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Comparative hemodynamic actions of ANF-related peptides in conscious sheep
Life Sciences, Vol. 45, pp. 2303-2312 Printed in the U.S.A.
Pergamon Press
COMPARATIVE HEMODYNAMIC ACTIONS OF ANF-RELATED PEPTIDES IN CONSCIOUS SHEEP
David G. Parkes, John P. Coghlan and Bruce A. Scoggins Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne,Parkville, Australia. 3052. (Received in final form October 5, 1989) Summary The rapid hemodynamic effects of several N- and C-terminal deleted fragments of ANF, a potent ANF analogue and the recently characterised brain natriuretic peptide (BNP) were investigated in conscious sheep, and compared to the rapid hemodynamic actions of ANF 1-28. The hypotensive potency of all peptides studied was as follows: ANF 1-28 = PL058 > ANF 5-27 = ANF 5-28 = BNP >ANF 7-28 > ANF 13-28 = ANF 5-25. All peptides reduced blood pressure via a decrease in total peripheral resistance, excluding ANF 5-25 and 1328 which were without effect on any parameter measured. These changes were associated with reflex increases in both heart rate and cardiac output, and a slight reduction in stroke volume. The duration of hypotensive/vasodilator action of ANF 1-28, 5-27, 5-28, 7-28 and BNP was approximately 3-4 minutes, whereas that of PL058 was 7-8 minutes. In conclusion, amino acid deletions from the Cand N-terminal of the ANF molecule reduced the hypotensive/ vasodilator potency of the peptide in conscious sheep. BNP produced similar rapid hemodynamic changes to ANF 1-28, suggesting that the two peptides may co-regulate blood pressure and possibly body fluids to promote fluid and cardiovascular homeostasis. The molecular form of ANF circulating in blood is known to be the 28 amino acid peptide (i), however many studies have used shorter fragments of ANF to examine the physiological effects of the hormone in vivo(2). Several investigators have compared the in vitro potency of different ANF fragments on relaxation of vascular smooth muscle (3-6), increases in cyclic GMP production (5,7), inhibition of aldosterone secretion from the adrenal gland (8) and variations in biologically active or "B" ANF receptor binding (4). However, few have considered the possibility of differing enzymatic degradation, or binding to ANF "clearance" (C) receptors (9,10). Because it seems possible that chain-length of ANF may have importance in determining the cardiovascular potency of the peptide, the present study compares the immediate hemodynamic actions of five C- or N-terminal deleted ANF fragments, together with a potent ANF analogue, with the responses to ANF 1-28. The sheep is very sensitive to the cardiovascular actions of ANF 1-28 when compared to other animals (ii), which suggests that this species is suitable to assess the relative potency of structural analogues of ANF in vivo. The cardiovascular actions of the recently characterised "brain natriuretic peptide" (BNP) (12) were also assessed. 0024-3205/89 $3.00 +.00 Copyright (c) 1989 Pergamon Press plc
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Methods
Conscious sheep (40-45 kg) were surgically prepared with carotid artery loops and oophorectomy under general anaesthesia. An electromagnetic flow (EMF) probe was placed around the ascending aorta during a second surgical procedure. The rapid hemodynamic actions produced by intravenous injection of five ANF fragments, an ANF analogue, together with brain natriuretic peptide (BNP) were examined in four conscious sheep. Peptides were injected intravenously via a jugular vein cannula inserted on the day prior to experimentation, at equimolar doses equivalent to 20~g and 100Dg of ANF 1-28 (Ser-Leu-Arg-Arg-Ser-
Cys-Phe-G~y-G~y-Arg-Met-Asp-Arg-I~e-G~y-A~a-G~n-Ser-G~y-Leu-G~y~Cys-Asn-Ser-Phe-
Arg-Tyr) i.e. 6.5 nmol and 32.5 nmol. Human ANF 5-25 (Ser5-Ser 5, atriopeptin i), ANF 5-27 ( S e ~ - A r g 27, atriopeptin ii), ANF 5-28 (Ser5-Tyr 28, atriopeptin iii), ANF 7-28 (CysT-Tyr28), ANF 13-28 (Aspl3-Tyr 28) and compound PL-058 (MprPhe-D-Ala-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-PheArg-Tyr) were obtained from Peninsula Laboratories (Belmont, CA, USA) and were shown to be chromatographically pure by HPLC. Porcine BNP (Asp-Ser-Gly-Cys-PheG•y-Arg-Arg-Leu-Asp-Arg-I•e-G•y-Ser-Leu-Ser-G•y-Leu-G•y-Cys-Asn-Va•-Leu-Arg-ArgTyr) was obtained from Auspep (Melbourne, Australia). Each peptide was injected at both doses into four sheep. Each experiment was performed on a separate day. The protocol comprised a fifteen minute control period, followed by injection of the peptide. Cardiovascular changes were recorded for 30 min after injection. Mean arterial pressure (MAP) and heart rat~ (HR) were measured continually using a pressure transducer connected to a Gould chart recorder. Cardiac output (CO) was measured every 30 seconds via the EMF probe connected to a Biotronex flowmeter. Data storage was made on an IBM Series/l computer. The EMF probe was calibrated against a thermodilution technique for measuring CO (13) within one week of experimentation. Enddiastolic aortic flow was taken to be zero flow, avoiding the problem of baseline shift. Stroke volume (SV) and calculated total peripheral resistance (CTPR) were derived from MAP, CO and HR. Results for time-course hemodynamic measurements are expressed as mean + standard error of the mean, and were analysed for changes from control by 2-way ANOVA with subsequent Student t-test. Comparisons were made between each peptide and ANF 1-28 by comparing the maximum change produced by each dose for each hemodynamic parameter. This was done by Student's unpaired t-test and changes were considered significant if p<0.05.
Results
Similar hemodynamic profiles were observed for ANF 1-28, 5-27, 5-28, 7-28, BNP and PLO58. Cardiovascular changes for peptide injection at 6.5 nmol are shown in Figure I. The changes in MAP, CTPR and SV produced by ANF 5-27, 5-28, 7-28 and BNP were sustained (p<0.05) for 3.2 + 0.4, 3.5 + 0,4, 3.3 + 0.3 and 3.6 + 0.4 min following injection. In all cases the initial increase in CO was followed by a decrease in CO 15 min after injection. This had returned to pre-injection values within 30 min of injection.
