Secretory form of atrial natriuretic polypeptide as cardiac hormone in humans and rats

To elucidate the secretory form of atrial natriuretic polypeptide from the atrium, the molecular form of atrial natriuretic polypeptide in the perfusate from the isolated beating rat heart and in plasma taken at the coronary sinus of 10 patients during cardiac catheterization has been investigated using high performance gel permeation chromatography and reverse phase high performance liquid chromatography coupled with radioimmunoassay for atrial natriuretic polypeptide. Atrial natriuretic polypeptide in the perfusate from the rat heart showed a single peak eluting at the position of a low molecular weight form of atrial natriuretic polypeptide, without any detectable amounts of atrial natriuretic polypeptide with high molecular weights. The major component of atrial natriuretic polypeptide in the rat heart perfusate co-migrated with rat alpha-atrial natriuretic polypeptide in reverse phase high performance liquid chromatography. In 9 out of 10 patients atrial natriuretic polypeptide in plasma taken at the coronary sinus revealed a single peak of atrial natriuretic polypeptide emerging at the position of human alpha-atrial natriuretic polypeptide in gel filtration. Only one plasma sample had a small quantity of high molecular weight forms with the predominant low molecular weight form of atrial natriuretic polypeptide. The major component of atrial natriuretic polypeptide in the plasma extract from the coronary sinus was identified with human alpha-atrial natriuretic polypeptide. These results indicate that alpha-ANP, a 28-amino acid polypeptide, is secreted as a cardiac hormone into the coronary blood stream from the atrium.     

Alpha-human atrial natriuretic polypeptide is released from the heart and circulates in the body

In order to clarify whether or not atrial natriuretic polypeptides are hormones in man, we have measured plasma alpha-human atrial natriuretic polypeptide (alpha-hANP)-like immunoreactivity (alpha-hANP-LI) with or without extraction procedure. alpha-hANP-LI was detected in plasma extracts from all 5 normal subjects and 7 patients with heart diseases. The alpha-hANP-LI concentration in normal peripheral plasma was 37.7 +/- 7.0 pg/ml (mean +/- SE). Plasma concentrations of alpha-hANP-LI in the coronary sinus obtained by cardiac catheterization were 3 to 10 times higher than those in the peripheral vein, inferior vena cava, right atrium, pulmonary artery and aorta. High performance gel permeation chromatography coupled with a radioimmunoassay (RIA) for alpha-hANP revealed that alpha-hANP-LI in normal peripheral plasma eluted at the position corresponding to that of authentic alpha-hANP without detectable amounts of high molecular weight forms. alpha-hANP-LI extracted from plasma taken from the coronary sinus of two patients also showed a single peak of alpha-hANP-LI co-eluting with alpha-hANP. In contrast, not only alpha-hANP but gamma-hANP and beta-hANP, high molecular weight forms, were present in the human atrial tissue. These results indicate that alpha-hANP is the predominant form of alpha-hANP-LI in human plasma and that this form generated in the atrial cardiocytes is preferentially released from these cells and circulates in the body.     

A potential diagnostic reagent for bovine cysticercosis

A fraction of larval Taenia hydatigena cyst fluid was shown to have high sensitivity and specificity in the enzyme-linked immunosorbent assay (ELISA) for the detection of bovine antibodies to the heterologous parasite Taenia saginata. This antigenically active lipoprotein fraction was isolated by ultracentrifugal density flotation using either ammonium sulfate (specific gravity = 1.231 g per ml) or NaCl/KBr (specific gravity = 1.225 g per ml), followed by ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that this fraction was composed of high molecular weight (65,000 to 77,000 Mr) and low molecular weight (9,500 to 16,000 Mr) proteins. Electrophoresis under non-denaturing conditions in either acrylamide (5%) or agarose (1%) resulted in 1 major diffuse band staining for both protein and lipid. The high and low molecular weight proteins observed on SDS-PAGE under reducing conditions could not be resolved by gel filtration chromatography and emerged as a single lipoprotein peak. This T. hydatigena cyst fluid fraction appears promising as a diagnostic reagent in the ELISA for bovine cysticercosis.     

