Journal of Applied Physiology Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 94: 60-68, 2003. First published September 20, 2002; doi:10.1152/japplphysiol.00269.2002
8750-7587/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
94/1/60    most recent
00269.2002v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miyauchi, T.
Right arrow Articles by Matsuda, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miyauchi, T.
Right arrow Articles by Matsuda, M.
Vol. 94, Issue 1, 60-68, January 2003

Exercise causes a tissue-specific change of NO production in the kidney and lung

Takashi Miyauchi1, Seiji Maeda2, Motoyuki Iemitsu1, Tsutomu Kobayashi1, Yoshito Kumagai3, Iwao Yamaguchi1, and Mitsuo Matsuda2

1 Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, 2 Department of Sports Medicine, Institute of Health and Sport Sciences, and 3 Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) is produced in the vascular endothelium and is a potent vasodilator substance that participates in the regulation of local vascular tone. Exercise causes peculiar changes in systemic and regional blood flow, i.e., an increase of systemic blood flow and a redistribution of local tissue blood flow, by which the blood flow is greatly increased in the working muscles, whereas it is decreased in some organs such as the kidney and intestine. Thus we hypothesized that exercise causes a tissue-specific change of NO production in some internal organs. We studied whether exercise affects expression of NO synthase (NOS) mRNA and protein, NOS activity, and tissue level of nitrite/nitrate (stable end products of NO) in the kidneys (in which blood flow during exercise is decreased) and lungs (in which blood flow during exercise is increased with the increase of cardiac output) of rat. Rats ran on a treadmill for 45 min at a speed of 25 m/min. Immediately after this exercise, kidneys and lungs were quickly removed. Control rats remained at rest during this 45-min period. Expression of endothelial NOS (eNOS) mRNA in the kidneys was markedly lower in exercise rats than in control rats, whereas that in the lungs was significantly higher in exercise rats than in control rats. Western blot analysis confirmed down- and upregulation of eNOS protein in the kidney and lung, respectively, after exercise. On the other hand, neither expression of neuronal NOS (nNOS) mRNA and nNOS protein nor inducible NOS (iNOS) mRNA and iNOS protein in the kidneys and lungs differed between exercise and control rats. NOS activity in the kidney was significantly lower in exercise rats than in control rats, whereas that in the lung was significantly higher in exercise rats than in control rats. On the other hand, the iNOS activity in the kidneys and lungs did not differ between exercise rats and control rats. Tissue nitrite/nitrate level in the kidneys was markedly lower in exercise rats, whereas that in the lungs was significantly higher in exercise rats. The present results show that production of NO is markedly and tissue-specifically changed in the kidney and lung by exercise.

treadmill running; nitric oxide synthase mRNA; tissue nitrite/nitrate level; nitric oxide synthase activity; redistribution of blood flow


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL KNOWN THAT NITRIC OXIDE (NO) is produced in the vascular endothelial cells and shows a potent vasodilator effect (29, 35). NO is produced from L-arginine by the action of NO synthase (NOS) in the vascular endothelium (34, 36). There are at least three isozymes of NOS consisting of endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) (14). NO is produced under normal physiological conditions by the vascular endothelium lining all blood vessels. Furthermore, mechanical interactions (e.g., increased shear stress) between blood flow and the vascular endothelium can evoke the changes of production of NO in isolated blood vessels and in cultured endothelial cells (3, 18, 27, 40). Exercise causes an increase in systemic blood flow and marked changes of tissue blood flow, in which the blood flow is greatly increased in the working muscles, whereas it is decreased in some internal organs, such as the kidney and intestine (1, 5, 6, 19, 20, 22, 30, 31, 33, 47). It has been demonstrated that NO-mediated vasodilation contributes to the blood flow responses in working muscles during exercise (9). This finding suggests that the production of NO is increased in the circulation of working muscles by exercise. However, it is not known whether exercise affects the production of NO in internal organs such as the kidney and lung.

Exercise results in a significant redistribution of tissue blood flow, in which the blood flow is greatly increased in the working muscles, whereas it is decreased in the splanchnic circulation (such as in the kidneys and intestines) (1, 5, 6, 19, 20, 22, 30, 31, 33, 47). Although it has been considered that the exercise-induced redistribution of blood flow is partly caused by the increased activity of the sympathetic nerve system (2) and multiple local metabolic factors (21), the precise mechanisms are not known. We previously reported that endothelin (ET)-1, endothelium-derived vasoconstrictor substance, is involved in the exercise-induced changes in the distribution of blood flow by a tissue-specific enhancement of ET-1 production (24, 26). Although this finding suggests that vascular endothelial cells are involved in the exercise-induced redistribution of blood flow by enhancing the production of the endothelium-derived vasoconstrictor ET-1, the roles of endothelium-derived relaxing factors, such as NO, in exercise-induced physiological responses remain to be investigated.

