Isolation and sequence determination of peptide components of atrial natriuretic factor

437

160

A 26 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) relaxed isolated rabbit aortic segments in which the endothelium was either intact or functionally destroyed. The relaxations were temporally associated with increases in levels of cGMP with no change in the levels of cAMP. The ANF-induced increases in cGMP were also observed in aortic segments pretreated with calcium-free buffer or the cGMP phosphodiesterase inhibitor M&B 22,948. Qualitatively similar results were obtained for sodium nitroprusside. ANF selectively activated particulate guanylate cyclase, having no effect on the soluble form of the enzyme. Thus, the direct (endothelium-independent) vasodilator effect of ANF may be mediated via increased tissue levels of cGMP. ANF appears to increase vascular cGMP levels by activation of particulate guanylate cyclase.Mammalian atria contain several peptides that may play an important role in regulating electrolyte balance and vascular resistance (1, 2). The elucidation of the amino acid sequence of these bioactive peptides (3-6) has led to the production of synthetic analogs (5), which will allow a more precise characterization of the pharmacology of atrial natriuretic factor (ANF). Recently, using one such synthetic peptide (26 amino acid fragment), the vasodilator profile of ANF was assessed and found to be qualitatively similar to the profile for sodium nitroprusside but not for other commonly used vasodilators, such as adenosine, papaverine, or nifedipine (7). The nitrovasodilators are thought to induce smooth muscle relaxation through activation of guanylate cyclase leading to the formation of cGMP (8-10). Furthermore, the relaxation and increase in the levels of cGMP in vascular smooth muscle produced by nitrovasodilators are independent of the vascular endothelium, in contrast to similar changes produced by endothelium-dependent vasodilators, such as acetylcholine (11-14). Thus, the ability of ANF to activate guanylate cyclase and to alter the levels of cGMP and cAMP was investigated and compared to the actions of the nitrovasodilator sodium nitroprusside. METHODSMale New Zealand White rabbits (2-2.5 kg) were stunned by a blow to the head and exsanguinated. The thoracic aorta was removed and quickly placed in physiological salt solution of the following composition: 130 mM NaCl/4.7 mM KCI/1.18 mM KH2PO4/1.17 mM MgSO4/1.6 mM CaCl2/14.9 mM NaHCO3/11.0 mM dextrose/0.026 mM EDTA, equilibrated with 95%02/5% CO2 (pH 7.2). Vessels were cleaned of extraneous tissue under a dissecting microscope taking care not to damage the lumenal surface. However, in a few aortas, the endothelium was functionally destroyed by rubbing the lumen with a wooden stick (15). Ring segments (4-5 mm long) were cut from each aorta and mounted in glass tissue baths (50 ml) for monitoring changes in isometric force. Aortic rings were stretched to a resting force (2.0 g), which allowed for optimal levels of contractile force to be generated. Tissues were washed with fresh oxygenated physiological salt solut...

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confidence: 99%

Atrial natriuretic factor elicits an endothelium-independent relaxation and activates particulate guanylate cyclase in vascular smooth muscle.

Winquist¹,

Faison²,

Waldman³

et al. 1984

437

160

“…These include the atrial natriuretic factors (ANF) (1)(2)(3)(4)(5)(6)(7)(8), atrial natriuretic polypeptides (ANP) (9,10), auriculins (9), atriopeptins (11)(12)(13), cardionatrins (14)(15)(16), and cardiodilatin (5,17). As revealed by cDNA analysis, all of these peptides are likely to be derived from a multihormone precursor (18)(19)(20)(21)(22)(23)(24)(25).…”

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confidence: 99%

“…As revealed by cDNA analysis, all of these peptides are likely to be derived from a multihormone precursor (18)(19)(20)(21)(22)(23)(24)(25). Strong sequence homology between rat and human ANP demonstrates the conservation of amino acid composition during mammalian evolution (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). These various peptides modulate blood-pressure homeostasis, aldosterone production, and extracellular fluid volume (for review, see refs.…”

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confidence: 99%

Characterization and distribution of receptors for the atrial natriuretic peptides in mammalian brain.

