Molecular aspects of atrial natriuretic peptides

Peptides possessing both natriuretic and smooth muscle relaxant activities have been isolated from heart atria and their structures have been determined. The peptides designated ANP (atrial natriuretic peptide) regulate salt and water balance and blood pressure. The scope of this article is to provide a summary of recent research developments directed towards understanding the molecular nature of atrial natriuretic peptides.     

Central natriuretic peptides regulation of peripheral atrial natriuretic factor release

Atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP) receptors have been described in encephalic areas and nuclei related to the regulation of cardiovascular as well as sodium and water homeostasis. Stimulation of the anterior ventral third ventricular region of the brain modifies plasma ANF concentration, suggesting the participation of the central nervous system in the regulation of circulating ANF. The aim of this work was to study the effect of centrally applied ANF or CNP on plasma ANF. Normal and blood volume expanded rats (0.8 ml isotonic saline/100 g body weight) were intra cerebralventricularly injected with 1, 10 or 100 ng/μl/min ANF. Blood volume expanded animals were also centrally injected with the same doses of CNP. Blood samples were collected at 5 and 15 min. after intracerebralventricular administration of either ANF or CNP. Centrally applied ANF did not affect circulating ANF in normal blood volume rats. In blood volume expanded animals both ANF (1, 10 or 100 ng/μl/min) and CNP (1 ng/μl/min) decreased plasma ANF concentration after 15 min. Moreover, CNP (10 and 100 ng/μl/min) lowered circulating ANF levels not only at 15 min but also at 5 min. Neither ANF nor CNP elicited any change in mean arterial pressure and heart rate in normal and blood volume expanded rats. These results suggest the existence of a central regulation exerted by natriuretic peptides on circulating ANF levels. Furthermore, this is the first study reporting an effect on plasma ANF induced by centrally applied CNP.     

Effects of atrial natriuretic peptides and furosemide in conscious dogs

Effects of ANF(8-33) and Auriculin A on renal variables were investigated in conscious water-diuretic dogs. The two substances were injected intravenously (1.08 micrograms/kg in 3 min) or ANF(8-33) was infused (0.2 microgram/kg X min in 20 min). The effects were compared to those of an equinatriuretic dose of furosemide (1.0 microgram/kg X min). Injections caused increases in sodium excretion, diuresis, and osmolar clearance. No significant change in exogenous creatine clearance (CCREA) occurred. Infusion of ANF(8-33) decreased blood pressure by 14% (P less than 0.01) and increased sodium excretion by a factor of 10 (P less than 0.01). The natriuresis was a function of increases in diuresis and urinary sodium concentration, the latter by a factor of 6 (P less than 0.01). Diuresis and free-water clearance (CH2O) increased by 60% (P less than 0.01), but urine osmolality did not change significantly. After the infusion a significant decrease in PAH clearance (CPAH) (P less than 0.01) was observed. Filtration fraction (FF) did not change. The furosemide natriuresis appeared later than that of ANF without significant deviations in diuresis, CH2O, CCREA, CPAH, and FF; urine osmolality increased by 35% (P less than 0.01). The effects of ANF(8-33) differ from those of furosemide in several ways. First, the onset of natriuresis is faster, second, the natriuresis is associated by marked increases in diuresis and free-water clearance but not in urine osmolality; and third, natriuresis is followed by a reduction in renal blood flow. The rapid natriuresis of ANF can occur without changes in glomerular filtration rate.     

Identification of new atrial natriuretic peptides in frog heart

It has been observed that mammalian atrial natriuretic peptide (ANP)-like immunoreactivity is found in frog heart, but to date the natriuretic factors have not yet been identified. Isolation from bull-frog heart extract was performed mainly by immunoaffinity chromatography on a column linked with anti-hANP IgG. From the low molecular weight fraction, 24- and 21-amino acid peptides were purified to homogeneity. Both peptides were found to elicit diuretic-natriuretic as well as vasorelaxant activity, and were named "frog ANP-24" and "frog ANP-21", respectively. Complete amino acid sequences of the peptides were established by microsequencing and confirmed by syntheses. Frog ANP-21 was identified as an N-terminally three amino acid deleted form of frog ANP-24. Remarkable sequence homology was observed between frog ANP and mammalian ANP, especially in the regions flanked by two half-cystine residues.     

