Regulator), Peptides, 39 (1992) 67-81
67
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00
REGPEP 01174
Right atrial predominance of atrial natriuretic peptide secretion in isolated perfused rat atria K y u n g H w a n Seul, K y u n g W o o Cho and Suhn H e e Kim Department of Physiology, Jeonbug National University, Medical School, Jeonju (South Korea) (Received 23 September 1991; revised version received 6 February 1992; accepted 7 February 1992)
Key words: Atrial natriuretic peptide; ANF; Distension-reduction; Stretch; Secretion; Atria
Summary In order to investigate the regulatory mechanism for the atrial release of atrial natriuretic peptide (ANP), a perfused rabbit atrial model was devised. In the present experiments, the effect of a reduction in atrial distension on the immunoreactive ANP (irANP) secretion was investigated and compared in the perfused right and left atria of rats. Elevations in right and left atrial pressure resulted in proportional increases in the volume of atrial distension-reduction which was larger in the right than in the left atria. The basal rate of irANP secretion was higher in the right than in the left atria. Increases in the volume of atrial distension-reduction resulted in proportional increases in irANP secretion in both atria. Increment in irANP secretion in response to a reduction in atrial distension was significantly higher in the right than in the left atria. Higher rate of irANP secretion in response to unit volume change was observed in the right atria. Increases in the volume of atrial distension-reduction resulted in accentuated irANP responses in the right atrium. IrANP content was significantly higher in the right than in the left atria. The results suggest that the right atrium is a predominant site in ANP secretion in rats.
Correspondence: K.W. Cho, Department of Physiology, Jeonbug National University, Medical School, 220, Keum-Am-Dong-San, Jeonju 560-180, South Korea.
68 Introduction
Atrial myocytes contain a family of biologically active peptides stored in specific atrial granules [1]. These peptides, termed atrial natriuretic peptides (ANPs), have potent natriuretic, diuretic and vascular smooth muscle relaxant activities. Atrial distension increases ANP secretion, both in vitro, in heart-lung preparations [2,3], modified Langendorff hearts [4,5] and isolated atria [6-9], and in vivo [4,1012]. The specific mechanism for the regulation of ANP secretion has not yet been elucidated. Recently, it has been shown that the atrial secretion of ANP is caused by exocytotic extrusion of atrial granule contents into extracellular space [13,14] and it was also suggested that the cytoplasmic microtubules may be involved in intracellular translocation of atrial secretory granules [ 15]. It has long been considered that the left atrium works as a volume receptor in the body fluid homeostasis through control of the secretion of antidiuretic hormone [16]. Brennan et al. [17] claimed that the right atrium may be an another control limb working with a different mechanism from the left atrium for the regulation of body fluid and electrolyte balance. This means that the right and the left atria may have different control mechanisms for the regulation of body fluid volume homeostasis. There have been controversial suggestions concerning the predominant atrium in ANP secretion in in vivo and in vitro experiments. Wong et al. [ 18,19] presented data showing a right atrial predominance in the basal rate of ANP secretion from the isolated rat atria. Bilder et al. [6] and Naruse et al. [20], however, presented no differences in basal and/or stimulated ANP secretions in both isolated atria in rats. Katsube et al. [10] and Garcia et al. [21] showed that the right atrium is more important in the regulation of ANP secretion in vivo in rats. This is consistent with the original report of Marie et al. [22], who observed predominant changes in specific atrial granules in the right atria by salt and water restriction. The situation was complicated by the findings observed in different animal species. Synhorst et al. [23] reported a more ANP secretion from the left atrium of Langendorff heart in rabbits. Metzler et al. [12] showed no difference in ANP secretion in both atria to cause increased plasma levels of ANP in conscious dogs. Some reports [24-27] presented a similar share of both atria in the control of ANP secretion, and other reports [28,29] represented a predominance of the left atrial role for the ANP secretion in humans. Still there have been no definitive data on the predominant atria in ANP secretion. Recently, we have demonstrated that the increase in ANP secretion in response to atrial distension is caused by a reduction in atrial distension rather than by the distension per se [30-32], and that the ANP secretion is regulated by sequential mechanism, i.e., the release of ANP into the intercellular space and then the translocation by an atrial contraction into the atrial lumen of extracellular fluid with ANP released [33] in the newly devised perfused rabbit atria. To test the hypothesis that the right atrium is the predominant site in atrial ANP secretion, we studied the effect of the mechanical distension-reduction stimuli on ANP secretion in the isolated perfused rat atria.
