Polarity of A2b adenosine receptor expression determines
characteristics of receptor desensitization
Shanthi V.
Sitaraman1,2,
Mustapha
Si-Tahar1,
Didier
Merlin1,
Gregg R.
Strohmeier3, and
J. L.
Madara1
1 Epithelial Pathobiology Unit, Department of
Pathology and Laboratory Medicine, Emory University School of
Medicine and 2 Division of Digestive Diseases,
Emory University School of Medicine, Atlanta, Georgia 30322; and
3 Pulmonary Center, Boston University Medical
Center, Boston, Massachusetts 02118
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ABSTRACT |
It is not known
if, in polarized cells, desensitization events can be influenced by the
domain on which the receptor resides. Desensitization was induced by
5'-(N-ethylcarboxamido)adenosine (NECA) and was
quantitated by measurement of short-circuit current (Isc) in response to adenosine. NECA added
to either the apical or basolateral compartments rapidly desensitized
receptors on these respective domains. Although apical NECA had no
effect on the basolateral receptor stimulation, basolateral NECA
induced a complete desensitization of the apical receptor. We
hypothesized that desensitization of apical receptor by basolateral
desensitization could relate to a trafficking step in which A2b
receptor is first targeted basolaterally upon synthesis and transported
to the apical surface via vesicular transport/microtubules. Because
desensitization is associated with downregulation of receptors, apical
adenosine receptor can thus be affected by basolateral desensitization. Both low temperature and nocodazole inhibited Isc
induced by apical and not basolateral adenosine. In conclusion:
1) a single receptor subtype, here modeled by the A2b receptor,
differentially desensitizes based on the membrane domain on which it is
expressed, 2) agonist exposure on one domain can result in
desensitization of receptors on the opposite domain, 3)
cross-domain desensitization can display strict polarity, and
4) receptor trafficking may play a role in the
cross-desensitization process.
intestinal; chloride secretion; G protein-coupled receptor
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INTRODUCTION |
ADENOSINE IS AN IMPORTANT mediator of various
physiological responses including muscle tone, neuronal firing, immune
function, and secretion of various hormones and cytokines (24, 27, 33). In addition to its role in the regulation of these physiological processes, adenosine is released during inflammatory conditions and
acts as a paracrine factor with diverse effects on a variety of organ
systems including cardiovascular, nervous, urogenital, respiratory, and
digestive systems (31, 33, 37). Adenosine release acts as an autocoid
by interacting with the adenosine receptor belonging to the family of
seven transmembrane G protein-coupled group of cell surface receptors
(27, 31). Biochemical and molecular cloning studies have
demonstrated the existence of four adenosine receptor subtypes
designated A1, A2a, A2b, and A3 (12, 18, 24, 27, 31). In the intestine,
where neutrophil transmigration into the lumen is characteristic of the
active phase of many intestinal disorders including inflammatory bowel
disease, adenosine has been shown to mediate electrogenic chloride
secretion, the event underlying secretory diarrhea (2, 3, 19, 23). With
the use of molecular, pharmacological, and biochemical approaches we
characterized the intestinal adenosine receptor as the A2b subtype in
both T84 cells, a model intestinal epithelial cell line, and intact
human intestinal epithelia (36). Furthermore, the A2b receptor appears
to be the only adenosine receptor present in T84 cells. In these cells,
A2b is functionally coupled to G
s, and the stimulation
of apical or basolateral surface with adenosine results in increased
cAMP in a polarized manner (36).
Prolonged exposure of G protein-coupled receptors to an agonist results
in a decrease in receptor responsiveness, a process termed
desensitization. This agonist-induced desensitization or refractoriness is a universal feature of G protein-coupled receptors (5, 13). Several studies have indicated that, for many
receptors, including the adenosine receptors (7, 8, 16, 25, 26, 28-30), desensitization can be divided into two temporally and mechanistically distinct phases: 1) short-term exposure to
agonist resulting in uncoupling of the receptor from G proteins and
mediated by receptor phosphorylation, and 2) long-term agonist
exposure resulting in receptor downregulation and mediated by
internalization of the receptor and/or decreased receptor synthesis.
Although much is known about the phenomenon of desensitization, it is
not known if, in polarized cells, desensitization events can be
influenced by the domain on which the receptor resides. Because
polarized epithelial cells such as T84 cells are able to seal the
apical from the basolateral domains, potential cross-domain receptor desensitization can be studied using these cells. The T84 monolayers used for these studies have high electrical resistance
(1,200-1,500
· cm2), as is
typical for this cell line (10). Such severe restriction on passive
permeation of small hydrophilic solutes permits sidedness of responses
to be clearly separated (9). We have made use of the apical and
basolateral expression of the A2b receptor in T84 cells to examine
whether biological events determining receptor desensitization might be
dictated by the membrane domain on which the receptor is expressed.
