Suppressed impact of nitric oxide on renal arteriolar function in rats with chronic heart failure

Hideki Ikenaga, Naohito Ishii, Sean P. Didion, Kun Zhang, Kurtis G. Cornish, Kaushik P. Patel, William G. Mayhan, and Pamela K. Carmines

Department of Physiology and Biophysics, University of Nebraska College of Medicine, Omaha, Nebraska 68198-4575

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We performed experiments to test the hypothesis that experimental heart failure (HF) is associated with altered nitric oxide (NO)-dependent influences on the renal microvasculature, including diminished modulation of constrictor responses to ANG II. Eight to ten weeks after inducing HF in rats by coronary artery ligation, we administered enalaprilat to suppress ANG II synthesis and studied renal arteriolar function using the in vitro blood-perfused juxtamedullary nephron technique. In kidneys from sham-operated rats, NO synthase inhibition [100 µM Nomega -nitro-L-arginine (L-NNA)] reduced afferent arteriolar diameter by 4.1 ± 0.6 µm and enhanced ANG II responsiveness (10 nM ANG II decreased afferent diameter by 10.1 ± 1.4 µm before and 12.8 ± 1.6 µm during L-NNA treatment; P < 0.05). In kidneys from HF rats, L-NNA did not alter afferent arteriolar baseline diameter or ANG II responsiveness (10 nM ANG II decreased diameter by 12.5 ± 1.5 µm before and 12.5 ± 2.3 µm during L-NNA). The effects of L-NNA on efferent arteriolar function were also abated in HF rats. In renal cortex of HF rats, NO synthase activity was decreased by 63% and superoxide dismutase activity was diminished by 39% relative to tissue from sham-operated rats. Urinary nitrate/nitrite excretion was also reduced in HF rats. Thus both diminished synthesis and augmented degradation are likely to contribute to a decreased renal microvascular impact of endogenous NO during chronic HF, the consequences of which include loss of NO-dependent modulation of ANG II-induced vasoconstriction.

angiotensin II; myocardial infarction; nitric oxide synthase; renal microvasculature; superoxide dismutase

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IN BOTH PHYSIOLOGICAL AND pathophysiological states, ANG II influences sodium and water balance in part through its effects on glomerular and tubular function. The effect of ANG II on glomerular function is complex, involving the effects on mesangial cells as well as a subtle balance between the vasoconstrictor effects of the peptide on afferent and efferent arterioles. Several physiological antagonists modify the effects of ANG II on these two crucial microvascular segments. In particular, nitric oxide (NO) derived from endothelial and tubular (macula densa) sources modulates the glomerular arteriolar effects of ANG II under normal conditions (16, 18, 22). In turn, tissue ANG II levels appear to modulate renal NO production and/or its impact on glomerular arteriolar tone (30, 33). Thus glomerular arteriolar tone under normal conditions is determined in part by a complex interaction between the vasodilator influence of NO and the vasoconstrictor influence of ANG II.

Activation of the renin-angiotensin system normally occurs in volume-depleted states; however, inappropriate activation of the renin-angiotensin system is likely to contribute to the sodium retention that is characteristic of chronic heart failure (HF) (39). Indeed, angiotensin-converting enzyme (ACE) inhibitors are now widely recognized to improve renal hemodynamics and excretory function in HF. Recent studies have indicated that HF is also associated with endothelial dysfunction. Although some reports suggest an increased cardiovascular impact of NO (15, 41), most evidence indicates that HF is associated with a suppressed impact of NO on basal vascular tone (25) and/or diminished responses to endothelium-dependent vasodilators (8, 24, 26). Loss of NO-dependent modulation also appears to contribute to exaggerated peripheral vasoconstrictor responses to norepinephrine, ANG II, and vasopressin during HF (7, 39).

Even though accumulating evidence indicates that diminished NO-dependent vascular regulation promotes peripheral vasoconstriction in HF, this phenomenon is not uniformly evident in all vascular beds (3, 34). The few studies that have evaluated the impact of NO on renal hemodynamics in HF have yielded conflicting evidence. Drexler and co-workers (10) observed normal renal vasoconstrictor responses to NO synthase (NOS) inhibition 8 wk after myocardial infarction in the rat, and acetylcholine and nitroglycerine have been reported to evoke substantial renal vasodilation in patients with HF (12). However, Abassi et al. (2) recently reported attenuated NO-mediated renal vasodilator responses in rats with high-output HF induced by aortocaval fistula. Because the potential for systemic and/or neural influences on renal function complicates interpretation of these results, the functional impact of NO in the renal vasculature in HF remains uncertain. In light of the normal interplay between NO and ANG II as potent determinants of renal arteriolar tone and sodium excretion, experiments were performed to test the hypothesis that HF is associated with diminished NO-dependent modulation of renal arteriolar constrictor responses to ANG II. Additional studies addressed whether the observed changes in microvascular function reflect an alteration in NO synthesis and/or degradation.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Production of Myocardial Infarction

The Institutional Animal Care and Use Committee of the University of Nebraska Medical Center approved all procedures utilized in this study. Male Sprague-Dawley rats weighing 200-250 g were assigned randomly to one of two groups: HF and sham operated. HF was produced by left coronary artery ligation, using a method similar to that originally described by Johns and Olson (23). Briefly, the animals were anesthetized with either pentobarbital sodium (50 mg/kg ip) or methohexital sodium (50 mg/kg ip), intubated, and ventilated using a Harvard rodent respirator. A left thoracotomy was performed in the fifth intercostal space, and 6-0 suture was used to place a ligature around the left main coronary artery ~2 mm from the aorta, between the pulmonary artery outflow tract and the left atrium. This suture was tied securely in HF rats but was pulled through in the sham-operated rats. The heart was returned to its original position and the thorax was closed. The air within the thorax was removed, allowing the rats to resume spontaneous respiration. The rats were allowed to recover from anesthesia and were caged individually in an environment with ambient temperature maintained at 22°C and humidity at 30-40%. Analgesics (Nubain-Stadol, 1 ml/kg sc) were administered on each of the first 2 days after the surgery. Laboratory chow (Purina) and tap water were available ad libitum. Eight to ten weeks after the production of HF, the animals were subjected to acute experiments.

