(Received for publication, August 15, 1994; and in revised form, November 15, 1994)
From the
Adenosine is thought to be a major effector in immunological
stimulation of Cl secretion in intestinal epithelia.
Previous studies indicate that both apical and basolateral domains of
intestinal epithelial cells possess functionally defined adenosine
receptors. However, it is unclear whether the same receptor subclass is
expressed, what the receptor subclass(es) is, or how the receptors
signal the Cl
secretory response. We now characterize
the intestinal epithelial adenosine receptor subtype using the model
epithelium, T84. Both apical and basolateral adenosine receptor agonist
response profiles revealed a hierarchy (ED
) of
5`-(N-ethylcarboxamido)adenosine > adenosine >
CGS-21680. Similarly, inhibition studies revealed identical ID
hierarchies for apical and basolateral antagonism by xanthine
amine congener > 1,3-diethyl-8-phenylxanthine > aminophylline.
Analyses of both agonist and antagonist pharmacological hierarchies in
Chinese hamster ovary cells stably expressing the A
receptor revealed these same hierarchies. Northern blots
performed on RNA extracted from polarized T84 monolayers demonstrated
no detectable message for A
or A
adenosine
receptor, but strong hybridization was detected for the A
adenosine receptor. Subsequent Northern blots of RNA prepared
from human alimentary tract revealed that A
adenosine
receptor message was heavily expressed throughout the colon, in the
appendix, and more modestly expressed in the small intestine (ileum).
Analyses of cAMP generation in T84 cells in response to adenosine
indicated that the basolateral A
receptor elicits
Cl
secretion through this signaling pathway.
Stimulation of Cl
secretion through the apical
A
receptor exhibited relatively small but significant
increases in cAMP compared with basolateral stimulation. The protein
kinase A inhibitor H-89, used at concentrations that did not affect
short circuit current responses to the Ca
-mediated
agonist carbachol, effectively inhibited short circuit current elicited
by either apical or basolateral adenosine. These data suggest that the
major intestinal epithelial adenosine receptor is the A
subclass, which is positively coupled to adenylate cyclase. Such
observations have potentially important implications for the treatment
of diarrheal diseases.
The purine nucleoside adenosine regulates ion transport in a
variety of epithelia. For example, adenosine elicits electrogenic
Cl secretion in a variety of
epithelia(1, 2, 3) . In intestinal epithelia,
this Cl
secretory pathway results in movement of
isotonic fluid into the lumen, a process that naturally serves to
hydrate the mucosal surface but, in the extreme, produces secretory
diarrhea(4, 5) . Up-regulation of this secretory
mucosal flush often parallels active inflammatory responses elicited by
lumenal pathogens and, by reducing the duration of colonization by
these pathogens, serves as a crude form of mucosal
defense(4, 6, 7) . We have recently shown
that both polymorphonuclear leukocytes (PMN) (
)and
eosinophils, when activated, release a soluble agonist that directly
stimulates electrogenic Cl
secretion by intestinal
epithelial cells(8, 9, 10) . Subsequent
studies have shown that this PMN-eosinophil-derived secretagogue is
5`-AMP, which is rapidly converted to adenosine at the epithelial
surface via the ectoenzyme, ecto-5`-nucleotidase(11) . In
addition to release of 5`-AMP by PMN and eosinophils, release of
adenosine by mast cells is also thought to be a key mediator of mucosal
inflammation(1, 12, 13) . These data support
the notion that adenosine serves as a major direct-acting secretagogue
in a variety of inflammatory states.