It has been demonstrated that endogenously generated NO contributes to vascular tonus in working muscles during exercise (9). However, it is unclear how exercise affects the production of NO in internal organs. To answer this question, the present study was designed to investigate whether exercise affects expression of NOS mRNA and protein, NOS activity, and tissue level of nitrite/nitrate (NOx; i.e., levels of NOx measured as stable end products of NO) in the internal organs, such as the kidney and lung. Exercise results in a marked decrease in renal blood flow (6, 19, 20, 22, 45), whereas it increases cardiac output and thereby increases pulmonary blood flow. Because NO is a potent vasodilator substance that participates in the regulation of local vascular tone (38), we hypothesized that production of NO in the kidney (in which the blood flow during exercise is decreased) would be diminished during an acute bout of exercise, whereas that in the lung (in which the blood flow during exercise is increased) would be augmented during an acute bout of exercise. Therefore, we investigated the production of NO in the kidneys and in the lungs of rat immediately after exercise. In the present study, the rats performed treadmill running for 45 min at a speed of 25 m/min. Immediately after this exercise, the kidneys and lungs were removed, and expressions of mRNAs and proteins for the three isoforms of NOS (eNOS, nNOS, iNOS), NOS activity, and tissue NOx level in these organs were determined.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and protocol. Fourteen male Wistar rats (7 wk old) were obtained from Clea Japan (Tokyo, Japan) and cared for according to the Guiding Principles for the Care and Use of Animals based on the Helsinki Declaration, 1964. 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 4-wk period until they were capable of running at a speed of 25 m/min for 45 min at no incline (0% grade). Running time and speed of the treadmill were gradually increased in 4 wk from 10 min at 10 m/min to 45 min at 25 m/min. Systolic arterial pressure and heart rate of the animals were measured with a tail-cuff sphygmomanometer (model PS-100, Riken Kaihatsu, Kanagawa, Japan) on the day before the experiment. Body weight of the animals was also measured on the day before the experiment. On the day of the experiment, rats were randomly divided into two groups. In one group, seven animals ran on a treadmill (0% grade) for 45 min at a speed of 25 m/min (exercise group). Shepherd and Gollnick (43) reported that this intensity (25 m/min) of treadmill running in rats is ~78% of maximal oxygen consumption. Furthermore, the present condition of exercise in rats, i.e., intensity at 25 m/min and duration of 45 min, results in a significant redistribution of tissue blood flow, in which blood flow is greatly increased in the active muscles and decreased in the kidney (6, 19, 20, 22). The other seven animals remained at rest during this 45-min period (control group).

Immediately after removal from the treadmill, animals in the exercise group were anesthetized with diethyl ether. After the animals were anesthetized, a blood sample was collected from the heart, and the lungs and kidneys were quickly removed, rinsed in ice-cold saline, weighed, and frozen in liquid nitrogen. The plasma and tissue samples were stored at -80°C for determination of plasma epinephrine concentration by radioenzymatic assay, expression levels of mRNA of three isoforms of NOS (eNOS, nNOS, iNOS) by RT-PCR analysis, NOS (eNOS, nNOS, iNOS) protein expression by Western blot analysis, NOS activity, and tissue NOx level. Control animals were killed ~24 h after their last bout of exercise, i.e., at the same time point as the exercised rats.

RT-PCR to determine levels of eNOS, nNOS, and iNOS mRNA in the kidneys and lungs. The expressions of eNOS, nNOS, and iNOS mRNA in the kidneys and lungs were analyzed by RT-PCR. The expression of GAPDH mRNA was determined as an internal control. Semiquantitative RT-PCR by using ethidium bromide gels was performed according to the method our laboratory described previously (10, 24, 25). The quantification of DNA by densitometric values from ethidium bromide gels was according to usual methods (42). Because the amount of fluorescence is proportional to the mass of DNA, the quantity of each PCR product was estimated by comparing the fluorescent yield of the sample with that of a series of standard DNA (42).

Total tissue RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with Isogen (Nippon Gene, Toyama, Japan) according to the method described in our laboratory's previous papers (10, 15, 24, 25). The tissue was homogenized in Isogen (100 mg tissue/1 ml Isogen) with a Polytron tissue homogenizer (model PT10SK/35, Kinematica, Lucerne, Switzerland). The chloroform extraction, isopropanol precipitation, and 75% (vol/vol) ethanol washing of the precipitated RNA were subsequently performed. The obtained RNA was resolved in pyrocarbonic acid diethyl ester-treated water and treated with DNase I (Takara, Shiga, Japan) and extracted again by Isogen to eliminate the genomic DNA. RNA concentration was determined spectrophotometrically at 260 nm.

Total tissue RNA (10 µg) was primed with 0.05 µg of oligo(dT) 12-18 and reverse transcribed by avian myelloblastosis virus reverse transcriptase by using a first-strand cDNA synthesis kit (Life Sciences). The reaction was performed at 43°C for 60 min.

The cDNA was diluted in a 1:10 ratio, and 1 µl was used for PCR. Each PCR reaction contained 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, 0.5 µM of each gene-specific primer, and 0.025 U/µl Taq polymerase (Takara, Shiga, Japan). The gene-specific primers were synthesized according to the published cDNA sequences for each of the following: eNOS (12), nNOS (44), iNOS (7), and GAPDH (46). The sequences of the oligonucleotides were as follows: eNOS (sense) 5'-CTGGCAAGACCGATTACACGAC-3'; eNOS (antisense) 5'-GTCCTCACCGCCTTTTCCAG-3'; nNOS (sense) 5'-AATGGAGACCCCCCTGAGAAC-3'; nNOS (antisense) 5'-TCCAGGAGGGTGTCCACCGC-3'; iNOS (sense) 5'-GTACATGGGCACCGAGATTG-3'; iNOS (antisense) 5'-CTACTACTACCAGATCGAGCC-3'; GAPDH (sense) 5'-GCCATCAACGACCCCTTCATTG-3'; GAPDH (antisense) 5'-TGCCAGTGAGCTTCCCGTTC-3'.

