Biological effects of rat iso-atrial natriuretic peptide and brain natriuretic peptide are indistinguishable from each other D. A. WELE Bepartnzenf of Anatomy9 Queen's Universitgi Kingston, gbnt.> Canada K7E 3N6
B. M. BENNETT Department of Pharrmcobgy and Toxicology, Queen S University, Kingston, Ont., Canada K7L 3N6
D. B. JENNINGS Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by 67.193.101.86 on 01/07/15 For personal use only.
Department sf Pbaysiology, Queen's University, Kingston, Ont., Cansdu K7E 3 N 6
I. R. SAWDA AND I?.G. F L Y N N ~ Department sf Biochemistry, Queen's University, Kingston, Ont., Canada K7L 31% AND
S. C . PAN^ Department of Anatomy, &rceen9s University, Kings~osa,Ont., Canada K7L 3NQ Received May 27, 1992 WHGLE,D. A., BENNETT,B. M., JENNIWGS, D. B., SARDA, I. R., FLYNN,T. G., and PANG,S. C. 1992. Biological effects of rat iso-atrial natriuretic peptide and brain natriuretic peptide are indistinguishable from each other. Can. J . Physiol. PharmacoI. '70: 1525- 1528. Rat brain natriuretic peptide (rBNP) and iso-atrial natriuretic peptide (iso-rANP) were discovered independently by two research laboratories. They are considered to be members of the B-type natriuretic peptides. Except for the Gln/Leu substitution at position 44, the amino acid sequence of iso-rANP is identical with that of the G-terminal45 amino acids of rat pro-BNP and with the 5-kDa cardiac peptide from rat atria. To determine whether this amino acid substitution can modifgr the known bislogical effects of rBNP and iso-rANP, the present investigation examined the cardiovascular and renal responses, vassrelaxant effect, receptor binding characteristics, and cyclic GMP production by the two peptides in relation to that of rat atrial natriuretic peptide (rANP). Results indicate that rBNP and iso-rANP are indistinguishable from each other in terms of these known biological activities sf atrial natriuretic peptide. We therefore conclude that rBNP and iso-rANP are identical peptides and that the amino acid substitution at position 44 represents a polymorphic form of the rat B-type natriuretic peptide. Key words: atrial natriuretic peptide, brain natriuretic peptide, cardiovascular response, vasorelaxation, cyclic GMP, receptor binding. WICLE,B. A., BENNETT, B. M., JBNNINGS, D. B., SARBA,I. R., PLYNN,T. 6., et PANG,S. C. 1992. Biological effects of rat iso-atrial natriuretic peptide and brain natriuretic peptide are indistinguishable from each other. Can. J. Physiol. Pharmacol. 78 : 1525- 1528. L9iso-peptidenatriurttique auriculaire (iso-PNAr) et le peptide natriuretique cerebral (PNCr) de rat ont Cte dCcouverts dans deux laboratoires de recherche diffkrents. Hls font partie de la famille des peptides natriurktiques de type B. Plutat que d'observer la substitution Gln/Leu en position 44, on constate que la sequence des acides arninCs de I'iso-PNAr est identique B celle des acides amines en position 45 C-terminale du pro-PNC et B celle du peptide cardiaque 5-kDa des esreillettes de rat. Pour determiner si cette substitution d'acides aminks pourrait modifier les effets biologiques connus du PNCr et de l'isoPNAr, la prksente ktude a examin6 les reponses renales et cardiovasculaires, l'effet vasorelaxant, les caraetCristiques de fixation aux recepteurs et la production de GMP cyclique par Bes deux peptides par rapport h ceux du PNAr. Les rksultats indiquent que le PNCr et l'iso-PNAr sont indiffkrenciables du point de vue de ces effets biologiques connus du peptide riatriurktique auriculaire. Ainsi, nous concluons que le PNCr et l'iso-PNAr sont des peptides identiques et que la substitution des acides aminks en position 44 represente une forme polymorphe du peptide natriurktique de type B du rat. Mots cbb : peptide natriurktique auriculaire, peptide natriuretique cCrCbral, rkponse cardiovasculaire, vasorelaxation, GMP cyclique, fixation au rkcepteur. [Traduit par la rkdactinn]
It is now well established that the mammalian heart is an endocrine organ and that atrial natriuretic peptide (ANP) is synthesized, stored, and secreted by atrial rnyocytes (Cantin and Genest 1985). A N P induces a number of cardiovascular and renal responses upon its release into the circulation, including natriuresis , diuresis, and hypotension (de Bold eb al. 198I). ANP exerts its effect, at least in part, by direct activation of receptor-linked particulate guanylate eyclase to produce cyclic G M P (cGMP) (Waldrnan et al. 1984; Hamet et al. 1986). In blood vessels, the increase in cGMP level correlates 'Authors for correspondence. Printed in Canada ! lmprime au Canada
