. Patch-clamp
studies have indicated that both phosphatase inhibitors and
3-isobutyl-1-methylxanthine (IBMX) can induce
Cl
secretion through the
G551D mutant protein. We have now assessed whether these agents can
induce Cl
secretion in
cftrG551D mutant
mice. No induction of Cl
secretion was seen with the alkaline phosphatase inhibitors
bromotetramisole or levamisole in either the respiratory or intestinal
tracts of wild-type or
cftrG551D mice.
In contrast, in G551D intestinal tissues, IBMX was able to produce a
small CFTR-related secretory response [means ± SE: jejunum,
1.8 ± 0.9 µA/cm2,
n = 7; cecum, 3.7 ± 0.8 µA/cm2,
n = 7; rectum (in vivo),
1.9 ± 0.9 mV, n = 5]. This
was approximately one order of magnitude less than the wild-type
response to this agent and, in the cecum, was significantly greater
than that seen in null mice
(cftrUNC). In
the trachea, IBMX produced a transient
Cl
secretory response (37.3 ± 14.7 µA/cm2,
n = 6) of a magnitude similar to that
seen in wild-type mice (33.7 ± 4.7 µA/cm2,
n = 9). This response was also present
in null mice and therefore is likely to be independent of CFTR. No
effect of IBMX on Cl
secretion was seen in the nasal epithelium of
cftrG551D mice.
We conclude that IBMX is able to induce detectable levels of
CFTR-related Cl
secretion
in the intestinal tract but not the respiratory tract through the G551D
mutant protein.
mouse model; 3-isobutyl-1-methylxanthine; cystic fibrosis transmembrane conductance regulator
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CYSTIC FIBROSIS (CF) pathology is a consequence of
mutations in the CF transmembrane conductance regulator (CFTR) protein that functions as an apical membrane
Cl channel (11).
CFTR-mediated Cl
secretion
is markedly reduced in CF, any residual function being dependent on
which of the over 400 mutations are considered. Recently, mouse models
of CF have become available, including those for two specific human
mutations,
F508 (6, 28, 30) and G551D (7), the latter representing
~3% of mutant alleles (14). The G551D mutation results in a channel
that is localized to the apical membrane but does not allow normal
rates of Cl
secretion in
response to ATP binding at the first nucleotide-binding fold (29).
Increasing CFTR regulatory domain phosphorylation makes available the
second nucleotide-binding fold domain for further ATP binding and
increases channel open probability (17). Thus therapeutic strategies
could be devised that might activate CFTR by increasing the probability
of phosphorylation of those molecules reaching the apical membrane.
Two principal strategies are available: reducing phosphatase activity
or reducing adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiester bond hydrolysis (12, 19). Some of the phosphatases associated with CFTR function have been suggested to include protein phosphatase (PP)-2A (5), PP-2C (16), and alkaline phosphatase (4, 25).
No evidence for the involvement of PP-2B has been shown to date (15).
With respect to CFTR mutant protein, patch-clamp studies have
demonstrated that the alkaline phosphatase inhibitor bromotetramisole
could induce Cl secretion
through the
F508 protein in precooled simian virus 40-transformed
human airway epithelial NP34 cells (4). The mutant CFTR processing is
temperature sensitive (8); cooling results in correctly localized
protein. However, no Cl
secretion was seen in
F508 airway cells tested with PP-1 and PP-2A
inhibitors in combination with the general phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX) (13). In this latter study, the
cells were not precooled, thus resulting in mislocalization of the
channel protein. Phosphatase inhibition is likely to be more successful
in the case of mutated CFTR that does reach the apical membrane. In
keeping with this, a recent patch-clamp study has described the
induction of Cl
secretion
in G551D cells using bromotetramisole (4).
