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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Center for Clinical Pharmacology (E.K.J., Z.M., C.Z., R.K.D.) and Departments of Medicine (E.K.J., Z.M., C.Z., R.K.D.) and Pharmacology (E.K.J.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and Clinic for Endocrinology, Department of Obstetrics and Gynecology, University Hospital Zurich, Zurich, Switzerland (R.K.D.)
Received July 16, 2003; accepted September 9, 2003.
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Abstract |
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,
-methylene-adenosine-5'-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In CD cells in culture, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine, and these changes were also modulated by the aforementioned inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. In conclusion, the extracellular cAMP-adenosine pathway is an important source of adenosine in CDs.
); 2) in A6 cells, a model system for transport in the CD and in collecting ducts adenosine receptor agonists alter ion transport (Lang et al., 1985; Yagil, 1990; Schwiebert et al., 1992; Edwards and Spielman, 1994; Yagil et al., 1994; Ma and Ling, 1996; Hoenderop et al., 1998; Macala and Hayslett, 2002); and 3) administration of selective A1 receptor antagonists increases sodium excretion without enhancing potassium excretion (Kuan et al., 1993), a pharmacological profile consistent with a diuretic action in CDs. Although endogenous adenosine significantly modulates transport in CDs, the sources of endogenous adenosine that mediate this effect are unknown.
The extracellular cyclic AMP-adenosine pathway is defined as cAMP efflux from cells during activation of adenylyl cyclase followed by the extracellular conversion of cAMP to adenosine by the serial actions of ectophosphodiesterase (ecto-PDE) and ecto-5'-nucleotidase (ecto-5'-NT) (Jackson and Dubey, 2001). This mechanism of extracellular adenosine production provides hormonal control of adenosine levels in the cell-surface biophase in which adenosine receptors reside. Tight coupling of the site of adenosine production to the site of adenosine receptors permits a low-capacity mechanism of adenosine biosynthesis to have a large impact on adenosine receptor activation (Jackson and Dubey, 2001). There is now substantial evidence for a physiological role of the extracellular cyclic AMP-adenosine pathway in glomerular mesangial cells, renal and nonrenal vascular smooth muscle cells, cardiac fibroblasts, cerebral microvessels, cerebral cortex, hippocampus, adipocytes, and hepatocytes (Jackson and Dubey, 2001).
An important, yet untested, hypothesis is that the extracellular cAMP-adenosine pathway is a significant source of adenosine for CD epithelial cells. Activation of some types of hormone receptors—for example, dopamine type 1 receptors (Felder et al., 1993), 1-adrenoceptors (Hanson and Linas, 1995), and parathyroid hormone receptors (Reshkin et al., 1991)—residing on apical and/or basolateral membranes of tubular epithelial cells stimulates adenylyl cyclase and increases intracellular levels of cAMP. Egress of cAMP into the tubular lumen (across the apical membrane) and/or interstitial space (across the basolateral membrane) during activation of adenylyl cyclase could result in adenosine formation provided that CD epithelial cells transport cAMP and express ectophosphodiesterase and ecto-5'-nucleotidase.
To test the hypothesis that the extracellular cAMP-adenosine pathway exists in CD epithelial cells, we examined the ability of freshly isolated CDs and CD epithelial cells in culture to convert exogenous cAMP to AMP and adenosine. We also investigated whether stimulation of adenylyl cyclase increases extracellular levels of cAMP, AMP, and adenosine. Finally, we examined whether 3-isobutyl-1-methylxanthine [IBMX; a PDE inhibitor that penetrates cell membranes (Beavo and Reifsnyder, 1990)], ,-methyleneadenosine-5'-diphosphate [AMPCP; an ecto-5'-NT inhibitor (Zimmermann, 1992)], and 1,3-dipropyl-8-p-sulfophenylxanthine [DPSPX; a xanthine that inhibits only ecto-PDE because it is restricted to the extracellular compartment and cannot gain access to intracellular PDE (Tofovic et al., 1991)] alter the conversion of exogenous and endogenous cAMP to AMP and adenosine.
