©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The A Adenosine Receptor Mediates cAMP Responses to Adenosine Receptor Agonists in Human Intestinal Epithelia (*)

(Received for publication, August 15, 1994; and in revised form, November 15, 1994)

Gregg R. Strohmeier (1)(§) Steven M. Reppert (2) Wayne I. Lencer (3) James L. Madara (1)

From the  (1)Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women's Hospital, the (2)Department of Pediatrics, Massachusetts General Hospital and (3)Children's Hospital, and the Departments of Pathology and Pediatrics, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(1) 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.


INTRODUCTION

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) (^1)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(1), A, A, A(3)), 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.


MATERIALS AND METHODS

Cell Culture

Approximately 10^3 monolayers were used for these studies. Confluent monolayers of the human intestinal epithelial cell line T84 were grown on collagen-coated permeable supports and maintained until steady-state resistance was achieved, as previously described(27) . The configuration of the majority of monolayers used was identical to that previously developed for a microassay(28) . Measurements of transepithelial resistance, voltage, and short-circuit current (Isc) were performed in Hanks' balanced salt solution (HBSS) using standard biophysical techniques as previously described(9, 27, 28) . Studies of cAMP generation were performed both using 0.33- and 5-cm^2 inserts. Chinese hamster ovary cells (CHO) transfected with the mouse A receptor were grown in 24-well plates in the presence of positive selection (geneticin) as described(19) . For assays of Ado responses, CHO cells were washed and refed with media lacking geneticin 2 days prior to use, grown 2 days, and then washed with HBSS. Responses to adenosine analogs were analyzed as described below.

cAMP Measurement

Measurements of cAMP were performed on ethanol extracts of cells obtained from monolayers grown on permeable supports, using a radioimmunoassay kit as directed by the supplier (DuPont NEN). Briefly, after monolayers were washed with HBSS and incubated 10 min, base line Isc readings were taken, and then agonists were added by the addition of 10% volume buffer containing agonist. For antagonist studies, the added 10% volume contained both adenosine and specific antagonist. The dose-response curves for antagonist inhibition of adenosine cAMP responses were performed using 100 µM adenosine (a dose at which cAMP increases 1 order of magnitude above base line). 5 min after stimulation, short circuit responses were acquired to verify the level of Cl secretion. At 10 min, reservoirs were aspirated from the monolayers, and 4 °C HBSS was added to stop the reaction. Quickly, the filters on which monolayers rested were cut from the plastic supports and placed in eppendorfs containing extract buffer (66% ethanol, 33% HBSS) at 4 °C. Where indicated, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (1 mM, Sigma) was included in the extract buffer. Monolayers were compacted and centrifuged, and an aliquot (100 µl) was withdrawn for the radioimmunoassay.

Molecular Analyses

Northern blot analysis was performed on poly (A) RNA isolated from six 5-cm^2 monolayers of T84 cells carefully scraped off the filters in cold HBSS and concentrated by low speed centrifugation. Primary tissue was acquired from the frozen section room of Brigham and Women's Hospital, mucosa stripped of the underlying muscularis, rapidly frozen on aluminum foil set on dry ice, and stored until use. Total RNA was isolated using the guanidium-thiocyanate method, and poly(A) RNA was isolated using oligo(dT)(29) . 5 µg of poly(A) RNA were fractionated on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (GeneScreen, DuPont NEN). Blots were hybridized with the requisite AdoR cDNA P-labeled by the method of random priming (specific activity > 10^9 cpm/µg). Hybridization reactions were performed in 50% formamide, 1 M NaCl, 1% SDS, 10% dextran sulfate, and denatured salmon sperm (100 µg/ml) at 42 °C overnight. The final wash of blots was 0.2 times SSC (1 times SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and 1% SDS at 65 °C for 40 min. Blots were exposed to x-ray film with an intensifying screen at -80 °C for 3 days.

Reverse Transcriptase-PCR Probing for AdoR Subclass Expression

Poly (A) RNA was prepared from T84 cells using established methods. 2 µg of the RNA were primed with oligo(dT) and reverse transcribed with avian myeloblastosis virus reverse transcriptase (Promega). The first strand cDNA was subjected to two rounds of 30 cycles each of PCR amplification with 1 µg of primer A (TCAGAATTCTA(T/C)ATGGTITACT(A/T)(C/T)AA(C/T)TT(C/T)TT) and primer B (TTCAAGCTTGGIA(A/G)CCA(A/G)(C/G)(A/T)IA(A/G)IGC(A/G)AA). Each reaction cycle consisted of incubations at 94 °C for 1.5 min, 45 °C for 2 min, and 72 °C for 2 min with Ampli Taq DNA polymerase (Perkin-Elmer Corp.). The amplified DNA was digested with HindIII and EcoRI and separated on an agarose gel. A prominent DNA band of approximately 220 base pairs was apparent, which was subsequently used to prepare and sequence recombinant clones as previously described(19) .