Here, we report the characteristics of adenosine receptor
desensitization and the effect of polarity on the desensitization. We
demonstrate that a single receptor subtype, here modeled by the A2b
receptor, may differentially desensitize based on the membrane domain
on which it is expressed. Furthermore, agonist exposure on one domain
can result in desensitization of receptors on the opposite domain. In
the intestine, potential effects of desensitization of apical receptors
on basolateral receptors or vice versa might have therapeutic implications.
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MATERIALS AND METHODS |
Reagents.
All tissue culture supplies were obtained from Life Technologies (Grand
Island, NY). Adenosine and
5'-(N-ethylcarboxamido)adenosine (NECA) were obtained
from Research Biochemicals International (Natick, MA). IBMX, vasoactive
intestinal peptide (VIP), and nocodazole were obtained from Sigma (St.
Louis, MO). Forskolin and carbachol (an acetylcholine receptor agonist)
were from Calbiochem (La Jolla, CA).
Cell culture.
T84 cells were grown and maintained in culture as previously described
(19) in a 1:1 mixture of DMEM and Ham's F-12 medium supplemented with
penicillin (40 mg/l), ampicillin (8 mg/l), streptomycin (90 mg/l), and
5% newborn calf serum. Confluent stock monolayers were subcultured by
trypsinization. Experiments were done on cells plated for 7-8 days
on permeable supports of 0.33 cm2 in area (inserts). This
permits differentiation of cells and development of tight junctions
with high electrical resistance (1,200-1,500
· cm2). Inserts with rat tail
collagen-coated polycarbonate membrane filter (5-µm pore size;
Costar, Cambridge, MA) rested in wells containing media until
steady-state resistance was achieved, as previously described (23).
This permits apical and basolateral membranes to be separately
interfaced with apical and basolateral buffer, a configuration
identical to that previously developed for various microassays (9).
Short-circuit current measurements.
Inserts were rinsed and placed in Hanks' balanced salt solution (HBSS)
in a new 24-well plate containing 0.5 ml of HBSS in the serosal surface
and 0.2 ml of HBSS in the apical compartment. To determine currents,
transepithelial potentials, and resistance, a set up consisting of a
commercial voltage clamp (Bioengineering Department, University of
Iowa) was interfaced with an equilibrated pair of calomel electrodes
submerged in saturated KCl and a pair of Ag-AgCl electrodes submerged
in HBSS (20). For electrical determinations, agar bridges were used to
interface one calomel and one Ag-AgCl electrode on each side of the
monolayer incubated at 37°C, and a pulse of 25 µA of current was
passed across the monolayer. With the use of Ohm's law (V = I × R), the transepithelial resistance and
the short-circuit current (Isc) was calculated.
Statistical analysis.
Data are expressed as mean ± SD. Student's t-test or ANOVA
with Student-Newman-Keuls post hoc test were used to compare mean values as appropriate. P values < 0.05 were considered to
represent significant differences.
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RESULTS |
Desensitization of the A2b receptor by NECA.
An Isc response representing electrogenic chloride
secretion was elicited from T84 cells by adenosine or its analogs
applied to either apical or basolateral membrane (2, 3, 36). Either apical or basolateral adenosine (100 µM) rapidly stimulated
Isc response from T84 cells. Because the natural
ligand of the A2b receptor, adenosine, is degraded with time by
ectoadenosine deaminase, NECA, a nonmetabolizable analog of adenosine,
was used for desensitization studies. After exposure to NECA, cells
were washed, and desensitization was assessed by functional response as
measured by Isc 10 min after the addition of
adenosine [we have shown earlier that apical and basolateral
stimulation of T84 cells with adenosine results in maximal
Isc response at 10 min (36)].
Pretreatment of apical or basolateral domains with NECA (10 µM) for
12 h inhibited subsequent Isc response to adenosine
(100 µM) added to the same domain, indicating that apical or
basolateral receptors could be desensitized (Fig.
