Plasma levels of human atrial natriuretic peptide in patients with hypertensive diseases

Three types of antihuman atrial natriuretic peptide antiserum were obtained. From the study of cross-reactivity to human atrial natriuretic peptide fragments, it was suggested that antisera-1, -2, and -3 are mostly specific to 1-28, 5-25, and the ring structure, respectively. The estimated values of this hormone were significantly lower in the order of antisera-1, -2, and -3. Moreover, high performance liquid chromatographic study showed that various types of fragments of atrial natriuretic peptide exist in human plasma. These findings suggested that the highly specific antiserum to 1-28 human atrial natriuretic peptide such as antiserum-1 should be used to estimate the 1-28 human atrial natriuretic peptide levels in human plasma. From the study by using antiserum-1, it was concluded that the plasma human atrial natriuretic peptide increased in essential hypertensives, and in patients with primary aldosteronism, chronic renal failure, and malignant hypertension. Regarding the pathophysiological significance of increased plasma atrial natriuretic peptide, it is unlikely that this plays an important role in the etiology of essential hypertension or other hypertensive diseases, because the plasma level of this hormone is elevated in these patients. The increase of plasma atrial natriuretic peptide level in these patients should be considered to be a secondary or compensatory reaction to high blood pressure.     

Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.     

Superactive analogs of the atrial natriuretic peptide (ANP)

Three analogs of the atrial natriuretic peptide ANP(105-126), lacking the N-terminal exocyclic peptide segment and containing 2-mercaptoacetic acid, 3-mercaptopropionic acid or 4-mercaptobutyric acid in place of the cysteine residue in position 105 of the peptide sequence, were synthesized by the solid-phase method. The resulting des-amino analogs showed 2 to 4 times higher diuretic/natriuretic activity than the most active natural ANP and displayed a potent hypotensive effect as well. All three analogs were relatively less potent in various in vitro bioassays and in a binding assay, indicating that their high activities in vivo may be due to resistance to enzymatic degradation and to reduced non-specific tissue adsorption. These compounds not only will serve as useful pharmacologic tools but also represent prototypes for the development of further reduced-size ANP analogs.     

Free and bound forms of atrial natriuretic peptide (ANP) in rat plasma: preferential increase of free ANP in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP)

Free and bound forms of atrial natriuretic peptide (ANP) in rat plasma were analysed by gel permeation chromatography combined with a radioimmunoassay (RIA) for rat ANP (rANP). Gel permeation chromatography showed two immunoreactive peaks in rat plasma, one corresponding to alpha-rANP, rANP(99-126), and the other eluted at a high molecular weight, clearly different from gamma-rANP, rANP(1-126). The chromatographic profile of rat plasma after incubation with synthetic alpha-rANP demonstrated that the high molecular immunoreactivity had ANP-binding capacity. This bound form of ANP was almost totally excluded following extraction procedure, therefore, the immunoreactive ANP (ir-ANP) measured with the extraction assay was mainly free ANP. On the other hand, direct RIA may detect not only the free but also the bound form of ANP. Using both direct RIA and the extraction method, bound forms of plasma ANP in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) were compared to normotensive Wistar Kyoto rats (WKY). Bound forms of plasma ANP in 20-week-old SHR and SHRSP were significantly higher than that in age-matched WKY. The ratio of free/bound form of plasma ANP in SHR and SHRSP also significantly increased compared to WKY, indicating a preferential increase in free ANP in the plasma of these hypertensive rats. These findings suggest that a bound form of ANP may be present in rat plasma and that it may play some pathophysiological role in the hypertension of SHR and SHRSP. Increased free ANP in plasma may indicate a compensatory increase in ANP release in these hypertensive rats.     

Heart and plasma atrial natriuretic peptide (ANP) in response to long-term endurance training in rats.