with the vasorelaxant responses induced by A N P ( R a p p o r t et al. 1986). In recent years a number s f natriuretic peptides have been identified in mammalian systems. These new members s f the natriuretic peptide family include brain o r B-type natriuretic peptide (BNP) (Sudoh et a/. 1988) and C-type natriuretic peptide (CNP) (Furuya eb al. 1990). In 1989, our laboratory isslated another atrial natriuretic peptide from rat atria (Flynn eb al. 1989). Because of the sequence homology and similarity in physiological response to ANP, this new atrial peptide was designated iso-rANP. Except for the G l n l k u substitution at position 44, the amino acid sequence of iso-rANP is identical with that of the C-terminal45 m i n o acids of rat pro-BNP
CAN. J. PHYSIOL. PHARMACOL. VOL. 70, 1992
1526
(Kojima et aE. 1989) and with the 5-kDa cardiac peptide from rat atria (Aburaya et al. 1989). To determine whether this amino acid substitution can modify the known biological effects s f rBNP and iss-rANP, the present investigation examined the cardiovascular and renal responses, vasorelaxant effect, receptor binding characteristics, and cGMP production hy the two peptides in relation t o that of rANP.
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Methods Sources of atrial natrs'umticpeptides Rat ANP(,,-L26jwas obtained from IAF Biochemicals h c . (Laval, Que.). Rat iss-ANP(, was synthesized by solid-phase peptide synthesis using a semiautomatic apparatus (Labortec SP640 peptide synthesizer, Switzerland) and 9-fluorenylmethyloxycarbonyl(FMBC) amino acid chemistry. All peptides were purified to homogeneity by HPLC. Iodination of rANPe,-126) was performed by the chloramine T method as described previously (Sarda et al. 1989). Hodinated peptide was purified by MPLC, lyophilized, and dissolved in 8.01 M acetic acid before use. Rat BNP(,-,,) and porcine BNP were pnrchased from Peninsula Laboratories Hnc. (Belmont, Calif.).
-,,
Rat ba'oassajs Viral antigen-free male Sprague -Dawley rats (body weight 293 k 33 g) were used for bioasays. Experimental protocols were identical with those described previously by our laboratory (Jennings and Flynn 1989). In brief, rats were anesthetized with sodium pentobarbita1 (70 mg/kg; MTC Pharmaceuticals, Hamilton, Ont.) and infused with saline (0.9% NaCl). Catheters were placed in a femoral artery, the right external jugular vein, and the urinary bladder. After a 40-min control period, rats were given a (8.2-mL, bolus injection of peptides or vehicle into the jugular vein. Cardiovascular and urine volume measurements were carried out for 20 min, and then arterial blood was sampled to measure the hematocrit. V'osoreIauatisn assay Aortic strips were prepared from male Sprague - Dawley rats (250 -300 g) as previously described (Stewart er a!. 1989). Experiments were performed in a double-jacketed water bath in a Mrebs solution of the following composition (n~lgM):NaCl, 1 18; KC], 4.74; MgSB,, 1.18; KH,PO,, 1.18; CaC12. 2.5; NaHCO,, 24.9; and glucose, 10. The solution was aerated with 95 % O2 - 5 % C 0 2 and maintained at 37'C. Tissues were contracted submaximally with 0.1 ,uM phenylephrine and cumulative concentration -response curves for rANP, rBNP, and iso-rANP obtained. EC,, values were determined graphically from each concentration-response curve. Cell cu/ferae Rat mesenteric artery smooth muscle sells (VSMC) were derived from adult male Wistar rats (150-200 g) as previously described (Hamet et al. 1989). Cultures were maintained in Dulkcco's modified Eagle's medium (DME) supplemented with 8 mM Hepes and 10% (v/v) calf serum, a d kept in a 95 % air - 5 % C 0 2 humidified atmosphere at 37°C. Cells from passages 15-20 were used for experiments. Binding studies Binding assays were performed in 24-well culture plates, using a method similar to that of Leitman et ad. (1988). Briefly, confluent cell layers were washed twice with a binding buffer (DME) containing 61.05 % bovine serum albumin at 3 7 T . Iodinated ANP was suspended in binding buffer and incubated (about 50 800 cpm/well; specific activity 1200- 1500 mCi/rng (1 Ci = 37 GBq)) with varying concentrations of cold peptide. After incubation for 2 h at 4"C, cells were washed three times with cold binding buffer to remove unbound ligand. Cells were then dissociated with 0.5 m& of I % trypsin at 37°C for 15 rnin and collected. Each well was subsequently washed twice with 0.5 rnL of 1 M NaBH, and the resulting 1.5-mL volume was counted to estimate bound ligand. Nonspecific binding was determined in the presence of 1 pM unlabelled peptide and subtracted from each value.