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Figure 2 compares the hemodynamic changes produced by injection of all peptides at 32.5 nmol. ANF 5-27 injected at 32.5 nmol reduced MAP from 73 + 2 to 64 + 1 mmHg (p
ANF 5-28 injected at 32.5 nmol decreased blood pressure from 71 + 4 to 61 + 2 mmHg (p<0.001), associated with a reduction in CTPR from 13.9 + 0.4 to 10.7 0.7 mmHg/i/min (p<0.01). Cardiac output increased from 5.1 + 0.T to 5.7 + 0.11
1-28
5-25
5-27
5-25
5-27
5-28
7-28
13-28
PL058
7-28
13-28
PL058
BNP
-10] + - 15 j A
1-28
5-28
BNP
+
A
I
so
v
.
1-P,8
5-e5
5-~/'
.
5-26
.
7-28
.
1S-P.,8
PL058
BHP
=i= ~_
-i0]
.
-15 FIG 1 Comparison of the maximum changes in mean arterial pressure (MAP), calculated total peripheral resistance (CTPR), cardiac output (CO), heart rate (HR) and stroke volume (SV) wlth intravenous injection of ANF 1-28, 5-25, 5-27, 5-28, 7-28, 13-28, PLO58 and BNP at 6.5 nmol in four sheep. Results are shown as mean + s.e.m. Like symbols represent no significant difference between peptides
2306
1/min b/min
ANF-related
Fragments
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(p<0.01) together with an increase (p<0.05). Stroke volume decreased
Vol.
in heart slightly
45, No.
24,
1989
rate from 63 + 3 to 76 + 6 from 81 + 2 t~ 75 + 4 ml/beat
(p<0.05).
ANF 7-28 reduced blood pressure from 69 + 2 to 63 + 1 mmHg (p<0.Ol) associated with a decrease in CTPR from 14.7--+ 0.4 to T2.0 + 0.3 mmHg/I/i~ l (p<0.001). Cardiac output increased from 4.7-+ 0.2 to 5.3 + 0.31/min (p<~ '~) a s did heart rate from 58 + 6 to 68 + 4 b/min ~p<0.05). No change was seen ~ stroke volume. The changes in MAP, CTPR, CO and HR were sustained for 3.8 + 0 . 4 min. 1-28
5-25
5-27
5-28
-I0
7-28
13-28
PL058
#
BNP
+
+ -15
j * 1-28
* 5-25
@. ~?
5-27
5-28
*
*
7-28
13-28
PL058
BNP
PL058
BN-P
1.5
3"~
.
<= 0~
*
~~
0
L--[" ' 1-28
5-25
I "r" I 5-27
5-28
7-28
13-25
+
3°l <] ~
1o 0 1-28
5-25
5-27
5-26
7-28
13-28
PL058
BNP
5
+ i
~
--F"
i
_1o I
,
FIG 2 Comparison of the m a x i m u m changes in mean arterial pressure (MAP), calculated total peripheral resistance (CTPR), cardiac output (CO), heart rate (HR) and stroke volume (SV) with intravenous injection of ANF 1-28, 5-25, 5-27, 5-28, 7-28, 13-28, PLO58 and BNP at 32.5 nmol in four sheep. Results are shown as mean + s.e.m. Like symbols represent no significant difference between peptides.
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ANF-related Fragments in Sheep
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ANF 5-25 and ANF 13-28 injected at 32.5 nmol produced no significant change in any of the hemodynamic parameters measured. PL058 reduced blood pressure from 69 + 4 to 58 + 3 mmHg (p<0.01) together with a decrease in CTPR from 14.7 + 0.4 t~ i0.0 + 0~8 mmHg/i/min (p<0.001). Cardiac output increased from 4.7 $ 0.3 to 5.8 +-0.3 i/min (p<0.001), and heart rate increased from 63 + 4 t~ 87 + 8 b/min. Stroke volume was reduced from 75 + 2 to 67 + 3 ml/beat-(p<0.01).- Similar to the lower dose of PL058 injected~ the hemodynamic changes lasted for 7.4 + 0.6 min following injection of PLO58, shown in Figure 3. Injection of BNP at 32.5 nmol reduced blood pressure from 76 + i to 68 + 2 mmHg (p<0.O01), and this was associated with a decrease in CTPR from 14.2 + 0.3 to 11.4 + 0.4 mmHg/i/min (p<0.001). Cardiac output increased from 5.4 + 0.I to 6.0 ~ 0.3 i/min (p<0.001), together with an increase in heart rate from 61 + 2 to 75 + b/min (p<0.001). Stroke volume decreased from 89 + 2 to
PL058 32.5nmol
n=4
MAP 70I (mmHg) 60
CO
5
I
(l/min)
* •*
-]
HR 80I (b/min) 70 60
8°I
SV
70
(ml/beat)
so 50
14I
CTPR (mmHg/l/min)12
10
-lO
-s
o
s
lo
15
20
2s
30
Time (min)
FIG 3 Hemodynamic effects of 32.5 nmol injection of PLO58 in 4 sheep. Results are shown as mean + s.e.m. Statistics ('I)<0.05, + p<0.01, # p<0.001) compare grouped control values with experimental values. Mean arterial pressure (MAP), cardiac output (CO), heart rate (HR), stroke volume (SV) and calculated total peripheral resistance (CTPR) were measured.
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ANF-related
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Vol. 45, No. 24, 1989
81 + 5 ml/beat (p<0.05). These parameters had returned to pre-injection valves within 4.2 + 0.4 min of injection of BNP (Fig. 4), however cardiac output continued to fall below control values approximately I0 minutes after injeCtion and remained suppressed for 5-10 minutes. It had returned to control values within 30 minutes of injection of the peptide. The reduction in cardiac output 15 minutes after injection, of ANF 5-27, ANF 5-28, ANF 7-28, PLO58, BNP and ANF 1-28.
BNP 32.5 nmol M A P (mmHg)70
6
was a feature
n=4
TT
T
T
T
T
T
~
L
:1= CO (l/rain)
6 ¢
HR 70 I (b/min) 60 SV 100 f (ml/beat) 80
16 CTPR 14 (mmHg/l/min) 12 10
lo
6
lo
20 Time (min)
FIG 4 Hemodynamic effects of 32.5 nmol injection of BNP in 4 sheep. Results are shown as mean + s.e.m. Statistics (*p<0.05, + p<0.01, ~p<0.001) compare grouped control values with experimental values. Mean arterial pressure (MAP), cardiac output (CO), heart rate (HR), stroke volume (SV) and calculated total peripheral resistance (CTPR) were measured.