Presence of the atrial natriuretic peptide in human cerebrospinal fluid

Using a highly sensitive and specific radioimmunoassay (RIA) for detection of the atrial natriuretic peptide (ANP), the presence of alpha-human ANP (alpha-hANP) in human cerebrospinal fluid (CSF) was confirmed. Its concentration in CSF, 3.6 +/- 2.3 pg/ml, n = 16, mean +/- SD, was remarkably lower than that in the plasma (161.8 +/- 157.4, p less than 0.0001). The regression coefficient between these concentrations was 0.320 (p = ns). Gel permeation chromatography conducted in conjunction with RIA indicated ANP in CSF to be eluted at the position of a low molecular weight form corresponding to alpha-hANP. No high molecular weight form could be detected. But in the plasma, both low and high molecular forms were found to be present. It is thus evident that ANP is present in human CSF and its origin may possibly be the brain and not the atrium.     

Epicardial release of immunoreactive atrial natriuretic peptides in inside-out perfused rabbit atria

An easy and convenient isolated atrial perfusion technique was developed. The effect of stretch of the atrial subpericardial myocytes was investigated in the inside-out perfused rabbit atria. Graded distension of the inverted atria was induced by changing the elevation of the atrial catheter tip. Intra-luminal volume expansion resulted in an increase in release of immunoreactive atrial natriuretic peptides (irANPs). The response was volume, or pressure dependent. Distension-induced release of irANPs occurred at the reduction of the distension. IrANPs in epicardial perfusate showed both high and low molecular weights. The major peak of irANP was observed at the corresponding fraction to the rat ANP-(1-28) in the Sephadex G-50 gel chromatography. The data suggest that the epicardial release of irANP is stretch-induced response and that the release may be involved in the regulation of cardiac function.     

Existence of atrial natriuretic polypeptide in kidney

Using a specific radioimmunoassay (RIA) for alpha-atrial natriuretic polypeptide (alpha-ANP), we have demonstrated the presence of alpha-rat ANP-like immunoreactivity (alpha-rANP-LI) in the rat kidney which is considered to be a target organ for atrial natriuretic polypeptides released from the heart. Most of alpha-rANP-LI was localized in the cortex. High performance gel permeation chromatography coupled with the RIA revealed that renal alpha-rANP-LI was eluted at the position of a low molecular weight form corresponding to alpha-rANP without detectable amounts of high molecular weight forms. This is in contrast to the observation that gamma-rANP, a high molecular weight form of 13k daltons, is the dominant form of alpha-rANP-LI in the rat atrium. In water-deprived rats, the concentration and content of alpha-rANP-LI in the kidney showed a significant decrease compared with control rats. In addition, the alpha-rANP-LI concentration and content in this organ revealed a substantial decrease after perfusion with physiological saline. These results indicate the existence of atrial natriuretic polypeptide (ANP) in the kidney and suggest that part of renal ANP may originate from the heart.     

Kallikrein activation of a high molecular weight atrial peptide

Mammalian atrial extracts contain bioactive peptides that exert profound effects upon renal function and isolated smooth muscle preparations. Gel filtration chromatography of rat atrial extract separates the activity into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. Mild proteolytic treatment (trypsin 1 U/ml) of the high molecular weight fraction enhances the smooth muscle relaxant activity of this fraction and concomitantly reduces the apparent molecular weight of this fraction to less than 10,000. In this report we show that urinary and submaxillary kallikrein enhances the activity of rat atrial extracts in a similar fashion. Pretreatment of the high molecular weight fraction with either kallikrein (1 microgram/ml) enhances the smooth muscle relaxant activity of this fraction. Similar treatment of the low molecular weight fraction had no effect. The enhancement of the bioactivity of the high molecular weight substance(s) by the kallikreins was abolished by aprotinin but was unaffected by soybean trypsin inhibitor. These results suggest that exogenous addition of tissue kallikrein activates a high molecular weight peptide by limited proteolysis. Analysis of the kallikrein-treated high molecular weight peptide fraction by gel filtration indicates that the biological activity comigrates with the low molecular weight peptides present in the original atrial extract.     