Systemic and Renal Vascular Function Studies

Under pentobarbital sodium anesthesia (50 mg/kg ip), a short (~10 mm) tapered polyethylene catheter connected to a 2-Fr catheter-tip Millar pressure transducer was introduced into the right carotid artery. The catheter was used first for measuring systemic arterial pressure and heart rate and then was advanced into the left ventricle for monitoring left ventricular end-diastolic pressure (LVEDP).

After cardiovascular assessment, rats were treated with the converting-enzyme inhibitor enalaprilat (2 mg ia) to suppress endogenous ANG II formation. Thirty minutes later, a laparotomy was performed and the left kidney was removed. A cannula introduced via the superior mesenteric artery into the right renal artery allowed renal perfusion with Tyrode solution containing 52 g/l dialyzed bovine serum albumin and a mixture of L-amino acids, including 1.8 mM L-arginine (33). The renal vein was incised to drain the perfusate, while blood was collected via the carotid cannula into a heparinized syringe. The kidney was removed and cut longitudinally to expose the pelvic cavity, leaving the papilla intact within the dorsal two-thirds of the organ. Renal perfusion was maintained throughout the dissection procedure needed to reveal the tubules, glomeruli, and related vasculature of juxtamedullary nephrons. Tight ligatures were placed around the most distal accessible segments of the large arterial branches that supply the exposed microvasculature.

Blood collected from the rat was processed to remove leukocytes and platelets. The resulting blood perfusate was stirred continuously in a closed reservoir that was pressurized by a 95% O2-5% CO2 tank, an arrangement that provided both oxygenation and the driving force for in vitro perfusion of the dissected kidney. Perfusion pressure was measured at the cannula tip in the renal artery using a P23XL transducer (Gould, Oxnard, CA) connected to a polygraph (Grass Instruments, Quincy, MA), and was maintained at 110 mmHg throughout the experiment. The tissue was warmed to 37°C, and its surface was continuously bathed with Tyrode solution containing 10 g/l bovine serum albumin. All vasoactive agents were applied to the tissue by addition to this bathing solution.

The tissue was transilluminated on the fixed stage of a Nikon Optiphot microscope equipped with a water-immersion objective (×40, numerical aperture 0.55). Video images of the microvessels were generated by a Newvicon camera (Dage-MTI, Michigan City, IN), passed through a time-date generator and an image enhancer, and displayed at a magnification of ×1,400 on a high-resolution monitor (Conrac Display Systems, Covina, CA). The video signal was recorded simultaneously on videotape using a SuperVHS videocassette recorder (Panasonic).

Completion of the microdissection procedure was followed by a 15-min equilibration period, during which a single afferent or efferent arteriole was selected for study on the basis of visibility and acceptable blood flow. Only arterioles with rapid flow of erythrocytes were studied, and vessels were rejected on the basis of inadequate flow if the passage of single erythrocytes could be discerned. Afferent arteriolar diameter was monitored at sites >100 µm upstream from the glomerulus and >50 µm downstream from the interlobular artery. Efferent arterioles were studied at sites within 220 µm of the glomerulus, before their first branching point.

The experimental protocol was designed to monitor arteriolar diameter at a single measurement site under several experimental conditions. Responses to increasing concentrations of ANG II (Sigma Chemical, St. Louis, MO) were assessed by sequential exposure of the tissue surface to the following bathing solutions: 1) Tyrode solution alone (10 min); 2) Tyrode solution containing increasing concentrations of ANG II (0.1, 1, and 10 nM; 3 min at each concentration); and 3) Tyrode solution alone (10 min). Each arteriole was subjected to this treatment sequence both before and during endogenous NOS inhibition. NOS inhibition was achieved by addition of 100 µM Nomega -nitro-L-arginine (L-NNA; Aldrich Chemical, Milwaukee, WI) to the bathing solution, as described previously (33). Juxtamedullary afferent arteriolar diameter responses to 100 µM L-NNA are apparent within 5 min, maximal at 10 min, and stable for more than 30 min thereafter (18). Thus, for the present study, L-NNA treatment was begun 15 min before the second ANG II exposure sequence and continued for the duration of the experiment. We previously confirmed that imposition of two consecutive ANG II exposure sequences, according to this protocol (but in the absence of L-NNA), evokes indistinguishable juxtamedullary arteriolar diameter responses, even when using peptide concentrations 10-fold higher than those employed in the present study (4).

Microvessel inside diameters were determined from videotaped images utilizing a digital image-shearing monitor (model 908; IPM, San Diego, CA). This device was calibrated using a stage micrometer (smallest division = 2 µm) and yielded diameter measurements reproducible to within ~0.5 µm. Microvessel diameter was measured at 12-s intervals from a single site along the vessel length. The average diameter during the final minute of each treatment period was utilized for statistical analysis.

Biochemical Studies

Sham-operated and HF rats were housed in metabolic cages (Nalge, Rochester, NY) for 2 days immediately before the terminal experiment. The total volume of urine collected during the final 24-h period was centrifuged (10 min at 3,000 rpm) to remove sediment and was stored at -70°C until measurement of nitrate plus nitrite (NOx) concentration. The rats were then anesthetized with pentobarbital sodium (50 mg/kg ip) and tracheotomized to facilitate spontaneous respiration. The left carotid artery was cannulated, a blood sample (0.5-1.0 ml) was collected into a heparinized syringe, and the plasma was stored at -70°C until measurement of NOx concentration. After flushing the kidneys with ice-cold isotonic saline from the abdominal aorta, we quickly removed both kidneys. The right kidney was used for assay of NOS activity, and the left kidney was used for assay of superoxide dismutase (SOD) activity. Each kidney was hemisected, and the medullary tissue was removed and discarded.