Synthesis and CD structural studies of CD52 peptides and glycopeptides

The syntheses of five natural and N-terminal acetylated peptides and glycopeptides of the CD52 antigen are described. Solid phase peptide synthesis was employed in the construction of the target compounds from Fmoc-protected commercial amino acids and synthetic glycan-asparagine conjugates. Circular dichroism studies of the synthetic targets showed that they exist as random coils in solution, and no significant change in secondary structure was observed when the CD52 peptide was either acetylated at the N-terminus or glycosylated at the Asn³ residue with a disaccharide or a fucose-containing branched trisaccharide.     

Bioactive atrial natriuretic factor-like peptides in rat anterior pituitary

The presence of biologically active atrial natriuretic factor (ANF)-like peptides was demonstrated in rat anterior pituitary. ANF-like immunoreactivity was detected in rat anterior pituitary by specific radioimmunoassay and was extracted from rat anterior pituitary homogenates by heat-activated Vycor glass beads; extracts were purified by reverse-phase high performance liquid chromatography. Two peaks containing ANF immunoreactive material were obtained. The first peak was eluted from the C18 mu Bondapak column at a position similar to the 28-amino acid carboxy terminal peptide (Ser99-Tyr126)-ANF of prohormone. The second peak had the same pattern of elution as the 126-amino acid prohormone, (Asn1-Tyr126)-ANF. The biological activity of the smaller molecular weight peptide (28 amino acid) was assessed by its inhibitory effect on 10(-8) M ACTH-stimulated aldosterone secretion in rat zona glomerulosa cell suspension. This ANF-like material also displaced I125-labelled ANF from rat glomerular receptors with a potency similar to synthetic (Arg101-Tyr126)-ANF. Immunocytochemical localization revealed a distribution of ANF-stained cells similar in pattern and location to that of gonadotrophs. These results suggest the existence of biologically active ANF-like peptides and ANF prohormone within the anterior pituitary. However, their role remains to be elucidated.     

Molecular forms of atrial natriuretic peptides in dog atrium and plasma

The level of immunoreactive ANP (iANP) as determined by radioimmunoassay, exhibits a fairly even distribution throughout the canine atria. However, the maximal concentration was found in the appendages of both the left and right atrium, and the level was significantly higher on the left side in all analyzed localizations. Reverse-phase HPLC of atrial extracts, coupled with RIA, revealed the presence of three iANP fractions. The dominant fraction (65% of iANP) has an estimated molecular weight 16,300, showing that it corresponds to the hormone precursor. By contrast, in the plasma extract, 92% of iANP coelutes with synthetic human ANP99-126, thus confirming the structural identity of dog and human ANP.     

Endothelin stimulates release of atrial natriuretic peptides in vitro and in vivo

The effect of endothelin (END) on the release of atrial natriuretic peptides (ANP) was studied in isolated rat atria and in conscious rats. END stimulates the ANP release in vitro in a dose-dependent manner. An increase in ANP plasma levels and cyclic GMP plasma levels was also observed in conscious rats after injection of END. When a monoclonal antibody directed against ANP was injected together with END the increase in cyclic GMP was completely blocked. From this study it is concluded that END is a potent secretagogue for ANP both in vitro and in vivo.     

Acute stress increases plasma concentrations of atrial natriuretic peptides

5 min exposure of inbred Maudsley Reactive male rats to intermittent foot-shock resulted in an approximate doubling of plasma atrial natriuretic peptides ANP (Control grp mean = 62.12 +/- 8.74; Stressed grp mean = 128.70 +/- 26.63 pg/ml) and 25 min exposure resulted in a three-fold increase (Stressed grp mean = 187.88 +/- 39.24 pg/ml). In the second experiment exposure of genetically heterogeneous Wistar male rats to 15 min of intermittent foot-shock produced a 10-fold increase in plasma ANP (Control grp mean = 45.76 +/- 6.05; Stressed grp mean = 471.20 +/- 58.49 pg/ml). The magnitude of the increase in plasma ANP produced by acute stress is as large as the increase caused by volume expansion and administration of various pharmacological agents and therefore delineation of biological role of ANP must take account of its potential role as a stress-hormone.     