The biological and immunological properties of fractionated atrial extracts from young and old rats

We have previously reported that the biological activity of rat atrial extract declines with age. The present study was undertaken to further evaluate the natriuretic, hypotensive and immunological properties of fractionated and HPLC purified atrial extracts prepared from young and old rats. Acetic acid extracts were prepared and subsequently fractionated by gel permeation chromatography. The high (greater than 10,000 daltons) and low (less than or equal to 10,000 daltons) molecular weight fractions were collected, lyophilized and assayed. Radioimmunoassay competitive binding curves of the initial and fractionated extracts were parallel (p greater than 0.05) to the synthetic ANP standard. No differences in parallelism (p greater than 0.05) were observed in the natriuretic activity of the initial extracts, the low molecular weight (LMW) fractions from both age groups, the 290 day high molecular weight (HMW) fraction or the synthetic ANP standard. However, the natriuretic activity of the 15 day HMW fraction was significantly attenuated compared to the other treatment groups (p less than 0.05). The initial 15 day extract was also significantly more hypotensive than the 290 day extract (p less than 0.05). HMW extracts were subjected to HPLC and the resulting immunoreactive ANP peak was reassayed. Based on SDS-PAGE and immuno blot analysis, the HPLC purified fraction was found to contain only immunoreactive proANP. Subsequent bioassay revealed greater hypotension and reduced natriuretic activity in the 15 day proANP fraction in comparison to a similarly prepared extract from older animals. Thus, we conclude that qualitative differences in the biological properties of atrial extracts may be ascribable to age-related changes in the composition of proANP or to other undefined biologically active atrial substance(s).     

Nature of atrial natriuretic polypeptide in rat brain

Using reverse phase high performance liquid chromatography (RP-HPLC) coupled with two radioimmunoassays for atrial natriuretic polypeptide (ANP) with different specificities, we investigated the nature of alpha-rat ANP-like immunoreactivity (alpha-rANP-LI) with a low molecular weight in the rat brain. Two major peaks with alpha-rANP-LI in the extract from rat whole brains were eluted in the vicinity of the elution position of synthetic alpha-rANP, a 28-amino acid polypeptide. These two components co-migrated with synthetic alpha-rANP (4-28) and alpha-rANP (5-28), respectively. The peak corresponding to alpha-rANP (4-28) was the highest, and only a little alpha-rANP-LI was detected at the elution position of alpha-rANP. An identical profile in RP-HPLC was also observed in the extract from the rat hypothalamus. These results indicate that the major components of alpha-rANP-LI with a low molecular weight in the rat brain are alpha-rANP (4-28) and alpha-rANP (5-28).     

Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells.

DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.     

Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

ADP-ribosylation of isolated rat islets of Langerhans

A rapid and reproducible radioimmunoassay method was developed for rat atrial natriuretic factor (ANF)-IV. The method is also applicable to human atrial peptide. ANF was detected in rat hypothalamus (5.03 pmoles/g tissue), right (86.8 pmoles/mg tissue) and left atria (52.5 pmoles/mg tissue), and plasma (156 fmoles/ml). After high salt intake immunoreactive ANF in atria and plasma increased significantly, while a significant decrease was observed in hypothalamus. Gel chromatography revealed high and low molecular weight ANF in atria and hypothalamus while only a low molecular weight form was found in plasma.     

Locally different role of atrial natriuretic peptide (ANP) in the pericardial fluid

