Department of Medicine, University of California San Diego School of Medicine, San Diego, California 92103-8414
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ABSTRACT |
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IFN- inhibits intestinal
Cl
secretion, in part via downregulation of CFTR and
Na+-K+-ATPase activity and expression, but the
proximal signaling events were unknown. We have shown that transforming
growth factor-
(TGF-
) inhibits calcium-activated Cl
secretion, and effects of IFN-
in other systems are mediated via EGF
family members. We tested whether IFN-
inhibits Cl
secretion via EGF receptor (EGFr) activation. IFN-
increased tyrosine phosphorylation in T84 cells at 24 h, including the EGFr. IFN-
also increased cell-associated pro-TGF-
, as well as free TGF-
in the bathing media. However, whereas IFN-
significantly inhibited carbachol-induced Cl
secretion, neither
neutralizing antibodies to TGF-
nor an EGFr inhibitor (1 µM
tyrphostin AG 1478) were able to reverse this inhibitory effect. AG
1478 also failed to reverse IFN-
-induced tyrosine phosphorylation of
the EGFr, but receptor phosphorylation was attenuated by both the
neutralizing antibody to TGF-
and PP2, a Src kinase inhibitor.
Moreover, PP2 reversed the inhibitory effect of IFN-
on
Cl
secretion. In total, our findings suggest an increase
in functional TGF-
and activation of the EGFr in response to
IFN-
. The release of TGF-
and intracellular Src activation likely
combine to mediate EGFr phosphorylation, but only Src appears to
contribute to the inhibition of transport. Nevertheless, because
TGF-
plays a role in restitution and repair of the intestinal
epithelium after injury, we speculate that these findings reflect a
feedback loop whereby IFN-
modulates the extent of cytokine-induced
intestinal damage.
chloride secretion; cytokines; inflammation; growth factors; mucosal injury
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INTRODUCTION |
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CHLORIDE SECRETION ACROSS intestinal epithelial cells is regulated by various paracrine, neurocrine, and endocrine factors (4). Such factors bind specific receptors on the epithelial cell surface and thereby stimulate elevations in the levels of various second messengers, such as cyclic nucleotides and calcium. These second messengers activate the transport machinery that comprises the chloride secretory pathway (4). This process is under tight control, and breakdown can lead to overexpression of secretion, resulting in diarrhea. Whereas the mechanisms responsible for initiating the secretory response have been well described, the negative signaling pathways are only now being worked out.
Our laboratory has identified various inhibitory pathways capable of
regulating the extent of the secretory response. We have shown that
pretreatment of T84 human colonic epithelial cells with muscarinic
agonists can render the monolayer refractory to subsequent stimulation
by other calcium-dependent secretagogs. The generation of the
D-isomer of inositol 3,4,5,6-tetrakisphosphate [D-Ins(3,4,5,6)P4], initiation
of the influx of extracellular calcium, and activation of PKC and MAPK
have all been implicated in mediating these muscarinic effects
(3, 17). We have also demonstrated that epidermal growth
factor (EGF) is able to inhibit calcium-activated chloride secretion in
T84 cells (31) via modification of potassium channel
function secondary to activation of the phosphatidylinositol 3-kinase
pathway (32). This finding is important in that EGF and
another EGF receptor (EGFr) ligand produced by intestinal epithelial
cells, transforming growth factor- (TGF-
), have also been shown
to possess various healing functions in the gastrointestinal tract,
including control of acid, bicarbonate, and mucus secretion, control of
gastrointestinal blood flow, the initiation of epithelial/mucosal restitution, and protection of the mucosa (30).
Upregulation of TGF-
levels and the EGFr also occurs in the
gastrointestinal tract in response to injury and inflammation (3,
7, 23, 28). This may suggest that receptor tyrosine kinase
pathways serve an important role in countering the inflammatory
response and initiating the healing process.
