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 gene for rat atrial natriuretic factor

Atrial natriuretic factor (ANF), a peptide hormone recently isolated from heart atria, appears to play an important role in the regulation of extracellular fluid volume and blood pressure. Indeed, natural and synthetic ANF rapidly and markedly stimulate natriuresis and diuresis and produce smooth muscle relaxation. Consistent with the hypothesis that ANF is a novel hormone, it was recently shown that ANF is present in circulation, and high affinity membrane receptors specific for ANF have been described in renal, vascular, and adrenal tissues. These important biological activities suggest that conditions like hypertension could be associated with defective ANF gene expression. We and others have shown by cDNA cloning that ANF is part of a larger precursor, pro-natriodilatin (PND). We now describe the isolation and structural analysis of the rat PND gene. Southern blot analysis of rat genomic DNA suggests the presence of a single PND gene per haploid genome. The PND coding sequences are interrupted by two short introns. A long alternating purine-pyrimidine tract (GT)9GATG(GT)27 is found 111 base pairs downstream of the polyadenylation site; such sequences could adopt Z-DNA configuration and they have been associated with sequences that appear very active in intergenic recombination. Comparison of the rat and human PND genomic sequences shows highest homology in 5'-flanking as well as in coding sequences. The rat PND gene will be a useful model to study the physiology and pathology of this important regulator of the cardiovascular system.     

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.     

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.     

Discovery of atrial natriuretic factor in the brain: Its characterization and cardiovascular implication

1. We have devised a radioimmunoassay for atrial natriueretic factor (ANF). Its application to rat brain extract led to the discovery of ANF in the brain. In addition to the hypothalamus and the pontine medullary region, it was widely distributed. 2. ANF in the brain is stored in a low molecular weight form, in contrast to pro-ANF in the atria. Thus, the processing of pro-ANF in the bran neuronal cells is different from that in the atria. 3. ANF was found in the anterior and posterior lobes of the pituitary, the peripheral ganglia, adrenergic neurons, and the adrenal medulla. 4. Brain ANF suppressed stimulated dipsogenesis, basal and stimulated vasopressin release, and angiotensin II-stimulated pressor effects. 5. ANF in the peripheral neuronal system inhibits catecholamine synthesis and release. Thus, central ANF functions to reduce the peripheral fluid volume and vascular tone in concert with the peripheral ANF.     

Radioreceptor assay for atrial natriuretic factor

Interest in accurate measurement of atrial natriuretic factor (ANF) in biological fluids and various tissues has been stimulated by recent data indicating the possible role of ANF in the homeostasis of salt and water. The presence of high-affinity binding sites for ANF in rat glomeruli has allowed us to develop a rapid, sensitive, and simple radioreceptor assay (RRA). A saturable high-affinity binding site on the membranes of rat glomeruli has been characterized by a dissociation constant of 33 pM and binding capacity of 396 fmol/mg protein. Rat plasma extracts or atrial homogenates or standards were incubated with radioiodinated ANF and a preparation of rat glomerular membranes. The receptor-bound and free radioactivity were separated by filtration on Whatman GF/C paper after 1 h incubation at room temperature. The sensitivity of the RRA was 2.08 fmol. The effective concentration of standard ANF that displaced 50% of labeled receptor-bound ANF (EC50) was 43.3 +/- 2.6 fmol/ml (n = 7). Both intra- and interassay coefficients of variation were smaller than 11%. This RRA assay has been compared with radioimmunoassay (RIA). High correlations for 19 plasma extracts and 34 atrial homogenates (r = 0.973 and r = 0.954, respectively) tested by RRA and RIA were obtained. This good correlation between the two methods suggests that the immunoreactive material found in rat plasma and atrial homogenates also displays biological activity.     

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.     

Prostatic kallikreins: biochemistry and physiology

This review describes and compares the properties of seven individual kallikreins present in the prostate of four mammalian species. The first kallikrein discovered in prostate was the one of guinea-pig. That protein has kininogenase activity like classical kallikreins. The rat prostate expresses two different kallikreins, S3 and P1, whose physiological functions remain to be determined precisely. In man, prostate-specific antigen (PSA) is an abundant secretory protein. It is currently used as a prostate cancer marker. The human prostate may also contain renal/pancreatic kallikrein and human glandular kallikrein-1 (hGK-1). Arginine esterase secreted by dog prostate is probably the most abundant kallikrein. It has no known physiological substrate.     

Radioimmunoassay of atrial natriuretic factor: Human plasma levels

A simple and sensitive radioimmunoassay procedure has been developed for the determination of atrial natriuretic factor (ANF) in human plasma. The rabbit antiserum was obtained from a commercial source. ANF was extracted from plasma using an octadecasilyl silica cartridge with a recovery of 78.7%. HPLC of the plasma extract showed the presence of one immunoreactive peak of ANF correponding to its low molecular weight form. Plasma ANF in humans increased from 8.0 ± 2.2 in upright position to 20.0 ± 5.9 fmol/ml (n = 6) in downward position (p < 0.005).     