Pericardial fluid (PF) contains several vasoactive agents in higher concentrations than venous plasma (VP). However, with human atrial natriuretic peptide (ANP) controversial data have been reported in earlier studies performed on a limited number of patients (less than 20). The present study was designed to characterize the ANP levels in human PF and cardiac tissues, and to ascertain whether myocardial ischemic state is a major factor in determining ANP production of the human heart. In a total of 316 consecutive patients undergoing open heart surgery ANP levels in VP, PF, atrial and ventricular tissues were measured by radioimmunoassay and analyzed by high-performance liquid chromatography (HPLC). The data are presented as median and 25th-75th percentiles. Our results showed ANP concentration [ANP] of PF significantly exceeded that of VP and [ANP] in the atrial tissue was significantly higher than in the ventricular tissue (p < 0.001). In patients without myocardial ischemia (valvular heart disease) [ANP] in the PF was 258.3 (189.9-342.5) pg/ml, in the VP 28.4 (11.7-57.6) pg/ml and 151.7 (78.4-447.6) ng/mg in the atrial, 0.4 (0.2-1.6) ng/mg in the ventricular tissue. The corresponding values for patients with coronary artery disease were 208.1 (153.8-318.9) pg/ml in the PF, 19.8 (9.4-27.9) pg/ml in the VP, 129.6 (66.5-455.0) ng/mg in the atrial and 1.0 (0.1-1.8) ng/mg in the ventricular tissue. The ventricular tissue levels correlated to the atrial tissue levels (r = 0.317; p < 0.05). Great difference (p < 0.001) was found in the atrial tissue levels between females [414.6 (119.7-734.4) ng/mg] and males [105.4 (65.3-204.2) ng/mg]. In HPLC analysis the majority of the pericardial fluid and tissue ir-ANP coeluted with human ANP [99-126]. In conclusion, [ANP] in PF of cardiosurgical patients is higher by an order of magnitude than in VP. Intrapericardial ANP may reflect the peptide concentration in the myocardial interstitium and may represent a paracrine regulatory mechanism, which seems independent of ANP-induced putative antiischemic influences.     

Digitalis Does not Improve Left Atrial Mechanical Dysfunction After Successful Electrical Cardioversion of Chronic Atrial Fibrillation

This study was designed to investigate whether administration of digitalis could improve mechanical function of left atrial appendage (LAA) and left atrium prospectively in patients with atrial stunning. Fifty-four consecutive patients in whom atrial stunning was observed immediately after cardioversion of chronic atrial fibrillation (AF) were randomized into digitalis or control group for 1 week following cardioversion. Transthoracic echocardiography (TTE) and transesophageal echocardiography (TEE) were performed prior to, immediately following, 1 day after and 1 week after cardioversion to measure transmitral flow velocity and LAA flow velocity. Electrical cardioversion of AF elicited significantly slower left atrial appendage peak emptying velocity (LAA-PEV) and peak filling velocity (LAA-PFV) immediately following cardioversion in both groups. 1 day post cardioversion, there were no significant differences in transmitral E wave, A wave, E/A ratio, LAA-PEV, LAA-PFV or left atrial appendage ejection fraction (LAA-EF) between digitalis and control groups. 1 week post cardioversion, no significant differences were found in transmitral E wave, A wave, E/A ratio, LAA-PEV, LAA-PFV or LAA-EF between the two groups. The occurrence rates of spontaneous echo contrast were not significantly different between digitalis and control groups one day and one week post cardioversion. In conclusion, digitalis did not improve left atrial and appendage mechanical dysfunction following cardioversion of chronic AF. Digitalis did not prevent the development of spontaneous echo contrast in left atrial chamber and appendage. This may be due to the fact that digitalis aggravates intracellular calcium overload induced by chronic AF and has a negative effect on ventricular rate.     

Distribution and molecular forms of brain natriuretic peptide in porcine heart and blood

Although brain natriuretic peptide (BNP) is a novel natriuretic peptide originally identified in porcine brain, recent investigation has verified the presence of BNP in porcine heart. In order to identify BNP as a circulating hormone, we analyzed the regional distribution and molecular form of immunoreactive (ir-) BNP in heart and blood. Tissue concentration of ir-BNP was high in atrium, but low in ventricle, in a manner similar to that of atrial natriuretic peptide (ANP). However, the concentration of ir-BNP in atrium was only about 1/50 that of ir-ANP. In plasma, ir-BNP was found at a concentration of 1-3 fmol/ml, which was about 1/20 that of ir-ANP. Both ir-BNP and ir-ANP were present as low molecular weight forms. Three forms of ir-BNP of about 3K daltons, including BNP-26, BNP-29 and BNP-32, are thought to circulate in blood.     

