Europratt

Juunral of Phmracolug_s.

202 (1991) 73-79

Elsevier Science Publishers B.V. All rights

,B I991 ADONIS

73

reserved 0011-2999/91,‘$03.50

001429999100601A

EJP 52016

Toshihiro Kita, Osamu Kida, Naoto Yokota, Tanenao Eto, Naoto Minamino 2, Kenji Kangawa ‘, Hisayuki Matsuo ’ and Kenjiro Tanaka First Deparlment

of hrernal

Medicine and

’ Departmer~t of Biochnism:

and ’ National

Cardiol,ascular

Cmsr

Miyazaki

Medical College, Kiyotake. Miyazaki

889.16. Japan

Research institute. Suita 565, Japan

Received 22 May 1991. accepted 18 June 1991

Rat brain natriuretic peptidc-45 (rat BNP-45) has recently been isolated from rat heart and shown to be a circulating form of rat BNP. We investigated the effects of rat BNP-45 in anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and compared them with those of rat cu-atrial natriuretic peptidc (u-ANP). BNP-45 was a potent natriuretic and hypotensive agent in both strains. The effects were comparable with those of (u-ANP and were far greater than those of porcine BNP-26 reported previously. In SHR blood pressure decreased more than in WKY following injection of the highest dose (2.0 nmol/kg) of BNP-45 or n-ANP. However, WKY were more susceptible than SHR to BNP-45 for diuresis, natriuresis and urinary cGMP excretion. Moreover, a high dose of BNP-45 led to a prolonged lowering of blood pressure and urinary cGMP excretion compared to n-ANP, and these features were prominent in WKY. BNP-45 disappeared more slowly than a-ANP when the two peptide (2.0 pg) were injected i.v. in WKY. Thus, rat BNP-45 and (u-ANP had comparable hypotensive and natriuretic p0ienc.y: however, the action and plasma half-lift of rat BNP-45 were more prolonged. BNP (brain natriurctic pcptidc); ANP (atrial natriuretic

pcptidc); Blood pressure;

1. Introduction Sudoh et al. (1988) discovered ‘brain natriurctic peptide’ (BNP) in porcine brain, and greater concentrations of BNP were found in porcine heart (Aburaya et al., 1989b). They established the existence of two families of natriuretic peptides, atrial natriuretic peptide (ANP) and BNP, in the central nervous system and peripheral organs (Aburaya et al., 1989b: Minamino et al., 1988; Sudoh et al., 1988; Ueda ct al., 1988). I? has already been reported that BNP is stored in the heart and secreted into the cardiac perfusate in pigs (Saito et al., 1989) and rats (Ogawa et al., 1990). Recently. a 45amino acid pcptide belonging to the BNP family was identified in rat heari and designated rat BNP-45 (Aburaya et al., 1989a; Kambayashi et al., 1989). Rat BNP-45 has a distinctly different sequence from porcine BNP (Aburaya et al., 1989a), and its distribution is also different: high concentrations of rat BNP-45 have been found only in the heart (Aburaya et

Natriuresis:

Metabolic

clearance

rate; (Rat)

al., 1989~). These findings strongly suggest that rat BNP-45 may function as a circulating hormone. increased plasma BNP has been reported in various human diseases, such as heart failure, chronic renal failure and hypertension (Mukoyama et al., 1990a, b). This fact suggests the possibility that not only AMP but also BNP regulates the water-elcctrolytc balance and blood pressure. As we have already reported, porcine BNP and ANP have similar biological activities in normal (Sudoh et al., 1988) and hypertensive rats (Kita et al., 1989a, b). However, the effects of rat BNP-45 in hypertensive rats are not yet known. Therefore, to study the properties of rat BNP-45 we investigated the effects of rat BNP-45 in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and compared them with the effects of rat CX-ANP.

2. Materials and methods 2.1. Animals Male SHR and WKY (20- to 25week-old) were purchased from Charles River Inc. (Atsugi, Japan). The rats wcrc housed in a temperature- and humidity-

c~~~~tr~~~led e~vjr~~nrnc~t and maint~~ined on standard rat chow ~N~h~~n CREA CE-2, Tokyo. Japan; 135 pmol Na,igl and tap water ad lihitum for at least one week prior to the experiment.

