0013-7227/90/1261-0587$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Endothelin Stimulates Basal and Stretch-Induced Atrial Natriuretic Peptide Secretion from the Perfused Rat Heart* PENTTI MANTYMAA, JUHANI LEPPALUOTO, AND HEIKKI RUSKOAHO Departments of Pharmacology and Toxicology and Physiology (J.L.), University of Oulu, Oulu, Finland
ABSTRACT. To examine the role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release, the effects of endothelin (ET), a putative endogenous agonist for voltage-dependent Ca2+ channels on basal and atrial stretchstimulated ANP release as well as on hemodynamic parameters (perfusion pressure, heart rate, contractile force) in isolated perfused rat hearts were studied. Infusion of ET (0.9 X 10"9-2.3 x 10"9 M) alone for 30 min caused a dose-dependent sustained increase in the perfusate immunoreactive ANP (IR-ANP) concentration and coronary vasoconstriction. An initial inotropic response with a later decrease in the contractile force in response to ET was observed, while heart rate remained unchanged. A phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase-C activity, at a dose of 4.6 x 10'8 M caused a gradual, slowly progressive increase in perfusate IR-ANP levels and a more rapid increase in perfusion pressure. ET, when infused in combination with TPA, enhanced IR-ANP secretion induced by the phorbol ester. When hearts from spontaneously hypertensive rats (SHR) were examined, the vasoconstrictor response to infusion of ET was greater than that in the
A
TRIAL myocytes synthesize, store, and release atrial natriuretic peptide (ANP), a peptide hormone that has potent natriuretic, diuretic, and vasodilating properties and inhibits renin, aldosterone, and vasopressin secretion (1-4). Atrial wall stretch is a major factor in regulation of the release of ANP (5-7); however, the precise cellular mechanisms linking mechanical distension to hormonal release are unknown. ANP is stored in granules, which suggests that its secretion is regulated rather than constitutive (8). By analogy to other secretory systems in which release of hormones and peptides are regulated, secretion may involve a change in the level of a cytoplasmic messenger, such as calcium (9). We have reported (10) that the Ca2+ ionophore A23187, which introduces free calcium into the cell, and phorbol esters,
normotensive Wistar-Kyoto (WKY) rats. Infusion of eguipressor doses of ET increased the release of IR-ANP in WKY rats, but had no effect on perfusate IR-ANP levels in SHR. To examine effects of ET on stretch-stimulated ANP release, the modified perfused rat heart preparation that enabled the stepwise distension of the right atrium was used. The increase in right atrial pressure (2.65 ± 0.13 mm Hg) was accompanied by an increase in the perfusate IR-ANP concentration (from 8.3 ± 1.1 to 13.9 ± 2.0 ng/5 min; P < 0.05; n = 15). The increase in right atrial pressure during the ET infusions resulted in a significantly greater increase in the perfusate IR-ANP concentration than vehicle alone. The calculated ANP increase corresponding to the 2-mm Hg increase in the right atrial pressure was 1.52-fold in the control group and 1.74-fold when 1.9 x 10"9 M ET was infused (P < 0. 05). This study shows that ET stimulates both basal and atrial stretch-stimulated ANP secretion from the isolated perfused heart and suggests that ET is involved in the regulation of stretch-induced ANP release. The results further confirm the potent vasoconstrictor and cardiac effects of ET. (Endocrinology 126: 587-595, 1990)
which mimic the action of diacylglycerol by acting directly on protein kinase-C, both increase ANP secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel activators, such as Bay K8644, and to agents that increase cAMP, such as forskolin (11). These results suggest a possible role for calcium-activated protein kinase-C in the regulation of ANP secretion. Endothelin-1 (ET-1) is an endothelial cell-derived vasoconstrictor peptide, which was originally isolated and seguenced from the culture medium of porcine aortic endothelial cells (12-15). This 21-amino acid residue peptide has been shown to be a potent constrictor of vascular smooth muscle in vitro and is dependent on extracellular calcium and inhibited by low concentrations of the voltage-dependent Ca2+ channel antagonists (12, 16-17). In cultured vascular smooth muscle cells, ET transiently increases intracellular Ca2+ concentrations with a sustained plateau phase (16). This study was designed to examine whether the peptide affects basal and atrial stretch-induced ANP secretion in vitro. The
Received July 20,1989. Address all correspondence and requests for reprints to: Heikki Ruskoaho, M.D., Department of Pharmacology and Toxicology, University of Oulu, Kajaanintie 52 D, SF-90220 Oulu, Finland. * This work was supported by the Academy of Finland (Helsinki, Finland), the Sigrid Juselius Foundation (Helsinki, Finland), and the Paulo Foundation (Helsinki, Finland).
