0013-7227/90/12712-0119$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 1 Printed in U.S.A.
Endothelin: A Potent Stimulus of Atrial Natriuretic Peptide Secretion by Superfused Rat Atria and Its Dependency on Calcium* RICK J. SCHIEBINGER AND CELSO E. GOMEZ-SANCHEZ Department of Internal Medicine, Wayne State University, Detroit, Michigan 48201; Veterans Administration Medical Center, Allen Park, Michigan 48101; and Department of Internal Medicine (C.E.GS.), University of South Florida and James A. Haley Veterans Administration Medical Center, Tampa, Florida 33612
ABSTRACT. Endothelin, a hormone secreted by endothelial cells, has potent vasoconstrictive properties. Due to its potential paracrine nature, we examined the effect of endothelin-I on atrial natriuretic peptide (ANP) secretion in vitro. Isolated superfused rat left atria, paced at 2 Hz, were used for study. Endothelin (1-100 nM) increased ANP secretion in a dosedependent manner from 1.6- to 6.7-fold above baseline. Spontaneously beating right atria increased ANP secretion by 2.3fold in response to 10 nM endothelin without a change in beat frequency. However, the right atrial ANP secretory response was less than the 3.8-fold increase seen by left atria, and the time to peak response was slower. The calcium dependency of endothelin-stimulated ANP secretion was examined using paced left atria. The dependency of endothelin-stimulated secretion on calcium influx was examined by lowering the superfusate calcium from 1.8 to 0.2 mM. The ANP secretory response to 10 nM endothelin was reduced by 65% with 0.2 mM calcium. Influx of calcium through voltagedependent calcium channels was examined by superfusion with 50 /xM nitrendipine. Nitrendipine decreased endothelin-stimu-
lated ANP secretion by 51% without affecting endothelin binding. The role of intracellular calcium release from the sarcoplasmic reticulum (SR) was examined by superfusion with 1 /*M ryanodine, an inhibitor of SR calcium release. Ryanodine had no effect on endothelin-stimulated ANP secretion. We conclude: 1) Endothelin is a potent stimulus of ANP secretion in vitro. 2) The relative secretory response of right atria to endothelin expressed as a function of basal secretion is less and the time to peak secretion delayed relative to left atria. 3) Enhanced calcium influx, primarily through voltage-dependent calcium channels, plays a significant role in endothelinstimulated secretion. 4) Release of intracellular calcium from the SR does not participate in the secretory response. 5)Part of the stimulatory signal appears to be independent of calcium influx or intracellular calcium release. Thus, endothelin may be an important secretagogue or modulator of ANP secretion in vivo; however, its physiological role in regulating ANP secretion in vivo remains to be determined {Endocrinology 127: 119-125, 1990)
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influence ANP secretion. Since cardiac cells possess high affinity endothelin receptors (12,13) and endothelin has a positive inotropic effect on human and rat atria (14, 15), we examined the effect of endothelin on ANP secretion by isolated, superfused, paced rat left atria and spontaneously beating right atria. We found endothelin to be a potent stimulator of ANP secretion. We also studied the calcium dependency of endothelinstimulated ANP secretion. In vascular smooth muscle, endothelin-stimulated vasoconstriction is associated with an increase in calcium influx and efflux, a transient rise in cytosolic calcium, and formation of inositol phosphates and diacylglycerol, suggesting activation of phospholipase C and the phosphoinositide pathway (16-23). The initial rise in cytosolic calcium appears to result from release of calcium from intracellular stores since removal of extracellular calcium has only a minimal affect on this response (16-18). The contractile response of vascular smooth muscle to endothelin is partially
ATURALLY occurring hormones and factors that modify or regulate atrial natriuretic peptide (ANP) secretion continue to be explored. It has been reported that norepinephrine, epinephrine, vasopressin, acetylcholine, calcitonin gene-related peptide, and stretch increase ANP secretion in vitro (1-10). A new hormone, endothelin, which is secreted by endothelial cells, has recently been discovered (11), Peptide hormones released from endothelial cells could potentially Received March 8, 1990. Address reprint requests to: Rick J. Schiebinger, M.D., Division of Endocrinology and Hypertension, Wayne State University School of Medicine, University Health Center 4H, 4201 St. Antoine, Detroit, Michigan 48201. * This work was supported by Veterans Administration Medical Research Funds, an American Heart Association Grant-in-Aid (88 1253), and NIH R01 HL 42209. Presented in abstract form at the Council for High Blood Pressure Research 43rd Annual Fall Conference and Scientific Session, September 26-29,1989, Cleveland, Ohio.