Postmortem stability of the rat atrial natriuretic peptide in blood and atrial tissue

The postmortem stability of the rat Atrial Natriuretic Peptide (ANP) has been studied as a necessary and previous step to be applied in the forensic field as a postmortem marker. This peptide--whose extreme sensitivity to slight changes in blood volume is well known--could have great importance in thanatochemistry to establish a correct diagnosis when macroscopical observations and classical parameters are not conclusive or cannot be employed. The results show high stability in atrial tissue, where values are similar from 0 hours (108.99 pm/mg) to 8 hours (109.41 pm/mg) and decrease uniformly until 15 pm/mg) at 32 hours, time of our last determination. Blood ANP showed similar stability from 0 h (105.43 pg/ml) to 8 h (106.62 pg/ml).     

Role of atrial natriuretic peptides and neuropeptide Y in blood pressure regulation

Atrial natriuretic peptides (ANP) are released into the circulation in response to enhanced atrial stretching. These peptides not only have diuretic and natriuretic properties, but also exert a relaxing effect on the vasculature. Moreover, they antagonize the contractions induced by norepinephrine and angiotensin II. Neuropeptide Y (NPY) is also a vasoactive peptide. It is widely distributed throughout the central and peripheral nervous systems. NPY is coreleased with norepinephrine by perivascular nerve endings. At high concentrations, this peptide has a direct vasoconstrictor effect. In addition, it enhances the vascular effect of various agonists, including norepinephrine and angiotensin II. Both ANP and NPY have an inhibitory effect on renin secretion. This effect may have important implications for the role of these peptides in cardiovascular regulation.     

Effects of morphine and opioid peptides on plasma levels of atrial natriuretic peptide

The effect of opiate ligand administration on plasma levels of atrial natriuretic peptide (ANP) was studied in awake, freely moving Sprague-Dawley rats. Prior to and following the intracerebroventricular (icv) or central venous (iv) injection of morphine (MS), leu-enkephalin (Leu-enk), dynorphin (Dyn) or beta-endorphin (B-endor), plasma samples were obtained for measurement of ANP concentrations by radioimmunoassay. MS was 10 times more potent when given icv than when given iv to increase plasma ANP levels. Icv injection of Leu-enk decreased plasma ANP concentrations. Dyn and B-endor administration (iv or icv) did not alter the plasma concentration of ANP. These effects of MS and Leu-enk on plasma concentrations of ANP appear to be mediated through actions on the central nervous system. MS, Leu-enk, B-endor, and Dyn given icv, produced elevations of plasma norepinephrine (NE) and epinephrine (Epi) concentrations. When MS was given icv, mean Epi and NE plasma levels increased 10-50 times the increases noted with B-endor, Leu-enk and Dyn. A role of catecholamines in mediating MS-stimulated ANP release is supported by the observation that ganglionic blockade with chlorisondamine significantly attenuated the increase of plasma ANP levels. MS, but not B-endor, Leu-enk and Dyn, acts within the brain to increase plasma levels of ANP. MS-induced elevations of plasma ANP levels may be dependent on an intact autonomic nervous system.     

Phosphorylation of atrial natriuretic peptides by cyclic AMP-dependent protein kinase

Atrial natriuretic peptides refer to a family of related peptides secreted by atria that appear to have an important role in the control of blood pressure. The structure of these peptides shows the amino acid sequence Arg101-Arg102-Ser103-Ser104, which is a typical recognition sequence (Arg-Arg-X-Ser) for phosphorylation by cyclic AMP-dependent protein kinase. With this background, we tested two synthetic atrial natriuretic peptides (Arg101-Tyr126 and Gly96-Tyr126) as substrates for in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. The tested atrial natriuretic peptides were found to be substrates for the reaction. Sequence studies demonstrated that the site of phosphorylation was located, as expected, at Ser104. Kinetic studies demonstrate that both atrial natriuretic peptides are excellent substrates for cyclic AMP-dependent protein kinase. In particular, the longer peptide Gly96-Tyr126 exhibited an apparent Km value of about 0.5 microM, to our knowledge the lowest reported Km for a cyclic AMP-dependent protein kinase substrate. Preliminary studies to measure the biological activity of the in vitro phosphorylated atrial peptides indicate that these compounds are more effective than the corresponding dephospho forms in stimulating Na/K/Cl cotransport in cultured vascular smooth muscle cells.     

