Luminal adenosine stimulates chloride secretion through A1 receptor in mouse jejunum

Esam Ghanem,1 Cecilia Lövdahl,2 Elisabetta Daré,2 Catherine Ledent,3 Bertil B. Fredholm,2 Jean-Marie Boeynaems,3,4 Willy Van Driessche,5 and Renaud Beauwens1

1Department of Cell Physiology, Free University of Brussels, Brussels, Belgium; 2Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden; 3Institute of Interdisciplinary Research, Free University of Brussels, Brussels; 4Department of Medical Chemistry, Erasme Hospital, Free University of Brussels, Brussels; and 5Department of Physiology, Katholieke Universiteit Leuven, Leuven, Belgium

Submitted 2 August 2004 ; accepted in final form 27 December 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adenosine is known to stimulate chloride secretion by mouse jejunum. Whereas the receptor on the basolateral side is believed to be A2B, the receptor involved in the luminal effect of adenosine has not been identified. We found that jejuna expressed mRNA for all adenosine receptor subtypes. In this study, we investigated the stimulation of chloride secretion by adenosine in jejuna derived from mice lacking the adenosine receptors of A1 (A1R) and A2A (A2AR) or control littermates. The jejunal epithelium was mounted in a Ussing chamber, and a new method on the basis of impedance analysis was used to calculate the short-circuit current (Isc) values. Chloride secretion was assessed by the Isc after inhibition of the sodium-glucose cotransporter by adding phloridzin to the apical bathing solution. The effect of apical adenosine on chloride secretion was lost in jejuna from mice lacking the A1R. There was no difference in the response to basolaterally applied adenosine or to apical forskolin. Furthermore, in jejuna from control mice, the effect of apical adenosine was also abolished in the presence of 8-cyclopentyl-1,3-dipropylxanthine, a specific A1R antagonist. Responses to adenosine were identical in jejuna from control and A2AR knockout mice. This study demonstrates that A1R (and not A2AR) mediates the enhancement of chloride secretion induced by luminal adenosine in mice jejunum.

intestinal secretion; diarrhea; chloride channels


ADENOSINE IS AN ENDOGENOUS nucleoside that can regulate a large number of physiological and pathophysiological processes (4, 916, 18, 24, 25, 31). Adenosine is generated by hydrolysis of intra- or extracellular adenine nucleotides (11). Extracellular adenosine can either be taken up by cells by various transporters or it can interact with specific adenosine receptors. Four subtypes of such adenosine receptors have been cloned: A1, A2A, A2B, A3 (14). These receptors are coupled to G proteins, A2A/A2B to Gs, Golf, Gq/11, or G15/16 and the A1/A3 to Gi, Gq/11, or Go. Stimulation of A2A and A2B increases cAMP levels via activation of adenylyl cyclase, whereas activation of A1 and A3 results in decreased cAMP levels. In addition, stimulation of the phospholipase C pathway has been described after activation of A1, A2B, and A3 receptors (12, 13, 36).

Adenosine acts as a paracrine and/or autocrine mediator of chloride secretion in tracheal and intestinal epithelia (6, 37, 40). Intestinal secretion is driven by electrogenic chloride secretion by crypt cells; Cl ions then drive Na+ ions by electrical coupling, and water follows by osmotic coupling. The net amount of fluid secreted is substantially greater in the small intestine than in the colon, amounting to nearly 2 liters daily in humans because it has to buffer gastric, pancreatic, and biliary secretions within its lumen; yet it is balanced by the greater absorption capacity of the small intestine even in the absence of nutrients (15). In the colon, adenosine has been shown to regulate chloride secretion through occupancy of A2B receptors, which have been clearly demonstrated to occur on the basolateral membrane. This process may be relevant during inflammation, when extracellular adenosine production appears greatly increased (9, 24). However, much less is known about the receptors mediating chloride secretion when adenosine is applied apically (i.e., via the lumen), especially in the small intestine. We therefore decided to identify the adenosine receptor that is involved in the increase in secretion in response to luminal adenosine. To solve this problem, we elected to study jejunal chloride secretion in response to adenosine using mice genetically lacking a specific adenosine receptor subtype, i.e., either the A1 or the A2A receptor.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. Adenosine, phloridzin, indomethacin, forskolin, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) were purchased from Sigma (St. Louis, MO). The RNeasy kit was obtained from Qiagen (Hilden, Germany). The GeneAmp RNA PCR kit and the TaqMan Universal PCR master mix were purchased from Applied Biosystems (Applera, Stockholm, Sweden). Primers and probes were obtained from MWG-Biotech (Ebersberg, Germany). The rest of the chemicals were purchased from Sigma.

