Atrial Extracts Research Articles

Effect of Domperidone on Renal Response to Certain Atrial Natriuretic Substances in Rats

Anesthetized rats, maintained on an intravenous (1 ml/h) infusion of saline, were injected with either atrial extract from normal rats (AE), atriopeptin III (APIII, 24 amino acids) or atrial peptide (AP, 28 amino acids) as either a bolus injection (1 atrial equivalent of AE, 1 microgram APIII, or 1 microgram AP) or by constant infusion (1.7 ng APIII or AP/min for 30 min) in isotonic saline. Diuretic, natriuretic, and kaliuretic responses were determined by subtraction of baseline values from the renal response during the first 15 min after administration of the atrial natriuretic factor (ANF). The change in renal response observed in rats receiving saline and a bolus injection of an ANF was compared to the change in response observed in the same rats that received the dopaminergic antagonist, domperidone (0.2 microgram/kg, i.v.), and the same ANF. In the case of the renal response following infusion of an ANF, a paired comparison was made between domperidone-treated and untreated rats. Domperidone attenuated the renal response to AP, administered either as a bolus injection or a constant infusion, whereas domperidone significantly reduced renal response to APIII only following constant infusion. The renal response to a bolus injection of AE was not affected by the administration of domperidone. These findings suggest that the renal action of the putative circulating form of ANF, i.e. AP, is mediated via dopaminergic receptors, and that the route of administration may affect the mechanism of action of an ANF.

The presence of atrial natriuretic peptide in canine cerebrospinal fluid and its possible origin in the brain.

The presence of atrial natriuretic peptide (ANP) in canine cerebrospinal fluid (CSF) was clearly demonstrated and an attempt was made to determine its origin as either the brain or the atrium. The concentration of ANP in canine CSF was 0.78 +/- 0.37 pmol/l (n = 31) and showed no evident correlation with that in plasma (r = 0.12). Physiological doses of human alpha-ANP (alpha-hANP) were continuously infused intravenously into nine dogs, and ANP concentrations in CSF and plasma were examined six to eight times within a 120-min period following this. The ANP level in CSF was not influenced by the systemic administration of alpha-hANP up to 180 min. Only one low molecular weight peak corresponding to alpha-hANP could be obtained from the CSF samples, while both low and high molecular weight peaks were observed for plasma ANP by gel permeation chromatography. In the atrial and hypothalamic tissue extracts the same kinds of peaks were also evident. These results prove the presence of ANP in canine CSF and that it does not come from blood that has seeped across the blood-CSF barriers, but suggest that it may originate from the brain.

The heart as an endocrine gland

Two independent series of biomedical investigations have led to the discovery that the atria constitute a peptide-secreting endocrine gland. The first investigation is mainly morphological and started with the finding that mammalian atrial (but not ventricular) cardiocytes contain dense bodies. These dense bodies, later called specific granules, were found to be different from lysosomes; to be made up of proteins; and to incorporate both 3H-leucine and 3H-fucose in a pattern typical of peptide-secreting endocrine cells. The finding that rat atrial granulation varied with the sodium and water balance led to the crucial observation that atrial extracts have natriuretic and diuretic effects. In less than five years, this new natriuretic hormone has been purified, sequenced and synthesized, and its CDNA and gene have been cloned. The atrial natriuretic factor (ANF) gene has been assigned to the distal short arm of chromosome 1 in band 1P36, while the mouse gene is localized in chromosome 4. The native and synthetic hormones exert identical wide ranging effects (possibly through particulate guanylate cyclase stimulation and adenylate cyclase inhibition) on the kidney, blood vessels, adrenal cortex, and pituitary. Physiopathologic implications of the hormone in experimental hypertension, congestive heart failure, and expansion of blood volume are already beginning to emerge. Concurrently, the search for the function of natriuretic hormones or factors (through studies of negative pressure breathing, atrial distension experiments, head-out water immersion, expansion of blood volume, Na+/K+-ATPase inhibition, and parabiosis experiments in Dahl rats) has provided a general framework within which to interpret this new cardiac function.

