Rat Atrial Natriuretic Peptide Research Articles

Effects of elevation of ANP and its deficiency on cardiorenal function.

Atrial natriuretic peptide (ANP), encoded by Nppa, is a vasodilatory hormone that promotes salt excretion. Genome-wide association studies identified Nppa as a causative factor of blood pressure development, and in humans, ANP levels were suggested as an indicator of salt sensitivity. This study aimed to provide insights into the effects of ANP on cardiorenal function in salt-sensitive hypertension. To address this question, hypertension was induced in SSNPPA–/– (KO of Nppa in the Dahl salt-sensitive [SS] rat background) or SSWT (WT Dahl SS) rats by a high-salt (HS) diet challenge (4% NaCl for 21 days). Chronic infusion of ANP in SSWT rats attenuated the increase in blood pressure and cardiorenal damage. Overall, the SSNPPA–/– strain demonstrated higher blood pressure and intensified cardiac fibrosis (with no changes in ejection fraction) compared with SSWT rats. Furthermore, SSNPPA–/– rats exhibited kidney hypertrophy and higher glomerular injury scores, reduced diuresis, and lower sodium and chloride excretion than SSWT when fed a HS diet. Additionally, the activity of epithelial Na+ channel (ENaC) was found to be increased in the collecting ducts of the SSNPPA–/– rats. Taken together, these data show promise for the therapeutic benefits of ANP and ANP-increasing drugs for treating salt-sensitive hypertension.

Abstract MP06: Insights Into The Role Of Anp In Hfpef During Ss Hypertension

Background: Atrial natriuretic peptide ( ANP) is a hormone produced primarily in the heart, which induces vasodilation and natriuresis. We showed previously that a knockout of ANP in Dahl SS rat results in hypertension and HFpEF phenotype. There is a gap in knowledge regarding the role of inflammation and mitochondrial function in HFpEF that accompanies SSH. We hypothesize that the ANP deficiency in SSH leads to pressure overload, which affects cardiac inflammation and mitochondrial function. Methods: At 8 weeks of age, male wild type (SS WT ) and Nppa knockout (ANP knockout, SS Nppa-/- ) Dahl SS rats were placed on normal salt ( NS , 0.4% NaCl) or a high salt ( HS , 4% NaCl) for 21 days. Tissues were harvested for histological and Western analysis. TEM was used to obtain electron micrographs. EPR spectroscopy was performed in snap-frozen tissues post injection of POBN. 2-way ANOVA was employed for statistical comparison. Results: Heart-to-body-weight ratio was higher in SS Nppa-/- rats (p<0.001), on NS and HS diet ( p <0.001). SS Nppa-/- rats displayed increases in cardiac fibrosis following HS diet ( p <0.01). EPR demonstrated that SS Nppa-/- hearts exhibit a trend for increased lipid peroxidation compared to SS WT . Cardiac cytokine profile demonstrated an increase in IL-1α, IL-1β, L-6, IL-17, TNF-α, sICAM-1, MIP-1α, MIP-3α, in the SS WT rats fed a HS diet vs NS diet, and lower levels of these in the HS-diet fed SS Nppa-/- rats vs SS WT . HS diet was sufficient to induce increases in mitochondrial area ( p <0.001), and decreases in density ( p 0.001), and overall health score ( p <0.001) in both SS WT and SS Nppa-/- rats, but no genotype-specific differences were reported. We observed changes in fission/fusion proteins: Opa1 expression was elevated in SS Nppa-/- vs SS WT ( p <0.01), and Parkin expression was lower in all rats on a HS diet ( p <0.05). Conclusions: Although SS Nppa-/- rats develop a HFpEF phenotype, exacerbated cardiac fibrosis and higher lipid peroxidation vs SS WT rats, inflammation and overall mitochondrial damage are diminished in the hearts of these animals at day 21 of the HS diet. Further studies are needed to elucidate the mechanisms by which inflammation may affect the development of HFpEF during SS hypertension in a cell-specific and time-dependent manner.

BMP-2 alleviates heart failure with type 2 diabetes mellitus and doxorubicin-induced AC16 cell injury by inhibiting NLRP3 inflammasome-mediated pyroptosis.

