Recombinant expression of a secreted form of the atrial natriuretic peptide clearance receptor

A general structure for the atrial natriuretic peptide clearance receptor (ANP C-receptor) has been proposed based on hydropathicity analysis of the deduced amino acid sequence of this membrane protein (Fuller, F., Porter, J.G., Arfsten, A., Miller, J., Schilling, J., Scarborough, R.M., Lewicki, J.A., and Schenk, D.B. (1988) J. Biol. Chem. 263, 9395-9401). The ANP C-receptor is believed to possess a large amino-terminal extracellular domain (436 amino acids), a single hydrophobic transmembrane anchor (23 amino acids), and a short cytoplasmic tail (37 amino acids). As a means of testing the structure and proposed cellular orientation of this protein, we have employed the technique of in vitro mutagenesis to prepare a receptor mutant (anc-) lacking the transmembrane and cytoplasmic domains. Expression of this mutant in mammalian cells using a vaccinia virus vector results in secretion of a truncated soluble form of the ANP C-receptor which binds native ANP and synthetic ANP analogs with a specificity similar to that of the native ANP C-receptor. In contrast to the native ANP C-receptor that exists predominantly as a homodimer on the cell surface, the secreted receptor exists as a monomeric species. The results are consistent with the proposed structure of this receptor with the amino-terminal domain containing the ANP-binding site oriented extracellular to the plasma membrane. In addition, these data demonstrate that the receptor does not require association with the plasma membrane or its native dimeric configuration in order to bind ANP ligands with high affinity and specificity.     

Identification of a biologically active circulating form of rat atrial natriuretic factor

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.     

Small HDL form via apo A-I a complex with atrial natriuretic peptide

The goal of this study was to test the ability of small high density lipoproteins (small HDL) to bind human alpha-atrial natriuretic peptide (alpha-hANP), an amyloidogenic peptide whose involvement in cardiac pathologies is gaining increasing clinical evidence. After incubation of HDL with labeled ANP, the peptide associated to lipoprotein was detectable only in small HDL containing preparations. HDL-associated alpha-[(125)I]hANP was subjected to chromatography, electrophoresis, and autoradiography. The autoradiograph showed two radioactive bands, whose molecular weight was consistent with the chromatographic pattern. Immunoblotting showed the presence of apo A-I in both autoradiographic bands. The proteins of the main band were electroeluted, incubated with labeled ANP, and subjected to two-dimensional electrophoresis followed by autoradiography. The mass spectrometry and molecular weight analyses of the radioactive spot demonstrated the presence of an apo A-I dimer. This finding provided a novel solid evidence that small HDL via apo A-I dimer are involved in the ANP sequestration and thus may play a role in preventing amyloid fibril formation.     

Expression and secretion of biologically active human atrial natriuretic peptide in Saccharomyces cerevisiae

A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor. Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium. The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus. The secreted product was also folded correctly with respect to the single disulfide bond. However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material. The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.     

Release of atrial natriuretic peptide by atrial distension

A heterologous radioimmunoassay was used to measure the concentration of immunoreactive atrial natriuretic peptide (iANP) in plasma from the femoral artery of eight chloralose anaesthetized dogs. Mitral obstruction which increased left atrial pressure by 11 cmH2O increased plasma iANP from 97 +/- 10.3 (mean +/- SE) to 135 +/- 14.3 pg/mL. Pulmonary vein distension increased heart rate but did not increase plasma iANP. Bilateral cervical vagotomy and administration of atenolol (2 mg/kg) did not prevent the increase in iANP with mitral obstruction. Samples of blood from the coronary sinus had plasma iANP significantly higher than simultaneous samples from the femoral artery confirming the cardiac origin of the iANP. Release of iANP depends on direct stretch of the atrium rather than on a reflex involving left atrial receptors.     