PCR was carried out by using a PCR thermal cycler (model TP-3000, Takara). The cycle profile included denaturation for 15 s at 94°C, annealing for each suitable time at each suitable temperature, and extension for each suitable time at 72°C. The annealing time and temperature were set as follows: 15 s at 66°C for eNOS, 30 s at 62°C for nNOS, 15 s at 65°C for iNOS, and 15 s at 58°C for GAPDH. The extension time was set as follows: 30 s for eNOS and GAPDH and 45 s for nNOS and iNOS. The reaction cycles of PCR were performed in the range that demonstrated a linear correlation between the amount of cDNA and the yield of PCR products. PCR products were found to be of the expected size, as shown by 1.8% agarose gel electrophoresis for eNOS and 1.2% agarose gel electrophoresis for nNOS, iNOS, and GAPDH. In addition, the specificity of the amplified sequences was confirmed by restriction enzyme analysis and DNA sequencing. The DNA sequence of each amplicon was perfectly matched to each published sequence.

Semiquantitative analysis of PCR products. The amplified PCR products were electrophoresed on 1.8 or 1.2% agarose gels, stained with ethidium bromide, visualized by an ultraviolet transilluminator, and photographed. The photographs were scanned by CanoScan 600 (Canon, Tokyo, Japan), and quantification was performed by computer with MacBAS software (Fuji Film, Tokyo, Japan). The cDNA for the verification of the semiquantitative PCR analysis was prepared from each gene PCR product of rat cDNA. Each PCR product was purified, quantified, and used as positive-control cDNA. Each PCR product concentration was calculated by assuming that the mass of a nucleotide pair in DNA is 660 Da (41). The copy numbers were used to estimate the mass of sample DNA, the molecular weight of PCR product, and Avogadro's constant (6.02 × 1023). We performed semiquantitative PCR analysis to evaluate the expression level of eNOS, nNOS, iNOS, and GAPDH mRNA. To demonstrate that our semiquantitative PCR strategy was valid, serial dilutions of the each positive-control cDNA were amplified by PCR and quantified by scanner. When each RT-PCR analysis was independently performed in triplicate for each isozyme and each rat, similar results were obtained from these experiments. PCR reactions with no cDNA were carried out, and no amplicon was detected in each experiment.

Electrophoresis and immunoblot analysis for measurement of eNOS, nNOS, and iNOS proteins in the kidneys and lungs. Kidney and lung 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 (Piece, Rockford, IL) with BSA as a standard. The samples were followed by heat denatureation at 96°C for 5 min with beta -mercaptoethanol. Western blot analysis was performed according to the method our laboratory described previously (10). Briefly, each microsomal preparation was separated on a SDS-polyacrylamide gel (8%) and then transferred to polyvinylidene difluoride (Millipore, Tokyo, Japan) membranes at 1 mA/cm2 for 120 min. The membrane was then treated with a blocking buffer of 5% skim milk in PBS containing 0.05% Tween 20 (PBS-T) for 12 h at 4°C. The membrane was probed with monoclonal anti-eNOS, -nNOS, and -iNOS antibodies (Trasduction Laboratories, Lexington, KY; eNOS 1:2,500, nNOS 1:1,000, and iNOS 1:1,000 dilutions with blocking buffer) for 1 h at room temperature and washed five times with PBS-T and then incubated with anti-mouse immunoglobulin antibody, a horseradish peroxidase-conjugated F(ab')2 fragment from sheep (Amersham Life Science, Buckinghamshire, UK; eNOS, nNOS, and iNOS 1:2,500 dilutions with blocking buffer) for 1 h at room temperature. After this reaction, the membrane was washed six times with PBS-T. Finally, eNOS, nNOS, and iNOS were detected by ECL system (Amersham Life Science) and exposed to Hyper film (Amersham Life Science).

Measurement of NOS activity in kidneys and lungs. NOS activity was determined by the method of Knowles et al. (13) with a minor modification. Incubation mixtures (0.1 ml) consisted of kidney sample (300-400 µg of protein) or lung 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}. When calcium-dependent NOS [i.e., constitutive NOS (cNOS)] activity was assayed, 2 mM CaCl2 and 1 µg of calmodulin were added to the complete medium. When the calcium-independent NOS (i.e., iNOS) activity was assayed, 1.2 mM EDTA and 1 mM EGTA were added to the complete medium. After the sample solution (sample, valine, citrulline, 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 these conditions, NO production determined by citrulline formation was found to be linear with time and protein concentration. Citrulline formation was determined as described previously (17). 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), 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 radioactivity was determined by using a Beckman LS-600 scintillation counter.

Measurement of NOx level in kidneys and lungs. NOx level was determined according to the method described by Green et al. (8) with a minor modification. The tissues of the lungs and kidneys were homogenized with two 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, and 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 supernantant obtained was centrifuged at 105,000 g for 60 min at 4°C. The resulting soluble fraction was stored at -80°C until the NOS activity and NOx assay, and the pellet (microsomal fraction) was frozen under liquid nitrogen and stored at -80°C until the NOS 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 FAD, 10 µl of 3.5 mM NADPH, 90 µl of distilled water (DW), and 20 µl nitrate reductase (1.75 U/ml; Sigma Chemical, 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.

Measurement of plasma epinephrine concentration. Plasma epinephrine concentration was measured by using a radioenzymatic assay based on the method of Peuler and Johnson (37). Plasma samples from each animal were determined in triplicate. The plasma epinephrine values in triplicate were averaged for each rat.