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dependent on extracellular calcium (17, 24). Calcium channel blockers are reported to have a minimal to moderate inhibitory effect on contraction (17, 24-26). Thus, in vascular smooth muscle, stimulation with endothelin is associated with changes in cellular calcium, some of which contribute to the contractile response. Endothelin has a positive inotropic effect on atrial tissue, suggesting that endothelin increases calcium influx or the amount of calcium released from the sarcoplasmic reticulum (SR). We therefore examined the dependency of endothelin-stimulated ANP secretion on calcium influx, calcium influx through voltage-dependent calcium channels, and intracellular calcium release from the SR. We found endothelin-stimulated ANP secretion to be partially dependent on calcium influx primarily through voltage-dependent calcium channels. Release of calcium from the SR did not appear to play a role in the response. Thus, endothelin-stimulated ANP secretion is also partially dependent on calcium similar to its contractile response in vascular smooth muscle.
Materials and Methods Materials Materials were purchased from the following sources: human endothelin (endothelin-I), rat aANP, and ANP antibodies from Peninsula Laboratories (Belmont, CA); ryanodine from Progressive Agri-Systems (Wind Gap, PA); medium 199 from GIBCO (Grand Island, NY); and goat antibody to rabbit yglobulin from Calbiochem (San Diego, CA). Nitrendipine was a gift from Alexander Scriabine, Miles Institute of Preclinical Pharmacology (New Haven, CT). Experimental design Atrial superfusion experiments were performed as previously described (27). Briefly, rat left or right atria were initially stretched to 1.25 g without further adjustment. Left atria were initially paced at 1 Hz. After 30 min the pacing frequency was increased to 2 Hz. After an 85 min equilibration time, baseline sample collection commenced. This is designated as time 0 in the figures. Atria were superfused at a flow rate of 1.2 ml/min with modified medium-199 containing Earle's salts with 19 mM NaHCOa and 4 mM KC1 and gassed with 95% 02-5% C02. Experiments were performed at pH 7.4 at 37 C. Low superfusate calcium experiments were performed by lowering the calcium concentration from 1.8 to 0.2 mM 20 min before the addition of endothelin. Blocking agents were added to the superfusate 30 min after the atria were mounted and continued until the termination of the experiments. Both nitrendipine and ryanodine were added in ethanol, final concentration of 0.1% or 0.05%, respectively. All control atria were superfused with the same ethanol concentration as those receiving the inhibitor. The concentrations of test agents used in these experiments were chosen to have a maximal effect. Lowering the calcium concentration from 1.8 to 0.2 mM or superfusion with 10 nM nitrendipine or 1 fiM ryanodine has been shown to lower iso-
Endo• 1990 Vol 127 • No 1
proterenol-stimulated immunoreactive ANP (ANP-IR) secretion to baseline (28). Likewise superfusion with 0.2 mM calcium totally blocks stimulated ANP-IR secretion by phenylephrine (29) and by increasing the pacing frequency (30). Thus, similar concentrations of test agents used in this study have been shown to totally negate other stimuli of ANP-IR secretion. Atrial performance was monitored through an isometric force transducer connected to a Gould rectilinear oscillographic recorder. ANP-RIA ANP measurements were performed on timed fractions of the superfusate by RIA as previously described (27). Endothelin binding studies A crude rat atrial membrane fraction was prepared by Polytron homogenization of the atria in 250 mM sucrose, 1 mM EDTA, 50 mM Tris buffer, pH 7.4, at 4 C followed by differential centrifugation. The pellet formed from the 1,500 X g, 10 min spin was discarded, and the pellet obtained from the 200,000 X g, 30 min spin was used for the binding studies. These studies were performed with [125I]endothelin-I in the homogenization buffer plus 10 n%/m\ leupeptin at 37 C. The incubation time was 60 min. Membranes were separated by high speed centrifugation. The dissociation constant (Kd) and binding capacity were calculated utilizing the Statview II statistical program by Brain Power, Inc., (Calavazas, CA). Data analyses The results from the superfusion experiments are expressed as a percentage of the mean value of 7 baseline measurements taken between 0 and 15 min. The results in Fig. 2B are expressed as a percent of the net maximal increase in ANP secretion. In these studies the average of 7 baseline samples was first subtracted from all of the ANP measurements. The resulting values were divided by the highest stimulated value after the subtraction step. All the results are expressed as mean ± SE. Statistical analyses were performed using paired or unpaired t test and by multivariant analysis of variance (repeated measures) procedure of SPSS Inc. (Chicago, IL).