Long-term endurance training effects on heart and plasma ANP were investigated in male Wistar rats. Maximal O2 uptake (VO2max) was significantly higher in trained groups, when they are used as their own control. After 3, 4, and 5 weeks of endurance training, VO2max was respectively increased by 7.7% (p less than 0.05), 13.7% (p less than 0.01), and 18.4% (p less than 0.001). Plasma ANP and glomerular ANP receptor density showed no clear variations in trained rats. However, cardiac ANP content decreased significantly in left and right atrial tissues by 35-36% (p less than 0.05) after 5 weeks of training. ANP immunoreactivity was investigated to show the distribution of ANP within the atria. ANP was found in diffuse and granular forms. The diffuse pattern (immature ANP) disappeared in cardiocytes of trained rats, while the granular form persisted, especially in the left atrial tissue. These data suggest that chronic endurance training might cause a decrease in ANP synthesis with no change in ANP storage. Such results are in agreement with the hypothesis that the left atrium could be especially involved in long-term fluid volume control.     

Secretion of atrial natriuretic peptide (ANP) from fish atrial and ventricular myocytes in tissue culture

Primary cultures of atrial and ventricular myocytes (approx. 1 x 10(5) cells/culture) were prepared from adult teleost fish Gila atraria and maintained for 10 days. Immunoreactive atrial natriuretic peptide (ir-ANP) from fish atrial and ventricular cells was 3.9 and 2.8 ng/culture respectively, values not significantly different. Atriocytes from rat and mouse secreted comparable amounts of ANP which were not significantly different from atrial fish cultures (5.2 and 4.3 ng/culture). In contrast, their ventricular myocytes secreted only small quantities of ANP (0.8 and 0.3 ng/culture). When analyzed by reversed-phase HPLC, the media of both fish atrial and ventricular myocytes contained a peptide which exhibited properties similar to authentic human ANP (Ser 99-Tyr 126), suggesting a significant degree of sequence homology between fish and mammalian ANP. Fish ventricular cells, unlike normal mammalian ventricular cells, secrete substantial quantities of immunoreactive-ANP.     

Locally different role of atrial natriuretic peptide (ANP) in the pericardial fluid

Pericardial fluid (PF) contains several vasoactive agents in higher concentrations than venous plasma (VP). However, with human atrial natriuretic peptide (ANP) controversial data have been reported in earlier studies performed on a limited number of patients (less than 20). The present study was designed to characterize the ANP levels in human PF and cardiac tissues, and to ascertain whether myocardial ischemic state is a major factor in determining ANP production of the human heart. In a total of 316 consecutive patients undergoing open heart surgery ANP levels in VP, PF, atrial and ventricular tissues were measured by radioimmunoassay and analyzed by high-performance liquid chromatography (HPLC). The data are presented as median and 25th-75th percentiles. Our results showed ANP concentration [ANP] of PF significantly exceeded that of VP and [ANP] in the atrial tissue was significantly higher than in the ventricular tissue (p < 0.001). In patients without myocardial ischemia (valvular heart disease) [ANP] in the PF was 258.3 (189.9-342.5) pg/ml, in the VP 28.4 (11.7-57.6) pg/ml and 151.7 (78.4-447.6) ng/mg in the atrial, 0.4 (0.2-1.6) ng/mg in the ventricular tissue. The corresponding values for patients with coronary artery disease were 208.1 (153.8-318.9) pg/ml in the PF, 19.8 (9.4-27.9) pg/ml in the VP, 129.6 (66.5-455.0) ng/mg in the atrial and 1.0 (0.1-1.8) ng/mg in the ventricular tissue. The ventricular tissue levels correlated to the atrial tissue levels (r = 0.317; p < 0.05). Great difference (p < 0.001) was found in the atrial tissue levels between females [414.6 (119.7-734.4) ng/mg] and males [105.4 (65.3-204.2) ng/mg]. In HPLC analysis the majority of the pericardial fluid and tissue ir-ANP coeluted with human ANP [99-126]. In conclusion, [ANP] in PF of cardiosurgical patients is higher by an order of magnitude than in VP. Intrapericardial ANP may reflect the peptide concentration in the myocardial interstitium and may represent a paracrine regulatory mechanism, which seems independent of ANP-induced putative antiischemic influences.     

Autoradiography of atrial natriuretic peptide (ANP) receptors in the rat brain.

