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1Department of Physiology and Biophysics and Stress Physiology and Research Center, Georgetown University Medical Center, Washington, District of Columbia; and 2Faculty of Medicine, Technion, Haifa, Israel
Submitted 19 April 2007 ; accepted in final form 23 August 2007
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ABSTRACT |
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Y1 receptor; Y2 receptor; renal blood flow; cardiac hypertrophy
Studies have demonstrated that the majority of the cardiovascular responses to NPY administration are due to the activation of peripheral Y1 and Y2 receptors. The Y1 receptor is primarily a vascular receptor located on the smooth muscle cells of arteries and veins (23). Y1 receptor activation is responsible for the vasoconstriction and smooth muscle cell proliferation associated with systemic/local NPY administration (34). Synergistic or additive potentiation of other vasoconstrictors like NE and angiotensin II (ANG II) is also mediated through the Y1 receptor (23, 29). Y2 receptors are primarily found presynaptically on sympathetic, parasympathetic, and sensory nerves (25), and when activated, inhibit neurotransmitter release. Recently, however, Y2 receptors have also been found to function postsynaptically as a main receptor for NPY-mediated angiogenesis in peripheral ischemic conditions (13) and in tumors (10).
There are several reports linking NPY to CHF. Millar et al. (20) demonstrated that NPY stimulates hypertrophy of cardiomyocytes. In contrast, studies done by Allen et al. (1) showed beneficial effects of NPY on the kidneys; a diuretic and natriuretic action, even in the face of reduced renal perfusion. This finding has been supported by others showing that NPY administration decreases plasma renin activity in high-renin conditions like CHF and some forms of hypertension (22, 26, 31). Taken as a whole, these studies suggest that instead of potentiating the effects of vasoconstrictive agents like ANG II and NE, NPY may be acting to modulate excessive vasoconstriction and sodium retention seen in CHF, possibly through the Y2 receptor.
The objective of this study was to elucidate the changes in the NPY system in heart failure using an experimental model of high-output CHF. We examined the changes in plasma NPY, as well as the concentration of NPY in several different sympathetically innervated tissues during the course of CHF. We also determined the expression of the Y1 and Y2 receptor genes, and whether this pattern of expression might account for differences in cardiac and renal NPY function in heart failure.
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METHODS |
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30 s) with a pair of mini-clamps. At the midpoint between the two clamps, an 18-gauge needle was inserted through the wall of the aorta, advanced into the vena cava through the common wall between the vessels, and manipulated from side to side to widen the opening between the aorta and vena cava. The needle was then withdrawn, the aortic puncture was sealed with cyanoacrylate glue, clamps were removed, and abdominal incisions were closed. After the surgical procedure was completed (10 min), the rats were given 0.2 ml of 40,000 U/ml penicillin and placed under a heat lamp until they recovered. The rats were then placed in metabolic cages to measure sodium balance for the remainder of the study. An age- and weight-matched group of rats underwent sham operations (the same procedure except for the needle puncture) and served as controls. All protocols were approved by the Animal Care and Use Committees of Georgetown University and in accordance with National Institutes of Health (NIH) guidelines. Chronic metabolic balance studies. Rats housed in metabolic cages had their food intake, water intake, urine output, and fecal weight measured daily. Sodium intake was calculated by multiplying the concentration of the electrolytes in the rat chow by the total amount of food eaten per day. Urinary sodium was determined by flame photometer (IL-943, Instrumentation Labs). Daily fluid balance was determined by subtracting the daily urine volume from the amount of water the rats drank per day. Animals were monitored for 6–8 days to determine the severity of CHF. Rats with A-V fistula, which also demonstrated physical signs of CHF (i.e., lethargy and dyspnea) and had sodium excretion below 0.50 meq/day, were considered to be in decompensated (severe) heart failure. Rats excreting more than 0.50 meq/day of sodium were considered to be in compensated (moderate) heart failure. At the end of the 6- to 8-day period, the three groups were anesthetized, blood samples were taken via aortic puncture for determination of plasma NPY concentration, and a variety of sympathetically innervated organs (heart, spleen, adrenal medulla, kidneys) were removed and weighed for analysis of tissue NPY.