IFN- is a 45-kDa dimeric glycosylated protein whose production is
upregulated under a variety of pathological circumstances, such as
trauma, infection, cancer, and autoimmunity (6). As a
cytokine, it is thought to regulate and amplify the immune response, induce tissue injury, and mediate complications of the inflammatory response in the intestine, such as diarrhea and fibrosis
(25). However, cytokines also have a critical role in
suppressing inflammation and mediating repair and healing
(25). IFN-
has direct effects on epithelial cells, such
as changes in cell morphology, decreased barrier function, increased
TNF-
receptor and myosin heavy chain II molecule expression, and
decreased levels of proteins involved in transport and barrier function
(1, 5, 11, 15, 19, 29). IFN-
has also been shown to
inhibit chloride secretion (11), and downregulation of
CFTR, Na+-K+-2Cl
cotransporter
(NKCC1), and Na+-K+-ATPase expression
has been strongly implicated in this effect (15, 29, 35).
However, the signaling pathways that initiate this downregulation have
not been examined. Various cytokines, including IFN-
, are able to
induce the expression of growth factors in various cell types, and it
is believed that many of the effects of cytokines may be attributed to
these newly expressed growth factors (2, 14, 27).
Therefore, the goal of our study was to determine whether IFN-
is
able to activate the EGFr and/or increase levels of functional TGF-
and inhibit chloride secretion in intestinal epithelial cells via these
downstream signals.
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MATERIALS AND METHODS |
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Materials.
Carbachol and tyrphostin AG 1478 (Sigma, St. Louis, MO), IFN- (R&D
Systems, Minneapolis, MN), TGF-
(Santa Cruz Biotechnology, Santa
Cruz, CA), polyclonal rabbit anti-TGF-
(Chemicon, Temecula, CA),
monoclonal mouse antiphosphotyrosine and anti-EGFr (Upstate Biotechnology, Lake Placid, NY), PP2, and TGF-
ELISA kit
(Calbiochem, La Jolla, CA) were obtained from the sources indicated.
All other reagents were of at least reagent grade and were obtained commercially.
Cells.
Methods for the maintenance of T84 cells for use in transepithelial
electrolyte transport studies have been described previously (12). In brief, T84 cells were grown in DMEM/Ham's
F-12 (JRH, Lenexa, KS), with the addition of 5% newborn calf
serum. Cells were passaged by trypsinization. For the measurement of
chloride secretion, 2.5 × 105 cells were seeded onto
12-mm Millicell-HA Transwells (Millipore, Bedford, MA). For experiments
involving immunoprecipitation and Western blotting, and for the TGF-
ELISA, 106 cells were seeded onto 30-mm Millicell-HA
Transwells. All cells were cultured for 7-10 days before use.
Chloride secretion.
Chloride secretion was measured as short-circuit current
(Isc) across monolayers of T84 cells, mounted in
Ussing chambers (0.6-cm2 window area) modified for use with
cultured cells (12). Isc (normalized to µA/cm2) was used to quantitate both basal
transepithelial chloride secretion and that induced by
calcium-dependent secretagogs. T84 cells secrete chloride in response
to various calcium-mobilizing agonists, and the resulting changes
in Isc are wholly reflective of chloride secretion (13). Isc measurements
were carried out in Ringer solution containing (in mM) 140 Na+, 5.2 K+, 1.2 Ca2+, 0.8 Mg2+, 119.8 Cl, 25 HCO
Immunoprecipitation and Western blotting.
T84 cells were treated with IFN- on the basolateral surface for the
indicated periods of time. On the day of the experiment, cells were
washed three times with ice-cold PBS. Ice-cold lysis buffer was then
added (consisting of PBS, 1% Nonidet P-40, 1 mM NaVO4, 1 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride), and
the cells were incubated at 4°C for 30 min. Cells were then scraped
into microcentrifuge tubes and centrifuged at 10,000 rpm for 10 min to
remove insoluble material, and an aliquot was removed from each sample
to determine protein content (Bio-Rad protein assay). Samples were then
adjusted with lysis buffer such that each sample had an identical
protein concentration. For the determination of tyrosine-phosphorylated
EGFr, 5 µg of monoclonal anti-EGFr was added to each sample and
allowed to incubate on ice for 60 min. This was followed by the
addition of 50 µl of a 1:1 mixture of protein A-Sepharose and water,
and samples were placed on a rotating platform at 4°C for 60 min.
