Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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Adenosine release
from mucosal sources during inflammation and ischemia activates
intestinal epithelial Cl
secretion. Previous data suggest that
A2b receptor-mediated
Cl
secretory responses may
be dampened by epithelial cell nucleoside scavenging. The present study
utilizes isotopic flux analysis and nucleoside analog binding assays to
directly characterize the nucleoside transport system of cultured T84
human intestinal epithelial cells and to explore whether adenosine
transport is regulated by secretory agonists, metabolic inhibition, or
phorbol ester. Uptake of adenosine across the apical membrane displayed characteristics of simple diffusion. Kinetic analysis of basolateral uptake revealed a Na+-independent,
nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system
with low affinity but high capacity for adenosine. NBTI binding studies
indicated a single population of high-affinity binding sites
basolaterally. Neither forskolin,
5'-(N-ethylcarboxamido)-adenosine, nor metabolic inhibition significantly altered adenosine transport. However, phorbol 12-myristate 13-acetate significantly reduced both
adenosine transport and the number of specific NBTI binding sites,
suggesting that transporter number may be decreased through activation
of protein kinase C. This basolateral facilitated adenosine transporter
may serve a conventional function in nucleoside salvage and a novel
function as a regulator of adenosine-dependent
Cl
secretory responses and
hence diarrheal disorders.
purinergic receptors; chloride channels; inflammation; ischemia; intestinal secretion
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INTRODUCTION |
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CLASSICALLY, THE NUCLEOSIDE adenosine is viewed as a central regulatory molecule that serves to balance cellular oxygen supply and demand during metabolic stress. According to this paradigm, adenosine (the product of sequential dephosphorylation of ATP) is released by cells into the extracellular space during hypoxia, ischemia, or exercise. Among the three known classes of adenosine receptors (A1, A2, and A3) (8, 20, 30, 34), the two major classes of adenosine receptors (A1 and A2) primarily interact with locally released adenosine to effect physiological changes that restore energy balance. In ischemic heart, for example, binding of adenosine to inhibitory A1 receptors in cardiac myocytes decreases energy demand, whereas binding to stimulatory A2 receptors in vascular smooth muscle cells leads to vasodilatation and consequently increased oxygen supply (7). This feedback arrangement has been shown to apply to numerous organs and cell types, including brain, skeletal muscle, kidney, and adipocytes (1, 4, 5, 7).
However, in mammalian intestine, adenosine appears to act in novel
fashion. We reported that the adenosine released during ischemia in model T84 intestinal epithelial cells, rather than decreasing energy-requiring processes such as active ion transport, paradoxically increases cell work in the form of electrogenic chloride
ion (Cl) secretion (25,
42). This unusual secretory response to metabolic stress bears a
striking resemblance to the early luminal fluid losses and diarrhea
associated with clinical mesenteric ischemia and appears to be
mediated through autocrine activation of
A2b membrane receptors that are
positively coupled to adenosine 3',5'-cyclic monophosphate
(cAMP)-dependent Cl
secretory pathways (40, 41). Thus, unlike its usual metabolic feedback
role, adenosine released by intestinal epithelia may "feed
forward" to activate the cellular machinery for salt and water
transport (25).
Adenosine is produced during normal cellular metabolism (7, 42), and
adenosine and its precursors are released from damaged cells and
discharged from mast cells and platelets (2, 3, 16, 22, 26). Moreover,
nucleosides are absorbed luminally from dietary sources. Thus, under a
variety of pathological and nonpathological conditions, substantial
quantities of adenosine may accumulate in the extracellular space in
the vicinity of secretory intestinal crypt epithelial cells. The
intestine is normally a proabsorptive organ, and secretion is generally
a tightly regulated process. This implies that intestinal epithelial
cells likely possess a control mechanism that dampens secretory
responsiveness to locally released adenosine under normal
circumstances. In metabolically intact T84 cells, we showed that
constitutive release of adenosine under nonischemic conditions is
unmasked by treatment with the conventional inhibitors of nucleoside
transport dipyridamole and nitrobenzylthioinosine (NBTI) and with
iodotubercidin, an inhibitor of adenosine kinase (42). Moreover,
extracellular adenosine accumulation induced by these agents elicited a
Cl secretory response that
was prevented by adenosine receptor blockade. These indirect data
suggested the presence of an adenosine transporter, which scavenges
extracellular adenosine and limits
A2b receptor activation, thereby
limiting the potential for autocrine activation of Cl
secretion. Such a transporter could serve as at least one mechanism by
which excessive adenosine-elicited secretion is prevented under nonischemic conditions.