69
Materials and Methods
Isolated perfused atrial preparation Male Wistar rats, weighing 200-250 g, were killed by decapitation. Isolated perfused atrial preparation was made by the method described previously [30-32]. Briefly, the hearts were rapidly removed and placed in oxygenated warm saline. Both atria were dissected from a rat and were separately perfused simultaneously. The number of animals used was 10. Additionally, five rats were subjected to repetitive stimulation of the right atrium in irANP responses. Another seven rats were used to verify the time sequence of irANP secretion in response to atrial distension. The right atria were carefully fixed not to contain sinus nodal area in the working part of preparation to be silent as the left atria [6,19]. Thereby, spontaneous sinus nodal activity was excluded. A Tygon cannula (3.0 mm outer diameter), made of two smaller catheters sealed water-tight with silicone glue inside, was inserted into the atrium through the atrioventricular orifice by about 1.5 mm. The atrial cannula was tied by three to four times. One of the two catheters located in the atrium, the tip of which was adjacent to the auricular apex, was for the in-flow to, and another was for the out-flow from the atrium. The cannulated atrium was transferred into the buffer containing constanttemperature (36.5°C) organ chamber. The cannulated atrium was fitted in the organ chamber, and fixed with a water-tight silicone rubber cap. The tip of the perfusion catheter was connected to a peristaltic pump. The atrium was immediately perfused with Krebs-Henseleit bicarbonate buffer solution to the atrial lumen. The composition of the Krebs-Henseleit bicarbonate buffer in mM was as follows: NaC1, 118; KC1, 4.7; CaCI 2, 2.5; KH2PO 4, 1.2; MgSO4, 1.2; NaHCO3, 25; glucose, 10.0 and bovine serum albumin, 0.1 ~o. The pericardial space was not perfused. Instead, the pericardial medium was oxygenated by silicone tubing coils located inside the organ chamber, which was connected to the gas chamber. The pericardial space of the organ chamber was sealed water-tight with one out-fit which was connected with a calibrated microcapillary tube (1.3 mm inner diameter). The calibrated microcapillary tube was siliconized. The calibrated microtube was located in a horizontal position at the level of atrium. Atrial volume changes were monitored by measuring the length of the calibrated microtube occupied by buffer solution. A peristaltic pump was used to maintain perfusion flow constant at 0.25 ml/min. The perfusate was equilibrated with an O2-CO2 mixture by passing silicone tubing into the gas mixing chamber (pO2=486.4+20.7 mmHg, p C O 2 = 40.3 + 1.1 mmHg, pH 7.406 + 0.014). The perfusion pressure was recorded on a Physiograph via the atrial in-flow catheter connected to a pressure transducer through the experiments.
Protocols The atria were perfused for 30 min to stabilize irANP secretion rate in baseline distension, which was maintained by the out-flow of the catheter tip being located at the level of the atrium. The perfusate was collected in siliconized tubes containing 40 lal of 0.1 M acetic acid and 10 lal of 376 mM phenylmethylsulfonyl fluoride containing 0.1 ~o bovine serum albumin at 2 min intervals at 4 ° C. After three collection periods at the baseline distension, atrial distension was induced by changing the elevation of
70 the out-flow catheter tip by 2, 4, 6, 10, 16 and 22 cmH20 above atrium. Every 2 min of atrial distension was followed by a reduction in atrial volume for 10 rain. Reduction in atrial volume was conducted by lowering the elevation of the out-flow catheter tip. In another series of experiments, seven left atria were used to verify' the fact that the secretion of irANP occurs at the reduction of atrial distension. After two collection periods at the baseline distension, atrial distension was induced by 10 cmH,O pressure elevations. The atrium was distended for periods of either 2 or 8 rain followed by a reduction. At the end of the experiments the in-flow catheter was connected by Hamilton microsyringe to validate the microcapillary volume checking system. The measuring system was also checked by weighing the collected perfused volume. After finishing the experiments atrial tissues were processed as described below.
