1 Department of Pathology and 2 Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia 30322; 3 Combined Program in Pediatric Gastroenterology and Nutrition, Department of Medicine, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115; and 4 Department of Pathology, University of California College of Medicine, Irvine, California 92717
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ABSTRACT |
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Opening of
anion-conductive pathways in apical membranes of secretory cells lining
mucosal surfaces is a critical step in salt and water secretion and,
thus, hydration of sites including airway and intestine. In intestine,
Paneth cells are positioned at the base of the secretory gland (crypt)
and release defensin peptide, in mice termed cryptdins, into the crypt
lumen. Because at least some defensins have been shown to form
anion-conductive channels in phospholipid bilayers, we tested whether
these endogenous antimicrobial peptides could act as soluble inducers
of channel-like activity when applied to apical membranes. To directly
evaluate the possibility of cryptdin-3-mediated apical anion
conductance (Gap), we have utilized amphotericin
B to selectively permeabilize basolateral membranes of electrically
tight monolayers of polarized human intestinal secretory epithelia (T84
cells), thus isolating the apical membrane for study. Cryptdin-3
induces Gap that is voltage independent
(Gap = 1.90 ± 0.60 mS/cm2) and exhibits ion selectivity contrasting to that
elicited by forskolin or thapsigargin (for cryptdin-3,
Cl
= gluconate; for forskolin and thapsigargin,
Cl
gluconate). We cannot exclude the possibility that
the macroscopic current induced by cryptdin could be the sum of cation
and Cl
currents. Cryptdin-3 induces a current in
basolaterally permeabilized epithelial monolayers derived from airway
cells harboring the
F508 mutation of cystic fibrosis (CF;
Gap = 0.80 ± 0.06 mS/cm2), demonstrating that cryptdin-3 restores anion
secretion in CF cells; this occurs independently of the CF
transmembrane conductance regulator channel. These results support the
idea that cryptdin-3 may associate with apical membranes of
Cl
-secreting epithelia and self-assemble into conducting
channels capable of mediating a physiological response.
intestinal epithelium; conducting channels; amphotericin B
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INTRODUCTION |
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PANETH CELLS RESIDE
at the base of small intestinal crypts and release granule contents
into the crypt lumen (17). Residency at the crypt base
places Paneth cells in a position to affect the biology of adjacent
epithelial cells lining the intestinal crypt, including the intestinal
stem cells and the cell type making up the bulk of the tubular crypt,
i.e., the "undifferentiated" Cl-secreting crypt cell
(1).
Along with other soluble proteins, mature human Paneth cells
secrete antimicrobial -defensins, termed cryptdins (17, 18, 21, 23). Mouse and human Paneth cell
-defensins exhibit
selective activity against several microorganisms
(21). These antimicrobial activities derive from the
ability of
-defensins to permeabilize microbial membranes by
self-assembly into anion-conductive channels (3, 4, 7,
24). Many known antimicrobial peptides (e.g., cecropin,
magainin, and dermaseptin) exhibit cytotoxicity against a diverse array
of microorganisms but do not display cytotoxicity against normal
eukaryotic cells (3, 6, 12-13, 22).
We recently found that cryptdins-2 and -3 interact with apical
membranes of human intestinal T84 cells to induce a physiological Cl secretory response (11). The secretory
response induced by cryptdin-3 appears to correlate with
permeabilization of the apical membrane, as judged by use of
membrane-impermeant solutes; however, activation of the endogenous
cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel
(or an endogenous Ca2+-regulated channel) could not be
ruled out. These data support the interpretation that the cryptdin-3
effect was not mediated by endogenous CFTR and included the fact that
cryptdin-3 did not induce detectable elevations of intracellular cAMP
or cGMP and did not activate apical membrane adenosine receptors
(11). On the basis of these results, we hypothesized that
cryptdin-3 may act as a novel paracrine regulator of intestinal
secretion by forming anion-conductive channels within the apical
membrane of neighboring Cl
-secreting crypt epithelial cells.
