Truncated atrial natriuretic factor analogs retain full agonist activity.

The synthesis, receptor binding, and agonist activity of a series of truncated atrial natriuretic analogs (ANF) are described. These analogs incorporate two portions of the native 28 amino peptide, the eight amino acids C-terminal to Cys7, and two amino acids from the C-terminus (phenylalanine and arginine), into disulfide-bonded cyclic peptides. The inclusion of the C-terminal amino acids converted the ANF analogs from receptor ligands to full agonists, as measured by several methods, including the stimulation of cGMP biosynthesis in endothelial cells, inhibition of aldosterone biosynthesis in rat adrenal cells, and natriuretic-hypotensive activity in vivo. The most potent analogs have cyclohexylalanine (Cha) at position 8. The lead compound (Arg6,Cha8 ANF 6-15 Phe-Arg-Cys-NH2) is a tridecapeptide that integrates the C-terminal amino acids inside the disulfide ring. This peptide, designated as A-68828, has a binding affinity of IC50 = 120 nM, approximately 1/400 of ANF 1-28. However, this analog, in vivo, is only slightly less natriuretic (1/20-1/50) than ANF 1-28. Unlike the native peptide, A-68828 is only mildly hypotensive and at the highest concentration tested reduced blood pressure less than 15 mmHg (1 mmHg = 133.322 Pa). A-68828 inhibited ACTH-induced aldosterone release to a greater extent than ANF 1-28: 100 vs. 50%. The selective natriuretic activity of A-66828, relative to ANF, suggests clinical utility for the treatment of acute renal failure.     

Amino- and carboxy-terminal PEST domains mediate gastrin stabilization of rat L-histidine decarboxylase isoforms

Control of enzymatic function by peptide hormones can occur at a number of different levels and can involve diverse pathways that regulate cleavage, intracellular trafficking, and protein degradation. Gastrin is a peptide hormone that binds to the cholecystokinin B-gastrin receptor and regulates the activity of L-histidine decarboxylase (HDC), the enzyme that produces histamine. Here we show that gastrin can increase the steady-state levels of at least six HDC isoforms without affecting HDC mRNA levels. Pulse-chase experiments indicated that HDC isoforms are rapidly degraded and that gastrin-dependent increases are due to enhanced isoform stability. Deletion analysis identified two PEST domains (PEST1 and PEST2) and an intracellular targeting domain (ER2) which regulate HDC protein expression levels. Experiments with PEST domain fusion proteins demonstrated that PEST1 and PEST2 are strong and portable degradation-promoting elements which are positively regulated by both gastrin stimulation and proteasome inhibition. A chimeric protein containing the PEST domain of ornithine decarboxylase was similarly affected, indicating that gastrin can regulate the stability of other PEST domain-containing proteins and does so independently of antizyme/antizyme inhibitor regulation. At the same time, endoplasmic reticulum localization of a fluorescent chimera containing the ER2 domain of HDC was unaltered by gastrin stimulation. We conclude that gastrin stabilization of HDC isoforms is dependent upon two transferable and sequentially unrelated PEST domains that regulate degradation. These experiments revealed a novel regulatory mechanism by which a peptide hormone such as gastrin can disrupt the degradation function of multiple PEST-domain-containing proteins.     

Synthesis and activity of partial retro-inverso modified atrial natriuretic factor analogs

The synthesis and pharmacological activity of partial retro-inverso modified rat atrial natriuretic factor (rANF) analogs is described. The route to these compounds utilized a combination of solution and solid-phase methods. The analogs prepared all contain a reversed amide bond (psi[NHCO]) at the Ser 25 to Phe26 linkage. This bond has been suggested to play a key role in the metabolic inactivation of ANF. The analogs are of comparable potency to the endogenous peptide rANF1-28 in binding to cultured rat vascular smooth muscle cells, in relaxing serotonin contracted rabbit aortic rings, and as natriuretic/diuretic agents in anesthetized rats. None of the peptides has an extended duration of action in vivo.     