Mice. A1 and A2A knockout mice were generated as previously described (18, 25). Experiments were performed on adult male mice (2–3 mo old, 30–35 g body wt). Heterozygous A1 mice were of a mixed 129OlaHsd/C57Bl6 background. A2A heterozygous mice were backcrossed on a CD1 background for >20 generations before being intercrossed to generate the experimental wild-type and knockout mice. Mice were genotyped using PCR. Animals were maintained under controlled environmental conditions (12:12-h light-dark cycle, 21°C, 60% relative humidity, food and water ad libitum). The number of animals was kept to a minimum, and all efforts were made to avoid animal suffering. Experiments were carried out in strict accordance with both the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1996, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205] and the European Community regulations for animal use in research (CEE no. 86/609). The Regional Ethics Committee for Animal Research approved all animal studies.

Tissue isolation. mRNA expression studies were performed using pooled tissue obtained by scraping from the jejunum of nine wild-type and nine A1 receptor knockout male mice. The mice were CO2 anesthetized before intestine removal.

RNA extraction and cDNA synthesis. RNA was isolated from the tissue using the reagents and protocols included in the Qiagen RNeasy kit. The tissue was placed in the Qiagen lysis buffer and homogenized by using a rotor-stator homogenizer at full speed for 2 min and then centrifuged for 3 min at 8,000 g. The supernatant was further homogenized by using a syringe and needle before RNA extraction with the Qiagen columns. RNA quantity and quality were determined spectrophotometrically at 260 and 280 nm with a Biophotometer (Eppendorf, Hamburg, Germany). The cDNA synthesis was performed with the GeneAmp RNA PCR kit using random hexamers and the MuLV reverse transcriptase.

Real-time RT-PCR. Semiquantitative real-time RT-PCR (17) was performed by using {beta}-actin and subtype-specific (A1, A2A, A2B and A3) adenosine receptor primers and probes synthesized according to sequences previously published (7) in an ABI Prism 7000 Sequence Detector System (Applied Biosystems). The real-time RT-PCR reactions were performed by using the TaqMan Universal PCR master mix and probes labeled at the 5'-end with the reporter dye 6-carboxy-fluorescein and at the 3'-end with the quencher dye 6-carboxytetramethyl-rhodamine. The passive reference dye ROX was included in the master mix to adjust automatically for background variability due to pipetting errors as well as differences in detection sensitivity across the plate. The four adenosine receptor (AR) subtypes and {beta}-actin were analyzed in parallel using the same amount of cDNA (20 ng) for each reaction. The RT-PCR reactions were performed in triplicates. Values are expressed as the difference in the number of cycles to reach the detection threshold (Ct = cycle at threshold), using {beta}-actin as reference ({Delta}Ct = CtAR - Ct{beta}-actin).

Measurements of short-circuit current. Mice aged 2–5 mo were anesthetized with pentobarbital sodium, and the midportion of jejunum was dissected 2 cm after the ligament of Treitz. The jejunal mucosa was washed to remove all intestinal content using Krebs- bicarbonate-Ringer solution of the following composition (in mM) 140 Na+, 120 Cl, 5.2 K+, 1.2 Mg2+, 1.2 Ca2+, 2.4 HPO42–, 0.4 H2PO4, 25 HCO3, and 11.5 glucose. The jejunum was opened longitudinally along the mesenteric border, and under a dissecting stereomicroscope, the epithelium was stripped out of the subepithelial tissue, stretched, and sealed onto the basolateral side to a fixation ring with an opening diameter of 3 mm, using Histoacryl glue applied to the periphery of the plastic ring. The ring was mounted between the two halves of an Ussing chamber (exposed surface area: 0.8 mm2) with each compartment having a volume of 2 ml. The solution was continuously flowing from a reservoir where it was equilibrated with gas (95% O2-5% CO2) and maintained at 37°C. Small Ag-AgCl electrodes connected to the bathing solution via short agar bridges were used for passing current and measuring the potential difference. Chloride secretion by the jejunum was evaluated from the short-circuit current (Isc) measurements and is expressed in µA/cm2. Therefore, the direct-current value of the transepithelial voltage was clamped to zero with a fast voltage-clamp device. The gain of the voltage amplifier was 50 and the current amplifier had a sensitivity of 50 mV/µA.