Renal and hormonal responses to atrial natriuretic peptide and turtle atrial extract in the freshwater turtle, Amyda japonica

AbstractThe effects of synthetic atrial natriuretic peptide and turtle atrial extract on cardiovascular and renal functions were examined in the freshwater turtle, Amyda japonica.Both the atrium and ventricle of the turtle heart contained an immunoreactive atrial natriuretic peptide. Synthetic rat atrial natriuretic peptide (atriopeptin III) and turtle atrial extract caused a dose‐dependent decrease in mean arterial blood pressure. In turtles that received either atriopeptin III or turtle atrial extract, no significant changes in renal function were observed. However, furosemide was effective in altering renal function. Levels of aldosterone in plasma decreased after 2 hours and increased after 24 hours following injection of atriopeptin III, although the plasma renin concentration did not change.We suggest that the heart of the freshwater turtle may contain an immunoreactive atrial natriuretic peptide and that the atrial natriuretic peptide is more important in the regulation of blood pressure than in the regulation of renal function.

Stretch-induced reduction in atrial content of natriuretic factor is locally mediated.

Two groups of male Wistar rats were chronically implanted with small inflatable balloons at the right superior vena caval (SVC)-atrial junction. The balloons were inflated for 60 min in one-third of the rats in each group; the remainder served as noninflated controls. Extract was prepared from the right atria of the first and from the left atria of the second group and bioassayed in male Wistars to determine the content of atrial natriuretic factor (ANF). The bioassay rats received either two injections of sham extract (control) or an initial injection (I1) of sham extract followed by an injection (I2) of balloon-inflated extract (experimental). For right atrial extract, the incremental response to the experimental injection (I2/I1) was significantly less than the response to the control injection (experimental, 1.15 +/- 0.09 mueq/min, n = 14; control, 1.59 +/- 0.15 mueq/min, n = 13; P less than 0.01). However, in the case of left atrial extract, there was no such difference between the control group (1.35 +/- 0.13 mueq/min, n = 19) and the experimental group (1.25 +/- 0.11 mueq/min, n = 11; Student's t test). We conclude that right atrial distension causes release of ANF from the right atrium, but not the left, and that this release is probably locally mediated.

The atrial natriuretic factor: its physiology and biochemistry.

In the history of medical research, few advances have been more rapid — in all aspects — than those in the research on the atrial natriuretic factor since the original observation by de Bold et al. in 1981 of the marked natriuresis, diuresis, and vasorelaxation following the i. v. administration of crude atrial extracts. The atrial factor responsible for these findings has been isolated and sequenced, the cDNA coding for ANF has been cloned, and the gene has been localized on the chromosomal map. Most of its biological activities have been determined, and these clearly provide a balance to the activities of the reninangiotensin system. Many points remain to be elucidated, such as the role of ANF in patients with essential hypertension with congestive heart failure; the participation of ventricular ANF in pathological states such as hypertension and congestive heart failure; the interplay of ANF and angiotensin II in brain regions involved in the regulation of sodium, water, and blood pressure such as the AV3V region and the subfornical organ; the role of ANF in the ciliary bodies of the eyes; the relationship of particulate guanylate cyclase stimulation and adenylate cyclase inhibition with vasorelaxation; the neuromodulatory role of ANF in neural transmission; and many others. Fundamental questions remain to be answered and offer a field for innovative researches.

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 ANP 99–126, thus confirming the structural identity of dog and human ANP.

A potent hypotensive factor in chicken left ventricle.

Chicken artria and ventricles both have membrane-bound granules which resemble those containing atriopeptin (ANP) in mammals. However, nothing is known about the contents of the avian granules. A previous study in chickens showed that although extracts of whole chicken heart or synthetic rat ANP both caused profound hypotension, ANP caused both natriuresis and diuresis, while chicken heart extract did not. The present study sought to locate the region(s) of chicken heart containing the hypotensive activity, and to observe the effect on sodium and water excretion and blood pressure in rats. Acid extracts of either atrium, either ventricle, ventricular septum, skeletal muscle, and liver were identically prepared from chickens and rats. Extracts were adjusted to the same protein concentration and injected (0.15 ml/kg) into anesthetized Single Comb White Leghorn roosters. Mean arterial pressure (MAP) and the time for recovery were measured. The most potent extract from chicken hearts was from the left ventricle (-38 +/- 1 mm Hg, 149 +/- 9 sec to recover). All other extracts (including right ventricle) produced only small (10-20 mm Hg), short-lived (20-30 sec) decreases in MAP. In contrast, only rat atrial extracts evoked long-lasting hypotension (greater than 40 mm Hg, recovery time greater than 200 sec). A 30-min infusion of the most potent chicken extract (left ventricle, CLV) into rats produced a small but significant natriuresis and diuresis compared to the vehicle time control (P less than 0.05) and the hypotensive response to bolus injection was about one-third that seen in the chicken. The location of potent spasmolytic activity primarily in chicken left ventricle, the different avian renal responses to chicken heart extract and synthetic rat ANP (5), and the weak diuretic, natriuretic, and hypotensive effects of CLV extract in rats all suggest that the chicken heart substance may be different from mammalian ANP.

