Vo1.173, No. 2,1990
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 599-605
December 14,1990
BIOLOGICAL C H A R A C T E R I Z A T I O N OF H U M A N B R A I N N A T R I U R E T I C P E P T I D E (BNP) A N D R A T B N P : S P E C I E S - S P E C I F I C A C T I O N S OF B N P Yoshikazu Kambayashil, Kazuwa Nakao2, Hiromi Kimural, Tomoji Kawabatal, Masuhisa N a k a m u r a l *, Ken Inouyel, Nobuo Yoshidal, and ttiroo Imura2
1 Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan 2 Second Division, Department of Medicine, Kyoto University School of Medicine, Shogo-in, Kyoto 606, Japan Received October 4, 1990
S u m m a r y : We examined the diuretic-natriuretic activities of rat BNP and h u m a n BNP in anesthetized rats in vivo and their vasorelaxant activities for rat thoracic aorta and porcine coronary artery in vitro. Rat BNP was almost equipotent to rat ANP in diuresis and natriuresis with relative potencies of 1.6 and 2.5, respectively, while h u m a n BNP exerted no significant activity. Rat ANP, rat BNP and h u m a n BNP relaxed PGF2a-contracted rat aortic strips with IC50 values of 0.62, 0.64 and 12.1 nM, respectively, while they relaxed PGF2a-contracted porcine coronary arteries with IC50 values of 0.04, 1.10 and 0.02 nM, respectively. These results strongly suggest that the biological action of BNP is species-specific. ©199oAca~emio Press,
Inc.
Since the discovery of brain natriuretic peptide (BNP) in porcine brain, it has been thought that BNP and atrial natriuretic peptide (ANP) form a peptide family involved in the regulation of water-electrolyte balance [1]. Recently a third member of the family called C-type natriuretic peptide (CNP) has been isolated from porcine brain and its 22-amino acid sequence has been determined [2]. Subsequent to porcine BNP, the isolation and sequence determination of rat BNP [3, 4] and h u m a n BNP [5, 6] were reported. The amino acid sequence of dog BNP has also been elucidated by cDNA sequencing [7]. As shown in Fig. 1, the amino acid sequences of BNPs v a r y from species to species, while those of
To whom correspondence should be addressed.
Abbreviations: ANP, atrial natriuretic peptide; t3NP, brain natriuretic peptide; PGF2a, prostaglandin F2a.
599
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
V o l . 173, N o . 2, 1 9 9 0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
HumanANP
S g RRISISICFGIGRMIDRqGAQISlGIg GClNS F I Y
Rat BNP SQDSAF R I QE a L RNS - K-MA H SlSlSlC FGIQK IIDR IIGAVlSlRILG ClDG L LF HumanBNP SPK-MVQGISlGICFGIR KMIDR IJS S SlSlGI'GClKV L RH PorcineBNP SPKTM- RDISlGICFGIR R 'IDR qGS LISlGILGClNVL RY
DogBNP
SPK-MMHKISIGICFG~RRLIDRIIGSLISIGILGClNVL KY
Figure 1. Amino acid sequences ofnatriuretic peptides. The representative low-molecular-weight forms of rat ANP, human ANP, rat BNP, human BNP and porcine BNP are shown in one-letter symbols. Although the precise form of dog BNP is still undetermined, the inferred low-molecular-weight form is shown [7]. Boxed amino acid residues are conserved among these species. m a m m a l i a n ANPs are fully conserved except for a single amino acid residue at position 12. In spite of the large difference in the sequence, porcine BNP and rat ANP were reported to be equipotent in natriuresis in the rat [1] and to exhibit similar central actions [8-10] by sharing common receptors [11]. On the other hand, the structural difference of non-mammalian ANPs (chicken [12], frog [13] and eel [14]) from mammalian ANPs seems to be reflected on their low natriuretic and vasorelaxant activities when examined in the rat. Thus we tried to see how BNPs act on different species. In the present study, we investigated rat BNP and h u m a n BNP in terms of their vasorelaxant activity in rat and porcine tissues and their diuretic-natriuretic activities in anesthetized rats. As a result, we found t h a t h u m a n BNP and rat BNP act with different potencies on the vascular beds of different species, unlike ANPs which act on any species equally. The present paper describes this comparative pharmacology of BNP in some detail. MATERIALS AND METHODS Materials: Rat ANP and porcine BNP were purchased from Peptide Institute, Osaka, Japan. Rat BNP, a 45-amino-acid peptide, and h u m a n BNP, a 