Tme (rnin)
FIG. 1. Effect of atrial peptides on mean arterial blood pressure (upper panel), heart rate (middle panel), and urine flow (lower panel). Values are percent changes f SEM far mean arterial pressure, heart rate, and urine flow following bolus injection of 0.8 pmsl of peptide (or vehicle, i.e., control) into bioassay rats at 0 time. All three atrial peptides induced hypotension, bradycardia, and diuresis. rBNP, iso-rANB, and rANP were equipotent in their ability to induce cardiovascular and urinary responses (rANP, n = 12; rBNP, n = 6; iso-rANP, ra == 15; vehicle, n = 16).
cGMP radioimrnunsassa~~ To determine extracellular and lntracellular cGMP responses, cells were cultured until confluent in 35-rnm dishes. Cells were washed twice and left in DME for stimulation with the indicated concentrations of peptide. Parallel dishes were retained for cell counting by haemocytometer. After 2 h stimulation at 37"C, culture medium (2 mL) was collected into 1 mL of 30%trichloroacetic acid (TCA) to estimate extracellular egression of cGMP. Intracellular production of cGMB was sampled by scraping the remaining cell layer off the dish with two I-mE volumes of 10% TCA. Samples were ether extracted to remove the TCA and quantitated for cGMP content by radioimmunoassay (Steiner et al. 1972).
Results and diseasssion Although the original amino acid sequence of isa-rANP reported a Gln residue in position 44 (Flynn et a%.1989), subsequent analysis sf the complementary D N A (cDNA) sequence for rBNP has shown a Leu in the penultimate position (Kojima et a!. 1989). Significant controversy has ensued as to whether these are the same peptide. Although a leucine residue was observed at position 44 during the initid purification of iso-rANP from 900 rat atria, the yield of phenylthiohydantoin-Leu was much less than that of phenylthiohydantoin-GBn, and we therefore placed Gln instead of Leu at this position of the sequence (Flynn et al. 1989). Our analy-
E 527
BRIEF REPORTS 1 RAPPORTS BRBFS
a V-V
pBNP
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8-&I rWNP @-Q
rBNP
A-A
iso-rANP
I
r
I
I 1
I
I
I
I
FIG.2. Effect of natriuretic peptides on relaxation of isolated rat aortic strips. Tissues were contracted with 0.1 p M phenylephrine and cumulative concentration - response curves obtained for rBNP, rANP, and iso-rANP. The EC,, values for relaxation were 15 f 2, 13 f 4, and 19 & 6 nM for rBNP, rANP, and iso-rANP, respectively. Each value represents the mean -b SB (n = 7).
Control
A-A
iso-rANP
@--@
rBNP
10
92
41
10
9
7
8
-10g [PEPTIDE]
NSB
(MI
FIG. 3. Displacement of "51-labelled rANP from cultured rat mesenteric VSMC with udabelled iso-rANP,, -,5,, rBNP, and rANP(,-,2,,. As evident from this figure, the concentrations of rBNP and iso-rANPf,-,,, required to displace labelled rANP were about fourfold higher than that of rANP(wNo detectable differEach value on ence was present between rBNP and iss-rANP(, the curve represents the mean of two experiments, each performed in duplicate. NSB, nonspecific binding.