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Materials and Methods |
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Culture of CD Epithelial Cells. Freshly isolated CDs were washed in phosphate-buffered saline (PBS) without calcium and magnesium and incubated for 15 min with collagenase type IV (1 mg/ml in 5 ml of DMEM F12) in a shaking water bath at 4°C. Ten milliliters of DMEM F12 with 10% fetal calf serum (FCS) was added, and the sample was centrifuged. Pellets were resuspended in DMEM F12 with 10% FCS, and 1 ml of the suspension was added to 75-cm2 flasks. Before adding cells, culture flasks were preconditioned by incubating with FCS for 30 min. The medium was changed after 2 days. After 4 days the cells were detached with trypsin/EDTA, washed, and plated with DMEM F12 with 10% FCS. All experiments were performed in CD cells in the first passage.
Confirmation of Identity of CDs. The identity of freshly isolated CDs was confirmed by Western blotting and by RT-PCR. With regard to Western blotting, we examined the putative CD segments for: 1) lack of expression of thiazide-sensitive cotransporter (TSC), a protein expressed specifically in distal convoluted tubules (DCTs) (Knepper and Brooks, 2001); 2) lack of expression of bumetanide-sensitive cotransporter type 1 (BSC-1), a protein expressed specifically in TALs (Knepper and Brooks, 2001); 3) lack of expression of aquaporin-1, a protein highly expressed in PCTs (Nielsen et al., 2002) but not expressed in CDs (Nielsen et al., 2002); and 4) the expression of aquaporin-2, a protein expressed specifically in CDs (Nielsen et al., 2002). In these experiments we included in the Western blots positive controls for aquaporin-1 [purified (see above) freshly isolated PCTs], BSC-1 [purified (see above) freshly isolated TALs], and TSC (impure isolated tubules obtained as a by-product of the purification procedure described above that are enriched in DCTs).
With regard to RT-PCR-based identification of freshly isolated CDs, we compared the expression of mRNA for A1, A2A, A2B and A3 receptors in freshly isolated CDs, TALs, and PCTs. In this regard, A1 receptors are more highly expressed in CDs compared with other nephron segments (Smith et al., 2001) and it is likely that CDs express a unique profile of adenosine receptors relative to other nephron segments.
Since the CD cells in culture were derived from highly purified freshly isolated CDs, it was unnecessary to characterize those cells as stringently as the freshly isolated CDs. Nonetheless, we performed Western blots on CD cells in culture for aquaporin-2, BSC-1, and TSC.
Western Blotting. Electrophoresis, Western blotting, and immunostaining were performed as previously described (Jackson et al., 2002). Antibodies (anti-BSC-1, anti-TSC, anti-aquaporin-1, and anti-aquaporin-2) were kindly provided by Dr. Mark Knepper.
Extraction of RNA and RT-PCR. Extraction of RNA and RT-PCR was performed as previously described (Jackson et al., 2002) and using the primer sequences listed in Table 1.
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Studies in Freshly Isolated CDs. Freshly isolated CDs (approximately 0.2 mg) were incubated in capped tubes in 0.3 ml of L-15 medium in a shaking water bath at 37°C without or with exogenous cAMP in the absence of and presence of IBMX (1 mmol/l), AMPCP (0.1 mmol/l), and DPSPX (1 mmol/l). The concentrations of various inhibitors were based on our previously published experience with inhibition of exogenous cAMP metabolism in cultured cardiac fibroblasts and vascular smooth muscle cells (Dubey et al., 1996, 2000). Just before adding the CDs to the L-15 medium, the medium was bubbled with 100% oxygen. Just after adding the isolated CDs, the head space of the capped tubes was flushed with 100% oxygen. After 20 min of incubation the isolated CDs were centrifuged and the supernatant was collected, transferred immediately into ice-cold tubes, and frozen at –70°C until purine levels were measured. The pellet was solubilized in 0.5 N NaOH, and protein content was measured by the copper bicinchoninic acid method.