NOS assay. Total NOS activity in renal cortex was determined as the rate of L-citrulline formation from radiolabeled L-arginine, using a modification of the method described by Moridani and Kline (29). The renal cortex was weighed, minced, and homogenized (Polytron model PT 10/35; Kinematica, Switzerland) in ice-cold homogenization buffer containing (in mM) 10 HEPES, 320 sucrose, 0.1 EDTA, 1 dithiothreitol, 0.1 soybean trypsin inhibitor, 0.1 aprotinin, and 1 phenylmethylsulfonyl fluoride, pH 7.40. Particulate NOS was released from membranes in the homogenate by 20-min treatment at 4°C with 0.2 vol (vol/wt) of 200 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). After centrifugation for 20 min at 10,000 g (4°C), endogenous L-arginine was removed from the supernatant by suspension in Dowex 50WX8-400 (1:1 Dowex:water; Sigma). After the suspension was allowed to settle (10 min at 4°C), 20 µl of the supernatant were added to 50 µl of incubation buffer (pH 7.2). The incubation buffer was composed of 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 5 mM L-valine, 2 µM (6R)-5,6,7,8-tetrahydrobiopterin, 1 mM beta -NADPH, 0.5 µM calmodulin, 20 µM L-arginine, and 0.2 µM L-[2,3,4,5-3H]arginine (12.6 µCi/ml assay buffer, 63 Ci/mmol specific activity; Amersham Life Science, Little Chalfont, UK). Samples were incubated for 30 min (37°C), after which the reaction was terminated by the addition of 500 µl suspended Dowex in water (to bind the remaining L-arginine). An additional volume of water (1 ml) was added to the samples, which were then allowed to settle for 20 min at room temperature. The supernatant was added to liquid scintillation cocktail (ScintiSafe Plus 50%; Fisher Scientific, Pittsburgh, PA) for counting of radiolabeled products (LS6500 Multipurpose Scintillation Counter; Beckman Instruments, Fullerton, CA). Total NOS activity of each background-corrected sample was determined as the difference between the radioactivity with or without 300 µM L-NNA as an inhibitor of NOS. Preliminary experiments confirmed that the effect of L-NNA was indistinguishable from that of 300 µM NG-monomethyl-L-arginine (L-NMMA). NOS activity was expressed as nanomoles L-[3H]citrulline formed per hour per gram protein.

SOD assay. Renal cortical tissue was weighed, minced, and homogenized (Polytron) in ice-cold sucrose (250 mM). The resulting crude homogenate was centrifuged for 10 min at 8,500 g (4°C) and the supernatant was stored at -70°C until assay for SOD activity. The thawed supernatant was extracted in ice-cold ethanol:chloroform (62.5:37.5), mixed, and centrifuged for 10 min at 3,000 g (4°C). A commercially available kit (BIOXYTECH SOD-525; OXIS International, Portland, OR) was used to measure SOD activity in the aqueous upper layer of the extract. The SOD-525 assay is based on the SOD-mediated increase in the rate of autoxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzol[c]fluorene to yield a chromophore with maximum absorbance at 525 nm. Each sample was pretreated with 1,4,6-trimethyl-2-vinylpyridinium trifluoromethanesulfonate, which scavenges interfering mercaptans (e.g., glutathione) via a very rapid alkylation reaction. For comparison purposes, SOD-525 activity was also measured in standard solutions of SOD purified from bovine erythrocytes (3,400 U/ml determined using the xanthine-xanthine oxidase reaction)(Sigma). The results indicate that one SOD-525 activity unit (defined as the activity that doubles the autoxidation background in the SOD-525 kit) is the equivalent of 24.3 units measured using the xanthine-xanthine oxidase reaction (defined as the activity that reduces the rate of reduction of cytochrome c by 50%).

NOx and protein assays. NOx concentrations in plasma and urine were measured using the ozone-chemiluminescence method (model 280 NO analyzer; Sievers Instruments, Boulder, CO). Protein content in tissue samples was measured using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Cardiac Histology

Immediately after the kidneys were harvested for use in functional or biochemical studies, the heart and lungs were removed, blotted, and weighed. The heart was fixed by immersion in 10% buffered formaldehyde, embedded in paraffin, and cut into three transverse slices from base to apex. The central slice was subjected to graded dehydration with ethanol, embedded in paraffin, and cut into 10-µm-thick sections, which were stained with phosphotungstic acid-hematoxylin and mounted on glass slides. Cardiac sections were projected onto paper, and the outline of the tissue was diagrammed. The scale drawing thus obtained was utilized for measuring left ventricular outer (epicardial) and inner (endocardial) circumferences, as well as the arc length of the infarcted region. The infarcted fraction of the left ventricular wall was calculated on the basis of these measurements. For each rat, the infarct size determined from three representative sections was averaged and utilized as the infarcted fraction of the left ventricle.

Statistical Analyses

Within each group, comparisons were made by ANOVA for repeated measures or by Friedman repeated-measures ANOVA on ranks, as appropriate, followed by the Newman-Keuls test. Simple comparisons between groups employed the unpaired t-test or Mann-Whitney rank sum test, as appropriate. All statistical computations were performed utilizing the SigmaStat software package (Jandel Scientific, San Rafael, CA). P < 0.05 was considered statistically significant. Results are expressed as means ± SE.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Table 1 summarizes the salient characteristics of sham-operated and HF rats utilized in the present study. For renal microvascular function experiments, any rats subjected to coronary artery ligation that failed to display LVEDP values >= 10 mmHg were excluded from the study. Accordingly, the rats retained in the chronic HF group exhibited significantly elevated LVEDP, as well as lower mean arterial pressure and pulse pressure, than was observed in sham-operated rats. Moreover, wet weights of both the heart and lungs were significantly elevated in chronic HF rats. These changes in organ weight remained evident when factored by body weight. For biochemical studies (in which hemodynamic measurements were not performed), rats subjected to coronary artery ligation were excluded from the study if histological assessment failed to provide evidence of an infarct. As detailed in Table 1, the HF rats retained in the study (for hemodynamic and biochemical measurements) displayed infarcts comprising ~40% of total left ventricular circumference, whereas no histological evidence of myocardial infarct was apparent in hearts from sham-operated rats.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Characteristics of chronic heart failure rats (8-10 wk) and sham-operated rats

Microvascular Function Studies

The total length of the afferent arterioles included in this study averaged 722 ± 104 µm (n = 18). In sham-operated rats, afferent arteriolar diameter measurements were made at sites located 261 ± 64 µm upstream from the glomerulus, whereas afferent arteriolar diameter was measured at sites located 378 ± 98 µm from the glomerulus in kidneys from HF rats [not significant (NS)]. There was no difference in baseline afferent arteriolar lumen diameter between kidneys from sham-operated (23.3 ± 0.8 µm, n = 9) and HF rats (23.9 ± 1.3 µm, n = 9). Efferent arteriolar function was monitored at sites located 120 ± 20 µm downstream from the glomerulus in sham-operated rats and 130 ± 20 µm from the glomerulus in HF rats. Baseline efferent arteriolar lumen diameter did not differ significantly between sham-operated (22.1 ± 1.2 µm, n = 10) and HF rats (24.3 ± 2.4 µm, n = 8).