,,.
-,,.
%isof the gene sequence for iso-rANP, however, also revealed coding for Leu at position 44 (Roy and Flynm 1990), and there exists a possibility that the Gln/Eeu substitution at this position may represent a polymorphic form of this peptide in rats. To investigate the biological activity o f rBNP and iso-rANP, we have compared the two peptides on the basis of h o w n biochemical, pharmacological, and physioBogical activities for natriaretic peptides. In bioassay experiments in anesthetized intact rats, cardiovascular and urine flow measurements were comparable during the control periods. We chose to administer a Q.8-pmol dose sf peptide since this dose of rANP produced maximal urinary responses Vennings and Flynn 1989). The effects of the three peptides on mean arterial pressure, heart rate, and urine flow of bioassay rats in relation to control (vehicle) are illustrated in Fig. 1. At a dose of 0.8 prnol, eardiovascula~and urine responses for the three peptides were similar; there were no significant differences
8
7
6
FIG.4.Extracellular (upper panel) and intracelluliar (lower panel) accumulation of cGMP in rat n~esentericVSMC in response to porcine BNP (pBNP), rANP rBNP, and iso-rANP,, (Upper panel) The extracellular accumulation of cGMP as induced by atrial peptides (lO-y M) was similar with rBNP and iso-rANP,,-,,,, but was significantly lower than that induced by rANP(,-,,,, and pBNP (one-way ANOVA).Each value on the curve represents: the mean f SD of three experiments, each performed in duplicate. (Lower panel) At 10-' M, the intracellular cGMP accumulation induced by pBNP and rANP,,,was significantly higher than that induced by rBNP Similar levels of intracellular cGMP were induced and iso-rANP(, by rBNP and ~ S O - ~ A N-45). P(~
,,- ,,,
control
9
-,,.
,, -,,.
between responses at any of the time periods. While injection of iso-rANP and rBNP had no effect on hematocrit, injection of rANP significantly lowered hematocrit compared with the other three groups ( p < 0.05, data not shown). This latter finding was consistent with previous results (Jennings and Flynn 1989). In isolated rat aortae, all three peptides elicited csncentratisn-dependent relaxation respnses (Fig. 2). The potency for relaxation by the three peptides was of similar magnitude, with no statistically significant differences in the Ee5()values for relaxation. The maximal relaxation response at the highest dose tested (180 nM) was the same for all three natriuretic peptides . Receptor binding studies using cultured sat mesenteric VSMC indicated that rBNP and ~ S O - ~ A N Pcompete ( ~ - ~ ~ with ) equd affinity for the same receptor population in this cell type. As shown in Fig. 3, rANP displaces labelled rANP with an ICSOvalue sf 2.1 x 1W9 M, while rBNP and iso-rANP do the same at an identical but higher ICso value of 7.9 x 164-%E%%. The BIBo ratios for rANP were significantly differfrom both iso-rANP and rBNP at ent (one-way ANOVA) and M unlabelled peptide, but n o significant difference was detectable between rBNP and iso-rANP. As illustrated in
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CAN. J. PHYSIOL. PWARMACOL. VOL. 70, 1992
Fig. 4, the extracellular and intracellular accumuBation sf cGMP in cultured rat mesenteric VSMC induced by natriuretic peptides showed no significant difference between rBNP and iso-rANP. These data indicate that both rBNP and iso-rANP are capable sf activating the guanylate cyclase linked receptor system at high doses (1V4M), and do so with similar efficacy. However, the concentration s f iss-rANP and rBNP required to elicit a cGMP response was significantly higher than that sf porcine BNP @BNP), a related natriuretic peptide with different amino acid sequence. This would suggest that rBNP and iss-rANP are not likely to be the endogenous Bigands for the guanylate cyclase linked B-receptor subtype prevalent in cultured rat mesenteric VSMC. These results demonstrate that despite the single amino acid residue substitution in rBNP and iso-rANP, the two peptides are virtually identical within the limits sf experimental variation in their ability to induce the characteristic bischemicd, pharmacological, and physiological responses of atrial watriuretic pepaides. The original amino acid sequence s f iso-rANP may represent a polymorphic form of the rat B-type natriuretic peptide, but our results indicate that both iso-rANP and rBNP are in essence the same peptide.