Two major classes of nucleoside transport systems have been described in mammalian tissues (31, 32). The first class is the Na+-dependent transporters, which have been identified in brush-border membranes of absorptive renal tubules and mammalian ileal enterocytes; these are termed concentrative transporters because of their ability to accumulate substrate intracellularly against a concentration gradient (6, 35, 45). Several examples of Na+-dependent transporters have been recently cloned (17, 27). These systems characteristically exhibit relative insensitivity to inhibition by the nucleoside analog NBTI. A second and more widely distributed class of nucleoside transporter is present in numerous nonepithelial cell types and is characterized by Na+ independence. These equilibrative transporters display kinetic properties consistent with facilitated diffusion and display a range of sensitivities to inhibition by NBTI (19, 32).
In nonepithelial systems, the major role of these facilitated
transporters is nucleoside salvage to conserve intracellular energy
pools. We have speculated that an adenosine transporter in intestinal
epithelia would additionally serve to maintain local concentrations of
adenosine below the activation threshold for the low affinity
A2b receptor. Thus this
transporter may negatively regulate adenosine-dependent epithelial
secretion, and changes in the functional expression of this putative
"off-switch" could modulate diarrheal responsiveness of the
intestinal tract to extracellular adenosine. However, the presence of
this transporter has yet to be firmly established, and the possibility
of regulated expression of adenosine transport in secretory intestinal
epithelia has not been addressed. In this study, we develop direct
evidence for the presence of a basolateral membrane nucleoside
transporter in model intestinal epithelial cells and define the kinetic
properties of this transporter in the context of other known nucleoside
transport systems. Additionally we examine whether this adenosine
transport can be altered by 1)
Cl secretory agonists [cAMP and
5'-(N-ethylcarboxamido) adenosine (NECA)],
2) metabolic inhibitors (oligomycin
plus 2-deoxyglucose, iodoacetate), or
3) protein kinase C (PKC) activation
[phorbol 12-myristate 13-acetate (PMA)].
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METHODS |
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Cell culture and buffers. Human colonic epithelial cells, T84 cells, were maintained in culture as previously described and grown to confluence on collagen-coated permeable filters mounted on supports for 12-well culture dishes from Costar (Cambridge, MA) (13, 24, 25, 42). Cells were fed the day before experiments. Experiments were carried out in a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered Ringer solution (HPBR) containing (in mM) 135 NaCl, 5.0 KCl, 3.33 NaH2PO4, 0.83 Na2HPO4, 1.0 CaCl2, 1.0 MgCl2, 10 glucose, and 5.0 HEPES, pH 7.4 at 37°C. Na+-free buffer contained (in mM) 5.0 HEPES, 5.0 KH2PO4, 1.0 MgCl2, 1.0 CaCl2, 140 N-methyl-D-glucamine (NMG), and 10 glucose, pH 7.4.
Adenosine uptake studies. Confluent monolayers grown on 1.0 cm2 supports were washed, preincubated in HPBR for 10 min, and then incubated for 1 min in HPBR containing 1.0 µCi/ml [3H]adenosine either apically or basolaterally as specified. The final concentration of adenosine was 1.0 µM to 1.0 mM. The membrane supports were then removed from incubation medium and dipped 10 times rapidly in three successive beakers of "stop-cold" solution (100 mM MgCl2, 10 mM tris(hydroxymethyl)aminomethane-HCl, pH 7.4 at 0°C). Each filter was subsequently excised and placed in a vial. Econofluor (3.0 ml; Packard, Meriden, CT) was added before measurement of radioactivity by a Packard 1600 TR (Packard) liquid scintillation counter. Spectrophotometric determination of protein content of several representative monolayer filters was done using a commercially available bicinchoninic acid assay (Pierce Biochemicals, Rockford, IL) with bovine serum albumin as standard.