Samples were then centrifuged to pellet the protein
A-Sepharose-antibody-antigen complex, and the complex was washed three
times with cold lysis buffer followed by three more washes with cold
PBS. The beads were then resuspended in gel loading buffer (50 mM Tris,
pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.2% bromophenol blue, 20%
glycerol). For the determination of total tyrosine-phosphorylated
proteins and membrane-bound TGF-
, the samples were mixed 1:1 with
double-strength gel loading buffer. All samples were placed in boiling
water for 5 min and then loaded on a 7.5% polyacrylamide gel to
resolve proteins (15% gel for TGF-
determination). The proteins
were transferred from the gel onto a polyvinylidene difluoride membrane
(DuPont-New England Nuclear, Boston, MA). The membrane was then blocked
with a 1% skim milk solution in PBS for 30 min, followed by further
incubation of the membrane with a 1% skim milk solution containing 5 µg monoclonal antiphosphotyrosine or 10 µl polyclonal anti-TGF-
for 60 min. This was followed by three 15-min washes with wash buffer
(1% skim milk, 0.5% BSA, 0.2% Tween 20, in PBS). After washes, 2 µl of secondary antibody [goat-anti-mouse or goat-anti-rabbit IgG conjugated to alkaline phosphatase (Clontech, Palo Alto, CA)] were
added to incubate for an additional 30 min. This was followed by three
more washes with wash buffer. The membrane was then treated with a
chemiluminescent solution according to the manufacturer's directions
(Clontech) and exposed to film. Densitometric analysis of the blot was
performed using a digital imaging system.
TGF- ELISA.
Enzyme-linked assays (ELISA) for TGF-
in basolateral and apical
supernatants from T84 cells grown on Nuclepore Transwells were
performed using a commercial kit (Calbiochem) according to the
manufacturer's instructions. In brief, the kit utilized rabbit polyclonal anti-TGF-
bound to microtiter plates as the capturing antibody and biotinylated polyclonal rabbit anti-TGF-
as a
second-step antibody. This was then followed by the addition of
streptavidin-horseradish peroxidase (HRP) as the reporter enzyme. Bound
HRP was visualized using O-phenylenediamine and measured
colorimetrically at an absorbance of 490 nm using a microtiter reader.
Statistical analysis. ANOVA with Student-Newman-Keuls post hoc test was used to compare mean values where appropriate. P values <0.05 were considered significant. All data are expressed as means ± SE for a series of experiments.
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RESULTS |
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Treatment with IFN- results in phosphorylation of the EGFr.
Binding of IFN-
to its receptor has been shown to activate a
tyrosine kinase cascade in various cell types, leading to the tyrosine
phosphorylation of various proteins (18). Our first step
therefore was to determine whether IFN-
had a similar effect on T84
cells and, specifically, whether it is able to tyrosine-phosphorylate high-molecular-mass transepithelial resistance proteins in the 170-kDa range, the molecular mass of the EGFr (33). Figure
1 shows the time course of effects of
IFN-
on tyrosine phosphorylation of various proteins in T84 cells. A
concentration of 100 ng/ml IFN-
was chosen based on prior studies
(11, 15, 19, 29) of effects of this cytokine on T84 cells.
Under nonstimulated conditions, T84 cells display a low level of
tyrosine phosphorylation. On stimulation with IFN-
, there is a
progressive increase in tyrosine phosphorylation of various proteins,
particularly in the 170- to 200-kDa range, with the peak of
phosphorylation around the 12- to 24-h time points. To determine
whether IFN-
stimulation of T84 cells does lead to EGFr tyrosine
phosphorylation, IFN-
-treated T84 cell lysates were
immunoprecipitated with antibodies to the EGFr, and the subsequent
Western blots were probed with antiphosphotyrosine. Figure
2 shows that 100 ng/ml IFN-
was able
to tyrosine-phosphorylate the EGFr in T84 cells in a time-dependent
fashion, with the peak of phosphorylation occurring at 24 h. On
the basis of these findings, we focused the rest of our studies on the
24-h time point.