Statistics. Values are expressed as means ± SE. To evaluate differences between the control group and the exercised group, Student's t-test for unpaired values was used. P < 0.05 was accepted as significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There were no significant differences between the control and the exercise rats in systolic arterial pressure (125 ± 3 vs. 122 ± 3 mmHg) or heart rate (379 ± 16 vs. 382 ± 7 beats/min) on the day before the animals were killed. There was no significant difference in body weight between the two groups (329 ± 6 vs. 334 ± 7 g). Neither the kidney wet weight nor the lung wet weight differed significantly between the two groups (Table 1). The kidney and lung weight mass indexes for body weight did not differ between the two groups (Table 1).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Tissue weights and plasma epinephrine concentration in exercised and control rats

Immediately after the 45-min exercise or rest period, the plasma concentration of epinephrine was significantly greater in the exercise group than in the control group (Table 1). Thus the plasma epinephrine concentration was increased by the acute exercise.

To semiquantitatively determine alterations in gene expression of NOS isozymes by exercise, the relationship between the amount of cDNA and the yield of PCR products was examined. There was a linear correlation between the initial amount of GAPDH cDNA and the yield of PCR products (r = 0.999). In the cases of eNOS, nNOS, and iNOS, the yield of PCR products was also in proportion to the initial amount of cDNA (r = 0.996, 0.997, and 0.999, respectively). These relationships in control rats were similar to those in exercise rats.

Under these conditions, the expression of eNOS mRNA in the kidneys was markedly lower in the exercise rats than in the control rats (Fig. 1A). However, neither the expression of nNOS mRNA nor iNOS mRNA in the kidneys differed significantly between the two groups (Fig. 1, B and C). In the lungs, the expression of eNOS mRNA was significantly higher in the exercise rats than in the control rats (Fig. 2A). However, neither the expression of nNOS mRNA nor iNOS mRNA in the lungs differed significantly between the two groups (Fig. 2, B and C). The changes in expression of nonnormalized eNOS, nNOS, and iNOS mRNA by GAPDH mRNA in the kidneys and lungs were similar to normalized mRNA expression by GAPDH mRNA. These findings suggest that the isozyme-specific down- or upregulation of NOS mRNA in internal organs was caused by exercise.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 1.   Expression of endothelial nitric oxide synthase (eNOS) mRNA (A), neuronal nitric oxide synthase (nNOS) mRNA (B), and inducible nitric oxide synthase (iNOS) mRNA (C) in the kidneys of exercised (n = 7) and control rats (n = 7). Top: typical examples of the RT-PCR analysis are shown for the levels of eNOS, nNOS, iNOS, and GAPDH mRNA. We studied the expression of GAPDH mRNA as an internal control. Bottom: results of the statistical analysis of the levels of expressions of eNOS, nNOS, and iNOS mRNA by a densitometer. Photos of PCR products were scanned by densitometer, and eNOS mRNA-, nNOS mRNA-, and iNOS mRNA-to-GAPDH mRNA ratios were calculated. Thus the values of expressions of eNOS, nNOS, and iNOS mRNA were normalized by that of GAPDH mRNA. Data are means ± SE. NS, not significant.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 2.   Expression of eNOS mRNA (A), nNOS mRNA (B), and iNOS mRNA (C) in the lungs of exercised (n = 7) and control rats (n = 7). Top: typical examples of the RT-PCR analysis are shown for the levels of eNOS, nNOS, iNOS, and GAPDH mRNA. We studied the expression of GAPDH mRNA as an internal control. Bottom: results of the statistical analysis of the levels of expressions of eNOS, nNOS, and iNOS mRNA by a densitometer. Photos of the PCR products were scanned by densitometer, and eNOS mRNA-, nNOS mRNA-, and iNOS mRNA-to-GAPDH mRNA ratios were calculated. Thus the values of expressions of eNOS, nNOS, and iNOS mRNA were normalized by that of GAPDH mRNA. Data are means ± SE.

Figure 3 shows the representive film of immunoblotting for eNOS, nNOS, and iNOS protein expression in the kidneys with or without acute exercise. The expression level of eNOS protein in the kidneys was lower in the exercise group than in the control group (Fig. 3). On the other hand, neither the expression of nNOS protein nor iNOS protein in the kidneys differed between the two groups (Fig. 3). Figure 4 shows the representive film of immunoblotting for eNOS, nNOS, and iNOS protein expression in the lungs with or without acute exercise. The expression level of eNOS protein in the lungs was higher in the exercise group than in the control group (Fig. 4). On the other hand, neither the expression of nNOS protein nor iNOS protein in the lungs differed between the two groups (Fig. 4). These findings suggest that change in eNOS mRNA expression is accompanied with change in eNOS protein expression in the rats after exercise.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   Typical examples of expression of eNOS, nNOS, and iNOS protein in the kidneys of exercised (n = 3) and control rats (n = 3) by Western blot analysis. Arrows indicate the immunoblot band for eNOS protein (135 kDa), nNOS protein (155 kDa), and iNOS protein (130 kDa).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4.   Typical examples of expression of eNOS, nNOS, and iNOS protein in the lungs of exercised (n = 3) and control rats (n = 3) by Western blot analysis. Arrows indicate the immunoblot band for eNOS protein (135 kDa), nNOS protein (155 kDa), and iNOS protein (130 kDa).