Studies in CD Epithelial Cells in Culture. Monolayers of CD cells in culture were grown in 12-well culture plates as described (see above). On the day of the experiment, the CD cells were washed with PBS and then incubated (37°C under 5% carbon dioxide in a cell incubator) with various treatments. Some CD cells were incubated for 0, 5, or 30 min with either 0, 10, 30, or 100 µmol/l of cAMP in PBS. In cells treated with 0, 10, 30, or 100 µmol/l of cAMP for "0" min, the PBS containing cAMP was added to the cells and then immediately removed. Other CD cells were treated with 100 µmol/l of cAMP for 30 min in the presence and absence of IBMX, DPSPX, and AMPCP. In addition, some CD cells were treated for 30 min with stimulators of adenylyl cyclase and in the presence or absence of IBMX, DPSPX, and AMPCP. Adenylyl cyclase was stimulated by treating the cells with a mixture of forskolin (10 µmol/l, a direct activator of adenylyl cyclase); parathyroid hormone (1 µmol/l); isoproterenol (1 µmol/l, a -adrenoceptor agonist); and fenoldapam (10 µmol/l, an agonist of dopamine type 1 receptors). In our cell culture system we did not know which receptors were both expressed and coupled to stimulate adenylyl cyclase. Therefore, we used the strategy of adding several agonists of adenylyl cyclase simultaneously. At the end of the specified incubation time, the conditioned PBS was collected, transferred immediately into ice-cold tubes, and frozen at –70°C until purine levels were measured. At the end of the experiment cells were solubilized in 0.5 N NaOH and protein content was measured by the copper bicinchoninic acid method.
Analysis of Purines. In experiments using exogenous cAMP, purines were measured by HPLC using UV absorption as previously described (Jackson et al., 1996). In experiments with adenylyl cyclase activators, cAMP and adenosine concentrations were measured by HPLC using fluorescence detection (a higher sensitivity relative to the UV method) as previously described (Jackson et al., 1996), and AMP was measured by HPLC using UV absorption.
Statistical Analysis. Data were analyzed by 1-factor or 2-factor analysis of variance followed by a Fisher's least significant difference test. The criterion of significance was P < 0.05. Results are presented as means ± S.E.M.
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; Nielsen et al., 2002).
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Figure 2, A–D are ethidium bromide-stained agarose gels of RT-PCR products. Freshly isolated CDs yielded a strong signal for the 790-base pair amplicon expected for the A1 receptor mRNA, whereas freshly isolated PCTs and TALs did not (Fig. 2A). Freshly isolated CDs also provided a signal for the 615-base pair amplicon expected for the A2A receptor mRNA; however, the signal was stronger for freshly isolated PCTs (Fig. 2B). Freshly isolated CDs did not demonstrate expression of the A2B receptor (1281-base pair amplicon) or A3 receptor (640-base pair amplicon) mRNA (Fig. 2, C and D, respectively), whereas freshly isolated PCTs clearly expressed A3 receptor mRNA. This expression profile was consistent with the conclusion that the freshly isolated tubules used in the present study were CDs, since CDs are known to express more A1 receptors than other nephron segments (Smith et al., 2001) and since the profile of expression of adenosine receptors was unique compared with the other nephron segments.
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Figure 3A illustrates a light micrograph of CD cells in culture. Figure 1, B–D show Western blots using antibodies against aquaporin-2, BSC-1, and TSC, respectively. A clear signal for aquaporin-2, but not BSC-1 or TSC, was detected in CD cells in culture. This expression profile was consistent with the conclusion that the cells in culture used in the present study were indeed CD cells (Knepper and Brooks, 2001; Nielsen et al., 2002).