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ANF-related Fragments in Sheep
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Discussion
This study has shown that three of the ANF fragments - ANF 5-27, ANF 5-28, ANF 7-28, plus the potent ANF analogue, PL058, all produced similar rapid cardiovascular effects to those seen with ANF 1-28 in conscious sheep. The novel brain peptide, BNP also had similar actions to ANF 1-28, however ANF 13-28, which lacks the Cys7-Cys23 disulphide bond and ANF 5-25 were without biological activity at the doses studied. The immediate fall in blood pressure produced by the active peptides was mediated by a vasodilator action, and this was associated with reflex increase~ in both heart rate and cardiac output i.e. an identical immediate hemodynamic profile to that observed with ANF 1-28 (14). The duration of hypotensive response with ANF 1-28, 5-27, 5-28, 7-28 and BNP was approximately 3-4 minutes. A similar study in conscious dogs (15) where ANF 5-28 was injected, showed that the changes in blood pressure were associated with decreases in end-diastolic pressure and left atrial pressure, as well as an increase in left ventricular pressure and left ventricular dP/dt, mediating the increase in cardiac output. The relative potency of the peptides studied in producing hemodynamic changes in vivo has not been previously reported. The present study shows that amino acid deletions from the N-terminal of ANF 1-28 reduce both hypotensive and vasodilator potency of the peptide in sheep, as seen with ANF 5-28 and ANF 7-28. Increases in cardiac output were similar for these three peptides, as well as for ANF 5-27, although ANF 1-28 produced a greater reflex increase in heart rate. This was probably related to the greater hypotensive action of ANF 1-28. However, because only two doses of each peptide were studied, care must be taken when interpreting dose-dependent changes produced by all peptides. Further deletions from the C-terminal as seen with ANF 5-25 completely abolished the hypotensive effects of the peptide. Recent reports have proposed that atriopeptin I, or ANF 5-25, is a pure agonist for the ANF C-receptor, as it produces vasorelaxation without increasing levels of cyclic GMP in vivo (16). Hence, any physiological effects seen with administration of this peptide in vivo could be mediated via elevated endogenous ANF 1-28, as a result of decreased clearance through binding to the C-receptor. However, studies have shown that ANF (5-27) relaxes smooth muscle in vitro (3,7) which suggests that the peptide binds to a receptor (possibly non-guanylate cyclase linked) which mediates its biological effects. The structural analogue of ANF (PLO58) was selected for study on the basis of work performed by Mogganam (17) showing the peptide to have enhanced natriuretic and diuretic properties when administered to rats. In the present study, PLO58 decreased blood pressure to a similar extent when compared to ANF 1-28, although changes in cardiac output, heart rate and total peripheral resistance were of a greater magnitude. Furthermore, the duration of hemodynamic changes with PLO58 was approximately twice that of ANF 1-28 (Fig. 3), which suggests slower metabolism possibly due to removal of the site of peptide bond cleavage for specific degradative enzymes. It may also be feasible that PLO58 shows decreased binding to the ANF C-receptor (18). However, at present, no evidence is available to support this hypothesis. The recently characterised porcine BNP (12) exhibits 57% homology with human ANF 1-28, mainly at the N-terminal to mid-region of the peptide. To date, little data has been published regarding the physiological actions
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1989
of BNP. The present study showed no difference between the rapid hemodynamic actions of porcine BNP and human ANF 1-28, which suggests that the amino acids common to both peptides may be important in mediating the binding to the respective vascular receptors, if indeed vascular BNP receptors exist. It was originally believed that localisation of BNP was restricted to regions of the central nervous system (12), however, recent studies have shown the highest concentration of peripheral BNP is within the cardiac atria (i-2% of brain concentration) (19). It is therefore possible that BNP may function not only as a neuropeptide, but also as a circulating hormone. A study by Inui and coworkers (20) demonstrated that BNP stimulates cyclic GMP production in kidney epithelial cells, and this effect was equipotent to ANF 1-28. They concluded that both peptides may share the same receptor. The duration of hemodynamic action of ANF 1-28, 5-27, 5-28, 7-28 and BNP was approximately 3-4 minutes. Recently, it was suggested that metabolism of smaller ANF fragments e.g. ANF 5-28 and ANF 5-27, may vary from that for ANF 1-28 (9). Cleavage at different sites of the peptides may influence duration of activity, and this was shown to be more obvious for atrial peptides with Cterminal amino acid deletions (3,4). Figure 2 shows that the relative hypotensive potency was : ANF 1-28 = PL058 > ANF 5-27 = ANF 5-28 = BNP > ANF 7-28 > ANF 13-28 = ANF 5-25. This correlated strongly with the vasodilator potency of all peptides, however changes in both cardiac output and heart rate were similar for ANF 1-28, 5-27, 5-28, 7-28 and BNP. PL058 produced the greatest change in total peripheral resistance, cardiac output and heart rate. Several studies have reported that the C-terminal portion of the ANF molecule may modulate the binding of these peptides to their receptors, since small differences greatly reduced activity (3,8). In contrast, these published studies suggested that the N-terminal seems to be much less important in this modulation of receptor binding of ANF (3,8). The present study shows that amino acid deletions from the Nterminal of ANF 1-28 to Met 12 reduces the vasoactive potency of the peptide in vivo. Hence, caution is required when assuming these fragments provide a good representation of the physiological actions of ANF 1-28 in conscious animals, as suggested in previous studies on the peptide (2). The study by Rapoport and coworkers (5) showed that ANF 1-28 and ANF 5-28 were equipotent in producing rabbit aortic relaxation and increasing cyclic GMP production. Hollemann reported that ANF 5-27 relaxed smooth muscle to the same extent as ANF 1-28 and 5-28, but had no effect on cyclic GMP production (7). Hence, they questioned the relationship between vasodilatation and increased cyclic GMP levels, and suggested that their data support the concept of multiple biologically active ANF receptor types. This supported the study by Budzik and coworkers (6) who also dissociated ANF-induced vasorelaxation from cyclic GMP production in histamine contracted rabbit aorta. Whether the ANF fragments examined in the present study all increase cyclic GMP levels to a similar extent remains to be determined. The in vitro potency of linear fragments of ANF i.e. ANF 13-28, has been reported to by approximately 0.1% that of ANF 1-28 (8). This indicates that the Cys7-Cys23 disulphide bridge stabilizes the biologically active confirmation of the peptides and is an absolute requirement for vascular and renal effects. This is consistent with the present study in sheep which demonstrated that ANF 13-28 had no hemodynamic effects in sheep following intravenous injection at the doses studied. The reduction in cardiac output observed approximately 15 minutes after injection with the biologically active peptides studied is consistent with
Vol. 45, No. 24, 1989
ANF-related Fragments in Sheep
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the hemodynamic effects of sustained infusion in sheep (21). ANF produces a time-dependent effect on the cardiovascular system in sheep, characterised by an immediate reduction in blood pressure mediated by vasodilatation. Over the next 24 hours ANF reduces CO and blood volume, associated with an increase in CTPR, however after 5 days infusion the fall in MAP becomes greater and is again mediated by a reduction in CTPR (11,21). In conclusion, the results from the present study indicate that amino acid deletions from the C- and N-terminals of ANF 1-28 reduced the immediate hypotensive activity of the peptide via reduced vasodilatation. Deletion of the Cys7-Cys23 disulphide bridge (ANF 13-28) abolished hemodynamic activity of ANF in the sheep. The recently characterised BNP produced similar rapid hemodynamic effects when compared to ANF, however whether the two peptides bind to the same receptor or possess individual receptor populations remains unclear. Accordingly, the occurrence of BNP together with ANF in mammals strongly suggests the possibility that many of the physiological functions so far thought to be mediated by ANF may be regulated through a dual mechanism involving both ANF and BNP. Further studies observing the short- and longterm physiological actions of the two peptides are required to elucidate the interrelationship and relative importance of ANF and BNP in certain pathophysiological situations. Acknowledgements These studies were supported by grants from the National Health and Medical Research Council of Australia, and the National Heart Foundation of Australia. REFERENCES i.
