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1Department of Nephrology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City; and 2Department of Pharmacology, Facultad de Medicina, Universidad Autonoma de San Luis Potosi, San Luis Potosí, Mexico
Submitted 14 March 2007 ; accepted in final form 8 October 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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renal tissue adenosine; angiotensin II-mediated hypertension; kidney; adenosine receptors
Taking into account the evidence mentioned above, local renal adenosine concentrations could be the factor that might contribute to the interaction with ANG II, and this mechanism has been partially studied (24). In this context, 5'-nucleotidase (5'-ND) and adenosine deaminase (ADA) are the most important enzymes involved in adenosine metabolism; changes in the activity or the expression of these enzymes may modify the local concentration of adenosine (19), which may result in predominant activation of the A1 adenosine receptors and, as a consequence, influence the effect of ANG II.
Since the studies examining the effect of adenosine on renal blood flow in the whole kidney have been performed during acute systemic administration of adenosine and infusion and/or blockade of the renin-angiotensin system (10, 14, 17, 44), it is not clear whether differences of ANG II levels in the milieu could explain the different responses of kidneys to adenosine administration. Therefore, an animal model consisting of transient administration of ANG II, during several days, which can achieve full systemic and local effects of ANG II on the kidney (11, 22), is appropriate to study the interaction between ANG II and adenosine. Therefore, we decided to study whether the ANG II-adenosine interaction was observed in ANG II-induced hypertension.
The aim of this study was to assess the effects of a temporal infusion of ANG II on generation of tissue and interstitial adenosine in the kidney, to get an insight into the intrinsic interaction between both vasoactive compounds. For this reason, we studied 1) the participation of the extracellular nucleotide hydrolysis on the renal synthesis of adenosine, 2) the effects of blockade of ANG II and adenosine receptors on renal hemodynamics, 3) the effect of the elevated ANG II milieu on the activities of 5'-ND and ADA, as well as on their protein and mRNA expression, and finally, 4) the effect of the temporal infusion of ANG II in the expression of adenosine receptors in the kidney.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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,β-methyleneadenosine-5'-diphosphate (,β-mADP)] on renal adenosine content, two additional groups of rats (n= 9 per group) were studied: one group of rats received 435 ng·kg–1·min–1 ANG II plus vehicle, and the other group received 435 ng·kg–1·min–1 ANG II and an acute intra-aortic infusion of vehicle followed by a solution of ,β-mADP (0.00105 mg·kg–1·min–1) 14 days later. Third, to test the contribution of adenosine receptors or adenosine receptor activation on the renal vasoconstriction induced by ANG II, another two additional groups of rats (n = 9 per group) were studied: one group of rats received 435 ng·kg–1·min–1 ANG II plus vehicle, and the other group received 435 ng·kg–1·min–1 ANG II plus intra-aortic acute infusion of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A1-receptor blocker (10 µg·kg–1·min–1). In previous studies we found that this dose of DPCPX was sufficient to effectively inhibit A1-dependent adenosine responses in the normal kidney (12). All animals were kept on a normal diet, with free access to food and water. Care, use, and treatment of all animals in this study were in strict agreement with the guidelines of the animal facilities from the Instituto Nacional de Cardiología Ignacio Chavez.
Blood pressure measurements. Systolic blood pressure (SBP) measurements were performed in conscious, restrained rats by tail-cuff plethysmography (Narco Biosystems, Austin, TX). The rats were conditioned twice before the blood pressure was measured at a basal period, every week.
Proteinuria. The rats were placed in metabolic cages with water and food ad libitum, and urine was collected for a 24-h period; collections were taken during a basal period and every week before the kidneys were obtained. The samples were used for determination of proteinuria. Urinary protein excretion was measured using the trichloroacetic acid assay, with bovine serum albumin as the protein standard (18).