Figure 1 illustrates the impact of NO synthesis inhibition (100 µM L-NNA) on baseline arteriolar diameters. In kidneys harvested from sham-operated rats, addition of L-NNA to the bathing solution significantly reduced lumen diameters of juxtamedullary afferent (-4.1 ± 0.6 µm) and efferent arterioles (-3.3 ± 0.5 µm). In kidneys from HF rats, however, L-NNA failed to evoke significant reductions in arteriolar diameter (afferent arteriole -1.6 ± 0.7 µm, efferent arteriole -2.6 ± 1.3 µm). Thus L-NNA had a smaller constrictor effect on renal juxtamedullary arterioles in HF rats than in sham-operated rats.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of Nomega -nitro-L-arginine (L-NNA) on afferent and efferent arteriolar lumen diameters in kidneys from sham-operated rats (filled bars) and rats with heart failure (HF; open bars). Data are means ± SE. * P < 0.05 vs. baseline diameter. dagger  P < 0.05 vs. response in sham-operated rats.

Figure 2 shows the effect of L-NNA on afferent arteriolar vasoconstrictor responses to ANG II. In kidneys from sham-operated rats studied under control (untreated) conditions, addition of 1 and 10 nM ANG II to the bathing solution significantly reduced afferent arteriolar lumen diameter by 4.6 ± 1.2 and 10.1 ± 1.4 µm, respectively. During L-NNA treatment, ANG II-induced afferent vasoconstriction was significantly enhanced in kidneys from sham-operated rats, with 1 and 10 nM ANG II decreasing lumen diameter by 5.8 ± 0.9 and 12.8 ± 1.6 µm, respectively (P < 0.05 vs. control responses for both). Thus L-NNA treatment caused ~30% augmentation of afferent arteriolar diameter response to 10 nM ANG II in kidneys from sham-operated rats. In kidneys from HF rats, ANG II also evoked concentration-dependent reductions in afferent arteriolar diameter. Under control (untreated) conditions, these responses averaged -5.2 ± 0.8 µm (1 nM, P < 0.05 vs. baseline) and -12.5 ± 1.5 µm (10 nM, P < 0.05 vs. baseline). Although control responses to ANG II were somewhat larger in HF rats than in sham-operated rats, these differences did not achieve statistical significance. L-NNA failed to significantly enhance ANG II-induced afferent vasoconstriction in HF rats, with 1 and 10 nM ANG II constricting vessels by 5.2 ± 1.2 and 12.5 ± 2.3 µm, respectively (NS vs. control responses). Thus, although L-NNA treatment augmented afferent arteriolar diameter responses to ANG II in kidneys from sham-operated rats, L-NNA did not alter this response in kidneys from HF rats.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of L-NNA on afferent arteriolar responses to exogenous ANG II. A: responses in kidneys from sham-operated rats. B: responses in kidneys from HF rats. ANG II concentration-response relationships were generated in same vessels before (untreated, filled symbols) and during exposure to 100 µM L-NNA (open symbols). Data are means ± SE. * P < 0.05 vs. baseline diameter. dagger  P < 0.05 vs. untreated response.

The effect of L-NNA on efferent arteriolar vasoconstrictor responses to ANG II is summarized in Fig. 3. In sham-operated rats studied under control (untreated) conditions, 1 nM ANG II reduced efferent arteriolar diameter by 3.6 ± 1.1 µm (P < 0.05 vs. baseline) and 10 nM ANG II decreased diameter by 9.4 ± 1.5 µm (P < 0.05 vs. baseline). During subsequent L-NNA treatment, the same concentrations of ANG II reduced efferent arteriolar diameter by 4.8 ± 1.1 and 10.7 ± 1.5 µm, respectively (P < 0.05 vs. control responses for both). Thus L-NNA evoked a significant (~20%) amplification of the efferent diameter response to 10 nM ANG II. In contrast, L-NNA failed to enhance ANG II-induced efferent arteriolar vasoconstriction in kidneys from HF rats at any peptide concentration examined. Under control conditions, 1 and 10 nM ANG II reduced efferent arteriolar lumen diameter in kidneys from HF rats by 4.1 ± 1.5 and 12.0 ± 2.3 µm (both P < 0.05 vs. baseline for both). During subsequent L-NNA treatment, the same peptide concentrations reduced efferent diameter by 4.5 ± 1.0 and 11.5 ± 2.0 µm, respectively (NS vs. control responses). Thus, although efferent arteriolar diameter responses to ANG II were potentiated by L-NNA in kidneys from sham-operated rats, no alteration of the efferent arteriolar ANG II response was observed after L-NNA treatment of kidneys from HF rats.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of L-NNA on efferent arteriolar responses to exogenous ANG II. A: responses in kidneys from sham-operated rats. B: responses in kidneys from HF rats. ANG II concentration-response relationships were generated in same vessels before (untreated, filled symbols) and during exposure to 100 µM L-NNA (open symbols). Data are means ± SE. * P < 0.05 vs. baseline diameter. dagger  P < 0.05 vs. untreated response.

Biochemical Studies

During the 24 h before tissue was harvested for biochemical studies, urine flow did not differ between sham-operated and HF rats (1.07 ± 0.23 and 1.00 ± 0.21 ml/h, respectively). Plasma NOx levels were also similar between groups (9 ± 1 nmol/ml for sham-operated vs. 10 ± 2 nmol/ml for HF rats). However, urinary NOx excretion (UNOxV) was markedly lower in HF rats (16 ± 4 nmol/h; n = 5) than in sham-operated rats (198 ± 60 nmol/h; n = 6; P < 0.05). NOS and SOD activities in renal cortex of sham-operated and HF rats are shown in Fig. 4. Cortical NOS activity in kidneys from HF rats averaged 37% of that observed in sham-operated rats (P < 0.001). Renal cortical SOD activity was also reduced in HF rats, averaging 61% of that observed in kidneys from sham-operated rats (P < 0.01). Thus both NOS and SOD activities were suppressed in kidneys from HF rats. The ratio of NOS-to-SOD activity was calculated for each rat as a crude indicator of the relative activities of primary NO synthesis and degradation pathways. The NOS-to-SOD ratio averaged 0.13 ± 0.01 in sham-operated rats and 0.07 ± 0.02 in HF rats (P < 0.05), indicating that activities of these enzymes did not decline in a parallel manner.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4.   Renal cortical nitric oxide synthase (NOS) activity (A) and superoxide dismutase (SOD) activity (B) in sham-operated (filled bars; n = 6) and HF rats (open bars; n = 5). Data are means ± SE. ** P < 0.01 vs. sham-operated rats.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Coronary artery ligation in the rat produces myocardial infarction characterized by noticeable loss of cardiac cells and thinning of the ventricular wall, which results in left ventricular dysfunction. In this experimental model of HF, elevations in LVEDP correlate directly with the severity of the infarction. Rats with infarcts involving 31-46% of the ventricular wall have reduced peak flow indexes and reduced developed pressure (13), whereas rats with infarcts exceeding 46% display overt signs of congestive heart failure with LVEDP exceeding 20 mmHg (13). The HF rats included in the present study displayed infarcts involving ~40% of the ventricular wall, with accompanying elevations in LVEDP to ~17 mmHg. Thus the rats in the HF group had moderate to large left ventricular infarcts and decreased cardiac contractile function, as well as increased lung wet weight (consistent with pulmonary edema, although dyspnea was not evident).