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.     

Plasma and atrial content of atrial natriuretic factor in cardiomyopathic hamsters

Immunoreactive atrial natriuretic factor (IR-ANF) was measured in plasma and atrial extracts from normal and cardiomyopathic Syrian golden hamsters. Plasma IR-ANF was increased from 84.8 ± 9.8 pg/ml(n = 17) to 234 ± 23 (n = 25; P<.0001) in hamsters with moderate failure, and to 1085 ± 321 pg/ml (n = 10; P<.02) in animals with severe failure. Plasma IR-ANF increased with increased atrial hypertrophy. Atrial IR-ANF content was essentially the same in normal animals and in those with moderate heart failure (3.06 ± 0.28 vs 3.17 ± 0.19 μg/100 g body wt., P<.001) and lower in the majority of those with severe failure (1.82 μg/100 g body wt., P<.001). The elevations of IR-ANF in plasma are similar to those seen in patients with congestive heart failure. Our studies do not support bioassay results showing a deficiency of atrial ANF content as being important in the congestive heart failure associated with cardiomyopathy in the hamster.     

Three immunologically similar atrial natriuretic factor receptors

One of the atrial natriuretic factor (ANF) receptors is a 180 kDa protein (180 kDa mGC) which possesses the extraordinary characteristic of being bifunctional: it is both a receptor and a guanylate cyclase. In addition to the 180 kDa mGC, there exists another 120–130 kDa protein which is also bifunctional and a 120 kDa disulfide-linked dimeric cell surface protein that is an ANF receptor, but is not a part of guanylate cyclase. A fundamental question that needs to be resolved is: Are these three apparently biochemically distinct ANF receptors structurally similar? With the aid of affinity crosslinking techniques, a highly specific antibody to the 180 kDa mGC, and GTP-affinity techniques, we now demonstrate the presence of three immunologically similar proteins in rat adrenal gland and testes. These proteins migrate as 180 kDa, 130 kDa and 65 kDa under denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis and specifically bind ANF, raising one or more of the following possibilities about their relationships: 1) Degradation of 180 kDa to 130 kDa and 65 kDa occurs during purification; 2) 180 kDa bears a precursor-product relationship with 130 kDa and 65 kDa, suggesting the role of a protease in the processing procedure; 3) these proteins are a result of gene splicing; or 4) they are the products of three separate, but very closely related genes.     

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.     

Biochemical mechanisms of atrial natriuretic factor action

Since atrial natriuretic factor (ANF) is a natriuretic and vasodilatory hormone, its mechanisms of action expectedly involve so-called negative pathways of cell stimulation, notably cyclic nucleotides. Indeed, the guanylate cyclase-cyclic GMP (cGMP) system appears to be the principal mediator of ANF's action. Specifically, particulate guanylate cyclase, a membrane glycoprotein, transmits ANF's effects, as opposed to the activation of soluble guanylate cyclase such agents as sodium nitroprusside. The stimulation of particulate guanylate cyclase by ANF manifests several characteristics. One of them is the functional irreversibility of stimulation with its apparent physiological consequences: the extended impact of ANF on diuresis and vasodilation in vivo lasts beyond the duration of increased plasma ANF levels and is accompanied by a prolonged elevation of cGMP. Another characteristic is the parallelism between guanylate cyclase stimulation and increases of cGMP in extracellular fluids. cGMP egression appears to be an active process, yet its physiological implications remain to be uncovered. In heart failure, cGMP continues to reflect augmented ANF levels, suggesting that in this disease, the lack of an ANF effect on sodium excretion is due to a defect distal to cGMP generation. In hypertension, where ANF levels are either normal or slightly elevated, probably secondary to high blood pressure, the ANF responsiveness of the particulate guanylate cyclase-cGMP system, the hypotensive effects, diuresis and natriuresis are exaggerated. The implications of this exaggerated responsiveness of the ANF-cGMP system in the pathophysiology of hypertension and its potential therapeutic connotations remain to be evaluated.     

The actions of atrial natriuretic factor on the vascular wall

The actions of atrial natriuretic factor (ANF) on the vascular wall are diverse and show a profound regional heterogeneity. ANF is a potent relaxant of aortic smooth muscle, a response which is associated with activation of particulate guanylate cyclase and elevation in tissue levels of cyclic GMP. However, many large and small muscular arteries and most veins are unresponsive to the peptide. The regional vascular heterogeneity may be due to an altered distribution of high affinity receptors and (or) alterations in the coupling of receptor activation to elevations in cyclic 3',5'-guanosine monophosphate (cGMP). Species differences exist in the structural requirements for receptor activation as well as the effects of infused ANF on peripheral resistance. Although the relaxation to ANF in vitro does not require an intact endothelium, endothelial cells contain multiple receptor subtypes for ANF. Differences amongst tissues and (or) species in the receptor profile for ANF may, in part, explain some of heterogeneity in responsiveness to ANF.