Kidney content of adenosine. To determine the renal tissue adenosine content, we used separate groups of 10 animals each with the same 435, 260, and 130 ng·kg–1·min–1 infusion of ANG II. The animals were anesthetized with pentobarbital sodium (30 mg·kg–1·min–1), the left kidney was exposed, and the whole organ was frozen in situ with a modified Wollemberger clamp precooled in liquid nitrogen. The time of the freezing procedure was <3 s (33, 34). Samples were stored at –80°C for subsequent adenosine determination. Tissue extraction of adenosine was performed by homogenizing 0.5 g of frozen renal tissue in five volumes of ice-cooled 0.4 M HClO4. The homogenates were then centrifuged at 9,000 rpm for 5 min at 4°C. The pH of the supernatant was kept constant at 8–9 for adenosine determination. Adenosine was measured by reverse-phase high-performance liquid chromatography (HPLC), according to the method of Hammer et al. (16). Separation of the compound was achieved using a C18 octadecylsilane column and a binary gradient containing buffer A [30 mM KH2PO4 and 7.5 mM tetrabutylammonium dihydrogen phosphate (TBA), pH 5.45] and buffer B [30 mM KH2PO4 and 7.5 mM TBA, pH 7.0 in 50% (vol/vol) acetonitrile]. Samples were filtered and measured directly into a Hewlett-Packard series 1100 HPLC, running an isocratic method. Adenosine was detected by an absorbance change at 255 nm, and the adenosine peak was identified and quantified by comparing it with retention times and peak areas of the known standards.
Renal interstitial concentration of adenosine. To determine the renal adenosine concentration, we used separate groups of animals (n = 9 rats per group). The renal adenosine concentration was determined in the renal cortex through a microdialysis technique, according to the method described by Nishiyama et al. (31) and Aki et al. (1). On day 14 after the beginning of the ANG II infusion, the animals were anesthetized with pentobarbital sodium (30 mg/kg ip) and supplementary doses were instilled as required. The rats were placed on a thermoregulated table, and the temperature was maintained at 37°C. Polyethylene tubing was used to catheterize the trachea (PE-240), jugular veins and femoral artery (PE-50), and the left ureter (PE-10); an intra-aortic catheter was introduced to reach 4–5 mm beyond the left renal artery for infusion of vehicle or antagonists. The left kidney was exposed and placed in a Lucite holder, covering the kidney surface with Ringer solution. Mean arterial pressure (MAP) was continuously monitored with a pressure transducer (model p23 LX; Gould, Hato Rey, PR) and recorded on a polygraph (Grass Instruments, Quincy, MA).
The rats were maintained euvolemic by infusion of 10 ml/kg body wt of isotonic rat plasma during surgery, followed by an infusion of 0.9% sodium saline solution at a rate of 1.2 ml/h. A microdialysis probe was gently implanted into the renal cortex. The probe was connected to a Harvard pump (Harvard Apparatus, Houston, MA) to perfuse a phosphate sodium solution, pH 7.4, containing 10 µM dihydrochloride hydrate (A134974), an inhibitor of adenosine kinase, as well as 100 µM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an ADA inhibitor, at a rate of 1.5 µl/min. The dialysates were collected in a chilled tube over a 90-min sampling period and were analyzed to determine the concentration of adenosine. The samples were stored at –70°C before analysis. After surgery, the preparation was equilibrated for 90 min to allow the stabilization of the interstitial adenosine concentration before the collection of samples was begun. Additional studies were performed to evaluate the effect of the blockade of 5'-ND on interstitial adenosine by infusing ,β-mADP at 0.00105 mg·kg–1·min–1 into the aorta (6, 23). The dose used induced the smaller changes in MAP and urinary flow compared with several tested doses (data not shown), including the 0.035 mg·kg–1·min–1 previously described (6).