Chronic heart failure (CHF) and diabetes mellitus are associated with morbidity and mortality. CHF and diabetes generally simultaneously occur, resulting in adverse outcomes. Diabetes complicates cardiomyopathy and exacerbates heart failure conditions. An increase in natriuretic peptides, including atrial natriuretic peptide (ANP), and another endsogenously generated peptide, brain natriuretic peptide (BNP), serves an essential role in CHF. The aim of this study was to explore the molecular regulation between bone morphogenetic protein-2 (BMP-2) and ANP or BNP in diabetes-associated cardiomyopathy. In total, 25 serum samples were collected from patients with CHF with or without type 2 diabetes mellitus to compare with 25 controls. Cardiomyopathy and hyperglycemia were induced in rats by doxorubicin and streptozotocin, respectively. AC16 cells were used to study molecular mechanisms. BMP, ANP and BNP concentration in patients and rats were measured by ELISA. Flow cytometry was performed to analyze cell pyroptosis and ROS production. Reverse transcription-quantitative PCR and western blotting were used to examine mRNA and protein expression of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), pro-caspase-1, caspase-1 (p20) and gasdermin D. BMP-2 was negatively correlated with ANP and BNP in CHF patients with type 2 diabetes mellitus. Similar results were obtained in rats and AC16 cells. BMP-2 decreased the NLRP3 inflammasome activation and cell pyroptosis. The present study found evidence that the cardioprotective effects of BMP-2 act through ANP and BNP both in vivo and in vitro. BMP-2 inhibits inflammasome formation. The results suggested that BMP-2 may serve as a novel therapeutic target for the treatment of diabetic heart conditions.

Effect of endostatin overexpression on angiotensin II-induced cardiac hypertrophy in rats.

Background:Endostatin, a biologically active fragment of collagen XVIII, has been observed in patients with ischemic heart disease. The aim of the present study was to investigate whether endostatin overexpression could attenuate cardiac hypertrophy by inhibiting the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling pathway.Methods:This study was examined in vivo in rats and in vitro in primary neonatal rat cardiomyocytes treated with angiotensin (Ang) II to model cardiac hypertrophy. Twenty-four male Sprague-Dawley rats were randomized into adenovirus (Ad)-green fluorescent protein, Ang II, Ad-endostatin, and Ang II + Ad-endostatin groups (n = 6 in each group). Four weeks later, all the rats were weighed and sacrificed after transthoracic echocardiography. Cardiac function was evaluated by transthoracic echocardiography, cardiomyocyte size was evaluated by hematoxylin-eosin staining. Levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were evaluated by quantitative reverse-transcription polymerase chain reaction or Western blotting, PKA level was evaluated by Western blotting, and cAMP level was evaluated by enzyme-linked immunosorbent assay. Statistical significance among multiple groups was evaluated by one-way analysis of variance.Results:Endostatin overexpression reduced the increases in left ventricle (LV) mass (P = 0.0063), LV mass/body weight (BW) (P = 0.0013), interventricular septal thickness (IVS) in diastole (P = 0.0013), IVS in systole (P = 0.0056), left ventricular posterior wall thickness (LVPW) in diastole (P = 0.0291), LVPW in systole (P = 0.0080), heart weight (HW) (P = 0.0138), HW/BW (P = 0.0001), and HW/tibial length (P = 0.0372) in Ang II-treated rats. In addition, endostatin overexpression reduced cardiomyocyte cross-sectional area expansion, and reduced the levels of ANP and BNP in Ang II-treated rats (P = 0.0251 and 0.0477 for messenger RNA [mRNA]), and primary neonatal rat cardiomyocytes (P = 0.0188 and P = 0.0024 for mRNA; P = 0.0023 and 0.0013 for protein, respectively). Additionally, endostatin overexpression reduced the increase of cAMP (P = 0.0054) and PKA (P = 0.0328) levels in cardiomyocytes treated with Ang II. Treatment with cAMP reversed the effects of endostatin overexpression on ANP (P = 0.0263) and BNP (P = 0.0322) levels in cardiomyocytes induced by Ang II.Conclusion:Endostatin overexpression could alleviate cardiac hypertrophy by inhibiting the cAMP-PKA signaling pathway.

