1Center for Tsukuba Advanced Research Alliance, 2Institute of Health and Sport Sciences, 3Institute of Clinical Medicine, and 4Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan
Submitted 19 August 2003 ; accepted in final form 1 December 2003
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ABSTRACT |
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endothelin-1; nitric oxide; cross talk
There are three isopeptides of ET (ET-1, ET-2, and ET-3) (10, 19, 23) and two subtypes of ET receptors (ETA and ETB receptors) (1, 24). Both of these receptors are active in mediating a variety of biological actions through their G protein-coupled signal transduction pathway (1, 24). The activation of ETA receptors on smooth muscle and mesangial cells triggers contraction, whereas the activation of ETB receptors on endothelial cells causes release of nitric oxide (NO) (19, 23). Both ET receptors are widely distributed and are found in the kidney (23). It has been reported that ET-1 infusion exerts renal cortical vasoconstriction and renal medullary vasodilation (5).
The vascular endothelial cells play an important role in the regulation of vascular tonus by producing vasoactive substances (19, 20, 23, 25). ET-1 is a potent vasoconstrictor peptide produced by vascular endothelial cells (19, 23, 25). Furthermore, vascular endothelial cells also produce NO, which is a potent vasodilator substance (20). There are interactions between ET-1 and NO. It has been demonstrated that ET-1 depresses NO synthase (NOS) activity of cultured cells in vitro (9). However, it is unclear whether endogenous ET-1 affects NOS activity and/or NO production in vivo. We previously reported that exercise causes an increase of ET-1 production in the kidney (17), whereas production of NO in the kidney is decreased by exercise (18). Because ET-1 affects the NO system in vitro (2, 9), we consider that the increase of endogenous ET-1 may participate in the decrease of renal blood flow through two pathways, i.e., vasoconstrictive action and action of attenuating NO production. Then, we hypothesized that an increase of ET-1 production in kidney during exercise contributes to exercise-induced decrease of NO production in the kidney, and we investigated whether administration of an ETA receptor antagonist attenuates the decreases of NOS activity and NO production in kidney during exercise. In the present study, rats performed treadmill running for 30 min after pretreatment with an ETA receptor antagonist (TA-0201, 0.5 mg/kg) or vehicle. The ETA receptor antagonist TA-0201 has high affinity for ETA receptors and potent ETA antagonistic action (7). In the binding study in which human cloned ET receptors were used, TA-0201 showed 3,000-fold higher affinity for ETA receptors than for ETB receptors (7). Thus TA-0201 shows higher affinity and selectivity for ETA receptors. In the first series of the study, we confirmed that the magnitude of decrease in the blood flow to kidney during this exercise was attenuated by the administration of the ETA receptor antagonist TA-0201. In the second series of the study, immediately after this exercise, under pretreatment with ETA receptor antagonist TA-0201 or vehicle, the kidney was removed, and NOS activity, endothelial NOS (eNOS) protein, and tissue NOx, the stable end product of NO, i.e.,
concentrations in the kidney were determined.
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METHODS |
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Male Wistar rats (6 wk old) were obtained from the Institute for Animal Reproduction (Ibaraki, Japan) and cared for according to Guiding Principles for the Care and Use of Animals, based on the Helsinki Declaration of 1964. The rats were maintained on a 12:12-h light-dark cycle and received food and water ad libitum. All rats were familiarized with running on a motor-driven treadmill 5 days/wk, over a 2-wk period, until they were capable of running at a speed of 30 m/min for 30 min. The running time and speed of the treadmill were gradually increased in 2 wk from 10 min at 10 m/min to 30 min at 30 m/min.
First Series of the Study
Experimental protocol. Blood flow in the kidney was measured using the microsphere technique. At the end of the training period, 12 rats were anesthetized with pentobarbital sodium (initial dose 35 mg/kg ip, supplemental doses administered as required) and instrumented with two chronically implanted polyurethane catheters. Surgical procedures were performed according to a method we have previously described (16) and the method of Laughlin et al. (15), with minor modification. One of the implanted catheters was placed in the ascending aorta via the right carotid artery (microsphere infusion catheter). The other catheter was placed in the descending aorta via the left renal artery (reference withdrawal catheter). The left kidney was then removed (15, 16). The catheters were passed subcutaneously and exteriorized in the dorsal cervical region. They were flushed daily and filled with heparinized (500 U/ml) 50% glucose solution.