We now test this idea by defining the biophysical characteristics of
cryptdin-3-induced channels. To do so, we electrically isolate the
apical membrane of intact T84 cell monolayers by selectively permeabilizing basolateral membranes with amphotericin B. Our data show
that cryptdin-3 induces apical membrane anion channels that display
biophysical features fundamentally different from those displayed by
endogenous apical membrane cAMP- and Ca2+-dependent
Cl channels. We also find that cryptdin-3 induces
Cl
secretion in CF (JME/CF15) cells. JME/CF15
cells do not contain the functional cAMP-dependent Cl
channel CFTR (5, 19). Thus cryptdin-3 induces a novel
apical conductance(s) (Gap) in human intestinal
and tracheal epithelial cell lines. These data are consistent with the
idea that cryptdin-3 associates with apical membranes of
Cl
-secreting epithelia and self-assembles into conducting
channels capable of mediating a physiological secretory response.
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METHODS |
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Cell culture. T84 cells (American Type Culture Collection), a human colonic carcinoma cell line that functionally and morphologically resembles crypt intestinal epithelia, were grown as confluent monolayers in a 1:1 mixture of Dulbecco's Vogt modified Eagle's medium and Ham's F-12 medium supplemented with 15 mM HEPES buffer (pH 7.5), 14 mM NaHCO3, 40 µg/ml penicillin, 90 µg/ml streptomycin, and 5% newborn calf serum. Monolayers were subcultured every 7 days by addition of 0.1% trypsin and 0.9 mM EDTA in Ca2+/Mg2+-free PBS and grown on collagen-coated permeable supports (area 0.3 cm2, pore size 0.4 µm). All experiments were performed using cells between passages 65 and 92.
Immortalized JME/CF15 cells from CF patients (Cryptdin-3 purification and synthesis. Synthetic, folded, and oxidized cryptdin-3 was prepared as described previously (21). Synthetic and natural cryptdin-3 peptides were shown to have identical physiochemical and antimicrobial characteristics (21).
Transepithelial current measurement.
Studies were carried out at 37°C with the use of confluent monolayers
plated on collagen-coated permeable supports and examined 7-16
days later, as previously described (16). Before all
studies, inserts were washed with HCO3-free medium
warmed to 37°C and transferred to new 24-well tissue culture plates
containing the experimental medium. To determine currents,
transepithelial potentials, and conductances, a commercial voltage
clamp (Bioengineering Department, University of Iowa, Iowa City, IA)
was interfaced with equilibrated pairs of calomel electrodes submerged
in saturated KCl and with paired Ag-AgCl electrodes submerged in the
experimental medium maintained at constant temperature (37°C). Apical
and basolateral experimental medium volume remained constant during the
course of the experiment, and the electrodes were stably placed on
either side of the monolayers. Positive currents correspond to anion
secretion/cation absorption, i.e., a lumen-negative potential under
open-circuit conditions. Before each experiment, a blank filter was
used to compensate for the fluid resistance and the resistance of the
filter. In some experiments, transepithelial voltage, short-circuit
current (Isc), and conductance were continuously
recorded with the aid of an analog-to-digital converter (MacLab, Word
Precision Instruments) and a microcomputer.
Measurement of conductance(s) of the apical plasma membrane. To evaluate the ion conductance of the isolated apical plasma membrane, the polyene ionophore amphotericin B (8) was added to the basolateral solution at 100 µM, the lowest concentration that gave a maximal change in steady-state conductance (16). Under these conditions, because of its requirement for cholesterol, amphotericin B permeabilizes only the plasma membrane domain in direct contact with the amphotericin B solution, as previously demonstrated (9, 10, 15, 16). As a result, the other plasma membrane becomes rate limiting for overall, transepithelial ion transport, and "stimulus"-dependent changes of its ion conductances can be assessed as Isc due to ion gradients or as transepithelial conductances changes.
Basolateral membrane in our case incorporates the ionophore, thus electrically isolating the opposing (apical) plasma membrane. After addition of amphotericin B (100 µM, submucosal solution), tissue conductance increased from 0.6 ± 0.08 to 2.8 ± 0.50 mS/cm2 (n = 50) over the course of the experiment. To study the Cl ![]() |
RESULTS |
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Cryptdin-3 increases Gap in basolaterally permeabilized
T84 cell monolayers.