2. 3. 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14.
A.SUGAWARA, K.NAKAO, N.MORII, M. SAKAMOTO, M.SUDA, M.SHIMOKURA, Y.KISO, M.KIHARA, Y.YAMORI, K.NiSHIMURA, J. SONEDA, T.BAN & H.IMURA. Biochem Biophys Res Commun 1 2 9 : 4 3 9 - 4 4 6 (1985). N.C.TRIPPODO, F.E.COLE, A.A.MACPHEE & B.L.PEGRAM. J Lab Clin Med 109: 112-119 (1987). R.GARCIA, G.THIBAULT, N.G.SEIDAH, C.LAZURE, M. CANTIN, J.GENEST & M. CHRETIEN. Biochem Biophys Res Commun 126: 178-184 (1985). R.J.WINQUIST, M.A.NAPIER, R.L.VANDLEN, K.ARCURI, M.E.KEEGAN, E.P. FAISON & E.P.BASKIN. Clin Exp Hypertens A 7 : 8 6 9 - 8 8 4 (1985). R.M.RAPOPORT, R.J.WINQUIST, E.P.BASKIN, E.P.FAISON, S.A.WALDMAN & F.MURAD. Eur J Pharmacol 120: 123-126 (1986). • G.P.BUDZIK, S.L.FIRESTONE, E.N.BUSH, P.J.CONNOLLY, T.W.ROCKWARY, V.K. SARIN & W.H.HOLLEMAN. Biochem Biophys Res Commun 144: 422-431 (1987). W.H.HOLLEMAN, E.N.BUSH, E.M.DEVINE, S.L.FIRESTONE, T.W.ROCKWAY, V.K. SARIN & G.P.BUDZIK. Drug Devel 12: 109-117 (1988). P.W. SCHILLER, F.BELLINI, G.DIONNE, L.A.MAZIACK, R.GARCIA, A.DELEAN & M. CANTIN. Biochem Biophys Res Commun 1 3 8 : 8 8 0 - 8 8 6 (1986). R.M. SCARBOROUGH. In: Advances in Atrial Peptide Research. B.M.Brenner & J.H.Laragh (eds) Raven Press, New YOrk (1989). J.KOEPKE, L.TYLER, A.TRAPANI, P.BOVY, K.SPEAR, G.OLINS & E.H.BLAINE. J Pharmacol Exp Ther (in press) (1989). D.G.PARKES, J.P.COGHLAN, J.G.McDOUGALL & B.A.SCOGGINS. Clin Exp Hypertens A 9 : 2 1 4 3 - 2 1 5 6 (1987). T.SUDOH, K.KANGAWA, N.MINAMINO & H.MATSUO. Nature 3 3 2 : 7 8 - 8 0 (1988). B.A.SCOGGINS, K.J.S.ALLEN, J.P.COGHLAN, D.A.DENTON, W.F.GRAHAM, T.J.HUMPHREY & J.A.WHITWORTH. Clin Sci 5 7 : 3 3 5 5 - 3 3 6 5 (1979). D.G.PARKES, J.P. COGHLAN, J.G.McDOUGALL & B.A.SCOGGINS. Clin Exp. Pharmacol Physiol 15: 67-71 (1988).
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15. 16.
17. 18. 19. 20. 21.
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J.T.SHAPIRO, V.M.DELEONARDIS, P.NEEDLEMAN & T.H.HINTZE. Am J Physiol 251: 1292-1297 (1986). T.MAACK, M. SUZUKI, F.A.ALMEIDA, A.OWADA, R.M. SCARBOROUGH, J.A.LEWICKI & D.R.NUSSENZWEIG. Proc Symp on ANP. 8th Intern Congr of Endocrinol, Kyoto, 0-16 (1988). H.D.MOGGANAM, D.CHANG, J.K.CHANG, CW.XIE, X.R.LI, D.L.SONG & J.TANG In: Biologically Active Atrial Peptides. p. 203-206 (1987). T.MAACK, M. SUZUKI, F.A.ALMEIDA, D.NUSSENZVEIG, R.M.SCARBOROUGH, G.A.MCENROE & J.A. LEWICKI. Science 2 3 9 8 : 6 7 5 - 6 7 8 (1987). N.MINAMINO, S.UEDA, M.ABURAYA, T.SUDOH, K.KANGAWA & H.MATSUO. Proc Symp on ANP. 8th Intern Congr Endocrinol. 0-5 (1988). K.INUI, T.IWATA, K.NAKAO, H.IMURA, H.MATSUO & R.HORI. Proc. ANP Symp of 8th Intern Congr Endocrinol. 0-11 (1988). D.G.PARKES, J.P.COGHLAN, J.G.MCDOUGALL & B.A.SCOGGINS. In: Biological and Molecular Aspects of Atrial Factors. P. Needleman (ed) Allan R. Liss, Inc., New York p. 185-204 (1988).