Impedance analysis. The transepithelial resistance of jejunum is rather small (10–20 {Omega}·cm2), whereas the resistance of the bathing solution between the voltage electrodes in series with the epithelium is at least 40–50 {Omega}·cm2. Consequently, Isc will be underestimated and transepithelial resistances will be dominated by the solution of the bath. Therefore, we applied impedance analysis to discriminate between the resistance of the tissue and that of the bathing solution. These parameters were used to correct the Isc values. The electrical equivalent circuit of the cell membrane consists of a capacitor (lipid phase) in parallel with a resistor (ion conductance). The apical, as well as the basolateral, membranes can be represented by such a resistance-capacitance (RC) network. Figure 1A shows the equivalent circuit of the epithelium where Ra and Ca are the resistance and capacitance of the apical membrane, respectively. The basolateral membrane is represented by Rb and Cb. Both membranes, and thus the RC networks, are arranged in series and shunted by a conductive pathway residing in the paracellular pathway (41). Because the paracellular pathway of an epithelium-like small intestine has a very high conductance, the two-membrane model can be represented by a single lumped RC circuit, consisting of the transepithelial resistance (RT) in parallel with the transepithelial capacitance (CT) (Fig. 1B). The resistance of the solution between the voltage electrodes (Rs) resides in series with the epithelial RC model. Due to the heterogeneity of the preparation, the presence of the lateral interspace and the dielectric dispersion in the lipid membranes the electrical impedance [Z(f)] is a complex function that can be approximated by the Cole-Cole equation

where j represents the imaginary unit and f the frequency. The number {alpha} (<1) describes the nonideal RC behavior of the epithelium. The representation in a Nyquist plot of the impedance function of the ideal RC circuit as depicted in Fig. 1B results in a semicircle with its center on the real axis. On the other hand, due to the fact that {alpha} is < 1, the center of the semicircle will be depressed. Figure 1C illustrates an impedance function calculated with parameters that resemble the experimental values obtained with the intestinal preparation. Figure 1C also illustrates the method we used to determine the Rs as well as the RT with the sine wave method that was implemented to record the impedance function of the epithelium. Hardware used for this purpose was based on two digital signal processing (DSP) boards. One DSP board was used to record the total resistance across the epithelium and bathing solution (RTS = Rs + RT) together with the Isc. Therefore, a 1-Hz sine wave was applied to the tissue. Transepithelial current and voltage changes caused by this sine wave were sampled to calculate Isc and RTS. Isc was calculated from the mean value of the data recorded during a sine wave period. To calculate RTS, we determined the amplitudes of the current (IT) and voltage (VT) sine waves by regression analysis and calculated RTS as VT/IT. This procedure enabled us to update the RTS values every 7 s. Simultaneously, with the second DSP board, the CT and Rs were determined by applying five high-frequency sine waves in the range of 2 to 16 kHz. From the current and voltage response to the high-frequency sine waves, we calculated the impedance of the epithelium and bathing solution arranged in series. At high frequencies, the impedance of the equivalent circuit in Fig. 1 equals the Rs. Therefore, Rs was calculated by extrapolation of the real part of impedance data to infinite frequency. RT was calculated by subtracting Rs from the RTS values obtained with the 1-Hz sine wave. Low- and high-pass filters were used to avoid interference between the high- and low-frequency sine waves used for the Rs and RTS measurements, respectively. Isc values were corrected by dividing the recorded values by RT/RTS. Data obtained from four records were averaged.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1. Equivalent circuit of the jejunum. A: two-membrane model of the epithelium. Electrical properties of the apical and basolateral membranes are represented by a resistance (R) in parallel with a capacitor (C). Apical membrane: Ra and Ca, basolateral membrane: Rb and Cb. B: lumped model of the transepithelium (RT and CT) and series resistance (Rs = Rsol + Rsub), where Rsol represents the resistance of the solution in between the voltage electrodes, and Rsub is the subepithelial resistance. C: impedance function calculated RT = 20 {Omega}, Rs = 40 {Omega}, and CT = 4 µF and {alpha} = 0.95. The impedance is represented in a Nyquist plot. R represents the real or resistive part of the impedance. X is the imaginary part or reactance. Due to the distribution of the capacitance in the lateral interspace and dielectric dispersion, the center of the semicircle is depressed.

