1 Children's Hospital Oakland
Research Institute, Cystic fibrosis (CF) affects a number of
epithelial tissues, including those in the gastrointestinal
tract. The goal of this review is to summarize data
related to regulation of the protein product of the CF gene, CF
transmembrane conductance regulator (CFTR), by a variety of small
molecules. There has been a surge of interest in discovering small
molecules that could be exogenously added to cells and tissues to
regulate CFTR and could potentially be used alone or in combination
with genetic approaches for therapy in CF. We will discuss the apparent
mechanisms of action of genistein, milrinone,
8-cyclopentyl-1,3-dipropylxanthine, IBMX, and NS-004; several of which
appear to interact directly with one or both nucleotide binding domains
of CFTR. We also discuss how HCO
cystic fibrosis; cystic fibrosis transmembrane conductance
regulator; pharmacology; epithelial transport; chloride
secretion
CYSTIC FIBROSIS (CF) is the most common fatal genetic
disease of the Caucasian population, with an incidence of 1 in 2,500 live births and a carrier frequency of ~1 in 25. The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) (30), which functions as a cAMP-activated
Cl Thus CFTR plays an important role in intestinal secretion. This
secretion is controlled by multiple hormones and nerve cell transmitters, which will couple to the activation of CFTR as well as
the other ion channels and transporters that are required to generate
transepithelial secretion. Phosphorylation of CFTR by protein kinases
(PK) and dephosphorylation by protein phosphatases (PP) is considered
the major way by which CFTR
Cl In addition to phosphorylation/dephosphorylation and ATP binding and
hydrolysis, investigators have searched for exogenous compounds that
are potential therapeutic activators of the mutant CFTR protein found
in CF patients. Figure 1 shows the chemical structures of several such small molecules that will be discussed in
this review. Some of these small molecules appear to bind directly to
one or both of the NBDs of CFTR and, if CFTR has been
prephosphorylated, increase the open probability of the channel. In the
first portion of this review, PHARMACOLOGICAL
REGULATION OF CFTR BY SMALL MOLECULES, we will discuss
work showing that genistein, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), IBMX and other xanthines, and NS-004 all seem to
operate this way. In addition, we will discuss the
phosphodiesterase (PDE) inhibitor milrinone, which also stimulates CFTR
but through a mechanism that is less well characterized.
ABSTRACT
Top
Abstract
Introduction
References
3 interacts with CFTR as both a permeating anion and a potential regulator of Cl
permeation
through the CFTR ion channel. It is likely that there are complicated
interactions between Cl
and
HCO
3 in the secretion of both ions
through the CFTR and the anion exchanger in intestinal cells, and these may yield a role of CFTR in regulation of intestinal
HCO
3 secretion as well as of intra-
and extracellular pH.
INTRODUCTION
Top
Abstract
Introduction
References
channel. At the cellular
level, CFTR dysfunction results in defective cAMP-regulated
Cl
conductance, primarily
in cells of epithelial origin (27). Although lung disease is the
primary cause of mortality in CF patients, a significant proportion of
the morbidity can be directly attributed to gastrointestinal
complications. The duodenum, jejunum, ileum, and colon express high
levels of CFTR mRNA (30). Immunocytochemical analysis also demonstrated
high CFTR protein expression at the luminal surfaces along the
intestine (7). The small intestine of CF patients exhibits decreased
Cl
and fluid secretion that
results in meconium ileus (lower water content and higher viscosity
compared with non-CF patients) in ~10% of all CF newborns and
accumulation of mucus and intestinal obstructions [primarily in
ileocecum and large intestine in >20% of adult patients (see Ref.
13)]. Intestinal pathophysiology appears to be the hallmark of
recently developed transgenic CF mouse models. The ileocecum and large
intestine appear to be the most common sites of intestinal blockade,
whereas jejunal obstructions occur less frequently (13).
channel activity is
physiologically regulated. In addition, the normal gating cycle (both
opening and closing) of CFTR requires ATP binding and hydrolysis at the
two nucleotide binding domains (NBDs) of CFTR (see Ref. 11). ATP
concentration is likely maintained constant in cells and is therefore
not a significant physiological regulator.