Rosiglitazone treatment restores renal responsiveness to atrial natriuretic peptide in rats with congestive heart failure.

The thiazolidinedione (TZD) class of Peroxisome proliferator‐activated receptor gamma agonists has restricted clinical use for diabetes mellitus due to fluid retention and potential cardiovascular risks. These side effects are attributed in part to direct salt‐retaining effect of TZDs at the renal collecting duct. A recent study from our group revealed that prolonged rosiglitazone (RGZ) treatment caused no Na+/H2O retention or up‐regulation of Na+ transport‐linked channels/transporters in experimental congestive heart failure (CHF) induced by surgical aorto‐caval fistula (ACF). The present study examines the effects of RGZ on renal and cardiac responses to atrial natriuretic peptide (ANP), Acetylcholine (Ach) and S‐Nitroso‐N‐acetylpenicillamine (SNAP‐NO donor). Furthermore, we assessed the impact of RGZ on gene expression related to the ANP signalling pathway in animals with ACF. Rats subjected to ACF (or sham) were treated with either RGZ (30 mg/kg/day) or vehicle for 4 weeks. Cardiac chambers pressures and volumes were assessed invasively via Miller catheter. Kidney excretory and renal hemodynamic in response to ANP, Ach and SNAP were examined. Renal clearance along with cyclic guanosine monophosphate (cGMP), gene expression of renal CHF‐related genes and ANP signalling in the kidney were determined. RGZ‐treated CHF rats exhibited significant improvement in the natriuretic responses to ANP infusion. This ‘sensitization’ to ANP was not associated with increases in neither urinary cGMP nor in vitro cGMP production. However, RGZ caused down‐regulation of several genes in the renal cortex (Ace, Nos3 and Npr1) and up‐regulation of ACE2, Agtrla, Mme and Cftr along down‐regulation of Avpr2, Npr1,2, Nos3 and Pde3 in the medulla. In conclusion, CHF+RGZ rats exhibited significant enhancement in the natriuretic responses to ANP infusion, which are known to be blunted in CHF. This ‘sensitization’ to ANP is independent of cGMP signalling, yet may involve post‐cGMP signalling target genes such as ACE2, CFTR and V2 receptor. The possibility that TZD treatment in uncomplicated CHF may be less detrimental than thought before deserves additional investigations.

Gallic acid increases atrial natriuretic peptide secretion and mechanical dynamics through activation of PKC

AimsGallic acid (GA) protects against myocardial ischemia-reperfusion (I/R) injury, prevents cardiac hypertrophy and fibrosis, and has anti-inflammatory activity in the heart. However, its effects in regulating atrial natriuretic peptide (ANP) secretion are unknown. The aim of this study was to determine the function of GA in regulating ANP secretion and atrial dynamics in rat atria. Key findingsGA (0.01, 0.05, and 0.1μmol/L) significantly increased atrial ANP secretion and induced positive inotropy dose-dependently. GA (0.1μmol/L) also increased plasma level of ANP and hemodynamics in rats. These effects were accompanied by upregulation of atrial protein kinase C subtypes β and ε (PKCβ and PKCε), which was completely blocked by LY333531 and EAVSLKPT, antagonists of protein PKCβ and PKCε, respectively. GA-induced ANP secretion was also attenuated by Gö6983 but not rottlerin, antagonists of PKCα and PKCδ, and the positive inotropy was reversed by Gö6983. U-73122, a phospholipase C (PLC) antagonist, mitigated the effects of GA on ANP secretion and mechanical dynamics and downregulated Phospho-PLCβ at Ser537 (pPLCβ S537), Phospho-PLCβ at Ser1105 (pPLCβ S1105), PKCβ and PKCε levels, whereas KN62, an inhibitor of Ca2+/calmodulin-dependent kinase II, was not modified the GA-induced ANP secretion and suppressed GA-induced mechanical dynamics. SignificanceGA promotes ANP secretion and effects positive inotropy with regard to mechanical dynamics through the activation of PLC-PKC signaling in rat atria.

