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Atriopeptin and spontaneous hypertension in rats
Life Sciences, Vol. 38, pp. 1035-1039 Printed in the U.S.A.
Pergamon Pres~
ATRIOPEPTIN AND SPONTANEOUS HYPERTENSION IN RATS C.W.Xie, D~L.Song, J.F.Ding, J.K.Chang*, D.Chang* and J.Tang Lab of Cardiopulmonary Endocrinology, Beijing Medical University, Beijing 100083, CHINA and *Peninsula Labs, Inc., Belmont, CA 94002, USA
(Received in final form January 2, 1986)
SUMMARY The involvement of atriopeptin in hypertension was investigated in spontaneously hypertensive rats(SHR). It was found that intravenous injection of atriopeptin III ( 20 - 80 nmol/kg ) markedly decreased the mean arterial pressure in anesthetized SHR in a dose dependent manner. The heart rate was not significantly affected. The contents of atriopeptin immunoreactive material in the rat atrium and plasma were measured with radioimmunoassay. Both the atrium and plasma contents of a t r i o p e p tin immunoreaetive material were found to be significantly higher in SHR than in the normotensive control Wistar Kyoto (~,~Y) , indicating an increase in the biosynthesis and release of atriopeptin in SHR. Whether this change was a compensatory response induced by hypertension remains to be investigated.
A number of atrial peptides, isolated from mammalian atria and variously named atriopeptin, cardionatrin and atrial natriuretic factor, have been purified and sequenced(l-6). Physiological experiments have demonstrated that atrial peptides possess natriuretic,diuretic,vasorelaxant and depressor activities (4,7-9), suggesting a possible involvement of atrial peptides in the control of circulatory functions and water-electrolyte balance. To date, however, little or nothing is known of their roles in pathology. In the present study,atriopeptin involvement in hypertension mechanism was investigated in spontaneously hypertensive rats ( SIIR ). The antihypertensive effect of atriopeptin 151 was observed and plasma and atrium levels of atriopeptin immunoreactivity were measured with a sensitive and specific radioimmunoassay. MATERIALS AND METHODS ~iale and female SIIR and the matched control WKY weighing 230 -270 were used in all experiments. A catheter was placed in the left carotid artery and attached to a pressure transducer and recorder to measure the mean arterial pressure(MAP) and the heart rate (HR) of the rats anesthetized with urethane(Ig/kg). Atriopeptin III, synthesized 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
1036
Atriopeptin and Spontaneous Hypertension
Vol. 38, No. ii, 1986
by standard solid phase method at Peninsula Labs (Belmont, CA), was dissolved in 0.1 ml of sterile normal saline and injected in a bolus into a cannulated jugular vein. Dose response was observed in the same animals given multiple injections at 2 hour intervals when the basal level of MAP and HR could be resumed . The loss of body fluid in the rats was compensated by a continuous infusion of normal saline (Iml/hr) into jugular vein with a peristaltic pump. Rats were killed by d e c a p i t a t i o n for m e a s u r e m e n t of immun o r e a c t i v e a t r i o p e p t i n in the atrium and plasma. The atria were dissected and homogenized in I ml of ice cold I N acetic acid . The h o m o g e n a t e s were heated at 95°C for 10 minutes and centrifuged at 12000 x g for 20 minutes. The supernatants were lyophilized, redissolved in distilled water, and r a d i o i m m u n o a s s a y e d . The blood samples were collected during d e c a p i t a t i o n into heparinized polyethylene tubes containing a p r o t i n i n (200 KIU/ml). The plasma was separated from the blood by c e n t r i f u g a t i o n at 1700 × g at 4°C for 30 minutes and stored at -20°C. Before radioimmunoassay a n o t h e r c e n t r i f u g a t i o n ( 4 O O O 0 x g , 4 ° C , 3 0 minutes ) was given to p r e c i p i t a t e the protein. The supernatants were assayed for a t r i o p e p t i n content. A plasma blank solution was prepared from the plasma without adding aprotinin, which was incubated at 37°C for 24 hours and then centrifuged as the sample. A radioimmunoassay was performed by incubating standard a t r i o p e p t i n III or a sample (50 ~I) for 17-24 hours at 4°C with a n t i s e r u m and 1251-atriopeptin (8000-10000 cpm) in 450 ~i of 0.1 M sodium phosphate buffer (pH 7.4). ',~en plasma samples were measured, the plasma blank solution (50 ~i) was added to each standard curve tube instead of the same volume of the buffer to correct the protein c o n c e n t r a t i o n and other interfering materials in the plasma. Separation of bound and free 1251-1abeled tracer was achieved by the addition of 2OO~I