Inhibition of aldosterone synthesis by atrial natriuretic factor

Atrial natriuretic factor (ANF) inhibits basal and stimulated aldosterone synthesis in adrenal glomerulosa cells. ANF probably acts through specific membrane receptors. Alterations in cyclic GMP and cyclic AMP levels do not account for ANF's inhibitory effect. ANF does not block angiotensin II (AngII) receptors nor does it interfere with phosphoinositide metabolism or calcium movements stimulated by adrenal agonists. ANF does not inhibit protein synthesis nor does it work by inhibiting NA+,K+-ATPase or depleting cell potassium. ANF decreases conversion of endogenous cholesterol to pregnenolone, the step stimulated by adrenocorticotropin and AngII. ANF does not affect the conversion of 20-alpha-hydroxycholesterol, which easily penetrates mitochondrial membranes to the site of the cholesterol side-chain cleavage enzyme. These results suggest that ANF inhibits the ability of endogenous cholesterol to reach or interact with the side-chain cleavage enzyme. ANF does not act like a calcium channel-blocking agent. However, ANF is less effective at high-calcium concentrations, which suggests that it may inhibit a step that calcium stimulates. Understanding ANF action will probably require identification of the specific biochemical changes (mediators) that it induces. Parallel efforts to understand how other agents stimulate steroidogenesis (particularly in the areas of protein synthesis, protein phosphorylation, and cholesterol movements) will further this understanding.     

Synthesis and biological activity profiles of atrial natriuretic factor (ANF) analogs

Several analogs of the atrial natriuretic factor (ANF) were synthesized by the solid-phase method using the acetamidomethyl (Acm) group for sulfhydryl protection. The compounds were tested in a receptor binding assay using bovine adrenal zona glomerulosa cell membranes and in the rat diuresis/natriuresis assay. Substitution of tyrosine in position 116 of ANF(101-126) and of the analog [3-Mpr105]ANF(105-126)(3-Mpr = 3-mercaptopropionic acid) did not alter the biological activity profiles and, therefore, these two analogs in radioiodinated form will be useful for enzymatic degradation and clearance studies. Replacement of 3-mercaptopropionic acid with 2-mercaptopropionic acid in [3-Mpr105]ANF(105-126) resulted in an analog with very low potency in both assay systems, presumably as a consequence of the steric bulk and/or local conformational restriction produced by the methyl group attached to the alpha-carbon in position 105. The analog [3-Mpr105,Nva109]ANF(105-126)(Nva = norvaline) showed very low affinity in the receptor binding assay but displayed considerable diuretic/natriuretic activity. The obtained biological activity profiles suggest that in comparison with other ANF peptides the des-amino ANF(105-126) analogs may have a somewhat longer half-life in vivo, or alternatively, may indicate a more complex situation of ANF receptor or binding site heterogeneity.     

Pharmacological profile of 8-amino octanoic acid substituted atrial natriuretic factor analogs

A series of six analogs of rat atrial natriuretic factor have been prepared by the solid-phase method. The modified analogs contain 8-amino octanoic acid (a simple alkyl spacer) in place of selected tripeptides. Binding affinity to cultured aortic smooth muscle cell membranes suggests that the sequence Arg11-Gly16 is important for binding. Vasorelaxant activity on serotonin contracted rabbit aortic rings indicates that the Phe8-Gly16 sequence must be present for vasorelaxation. In anesthetized rats, the natriuretic and diuretic effects of an IV bolus dose correlate with in vitro vasodilatory activity. The alkyl spacer approach provides a facile method to quickly determine key regions of a large peptide involved in molecular recognition.     

Ozone increases amino- and carboxy-terminal atrial natriuretic factor prohormone peptides in lung,heart, and circulation

Ozone can cause pulmonary edema and simultaneously decrease blood pressure. Atrial natriuretic peptides may mediate both of these effects in that they increase pulmonary capillary permeability resulting in edema formation and are potent vasodilating peptides. To examine this possibility, the lungs of Fischer 344 rats were exposed to ozone (0.5 ppm) for 8 hours which resulted in a three- to fourfold increase in atrial natriuretic peptides. Ozone also increased atrial natriuretic peptides in the heart two- to fivefold from 266 ± 25, 226 ± 22, and 288 ± 40 ng/g (room air) to 716 ± 26, 471 ± 14, and 1473 ± 235 ng/g recognized by the proANFs 1–30 and 31–67 and atrial natriuretic factor radioimmunoassays, respectively. Ozone also doubled the concentrations of proANFs 1–30, 31–67, and 1–98 and ANF in the circulation. This study demonstrates that ozone increases atrial natriuretic peptides within the heart, lung, and circulation, suggesting that atrial natriuretic peptides may mediate the decreased blood pressure and pulmonary edema observed with ozone exposure. Since the proANF 31–67 radioimmunoassay exclusively recognizes the ANF prohormone within the heart, this study further indicates that ozone can increase the synthesis of the ANF prohormone.     