Reverse-phase high-performance liquid chromatography proliles Atrial perfusates during basal and stimulated periods were pooled and extracted separately. Tissue samples were put into 0.1 M cold acetic acid containing aprotinin (200 KIU/ml) and phenylmethylsulfonyl fluoride (0.6 raM). Tissue samples were weighed and boiled for 10 rain to inactivate proteolytic enzymes followed by homogenizing with Polytron homogenizer. The homogenate was centrifuged at 10,000 g for 15 rain at 4°C and the supernatant was subjected to the measurement of atrial content ofirANP and to the application for the H P L C after extraction. The irANP in both perfusates and atrial tissues was extracted with Sep-Pak C18 cartridges (Waters Associates, Milford, MA, USA) and was subjected to reverse-phase high-performance liquid chromatography (HPLC) on a C-18 uBondapak column (Waters Associates) as described previously [34]. Elution was performed with linear gradient of 20 to 60"o acetonitrile in 0.13 o trifluoroacetic acid for 40 rain at a rate of 1 ml/min. 1-min fractions were collected.
R adioimmunoassav The irANP in the perfusate, tissue homogenate and eluted samples was measured by radioimmunoassay as described previously [30-32]. The radioimmunoassay was performed in Tris-acetate buffer (0.1 M, pH 7.40), containing 0.2°~, neomycin, 1.0 mM EDTA, 50 benzoyl arginine ethyl ester units/ml soybean trypsin inhibitor, 200 KIU/ ml aprotinin, 0.4 mg/o o~ phenylmethylsulfonyl fluoride, 0.0230 sodium azide and 1'!,, bovine serum albumin. The irANP in the perfusate and tissue samples was measured by direct method. The volume of sample used for radioimmunoassay was 50 ral and the total assay volume was 300 ~tl. Standard, perfusate or tissue samples were incubated with anti-ANP antibody for 24 h at 4°C. Following the additional incubation with [125I ]ANP for 24 h at 4 ° C, separation of free from antibody-bound tracer was achieved by adding 1.0 ml of Dextran-charcoal suspension. Anti-ANP antibody' equally reacted with atriopeptins I, II, III and rat-ANP-(1-28). No cross-reactivity was observed with atrial peptide fragment (18-28, rat). Serial dilutions of the perfusate and tissue homogenate inhibited the binding of [125I]ANP to the antibody in parallel with the standard curve. Radioimmunoassay for irANP was done on the day of experiments and all samples in an experiment were analyzed in a single assay. Non-specific binding was less than 30Jo. The 50°o intercept was at 26.6 + 2.9 pg/tube (t7 = 11). The intra-
71
and interassay coefficients of variation were 6.3 (n = 9) and 7.8 ~o (n = 11), respectively. The secreted amount of irANP, in ng irANP secreted/2 min, was calculated by multiplying the perfusate volume/2 min with the irANP concentration/ml.
Statistics Statistical significance of difference was tested using Student's t-test and was defined as a P-value of less than 0.05. The results were given as mean + S.E.M.
Results
Changes in the volume of atrial distension-reduction by pressure elevation Atrial pressure changes from 2 to 22 cmH20 resulted in proportional increases in the volume of atrial distension-reduction in both right and left isolated perfused atrial preparations (Fig. 1). Increases in right atrial pressure from 2 to 22 cmH20 resulted in increases in the volume changes of atrial distension-reduction from 19.9 + 2.8 lal (1.64_+ 0.27 ~tl/mg) to 56.2 + 3.5 ~tl (4.51 _+0.32 ~tl/mg), respectively. Increases in left
6
i-- ill*
--
GI
i l i l i!l
F**-I
5
E
I1
Ill
i
r-"--- * - " - 7
-
4-
-i, A
~ 2
,n,e,,eO Vo,.