Studies performed using
intestinal mucosal sheets or cultured human intestinal epithelial cells
such as T84 cells, which appropriately model Cl secretion, indicate that adenosine is an effective secretagogue
whether placed apically or basolaterally(11, 14) . The
presence of apical receptors on these polarized epithelial cells is
conceptually important. Inflammatory cells (PMN and eosinophils)
respond to lumenal pathogens by migrating across the
epithelium(7, 15, 16) . Once in the lumen,
paracrine release of 5`-AMP permits efficient conversion to adenosine,
since the glycosylphosphatidylinositol-linked ectoenzyme is expressed
in polarized fashion on the apical membrane of
enterocytes(11) . Thus, engagement of the apical
5`-AMP-adenosine signaling pathway subsequent to transepithelial
migration facilitates coupling of two defenses: (i) translocation of
inflammatory cells into the threatened lumenal compartment and (ii)
subsequent volume flush of the mucosal surface. However, it is unclear
which adenosine receptor subclass(es) are expressed by intestinal
epithelial cells, whether apical and basolateral membranes express
different receptor subtypes and how the specific subclass of adenosine
receptor(s) expressed are coupled to signaling cascades that permit
activation of electrogenic Cl
secretion. Several
subclasses of adenosine receptors (AdoR) exist (A
,
A
, A
, A
), all of which are
examples of guanine nucleotide-coupled
receptors(17, 18, 19, 20, 21, 22, 23) .
Each AdoR subclass appears to exhibit restricted tissue expression and
unique pharmacokinetics (17, 18, 19, 24) . Studies using
mammalian expression systems suggest that all of these adenosine
receptors may couple to adenylate cyclase (17, 18, 19, 20, 21, 23, 24) .
In contrast, studies of Ado-elicited intestinal Cl
secretion indicated that the AdoR signaling pathway in this
epithelium does not appear to involve a cAMP, cGMP, or an intracellular
calcium signal(8, 11, 25, 26) .
Here, we report that the polarized human intestinal epithelial cell
line, T84, which serves as a predictive and widely used model for the
study of regulated intestinal Cl secretion,
exclusively expresses the recently cloned A
adenosine
receptor subclass. The basolateral A
receptor is
positively coupled to adenylate cyclase as assessed by measurement of
intracellular cAMP. Signaling through the apical receptor by adenosine
also elicits a dose-dependent increase in intracellular cAMP, which is
significantly smaller than that observed in cells stimulated with
basolateral adenosine. However, specific inhibition of protein kinase A
confirms that both apical and basolateral adenosine receptor signal
transduction is mediated by cAMP. Lastly, analyses of A
message in mucosal samples from along the human alimentary tract
suggest that the A
receptor is richly expressed at many
sites, particularly in the colon. These data may have important
implications for structuring new therapies for diarrheal disease
associated with intestinal inflammation in humans.
Figure 1:
Isc time
course and dose response of T84 cells to NECA. Both panels display the
T84 Isc response to apical () or basolateral (
) stimulation
with 1 µM NECA. The upper panel (A)
illustrates the time course of T84 Isc response to NECA, while the bottompanel (B) represents the dose
response. Each point represents the mean of at least four
experiments performed in duplicate or
triplicate.
Figure 2:
Isc dose-response curves for adenosine
agonists. Apical (leftpanel) and basolateral (rightpanel) Isc stimulation of T84 cells by NECA
(), Ado (
), and CGS-21680 (
) were performed in
duplicate, with each point representing the mean of at least
four experiments. Data are presented as the percent of the maximal Isc
response observed in each experiment.
Figure 3:
Isc response in the presence of adenosine
uptake inhibitors. Basolateral adenosine () was added to T84
cells previously incubated with the Ado uptake inhibitors
4-nitrobenzyl-6-thioguanosine (
) or 4-nitrobenzyl-6-thioinosine
(
), or with the phosphodiesterase inhibitor IBMX (
) or
with DPM (
), which possesses both Ado uptake and
phosphodiesterase inhibitory activity. All inhibitors were diluted
10
in HBSS
and 10% volume added to the cells to
initiate incubation. Data are the average of two experiments performed
in duplicate.
The AdoR
antagonist inhibition profiles (inhibition of response elicited by the
Ado ED dose for the apical or basolateral stimulation, 1
and 10 µM, respectively) are shown in Fig. 4and
summarized in Table 1. Again, both the apical and basolateral
receptors demonstrated identical antagonist hierarchies (XAC > DPX
> AM).