 
Chloride secretory response to adenosine. To record the chloride current component only, 1 mM phloridzin was added to the apical bathing solution to abolish the current related to the operation of the Na-glucose cotransporter. Because the stripping of jejunal mucosa may induce a release of prostaglandins that could themselves increase chloride secretion, an inhibitor of prostaglandin synthesis, indomethacin (100 µM), was added to both bathing solutions. Thus before testing of the chloride secretory response to adenosine, all tissues were first exposed to phloridzin and to indomethacin. Adenosine and the A1 receptor antagonist DPCPX (100 nM), were added to the apical or basolateral solution, depending on the experimental protocol. Forskolin (10 µM) was always added to the basolateral bathing solution. The increase in SCC (expressed in µA/cm2) was calculated as the difference between the basal current and the peak current obtained within 15 min of adenosine or forskolin addition.

Statistics. Data are expressed as means ± SE. Statistical analysis was performed by using Student's unpaired t-test.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression of adenosine receptors in mouse jejunum. The expression of adenosine receptors in the jejunum was assessed by semiquantitative real-time PCR analysis. In jejunum of wild-type mice, the abundance of adenosine receptor mRNA was characterized by the following rank order: A2B >> A2A = A3 > A1. The A1 knockout jejuna expressed levels of A2A, A2B, and A3 similar to those found in wild-type tissue (Fig. 2), but, as expected, no A1 mRNA. The difference between A2B and A2A mRNA is approximately sevenfold. The difference between A2B and A1 mRNA is ~34-fold.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2. Quantification of the mRNA expression level of adenosine receptor (AR) subtypes in the jejunum by real-time RT-PCR. Values ({Delta}Ct = CtAR – Ct{beta}-actin) indicate the difference in the number of cycles necessary to reach the detection threshold, using {beta}-actin as reference. Ct, cycle at threshold; WT, wild type; KO, knockout.

 
Role of adenosine A1 receptors. The addition of adenosine to the apical bathing solution increased Isc in jejuna derived from control mice, although rather high concentrations were required (Fig. 3). That chloride secretion fully accounts for Isc in the presence of phloridzin was attested by its fall when both solutions were made chloride free by isosmotic replacement by gluconate (Fig. 4). The adenosine-mediated response was not seen in jejuna from mice lacking A1 receptors (Fig. 3, Table 1). This increase in Isc was therefore ascribed to occupancy of apical A1 receptors. This was further supported by the fact that the A1 receptor antagonist DPCPX abolished the stimulation of chloride secretion. Forskolin elicited a substantially larger response than adenosine, but the pattern was similar in jejuna from both groups of mice (Fig. 3). There was no statistically significant difference in adenosine-induced Isc between the two groups (Table 1). Furthermore, the response to forskolin was similar regardless of the previous addition of adenosine (data not shown), implying no synergistic effect. On the other hand, the addition of adenosine to the basolateral side of the jejunum increased the Isc response similarly in both groups of mice (control and A1 receptor knockout), thus eliminating a role for A1 receptor in this latter secretory response (Table 1).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Effect of apical addition of adenosine (ado) on short-circuit current (Isc) across jejunal mucosa from either control (A1+/+; A) or A1 receptor knockout (A1–/–; B) mice. Adenosine was sequentially increased from 10 to 1,000 µM. Representative experiment of n = 5. Phlor, phloridzin; indo, indomethacin; fsk, forskolin.

 


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 4. Effect of removal of chloride on Isc across jejunal mucosa from control mice. Chloride was isosmotically replaced by gluconate (Cl).

 

View this table:
[in this window]
[in a new window]
 
Table 1. Effect of adenosine on chloride secretion by jejunal mucosa from A1 receptor knockout (A1–/–) or control (A1+/+) mice

 
Role of adenosine A2A receptors. Separate addition of adenosine to either the apical or basolateral side of jejuna from controls and from mice lacking the A2A receptor, induced identical increase in Isc (Table 2). Thus we find no role for the A2A receptor in this secretory response. The response to forskolin was always large and identical in both groups, in keeping with the important role of the cystic fibrosis transmembrane conductance regulator in jejunal crypts.