1). As seen in
Fig. 1, the exposure of the apical compartment to NECA inhibited
subsequent Isc response to apical adenosine by
~60%, and the exposure of the basolateral compartment to NECA
inhibited subsequent Isc response to basolateral
adenosine by ~50% compared with cells treated with adenosine
alone. In contrast, apical desensitization with NECA did
not affect the Isc elicited by basolateral
adenosine. Surprisingly, basolateral desensitization with NECA for 12 h
abrogated the Isc response elicited by apical
adenosine stimulation. Isc response after
basolateral NECA exposure was ~10% maximal response, which was not
significantly different from untreated cells. NECA-treated cells
(apical or basolateral) without subsequent stimulation with adenosine
had an Isc of 9.0 ± 3.0 µA/cm2 and
untreated cells had an Isc of 4.0 ± 0.2 µA/cm2.

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Fig. 1.
Desensitization of apical or basolateral A2b receptor. T84 cells were
treated with 5'-(N-ethylcarboxamido)adenosine
[NECA; 10 µM, apical (Ap) or basolateral (Bs)] for a
period of 12 h. Cells were washed with Hanks' balanced salt solution
(HBSS), equilibrated at 37° for 20 min, and stimulated with
adenosine (100 µM Ap or 100 µM Bs). Short-circuit current
(Isc) was measured 10 min poststimulation. Data are
represented as percentage of maximal response of
Isc-induced apical or basolateral adenosine
stimulation. In µA/cm2: unstimulated, 9.0 ± 2.8; Ap
adenosine (Ado), 70.0 ± 4.0; Bs Ado, 82.0 ± 9.8; Ap NECA + Ap Ado, 28.0 ± 3.0; Bs NECA + Bs Ado, 39.0 ± 6.0. Data represent
the responses observed in 4 separate experiments plotted as mean ± SD, n = 6 per treatment group. # Significantly
different from cells treated with adenosine P < 0.001 by
ANOVA; NS, not significantly different from Ap NECA + Ap Ado;
** significantly different from Ap NECA + Bs Ado, P < 0.001.
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Time course of desensitization.
We next studied the time course of desensitization of apical and
basolateral adenosine receptors. Figure
2A shows that apical NECA inhibited
subsequent Isc responses to apical adenosine. This inhibition began within 20 min and plateaued at 2-3 h after NECA treatment. A similar time course of desensitization was observed with
basolateral NECA pretreatment and subsequent stimulation with
basolateral adenosine (i.e., maximum Isc inhibition
of 60% ± 3% compared with basolateral adenosine treatment alone was
seen 2-3 h after exposure to basolateral NECA). Figure 2B
shows that the inhibition of Isc response to apical
adenosine by pretreatment with basolateral NECA began around 3 h, and
>90% inhibition of apical adenosine response occurred between 6 and
12 h. Desensitization of the apical receptor by exposure to NECA did
not inhibit subsequent Isc response to basolateral
adenosine at any time.

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Fig. 2.
Time course of adenosine receptor desensitization. T84 cells were
exposed to apical or basolateral NECA (10 µM) at the various times
indicated. Cells were subsequently washed with HBSS, equilibrated at
37° for 20 min, and stimulated with adenosine (100 µM apical and
100 µM basolateral). Isc before NECA washout
varied depending on the time of exposure to NECA. 20 min: Ap NECA, 10 ± 2; Bs NECA, 11 ± 3; 1 h: Ap NECA, 7.1 ± 1; Bs NECA, 5.4 ± 0.9; 2 h: Ap NECA, 5.8 ± 2; Bs NECA, 3 ± 0.5; 3-12 h: values
ranged from 4.8 to 5.2 ± 2 for both apical and basolateral NECA.
Isc was measured 10 min after stimulation with
adenosine. Experiment was repeated at least 4 times and data from 1 representative experiment are plotted as mean ± SD, n = 3 per
treatment group. A: cells were desensitized apically or
basolaterally and stimulated with apical or basolateral adenosine,
respectively (i.e., stimulation added to the same domain as
desensitization). B: cells were desensitized apically or
basolaterally and stimulated with basolateral or apical adenosine,
respectively (i.e., stimulation added to opposite domain as
desensitization). Significantly different from cells stimulated with
adenosine alone, * P < 0.05, ** P < 0.001 by ANOVA.
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Recovery from desensitization.