Figure 4:
Isc dose-response curves for adenosine
antagonists added apically or basolaterally to T84 cells. Apical (leftpanel) and basolateral (rightpanel) antagonists were dissolved in HBSS buffer containing an
ED
dose of adenosine (1
µM apical or 10 µM basolateral final Ado
concentrations) and added to cells, and Isc responses were acquired.
Each point represents the mean of at least three separate
experiments performed in duplicate. The antagonists used were XAC
(
), DPX (
), and AM (
). Data are presented as percent
of the Isc response to Ado alone for that
experiment.
The above data suggested that the same receptor subclass
was expressed apically and basolaterally and, by comparison to
published antagonist/agonist
hierarchies(17, 18, 19) , that the A receptor, recently cloned from rat brain and highly expressed in
rat colon, might underlie these responses. Thus, we analyzed
agonist/antagonist hierarchies in CHO cells stably transfected with the
A
AdoR and compared the results with those obtained from
T84 cells. Responses in CHO cells were measured as cAMP generation
since it has previously been shown that this receptor is positively
coupled to adenylate cyclase. As shown in Fig. 5, the
pharmacology of the expressed A
receptor in CHO cells
mirrored that of the apical and basolateral receptors of T84 cells
(NECA > Ado > CGS-21680 for agonist ED
values and
XAC > DPX > AM for antagonist ID
values).
Figure 5:
cAMP dose-response curves for Ado agonists
and antagonists from CHO cells stably transfected with the A AdoR. The leftpanel illustrates the agonist
stimulation of CHO cells by NECA (
), Ado (
), and CGS-21680
(
) added to cells in HBSS
buffer, while the rightpanel shows the antagonist inhibition of CHO
cell cAMP responses elicited by 10 µM Ado (antagonists
dissolved in HBSS
containing Ado and then added to
cells). Antagonists used were XAC (
), DPX (
), and AM
(
). In both panels, each point represents the
mean of three separate experiments performed in duplicate. Data are
presented as percent of the maximal cAMP response observed in each
experiment.
Figure 6:
Expression of adenosine receptor subclass
in T84 cells. Poly(A) RNA (20 µg) isolated from
T84 cells was hybridized with specific
P-labeled cDNA
probes for the A1, A2a, and A2b adenosine receptors. Darkbands represent specific hybridization signals, with a
myosin loading control designated by the arrow. RNA size
markers (Life Technologies, Inc.) are shown in the leftcolumn. Kb,
kilobases.
We next
determined whether the A receptor was expressed in the
human intestine. Previous in situ hybridization studies of rat
intestine have suggested that the epithelium represents the major site
within this tissue in which the AdoR is expressed. (
)As
shown in Fig. 7, the A
receptor appeared to be
expressed at many levels of the human alimentary tract including the
esophagus, gastric antrum, weakly in the small intestine, and heavily
throughout the colon. Again, two transcripts comparable in size with
those noted in T84 cells were present.
Figure 7:
Northern blot analysis of the A adenosine receptor expression in eight different human tissues.
Poly(A)
RNA (20 µg) isolated from rapidly frozen
natural human tissues acquired through surgery was hybridized with a
P-labeled A2b adenosine receptor cDNA probe. Darkbands represent specific hybridization signals. RNA size
markers (Life Technologies, Inc.) are shown in the leftcolumn. Kb,
kilobases.
Figure 8:
cAMP dose-response curves for adenosine
agonist stimulation in T84 cells. Agonists were added apically (leftpanel) or basolaterally (rightpanel), the reaction was allowed to run 10 min, and then
cells were lysed and the generated cAMP concentrations were analyzed.
Each point represents the mean of at least three separate
experiments performed in duplicate. Agonists used were NECA (),
Ado (
), and CGS-21680 (
). Data are presented as percent of
the Isc response to Ado alone for that
experiment.
Figure 9:
cAMP dose-response curves for adenosine
antagonist inhibition of basolateral Ado stimulation in T84 cells.
Antagonists were dissolved in HBSS buffer containing
100 µM Ado (final concentration), the response was allowed
to proceed for 10 min; then cells were lysed, and the generated cAMP
concentrations were analyzed. Each point represents the mean
of at least three separate experiments performed in duplicate.