Reagents

All tissue culture supplies were obtained from Life Technologies, Inc., and cAMP radioimmunoassay kits were from DuPont NEN. AdoR agonists and antagonists and Ado uptake inhibitors were purchased from Research Biochemicals Inc. (Natick, MA), except adenosine. H-89 was obtained from Seikagaku America, Inc. (Rockville, MD). All other reagents were obtained from Sigma and Calbiochem.


RESULTS

Adenosine Receptor Stimulation

A Isc representing electrogenic Cl secretion may be elicited from T84 cells by the application of adenosine or its analogs to either the apical or basolateral membrane(11, 13, 14) . Fig. 1shows that both apical and basolateral NECA, a non-metabolizable AdoR agonist, rapidly stimulate a Isc response from T84 cells (dIsc/dt; peak Isc = 70.75 ± 8.85 µA/cm^2 for apical and 69.92 ± 6.17 µA/cm^2 for basolateral stimulation). The NECA-elicited increases in Isc for apical and basolateral stimulation show nearly identical time constants (apical 25.53 µA/cm^2/min and basolateral 24.56 µA/cm^2/min) and dose dependence (ED = 0.16 ± 0.03 µM and 0.06 ± 0.02 µM, respectively) (Fig. 1B). The T84 monolayers used for these studies had high electrical resistance (800-1200 ohmbulletcm^2), as is typical for this cell line (27) . Such severe restriction on the passive permeation of small hydrophilic solutes permits sidedness of responses to be clearly separated since agonists will not diffuse across the monolayer to activate receptors on the opposing membrane. Thus, these data confirm the presence of adenosine receptors on both apical and basolateral membranes and suggest that there is little difference in signaling from apical or basolateral membrane receptors when stimulated by NECA.


Figure 1: Isc time course and dose response of T84 cells to NECA. Both panels display the T84 Isc response to apical () or basolateral (bullet) 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.



T84 AdoR Subclass (Pharmacological Properties)

We next used pharmacologic approaches to characterize the properties of apically and basolaterally expressed adenosine receptors. In Fig. 2, the T84 Isc dose responses elicited by three AdoR agonists are shown. Either apical or basolateral stimulation with Ado, CGS-21680, or NECA induced a dose-dependent Isc response in T84 cells, with the hierarchy for the ED doses being identical for both apical and basolateral stimulation (NECA > Ado > CGS-21680, Fig. 2and Table 1). The dose dependences for NECA-induced secretion were identical for apical and basolateral receptors, and the secretory responses to four other agonists revealed only modest differences between ED values for apical versus basolateral stimulation (Table 1). However, T84 cells were 10-fold more sensitive to authentic adenosine applied to apical rather than basolateral cell surfaces (Fig. 2, A and B, respectively; apical 0.63 ± 0.16 µM EDversus basolateral 7.78 ± 1.77 µM ED). This difference in Ado-elicited Isc appears attributable to Ado uptake by the basolateral membrane, resulting in a shift of the dose-response curve to the left. Inhibition of the basolateral Ado uptake process by dipyridamole (DPM), 4-nitrobenzyl-6-thioguanosine, or 4-nitrobenzyl-6-thioinosine all shifted the basolateral Ado dose curve to the left (Fig. 3). The ED values of the resulting Ado dose-response curves in the presence of these inhibitors was 1 µM, 1.6 µM, and 1 µM, respectively, values approximating that observed for apical stimulation. Since one of the inhibitors of Ado uptake, DPM, may also exhibit phosphodiesterase inhibitory effects, IBMX was used as a control to show that the DPM effect on the dose-response curve could not be explained by phosphodiesterase inhibition. None of these inhibitors altered the apical Ado Isc response (not shown). Lastly, the apical Ado dose-response curve and the leftward shifted basolateral Ado + Ado uptake inhibitor dose-response curves not only overlay each other but are highly similar to the Ado dose-response curves measured in CHO cells transfected with the same Ado receptor subtype, which leads to the T84 cell responses (see below). Thus, such data indicate that the relative insensitivity of the basolateral response to Ado is due to depletion of the basolateral microenvironment due to the activity of basolaterally polarized adenosine uptake.


Figure 2: Isc dose-response curves for adenosine agonists. Apical (leftpanel) and basolateral (rightpanel) Isc stimulation of T84 cells by NECA (bullet), 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 (box) or with DPM (bullet), which possesses both Ado uptake and phosphodiesterase inhibitory activity. All inhibitors were diluted 10times 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 (bullet), 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 (bullet), 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 (bullet), 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.