For the study of the effect of various agents on adenosine transport, the cells were first preincubated in HPBR containing these compounds for specified durations, and then adenosine transport was measured as previously described by adding [3H]adenosine. Unless otherwise specified, these agents were present during the transport measurement. For determination of uptake dependence on Na+, monolayers were incubated with 1.0 µCi/ml [3H]adenosine (final adenosine concentration of 10 µM) with or without 2.0 µM NBTI in the above-mentioned Na+-free buffer basolaterally. For NBTI inhibition studies, monolayers were incubated in basolateral HPBR containing graded concentrations of NBTI (0-1.0 µM) for 20 min. One-minute adenosine uptake was then measured using 1.0 µM adenosine in the presence of NBTI.[3H]NBTI binding assay. We preincubated 1-cm2 supports containing T84 monolayers in HPBR for 10 min before the initiation of equilibrium binding assays. Supports were then transferred to basolateral solution containing varying concentrations (0.15-10 nM) of [3H]NBTI in the presence and absence of 10 µM excess nonlabeled NBTI. After incubation at 37°C for 20 min each filter was excised, and radioactivity was determined as previously described.
Statistical analysis. Data are expressed as means ± SE. Student's t-test and analysis of variance (ANOVA) were performed for paired variates and multiple variates, respectively, when appropriate, and P < 0.05 was considered significant. Linear curve fitting was by the least-squares method.
Materials. [2,8-3H]adenosine was purchased from Moravek Biochemicals (Brea, CA) and ICN Pharmaceuticals (Irvine, CA). [3H]NBTI was from Du Pont (Wilmington, DE). All other reagents were from Sigma Chemical (St. Louis, MO).
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RESULTS |
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Adenosine uptake in T84 monolayers.
Adenosine uptake was measured as a function of time (Fig.
1). Both apical and basolateral
[3H]adenosine uptake
was linear for 3 min (R = 0.982 and
0.999, respectively). Therefore, all subsequent uptake studies were
performed for a 1-min incubation. One-minute uptake was 13-fold higher
across the basolateral membrane than the apical membrane (9.96 ± 0.79 vs. 0.75 ± 0.045 pmol · min1 · mg
protein
1, respectively, for
extracellular adenosine concentration of 1.0 µM,
n = 6, P < 0.001), consistent
with our earlier report (42). Adenosine uptake was then measured
against graded concentrations of adenosine. Uptake of adenosine across
the apical surface of the monolayers displayed linear, nonsaturable
behavior in concentrations up to 1 mM (data not shown), indicating that
adenosine movement across this membrane occurs by simple diffusion
alone.
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NBTI inhibition studies. To study the sensitivity of adenosine transport to NBTI, adenosine uptake was measured in the presence of different concentrations of NBTI at a constant 1.0 µM extracellular adenosine concentration. NBTI inhibited basolateral adenosine uptake in a dose-dependent manner, as shown in Fig. 3. Inhibition was observed with a 50% inhibitory concentration (IC50) of 1.55 ± 0.73 nM (result of 3 separate experiments performed in triplicate). Approximately 20% of adenosine transport was not inhibitable at apparent saturating concentrations of NBTI (1.0 µM). This portion of uptake probably represents the simple diffusion component of adenosine movement across the basolateral membrane.
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Na+
independence of adenosine transport in T84 cells.
To explore the Na+ dependence of
basolateral adenosine transport, extracellular
Na+ was replaced with NMG (Fig.
4). There was minor reduction of adenosine
uptake in the absence of Na+, and
this was statistically insignificant (49.5 ± 1.7 vs. 46.3 ± 0.9 pmol · min1 · mg
protein
1 for control vs.
Na+ free, respectively, at 10 µM
adenosine, n = 15, P = 0.097). The residual adenosine
transport insensitive to 2.0 µM NBTI inhibition in the absence of
Na+ was the same as that observed
in the presence of Na+ (5.84 ± 0.30 vs. 4.96 ± 0.40 pmol · min
1 · mg
protein
1 for control vs.
Na+ free, respectively, at 10 µM
adenosine and 2.0 µM NBTI, n = 15, P = 0.090). Approximately 12% of the
total uptake in the presence of
Na+ was insensitive to NBTI
inhibition (5.84 ± 0.30 vs. 49.5 ± 1.7 pmol · min
1 · mg
protein
1 at 2.0 µM NBTI,
n = 15, P < 0.0001) and may represent the
nonsaturable portion of the total uptake (estimated nonsaturable uptake
from Fig. 2A is ~4.15
pmol · min
1 · mg
protein
1 at 10 µM
adenosine). Thus the adenosine transport system in T84 cells appears to
consist predominantly of
Na+-independent transporters.
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Equilibrium binding study of
[3H]NBTI to T84 cells.
