© 1997 American Heart Association, Inc.
Articles |
Combined Inhibition of Endothelin and Angiotensin II Receptors Blocks Volume Load–Induced Cardiac Hormone Release
the Departments of Physiology (H.L., O.V.) and Pharmacology and Toxicology (H.R.), Biocenter Oulu, University of Oulu (Finland).
Correspondence to Heikki Ruskoaho, MD, PhD, Department of Pharmacology and Toxicology, University of Oulu, Kajaanintie 52 D, FIN-90220 Oulu, Finland. E-mail heikki.ruskoaho@oulu.fi
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Abstract |
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Volume expansion has been shown to increase plasma atrial natriuretic peptide (ANP) levels, but the precise role of paracrine and autocrine factors in stretch-induced cardiac hormone release is not clear. In the present study, we report the effects of endothelin (ET) and angiotensin receptor (AT receptor) antagonists on baseline and atrial stretch–induced immunoreactive ANP (IR-ANP) and immunoreactive N-terminal ANP (IR-NT-ANP) release in vivo by using BQ-123 (ETA receptor antagonist), bosentan (ETA and ETB receptor antagonist), and losartan (AT1 receptor antagonist). Intravenous administration of BQ-123 had no significant effect on baseline hemodynamics in conscious rats, whereas bosentan (10 mg/kg) and losartan (10 mg/kg) decreased slightly (4 to 7 mm Hg, P<.05 to .001) the mean arterial pressure. Both the ETA receptor antagonist BQ-123 and ETA/ETB receptor antagonist bosentan decreased plasma ANP and NT-ANP responses to volume load (P<.05 to .001), whereas the AT1 receptor antagonist losartan had no significant effect on this response. The relative increase in plasma IR-ANP corresponding to a 3 mm Hg increase in right atrial pressure was 2.7-fold in the vehicle-treated group. BQ-123 (0.3 and 1.0 mg/kg) decreased this response 2.5- and 2.1-fold (P<.05); bosentan (3 and 10 mg/kg), 1.7-fold (P<.001) and 1.9-fold (P<.05); and bosentan (10 mg/kg)+losartan (10 mg/kg), 1.6-fold (P<.001). The responses in plasma IR-NT-ANP decreased simultaneously. These results indicate that combined inhibition of ETA/B and AT1 receptors almost completely blocks ANP response to acute volume load. Therefore, our study shows that endogenous paracrine and/or autocrine factors liberated in response to atrial wall stretch rather than myocyte stretch itself are responsible for the activation of ANP peptide secretion in response to acute volume load. Our results also show that ETA receptors are more important in the regulation of mechanical stretch–induced changes in cardiac hormone secretion than AT1 receptors.
Key Words: volume load • atrial natriuretic peptide • N-terminal atrial natriuretic peptide • endothelin • angiotensin
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Introduction |
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Atrial natriuretic peptide and BNP are cardiac hormones involved in the regulation of blood pressure and fluid homeostasis.1 2 3 The bulk of ANP is synthesized in the atria of the normal adult heart, whereas BNP is produced by both the atria and ventricles. The major determinant of ANP secretion is atrial wall stretch.3 The primary sensors for stretch-dependent ANP secretion may be cardiac myocytes or other cell types, including endothelial and endocardial cells as well as fibroblasts. However, it has not been established whether wall stretch acts directly or via local factors such as ET-1 and Ang II liberated in response to distension.
