General Cardiology, Basic Research
Cardiology 1992;80:106-111
Department of Medicine, University of British Columbia, Vancouver, Canada
Changes in Atrial Natriuretic Factor Processing and Secretion with Age
KeyWords
Abstract
ANF Secretion Processing Molecular forms Age
The effect of age on atrial natriuretic factor (ANF) metabolism in the rat was studied. Plasma ANF levels rose with age. A positive correlation was found between age and plasma ANF levels. Molecular forms of ANF were also examined. a-ANF and y-ANF were present in the circulation. The ratio of yANF/a-ANF increased with age. The atria were isolated and perfused to determine the ANF secretion rate. The sponta neous release of ANF decreased with age. The released ANF mainly consisted of a-ANF with a small amount of y-ANF. The ratio of y-ANF/a-ANF in the effluent increased with age. ANF concentrations in the atria varied with age. but the molecular forms did not. y-ANF was the main form found in the atria. These results suggest that ANF secretion as well as post-transcriptional processing is altered with age.
Introduction
ANF occurs. Conversion may take place either during or immediately after release. Isolated perfused heart studies showed that the main form of ANF in the effluent was aANF. This suggested that processing occurred in the heart. Recent studies have shown that plasma ANF levels are elevated with age [6-8]. It was also shown that expansion of extracellular fluid volume caused a greater increase in plasma ANF concentrations in the elderly
Atrial natriuretic factor (ANF) is a potent natriuretic, diuretic and hypotensive polypep tide hormone synthesized and secreted by mammalian atrial myocyte [1, 2], The main form of ANF in the rat atria is the 126-amino acid precursor y-ANF [3], ANF found in the plasma is mainly the 28-amino acid a-ANF with a small amount of y-ANF [4, 5]. It is not clear where the conversion of y-ANF to a-
Received: August 15, 1991 Accepted: August 22, 1991
Dr. Norman L.M. Wong Department o f Medicine U ni versity Hospital - U BC Site 2211 Wesbrook Mall Vancouver, B.C. V6T 2B5 (Canada)
© 1992 S. Karger AG. Basel 0008-6312/92/ 0802-010652.75/0
Downloaded by: Vanderbilt University Library 129.59.95.115 - 1/14/2019 3:20:12 AM
Jonathan Kao Eric F.C. Wong Norman L.M. Wong
than in young men [7], In addition, intrave nous administration of a-ANF produced ex aggerated hypotensive, diuretic, natriuretic and calciuretic responses in the elderly [9], These reports suggest changes in ANF homeo stasis in the aged individuals. The aim of the present study was to exam ine the effect of age on ANF homeostasis.
Methods One- to 20-month-old male Wistar rats were stud ied. The animals were anesthetized with sodium pen tobarbital (25 mg/kg i.p.). A small blood sample was taken from the abdominal aorta for the determination of plasma ANF. The blood was taken into a prcehilled EDTA tube. To determine ANF secretion rate the right atrium was rapidly removed and superfused in a modi fied Langendorff apparatus as described before [10]. The atria were superfused with Tyrode solution at 0.5 ml/min for 60 min. to stabilize the hormone secre tion rate before the effluents were collected at 5-min intervals for 100 min. The effluent was collected with a fraction collector (LKB Bromma, Sweden) and stored at - 2 0 °C until assayed. Plasma ANF and ANF con tents in the atria were measured as before [ 10]. Reverse-phase high-performance liquid chroma tography was used for the determination of the molec ular forms of ANF found in plasma, effluent and atrial tissue. A Whatman Partisil ODS-3 CIS column was used. The column was first equilibrated with 10% ace tonitrile (CH'.CN) in 0.1 % trifluoroacetic acid (TFA). The gradient elution was carried out over 55 min using a linear gradient (slope 0.4%/min) of 19-40% CHjCN in 0.1 % TFA for the first 24 min and then at a constant
A§c
HPLC and RIA One month Three months Ten months Twenty months
Results
The effect of age on plasma ANF levels is shown in table I and illustrated in figure 1. Plasma ANF rose with age. A significant posi tive correlation was found between age and plasma ANF. These results suggest that age caused an elevation in plasma ANF levels.