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Studies in Freshly Isolated CDs. Figure 4 illustrates the concentration-dependent (0–100 µmol/l) conversion of exogenous cAMP to AMP (Fig. 4A), adenosine (Fig. 4B), and inosine (Fig. 4C) in freshly isolated CDs. Addition of increasing concentrations of cAMP to CDs caused corresponding increases in the levels of AMP, adenosine, and inosine (a metabolite of adenosine) in the medium.
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Figure 5 summarizes the effects of inhibitors of PDE (IBMX), ecto-PDE (DPSPX), and ecto-5'-NT (AMPCP) on conversion of exogenous cAMP (30 µmol/l) to AMP (Fig. 5A), adenosine (Fig. 5B), and inosine (Fig. 5C) in freshly isolated CDs. IBMX and DPSPX decreased levels of AMP, adenosine, and inosine in the medium, and AMPCP decreased levels of adenosine and inosine, but increased levels of AMP.
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Studies in CD Epithelial Cells in Culture. Figure 6 illustrates conversion of exogenous cAMP to AMP (Fig. 6A) and adenosine (Fig. 6B) as a function of time (0, 5, and 30 min) and cAMP concentration (0, 10, 30, and 100 µmol/l). Addition of increasing concentrations of cAMP to CD cells caused corresponding increases in the levels of AMP in the medium. The increase in AMP after 30 min of incubation with cAMP was approximately the same as that obtained after 5 min of incubation, indicating rapid attainment of a steady state with respect to AMP. In contrast, adenosine levels in the medium continued to increase with time as adenosine accumulated in the medium. Inosine levels in the medium were below the detection limit of our assay. As with freshly isolated CDs, the accumulation of AMP in the medium after addition of exogenous cAMP was concentration-dependent; however, the formation of adenosine appeared to saturate at concentrations of cAMP less than 30 µmol/l.
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Figure 7 summarizes the effects of inhibitors of PDE (IBMX), ecto-PDE (DPSPX), and ecto-5'-NT (AMPCP) on conversion of exogenous cAMP (100 µmol/l) to AMP (Fig. 7A) and adenosine (Fig. 7B) in CD cells in culture. Inosine levels in the medium were below the detection limit of our assay. IBMX and DPSPX decreased levels of AMP and adenosine in the medium and AMPCP decreased levels of adenosine in the medium, but tended to increase levels of AMP.
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CD cells in culture were markedly responsive to activators of adenylyl cyclase (Fig. 8). In this regard, stimulation of adenylyl cyclase increased extracellular levels of cAMP, particularly in the presence of IBMX, DPSPX, and AMPCP (Fig. 8, B–D, respectively). Stimulation of adenylyl cyclase also increased extracellular levels of AMP (Fig. 8E), a response that was blocked by IBMX (Fig. 8F) and DPSPX (Fig. 8G). AMPCP increased baseline levels of AMP and augmented the increase in AMP induced by stimulation of adenylyl cyclase (Fig. 8H).
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As shown in Fig. 9A, stimulation of adenylyl cyclase in CD cells approximately doubled the extracellular levels of adenosine, and this response was abolished by IBMX (Fig. 9B) and DPSPX (Fig. 9C). AMPCP interfered with the fluorescence-based HPLC assay for adenosine, so we were unable to measure adenosine levels in CD cells treated with AMPCP.
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Discussion |
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). The intracellular ATP pathway is activated when energy demand exceeds energy supply and is mediated by intracellular dephosphorylation of ATP to adenosine (Schrader et al., 1977; Lloyd and Schrader, 1993; Decking et al., 1997). The extracellular ATP pathway is activated by the release of adenine nucleotides from a variety of cell types and is characterized by the metabolism of adenine nucleotides by ectoenzymes (ecto-ATPases, ecto-ADPases, and ecto-5'-NT) that convert ATP to adenosine (Schwiebert and Kishore, 2001). This latter pathway is engaged when adenine nucleotides are released during sympathoadrenal activation, platelet aggregation and activation of cells by clotting factors, neutrophil interactions, and catecholamines. The fourth mechanism of adenosine production is the cAMP-adenosine pathway, which involves conversion of cAMP to adenosine (Jackson and Dubey, 2001).