The role of Atrial Natriuretic Peptide in the development of renal and cardiac damage in a Dahl SS rat model

Atrial natriuretic peptide (ANP), encoded by the Nppa gene, is an osmoregulatory hormone that promotes salt excretion. A GWAS identified ANP as a causative factor of blood pressure development, and in humans ANP levels are suggested as an indicator of salt‐sensitivity. Recently, Flister et al. (Genome Res, 2013) generated a knockout of Nppa in the Dahl Salt‐Sensitive (SS) rat background (further referred to as the SSNPPA−/− rat). The aim of the current study was to provide mechanistic insights into the effects of ANP deficiency on renal and cardiac function in the Dahl SS rats.In this study hypertension was induced in SSNPPA−/− and control Dahl SS rats by placing them on a high salt diet (HS, 4% NaCl) for 21 days. A combination of in vivo techniques (blood pressure monitoring and GFR measurements) with ex vivo imaging, electrophysiological, and biochemical methods were used to test the role of ANP deficiency in the development of SS hypertension. Immunohistochemical analysis was employed to assess renal and cardiac damage.Chronic i.v. infusion of ANP in wild type SS rats (100 ng/kg/day) attenuated the salt‐induced increase in blood pressure in the wild type SS rats, whereas SSNPPA−/− rats demonstrated a significantly higher mean arterial pressure compared to SS controls when the rats were fed a HS diet: on day 21 of diet MAP was 185.8 ± 9 mmHg in SSNPPA−/− rats compared to 144.6 ± 4 mmHg in SS controls. Additionally, SSNPPA−/− rats showed exacerbated kidney damage as revealed by intensified kidney hypertrophy, higher glomerular injury scores, and increased renal arterial hypertrophy. GFR was found to be reduced in the control SS rats fed a HS diet compared to LS diet, however significance was not reached in the SSNPPA−/− group. SSNPPA−/− rats did exhibit reduced diuresis, as well as a trend for lower sodium and chloride excretion when fed a HS diet. The activity of Epithelial Sodium Channel (an important contributor to the development of SS hypertension) was found to be increased in the cortical collecting ducts of the SSNPPA−/− rats. Additionally, SSNPPA−/− rats fed a HS diet showed intensified cardiac damage compared to SS controls, as demonstrated by higher heart to body weight ratio, and a dramatically elevated interstitial fibrosis. However, ultrasound echocardiography revealed that although SSNPPA−/− rats have cardiac remodeling, they do not exhibit heart failure.Therefore, ANP is major regulator in the development of SS hypertension, and its deficiency leads to more severe cardiac and kidney damage. Further work will be devoted to discovering specific ANP‐associated molecular pathways that mediate its effects in SS hypertension, and assessing the therapeutic benefits of ANP for treating cardiovascular diseases.Support or Funding InformationMCW RAC Pilot Grant (DVI) and NIH HL10880 (AS)

Effect of acupuncture on the genetic expression of myocardial endothelin-1 and atrial natriuretic peptide in rats with stress-induced prehypertension

ObjectiveTo explore the protective effect of acupuncture against myocardial injury in rats with stress-induced prehypertension (SIPH) by observing the genetic expression of myocardial endothelin-1 (ET-1) and atrial natriuretic peptide (ANP). MethodsThirty-six Wistar rats were randomized into three groups: the control group, model group, and model + acupuncture (AP) group (n = 12 rats per group). During the 11-day modeling period, the model group and the model + AP group experienced plantar electric stimulation in combination with noise exposure, and daily acupuncture intervention was applied simultaneously in the model + AP group; the control group did not experience modeling or acupuncture. Systolic pressure (SP) was measured the day before modeling began, and on the 3rd, 5th, 7th, 9th, and 11th day after modeling initiation. On the 12th day, histopathological observation of the left ventricle was made with hematoxylin-eosin staining and quantitative genetic expression of myocardial ET-1, and ANP was tested by quantitative real-time polymerase chain reaction (PCR). ResultsSP was higher in the model group than the control group on the 3rd, 5th, 7th, 9th, and 11th days (all P < .01). SP in the model + AP group was lower than that in the control group on the 5th and 7th days (respectively, P = .008, P = .002) and on the 9th and 11th days (P = .029, P = .039). Hematoxylin-eosin staining showed normal myocardial cellular structure in the control group. The model group showed disordered arrangement of cardiac cells with morphological changes in some nuclei, including enlargement or dissolution; there was also infiltration of inflammatory cells and proliferation of collagen fibers. In the model + AP group, most of the myocardial cells were normally structured, and only part of the cells had morphological changes with enlarged nuclei or dissolution. Real-time PCR showed that expression of ET-1 and ANP mRNA in the model group was greater than the control group (respectively, P = .024, P = .000101). The model + AP group had lower expression of ET-1 and ANP mRNA compared with the model group (respectively, P = .033, P = .043). ConclusionAcupuncture may lower blood pressure and downregulate the genetic expression of myocardial ET-1 and ANP in SIPH rats, suggesting a protective effect of acupuncture against myocardial damage.