To test the idea that cryptdin-3 may induce Cl secretion
in secretory epithelia by self-assembling into novel anion-conducting channels, we studied confluent monolayers of T84 cells in which the
basolateral membranes had been selectively permeabilized with amphotericin B. Initial experiments were performed in the presence of
an apically directed 137 mM Cl
gradient, with
Cl
representing the major membrane-permeant ion (Table
1; medium A, basolateral;
medium B, apical). Under these conditions with the
transepithelial voltage clamped to 0 mV, baseline
GCl(ap) was 3.10 ± 0.40 mS/cm2
(n = 3). Figure 1 shows
that addition of 50 µg/ml cryptdin-3 stimulated
GCl(ap) by 1.40 ± 0.6 mS/cm2
(n = 3). Peak conductances were observed after
~10-min incubations and returned to baseline within 30 min. These
data demonstrate that cryptdin-3 increases Gap
of T84 cell monolayers in the absence of an applied membrane potential
and in a nonsustained manner. The characteristics of the
cryptdin-3-induced GCl(ap) contrast with those
of endogenous GCl(ap) induced using forskolin or
thapsigargin as secretory agonists (16).
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Biophysical characteristics of cryptdin-3-induced Gap.
To distinguish between anion transport mediated by exogenous
cryptdin-3-based channels and that mediated by endogenous apical membrane cAMP- and Ca2+-dependent Cl
channels, the voltage dependency and size selectivity of
cryptdin-induced anion conductances were defined. In these experiments,
basolaterally permeabilized T84 cell monolayers were studied in
symmetrical buffers containing Cl
as the major
membrane-permeant ion.
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Cryptdin-3 activates Gap in epithelial cells deficient
in functional CFTR.
To further distinguish between Gap mediated by
cryptdin-3-based channels and Gap mediated by
the endogenous cAMP-dependent Cl channel CFTR, we used
the airway cell line JME/CF15 containing CFTR with the inactivating
F508 mutation. These epithelial cells, derived from a patient with
CF, grow in monolayer culture with high transepithelial resistance but
do not exhibit cAMP-dependent Cl
secretion because of the
inactivating mutation in CFTR (5).
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DISCUSSION |
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Members of the cryptdin family display an amphipathic structure constrained by three disulfide bridges. Several studies indicate that these peptides act by altering the permeability of biological membranes. However, the mechanism of the membrane permeabilization is not well understood.
In the present study we found that cryptdin-3, at concentrations (50 µg/ml) comparable to those at which they exert antimicrobial effects
(22), increased Gap in
basolaterally permeabilized T84 cell monolayers. The reversible effect
of cryptdin-3 could be that cryptdin channels could be removed from the
apical membrane by partitioning into the aqueous phase or by
endocytosis. More specifically, cryptdin-3 induced voltage-independent
Gap in apical membranes of T84 cells. In
previous studies on artificial bilayers (7), human
-defensin human neutrophil peptide-1 (HNP-1) induced voltage-dependent ionic conductances. This discrepancy may reflect differences in the lipid composition of T84 cell membranes vs. phospholipid bilayers, presence or absence of phosphates, divalent cations, other factors present in T84 cells but not in bilayers, or
structure-function differences in monomeric cryptdin-3 and dimeric
HNP-1. Alternatively, a voltage-dependent conformation in T84 cells
could be short lived and progress rapidly to a voltage-independent conductance.
The increase of Gap by cryptdin-3 in the
basolaterally permeabilized T84 cells could be the result of
1) formation of novel conductive channels by this peptide or
2) the peptides (cryptdin-3) perturbing lipid organization
and, thereby, enhancing endogenous apical membrane channels (CFTR or
Ca2+-dependent Cl channel). The present data
show that 1) the combination of forskolin and cryptdin-3
induces an increase in conductance greater than that stimulated by
forskolin alone; 2) cryptdin-3, but not thapsigargin, stimulates Gap at 0 mV; and 3)
cryptdin-3 does not increase cAMP, cGMP (11), or
Ca2+ (data not shown) in intact cells. It is unlikely that
cryptdin-3 activates volume-sensitive channel(s) (volume-sensitive
Cl
channels are Ca2+ and voltage dependent),
since channels formed by cryptdin-3 are Ca2+ and voltage
independent. These results, in combination with a recent study
(25) demonstrating that cryptdin-3 forms anion-selective channels in cytoplasmic membranes of human embryonic kidney cells, suggest that, as reported for human myeloid defensins, it is likely that cryptdin-3 self-assembles into anion-conducting channel(s) by
interacting with the apical plasma membrane of T84 cells.