To study the impact of unilateral nephrectomy, we compared serum creatine phosphokinase (CPK) levels with the settings of unilateral nephrectomy (n = 5), sham operation (rats had both kidneys: n = 5), and noninvasive rats (n = 5). There were no significant differences among the three groups in serum CPK levels (unilateral nephrectomy: 153.2 ± 18.0; sham operation: 155.0 ± 8.6; noninvasive rats: 154.2 ± 23.8 IU/l).
The rats were allowed to recover from the surgery for 2 days (15, 16). On the day of the experiment, they were randomly divided into two groups. In one group, TA-0201 (7), an ETA receptor antagonist, was administered before exercise (n = 6). In the other group, vehicle (saline) was administered before exercise (n = 6). Blood flow in the kidney before and during exercise was determined using the stable isotope-labeled microsphere technique. On the day of blood flow measurement, the animal was placed on the treadmill, and all catheters and instruments were connected. Arterial blood pressure and heart rate were monitored. After the rat showed a relatively constant arterial blood pressure and heart rate, ETA receptor antagonist TA-0201 (donated by Tanabe Seiyaku, Saitama, Japan) or vehicle was infused into the rat through the ascending aorta (0.5 mg/kg). The intravenous administration of TA-0201 at a lower dose of 0.1 mg/kg significantly inhibited the pressor response to exogenous big ET-1 (1 nmol/kg iv) (7). TA-0201 has high affinity for ETA receptors and potent ETA antagonistic action (7). After the administration of TA-0201 or vehicle, the first infusion of microspheres (into the ascending aorta) was performed to measure preexercise (resting condition) blood flow. The rat then performed treadmill running (0% grade) for 30 min at a speed of 30 m/min. After the rat had exercised for 30 min, a second species of microspheres was infused for measurement of regional blood flow during exercise.
Upon completion of the exercise protocol, each rat was given pentobarbital sodium (50 mg/kg) via the right carotid artery catheter for anesthesia. After anesthetization, the right kidney was removed. The tissue samples were blotted, weighed, and placed immediately into sample vials (BioPAL, Wellesley, MA).
Blood flow measurements. Renal blood flow was measured using the microsphere technique, an established method for repeated measuring of blood flow before and during exercise (3, 13-16). Stable isotope-labeled microspheres (198Au and 153Sm; BioPAL) of 15 µm diameter were used for this study. Stable isotope-labeled and standard radiolabeled microspheres give similar measurements of regional myocardial blood flow over a wide range of flow with a high linear correlation (r = 0.95-0.99) (22). Therefore, stable isotope-labeled microspheres show comparable data to those of radioactive microspheres. The microsphere suspensions consisted of 5 x 106 spheres in saline solution containing 0.05% polyoxyethylenesorbitan monooleate (Tween 80) and 0.01% Thimerosal. The suspensions were mixed and vortexed thoroughly before infusion. Each microsphere infusion was performed as follows. The stable isotope-labeled microsphere suspension was slowly injected into the aortic catheter, followed by infusion of warm (37°C) 0.85% NaCl. Simultaneously, a 1.5-min reference blood sample was drawn at a rate of 0.8 ml/min from the renal arterial catheter by means of an infusion-withdrawal pump (CFV-2100; Nihon Koden, Tokyo, Japan). The total blood withdrawal amount was replaced by the infusion of microsphere suspension. It has been demonstrated that aortic infusions of microspheres in rats result in microsphere-blood mixing that is as good as left ventricular infusions (15). It has also been reported that there was no significant difference between right and left renal flows with aortic microsphere infusion (15, 16). Therefore, we consider that microsphere infusion into the ascending aorta via the right carotid artery in the present study results in adequate microsphere mixing in the aortic arch.