Antagonists used were XAC (
), DPX (
), and AM (
).
Data are presented as percent of the Isc response to Ado alone for that
experiment.
Figure 10: Correlation between T84 cAMP and Isc responses to forskolin or adenosine stimulation. Basolateral stimulation of T84 cells was elicited by forskolin (leftpanel) or adenosine (rightpanel), and the Isc (curves) and cAMP (bars) responses were analyzed. All points were performed in duplicate or triplicate using IBMX in the ethanol extract buffer and represent the responses observed in four separate experiments. Data are presented as the mean percent of the maximal Isc response observed in each experiment on the leftaxes, and the picomoles of cAMP-generated/monolayer of T84 cells are indicated in a representative experiment on the rightaxes.
In contrast, cAMP generation following apical exposure to Ado
was less clearly related to the Isc response (Fig. 11B). While significant and progressive increases
in cAMP generation could be measured in the Ado concentration range of
3 10
to 10
M (roughly corresponding to the range of ED
saturation), no significant increase in cAMP generation was
measurable at the Ado concentration corresponding to the
ED
. In addition, the cAMP responses observed for the dose
range corresponding to ED
-ED
were
small (<10%) compared with those observed in this same area of the
dose-response curves for forskolin or basolateral Ado ( Fig. 10and Fig. 11). Therefore, to further test whether
the cAMP-dependent protein kinase mediates Isc in response to both
apical and basolateral adenosine stimulation, the Isc responses to Ado,
forskolin, or carbachol were assessed in the presence of the protein
kinase A-specific antagonist H-89. As shown in Fig. 12, the
cAMP-mediated response to forskolin and both the apical or basolateral
responses to Ado were comparably inhibited by increasing doses of H-89.
In contrast, Isc responses to the calcium-mediated agonist carbachol
were unaffected by the identical doses of H-89. These results, coupled
with the data presented above relating the Isc and cAMP dose curves for
Ado, strongly suggest that both apical and the basolateral
AdoR-mediated Isc responses signal via the cAMP-protein kinase A
pathway.
Figure 11: Comparison between apical and basolateral Ado stimulation of T84 cAMP and Isc responses. Adenosine stimulation of T84 cells was elicited basolaterally (leftpanel) or apically (rightpanel), and the Isc (curves) and cAMP (bars) responses were analyzed. All points were performed in duplicate using IBMX in the ethanol extract buffer and represent the responses observed in four separate experiments. Data are presented as the mean percent of the maximal Isc response observed in each experiment on the leftaxes, and the picomoles of cAMP-generated/monolayer of T84 cells are indicated in a representative experiment on the rightaxes.
Figure 12:
Inhibition of cAMP-dependent protein
kinase. Adenosine stimulation of T84 cells was elicited apically
() or basolaterally (
) with adenosine or by forskolin
(
) or carbachol (
), and the Isc responses were analyzed
in the presence of increasing doses of protein kinase A-specific
inhibitor H-89. All points are the average of two experiments
performed in duplicate and are presented as the mean percent of the
control Isc response observed in each experiment on the leftaxes.
These studies show that intestinal epithelial cells, as
modeled by the human cell line T84, express both apical and basolateral
AdoRs that pharmacologically behave as A adenosine
receptors. Northern blots of T84 mRNA using subclass-specific probes
reveal strong expression of the A
AdoR but no detectable
expression of the A
or A
subclasses of
receptor. Furthermore, reverse transcriptase-PCR did not identify any new AdoR subtypes in T84 cells. The strong expression of the
A
receptor by natural colonic tissue confirms that the
A
subclass of AdoR is expressed in the natural tissue as
well as the widely utilized T84 model. Lastly, we show that adenosine
signals Cl
secretion via cAMP in T84 cells.