Results The effect of endothelin on ANP-IR secretion by rat left atria paced at 2 Hz is shown in Fig. 1. Endothelin at concentrations ranging from 1-100 nM induced a rapid rise in ANP-IR secretion. ANP-IR secretion rose maximally to 1.6-, 3.2-, and 6.7-fold above baseline at 1, 10, and 100 nM, respectively. Maximal ANP-IR secretion at 100 nM, however, was not sustained. Developed tension rose 1.7 ± 0.2, 2.6 ± 0.4, and 4.8 ± 0.5-fold above baseline at 1, 10, and 100 nM endothelin, respectively (Table 1). Resting tension did not change. Thus, endothelin has positive inotropic properties and is a potent stimulator of ANP-IR secretion. In spontaneously beating right atria, superfusion with
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 09:55 For personal use only. No other uses without permission. . All rights reserved.
ENDOTHELIN AND ANP SECRETION
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FIG. 1. Dose-dependent ANP-IR secretion in response to endothelin. Left atria paced at 2 Hz were continuously superfused with none (A, n = 7), 1 nM (O, n = 6), 10 nM (•, n = 6), or 100 nM (A, n = 5) endothelin from 15 (arrow) to 60 min. Results are expressed as a percent of basal secretion defined as the mean value obtained between 0-15 min. Basal ANP-IR secretion was 249 ± 49, 257 ± 23, 350 ± 63, and 228 ± 37 pg/ ml for none, 1, 10, and 100 nM endothelin, respectively. TABLE 1. Effect of endothelin on developed and resting tension by rat left atria paced at 2 Hz Developed tension (g)
Resting tension (g)
None 1 nM
0.10 ± 0.02 0.16 ± 0.02°
0.32 ± 0.05 NC6
None 10 nM
0.12 ± 0.02 0.31 ± 0.05°
0.37 ± 0.05
None 100 nM
0.07 ± 0.01 0.33 ± 0.09c
0.35 ± 0.05
10
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TIME (min)
Endothelin
(X 250 O LJ 200 if) (£ 150
Number of atria
NC NC
0
P < 0.02 relative to baseline. * NC, No change. c P < 0.04.
10 nM endothelin increased ANP-IR (Fig. 2A) without significantly affecting the beat frequency (4.0 ± 0 . 1 vs. 3.8 ± 0.2 Hz after addition of endothelin). The maximal fold increase in the ANP-IR secretory response of right atria was less than that achieved by left atria paced at 4 Hz (2.4 ± 0.3 us. 3.3 ± 0.2-fold, P < 0.03). However, basal ANP-IR secretion by right atria was greater than that of left atria (677 ± 74 vs. 240 ± 27 pg/ml). Thus, even though the maximal fold increase in ANP-IR secretion by right atria was less than in left atria, the absolute maximal rise in ANP-IR secretion was greater in right atria, 880 ± 133 us. 530 ± 74 pg/ml (P = 0.05). The pattern of the ANP-IR secretory response of right atria also differed from that of left atria (Fig. 2B). The maximal right atrial response to endothelin was delayed relative to that of left atria. The delay of right atria to reach the peak secretory response was associated with a longer time interval to reach a maximal rise in developed tension, 23.3 ± 2.0 min vs. 13.2 ± 0.9 min for left atria (P < 0.01). Right atrial developed tension rose from 0.16 ±
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TIME (min) FIG. 2. Effect of endothelin on ANP-IR secretion by spontaneously beating right atria. A, Spontaneously beating right atria (O, n = 5) and left atria paced at 4 Hz (•, n = 5) were continuously superfused with 10 nM endothelin from 15 (arrow) to 60 min. Right atria that did not beat spontaneously (n = 3) and left atria (n = 3) were paced at 4 Hz and used as controls (A). Results are expressed as in Fig. 1. Basal ANPIR secretion was 389 ± 86, 677 ± 74, and 240 ± 27 pg/ml for control, right, and left atria, respectively. B, Comparison of the normalized response of right (O) and left (•) atria to 10 nM endothelin. The data from panel A are plotted as a percent of the net maximal rise in ANPIR secretion in response to 10 nM endothelin. The response curves are significantly different (P < 0.001).