Direct chemiluminescent enzyme immunoassay for atrial natriuretic peptide in mammalian plasma using a PEGylated antibody

Atrial natriuretic peptide (ANP) is a peptide hormone that is synthesized and secreted by cardiac tissues and plays a pivotal role in maintaining cardiovascular homeostasis. Clinically, ANP is used as a marker of cardiovascular diseases, including heart failure. Although multiple ANP assays are currently available, a more sensitive assay is required for the direct measurement of plasma ANP where there is limited plasma availability, especially in mouse experiments. In the current study, we developed a plate-based sandwich chemiluminescent enzyme immunoassay for the measurement of plasma ANP in rats and mice without the need for prior extraction. To minimize nonspecific binding, we performed a single-step PEGylation procedure targeting the immobilized antibody, which markedly improved the assay’s sensitivity and linearity. The linear range was 0.1 to 250pM, and the minimum detection limit was 0.13pM, 5-fold lower than the lowest value of the commercially available kits. ANP was directly measured in plasma samples without detectable cross-reactivity with B- and C-type natriuretic peptides. The accuracy of the assay was confirmed by spike recovery tests and dilution tests and by comparison with a conventional radioimmunoassay. Based on the species cross-reactivity, this assay can be used to measure human ANP.

Restoration of Renal Responsiveness to Atrial Natriuretic Peptide in Experimental Nephrotic Syndrome by Albumin Infusion

Background: The natriuretic/diuretic response to atrial natriuretic peptide (ANP), an important regulator of water and Na⁺ balance, is markedly attenuated in nephrotic syndrome (NS). It has been suggested that the diminished renal responsiveness to ANP may contribute to the pathogenesis of salt retention and edema formation in NS. However, the mechanisms underlying the renal hyporesponsiveness to ANP remain largely unknown. Methods: The acute effects of exogenous infusion of ANP (5 µg/kg + 10 µg/kg/h) were studied by clearance methodology in control rats, hypoalbuminemic rats with Adriamycin (ADR)-induced NS and in ADR-treated rats infused with hyperoncotic albumin sufficient to restore plasma albumin to normal levels. Results: Administration of ANP to control rats resulted in a significant increase in urinary flow rate, absolute rate of sodium excretion (+456%) and glomerular filtration rate (GFR). Mean arterial blood pressure decreased following infusion of the peptide. In the nephrotic rats, baseline GFR and Na⁺ excretion were significantly lower than in the control animals, and the renal effects of ANP were markedly blunt compared to the control animals. In contrast, the hypotensive effect of ANP in the ADR-treated rats was largely preserved. Infusion of hyperoncotic albumin prior to ANP administration reversed the decrease in baseline GFR and Na⁺ excretion and completely restored the renal effects of ANP in the nephrotic rats. Conclusion: These findings indicate that renal hyporesponsiveness to ANP in rats with ADR-induced NS is a reversible phenomenon that appears to be of functional origin rather than reflecting permanent cellular damage.

Concerted Action of ANP and Dopamine D1-Receptor to Regulate Sodium Homeostasis in Nephrotic Syndrome