32-amino-acid peptide, were synthesized by the solid-phase method using an automatic peptide synthesizer (Applied Biosystems Inc., Model 430 A). Vasorelaxant activity: Rat aortic strips (2 m m x 5 mm, from male Sprague-Dawley rats) or porcine coronary arterial strips (2 mm x 10 mm) were mounted on stainlesssteel hooks under 1 g (for rat aorta) or 2 g resting tension (for porcine coronary artery). The strips were equilibrated in a modified Locke-Ringer solution at 37°C containing 120 mM NaC1, 5.4 mM KC1, 2.2 mM CaC12, 1.0 mM MgC12, 25 mM NaHCO3 and 5.5 mM glucose, gassed with 95% 02 and 5% CO2. After contraction of the strips with about 1 pM PGF2a, peptide solution was added cumulatively. At the end of the experiment, 0.1 mM papaverine hydrochloride was added for normalization. Diuretic-natriuretic activities: Male Sprague-Dawley rats (300-350 g) were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). Saline was infused at 0.1 ml/min/kg through a femoral vein catheter and peptide solution (100 pl) was added intravenously. Urine was collected at 5-min intervals from the bladder through a polyethylene tube. The concentration of sodium ion was measured by flame photometry. Statistics: Tukey's test was used for judgement of statistical significance [15]. 600
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS Diuretic and n a t r i u r e t i c activities of r a t B N P and h u m a n B N P in the r a t Table 1 s u m m a r i z e s the d o s e - d e p e n d e n t i n c r e a s e s in u r i n e flow r a t e a n d sodium excretion r a t e induced by r a t ANP, r a t B N P and h u m a n B N P in anesthetized rats. The r e l a t i v e potencies of r a t B N P to r a t A N P in diuresis and n a t r i u r e s i s were e s t i m a t e d as 1.6 and 2.5, respectively, by the 4-point parallel line assay [16] based on the responses a t doses i nmol/kg and 3 nmol/kg. H u m a n B N P caused m u c h less responses t h a n r a t B N P even a t 10 times h i g h e r doses. The time courses of the u r i n e flow and sodium excretion rates upon a d m i n i s t r a t i o n of r a t A N P (3 nmol/kg), r a t B N P (3 nmol/kg) or h u m a n B N P (30 nmol/kg) are shown in Fig. 2. R a t A N P and r a t B N P e q u a l l y caused t r a n s i e n t increases in u r i n e and sodium o u t p u t s and the significant increases lasted for a b o u t 15 and 20 rain, respectively.
On the o t h e r
hand, h u m a n B N P caused no significant increase in the u r i n e flow r a t e and induced only a slight, even t h o u g h significant, increase in the sodium excretion r a t e for a t least 10 min. V a s o r e l a x a n t a c t i v i t y of r a t B N P and h u m a n B N P in r a t thoracic aorta As shown in Fig. 3A, r a t B N P exhibited almost the same v a s o r e l a x a t i o n profile as t h a t of r a t ANP, while h u m a n B N P was only slightly active in isolated r a t aorta. P o r c i n e B N P also s e e m e d to be less active t h a n r a t B N P , e v e n t h o u g h t h e i r difference was not significant. The IC50 value, the concentration r e q u i r e d to induce h a l f the m a x i m u m relaxation, of h u m a n B N P is significantly h i g h e r t h a n those of the other peptides (Table 2). The r e l a t i v e potencies of r a t B N P and h u m a n B N P to r a t A N P are 0.97 and 0.05, respectively, w h e n expressed as reciprocal ratios of the ICs0 v a l u e s (Table 2). Table 1. DIURETIC AND NATRIURETIC ACTIVITIES OF HUMAN BNP, RAT BNP AND RAT ANP IN ANESTHETIZED RATS Peptide Rat ANP
Rat BNP Human BNP
Dose (nmol/kg) 1 3 10 1 3 10 30
n 5 5 4 5 5 7 4
AU (pl/min) 29 + 14 39 + 9 43 + 20 25 + 4 57 + 7a,b 9 -4-_ 3a 6 --4- 2b
AUNa (pEq/min) 5.5 + 1.6a,b 6.8 + 1.1 7.0 --t- 3.1 5.8 +_ 0.8 12.0 ± 1.6c,d,e 1.1 - 0.7d 0.4 _--4-0.1e
Results are expressed as mean ± SEM. AU and AUNa m e a n the increases in urine flow rate and sodium excretion rate, respectively. They were calculated as the differences between the values averaged during the pre-administration period (30 min) and postadministration period (30 min). The paired values with a common superscript a, b, c, d or e are significantly different from each other (a, b, c: p < 0.05; d, e: p < 0.01, Tukey's test). 601
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 599-605