0.04 to 0.26 ± 0.05 g (P < 0.01), and left atrial developed tension rose from 0.09 ± 0.01 to 0.20 ± 0.01 g (P < 0.01). The relative increase in developed tension by right atria was less than that in left atria 1.7 ± 0.1 vs. 2.2 ± 0.1-fold (P < 0.02), respectively. Resting tensions were 0.42 ± 0.06 and 0.34 ± 0.02 g for right and left atria, respectively. Again, resting tension did not change with the addition of endothelin. Thus, endothelin increases ANP-IR secretion by spontaneously beating right atria without chang-
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ing the beat frequency. To determine whether calcium influx was necessary for endothelin stimulation, the superfusate calcium concentration was lowered from 1.8 to 0.2 mM 20 min before the addition of 10 nM endothelin to left atria paced at 2 Hz. ANP-IR secretion rose 2.2 ± 0.1-fold in the presence of 0.2 mM calcium vs 4.4 ± 0.4-fold with 1.8 mM calcium (Fig. 3). Thus, the secretory response to endothelin with 0.2 mM calcium was 35% of that achieved with 1.8 mM calcium. Developed tension was less than 0.01 g by atria superfused with 0.2 mM calcium. No detectable change in developed tension was observed after the addition of endothelin. These results suggest that calcium influx plays a significant role in endothelin-stimulated ANPIR secretion. The dependency of endothelin-stimulated ANP on calcium influx through voltage-dependent calcium channels was studied next. Left atria paced at 2 Hz were superfused with 50 nM nitrendipine for 70 min before stimulation with 10 nM endothelin. Nitrendipine blunted endothelin-stimulated secretion by 51% (Fig. 4). Endothelin-stimulated ANP-IR secretion rose 2.9 ± 0.3-fold in the presence of nitrendipine and 4.8 ± 0.4-fold in its absence. Developed tension rose from 0.06 ± 0.01 to 0.13 ± 0.02 g (2.1 ± 0.1-fold) with nitrendipine and from 0.11 ± 0.01 to 0.35 ± 0.04 g (3.1 ± 0.3-fold) without nitrendipine after stimulation with endothelin. Thus, there was a reduction of 46% in the fold increase in developed tension with nitrendipine (P < 0.01). Nitrendipine did not inhibit endothelin binding to atrial membranes. The Kd and maximal binding capacity without and with 50 nM nitrendipine were 0.62 vs. 0.50 nM and 671 vs. 628 fmol/mg protein (n = mean of two experiments). These observations suggest that increased calcium influx
Endo • 1990 Vol 127 • No 1
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TIME (min) FlG. 4. Effect of nitrendipine on endothelin-stimulated ANP-IR secretion. Left atria paced at 2 Hz were superfused with 50 (iM nitrendipine alone (A, n = 4), 50 j*M nitrendipine plus 10 nM endothelin (•, n = 7), or vehicle plus 10 nM endothelin (O, n = 7). Results are expressed as in Fig. 1. Basal ANP-IR secretion: 146 ± 30, 253 ± 41, and 359 ± 47 pg/ml for nitrendipine alone, nitrendipine plus endothelin, and vehicle plus endothelin, respectively.