587
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Endo • 1990 Voll26»Nol
ET AND ANP RELEASE
588
results demonstrate that ET, in addition to atrial stretch, is a potent stimulus for ANP release and that stretch of the myocytes appears to be positively modulated by ET.
Protocol l Infusion of vehicle or drugs
Materials and Methods Animals
60
Male Wistar rats (weighing 250-350 g), 6-8 months old, spontaneously hypertensive rats (SHR; mean age, 7.1 ± 0.1 months; weighing 330-420 g) of the Okamoto-Aoki strain, and Wistar-Kyoto (WKY; 7.1±0.1 months old, weighing 350-440 g) rats from the Department of Pharmacology colony at the University of Oulu were used. WKY and SHR strains were originally obtained from Mollegaards Avslaboratorium (Skensved, Denmark). The rats were housed in plastic cages in a room with controlled 40% humidity and a temperature of 22 C. A 0600-1800 h light/1800-0600 h dark cycle was maintained. The experimental protocols were approved by Committee for Animal Experimentation of the University of Oulu. Isolated perfused heart preparation The rat isolated perfused heart preparation used in this study was similar to that previously described (18). Twenty minutes after being anticoagulated with heparin (500 IU/kg BW, ip), the rats were decapitated. The abdominal cavity was immediately opened, the diaphragm was transected, and lateral incisions were made along both sides of the rib cages. The anterior chest wall was retracted, and the heart was cooled with perfusion fluid (4-10 C). The aorta was cannulated superior to the aortic valve, and retrograde perfusion was begun with a modified Krebs-Henseleit bicarbonate buffer, pH 7.40, eguilibrated with 95% O2-5% CO2 at 37 C. The final concentrations of the salts in the buffer (millimoles per liter) were: NaCl, 113.8; NaHCO3, 22.0; KC1, 4.7; KH2PO4, 1.2; MgSO4 • 7H2O, 1.1; CaCl2-2H2O, 2.5; and glucose, 11. Variations in perfusion pressure caused by changes in coronary vascular resistance were recorded on a Grass polygraph (model 7DA, Grass Instruments, Quincey, MA) with a pressure transducer (model MP-15, Micron Instruments, Los Angeles, CA) situated on a side-arm of the aortic cannula. The isometric force of contraction was recorded by a strain gauge transducer (model FTO3, Grass Instruments) connected to the Grass polygraph. The output was damped to give a mean contractile force. The hearts were submitted to a resting tension of 2 g. Heart rate was counted from contractions by the Grass tachograph. During the equilibration period (60 min) they were perfused by a peristaltic pump (Ismatec 5A801, Ismatec Instruments, Zurich, Switzerland) at a flow rate of 7.5 ml/min. To study the vasoconstrictor effect of ET, the vasculature was dilated by decreasing the perfusion rate to 5 ml/min before experiments began. Experimental design Investigations on the isolated perfused heart preparation followed three protocols (Fig. 1). In protocol 1, a 10-min control period was followed by the addition of vehicle, ET, or 12-0tetradecanoyl-phorbol-13-acetate (TPA) alone, or ET in com-
Time (min) Protocol 2 Infusion of vehicle or endothelin Endothelin O.9xio"9
WKY
10
Endothelin 3.6xlO"
SHR
-X0
Endothelin 2.7xl0"9 Endothelin 0.9xl0~9
0 Time (min)
Protocol 3 Infusion of vehicle or endothelin
-10
o Time (min)
FlG. 1. Experimental protocols.