Recently, it has been suggested that endothelial cells may, to some extent, store ET,4 5 and when endothelial cells in culture are stretched, ET-1 can be released rapidly.4 ET-1 is a potent secretagogoue for ANP,6 7 and this response has been shown to be mediated by ETA receptors in cultured rat atrial myocytes.8 ET enhances right atrial stretch-induced ANP release in isolated perfused rat heart preparation,9 suggesting that ET may be involved in the regulation of ANP release. On the other hand, the local renin-angiotensin system has been proposed to act as a mediator of stretch-induced adaptative responses in cardiac myocytes and cardiac fibroblasts.10 11 Immunoreactive Ang II has been localized in secretory granules in ventricular myocytes, and acute stretching of cardiac myocytes in vitro releases Ang II.12 However, findings on the actions of Ang II on ANP release are contradictory, and it is not clear whether its effects are direct or secondary to hemodynamic changes.3
The aim of the present study was to examine whether mechanical wall stretch or factors such as ET and Ang II liberated in response to stretch are responsible for the regulation of natriuretic peptide secretion from the heart. Thus, we determined the effects of selective ET and AT receptor antagonists on both baseline and atrial stretch-induced IR-ANP and IR-BNP release in conscious rats by using BQ-123 (an ETA receptor antagonist),13 14 bosentan (ETA and ETB receptor antagonists),15 and losartan (AT1 receptor antagonist).16 17 18 Measurement of plasma concentrations of IR-NT-ANP, which is cosecreted with ANP in equimolar amounts but is not subject to effective enzymatic degradation and receptor binding,19 20 was used to characterize the endogenous secretion of the peptides. Our results show that autocrine/paracrine secretion of ET plays a critical role in the acute volume load–induced ANP secretion in conscious rats, whereas the influence of circulating and locally produced Ang II on atrial peptide secretion during acute volume load is small.
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Materials and Methods |
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Animals
Male Sprague-Dawley rats (weighing 250 to 350 g) from the colony of the Centre of Experimental Animals at the University of Oulu, Finland, were used. The rats were housed in plastic cages in a room with a controlled humidity of 40% and a temperature of 22°C. A 12-hour light and 12-hour dark environmental light cycle was maintained. The experimental design was approved by the Animal Experimentation Committee of the University of Oulu.
Chronically Instrumented Rats
Under chloral hydrate (300 to 400 mg/kg IP) anesthesia, a PE-60 catheter was placed into the abdominal aorta through the left femoral artery for the measurement of blood pressure and heart rate and for collection of blood samples, as previously described.21 PE-50 catheters were inserted into the right atrium through the jugular vein for measurement of right atrial pressure and into the femoral vein for administration of drugs. All catheters were exteriorized behind the neck, filled with a heparinized (500 IU/mL) saline solution, and plugged with a stainless pin. After operation, rats were housed individually in the experimental cages and had free access to food and water.
The day after the operation, the arterial and right atrial catheters were attached to pressure transducers (model MP-15, Micron Instruments) and a polygraph (model 7E, Grass Instruments) for recording of mean arterial pressure, heart rate, and right atrial pressure. The venous catheter was connected to a syringe or an infusion pump (B. Braun Perfusor ED, Braun Melsungen AG) for administration of vehicle or drugs. The animals were left undisturbed for 30 minutes to become acclimatized to the laboratory before the recording of hemodynamic variables in the conscious freely moving rats was begun.
Experimental Design in Conscious Rats
Mean arterial pressure, heart rate, and right atrial pressure were measured for 25 minutes before 1.0 mL blood was withdrawn from the arterial catheter for measurement of basal plasma IR-ANP, IR-NT-ANP, and IR-BNP levels (B-5). An equal volume of blood from a donor rat was then infused. Donor blood was obtained from conscious rats, to which this volume was replaced by 0.9% NaCl. The baseline hemodynamic measurements were made 5 minutes later, when mean arterial pressure, heart rate, and right atrial pressure were stabilized near the control values.
We first studied the effect of selective ET and AT receptor antagonists on baseline natriuretic peptide release in conscious rats. In protocol A, BQ-123 at a concentration of 1.0 mg/kg, bosentan at a concentration of 10 mg/kg, or vehicle (0.9% NaCl) was administered intravenously as a bolus injection. Injection volume was 0.1 mL/100 g body wt. In protocol B, losartan at a concentration of 10 mg/kg or vehicle was administered intravenously as a bolus injection, similar to that described above. In protocols A and B, blood samples were obtained 10, 17, and 21 minutes after the administration of vehicle or drug.