Figure 5 shows the concentration dependence
of specific basolateral membrane
[3H]NBTI binding,
where membrane-associated binding is plotted against the equilibrium
free concentration of the inhibitor. There was saturable specific
[3H]NBTI binding and
when transformed for Scatchard analysis, a highly linear relationship
(R = 0.995) was seen, indicating a single population of high-affinity NBTI binding sites with an apparent
dissociation constant
(Kd) value of
3.11 ± 1.09 nM and a maximal binding
(Bmax) of 0.311 ± 0.021 pmol/mg protein (result of 2 experiments performed in
triplicate). The calculated turnover number for the transport system
operating at Vmax
(491 pmol · min1 · mg
protein
1) is ~26
molecules · transporter
1 · s
1
and is consistent with several reported values for the turnover rate in
other transporters (31, 38, 39). Taken together with the results
presented above, the basolateral adenosine transporter in T84 cells
appears to be Na+ independent and
NBTI sensitive, consistent with so-called "equilibrative" facilitated nucleoside transporters identified in other systems.
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Nonregulation of adenosine transport in T84 intestinal epithelial
cells by secretory agonists and chemical hypoxia.
We next examined the possibility that the facilitated adenosine
transporter itself displays the capacity for regulation. We found that
neither the cAMP receptor agonist forskolin (10 µM) nor the
nontransported adenosine A2b
receptor agonist NECA (1.0 µM) affected adenosine transport at two
relevant concentrations of adenosine (10 and 100 µM) as seen in Fig.
6A.
Long-term (48 h) exposure to NECA, which functionally downregulates
adenosine-elicited Cl
secretion (3), was also without effect on basolateral adenosine transport (data not shown), further demonstrating the independence of
adenosine transport and adenosine surface receptor responses. Similarly, adenosine uptake was not affected by metabolic inhibition using oligomycin A plus 2-deoxyglucose or iodoacetate, as seen in Fig.
6B. This treatment decreased cellular
ATP levels to ~20% of initial levels within 30 min, as determined by
chemiluminescence (data not shown). Shorter incubation periods with
above ischemia-inducing agents (10 and 15 min), during which
time formation of cellular adenosine may be high, also did not affect
adenosine uptake (data not shown).
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Downregulation of basolateral adenosine transport by phorbol ester.
In contrast to the above results, a significant decrease in adenosine
transport was induced by PMA (100 nM). As seen in Fig. 7A, after
4 h PMA inhibited ~37% of total adenosine uptake at varying
concentrations of extracellular adenosine (42, 33, and 37% inhibition
for extracellular adenosine concentration of 0.05, 0.1, and 0.5 mM,
respectively, n = 4 for each group,
P < 0.0001 for all 3 concentrations). Shorter incubation periods (30 min) resulted in
similar reductions in adenosine transport (data not shown), suggesting
that these effects may be due to activation rather than downregulation
of PKC. Detailed kinetic analysis of adenosine transport (Fig. 7,
B and
C) revealed that 100 nM PMA significantly increased
Km (219 ± 19 vs. 332 ± 5.9 µM for control vs. PMA, respectively,
n = 3, P < 0.005) and reduced
Vmax (410 ± 46 vs. 254 ± 15 pmol · min1 · mg
protein
1 for control vs.
PMA, respectively, n = 3, P < 0.05), suggesting that activation of PKC decreases adenosine
transport by reducing the number of basolateral adenosine transporters
and reducing transporter affinity for its substrate.
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DISCUSSION |
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These data demonstrate the presence of a
Na+-independent facilitated
adenosine transport system within the basolateral membrane domain of
model intestinal crypt epithelial cells. Equilibrative adenosine
transporters in neural systems display higher affinity transporters
with typical Km
values in the range of 1-10 µM, whereas peripheral systems tend
to be characterized by broader substrate specificity, with
Km values for
adenosine of 100-1,000 µM. The observed
Km value of 114 µM for adenosine transport in T84 intestinal epithelial cells is
comparable to nucleoside transporters in peripheral tissues and is thus
of low affinity (10, 15). Other investigators have identified high
affinity (Km
~17 µM), Na+-dependent
nucleoside transport in the apical membrane of mammalian absorptive
enterocytes (6), but this process does not appear to be a major
component of the adenosine transport in the
Cl-secreting T84 cell line.
This suggests the possibility that expression of
Na+-coupled adenosine transporter
may be regulated along the crypt-villus axis, similar to a number of
other Na+-coupled transporters.