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Vol. 127, No. 1 Printed in U.S.A.
Endothelin: A Potent Stimulus of Atrial Natriuretic Peptide Secretion by Superfused Rat Atria and Its Dependency on Calcium* RICK J. SCHIEBINGER AND CELSO E. GOMEZ-SANCHEZ Department of Internal Medicine, Wayne State University, Detroit, Michigan 48201; Veterans Administration Medical Center, Allen Park, Michigan 48101; and Department of Internal Medicine (C.E.GS.), University of South Florida and James A. Haley Veterans Administration Medical Center, Tampa, Florida 33612
ABSTRACT. Endothelin, a hormone secreted by endothelial cells, has potent vasoconstrictive properties. Due to its potential paracrine nature, we examined the effect of endothelin-I on atrial natriuretic peptide (ANP) secretion in vitro. Isolated superfused rat left atria, paced at 2 Hz, were used for study. Endothelin (1-100 nM) increased ANP secretion in a dosedependent manner from 1.6- to 6.7-fold above baseline. Spontaneously beating right atria increased ANP secretion by 2.3fold in response to 10 nM endothelin without a change in beat frequency. However, the right atrial ANP secretory response was less than the 3.8-fold increase seen by left atria, and the time to peak response was slower. The calcium dependency of endothelin-stimulated ANP secretion was examined using paced left atria. The dependency of endothelin-stimulated secretion on calcium influx was examined by lowering the superfusate calcium from 1.8 to 0.2 mM. The ANP secretory response to 10 nM endothelin was reduced by 65% with 0.2 mM calcium. Influx of calcium through voltagedependent calcium channels was examined by superfusion with 50 /xM nitrendipine. Nitrendipine decreased endothelin-stimu-
lated ANP secretion by 51% without affecting endothelin binding. The role of intracellular calcium release from the sarcoplasmic reticulum (SR) was examined by superfusion with 1 /*M ryanodine, an inhibitor of SR calcium release. Ryanodine had no effect on endothelin-stimulated ANP secretion. We conclude: 1) Endothelin is a potent stimulus of ANP secretion in vitro. 2) The relative secretory response of right atria to endothelin expressed as a function of basal secretion is less and the time to peak secretion delayed relative to left atria. 3) Enhanced calcium influx, primarily through voltage-dependent calcium channels, plays a significant role in endothelinstimulated secretion. 4) Release of intracellular calcium from the SR does not participate in the secretory response. 5)Part of the stimulatory signal appears to be independent of calcium influx or intracellular calcium release. Thus, endothelin may be an important secretagogue or modulator of ANP secretion in vivo; however, its physiological role in regulating ANP secretion in vivo remains to be determined {Endocrinology 127: 119-125, 1990)
N
influence ANP secretion. Since cardiac cells possess high affinity endothelin receptors (12,13) and endothelin has a positive inotropic effect on human and rat atria (14, 15), we examined the effect of endothelin on ANP secretion by isolated, superfused, paced rat left atria and spontaneously beating right atria. We found endothelin to be a potent stimulator of ANP secretion. We also studied the calcium dependency of endothelinstimulated ANP secretion. In vascular smooth muscle, endothelin-stimulated vasoconstriction is associated with an increase in calcium influx and efflux, a transient rise in cytosolic calcium, and formation of inositol phosphates and diacylglycerol, suggesting activation of phospholipase C and the phosphoinositide pathway (16-23). The initial rise in cytosolic calcium appears to result from release of calcium from intracellular stores since removal of extracellular calcium has only a minimal affect on this response (16-18). The contractile response of vascular smooth muscle to endothelin is partially
ATURALLY occurring hormones and factors that modify or regulate atrial natriuretic peptide (ANP) secretion continue to be explored. It has been reported that norepinephrine, epinephrine, vasopressin, acetylcholine, calcitonin gene-related peptide, and stretch increase ANP secretion in vitro (1-10). A new hormone, endothelin, which is secreted by endothelial cells, has recently been discovered (11), Peptide hormones released from endothelial cells could potentially Received March 8, 1990. Address reprint requests to: Rick J. Schiebinger, M.D., Division of Endocrinology and Hypertension, Wayne State University School of Medicine, University Health Center 4H, 4201 St. Antoine, Detroit, Michigan 48201. * This work was supported by Veterans Administration Medical Research Funds, an American Heart Association Grant-in-Aid (88 1253), and NIH R01 HL 42209. Presented in abstract form at the Council for High Blood Pressure Research 43rd Annual Fall Conference and Scientific Session, September 26-29,1989, Cleveland, Ohio.