Acknowledgements This project was supported by grants from the Medical Research Council of Canada and the Faculty of Medicine and the Principal's Development Fund, Queen's University. The technical assistance of Maureen St. James and Barbara Lawson and the secretarial work of Dianne Snider were much appreciated. The authors also thank Dr. Seon Shin of the Department of Physiology for the use s f his gamma counter. Aburaya, M., Hino, J., Minamino, N., Kangawa, K., and Matsuo, H. 1989. Isolation and identification of rat brain natriuretie peptides in cardiac atrium. Biochem. Biophys. Res. Cornmun. 163: 226-232. Cantin, M., and Genest, J. 1985. The heart and the atrial natriuretic factor. Endocr. Rev. 6 : 107- 127. de Bold, A. J., Borenstein, H. B., Veress, A. T., and Sonnenberg, PI. 1981. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rdts. Life Sci. 28: 89-94. FBynn, T. G., Brar, A., Trernblay, L., Sarda, I., Lyons, $I., and Jennings, D. B. 1989. Isolation and characterization of iso-rANP, a new natriuretic peptide from rat atria. Biockem. Biophys. Res. Commun. 161: 8369-837. Fumya, M., Takehisa, M . , Minarnitake, Y., Kitajima,Y.,
Hayashi,Y., Ohnuma, N., Hshihara, T., Minamino, N., Kangawa, K., and Matsuo, H. 1990. Novel natriuretic pegtide, CNP, potently stimulates cyclic GMP production in rat cultured vascular smooth muscle cel8s. Biochem. Biophys. Res. Comrnun. 170: 2631 -208. Hamet, P., Tremblay, J., Pang, So C., Shherska, R., Schiffrin, E. L., Garcia, R., Cantin, M., Genest, J., Balmour, R., Ervin, F. R., Martin, S., and Goldwater, R. 1986. Cyclic GMP as mediator and biological marker of atrial natriuretic factor. S . Hypertens. 4(Suppl.): S49 - S56. Hamet, P., Pang, S. C., and Tremblay, S. 1989. Atrial natriuretic factor-induced egression of cyclic guanosine 3':s'-monophosphate in cultured vascular smooth muscle and endothelial cells. J . Biol. Chem. 264: 12 364 - 12 369. Jennings, D. B., and Flynn, T. G. 1989. Cardiovascular and renal effects of iso-rANP, a second natriuretic peptide from rat atria. Can. J. Physiol. Pharmacol. 6 7 11372- 1379. Kojima, M., Minamino, N., Kangawa, K., and Matsuo, H. 1989. Cloning and sequence analysis of cDNA encoding a precursor for rat brain natriuretic peptide. Biwhem. Biophys. Res. Commun. 158: 1420- 1426. Eeitman, D. C., Andresen, J. W., Catalano, R. M., Waldman, S. A . , Tuan, J. J., and Murad, F. 1988. Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cycrase activity in cultured cells. J. Biol. Chern. 263: 3720 -3728. Rapport, R. M.,Winquist, R. S., Baskin, E. P., Faison, E. P., Waldrnan, S. A., and Murad, F. 1986. Effects of atriopeptins on relaxation and cyclic GMP levels in rat and rabbit aortas. Eur. J. Pharrnacol. 120: 123- 126. Roy, R. N., and FIynn, T. G. 1990. Organization of the gene for isorANP, a rat B-type natriuretic peptide. Biochem. Biophys. Res. Commun. 871: 416 -423. Sarda, I. W., de Bold, M. L., and de Bold. A. S. 1889. Optimization of atrial natriuretic factor radioimmamsassay. Clin. Biochem. 22: 11- 15. Steiner, A. L.,Parker, C. W., and Kipnis, D. M. 1972. bdiesimmunoassay for cyclic nucleotides. 61. Adenosine 3 ' ,5 '-monophessphate and guanosine 3',5'-monophosphate in mammalian tissues and body fluids. J. Biol. Chem. 249: 1106-1113. Stewart, D. H., Hayward, L. D., and Bennett, B. M. 1989. Differential bistransfomation of the enantiomers of isoidide dinitrate in isolated rat aorta. Can. J. PhysioB. Pharrnacol. 67: I403 - 1408. Sredoh, T., Kangawa, K., Minarnino, N., and Matsuo, H. 1988. A new natriuretic peptide in porcine brain. Nature (London): 332: 78-81. Waldman, S . A., Rapoport, R. M . , and Maarad, F. 1984. Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues. J. Biol. Chem. 259: 14 332 14 334.