Quirion¹,

Dalpé²,

Dam³

1986

149

Various atrial natriuretic peptides have been isolated recently from secretory-like granules present in the myocytes of mammalian atria. These include the atrial natriuretic factors (ANF) (1-8), atrial natriuretic polypeptides (ANP) (9, 10), auriculins (9), atriopeptins (11-13), cardionatrins (14-16), and cardiodilatin (5, 17). As revealed by cDNA analysis, all of these peptides are likely to be derived from a multihormone precursor (18)(19)(20)(21)(22)(23)(24)(25). Strong sequence homology between rat and human ANP demonstrates the conservation of amino acid composition during mammalian evolution (1-17). These various peptides modulate blood-pressure homeostasis, aldosterone production, and extracellular fluid volume (for review, see refs. 7 and 26) by acting as a circulating hormone stimulated by volume loading (27). Recently, the presence of ANP or ANF-like immunoreactivity (28,29) in rat brain has been demonstrated. We also provided preliminary evidence for the existence of ANF binding sites in rat brain, using an autoradiographic technique (30). However, essential saturation analysis and ligand selectivity pattern were not obtained because of the low density of sites in most regions of the rat brain.Here we report on the characterization and differential autoradiographic distribution of ANP receptor binding sites in various mammalian brains. In the guinea pig brain, 125I-labeled ANP (12-I-ANP) binds with high affinity (Kd between 0.02 and 0.08 nM) to receptor sites that are mainly concentrated in the olfactory bulb, subfornical organ, paraventricular, paratenial, paracentral, and centrolateral nuclei of the thalamus, hippocampus, medial geniculate nucleus, and lobules 9 and 10 of the cerebellum. The high density ofANP binding sites in these various regions suggests the possible existence of a family of brain-heart peptides that could act as neurotransmitters/neuromodulators, in analogy to the well-known brain-gut peptides. MATERIALS AND METHODSIn preliminary experiments, we observed that the guinea pig cerebellum and thalamus/hypothalamus area were enriched in 1251-ANP binding sites. In other regions such as the striatum, cortex, and brain stem, it was not possible to perform appropriate receptor binding studies (saturation, ligand selectivity pattern) because ofthe low densities ofsites in these areas. Thus, we used those first two brain regions to further characterize those sites. Male guinea pigs (500 g) were decapitated, and the thalamus/hypothalamus area and the cerebellum were rapidly dissected out on ice before homogenization in 10 vol of 150 mM Tris HCl buffer (pH 7.4 at 4°C) containing 120 mM NaCl and 5 mM KCl using a Brinkmann polytron at setting 6 for 20 sec. Homogenates were then centrifuged for 10 min at 49,000 x g. Supernatants were discarded, and pellets were resuspended in 50 mM Tris'HCl buffer (pH 7.4 at 4°C) containing 300 mM KCl and 10 mM Na2EDTA before incubation on ice for 30 min with gentle agitation. After centrifugation as above, pellets were resuspended in 20 vol of 50 mM Tris HCl b...

“…Recently, a family of peptides has been isolated from acidic extracts of rat (10,11,(19)(20)(21)(22) and human (23) atria and sequenced independently by several laboratories. From rat atria, the longest peptide that has been reported contains 33 amino acids, 1, and was isolated as the free COOH-terminal tyrosine acid (20,21). Shorter rat ANF peptides are truncated at either the NH2 or COOH terminus.…”

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confidence: 99%

Specific membrane receptors for atrial natriuretic factor in renal and vascular tissues.

Napier¹,

Vandlen²,

Albers-Schönberg³

et al. 1984

230

Membranes from rabbit aorta and from rabbit and rat kidney cortex possess high-affinity (Kd = 10-10 M) specific binding sites for atrial natriuretic factor (ANF). Similar high-affinity sites are present in an established cell line from pig kidney, LLC-PK,. Results of fractionation studies indicate that the receptors are localized in the plasma membrane of these tissues. The binding is time-dependent and saturable. An excellent quantitative correlation was found between the affinity of synthetic ANF and analogs of intermediate activity to aorta membranes and the half-maximal concentration needed for relaxation of rabbit aorta rings contracted by addition of serotonin. Furthermore, the binding affinity of the receptor in kidney membranes is consistent with the concentration required for in vivo natriuresis in the rat. Biologically inactive synthetic ANF fragments and other peptide hormones such as angiotensin II and vasopressin do not significantly inhibit binding. These data suggest that the receptors for ANF in vascular and renal tissues are responsible for mediating the physiological actions of this peptide in these target tissues.A possible role for the cardiac atria in the regulation of extracellular fluid volume and electrolyte concentration has been shown by the induction of sodium and water excretion in response to changes in intraatrial pressure and stretch of the atrial wall (1). The cardiac atria possess granules that have the appearance of secretory granules (2-4) and whose number can be altered by manipulation of the water-electrolyte balance in experimental animals (5). Crude extracts of atria from several species have been shown to possess potent diuretic and natriuretic activity when given intravenously to rats (6). Subcellular fractionation (7) and immunocytochemistry (8) indicated that these granules are storage sites for an atrial natriuretic factor (ANF). Furthermore, in vitro, atrial extracts were shown to be potent vasorelaxants (9-14). As little as 0.0006 atrial equivalent per milliliter gave 50% relaxation of precontracted aorta tissues (12). In vivo, ANF caused lowering of mean arterial blood pressure in normal and hypertensive animals (6,15,16). Recently, a family of peptides has been isolated from acidic extracts of rat (10,11,(19)(20)(21)(22) and human (23) atria and sequenced independently by several laboratories. From rat atria, the longest peptide that has been reported contains 33 amino acids, 1, and was isolated as the free COOH-terminal tyrosine acid (20,21). Shorter rat ANF peptides are truncated at either the NH2 or COOH terminus.5 10 H-Leu -Ala-Gly-Pro -Arg-Ser -Leu-Arg-Arg-Ser-Ser-15 20Cys-Phe-Gly-Gly-Arg-Ile -Asp-Arg-Ile -Gly-Ala--30Gln -Ser-Gly-Leu-Gly-Cys -Asn-Ser-Phe-Arg-Tyr-OH 1 A peptide consisting of residues 8-33, designated sANF, has been synthesized (21, 24) and shown to possess full biological activity (12,16,21,24). The IC50 of sANF for relaxation of precontracted rabbit aorta rings is 550 pM (12). The amount of sANF required for half-maximal natriuresis wh...