Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells

The effects of atrial natriuretic factor (ANF) on phosphoinositide hydrolysis were examined in preparations of cultured bovine aortic smooth muscle cells. In homogenates or particulate fractions from cultured bovine aortic smooth muscle cells, ANF and atriopeptin I increased the formation of inositol phosphates and GTPase activity. The effects on inositol phosphates were markedly enhanced with guanosine 5'[gamma-thio]triphosphate. Both atrial peptides also stimulated the formation of diacylglycerol in intact cultured cells. In these experiments, atriopeptin I was about 10-fold more potent than ANF. These studies indicate that atrial peptides have stimulatory effects on phosphoinositide hydrolysis which are mediated through a guanine nucleotide regulatory protein. The greater potency of atriopeptin I on GTPase activity and the accumulation of inositol phosphates suggests that the nonguanylate cyclase-coupled receptor for ANF (ANF-R2) mediates the stimulatory effects of ANF on phosphoinositide hydrolysis through a guanine nucleotide regulatory protein.     

Effect of chronic treatment with deoxycorticosterone acetate on content of a natriuretic substance in atria of rats

Chronic (72 days) administration of deoxycorticosterone acetate (DOCA), with or without saline as the sole drinking fluid, depleted atria of rats of their diuretic and natriuretic activities. Chronic ingestion of saline as the sole drinking fluid did not affect the diuretic, natriuretic, and kaliuretic activities of atria compared with those of rats receiving water to drink. Since systolic blood pressure of the DOCA-treated group did not differ significantly from that of the untreated control group, the decrease in potency of atrial extract from DOCA-treated rats most likely occurred in response to increases in extracellular and vascular volumes. The ability of DOCA to decrease diuretic and natriuretic activities of atria was dose dependent. The decreased activities of the atria of DOCA-treated rats could reflect an increased production and turnover of atrial natriuretic factor. Additional studies revealed an increased diuretic and natriuretic responsiveness of DOCA-treated recipients to atrial extract from untreated rats. Thus, the results of these studies suggest that chronic treatment with DOCA reduced the natriuretic and diuretic potencies of atrial extract and increased renal responsiveness to it.     

The elucidation of the structure of atrial natriuretic factor, a new peptide hormone

The benchmark experiments of Adolfo de Bold and Harald Sonnenberg revealed that heart atria contained a substance or substances (atrial natriuretic factor) which when injected into rats caused a profound diuresis, natriuresis, and fall in blood pressure. Acid extraction and purification of atrial natriuretic factor resulted initially in the purification of a low molecular weight peptide containing a disulfide bond. This peptide was named cardionatrin I. Amino acid sequencing of less than 1 nmol of cardionatrin I revealed it to be a 28-residue peptide with the following structure: (sequence; see text) The position of the disulfide bond was verified by a radioactive method. From the sequence of complementary DNA for atrial natriuretic factor, the 28-residue peptide was shown to be the C-terminal portion of a larger protein called pro-atrial natriuretic factor. The discovery and characterization of atrial natriuretic factor substantiated the idea that the heart atria serve in an endocrine capacity.     

Mechanisms of release and renal tubular action of atrial natriuretic factor

Inasmuch as atrial natriuretic factor (ANF) is apparently involved causally in the renal response to acute hypervolemia, it became of interest to study cellular mechanisms of release and renal tubular action. To study release mechanisms, freshly excised rat heart atria were incubated in vitro. Activation of the cellular adenylate cyclase system by either beta-adrenergic stimulation or the vasopressin analog deamino-8-D-arginine vasopressin did not result in ANF release. By contrast, activation of the polyphosphoinositide system by alpha-adrenergic stimulation or stimulation of the V1-type vasopressin receptors, and by a calcium ionophore or active phorbol ester, significantly increased natriuretic activity in the medium and reduced it in tissue. It is concluded, therefore, that activation of this latter system is the mechanism for ANF secretion from atrial myocytes. To test the effect of ANF on tubular transport in the medullary collecting duct, microcatheterization was used in rats before and during i.v. infusion of synthetic atrial peptide (23 amino acids). It was found that tubular delivery of salt to this part of the nephron was increased, and that reabsorption in the duct itself was reduced. In control experiments, increased delivery was associated with proportionately increased reabsorption, which demonstrated glomerulotubular balance in the nephron segment under normal conditions. The natriuretic effect of ANF, therefore, was not caused solely by enhanced tubular load, but included specific inhibition of duct sodium reabsorption as an essential feature of the renal response.     