An alternative strategy for increasing phosphorylation and hence
Cl secretion is to reduce
cAMP phosphodiester bond hydrolysis. Thus IBMX used at high
concentrations has been shown to induce
Cl
secretion in
Xenopus oocytes transfected with the
F508 cRNA (10) (
F508 protein is known to reach the apical
membrane in Xenopus oocytes). However,
in a further study, no secretion could be produced either in cell lines
derived from
F508 patients or by direct in vivo assessment (13). As
for phosphatase inhibition, it is likely that such a strategy may be
more successful in the case of a mutated protein reaching the apical
membrane in mammalian cells. Thus IBMX [again at high
concentrations (10)] has been shown to induce a level of
Cl
secretion in G551D cells
in vitro (4).
These cellular studies suggest that agents such as bromotetramisole and
IBMX may be useful clinically in certain CF mutations. An important
next step is to assess their effect in animal models. With the
generation of the specific G551D mutant mouse, we have now assessed
whether alkaline phosphatase inhibitors or IBMX can induce
Cl secretion in the airways
or intestinal tract of these animals. Because levamisole, a compound
related to bromotetramisole (26), is already in clinical use (1), we
also assessed the effect of this agent.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
Mice (15-35 g) were housed in a temperature-controlled room (21°C) with food (Special Diet Services, Witham, Essex, UK) and water freely available. MF1 mice were obtained from Harlan. cftrG551D (7) and cftrUNC null (22) mice (producing no CFTR) were generated as previously described and housed on pine shavings. G551D mRNA levels are ~50% of that of wild-type (+/+) levels when assessed in the lung, small intestine, kidney, and testes (7).In Vivo Measurement
Both these and the in vitro measurements (below) have previously been extensively described (24). Briefly, the reference electrode was placed subcutaneously in a hindlimb of the anesthetized animals [Avertin (tribromoethanol, 0.43 g/kg intraperitoneally)] and connected via N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Krebs buffer of composition (in mM) 140 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.4) to a calomel half cell. This was in turn connected to a hand-held computer attached to a preamplifier containing a low-pass signal-averaging filter with a time constant of 0.5 s (Logan Research, Sussex, UK). The exploring electrode consisted of a double-lumen polyethylene tube (outside diameter ~0.5 mm for nose, 1 mm for rectum), similarly connected to the logging device. Drugs were perfused onto the relevant epithelium at a rate of 40 µl/min. In some cases (in the nasal epithelium), a low-ClIn Vitro Measurements
After in vivo measurements, animals were killed by sectioning of the dorsal aorta, and tracheal, jejunal, and cecal segments were mounted in Ussing chambers of aperture area 0.28 cm2 (intestine) and 0.03 cm2 (airway), under short-circuit conditions. If multiple tissues were obtained from one animal, the mean value was used, so that n refers to the number of animals. Tissues were circulated with Krebs-Henseleit solution of molar composition (in mM) 145 Na+, 126 ClProtocols
Drugs were principally assessed in vitro; additional in vivo studies were performed as indicated. Dose-ranging studies were carried out in +/+ animals; selected concentrations were subsequently assessed in mutant mice.In vitro. All tissues were pretreated with amiloride (10 µM, mucosal) after baseline recordings. Subsequently, either bromotetramisole or levamisole (each bilateral) or IBMX (mucosal) was added. This was followed in the trachea by the addition of forskolin (10 µM, mucosal) and subsequently ATP (100 µM, mucosal), in the intestinal tract by forskolin (10 µM, mucosal), and in the small intestine only by glucose (10 mM, mucosal) and phloridzin (200 µM, mucosal). Glucose and phloridzin (200 µM) were used to, respectively, stimulate and inhibit Na+-glucose cotransport. This response is useful as an indicator of viability in CF intestinal tissues.
In vivo.
Both nasal and rectal tissues were pretreated with amiloride (100 µM)
following baseline recordings. Subsequent perfusion included IBMX (1 mM) followed by forskolin (10 µM) and, in the nose, ATP (100 µM).
For nasal perfusion all drugs subsequent to amiloride were in a
low-Cl-containing solution.