Microdialysis probe. The dialysis membrane was made of polysulfone fiber, measuring 15 mm in length with a 0.20-mm outer diameter and a 33,000-Da transmembrane diffusion cutoff. Steel needles were inserted in both sides of the polysulfone fiber. The efficiency of the microdialysis probe was determined as follows: the probe was placed in a beaker containing an isotonic saline solution to which different quantities of adenosine were added. We perfused the probes with 250, 500, and 750 µM adenosine. The dialysate was collected, and the recovery of adenosine was calculated by dividing the concentration in the dialysate by the concentration in the medium. At a perfusion rate of 1.5 µl/min, the relative equilibrium rate of adenosine was 67.4 ± 2.2%, which did not vary with time (data not shown). In vivo calibrations were performed from 0 to 180 min to test the stability of the adenosine concentrations, according to the method described by Aky et al. (1). Thirty minutes after the implantation of the probe, the concentrations of adenosine fell from 101.9 ± 29.26 to 31.4 ± 4.0 nM and remained stable up to 160 min (nearly 29 nM). To determine whether adenosine derived from tubular fluid contaminated the dialysate, we used polyfructosan (Inutest; Laevosan-Gesellschafft, Linz, Austria) as a marker of tubular fluid as previously described (31); in three rats, concentrations of polyfructosan were 1.09 ± 0.12 mg/ml in plasma, 188.60 ± 12.95 mg/ml in urine, and 0.048 ± 0.01 mg/ml in the dialysate, indicating that adenosine derived from tubular fluid did not contaminate the dialysate (1, 3).
Adenosine fluorometric HPLC determination. Adenosine in the form of ethenoadenosine was measured in the dialysate samples by HPLC using a fluorescence detector. The determination was performed according to the method of Zhang et al. (47) with minor modifications carried out in our laboratory as follows: the derivatization procedure was initiated with 75 µl of the dialysate mixed with 15 µl of 1 M ZnSO4·7H2O plus 30 µl of a saturated solution of Ba(OH)2·8H2O. Samples were centrifuged at 13,000 rpm for 10 min at 4°C. The precipitate was withdrawn and discarded, and 100 µl of supernatant were mixed with 10 µl of 6 M chloroacetaldehyde. This preparation was incubated at 80°C for 1 h. The derivatives obtained were separated in two in-serial-connected reverse-phase Zorbax SB (C18) columns by an isocratic elution (1, 30). The mobile phase consisted of 90:10% (vol/vol) 10 mM phosphate buffer, pH 3.5, and methanol, respectively. Measurements were performed in a HPLC system (1100 series; Agilent Tech). Twenty microliters of this reaction were injected at a 1 ml/min flow rate. Fluorescence was measured at a 280-nm excitation wave and 380-nm emission waves. To determine recovery (85 ± 9%), we prepared known concentrations of adenosine (5, 10, 25, 50, 125, and 500 nM) by a reaction with chloroacetaldehyde as described above. Previously, a N6-ethenoadenosine standard curve was prepared. When derivatized samples were eluted, every four tubes with known standard samples (25 or 125 nM) were also intercalated in between to determine the detection limit (3.63 nM) and precision (3.6 and 9.2% for low and high intra-assay concentrations; 5.9 and 2.4% for low and high interassay concentrations). On account of the results obtained, only samples from animals that received 425 ng·kg–1·min–1 ANG II were used for the remainder of the study.
Micropuncture studies. The surgical preparation of the animals for micropuncture studies was identical to that described for the microdialysis studies. After the surgery, an infusion of 10% polyfructosan in 0.9% sodium saline solution was administered at a 2.5 ml/h rate. After 60 min, seven timed samples of proximal tubular fluid were obtained to determine flow rate and polyfructosan concentration. Intratubular hydrostatic pressure under free-flow and stop-flow conditions and peritubular capillary pressures were measured in other proximal tubules with a servo-null device (Servo-Nulling Pressure System; Instrumentation for Physiology and Medicine, San Diego, CA) as previously described (2, 8). MAP was continuously monitored with a pressure transducer (model p23 LX; Gould) and recorded on a polygraph (Grass Instruments). Blood samples were taken periodically every 45–60 min and replaced with blood from a normal donor rat. Polyfructosan was measured in plasma samples. Glomerular colloid osmotic pressure was estimated in protein from blood taken from the femoral artery and from the surface of the efferent arterioles.
Polyfructosan concentrations were determined using the technique of Davidson et al. (7). The concentration of tubular polyfructosan was measured using the method of Vurek and Pegram (43). The protein concentration in the efferent samples was determined using the method of Viets et al. (42).