Quantitative autoradiography was used to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat brain and to study their regulation. Peptide receptors are selectively located to circumventricular organs outside the blood brain barrier, such as the subfornical organ, and to brain areas involved in fluid and cardiovascular regulation. Dehydration, either by water deprivation of normal rats, or chronic dehydration present in homozygous Brattleboro rats lacking vasopressin, results in large increases in ANP binding in receptor number in the subfornical organ. In the deoxycorticosterone acetate (DOCA)-salt hypertensive model, only salt treatment, but not DOCA alone or the combination of DOCA-salt, increased the ANP receptor number in the subfornical organ and the choroid plexus. Both young and adult genetically hypertensive rats have a greatly decreased ANP receptor number in the subfornical organ and the choroid plexus. Selective displacement with an inactive analog lacking the disulfide bond (ANP 111-126) suggests that genetically hypertensive rats may lack C (clearance) atrial natriuretic peptide receptors. Our results implicate brain atrial natriuretic peptide receptors in the central response to alterations in fluid regulation and blood pressure.     

Receptors for atrial natriuretic peptide (ANP) and regulation of thyroglobulin secretion by ANP in human thyroid cells

Specific binding sites for atrial natriuretic peptide (ANP) were identified and characterized in primary cultures of human thyroid cells. Saturation analysis using [125I] alpha rat ANP as the ligand showed a single class of high affinity binding (Kd = 0.2 nM) which was inhibited by atriopeptin I and the alpha -human form of ANP, but not by a C-terminal fragment of the peptide. The number of ANP binding sites in these cultures was not altered by the thyroid hormone concentration of the medium. In a dose-response experiment, thyro-globulin secretion was significantly reduced in the presence of 0.01 nM ANP and was maximally reduced (to 25% of control value) with 10 nM ANP. Cyclic GMP production was increased threefold in the presence of 100 nM ANP, but was unchanged with lower doses (0.01 and 0.1 nM) of the peptide. The finding of receptors in thyroid follicular cells suggests a hitherto unrecognized role of ANP in the thyroid gland.     

The decline of atrial natriuretic peptide (ANP) gene expression in older rats and the effects of ginsenoside on ANP gene expression.

1. The levels of atrial natriuretic peptide(ANP) gene expression in rat atria at 2-3 and 14-18 months of age and the effects of ginsenosides on r-ANP gene expression by determining the concentration of ANP-mRNA were investigated. The male and female rats were abdominally (i.p.) injected with aqueous solution of ginsenosides prepared from ginseng stems and leaves (G-PSL) and ginseng roots (G-PR), 50 mg/kg body wt, once a day for 7 days. Atria total RNA was extracted by the cold phenol method. The ANP-mRNA contents were determined using the Northern blot and dot blot hybridization technique with alpha-32*P-labelled r-prepro-ANP-cDNA probe. 2. The ANP-mRNA contents of 14-18 month rats were remarkably less than that of 2-3 month rats. The levels of male and female rats' atria at 14-18 months were about 15 and 60%, the content of male and female rats at 2-3 months respectively. 3. G-PSL and G-PR increased the ANP-mRNA content of male rats at 14-18 months 1- and 2-fold, respectively, whereas G-PSL and G-PR decreased the ANP-mRNA content in male rats of age 2-3 months. 4. These results revealed that the ANP gene expression declined during ontogenic ageing development and ginsenosides possessed anti-ageing effects in the heart endocrineous function aspect.     

Continuos intravenous infusion of atrial natriuretic peptide (ANP) prevented liver fibrosis in rat

The atrial natriuretic peptide (ANP) are used as the acute heart failure treatment in clinical and reported the suppression of fibrosis in the heart, lung recently. The aim of this study was to analyze the suppressive effect of liver fibrosis about ANP. In vitro, rat hepatic stellate cell line (HSC-T6) were treated with ANP. In vivo, Wister rats were injected with dimethylnitrosamine (DMN) twice a week via intra-peritoneal for 4 weeks. ANP group was given by continuance intravenous dosage system used 24 h infusion pump for 3 weeks after 1 week of DMN administration. In vitro, ANP suppressed α-SMA expression and was inhibited the growth of HSC, and reduced the expression of type 1 procollagen, TIMP-1, -2 expression. In vivo, The ANP group showed lower serum AST, ALT, HA level. Liver fibrosis was suppressed by ANP. ANP also decreased gene expression of type 1 procollagen, TIMP-1, -2 and α-SMA, TGF-β1 expression. Our results showed that continuous ANP infusion has the specific capacity of inhibiting HSC activation and protecting hepatocytes and the useful capacity to suppress the liver fibrosis.     