The cAMP-adenosine pathway is most amenable to physiological modulation by hormones (Jackson and Dubey, 2001). This mechanism has both intracellular and extracellular locations. Inside the cell, conversion of cAMP to AMP and AMP to adenosine is catalyzed via cytosolic PDE and cytosolic 5'-nucleotidase, respectively. In this case, adenosine thus formed reaches the extracellular space by way of facilitated transport. However, the biological significance of the intracellular cAMP-adenosine pathway is limited by the competition of cytosolic 5'-nucleotidase and adenylate kinase for AMP and by the competition of transport mechanisms with adenosine kinase for adenosine. Thus, the extracellular limb of the cyclic AMP-adenosine pathway is probably more important.
Many cell types express ecto-5'-NT, an enzyme that is tethered to the extracellular face of the plasma membrane via a lipid-sugar linkage and that efficiently metabolizes AMP to adenosine (Zimmermann, 1992). Since activation of adenylyl cyclase stimulates egress of cAMP into the extracellular space (Rindler et al., 1978; Barber and Butcher, 1981), provided sufficient levels of ecto-PDE exist, activation of adenylyl cyclase would lead to the extracellular metabolism of cAMP to AMP and hence to adenosine. Inasmuch as these biochemical processes would take place in a highly localized environment, adenosine formed by the extracellular cAMP-adenosine pathway would act in an autocrine/paracrine fashion to amplify, inhibit, and/or expand the local response to hormonal stimulation of adenylyl cyclase. In this regard, modest increases in cAMP biosynthesis could give rise to significant concentrations of adenosine at the cell surface.
The vascular compartment of the kidney clearly expresses an extracellular cAMP-adenosine pathway. For example, infusion of cAMP into the isolated rat kidney causes a concentration-dependent enhancement in the renal secretion rates of AMP, adenosine, and inosine, and the increases in AMP and adenosine secretion are attenuated by IBMX (PDE inhibitor) and DPSPX (ecto-PDE inhibitor) (Mi and Jackson, 1995). Moreover, AMPCP (ecto-5'-NT inhibitor) blocks the increases in adenosine, but not AMP, induced by cAMP in the isolated rat kidney (Mi and Jackson, 1995). Cultured preglomerular vascular smooth muscle cells (Jackson et al., 1997) and freshly isolated preglomerular microvessels (Jackson and Mi, 2000) also express the cAMP-adenosine pathway.
The results of the present study support the conclusion that renal epithelial cells in the CD express an extracellular cAMP-adenosine pathway. In this regard, addition of exogenous cAMP to freshly isolated CDs or CD cells in culture results in the synthesis of AMP and adenosine. Moreover, the conversion of exogenous cAMP to AMP and adenosine is blocked by inhibition of PDE (with IBMX) and ecto-PDE (with DPSPX). Also, inhibition of ecto-5'-NT (with AMPCP) blocks the conversion of exogenous cAMP to adenosine while enhancing the accumulation of AMP.
In the present study we examined the conversion of exogenous cAMP to extracellular purines. Since cAMP is hydrophilic and does not penetrate lipid bilayers, the conversion of exogenous cAMP to extracellular adenosine most likely represents the results of transformations in the extracellular compartment. This conclusion is corroborated by the results with AMPCP and DPSPX. AMPCP inhibits ecto-5'-NT, but not cytosolic 5'-NT (Zimmermann, 1992), and DPSPX does not penetrate cell membranes (Tofovic et al., 1991). Thus, the ability of these inhibitors to attenuate the conversion of exogenous cAMP to extracellular adenosine and inosine is strong evidence for an extracellular site of cAMP metabolism. The ability of AMPCP to increase extracellular AMP levels is also consistent with an extracellular site of cAMP conversion to AMP. In this regard, if cAMP were converted to AMP intracellularly, inhibition of ecto-5'-NT would not prevent the further metabolism of AMP to adenosine and would not cause the accumulation of extracellular AMP.