The experimental method for using stable isotope-labeled microspheres is identical to the traditional radioactive microsphere method, differing only in the assay of microspheres (22). The assay of stable isotope-labeled microspheres was performed according to the method described by Reinhardt et al. (22). Calculation of absolute and relative regional blood flows measured by stable labeled microspheres was performed according to the method described by Heyman et al. (6).
Second Series of the Study
Experimental protocol. At the end of the training period, each rat was anesthetized with pentobarbital sodium (initial dose 35 mg/kg ip, supplemental doses administered as required) and instrumented with a chronically implanted polyurethane catheter. This catheter was placed in the cervical vein. The catheter was passed subcutaneously and exteriorized in the dorsal cervical region. This was flushed daily and filled with heparinized (500 U/ml) 50% glucose solution.
The rats were allowed to recover from the surgery for 2 days. On the day of the experiment, the rats were randomly divided into three groups. In one group, TA-0201 (7), an ETA receptor antagonist, was administered before exercise, and animals performed treadmill running (0% grade) for 30 min at a speed of 30 m/min (TA-0201-treated exercise group; n = 12). In a second group, vehicle (saline) was administered before exercise, and animals performed treadmill running (0% grade) for 30 min at a speed of 30 m/min (vehicle-treated exercise group; n = 12). In a third group, vehicle (saline) was administered, and animals remained at rest for 30 min (vehicle-treated sedentary group; n = 8). After the animal was placed on the treadmill, TA-0201 (0.5 mg/kg) or vehicle was infused into the rat through the cervical vein. After the administration of TA-0201 or vehicle, the rat performed treadmill running for 30 min at a speed of 30 m/min. The animals in the vehicle-treated sedentary group remained at rest for 30 min on the treadmill.
Immediately after removal from the treadmill, the rats were anesthetized with diethyl ether. After anesthetization, the kidney was quickly removed, weighed, and frozen in liquid nitrogen. The tissue samples were stored at -80°C for measurement of NOS activity, determination eNOS protein expression by Western blot analysis, and measurement of tissue NOx level.
Measurement of NOS activity in kidney. NOS activity was determined by the method of Knowles and Moncada (11), with a minor modification. Incubation mixtures (0.1 ml) consisted of kidney sample (300-400 µg of protein), 50 mM valine (an inhibitor for arginine), 1 mM citrulline, 20 mM HEPES (pH 7.4), and complete medium {20 nM [2,3-3H]arginine, 50 µM arginine, 100 µM NADPH, 10 µM (6R)-5,6,7,8-tetrahydro-L-biopterin, 2 mM CaCl2, and 1 µg of calmodulin}. After the sample solution (sample, valine, citrulline, and HEPES) was preincubated at 37°C for 5 min, reactions were initiated by addition of the complete medium. Incubation was carried out at 37°C for 10 min; under this condition, NO production determined by citrulline formation was found to be linear with time and protein concentration. Citrulline formation was determined as described previously (12). Briefly, reaction was terminated by the addition of 10 µl of 20% perchloric acid. After each mixture was centrifuged at 10,000 g for 5 min, a portion (80 µl) of the supernatant was added to 2 ml of cold stop buffer [20 mM sodium acetate buffer (pH 5.5)-1 mM citrulline-2 mM EDTA-0.2 mM EGTA], and a portion (2 ml) of the mixture was applied to a column packed with AG50W-X8 resin (1 ml, Bio-Rad Laboratories, Hercules, CA), which had been extensively equilibrated with the stop buffer, and then the column was washed with 2 ml of water. A sample (1 ml) of the collected eluent was mixed with 5 ml of the scintillation cocktail, and the radioactivity was determined using a Beckman LS-600 scintillation counter.