67
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00
REGPEP 01174
Right atrial predominance of atrial natriuretic peptide secretion in isolated perfused rat atria K y u n g H w a n Seul, K y u n g W o o Cho and Suhn H e e Kim Department of Physiology, Jeonbug National University, Medical School, Jeonju (South Korea) (Received 23 September 1991; revised version received 6 February 1992; accepted 7 February 1992)
Key words: Atrial natriuretic peptide; ANF; Distension-reduction; Stretch; Secretion; Atria
Summary In order to investigate the regulatory mechanism for the atrial release of atrial natriuretic peptide (ANP), a perfused rabbit atrial model was devised. In the present experiments, the effect of a reduction in atrial distension on the immunoreactive ANP (irANP) secretion was investigated and compared in the perfused right and left atria of rats. Elevations in right and left atrial pressure resulted in proportional increases in the volume of atrial distension-reduction which was larger in the right than in the left atria. The basal rate of irANP secretion was higher in the right than in the left atria. Increases in the volume of atrial distension-reduction resulted in proportional increases in irANP secretion in both atria. Increment in irANP secretion in response to a reduction in atrial distension was significantly higher in the right than in the left atria. Higher rate of irANP secretion in response to unit volume change was observed in the right atria. Increases in the volume of atrial distension-reduction resulted in accentuated irANP responses in the right atrium. IrANP content was significantly higher in the right than in the left atria. The results suggest that the right atrium is a predominant site in ANP secretion in rats.
Correspondence: K.W. Cho, Department of Physiology, Jeonbug National University, Medical School, 220, Keum-Am-Dong-San, Jeonju 560-180, South Korea.
68 Introduction
Atrial myocytes contain a family of biologically active peptides stored in specific atrial granules [1]. These peptides, termed atrial natriuretic peptides (ANPs), have potent natriuretic, diuretic and vascular smooth muscle relaxant activities. Atrial distension increases ANP secretion, both in vitro, in heart-lung preparations [2,3], modified Langendorff hearts [4,5] and isolated atria [6-9], and in vivo [4,1012]. The specific mechanism for the regulation of ANP secretion has not yet been elucidated. Recently, it has been shown that the atrial secretion of ANP is caused by exocytotic extrusion of atrial granule contents into extracellular space [13,14] and it was also suggested that the cytoplasmic microtubules may be involved in intracellular translocation of atrial secretory granules [ 15]. It has long been considered that the left atrium works as a volume receptor in the body fluid homeostasis through control of the secretion of antidiuretic hormone [16]. Brennan et al. [17] claimed that the right atrium may be an another control limb working with a different mechanism from the left atrium for the regulation of body fluid and electrolyte balance. This means that the right and the left atria may have different control mechanisms for the regulation of body fluid volume homeostasis. There have been controversial suggestions concerning the predominant atrium in ANP secretion in in vivo and in vitro experiments. Wong et al. [ 18,19] presented data showing a right atrial predominance in the basal rate of ANP secretion from the isolated rat atria. Bilder et al. [6] and Naruse et al. [20], however, presented no differences in basal and/or stimulated ANP secretions in both isolated atria in rats. Katsube et al. [10] and Garcia et al. [21] showed that the right atrium is more important in the regulation of ANP secretion in vivo in rats. This is consistent with the original report of Marie et al. [22], who observed predominant changes in specific atrial granules in the right atria by salt and water restriction. The situation was complicated by the findings observed in different animal species. Synhorst et al. [23] reported a more ANP secretion from the left atrium of Langendorff heart in rabbits. Metzler et al. [12] showed no difference in ANP secretion in both atria to cause increased plasma levels of ANP in conscious dogs. Some reports [24-27] presented a similar share of both atria in the control of ANP secretion, and other reports [28,29] represented a predominance of the left atrial role for the ANP secretion in humans. Still there have been no definitive data on the predominant atria in ANP secretion. Recently, we have demonstrated that the increase in ANP secretion in response to atrial distension is caused by a reduction in atrial distension rather than by the distension per se [30-32], and that the ANP secretion is regulated by sequential mechanism, i.e., the release of ANP into the intercellular space and then the translocation by an atrial contraction into the atrial lumen of extracellular fluid with ANP released [33] in the newly devised perfused rabbit atria. To test the hypothesis that the right atrium is the predominant site in atrial ANP secretion, we studied the effect of the mechanical distension-reduction stimuli on ANP secretion in the isolated perfused rat atria.