Additional evidence for a functional extracellular cAMP-adenosine pathway is provided by the results with stimulation of adenylyl cyclase in CD cells in culture. Consistent with our hypothesis, activation of adenylyl cyclase causes accumulation of cAMP, AMP, and adenosine in the extracellular medium. As expected, the increase in extracellular cAMP induced by stimulation of adenylyl cyclase is augmented by inhibition of PDE and ecto-PDE, whereas the increase in AMP and adenosine is blocked by inhibition of PDE and ecto-PDE. Moreover, the increase in AMP is enhanced by inhibition of ecto-5'-NT. This indicates that CD cells can 1) synthesize large amounts of cAMP; 2) transport intracellular cAMP to the extracellular compartment; 3) convert extracellular cAMP to AMP; and 4) convert extracellular AMP to adenosine.
Importantly, inhibition of ecto-5'-NT augments the ability of stimulation of adenylyl cyclase to increase extracellular levels of cAMP. As demonstrated in the present study, CDs express high levels of adenosine A1 receptors, and it is well known that activation of A1 receptors inhibits adenylyl cyclase. Thus the ability of AMPCP to enhance the cAMP response suggests that A1 receptors are activated by endogenous adenosine to restrain adenylyl cyclase activity. Along these same lines, DPSPX is both an adenosine receptor antagonist (Tofovic et al., 1991) and an ecto-PDE inhibitor. Therefore, the enhanced cAMP response in the presence of DPSPX may be due to a combination of factors, i.e., inhibition of the degradation of extracellular cAMP, inhibition of adenosine formation, and blockade of A1 receptors.
The ability of CDs to support a cAMP-adenosine pathway has important implications for the role of adenosine in modulating transport in CDs. Activation of several types of hormone receptors in the kidney epithelium stimulates adenylyl cyclase and increases intracellular levels of cAMP (Reshkin et al., 1991; Felder et al., 1993; Hanson and Linas, 1995). This would increase the egress of cAMP across the apical membrane into the tubular lumen and across the basolateral membrane into the renal interstitial space (Rindler et al., 1978; Barber and Butcher, 1981). Conversion of cAMP to adenosine by the extracellular cAMP-adenosine pathway on the external face of apical and basolateral membranes of epithelial cells in CDs would provide adenosine in the local environment of adenosine receptors, which would modulate the rate of tubular transport.
Exogenous cAMP added to monolayers of CD cells in culture would gain access mostly to the apical membrane. Thus the appearance of AMP and adenosine in the medium very quickly after adding exogenous cAMP to cultured CD cells indicates that apical membranes of CD cells express a functional extracellular cAMP-adenosine pathway. In contrast to monolayers of CD cells in culture, exogenous cAMP added to freshly isolated CDs would gain access first to the basolateral membrane, and then diffuse more slowly via the partially open ends of the tubules to gain access to the apical membranes. In the present study, the appearance of AMP and adenosine after addition of exogenous cAMP to freshly isolated CDs was delayed by approximately 15 min (data not shown). This suggests that even in freshly isolated CDs, it is the apical membrane that converts cAMP to adenosine. However, this inference must be evaluated by additional studies. A recent study in A6 distal nephron cells indicates that A1 receptors on both the apical and basolateral membranes stimulate sodium transport (Macala and Hayslett, 2002); therefore, regardless of whether the conversion of cAMP to adenosine is restricted to the apical membrane or occurs on both sides of CD cells, the adenosine so formed would have access to functional receptors.