B. M. BENNETT Department of Pharrmcobgy and Toxicology, Queen S University, Kingston, Ont., Canada K7L 3N6
D. B. JENNINGS Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by 67.193.101.86 on 01/07/15 For personal use only.
Department sf Pbaysiology, Queen's University, Kingston, Ont., Cansdu K7E 3 N 6
I. R. SAWDA AND I?.G. F L Y N N ~ Department sf Biochemistry, Queen's University, Kingston, Ont., Canada K7L 31% AND
S. C . PAN^ Department of Anatomy, &rceen9s University, Kings~osa,Ont., Canada K7L 3NQ Received May 27, 1992 WHGLE,D. A., BENNETT,B. M., JENNIWGS, D. B., SARDA, I. R., FLYNN,T. G., and PANG,S. C. 1992. Biological effects of rat iso-atrial natriuretic peptide and brain natriuretic peptide are indistinguishable from each other. Can. J . Physiol. PharmacoI. '70: 1525- 1528. Rat brain natriuretic peptide (rBNP) and iso-atrial natriuretic peptide (iso-rANP) were discovered independently by two research laboratories. They are considered to be members of the B-type natriuretic peptides. Except for the Gln/Leu substitution at position 44, the amino acid sequence of iso-rANP is identical with that of the G-terminal45 amino acids of rat pro-BNP and with the 5-kDa cardiac peptide from rat atria. To determine whether this amino acid substitution can modifgr the known bislogical effects of rBNP and iso-rANP, the present investigation examined the cardiovascular and renal responses, vassrelaxant effect, receptor binding characteristics, and cyclic GMP production by the two peptides in relation to that of rat atrial natriuretic peptide (rANP). Results indicate that rBNP and iso-rANP are indistinguishable from each other in terms of these known biological activities sf atrial natriuretic peptide. We therefore conclude that rBNP and iso-rANP are identical peptides and that the amino acid substitution at position 44 represents a polymorphic form of the rat B-type natriuretic peptide. Key words: atrial natriuretic peptide, brain natriuretic peptide, cardiovascular response, vasorelaxation, cyclic GMP, receptor binding. WICLE,B. A., BENNETT, B. M., JBNNINGS, D. B., SARBA,I. R., PLYNN,T. 6., et PANG,S. C. 1992. Biological effects of rat iso-atrial natriuretic peptide and brain natriuretic peptide are indistinguishable from each other. Can. J. Physiol. Pharmacol. 78 : 1525- 1528. L9iso-peptidenatriurttique auriculaire (iso-PNAr) et le peptide natriuretique cerebral (PNCr) de rat ont Cte dCcouverts dans deux laboratoires de recherche diffkrents. Hls font partie de la famille des peptides natriurktiques de type B. Plutat que d'observer la substitution Gln/Leu en position 44, on constate que la sequence des acides arninCs de I'iso-PNAr est identique B celle des acides amines en position 45 C-terminale du pro-PNC et B celle du peptide cardiaque 5-kDa des esreillettes de rat. Pour determiner si cette substitution d'acides aminks pourrait modifier les effets biologiques connus du PNCr et de l'isoPNAr, la prksente ktude a examin6 les reponses renales et cardiovasculaires, l'effet vasorelaxant, les caraetCristiques de fixation aux recepteurs et la production de GMP cyclique par Bes deux peptides par rapport h ceux du PNAr. Les rksultats indiquent que le