View this table:
[in this window]
[in a new window]
 
Table 2. Effect of adenosine on chloride secretion by jejunal mucosa from A2A receptor knockout (A2A–/–) or control (A2A+/+) mice

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have confirmed and extended previous reports on the distribution of adenosine receptors in the intestine (6). We show that mRNA for all four adenosine receptors is present in jejunal mucosa. The most abundant was A2B receptor mRNA, which is compatible with previous reports suggesting that A2B receptors mediate changes in gut epithelial chloride conductance (40). However, we have no direct proof that receptor protein of A2A, A2B, or A3 is present in mouse jejunum, but this is likely because A2B receptors are present on practically all cells, and A2A and A3 are present on vascular endothelium and/or cells of the immune system (1216, 24), both of which are obviously present in jejunum.

Extracellular occurrence of adenosine (either by generation or release) is encountered in several ischemic or stressed conditions and mediates protective effects in heart, brain, kidney, skeletal muscle, and adipose tissue. Because of these protective properties, adenosine is sometimes called a "retaliatory metabolite" (16, 31). Although this has not been extensively studied in the intestine, there is evidence that adenosine is biologically important there also. In ischemic and inflammatory conditions, polymorphonuclear leukocytes can migrate across the intestinal mucosa (32) and release 5' adenosine monophosphate (26) that is degraded into adenosine at the apical pole by an ecto-5'-nucleotidase (often referred to as CD73), anchored within the luminal membrane by a glycosylphosphoinositol linkage (39). This enzyme is also upregulated during hypoxia and ischemia (38). The secretory response to adenosine can also be considered beneficial because it will flush the mucosa and eliminate pathogens (hence preventing colonization). Strohmeier et al. (40) have characterized the chloride secretory response to adenosine added to either side in T84 colonic epithelia. By RT-PCR, only adenosine A2B receptor was found to be significantly expressed, implying that it mediates all the effects of adenosine on chloride secretion at both sides of the epithelium (4). This conclusion may hold for human or mouse colon, but certainly cannot be extended to small intestine, because we definitely show that A1 receptors are important there.

In the mouse jejunum, the A1 receptor mRNA appeared to be least expressed of all subforms. If A1 receptors are predominantly expressed in crypt cells, this would explain the low abundance. Indeed, our preliminary evidence is compatible with such a distribution (Lövdahl C, Daré E, and Beauwens R, unpublished data). As expected, the expression of A1 receptor mRNA was completely lost in the A1 receptor null mice. None of the other receptor mRNAs were affected, however. This tallies with previous data indicating that adenosine receptors do not alter the expression of each other to any appreciable extent (14). The luminal response to adenosine was absent in jejuna from A1 receptor knockout mice. Furthermore, the luminal response to adenosine observed in jejuna from control mice was also abolished by the specific A1 receptor antagonist DPCPX in agreement with previous findings (8, 27, 28, 34). The intracellular signaling pathway leading to increase in chloride secretion from A1 receptor occupancy is clearly unknown at the present time. In the jejunum, the CFTR appears as the only chloride channel present in the apical membrane (21, 23), whereas in colonic cells, calcium-activated chloride channels also appear to play a role in chloride secretion (3). The CFTR can be activated by mechanisms other than an increase in cAMP (21). In particular, it has been shown in several systems that increased intracellular calcium leads to increased trafficking and insertion of CFTR molecules into the membrane (1, 5, 10). In the jejunum, such a mechanism may explain the stimulation of chloride secretion by apical ATP and UTP that occurs via activation of the Gq-coupled P2Y4 receptor (33) and that is abolished in CFTR–/– mice (22). The A1-mediated chloride current induced by luminal adenosine might involve a similar mechanism because in airway epithelial cells, A1 agonists activate chloride secretion via mobilization of intracellular calcium (35). Other mechanisms also deserve consideration in light of the recent elegant studies of the group of Barrett and colleagues (2, 19), who established that transactivation of the EGF receptor (EGFR) is a new mechanism controlling intestinal chloride secretion with downstream effectors involving either phosphatidylinositol 3-kinase (PI3-kinase) or MAPK. For instance, the full chloride secretory response to VIP in T84 monolayers involves activation of PI3-kinase through EFGR transactivation by a typical Gs-coupled receptor (2). On the other hand, transactivation of EGFR via Gq-coupled receptors would decrease chloride secretion in T84 epithelial cells as a result of activation of MAPK (19). However, the coupling to either downstream effector may not be so straightforward as the occupancy of Gq-coupled receptor (e.g., acetylcholine and adenosine A1 receptor) in rabbit hearts leads to PI3-kinase activation through EGFR transactivation (20). Finally, the coupling of adenosine receptors to MAPK has been established (36), but its potential role in modulating chloride secretion remains to be examined.