血管钠肽、 C型钠尿肽和心房钠尿肽舒血管作用的对比

本实验采用离体血管灌流方法,观察和比较血管钠肽（ＶＮＰ）,Ｃ型钠尿肽（ＣＮＰ）和心房钠尿肽（ＡＮＰ）对大鼠肺动脉,腹主动脉和腹腔静脉的舒张作用。．结果表明,ＶＮＰ,ＣＮＰ和ＡＮＰ对离体大鼠的保留内皮与去内皮的肺动脉,腹主动脉和腹腔静脉均有浓度依赖性舒张作用。     

Brain natriuretic peptide is cosecreted with atrial natriuretic peptide from porcine cardiocytes

Using primary cultures of atrial cardiocytes from neonatal pig, the secretion brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP)-like immunoreactivities (LI) was studied in vitro. Porcine cardiocytes time-dependently secreted both BNP-LI and ANP-LI into medium under a serum-free condition, although the amount of BNP-LI secreted was about one-third that of ANP-LI. Phorbol ester and Ca2+ ionophore had less stimulatory effects on secretion of BNP-LI than that of ANP-LI. Reverse-phase HPLC of the conditioned medium revealed a single major BNP-LI component corresponding to synthetic porcine BNP(1-26). These data suggest that a small molecular weight form BNP, possibly BNP(1-26), is cosecreted with ANP from porcine cardiocytes.     

重组人心房利钠肽 (ANP) 的原核表达、纯化及鉴定

为提高重组人心房利钠肽(Atrial natriuretic peptide,ANP)的表达量,将3个ANP通过赖氨酸(Lysine,K)串联,并构建相对应的重组表达载体p ET28a(+)/ANP3。转染大肠杆菌进行诱导表达,目的蛋白约占菌体总蛋白的60%。经过包涵体变复性,赖氨酸酶(Lys-C)和羧肽酶(CPB)水解,以及一系列层析纯化,每升培养液可获得约16 mg的ANP蛋白。最终,纯化后的ANP经UPLC及Tricine SDS-PAGE鉴定,纯度大于90%,LC-MS鉴定显示其分子量为3 080 Da,且为二硫键正确形成的ANP单体,通过ELISA试剂盒检测,其具有和参比品一致活性。本研究为ANP的大规模制备打下了基础。同时,所采用的串联表达技术也为其他多肽类药物的重组表达提供了新的思路。     

Effects of atrial natriuretic peptide in the gut

The intestinal tract is a target organ for atrial natriuretic peptide (ANP), characterized by various biologic activities, immunoreactivity, as well as specific binding sites for ANP. A review of previous studies reveals that ANP is an important regulator of water and nutrient intake, which acts via multiple signaling pathways including activation of guanylyl cyclase to produce its biologic responses. As a regulator, the peptide locally controls hydrosaline balance and acute systemic effects. Therefore, ANP could also act as a local mediator or paracrine effector of intestinal function.     

Hypoxia reduces atrial natriuretic peptide clearance receptor gene expression in ANP knockout mice

We tested the hypotheses that hypoxic exposure is associated with exacerbated pulmonary hypertension and right ventricular (RV) enlargement, reduced atrial natriuretic peptide (ANP) clearance receptor (NPR-C) expression, and enhanced B-type natriuretic peptide (BNP) expression in the absence of ANP. Male wild-type [ANP(+/+)], heterozygous [ANP(+/-)], and homozygous [ANP(-/-)] mice were studied after a 5-wk hypoxic exposure (10% O(2)). Hypoxia increased RV ANP mRNA and plasma ANP levels only in ANP(+/+) and ANP(+/-) mice. Hypoxia-induced increases in RV pressure were significantly greater in ANP(-/-) than in ANP(+/+) or ANP(+/-) mice (104 +/- 17 vs. 45 +/- 10 and 63 +/- 7%, respectively) as were increases in RV mass (38 +/- 4 vs. 26 +/- 5 and 29 +/- 4%, respectively). NPR-C mRNA levels were greatly reduced in the kidney, lung, and brain by hypoxia in all three genotypes. RV BNP mRNA and lung and kidney cGMP levels were increased in hypoxic mice. These findings indicate that disrupted ANP expression worsens hypoxic pulmonary hypertension and RV enlargement but does not alter hypoxia-induced decreases in NPR-C and suggest that compensatory increases in BNP expression occur in the absence of ANP.     

Evaluation of atrial natriuretic peptide and brain natriuretic peptide in atrial granules of rats with experimental congestive heart failure.