It should be emphasized that this study applies only to the collecting duct. In this regard, it is highly likely that there are multiple sources of adenosine in the kidney and the sources of adenosine may differ quantitatively from site to site along the nephron. For example, recent evidence (Bell et al., 2003) indicates the release of ATP from basolateral membranes of macula densa cells. Released ATP could mediate signaling through P2 receptors, but it could also be another example of adenosine production from the hydrolysis of extracellular nucleotides playing a regulatory role, in this instance mediating tubuloglomerular feedback.
In summary, the present study indicates that inner medullary collecting ducts express an extracellular cAMP-adenosine pathway that provides adenosine to the cell surface whenever adenylyl cyclase is activated. It is conceivable that the extracellular cAMP-adenosine pathway importantly contributes to the regulation of epithelial transport in inner medullary collecting ducts.
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Footnotes |
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,-methyleneadenosine-5'-diphosphate; DPSPX, 1,3-dipropyl-8-p-sulfophenylxanthine; PCT, proximal convoluted tubule; TAL, thick ascending limb; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; RT-PCR, reverse transcription-polymerase chain reaction; TSC, thiazide-sensitive cotransporter; DCT, distal convoluted tubule; BSC-1, bumetanide-sensitive cotransporter type 1; HPLC, high-performance liquid chromatography. Supported by National Institutes of Health Grant 55314.
Address correspondence to: Dr. Edwin K. Jackson, Center for Clinical Pharmacology, University of Pittsburgh School of Medicine, 623 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261. E-mail: edj+{at}pitt.edu
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 T. Rieg, K. Pothula, J. Schroth, J. Satriano, H. Osswald, J. Schnermann, P. A. Insel, R. A. Bundey, and V. Vallon Vasopressin regulation of inner medullary collecting ducts and compensatory changes in mice lacking adenosine A1 receptors Am J Physiol Renal Physiol, March 1, 2008; 294(3): F638 - F644. [Abstract] [Full Text] [PDF]
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 V. Vallon, C. Miracle, and S. Thomson Adenosine and kidney function: Potential implications in patients with heart failure Eur J Heart Fail, February 1, 2008; 10(2): 176 - 187. [Abstract] [Full Text] [PDF]
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V. Vallon P2 receptors in the regulation of renal transport mechanisms Am J Physiol Renal Physiol, January 1, 2008; 294(1): F10 - F27. [Abstract] [Full Text] [PDF]
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E. K. Jackson, J. Ren, L. C. Zacharia, and Z. Mi Characterization of Renal Ecto-Phosphodiesterase J. Pharmacol. Exp. Ther., May 1, 2007; 321(2): 810 - 815. [Abstract] [Full Text] [PDF]
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E. K. Jackson, Z. Mi, L. C. Zacharia, S. P. Tofovic, and R. K. Dubey The Pancreatohepatorenal cAMP-Adenosine Mechanism J. Pharmacol. Exp. Ther., May 1, 2007; 321(2): 799 - 809. [Abstract] [Full Text] [PDF]
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E. K. Jackson, Z. Mi, and R. K. Dubey The Extracellular cAMP-Adenosine Pathway Significantly Contributes to the in Vivo Production of Adenosine J. Pharmacol. Exp. Ther., January 1, 2007; 320(1): 117 - 123. [Abstract] [Full Text] [PDF]
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 V. Vallon, B. Muhlbauer, and H. Osswald Adenosine and kidney function. Physiol Rev, July 1, 2006; 86(3): 901 - 940. [Abstract] [Full Text] [PDF]
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E. K. Jackson, L. C. Zacharia, M. Zhang, D. G. Gillespie, C. Zhu, and R. K. Dubey cAMP-Adenosine Pathway in the Proximal Tubule J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1219 - 1229. [Abstract] [Full Text] [PDF]
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 T. Pawelczyk, M. Grden, R. Rzepko, M. Sakowicz, and A. Szutowicz Region-Specific Alterations of Adenosine Receptors Expression Level in Kidney of Diabetic Rat Am. J. Pathol., August 1, 2005; 167(2): 315 - 325. [Abstract] [Full Text] [PDF]
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