View larger version (14K):
[in a new window]
Fig. 1.
Chemical structures of compounds discussed in text. DPCPX,
8-cyclopentyl-1,3-dipropylxanthine.
In the second portion of this review,
HCO3
PERMEATION AND POSSIBLE REGULATION OF CFTR-MEDIATED CL
CURRENTS, we will
discuss the question of HCO
3 permeation and potential regulation of CFTR. This has been a
controversial area of research. Most secretory epithelia secrete both
Cl
and
HCO
3, and CFTR seems to be involved in a critical way that is still poorly understood. We will discuss data
relevant to the question of whether the key role of CFTR is to conduct
HCO
3 itself or to play some sort of
permissive role in HCO
3 secretion
through an adjacent anion exchanger that requires
Cl
at the cell's luminal
aspect to exchange for cellular HCO
3. Alternatively, CFTR could also be required to keep cellular
Cl
concentration low enough
that Cl
enters the cell on
the exchanger and exchanges for cellular
HCO
3. We will also discuss the
possibility that HCO
3 may regulate the
activity of CFTR by altering the permeation of Cl
due to the multi-ion
pore behavior of CFTR.
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PHARMACOLOGICAL REGULATION OF CFTR BY SMALL MOLECULES |
---|
Genistein. Genistein is a member of the large class of naturally occurring flavonoids. Some flavonoids are more potent than genistein in activating CFTR (19), but we will focus on genistein because it has been investigated the most thoroughly. Genistein is a potent activator of CFTR (e.g., Ref. 20, 22), but the activating mechanism has been debated. Genistein did not seem to operate through activation of protein kinase A (PKA), PKC, or PKG, because there were no increases in intracellular cAMP, Ca2+, or cGMP concentrations (21, 32). Genistein is a well-known tyrosine kinase (PTK) inhibitor, and two other PTK inhibitors (tyrphostin 47 and tyrphostin B42) also stimulated CFTR, and genistein-stimulated channel activity was blocked by the tyrosine phosphatase inhibitor vanadate (21, 32). However, several other PTK inhibitors (tyrphostin A23, tyrphostin A51, erbstatin analog, and herbimycin) failed to stimulate CFTR (20). Genistein also stimulated CFTR equally well when GTP (a poor substrate for tyrosine kinases) was used as a replacement for ATP (10). Some results suggested that genistein activated CFTR by blocking a PP: inactivation rates of CFTR currents on cAMP removal were markedly decreased by genistein (20), and CFTR was phosphorylated on the same amino acids by both genistein (which did not raise cAMP concentration) and forskolin (which did) (29).
More recent data have indicated that the most likely explanation for the stimulatory effect of genistein was through direct binding to an NBD of phosphorylated CFTR. Genistein did not directly open CFTR channels, because genistein did not stimulate CFTR in ATP- or cAMP-depleted monolayers (22). Similarly, genistein activation of CFTR in excised patches required both ATP and prior phosphorylation by PKA (10). In addition, genistein stimulated phosphatase-resistant, thiophosphorylated CFTR, even in the presence of PKI (to block PKA) or VO4 (to block PP) (10, 39). Kinetic analysis indicated that a genistein concentration of <35 µM induced prolonged openings of CFTR (similar to the effect of a nonhydrolyzable ATP analog), whereas a genistein concentration of >35 µM caused prolonged closed times (38). An attractive model for stimulatory and inhibitory effects of genistein on CFTR is that of Gadsby et al. (11) for CFTR gating. This model (11) proposes that ATP binding and hydrolysis at NBD1 control channel opening and closing at low phosphorylation levels, and once ATP hydrolysis at NBD1 has opened a highly phosphorylated CFTR channel, ATP binding to NBD2 can stabilize the open state. Hydrolysis of the nucleotide at NBD2 terminates the stabilization. Agents that interfere with ATP hydrolysis at NBD2 [e.g., 5'-adenylylimidodiphosphate (AMP-PNP) or pyrophosphate] induce prolonged burst opening. Genistein is a competitive inhibitor with ATP for binding to PTK (inhibitor constant, 13.7 µM) and to other ATP-binding proteins, and crystal structure analysis of the Src family tyrosine kinase Hck revealed that the structurally related flavone and CFTR activator quercetin (19) localized to the binding site for the adenine ring of AMP-PNP or ATP (34). When considering the sequence homology shared by Walker A-type-binding motifs of PTK and NBDs of CFTR, it therefore seems possible that genistein competes for ATP-binding sites on CFTR, possibly at NBD2 (10), thereby slowing down ATP hydrolysis and preventing CFTR channel closure. It is unlikely that genistein activates CFTR by binding to a Src kinase, because this would require activation of a tyrosine kinase and genistein blocks tyrosine kinases. It might also be proposed that higher concentrations of genistein inhibit CFTR by competing with ATP at NBD1 (which may have a lower affinity for genistein binding) and prevent CFTR from opening.NS-004.