Proximal single nephron glomerular filtration rate (SNGFR), intratubular pressure during free-flow and stopped-flow (SFP) conditions (after the tubular lumen was blocked with a long oil column), glomerular capillary hydrostatic pressure (PGC), peritubular capillary pressure, afferent oncotic pressure (
A), efferent oncotic pressure, glomerular capillary hydrostatic pressure gradient, single-nephron filtration fraction, single-nephron glomerular blood and plasma flow, afferent and efferent resistances, and ultrafiltration coefficient (Kf) were all calculated according to equations given elsewhere (2, 7). PGC was estimated using the stopped-flow method according to the following equation: PGC = SFP + A (2, 7).
Separation of cytosolic and membrane fractions of the renal cortex. On day 14, the rats were anesthetized with pentobarbital sodium (30 mg/kg), and the kidneys were exposed and removed, placed in cold sodium phosphate buffer solution, and dissected under ice into cortex and medulla. The tissues were frozen in liquid nitrogen and stored at –80°C for subsequent determination of 5'-ND and ADA activity as well as mRNA and protein expression and the expression of 5'-ND, ADA, and A1, A2a, A2b, and A3 receptors. The renal cortex was homogenized in glycerol buffer containing 25% (vol/vol) glycerol, 0.1 mM dithiothreitol, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 5 µM leupeptin, 2 µM pepstatin, and 50 mM HEPES, pH 7, at 4°C with a polytron (PT 2100; Kinematica) using four bursts of 30 s each at 7,000 rpm with a 2-min interval between bursts. The homogenate was then centrifuged at 100,000 gfor 1 h at 4°C. The supernatant was removed from each tube, and the pellets were resuspended in fresh glycerol buffer (6).
Assay of 5'-ND activity.
Cytosolic and ecto-5'-ND activity was measured according to the method of Wu et al. (46). 5'-ND was then electrophoretically separated in a 0.5-mm agarose gel prepared with 25 mM Tris-glycine buffer, pH 9.0, using a horizontal electrophoresis system. Electrophoresis was carried out in 50 mM Tris-glycine buffer, pH 9.0, at 20 V/cm across the gel for 2.5 h at 4°C. After electrophoresis, the gel was incubated in the reaction solution containing 50 mM Tris-maleate buffer, pH 7.0, 1 mM adenosine 5'-monophosphate (AMP), 2 mM Pb(NO3)2, and 50 mM Mn(NO3)2 (at 37°C for 3 h). Control gels were incubated in a reaction solution with the substrate AMP omitted. To verify the specificity of the reaction, we added 5 µM ,β-mADP to the reaction solution. After incubation, the gel was rinsed with distilled water and the bands corresponding to the enzyme reaction products were visualized with a 2% Na2S solution. After being washed, the gel was dried. The intensity of the enzyme reaction bands on the gel was measured in a densitometer (GS-800; Bio-Rad, Hercules CA). The activity of the 5'-ND was estimated by comparing the densitometric units of unknown samples with those of purified 5'-ND (2 mU) in the same gel.
Assay of ADA activity. Cytosolic and ecto-ADA activity was measured according to the method described by Wu et al. (46). Isoelectric focusing was carried out in 0.5-mm thick gels containing 4.85% acrylamide, 0.15% bisacrylamide, 2% (vol/vol) preblended ampholytes, pH 3.5–9.5 (Bio-Rad), 300 mM sucrose, and 2 µM riboflavin. The electrode solutions were composed of 150 mM acetic acid for the anode and 150 mM ethanolamine for the cathode. The samples were electrically focused at 150 V/cm at 4°C for 3 h. Immediately after isoelectric focusing, the gel was overlaid with 1% agar-Noble gel mixture containing 1.5 mM adenosine, 0.2 mM tetrazolium salt (MMT), 0.3 mM phenazine methosulfate, 0.3 units of xanthine oxidase, and 3 units of nucleoside phosphorylase in 0.1 mM sodium phosphate buffer, pH 5.0. After incubation for 2 h at 37°C, a blue band representing the ADA activity was apparent. Control gels were incubated with an overlay gel with the substrate adenosine omitted. A selective inhibitor of ADA, EHNA, was added to the overlay gel mixture in additional gel incubations to demonstrate the specificity of the reaction. The activity of ADA was estimated by measuring the intensity of gel bands and comparing it with that of purified ADA (1 mU) on the same gel.