Drug infusion studies. In a separate experimental group, rats underwent either A-V fistula or sham surgery. The rats were then placed in metabolic balance and monitored for 5 days. At the end of the observation period, rats were anesthetized and prepared for acute studies. Rats in severe heart failure did not tolerate acute surgery and thus, the drug infusion studies were only performed on COMP A-V fistula rats. Male control (n = 5) and A-V fistula (n = 15) rats were anesthetized with 100 mg/kg ip inactin and a tube was inserted into the trachea to promote spontaneous breathing. Catheters (PE-50) were placed in the carotid artery (for blood pressure measurement), jugular vein (for infusion of 2% inulin), femoral vein (for saline, NPY, Y1 agonist, or Y2 agonist infusion), and bladder (for urine collection). The renal artery and vein were carefully separated without affecting the renal nerves and a Doppler flow probe (Valpey-Fisher) was placed around the renal artery for renal blood flow (RBF) measurement using a pulsed Doppler flowmeter (Department of Bioengineering, University of Iowa) (35). Inulin (2% in isotonic saline) was infused throughout the study at 2.0% of body wt/h to replace fluid lost due to surgical manipulations and to ensure adequate urine production. Rats were allowed to equilibrate for 60 min before the experiment. Baseline mean arterial pressure (MAP) and RBF were measured and normal saline (through femoral catheter) was infused for 30 min. Following the saline, either NPY (200 or 400 ng/min), Y1 agonist {[Leu31, Pro34]-NPY, 200 or 400 ng/min}, or Y2 agonist [NPY (3–36), 700 or 1,400 µg/min iv] was infused for 30 min. RBF was recorded every 5 min, and average RBF was calculated for the 30-min period with values printed out using the Buxco system (Buxco Biosystem XA version 2.10, Buxco Electronics, Wilmington, NC).
Plasma and tissue processing for determination of NPY immunoreactivity.
Tissue (50 mg) and plasma samples from the sham, compensated (COMP), and decompensated (DECOMP) rats were placed in Eppendorf tubes containing 2 ml of 1 M acetic acid, placed in a 100°C water bath for 20 min, and then sonicated. Ten microliters of the tissue/acetic acid solution were removed from each sample to determine protein concentration and the remainder was spun at 20,000 g for 60 min. The supernatant was removed and placed in (12 x 75 mm) polypropylene tubes while the pellet was washed with 500 µl of 1 M acetic acid and respun for 20 min. The second supernatant was added to the first; the samples were dried in a high-speed vacuum and stored at 4°C until used for NPY radioimmunoassay.
Northern blot analysis. Tissue samples (0.5 g) were homogenized in a guanidine isothiocyanate/β-mercaptoethanol solution and RNA was purified by overnight ultracentrifugation in CsCl at 32,000 rpm at 24°C. The RNA pellet was suspended in a 0.3 M sodium acetate solution, precipitated overnight using cold ethanol, and resuspended in Tris·EDTA (ethylenediaminetetraacetic acid) (TE) buffer.
Ten micrograms of total RNA were separated by electrophoresis on a 1.2% denaturing agarose gel with formaldehyde, transferred to a positively charged nylon membrane by capillary blotting, and fixed by exposure to UV light for 1 min. The membranes were prehybridized in buffer containing 50% formamide, 5x SSC, 5x Denhart's solution, 1% SDS, 100 µg/ml of salmon sperm DNA, and 5% Dextran sulfate at 42°C for 1 h. Hybridization was carried out at 42°C overnight in prehybridization buffer supplemented with cDNA probe labeled with [32P] using Prime-a-Gene labeling kit (Promega). The Y1 receptor probe, provided by Dr. D. Larhammar, was a 1.4-kb XhoI-EcoRI fragment of human Y1 receptor cDNA containing part of the COOH-terminal coding region and 3'-untranslated region (11). The Y2 receptor mRNA was detected using cDNA probe containing the entire reading frame of the human Y2 receptor (24). After hybridization, membranes were washed and exposed to the Kodak X-ray film overnight at –70°C. Northern blots were scanned using a digital camera and density of the bands was quantified using NIH Image software.
Statistical analysis. The SigmaStat and one-way repeated-measures ANOVA with post hoc Dunnett's t-test were used for all statistical analysis (P < 0.05 was considered significant). Data are means ± SE for indicated number of n, and graphed using SigmaStat, Microsoft Excel, GraphPad Prism, and Origin 5.0.
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RESULTS |
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Cardiac hypertrophy. The average total wet heart weight in rats from the sham, COMP, DECOMP, and PFS rat groups is shown in Fig. 1C. Heart weight in the COMP and DECOMP groups was significantly increased compared with either of the control groups (P < 0.01). Total heart weight-to-body weight ratio was used as a marker for relative cardiac hypertrophy, and only DECOMP rats demonstrated a significant increase in heart weight-to-body weight ratio compared with the sham control and COMP groups (P < 0.05; Fig. 1C).