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IFN- increases expression of the transmembrane and soluble forms
of TGF-
.
Previous studies (2, 14, 27) have shown that various
cytokines, including IFN-
, are able to increase the expression of
several growth factors. We therefore examined whether IFN-
was able
to induce the expression and release of a ligand able to activate the
EGFr. We focused on TGF-
because various studies (3, 23,
28) have demonstrated that this EGFr ligand is upregulated in
the intestine in response to inflammation and damage. TGF-
is initially expressed as a 20-kDa membrane-bound pro-TGF-
precursor
protein, which on appropriate stimulation is proteolytically cleaved
and released as a 6.0-kDa soluble form (26). As the membrane-bound precursor can itself function as a ligand for the EGFr
(20), we first wanted to determine whether IFN-
was
able to increase the content of this protein in T84 cells. Figure
3 shows the effects of 24-h IFN-
on
the levels of membrane-bound TGF-
. T84 cells display basal levels of
a 20-kDa protein that is detected with antibodies to TGF-
on Western
blot. This is likely pro-TGF-
. On stimulation with IFN-
, there is
a significant increase in the levels of this protein. These findings
demonstrate that IFN-
is able to increase protein synthesis of this
growth factor in T84 cells and in a time frame consistent with EGFr
phosphorylation.
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Activation of the EGFr does not mediate IFN- inhibition of
chloride secretion in T84 cells.
TGF-
has been shown to have many effects on the gastrointestinal
system including control of various secretory processes (30). To determine whether the ability of IFN-
to
release TGF-
was involved in the effect of this cytokine on chloride
secretion, we conducted control studies to determine whether the
effects of exogenously added TGF-
could be reversed. Figure
5 shows the effects of a 15-min
pretreatment with 50 ng/ml TGF-
, added to the basolateral surface of
T84 cells, on carbachol-induced chloride secretion. In these studies,
carbachol increased Isc by 42.0 ± 8.7 µA/cm2, which was reduced to 14.7 ± 1.7 µA/cm2 in the presence of TGF-
(n = 3, P < 0.01). This inhibitory effect was reversed by 24-h
coincubation of TGF-
with 5 µg of rabbit polycolonal anti-TGF-
before its addition to T84 cells. Under these conditions, the response
to carbachol returned to 32.7 ± 1.8 µA/cm2. The
neutralizing antibody by itself had no significant effect on the
response to carbachol. These data suggest that an antibody to
TGF-
is able to block the effect of a large dose of exogenous TGF-
and that the antibody is stable for 24 h.
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DISCUSSION |
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In the present study, we investigated the mechanism by which
IFN- is able to inhibit calcium-activated chloride secretion in
intestinal epithelial cells. We focused on the possible role of growth
factors as mediators of the inhibitory effect, because the literature
supports such a hypothesis: 1) in the setting of gastrointestinal inflammation, various growth factors, including TGF-
, have been shown to be upregulated (3, 23, 28);
2) we have previously demonstrated that EGF is rapidly able
to inhibit calcium-activated chloride secretion in T84 cells
(31); 3) Asano et al. (2) have
shown that IFN-
is able to induce expression of TGF-
in human
bronchial epithelial cells; and 4) Prenzel et al.
(24) initially showed that, in response to G
protein-dependent ligands, at least the membrane-bound precursor to
heparin-binding EGF, a member of the EGF family of peptides, is
cleaved, releasing soluble EGFr ligand into the media and subsequently
activating the EGFr. More recent studies (21) from our own
laboratory have likewise indicated that carbachol can evoke TGF-
release from T84 cells. Thus we hypothesized that the inhibitory
mechanism evoked by IFN-
involved activation of the EGFr via
increased expression and/or release of TGF-
and that this ligand
might then mediate the inhibitory effect of IFN-
on chloride
secretion via an autocrine TGF-
/EGFr loop.