The second part of our study established that A2A adenosine receptors do not appear to play any significant role in chloride secretion, at least in mice, because Isc increased to a similar extent in jejuna from control and A2A receptor knockout mice. This does not rule out that A2A receptors could play a role in ion transport in vivo, because these receptors do influence blood flow.

The basolateral adenosine receptor controlling chloride secretion could not be identified in the present study but is likely of the A2B type (40). Coexistence of different subtypes of adenosine receptors within the same tissue is known to occur in several cells of the human jejunum, including mucosa, myenteric neuron, and muscle layers (6), as well as in guinea pig small intestine muscle cells (29, 30). Thus A2B adenosine receptor null mice are now required not only to establish the role of A2BR in the response to basolateral adenosine but also to completely rule out any participation of A2BR in the response to apical adenosine.

In conclusion, generation of adenosine into the lumen of the jejunum induces a chloride secretory response that is mediated by A1 adenosine receptor.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
E. Dare was supported by Hjärnfonden, and C. Lövdahl was supported by the Wallenberg Foundation. This work was supported in Belgium by the Fonds National de la Recherche Scientifique Médicale, the Foundation Alphonse et Jean Forton, the Action de Recherche Concertée de la Communauté Française de Belgique, and the Fonds voor Wetenschappelijke Onderzoek Vlaanderen (Grant 0277.03), and in Sweden by ke Wibergs Stiftelse, Tore Nilsons Stiftelse, and funds from Karolinska Institutet.