Atrial natriuretic factor: atrial conversion of high to low molecular weight forms

The atrial natriuretic factor elutes by gel filtration in high and low molecular weight fractions. Extraction and elution of rat atria in 1.0 M acetic acid yielded a predominance of the high molecular weight form(s); whereas when these procedures were carried out in 0.1 M acetic acid, there was a predominance of the low molecular weight forms. When partially purified high molecular weight natriuretic activity was eluted in 0.1 M acetic acid, the high molecular weight form(s) remained intact. When partially purified high molecular weight natriuretic activity was mixed with crude atrial extract in 0.1 M acetic acid, there was an apparent conversion to the low molecular weight forms. Extraction of rat atria in boiling 0.1 M acetic acid blocked this conversion. It is concluded that rat atria contain a heat labile factor that converts high molecular weight natriuretic activity to the low molecular weight forms.     

Basal and stimulated ANF secretion: role of tissue preparation

Our previous results showed that addition of agonists, such as vasopressin and angiotensin, added to incubation medium with freshly excised rat atria caused marked release of atrial natriuretic factor (ANF). This release was in the form of prohormone rather than active peptide. Since others had difficulty reproducing these findings, in the present study we investigated ANF release with and without angiotensin addition in two sets of atrial tissue. In the first, tissue was blotted and carefully cleaned as previously described; in the second, atrial tissue was placed into incubation medium without prior preparation. ANF activity in the medium was measured by radioimmunoassay and receptor assay. Using the immunoassay, basal release of ANF was threefold greater from prepared vs. nonprepared atrial tissue; significant stimulation by angiotensin was seen only in the prepared atria. ANF release measured by radioreceptor assay was 1/5-1/10 of that measured by immunoassay. Taking the difference between the two measurements as an index of prohormone secretion, the results confirm that both basal and stimulated release was primarily in the form of proANF. Scanning electron microscopy revealed that cleaning of the atria had removed the endocardial lining of the tissue. The results thus indicate that an intact endocardium can prevent agonist-induced proANF secretion, suggesting that this tissue may be an important modulator of plasma ANF levels.     

Monoclonal antibody-directed immunopurification and identification of cytochromes P-450

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.     

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.     

Responses of vasoactive hormones in congestive cardiac failure

Congestive cardiac failure causes activation of various neurohumoral responses that increase total peripheral resistance and promote salt and water retention. These effects increase blood pressure and organ perfusion in the short term, but ultimately cause further cardiac decompensation by increasing ventricular afterload and cardiac work. The role of the renin-angiotensin-aldosterone system and the catecholamines is partially understood, and blockade of these systems as a treatment of heart failure is now established. The role of vasopressin in heart failure is more controversial, but there is now compelling evidence that vasopressin may have important vasoconstrictor actions in addition to its fluid retaining properties. Atrial natriuretic factor is a newly described cardiac hormone released from the atrium. Atrial natriuretic factor causes natriuresis, diuresis, vasodilatation, suppression of thirst, and suppression of both renin and aldosterone. These actions largely counteract the effects of the renin-angiotensin system and vasopressin. Plasma atrial natriuretic factor has been reported to be markedly elevated in human and experimental heart failure, and may act to limit the neurohumoral response to reduced cardiac output. This review summarizes our understanding of the vasoactive hormones and reports experimental evidence supporting a pathophysiological role for vasopressin and atrial natriuretic factor in congestive cardiac failure.     

cis-dominance of rat atrial natriuretic factor gene regulatory sequences in transgenic mice.

We have produced transgenic mice that express the prokaryotic marker protein chloramphenicol acetyltransferase under the control of regulatory sequences derived from the rat atrial natriuretic factor gene. The transgene, which contains 2.4 kilobases of the rat atrial natriuretic factor gene regulatory region, was found to direct 4000-fold more chloramphenicol acetyltransferase expression in adult atria than in ventricles. Low-level activity was also detected in the hypothalamus, demonstrating that these sequences contain the signals necessary for cardiac and central nervous system expression of the hormone atrial natriuretic factor. Developmental analyses showed early, high-level transgene expression in fetal atrial and ventricular tissues but marked reduction of ventricular transgene expression following birth. Further, the developmental expression patterns of the endogenous murine atrial natriuretic factor gene and rat transgene were found to be quite distinct. Although both the rat and mouse atrial natriuretic factor genes are activated early in embryogenesis, perinatal ventricular expression appears to differ in these two rodent species. The transgene is expressed in a pattern analogous to the neonatal rat rather than the endogenous murine gene. These studies demonstrate that the cis-acting signals required for correct tissue specificity and developmental regulation of the rat atrial natriuretic factor gene are encoded in this 2.4-kilobase fragment and that these sequences act in a dominant fashion.     