Next, we determined the effect of BQ-123, bosentan, and losartan on volume load–induced ANP release in conscious rats (protocol C). BQ-123 (0.3 and 1.0 mg/kg), bosentan (3 and 10 mg/kg), losartan (3 and 10 mg/kg), or 0.9% saline was administered intravenously as a bolus injection. A second blood sample (B10) was obtained 10 minutes after the administration of vehicle or drugs. Five minutes later (ie, at time=15 minutes), right atrial pressure was acutely increased by infusing 6.2±0.1 mL physiological saline solution per rat over 1 minute to vehicle- and drug-treated animals so that an identical degree of stretch (ie, increase in right atrial pressure, >4 mm Hg) was observed. A third blood sample for plasma natriuretic peptide measurements was taken at 1 minute (B17) and a fourth at 5 minutes (B21) after volume expansion. Finally, effects of combined treatment with losartan and bosentan on plasma IR-ANP and IR-NT-ANP were studied. Losartan (10 mg/kg) and bosentan (10 mg/kg) were administered intravenously as a bolus injection. Blood samples were collected at -5, 10, 17, and 21 minutes, and volume load was induced as above described.
In order to validate the doses of drugs used, we studied the effects of ET and AT receptor antagonists on hemodynamic and hormonal responses evoked by intravenously infused big ET-1 and Ang II, respectively. Big ET-1 (1 nmol/kg IV) was given at the end of the experiments (at time=25 minutes) as a bolus injection, and blood samples were collected 10 minutes (B35; ie, at time=35 minutes) and 30 minutes (B55; ie, at time=55 minutes) later (protocol A). Ang II infusion (0.5 µg/kg per minute, 1.2 mL/min IV) was also started (time=25 minutes), and fifth and sixth blood samples were collected 10 minutes (B35; ie, at time=35 minutes) and 30 minutes (B55; ie, at time=55 minutes) later (protocol B).
All samples were taken into precooled tubes containing 1.5 mg EDTA per 1 mL blood, kept on ice, and immediately centrifuged (2000g, 10 minutes, 4°C). Plasma was stored at -20°C until assayed by RIA.
Assay of IR-NT-ANP in Plasma
NT-ANP was determined directly from plasma samples by RIA, as previously described.22 Briefly, the plasma samples in duplicates of 25 µL were incubated with the rabbit antiserum (200 µL; final dilution, 1:40 000) and 125I-labeled human Tyr0-pro-ANP-(79-98) (200 µL, 10 000 cpm) overnight at 4°C. The bound and free fractions were separated with double antibody in the presence of polyethylene glycol. Synthetic human pro-ANP-(79-98) was used as standard. This, as well as purified human and rat pro-ANP-(1-126), was recognized with similar avidity, whereas the antiserum did not recognize human or rat ANP-(99-126), rat BNP, rat C-type natriuretic peptide, ETs, or Ang II (cross-reactivity, <0.1%). The sensitivity of the assay was 0.03 nmol/L, and the within- and between-assay coefficients of variation were <10% and <15%, respectively. The 50% displacement of the standard curve was at 0.4 nmol/L.
Assay of IR-ANP and IR-BNP in Plasma
IR-ANP and IR-BNP levels were measured by RIA from the extracted plasma samples, as previously described.23 24 25 Briefly, the plasma samples (0.5 mL) were extracted by SepPak C18 cartridges, and the eluates were redissolved in 500 µL RIA buffer. The extracted samples were incubated in duplicates of 100 µL with 100 µL of the specific rabbit ANP antiserum (final dilution, 1:200 000) or the rabbit BNP antiserum (final dilution, 1:50 000) at 4°C. Synthetic rat ANP-(99-126) and rat BNP-(51-95) were used as standards. After incubation for 48 hours, 125I-labeled rat ANP-(99-126) (100 µL, 10 000 cpm) or Tyr0-BNP-(51-95) (100 µL, 10 000 cpm) was added. After another 24-hour incubation at 4°C, the immunocomplexes were precipitated with double antibody in the presence of polyethylene glycol. The sensitivities of the ANP and BNP assays were 1.0 fmol per tube, and the within- and between-assay coefficients of variation were <10% and <15%, respectively. The 50% displacements of the standard curves were at 10 and 12 fmol per tube in the ANP and BNP assays, respectively. The BNP peptides and antiserum were generously supplied by Dr Kazuwa Nakao, Kyoto (Japan) University School of Medicine.