The substituted benzimidazolone NS-004 has effects that are quite
similar to those exhibited by genistein. NS-004 activated CFTR (at 0.1 to 1 µM) and F508-CFTR (at >10 µM) without raising cAMP
concentration and also in the presence of a PKA inhibitor (12), but it
was necessary to pretreat cells with forskolin before patch excision to
observe CFTR channel activation by NS-004. In addition, the drug caused
active CFTR channels to increase their activity, but it had no effect
when added after channel rundown, indicating that NS-004 required
phosphorylated CFTR. NS-004 did not inhibit in vitro PP activity. Thus,
although NS-004 was thought to operate by directly opening CFTR (12),
the drug may use a mechanism similar to that of genistein to increase
open probabilities of phosphorylated, active channels without affecting either PK or PP.
Milrinone.
The cardiotonic drug milrinone is an inhibitor of cGMP-inhibited PDE (a
class III PDE) and was selected from several drugs inhibiting PDE
isozymes (23). Because PDE inhibitors raise cellular cAMP concentration
and CFTR is activated by increases in cAMP concentration, it has been
assumed that milrinone (and also IBMX, see below) stimulate CFTR by
raising cAMP. However, the exact mechanisms have not been determined.
Milrinone activated both normal and F508-CFTR in transformed nasal
epithelial cells (24), and a combination of forskolin and milrinone
(but not milrinone alone) increased the potential difference across
nasal epithelium of
F508-CFTR mice in vivo but not in transgenic
mice lacking CFTR (24). Milrinone activated CFTR even when cells had
been maximally stimulated with forskolin or some other cAMP agonist, indicating that the drug likely had other effects than to stimulate production of cAMP.
IBMX, DPCPX, and other xanthine derivatives. IBMX is an inhibitor of PDE with broad specificity. Despite its broad use as a PDE inhibitor, it seems unlikely that IBMX works solely via this mechanism. IBMX activated CFTR even when cells had been maximally stimulated with forskolin or some other cAMP agonist (23). Although other substituted xanthine derivatives activated CFTR, there was no correlation between effects on CFTR and cellular cAMP and ATP concentrations (4). We have also found, through use of a fluorescence assay for PKA activity (as opposed to measurements of cAMP concentration using RIA), that forskolin caused fibroblasts to raise cAMP concentration to such a high level that PKA activity was saturated (by maximally binding cAMP), and IBMX (which indeed raised cAMP concentration) had no further effect to increase PKA activity (B. Eckert and T. E. Machen, unpublished observations). It therefore seems likely that the stimulatory effects of IBMX on CFTR are due to combined effects to raise cAMP (when PKA has not been saturated) and also to block PP or directly bind to CFTR.
The xanthine derivative DPCPX is an adenosine A1 antagonist that may have some selectivity forPharmacological stimulation of mutant CFTR and therapeutic
potential.