    ACKNOWLEDGMENTS
 
We thank Prof. Brun Ulfhake for kindly letting us use the ABI Prism 700 Sequence Detector System.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. Beauwens, Dept. of Cell Physiology, Université Libre de Bruxelles, Campus Erasme CP 611, Rm. E1.6.214, Route de Lennik, 808, B 1070 Brussels, Belgium (E-mail: renbeau{at}ulb.ac.be)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Atia F, Zeiske W, and Van Driessche W. Secretory apical Cl channels in A.6 cells: possible control by cell Ca2+, and cAMP. Pflügers Arch 438: 344–353, 1999.[CrossRef][ISI][Medline]
  2. Bertelsen LS, Barrett KE, and Keely SJ. Gs protein-coupled receptor agonists induce transactivation of the epidermal growth factor receptor in T84 cells: implications for epithelial secretory responses. J Biol Chem 279: 6271–6279, 2004.[Abstract/Free Full Text]
  3. Barrett KE and Keely SJ. Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annu Rev Physiol 62: 535–572, 2000.[CrossRef][ISI][Medline]
  4. Bucheimer RE and Linden J. Purinergic regulation of epithelial transport. J Physiol 555: 311–321, 2004.[Abstract/Free Full Text]
  5. Cantiello HF, Prat AG, Reisin IL, Ercole LB, Abraham EH, Amara JF, Gregory RJ, and Ausiello DA. External ATP and its analogs activate the cystic fibrosis transmembrane conductance regulator by a cyclic AMP-independent mechanism. J Biol Chem 269: 11224–11232, 1994.[Abstract/Free Full Text]
  6. Christofi FL, Zhang H, Yu JG, Guzman J, Xue J, Kim M, Wang YZ, and Cooke HJ. Differential gene expression of adenosine A1, A2a, A2b, A3 receptors in the human enteric nervous system. J Comp Neurol 439: 46–64, 2001.[CrossRef][ISI][Medline]
  7. Chunn JL, Young HW, Banerjee SK, Colasurdo GN, and Blackburn MR. Adenosine-dependent airway inflammation and hyperresponsiveness in partially adenosine deaminase-deficient mice. J Immunol 167: 4676–4685, 2001.[Abstract/Free Full Text]
  8. Clancy JP, Ruiz FE, and Sorscher EJ. Adenosine and its nucleotides activate wild-type and R117H CFTR through A2B receptors coupled pathway. Am J Physiol Cell Physiol 276: C361–C369, 1999.[Abstract/Free Full Text]
  9. Cronstein BN. Adenosine, an endogenous anti-inflammatory agent. J Appl Physiol 76: 5–13, 1994.[Abstract/Free Full Text]
  10. Cuffe JE, Bielfeld-Ackermann A, Thomas J, Leipziger J, and Korbmacher C. ATP stimulates Cl secretion and reduces amiloride-sensitive Na+ absorption in M-1 mouse cortical collecting duct cells. J Physiol 524: 77–90, 2000.[Abstract/Free Full Text]
  11. Dunwiddie TV and Fredholm BB. Adenosine neuromodulation. In: Purinergic Approaches in Experimental Therapeutics, edited by Jacobson KA and Jarvis MF. New York: Wiley-Liss, 1997, p. 359–382.
  12. Feoktistov I and Biaggioni I. Adenosine A2B receptors. Pharmacol Rev 49: 381–402, 1997.[Abstract/Free Full Text]
  13. Fredholm BB. Adenosine receptors. In: Understanding G Protein-Coupled Receptors and Their Role in the CNS, edited by Pangalos MN and Davies CH. Oxford: Oxford University Press, 2002, p. 191–204.
  14. Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, and Linden J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev 53: 527–552, 2001.[Abstract/Free Full Text]
  15. Gawenis LR, Hut H, Bot AG, Shull GE, de Jonge HR, Stien X, Miller ML, and Clarke LL. Electroneutral sodium absorption and electrogenic anion secretion across murine small intestine are regulated in parallel. Am J Physiol Gastrointest Liver Physiol 287: G1140–G1149, 2004.[Abstract/Free Full Text]
  16. Hasko G and Cronstein BN. Adenosine: an endogenous regulator of innate immunity. Trends Immunol 25: 33–39, 2004.[CrossRef][ISI][Medline]
  17. Heid CA, Stevens J, Livak KJ, and Williams PM. Real time quantitative PCR. Genome Res 6: 986–994, 1996.[Abstract]
  18. Johansson B, Halldner L, Dunwiddie TV, Masino SA, Poelchen W, Gimenez-Llort L, Escorihuela RM, Fernandez-Teruel A, Wiesenfeld-Hallin Z, Xu XJ, Hardemark A, Betsholtz C, Herlenius E, and Fredholm BB. Hyperalgesia, anxiety and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor. Proc Natl Acad Sci USA 98: 9407–9412, 2001.[Abstract/Free Full Text]
  19. Keely SJ, Calandrella SO, and Barrett KE. Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T(84) cells is mediated by intracellular Ca2+, PYK-2, and p60(src). J Biol Chem 275: 12619–12625, 2000.[Abstract/Free Full Text]
  20. Krieg T, Qin Q, McIntosh EC, Cohen MV, and Downey JM. ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases. Am J Physiol Heart Circ Physiol 283: H2322–H2330, 2002.[Abstract/Free Full Text]
  21. Kunzelmann K and Mall M. Electrolyte transport in the mammalian colon: mechanisms and implications for disease. Physiol Rev 82: 245–289, 2002.[Abstract/Free Full Text]