Figure 5 shows the NOS activity in the kidney in rats with or without exercise. NOS activity in the kidneys was significantly lower in the exercise group than in the control group (Fig. 5A). The cNOS activity in the kidney was significantly lower in the exercise group than in the control group (Fig. 5B), whereas iNOS activity in the kidney did not differ significantly between the two groups (Fig. 5C). Figure 6 shows NOS activity in the lung in rats with or without exercise. NOS activity in the lung was significantly higher in the exercise group than in the control group (Fig. 6A). The cNOS activity in the lung was significantly higher in the exercise group than in the control group (Fig. 6B), whereas iNOS activity in the lung did not differ significantly between the two groups (Fig. 6C).


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 5.   Nitric oxide synthase (NOS) activity (A), constitutive NOS (cNOS) activity (B), and iNOS activity (C) in the kidneys of exercised (n = 7) and control rats (n = 7). Data are means ± SE.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 6.   NOS activity (A), cNOS activity (B), and iNOS activity (C) in the lungs of exercised (n = 7) and control rats (n = 7). Data are means ± SE.

NOx level in the kidneys after acute exercise was markedly lower in exercise rats than in control rats, whereas NOx level in the lungs was significantly higher in exercise rats than in control rats (Fig. 7). Taken together, these findings suggest that the production of NO was tissue-specifically changed in the kidneys and lungs by acute exercise.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 7.   Tissue nitrite/nitrate (NOx) level in the kidneys (A) and lungs (B) of exercise (n = 7) and control rats (n = 7). Data are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we determined NO production in the kidneys and lungs of rats after acute exercise. Expression of eNOS mRNA in the kidneys was markedly lower in exercise rats than in control rats, whereas, in the lungs, it was significantly higher in exercise rats than in control rats. Western blot analysis confirmed down- and upregulation of eNOS protein in the kidney and lung, respectively, after exercise. Furthermore, NOS activity in the kidneys was significantly lower in exercise rats than in control rats, whereas NOS activity in the lung was significantly higher in exercise rats than in control rats. Tissue NOx levels in the kidneys were also markedly lower in exercise rats, whereas tissue NOx levels in the lungs were significantly higher in exercise rats. The present results show for the first time that production of NO is markedly changed tissue specifically in the kidneys and lungs by exercise.

The only known source of endogenous NOx in mammalian tissues is considered to be generated through the conversion of L-arginine to L-citrulline by NOS. NO is easily oxidized to nitrite, which is then converted to nitrate, when it reacts with hemoglobin (32). In this study, we examined the tissue level of NOx to estimate NO production after exercise. Tissue NOx level in the kidneys was markedly lower in exercise rats than in control rats, whereas, in the lungs, it was significantly higher in exercise rats than in control rats. There are three isozymes of NOS, consisting of eNOS, nNOS, and iNOS. In the present study, the levels of eNOS mRNA and eNOS protein in the kidneys were markedly lower in exercise rats than in control rats, whereas those levels in the lungs were significantly higher in exercise rats than in control rats. On the other hand, neither the levels of nNOS mRNA and nNOS protein nor iNOS mRNA and iNOS protein in the kidneys and lungs differed between the two groups. Furthermore, NOS activity in the kidneys was significantly lower in exercise rats than in control rats, whereas NOS activity in the lungs was significantly higher in exercise rats than in control rats. The cNOS activity in the kidneys was also significantly lower in exercise rats than in control rats, whereas, in the lungs, it was significantly higher in exercise rats than in control rats. On the other hand, iNOS activity in the kidneys and lungs did not differ significantly between the two groups. We therefore suspect that the change of NOS activity in the kidneys and lungs induced by exercise is attributed to a change of eNOS activity. Taken together, it is considered that the change of NOx level in the kidneys and lungs induced by exercise is attributed to a change of eNOS level, which was also accompanied with a similar change of eNOS activity.

It has been reported that hemodynamic shear stress increases production of NO from the vascular endothelium (3, 18, 27, 40). Exercise results in a marked decrease in renal blood flow, an increase in cardiac output, and, hence, a great increase in pulmonary blood flow. The different alterations in blood flow between the kidney and lung by exercise might cause a difference in the levels of shear stress on vascular endothelial cells of the kidney and lung. Therefore, it is possible that a difference in the level of shear stress on vascular endothelial cells between the kidney and lung causes the difference in NO production. It has been shown that, without increases in sympathetic activity, little reduction in renal blood flow occurs during exercise (28). In the kidney, the reduced NO production could likely amplify the reductions in blood flow induced by the increases in sympathetic tone.

Blockade of basal NO synthesis can potentiate responses to sympathetic nerve stimulation (23). Therefore, it is considered that interaction exists between the NO system and the sympathetic nervous system. In the present study, NO production in the kidneys was significantly decreased by acute exercise. Therefore, in blood vessels in the kidneys, it is possible that the decrease in NO production potentiates sympathetic nervous system-induced vasoconstriction. Because exercise results in a marked decrease in renal blood flow, the decrease in NO in the kidneys may cause vasoconstriction through depression of its vasodilative action and/or by enhancing the vasoconstriction to sympathetic nerve stimulation. Thus such a decrease in NO in the kidneys would contribute to maximize the decrease in blood flow in the kidneys, thereby contributing to the redistribution of blood flow during exercise. On the other hand, several studies have shown that the production of NO is considered to be regulated by several endogenous substances. ET-1 is a potent vasoconstrictor peptide produced by vascular endothelial cells (39, 48). It has also been reported that ET-1 inhibits NOS activity in vascular smooth muscle cells (11). In our laboratory's previous study, expression of ET-1 mRNA in the kidneys was markedly increased after exercise (24). Therefore, it is possible that the increase in ET-1 production in the kidney by exercise (24) partly contributes to a tissue-specific reduction in NO production by directly inhibiting NOS activity in the kidney after exercise, as was observed in the present study. Furthermore, it has been reported that NO inhibits the production of ET-1 in vascular endothelium (39). Therefore, it is also possible that the increase in ET-1 production in the kidney by exercise (24) is partly attributed to a tissue-specific reduction in NO production in the kidney after exercise, as was observed in the present study. Taken together, during exercise, there may be interactions among NO, sympathetic nervous system, and endothelium-derived vasoconstricting factors, such as ET-1, in the regulation of blood flow in the internal organs. However, the precise physiological interactions of those factors during exercise remain to be elucidated.