Pergamon Press
COMPARATIVE HEMODYNAMIC ACTIONS OF ANF-RELATED PEPTIDES IN CONSCIOUS SHEEP
David G. Parkes, John P. Coghlan and Bruce A. Scoggins Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne,Parkville, Australia. 3052. (Received in final form October 5, 1989) Summary The rapid hemodynamic effects of several N- and C-terminal deleted fragments of ANF, a potent ANF analogue and the recently characterised brain natriuretic peptide (BNP) were investigated in conscious sheep, and compared to the rapid hemodynamic actions of ANF 1-28. The hypotensive potency of all peptides studied was as follows: ANF 1-28 = PL058 > ANF 5-27 = ANF 5-28 = BNP >ANF 7-28 > ANF 13-28 = ANF 5-25. All peptides reduced blood pressure via a decrease in total peripheral resistance, excluding ANF 5-25 and 1328 which were without effect on any parameter measured. These changes were associated with reflex increases in both heart rate and cardiac output, and a slight reduction in stroke volume. The duration of hypotensive/vasodilator action of ANF 1-28, 5-27, 5-28, 7-28 and BNP was approximately 3-4 minutes, whereas that of PL058 was 7-8 minutes. In conclusion, amino acid deletions from the Cand N-terminal of the ANF molecule reduced the hypotensive/ vasodilator potency of the peptide in conscious sheep. BNP produced similar rapid hemodynamic changes to ANF 1-28, suggesting that the two peptides may co-regulate blood pressure and possibly body fluids to promote fluid and cardiovascular homeostasis. The molecular form of ANF circulating in blood is known to be the 28 amino acid peptide (i), however many studies have used shorter fragments of ANF to examine the physiological effects of the hormone in vivo(2). Several investigators have compared the in vitro potency of different ANF fragments on relaxation of vascular smooth muscle (3-6), increases in cyclic GMP production (5,7), inhibition of aldosterone secretion from the adrenal gland (8) and variations in biologically active or "B" ANF receptor binding (4). However, few have considered the possibility of differing enzymatic degradation, or binding to ANF "clearance" (C) receptors (9,10). Because it seems possible that chain-length of ANF may have importance in determining the cardiovascular potency of the peptide, the present study compares the immediate hemodynamic actions of five C- or N-terminal deleted ANF fragments, together with a potent ANF analogue, with the responses to ANF 1-28. The sheep is very sensitive to the cardiovascular actions of ANF 1-28 when compared to other animals (ii), which suggests that this species is suitable to assess the relative potency of structural analogues of ANF in vivo. The cardiovascular actions of the recently characterised "brain natriuretic peptide" (BNP) (12) were also assessed. 0024-3205/89 $3.00 +.00 Copyright (c) 1989 Pergamon Press plc
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ANF-related Fragments in Sheep
Vol. 45, No. 24, 1989
Methods
Conscious sheep (40-45 kg) were surgically prepared with carotid artery loops and oophorectomy under general anaesthesia. An electromagnetic flow (EMF) probe was placed around the ascending aorta during a second surgical procedure. The rapid hemodynamic actions produced by intravenous injection of five ANF fragments, an ANF analogue, together with brain natriuretic peptide (BNP) were examined in four conscious sheep. Peptides were injected intravenously via a jugular vein cannula inserted on the day prior to experimentation, at equimolar doses equivalent to 20~g and 100Dg of ANF 1-28 (Ser-Leu-Arg-Arg-Ser-
Cys-Phe-G~y-G~y-Arg-Met-Asp-Arg-I~e-G~y-A~a-G~n-Ser-G~y-Leu-G~y~Cys-Asn-Ser-Phe-
Arg-Tyr) i.e. 6.5 nmol and 32.5 nmol. Human ANF 5-25 (Ser5-Ser 5, atriopeptin i), ANF 5-27 ( S e ~ - A r g 27, atriopeptin ii), ANF 5-28 (Ser5-Tyr 28, atriopeptin iii), ANF 7-28 (CysT-Tyr28), ANF 13-28 (Aspl3-Tyr 28) and compound PL-058 (MprPhe-D-Ala-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-PheArg-Tyr) were obtained from Peninsula Laboratories (Belmont, CA, USA) and were shown to be chromatographically pure by HPLC. Porcine BNP (Asp-Ser-Gly-Cys-PheG•y-Arg-Arg-Leu-Asp-Arg-I•e-G•y-Ser-Leu-Ser-G•y-Leu-G•y-Cys-Asn-Va•-Leu-Arg-ArgTyr) was obtained from Auspep (Melbourne, Australia). Each peptide was injected at both doses into four sheep. Each experiment was performed on a separate day. The protocol comprised a fifteen minute control period, followed by injection of the peptide. Cardiovascular changes were recorded for 30 min after injection. Mean arterial pressure (MAP) and heart rat~ (HR) were measured continually using a pressure transducer connected to a Gould chart recorder. Cardiac output (CO) was measured every 30 seconds via the EMF probe connected to a Biotronex flowmeter. Data storage was made on an IBM Series/l computer. The EMF probe was calibrated against a thermodilution technique for measuring CO (13) within one week of experimentation. Enddiastolic aortic flow was taken to be zero flow, avoiding the problem of baseline shift. Stroke volume (SV) and calculated total peripheral resistance (CTPR) were derived from MAP, CO and HR. Results for time-course hemodynamic measurements are expressed as mean + standard error of the mean, and were analysed for changes from control by 2-way ANOVA with subsequent Student t-test. Comparisons were made between each peptide and ANF 1-28 by comparing the maximum change produced by each dose for each hemodynamic parameter. This was done by Student's unpaired t-test and changes were considered significant if p<0.05.
Results
Similar hemodynamic profiles were observed for ANF 1-28, 5-27, 5-28, 7-28, BNP and PLO58. Cardiovascular changes for peptide injection at 6.5 nmol are shown in Figure I. The changes in MAP, CTPR and SV produced by ANF 5-27, 5-28, 7-28 and BNP were sustained (p<0.05) for 3.2 + 0.4, 3.5 + 0,4, 3.3 + 0.3 and 3.6 + 0.4 min following injection. In all cases the initial increase in CO was followed by a decrease in CO 15 min after injection. This had returned to pre-injection values within 30 min of injection.