The edema formation in nephrotic syndrome (NS) is associated with a blunted response to atrial natriuretic peptide (ANP). The natriuretic effects of ANP have been related to renal dopamine D1-receptors (D1R). We examined the interaction between ANP and renal D1R in rats with puromycin aminonucleoside-induced NS (PAN-NS). Urinary sodium, cyclic guanosine monophosphate (cGMP) excretion, and D1R protein expression and localization in renal tubules were evaluated in PAN-NS and control rats before and during volume expansion (VE). The effects of zaprinast (phosphodiesterase type 5 inhibitor), alone or in combination with Sch-23390 (D1R antagonist), were examined in both groups. The increased natriuresis and urinary cGMP excretion evoked by acute VE were blunted in PAN-NS despite increased levels of circulating ANP. This was accompanied in PAN-NS by a marked decrease of D1R expression in the renal tubules. Infusion of zaprinast in PAN-NS resulted in increased urinary excretion of cGMP and sodium to similar levels of control rats and increased expression of D1R in the plasma membrane of renal tubular cells. Combined administration of Sch-23390 and zaprinast prevented natriuresis and increased cGMP excretion induced by zaprinast alone. We conclude that D1R may play a major role in the ANP resistance observed in PAN-NS.

Synthesis of atrial natriuretic peptide in the rabbit inner ear

Atrial natriuretic peptide (ANP) exhibits natriuretic, diuretic, and vasorelaxant activity, maintaining fluid and electrolyte homeostasis. Although ANP is mainly synthesized and secreted in/from atrium, ANP has been found in various tissues including the rat inner ear. The aim of this study was to identify the synthesis of ANP in lateral cochlear wall tissues and the presence of ANP in perilymph using rabbits. In vivo study using a rabbit model. Expression of ANP in the rabbit inner ear tissue and the presence of perilymph were examined by radioimmunoassay and polymerase chain reaction using an ANP polyclonal antibody and rat ANP primers, respectively. Characteristics of ANP and pro-ANP present in the inner ear were also evaluated by high-performance liquid chromatography (HPLC), comparing to rat cardiac ANP and pro-ANP. Immunoreactive ANP (ir-ANP) was found in the perilymph and lateral cochlear wall tissue of rabbits. The levels of ir-ANP in the perilymph were 5 to 16 ng/mL. Elusion profiles of HPLC showed two main peaks that were exactly matched with rat cardiac ANP and pro-ANP. Expression of ANP mRNA was also detected. Our findings suggest that ANP and its precursor protein are synthesized in the ear tissues and secreted to perilymph. This hormone may play a role in control of water and/or ion homeostasis of the fluids in the ear that are responsible for normal hearing.

Rosiglitazone improves the natriuretic response to atrial natriuretic peptide in rats with experimental congestive heart failure: possible involvement of a post-guanylate cyclase mechanism

Background Congestive heart failure (CHF) in patients and experimental animals is characterized by renal retention of salt and water and a blunted natriuretic/diuretic response to atrial natriuretic peptide (ANP). Recently, we reported that chronic treatment with the PPARgagonist rosiglitazone (RGZ) improved the natriuretic and diuretic responses to extracellular fluid volume expansion in rats with aorto-caval fistula, an experimental model of volume-overload CHF [1]. In the present study we explored whether RGZ improves also the natriuretic/diuretic response to ANP in rats with experimental CHF. In addition, we evaluated the effects of the drug on ANP-mediated cGMP signalling in the kidney.

Glucocorticoids Improve Renal Responsiveness to Atrial Natriuretic Peptide by Up-Regulating Natriuretic Peptide Receptor-A Expression in the Renal Inner Medullary Collecting Duct in Decompensated Heart Failure

In heart failure, the renal responsiveness to exogenous and endogenous atrial natriuretic peptide (ANP) is blunted. The mechanisms of renal hyporesponsiveness to ANP are complex, but one potential mechanism is decreased expression of natriuretic peptide receptor-A (NPR-A) in inner medullary collecting duct (IMCD) cells. Newly emerging evidence shows that glucocorticoids could produce potent diuresis and natriuresis in patients with heart failure, but the precise mechanism is unclear. In the present study, we found dexamethasone (Dex) dramatically increased the expression of NPR-A in IMCD cells in vitro. The NPR-A overexpression induced by Dex presented in a time- and dose-dependent manner, which emerged after 12 h and peaked after 48 h. The cultured IMCD cells were then stimulated with exogenous rat ANP. Consistent with the findings with NPR-A expression, Dex greatly increased cGMP (the second messenger for the ANP) generation in IMCD cells, which presented in a time- and dose-dependent manner as well. In rats with decompensated heart failure, Dex dramatically increased NPR-A expression in inner renal medulla, which was accompanied by a remarkable increase in renal cGMP generation, urine flow rate, and renal sodium excretion. It is noteworthy that Dex dramatically lowered plasma ANP, cGMP levels, and left ventricular end diastolic pressure. These favorable effects induced by Dex were glucocorticoid receptor (GR)-mediated and abolished by the GR antagonist 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU486). Collectively, glucocorticoids could improve renal responsiveness to ANP by up-regulating NPR-A expression in the IMCD and induce a potent diuretic action in rats with decompensated heart failure.

Caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion

Caveolae may act as mechanosensors and function as binding sites for calcium ions. The intracaveolar localization of atrial natriuretic peptide (ANP) derived from the direct interaction of atrial granules with caveolae has been demonstrated. The aim of this study was to define the effect of caveolae on ANP secretion induced by stretch and angiotensin II. The isolated perfused beating atria from Sprague-Dawley rats were used. To disrupt caveolae, 10mM methyl-β-cyclodextrin (MbCD) was applied for 1h and the number of caveoli were markedly decreased. MbCD increased basal ANP secretion and atrial diastolic pressure. The molecular profile of ANP in perfusate from control atria showed mainly one major peak corresponded to synthetic ANP whereas that from MbCD-treated atria showed two major immunoreactive peaks corresponded to synthetic rat ANP and proANP. High atrial stretch induced by elevating the height of outflow catheter from 5cmH2O to 7.5cmH2O increased atrial contractility and ANP secretion. The response of ANP secretion to high stretch was attenuated in MbCD-pretreated atria. Pretreatment with MbCD abolished angiotensin II-induced suppression and losartan-induced stimulation of ANP secretion. However, the effect of angiotenisin (1-7) on ANP secretion was not altered by MbCD treatment. The expression of angiotensin II type 1 receptor protein was reduced by MbCD treatment. These data suggest that caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion and relate to the processing of proANP.

Effects of urotensin II on hypoxia‐induced ANP secretion in rats

Urotensin II (UII) is a vasoactive peptide with many potent effects in the cardiovascular system through a G‐protein‐coupled receptor termed UT. The synthesis and secretion of atrial natriuretic peptide (ANP) are increased during hypoxic condition and plays an important role in adaptation to hypoxia and in the pathogenesis of cardiopulmonary diseases. The present study was to investigate the effects of human UII (hUII) on ANP secretion in hypoxia condition in vitro and in vivo experiments. Hypoxia caused an increase in ANP secretion compared to normoxia in isolated perfused beating rat atria. hUII (10‐10 M) decreased hypoxia‐induced ANP secretion whereas hUII receptor antagonist, urantide (10‐6 M), increased it. However, there were no significant changes in atrial contractility, stroke volume, and extracellular fluid (ECF) translocation by hUII in hypoxia condition. The pretreatment with urantide attenuated the effect of hUII on hypoxia‐induced ANP secretion. Monocrotaline‐treated (MCT, 60mg/kg, sc., 4weeks) rats increased UII level in plasma. In vivo experiment, hUII (5*10‐6 M injection plus 2.5*10‐6 M infusion for 15 min) was infused into MCT‐treated and control rats anesthetized with ketamine. hUII increased blood pressure and heart rate in both groups without significant difference. Interestingly, intravenous infusion of hUII decreased plasma concentration of ANP in MCT‐treated rats whereas hUII increased plasma concentration of ANP in control rats. The ANP generation may protect against the hypoxia and pulmonary hypertension. These results suggest that hUII may play a pathophysiological role in the regulation of ANP secretion in hypoxic condition. Supported by the KOSEF (R13‐2008‐005‐00000‐0).

Circadian Rhythms and Effects of Anesthesia on Plasma Natriuretic Peptide Levels in Rats

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are circulating hormones secreted predominantly in patients with hypertension or congestive heart failure. To obtain background data on plasma ANP and BNP levels in rats, we investigated the circadian rhythms and effects of anesthesia on these peptides. To determine the circadian rhythms, plasma samples from thirty rats were collected by non-anesthesia (decapitation) at six time points every fourth hour. To determine the effects of anesthesia, plasma samples from thirty-two rats were collected under diethyl ether, pentobarbital or urethane anesthesia. The plasma ANP and BNP levels were determined using a radioimmunoassay. The plasma ANP levels were high from the evening to early morning, while the plasma BNP levels were relatively low at 2:30 AM. The difference in the BNP levels was statistically significant. The plasma BNP levels were relatively high when the rats were anesthetized using urethane. These results suggest that blood collection should be performed between 10:30 AM to 2:30 PM to determine plasma ANP and BNP. The use of pentobarbital is also recommended for toxicological studies in rats.