Exercise causes a marked decrease in renal blood flow (6, 19, 20, 22, 45). The exercise-induced decrease in renal blood flow results in a below normal physiological condition in the kidney (6, 19, 20, 22, 45). In the present study, it is possible that blood flow is decreased in the kidney below normal physiological condition. In this condition in the present study, expression of eNOS mRNA in the kidney was markedly lower in exercise rats than in control rats. Therefore, it is considered that expression of eNOS mRNA is decreased in the kidneys, when renal blood flow decreases and shear stress falls way below normal physiological condition. Indeed, hemodynamic shear stress appears to affect the production of endothelium-derived vasoconstrictive substances because it has been reported that low levels of shear stress stimulate and higher levels of shear stress depress production of ET-1, an endothelium-derived vasoconstrictor, in cultured vascular endothelial cells (16).

The present study has the following study limitations. First, all rats (control and exercise rats) were familiarized with running on a treadmill over a 4-wk period. Delp and Laughlin (4) reported that the expression of eNOS protein in aorta increased with 4- to 10-wk trained exercise rats. In the present study, rats have performed a 4-wk exercise training program; therefore, NO production results could be different if the entire untrained rat group had been used, i.e., the response to exercise may depend highly on training status. However, because both control and exercise rats performed the same exercise training (familiarization to treadmill running), it is considered that the alterations of NO production in the kidneys and lungs in the present study were caused at least by acute exercise but not by exercise training. Second, exercise results in a significant redistribution of tissue blood flow, in which the blood flow is greatly increased in the working muscle and decreased in splanchnic circulation (1, 5, 6, 19, 20, 22, 30, 31, 33, 47). On the other hand, it is generally accepted that the brain shows no change in blood flow during exercise. Although the brain is control tissue and the working muscles are positive control tissues relating to changes in blood flow during exercise, we did not investigate the production of NO in these tissues. Therefore, it is unclear whether exercise affects the production of NO in these tissues. Third, the lungs exhibit one-eighth the resistance of systemic circulation and increase blood flow mainly through capillary recruitment and distension. It is unclear how much the resistance in the lungs get lower with increased NO production during exercise. Further studies are needed to determine whether application of L-NMMA/L-NAME affects the exercise-induced change in pulmonary blood flow.

In summary, we have demonstrated for the first time that the expression of eNOS mRNA in the kidneys was markedly lower in exercise rats than in control rats, whereas, in the lungs, it was significantly higher in exercise rats than in control rats. Western blot analysis confirmed down- and upregulation of eNOS protein in the kidney and lung, respectively, after exercise. Furthermore, NOS activity in the kidneys was significantly lower in exercise rats than in control rats, whereas NOS activity in the lung was significantly higher in exercise rats than in control rats. We also demonstrated for the first time that the tissue NOx level in the kidneys was markedly lower in exercise rats, whereas, in the lungs, it was significantly higher in exercise rats. These findings suggest that production of NO is markedly and tissue-specifically changed in the kidneys and lungs by acute exercise.


    ACKNOWLEDGEMENTS

This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (nos. 00006132, 00006781, 11480003, 11557047, 12470147, and 12670646), a grant from University of Tsukuba Research Projects, and a grant from the Miyauchi project of Tsukuba Advanced Research Alliance in University of Tsukuba.


    FOOTNOTES

Address for reprint requests and other correspondence: T. Miyauchi, Cardiovascular Division, Dept. of Internal Medicine, Institute of Clinical Medicine, Univ. of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan (E-mail: t-miyauc{at}md.tsukuba.ac.jp).

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.

September 20, 2002;10.1152/japplphysiol.00269.2002

Received 29 March 2002; accepted in final form 12 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Armstrong, RB, Delp MD, Goljan EF, and Laughlin MH. Distribution of blood flow in muscles of miniature swine during exercise. J Appl Physiol 62: 1285-1298, 1987[Abstract/Free Full Text].

2.   Blair, DA, Glover WE, and Roddie IC. Vasomotor responses in the human arm during leg exercise. Circ Res 9: 264-274, 1961[Abstract/Free Full Text].

3.   Busse, R, Mülsch A, Fleming I, and Hecker M. Mechanisms of nitric oxide release from the vascular endothelium. Circulation 87, Suppl 5: S18-S25, 1993.

4.   Delp, MD, and Laughlin MH. Time course of enhanced endothelium-mediated dilation in aorta of trained rat. Med Sci Sports Exerc 29: 1454-1461, 1997.

5.   Fixler, DE, Atkins JM, Mitchell JH, and Horwitz LD. Blood flow to respiratory, cardiac, and limb muscles in dogs during graded exercise. Am J Physiol 231: 1515-1519, 1976[Abstract/Free Full Text].