December 14,1990
BIOLOGICAL C H A R A C T E R I Z A T I O N OF H U M A N B R A I N N A T R I U R E T I C P E P T I D E (BNP) A N D R A T B N P : S P E C I E S - S P E C I F I C A C T I O N S OF B N P Yoshikazu Kambayashil, Kazuwa Nakao2, Hiromi Kimural, Tomoji Kawabatal, Masuhisa N a k a m u r a l *, Ken Inouyel, Nobuo Yoshidal, and ttiroo Imura2
1 Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan 2 Second Division, Department of Medicine, Kyoto University School of Medicine, Shogo-in, Kyoto 606, Japan Received October 4, 1990
S u m m a r y : We examined the diuretic-natriuretic activities of rat BNP and h u m a n BNP in anesthetized rats in vivo and their vasorelaxant activities for rat thoracic aorta and porcine coronary artery in vitro. Rat BNP was almost equipotent to rat ANP in diuresis and natriuresis with relative potencies of 1.6 and 2.5, respectively, while h u m a n BNP exerted no significant activity. Rat ANP, rat BNP and h u m a n BNP relaxed PGF2a-contracted rat aortic strips with IC50 values of 0.62, 0.64 and 12.1 nM, respectively, while they relaxed PGF2a-contracted porcine coronary arteries with IC50 values of 0.04, 1.10 and 0.02 nM, respectively. These results strongly suggest that the biological action of BNP is species-specific. ©199oAca~emio Press,
Inc.
Since the discovery of brain natriuretic peptide (BNP) in porcine brain, it has been thought that BNP and atrial natriuretic peptide (ANP) form a peptide family involved in the regulation of water-electrolyte balance [1]. Recently a third member of the family called C-type natriuretic peptide (CNP) has been isolated from porcine brain and its 22-amino acid sequence has been determined [2]. Subsequent to porcine BNP, the isolation and sequence determination of rat BNP [3, 4] and h u m a n BNP [5, 6] were reported. The amino acid sequence of dog BNP has also been elucidated by cDNA sequencing [7]. As shown in Fig. 1, the amino acid sequences of BNPs v a r y from species to species, while those of
To whom correspondence should be addressed.
Abbreviations: ANP, atrial natriuretic peptide; t3NP, brain natriuretic peptide; PGF2a, prostaglandin F2a.
599
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
V o l . 173, N o . 2, 1 9 9 0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
HumanANP
S g RRISISICFGIGRMIDRqGAQISlGIg GClNS F I Y
Rat BNP SQDSAF R I QE a L RNS - K-MA H SlSlSlC FGIQK IIDR IIGAVlSlRILG ClDG L LF HumanBNP SPK-MVQGISlGICFGIR KMIDR IJS S SlSlGI'GClKV L RH PorcineBNP SPKTM- RDISlGICFGIR R 'IDR qGS LISlGILGClNVL RY
DogBNP
SPK-MMHKISIGICFG~RRLIDRIIGSLISIGILGClNVL KY
Figure 1. Amino acid sequences ofnatriuretic peptides. The representative low-molecular-weight forms of rat ANP, human ANP, rat BNP, human BNP and porcine BNP are shown in one-letter symbols. Although the precise form of dog BNP is still undetermined, the inferred low-molecular-weight form is shown [7]. Boxed amino acid residues are conserved among these species. m a m m a l i a n ANPs are fully conserved except for a single amino acid residue at position 12. In spite of the large difference in the sequence, porcine BNP and rat ANP were reported to be equipotent in natriuresis in the rat [1] and to exhibit similar central actions [8-10] by sharing common receptors [11]. On the other hand, the structural difference of non-mammalian ANPs (chicken [12], frog [13] and eel [14]) from mammalian ANPs seems to be reflected on their low natriuretic and vasorelaxant activities when examined in the rat. Thus we tried to see how BNPs act on different species. In the present study, we investigated rat BNP and h u m a n BNP in terms of their vasorelaxant activity in rat and porcine tissues and their diuretic-natriuretic activities in anesthetized rats. As a result, we found t h a t h u m a n BNP and rat BNP act with different potencies on the vascular beds of different species, unlike ANPs which act on any species equally. The present paper describes this comparative pharmacology of BNP in some detail. MATERIALS AND METHODS Materials: Rat ANP and porcine BNP were purchased from Peptide Institute, Osaka, Japan. Rat BNP, a 45-amino-acid peptide, and h u m a n BNP, a 32-amino-acid peptide, were synthesized by the solid-phase method