Drugs were dissolved in water (bromotetramisole, levamisole),
Krebs-Henseleit (amiloride, ATP), or ethanol (forskolin, IBMX) and were
added in dilutions of 1:100 or greater. Phloridzin was dissolved in
water. Drugs were obtained from Sigma-Aldrich (Poole, UK).
Statistics
All group comparisons were made using the Mann-Whitney U test, and significance of an individual response was assessed using the Wilcoxon signed rank test. Data are expressed as means ± SE. The null hypothesis was rejected at P < 0.05. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bromotetramisole
Trachea. In +/+ tissues, bromotetramisole produced no stimulation of current at any concentration from 100 nM to 1 mM. No effect was seen on the subsequent forskolin or ATP responses, except at the highest concentration (1 mM), which produced a significant reduction in the response to both agents (Fig. 1A). As in the +/+ tissues, no stimulation of current was seen in the G551D tracheae, and again bromotetramisole (1 mM) effectively abolished the subsequent forskolin and ATP responses (Fig. 1A).
|
Intestinal tract.
In +/+ tissues, bromotetramisole produced no consistent change in
baseline currents at 1 µM-1 mM in either the jejunum (Fig. 1B) or cecum (Fig.
1C). The subsequent response to
forskolin was significantly reduced in both tissues at the highest
concentration (1 mM) of bromotetramisole. The same profile was seen in
the cftrG551D
mice, with no induction of
Cl secretion and a complete
abolition of the subsequent response to forskolin.
Levamisole
Trachea. Levamisole (100 nM-1 mM) produced no significant change in baseline Isc and no effect on the subsequent response to forskolin in +/+ mice. Similar results were seen in cftrG551D mice. The subsequent responses to ATP were significantly reduced in G551D tissues (Fig. 2A).
|
Intestinal tract.
In +/+ mice, levamisole produced no significant effect on the baseline
Isc in either the
jejunum (Fig. 2B) or cecum (Fig. 2C) at any concentration assessed
(100 nM-1 mM). In keeping with this, there was also no significant
alteration in the subsequent response to forskolin. Again, in
cftrG551D mutant
mice levamisole did not induce
Cl secretion or enhance the
subsequent response to forskolin (Fig. 2,
B and
C). It is worth noting that the
responses in both genotypes to bromotetramisole and levamisole in all
tissues were characterized by marked variability, albeit at low
absolute levels of response.
IBMX
Trachea.
In +/+ tissues, IBMX produced a dose-related increase in
Isc, maximal at 1 mM. The maximal level of stimulation (33.7 µA/cm2) was similar to that
seen following addition of forskolin (10 µM; 39.2 µA/cm2) in the absence of
IBMX. Furthermore, this stimulation by IBMX (1 mM) was associated with
a complete abolition of the subsequent forskolin response (Fig.
3A).
Thus, as previously described, it is very likely that the response to
IBMX is mediated principally through a cAMP pathway (13). Because very
high concentrations of IBMX (5 mM) have been reported to induce
Cl secretion in CF tissues,
we also assessed the effect of this concentration on +/+ tissues
(n = 4). A lower level of stimulation of Isc was seen
in comparison with addition of 1 mM, whereas the subsequent response to
forskolin was completely abolished and the ATP response significantly
diminished. Because of these effects, further studies were carried out
at a concentration of 1 mM.
|
|
|
Jejunum. The +/+ tissue showed a dose-related (1 µM-1 mM) increase in baseline current, with a maximal response very similar to that produced by forskolin (10 µM) in the absence of IBMX (Fig. 6A). A reciprocal reduction in the subsequent forskolin response was seen over the same concentration range. The cftrG551D response to IBMX (1 mM) was 1.8 ± 0.9 µA/cm2 (n = 7), and the response to forskolin alone was 0.8 ± 0.9 µA/cm2 (n = 7; Fig. 6A). For comparison, the response to IBMX in the cftrUNC mice was 0.6 ± 0.2 µA/cm2 (n = 3; not significant compared with cftrG551D), and the response to forskolin alone was 0.5 ± 0.3 µA/cm2 (n = 6; not significant compared with cftrG551D). In both types of CF mice, forskolin produced no stimulation of the Isc when added after IBMX. Representative tracings are shown in Fig. 6B.
|
Cecum.