The natriuretic peptides are believed to play an important role in the pathophysiology of congestive heart failure (CHF). We utilized a quantitative cytomorphometric method, using double immunocytochemical labeling, to assess the characteristics of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in atrial granules in an experimental model of rats with CHF induced by aortocaval fistula. Rats with CHF were further divided into decompensated (sodium-retaining) and compensated (sodium-excreting) subgroups and compared with a sham-operated control group. A total of 947 granules in myocytes in the right atrium were analyzed, using electron microscopy and a computerized analysis system. Decompensated CHF was associated with alterations in the modal nature of granule content packing, as depicted by moving bin analysis, and in the granule density of both peptides. In control rats, the mean density of gold particles attached to both peptides was 347.0 +/- 103.6 and 306.3 +/- 89.9 gold particles/microm2 for ANP and BNP, respectively. Similar mean density was revealed in the compensated rats (390.6 +/- 81.0 and 351.3 +/- 62.1 gold particles/microm2 for ANP and BNP, respectively). However, in rats with decompensated CHF, a significant decrease in the mean density of gold particles was observed (141.6 +/- 67.3 and 158.0 +/- 71.2 gold particles/microm2 for ANP and BNP, respectively; p<0.05 compared with compensated rats, for both ANP and BNP). The ANP:BNP ratio did not differ between groups. These findings indicate that the development of decompensated CHF in rats with aortocaval fistula is associated with a marked decrease in the density of both peptides in atrial granules, as well as in alterations in the quantal nature of granule formation. The data further suggest that both peptides, ANP and BNP, may be regulated in the atrium by a common secretory mechanism in CHF.     

High-level expression of alpha-human atrial natriuretic peptide from multiple joined genes in Escherichia coli

A method is described which allows alpha-human atrial natriuretic peptide to be synthesized in stable form and with high yield in Escherichia coli. In the final expression system, eight copies of the synthetic alpha-hANP gene were linked in tandem, separated by codons specifying a 4-amino-acid (aa) linker with lysine residues flanking the authentic N and C termini of the 28-aa hormone. This sequence was in turn joined to the 3' end of a fragment containing the lac promoter and a leader sequence coding for the first seven N-terminal amino acids of beta-galactosidase. The expressed multidomain protein accumulated intracellularly into stable inclusion bodies and was easily purified by urea extraction of the insoluble cell fraction. The purified protein was cleaved into monomers by digestion with endoproteinase Lys-C, trimmed to expose the authentic C terminus by digestion with carboxypeptidase-B and a single disulfide bond was formed by gentle oxidation with potassium ferricyanide. The fully processed recombinant peptide was shown by reverse phase liquid chromatography to be indistinguishable from the chemically synthesized standard alpha-hANP in both the reduced and in the folded form.     

Effect of exercise on gene expression of atrial natriuretic peptide receptor of kidney

To study the effect of exercise on gene expression of natriuretic peptide receptors (NPRs) in the kidney, with in situ hybridization and the computerized image analysis, we investigated the alterations of gene expression of NPRs on the animal model of exercise training of different intensity. We found that after exercise training of different intensity, renal NPR-A mRNA and NPR-C mRNA expression showed different changes, the expression of NPR-A mRNA upregulated and NPR-C mRNA downregulated in the kidney. With the increase exercise intensity, change in NPR-A mRNA expression was insignificant, but downregulation in NPR-C mRNA expression was more significant. The result suggested that the effect of exercise on renal NPRs mRNA expression was mainly on the modulation level of NPR-C mRNA, it could reduce the clearance rate of ANP, increase the level of ANP, and enhance the biological effect of ANP on the kidney and regulative action of kidney in exercise.     

Immunoreactive atrial natriuretic peptide in the thyroid gland

Human thyroid follicles and primary cell cultures derived from them demonstrated atrial natriuretic peptide (ANP)-like immunoreactivity when stained with a monoclonal antibody raised against rat alpha-ANP (ANP 1-28). In thyroid sections the staining was most intense in the tall cuboidal epithelium of small follicles. The intracellular distribution of immunoreactive (ir)-ANP in primary cultures of thyroid follicular cells consisted of discrete granules with a largely perinuclear distribution. The granule density increased with time in culture but was unaffected by exogenous ANP, suggesting an intrinsic synthesis of the immunoreactivity. Thyroid stimulating hormone (TSH; thyrotropin) failed to alter the distribution of ir-ANP after either short-term (6 h) or long-term (1-12 day) exposure. Epinephrine or norepinephrine treatment, however, caused a reduction in the ir-ANP granularity compared with controls in what might represent a stimulated release of the immunoreactivity. The present results suggest that the peptide ANP coexists with thyroid hormones in follicular cells and that the two endocrine activities might be under separate control mechanisms.