Renal hemodynamic and natriuretic effects of atrial natriuretic factor

In this article we review the renal hemodynamic and excretory actions of atrial natriuretic factor (ANF) and consider some of the mechanisms of its vascular and natriuretic effects. ANF leads to a marked, sustained, and parallel increase in whole-organ and superficial single-nephron glomerular filtration rate (GFR) while mean blood pressure is decreased and renal blood flow (RBF) is unchanged or even decreased. The increase in GFR is caused by an efferent arteriolar vasoconstriction or by a combination of afferent vasodilation and efferent vasoconstriction. ANF also leads to a decrease in the hypertonicity of the innermedullary interstitium. Together with the increase in GFR, this phenomenon accounts wholly or in great part for the ANF-induced natriuresis. The overall renal vascular effects of ANF are complex and may tentatively be conceptualized as a behavior of a functional partial agonist: slight vasoconstriction in vasodilated kidneys, no sustained effects on the vascular resistance in normal kidneys, and vasodilation in vasoconstricted kidneys. The vasoconstrictor effect of ANF may be direct or indirect and depends on extracellular calcium whereas the antagonist effect likely results from alterations in intracellular calcium homeostasis. The data raise the perspective that ANF is not only a powerful natriuretic substance but has the potential of being an important modulator of GFR and RBF in intact animals.     

Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.     

A decade of atrial natriuretic factor research.

Present views on the biological significance of atrial natriuretic factor (ANF) relate this polypeptide hormone to the regulation of blood pressure and volume through its modulating effects on renal function, on blood vessel tone and permeability, and on the renin-angiotensin-aldosterone system. Although very important advances in the understanding of ANF have been made over the decade since its discovery, some fundamental facts about ANF biosynthesis and release remain to be elucidated. Stretch-induced enhancement of ANF release appears as the most significant mechanism underlying the endocrine response of the atria to acute volume load. This response decays over a period of minutes, indicating that chronic stimulation of ANF release involves mechanisms different from, or in addition to, those acting during acute stretch-stimulated release. In neither acute nor chronic conditions are the cellular or molecular mechanisms underlying ANF release understood. To better understand long-term stimulation of ANF release, we have conducted extensive in vitro testing of several hormones and neurotransmitters to determine their ability to modify ANF release. From these studies, clear-cut evidence of ANF stimulation was obtained with the vasopressor peptide endothelin. Investigations on the cell and molecular biology of cardiac muscle development and hypertrophy have shown that ANF is involved in cardiac growth. The role played by ANF in these processes is now being determined, but this is one line of evidence that suggests that this hormone, together with other natriuretic peptides, may have autocrine or paracrine functions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood-brain barrier and atrial natriuretic factor

In brain, binding sites for atrial natriuretic factor (ANF) have been characterized in areas such as circumventricular organs that lack the tight capillary endothelial junctions of the blood-brain barrier and therefore are exposed to circulating peptides. Since atrial natriuretic factor acts directly on vascular endothelium and has been proposed to be actively involved in blood pressure regulation and fluid homeostasis, it is interesting to know whether ANF receptors exist on brain capillaries that constitute the blood-brain barrier and participate in the constant fluid exchange between blood and brain. The present paper reports recent evidence of the presence of ANF receptors located on the structure. It assesses the specific binding of 125I-labelled ANF on bovine brain microvessel preparations and its coupling with a guanylate cyclase system. The potential physiological role of ANF on brain microcirculation and blood-brain barrier functions is discussed.     