of d e x t r a n - c o a t e d charcoal solution and centrifuged at 17OOxg for 10 minutes. The 1251a t r i o p e p t i n in the supernatant was measured in a gamma counter. The a n t i s e r u m was raised against a t r i o p e p t i n I, which shows IOO% cross reactivity with atriopeptin III and ~-human atrial n a t r i u r e t i c polypeptide ( ~ - h A N P ) and dose not cross-react with methionine enkephalin, cholecystokinin, dynorphin A ( 1-13 ), bradykinin, substance P, or ~ - e n d o r p h i n ( 1 0 ) . The a n t i s e r u m was used at a final dilution of 1:20000 giving a p p r o x i m a t e l y 40% ~ binding of the added tracer in the absence of "cold"atriopeptin. RESULTS The time-course and d o s e - r e s p o n s e relation of the hypotensive action of a t r i o p e p t i n III is shown in Fig.1. The basal levels of MAP and HR were 175+17 mmHg and 340+18 beats/min, respectively. Intravenous injection of a t r i o p e p t i ~ III (20-80 nmol/kg) lowered MAP in a d o s e - d e p e n d e n t manner. The decrease in ~AP lasted more than I hour with peak change occurring about 5 minutes after injection. B r a d y c a r d i a c response was found in a p p r o x i m a t e l y I/3 of the animals after injection, but no statistical significance was shown in the change of HR before and after injection.
Vol. 38, No. ii, 1 9 8 6
w
Atriopeptln and Spontaneous Hypertension
1037
D.Il
i
0
i
~
|
FIG.
4~0
i
i
60rain
I
Effect of atriopeptin III (AP III) on m e a n a ~ e r i a l pressure (MAP) and heart rate (HR) of SHR. Dosedependent responses were obtained in the same animals given intravenously 2 0 ( o ) , 4 0 ( @ ) and 80 ( . ) nmol/kg of AP III (injection intervals of 2
hours).
The contents of atriopeptin immunoreactive material (atriopeptin IR) in the rat atrium and plasma were measured. The results are shown in Table I. Atriopeptin IR contents in SHR were significantly higher than those in WKY. There was no significant correlation between the level of blood pressure with atriopeptin IR level in the atrium or plasma of SHR. TABLE I Atrium (pmol/mg protein)
Plasma (pmol/ml)
SHR
1.21 + 0.10"
0.36 + 0.07*
WKY
0.86 + 0.09
0.16 + 0.06
TABLE I: *P <0.05.
Change of atriopeptin IR in SHR.
N = 8,
DISCUSSION It has been reported that intravenous injection of synthetic atrial peptides induced hypotensive response in normotensive rats. Atriopeptin III and ~ - h A N P were more active than atriopeptin I and II(8). The data presented here show that synthetic atriopeptin III had a potent depressor effect also in h y p e r t e n sive rats. Consistent with early results, The heart rate was not affected significantly by atriopeptin, which indicated that the fall in blood pressure was not due to the decrease in heart rate.
1038
Atriopeptin and Spontaneous Hypertension
Vol. 38, No. ii, 1986
The depressor effect occured almost immediately after injection, so that it is infered that the direct vasorelaxant activity (3) and decrease in stroke volume(11) induced by atriopeptin seem to be responsible for the hypotensive response of SHR. A possible hypovolemia induced by diuresis and natriuresis after injection may be also involved in the depressor mechanism of atriopeptin, because the potency order of atrial peptides in depressor activity was quite similar to that in diuretic and natriuretic activities(8). In order to understand the effect of atriopeptin in the mechanism of hypertension, we measured the atriopeptin contents in the atrium and plasma. It was found that SHR had a higher content of atriopeptin than },~Y both in the atrium and in the plasma. This result, which indicates an over-activation of the atriopeptin system with increased biosynthesis and release in hypertensive rats, is contrary to the result recently found in man where an obvious decrease in plasma level of atrio~eptin was found in patients with sever essential hypertension(12). Such a conflict suggests that atriopeptin involvement in hypertension pathophysiology may be considerably complicated and may depend on the species, type, severity and duration of hypertension. We suggest that the high level of atriopeptin in the plasma and atrium of SHR seems to be a compensatory response induced by b@pertension and leaves open the possibility that different mechanisms of action occur in the two species, humans and rats. Although the significance of increased atriopeptin IR contents in SHR remains obscure because no good correlation was found between the MAP level and atriopeptin IR contents, the potent antihypertensive effect of atriopeptin on SHR suggests that the application of atriopeptins or their analogs may be a new way for clinical treatment of hypertension. REFEREN CES I.