AdoR Subclass mRNA in T84 and Natural Human Intestine

To verify that A receptors are expressed on T84 cells, we next performed Northern blots using specific cDNA probes for the A(1), A, and A AdoR. As shown in Fig. 6, only A-specific cDNA hybridized with mRNA from T84 cells, revealing two transcripts of approximately 2.4 and 1.7 kilobases. To examine whether additional AdoR family members may be expressed in T84 cells, reverse transcriptase-PCR was used employing a set of degenerate oligonucleotides. Primers were based on regions of the fifth and sixth transmembrane domains that are conserved among the A(1), A, and A AdoR cDNAs. Fragments of each of the three AdoR cDNAs were amplified. No other AdoR-like fragments were present, indicating the absence of expression of novel adenosine receptors that share the general homologies of this receptor family (data not shown).


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. (^2)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.



Ado- and Ado Analog-mediated cAMP Generation in T84 Cells

We next examined whether the A receptor was positively coupled to adenylate cyclase in the highly polarized T84 cell, as it is in the unpolarized CHO cell(18) . As shown in Fig. 8, all three agonists studied (Ado, CGS-21680, and NECA) elicited a cAMP response in T84 cells both apically (Fig. 8A) and basolaterally (Fig. 8B). The rank order of potency for the agonist cAMP ED values was the same as that observed for the Isc response, that is, NECA > Ado > CGS-21680 (see also Table 1), both apically and basolaterally. Strikingly, however, the ability to generate cAMP in response to apical versus basolateral agonists differed. As seen in Fig. 8, basolateral agonists were able to generate cAMP signals in 10-30-fold excess of the signals generated by apically applied agonists. The pharmacology of antagonist inhibition of the basolateral Ado-elicited cAMP responses was also examined. Fig. 9shows that the antagonist ID profile in T84 cells stimulated with 100 µM Ado added basolaterally, a dose chosen for its large cAMP increase in the control cells, had a pharmacological hierarchy of XAC > DPX > AM. The order of the antagonist inhibition is identical to that shown for inhibition of Isc response in T84 cells (Fig. 4, Table 1) and inhibition of cAMP generation in transfected CHO cells (Fig. 5).


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 (bullet), 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 (bullet), and AM (). Data are presented as percent of the Isc response to Ado alone for that experiment.



Relationship between A-generated cAMP and Isc Responses

It has been previously suggested that Ado analog-mediated Cl secretion in T84 cells is not linked to cAMP generation(8, 11, 26) . Thus, we defined the relationship between Ado and adenosine analog-mediated cAMP generation and the Isc response. A key aspect in considering such relationships, as pointed out by comparing the data in Fig. 2and Fig. 8, is that the ability of T84 cells to secrete Cl, as manifested by Isc, saturates well before the ability of the cell to generate cAMP. Therefore, using forskolin as a positive control for cAMP-mediated Isc, we focused on the physiologically relevant (for Isc) dose range and found that detectable and graded increments in cAMP occur with ascent through the Isc dose-response curve (Fig. 10). However, the cAMP response continues to rise beyond the point where the Isc response is saturated. Such subsequent rises in cAMP generation, which are non-physiological with regard to the already maximal Isc response, dwarf the cAMP responses that occur in the agonist concentration range for stimulated Isc (Fig. 10A). This point is readily apparent by comparing the cAMP responses from Fig. 8B (which approach 10^3 pmol cAMP/10^6 cells after basolateral exposure to agonists) with those from Fig. 10B (which, in the range of the Isc response, are less than 10^2 pmol cAMP/10^6 cells). In response to basolateral adenosine, a graded increase in cAMP was observed within the range of the doses required to elicit a secretory response. Moreover, the quantity of cAMP generated in response to agonist concentrations within the dose-response curve was similar between forskolin and basolateral adenosine. These data strongly suggest that the basolateral A receptor is positively linked to adenylate cyclase. Ligand binding to the basolateral receptor then elicits a Isc response via cAMP.


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 times 10 to 10M (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 (bullet) 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.




DISCUSSION

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(1) 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.

Signaling of ClSecretion through the AAdoR

Previous studies of adenosine-elicited Cl secretion in T84 cells (elicited by apical (neutrophil-derived secretagogue(8) ) or basolateral + apical (Ado and analogs(26) ) stimulation) have suggested that this receptor does not signal through cAMP. For example, others have found that the dose-response curve of cAMP generation, elicited by the Ado analog NECA, is shifted 1 order of magnitude to the right of that for Isc generation(26) . Two pieces of information from the current study help explain this. First, the cAMP responses to basolateral addition of adenosine over the physiologically relevant dose range are small compared to those obtained at doses above saturation. To observe such small changes, a phosphodiesterase inhibitor was needed in the original lysis buffer (data not shown). Second and more importantly, however, it appears that the ability of T84 cells to generate cAMP far exceeds the concentration of cAMP needed to saturate the Cl secretory response. Thus, even with the known cAMP-mediated Cl secretagogue forskolin(4) , the cAMP signals generated throughout the Cl secretory dose-response curve are dwarfed by those that follow saturation of the secretory response, thus shifting the ED by at least a magnitude. Since it appeared that the size of the Ado-elicited cAMP signal throughout the secretory dose-response curve is comparable with that throughout the forskolin dose-response curve, the response elicited by the basolateral A receptor is well accounted for by an A adenylate cyclase-cAMP signaling pathway. This fits well with reports that the physiological response profile, based on the additive and/or potentiating interactions of adenosine analogs with other secretagogues, was identical to that expected for a cAMP-based response (8, 26) .