bination with TPA to the aortic perfusate as a continuous infusion via an infusion pump (B. Braun Perfuser ED, Braun Melsungen AG, Melsungen, West Germany) at an infusion rate of 0.5 ml/min for 30 min. The doses of ET used were 0.9 X 10" 9 and 2.3 X 10'9 M. Concentrations above 3 X 10"9 M were not studied, because preliminary dose-response studies showed that ET may cause arrhythmias or cardiac arrest at these doses. The experiments in hearts from SHR and WKY rats consisted of three consecutive infusions (protocol 2; Fig. 1). After a 10-min control period, vehicle or ET was infused at a rate of 0.5 ml/min for 60 min; the dose of ET was increased after 30 min of infusion. The administration of lower doses of ET into the hearts of SHR (Fig. 1) allowed us to study the effect of ET at equipressor doses on ANP release in both strains, since preliminary experiments revealed that ET had more potent vasoconstrictor actions in the SHR than in the WKY rats. In protocol 3, the effect of ET on atrial stretch-induced IRANP secretion was examined using our modified perfused rat heart preparation (6). Right atrial pressure was recorded on a Grass polygraph via a cannula (PE-60) in the inferior vena cava connected to a pressure transducer (model MP-15, Micron Instruments). A glass cannula was inserted into the pulmonary artery for the collection of perfusate. Right atrial pressure was kept constant at the desired level by adjusting the level of the pulmonary artery cannula tip. After a 10-min control period (Fig. 1) a continuous infusion of vehicle or ET was made via the aortic perfusion cannula at a rate of 0.5 ml/min for 35 min.
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ET AND ANP RELEASE Atrial stretch was superimposed for 5 min after 25-min perfusion by elevating the level of the pulmonary artery cannula tip. In protocols 2 and 3, heart rate was increased 15-20% above the spontaneous beating rate by using Harvard Apparatus Stimulator model 345 (Harvard Apparatus, Millis, MA). The coronary venous effluents were collected at either 1 (protocol 3)- or 2-min intervals, placed immediately on dry ice, and stored at —20 C until assayed. Control experiments were run with the solvents dimethylsulfoxide and 0.9% saline. Addition of an appropriate concentration of each caused no significant changes in immunoreactive ANP (IR-ANP) in the perfusate. Assay of IR-ANP in perfusate For the ANP RIA the unextracted perfusate samples were incubated in duplicates of 100 /A with 100 /xl of the middle specific rabbit ANP antiserum developed by Vuolteenaho (19, 20) in a final dilution of 1:25,000. Synthetic rat ANP-(l-28), ranging from 0-1,250 pg/tube, was incubated as standard. The ANP tracer was rat [125I]ANP-(l-28) from Amersham (Buckinghamshire, United Kingdom). After incubation for 48 h at 4 C, the immunocomplexes were precipitated with sheep antiserum against rabbit 7-globulin in the presence of 500 fi\ 1.2 M ammonium sulfate, pH 7, followed by centrifugation at 3,000 x g for 40 min. The sensitivity of the assay was 0.8 pg/tube. The 50% displacement of the standard curve occurred at 20 pg/ tube. The inter- and the intraassay variations were 14% and 5%, respectively. Serial dilutions of perfusate showed parallelism to the synthetic ANP standard. Materials Drugs used in this study were: ET-1 (Peninsula Laboratories, Inc., Belmont, CA) and the phorbol esters TPA (Sigma Chemical Co., St. Louis, MO), synthetic rat ANP-(l-28) (Sigma Chemical Co.), and heparin (Medica, Helsinki, Finland). Other chemicals were obtained from Sigma. The phorbol ester was dissolved in dimethylsulfoxide, and ET in 0.9% saline. The final concentration of each solvent was less than 0.03%. Data analysis The results are expressed as the mean ± SEM. The data were analyzed with two- or one-way analysis of variance (ANOVA). For the comparison of statistical significance between groups, Student's t test for paired or unpaired data was used. For multiple comparison, one-way ANOVA followed by the Bonferroni t test was used. Differences at the 95% level were considered significant.