Molecular distribution of zinc in biliary and pancreatic secretions

Rat bile and pancreatic fluid were examined for the presence of low molecular weight zinc complexes. Fluids were collected separately by cannulation, and zinc distribution in collected samples was analyzed by gel filtration on Sephadex G-50. Most of the zinc in bile was associated with low molecular weight zinc complexes; only a small amount of zinc was present in the high molecular weight fraction. In contrast, pancreatic secretions did not contain low molecular weight zinc complexes, but there were considerable amounts of zinc bound to high molecular weight compounds. The addition of zinc to bile resulted in an increased amount of zinc in the low molecular weight fraction, while the addition of zinc to pancreatic fluid resulted primarily in an increase in zinc bound to the high molecular weight components. Like pancreatic fluid, homogenates of pancreatic tissue had no low molecular weight zinc complex. In rats whose bile and pancreatic fluid were removed and not returned into the intestine, the amount of zinc bound to low molecular weight complexes in intestinal homogenates was reduced. This alteration of the molecular distribution of zinc in intestinal homogenates by removal of bile and pancreatic fluid suggests the potential importance of low molecular weight zinc complexes for zinc homeostasis.     

Enhanced accumulation of pericardial fluid adenosine and inosine in patients with coronary artery disease.

Adenosine and inosine are believed to have cardioprotective effects. However, little is known about their possible role in the metabolic autoregulation of human coronaries and in pathologic conditions with supply/demand imbalance of the heart such as coronary artery disease. Since these low molecular weight nucleosides freely diffuse through the monolayer of the visceral pericardium, adenosine and inosine concentrations in pericardial fluid may well reflect the conditions in cardiac interstitium. The pericardial fluid and systemic venous blood adenosine and inosine concentrations were measured in 98 human subjects undergoing heart surgery for coronary artery disease or valvular heart disease. Adenosine and inosine concentrations were measured by HPLC with UV detection. In subjects with coronary artery disease pericardial fluid nucleoside concentrations were significantly higher than in patients with valvular heart disease (adenosine: 1545 (996-3146) nmol/L [median (25th-75th quartiles)] vs. 738 (390-2527) nmol/L, P<0.01; inosine: 658 (321-1331) nmol/L vs. 347 (159-1037) nmol/L, P<0.05), while in both patient groups pericardial fluid nucleoside concentrations were higher by an order of magnitude than in venous plasma. Our results show the enhanced release of adenosine and inosine by the ischemic myocardium as a marker of supply/demand imbalance and support the hypothesis that these cardiac nucleosides may have an important role in the adaptation of coronary blood flow in human coronary artery disease.     

Variability of the Left Atrial Appendage in Human Hearts

Atrial fibrillation increases the risk of thrombus formation. It is commonly responsible for cerebral stroke whereas less frequently for pulmonary embolism. The aim of the study was to describe the morphology of the left atrial appendage in the human heart with respect to sex, age and weight. Macroscopic examination was carried out on 100 left appendages taken from the hearts of the patients aged 18–77, both sexes. All hearts preserved in 4% water solution of formaldehyde carried neither marks of coronary artery disease nor congenital abnormalities. Three axes of appendage orientation were performed. After the appendage had been cut off, morphological examination was performed in long and perpendicular axes. Measurements of the appendages were taken from anatomical specimens and their silicone casts. We classified the left atrial appendage into 4 morphological groups according to the number of lobes. Most left atrial appendages in female population were composed of 2 lobes. In the male group typically 2 or 3-lobed appendages were observed. The mean left atrial appendage orifice ranged from 12.0 to 16.0 mm and the most significant difference in the orifices between males and females was observed in LAA type 2 (about 3.3 mm). A smaller orifice and narrower, tubular shape of the LAA lobes could explain a higher risk of thrombus formation during nonvalvular atrial fibrillation in women. Knowledge of anatomical variability of the LAA helps diagnose some undefined echoes in the appendage during transesophageal echocardiographic examination.     

Immunoreactive atrial natriuretic factor (IR-ANF) in human plasma

A direct radioimmunoassay for ANF in human plasma was developed. A synthetic alpha-human atrial peptide (Ser 99-Tyr 126) was used for preparation of the iodinated tracer and the standards. The sensitivity of the method is 1.9 pg/ml. Concentration of immunoreactive ANF (IR-ANF) in plasma of 59 clinically normal subjects was 65.3 +/- 2.5 pg/ml (mean +/- SE). In two patients who underwent atrial pacing an increase of about 100 percent in circulating IR-ANF was observed. IR-ANF was extracted from human plasma by Vycor glass and purified by HPLC. The main immunoreactive isolated peak contained a low molecular weight peptide.     

Atriopeptin release from the isolated perfused rabbit heart

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (< 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.