Materials
Big ET-1 and Ang II were purchased from Peninsula Laboratories, and heparin was from Leiras. BQ-123 was generously supplied by Dr Mitsuo Yano from Banyu Pharmaceutical Co, Ltd; bosentan (Ro 47-0203), by Dr Martine Clozel from F. Hoffmann–La Roche Ltd; and losartan (DuP 753), by Dr Ronald D. Smith from The DuPont Merck Pharmaceutical Co. For intravenous injections, big ET-1, Ang II, and BQ-123 were dissolved in 0.9% NaCl solution, whereas bosentan and losartan were dissolved in water. Unless otherwise stated, all the other chemicals were obtained from Sigma Chemical Co.
Statistical Analysis
The results are expressed as mean±SEM. The data were analyzed with one- or two-way ANOVA. For the comparison of statistical significance between two groups, Student's t test for paired and unpaired data was used. For multiple comparisons, one-way ANOVA followed by Bonferroni's t test was used. Differences at the 95% level were considered significant.
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Results |
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Effects of ET and AT Receptor Antagonists on Hemodynamics and Plasma ANP and NT-ANP Levels in Conscious Rats
We first studied the effect of selective ET and AT receptor antagonists on baseline natriuretic peptide release in conscious rats. The basal mean arterial pressure, as measured directly in conscious chronically cannulated rats, was 117±1 mm Hg, the heart rate was 400±2 bpm, and the right atrial pressure was 0.1±0.1 mm Hg (n=109). Intravenous administration of BQ-123 did not have any significant effects on the mean arterial pressure, heart rate, or right atrial pressure (Table 1
). Bosentan, in a dose of 10 mg/kg, slightly decreased the mean arterial pressure by 3% (P<.05) but had no significant effect on the heart rate. Administration of 10 mg/kg losartan decreased the mean arterial pressure by 6% (P<.001) and increased the heart rate by 10% (P<.001). When bosentan (10 mg/kg) and losartan (10 mg/kg) were administered together, the mean arterial pressure decreased significantly by 11% (P<.01), and the heart rate increased by 13% (P<.001).
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The baseline plasma concentrations of IR-ANP and IR-NT-ANP before administration of vehicle or drugs were 56±2 pmol/L and 1.01±0.39 nmol/L, respectively (n=109). Plasma IR-ANP and IR-NT-ANP had a tendency to decrease in all groups after administration of vehicle or drugs (Table 2). However, no statistically significant differences in the change of plasma IR-ANP or IR-NT-ANP concentrations were seen between the groups (Student's t test for unpaired data) (Table 2).
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Effects of Volume Load on Hemodynamics and Plasma ANP and NT-ANP Levels in Conscious Rats Pretreated With ET and AT Receptor Antagonists
Next, we determined the effect of BQ-123, bosentan, and losartan on volume load–induced ANP release in conscious rats. Acute volume expansion with 0.9% saline increased the right atrial pressure by 4.4±0.1 mm Hg (1 minute after volume load, P<.001) and slightly decreased heart rate (14±5 bpm, 5 minutes after volume load, P<.05) in the vehicle-treated group, while the mean arterial pressure remained unchanged (Table 3). After the administration of BQ-123, bosentan, or losartan, changes in all hemodynamic variables in response to volume expansion were similar to those seen after vehicle administration (Table 3). In conscious animals with indwelling catheters, volume expansion 15 minutes after the vehicle infusion resulted in a 3.5-fold increase in plasma IR-ANP concentrations 1 minute after volume load (from 51±4 to 177±17 pmol/L, P<.001) and a 1.8-fold increase in plasma IR-NT-ANP concentrations 5 minutes after volume load (from 0.82±0.11 to 1.44±0.13 nmol/L, P<.01) (Fig 1).
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Administration of ET receptor antagonists decreased the volume expansion–induced atrial peptide release. Acute saline infusion resulted in a significantly smaller increase in plasma IR-ANP levels in conscious rats infused with 0.3 and 1.0 mg/kg BQ-123 than in control rats (2.9-fold increase, F=4.4, and P<.05 for 0.3 mg/kg BQ-123 versus vehicle; 2.1-fold increase, F=8.0, and P<.01 for 1.0 mg/kg BQ-123 versus vehicle) (Fig 1
, top left). Similarly, both doses of bosentan significantly attenuated the increase in plasma IR-ANP levels produced by volume expansion (2.1-fold increase, F=8.1, and P<.01 for 3 mg/kg bosentan versus vehicle; 2.2-fold increase, F=7.5, and P<.01 for 10 mg/kg bosentan versus vehicle) (Fig 1, top right).