To study the effect of polarity on the pattern of resensitization, T84
monolayers were desensitized with NECA for 12 h, washed, and then
stimulated with adenosine at various time intervals. As
seen in Fig. 3A, monolayers
pretreated with apical NECA were able to elicit maximal
Isc response to apical adenosine stimulation (compared with control monolayers treated with apical adenosine alone)
within 6 h of NECA washout. Similar time course of recovery was seen in
cells pretreated with basolateral NECA and then stimulated with
basolateral adenosine. In contrast, as shown in Fig. 3B, the
desensitization of apical adenosine receptors by basolateral NECA
showed only a partial recovery. Apical adenosine stimulation resulted
in only 60% ± 2% maximal response compared with apical adenosine
alone even 6 h after NECA washout. Full recovery to apical adenosine
response occurred around 12 h after NECA washout.

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Fig. 3.
Time course of recovery from adenosine receptor desensitization. T84
cells were exposed to apical or basolateral NECA (10 µM) for 12 h.
Cells were subsequently washed with HBSS and stimulated with adenosine
(100 µM apical and 100 µM basolateral) at the times indicated.
Isc was measured 10 min after stimulation with
adenosine. Experiment was repeated at least 3 times, and data were
plotted as mean ± SD, n = 3 per treatment group. A:
cells desensitized apically or basolaterally were stimulated with
apical and basolateral adenosine, respectively. B: cells
desensitized apically or basolaterally were stimulated with basolateral
and apical adenosine, respectively. * Significantly different from
cells stimulated with adenosine alone, P < 0.001 by
ANOVA.
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Heterologous desensitization.
Because heterologous desensitization has been reported for other G
protein-coupled receptors such as chemoattractant receptors of
neutrophils (1) and adenosine receptors in pulmonary epithelial cells
(14), we next sought to determine whether the desensitization of
adenosine receptors affected signaling of other receptors associated with chloride secretion. For this purpose, we used VIP
and carbachol, which elicit Isc via cAMP- and
calcium-dependent signaling pathways, respectively (6, 11). Monolayers
were desensitized with apical or basolateral NECA for 12 h, washed, and
then stimulated with VIP (2 nM, basolateral) or carbachol (10 µM,
basolateral). Figure 4 shows that
VIP-induced Isc was not affected by apical or
basolateral NECA pretreatment. Similarly, Isc
induced by carbachol was not affected by apical or basolateral NECA
pretreatment. In addition, Isc induced by the
direct activation of G
s protein by cholera toxin (20 nM) (17) was
not affected by apical or basolateral NECA treatment under conditions
where the Isc induced by adenosine is inhibited
(Fig. 4). The addition of forskolin, a direct activator of adenylate
cyclase, elicited the usual substantial increase in
Isc in the desensitized cells, suggesting that no
form of desensitization of A2b receptors interferes with the adenylate
cyclase-dependent signaling pathway (in µA/cm2): Fsk
alone, 62.2 ± 3.9; Ap NECA + Fsk, 57.8 ± 5.0; Bs NECA + Fsk, 78.7 ± 3.1; unstimulated, 4.1 ± 1.0. These results suggest that
desensitization observed here is specific to the adenosine receptor,
and heterologous desensitization as reported for other adenosine
receptors does not occur.

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Fig. 4.
Effect of adenosine receptor desensitization on other receptors. T84
cells were exposed to apical and basolateral NECA (10 µM) for 12 h.
Cells were washed and stimulated with vasoactive intestinal peptide
(VIP; 2 nM basolateral), carbachol (10 µM basolateral), or cholera
toxin (C.T.; 20 nM). Isc was measured 10 min after
stimulation with VIP, 3 min after carbachol, and 1 h after cholera
toxin. Unstimulated cells had an Isc of 1.9 ± 0.2 µA/cm2. Isc induced by VIP,
carbachol, and cholera toxin were significantly different from
unstimulated cells, P < 0.001. Experiment was repeated at
least 3 times, and data from 1 representative experiment were plotted
as mean ± SD, n = 6 per treatment group.
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Effect of low temperature or nocodazole on adenosine-induced
Isc.