Western blot analysis for 5'-ND, ADA, and adenosine receptors. Forty micrograms of protein were treated with 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was then washed and probed with 1:1,000 polyclonal antibody against ecto-5'-ND, ADA (Santa Cruz Biotechnology, Santa Cruz, CA), and A1, A2a, A2b, or A3 receptors (Alpha Diagnostics, San Antonio, TX) and 1:1,000 goat horseradish peroxidase-labeled anti-rabbit IgG. Finally, enhanced chemiluminescence (ECL) detection solution was added, and a Kodak Omat film was exposed to the membrane. Each membrane was stripped of bound antibody and reprobed with anti-β-actin on the same membrane for quantitative comparison. The protein concentration was measured using the Lowry method (26).
RT-PCR for 5'-ND, ADA, and adenosine receptors. Total RNA was isolated using TRIzol (Invitrogen Life Technologies, Carlsbad, CA). Total RNA was converted into cDNA using a SuperScript II reverse transcriptase kit (Invitrogen Life Technologies). PCR was performed in a PTC-100 (MJ Research, South San Francisco, CA) for 35 cycles at 94°C for 1 min, 64°C for 1 min, and 72°C for 1 min, followed by a 10-min extension at 72°C. The primers utilized were the following: 5'-ND, sense 5'-ccgcaaggaagaacccaacgtact-3' and antisense 5'-ctggatttgagaggaagggggttt-3'; ADA, sense 5'-ccctgatgcagctgaacgagatca-3' and antisense 5'-ggcgcagctcattcaagatcatca -3'; A1 receptor, sense 5'-caacttcttcgtctgggtgctgc-3' and antisense 5'-cttcatcgatgggaggcttaggc-3'; A2a receptor, sense 5'-catcctctcccacagcaactcc-3' and antisense 5'-ggggcaaactctgaagaccatg-3'; A2b receptor, sense 5'-gctgctgccctgtgaagtgtc-3' and antisense 5'-aagtcccggttcctgtaggca-3'; A3 receptor, sense 5'-gctgttggggtgctggtcatac-3' and antisense 5'-atgacaaccagggggatgagga-3'; and β-actin, sense 5'-gaaatcgtgcgtgacattaaag-3'. The final PCR products were 350, 489, 404, 420, 397, 401, and 511 bp in size, respectively. The bands were analyzed with an electrophoresis documentation and analysis system (model EDAS 290; Kodak).
Chemicals.
ANG II, bovine serum albumin, A134974, adenosine, N6-ethenoadenosine, ADA, 5'-ND, AMP, ,β-mADP, xanthine oxidase, nucleoside phosphorylase, lead nitrate, manganous nitrate, sodium sulfide, ethanolamine, tetrazolium salt MMT, phenazine methosulfate, ZnSO4·7H2O, Ba(OH)2·8H2O, and DPCPX were purchased from Sigma. TBA was obtained from ICN Biomedicals (Aurora, OH). Antibodies against A1, A2a, A2B, and A3 adenosine receptors were purchased from Alpha Diagnostic. Zwittergent 3-14 was obtained from Calbiochem (La Jolla, CA). ECL detection solution was obtained from Amersham Biosciences (Little Chalfont, UK). Ampholytes, agarose, mercaptoethanol acrylamide, and bisacrylamide were purchased from Bio-Rad. Polyfructosan was from Inutest. The rest of the compounds were of analytical grade and were purchased from several companies.
Statistical analysis. Results are means ± SE. The significance of differences within and between groups was evaluated by ANOVA followed by Tukey's post hoc test for multiple groups. Differences with P < 0.05 were considered statistically significant.
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RESULTS |
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Renal content of adenosine. The content of adenosine in renal tissue increased significantly in rats with 435 ng·kg–1·min–1 ANG II infusion compared with the sham group (44.17 ± 4.63 vs. 28.6 ± 2.18 nmol/g wet tissue, P < 0.001), the 260 ng·kg–1·min–1 ANG II group (31.09 ± 1.63 nmol/g wet tissue), and the 130 ng·kg–1·min–1 ANG II group (28.94 ± 1.69 nmol/g wet tissue), respectively; however, the 260 and 130 ng·kg–1·min–1 ANG II groups were not different from the sham control.