Vol. 45, No. 24, 1989
ANF-related
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2305
Figure 2 compares the hemodynamic changes produced by injection of all peptides at 32.5 nmol. ANF 5-27 injected at 32.5 nmol reduced MAP from 73 + 2 to 64 + 1 mmHg (p
ANF 5-28 injected at 32.5 nmol decreased blood pressure from 71 + 4 to 61 + 2 mmHg (p<0.001), associated with a reduction in CTPR from 13.9 + 0.4 to 10.7 0.7 mmHg/i/min (p<0.01). Cardiac output increased from 5.1 + 0.T to 5.7 + 0.11
1-28
5-25
5-27
5-25
5-27
5-28
7-28
13-28
PL058
7-28
13-28
PL058
BNP
-10] + - 15 j A
1-28
5-28
BNP
+
A
I
so
v
.
1-P,8
5-e5
5-~/'
.
5-26
.
7-28
.
1S-P.,8
PL058
BHP
=i= ~_
-i0]
.
-15 FIG 1 Comparison of the maximum changes in mean arterial pressure (MAP), calculated total peripheral resistance (CTPR), cardiac output (CO), heart rate (HR) and stroke volume (SV) wlth intravenous injection of ANF 1-28, 5-25, 5-27, 5-28, 7-28, 13-28, PLO58 and BNP at 6.5 nmol in four sheep. Results are shown as mean + s.e.m. Like symbols represent no significant difference between peptides
2306
1/min b/min
ANF-related
Fragments
in Sheep
(p<0.01) together with an increase (p<0.05). Stroke volume decreased
Vol.
in heart slightly
45, No.
24,
1989
rate from 63 + 3 to 76 + 6 from 81 + 2 t~ 75 + 4 ml/beat
(p<0.05).
ANF 7-28 reduced blood pressure from 69 + 2 to 63 + 1 mmHg (p<0.Ol) associated with a decrease in CTPR from 14.7--+ 0.4 to T2.0 + 0.3 mmHg/I/i~ l (p<0.001). Cardiac output increased from 4.7-+ 0.2 to 5.3 + 0.31/min (p<~ '~) a s did heart rate from 58 + 6 to 68 + 4 b/min ~p<0.05). No change was seen ~ stroke volume. The changes in MAP, CTPR, CO and HR were sustained for 3.8 + 0 . 4 min. 1-28
5-25
5-27
5-28
-I0
7-28
13-28
PL058
#
BNP
+
+ -15
j * 1-28
* 5-25
@. ~?
5-27
5-28
*
*
7-28
13-28
PL058
BNP
PL058
BN-P
1.5
3"~
.
<= 0~
*
~~
0
L--[" ' 1-28
5-25
I "r" I 5-27
5-28
7-28
13-25
+
3°l <] ~
1o 0 1-28
5-25
5-27
5-26
7-28
13-28
PL058
BNP
5
+ i
~
--F"
i
_1o I
,
FIG 2 Comparison of the m a x i m u m changes in mean arterial pressure (MAP), calculated total peripheral resistance (CTPR), cardiac output (CO), heart rate (HR) and stroke volume (SV) with intravenous injection of ANF 1-28, 5-25, 5-27, 5-28, 7-28, 13-28, PLO58 and BNP at 32.5 nmol in four sheep. Results are shown as mean + s.e.m. Like symbols represent no significant difference between peptides.
Vol. 45, No. 24, 1989
ANF-related Fragments in Sheep
2307
ANF 5-25 and ANF 13-28 injected at 32.5 nmol produced no significant change in any of the hemodynamic parameters measured. PL058 reduced blood pressure from 69 + 4 to 58 + 3 mmHg (p<0.01) together with a decrease in CTPR from 14.7 + 0.4 t~ i0.0 + 0~8 mmHg/i/min (p<0.001). Cardiac output increased from 4.7 $ 0.3 to 5.8 +-0.3 i/min (p<0.001), and heart rate increased from 63 + 4 t~ 87 + 8 b/min. Stroke volume was reduced from 75 + 2 to 67 + 3 ml/beat-(p<0.01).- Similar to the lower dose of PL058 injected~ the hemodynamic changes lasted for 7.4 + 0.6 min following injection of PLO58, shown in Figure 3. Injection of BNP at 32.5 nmol reduced blood pressure from 76 + i to 68 + 2 mmHg (p<0.O01), and this was associated with a decrease in CTPR from 14.2 + 0.3 to 11.4 + 0.4 mmHg/i/min (p<0.001). Cardiac output increased from 5.4 + 0.I to 6.0 ~ 0.3 i/min (p<0.001), together with an increase in heart rate from 61 + 2 to 75 + b/min (p<0.001). Stroke volume decreased from 89 + 2 to
PL058 32.5nmol
n=4
MAP 70I (mmHg) 60
CO
5
I
(l/min)
* •*
-]
HR 80I (b/min) 70 60
8°I
SV
70
(ml/beat)
so 50
14I
CTPR (mmHg/l/min)12
10
-lO
-s
o
s
lo
15
20
2s
30
Time (min)
FIG 3 Hemodynamic effects of 32.5 nmol injection of PLO58 in 4 sheep. Results are shown as mean + s.e.m. Statistics ('I)<0.05, + p<0.01, # p<0.001) compare grouped control values with experimental values. Mean arterial pressure (MAP), cardiac output (CO), heart rate (HR), stroke volume (SV) and calculated total peripheral resistance (CTPR) were measured.
2308
ANF-related
Fragments
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Vol. 45, No. 24, 1989
81 + 5 ml/beat (p<0.05). These parameters had returned to pre-injection valves within 4.2 + 0.4 min of injection of BNP (Fig. 4), however cardiac output continued to fall below control values approximately I0 minutes after injeCtion and remained suppressed for 5-10 minutes. It had returned to control values within 30 minutes of injection of the peptide. The reduction in cardiac output 15 minutes after injection, of ANF 5-27, ANF 5-28, ANF 7-28, PLO58, BNP and ANF 1-28.
BNP 32.5 nmol M A P (mmHg)70
6
was a feature
n=4
TT
T
T
T
T
T
~
L
:1= CO (l/rain)
6 ¢
HR 70 I (b/min) 60 SV 100 f (ml/beat) 80
16 CTPR 14 (mmHg/l/min) 12 10
lo
6
lo
20 Time (min)
FIG 4 Hemodynamic effects of 32.5 nmol injection of BNP in 4 sheep. Results are shown as mean + s.e.m. Statistics (*p<0.05, + p<0.01, ~p<0.001) compare grouped control values with experimental values. Mean arterial pressure (MAP), cardiac output (CO), heart rate (HR), stroke volume (SV) and calculated total peripheral resistance (CTPR) were measured.