A separate group of COMP animals was used for the acute infusion studies (DECOMP could not survive acute surgery). After the equilibration period, baseline MAP and RBF were measured in sham and A-V fistula (COMP only) groups. Both MAP (Fig. 1D) and RBF (Fig. 1E) were significantly decreased in the compensated A-V fistula rats compared with sham (P < 0.01).
Plasma and tissue NPY. The average immunoreactive NPY plasma concentrations in control and A-V fistula rats are shown in Fig. 2A. Interestingly, plasma NPY was 80% higher in all A-V fistula rats, regardless of severity of CHF, compared with control animals (P < 0.04). This is in sharp contrast to the observed changes in NPY in key tissues, which appear to mirror severity of heart failure.
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The heart and the kidneys exhibited differential responses to heart failure. Figure 2C compares the atrial tissue levels of NPY between sham, COMP, and DECOMP rats. Both groups of rats in heart failure demonstrated significant reductions in atrial NPY concentration compared with sham rats, with the greatest decrease in tissue NPY found in the DECOMP rats (P < 0.05 vs. sham and COMP). In contrast, ventricular and renal NPY content was significantly reduced in DECOMP but not COMP rats, compared with sham (P < 0.05; Fig. 2, D and G). In spleen and adrenal glands from both COMP and DECOMP rats, there were similar reductions in NPY concentration, compared with tissues from sham rats (P < 0.03 vs. sham for both tissues; Fig. 2, E and F). There was no difference in NPY concentration in the renal arteries among the three groups (Fig. 2H).
Changes in NPY-Y1 receptor gene expression. Figure 3 shows the changes in the mRNA concentration of the NPY-Y1 receptor in the hearts and kidneys of A-V fistula rats. Given the relatively low yield from RNA isolation, whole heart tissue was processed for mRNA analysis, rather than separate processing for the atria and ventricles. NPY-Y1 receptor gene expression was reduced >50% in the hearts of rats with A-V fistula compared with the sham controls (P < 0.05; Fig. 3A). Similar reductions in Y1 receptor mRNA were seen in the kidney tissue of rats with A-V fistula (P < 0.05 vs. sham; Fig. 3B).
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The effects of NPY on RBF are depicted in Fig. 5C. NPY significantly reduced RBF in both sham (P < 0.001 vs. saline) and the A-V fistula group (P < 0.001 vs. saline).
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DISCUSSION |
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The A-V fistula model of CHF exhibits cardiomegaly, depressed MAP and RBF, and produces both moderate (COMP, compensate and excrete sodium) and severe (DECOMP, retain sodium and expire) disease. The high-output CHF model used in this set of experiments revealed the changes in the NPY system, similar to those reported in human studies (15).
Our analysis began with a characterization of this model of heart failure. As expected, two forms of disease were demonstrated, differentiated by the level of urinary Na+ excretion (Fig. 1A). In general, rats subjected to an A-V fistula demonstrated an increase in total heart weight, a decrease in MAP and RBF (Fig. 1, C–E), an increase in plasma NPY concentrations, and a decrease in peptide levels in almost all major sympathetically innervated organs (Fig. 2). This, most likely, reflected a marked overall increase in sympathetic activity, known to accompany CHF (17). As hypothesized with heart failure, the discrepancy in NPY levels in plasma and tissue may be attributed to the potential exhaustion in NPY-producing capabilities in each organ, which may have released their NPY into the systemic circulation as a result of sympathetic hyperactivity (18). This resulted in a high plasma NPY levels compared with decreased NPY concentrations in all organs including adrenal glands.
The present studies confirmed and extended previous reports by demonstrating a differential pattern of expression of NPY and its major receptors, Y1 and Y2, which may have important implications in the progression of CHF. Interestingly, only the DECOMP group displayed a decrease in ventricular NPY levels (Fig. 2D), consistent with what was reported for CHF patients (2, 15, 16): this was also observed in the kidney (Fig. 2G). In addition, our study demonstrated a negative association between cardiac NPY concentration and the severity of heart failure (Fig. 2, C and D). Unlike previously reported observations, however, our studies included an examination of renal NPY content in healthy and CHF animals.
With regard to the kidney, high renin levels have been associated with the development of severe CHF. Interestingly, only rats in severe heart failure demonstrated a reduction in kidney NPY concentration (Fig. 2G). Since renal NPY depletion, like that in the heart, may indicate excessive NPY release from hyperactive sympathetic nerves, these data suggest that renal NPY may contribute to the severity of heart failure in these rats. However, previous studies showed NPY to reduce the release of renin in high-renin states like CHF (22, 31). Furthermore, Winaver et al. (30) showed that rats in severe heart failure display markedly increased plasma renin-angiotensin levels, while rats in moderate heart failure do not. Thus we hypothesize that while increased release of NPY in the kidneys may be beneficial, rats that exhausted their renal stores of NPY are unable to inhibit renin release, allowing for increased ANG II to further impair cardiovascular function.