In +/+ tissues, IBMX produced a dose-related increase in
Isc, again with
maximal response similar to that produced by forskolin alone in this
tissue (Fig.
7A).
This was accompanied by a reciprocal reduction in the response to the
subsequent addition of forskolin (Fig.
7A). The response to IBMX in
cftrG551D
animals was 3.7 ± 0.8 µA/cm2
(n = 7), and the response to forskolin
alone was 1.9 ± 0.5 µA/cm2.
For comparison, the response to IBMX in the
cftrUNC mice was
0.6 ± 0.4 µA/cm2
(n = 3;
P < 0.05 compared with
cftrG551D),
and the response to forskolin alone was 0.2 ± 0.3 µA/cm2
(n = 6;
P < 0.01 compared with
cftrG551D). In
both types of CF mice, the subsequent response to forskolin was
abolished. Representative tracings are shown in Fig.
7B.
|
Rectum.
After amiloride pretreatment, in
cftrG551D mice,
IBMX produced a response of 1.9 ± 0.9 mV
(n = 5) and forskolin alone produced a
reduction of 2.1 ± 0.3 mV
(n = 7). The forskolin produced no stimulation of the potential difference when added after IBMX. Representative tracings are shown in Fig.
8, A and
B.
|
Nasal cavity.
After amiloride pretreatment, IBMX and forskolin increased the
post-low-Cl baseline
potential difference of
cftrG551D
animals (2.5 ± 1.4 mV, n = 4).
This effect was similar in magnitude to that produced by forskolin (10 µM) alone (2.9 ± 1.1 mV, n = 6).
A high dose (5 mM) of IBMX (10) was used for these nasal experiments in
the light of previous reports of activation of Cl
transport through G551D
channels by these doses (5 mM) of IBMX. Representative tracings are
shown in Fig. 8, C and
D.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study demonstrates that in
cftrG551D mice
two agents with alkaline phosphatase-inhibitory activity do not induce
Cl secretion in either the
respiratory or intestinal tracts. The phosphodiesterase inhibitor IBMX
is able to produce a very small response, likely mediated via CFTR,
throughout the intestinal tract. No induction of CFTR-mediated
Cl
secretion by IBMX could
be measured in the respiratory tract. However, IBMX appeared to
activate an alternate, possibly
Ca2+-related,
Cl
secretory pathway in the
trachea.
As discussed above, evidence exists to suggest the involvement of a
number of phosphatases in the regulation of CFTR. To our knowledge,
this has never been assessed in intact tissues or in vivo. We were
unable to demonstrate induction of
Cl secretion by
bromotetramisole, either in the respiratory or intestinal tract of +/+
animals, assessed at a wide range of concentrations. In addition, no
evidence was seen for activation of
Cl
secretion in G551D
tissues at a concentration (1 mM) previously shown to activate
Cl
currents through the
G551D protein (4).
Evidence for an effect of bromotetramisole was seen in +/+ intestinal
tissues, where addition of the active isomer significantly inhibited
the response to the subsequent addition of forskolin; this was not seen
following addition of the inactive isomer (data not shown). We also
assessed the effect of levamisole, an alkaline phosphatase inhibitor
related to bromotetramisole, already in clinical use [for the
treatment of roundworm infection (1)], and therefore a potential
therapeutic agent for CF. Again no stimulation of basal
Cl secretion was seen in
any tissue. Furthermore, this agent did not alter the subsequent
response to forskolin. Whether this lack of effect relates to a lower
potency for phosphatase inhibition cannot be determined by this study.