In both BQ-123–pretreated and bosentan-pretreated groups, the secretion rate of endogenous ANP, as assessed by changes in plasma IR-NT-ANP concentrations in response volume expansion, differed significantly from the rate in vehicle-treated groups. IR-NT-ANP increased 1.4-fold in response to volume load in rats infused with 1.0 mg/kg BQ-123, and this secretory response was significantly smaller compared with that observed in the vehicle group (F=5.6, P<.01) (Fig 1, bottom left). Both doses of bosentan significantly decreased the response of IR-NT-ANP to volume load (F=4.2 and P<.05 for 3 mg/kg bosentan versus vehicle; F=4.55 and P<.05 for 10 mg/kg bosentan versus vehicle) (Fig 1, bottom right).
On the other hand, administration of the AT receptor antagonist losartan had no statistically significant effect on volume expansion–induced ANP release. Acute volume loading increased the plasma IR-ANP concentration 3.3-fold (F=0.7, P=NS) in rats pretreated with 3 mg/kg losartan and 3.4-fold (F=1.8, P=NS) in rats pretreated with 10 mg/kg losartan (Fig 2, top left). IR-NT-ANP increased 1.6-fold in response to volume load after 3 mg/kg and 1.3-fold after 10 mg/kg losartan (Fig 2, bottom left). Although the effect in rats pretreated with 10 mg/kg losartan appears to be rather similar to the effect after 10 mg/kg bosentan, neither dose of losartan statistically significantly changed the response of IR-NT-ANP to volume load (F=2.6 and P=NS for 3 mg/kg losartan versus vehicle; F=2.4 and P=NS for 10 mg/kg losartan versus vehicle).
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Combined treatment with bosentan (10 mg/kg) and losartan (10 mg/kg) markedly decreased the response of IR-ANP to volume load (F=14.1 and P<.001 for bosentan+losartan versus vehicle). Only a 1.8-fold increase in plasma IR-ANP was seen (Fig 2, top right), and IR-NT-ANP did not increase in rats pretreated with bosentan+losartan in response to acute volume expansion (F=8.2 and P<.01 for bosentan+losartan versus vehicle group) (Fig 2, bottom right). There were no significant differences in plasma IR-ANP (F=2.4, P=NS) and IR-NT-ANP (F=1.8, P=NS) responses to volume load between the losartan (10 mg/kg)+bosentan (10 mg/kg)–treated group and the bosentan (10 mg/kg)–treated group. However, the effect of bosentan+losartan appears more marked than that of bosentan alone. Although after bosentan alone IR-ANP is still significantly increased by stretch, this is not the case after combined treatment with bosentan and losartan (see Fig 1, top right, and Fig 2, top right). Thus, it appears that Ang II may play a small role in stretch-induced ANP release, which is somehow unmasked by treatment with bosentan.
Relationship Between Changes in Right Atrial Pressure and Plasma ANP and NT-ANP Levels During Acute Volume Expansion
To further analyze the effects of ET and AT receptor antagonists on plasma atrial peptide concentrations, the relative increase in plasma hormone levels in response to volume load was correlated with changes in the right atrial pressure, eg, the degree of right atrial stretch. For plasma IR-ANP concentration, a 1-minute value was selected in order to plot the data as a function of change in right atrial pressure, because the maximal response of IR-ANP was seen at the blood sample taken 1 minute after acute volume expansion. The maximal increase in plasma NT-ANP levels was noted 5 minutes after volume load; therefore, this value was used to plot the data as a function of change in right atrial pressure. The relative increase in plasma IR-ANP corresponding to the 3 mm Hg increase in the right atrial pressure in the vehicle-treated group was 2.7-fold (Fig 3). In rats pretreated with BQ-123, bosentan, or bosentan+losartan, the relation in the changes in plasma IR-ANP and right atrial pressure showed a reduced IR-ANP response to volume load, whereas in the losartan-pretreated rats, no statistically significant change was seen (Fig 3). Thus, at an identical degree of right atrial stretch, the increase in plasma ANP concentration was smaller in ET receptor antagonist–treated rats. The relative increase in plasma IR-NT-ANP corresponding to the 3 mm Hg increase in the right atrial pressure in the vehicle-treated group was 1.6-fold (Fig 4). In all ET receptor antagonist–treated groups, the relation in the changes in plasma IR-NT-ANP and the right atrial pressure showed a reduced IR-NT-ANP response to volume load (Fig 4). The relative increase corresponding to the 3 mm Hg increase in the right atrial pressure was only 1.1-fold in the bosentan+losartan–treated group, showing that the combined inhibition of ET and AT receptors almost completely blocked volume load–induced NT-ANP release.