Renal interstitial content of adenosine.
When the interstitial concentration of adenosine was measured using microdialysis, a significant increase on interstitial adenosine was observed. The adenosine concentrations in the renal cortex were 31.40 ± 4 nM in sham rats, 62.0 ± 8.4 nM in the 435 ng·kg–1·min–1 ANG II group (P < 0.001), 27.0 ± 3.7 nM in the 260 ng·kg–1·min–1 ANG II group, and 27.8 ± 2.0 nM in the 130 ng·kg–1·min–1 ANG II group (Fig. 1). Since the renal tissue content and interstitial concentration of adenosine only increased with the dose of 435 ng·kg–1·min–1, the rest of the experiments were performed using this dose. The blockade of ecto-5'-ND with ,β-mADP decreased adenosine from 70.86 ± 3.1 to 33.43 ± 4.9 nM (P < 0.001).
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DISCUSSION |
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In this study when adenosine tissue content and interstitial concentrations were measured, only the 435 ng·kg–1·min–1 dose significantly increased both, suggesting that ANG II by some mechanism was able to modify the renal adenosine concentrations; in this regard, the relevance of local adenosine concentrations has been suggested by Kost et al. (24), who did not find elevation of adenosine plasma concentrations in several ANG II-dependent hypertensive models such as 2-kidney, 1-clip, aortic ligated, and ANG II infused, but ANG II induced elevation in the isolated perfused lung, which is in agreement with our findings (4). It should be mentioned that the control adenosine values we obtained in microdialysis were lower than those reported by Zou et al. (47) and Aki et al. (1). These differences can be attributed to the fact that ATP, ADP, and AMP were extracted from the sample (47) and that we were able to clearly separate the adenosine and inosine peaks, which was not achieved by Aki et al. (1) and Zou et al. (47); nevertheless, the adenosine levels we determined were similar to those obtained by Jackson et al. (20) using mass spectrometry. In addition, the values obtained for the renal tissue content of adenosine were slightly higher than those reported by Osswald et al. (34) following the same technique. However, it is interesting that adenosine increased to a similar extent in the tissue as in the interstitium, indicating the consistency of our results.
It has been proposed that the renal vasoconstriction and reduced glomerular filtration rate observed in this model result from a fall in glomerular plasma flow and Kf (11), as a consequence of the ANG II administration. Indeed, our results obtained in the micropuncture studies clearly demonstrate the vasoconstriction after 14 days of treatment with ANG II (Table 1). In this regard, Zou et al. (49) clearly demonstrated that after 14 days of treatment, ANG II was elevated in the kidney; the renal concentration of ANG II may be attributed to accumulation of the exogenous peptide, but endogenous angiotensin was also elevated. Since adenosine tissue content and interstitial concentration of adenosine are elevated, the interaction between both vasoactive compounds is certain. In addition, when we infused the specific A1 adenosine receptor blocker DPCPX, the blood pressure remained unchanged, and renal hemodynamic alterations, induced by ANG II, were completely reversed. Only the glomerular capillary pressure remained elevated during the infusion of the A1 receptor antagonist, suggesting an impairment of renal autoregulation, which allowed the transmission of the systemic pressure to the glomeruli. However, the extent of adenosine receptor participation in the renal vasoconstriction by temporal ANG II infusion has been clearly demonstrated.
Considering the results obtained, it was reasonable to assume that the renal vasoconstriction induced by ANG II could be responsible for renal hypoxia and increased production of adenosine. Ectoenzymes (ecto-ATPases, ecto-ADPases, and ecto-5'-ND) degrade adenine nucleotides to adenosine in the extracellular compartment from renal sympathetic nerve terminals, intrarenal platelets, renal endothelial cells, renal vascular smooth muscle cells, and/or renal epithelial cells (13, 20). In this regard our microdialysis results from experiments in which ,β-mADP was infused clearly demonstrate that the inhibition of 5'-ND reduced adenosine concentrations by 47%, supporting an important role of extracellular adenosine generation from locally released nucleotides for the adenosine-ANG II synergism; however, an increase in intracellular adenosine cannot be discarded, since the renal tissue adenosine content was also elevated.