Age-related differences in the natriuretic and hypotensive properties of rat atrial extracts.

The natriuretic, hypotensive, and immunological properties of atrial extracts (AE) prepared from male and female, prepubertal, adult, and old rats were evaluated. Groups of animals were killed at ages 15, 25, 32, 39, 46, 56, and 290 days of age, and the atria were homogenized and extracted in 1.0 M acetic acid. Boiled, lyophilized extracts were stored and subsequently evaluated by bioassay and RIA. No sex differences (P greater than 0.05) were observed in the natriuretic, hypotensive, or immunoreactive properties of AE from rats of identical age. On the basis of bioassay determinations, no significant differences (P greater than 0.05) were observed in the natriuretic activity of AE prepared from rats of different ages. In contrast, AE-induced hypotension was significantly lower in bioassay rats receiving AE from 290-day-old rats than in rats infused with AE from 15-, 39-, or 56-day-old animals. Despite this reduced hypotensive effect, significant differences in RIA or bioassay parallelism were not observed between the tissue extracts and a synthetic atrial natriuretic factor (ANF) standard. Estimation of ANF content revealed that the quantity of ANF per heart as well as per microgram of DNA rose steadily with age. Two independent methods of ANF quantification (i.e. rat bioassay and RIA) yielded different estimates of atrial ANF content except at 15 days of age where the bioassay/RIA ratio was approximately 0.95. At 39, 56, and 290 days of age the ratios were 0.51, 0.44, and 0.53, respectively. A significant age-dependent decline in biological activity was noted when the hypotensive and natriuretic responses were normalized to an equivalent quantity of immunoreactive ANF. On the basis of these findings, we conclude that significant age-dependent differences exist in atrial ANF content and, more importantly, in the biological and immunological activity of rat AE. Since the composition of AE is not well defined, analysis of biological activity solely on the basis of RIA determinations may be insufficient to adequately evaluate the physiological properties associated with these extracts.

Effect of atrial natriuretic peptides on renal medullary solute gradients.

Studies were performed to examine the effect of the atrial natriuretic peptides on the solute gradient of the renal medulla. Experiments were performed in anesthetized male Sprague-Dawley rats given an infusion of either a purified atrial extract (n = 13), atrial natriuretic peptide 8-33 (1 microgram prime followed by 100 ng/min) (n = 8), furosemide (75 micrograms prime and 7.5 micrograms/min, n = 3), or vehicle alone (n = 17). Kidneys were removed 5 min after beginning the infusion, except in four animals, which were infused with atrial extract for 15 min before kidney removal. Both synthetic atrial peptides and an atrial extract result in rapid dissipation of the medullary gradient for urea, sodium, and total solute. No additional loss of solute was apparent at 15 min compared with 5 min. Furosemide caused equally rapid dissipation of the solute gradient at an approximately equidiuretic dose. The fall in tissue concentrations of urea and sodium was attributable to a loss of solute rather than addition of water. Administration of all three agents was associated with an increase in urinary urea excretion, which peaked earlier than the natriuresis. Urinary losses appear to account for a substantial portion of the loss of medullary solute.

On the shoulders of giants: the discovery of atrial natriuretic factor.