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, n=16), 0.3 mg/kg BQ-123 (
, n=8), and 1 mg/kg BQ-123 (
, n=8); for panel B, vehicle (
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Effects of ET and AT Receptor Antagonists on Hemodynamic and Hormonal Responses to Big ET-1 and Ang II in Conscious Rats
To validate the doses of drugs used, we studied the effects of ET and AT receptor antagonists on hemodynamic and hormonal responses evoked by intravenously infused big ET-1 and Ang II, respectively. Big ET-1 (1 nmol/kg) increased the mean arterial pressure maximally from 116±4 to 149±3 mm Hg (P<.001) and decreased the heart rate from 406±4 to 353±8 bpm (P<.001); right atrial pressure remained unchanged (Fig 5, bottom left). Bosentan (10 mg/kg) completely blocked the increase in the mean arterial pressure (F=21.73, P<.001) and attenuated the decrease in the heart rate (F=9.02, P<.001) produced by big ET-1 infusion. BQ-123 (1.0 mg/kg), administered intravenously as a bolus injection, also attenuated the increase in mean arterial pressure induced by big ET-1 infusion (F=5.58, P<.01), whereas it did not significantly affect the decrease in heart rate (F=2.33, P=NS) (Fig 5, middle left). Big ET-1 (1 nmol/kg) increased plasma IR-ANP concentrations from 67±8 to 159±30 pmol/L (P<.01). BQ-123 and bosentan blocked this effect of big ET-1 on plasma IR-ANP (P<.001 versus control group). Plasma IR-ANP increased only from 55±10 to 76±25 pmol/L in the BQ-123–treated group and from 54±4 to 60±6 pmol/L in the bosentan-treated group.
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, n=11), 1 mg/kg BQ-123 (
, n=8), and 10 mg/kg bosentan (
Ang II infusion (0.5 µg/kg per minute) increased the mean arterial pressure maximally by 43% (from 120±3 to 171±5 mm Hg, P<.001), decreased the heart rate by 23% (from 411±9 to 316±5 bpm, P<.001), and increased the right atrial pressure by 12% (from -0.1±0.2 to 1.2±0.2 mm Hg, P<.001) (Fig 5, right). Losartan (10 mg/kg) blocked the increase in the mean arterial pressure and right atrial pressure as well as the decrease in the heart rate during infusion of Ang II (for mean arterial pressure, F=33.3 and P<.001; for heart rate, F=12.4 and P<.001; and for right atrial pressure, F=5.80 and P<.001). The mean arterial pressure increased by 4%, heart rate decreased by 7%, and right atrial pressure showed no increase in the losartan (10 mg/kg)–pretreated group. Ang II infusion increased plasma IR-ANP from 47±5 to 301±53 pmol/L (P<.001), and this effect was blocked by losartan. Plasma IR-ANP levels increased from 56±5 to only 65±4 pmol/L in the losartan (10 mg/kg)–pretreated group (P<.001 versus control group).
Effects of Volume Load and Ang II on Plasma BNP Concentrations
In contrast to the marked increase in plasma ANP levels, acute volume expansion did not significantly affect plasma IR-BNP concentrations in conscious animals (32±4 versus 28±4 pmol/L before volume expansion, n=6). Thus, we did not further evaluate the role of ET and Ang II in the regulation of plasma BNP levels during volume load. Even though Ang II infusion markedly increased plasma IR-ANP concentrations, Ang II infusion did not have any significant effect on plasma IR-BNP concentration (32±2 before and 47±6 pmol/L after 30 minutes of Ang II infusion).