The primary goal of the present study was to assess the impact of HF on NO-dependent regulation of the renal microvascular function, with emphasis on the modulatory impact of NO on ANG II-induced constrictor responses. This was achieved through use of the in vitro blood-perfused juxtamedullary nephron technique (6), an experimental setting that provides several practical advantages over in vivo clearance or micropuncture studies. First, renal microvascular function is assessed by direct videomicroscopic methods during perfusion of the tissue at a constant renal arterial pressure. Second, the in vitro setting removes systemic and neural influences that typically confound interpretation of the results when vasoactive agents are administered in vivo by intravenous infusion. Third, because vasoconstrictor responses to ANG II are similar in magnitude when the peptide is applied via the bath or perfusate in this experimental setting (21), the present studies employed the technically simple method of adding ANG II and L-NNA to the bathing solution. This avenue for application of vasoactive agonists at known concentrations provides access to the site of action without dependence on renal blood flow. The limitations of the perfused juxtamedullary nephron technique have been considered in detail previously (35) and include the unknown status of endogenous vasoactive modulators. In particular, the processing of perfusate blood and the acute enalaprilat treatment employed to reduce endogenous ANG II levels can be expected to elevate kinin levels. Because neither the arteriolar impact of endogenous kinins nor the mechanism by which these effects arise (NO, prostaglandins, etc.) have been evaluated in perfused juxtamedullary nephrons, the nature and extent to which kinin accumulation complicates the responses observed in the present study remain uncertain. The possibility for interactive vascular responses between the series-arranged afferent and efferent arteriolar segments might also be anticipated to complicate the data interpretation; however, such a phenomenon is not likely to represent a serious limitation because efferent arteriolar diameter can be regulated independently of alterations in afferent arteriolar caliber in this unique experimental setting (5, 19, 40).

Micropuncture studies have documented reduced glomerular plasma flow associated with increased afferent and (more prominently) efferent arteriolar resistance in HF (17, 32); however, in the present study arteriolar baseline diameters were similar in kidneys from HF and sham-operated rats. This phenomenon likely reflects the fact that these in vitro studies were performed in tissue acutely treated with enalaprilat. This treatment regimen reduces ANG II levels in the perfusate blood to undetectable levels and decreases renal ANG II levels by ~60% (unpublished observations). Thus, in contrast to in vivo micropuncture studies in which glomerular dynamics in HF rats are studied while the rats are under the influence of an activated renin-angiotensin system (36), the present study was performed under conditions characterized by reduced endogenous ANG II levels. This situation has obvious advantages in allowing assessment of vasoconstrictor responses to exogenous ANG II but does not provide a reliable reflection of basal arteriolar tone, which would be evident in vivo. However, in vivo micropuncture studies indicate that ACE inhibition in HF rats returns afferent and efferent arteriolar resistances to values not different from normal (17). This situation is consistent with similar arteriolar baseline diameters observed in enalaprilat-treated kidneys from sham-operated and HF rats in the present study.

Accumulating evidence indicates that a sufficient quantity of endogenously synthesized NO is tonically available in vivo to affect pre- and postglomerular arteriolar resistance in the rat kidney (31). The impact of NOS inhibition on in vitro blood-perfused juxtamedullary arteriolar diameter and vascular responsiveness has also been well characterized. The potent and specific NOS inhibitor L-NNA (20) blocks the juxtamedullary afferent arteriolar dilator response to acetylcholine while maintaining responsiveness to sodium nitroprusside (33). Moreover, this agent elicits concentration-dependent diameter reductions in juxtamedullary afferent and efferent arterioles (18, 33), indicating that endogenous NO exerts a vasodilator impact on baseline tone under our experimental conditions. Accordingly, the NO-dependent component of baseline tone can be quantified in our experimental setting by assessing the influence of NO synthesis inhibition on arteriolar caliber. This approach was exploited in the present study to examine the role of NO in determining renal arteriolar tone in HF. These experiments employed a concentration of L-NNA (100 µM) that is maximally effective in decreasing baseline diameters of both afferent and efferent arterioles studied using the in vitro blood-perfused juxtamedullary nephron technique (33). The data reveal attenuated afferent and efferent arteriolar constrictor responses to L-NNA in rats with HF, indicative of a diminished tonic vasodilator impact of NO on the renal microvasculature in HF. The impact of NOS inhibition on the peripheral vasculature has been variably reported to be increased, normal, or decreased in experimental HF as well as in patients with HF (3, 10, 11, 25, 28). These disparities likely reflect functional heterogeneity between vascular beds, as well as the different models or stages of HF (low- vs. high-output and compensated vs. decompensated) or the specific experimental approach employed (in vitro vs. in vivo, conscious vs. anesthetized animals, etc.). To our knowledge, only one previous report has assessed renal vasoconstrictor responses to NOS inhibition in HF, with the results failing to reveal significant alterations in the response to L-NMMA 8 wk after myocardial infarction in the conscious rat (10). In contrast, results of the present study indicate an attenuated juxtamedullary arteriolar contractile response to L-NNA in the same model of HF. This discrepancy likely reflects the fact that the present study was conducted in the absence of the potentially complicating systemic and neural factors that are retained in conscious animal studies. However, we cannot discount the possibility that the tonic vasodilator impact of NO on the juxtamedullary nephron microvasculature is preferentially suppressed in HF, with the mid- and superficial cortical nephron populations relatively unaffected.