However, Na+-dependent transport
has been reported to be present in monolayers of undifferentiated,
cryptlike IEC-6 cells (18). Betcher and co-workers (6) described
Na+-independent adenosine
uptake in rabbit ileal basolateral membrane vesicles and suspected the
presence of facilitated transport in absorptive enterocytes but
postulated that facilitated transport served as the exit rather than
the entry pathway for transcellular adenosine movement, in analogy to
the absorption of glucose and various amino acids.
Facilitated adenosine transporters are an integral component of the
nucleoside salvage pathways in mammalian cells; this is a critical
biochemical process in intestinal epithelial cells, which are
particularly deficient in the ability to synthesize nucleosides de novo
(20, 22). Brush-border
Na+-dependent transporters allow
scavenging of luminal nucleosides from dietary sources, and recent
molecular evidence from Patil and Unadkat (29) indicates the presence
of two cloned isoforms (N1 and N2) and the absence of
Na+-independent transport in
brush-border membrane vesicles from human ileum (29). Nucleoside
scavenging from the subepithelial compartment is likely to occur
through the facilitated transport system we have characterized in the
present report. The uptake capacity of this transporter appears large
as evidenced by its substantial
Vmax of 491 pmol · min1 · mg
protein
1.
Human erythrocytes, rat cardiac myocytes, and bovine adrenal endothelial cells have adenosine transport systems that are inhibited by low concentrations (IC50 <10 nM) of NBTI (10, 19, 38), whereas other cells (for example, Walker 256 carcinosarcoma and Bovikoff hepatoma N1S1-67) possess systems that require higher concentrations of NBTI (IC50 >1.0 µM) for inhibition (28). High-affinity binding of NBTI to the specific membrane sites seems to correlate with inhibition of nucleoside transport (9). We observed an IC50 of <2 nM in T84 cells, consistent with the measured high-affinity binding of NBTI (Kd ~3 nM). This high sensitivity of the adenosine transporter to NBTI inhibition in T84 cells additionally indicates that this is predominantly a facilitated-diffusion process.
The presence of this basolaterally restricted low-affinity but
high-capacity NBTI-sensitive facilitated nucleoside transporter in T84
cells appears to account for effective scavenging of physiologically relevant amounts of extracellular adenosine released during normal cellular metabolism (42). According to the data of Strohmeier et al.
(41), A2b receptor activation in
T84 cells requires an extracellular concentration of adenosine
>106 M for either cAMP
generation or short-circuit current
(Isc) response with a 50% effective dose
(Isc) of 7.78 µM. These values, although slightly lower than the
observed Km (114 µM) of our transport system, appear to fall within physiologically
operative concentrations of the transporter. The data of Strohmeier et
al. (41) further support this hypothesis that the transporter may keep
the extracellular adenosine concentration below the threshold of
A2b receptor activation. In that
study, Cl
secretory
(Isc) responses
elicited by basolateral extracellular adenosine were enhanced 10- to
15-fold by adenosine transport inhibitors, suggesting that effective
removal of local extracellular adenosine is mediated by this nucleoside
transport system. The work of Dobbins et al. (14), also attests to the
potential biological relevance of this transporter; these investigators
demonstrated that dipyridamole enhanced the
Cl
secretory response to
exogenous adenosine in mammalian ileum. The adenosine scavenging system
thus may represent a mechanism for limiting adenosine-dependent
activation of Cl
secretion,
and hence diarrhea. However, in acute ischemic conditions, where
relatively large quantities of adenosine are released into the
extracellular space, the capacity of this tranport system may be
overwhelmed and thus insufficient to prevent stimulation of the
A2b receptors.