Investigations culminating at the beginning of this century clearly established that the cardiac muscle cell (cardiocytes) is differentiated for excitation, conduction, and contraction. All of the physiology and pathophysiology of the heart was developed subsequently based on this concept. However, morphological investigations in the mid 1950s suggested a secretory function for mammalian atrial cardiocytes. These cells contain storage granules, the specific atrial granules, which resemble granules found in polypeptide hormone-producing cells. The development of techniques for the study of these granules using a combined biochemical-morphological approach during the 1970s defined their general chemical nature and their behaviour under different experimental conditions. Because the number of atrial granules change dramatically following upsets of water and electrolyte balance, atrial muscle extracts were tested for effects on kidney function. In 1981, it was reported that atrial extracts contain a natriuretic factor (ANF) capable of inducing massive diuresis, increases in hematocrit, and lowering of blood pressure. It was demonstrated soon thereafter that ANF is stored within specific atrial granules. More recent work has defined ANF as a polypeptide hormone that appears to modulate or antagonize the renin-angiotensin-aldosterone system. Current work attempts to define the physiological and pathophysiological role for ANF as well as possible therapeutic uses.

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.

Decreased ANF atrial content and vascular reactivity to ANF in spontaneous and renal hypertensive rats

The relationship between ANF activity and hypertension was determined by measuring ANF atrial content and vascular reactivity in two different models: spontaneous hypertensive rats (SHR) and renal hypertensive rats (RHR). Atrial extracts and aortic strips were prepared from hypertensive and normotensive animals. Relaxant activities of extracts, synthetic ANF and nitroglycerin were assayed on superfused aortic strips previously contracted by norepinephrine. ANF atrial content was statistically significantly lower in both models of hypertension, presumably by increased ANF release into the circulation which results in depletion of tissue storage sites. Vascular subsensitivity to ANF and nitroglycerin was found in both models of hypertension. Diminished ANF vascular reactivity in hypertension could be due to receptor down-regulation and/or to a decrease in the ability of cGMP to induce relaxation.

Potentiating effect of aldosterone on the diuretic action of atrial extract.

The typical stimulatory effect of a rat heart atrial extract on urinary water, sodium, potassium and kallikrein excretion is significantly increased by a previous administration of aldosterone (0.5 microgram/100 g b. wt) in the rat.

Partial characterization of atrial natriuretic factor in the hearts of Dahl salt-sensitive and salt-resistant rats.

Atria of adult Dahl salt-sensitive rats contain more atrial natriuretic factor (ANF) than those of Dahl salt-resistant rats, as measured by RIA. This strain difference was not seen at 15 days of age, but was observed at and after 30 days of age. Neither a sodium-deficient diet nor an 8% NaCl diet started as early as 2 weeks of age altered this strain difference, although high dietary NaCl was associated with reduced atrial ANF concentration. In addition, no qualitative differences were found in the S and R ANF precursor, as assessed by 1) in vitro translation of atrial mRNA followed by immunoprecipitation of the ANF precursor and polyacrylamide gel electrophoresis, and 2) fractionation of S and R atrial extracts by reverse phase HPLC. These findings support the view that the S vs. R strain difference in atrial ANF concentration is more likely to arise from genetic control of regulatory phenomena than from alterations in the structural part of the ANF gene.

Human atrial natriuretic peptide.

Atrial peptides have been the subject of increasing attention since de Bold demonstrated that atrial extracts, when administered to anaethetized rats, induced a potent natriuretic and diuretic response (de Bold, 1979; de Bold et al., 198 1). Within 3 years, specific peptides with natriuretic and vasorelaxant activities were isolated, identified and synthesized (Flynn et al., 1983; Kangawa & Matsuo, 1984) to allow studies in experimental animals, including man. These studies have lead to the concept that atrial peptides, secreted in response to increased atrial pressure, are specific hormones with a wide range of renal, haemodynamic and endocrine actions which serve to regulate circulatory volume and blood pressure. Since many aspects of atrial peptide chemistry and physiology in experimental animals have been recently reviewed (Cantin & Genest, 1985; Ballerman & Brenner, 1985; Flynn & Davies, 1985; Needleman et al., 1985; Needleman & Greenwald, 1986), the focus of this review is atrial natriuretic peptide in man.

A rapid bioassay for quantification of atrial natriuretic polypeptides.

A quantitative bioassay for the detection and quantification of atrial natriuretic polypeptides (ANPs) was developed in a pentobarbital-anesthetized rat. Ten percent mannitol in 0.9% saline was infused to achieve stable diuresis. The conductivity of the urine, urine flow, and blood pressure were continuously recorded. A bolus injection of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) elicited dose-dependent increases in the urine conductivity, sodium excretion, and urine volume. Changes in the urine conductivity correlated significantly with the increase in sodium excretion. By use of changes in urine conductivity, biological ANP activity of crude rat atrial extract was determined. Atrial contents of ANP in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were 25.5 +/- 1.2 microgram per atrium (n = 4) and 25.1 +/- 0.8 (n = 5) in euhydration. They were increased to 27.0 +/- 1.1 micrograms (n = 4) and 29.3 +/- 1.3 (n = 5, P less than 0.05), after 5-day water deprivation, respectively. This assay procedure provides a good tool for rapid and quantitative determination of ANPs.