In addition to its impact on basal tone within the kidney, NO acts as a physiological antagonist of ANG II. Numerous studies have revealed that NOS inhibition augments renal vasoconstrictor responses to ANG II (31). Accordingly, juxtamedullary microvascular contractile responses to exogenous ANG II are exaggerated during treatment with 100 µM L-NNA (18), indicating that endogenous NO blunts ANG II-induced constriction of afferent and efferent arterioles. In the present study, this phenomenon was evident in kidneys from sham-operated rats. If the renal microvascular impact of NO is abated during HF, one would predict that ANG II-induced vasoconstriction would be unaffected by NOS inhibition in rats with HF. Indeed, in both afferent and efferent arterioles from HF rats, L-NNA treatment failed to enhance ANG II-induced vasoconstriction. Thus, in addition to the diminished impact of NO on basal arteriolar tone, NO modulation of agonist-induced vasoconstriction is abated in kidneys from rats with HF.

We recently reported that third-order arterioles of spinotrapezius muscle exhibit exaggerated responses to both ANG II and vasopressin in HF rats and that these responses did not exhibit normal sensitivity to L-NMMA (7). Thus, in two vascular beds (kidney and skeletal muscle) known to be targets of compensatory vasoconstriction during HF, diminished NO modulation of agonist-induced vasoconstriction may contribute to the increase in vascular resistance. Mild HF activates the intrarenal renin-angiotensin system, whereas severe HF increases circulating levels of both ANG II and vasopressin (14, 36). Because NO normally tempers vasoconstrictor responses to both of these agents in kidney and skeletal muscle (7, 18), the functional consequences of elevated ANG II and vasopressin levels in HF are likely magnified by the diminished modulatory impact of NO.

Before L-NNA treatment, ANG II responsiveness was not significantly exaggerated in HF rats compared with sham-operated controls, although this tendency was evident in both afferent and efferent arterioles. This observation at first seems somewhat surprising given the loss of the tempering influence of NO in HF rats. However, renal ANG II receptors may be downregulated because of chronic elevations in endogenous ANG II levels, downstream signaling events may be altered by the HF state, or activation or other compensatory mechanisms (e.g., vasodilator prostaglandins) may counter ANG II-induced vasoconstrictor responses in kidneys from HF rats. In addition, kinin accumulation during ACE inhibition may also temper vasoconstrictor responses in HF kidneys under our in vitro experimental conditions. Further studies are necessary to discern the extent to which these phenomena represent determinants of renal arteriolar ANG II responsiveness in HF.

To determine whether the reduced renal microvascular impact of NO reflects an alteration in NO production, urinary NOx excretion and renal cortical NOS activity were measured in a separate group of rats not subjected to acute enalaprilat treatment. The results indicate that both NOx excretion and renal cortical NOS activity are reduced 8 wk after myocardial infarction in the rat compared with sham-operated controls. These observations suggest that a reduction in renal NO synthesis occurs in this low-output model of HF. The decline in cortical NOS activity was evident in the presence of excess exogenous substrate (L-arginine) and relevant cofactors, suggesting that alterations in NOS protein levels may underlie the reduction in enzyme activity. Although the NOS assay employed in the present study provided exogenous Ca2+ and calmodulin, no specific attempt was made to distinguish specific involvement of constitutive (Ca2+ dependent) vs. inducible (Ca2+ independent) NOS isoforms. However, because activity of the inducible NOS isoform is not prominent in normal kidney, the reduction in measured NOS activity during HF likely reflects changes in the activity of constitutive NOS isoforms. Both endothelial and neuronal isoforms are normally present in the renal cortex, and the NO produced by both isoforms has been implicated in the control of juxtamedullary arteriolar resistance (16); hence, decreased activity of either isoform could be responsible for the functional alterations detected in the renal microvasculature of HF rats. Renal expression of specific NOS isoforms has not yet been assessed in the postmyocardial infarction model of HF in the rat. However, marked reductions in endothelial NOS protein, mRNA expression, and NO3 production have been reported in thoracic aortas from dogs with pacing-induced HF (37). In contrast, rats with aortocaval fistula display normal baseline and agonist-stimulated NOx excretion as well as normal or increased renal expression of endothelial NOS (1, 2), indicating that reduced renal NO synthesis does not underlie impaired NO-dependent vasodilator responsiveness in this high-output HF model. These discrepant observations raise the possibility that the different hemodynamic status of high- and low-output models of HF results in disparate effects on renal NOS activity. The ability of acute changes in arterial pressure to alter urinary NOx excretion in the rat, even in the absence of apparent changes in hormonal and neural factors (38), lends credence to this as-yet-unexplored postulate. Nevertheless, the results of the present study support the contention that reduced NOS activity contributes to a decline in NO-dependent regulation of the renal microvasculature 8 wk after myocardial infarction in the rat.

Because HF represents a state of oxidative stress (27), reduced ambient NO levels in HF may also result from superoxide anion accumulation. In spinotrapezius muscle of rats with HF, NO-dependent vasodilation can partially be restored by acute treatment with SOD (9), implicating exaggerated NO degradation by superoxide anion. The results of the present study reveal a decline in renal cortical SOD activity in rats with HF. Although both NOS and SOD activities were diminished in the renal cortex of HF rats, HF appeared to provoke an imbalance between NO synthesis (~60% decrease in NOS activity) and tissue antioxidant capability (~40% decrease in SOD activity). The substantial reduction in NOS activity can be expected to decrease ambient NO levels in HF; however, this situation would be exacerbated by diminished SOD activity because accumulating superoxide anions should further diminish NO levels through the rapid formation of peroxynitrite. Thus the present data are consistent with the postulate that both decreased NO synthesis and (perhaps to a lesser extent) accelerated NO degradation may curtail the renal microvascular impact of endogenous NO in rats with experimental HF.

In summary, experiments were performed to explore the impact of NO on basal and ANG II-dependent regulation of the renal microvasculature in rats studied 8 wk after induction of HF by coronary artery ligation. The data reveal attenuated afferent and efferent arteriolar vasoconstrictor responses to NOS inhibition in HF rats, indicating that the basal impact of endogenous NO on the renal microvasculature is reduced under these conditions. NOS and SOD activities in renal cortical homogenates were reduced in HF, suggesting that both decreased NO production and its accelerated breakdown may contribute to a decline in ambient NO levels. Moreover, ANG II-induced arteriolar vasoconstrictor responses were exaggerated by NOS inhibition in kidneys from sham-operated rats but were unaffected by NOS inhibition in rats with HF. Thus the normal modulatory impact of NO on ANG II-induced vasoconstriction is not evident in HF. In light of the fact that NO acts as an important physiological antagonist of ANG II within the kidney, reduced intrarenal NO levels can be expected to exacerbate the functional consequences of an activated renin-angiotensin system in HF.