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dependent on extracellular calcium (17, 24). Calcium channel blockers are reported to have a minimal to moderate inhibitory effect on contraction (17, 24-26). Thus, in vascular smooth muscle, stimulation with endothelin is associated with changes in cellular calcium, some of which contribute to the contractile response. Endothelin has a positive inotropic effect on atrial tissue, suggesting that endothelin increases calcium influx or the amount of calcium released from the sarcoplasmic reticulum (SR). We therefore examined the dependency of endothelin-stimulated ANP secretion on calcium influx, calcium influx through voltage-dependent calcium channels, and intracellular calcium release from the SR. We found endothelin-stimulated ANP secretion to be partially dependent on calcium influx primarily through voltage-dependent calcium channels. Release of calcium from the SR did not appear to play a role in the response. Thus, endothelin-stimulated ANP secretion is also partially dependent on calcium similar to its contractile response in vascular smooth muscle.
Materials and Methods Materials Materials were purchased from the following sources: human endothelin (endothelin-I), rat aANP, and ANP antibodies from Peninsula Laboratories (Belmont, CA); ryanodine from Progressive Agri-Systems (Wind Gap, PA); medium 199 from GIBCO (Grand Island, NY); and goat antibody to rabbit yglobulin from Calbiochem (San Diego, CA). Nitrendipine was a gift from Alexander Scriabine, Miles Institute of Preclinical Pharmacology (New Haven, CT). Experimental design Atrial superfusion experiments were performed as previously described (27). Briefly, rat left or right atria were initially stretched to 1.25 g without further adjustment. Left atria were initially paced at 1 Hz. After 30 min the pacing frequency was increased to 2 Hz. After an 85 min equilibration time, baseline sample collection commenced. This is designated as time 0 in the figures. Atria were superfused at a flow rate of 1.2 ml/min with modified medium-199 containing Earle's salts with 19 mM NaHCOa and 4 mM KC1 and gassed with 95% 02-5% C02. Experiments were performed at pH 7.4 at 37 C. Low superfusate calcium experiments were performed by lowering the calcium concentration from 1.8 to 0.2 mM 20 min before the addition of endothelin. Blocking agents were added to the superfusate 30 min after the atria were mounted and continued until the termination of the experiments. Both nitrendipine and ryanodine were added in ethanol, final concentration of 0.1% or 0.05%, respectively. All control atria were superfused with the same ethanol concentration as those receiving the inhibitor. The concentrations of test agents used in these experiments were chosen to have a maximal effect. Lowering the calcium concentration from 1.8 to 0.2 mM or superfusion with 10 nM nitrendipine or 1 fiM ryanodine has been shown to lower iso-
Endo• 1990 Vol 127 • No 1
proterenol-stimulated immunoreactive ANP (ANP-IR) secretion to baseline (28). Likewise superfusion with 0.2 mM calcium totally blocks stimulated ANP-IR secretion by phenylephrine (29) and by increasing the pacing frequency (30). Thus, similar concentrations of test agents used in this study have been shown to totally negate other stimuli of ANP-IR secretion. Atrial performance was monitored through an isometric force transducer connected to a Gould rectilinear oscillographic recorder. ANP-RIA ANP measurements were performed on timed fractions of the superfusate by RIA as previously described (27). Endothelin binding studies A crude rat atrial membrane fraction was prepared by Polytron homogenization of the atria in 250 mM sucrose, 1 mM EDTA, 50 mM Tris buffer, pH 7.4, at 4 C followed by differential centrifugation. The pellet formed from the 1,500 X g, 10 min spin was discarded, and the pellet obtained from the 200,000 X g, 30 min spin was used for the binding studies. These studies were performed with [125I]endothelin-I in the homogenization buffer plus 10 n%/m\ leupeptin at 37 C. The incubation time was 60 min. Membranes were separated by high speed centrifugation. The dissociation constant (Kd) and binding capacity were calculated utilizing the Statview II statistical program by Brain Power, Inc., (Calavazas, CA). Data analyses The results from the superfusion experiments are expressed as a percentage of the mean value of 7 baseline measurements taken between 0 and 15 min. The results in Fig. 2B are expressed as a percent of the net maximal increase in ANP secretion. In these studies the average of 7 baseline samples was first subtracted from all of the ANP measurements. The resulting values were divided by the highest stimulated value after the subtraction step. All the results are expressed as mean ± SE. Statistical analyses were performed using paired or unpaired t test and by multivariant analysis of variance (repeated measures) procedure of SPSS Inc. (Chicago, IL).