Stimulation of phosphatidylinositol metabolism in atrial and ventricular myocytes

Receptor-stimulated hydrolysis of inositol phospholipids was studied in atrial and ventricular myocytes isolated from guinea-pigs. Membrane phospholipids were labelled with [3H] inositol and their conversion to [3H] labelled inositol phosphate was measured in the presence of Li+ (10 mM). In the absence of added stimulatory hormones or neurotransmitters, little inositol phosphate accumulation was observed. Acetylcholine and carbachol stimulated inositol phosphate accumulation with a maximum of more than 12 times the unstimulated values in atrial myocytes and 7 times in ventricular myocytes. The EC50 values and 95% confidence limits for acetylcholine and carbachol were 0.9 microM (0.2 - 5.3) and 8.8 microM (6.3 - 11.8) in atria and 0.6 M (0.5 - 0.8) and 10.0 M (1.8 - 55.9) in ventricles, respectively. Oxotremorine was a partial agonist in stimulating inositol phosphate accumulation in both atrial and ventricular myocytes. The vasoactive peptides angiotensin II and vasopressin also stimulated inositol phosphate accumulation but the maximum effect was lower than that mediated through muscarinic receptors. However, the adenosine analogues, L-N6-phenylisopropyladenosine and 5'N-ethylcarboxamidoadenosine which, like muscarinic agonists depress cardiac contractility, did not affect inositol phosphate accumulation at concentrations up to 10(-4)M.     

Immuno-electron microscopy of atrial natriuretic factor secretory pathways in atria and ventricles of control and cardiomyopathic hamsters with heart failure

Summary The secretory pathways of atrial natriuretic factor have been investigated in atrial and ventricular cardiocytes of control and cardiomyopathic Syrian hamsters in severe congestive heart failure with four antibodies: a monoclonal antibody (2H2) against rat synthetic atrial natriuretic factor (101–126), which is directed against region 101–103 of rat atrial natriuretic factor (99–126), and polyclonal, affinity-purified antibodies produced in rabbits against synthetic C-terminal atrial natriuretic factor (101–126), synthetic N-terminal atrial natriuretic factor (11–37) or the putative cleavage site of atrial natriuretic factor (98–99): atrial natriuretic factor (94–103). Application of the immunogold technique on thin frozen sections (immunocryoultramicrotomy) revealed an identical picture with the four antibodies. In atria of both control and cardiomyopathic hamsters where atrial natriuretic factor secretion is regulated, the atrial natriuretic factor propeptide travels, uncleaved, from the Golgi complex to immature and mature secretory granules. In ventricles of control hamsters, where secretion is constitutive, the atrial natriuretic factor propeptide travels from the Golgi complex to secretory vesicles. In the ventricles of hamsters with severe congestive heart failure, the Golgi complex is larger, secretory vesicles more abundant and a few secretory granules are present in 20% of cardiocytes. Here again, the peptide travels uncleaved in all these pathways. These results reveal the pathways of secretion of atrial natriuretic factor in atrial and ventricular cardiocytes and indicate that the propeptide is not cleaved intracellularly.Supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Research Group on Hypertension, by the Canadian Heart Foundation and the Pfizer Company (England)     

Inhibitory roles of the hypothalamic atrial natriuretic polypeptide on the vasopressin release in the sodium-loaded rats

Implication of the brain atrial natriuretic polypeptide on the vasopressin release was investigated using rats fed with a high-sodium containing diet. Sodium loading increased not only the blood pressure but also the urinary output of vasopressin significantly. The plasma vasopressin concentration increased about 10 times after the intracerebroventricular injections of angiotensin II. Thereby, magnitude of the response was significantly smaller in the rat fed with a high sodium diet than in rats with the regular-diet. The hypothalamic content of both vasopressin and atrial natriuretic polypeptide was significantly larger in the high-salt group than the regular-salt. The intraventricular injections of atrial natriuretic polypeptide abolished the vasopressin release induced by the intraventricular injections of angiotensin II. These results indicate that the vasopressin production in the hypothalamus is increased, but the release is relatively suppressed in the sodium-loaded rats, and that increased hypothalamic atrial natriuretic polypeptide is involved in the suppression of the vasopressin release and in decreasing their sodium appetite to avoid the high sodium environment.     