    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical assistance of Rachel W. Fallet, Angela Werth, and Phyllis Anding.

    FOOTNOTES

Merck, Sharp and Dohme Research Laboratories (Rahway, NJ) provided enalaprilat for use in these studies.

This work was supported by the Univ. of Nebraska College of Medicine and a Grant-In-Aid from the American Heart Association (96006840). The Nebraska Affiliate of the American Heart Association provided fellowship support for H. Ikenaga (postdoctoral, 9504603S) and S. Didion (predoctoral, 9504027S and 9604016S).

Present addresses: H. Ikenaga, Third Department of Internal Medicine, Otawara Redcross Hospital, 2-7-3 Sumiyoshi-cho, Otawara, Tochigi 324, Japan; N. Ishii, Dept. of Clinical Chemistry, Kitasato University School of Allied Health Sciences, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan; S. P. Didion, Cardiovascular Center, Dept. of Internal Medicine, Univ. of Iowa, 2000 Medical Laboratories, Iowa City, IA 52242; K. Zhang, Dept. of Pharmacology, Univ. of Texas Health Sci. Center, 7703 Floyd Curl Dr., San Antonio, TX 78284.

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. §1734 solely to indicate this fact.

Address for reprint requests: P. K. Carmines, Dept. of Physiology & Biophysics, Univ. of Nebraska College of Medicine, 984575 Nebraska Medical Center, Omaha, NE 68198-4575.

Received 30 January 1998; accepted in final form 1 October 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Abassi, Z., K. Gurbanov, I. Rubinstein, O. S. Better, A. Hoffman, and J. Winaver. Regulation of intrarenal blood flow in experimental heart failure: role of endothelin and nitric oxide. Am. J. Physiol. 274 (Renal Physiol. 43): F766-F774, 1998[Abstract/Free Full Text].

2.   Abassi, Z. A., K. Gurbanov, S. E. Mulroney, C. Potlog, T. J. Opgenorth, A. Hoffman, A. Haramati, and J. Winaver. Impaired nitric oxide-mediated renal vasodilation in rats with experimental heart failure: role of angiotensin II. Circulation 96: 3655-3664, 1997[Abstract/Free Full Text].

3.   Baggia, S., K. Perkins, and B. Greenberg. Endothelium-dependent relaxation is not uniformly impaired in chronic heart failure. J. Cardiovasc. Pharmacol. 29: 389-396, 1997[Medline].

4.   Carmines, P. K. Segment-specific effect of chloride channel blockade on rat renal arteriolar contractile responses to angiotensin II. Am. J. Hypertens. 8: 90-94, 1995[Medline].

5.   Carmines, P. K., and L. G. Navar. Disparate effects of Ca channel blockade on afferent and efferent arteriolar responses to ANG II. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F1015-F1020, 1989[Abstract/Free Full Text].

6.   Casellas, D., and L. G. Navar. In vitro perfusion of juxtamedullary nephrons in rats. Am. J. Physiol. 246 (Renal Fluid Electrolyte Physiol. 15): F349-F358, 1984[Abstract/Free Full Text].

7.   Didion, S. P., P. K. Carmines, H. Ikenaga, and W. G. Mayhan. Enhanced constrictor responses of skeletal muscle arterioles during chronic myocardial infarction. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H1502-H1508, 1997[Abstract/Free Full Text].

8.   Didion, S. P., and W. G. Mayhan. Effect of chronic myocardial infarction on in vivo reactivity of skeletal muscle arterioles. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H2403-H2408, 1997[Abstract/Free Full Text].

9.   Didion, S. P., and W. G. Mayhan. Impaired endothelium-dependent responses of skeletal muscle arterioles during chronic heart failure: role of oxygen radicals (Abstract). FASEB J. 11: A44, 1997.

10.   Drexler, H., E. Hablawetz, W. Lu, U. Riede, and A. Christes. Effects of inhibition of nitric oxide formation on regional blood flow in experimental myocardial infarction. Circulation 86: 255-262, 1992[Abstract].

11.   Drexler, H., D. Hayoz, T. Munzel, B. Hornig, H. Just, H. R. Brunner, and R. Zelis. Endothelial function in chronic congestive heart failure. Am. J. Cardiol. 15: 1596-1601, 1992.

12.   Elkayam, U., G. Cohen, H. Gogia, A. Mehra, J. V. Johnson, and P. A. N. Chandraratna. Renal vasodilatory effect of endothelial stimulation in patients with chronic congestive heart failure. J. Am. Coll. Cardiol. 28: 176-182, 1996[Medline].

13.   Fletcher, P. J., J. M. Pfeffer, M. A. Pfeffer, and E. Braunwald. Left ventricular diastolic pressure-volume relations in rats with healed myocardial infarction: effect on systolic function. Circ. Res. 49: 618-626, 1981[Abstract].

14.   Goldsmith, S. R., G. S. Francis, A. W. Cowley, Jr., T. B. Levine, and J. N. Cohn. Increased plasma arginine vasopressin levels in patients with congestive heart failure. J. Am. Coll. Cardiol. 1: 1385-1390, 1983[Medline].

15.   Habib, F., D. Dutka, D. Crossman, C. M. Oakley, and J. G. F. Cleland. Enhanced basal nitric oxide production in heart failure: Another failed counter-regulatory vasodilator mechanism. Lancet 344: 371-373, 1994[Medline].

16.   Ichihara, A., E. W. Inscho, J. D. Imig, and L. G. Navar. Neuronal nitric oxide synthase modulates rat renal microvascular function. Am. J. Physiol. 274 (Renal Physiol. 43): F516-F524, 1998[Abstract/Free Full Text].

17.  Ichikawa, I., T. Yoshioka, A. Fogo, and V. Kon. Role of angiotensin II in altered glomerular hemodynamics in congestive heart failure. Kidney Int. 38, Suppl. 30: S123-S126, 1990.

18.   Ikenaga, H., R. W. Fallet, and P. K. Carmines. Basal nitric oxide production curtails arteriolar vasoconstrictor responses to ANG II in rat kidney. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F365-F373, 1996[Abstract/Free Full Text].