PNCr et l'iso-PNAr sont indiffkrenciables du point de vue de ces effets biologiques connus du peptide riatriurktique auriculaire. Ainsi, nous concluons que le PNCr et l'iso-PNAr sont des peptides identiques et que la substitution des acides aminks en position 44 represente une forme polymorphe du peptide natriurktique de type B du rat. Mots cbb : peptide natriurktique auriculaire, peptide natriuretique cCrCbral, rkponse cardiovasculaire, vasorelaxation, GMP cyclique, fixation au rkcepteur. [Traduit par la rkdactinn]
It is now well established that the mammalian heart is an endocrine organ and that atrial natriuretic peptide (ANP) is synthesized, stored, and secreted by atrial rnyocytes (Cantin and Genest 1985). A N P induces a number of cardiovascular and renal responses upon its release into the circulation, including natriuresis , diuresis, and hypotension (de Bold eb al. 198I). ANP exerts its effect, at least in part, by direct activation of receptor-linked particulate guanylate eyclase to produce cyclic G M P (cGMP) (Waldrnan et al. 1984; Hamet et al. 1986). In blood vessels, the increase in cGMP level correlates 'Authors for correspondence. Printed in Canada ! lmprime au Canada
with the vasorelaxant responses induced by A N P ( R a p p o r t et al. 1986). In recent years a number s f natriuretic peptides have been identified in mammalian systems. These new members s f the natriuretic peptide family include brain o r B-type natriuretic peptide (BNP) (Sudoh et a/. 1988) and C-type natriuretic peptide (CNP) (Furuya eb al. 1990). In 1989, our laboratory isslated another atrial natriuretic peptide from rat atria (Flynn eb al. 1989). Because of the sequence homology and similarity in physiological response to ANP, this new atrial peptide was designated iso-rANP. Except for the G l n l k u substitution at position 44, the amino acid sequence of iso-rANP is identical with that of the C-terminal45 m i n o acids of rat pro-BNP
CAN. J. PHYSIOL. PHARMACOL. VOL. 70, 1992
1526
(Kojima et aE. 1989) and with the 5-kDa cardiac peptide from rat atria (Aburaya et al. 1989). To determine whether this amino acid substitution can modify the known biological effects s f rBNP and iss-rANP, the present investigation examined the cardiovascular and renal responses, vasorelaxant effect, receptor binding characteristics, and cGMP production hy the two peptides in relation t o that of rANP.
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by 67.193.101.86 on 01/07/15 For personal use only.
Methods Sources of atrial natrs'umticpeptides Rat ANP(,,-L26jwas obtained from IAF Biochemicals h c . (Laval, Que.). Rat iss-ANP(, was synthesized by solid-phase peptide synthesis using a semiautomatic apparatus (Labortec SP640 peptide synthesizer, Switzerland) and 9-fluorenylmethyloxycarbonyl(FMBC) amino acid chemistry. All peptides were purified to homogeneity by HPLC. Iodination of rANPe,-126) was performed by the chloramine T method as described previously (Sarda et al. 1989). Hodinated peptide was purified by MPLC, lyophilized, and dissolved in 8.01 M acetic acid before use. Rat BNP(,-,,) and porcine BNP were pnrchased from Peninsula Laboratories Hnc. (Belmont, Calif.).