Renal response to atrial peptides is reduced in experimental nephrosis.

The aim of this study was to evaluate the renal response to atrial extracts (AE) and synthetic atrial natriuretic factor (ANF) in control rats and in rats with experimental nephrotic syndrome (NS). NS was obtained by a single intravenous injection of adriamycin (7.5 mg/kg). Bolus injection of AE from normal or NS rats resulted in marked increase of diuresis and natriuresis in bioassay control rats (AE from normal rats, urine flow rate, 14.87 +/- 2.94 to 186.18 +/- 55.86 microliters/min; Na excretion, 0.68 +/- 0.26 to 21.80 +/- 5.45 mu eq/min; AE from NS, urine flow rate, 13.49 +/- 4.30 to 167.14 +/- 51.44 microliters/min; Na excretion, 0.98 +/- 0.57 to 20.71 +/- 9.76 mu eq/min). In contrast, blunted diuretic (from 11.26 +/- 3.05 to 65.20 +/- 27.30 microliters/min) and natriuretic (from 0.58 +/- 0.15 to 4.52 +/- 1.59 mu eq/min) effect was observed when AE were injected in rats with NS. Injection of the vehicle in which AE were dissolved or ventricular extracts did not increase urinary flow rate or Na excretion in both control and NS animals. Bolus injection of synthetic ANF (Arg-101-Tyr-126) induced marked diuretic and natriuretic response in control but not in NS rats. Similar results were obtained when AE were infused by constant infusion in control or in NS bioassay rats. AE given by constant infusion induced comparable increase in glomerular filtration rate (GFR) over basal values both in control and NS animals (controls, 39%; NS rats, 40%).(ABSTRACT TRUNCATED AT 250 WORDS)

Development, validation and application of an enzyme immunoassay (EIA) of atriopeptin

A rapid, convenient, and sensitive enzyme immunoassay (EIA) for atriopeptin (AP) has been developed. The tracer-ligand for the assay is the 24-amino acid peptide, AP24, which has been covalently coupled to the tetrameric form of acetylcholinesterase (AChE) (EC 3.1.1.7). Tracer, unknown, and primary antibody are incubated in a 96-well microtiter plate precoated with secondary antibody. After washing, a colorimetric reaction is used to measure acetylcholinesterase activity. A direct linear correlation was obtained when comparing the conventional radioimmunoassay and the EIA by using the same primary antibody to assay: plasma samples (rat or human), HPLC column fractions, or atrial extracts. Besides being technically much less demanding and not requiring the use of the radioisotopes, the EIA is more sensitive than the radioimmunoassay and thereby lends itself to a "flash" same-day assay of samples.

Atrial natriuretic peptide inhibits angiotensin-stimulated proximal tubular sodium and water reabsorption.

The discovery that atrial extracts have potent diuretic and natriuretic properties revealed a possible endocrine function of the heart in the regulation of extracellular fluid volume. Since that first report intense research activity has been directed towards determining the mechanism of action of the active atrial natriuretic polypeptides (ANP) found in these extracts. Despite these efforts it remains controversial whether the renal actions of ANP are exerted solely on the process of glomerular filtration or involve additional direct actions on tubular transport. We have investigated the possibility that atrial natriuretic polypeptides may induce natriuresis by suppression of proximal tubular sodium and water reabsorption. Using shrinking split-drop micropuncture combined with simultaneous capillary perfusion in anaesthetized rats we report that 20 nanomolar alpha-rANP (the main component of ANP in rat plasma) added to the peritubular fluid had no direct effect on proximal fluid uptake whereas picomolar angiotensin II had a marked stimulatory action as reported. The stimulatory effect of angiotensin II on fluid reabsorption was inhibited by peritubular ANP at physiological concentrations and abolished by higher concentrations of ANP. Thus at physiological concentrations ANP acts within the kidney to decrease proximal reabsorption by inhibition of angiotensin-stimulated sodium and water transport.