The concentration of atrial natriuretic peptide (ANP) is decreased in plasma but not in atria in hypophysectomised rats

To study the role of the pituitary gland in the release of Atrial Natriuretic Peptide (ANP) plasma and atrial concentrations were measured both in intact and in hypophysectomized rats. The plasma concentration of ANP (pg/ml) was significantly (p less than 0.01) decreased from 143 +/- 35 to 82 +/- 29 (mean +/- SD, n) while the tissue concentration (ng/wet tissue mg) remained unchanged, 192 +/- 46 and 194 +/- 39, respectively. The total atrial amount of ANP (ug) was, however, significantly (p less than 0.01) decreased from 29.7 +/- 7.8 to 17.0 +/- 3.3 after hypophysectomy. In intact animals, a volume load (1.1ml/100 body weight g 0.9% NaCl) resulted in 2-fold (p less than 0.001) increase in the plasma ANP levels whereas similar load had no effects on plasma ANP levels in hypophysectomized animals. In both groups, the right atrial pressure was increased from about 2 to about 6 mmHg. We conclude that in the absence of pituitary gland the right atrial pressure and the atrial ANP concentration do not change but plasma ANP levels and the response to volume stimulus are attenuated.     

A linear analog of atrial natriuretic peptide (ANP) discriminates guanylate cyclase-coupled ANP receptors from non-coupled receptors

Atrial natriuretic peptide (ANP) contains a disulfide which is generally considered to be required for biological activity. A truncated linear ANP analog, des-Cys105,Cys121-ANP-(104-126) (referred to as analog I), that lacks the 2 cysteine residues of the parent peptide was synthesized. In competition binding studies using rabbit lung membranes, ANP-(103-126) and analog I displaced bound 125I-ANP-(103-126) from specific ANP binding sites 100 and 73%, respectively. The concentrations of ANP-(103-126) and analog I that produced 50% inhibition of radioligand binding to the membranes were 0.26 +/- 0.07 and 0.31 +/- 0.09 nM, respectively. Radioiodinated ANP-(103-126) and analog I were chemically cross-linked to binding sites on rabbit lung membranes, and the labeled membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. 125I-Analog I specifically labeled a 65,000-dalton protein and a 135,000-dalton protein which, under reducing conditions, dissociated into 65,000-dalton subunits. In contrast, 125I-ANP-(103-126) labeled specifically a nonreducible 135,000-dalton protein, in addition to the 65,000-dalton species and the reducible 135,000-dalton species. ANP-(103-126) (100 nM) stimulated rabbit lung particulate guanylate cyclase activity, whereas analog I, at the same concentration, had no effect on cyclic GMP production and did not antagonize the effect of ANP-(103-126). From these observations, we conclude that analog I is a selective ligand which binds to approximately 73% of the total ANP binding sites present in rabbit lung membranes. Unlike ANP-(103-126), analog I does not bind to the remaining 27% of the binding sites and does not activate guanylate cyclase. Binding to the cyclase-linked ANP receptor correlates with the specific labeling by 125I-ANP-(103-126) of the nonreducible 135,000-dalton membrane protein.     

A highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine

A highly sensitive radioimmunoassay has been established for measurement of human plasma and urine concentrations of atrial natriuretic peptide (ANP) and requires no extraction or concentration process such as Sep-Pak C-18 cartridge treatment. An antiserum was prepared from rabbits immunized with alpha-human ANP (alpha-hANP) coupled with bovine-thyroglobulin. The sensitivity of this method was 0.3 pg/tube of synthetic alpha-hANP utilized as authentic standard. Recovery of alpha-hANP spiked to plasma and urine was 97.7 +/- 15.4% and 97.1 +/- 9.5% (mean +/- SD), respectively. Plasma and urinary ANP concentrations versus assay data showed satisfactory linearity. In 124 healthy subjects, the plasma ANP-concentration was 31.7 +/- 12.0 pg/ml. Two different molecular forms of ANP in plasma and a single form in urine were found by gel permeation chromatography.     