However, at least from our data in the
cftrG551D mouse
model, neither of the phosphatase inhibitors studied appears to hold
promise as a therapeutic agent in CF. We cannot determine from this
study whether these alkaline phosphatase inhibitors reached the
intracellular surface, but studies have demonstrated their efficacy in
cultured epithelial monolayers (18).
We also tested IBMX, an agent having both phosphatase- and phosphodiesterase-inhibitory properties. Initial dosing experiments in +/+ mice suggested that nonspecific effects become apparent at 5 mM, and we therefore used 1 mM in subsequent experiments. Our study suggests that in the respiratory and intestinal tracts of +/+ mice this agent acts principally through a pathway in common with that activated by forskolin. Thus IBMX produced responses of a magnitude similar to those produced by forskolin and abolished the subsequent response to the latter in all tissues studied. These findings, and the marked differences in response to the phosphatase inhibitors, suggest that phosphodiesterase inhibition may be the predominant mode of action of IBMX in these tissues. This is in keeping with many studies demonstrating elevation of intracellular cAMP levels following addition of IBMX, in airway (13) as well as in a variety of other tissues (2, 20).
IBMX has been shown to induce
Cl secretion through
F508 mutant CFTR in two patch-clamp studies, but not when assessed
in monolayers or in humans in vivo (13). IBMX could also induce Cl
currents through the
G551D mutant protein, again when assessed by the patch-clamp technique
(4). In the present study, IBMX induced a very low level of
Cl
secretion in all three
regions of the G551D intestinal tract, whether studied in vitro
(jejunum and cecum) or in vivo (rectum). Because we wished to assess
the effect of IBMX both on the baseline Isc as well as on
the subsequent response to forskolin, we could not assess the effect of
bumetanide. However, we have previously demonstrated that the
forskolin-induced stimulation of
Isc in the murine
intestine (both +/+ and
cftrG551D) is
mediated principally through
Cl
(7, 24).
Furthermore, in all of the present studies, tissues were pretreated
with amiloride. Thus it is likely that the stimulation seen was related
to Cl. To assess whether
this response was solely mediated through CFTR, we also studied the
effect of IBMX in the intestinal tract of null mice. This response
provides a background against which to assess the effect of IBMX in
G551D tissues. In comparison with the null mice, we consistently saw a
small response in the G551D intestinal tract, although this only
reached significance in the cecum. The magnitude of this current
represents only approximately one-tenth of that induced in +/+ tissues,
but this should be taken in the context of a nonlinear relationship
between the magnitude of CFTR function and phenotypic outcome (9). Thus
we have previously shown that such low levels of cAMP-mediated
Cl
secretion correspond to
an increase in survival at 35 days of ~20% in CF mutant mice (7).
In contrast to these findings of very low levels of
Cl secretion induced by
IBMX in the intestinal tract of the
cftrG551D mice,
no evidence for Cl
secretion was seen in the nasal epithelium. This difference may reflect
the domination by CFTR of electrogenic
Cl
secretion in the
intestine. In contrast, nasal
Cl
secretion is difficult
to elicit both in mice and in humans. These phenotypic findings are
consistent with the very low levels of CFTR found in the respiratory
tract, the reduced efficiency of expression of CFTR mRNA in the
cftrG551D model,
and the low anticipated level of channel function of the G551D protein
in the apical membrane. Thus under these conditions any response may
have remained undetectable.