Atrial natriuretic factor: radioimmunoassay and effects on adrenal and pituitary glands

A simple and sensitive radioimmunoassay was developed for measurement of immunoreactive atrial natriuretic factor (IR-ANF) in rat and human plasma and in rat atria. The two atria contain about 20 micrograms ANF per rat. The right atrium contained 2.5 times more ANF than did the left. Ether anesthesia and morphine markedly increased IR-ANF in rat plasma. The concentration of IR-ANF in plasma of clinically normal human subjects was 65.3 +/- 2.5 pg/ml. Paroxysmal tachycardia and rapid atrial pacing significantly increased IR-ANF in human plasma. Two- to seven-fold higher concentrations were found in coronary sinus blood than in the peripheral circulation. In the plasma of rats and humans, circulating ANF is probably a small-molecular-weight peptide. ANF acts on the adrenal and the pituitary. ANF inhibits aldosterone secretion from rat zona glomerulosa and steroid secretion by bovine adrenal zona glomerulosa and fasciculata. ANF stimulated the basal secretion of arginine vasopressin (AVP) in vitro and inhibited KCl-stimulated release of AVP.     

Effects of atrial natriuretic factor on renal handling of water and electrolytes in rats

The intravenous injection of an extract of atrial myocardium into anesthetized rats during a hypotonic diuresis resulted in an increase in the renal excretion of water, sodium, potassium, calcium, magnesium, and phosphate. There was an increase in urine concentration which was probably a result of the secretion of vasopressin since it did not occur in Brattleboro (di/di) rats. A transient increase in glomerular filtration rate and renal plasma flow occurred during the first five minutes with a more sustained rise in filtration fraction. Injection of atrial extract also caused a partial inhibition of solute-free water formation in Brattleboro rats subjected to water diuresis and a partial inhibition of solute-free water reabsorption in rats subjected to maximal antidiuresis by infusing vasopressin. In neither case was the degree of inhibition as profound as that observed after injecting furosemide in a dose which caused a comparable natriuretic response. A large dose of furosemide blocked the natriuretic response to atrial extracts whereas, when a comparable level of sodium and water output was produced by massive infusions of saline, the natriuretic response to atrial extract was increased. It is suggested that atrial natriuretic factor might inhibit sodium transport in nephron segments beyond the medullary thick ascending limb. Furosemide might also act at the same tubular site or inhibit tubular secretion of the atrial natriuretic factor.     

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.     

Accumulation of messenger RNA of the atrial natriuretic factor in the rat left ventricle at the compensatory hypertrophy stage in pressure overload

The atria produce several peptides that have natriuretic and vasoactive properties, collectively called atrial natriuretic factor. All these peptides share a single messenger ribonucleic acid, the amount of which greatly increases in the rat left ventricle when the latter is submitted to chronic volume overload. Using the molecular hybridization technique and a desoxyribonucleic acid probe complementary to the atrial natriuretic factor messenger ribonucleic acid, we now report that a very important increase in the amount of this messenger ribonucleic acid is also observed in rat ventricle at at the compensatory stage of a pressure overload induced cardiac hypertrophy. This result suggests that the pressure overload hypertrophied rat ventricle also has the potential to itself regulate it's loading conditions via the regulation of extracellular fluid volume and vascular resistance.     

Inhibition of water-sodium intestinal absorption by an atrial extract

The rat atrium contains a diuretic and natriuretic factor which appears to inhibit the sodium reabsorption in the kidney tubules. We observed, in rats, that our atrial extract possesses a potent diuretic and natriuretic effect that was accompanied by an increased dextrose excretion. Similarly, extracts of rat atria, but not of ventricles, reduced intestinal absorption of water, sodium, and dextrose. The omission of sodium or dextrose in the perfusion fluid annulled this effect. These data suggest that the substance inhibiting the intestinal absorption of water and solutes is probably atrial natriuretic factor and that it acts on the sodium-dextrose cotransport mechanism.     

Atrial natriuretic peptide deposited as atrial amyloid fibrils

Deposition of amyloid in the atria is exceedingly common in the aging heart. We have extracted amyloid fibrils from atria and purified a major protein which had N-terminal amino acid sequence identical to that of atrial natriuretic peptide (ANP). Antisera to ANP and to the amyloid fibril protein both labelled atrial muscle cells and atrial amyloid in an identical way.