Disulfide bond structure of the atrial natriuretic peptide receptor extracellular domain: conserved disulfide bonds among guanylate cyclase-coupled receptors

The disulfide bond structure of the extracellular domain of rat atrial natriuretic peptide (ANP) receptor (NPR-ECD) has been determined by mass spectrometry (MS) and Edman sequencing. Recombinant NPR-ECD expressed in COS-1 cells and purified from the culture medium binds ANP with as high affinity as the natural ANP receptor. Reaction with iodoacetic acid yielded no S-carboxymethylcysteine, indicating that all six Cys residues in NPR-ECD are involved in disulfide bonds. Electrospray ionization MS of NPR-ECD deglycosylated by peptide-N-glycosidase F gave a molecular mass of 48377.5+/-1.6 Da, which was consistent with the presence of three disulfide bonds. Liquid chromatography MS analysis of a lysylendopeptidase digest yielded three cystine-containing fragments with disulfide bonds Cys(60)-Cys(86), Cys(164)-Cys(213) and Cys(423)-Cys(432) based on their observed masses. These bonds were confirmed by Edman sequencing of each of the three fragments. No evidence for an inter-molecular disulfide bond was found. The six Cys residues in NPR-ECD, forming a 1-2, 3-4, 5-6 disulfide pairing pattern, are strictly conserved among A-type natriuretic peptide receptors and are similar in B-type receptors. We found that in other families of guanylate cyclase-coupled receptors, the Cys residues involved in 1-2 and 5-6 disulfide pairs are conserved in nearly all, suggesting an important contribution of these disulfide bonds to the receptor's structure and function.     

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.     

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.     

A ring-reversed analog of atrial natriuretic peptide retains receptor binding, guanylate cyclase stimulation.

We have prepared an atrial natriuretic peptide analog, ANP[13-27][1-12], in which the connectivity of the disulfide-linked ring has been reversed by formally cleaving the ring and cyclizing the N- and C-terminal tails. This analog, which retains many of the spatial relationships of the native molecule, binds to both ANP-A and ANP-C receptor subtypes, and triggers the production of cyclic-GMP by ANP-A. ANP-C binding of ANP[13-27][1- 12] is roughly equipotent to that of ANP itself, although the ring cleavage falls within the putative ANP-C binding domain. ANP[13-27][1-8], a truncated analog in which much of this binding domain has been removed, surprisingly maintains a high affinity for ANP-C; however, this peptide has lost the ability to activate the ANP-A-linked guanylate cyclase.     

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.     

Biochemical studies of soluble atrial natriuretic peptide (ANP) receptors from rat olfactory bulb and vascular smooth muscle cells

1. Aim. The biochemical characteristics of atrial natriuretic peptide receptors (ANP-R) derived from rat vascular smooth muscle (A-10 cell line) and central nervous system (CNS; olfactory bulb) tissue were compared. 2. Method and Results. ANP-Rs from each source were solubilized with 40 to 65% efficiency utilizing the nonionic detergent Lubrol-PX. Upon solubilization, the ANP-R from each source maintained the ability to bind 125I-ANP (99-126) with a high affinity; Scatchard analysis indicated that the VSMC ANP-R displayed a Kd for the radioligand of approximately 10 pM, whereas the olfactory receptor possessed a Kd of about 165 pM. The Bmax values for the soluble VSMC and olfactory ANP-Rs were 285 and 30 fmol/mg protein, respectively. Competition binding studies indicated that the VSMC ANP-R bound ANP(99-126), ANP(103-126), and ANP(103-123) with similar affinities, whereas the olfactory ANP-R was much more sensitive to changes in the COOH-terminal structure of the competing peptide. The soluble ANP-Rs from VSMC and olfactory were chromatographically indistinguishable on phenyl-, DEAE-, and wheat germ agglutinin-agarose columns. However, the ANP-Rs could be distinguished using GTP-agarose; the olfactory ANP-R was capable of binding to the resin, whereas the VSMC ANP-R was not. 3. Conclusions. Coupled with other studies, these data suggest that the A10 VSMC ANP-R observed in this study may not be coupled to guanylate cyclase and may represent a receptor serving a clearance function, whereas a significant proportion of the olfactory CNS ANP-R appears to be associated with GTP-binding proteins, likely particulate guanylate cyclase, and probably represents a coupled form of the receptor.     