Interestingly, in the trachea, we observed a response to IBMX that did
not differ in magnitude from that seen in +/+ mice. However, several
features suggest that this response was not principally mediated
through CFTR. In +/+ animals, forskolin produces an increase in
Cl secretion that relates
principally to cAMP-mediated pathways (13, 24). However, approximately
one-third of this response is likely to relate to pathways mediated
through other second messenger systems, in particular, elevation of
intracellular Ca2+ (23). We
speculate that the response to IBMX was predominantly mediated through
this Ca2+ pathway in the G551D
trachea. In support of this view is our finding that both
cftrG551D and
null mice exhibited a different IBMX response profile that was
significantly more transient than that seen in the +/+ mice. In both
groups of mutant mice, there is an increased response to
Ca2+-mediated agonists such as
ATP. IBMX reduced the subsequent ATP response in these mutant mice more
than in +/+ animals, although this did not reach significance. Grubb et
al. (13) have suggested this relates to P2 receptor
antagonism by IBMX. In keeping with this, there was no effect of IBMX
on the subsequent response to the
Ca2+ ionophore A-23187 (Fig. 5).
Are there therapeutic implications for G551D CF subjects from this
study? Intestinal disease in CF subjects is associated in neonates with
an ~10% incidence of meconium ileus (27), this being some threefold
reduced in the G551D population (14). In addition, intestinal ion
transport is altered in CF adults (21), and they are prone to large
intestinal obstruction (meconium ileus equivalent). It is unlikely that
IBMX would find a role in the treatment of these conditions, even if
endoscopic application was feasible at any early stage. Agents such as
ATP or UTP that activate alternate
Ca2+-linked pathways have been
suggested as a potential new form of treatment for CF subjects. At
least in the
cftrG551D mouse
model, the magnitude of Cl
secretion produced by IBMX appears to be three- to fourfold less than
that produced by ATP. However, ATP was able to produce a response, if
somewhat reduced, following IBMX. Thus whether the actions of
nucleotides and IBMX are synergistic may deserve further attention.
Finally, other phosphodiesterase inhibitors such as theophylline and
milrinone are in routine clinical use. Preliminary data indicate that
these produce very similar responses to IBMX in +/+ tissues, and these
and others currently being identified (3) may therefore be worth
assessing in CF tissues.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful for assistance from members of the Biosciences Unit (National Heart and Lung Institute, London, UK) and from Dominic Lunn and Paul Lovelock (Centre for Molecular and Cellular Biology, Brisbane, Australia) and for the contribution of animal husbandry and genotyping by Sheila Webb (Medical Research Council Human Genetics Unit, Edinburgh, UK).
![]() |
FOOTNOTES |
---|
This study was supported by the Cystic Fibrosis Research Trust, the Association Française de Lutte Contra la Mucoviscidose (ARTEMIS project), the Medical Research Council (UK), and a Wellcome Trust Senior Clinical Fellowship (to E. W. F. W. Alton).
Address for reprint requests: E. W. F. W. Alton, Ion Transport Unit, National Heart and Lung Institute, Manresa Rd., London SW3 6LR, UK.
Received 9 April 1997; accepted in final form 6 November 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abdel-Meguid, M. S.,
K. Badr,
and
M. Saif.
The polyanthelmintic efficacy of levamisole.
J. Egyptian Med. Assoc.
58:
334-336,
1975.
2.
Abrahamsen, N.,
K. Lundgren,
and
E. Nishimura.
Regulation of glucagon receptor mRNA in cultured primary rat hepatocytes by glucose and cAMP.
J. Biol. Chem.
270:
15853-15857,
1995
3.
Beavo, J. A.
Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms.
Physiol. Rev.
75:
725-748,
1995
4.
Becq, F.,
T. J. Jensen,
X.-B. Chang,
A. Savoia,
J. M. Rommens,
L.-C. Tsui,
M. Buchwald,
J. Riordan,
and
J. W. Hanrahan.
Phosphatase inhibitors activate normal and defective cftr chloride channels.
Proc. Natl. Acad. Sci. USA
91:
9160-9164,
1994
5.
Berger, H. A.,
S. M. Travis,
and
M. J. Welsh.
Regulation of the cystic fibrosis transmembrane conductance regulator Cl channel by specific protein kinases and protein phosphatases.
J. Biol. Chem.
268:
2037-2047,
1993
6.