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Discussion |
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Although there is much evidence that volume load increases ANP secretion, the precise mechanisms linking stretch and cardiac hormone secretion are not known. The present study shows for the first time that endogenous paracrine and/or autocrine factors liberated in response to atrial wall stretch rather than myocyte stretch itself are responsible for the activation of ANP secretion during volume load. In conscious animals, the different stimuli themselves invoke a complex array of hemodynamic responses, neuronal reflexes, and changes in circulating humoral factors, and this makes the interpretation of results more difficult. Therefore, we have carefully documented changes in hemodynamic variables, especially in right atrial pressure, between different groups. Another complicating fact in these studies is that arterial or venous natriuretic peptide concentrations result from cumulative changes in the secretion rate, elimination rate, and distribution volume. We have used arterial blood samples in these studies, since compared with venous blood, arterial blood better reflects cardiac ANP release. We also measured plasma IR-NT-ANP concentrations concomitantly with plasma IR-ANP. The major storage form of ANP in myocytes is the 126–amino acid pro-ANP-(1-126), which is cleaved into the N-terminal fragment of pro-ANP (NT-ANP) and the C-terminal peptide ANP during or shortly after release.3 NT-ANP is cosecreted with ANP in response to atrial stretch both in vitro26 and in vivo.19 20 Since NT-ANP is not subject to effective enzymatic degradation and receptor binding and thus has a reduced clearance compared with the ANP,27 NT-ANP circulates at a concentration 10- to 20-fold higher than that of ANP. Therefore, plasma IR-NT-ANP levels reflect more closely the endogenous secretion of atrial peptides from the heart than plasma IR-ANP levels.
Two subtypes of ET receptors have been cloned, the ETA receptor mediating vasoconstriction and the ETB receptor mediating both vasoconstriction and vasodilatation.28 29 30 31 In the present study, the ETA receptor antagonist BQ-12313 had no effect on basal hemodynamics, whereas the new nonpeptide mixed ETA/ETB receptor antagonist bosentan15 32 decreased blood pressure slightly (3%), which agrees with previous studies.15 33 Since bosentan totally blocked pressor effects of big ET-1 and since BQ-123 was also effective, these results suggests that ETs do not have a major role in the acute regulation of blood pressure under basal conditions in conscious normotensive rats. Nevertheless, ET has been shown to play a role in the maintenance of blood pressure in rats with congestive heart failure.34 ET-1 has previously been shown to be a potent secretagogue for ANP in cultured rat atrial myocytes,7 35 in isolated perfused rat hearts,9 and in anesthetized and conscious rats.36 37 In our present study, however, ET receptor antagonists did not have any significant effect on basal ANP and NT-ANP concentrations in conscious rats, showing that ETs do not regulate baseline plasma concentrations of atrial peptides. This is in contrast to a recent study in which inhibition of ET action by passive immunization with an ET-1 antiserum reduced basal plasma ANP levels by 30% in anesthetized rats.38 These contradictory results may be explained by the use of different experimental models. In the anesthetized rats, the basal blood pressure was markedly lower than in our conscious rats. ET may thus have a more important role in the maintenance of cardiovascular function in anesthetized animals and in pathophysiological conditions.
The renin-angiotensin-aldosterone system has previously been suggested to play a minor role in the control of blood pressure in normotensive animals.16 17 18 In the present study, we found that losartan had a slight hypotensive effect on baseline hemodynamics. The highest dose of losartan (10 mg/kg) decreased blood pressure by 6%. Infusion of pharmacological doses of Ang II has previously been shown to increase plasma ANP concentrations, probably secondary to the changes in blood pressure and left atrial stretch.39 40 In the present study, intravenous administration of Ang II also markedly stimulated secretion of atrial peptides concomitantly with increased blood pressure and right atrial pressure, and all these effects of Ang II were inhibited by losartan. However, losartan did not have any significant acute effect on baseline plasma ANP or NT-ANP concentrations, suggesting that Ang II does not regulate baseline plasma atrial peptide concentrations in conscious rats.