T.G.FLYNN, M.L.DE BOLD and A.J.DE BOLD, Biochem. Biophys.Res. Commun., 117, 859-865 (1983). 2. K.KANGAWA, A.FUKUDA, N.MINAMINO, H.~ATSUO, ~iochem. ~iophys. Res. Commun., 119, 933-940 (1984). 3. K.S.MISONS, H.FUKUMI, R.T.GRAMMER and T. INAGAMI, Biochem . Biophys. Res. Commun., 119, 524-529 (1984). N.R.SIEGEL, K.F.POK, S. P. 4. M.G.CURRIE, D.M.GELLER, B.R. vOLE, '~ ~ ADAMS, S.R.EUBANKS, G.R.CALLUPPI and P.NEEDLEMAN, Science, 223, 67-69 (1984). 5. D.M.GELLER, H.G.CURRIE, K.WAKITANI, B.R.COLE, S.P.ADAMS, K.F. FOK, N.R.SIEGEL, S.R.EUBANKS, G.R.GALLUPPI and P.NEEDLEMAN , Biochem. Biophys. Res. Commun., 120, 333-338 (1984). 6. K.KANGAWA and H.MATSUO, Biochem. Biophys. Res. Commun., 118, 131-139 (1984). 7. A.J.DE BOLD, H.B.BORENSTEIN, A.T.VERESS and SONNENBERG, Life Sci., 28, 89-94 (1981). 8. J.TANG, R . J . ~ B B E R , D.CHANG, J.K.CHANG, J.KI&NG and E.T.}SI, Regul. Pept., ~, 53-59 (1984). 9. R.T.GRAMMER, H.FUKUMI, T.iNAGAMI, K.S.MISONO, Biochem. Biophys. Res. Commun., 116, 696-703 (1983). 10. J.TANG, H.FEI, C.W.XIE, M.Z.SUEN, J.S.HAN, R.J.V~BBER, D.CHANG and J.K.CHANG, Pepti@es, ~, 1173-1177 (1984).
Vol. 38, No. ii, 1986
Atriopeptin and Spontaneous Hypertension
1039
11. H.D.KLEINERT, M.VOLPE, M.J.F.CAMARGO, S.A.ATLAS, J.H.LARAGH and T.Y~IACK, Fed. Proe., 44, 1729 (1985). 12. J.TANG, D.L.SONG, M.Z.SUEN and C.W.XIE, Peptides, in press, ( 1986 ).
Pergamon Pres~
ATRIOPEPTIN AND SPONTANEOUS HYPERTENSION IN RATS C.W.Xie, D~L.Song, J.F.Ding, J.K.Chang*, D.Chang* and J.Tang Lab of Cardiopulmonary Endocrinology, Beijing Medical University, Beijing 100083, CHINA and *Peninsula Labs, Inc., Belmont, CA 94002, USA
(Received in final form January 2, 1986)
SUMMARY The involvement of atriopeptin in hypertension was investigated in spontaneously hypertensive rats(SHR). It was found that intravenous injection of atriopeptin III ( 20 - 80 nmol/kg ) markedly decreased the mean arterial pressure in anesthetized SHR in a dose dependent manner. The heart rate was not significantly affected. The contents of atriopeptin immunoreactive material in the rat atrium and plasma were measured with radioimmunoassay. Both the atrium and plasma contents of a t r i o p e p tin immunoreaetive material were found to be significantly higher in SHR than in the normotensive control Wistar Kyoto (~,~Y) , indicating an increase in the biosynthesis and release of atriopeptin in SHR. Whether this change was a compensatory response induced by hypertension remains to be investigated.