Vascular atrial natriuretic factor receptor subtypes are not independently regulated by atrial peptides

The regulation of the atrial natriuretic factor (ANF) receptor system in cultured rat vascular smooth muscle cells (RVSMC) was examined following long term pretreatment of these cells with rANF99-126 or with any one of a series of truncated and ring-deleted analogs. The latter analogs are reported to bind selectively the ANF-C or clearance receptor. Initial competition binding studies revealed that all analogs examined showed comparable apparent receptor binding affinities (Ki values did not differ by more than 10-fold). In contrast, the extent of interaction of the ANF analogs with the receptor pool coupled to particulate guanylate cyclase (the ANF-B receptor) was much more variable, with some ligands failing to stimulate cGMP production or particulate guanylate cyclase over the concentrations tested. Pretreatment of cells for 24 h with rANF99-126 or any of the truncated analogs that interact with the ANF-B receptor caused a dose- and time-dependent decrease in the number of ANF binding sites (99% of which are uncoupled in RVSMC) without any change in affinity. Examination of the binding activity following pretreatment of the cells with ANF suggested that the observed reduction in 125I-rANF99-126 binding capacity was not because of the retention of the peptide on its receptor. Furthermore, this down-regulation was associated with desensitization of particulate guanylate cyclase resulting in a decreased responsiveness of intracellular cGMP accumulation to ANF. In contrast, however, analogs selective for the ANF-C receptor pool failed to cause down-regulation or desensitization. These findings suggest that ANF-C receptors in RVSMC are not independently down-regulated by selective ligands but that nonselective analogs that down-regulate and desensitize the ANF-B receptor system can by some cooperative mechanism reduce the size of the predominant ANF-C receptor pool in these cells.     

Synthesis and presence of atrial natriuretic factor in rat ventricle

Rat heart ventricles contained immunoreactive atrial natriuretic factor (irANF) and mRNA for ANF. The size of ANF mRNA in the ventricle was identical with that of the atria. High performance gel filtration chromatography showed that 84% of ventricular irANF elutes at a position corresponding to the low molecular weight form of ANF (99-126) and 16% of irANF elutes at a position corresponding to the precursor form of ANF. The irANF content of the ventricles of spontaneously hypertensive rats was 3 times as much as that of Wistar Kyoto rats. These results suggest that ventricle synthesizes ANF in response to hypertension and processes in a manner different from that in atria.     

Synthesis and in vitro analysis of atrial natriuretic peptide-albumin conjugates

Atrial natriuretic peptide (ANP) is a clinically useful anti-hypertensive hormone. Maleimide derivatives of ANP have been synthesized and conjugated to cysteine-34 of human serum albumin. The conjugates were analyzed to assess their stability, receptor binding affinity and ability to stimulate guanylyl-cyclase activity in rat lung fibroblasts.     

Epicardial release of immunoreactive atrial natriuretic peptides in inside-out perfused rabbit atria

An easy and convenient isolated atrial perfusion technique was developed. The effect of stretch of the atrial subpericardial myocytes was investigated in the inside-out perfused rabbit atria. Graded distension of the inverted atria was induced by changing the elevation of the atrial catheter tip. Intra-luminal volume expansion resulted in an increase in release of immunoreactive atrial natriuretic peptides (irANPs). The response was volume, or pressure dependent. Distension-induced release of irANPs occurred at the reduction of the distension. IrANPs in epicardial perfusate showed both high and low molecular weights. The major peak of irANP was observed at the corresponding fraction to the rat ANP-(1-28) in the Sephadex G-50 gel chromatography. The data suggest that the epicardial release of irANP is stretch-induced response and that the release may be involved in the regulation of cardiac function.     

Synthesis and structural analysis of vinylogous peptides

A Z- or E-ethenyl group has been inserted between the -carbon andthe carboxyl group of the proline residue by stereoselective Hornersynthesis. The resulting vinylogous amino acid has been coupled with aminocompounds by classical methods, and model amino acid derivatives anddipeptides containing a Z- or E-CH=CMe group have been investigated insolution by ¹H-NMR and IR spectroscopy, and in the solid stateby X-ray diffraction. The E-ethenyl group gives rise to an openconformation and the Z-conformer to a folded structure with anintramolecular hydrogen bond closing a nine-membered pseudocycle.