Could this nucleoside transporter represent an endogenous regulatory site for adenosine-dependent intestinal secretory responses? The second part of this study attempts to address this question. Whether regulation can occur at the level of a nucleoside transporter has to date been studied most thoroughly in cultured chromaffin cells of bovine adrenal gland (11, 12, 36, 37, 43). Sen et al. (37) observed that effectors of secretion in these cells such as forskolin or other direct activators of protein kinase A (PKA) inhibit adenosine transport by decreasing the number of membrane transporters without affecting affinity (37). Subseqently, these investigators observed downregulation of transport sites mediated by various agonists for purinergic P2y-receptor (36). Conversely, the nontransported adenosine receptor agonist NECA, which does not induce granule secretion in these cells, increases adenosine transport capacity by upregulating the transport sites without altering affinity (11). In the present experiments, neither forskolin nor NECA (short- and long-term incubation) exerted a significant effect on T84 cell adenosine transport. Therefore, these regulatory mechanisms are likely to be cell tissue specific; in bovine adrenal endothelial cells adenosine transport is not regulated by PKA (38), and indeed in most systems evidence for regulation of facilitated adenosine transport has been difficult to demonstrate. Adenosine transport was also not altered by cellular energy depletion in these experiments, suggesting that the adenosine-dependent activation of secretion observed in metabolically stressed T84 cells is probably not associated with a decrease in adenosine reuptake capacity. We cannot entirely exclude the possibility that early in the course of the chemical hypoxia there could have been a period during which adenosine uptake was decreased. However, these data displayed enough scatter to render firm conclusions impossible, likely confounded by the combination of nonequilibrium conditions and trans-inhibition of facilitated adenosine uptake during intracellular adenosine formation.
PKC-dependent regulation of adenosine transport has been observed in
adrenal chromaffin cells (12), and we also observed that short-term
incubation with a PKC-activating phorbol ester significantly altered
adenosine transport. This effect appeared to reflect a decrease in
affinity for substrate and
Vmax of
transport. The NBTI binding experiments suggested that a reduction in
transporter number rather than a change in binding affinity for
nucleoside analog primarily accounted for the observed transport
decrease. There was a rather consistent quantitative relationship
between adenosine transport reduction (~37%) with the decrease in
transporter number (~35%). Whether the PMA-induced reduction in
Bmax is the result of internalization of functional transporters from the plasma
membrane or is a reflection of PKC-dependent deactivation of functional
transport sites without a change in total number of surface
transporters cannot be distinguished from the present experiments. Both
types of PKC-dependent regulatory mechanisms have been described in
adrenal chromaffin cells (12). Whereas the above data suggest that the
prosecretory effects of extracellular adenosine could be enhanced by
PMA due to reduced adenosine uptake from the extracellular space, we
could not demonstrate this in monolayers subjected to short-term (30 to
60 min) incubation with PMA. We previously showed that PMA alone exerts
an inhibitory effect on cAMP-elicited
Cl secretion in T84 cells
by decreasing the basolateral
Na+-K+-2Cl
cotransporter (23).
Additionally, others have found negative interactions of PMA with
apical cystic fibrosis transmembrane conductance regulator and
basolateral K+ channels (33,
44). Therefore PMA appears to have multiple, primarily
negative, effects on the secretory apparatus, thus making it difficult
to show an isolated stimulatory effect on
Cl
secretion by
downregulation of the nucleoside transport.
In summary, we have characterized the kinetics of adenosine transport
across the apical and basolateral surfaces of a polarized Cl secreting human
intestinal epithelial cell line. Uptake of adenosine across the apical
membrane was nonsaturable and consistent with simple diffusion.
Basolateral uptake studies revealed the presence of a carrier system
that obeyed Michaelis-Menten kinetics in addition to a diffusional
component. The results indicate the presence of a basolaterally
restricted Na+-independent
NBTI-sensitive facilitated adenosine tranporter. This transporter may,
as elsewhere, be important in nucleoside salvage; however, in this
model system and likely in native intestinal crypts, this transporter
may serve a novel function in limiting adenosine-dependent secretory
responses. The transport capacity of this system appears to be
modulated by agents affecting PKC but not by cAMP-dependent secretory
agonists or metabolic inhibition, although whether such regulation is
meaningful in terms of integrated Cl
secretory function and
its metabolic regulation remains to be determined. A deeper
understanding of the characteristics of this adenosine scavenging
system could lead to the development of novel antidiarrheal agents.
Because adenosine transporters in nongastrointestinal (e.g., cardiac)
systems already represent an attractive target for new pharmacotherapy
in ischemic disorders and because some agents already in use (e.g.,
dipyridamole) are directed at this site, the ability to functionally
discriminate among specific transporter subtypes may allow improved
drug design that limits potential for diarrheal side effects.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Training Grant GMO-780617 to E. C. Mun, National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48010 and DK-51630, and the George H. A. Clowes Memorial Career Development Award from the American College of Surgeons to J. B. Matthews.
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FOOTNOTES |
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Address for reprint requests: J. B. Matthews, Dept. of Surgery, Beth Israel Deaconess Hospital, 330 Brookline Ave., Boston, MA 02215.
Received 23 July 1997; accepted in final form 30 October 1997.
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