Atrial natriuretic peptide (ANP) in preeclampsia-like syndrome in a rat model.

The pathophysiology of preeclampsia has not been fully clarified. A variety of factors have been implicated with this disease including vasoactive peptides and hormones during the last 20 years. Inadequate generation of atrial natriuretic peptide (ANP) has been one of the mechanisms discussed as to possibly contribute to the development of hypertension. In human pregnancy multiple studies of ANP-plasma-concentration in normal or hypertensive pregnancies showed conflicting results. The complexity of the clinical findings of hypertension in pregnancy makes it very difficult to carry out comparative clinical and biochemical studies in humans. In an animal experience genetic as environmental influences could be excluded. Therefore, the present study shows an experimental preeclampsia-like syndrome in the rat by reduction of the utero-placental flow. We observed a significant increase of plasma ANP in pregnant rats with experimentally induced hypertension. Furthermore, our results suggest that the ventricles could be an important source of ANP gene expression.

Increased renal expression of nitric oxide synthase and atrial natriuretic peptide in rats with glycyrrhizic‐acid‐induced hypertension

The present study aimed to examine whether there is an altered regulation of local hormonal systems in the kidney following the treatment of glycyrrhizic acid (GA), the active ingredient in licorice. Male Sprague-Dawley rats were treated with GA for 3 weeks. The expression of mineralocorticoid receptor (MR) was determined in the kidney by immunoblotting and real-time polymerase chain reaction (PCR). The protein expression of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) was determined. The expression of atrial natriuretic peptide (ANP), natriuretic peptide receptor (NPR)-A and NPR-C was determined by real-time PCR. The activity of guanylyl cyclase was determined by the amount of cGMP generated in responses to sodium nitroprusside (SNP) or ANP. Following the GA treatment, systolic blood pressure was increased. The mRNA and protein expressions of MR were increased in the kidney. The protein expression of eNOS and iNOS was also increased. The expression of ANP mRNA was increased although that of NPR-A and NPR-C mRNA was not changed. The cGMP production provoked by either SNP or ANP was not changed. The increased expression of MR may contribute to GA-induced hypertension. The enhanced expression of NOS and ANP may play a compensatory role in GA-induced hypertension.

Natriuretic peptide binding sites in the gills of the pouched lamprey Geotria australis

Iodinated atrial natriuretic peptide (ANP) binding sites were examined in the gills and ventral aorta of the adult upstream-migrating lamprey Geotria australis using tissue section autoradiography, in vitro competition analysis and affinity cross-linking, while guanylate cyclase assays were performed on gill membranes of both adult and juvenile lampreys. A partial natriuretic peptide (NP) receptor sequence was amplified using reverse transcription/polymerase chain reaction (RT-PCR). The results indicated that there was specific NP binding to the aortic endothelium and to pillar cell regions in the axial plate and secondary lamellae. In competition studies, 50 % of NP binding was abolished by 4 nmol l-1 rat ANP, 35 nmol l-1 porcine C-type NP (CNP) and 45 nmol l-1 C-ANF (a truncated ANP). Affinity cross-linking followed by SDS-PAGE demonstrated two binding sites at 205 and 65 kDa under non-reducing conditions and at 85 and 65 kDa under reducing conditions. Guanylate cyclase assays demonstrated that, while no NP-stimulated GC activity occurred in adult lampreys, NP-stimulated enhancement of cyclic GMP accumulation was found in juveniles in fresh water and more particularly in salt water. RT-PCR amplified a 471 base pair fragment with 68 % amino acid sequence homology to the eel natriuretic peptide receptor D (NPR-D). This study suggests that NP binding sites in the adult gill and aorta are of an NPR-C/D type, whereas an additional GC-coupled site exists in juveniles.