using an automatic peptide synthesizer (Applied Biosystems Inc., Model 430 A). Vasorelaxant activity: Rat aortic strips (2 m m x 5 mm, from male Sprague-Dawley rats) or porcine coronary arterial strips (2 mm x 10 mm) were mounted on stainlesssteel hooks under 1 g (for rat aorta) or 2 g resting tension (for porcine coronary artery). The strips were equilibrated in a modified Locke-Ringer solution at 37°C containing 120 mM NaC1, 5.4 mM KC1, 2.2 mM CaC12, 1.0 mM MgC12, 25 mM NaHCO3 and 5.5 mM glucose, gassed with 95% 02 and 5% CO2. After contraction of the strips with about 1 pM PGF2a, peptide solution was added cumulatively. At the end of the experiment, 0.1 mM papaverine hydrochloride was added for normalization. Diuretic-natriuretic activities: Male Sprague-Dawley rats (300-350 g) were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). Saline was infused at 0.1 ml/min/kg through a femoral vein catheter and peptide solution (100 pl) was added intravenously. Urine was collected at 5-min intervals from the bladder through a polyethylene tube. The concentration of sodium ion was measured by flame photometry. Statistics: Tukey's test was used for judgement of statistical significance [15]. 600
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS Diuretic and n a t r i u r e t i c activities of r a t B N P and h u m a n B N P in the r a t Table 1 s u m m a r i z e s the d o s e - d e p e n d e n t i n c r e a s e s in u r i n e flow r a t e a n d sodium excretion r a t e induced by r a t ANP, r a t B N P and h u m a n B N P in anesthetized rats. The r e l a t i v e potencies of r a t B N P to r a t A N P in diuresis and n a t r i u r e s i s were e s t i m a t e d as 1.6 and 2.5, respectively, by the 4-point parallel line assay [16] based on the responses a t doses i nmol/kg and 3 nmol/kg. H u m a n B N P caused m u c h less responses t h a n r a t B N P even a t 10 times h i g h e r doses. The time courses of the u r i n e flow and sodium excretion rates upon a d m i n i s t r a t i o n of r a t A N P (3 nmol/kg), r a t B N P (3 nmol/kg) or h u m a n B N P (30 nmol/kg) are shown in Fig. 2. R a t A N P and r a t B N P e q u a l l y caused t r a n s i e n t increases in u r i n e and sodium o u t p u t s and the significant increases lasted for a b o u t 15 and 20 rain, respectively.
On the o t h e r
hand, h u m a n B N P caused no significant increase in the u r i n e flow r a t e and induced only a slight, even t h o u g h significant, increase in the sodium excretion r a t e for a t least 10 min. V a s o r e l a x a n t a c t i v i t y of r a t B N P and h u m a n B N P in r a t thoracic aorta As shown in Fig. 3A, r a t B N P exhibited almost the same v a s o r e l a x a t i o n profile as t h a t of r a t ANP, while h u m a n B N P was only slightly active in isolated r a t aorta. P o r c i n e B N P also s e e m e d to be less active t h a n r a t B N P , e v e n t h o u g h t h e i r difference was not significant. The IC50 value, the concentration r e q u i r e d to induce h a l f the m a x i m u m relaxation, of h u m a n B N P is significantly h i g h e r t h a n those of the other peptides (Table 2). The r e l a t i v e potencies of r a t B N P and h u m a n B N P to r a t A N P are 0.97 and 0.05, respectively, w h e n expressed as reciprocal ratios of the ICs0 v a l u e s (Table 2). Table 1. DIURETIC AND NATRIURETIC ACTIVITIES OF HUMAN BNP, RAT BNP AND RAT ANP IN ANESTHETIZED RATS Peptide Rat ANP
Rat BNP Human BNP
Dose (nmol/kg) 1 3 10 1 3 10 30
n 5 5 4 5 5 7 4
AU (pl/min) 29 + 14 39 + 9 43 + 20 25 + 4 57 + 7a,b 9 -4-_ 3a 6 --4- 2b
AUNa (pEq/min) 5.5 + 1.6a,b 6.8 + 1.1 7.0 --t- 3.1 5.8 +_ 0.8 12.0 ± 1.6c,d,e 1.1 - 0.7d 0.4 _--4-0.1e
Results are expressed as mean ± SEM. AU and AUNa m e a n the increases in urine flow rate and sodium excretion rate, respectively. They were calculated as the differences between the values averaged during the pre-administration period (30 min) and postadministration period (30 min). The paired values with a common superscript a, b, c, d or e are significantly different from each other (a, b, c: p < 0.05; d, e: p < 0.01, Tukey's test). 601
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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