A number of atrial peptides, isolated from mammalian atria and variously named atriopeptin, cardionatrin and atrial natriuretic factor, have been purified and sequenced(l-6). Physiological experiments have demonstrated that atrial peptides possess natriuretic,diuretic,vasorelaxant and depressor activities (4,7-9), suggesting a possible involvement of atrial peptides in the control of circulatory functions and water-electrolyte balance. To date, however, little or nothing is known of their roles in pathology. In the present study,atriopeptin involvement in hypertension mechanism was investigated in spontaneously hypertensive rats ( SIIR ). The antihypertensive effect of atriopeptin 151 was observed and plasma and atrium levels of atriopeptin immunoreactivity were measured with a sensitive and specific radioimmunoassay. MATERIALS AND METHODS ~iale and female SIIR and the matched control WKY weighing 230 -270 were used in all experiments. A catheter was placed in the left carotid artery and attached to a pressure transducer and recorder to measure the mean arterial pressure(MAP) and the heart rate (HR) of the rats anesthetized with urethane(Ig/kg). Atriopeptin III, synthesized 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
1036
Atriopeptin and Spontaneous Hypertension
Vol. 38, No. ii, 1986
by standard solid phase method at Peninsula Labs (Belmont, CA), was dissolved in 0.1 ml of sterile normal saline and injected in a bolus into a cannulated jugular vein. Dose response was observed in the same animals given multiple injections at 2 hour intervals when the basal level of MAP and HR could be resumed . The loss of body fluid in the rats was compensated by a continuous infusion of normal saline (Iml/hr) into jugular vein with a peristaltic pump. Rats were killed by d e c a p i t a t i o n for m e a s u r e m e n t of immun o r e a c t i v e a t r i o p e p t i n in the atrium and plasma. The atria were dissected and homogenized in I ml of ice cold I N acetic acid . The h o m o g e n a t e s were heated at 95°C for 10 minutes and centrifuged at 12000 x g for 20 minutes. The supernatants were lyophilized, redissolved in distilled water, and r a d i o i m m u n o a s s a y e d . The blood samples were collected during d e c a p i t a t i o n into heparinized polyethylene tubes containing a p r o t i n i n (200 KIU/ml). The plasma was separated from the blood by c e n t r i f u g a t i o n at 1700 × g at 4°C for 30 minutes and stored at -20°C. Before radioimmunoassay a n o t h e r c e n t r i f u g a t i o n ( 4 O O O 0 x g , 4 ° C , 3 0 minutes ) was given to p r e c i p i t a t e the protein. The supernatants were assayed for a t r i o p e p t i n content. A plasma blank solution was prepared from the plasma without adding aprotinin, which was incubated at 37°C for 24 hours and then centrifuged as the sample. A radioimmunoassay was performed by incubating standard a t r i o p e p t i n III or a sample (50 ~I) for 17-24 hours at 4°C with a n t i s e r u m and 1251-atriopeptin (8000-10000 cpm) in 450 ~i of 0.1 M sodium phosphate buffer (pH 7.4). ',~en plasma samples were measured, the plasma blank solution (50 ~i) was added to each standard curve tube instead of the same volume of the buffer to correct the protein c o n c e n t r a t i o n and other interfering materials in the plasma. Separation of bound and free 1251-1abeled tracer was achieved by the addition of 2OO~I of d e x t r a n - c o a t e d charcoal solution and centrifuged at 17OOxg for 10 minutes. The 1251a t r i o p e p t i n in the supernatant was measured in a gamma counter. The a n t i s e r u m was raised against a t r i o p e p t i n I, which shows IOO% cross reactivity with atriopeptin III and ~-human atrial n a t r i u r e t i c polypeptide ( ~ - h A N P ) and dose not cross-react with methionine enkephalin, cholecystokinin, dynorphin A ( 1-13 ), bradykinin, substance P, or ~ - e n d o r p h i n ( 1 0 ) . The a n t i s e r u m was used at a final dilution of 1:20000 giving a p p r o x i m a t e l y 40% ~ binding of the added tracer in the absence of "cold"atriopeptin. RESULTS The time-course and d o s e - r e s p o n s e relation of the hypotensive action of a t r i o p e p t i n III is shown in Fig.1. The basal levels of MAP and HR were 175+17 mmHg and 340+18 beats/min, respectively. Intravenous injection of a t r i o p e p t i ~ III (20-80 nmol/kg) lowered MAP in a d o s e - d e p e n d e n t manner. The decrease in ~AP lasted more than I hour with peak change occurring about 5 minutes after injection. B r a d y c a r d i a c response was found in a p p r o x i m a t e l y I/3 of the animals after injection, but no statistical significance was shown in the change of HR before and after injection.
Vol. 38, No. ii, 1 9 8 6
w
Atriopeptln and Spontaneous Hypertension
1037
D.Il
i
0
i
~
|
FIG.
4~0
i
i
60rain
I
Effect of atriopeptin III (AP III) on m e a n a ~ e r i a l pressure (MAP) and heart rate (HR) of SHR. Dosedependent responses were obtained in the same animals given intravenously 2 0 ( o ) , 4 0 ( @ ) and 80 ( . ) nmol/kg of AP III (injection intervals of 2
hours).
The contents of atriopeptin immunoreactive material (atriopeptin IR) in the rat atrium and plasma were measured. The results are shown in Table I. Atriopeptin IR contents in SHR were significantly higher than those in WKY. There was no significant correlation between the level of blood pressure with atriopeptin IR level in the atrium or plasma of SHR. TABLE I Atrium (pmol/mg protein)
Plasma (pmol/ml)
SHR
1.21 + 0.10"
0.36 + 0.07*
WKY
0.86 + 0.09
0.16 + 0.06
TABLE I: *P <0.05.