Colledge, W. H.,
B. S. Abella,
K. W. Southern,
R. Ratcliff,
C. Jiang,
S. H. Cheng,
L. J. MacVinish,
J. R. Anderson,
A. W. Cuthbert,
and
M. J. Evans.
Generation and characterisation of a F508 cystic fibrosis mouse model.
Nat. Genet.
10:
445-452,
1995[Medline].
7.
Delaney, S. J.,
E. W. F. W. Alton,
S. N. Smith,
D. P. Lunn,
R. Farley,
P. K. Lovelock,
S. A. Thomson,
D. A. Hume,
D. Lamb,
D. J. Porteous,
J. R. Dorin,
and
B. J. Wainwright.
Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype/phenotype correlations.
EMBO J.
15:
955-963,
1996[Abstract].
8.
Denning, G. M.,
M. P. Anderson,
J. F. Amara,
J. Marshall,
A. E. Smith,
and
M. J. Welsh.
Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive.
Nature
358:
761-764,
1992[Medline].
9.
Dorin, J. R.,
R. Farley,
S. Webb,
S. N. Smith,
E. Farini,
S. J. Delaney,
B. J. Wainwright,
E. W. F. W. Alton,
and
D. J. Porteous.
A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction.
Gene Ther.
3:
797-801,
1996[Medline].
10.
Drumm, M. L.,
D. J. Wilkinson,
L. S. Smit,
R. T. Worrell,
T. V. Strong,
R. A. Frizzell,
D. C. Dawson,
and
F. S. Collins.
Chloride conductance expressed by F508 and other mutant CFTRs in Xenopus oocytes.
Science
254:
1797-1799,
1991[Medline].
11.
Fuller, C. M.,
and
D. J. Benos.
CFTR!
Am. J. Physiol.
263 (Cell Physiol. 32):
C267-C286,
1992
12.
Gadsby, D. C.,
and
A. G. Nairn.
Regulation of CFTR channel gating.
Trends Biochem. Sci.
19:
513-518,
1994[Medline].
13.
Grubb, B. R.,
E. Lazarowski,
M. Knowles,
and
R. C. Boucher.
Isobutylmethylxanthine fails to stimulate chloride secretion in cystic fibrosis airway epithelia.
Am. J. Respir. Cell Mol. Biol.
8:
454-460,
1993[Medline].
14.
Hamosh, A.,
T. M. King,
B. J. Rosenstein,
M. Corey,
H. Levison,
P. Durie,
L.-C. Tsui,
I. McIntosh,
M. Keston,
D. J. H. Brock,
M. Macek,
D. Zemkova,
H. Krasnicanova,
V. Vavrova,
N. Golder,
M. J. Schwarz,
M. Super,
E. K. Watson,
C. Williams,
A. Bush,
S. M. O'Mahoney,
P. Humphries,
M. A. DeArce,
A. Reis,
J. Burger,
M. Stuhrmann,
J. Schmidtke,
U. Wulbrand,
T. Dork,
B. Tummler,
and
G. R. Cutting.
Cystic fibrosis patients bearing both the common missense mutation GlyAsp at codon 551 and the deltaF508 mutation are clinically indistinguishable from delta F508 homozygotes, except for decreased risk of meconium ileus.
Am. J. Hum. Genet.
51:
245-250,
1992[Medline].
15.
Hanrahan, J. W.,
F. Becq,
J. A. Tabcharani,
T. J. Jensen,
X.-B. Chang,
and
J. R. Riordan.
Use of phosphatase inhibitors as therapeutic agents to activate mutant CFTR.
Pediatr. Pulmonol. Suppl.
12:
152-153,
1995.
16.
Hwang, T.-C.,
M. Horie,
and
D. C. Gadsby.
Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl conductance in mammalian heart.
J. Gen. Physiol.
101:
629-650,
1993[Abstract].
17.
Hwang, T.-C.,
G. Nagel,
A. C. Nairn,
and
D. C. Gadsby.
Regulation of the gating of cystic fibrosis transmembrane conductance regulator Cl channels by phosphorylation and ATP hydrolysis.