69
Materials and Methods
Isolated perfused atrial preparation Male Wistar rats, weighing 200-250 g, were killed by decapitation. Isolated perfused atrial preparation was made by the method described previously [30-32]. Briefly, the hearts were rapidly removed and placed in oxygenated warm saline. Both atria were dissected from a rat and were separately perfused simultaneously. The number of animals used was 10. Additionally, five rats were subjected to repetitive stimulation of the right atrium in irANP responses. Another seven rats were used to verify the time sequence of irANP secretion in response to atrial distension. The right atria were carefully fixed not to contain sinus nodal area in the working part of preparation to be silent as the left atria [6,19]. Thereby, spontaneous sinus nodal activity was excluded. A Tygon cannula (3.0 mm outer diameter), made of two smaller catheters sealed water-tight with silicone glue inside, was inserted into the atrium through the atrioventricular orifice by about 1.5 mm. The atrial cannula was tied by three to four times. One of the two catheters located in the atrium, the tip of which was adjacent to the auricular apex, was for the in-flow to, and another was for the out-flow from the atrium. The cannulated atrium was transferred into the buffer containing constanttemperature (36.5°C) organ chamber. The cannulated atrium was fitted in the organ chamber, and fixed with a water-tight silicone rubber cap. The tip of the perfusion catheter was connected to a peristaltic pump. The atrium was immediately perfused with Krebs-Henseleit bicarbonate buffer solution to the atrial lumen. The composition of the Krebs-Henseleit bicarbonate buffer in mM was as follows: NaC1, 118; KC1, 4.7; CaCI 2, 2.5; KH2PO 4, 1.2; MgSO4, 1.2; NaHCO3, 25; glucose, 10.0 and bovine serum albumin, 0.1 ~o. The pericardial space was not perfused. Instead, the pericardial medium was oxygenated by silicone tubing coils located inside the organ chamber, which was connected to the gas chamber. The pericardial space of the organ chamber was sealed water-tight with one out-fit which was connected with a calibrated microcapillary tube (1.3 mm inner diameter). The calibrated microcapillary tube was siliconized. The calibrated microtube was located in a horizontal position at the level of atrium. Atrial volume changes were monitored by measuring the length of the calibrated microtube occupied by buffer solution. A peristaltic pump was used to maintain perfusion flow constant at 0.25 ml/min. The perfusate was equilibrated with an O2-CO2 mixture by passing silicone tubing into the gas mixing chamber (pO2=486.4+20.7 mmHg, p C O 2 = 40.3 + 1.1 mmHg, pH 7.406 + 0.014). The perfusion pressure was recorded on a Physiograph via the atrial in-flow catheter connected to a pressure transducer through the experiments.
Protocols The atria were perfused for 30 min to stabilize irANP secretion rate in baseline distension, which was maintained by the out-flow of the catheter tip being located at the level of the atrium. The perfusate was collected in siliconized tubes containing 40 lal of 0.1 M acetic acid and 10 lal of 376 mM phenylmethylsulfonyl fluoride containing 0.1 ~o bovine serum albumin at 2 min intervals at 4 ° C. After three collection periods at the baseline distension, atrial distension was induced by changing the elevation of
70 the out-flow catheter tip by 2, 4, 6, 10, 16 and 22 cmH20 above atrium. Every 2 min of atrial distension was followed by a reduction in atrial volume for 10 rain. Reduction in atrial volume was conducted by lowering the elevation of the out-flow catheter tip. In another series of experiments, seven left atria were used to verify' the fact that the secretion of irANP occurs at the reduction of atrial distension. After two collection periods at the baseline distension, atrial distension was induced by 10 cmH,O pressure elevations. The atrium was distended for periods of either 2 or 8 rain followed by a reduction. At the end of the experiments the in-flow catheter was connected by Hamilton microsyringe to validate the microcapillary volume checking system. The measuring system was also checked by weighing the collected perfused volume. After finishing the experiments atrial tissues were processed as described below.