When the activities and of 5'-ND and ADA were investigated, the cytosolic and membrane fractions were separated to evaluate the participation of both fractions. Despite the fact that no changes were observed in 5'-ND activity, ADA activity was significantly higher in the membrane fraction from the sham control group than in that from the ANG II-treated rats; a significant decrease of the enzyme activity was observed only in the membrane fraction from the ANG II-treated animals. When the expression of the enzymes was evaluated, there was a marked decrease in the mRNA despite the lack of changes in the protein of 5'-ND, suggesting an increased mRNA degradation or mRNA instability in the ANG II-treated animals; in contrast, both the protein and the mRNA levels of the ADA decreased. These data are in agreement with those for the concentrations of adenosine found in the tissue and for microdialysis determination in treated animals. In addition, since ecto- and intracellular enzymes were separated in our study, the changes in ecto-ADA suggest that the increase of interstitial adenosine concentrations could be attained through the decreased activity of ecto-ADA and an increased production of adenosine. In this regard, recent studies by Dietrich et al. (9) have demonstrated that adenosine can be synthesized in the extracellular compartment through the breakdown of ATP. The ATP is released from circulating erythrocytes when luminal O2 levels fall in the arterioles, as happens in the ANG II-mediated hypertensive model (44). Moreover, microvascular endothelial cells and basolateral membranes from the proximal tubule have a high activity of extracellular ecto-nucleotidase, which degrades adenine nucleotides to adenosine (19, 20). The adenosine increase in renal tissue, observed in the ANG II-induced hypertensive model, is probably the result of a significant increase of local nucleotides, with adenosine as a metabolite, and a reduction of the ecto-ADA, as we have demonstrated in this study.
Furthermore, the adenosine receptor expression was modified, since an imbalance between the receptors that mediate vasoconstriction (A1) and vasodilation (A2) could influence the renal ANG II-mediated vasoconstriction. When the adenosine receptors were evaluated, the A1 receptor protein did not change significantly in either the cortex or the medulla, indicating a lack of regulation by the high adenosine concentrations, which has not been observed in the acute model; the adaptation of adenosine receptors to chronic exposure to the agonist remains to be proved. In this regard, the A1 receptor requires a long time for desensitization, up to 6 days in rat adipocytes (35). In our study a slight but significant decrease in the high-affinity A2a receptor and no change in the low-affinity A2b receptors were observed, indicating downregulation of the former; this finding is in agreement with the observation in acute studies that A2a receptors show a fast response to high levels of the agonists (35). Normal expression of A1 receptor population, with a decrease in A2a receptors, associated with a high concentration of adenosine, can explain the increase in renal vasoconstriction in the presence of ANG II (11). This notion was further supported by the results obtained in the micropuncture studies; the acute blockade of A1 receptors with intra-aortic administration of the specific A1 antagonist DPCPX completely reverted the renal vasoconstriction induced by ANG II. These unexpected results clearly demonstrate the great contribution of adenosine in the renal vasoconstriction of the ANG II-infused hypertensive rat. We can attribute the vasodilatory effect of the blocker to an overriding effect of adenosine A2 receptors; the reason for this marked vasodilatory response to the specific adenosine A1 antagonist remains to be elucidated.
We also have demonstrated an increase in the expression of A3 receptors. In this regard, the physiological effect of A3 receptors in the kidney remains unclear. However, upregulation of these receptors has been associated with deleterious effects on renal function in the renal ischemia-reperfusion model (25). In the ANG II-mediated hypertensive model, they could have a similar effect, since ischemia has also been observed (44).
In conclusion, elevation of renal tissue content and interstitial concentration of adenosine contribute to the renal vasoconstriction observed in short-term ANG II-induced hypertension. Our studies suggest that the mechanism by which ANG II regulates 5'-ND expression may be posttranscriptional or posttranslational; ADA expression may be at the transcriptional level and at the posttranscriptional level for A2a and A3 receptors. The extracellular nucleotide degradation pathway as well as the decrease of ecto-ADA may be involved in this physiopathological condition, as well as an imbalance in A1 and A2 adenosine receptors.
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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.
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