19.   Inscho, E. W., K. Ohishi, and L. G. Navar. Effects of ATP on the pre- and postglomerular juxtamedullary microvasculature. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F886-F893, 1992[Abstract/Free Full Text].

20.   Ishii, K., B. Chang, J. F. Kerwin, Z.-J. Huang, and F. Murad. Nomega -nitro-L-arginine: a potent inhibitor of endothelium-derived relaxing factor formation. Eur. J. Pharmacol. 176: 219-223, 1990[Medline].

21.   Ito, S., J. Amin, Y. L. Ren, S. Arima, K. Abe, and O. A. Carretero. Heterogeneity of angiotensin action in renal circulation. Kidney Int. 52: S128-S131, 1997.

22.   Ito, S., S. Arima, Y. L. Ren, L. A. Juncos, and O. A. Carretero. Endothelium-derived relaxing factors/nitric oxide modulates angiotensin II action on the isolated perfused rabbit afferent but not efferent arteriole. J. Clin. Invest. 91: 2012-2019, 1993[Medline].

23.   Johns, T. N. P., and B. J. Olson. Experimental myocardial infarction. I. A method of coronary occlusion in small animals. Ann. Surg. 140: 675-682, 1954.

24.   Kiuchi, K., N. Sato, R. P. Shannon, D. E. Vatner, K. Morgan, and S. F. Vatner. Depressed beta -adrenergic receptor- and endothelium-mediated vasodilation in conscious dogs with heart failure. Circ. Res. 73: 1013-1023, 1993[Abstract].

25.   Kubo, S. H., T. S. Rector, A. J. Bank, L. Raij, M. D. Kraemer, P. Tadros, M. Beardslee, and M. D. Garr. Lack of contribution of nitric oxide to basal vasomotor tone in heart failure. Am. J. Cardiol. 74: 1133-1136, 1994[Medline].

26.   Kubo, S. H., T. S. Rector, A. J. Bank, R. E. Williams, and S. M. Heifetz. Endothelium-dependent vasodilation is attenuated in patients with heart failure. Circ. Res. 84: 1589-1596, 1991.

27.   McMurray, J., M. Chopra, I. Abdullah, W. E. Smith, and H. J. Dargie. Evidence of oxidative stress in chronic heart failure in humans. Eur. Heart J. 14: 1493-1498, 1993[Abstract].

28.   Mohri, M., K. Egashira, T. Tagawa, T. Kuga, H. Tagawa, Y. Harasawa, H. Shimokawa, and A. Takeshita. Basal release of nitric oxide is decreased in the coronary circulation in patients with heart failure. Hypertension 30: 50-56, 1997[Abstract/Free Full Text].

29.   Moridani, B. A., and R. L. Kline. Effect of endogenous L-arginine on the measurement of nitric oxide synthase activity in the rat kidney. Can. J. Physiol. Pharmacol. 74: 1210-1214, 1996[Medline].

30.   Murakami, K., K. Tsuchiya, M. Naruse, K. Naruse, H. Demura, J. Arai, and H. Nihei. Nitric oxide synthase I immunoreactivity in the macula densa of the kidney is angiotensin II dependent. Kidney Int. 52: S208-S210, 1997.

31.   Navar, L. G., E. W. Inscho, D. S. A. Majid, J. D. Imig, L. M. Harrison-Bernard, and K. D. Mitchell. Paracrine regulation of the renal microcirculation. Physiol. Rev. 76: 425-536, 1996[Abstract/Free Full Text].

32.   Numabe, A., T. Nishikimi, K. Komatsu, and E. D. Frohlich. Intrarenal hemodynamics in low- and high-output cardiac failure in rats. Am. J. Med. Sci. 308: 331-337, 1994[Medline].

33.   Ohishi, K., P. K. Carmines, E. W. Inscho, and L. G. Navar. EDRF-angiotensin II interactions in rat juxtamedullary afferent and efferent arterioles. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F900-F906, 1992[Abstract/Free Full Text].

34.   O'Murchu, B., V. M. Miller, M. A. Perrella, and J. C. Burnett, Jr. Increased production of nitric oxide in coronary arteries during congestive heart failure. J. Clin. Invest. 93: 165-171, 1994[Medline].

35.   Roman, R. J., P. K. Carmines, R. Loutzenhiser, and J. D. Conger. Direct studies on the control of the renal microcirculation. J. Am. Soc. Nephrol. 2: 136-149, 1991[Abstract].

36.   Schunkert, H., S.-S. Tang, S. E. Litwin, D. Diamant, G. Riegger, V. J. Dzau, and J. R. Ingelfinger. Regulation of intrarenal and circulating renin-angiotensin systems in severe heart failure in the rat. Cardiovasc. Res. 27: 731-735, 1993[Medline].

37.   Smith, C. J., D. Sun, C. Hoegler, B. S. Roth, X. Zhang, G. Zhao, X. B. Xu, Y. Kobari, K. Pritchard, Jr., W. C. Sessa, and T. H. Hintze. Reduced gene expression of vascular endothelial NO synthase and cyclooxygenase-1 in heart failure. Circ. Res. 78: 58-64, 1996[Abstract/Free Full Text].

38.   Suzuki, H., H. Ikenaga, K. Hishikawa, T. Nakaki, R. Kato, and T. Saruta. Increases in NO-2/NO-3 excretion in the urine as an indicator of the release of endothelium-derived relaxing factor during elevation of blood pressure. Clin. Sci. (Colch.) 82: 631-634, 1992[Medline].

39.   Teerlink, J. R., G. A. Gray, M. Clozel, and J.-P. Clozel. Increased vascular responsiveness to norepinephrine in rats with heart failure is endothelium dependent: dissociation of basal and stimulated nitric oxide release. Circulation 89: 393-401, 1994[Abstract].

40.   Veldkamp, P. J., P. K. Carmines, E. W. Inscho, and L. G. Navar. Direct evaluation of the microvascular actions of ANP in juxtamedullary nephrons. Am. J. Physiol. 254 (Renal Fluid Electrolyte Physiol. 23): F440-F444, 1988[Abstract/Free Full Text].

41.   Winlaw, D. S., G. A. Smythe, A. M. Keogh, C. G. Schyvens, P. M. Spratt, and P. S. Macdonald. Increased nitric oxide production in heart failure. Lancet 344: 373-374, 1994[Medline].


Am J Physiol Renal Physiol 276(1):F79-F87
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society