Results Protocol 1: effect of ET on hembdynamic variables and IR-ANP release from hearts of Wistar rats In our rat heart preparation (n = 35), the mean initial heart rate, the perfusion pressure, and the contractile force were 240 ± 3 beats/min, 28 ± 1 mm Hg, and 1.9 ± 0.1 g, respectively. The basal concentration of IR-ANP
589
in the perfusate was 231 ± 20 pg/ml (n = 35). Addition of ET (0.9 x 109-2.3 x 10 9 M) into the perfusate at a constant rate had no significant effect on heart rate (Fig. 2). The initial effect of ET (2.3 X 10 9 M) was to produce a small transient positive inotropic effect (2.02 ± 0.06 us. 1.86 ± 0.02 g before infusion; P < 0.05). The maximum increases were attained after 1-10 min of infusion. Contractile force decreased (F = 5.6 for drug and time interaction; P < 0.001) during continuous ET infusion compared to that during infusion of vehicle (Fig. 2). ET produced a rapid dose-dependent increase in perfusion pressure (0.9 X 10"9 M, F = 33.9 and P < 0.001; 2.3 X 10' 9 M, F = 111.7 and P < 0.001; F = 4.6 and P < 0.001 between the doses of ET) as soon as the infusion was started (Fig. 2). Infusion of ET caused a gradual increase in IR-ANP secretion into the perfusate (0.9 X 10"9 M, F = 14.9 and P < 0.001; 2.3 X 10"9 M, F = 15.5 and P < 0.001; Fig. 2). Infusion of 4.6 X 10 8 M of the phorbol ester TPA, alone (F = 4.1; P < 0.01) or in combination with ET (0.9 x 10"9 M; F = 8.8; P < 0.001) increased heart rate significantly (Fig. 3). Infusion of TPA also decreased contractile force (F = 37.6; P < 0.001). When ET was infused together with TPA, it transiently blocked the negative effect of TPA on contractile force by shifting the contractile force curve to the right (Fig. 3). Later, contractile force decreased in response to infusion (F = 30.1; P < 0.001). Addition of phorbol ester or phorbol ester combined with ET produced similar increases in perfusion pressure (F = 48.9 and P < 0.001, TPA us. vehicle; F = 101.3 and P < 0.001, ET and TPA vs. vehicle) as soon as the infusion was started. Addition of TPA to the perfusate did not cause any immediate change in IR-ANP secretion, but caused a gradual sustained increase in perfusate IR-ANP (F = 14.1 and P < 0.001, TPA vs. vehicle). If TPA was added to the perfusate together with ET (0.9 X 10"9 M), ET significantly enhanced the phorbol ester-induced increase in IR-ANP secretion (F = 4.5 and P < 0.001, endothelin and TPA vs. TPA; Fig. 3). Protocol 2: effect of ET on hemodynamics and IR-ANP release in WKY and SHR The mean initial heart rate was 250 ± 2 beats/min in WKY rats (n = 14) and 252 ± 2 beats/min in SHR (n = 14) and was held constant by pacing throughout the experiments. The perfusion pressures were 22 ± 1 and 26 ± 1 mm Hg (P < 0.01), and the contractile forces were 1.9 ± 0.1 and 2.0 ± 0.1 g in WKY and SHR, respectively. The basal concentration of IR-ANP in the perfusate of SHR hearts was 603 ± 67 pg/ml; in WKY hearts it was 385 ± 39 pg/ml (n = 14; P < 0.01). These basal IR-ANP levels in the perfusate were similar to
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ET AND ANP RELEASE
590
Endo • 1990 Vol 126 • No 1 CONTRACTILE FORCE
FIG. 2. Effect of ET on heart rate, contractile force, perfusion pressure, and release of IR-ANP in isolated perfused rat hearts. At 10 min, as indicated by the arrows, ET at a concentration of 2.3 x 10"9 M (A; n = 8) or 0.9 X 10"9 M (•; n = 6) or vehicle (O; n = 8) was added to the perfusion fluid for 30 min. IR-ANP secretion is expressed as picograms per ml perfusate, and each point is the mean ± SEM from six to eight separate experiments run on different isolated rat hearts.
30
40
PERFUSION PRESSURE
10
20 TIME (min)
0
10
180
30
10
20
30
PERFUSION PRESSURE
those we reported previously (21). Infusion of vehicle alone did not affect contractile force or perfusion pressure, but a significant decrease (60%; P < 0.05) was seen in the perfusate IR-ANP concentration toward the end of the infusion in both strains (Fig. 4). Contractile force increased in response to infusion of the lower dose of ET in WKY rats and decreased in both strains when the higher concentrations of ET were infused. ET caused a dose-dependent increase in perfusion pressure in both SHR and WKY rat hearts (Fig. 4). The changes in IRANP secretion evoked by ET infusions were different in SHR and WKY rats. The ANOVA comparing the effects of ET (2.7 X 10 § M) and vehicle in the WKY rats revealed a significant drug and time interaction (F = 4.16; P
Vol. 126, No. 1 Printed in U.S.A.