Vol. 45, No. 24, 1989
ANF-related Fragments in Sheep
2309
Discussion
This study has shown that three of the ANF fragments - ANF 5-27, ANF 5-28, ANF 7-28, plus the potent ANF analogue, PL058, all produced similar rapid cardiovascular effects to those seen with ANF 1-28 in conscious sheep. The novel brain peptide, BNP also had similar actions to ANF 1-28, however ANF 13-28, which lacks the Cys7-Cys23 disulphide bond and ANF 5-25 were without biological activity at the doses studied. The immediate fall in blood pressure produced by the active peptides was mediated by a vasodilator action, and this was associated with reflex increase~ in both heart rate and cardiac output i.e. an identical immediate hemodynamic profile to that observed with ANF 1-28 (14). The duration of hypotensive response with ANF 1-28, 5-27, 5-28, 7-28 and BNP was approximately 3-4 minutes. A similar study in conscious dogs (15) where ANF 5-28 was injected, showed that the changes in blood pressure were associated with decreases in end-diastolic pressure and left atrial pressure, as well as an increase in left ventricular pressure and left ventricular dP/dt, mediating the increase in cardiac output. The relative potency of the peptides studied in producing hemodynamic changes in vivo has not been previously reported. The present study shows that amino acid deletions from the N-terminal of ANF 1-28 reduce both hypotensive and vasodilator potency of the peptide in sheep, as seen with ANF 5-28 and ANF 7-28. Increases in cardiac output were similar for these three peptides, as well as for ANF 5-27, although ANF 1-28 produced a greater reflex increase in heart rate. This was probably related to the greater hypotensive action of ANF 1-28. However, because only two doses of each peptide were studied, care must be taken when interpreting dose-dependent changes produced by all peptides. Further deletions from the C-terminal as seen with ANF 5-25 completely abolished the hypotensive effects of the peptide. Recent reports have proposed that atriopeptin I, or ANF 5-25, is a pure agonist for the ANF C-receptor, as it produces vasorelaxation without increasing levels of cyclic GMP in vivo (16). Hence, any physiological effects seen with administration of this peptide in vivo could be mediated via elevated endogenous ANF 1-28, as a result of decreased clearance through binding to the C-receptor. However, studies have shown that ANF (5-27) relaxes smooth muscle in vitro (3,7) which suggests that the peptide binds to a receptor (possibly non-guanylate cyclase linked) which mediates its biological effects. The structural analogue of ANF (PLO58) was selected for study on the basis of work performed by Mogganam (17) showing the peptide to have enhanced natriuretic and diuretic properties when administered to rats. In the present study, PLO58 decreased blood pressure to a similar extent when compared to ANF 1-28, although changes in cardiac output, heart rate and total peripheral resistance were of a greater magnitude. Furthermore, the duration of hemodynamic changes with PLO58 was approximately twice that of ANF 1-28 (Fig. 3), which suggests slower metabolism possibly due to removal of the site of peptide bond cleavage for specific degradative enzymes. It may also be feasible that PLO58 shows decreased binding to the ANF C-receptor (18). However, at present, no evidence is available to support this hypothesis. The recently characterised porcine BNP (12) exhibits 57% homology with human ANF 1-28, mainly at the N-terminal to mid-region of the peptide. To date, little data has been published regarding the physiological actions
2310
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Vol.
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1989
of BNP. The present study showed no difference between the rapid hemodynamic actions of porcine BNP and human ANF 1-28, which suggests that the amino acids common to both peptides may be important in mediating the binding to the respective vascular receptors, if indeed vascular BNP receptors exist. It was originally believed that localisation of BNP was restricted to regions of the central nervous system (12), however, recent studies have shown the highest concentration of peripheral BNP is within the cardiac atria (i-2% of brain concentration) (19). It is therefore possible that BNP may function not only as a neuropeptide, but also as a circulating hormone. A study by Inui and coworkers (20) demonstrated that BNP stimulates cyclic GMP production in kidney epithelial cells, and this effect was equipotent to ANF 1-28. They concluded that both peptides may share the same receptor. The duration of hemodynamic action of ANF 1-28, 5-27, 5-28, 7-28 and BNP was approximately 3-4 minutes. Recently, it was suggested that metabolism of smaller ANF fragments e.g. ANF 5-28 and ANF 5-27, may vary from that for ANF 1-28 (9). Cleavage at different sites of the peptides may influence duration of activity, and this was shown to be more obvious for atrial peptides with Cterminal amino acid deletions (3,4). Figure 2 shows that the relative hypotensive potency was : ANF 1-28 = PL058 > ANF 5-27 = ANF 5-28 = BNP > ANF 7-28 > ANF 13-28 = ANF 5-25. This correlated strongly with the vasodilator potency of all peptides, however changes in both cardiac output and heart rate were similar for ANF 1-28, 5-27, 5-28, 7-28 and BNP. PL058 produced the greatest change in total peripheral resistance, cardiac output and heart rate. Several studies have reported that the C-terminal portion of the ANF molecule may modulate the binding of these peptides to their receptors, since small differences greatly reduced activity (3,8). In contrast, these published studies suggested that the N-terminal seems to be much less important in this modulation of receptor binding of ANF (3,8). The present study shows that amino acid deletions from the Nterminal of ANF 1-28 to Met 12 reduces the vasoactive potency of the peptide in vivo. Hence, caution is required when assuming these fragments provide a good representation of the physiological actions of ANF 1-28 in conscious animals, as suggested in previous studies on the peptide (2). The study by Rapoport and coworkers (5) showed that ANF 1-28 and ANF 5-28 were equipotent in producing rabbit aortic relaxation and increasing cyclic GMP production. Hollemann reported that ANF 5-27 relaxed smooth muscle to the same extent as ANF 1-28 and 5-28, but had no effect on cyclic GMP production (7). Hence, they questioned the relationship between vasodilatation and increased cyclic GMP levels, and suggested that their data support the concept of multiple biologically active ANF receptor types. This supported the study by Budzik and coworkers (6) who also dissociated ANF-induced vasorelaxation from cyclic GMP production in histamine contracted rabbit aorta. Whether the ANF fragments examined in the present study all increase cyclic GMP levels to a similar extent remains to be determined. The in vitro potency of linear fragments of ANF i.e. ANF 13-28, has been reported to by approximately 0.1% that of ANF 1-28 (8). This indicates that the Cys7-Cys23 disulphide bridge stabilizes the biologically active confirmation of the peptides and is an absolute requirement for vascular and renal effects. This is consistent with the present study in sheep which demonstrated that ANF 13-28 had no hemodynamic effects in sheep following intravenous injection at the doses studied. The reduction in cardiac output observed approximately 15 minutes after injection with the biologically active peptides studied is consistent with
Vol. 45, No. 24, 1989
ANF-related Fragments in Sheep
2311
the hemodynamic effects of sustained infusion in sheep (21). ANF produces a time-dependent effect on the cardiovascular system in sheep, characterised by an immediate reduction in blood pressure mediated by vasodilatation. Over the next 24 hours ANF reduces CO and blood volume, associated with an increase in CTPR, however after 5 days infusion the fall in MAP becomes greater and is again mediated by a reduction in CTPR (11,21). In conclusion, the results from the present study indicate that amino acid deletions from the C- and N-terminals of ANF 1-28 reduced the immediate hypotensive activity of the peptide via reduced vasodilatation. Deletion of the Cys7-Cys23 disulphide bridge (ANF 13-28) abolished hemodynamic activity of ANF in the sheep. The recently characterised BNP produced similar rapid hemodynamic effects when compared to ANF, however whether the two peptides bind to the same receptor or possess individual receptor populations remains unclear. Accordingly, the occurrence of BNP together with ANF in mammals strongly suggests the possibility that many of the physiological functions so far thought to be mediated by ANF may be regulated through a dual mechanism involving both ANF and BNP. Further studies observing the short- and longterm physiological actions of the two peptides are required to elucidate the interrelationship and relative importance of ANF and BNP in certain pathophysiological situations. Acknowledgements These studies were supported by grants from the National Health and Medical Research Council of Australia, and the National Heart Foundation of Australia. REFERENCES i.