We demonstrated that IFN- is able to increase both the expression
and release of TGF-
from T84 cells over a time frame consistent with
its ability to inhibit chloride secretion. However, the concentration of TGF-
found in the spent media from T84 cells treated with IFN-
is well below the IC50 for TGF-
to inhibit chloride
secretion (3.7 nM) (31). Therefore, whereas TGF-
levels
are elevated, it is unlikely that soluble TGF-
alone would activate
the EGFr sufficiently to inhibit chloride secretion. However, we also
demonstrated a significant increase in the amount of membrane-bound
pro-TGF-
present on T84 cells in response to treatment with IFN-
.
This is significant in that the membrane-bound precursor has been shown to possess biological activity (20) and thus can serve as
an additional source of ligand. Furthermore, it has been suggested that
the membrane-bound precursor may function as a means to concentrate the
growth factor within a defined area, thereby decreasing the effective
IC50 (20). Despite this, we were unable to
reverse the IFN-
-mediated inhibition of secretion using either
neutralizing antibodies to TGF-
or a specific inhibitor to the EGFr,
tyrphostin AG 1478, at maximal concentrations, although the
neutralizing antibodies at least could partially reduce (although not
abolish) IFN-
-stimulated EGFr phosphorylation. These findings
suggest that IFN-
mediates its inhibition of chloride secretion
utilizing pathways that are, at least in part, independent of
extracellular ligand activation of the EGFr.
Currently, it is believed that the inhibitory effect of IFN- on
chloride secretion is secondary to downregulation of the activity and
expression of transport proteins, including CFTR. However, this is
speculative at present because no studies have been performed to
determine whether the decrease in CFTR expression is directly
responsible for the inhibitory effect. Furthermore, Fish et al.
(15), utilizing the T84 cell model, have shown that decreases in CFTR protein do not parallel changes in chloride secretion
induced by IFN-
. Similarly, Sugi et al. (29) failed to
see an effect of IFN-
on CFTR expression, reporting instead a
reduction in Na+-K+-ATPase and the
Na+-K+-2Cl
cotransporter NKCC1.
Whereas they do not dismiss the possibility of CFTR downregulation as
an important factor in the inhibition of secretion by IFN-
, our
findings and those of others suggest that other pathways are likely to
be also involved. In our studies, the EGFr remained phosphorylated in
response to IFN-
despite the use of tyrphostin AG 1478, suggesting
that the signaling events for inhibition of secretion due to EGFr
activation may be still active. On the other hand, IFN-
-induced EGFr
phosphorylation could be blocked by an antibody to TGF-
or by the
Src inhibitor PP2. Our data therefore imply at least a partial role for
the EGFr in mediating the effect of IFN-
on secretion, perhaps
acting in concert with Src. We have shown that potassium channel
modification via PKC
is required for EGFr-mediated inhibition of
secretion (10). Therefore, future studies should emphasize
examination of the functional properties of the potassium channel to
better understand the possible role of the EGFr in the inhibitory
effect of IFN-
. It is also possible that Src and/or other signaling events linked to EGFr activity could negatively regulate
Na+-K+-ATPase activity, thereby accounting for
the effect reported by Sugi et al. (29).
The role of elevated TGF- in response to IFN-
in T84 cells is
currently unknown. TGF-
has been shown to disrupt tight junctions in
mammary epithelial cells as evidenced by a reduction in monolayer transepithelial electrical resistance, an increase in paracellular transport, and a redistribution of the ZO-1 tight junctional protein (8). These findings are in keeping with the fact that Sugi et al. (29) reported recently that IFN-
decreases ZO-1
protein expression in T84 cells. Similar responses have been previously ascribed to IFN-
on functional grounds (19). It is
therefore possible that TGF-
may be mediating these effects of
IFN-
. In our study, there was little, if any, change in
transepithelial resistance in response to IFN-
after 24 h (data
not shown). These findings are consistent with the findings of previous
studies (1, 29). As peak changes in transepithelial
resistance occur at 48-72 h of IFN-
treatment, examination of
this potential effect will require a longer time course than that used
in this study. TGF-
has also been shown to posses mitogenic and
restitutive properties in intestinal epithelial cells and has been
implicated in the healing process in the gastrointestinal tract in
response to inflammation (30). Various studies have shown
a significant increase in the levels of TGF-
in response to
inflammation and injury to the gastrointestinal tract (3, 23,
28). One can therefore speculate that cytokines increase TGF-
expression to limit the extent of inflammation-induced intestinal
damage and/or excessive secretion.