The most common mutation in CF, F508-CFTR, leads to a
trafficking-impaired protein that gets degraded in the cell (40). Some
mutations lead to trafficking-competent, but misfunctional, proteins
(e.g., G551D-CFTR; Ref. 40). It appears that mutants with different
misfunctions will need selective treatments. For example, genistein
failed to activate
F508-CFTR in CF bronchial epithelial cells (22),
but addition of genistein and cAMP agonists to NIH/3T3 cells
overexpressing
F508-CFTR caused a strong, synergistic activation
(18). Alternatively, when CF nasal epithelial cells were treated with
4-phenylbutyrate or low temperature to increase expression of
F508-CFTR in the membrane (31, 40), genistein was a potent stimulant
(9). In addition, the trafficking-competent G551D-CFTR mutant expressed
in fibroblasts was stimulated by the combination of genistein and
forskolin when forskolin alone was ineffective (9). These data indicate
that genistein targets CFTR mutants present in the plasma membrane.
Both NS-004 and, especially, DPCPX have also been shown to activate
F508-CFTR. IBMX produced a small, CFTR-related secretory response in
jejunum, cecum, and rectum from G551D mice but had no effect in the
nasal epithelium (36). NS-004 restored near normal channel activity from P574H-CFTR (a mild, trafficking-impaired mutation in NBD1) (3).
These results suggest that, in addition to their direct effects on
CFTR, these drugs may also have other effects that increase the number
of channel proteins found in the membrane. It is also important to
remember that there may be tissue-specific effects of the drugs,
because epithelial cells will require concomitant activity of
basolateral K+ channels to secrete
fluid efficiently.
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HCO![]() ![]() |
---|
Background: Role of CFTR in
HCO3 secretion across
intestine.
HCO
3 secretion is a key function that
occurs in the stomach, pancreas, and small and large intestine.
HCO
3 secretion is particularly
important in the duodenum to protect the intestinal mucosa against
damage from large amounts of acid from the stomach (1). Transepithelial
secretion of HCO
3 likely involves the
concerted activities of an apical anion channel, Cl
/HCO
3
exchange, HCO
3 uptake across the
basolateral membrane, and/or production of
HCO
3 by intracellular carbonic
anhydrase activity. The paracellular pathway may also contribute, since
a transepithelial gradient for HCO
3
favoring secretion occurs. Electrogenic HCO
3 secretion is activated by
secretagogues that increase intracellular cAMP, cGMP, or
Ca2+ concentrations (1), and this
HCO
3 secretion required the presence
of CFTR in all segments of the small intestine in mice (16, 17, 33).
However, the specific mechanism(s) involved remains a mystery. With the
use of the pH stat method in combination with inhibitors of anion
exchange and CFTR, recent work (5) with duodenum from normal and CFTR
knockout mice shows that CFTR may have contributed to
HCO
3 secretion in two ways: directly
by conducting HCO
3 into the lumen and
also indirectly by conducting
Cl
into the lumen, which
could be recycled back into the cell via anion exchange for
HCO
3. Because CFTR is expressed at
highest levels in the crypts (7), electrogenic
HCO
3 secretion is predicted to occur
primarily in this region. In contrast, CFTR is expressed at lower
levels and carbonic anhydrase activity is expressed at higher levels in
the villus epithelium, so CFTR-coupled Cl
/HCO
3
exchange activity may be primarily localized to the villus.
Does CFTR conduct HCO3?
Patch-clamp studies showed that HCO
3
was conducted through CFTR in NIH/3T3 cells recombinantly expressing wild-type CFTR (26), with a permeability ratio of
Cl
to
HCO
3 of 0.25. Similar
measurements have also been presented recently by Linsdell et al. (25),
who found a permeability ratio of
Cl
to
HCO
3 of 0.14. We also measured
CFTR-mediated permeation of HCO
3
across the apical membrane of Calu-3 monolayers, and conductance ratios
of cAMP-stimulated HCO
3 and
Cl
currents of
0.1-0.27 were obtained (22). Thus
HCO
3-to-Cl
permeability or conductance ratios of CFTR ranged from 0.10 to 0.27. A
role of CFTR's function as a HCO
3
conductor has also been suggested in CF models using a variety of
methods comparing wild-type CFTR-expressing cells with
F508-CFTR-expressing CF cells (22, 35) or wild-type with CFTR
knockout mice (CFTR
/
) (5, 13, 17, 33). These reports
suggested the possibility that a defect in
HCO
3 secretion through CFTR may
contribute to the pathophysiology of CF pulmonary disease (22, 35). In
contrast to these experiments that showed that CFTR conducts
HCO
3, Quinton and Reddy (28) (P. Quinton, personal communication) showed using microelectrode methods
that in intact sweat ducts HCO
3 was equally impermeant through CFTR as gluconate. This contradiction remains unresolved.