Electrophoresis and immunoblot analysis for measurement of eNOS protein in the kidney. Kidney microsomes were denatured by boiling for 5 min with SDS sample buffer (62.5 mM Tris·HCl buffer, pH 6.8, containing 25% glycerol and 2% SDS). Protein concentrations were determined by the bicinchoninic acid protein assay reagents (Pierce, Rockford, IL), with BSA as a standard. The samples were followed by heat denaturation at 96°C for 5 min with -mercaptoethanol. Western blot analysis was performed according to the method we described previously (8, 18). Briefly, each microsomal preparation was separated on an SDS-polyacrylamide gel (8%) and then transferred to polyvinylidene difluoride (PVDF; Millipore, Tokyo, Japan) membranes at 1 mA/cm2 for 120 min. After the membrane was treated with blocking buffer [5% skim milk in PBS containing 0.05% Tween 20 (PBS-T)] for 12 h at 4°C, it was probed with monoclonal anti-eNOS antibodies (Transduction Laboratories, Lexington, KY; 1:2,500 dilution with blocking buffer) for 1 h at room temperature, washed with PBS-T five times, and then incubated with anti-mouse immunoglobulin antibody, a horseradish peroxidase-conjugated F (ab')2 fragment from sheep (Amersham Life Science, Buckinghamshire, UK; 1:2,500 dilution with blocking buffer), for 1 h at room temperature. After this reaction, the membrane was washed with PBS-T six times. Finally, the eNOS was detected by the enhanced chemiluminescence system (Amersham Life Science) and exposed to Hyperfilm (Amersham Life Science).
Measurement of NOx level in kidney. NOx level was determined according to the method described by Green et al. (4), with a minor modification. Kidney tissues were homogenized with 2 volumes of 50 mM Tris·HCl (pH 7.4, 4°C)-0.1 mM EDTA-0.1 mM EGTA-0.5 mM dithiothreitol-1 µM pepstatin A-2 µM leupeptin-1 mM phenylmethylsulfonyl fluoride on ice with a Teflon homogenizer. The homogenate was centrifuged at 9,000 g for 20 min at 4°C, and the supernatant obtained was centrifuged at 105,000 g for 60 min at 4°C. The resulting soluble fraction was stored at -80°C until the NOx assay, and the pellet (microsomal fraction) was frozen under liquid nitrogen and stored at -80°C until the eNOS protein assay.
For determination of NOx level, 80 µl of each sample were incubated for 60 min at 25°C in a 270-µl incubation mixture containing 140 µl of 125 mM KPi (pH 7.5), 10 µl of 87.5 µM flavine adenine dinucleotide, 10 µl of 3.5 mM NADPH, 90 µl of distilled water (DW), and 20 µl of nitrate reductase (1.75 U/ml; Sigma, St. Louis, MO). The reaction was initiated by addition of the nitrate reductase to convert nitrite to nitrate. The reaction was terminated by addition of 0.8 ml of Griess reagent and 0.45 ml of DW. After each mixture was centrifuged at 14,000 g for 5 min, the supernatants obtained were determined spectrophotometrically at 542 nm.
Statistics
Data are expressed as means ± SE. To evaluate the differences in renal blood flow between times (before and during exercise) and treatments (administrations of TA-0201 and vehicle), Student's t-test for paired or unpaired values was used. To evaluate differences among the vehicle-treated sedentary, vehicle-treated exercise, and TA-0201-treated exercise groups, statistical analysis was carried out by analysis of variance followed by Fisher's protected least significant difference test for multiple comparisons. P < 0.05 was accepted as significant.
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RESULTS |
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Renal blood flow was markedly decreased by exercise, but the magnitude of the decrease after pretreatment with TA-0201 was significantly smaller than that after pretreatment with vehicle (Fig. 1). Therefore, we confirmed that ET-1-mediated vasoconstriction participates in the decrease of blood flow in the kidney during exercise.
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Second Series of the Study
Table 1 shows the body weight and kidney weight in the vehicle-treated sedentary, vehicle-treated exercise, and TA-0201-treated exercise groups. There were no significant differences in body weight among the three groups (Table 1). Neither the kidney wet weight nor the kidney weight mass index for body weight differed significantly among the three groups (Table 1).