Plasma and atrial levels of atrial natriuretic peptide (ANP) in pulmonary hypertensive rats

Immunoreactive atrial natriuretic peptide (IR-ANP) was measured in plasma and atrium of normal and monocrotaline induced pulmonary hypertensive rats (PH rats). In these animals, there was right ventricular hypertrophy and right ventricular systolic pressure was elevated. Fourteen days after a single dose of monocrotaline (40 mg/kg), plasma IR-ANP concentrations were significantly elevated (964.3 +/- 63.0 pg/ml vs. 521.0 +/- 81.9 pg/ml in controls, p less than 0.001). Tissue levels of IR-ANP in the right atrium in PH rats was significantly lower than those in the controls (45.1 +/- 3.9 ng/mg vs. 240.5 +/- 10.4 ng/mg, p less than 0.001), while there was no significant difference in tissue levels of atrial IR-ANP in the left atrium between the two groups. Thus, development of pulmonary hypertension led to an increase in release of ANP from the right atrium.     

Calcitonin gene-related peptide(CGRP) stimulates the release of atrial natriuretic peptide(ANP) from isolated rat atria

The effect of calcitonin gene-related peptide(CGRP) on the release of atrial natriuretic peptide(ANP) was studied in spontaneously beating, isolated rat atria. CGRP stimulated the ANP release in a dose-dependent manner. When the atria were incubated with a combination of phentolamine, propranolol, and atropine, these antagonists blocked neither the rise in ANP release nor the positive chronotropic and inotropic effects of CGRP. Therefore, we conclude that CGRP stimulates ANP release as well as cardiac contractility independently of adrenergic and cholinergic receptors.     

The three subtypes of atrial natriuretic peptide (ANP) receptors are expressed in the rat adrenal gland

Atrial natriuretic peptide (ANP) actions are mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANP receptor-A (or GC-A), -B (or GC-B) and -C (the so-called clearance receptor). In rat adrenal gland, the mRNA for each subtype was detected using ³⁵S-dUTP or digoxigenin-11-dUTP specific labeled probes, and in situ hybridization at light and electron microscopic levels respectively. The three subtypes were expressed the most abundantly in the zona glomerulosa. The amount of GC-A mRNA expression, quantified using macroautoradiography and densitometry, was higher than the amounts of GC-B mRNA and ANPR-C mRNA both in zona glomerulosa and medullary of adrenal gland. At electron microscopic level, the three subtypes of ANPR were revealed in glomerulosa cells. A noticeable signal was also present in the medullary area, especially for GC-A mRNA, in adrenaline-containing chromaffin cells. No signal was detected in noradrenaline-containing chromaffin cells. The subcellular localization of the three mRNAs is similar: in the cytoplasmic matrix and in the euchromatin of the nucleus in each cell of glomerulosa, and in the same compartments of the adrenaline-containing chromaffin cells. These data indicate that the adrenal gland is an important target tissue for ANP action both in glomerulosa cells and adrenaline-containing chromaffin cells. The mRNA expression levels were different for each ANPR subtype.     

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.     

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.     

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.     

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.     

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.     

Specific binding sites for atrial natriuretic peptide (ANP) in cultured mesenchymal nonmyocardial cells from rat heart

Specific binding sites for atrial natriuretic peptide (ANP) were studied in cultured mesenchymal nonmyocardial cells (NMC) from rat heart. Binding study using 125I-labeled synthetic rat (r) ANP revealed the presence of a single class of high-affinity binding sites for rANP in cultured NMCs derived from both atria and ventricles; the apparent dissociation constant (Kd) was approximately 0.2 - 0.3 nM and the number of maximal binding sites was approximately 190,000 - 300,000 sites/cell. rANP significantly stimulated intracellular cGMP formation of cardiac NMCs in a dose-dependent manner (1.6 X 10(-8) M - 3.2 X 10(-7) M). rANP had no effect on synthesis of prostaglandin I2 by cultured cardiac NMCs. The physiological significance of ANP action on cardiac tissue remains to be determined.