Plasma, pg/fraction
Perfusate, pg/fraction
a-ANF
a-ANF
y-ANF
143 ±0.8 27 ± 1 7.3±0.8 11 ±0.5
0.6 ±0.3 1.01 ±0.4 1.7 ±0.6 8 ±2**
y-ANF
8 ±2 58 ±5 14 ± 4 74 ±8 91 ±10** 23 ±6* 111± 7** 68 ±6**
* p < 0.01, ** p < 0.05, compared to 1 month.
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Table 1. a-ANF and y-ANF concentrations determined by
60% CH3CN in 0.1 % TFA for the remaining period. The column was maintained at 30 °C (Shimadzu model CTO-6A oven, Kyoto. Japan). The column pressure ranged from 90 to 120 kgf/cm-. The flow rate was maintained at I ml/min (Shimadzu model LC-6A pumps). The column effluent was monitored at 215 nm (Shimadzu model SPD-6AV spectrophotometric detector) and collected at 1-min intervals (LKB fraction collector, Bromma. Sweden). The effluents were iyophilized and then resuspended in RIA bulTcr before analyzing the ANF content by a radioimmu noassay method. Calibration was done against syn thetic a-ANF (Peninsula Laboratory. Belmont, Calif.. USA), synthetic |)-ANF (Peninsula Laboratory') and yANF purified from rat atria using the method of Kangawa ct al. [ 11 ]. ANF levels w'ere measured by radioimmunoassay. ANF antibody bought from Peninsula Laboratory was used. The sensitivity of the assay was at least 2 pg ANF/tube at more than 95% recovery'. Inter- and intra-assay variability was 10 and 6%, respectively. The results are expressed as means ± SEM. Linear regression analysis was used to determine correlations between variables. Statistical analysis was performed using analysis of variance and unpaired Student’s t test.
P lasm a IR -A N F (pg/m l)
Fig. 1. Relationship between plasma ANF levels and age. Data obtained from rats with various age showed a positive correlation be tween plasma ANF levels and age (y = 0.15x + 78. r = 0.59, p
Cardiology 1992;80:106-111
Department of Medicine, University of British Columbia, Vancouver, Canada
Changes in Atrial Natriuretic Factor Processing and Secretion with Age
KeyWords
Abstract
ANF Secretion Processing Molecular forms Age
The effect of age on atrial natriuretic factor (ANF) metabolism in the rat was studied. Plasma ANF levels rose with age. A positive correlation was found between age and plasma ANF levels. Molecular forms of ANF were also examined. a-ANF and y-ANF were present in the circulation. The ratio of yANF/a-ANF increased with age. The atria were isolated and perfused to determine the ANF secretion rate. The sponta neous release of ANF decreased with age. The released ANF mainly consisted of a-ANF with a small amount of y-ANF. The ratio of y-ANF/a-ANF in the effluent increased with age. ANF concentrations in the atria varied with age. but the molecular forms did not. y-ANF was the main form found in the atria. These results suggest that ANF secretion as well as post-transcriptional processing is altered with age.
Introduction
ANF occurs. Conversion may take place either during or immediately after release. Isolated perfused heart studies showed that the main form of ANF in the effluent was aANF. This suggested that processing occurred in the heart. Recent studies have shown that plasma ANF levels are elevated with age [6-8]. It was also shown that expansion of extracellular fluid volume caused a greater increase in plasma ANF concentrations in the elderly
Atrial natriuretic factor (ANF) is a potent natriuretic, diuretic and hypotensive polypep tide hormone synthesized and secreted by mammalian atrial myocyte [1, 2], The main form of ANF in the rat atria is the 126-amino acid precursor y-ANF [3], ANF found in the plasma is mainly the 28-amino acid a-ANF with a small amount of y-ANF [4, 5]. It is not clear where the conversion of y-ANF to a-
Received: August 15, 1991 Accepted: August 22, 1991
Dr. Norman L.M. Wong Department o f Medicine U ni versity Hospital - U BC Site 2211 Wesbrook Mall Vancouver, B.C. V6T 2B5 (Canada)
© 1992 S. Karger AG. Basel 0008-6312/92/ 0802-010652.75/0
Downloaded by: Vanderbilt University Library 129.59.95.115 - 1/14/2019 3:20:12 AM
Jonathan Kao Eric F.C. Wong Norman L.M. Wong
than in young men [7], In addition, intrave nous administration of a-ANF produced ex aggerated hypotensive, diuretic, natriuretic and calciuretic responses in the elderly [9], These reports suggest changes in ANF homeo stasis in the aged individuals. The aim of the present study was to exam ine the effect of age on ANF homeostasis.