Endothelin Stimulates Basal and Stretch-Induced Atrial Natriuretic Peptide Secretion from the Perfused Rat Heart* PENTTI MANTYMAA, JUHANI LEPPALUOTO, AND HEIKKI RUSKOAHO Departments of Pharmacology and Toxicology and Physiology (J.L.), University of Oulu, Oulu, Finland
ABSTRACT. To examine the role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release, the effects of endothelin (ET), a putative endogenous agonist for voltage-dependent Ca2+ channels on basal and atrial stretchstimulated ANP release as well as on hemodynamic parameters (perfusion pressure, heart rate, contractile force) in isolated perfused rat hearts were studied. Infusion of ET (0.9 X 10"9-2.3 x 10"9 M) alone for 30 min caused a dose-dependent sustained increase in the perfusate immunoreactive ANP (IR-ANP) concentration and coronary vasoconstriction. An initial inotropic response with a later decrease in the contractile force in response to ET was observed, while heart rate remained unchanged. A phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase-C activity, at a dose of 4.6 x 10'8 M caused a gradual, slowly progressive increase in perfusate IR-ANP levels and a more rapid increase in perfusion pressure. ET, when infused in combination with TPA, enhanced IR-ANP secretion induced by the phorbol ester. When hearts from spontaneously hypertensive rats (SHR) were examined, the vasoconstrictor response to infusion of ET was greater than that in the
A
TRIAL myocytes synthesize, store, and release atrial natriuretic peptide (ANP), a peptide hormone that has potent natriuretic, diuretic, and vasodilating properties and inhibits renin, aldosterone, and vasopressin secretion (1-4). Atrial wall stretch is a major factor in regulation of the release of ANP (5-7); however, the precise cellular mechanisms linking mechanical distension to hormonal release are unknown. ANP is stored in granules, which suggests that its secretion is regulated rather than constitutive (8). By analogy to other secretory systems in which release of hormones and peptides are regulated, secretion may involve a change in the level of a cytoplasmic messenger, such as calcium (9). We have reported (10) that the Ca2+ ionophore A23187, which introduces free calcium into the cell, and phorbol esters,
normotensive Wistar-Kyoto (WKY) rats. Infusion of eguipressor doses of ET increased the release of IR-ANP in WKY rats, but had no effect on perfusate IR-ANP levels in SHR. To examine effects of ET on stretch-stimulated ANP release, the modified perfused rat heart preparation that enabled the stepwise distension of the right atrium was used. The increase in right atrial pressure (2.65 ± 0.13 mm Hg) was accompanied by an increase in the perfusate IR-ANP concentration (from 8.3 ± 1.1 to 13.9 ± 2.0 ng/5 min; P < 0.05; n = 15). The increase in right atrial pressure during the ET infusions resulted in a significantly greater increase in the perfusate IR-ANP concentration than vehicle alone. The calculated ANP increase corresponding to the 2-mm Hg increase in the right atrial pressure was 1.52-fold in the control group and 1.74-fold when 1.9 x 10"9 M ET was infused (P < 0. 05). This study shows that ET stimulates both basal and atrial stretch-stimulated ANP secretion from the isolated perfused heart and suggests that ET is involved in the regulation of stretch-induced ANP release. The results further confirm the potent vasoconstrictor and cardiac effects of ET. (Endocrinology 126: 587-595, 1990)
which mimic the action of diacylglycerol by acting directly on protein kinase-C, both increase ANP secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel activators, such as Bay K8644, and to agents that increase cAMP, such as forskolin (11). These results suggest a possible role for calcium-activated protein kinase-C in the regulation of ANP secretion. Endothelin-1 (ET-1) is an endothelial cell-derived vasoconstrictor peptide, which was originally isolated and seguenced from the culture medium of porcine aortic endothelial cells (12-15). This 21-amino acid residue peptide has been shown to be a potent constrictor of vascular smooth muscle in vitro and is dependent on extracellular calcium and inhibited by low concentrations of the voltage-dependent Ca2+ channel antagonists (12, 16-17). In cultured vascular smooth muscle cells, ET transiently increases intracellular Ca2+ concentrations with a sustained plateau phase (16). This study was designed to examine whether the peptide affects basal and atrial stretch-induced ANP secretion in vitro. The
Received July 20,1989. Address all correspondence and requests for reprints to: Heikki Ruskoaho, M.D., Department of Pharmacology and Toxicology, University of Oulu, Kajaanintie 52 D, SF-90220 Oulu, Finland. * This work was supported by the Academy of Finland (Helsinki, Finland), the Sigrid Juselius Foundation (Helsinki, Finland), and the Paulo Foundation (Helsinki, Finland).