The ability of other anions to substitute for Cl in
transepithelial anion secretion has been determined in intact
monolayers of T84 cells. For example, in a previously reported study,
when Cl
is replaced by gluconate, the
cryptdin-3-dependent Isc was nearly abolished (11). Net secretion, however, reflects
the selectivities of two serial steps in transepithelial movement:
uptake at the basolateral membrane and exit across the apical membrane,
with the overall selectivity determined by the most restrictive step. The basolateral cotransport process responsible for anion uptake from
the serosal solution restricts influx of halides other than Cl
and determines the anion selectivity for secretion. In
contrast, in basolaterally permeabilized monolayers, the only
restrictive step for anion secretion is represented by the apical
plasma membrane. In the present study we find that when
Cl
is replaced by gluconate, the increase of
Gap by forskolin and thapsigargin is completely
ablated. In contrast, the Gap stimulated by
cryptdin-3 was only slightly reduced in the presence of gluconate. Furthermore, as previously reported and confirmed here, forskolin and
thapsigargin have no effect on gluconate current (16). In addition, the finding that, in the presence of gluconate as the major
charge carrier, the increase of Gap stimulated
by the combination of forskolin and cryptdin-3 is only partially
reduced is consistent with the residual current being a gluconate
current that is cryptdin-3 dependent. Interestingly, the
cryptdin-3 channels are more selective to Cl
than
HPO42
and Na+, showing its preference for
Cl
. We cannot exclude the possibility that the
macroscopic current induced by cryptdin could be the sum of the cation
and Cl
currents. The question of anion selectivity
induced by cryptdin-3 could be solved using methods such as patch clamp
and lipid bilayer. With the use of single-channel analysis, it has also
been recently reported that cryptdin-3 at low concentration (1 µg/ml)
may activate an anion-selective channel with unique features, including
a conductance of 15 pS (25). As for other defensins, the
selectivity of the channel(s) formed by cryptdin-3 could be dose
dependent (4, 25).
In the most common form of CF, the gene that encodes for CFTR anion
channel is mutated, resulting in a protein that is capable of
conducting Cl (19) but is absent from the
plasma membrane because of aberrant intracellular processing (2,
20). For example, in CF airway epithelia, Ca2+
stimulated Ca2+-sensitive (i.e., non-CFTR) Cl
channels, but the response to cAMP (via CFTR) is defective. Recently, many studies have been focused on methods to promote functional correction of cAMP-mediated Cl
transport in CF epithelial
cells. These studies have included pharmacological approaches aimed at
restoration of the amount of
F508-CFTR on the surface epithelium of
patients with CF as well as gene-targeting strategies to promote normal
Cl
secretion in epithelia (2). One logical
extension of our present study was to investigate the effect of
cryptdin-3 on CF cells. Using an airway CF cell line (JME/CF15), we
show that cryptdin-3 promotes a current in permeabilized CF monolayers.
These results show that cryptdin-3 induces a restoration of
Cl
secretion in CF cells that is probably not cAMP or
CFTR dependent.
In summary, our results demonstrate that cryptdin-3 induces
Gap with ion selectivity different from that
elicited by forskolin or thapsigargin. Cryptdin-3 is positively charged
at physiological pH and might bind to negatively charged phospholipid
membranes with the aid of electrostatic interactions, forming
channel(s) and permeabilizing the apical plasma membrane of normal and
CF phenotypes of epithelial cells, even in the absence of the
transmembrane potential. Such cryptdin-dependent channel formation is
capable of supporting electrogenic Cl secretion in cells
electrochemically poised for this key physiological event.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants DK-35932 (J. L. Madara and W. I. Lencer) and AI-22931 (M. E. Selsted) and Large Scale Biology (M. E. Selsted). This work was initiated with National Institute of Diabetes and Digestive and Kidney Diseases National Research Service Award DK-09800 (D. Merlin). D. Merlin is a recipient of a Career Development Award from the Crohn's and Colitis Foundation of America.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Merlin, Dept. of Pathology and Laboratory Medicine, Emory University, WMRB-2, Rm. 2329, 1639 Pierce Dr., Atlanta, GA 30322 (E-mail: dmerlin{at}emory.edu).
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 27 April 2000; accepted in final form 8 September 2000.
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