Plasma atrial natriuretic peptide in conscious rats with reduced renal mass

The effect of salt intake and reduction of renal mass (RRM) on plasma immunoreactive atrial natriuretic peptide (iANP) levels in conscious rats was studied. Rats were divided into RRM and sham-operated groups, and then further subdivided into groups infused with 1 or 6 mEq of sodium per day. Plasma urea nitrogen increased in the groups with RRM. Plasma sodium, sodium balance, and heart rate did not differ between the sham and RRM groups. Rats with RRM maintained on 1 mEq of sodium per day did not have an elevation of water intake, arterial pressure, or plasma iANP. Rats with RRM maintained on 6 mEq of sodium per day had significantly (P less than 0.05) elevated water intake, arterial pressure, and plasma iANP. Arterial pressure and plasma iANP were correlated (r = 0.800) for rats with RRM on either 1 or 6 mEq of sodium per day. Increased plasma iANP in the RRM group on 6 mEq per day was not caused by either RRM or high sodium alone; it was an effect of RRM plus high salt intake. The increase in plasma iANP in the RRM group may be caused by the increase in arterial pressure, possibly due to an increase in extracellular fluid volume. ANP may not be responsible for the sustained increase in fractional sodium excretion observed in RRM.     

Dissociation between plasma and atrial content of atrial natriuretic polypeptide (ANP) following sodium load in rats

To investigate the time course effect of sodium intake on release and synthesis of atrial natriuretic polypeptide (ANP), plasma and atrial content of ANP were measured in rats which had been fed either a high or a low salt diet for 1, 3, 7, 14 and 35 days. Plasma ANP in rats fed the high salt diet for one day was significantly higher than in those fed the low salt diet. However, there were no significant differences between the groups fed either the high or the low salt diet for 3 days or more. In contrast to the direction of change in plasma ANP, atrial content of ANP in rats fed the high salt diet for one day tended to be lower and was significantly lower in those fed for 3 and 7 days than in the low salt diet group, while there were no significant differences between both groups that were fed for 14 and 35 days. These results suggest that ANP is rapidly released into the circulation when sodium is loaded, however, the atrial storage of ANP remains depleted for about one week.     

Evolutionary implication of the absence of atrial natriuretic peptide (ANP) in euryhaline Oryzias fishes

The genus Oryzias contains nearly 20 species, including the Japanese medaka (Oryzias latipes). Because each species exhibits different adaptability to environmental salinity, Oryzias fishes offer unique opportunities for comparative studies. To understand the mechanisms of osmotic adaptation, we are studying the functional evolution of the natriuretic peptide (NP) family??a group of small peptide hormones involved in body fluid regulation??by using Oryzias fishes. Analysis of the Japanese medaka genome revealed that 7 NP subtypes, namely, Atrial NP (ANP), B-type NP (BNP), Ventricular NP (VNP), and 4?C-type NPs (CNP-1 through CNP-4) were generated from a CNP-4-like ancestral gene discovered in the cyclostomes before the ray-finned fish/lobe-finned fish divergence. This evolutionary history has been confirmed by the discovery of hidden NP genes in tetrapods. Through analyses of phylogenetic distribution of NP subtypes, we also found that specific losses of subtypes have occurred in each vertebrate lineage. For example, ANP is absent in the Japanese and Indian medaka and the flying fish, suggesting that loss of the ANP gene occurred after the divergence of Beloniformes from Cyprinodontiformes. This fact also supports the inclusion of Oryzias into Beloniformes as suggested by phylogenetic analysis using whole mitochondrial genome sequences. How Oryzias fishes have retained their euryhalinity with a reduced number of NPs is an interesting question. CNP-3, which is functionally flexible, may be a substitute for the lost cardiac NPs.