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The NOS activity in the kidney was significantly lower in the vehicle-treated exercise group than in the vehicle-treated sedentary group, whereas that in the TA-0201-treated exercise group was significantly higher than the vehicle-treated exercise group (Fig. 2). The expression of eNOS protein in the kidney was significantly lower in the vehicle-treated exercise group than in the vehicle-treated sedentary group, whereas that in the TA-0201-treated exercise group was significantly higher than in the vehicle-treated exercise group (Fig. 3). The NOx concentration in the kidney was markedly lower in the vehicle-treated exercise group than in the vehicle-treated sedentary group, whereas that in the TA-0201-treated exercise group was significantly higher than in the vehicle-treated exercise group (Fig. 4). Thus NOS activity, expression of eNOS protein, and tissue NOx concentration in the kidney were decreased by the acute exercise, whereas the exercise-induced decrease of NOS activity, expression of eNOS protein, and tissue NOx concentration in the kidney were attenuated by ETA receptor blockade.
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DISCUSSION |
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NO is produced from L-arginine by the action of NOS in vascular endothelium (21). There are at least three isozymes of NOS, eNOS, neuronal NOS (nNOS), and inducible NOS (iNOS) (11). In the present study, we examined the expression of eNOS protein, the activity of NOS, and the level of NOx in the tissue of the kidney to estimate the NO system after exercise. We previously reported that the levels of eNOS mRNA and eNOS protein in the kidney were markedly decreased by acute exercise, whereas neither the levels of nNOS mRNA and nNOS protein nor of iNOS mRNA and iNOS protein in the kidney differed before or after acute exercise (18). Then, we considered that the changes of NOx level and NOS activity induced by the exercise and/or TA-0201 administration might be attributed to a consistent change of eNOS protein.
Exercise results in a significant redistribution of tissue blood flow, by which the blood flow is greatly increased in the active muscles, whereas it is decreased in the splanchnic circulation (such as in the kidneys) (3, 13-16). We previously demonstrated that, in the rat, treadmill running for 30 min at a speed of 30 m/min, which was the same exercise condition in the present study, resulted in increased blood flow in active muscles and decreased blood flow in the kidney (16). In the present study, we also showed that renal blood flow was markedly decreased by exercise. We reported that exercise causes an increased production of ET-1, which has a potent vasoconstrictor effect (19, 20), in the kidney (17). Because the exercise-induced redistribution of tissue blood flow was significantly attenuated by the administration of ETA receptor blockade (16), it was suggested that ET-1-mediated vasoconstriction participates in the decrease of blood flow in visceral organs during exercise, thereby contributing to the increase of blood flow in active muscles during exercise. Furthermore, we recently revealed that exercise causes a decrease in production of NO, which shows a potent vasodilator effect (20) in the kidney (18). It has been reported that ET-1 depresses NOS activity of cultured cells in vitro (9). Therefore, it is possible to hypothesize that the increase of ET-1 production in the kidney during exercise contributes to the decreased production of NO in the kidney in vivo, thereby facilitating renal vasoconstriction to promote the increase of blood flow available to the active muscles.
The renal vasculature is heterogenous, and the regulation of the renal medullary microcirculation markedly differs from the cortical microcirculation. It has been reported that ET-1 infusion exerts renal cortical vasoconstriction (mediated through ETA receptors), whereas medullary blood flow increases (mediated by ETB receptors) (5). In the present study, the blood flow in the kidney was decreased by exercise, but the magnitude of the decrease after pretreatment with ETA receptor antagonist TA-0201 was significantly smaller than that after pretreatment with vehicle. Therefore, it is possible that the changes noted in renal blood flow predominantly reflect changing cortical blood flows.
The unilateral nephrectomy in the first series of this study did not have any apparent effect on the cardiovascular system, as evidenced by heart rate, arterial blood pressure, and exercise performance (13-16). Moreover, the experimental settings in the present study do not cause exercise-induced rhabdomyolysis.
In summary, we have demonstrated that NOS activity and NO production in the kidney were decreased by acute exercise, whereas the exercise-induced decreases of NOS activity and NO production in the kidney were reversed by ETA receptor blockade. We propose that there may be an interaction between the ET-1 production system and the NO production system in the regulation of renal blood flow during exercise, i.e., the increase of ET-1 production in the kidney during exercise may participate in the decrease of NO production during exercise, thereby contributing to the greater decrease in blood flow in the kidney and the more sufficient supply of blood flow to the active muscles.
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GRANTS |
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FOOTNOTES |
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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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REFERENCES |
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