  22. Lazarowski ER, Rochelle LG, O'Neal WK, Ribeiro CM, Grubb BR, Zhang V, Harden TK, and Boucher RC. Cloning and functional characterization of two murine uridine nucleotide receptors reveal a potential target for correcting ion transport deficiency in cystic fibrosis gallbladder. J Pharmacol Exp Ther 297: 43–49, 2001.[Abstract/Free Full Text]
  23. Leipziger J. Control of epithelial transport via luminal P2 receptors. Am J Physiol Renal Physiol 284: F419–F432, 2003.[Abstract/Free Full Text]
  24. Linden J. Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection. Annual Rev Pharmacol Toxicol 41: 775–787, 2001.[CrossRef][ISI][Medline]
  25. Ledent C, Vaugeois JM, Schiffmann S, Pedrazzini T, El Yacoubi M, Vanderhaegen JJ, Costentin J, Heath JK, Vassart G, and Parmentier M. Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor. Nature 388: 674–678, 1997.[CrossRef][ISI][Medline]
  26. Madara JL, Patapoff TW, Gillece-Castro B, Colgan SP, Parkos CA, Delp C, and Mrsny RJ. 5'-Adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithalial cell monolayers. J Clin Invest 91: 2320–2325, 1993.[ISI][Medline]
  27. Macala LJ and Hayslett JP. Basolateral and apical A1 adenosine receptors mediate sodium transport in cultured renal cells (A6) cells. Am J Physiol Renal Physiol 283: F1216–F1225, 2002.[Abstract/Free Full Text]
  28. McCoy DE, Schwiebert EM, Karlson KH, Spielman WS, and Stanton BA. Identification and function of A1 adenosine receptors in normal and cystic fibrosis human airway epithelial cells. Am J Physiol Cell Physiol 268: C1520–C1527, 1995.[Abstract/Free Full Text]
  29. Murthy KS and Makhlouf GM. Adenosine A1 receptor-mediated activation of phospholipase C-{beta}3 in intestinal muscle: dual requirement for alpha and beta gamma subunits of Gi3. Mol Pharmacol 47: 1172–1179, 1995.[Abstract]
  30. Murthy KS, McHenry L, Grider JR, and Makhlouf GM. Adenosine A1 and A2b receptors coupled to distinct interactive signaling pathways in intestinal muscle cells. J Pharmacol Exp Ther 274: 300–306, 1995.[Abstract]
  31. Newby AC. Adenosine and the concept of "retaliatory metabolites." Trends Biochem 9: 42–44, 1984.[CrossRef][ISI]
  32. Parkos CA, Colgan SP, Delp C, Arnaout MA, and Madara JL. Neutrophil migration across a cultured epithelial monolayer elicits a biphasic resistance response representing sequential effects on transcellular and paracellular pathways. J Cell Biol 117: 757–763, 1992.[Abstract]
  33. Robaye B, Ghanem E, Wilkin F, Fokan D, Van Driessche W, Schurmans S, Boeynaems JM, and Beauwens R. Loss of nucleotide regulation of epithelial chloride transport in the jejunum of P2Y4-null mice. Mol Pharmacol 63: 777–783, 2003.[Abstract/Free Full Text]
  34. Rubera I, Barriere H, Tauc M, Bidet M, Verheecke-Mauze C, Poujeol C, Cuilier B, and Poujeol P. Extracellular adenosine modulates a volume sensitive-like chloride conductance in immortalized rabbit DC1 cells. Am J Physiol Renal Physiol 280: F126–F145, 2001.[Abstract/Free Full Text]
  35. Rugolo M, Mastrocola T, Whorle C, Rasola A, Gruenert DC, Romeo G, and Galietta LJ. ATP and A1 adenosine receptor agonists mobilize intracellular calcium and activate K+ and Cl currents in normal and cystic fibrosis airway epithelial cells. J Biol Chem 268: 24779–24784, 1993.[Abstract/Free Full Text]
  36. Schulte G and Fredholm BB. Signalling from adenosine receptors to mitogen-activated protein kinases. Cell Signal 15: 813–827, 2003.[CrossRef][ISI][Medline]
  37. Sitaraman SV, Mustapha ST, Merlin D, Strohmeier GR, and Madara JL. Polarity of A2b adenosine receptors expression determines characteristics of receptor desensitization. Am J Physiol Cell Physiol 278: C1230–C1236, 2000.[Abstract/Free Full Text]
  38. Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK, Hansen KR, Thompson LF, and Colgan SP. Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest 110: 993–1002, 2002.[Abstract/Free Full Text]
  39. Strohmeier GR, Lencer WI, Patapoff TW, Thompson LF, Carlson SL, Moe SJ, Carnes DK, Mrsny RJ, and Madara JL. Surface expression, polarization, and functional significance of CD73 in human intestinal epithelia. J Clin Invest 99: 2588–2601, 1997.[Abstract/Free Full Text]
  40. Strohmeier GR, Reppert SM, Lencer WL, and Madara JL. The A2b adenosine receptor mediates cAMP response to adenosine receptor agonists in human intestinal epithelia. J Biol Chem 270: 2387–2394, 1995.[Abstract/Free Full Text]
  41. Van Driessche W, De Vos R, Jans D, Simaels J, De Smet P, and Raskin G. Transepithelial capacitance decrease reveals closure of lateral interspace in A6 epithelia. Pflügers Arch 437: 680–690, 1999.[CrossRef][ISI][Medline]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
288/5/G972    most recent
00346.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Ghanem, E.
Articles by Beauwens, R.
Articles citing this Article
PubMed
PubMed Citation
Articles by Ghanem, E.
Articles by Beauwens, R.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.