The rats were anesthetized by an i.p. injection of pentobarbital sodium (50 mg/kgl. Anesthesia was supplemented at 20-25 mg/kg per hour (Faraci, 1989). A polyethylene catheter (PE-250) WAS inserted into the rrachea to aid breathing. Mean blood pressure IMBPl and heart rate (HR) were m~nit~~red continuously by a right carotid artery catheter (PE-Sill connected to a Statham pressure transducer (model P231D, Gould, Saddle Brook. NJ). A PE-IO catheter was inserted into the right jugular vein for administration of both maintenance solution and peptides. The bladder was exposed through a suprapul~ic incisi~~n and catheterized for the C~~~~~CFi~~n of urine. Isotonic saline (20 pl/minl was infused throughout e esperiment. After equilibration for at least 60 min, urine was collected every IO min during a 20-min control period. Rat BNP-45 or rat ru-ANP (0. I, O.2.0.5. 1.0 and 2.0 nmol/kgI dissolved in saline containing 1% bacitracin (Sigma. St. Louis, M(S) was injected i.v.. and urine was collected continuously for three to six IO-min periods following each dose. There was a 30- to 60-min rest interval between each injection, to allow the urine ~~~lurn~to return to a steady baseline value. Urine vohtme was determined by weight. Urinary sodium and potassium were measured by flame photometry (model 20SD. Hitachi, Tokyo, Japan). The concentration of cyclic guanosine 3’S’-monophosphate tcGMP) in urine was measured by radioimmunoassay, using cGMP assay kit (Ametsham, Tokyo, Japan),

Another group of WKY (n = 10) was anesthetized by i.p. injection of pentobarhital sodium (SO mg/kgl and the trachea, jugular vein and carotid artery were catheterized. A bolus i.v. injection of rat BNP-45 or rat (u-ANP (2.0 pg, dissolved in I50 ~1 saline) was given, after which 300 gL1blood samples were drawn at 0.5, 1, 1.5, 2, 3, 5 and 10 min via the carotid artery catheter. The blood taken was replaced by an equal volume of saline. The remaining blood was finally taken at 30 min after rhe injection. Blood was collected with aprotinin (SO0 kiU/mll and EDTA-2Na (1 mg/ml) and centrifuged at 3000 x g for 10 min at 4 o C. Plasma was stored at -20°C until assayed. Plasma wa; loaded onto a Sep-Pak Cl8 cartridge, and ANP and BNP were extracted as described previously (Kate et al., 1988}. The concentrations of the peptides were measured by using specific radioimmunoassays for rat BNP-45

(Aburaya et al., 1989~1 and rat CX-ANP(Kate et al., 1(IX71 as described previously.

Rat BNP-45 was donated by Shionogi Research taboratorics (Osaka, Japan); the peptide was synthesized by solid-phase method, and its homogeneity was confirmed by reverse-phase high-performance liquid chromatography and amino acid analysis. Rat a-ANP (rat ANF-(99-126)) was purchased from Peptide Institute Inc. (Osaka, Japan).

All data are expressed as means It S.E.M. Dose-dependent relationships are expressed by maximal changes as compared to the mean of two pre-injection measurements. The time course study was evaluated with a one-way analysis of variance and significant differences were subsequently determined with Dunnett’s t-test. Unpaired t-tests were used for comparisons between SHR and WKY and between ANP and BNP. Values of P < 0.05 were considered statistically significant.

3. Results

Table 1 show basal values for MBP, HR, urinal volume i’UVl, and urinary excretion of sodium (UNaVl, potassium (UKVl and cGMP (UcGMPV), There were no sigmficant intrastrain differences between these parameters. MBP and HR were significantly higher in SHR than in WKY in both BNP-45 and ru-ANP groups. Figure 1 shows dose-response relationshjps for the hypotensive effect induced by rat BNP-45, rat ru-ANP and porcine BNP-26. At the highest dose, all three peptides lowered MBP more in SHR than in WKY. Porcine BNP-26 was less potent than the other peptides in both strains. Figure 2 shows the natriuretic effects of the three peptides in SHR and WKY. Rat BNP-45 and cu-ANP caused dose-dependent natriuresis in both strains. The natriuretic effect of porcine BNP-26 was weak. .4t 0.5 nmol/kg of BNP-45, WKY excreted more water and sodium than SHR (UV; 152.8 f 19.9 vs. 70.3 -C9.9 pl/min per kg, P < 0.01, UNaV; 25.86 & 3.81 vs. 11.33 f 1.68 ~Eq/min per kg, P < 0.05, for WKY and SHR, respectively). Urinary potassium was also greater in WKY than in SHR at 0.5 nmol/kg BNP-45 (8.91 f 0.37 vs. 5.91 t_ 0.84 pEq/min per kg, P < 0.01, for WKY and SHR, respzc!ivelyj. There were no such differences with a-ANP. The time courses of MBP in SHR and WKY are illustrated in fig, 3. T.V.administration of at least 0.5 nmol/kg rat BNP-45 was followed by a rapid dose-dc-