In addition to determining the NPY content in different tissues, this study also examined the mRNA expression of NPY-Y1 and NPY-Y2 receptors in the hearts and kidneys of rats with moderate and severe heart failure. mRNA of both the Y1 and Y2 receptor was altered in rats with CHF. Y1 mRNA was significantly reduced in both the hearts and kidneys of COMP and DECOMP rats, dependent on severity of CHF (Fig. 3). This was similar to what is known to occur with β1-adrenergic receptor mRNA in patients with CHF (3), suggesting that both may reflect receptor downregulation due to enhanced release of sympathetic neurotransmitters during CHF-induced sympathetic activation. Conversely, the expression of Y2 receptor mRNA was markedly increased in the heart and kidney of rats with CHF (Fig. 4). This suggests that while the effects of Y1 receptors are attenuated in both the heart and the kidney, at the same time, the effects of Y2 receptors are enhanced. Subsequently, we investigated whether these changes in receptor mRNA expression were associated with alterations in kidney function.
In a separate group of CHF rats, we studied the renal blood responses to specific NPY-Y1 and -Y2 receptor agonists. Animals that underwent an A-V fistula, whose basal RBF was lower than in control rats, exhibited an attenuated response to the RBF-lowering effects of Y1 agonist, [Leu31, Pro34]-NPY (Fig. 5A), presumably due to a downregulation of Y1 receptors. In contrast, the Y2 agonist (NPY3–36) tended to increase RBF (Fig. 5B) in CHF as well as in sham, suggesting functional Y2 receptors.
The differential changes in Y1 and Y2 receptor expression may be an important counterregulatory response to limit the excessive vasoconstriction seen in CHF by 1) a reduction in Y1 receptor-mediated vasoconstriction, direct and indirect via other vasoconstrictors such as NE or ANG II, 2) enhancement of the angiogenic effects of NPY via postsynaptic endothelial Y2 receptors (36) in peripheral and coronary vasculatures, and 3) an anti-adrenergic effect of NPY via presynaptic Y2 receptors to reduce NE release and hence, limit its deleterious activity in CHF. Although the expression of mRNA and protein level can be used as preliminary indicators of NPY receptor's functionality in CHF, further studies are needed to investigate NPY signaling pathways and functional consequences in CHF.
It is interesting to note that NPY itself decreased RBF in CHF kidney (Fig. 5C), even in the presence of increased Y2 receptor expression. A likely explanation for this is the involvement of another receptor type, the Y5 receptor. As reported by our group (14, 23), the Y5 receptor, which is induced by tissue ischemia and injury (12), has amplifying effects on Y1 receptor-mediated mitogenic effects of the peptide (23). Vascular hypertrophy has previously been shown to increase responsiveness of the vessels to vasoconstrictors via purely structural reasons (Folkov's theory) (5). Therefore, when NPY, a nonselective Y1, Y2, and Y5 agonist, was infused, it might have been still effective in reducing RBF in CHF kidney by utilizing its Y5 receptors, despite the significant decrease in Y1 receptor expression. Thus, an overall effect of NPY in the renal circulation in CHF may be a net effect of enhanced vasoconstriction, vascular hypertrophy, and increased blood flow via Y2 receptors, either due to vasodilation and increased angiogenesis.
An alternative or additional possibility is that the changes in NPY receptor populations in this model of CHF are also associated with alterations in NPY metabolizing enzymes, specifically dipeptidyl peptidase IV (DPPIV). It has been shown that NPY (3–36), the end product following cleavage of NPY by DPPIV, is an endogenous Y2/Y5 receptor-preferring ligand (8). If this enzyme is upregulated along with the Y2 receptors, as we previously showed in other ischemic states (13), then subjects with CHF would have an innate mechanism for controlling excessive NE and renin release. Overall, characterizing the role of DPPIV and other NPY receptors in CHF would be useful to determine NPY's role in CHF.
The heart failure-induced differential regulation of the NPY system suggests that NPY is an important neurovascular modulator in the development of CHF-related pathology. Further studies are needed to determine the factors contributing to whether NPY acts to compensate or exacerbate the development of the disease; as it is capable of deteriorating the conditions with excessive vasoconstriction and cardiac hypertrophy via Y1 receptors and enhancing the cardiovascular compensatory mechanisms, such as angiogenesis via Y2 receptors.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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