587
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Endo • 1990 Voll26»Nol
ET AND ANP RELEASE
588
results demonstrate that ET, in addition to atrial stretch, is a potent stimulus for ANP release and that stretch of the myocytes appears to be positively modulated by ET.
Protocol l Infusion of vehicle or drugs
Materials and Methods Animals
60
Male Wistar rats (weighing 250-350 g), 6-8 months old, spontaneously hypertensive rats (SHR; mean age, 7.1 ± 0.1 months; weighing 330-420 g) of the Okamoto-Aoki strain, and Wistar-Kyoto (WKY; 7.1±0.1 months old, weighing 350-440 g) rats from the Department of Pharmacology colony at the University of Oulu were used. WKY and SHR strains were originally obtained from Mollegaards Avslaboratorium (Skensved, Denmark). The rats were housed in plastic cages in a room with controlled 40% humidity and a temperature of 22 C. A 0600-1800 h light/1800-0600 h dark cycle was maintained. The experimental protocols were approved by Committee for Animal Experimentation of the University of Oulu. Isolated perfused heart preparation The rat isolated perfused heart preparation used in this study was similar to that previously described (18). Twenty minutes after being anticoagulated with heparin (500 IU/kg BW, ip), the rats were decapitated. The abdominal cavity was immediately opened, the diaphragm was transected, and lateral incisions were made along both sides of the rib cages. The anterior chest wall was retracted, and the heart was cooled with perfusion fluid (4-10 C). The aorta was cannulated superior to the aortic valve, and retrograde perfusion was begun with a modified Krebs-Henseleit bicarbonate buffer, pH 7.40, eguilibrated with 95% O2-5% CO2 at 37 C. The final concentrations of the salts in the buffer (millimoles per liter) were: NaCl, 113.8; NaHCO3, 22.0; KC1, 4.7; KH2PO4, 1.2; MgSO4 • 7H2O, 1.1; CaCl2-2H2O, 2.5; and glucose, 11. Variations in perfusion pressure caused by changes in coronary vascular resistance were recorded on a Grass polygraph (model 7DA, Grass Instruments, Quincey, MA) with a pressure transducer (model MP-15, Micron Instruments, Los Angeles, CA) situated on a side-arm of the aortic cannula. The isometric force of contraction was recorded by a strain gauge transducer (model FTO3, Grass Instruments) connected to the Grass polygraph. The output was damped to give a mean contractile force. The hearts were submitted to a resting tension of 2 g. Heart rate was counted from contractions by the Grass tachograph. During the equilibration period (60 min) they were perfused by a peristaltic pump (Ismatec 5A801, Ismatec Instruments, Zurich, Switzerland) at a flow rate of 7.5 ml/min. To study the vasoconstrictor effect of ET, the vasculature was dilated by decreasing the perfusion rate to 5 ml/min before experiments began. Experimental design Investigations on the isolated perfused heart preparation followed three protocols (Fig. 1). In protocol 1, a 10-min control period was followed by the addition of vehicle, ET, or 12-0tetradecanoyl-phorbol-13-acetate (TPA) alone, or ET in com-
Time (min) Protocol 2 Infusion of vehicle or endothelin Endothelin O.9xio"9
WKY
10
Endothelin 3.6xlO"
SHR
-X0
Endothelin 2.7xl0"9 Endothelin 0.9xl0~9
0 Time (min)
Protocol 3 Infusion of vehicle or endothelin
-10
o Time (min)
FlG. 1. Experimental protocols.