75 (nmol : kg)

0.1

I

0

0.2

0.5

I

I

1.0

I

2.0

I

0.1

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0.2

0.5

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1

-2

9 -

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8 : 5

-20

% 5

-30

SHR

-40

WKY rat (r-ANP

I

- so

rat CT-ANP Fig. I. Dose-response relarionships of the maximal reduction in mean blood pressure (MBP) following injection of rat BNP-45. rat I~-ANP and porcine BNP-26 in SHR and WKY. All data are means i S.E.M.

TABLE

I

Basal values for mean arterial pressure. heart rate. urinary output and urinar)’ cxcrrlion of sodium. potassium and cGMP in WKY and SHR. Values are means+ S.E.M.: n. no. of rats. Rat BNP--IS

Me-n arterial pressure (mm Hg) Hesrt rate fbeats/min) Urinary output (Ml/min per kg) Urinary sodium excretion (pEq/min per kg) Urinary potassium excretion (pEq/min per kg) Urinary cGMP excretion (pmol/min)

Rar n-ANP

WKY (n =X)

SHR (n = 7)

WKY (n = 8)

129 +I ‘06 +I1 I2.0 f 1.0 1).13* 0.13 2.Y I + O.JO 17.0 1_ 3.1

IX9 *Id ?.c7 +5 .’ 12.J +1).x 0.33 a 0. I 1 3.11hf 0.X 27.-I + I.4

129 +2 292 fll 12.3 _I 00

0.-e* 0.11l 2.5Y * 2x.0 i

t1.1x 2.6

” P < 0.01 when compared with values in WKY given BPJP-JS or n-ANP

rat BNP4 50

3

rat a-ANP

T

SHR

40

WKY

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3o

0

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0.2

0.5

1.0

2.0

0.1

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0.2

0.5

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I

1.0

2.0

(nmol I kg) Fig. 1. Dose-rcspon~ relationships of the maximal incrcasc in urinary sodium rxcrction (UNoV) following injection of rat BNP--lc. r31 n-ANP and porcine BNP-20 in SI(R and WKY. All Dada are means+S.E.M.

Time

(mini

(nmoi’kg

rat ENP.45 --

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--h

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--

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-

1.0

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-

raf

2.0

o.ANP

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--

2.0

in UcGM3V. which returned relatively quickly to the basal value. The effects of BNP-45 were more protonged in WKY than in SI-IR. The natriuresis elicited by 3.0 nmol/kg of rat BNP-45 also lasted for SO min in WKY, while the effect of an equimolar dose of n-ANP lasted only 20 min. Figure 5 illustrates the disappearance of the peptides after holus injection. Immunoreactive BNP-45 and (u-ANP disappeared from the circulation very quickly during the initial phase of clearance. with t,,z equal to 45 and 55 s for BNP-45 and a-ANP, respectively. The remaining BNP-45 disappeared more slowly than ru-ANP during the second phase ft,,,; 6.95 min vs. 4.03 min for BNP-45 and a-ANP, respectively).

increase

Figure 4 sholvs the time course of UcGMPV in SHR xx! WKY. Dose-dcpcndcnt incrcrtscv in IJcGMPV wcrc ohscwed in both strains. and the cffccts lasted over hf) min with 1.11and 2.0 nmol/kg of BNP-35, I~~ect~~)~of ru-ANP \%a5 ais0 f~)~~~3~ved hy a marked

500 (Rrn~iikg~

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400

300

200

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Time

10

30

tmin)

Fig. 5. Kinetics of exogenotrsly administered rat BNWJS and rat a-ANP (2.0 pg iv.1 in

WKY.

Therefore, there was a relatively high concentration of rat BNP-45 in the circulation 30 min after injection (BNP-45; 308.0 f 24.7 pg/ml vs. (u-ANP; 158.2 k 12.6 pg/ml, P < 0.01).