Another significant finding of our study was that tyrosine
phosphorylation of the EGFr occurred in response to IFN- treatment. This finding is consistent with other studies that show transactivation of the EGFr in other cell types. Both intra- and extracellular pathways
for EGFr transactivation have been studied and include release of
soluble ligand, the activation of G protein-coupled receptors, and the
activation of cytokine receptors (17, 24, 34). We show
that levels of TGF-
are increased in response to IFN-
, and our
group (17) has previously shown that T84 cells can undergo
activation of the EGFr via a G protein-coupled receptor pathway.
However, the inability of a specific inhibitor of the EGFr kinase to
reverse the tyrosine phosphorylation of the receptor suggests that
other mechanisms are involved in the response to IFN-
, as these
latter pathways, at least, are sensitive to tyrphostin AG 1478 (17). On the other hand, Yamauchi et al. (34)
have shown that growth hormone can tyrosine-phosphorylate the EGFr via
pathways independent of the receptor's intrinsic kinase activity. They
demonstrated a requirement for the cytosolic tyrosine kinase, janus
kinase 2 (Jak2), for tyrosine phosphorylation of the EGFr at the
Grb2 binding site, thus making the EGFr kinase activity dispensable. Such a pathway in T84 cells would predictably be insensitive to tyrphostin AG 1478. Because the IFN-
receptor is
similar to the growth hormone receptor in that they are both in the
class of cytokine receptors, neither possesses intrinsic tyrosine
kinase activity, and both are able to activate Jak2 (16), it is quite likely that activation of the EGFr occurs via this pathway.
Indeed, we show that the ability of IFN-
to induce EGFr phosphorylation is blocked by an inhibitor of the soluble tyrosine kinase Src. However, it should also be noted that we have recently shown that growth hormone at least activates EGFr phosphorylation in
T84 cells in a manner that is, in fact, sensitive to tyrphostin AG 1478 (9). This underscores the fact that the details of complex
signaling pathways may depend on the cell type studied, particularly
when cross talk among various signaling cascades is involved.
In conclusion, the present study demonstrates an increase in the
expression and release of TGF- and the phosphorylation of the EGFr
in response to IFN-
treatment of T84 cells. Although we were unable
to reverse the inhibitory effect of IFN-
on chloride secretion with
antibodies to TGF-
or the tyrosine kinase inhibitor tyrphostin
AG 1478, we were able to block inhibition by antagonizing Src activity.
Moreover, at the very least, our findings do expand on the various
roles of IFN-
in the gastrointestinal tract. We speculate that the
increase in functional growth factor and activation of the EGFr may
serve as a mechanism to restrict the extent of cytokine-induced
intestinal damage.
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ACKNOWLEDGEMENTS |
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We thank Glenda Wheeler for administrative support.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28305 (to K. E. Barrett). D. F. McCole is the recipient of a Research Fellowship Award from the Crohn's and Colitis Foundation of America. J. M. Uribe was the recipient of an American Digestive Health Foundation student research fellowship award while a medical student in the University of California San Diego School of Medicine.
Present address of J. M. Uribe: Dept. of Anesthesiology and Critical Care Medicine, Blalock 1415, The Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, MD 21287-4965.
Address for reprint requests and other correspondence: K. E. Barrett, Univ. of California San Diego Medical Center, 8414, 200 West Arbor Drive, San Diego, CA 92103-8414 (E-mail: kbarrett{at}ucsd.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.
July 11, 2002;10.1152/ajpgi.00237.2002
Received 25 February 2002; accepted in final form 26 June 2002.
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