In contrast to the basolateral receptor,
the signaling pathway linking the apical A receptor to the
Cl
secretory response seemed less straightforward
initially. In the presence of phosphodiesterase inhibitors in the lysis
buffer and throughout the cAMP assay, minor but significant increments
in cAMP were observed within the Isc dose-response curve following
apical stimulation (Fig. 11). However, such increments are not
apparent until the ED
dose for Isc generation has been
exceeded. On the surface, these data appear to negate a positive
coupling of apical A
with adenylate cyclase. However, the
dose-dependent increase in cAMP elicited by apically applied Ado and
the closely correlated pharmacology between apical agonist stimulation
of Isc and cAMP indicated a relationship between Isc and cAMP. Since
the Isc dose-response curve is shifted one log to the left for apical versus basolateral stimulation (see below), with the apical
response occurring rapidly, and since T84 monolayers severely restrict
apical to basolateral diffusion of solutes, the small cAMP increments
seen with addition of apical Ado at concentrations of 5-10
µM can almost certainly not be attributed to Ado leak to
the basolateral compartment with subsequent binding to the basolateral
receptor. Therefore, we further investigated the relationship between
cAMP and Isc in Ado-stimulated T84 cells using a cAMP-dependent protein
kinase inhibitor, H-89. While H-89 comparably inhibited forskolin,
apical Ado, and basolateral Ado-elicited Isc responses over the same
dose range, these concentrations of the inhibitor had little or no
effect upon the carbachol-stimulated Isc. These data strongly suggest
that, like A
receptors in CHO cells and basolateral
A
receptors in T84 cells, the apical A
receptor is also coupled to adenylate cyclase. If so, these data
imply that T84 cells express a small pool of adenylate cyclase on the
apical membrane in close proximity to apically localized cAMP-dependent
Cl
channels. Thus, signal transduction via apical
AdoRs may occur at low receptor occupancy (ED
> 10-fold
lower) via efficient coupling to adenylate cyclase, protein kinase A,
and Cl
conductance
pathways(30, 31, 32) . Such efficiency would
simply reflect the close spatial relation between the components of
this putative signal-transducing pathway.
Given these data above, it
seems that explanations for apical signaling by adenosine via a
cAMP-protein kinase A-independent pathway may not be necessary. Such
alternative explanations would have included the possibility that the
A receptor may be linked directly to Cl
channels via heterotrimeric GTPases. The serpentine receptor
family to which adenosine receptors belong are, as a paradigm, linked
heterotrimeric G-proteins(24) . Since the apical receptor
resides in the same membrane domain as the regulated Cl
channel and since it has been shown that G-proteins can directly
regulate the cAMP-responsive Cl
channel,
CFTR(33) , it is possible that subclasses of receptors within
this family could via a direct cAMP-independent pathway regulate
Cl
secretion. Additionally, Barrett and Bigby (34) have recently reported evidence of phospholipid remodeling
and arachidonic acid release as a consequence of exposure of T84 cells
to adenosine analogs. These observations raise the possibility that
signal transduction following apical stimulation might be influenced by
a lipid-derived mediator. Finally, the possibility exists that multiple
signaling pathways might be involved in mediating the apical Ado
response, thus permitting potentiation of responses as observed in
other cell types and
systems(35, 36, 37, 38) . While the
present study clearly indicates that cAMP-protein kinase A signaling is
crucial to the Isc response to signaled by apical adenosine, the
possibility that such small cAMP responses interact synergystically
with other mediator pathways (such as those possibilities outlined
above) are not ruled out by our findings.
In summary,
human intestinal epithelial cells and human colonic tissue contain
abundant adenosine receptor of the A subclass. Both the
apical and basolateral receptors of T84 cells appear to represent
A
receptors. The signaling pathway for both apical and
basolateral adenosine-mediated Cl
secretion is via
cAMP. Lastly, basolateral stimulation with the natural agonist is less
sensitive than that observed with apical agonist stimulation due to
polarized uptake of authentic adenosine. Apical adenosine is known to
stimulate intestinal secretion in natural as well as cultured
intestinal epithelium, and adenosine is held to be a potentially key
agonist associated with inflammatory cell infiltration of
mucosa(43, 44) . The expression of this unique
subclass of adenosine receptor at this site opens the vista of
lumenally directed, A
subclass-specific receptor
antagonists for the treatment of secretory diarrhea associated with
inflammation.