6.   Flaim, SF, Minteer WJ, Clark DP, and Zelis R. Cardiovascular response to aquatic and treadmill exercise in the untrained rat. J Appl Physiol 46: 302-308, 1979[Abstract/Free Full Text].

7.   Geng, YJ, Almqvist M, and Hansson GK. cDNA cloning and expression of inducible nitric oxide synthase from rat vascular smooth muscle cells. Biochim Biophys Acta 1218: 421-424, 1994[Medline].

8.   Green, LC, Wagner DM, Glogowski J, Skipper PL, Wishnok JS, and Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126: 131-138, 1982[ISI][Medline].

9.   Hirai, T, Visneski MD, Kearns KJ, Zelis R, and Musch TI. Effects of NO synthase inhibition on the muscular blood flow response to treadmill exercise in rats. J Appl Physiol 77: 1288-1293, 1994[Abstract/Free Full Text].

10.   Iemitsu, M, Miyauchi T, Maeda S, Yuki K, Kobayashi T, Kumagai Y, Shimojo N, Yamaguchi I, and Matsuda M. Intense exercise causes decrease in expression of both endothelial NO synthase and tissue NOx level in hearts. Am J Physiol Regul Integr Comp Physiol 279: R951-R959, 2000[Abstract/Free Full Text].

11.   Ikeda, U, Yamamoto K, Maeda Y, Shimpo M, Kanbe T, and Shimada K. Endothelin-1 inhibits nitric oxide synthesis in vascular smooth muscle cells. Hypertension 29: 65-69, 1997[Abstract/Free Full Text].

12.   Kawai, N, Bloch DB, Filippov G, Rabkina D, Suen HC, Losty PD, Janssens SP, Zapol WM, Monte de la S, and Bloch KD. Constitutive endothelial nitric oxide synthase gene expression is regulated during lung development. Am J Physiol Lung Cell Mol Physiol 268: L589-L595, 1995[Abstract/Free Full Text].

13.   Knowles, RG, Merrett M, Salter M, and Moncada S. Differential induction of brain, lung and liver nitric oxide synthase by endotoxin in the rat. Biochem J 270: 833-836, 1990[ISI][Medline].

14.   Knowles, RG, and Moncada S. Nitric oxide synthases in mammals. Biochem J 298: 249-258, 1994[ISI][Medline].

15.   Kobayashi, T, Miyauchi T, Sakai S, Kobayashi M, Yamaguchi I, Goto K, and Sugishita Y. Expression of endothelin-1, ETA and ETB receptors, and ECE and distribution of endothelin-1 in failing rat heart. Am J Physiol Heart Circ Physiol 276: H1197-H1206, 1999[Abstract/Free Full Text].

16.   Kuchan, MJ, and Frangos JA. Shear stress regulates endothelin-1 release via protein kinase C and cGMP in cultured endothelial cells. Am J Physiol Heart Circ Physiol 264: H150-H156, 1993[Abstract/Free Full Text].

17.   Kumagai, Y, Nakajima H, Midorikawa K, Homma-Takeda S, and Shimojo N. Inhibition of nitric oxide formation by neuronal nitric oxide synthase by quinones: nitric oxide synthase as a quinone reductase. Chem Res Toxicol 11: 608-613, 1998[ISI][Medline].

18.   Kuo, L, Davis MJ, and Chilian WM. Endothelium-dependent, flow-induced dilation of isolated coronary arterioles. Am J Physiol Heart Circ Physiol 259: H1063-H1070, 1990[Abstract/Free Full Text].

19.   Laughlin, MH, and Armstrong RB. Muscular blood flow distribution patterns as a function of running speed in rats. Am J Physiol Heart Circ Physiol 243: H296-H306, 1982[Abstract/Free Full Text].

20.   Laughlin, MH, and Armstrong RB. Rat muscle blood flows as a function of time during prolonged slow treadmill exercise. Am J Physiol Heart Circ Physiol 244: H814-H824, 1983[Abstract/Free Full Text].

21.   Laughlin, MH, and Armstrong RB. Muscle blood flow during locomotory exercise. Exerc Sport Sci Rev 13: 95-136, 1985[Medline].

22.   Laughlin, MH, Armstrong RB, White J, and Rouk K. A method for using microspheres to measure muscle blood flow in exercising rat. J Appl Physiol 52: 1629-1635, 1982[Abstract/Free Full Text].

23.   Liu, SF, Crawley DE, Evans TW, and Barnes PJ. Endogenous nitric oxide modulates adrenergic neural vasoconstriction in guinea-pig pulmonary artery. Br J Pharmacol 104: 565-569, 1991[ISI][Medline].

24.   Maeda, S, Miyauchi T, Kobayashi T, Goto K, and Matsuda M. Exercise causes tissue-specific enhancement of endothelin-1 mRNA expression in internal organs. J Appl Physiol 85: 425-431, 1998[Abstract/Free Full Text].

25.   Maeda, S, Miyauchi T, Sakai S, Kobayashi T, Iemitsu M, Goto K, Sugishita Y, and Matsuda M. Prolonged exercise causes an increase in endothelin-1 production in the heart in rats. Am J Physiol Heart Circ Physiol 275: H2105-H2112, 1998[Abstract/Free Full Text].

26.   Maeda, S, Miyauchi T, Sakane M, Saito M, Maki S, Goto K, and Matsuda M. Does endothelin-1 participate in the exercise-induced changes of blood flow distribution of muscles in humans? J Appl Physiol 82: 1107-1111, 1997[Abstract/Free Full Text].