Does CFTR contribute to cytosolic and/or extracellular pH
regulation?
If CFTR is permeant to HCO3, it is
expected that it should contribute to movements of
HCO
3 into and out of the cell and
thereby alter pH of the cell or extracellular fluids. In NIH/3T3 cells
expressing exogenous CFTR, we showed forskolin-stimulated changes of pH
that were absent in cells expressing
F508-CFTR (26). Data have also
been presented for biliary epithelial cells showing that CFTR may
regulate the activity of the anion exchanger and alter cytosolic pH
regulatory ability in these cells (14) In contrast, we have recently
compared cytosolic pH regulation in CF and CFTR-corrected nasal cells
and found no apparent differences (L. Lu, E. Wunderlich, and T. E. Machen, unpublished results). A possible explanation was that these
cells expressed
Na+/H+
and
Cl
/HCO
3
exchange and
Na+-HCO
3
cotransport, and these mechanisms were much more prominent than CFTR in
regulating cytosolic pH. Thus, in cells that express prominent activity
of transporters that regulate cytosolic pH, the conductive pathway for
HCO
3 across CFTR might be of minor
importance for cytosolic pH regulation. However, CFTR might still
contribute importantly to the accumulation of
HCO
3 in the poorly buffered
apical/luminal fluid.
Do anions regulate Cl permeation
through CFTR?
It has been well described that thiocyanate (SCN) is highly permeant
across CFTR, but when mixtures of
Cl
and SCN are used, the
conductance of CFTR decreases (37). This effect has been
interpreted in terms of multi-ion pore behavior of CFTR, i.e.,
interactions of anions within the pore can lead to anomalous effects.
Also, the halide anion I
both permeated and blocked
CFTR. We have similarly shown that both
F
and
HCO
3 added to the apical surface
reduced Cl
conductance of
CFTR in Calu-3 cells (Illek and Machen, unpublished observations). In
vivo both Cl
and
HCO
3 are present in the cytosol at
similar concentrations, and both are driven by the membrane potential into the pore of CFTR, suggesting that both ions may compete for common
binding sites in the permeation path, and thus affect the permeation of
each other. This could be particularly important for tissues that
secrete large amounts of HCO
3.
Summary: Role of CFTR in HCO3
secretion.
The exact role of CFTR in HCO
3
permeation under physiological conditions likely depends on the
relative expression of CFTR and other
HCO
3 channels, HCO
3 transporters and paracellular
permeability of HCO
3. In addition, the
cell-to-lumen electrochemical driving forces for
Cl
and
HCO
3 will determine ion movements
across the luminal cell membrane and tight junctions. CFTR appears to be important both as a HCO
3
conductance and also in some way as a direct or indirect regulator of
adjacent anion exchange, e.g., as a source of luminal
Cl
that may exchange for
cellular HCO
3 and/or as a
means of keeping cellular
Cl
concentration low enough
that the anion exchanger operates as an
HCO
3 secretion mechanism. Many
CFTR-expressing epithelia, including the intestine, pancreas, and
liver, exhibit different gradients of
Cl
and
HCO
3 along the length of the organ, so these relationships will also be affected by the anatomic structures.
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ACKNOWLEDGEMENTS |
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We apologize to those whose work we were unable to quote due to space limitations.
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FOOTNOTES |
---|
* Second in a series of invited articles on Genetic Disorders of Membrane Transport
Study in our laboratories has been supported by grants from the National Institutes of Health (R01-DK-51799), Cystic Fibrosis Research, Inc., the Cystic Fibrosis Foundation (Grant ILLEK96F0), and the Commercial Endowment of Children's Hospital Oakland.
Address for reprint requests: T. E. Machen, Dept. of Molecular and Cell Biology, Univ. of California, Berkeley, CA 94720.
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