Reverse-phase high-performance liquid chromatography proliles Atrial perfusates during basal and stimulated periods were pooled and extracted separately. Tissue samples were put into 0.1 M cold acetic acid containing aprotinin (200 KIU/ml) and phenylmethylsulfonyl fluoride (0.6 raM). Tissue samples were weighed and boiled for 10 rain to inactivate proteolytic enzymes followed by homogenizing with Polytron homogenizer. The homogenate was centrifuged at 10,000 g for 15 rain at 4°C and the supernatant was subjected to the measurement of atrial content ofirANP and to the application for the H P L C after extraction. The irANP in both perfusates and atrial tissues was extracted with Sep-Pak C18 cartridges (Waters Associates, Milford, MA, USA) and was subjected to reverse-phase high-performance liquid chromatography (HPLC) on a C-18 uBondapak column (Waters Associates) as described previously [34]. Elution was performed with linear gradient of 20 to 60"o acetonitrile in 0.13 o trifluoroacetic acid for 40 rain at a rate of 1 ml/min. 1-min fractions were collected.
R adioimmunoassav The irANP in the perfusate, tissue homogenate and eluted samples was measured by radioimmunoassay as described previously [30-32]. The radioimmunoassay was performed in Tris-acetate buffer (0.1 M, pH 7.40), containing 0.2°~, neomycin, 1.0 mM EDTA, 50 benzoyl arginine ethyl ester units/ml soybean trypsin inhibitor, 200 KIU/ ml aprotinin, 0.4 mg/o o~ phenylmethylsulfonyl fluoride, 0.0230 sodium azide and 1'!,, bovine serum albumin. The irANP in the perfusate and tissue samples was measured by direct method. The volume of sample used for radioimmunoassay was 50 ral and the total assay volume was 300 ~tl. Standard, perfusate or tissue samples were incubated with anti-ANP antibody for 24 h at 4°C. Following the additional incubation with [125I ]ANP for 24 h at 4 ° C, separation of free from antibody-bound tracer was achieved by adding 1.0 ml of Dextran-charcoal suspension. Anti-ANP antibody' equally reacted with atriopeptins I, II, III and rat-ANP-(1-28). No cross-reactivity was observed with atrial peptide fragment (18-28, rat). Serial dilutions of the perfusate and tissue homogenate inhibited the binding of [125I]ANP to the antibody in parallel with the standard curve. Radioimmunoassay for irANP was done on the day of experiments and all samples in an experiment were analyzed in a single assay. Non-specific binding was less than 30Jo. The 50°o intercept was at 26.6 + 2.9 pg/tube (t7 = 11). The intra-
71
and interassay coefficients of variation were 6.3 (n = 9) and 7.8 ~o (n = 11), respectively. The secreted amount of irANP, in ng irANP secreted/2 min, was calculated by multiplying the perfusate volume/2 min with the irANP concentration/ml.
Statistics Statistical significance of difference was tested using Student's t-test and was defined as a P-value of less than 0.05. The results were given as mean + S.E.M.
Results
Changes in the volume of atrial distension-reduction by pressure elevation Atrial pressure changes from 2 to 22 cmH20 resulted in proportional increases in the volume of atrial distension-reduction in both right and left isolated perfused atrial preparations (Fig. 1). Increases in right atrial pressure from 2 to 22 cmH20 resulted in increases in the volume changes of atrial distension-reduction from 19.9 + 2.8 lal (1.64_+ 0.27 ~tl/mg) to 56.2 + 3.5 ~tl (4.51 _+0.32 ~tl/mg), respectively. Increases in left
6
i-- ill*
--
GI
i l i l i!l
F**-I
5
E
I1
Ill
i
r-"--- * - " - 7
-
4-
-i, A
~ 2
,n,e,,eO Vo,.