Results The effect of endothelin on ANP-IR secretion by rat left atria paced at 2 Hz is shown in Fig. 1. Endothelin at concentrations ranging from 1-100 nM induced a rapid rise in ANP-IR secretion. ANP-IR secretion rose maximally to 1.6-, 3.2-, and 6.7-fold above baseline at 1, 10, and 100 nM, respectively. Maximal ANP-IR secretion at 100 nM, however, was not sustained. Developed tension rose 1.7 ± 0.2, 2.6 ± 0.4, and 4.8 ± 0.5-fold above baseline at 1, 10, and 100 nM endothelin, respectively (Table 1). Resting tension did not change. Thus, endothelin has positive inotropic properties and is a potent stimulator of ANP-IR secretion. In spontaneously beating right atria, superfusion with
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 09:55 For personal use only. No other uses without permission. . All rights reserved.
ENDOTHELIN AND ANP SECRETION
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FIG. 1. Dose-dependent ANP-IR secretion in response to endothelin. Left atria paced at 2 Hz were continuously superfused with none (A, n = 7), 1 nM (O, n = 6), 10 nM (•, n = 6), or 100 nM (A, n = 5) endothelin from 15 (arrow) to 60 min. Results are expressed as a percent of basal secretion defined as the mean value obtained between 0-15 min. Basal ANP-IR secretion was 249 ± 49, 257 ± 23, 350 ± 63, and 228 ± 37 pg/ ml for none, 1, 10, and 100 nM endothelin, respectively. TABLE 1. Effect of endothelin on developed and resting tension by rat left atria paced at 2 Hz Developed tension (g)
Resting tension (g)
None 1 nM
0.10 ± 0.02 0.16 ± 0.02°
0.32 ± 0.05 NC6
None 10 nM
0.12 ± 0.02 0.31 ± 0.05°
0.37 ± 0.05
None 100 nM
0.07 ± 0.01 0.33 ± 0.09c
0.35 ± 0.05
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(X 250 O LJ 200 if) (£ 150
Number of atria
NC NC
0
P < 0.02 relative to baseline. * NC, No change. c P < 0.04.
10 nM endothelin increased ANP-IR (Fig. 2A) without significantly affecting the beat frequency (4.0 ± 0 . 1 vs. 3.8 ± 0.2 Hz after addition of endothelin). The maximal fold increase in the ANP-IR secretory response of right atria was less than that achieved by left atria paced at 4 Hz (2.4 ± 0.3 us. 3.3 ± 0.2-fold, P < 0.03). However, basal ANP-IR secretion by right atria was greater than that of left atria (677 ± 74 vs. 240 ± 27 pg/ml). Thus, even though the maximal fold increase in ANP-IR secretion by right atria was less than in left atria, the absolute maximal rise in ANP-IR secretion was greater in right atria, 880 ± 133 us. 530 ± 74 pg/ml (P = 0.05). The pattern of the ANP-IR secretory response of right atria also differed from that of left atria (Fig. 2B). The maximal right atrial response to endothelin was delayed relative to that of left atria. The delay of right atria to reach the peak secretory response was associated with a longer time interval to reach a maximal rise in developed tension, 23.3 ± 2.0 min vs. 13.2 ± 0.9 min for left atria (P < 0.01). Right atrial developed tension rose from 0.16 ±
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TIME (min) FIG. 2. Effect of endothelin on ANP-IR secretion by spontaneously beating right atria. A, Spontaneously beating right atria (O, n = 5) and left atria paced at 4 Hz (•, n = 5) were continuously superfused with 10 nM endothelin from 15 (arrow) to 60 min. Right atria that did not beat spontaneously (n = 3) and left atria (n = 3) were paced at 4 Hz and used as controls (A). Results are expressed as in Fig. 1. Basal ANPIR secretion was 389 ± 86, 677 ± 74, and 240 ± 27 pg/ml for control, right, and left atria, respectively. B, Comparison of the normalized response of right (O) and left (•) atria to 10 nM endothelin. The data from panel A are plotted as a percent of the net maximal rise in ANPIR secretion in response to 10 nM endothelin. The response curves are significantly different (P < 0.001).