2. 3. 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14.
A.SUGAWARA, K.NAKAO, N.MORII, M. SAKAMOTO, M.SUDA, M.SHIMOKURA, Y.KISO, M.KIHARA, Y.YAMORI, K.NiSHIMURA, J. SONEDA, T.BAN & H.IMURA. Biochem Biophys Res Commun 1 2 9 : 4 3 9 - 4 4 6 (1985). N.C.TRIPPODO, F.E.COLE, A.A.MACPHEE & B.L.PEGRAM. J Lab Clin Med 109: 112-119 (1987). R.GARCIA, G.THIBAULT, N.G.SEIDAH, C.LAZURE, M. CANTIN, J.GENEST & M. CHRETIEN. Biochem Biophys Res Commun 126: 178-184 (1985). R.J.WINQUIST, M.A.NAPIER, R.L.VANDLEN, K.ARCURI, M.E.KEEGAN, E.P. FAISON & E.P.BASKIN. Clin Exp Hypertens A 7 : 8 6 9 - 8 8 4 (1985). R.M.RAPOPORT, R.J.WINQUIST, E.P.BASKIN, E.P.FAISON, S.A.WALDMAN & F.MURAD. Eur J Pharmacol 120: 123-126 (1986). • G.P.BUDZIK, S.L.FIRESTONE, E.N.BUSH, P.J.CONNOLLY, T.W.ROCKWARY, V.K. SARIN & W.H.HOLLEMAN. Biochem Biophys Res Commun 144: 422-431 (1987). W.H.HOLLEMAN, E.N.BUSH, E.M.DEVINE, S.L.FIRESTONE, T.W.ROCKWAY, V.K. SARIN & G.P.BUDZIK. Drug Devel 12: 109-117 (1988). P.W. SCHILLER, F.BELLINI, G.DIONNE, L.A.MAZIACK, R.GARCIA, A.DELEAN & M. CANTIN. Biochem Biophys Res Commun 1 3 8 : 8 8 0 - 8 8 6 (1986). R.M. SCARBOROUGH. In: Advances in Atrial Peptide Research. B.M.Brenner & J.H.Laragh (eds) Raven Press, New YOrk (1989). J.KOEPKE, L.TYLER, A.TRAPANI, P.BOVY, K.SPEAR, G.OLINS & E.H.BLAINE. J Pharmacol Exp Ther (in press) (1989). D.G.PARKES, J.P.COGHLAN, J.G.McDOUGALL & B.A.SCOGGINS. Clin Exp Hypertens A 9 : 2 1 4 3 - 2 1 5 6 (1987). T.SUDOH, K.KANGAWA, N.MINAMINO & H.MATSUO. Nature 3 3 2 : 7 8 - 8 0 (1988). B.A.SCOGGINS, K.J.S.ALLEN, J.P.COGHLAN, D.A.DENTON, W.F.GRAHAM, T.J.HUMPHREY & J.A.WHITWORTH. Clin Sci 5 7 : 3 3 5 5 - 3 3 6 5 (1979). D.G.PARKES, J.P. COGHLAN, J.G.McDOUGALL & B.A.SCOGGINS. Clin Exp. Pharmacol Physiol 15: 67-71 (1988).
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J.T.SHAPIRO, V.M.DELEONARDIS, P.NEEDLEMAN & T.H.HINTZE. Am J Physiol 251: 1292-1297 (1986). T.MAACK, M. SUZUKI, F.A.ALMEIDA, A.OWADA, R.M. SCARBOROUGH, J.A.LEWICKI & D.R.NUSSENZWEIG. Proc Symp on ANP. 8th Intern Congr of Endocrinol, Kyoto, 0-16 (1988). H.D.MOGGANAM, D.CHANG, J.K.CHANG, CW.XIE, X.R.LI, D.L.SONG & J.TANG In: Biologically Active Atrial Peptides. p. 203-206 (1987). T.MAACK, M. SUZUKI, F.A.ALMEIDA, D.NUSSENZVEIG, R.M.SCARBOROUGH, G.A.MCENROE & J.A. LEWICKI. Science 2 3 9 8 : 6 7 5 - 6 7 8 (1987). N.MINAMINO, S.UEDA, M.ABURAYA, T.SUDOH, K.KANGAWA & H.MATSUO. Proc Symp on ANP. 8th Intern Congr Endocrinol. 0-5 (1988). K.INUI, T.IWATA, K.NAKAO, H.IMURA, H.MATSUO & R.HORI. Proc. ANP Symp of 8th Intern Congr Endocrinol. 0-11 (1988). D.G.PARKES, J.P.COGHLAN, J.G.MCDOUGALL & B.A.SCOGGINS. In: Biological and Molecular Aspects of Atrial Factors. P. Needleman (ed) Allan R. Liss, Inc., New York p. 185-204 (1988).