Volume load has been shown to increase plasma ANP concentrations in vivo,41 and it is known that wall stretch and not pressure per se is a direct stimulator of ANP release.42 43 However, it is not known whether cardiac hormone secretion is due to direct effects on atrial myocytes or to the liberation of autocrine/paracrine factors, which could then influence hormone release from atrial secretory granules. Previously, it has been shown that passive immunization with an ET-1 antiserum decreases volume load–induced ANP secretion in anesthetized rats.38 However, because in anesthetized animals low blood pressure exaggerates vasoconstrictive effects and is known to influence ANP secretory response to several experimental manipulations,3 the role of endogenous ET in volume expansion–induced ANP release under physiological conditions remained unclear. In the present study, volume load–induced ANP and NT-ANP release was reduced in conscious rats by the ETA receptor antagonist BQ-123 and the combined ETA and ETB receptor antagonist bosentan but was not significantly affected by the Ang II receptor antagonist losartan. In addition, the combined inhibition of ET and Ang II receptors almost completely blocked volume load–induced NT-ANP release. These results show that autocrine/paracrine secretion of ET plays a critical role in the acute volume load–induced ANP secretion in conscious rats, whereas the influence of Ang II on atrial peptide secretion during acute volume load is small.
ETs may influence volume load–induced ANP release in vivo by several mechanisms. It has been suggested that in addition to endothelial cells, endocardial cells may release ET, which modulates the inotropic state of the heart.44 Volume load may influence the release of ET from these sources, which in turn may influence ANP release. Exogenous ET has been shown to enhance ANP response to atrial stretch in isolated perfused rat hearts9 and superfused rat left atrial preparations.45 Since in our present study the diminished ANP and NT-ANP response was seen as early as 1 minute after volume load, de novo synthesis of ET obviously cannot explain the cardiac hormone release. Recently, it has been suggested that endothelial cells may, to some extent, store ET.4 5 Furthermore, when endothelial cells in culture were stretched, actinomycin D or cycloheximide did not affect the acute (
20-minute) increase in ET-1 release, suggesting that endothelial cells contain preformed stores of ET-1 that can be released rapidly.4 Local wall stretch caused by volume load may, therefore, release the stored ET, which then stimulates ANP secretion from atrial secretory granules.
In the present study, both bosentan and BQ-123 were potent inhibitors of volume load–stimulated ANP and NT-ANP release. Thus, the effect of ET during volume load seems to be principally mediated by ETA receptors. By using quantitative autoradiography, it has been reported that ETA receptors constitute 
91% of the total population of ET receptors in human right atrial myocytes.46 Moreover, it has been shown that ETA but not ETB receptor antagonists inhibit ET-stimulated secretion of natriuretic peptides in cultured atrial myocytes.8 In another study, low doses of intravenously administered ET-1, which caused a brief vasodilatation in vivo, inhibited ANP secretion, whereas high doses with long-lasting vasoconstriction significantly increased plasma ANP levels.47 Although the results may have been caused by different hemodynamic responses to low and high doses of ET-1, they also suggest that stimulation of ETB receptors and release of NO may neutralize the effects of ETA receptor stimulation on ANP release. In conscious rats, nitric oxide appears to inhibit acute volume load–induced ANP release.48
In conclusion, mechanical forces alter the structure and function of many different cell types, including cardiac myocytes, smooth muscle cells, endothelial cells, and fibroblasts. The present study shows that ET, by stimulating ETA receptors, plays an important physiological role as a mediator of acute volume load–induced ANP secretion from atrial myocytes in conscious normotensive animals. In fact, endogenous autocrine/paracrine factors liberated in response to atrial wall stretch rather than direct stretch appear to be responsible for the activation of ANP peptide secretion in response to acute volume load, as evidenced by almost total blockade of ANP response during combined inhibition of ETA/B and AT1 receptors. These results also provide links for a pathophysiological role of autocrine factors in load-induced growth of cardiac muscle cells. ET itself is sufficient to cause cardiac myocyte hypertrophy49 and may thus be involved in mediating stretch-induced myocyte hypertrophy. In support of this, ETA receptor antagonist BQ-123 prevented the expression of transcriptional markers for cardiac hypertrophy, such as skeletal 
-actin and ANP mRNA, in the left ventricle in response to hemodynamic overload in vivo.50
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Medical Research Council of the Academy of Finland, Sigrid Juselius Foundation, Novo Nordisk Foundation, Aarne and Aili Turunen Foundation, Ida Montin Foundation, and Finnish Cultural Society. We thank Tuula Lumijarvi and Sirpa Rutanen for expert technical assistance.
Received May 20, 1996; accepted October 3, 1996.
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