One possible mechanism of desensitization of apical receptors as a late
consequence of basolateral receptor desensitization (and not vice
versa) could relate to a possible trafficking step in which the A2b
receptor is first targeted basolaterally upon synthesis. The receptor
could then be internalized and transported to the apical membrane
domain via vesicular transport/microtubules. Such trafficking has been
reported for the A1 receptor synthesis and trafficking in renal
epithelia (34). To test the hypothesis that a similar event occurs for
the intestinal A2b receptor, cells were incubated at 15-20°C
for 12 h to inhibit vesicular movement (17), and responses of cells to
apical or basolateral adenosine were subsequently tested. Under these
conditions, a selective inhibition of the apical response to adenosine
was observed while the basolateral response was preserved. As shown in
Fig. 5, incubating cells at
15-20°C inhibited apical response to adenosine by 54%, whereas the basolateral response remained 96% compared with control response (in µA/cm2): apical adenosine, 76.4 ± 7.2; low
temperature + apical adenosine, 41.2 ± 2.9; basolateral adenosine,
72.1 ± 4.0; low temperature/basolateral adenosine, 67.8 ± 4.2. Similarly, pretreatment of cells with nocodazole (33 µM equivalent to 10 µg/ml, 12 h), which disrupts microtubules by
preventing repolymerization of tubulin heterodimers (15), also
inhibited subsequent response to apical adenosine stimulation by 48%
(in µA/cm2): nocodazole + apical adenosine, 38.3 ± 4.4;
apical adenosine, 75.9 ± 6.8, whereas the response to basolateral
adenosine was not affected [93% of control response (in
µA/cm2): basolateral adenosine, 71.9 ± 3.9; nocodazole + basolateral adenosine, 66.6 ± 6.3]. Figure
6 shows the dose-response curve to apical
(Fig. 6A) and basolateral adenosine (Fig. 6B) after nocodozole treatment of cells. As seen in the figure, the dose response
to apical adenosine and not basolateral adenosine is blunted in cells
exposed to nocodozole. These observations are consistent with the
notion that apical A2b receptors traffic to the apical domain from the
basolateral domain by a process that is dependent upon
microtubule-mediated vesicular traffic.

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Fig. 5.
Effect of inhibition of vesicular movement/microtubule on
Isc response to adenosine. T84 cells were incubated
at low temperature (15-20°C), nocodazole (33 µM), or vehicle
for 12 h. Cells were washed with HBSS, equilibrated at 37° for 10 min, and stimulated with adenosine (100 µM apical and 100 µM
basolateral), and Isc was measured 10 min after
stimulation with adenosine. Experiment was repeated 3 times, and data
from 1 representative experiment were plotted as mean ± SD, n = 6 per treatment group.
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Fig. 6.
Effect of nocodazole on adenosine dose response. T84 cells were
treated with nocodazole (33 µM) for 12 h. Cells were washed and
stimulated with various doses of apical adenosine (A) or
basolateral adenosine (B). Isc was measured
10 min after stimulation with adenosine. Experiment was repeated at
least 2 times. Data were plotted as mean ± SD, n = 4 per
treatment group. Significantly different from cells treated with
vehicle + Ap adenosine treatment, * P < 0.05;
** P < 0.01 by ANOVA followed by Student-Newman-Keuls post
hoc test.
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 |
DISCUSSION |
Our study provides evidence that, in polarized epithelial cells,
agonist-induced desensitization of the natively expressed A2b receptor
on one domain is not only able to desensitize the receptor on the
opposite membrane domain but also induces a differential desensitization pattern, i.e., the basolateral receptor desensitization affects the apical receptor-induced Isc but not
vice versa. In addition, this study underlines the selectivity of A2b
receptor-mediated desensitization because neither the cAMP-mediated
Cl
secretion induced by agonists such as VIP,
cholera toxin, and adenylate cyclase activator, forskolin nor the
calcium-mediated Cl
secretion induced by carbachol
are affected by A2b receptor desensitization.
Attenuation of agonist-induced signaling is classically observed after
chronic stimulation of G protein-coupled receptors. Two patterns of
desensitization have been described: 1) a rapid event that
occurs after brief exposure to agonists (seconds to minutes) resulting
in the loss of agonist-induced functional response with no change in
receptor number or affinity for the ligand, and 2) a slower
event following long-term exposure to agonist (hours to days) that
results in the downregulation of the receptor caused by sequestration,
internalization, or decreased synthesis of the receptor (5, 13, 31,
32). Our results show that the A2b receptor is subject to both of these
patterns of desensitization albeit in a polarized manner. The
desensitization of the A2b receptor in the same membrane domain is
rapid and is totally reversible within 6 h of agonist washout.
Moreover, such desensitization reaches a plateau of 40-60%
maximal response within 3 h of exposure to NECA, and no further
desensitization occurs despite the presence of agonist. The rapidity
and reversible nature of desensitization of the A2b receptor in the
same membrane domain is suggestive of uncoupling of the A2b receptor
from G protein after receptor phosphorylation either by specific
receptor kinase (GRKs) or nonspecific phosphorylation of the receptor
(protein kinase A or C). Indeed, rapid termination of signaling by G
protein receptors is typically initiated by such phosphorylation events
catalyzed by either second messenger-activated kinases or G protein
receptor coupled kinases (GRKs), which in turn promotes high-affinity
binding of arrestins (13). Interestingly, recent studies on short-term
desensitization of A2 receptor in nonpolarized NG-108-15 (21) and
HEK-293 (22) cells have shown that GRK-2 and arrestin-2 are both
involved in the rapid desensitization process (21, 22).