bination with TPA to the aortic perfusate as a continuous infusion via an infusion pump (B. Braun Perfuser ED, Braun Melsungen AG, Melsungen, West Germany) at an infusion rate of 0.5 ml/min for 30 min. The doses of ET used were 0.9 X 10" 9 and 2.3 X 10'9 M. Concentrations above 3 X 10"9 M were not studied, because preliminary dose-response studies showed that ET may cause arrhythmias or cardiac arrest at these doses. The experiments in hearts from SHR and WKY rats consisted of three consecutive infusions (protocol 2; Fig. 1). After a 10-min control period, vehicle or ET was infused at a rate of 0.5 ml/min for 60 min; the dose of ET was increased after 30 min of infusion. The administration of lower doses of ET into the hearts of SHR (Fig. 1) allowed us to study the effect of ET at equipressor doses on ANP release in both strains, since preliminary experiments revealed that ET had more potent vasoconstrictor actions in the SHR than in the WKY rats. In protocol 3, the effect of ET on atrial stretch-induced IRANP secretion was examined using our modified perfused rat heart preparation (6). Right atrial pressure was recorded on a Grass polygraph via a cannula (PE-60) in the inferior vena cava connected to a pressure transducer (model MP-15, Micron Instruments). A glass cannula was inserted into the pulmonary artery for the collection of perfusate. Right atrial pressure was kept constant at the desired level by adjusting the level of the pulmonary artery cannula tip. After a 10-min control period (Fig. 1) a continuous infusion of vehicle or ET was made via the aortic perfusion cannula at a rate of 0.5 ml/min for 35 min.
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ET AND ANP RELEASE Atrial stretch was superimposed for 5 min after 25-min perfusion by elevating the level of the pulmonary artery cannula tip. In protocols 2 and 3, heart rate was increased 15-20% above the spontaneous beating rate by using Harvard Apparatus Stimulator model 345 (Harvard Apparatus, Millis, MA). The coronary venous effluents were collected at either 1 (protocol 3)- or 2-min intervals, placed immediately on dry ice, and stored at —20 C until assayed. Control experiments were run with the solvents dimethylsulfoxide and 0.9% saline. Addition of an appropriate concentration of each caused no significant changes in immunoreactive ANP (IR-ANP) in the perfusate. Assay of IR-ANP in perfusate For the ANP RIA the unextracted perfusate samples were incubated in duplicates of 100 /A with 100 /xl of the middle specific rabbit ANP antiserum developed by Vuolteenaho (19, 20) in a final dilution of 1:25,000. Synthetic rat ANP-(l-28), ranging from 0-1,250 pg/tube, was incubated as standard. The ANP tracer was rat [125I]ANP-(l-28) from Amersham (Buckinghamshire, United Kingdom). After incubation for 48 h at 4 C, the immunocomplexes were precipitated with sheep antiserum against rabbit 7-globulin in the presence of 500 fi\ 1.2 M ammonium sulfate, pH 7, followed by centrifugation at 3,000 x g for 40 min. The sensitivity of the assay was 0.8 pg/tube. The 50% displacement of the standard curve occurred at 20 pg/ tube. The inter- and the intraassay variations were 14% and 5%, respectively. Serial dilutions of perfusate showed parallelism to the synthetic ANP standard. Materials Drugs used in this study were: ET-1 (Peninsula Laboratories, Inc., Belmont, CA) and the phorbol esters TPA (Sigma Chemical Co., St. Louis, MO), synthetic rat ANP-(l-28) (Sigma Chemical Co.), and heparin (Medica, Helsinki, Finland). Other chemicals were obtained from Sigma. The phorbol ester was dissolved in dimethylsulfoxide, and ET in 0.9% saline. The final concentration of each solvent was less than 0.03%. Data analysis The results are expressed as the mean ± SEM. The data were analyzed with two- or one-way analysis of variance (ANOVA). For the comparison of statistical significance between groups, Student's t test for paired or unpaired data was used. For multiple comparison, one-way ANOVA followed by the Bonferroni t test was used. Differences at the 95% level were considered significant.