0.04 to 0.26 ± 0.05 g (P < 0.01), and left atrial developed tension rose from 0.09 ± 0.01 to 0.20 ± 0.01 g (P < 0.01). The relative increase in developed tension by right atria was less than that in left atria 1.7 ± 0.1 vs. 2.2 ± 0.1-fold (P < 0.02), respectively. Resting tensions were 0.42 ± 0.06 and 0.34 ± 0.02 g for right and left atria, respectively. Again, resting tension did not change with the addition of endothelin. Thus, endothelin increases ANP-IR secretion by spontaneously beating right atria without chang-
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ENDOTHELIN AND ANP SECRETION
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ing the beat frequency. To determine whether calcium influx was necessary for endothelin stimulation, the superfusate calcium concentration was lowered from 1.8 to 0.2 mM 20 min before the addition of 10 nM endothelin to left atria paced at 2 Hz. ANP-IR secretion rose 2.2 ± 0.1-fold in the presence of 0.2 mM calcium vs 4.4 ± 0.4-fold with 1.8 mM calcium (Fig. 3). Thus, the secretory response to endothelin with 0.2 mM calcium was 35% of that achieved with 1.8 mM calcium. Developed tension was less than 0.01 g by atria superfused with 0.2 mM calcium. No detectable change in developed tension was observed after the addition of endothelin. These results suggest that calcium influx plays a significant role in endothelin-stimulated ANPIR secretion. The dependency of endothelin-stimulated ANP on calcium influx through voltage-dependent calcium channels was studied next. Left atria paced at 2 Hz were superfused with 50 nM nitrendipine for 70 min before stimulation with 10 nM endothelin. Nitrendipine blunted endothelin-stimulated secretion by 51% (Fig. 4). Endothelin-stimulated ANP-IR secretion rose 2.9 ± 0.3-fold in the presence of nitrendipine and 4.8 ± 0.4-fold in its absence. Developed tension rose from 0.06 ± 0.01 to 0.13 ± 0.02 g (2.1 ± 0.1-fold) with nitrendipine and from 0.11 ± 0.01 to 0.35 ± 0.04 g (3.1 ± 0.3-fold) without nitrendipine after stimulation with endothelin. Thus, there was a reduction of 46% in the fold increase in developed tension with nitrendipine (P < 0.01). Nitrendipine did not inhibit endothelin binding to atrial membranes. The Kd and maximal binding capacity without and with 50 nM nitrendipine were 0.62 vs. 0.50 nM and 671 vs. 628 fmol/mg protein (n = mean of two experiments). These observations suggest that increased calcium influx
Endo • 1990 Vol 127 • No 1
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TIME (min) FlG. 4. Effect of nitrendipine on endothelin-stimulated ANP-IR secretion. Left atria paced at 2 Hz were superfused with 50 (iM nitrendipine alone (A, n = 4), 50 j*M nitrendipine plus 10 nM endothelin (•, n = 7), or vehicle plus 10 nM endothelin (O, n = 7). Results are expressed as in Fig. 1. Basal ANP-IR secretion: 146 ± 30, 253 ± 41, and 359 ± 47 pg/ml for nitrendipine alone, nitrendipine plus endothelin, and vehicle plus endothelin, respectively.
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