4. Discussion The major findings of the present study arc (1 f rat BNP-45 and ry-ANP showed similar pharmacological spectra in barth WKY and SHR, and their effects were far greater than those of porcine BNP-26 in the same animals . Ellcct\ of endrahclin and vnsopreshin on cerebral hklud vcsxh, Am. J. Phy\iot. 257. t17YY. Cicll;~i. M.. R.E. Dcuolf. L.B. Kintcr d R. Breuwkc5 III. I9Xh. The cffcct of atrid nalriurrtic factor cm hlwcl preswrc. heart rate. and renal functirm3 in conhcioub.spontrtncc~u~l~hyportsn+s rat*. (‘ire. Rc\. 50, 56.

Hirata. Y.. M. Shichiri. T. Emori. F. Marumct. K. Kangawa and fj. Matsuo, IYXX. brain n~triuretic peptidr interacts with atrial natriuretic peptide receptor in cultured rat vascular smooth muscle cells. FEBS Lett. 2.W. 415. Ksmhnyashi. Y.. K. Nakao. H. Itoh. K. Hosoda, Y. Saito. T. Yamada. M. Mukoyama. 11. Arili. C. Shirakami. S. Suga. Y. Ogawa. M. Jougasaki. N. .Minamino. K. K;mgawa. H. Matsur). K. lnouye and Il. Imum. IYXY. Isolation and sequence determinatioa uf rat cardiac natriuretie peptide. B&hem. Biophys. Res. C~~mmun. lft.7. 237. Kate. J.. 0. Kida. S. Naknmura. A. Sasaki. K. Kodatna and K. Tanaka. IYK7. Atrial natriurrtic polypeptide (ANP) in the development of spontrneously hypertensive ruts (SHRl and strokeprone SHR (SHRSP). Biochem. Biophys. Res. Commun. 113, 316. Katu. J.. 0. #ida. T. Kita. S. Nu~rnuru. A. Snsaki. K. Kodamn and K. Tanaka, IYXX. Free and hound forms of atrial nutriuretic peptide (ANP1 in rat plasma: Preferential increase of free ANP in spontaneously hypertensive ruts (SHRI and strokeprone STIR (SHRSP), Biochem. Biophys. Res. Commun. 152. 1084 Katsuhe. N.. D. Schwartz and P. Needleman. IY8h. Atriopeptin turnover: Quantitative relationship between in vivo changes in plasma levels and atrial ctmtrnt. J. Ph~rrn~c(~l. Exp. Ther. 23Y. 474. Kita. T.. 0. Kid:,. J. Kate. S. Nakamura. T. Eto. N. Minnmino. K. Kangawa, H. Matsuo and K. Tanaka. IYHYa. Natriuretic and hypotensive effects of brain natriuretic peptide (BNP) in spontaneously hypertensive rats. Life Sci. 4-t. 1541. Kita. T.. 0. Kida. J. K&o. S. Nakamura. T. Eta. N. M~nurnin~~.K. hngaWi#. H. Matsuo and K. Tanaka. IYXYh. N~triurctic and ‘- ...-. * hvnrrtensi-,* &:ct’; ,,f hr&, f::,rr;..-.-*;.. . peptide in 89”