Results Protocol 1: effect of ET on hembdynamic variables and IR-ANP release from hearts of Wistar rats In our rat heart preparation (n = 35), the mean initial heart rate, the perfusion pressure, and the contractile force were 240 ± 3 beats/min, 28 ± 1 mm Hg, and 1.9 ± 0.1 g, respectively. The basal concentration of IR-ANP
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in the perfusate was 231 ± 20 pg/ml (n = 35). Addition of ET (0.9 x 109-2.3 x 10 9 M) into the perfusate at a constant rate had no significant effect on heart rate (Fig. 2). The initial effect of ET (2.3 X 10 9 M) was to produce a small transient positive inotropic effect (2.02 ± 0.06 us. 1.86 ± 0.02 g before infusion; P < 0.05). The maximum increases were attained after 1-10 min of infusion. Contractile force decreased (F = 5.6 for drug and time interaction; P < 0.001) during continuous ET infusion compared to that during infusion of vehicle (Fig. 2). ET produced a rapid dose-dependent increase in perfusion pressure (0.9 X 10"9 M, F = 33.9 and P < 0.001; 2.3 X 10' 9 M, F = 111.7 and P < 0.001; F = 4.6 and P < 0.001 between the doses of ET) as soon as the infusion was started (Fig. 2). Infusion of ET caused a gradual increase in IR-ANP secretion into the perfusate (0.9 X 10"9 M, F = 14.9 and P < 0.001; 2.3 X 10"9 M, F = 15.5 and P < 0.001; Fig. 2). Infusion of 4.6 X 10 8 M of the phorbol ester TPA, alone (F = 4.1; P < 0.01) or in combination with ET (0.9 x 10"9 M; F = 8.8; P < 0.001) increased heart rate significantly (Fig. 3). Infusion of TPA also decreased contractile force (F = 37.6; P < 0.001). When ET was infused together with TPA, it transiently blocked the negative effect of TPA on contractile force by shifting the contractile force curve to the right (Fig. 3). Later, contractile force decreased in response to infusion (F = 30.1; P < 0.001). Addition of phorbol ester or phorbol ester combined with ET produced similar increases in perfusion pressure (F = 48.9 and P < 0.001, TPA us. vehicle; F = 101.3 and P < 0.001, ET and TPA vs. vehicle) as soon as the infusion was started. Addition of TPA to the perfusate did not cause any immediate change in IR-ANP secretion, but caused a gradual sustained increase in perfusate IR-ANP (F = 14.1 and P < 0.001, TPA vs. vehicle). If TPA was added to the perfusate together with ET (0.9 X 10"9 M), ET significantly enhanced the phorbol ester-induced increase in IR-ANP secretion (F = 4.5 and P < 0.001, endothelin and TPA vs. TPA; Fig. 3). Protocol 2: effect of ET on hemodynamics and IR-ANP release in WKY and SHR The mean initial heart rate was 250 ± 2 beats/min in WKY rats (n = 14) and 252 ± 2 beats/min in SHR (n = 14) and was held constant by pacing throughout the experiments. The perfusion pressures were 22 ± 1 and 26 ± 1 mm Hg (P < 0.01), and the contractile forces were 1.9 ± 0.1 and 2.0 ± 0.1 g in WKY and SHR, respectively. The basal concentration of IR-ANP in the perfusate of SHR hearts was 603 ± 67 pg/ml; in WKY hearts it was 385 ± 39 pg/ml (n = 14; P < 0.01). These basal IR-ANP levels in the perfusate were similar to
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ET AND ANP RELEASE
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Endo • 1990 Vol 126 • No 1 CONTRACTILE FORCE
FIG. 2. Effect of ET on heart rate, contractile force, perfusion pressure, and release of IR-ANP in isolated perfused rat hearts. At 10 min, as indicated by the arrows, ET at a concentration of 2.3 x 10"9 M (A; n = 8) or 0.9 X 10"9 M (•; n = 6) or vehicle (O; n = 8) was added to the perfusion fluid for 30 min. IR-ANP secretion is expressed as picograms per ml perfusate, and each point is the mean ± SEM from six to eight separate experiments run on different isolated rat hearts.
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those we reported previously (21). Infusion of vehicle alone did not affect contractile force or perfusion pressure, but a significant decrease (60%; P < 0.05) was seen in the perfusate IR-ANP concentration toward the end of the infusion in both strains (Fig. 4). Contractile force increased in response to infusion of the lower dose of ET in WKY rats and decreased in both strains when the higher concentrations of ET were infused. ET caused a dose-dependent increase in perfusion pressure in both SHR and WKY rat hearts (Fig. 4). The changes in IRANP secretion evoked by ET infusions were different in SHR and WKY rats. The ANOVA comparing the effects of ET (2.7 X 10 § M) and vehicle in the WKY rats revealed a significant drug and time interaction (F = 4.16; P