In contrast to the desensitization of the A2b receptor in the same
membrane domain, the down modulation of Isc
response to apical A2b receptor stimulation after desensitization of
basolateral A2b receptor is slow, complete (100% inhibition of apical
Isc response to adenosine), and only partially
reversible at 6 h after NECA washout. Complete recovery did not occur
until 12 h after NECA washout. On the other hand, NECA pretreatment of
the apical A2b receptor does not affect subsequent
Isc response to basolateral adenosine stimulation
at any time. Interestingly, Barrett et al. (2, 3) observed that, after
1 wk of culture with apical and basolateral NECA, the apical response
to adenosine was completely abolished, whereas a significant component
of the basolateral response to adenosine persisted. However, the
polarity of desensitization of the apical receptor by the basolateral
receptor was not observed, possibly because these investigators did not
assess the compartmentalization on the desensitization process. The
polarized effect of the basolateral A2b receptor on the apical receptor
may reflect distinct receptor proteins, differences in receptor
density, differences in linkages to postreceptor signaling mechanisms,
or compartmentalization of intracellular target proteins. Our
observation cannot be explained by the presence of different types of
adenosine receptor in the apical and basolateral membrane domain
because, using molecular and pharmacological approaches, we have
previously shown that A2b is the only known adenosine receptor subtype
and is present in both the apical and basolateral membrane domains of
T84 cells (36). Moreover, we and others have shown that both apical and basolateral A2b receptors have similar
Km for activation by various agonists (3, 36) and, therefore, it is unlikely that the ligand-receptor interaction on both surfaces is different (36). The
cAMP response of the apical and basolateral receptors in response to
agonist differs markedly (36). Whereas the basolateral A2b receptor
results in a severalfold increase in cAMP, the apical A2b receptor
results in a small but significant increase in cAMP for comparable
Isc response to the same dose of agonist. However, our data show that cAMP levels alone do not influence desensitization because dibutyryl cAMP does not induce desensitization of
apical or basolateral adenosine receptors. By Northern blot analysis, the levels of A2b receptor mRNA are not altered in monolayers treated
with NECA under conditions that produced maximal desensitization for
Isc (preliminary data). These data do not exclude
the possibility that receptor density may be affected by
posttranscriptional or posttranslational mechanisms.
A possible explanation of the polarized effect of the basolateral A2b
receptor on the apical Isc response is that the A2b receptor is first targeted to the basolateral domain upon synthesis and
then moves to the apical membrane via vesicular transport. If so,
desensitization of the basolateral A2b receptor resulting in
downregulation of the basolateral receptor would affect the subsequent
expression of the receptor on the apical domain. Consistent with this
hypothesis is our observation that the inhibition of vesicular movement
or microtubules, which affect intracellular trafficking, selectively
inhibit Isc induced by apical adenosine while not
affecting Isc induced by basolateral adenosine
exposure. These data suggest that the A2b receptor may first be
targeted basolaterally and thereafter transported to the apical domain, as has been documented for a subset of other apical membrane proteins including the adenosine A1 receptor (4, 34). Although the polarized
effect of low temperature and nocodazole on adenosine-induced Isc is consistent with our hypothesis, this
result could also be explained by the effect of these treatments on
chloride secretion that is independent of adenosine receptor
trafficking. We are in the process of developing antibodies to the A2b
receptor to explore these possibilities.
In conclusion, we have demonstrated that a single receptor subtype,
here modeled by the A2b receptor, may differentially desensitize based
on the membrane domain on which it is expressed, and agonist exposure
on one domain can result in desensitization of receptors on the
opposite domain. This occurs in a polarized fashion, i.e., desensitization of the basolateral A2b receptor causes desensitization of the apical A2b receptor and not vice versa.
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ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-47662 and DK-35932. S. Sitaraman is a recipient of Crohn's and Colitis Career Development Award. D. Merlin is a recipient of National Research Service Award DK-09800.
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FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Sitaraman,
Dept. of Medicine and Pathology, Room 2101, WMRB, 1639 Pierce Drive,
Atlanta, GA 30322 (E-mail: ssitar2{at}emory.edu).
Received 21 October 1999; accepted in final form 7 January 2000.
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