Proc. Natl. Acad. Sci. USA
91:
4698-4702,
1994[Abstract].
18.
Letellier, M.,
N. Briere,
G. E. Plante,
and
C. Petitclerc.
Phosphate transport and alkaline phosphatase in confluent MDCK cell monolayers.
Can. J. Physiol. Pharmacol.
65:
1151-1156,
1987[Medline].
19.
Mackintosh, C.,
and
R. W. Mackintosh.
Inhibitors of protein kinases and phosphatases.
Trends Biochem. Sci.
19:
444-448,
1994[Medline].
20.
Manolopoulos, V. G.,
M. M. Samet,
and
P. I. Lelkes.
Regulation of the adenylyl cyclase signaling system in various types of cultured endothelial cells.
J. Cell. Chem.
57:
590-598,
1995.
21.
O'Loughlin, E. V.,
D. M. Hunt,
K. J. Gaskin,
D. Stiel,
I. M. Bruzuszcak,
C. O. Martin,
C. Bambach,
and
R. Smith.
Abnormal epithelial transport in cystic fibrosis jejunum.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G758-G763,
1991
22.
Snouwaert, J. N.,
K. K. Brigman,
A. M. Latour,
N. N. Malouf,
R. C. Boucher,
O. Smithies,
and
B. Koller.
An animal model for cystic fibrosis made by gene targeting.
Science
257:
1083-1088,
1992[Medline].
23.
Smith, S. N.,
J. R. Dorin,
S. J. Delaney,
D. M. Geddes,
B. Wainwright,
D. J. Porteous,
and
E. W. F. W. Alton.
Tracheal bioelectric characteristics of CF mouse models (Abstract).
Pediatr. Pulmonol. Suppl.
12:
214,
1995.
24.
Smith, S. N.,
D. M. Steel,
P. G. Middleton,
F. M. Munkonge,
D. M. Geddes,
N. J. Caplen,
D. J. Porteous,
J. R. Dorin,
and
E. W. F. W. Alton.
Bioelectric properties of exon 10 insertional cystic fibrosis mouse: comparison with humans.
Am. J. Physiol.
268 (Cell Physiol. 37):
C297-C307,
1995
25.
Tabcharani, J. A.,
X.-B. Chang,
R. J. Riordan,
and
J. W. Hanrahan.
Phosphorylation-regulated Cl channel in CHO cells stably expressing the cystic fibrosis gene.
Nature
352:
628-631,
1991[Medline].
26.
Takahara, N.,
F. Herz,
R. M. Singer,
A. Hirano,
and
L. G. Koss.
Induction of alkaline phosphatase activity in cultured human intracranial tumor cells.
Cancer Res.
42:
563-568,
1982[Abstract].
27.
Tomashefski, J. F., Jr.,
C. R. Abramowsky,
and
B. B. Dahms.
The pathology of cystic fibrosis.
In: Cystic Fibrosis, edited by P. B. Davis. New York: Dekker, 1993, p. 435-489. (Lung Biol. Health Dis. Ser., vol. 64)
28.
Van Doorninck, J. H.,
P. J. French,
E. Verbeek,
R. H. P. C. Peters,
H. Morreau,
J. Bijman,
and
B. J. Scholte.
A mouse model for the cystic fibrosis F508 mutation.
EMBO J.
14:
4403-4411,
1995[Abstract].
29.
Welsh, M. J.,
and
A. E. Smith.
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell
73:
1251-1254,
1993[Medline].
30.
Zeiher, B. G.,
E. E. Zabner,
J. J. Smith,
A. P. Puga,
P. B. McCray,
M. R. Capecchi,
M. J. Welsh,
and
K. R. Thomas.
A mouse model for the delta-F508 allele of cystic fibrosis.
J. Clin. Invest.
96:
2051-2064,
1995[Medline].