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.

Polarization of the Secretory Response Elicited by the Natural Ligand

Using the adenosine analog NECA, others have previously shown that the Isc dose response was not significantly different between apical and basolateral stimulation, although it was noted that the size of the response to basolateral stimulation was modestly greater than that for apical stimulation(14) . We previously reported that a buffer conditioned by a neutrophil-derived secretagogue preferentially elicited Cl secretion from the apical membrane, with little evidence of basolateral secretory activity observable(8, 39) . When this neutrophil-derived agonist was later defined as 5`-AMP, it was also recognized that the concentrations present in solutions conditioned by activated neutrophils were in the low µM range. As shown here, adenosine in this concentration range is only an effective secretagogue when applied apically. Indeed, the ED for adenosine is 0.63 ± 0.16 µM apically but 7.78 ± 1.77 µM basolaterally. In contrast, the basolateral ED values for five other non-metabolized Ado analogs were 30-50% of those for apical stimulation. Such data imply a slightly greater sensitivity to basolateral as compared with apical stimulation for metabolically stable AdoR agonists. Thus, it is likely the greater apical sensitivity for the natural agonist reflects the presence of a catabolic pathway restricted to the basolateral domain. Such ``catabolism'' represents uptake of basolateral adenosine, a mechanism identified in other cell types(1, 2) , since inhibition of Ado uptake by three inhibitors shifted the basolateral adenosine dose curve to the left, closely approximating the apical dose curve. In addition, we have found that neither the adenosine deaminase inhibitor deoxycoformycin nor the 5`-nucleotidase inhibitor alpha,beta-methylene-ADP alters the ED shift observed between apical and basolateral adenosine stimulation (not shown).

T84 Cells and Natural Human Intestine Express the AAdoR (Therapeutic Implications)

Stehle et al.(18) recently used PCR-based approaches to clone a novel adenosine receptor subtype from rodent brain, which they classified as the A receptor based on its ligand binding characteristics and its apparent positive coupling to adenylate cyclase. Initial Northern blots of rat tissues indicated that the A receptor was expressed by the central nervous system and, in a restricted manner, in other organs. While no expression was detected in rodent liver, kidney, small intestine, or heart, expression in rodent large intestine, urinary bladder, and lung was found(18) . Adenosine is a known secretagogue in the ileum and colon (2, 3) and is the major direct-acting secretagogue released from stimulated neutrophils(8, 11) , although it is likely that other PMN products might also contribute to Cl secretion through indirect actions mediated by subepithelial cells(40) . Neutrophils release this secretagogue in the form of 5`-AMP, which is then converted by an ectoenzyme on the intestinal epithelial cell apical membrane to authentic adenosine(11) . During states of active intestinal inflammation, activated neutrophils accumulate in the colonic crypts, the site of electrogenic chloride secretion(41) , where the neutrophils have direct access to the apical membrane ectoenzyme as well as the apical AdoR(15, 42) . Indeed, such ``crypt abscesses'' are the major histological criteria used in evaluating active inflammatory intestinal disease(7, 42) . Since it appears that this apical receptor is of the A subtype and since free access to this receptor is afforded from the colonic lumen, an attractive form of therapy for Cl secretion related to active inflammation might be the lumenal application of non-metabolizable A-specific antagonists.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK-35932, DK-47662, DK-42125, and DK-48106. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Brigham and Women's Hospital, 20 Shattuck St., Thorn 1419, Boston, MA 02115. Tel.: 617-732-6528; Fax: 617-732-6796.

(^1)
The abbreviations used are: PMN, polymorphonuclear leukocytes; NECA, 5`-(N-ethylcarboxamido)adenosine; XAC, xanthine amine congener; DPX, 1,3-diethyl-8-phenylxanthine; AM, aminophylline; DPM, dipyridamole; CHO cells, Chinese hamster ovary cells; AdoR, adenosine receptor; HBSS, Hanks' balanced salt solution; Isc, short circuit current; IBMX, 3-isobutyl-1-methylxanthine; PCR, polymerase chain reaction.

(^2)
S. A. Rivkees and S. M. Reppert, unpublished observations.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Ray Frizzell for valuable discussions and thank John Lee and Charlene Delp-Archer for expert technical assistance.


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