27.   Martin, CM, Beltran-del-Rio A, Albrecht A, Lorenz RR, and Joyner MJ. Local cholinergic mechanisms mediate nitric oxide-dependent flow-induced vasorelaxation in vitro. Am J Physiol Heart Circ Physiol 270: H442-H446, 1996[Abstract/Free Full Text].

28.   Millard, RW, Higgins CB, Franklin D, and Vatner SF. Regulation of the renal circulation during severe exercise in normal dogs and dogs with experimental heart failure. Circ Res 31: 881-888, 1972[Abstract/Free Full Text].

29.   Moncada, S, Palmer RM, and Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109-142, 1991[ISI][Medline].

30.   Musch, TI, Friedman DB, Pitetti KH, Haidet GC, Stray-Gundersen J, Mitchell JH, and Ordway GA. Regional distribution of blood flow of dogs during graded dynamic exercise. J Appl Physiol 63: 2269-2277, 1987[Abstract/Free Full Text].

31.   Musch, TI, Haidet GC, Ordway GA, Longhurst JC, and Mitchell JH. Training effects on regional blood flow response to maximal exercise in foxhounds. J Appl Physiol 62: 1724-1732, 1987[Abstract/Free Full Text].

32.   Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB J 6: 3051-3062, 1992[Abstract].

33.   Norton, KI, Jones MT, and Armstrong RB. Oxygen consumption and distribution of blood flow in rats climbing a laddermill. J Appl Physiol 68: 241-247, 1990[Abstract/Free Full Text].

34.   Palmer, RMJ, Ashton DS, and Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333: 664-666, 1988[Medline].

35.   Palmer, RMJ, Ferrige AG, and Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524-526, 1987[Medline].

36.   Palmer, RMJ, Rees DD, Ashton DS, and Moncada S. L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun 153: 1251-1256, 1988[ISI][Medline].

37.   Peuler, JD, and Johnson GA. Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci 21: 625-636, 1977[ISI][Medline].

38.   Rubanyi, GM. Cardiovascular Significance of Endothelium-Derived Vasoactive Factors. Mount Kisco, NY: Futura, 1991, p. xi-xix, 3-81.

39.   Rubanyi, GM, and Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 46: 325-415, 1994[ISI][Medline].

40.   Rubanyi, GM, Romero JC, and Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol Heart Circ Physiol 250: H1145-H1149, 1986[Abstract/Free Full Text].

41.   Sambrook, J, and Russell DW. Molecular Cloning (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001, p. A6.5.

42.   Sambrook, J, and Russell DW. Molecular Cloning (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001, p. A8.23-A8.24.

43.   Shepherd, RE, and Gollnick PD. Oxygen uptake of rats at different work intensities. Pflügers Arch 362: 219-222, 1976[ISI][Medline].

44.   Shinyashiki, M, Kumagai Y, Nakajima H, Nagafune J, Homma-Takeda S, Sagai M, and Shimojo N. Differential changes in rats brain nitric oxide synthase in vivo and in vitro by methylmercury. Brain Res 798: 147-155, 1998[ISI][Medline].

45.   Tidgren, B, Hjemdahl P, Theodorsson E, and Nussberger J. Renal neurohormonal and vascular responses to dynamic exercise in humans. J Appl Physiol 70: 2279-2286, 1991[Abstract/Free Full Text].

46.   Tso, JY, Sun XH, Kao TH, Reece KS, and Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13: 2485-2502, 1985[Abstract/Free Full Text].

47.   Van Citters, RLV, and Franklin D. Cardiovascular performance of Alaska sled dogs during exercise. Circ Res 24: 33-42, 1969[Abstract/Free Full Text].

48.   Yanagisawa, M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411-415, 1988[Medline].

J APPL PHYSIOL 94(1):60-68
8750-7587/03 $5.00 Copyright © 2003 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
O. Yalcin, P. Ulker, U. Yavuzer, H. J. Meiselman, and O. K. Baskurt
Nitric oxide generation by endothelial cells exposed to shear stress in glass tubes perfused with red blood cell suspensions: role of aggregation
Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2098 - H2105.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
R. D. Moraes, G. Gioseffi, A. C. L. Nobrega, and E. Tibirica
Effects of exercise training on the vascular reactivity of the whole kidney circulation in rabbits
J Appl Physiol, August 1, 2004; 97(2): 683 - 688.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
S. Maeda, T. Miyauchi, M. Iemitsu, T. Tanabe, K. Goto, I. Yamaguchi, and M. Matsuda
Endothelin receptor antagonist reverses decreased NO system in the kidney in vivo during exercise
Am J Physiol Endocrinol Metab, April 1, 2004; 286(4): E609 - E614.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. E. Davis, I. M. Grumbach, T. Fukai, A. Cutchins, and D. G. Harrison
Shear Stress Regulates Endothelial Nitric-oxide Synthase Promoter Activity through Nuclear Factor {kappa}B Binding
J. Biol. Chem., January 2, 2004; 279(1): 163 - 168.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
O. K. Baskurt, O. Yalcin, S. Ozdem, J. K. Armstrong, and H. J. Meiselman
Modulation of endothelial nitric oxide synthase expression by red blood cell aggregation
Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H222 - H229.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
94/1/60    most recent
00269.2002v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miyauchi, T.
Right arrow Articles by Matsuda, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miyauchi, T.
Right arrow Articles by Matsuda, M.

HOME HELP FEEDBACK SUBSCRIPTIONS