z

Kojima. M.. N. Minamino. K. Kangaw;~and If. M;r~suo. IYXY. Cltrning and sequencr: analyGs of cDNA encoding a precursor for rut brain natriuretic peptidr. Biochem. Biophys. Res. I’ammun. 15% I420. Kurt&. K.. 0. Kida, K. Kangnwa. ti. Mutes and K. Txnxka. 1YX.i. Enh;tnced natriuretic and hypotcnsi\e responGvenes\ tr, rt-human atrial natriuretic polypeptide (rr-h,\NP! in S?!R. Clin. Eup. ilypert. A7. loY7. Maeda. T.. M. Nina. K. Shigematsu. M. Kuriharu. Y. Kataoka. K. Nakao, Il. Imurrt. ti. Matzo. ft. Tsuchi!;u%t and M. Owli. binding site\ in IYSHI. Specific I ‘-“l]hrain n~itriuretic peptiderat itnd pip kidneys. Eur. J. Phitrmuc4. l7h. 341. Marsh. EA., A.A. Seymour. A.13. llaley. M.A. Whinncry. M.A. Nilpier, H.F. Nutt and E.11. Blainc. IYH.5. Tienil alld l~h)t~d pressure responses to synthrtic iitrial uittriurctic factor 1115pcMttilneously hypertensive ritts. llyprrtensian 7. 3Xh. Matsuo. H. ;tnd It. Nekazato, 14X7. ~~t~lcc~ll;irttiolt~~l; ctf ;ttri;tl nutriuretic peptides. Endocrinol. Mrtab. c‘lin. North Am. I& -G. McGregor. A., M. Richards. E. Espincr. T. Yundlc und 11. Ikram. IYYO, hitin niltriurctic peptide administcrcd to man: action5 and met;lhr~lism. J. Clin. Endocrinol. Mct:th. 7tt. 1ItI?. ;md Min;m~ino. N.. K. K;mg;la;I and II. Mx:$x:. IYSX. Ix&tion identification of a high molecular weight brain n:lt:iurctic prptidc in porcine c;trdiac atrium. Rio&em. Bioph~\. Re>. ~.~~nlrnun.157. 402.

Mukoyama. M.. K. Nakao. Y. Saito. Y. Oga~. K. tios(jd;(. S. Sug.,. C. Shiruk~mi. M. Jougawki and tf. Imum. IYYOa. Ijumcrn brain natriuretic peptidr. a novel cardiac hormone. Lancrt i. 801. Mukoyama. M.. K. Nakao. Y. Saito, Y. Og~a. K. Hw)J,,. S. Supa. G. Shirakami. M. Jougasabi and H. Imura. IYYllh. Incrr~d human brain natriuretic pcptide in conpc\ti\e heart fa!i!urr. \. Engl. J. Med. .73.X 757. Nakao. K. and 11. lmura. IYYII. Biosynthesis. srcretion and rrccptor selectivity of human BNP (Ahstractf. Ottawa Symposium on Arrial Natriuretic Factor. Sarrllitr of the 13th lntcrnuti~inul Snciet)’ of Hypertensiun. Ottaua. Canada. p. I. Orhtenschluger. W.F.. D.A. Baron. H. Schemer and M.Ci. Currie. IYXY. Atrial and brain natriurrtic prptida share binding \itc\ m the kidney and heart. Eur. J. Pharmacol. IhI. 159. Ogasa. Y.. K. Nakacr. M. Mukoyama. G. Shirakami. Il. Itoh. K. Hosnda. Y. Saito. H. A& S. Supa. M. Ji~u~~~~kr.T. Yamada. Y. Kamhayashi. K. Inouye asd H. Imurcl. IYYtt. Rat brain nstriurctic peptide: tissue distribution and molecular form. Endocrinoktg) l’h _. 2’l.s __. Saito. Y.. K. Nakao. H. Itoh. T. Yamada. M. Muko~ama. Il. Arai. K. Hosoda. G. Shirakami. S. Su_ea. N. Minamino. K. Kango\\a. H. Matsuo and I-1. lmurs. IYXY. Brain natriuretic prptidr is a ntnrl cardiac hormone. Bil~chem. Biophys. Rrs. C~~rnnlun.1%. ihI). Szsaki. A.. 0. Kida. K. Kangswa. 13. M-f;lt~~: ad K. Tanaka. lYti5. C’ardil,supprrzsive effect of a-human atria1 natriuretic pc~l)pcptide Co-hANPI in spontaneously h!pertenGvc rat\. Eur. J. Phxmacol. I IS. Xl. Sauro. h1.D.. D.F. Fitzpatrick and R.G. Coffry. IYXS. Drtrctivc cyclic GMP ~ccumul~ti(~nin ~pontrtneot4~ hypsrtrnG\e rat itort~ in response to atria1 n~itriur~tic factor. Biochem. Pharmacrll. 5. ,,a” Sudoh. L . h. hang;rha. ii. hlm.tmino and II. hlathuo. IYSS. I\ nrrn n;Itriurctic prptide in porcine brain. Nature 322. 7s. Tatc)sma. Il.. J. tlino. N. hlinamin~l. K. Ksnga\\a. T. Opihar:t and 11.Matzo. IWI. Charactcri7crlion of immumlrcacti5e hram natriurctic prptdc in human c;:rdi:tc atrium. Bitt&em. Bi