Change of atriopeptin IR in SHR.
N = 8,
DISCUSSION It has been reported that intravenous injection of synthetic atrial peptides induced hypotensive response in normotensive rats. Atriopeptin III and ~ - h A N P were more active than atriopeptin I and II(8). The data presented here show that synthetic atriopeptin III had a potent depressor effect also in h y p e r t e n sive rats. Consistent with early results, The heart rate was not affected significantly by atriopeptin, which indicated that the fall in blood pressure was not due to the decrease in heart rate.
1038
Atriopeptin and Spontaneous Hypertension
Vol. 38, No. ii, 1986
The depressor effect occured almost immediately after injection, so that it is infered that the direct vasorelaxant activity (3) and decrease in stroke volume(11) induced by atriopeptin seem to be responsible for the hypotensive response of SHR. A possible hypovolemia induced by diuresis and natriuresis after injection may be also involved in the depressor mechanism of atriopeptin, because the potency order of atrial peptides in depressor activity was quite similar to that in diuretic and natriuretic activities(8). In order to understand the effect of atriopeptin in the mechanism of hypertension, we measured the atriopeptin contents in the atrium and plasma. It was found that SHR had a higher content of atriopeptin than },~Y both in the atrium and in the plasma. This result, which indicates an over-activation of the atriopeptin system with increased biosynthesis and release in hypertensive rats, is contrary to the result recently found in man where an obvious decrease in plasma level of atrio~eptin was found in patients with sever essential hypertension(12). Such a conflict suggests that atriopeptin involvement in hypertension pathophysiology may be considerably complicated and may depend on the species, type, severity and duration of hypertension. We suggest that the high level of atriopeptin in the plasma and atrium of SHR seems to be a compensatory response induced by b@pertension and leaves open the possibility that different mechanisms of action occur in the two species, humans and rats. Although the significance of increased atriopeptin IR contents in SHR remains obscure because no good correlation was found between the MAP level and atriopeptin IR contents, the potent antihypertensive effect of atriopeptin on SHR suggests that the application of atriopeptins or their analogs may be a new way for clinical treatment of hypertension. REFEREN CES I.
T.G.FLYNN, M.L.DE BOLD and A.J.DE BOLD, Biochem. Biophys.Res. Commun., 117, 859-865 (1983). 2. K.KANGAWA, A.FUKUDA, N.MINAMINO, H.~ATSUO, ~iochem. ~iophys. Res. Commun., 119, 933-940 (1984). 3. K.S.MISONS, H.FUKUMI, R.T.GRAMMER and T. INAGAMI, Biochem . Biophys. Res. Commun., 119, 524-529 (1984). N.R.SIEGEL, K.F.POK, S. P. 4. M.G.CURRIE, D.M.GELLER, B.R. vOLE, '~ ~ ADAMS, S.R.EUBANKS, G.R.CALLUPPI and P.NEEDLEMAN, Science, 223, 67-69 (1984). 5. D.M.GELLER, H.G.CURRIE, K.WAKITANI, B.R.COLE, S.P.ADAMS, K.F. FOK, N.R.SIEGEL, S.R.EUBANKS, G.R.GALLUPPI and P.NEEDLEMAN , Biochem. Biophys. Res. Commun., 120, 333-338 (1984). 6. K.KANGAWA and H.MATSUO, Biochem. Biophys. Res. Commun., 118, 131-139 (1984). 7. A.J.DE BOLD, H.B.BORENSTEIN, A.T.VERESS and SONNENBERG, Life Sci., 28, 89-94 (1981). 8. J.TANG, R . J . ~ B B E R , D.CHANG, J.K.CHANG, J.KI&NG and E.T.}SI, Regul. Pept., ~, 53-59 (1984). 9. R.T.GRAMMER, H.FUKUMI, T.iNAGAMI, K.S.MISONO, Biochem. Biophys. Res. Commun., 116, 696-703 (1983). 10. J.TANG, H.FEI, C.W.XIE, M.Z.SUEN, J.S.HAN, R.J.V~BBER, D.CHANG and J.K.CHANG, Pepti@es, ~, 1173-1177 (1984).
Vol. 38, No. ii, 1986
Atriopeptin and Spontaneous Hypertension
1039
11. H.D.KLEINERT, M.VOLPE, M.J.F.CAMARGO, S.A.ATLAS, J.H.LARAGH and T.Y~IACK, Fed. Proe., 44, 1729 (1985). 12. J.TANG, D.L.SONG, M.Z.SUEN and C.W.XIE, Peptides, in press, ( 1986 ).