Rat atrial natriuretic factor. Purification and vasorelaxant activity

The atrial natriuretic activity of rat heart has been found to exist in multiple forms. One of these factors has been purified to apparent homogeneity by a combination of gel filtration and high pressure liquid chromatography in two different systems and its amino acid composition determined. The purified active peptide is shown to have a molecular weight of approximately 3800. In addition, the vasorelaxant activity of rat atrium has been purified and found to cochromatograph with the natriuretic activity in all chromatographic systems employed. Thus, the vasorelaxant activity resides in the natriuretic factor. The existence of this new multifunctional peptide implies a higher level of complexity for cardiovascular control of blood volume and pressure.     

The physiology of atrial natriuretic factor

Following the discovery of the natriuretic effect of atrial extract, our laboratory attempted to dissect the possible physiological role of atrial natriuretic factor. Initial micropuncture experiments demonstrated that the reduction of tubular sodium reabsorption was localized in the medullary collecting duct, a nephron site in which sodium transport was known to be inhibited after acute hypervolemia. Partial removal of the endogenous source of atrial natriuretic factor was associated with a reduced renal response to hypervolemia, confirming that the factor is causally involved in acute sodium balance. In vitro incubation of atrial tissue was used to investigate mechanisms of release of atrial natriuretic factor. It was found that agonists known to activate the intracellular polyphosphoinositide system in other tissues were effective in releasing natriuretic activity from the atria into the incubation medium. To determine whether atrial natriuretic factor might play a role in hypertension, atrial natriuretic content was measured in spontaneously hypertensive rats and their normotensive controls. Hypertension was associated with increased content. Since the renal response to exogenous factor was not impaired in these animals, we suggested that the increased content might play a compensatory role. Our early studies thus indicated that atrial natriuretic factor was a previously unrecognized hormone involved in cardiovascular regulation.     

The cosecretional maturation of atrial natriuretic factor by primary atrial myocytes.

Cardiac myocytes store the 126-amino acid precursor of atrial natriuretic factor (pro-ANF), yet the mature, bioactive 28-amino acid peptide, ANF-(99-126), and the resulting N-terminal product, ANF-(1-98), are the forms of the hormone that are released by the heart and found in the circulation. Although previous studies have shown that the maturation of ANF takes place in the heart, it is not known whether it occurs in or on the myocyte concurrently with secretion, or whether cleavage takes place postsecretionally on either the myocyte surface or the surface of a nonmuscle cardiac cell. To address these questions, experiments were carried out in the present study using primary atrial cultures that had been prepared such that greater than 90% of the cells were myocytes. Reversed-phase and ion-exchange HPLC, coupled with immunoprecipitation of biosynthetically labeled ANF, showed that the stored peptide, pro-ANF, was cleaved between residues 98 and 99 such that ANF-(1-98) and (99-126) accumulated in the medium. Coupling biosynthetic labeling with timed secretion experiments showed that the extent of ANF processing was not dependent on the time after secretion; maximal levels of processing were observed at all secretion times examined. Additionally, the processing-competent myocyte-enriched cultures were unable to cleave exogenously added pro-ANF. These results indicate that the myocyte is the cell type responsible for pro-ANF maturation and that this cleavage event takes place cosecretionally.     

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

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.     

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.     

Atrial dynamics and release of atrial natriuretic factor in vivo.

In anaesthetized rabbits blood volume was altered by infusion and withdrawal of donor blood over the range of +60 to -40% of the blood volume. Right and left atrial pressures were measured and it was shown that sonomicrometry allowed adequate measurement of phasic changes in atrial dimensions. Plasma immunoreactive atrial natriuretic peptide concentration changed in a nonlinear fashion with changes in blood volume, and was linearly related to both peak systolic and peak diastolic right and left atrial wall stress. It was not possible to make the distinction between distension (diastolic stress) or tension (systolic stress) as the major determinant of ANF release in response to changes in blood volume.     

Inactivation of atrial natriuretic factor in blood

Tissue extracts of rat heart atria contain a family of peptides with natriuretic and vasorelaxant properties. It has been shown by others that inactivation of this atrial natriuretic factor may involve endogenous peptidases. The present experiments demonstrate that incubation in blood in vitro reduces the natriuretic activity of the factor. Specifically, inactivation was associated with a white cell/platelet fraction, indicating that these blood elements may play a physiological role in the metabolism of this new putative hormone.