Methods One- to 20-month-old male Wistar rats were stud ied. The animals were anesthetized with sodium pen tobarbital (25 mg/kg i.p.). A small blood sample was taken from the abdominal aorta for the determination of plasma ANF. The blood was taken into a prcehilled EDTA tube. To determine ANF secretion rate the right atrium was rapidly removed and superfused in a modi fied Langendorff apparatus as described before [10]. The atria were superfused with Tyrode solution at 0.5 ml/min for 60 min. to stabilize the hormone secre tion rate before the effluents were collected at 5-min intervals for 100 min. The effluent was collected with a fraction collector (LKB Bromma, Sweden) and stored at - 2 0 °C until assayed. Plasma ANF and ANF con tents in the atria were measured as before [ 10]. Reverse-phase high-performance liquid chroma tography was used for the determination of the molec ular forms of ANF found in plasma, effluent and atrial tissue. A Whatman Partisil ODS-3 CIS column was used. The column was first equilibrated with 10% ace tonitrile (CH'.CN) in 0.1 % trifluoroacetic acid (TFA). The gradient elution was carried out over 55 min using a linear gradient (slope 0.4%/min) of 19-40% CHjCN in 0.1 % TFA for the first 24 min and then at a constant
A§c
HPLC and RIA One month Three months Ten months Twenty months
Results
The effect of age on plasma ANF levels is shown in table I and illustrated in figure 1. Plasma ANF rose with age. A significant posi tive correlation was found between age and plasma ANF. These results suggest that age caused an elevation in plasma ANF levels.
Plasma, pg/fraction
Perfusate, pg/fraction
a-ANF
a-ANF
y-ANF
143 ±0.8 27 ± 1 7.3±0.8 11 ±0.5
0.6 ±0.3 1.01 ±0.4 1.7 ±0.6 8 ±2**
y-ANF
8 ±2 58 ±5 14 ± 4 74 ±8 91 ±10** 23 ±6* 111± 7** 68 ±6**
* p < 0.01, ** p < 0.05, compared to 1 month.
107
Downloaded by: Vanderbilt University Library 129.59.95.115 - 1/14/2019 3:20:12 AM
Table 1. a-ANF and y-ANF concentrations determined by
60% CH3CN in 0.1 % TFA for the remaining period. The column was maintained at 30 °C (Shimadzu model CTO-6A oven, Kyoto. Japan). The column pressure ranged from 90 to 120 kgf/cm-. The flow rate was maintained at I ml/min (Shimadzu model LC-6A pumps). The column effluent was monitored at 215 nm (Shimadzu model SPD-6AV spectrophotometric detector) and collected at 1-min intervals (LKB fraction collector, Bromma. Sweden). The effluents were iyophilized and then resuspended in RIA bulTcr before analyzing the ANF content by a radioimmu noassay method. Calibration was done against syn thetic a-ANF (Peninsula Laboratory. Belmont, Calif.. USA), synthetic |)-ANF (Peninsula Laboratory') and yANF purified from rat atria using the method of Kangawa ct al. [ 11 ]. ANF levels w'ere measured by radioimmunoassay. ANF antibody bought from Peninsula Laboratory was used. The sensitivity of the assay was at least 2 pg ANF/tube at more than 95% recovery'. Inter- and intra-assay variability was 10 and 6%, respectively. The results are expressed as means ± SEM. Linear regression analysis was used to determine correlations between variables. Statistical analysis was performed using analysis of variance and unpaired Student’s t test.
P lasm a IR -A N F (pg/m l)
Fig. 1. Relationship between plasma ANF levels and age. Data obtained from rats with various age showed a positive correlation be tween plasma ANF levels and age (y = 0.15x + 78. r = 0.59, p