-,,
Rat ba'oassajs Viral antigen-free male Sprague -Dawley rats (body weight 293 k 33 g) were used for bioasays. Experimental protocols were identical with those described previously by our laboratory (Jennings and Flynn 1989). In brief, rats were anesthetized with sodium pentobarbita1 (70 mg/kg; MTC Pharmaceuticals, Hamilton, Ont.) and infused with saline (0.9% NaCl). Catheters were placed in a femoral artery, the right external jugular vein, and the urinary bladder. After a 40-min control period, rats were given a (8.2-mL, bolus injection of peptides or vehicle into the jugular vein. Cardiovascular and urine volume measurements were carried out for 20 min, and then arterial blood was sampled to measure the hematocrit. V'osoreIauatisn assay Aortic strips were prepared from male Sprague - Dawley rats (250 -300 g) as previously described (Stewart er a!. 1989). Experiments were performed in a double-jacketed water bath in a Mrebs solution of the following composition (n~lgM):NaCl, 1 18; KC], 4.74; MgSB,, 1.18; KH,PO,, 1.18; CaC12. 2.5; NaHCO,, 24.9; and glucose, 10. The solution was aerated with 95 % O2 - 5 % C 0 2 and maintained at 37'C. Tissues were contracted submaximally with 0.1 ,uM phenylephrine and cumulative concentration -response curves for rANP, rBNP, and iso-rANP obtained. EC,, values were determined graphically from each concentration-response curve. Cell cu/ferae Rat mesenteric artery smooth muscle sells (VSMC) were derived from adult male Wistar rats (150-200 g) as previously described (Hamet et al. 1989). Cultures were maintained in Dulkcco's modified Eagle's medium (DME) supplemented with 8 mM Hepes and 10% (v/v) calf serum, a d kept in a 95 % air - 5 % C 0 2 humidified atmosphere at 37°C. Cells from passages 15-20 were used for experiments. Binding studies Binding assays were performed in 24-well culture plates, using a method similar to that of Leitman et ad. (1988). Briefly, confluent cell layers were washed twice with a binding buffer (DME) containing 61.05 % bovine serum albumin at 3 7 T . Iodinated ANP was suspended in binding buffer and incubated (about 50 800 cpm/well; specific activity 1200- 1500 mCi/rng (1 Ci = 37 GBq)) with varying concentrations of cold peptide. After incubation for 2 h at 4"C, cells were washed three times with cold binding buffer to remove unbound ligand. Cells were then dissociated with 0.5 m& of I % trypsin at 37°C for 15 rnin and collected. Each well was subsequently washed twice with 0.5 rnL of 1 M NaBH, and the resulting 1.5-mL volume was counted to estimate bound ligand. Nonspecific binding was determined in the presence of 1 pM unlabelled peptide and subtracted from each value.
Tme (rnin)
FIG. 1. Effect of atrial peptides on mean arterial blood pressure (upper panel), heart rate (middle panel), and urine flow (lower panel). Values are percent changes f SEM far mean arterial pressure, heart rate, and urine flow following bolus injection of 0.8 pmsl of peptide (or vehicle, i.e., control) into bioassay rats at 0 time. All three atrial peptides induced hypotension, bradycardia, and diuresis. rBNP, iso-rANB, and rANP were equipotent in their ability to induce cardiovascular and urinary responses (rANP, n = 12; rBNP, n = 6; iso-rANP, ra == 15; vehicle, n = 16).
cGMP radioimrnunsassa~~ To determine extracellular and lntracellular cGMP responses, cells were cultured until confluent in 35-rnm dishes. Cells were washed twice and left in DME for stimulation with the indicated concentrations of peptide. Parallel dishes were retained for cell counting by haemocytometer. After 2 h stimulation at 37"C, culture medium (2 mL) was collected into 1 mL of 30%trichloroacetic acid (TCA) to estimate extracellular egression of cGMP. Intracellular production of cGMB was sampled by scraping the remaining cell layer off the dish with two I-mE volumes of 10% TCA. Samples were ether extracted to remove the TCA and quantitated for cGMP content by radioimmunoassay (Steiner et al. 1972).
Results and diseasssion Although the original amino acid sequence of isa-rANP reported a Gln residue in position 44 (Flynn et a%.1989), subsequent analysis sf the complementary D N A (cDNA) sequence for rBNP has shown a Leu in the penultimate position (Kojima et a!. 1989). Significant controversy has ensued as to whether these are the same peptide. Although a leucine residue was observed at position 44 during the initid purification of iso-rANP from 900 rat atria, the yield of phenylthiohydantoin-Leu was much less than that of phenylthiohydantoin-GBn, and we therefore placed Gln instead of Leu at this position of the sequence (Flynn et al. 1989). Our analy-
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BRIEF REPORTS 1 RAPPORTS BRBFS
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rBNP
A-A
iso-rANP
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FIG.2. Effect of natriuretic peptides on relaxation of isolated rat aortic strips. Tissues were contracted with 0.1 p M phenylephrine and cumulative concentration - response curves obtained for rBNP, rANP, and iso-rANP. The EC,, values for relaxation were 15 f 2, 13 f 4, and 19 & 6 nM for rBNP, rANP, and iso-rANP, respectively. Each value represents the mean -b SB (n = 7).
Control
A-A
iso-rANP
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rBNP
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41
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9
7
8
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FIG. 3. Displacement of "51-labelled rANP from cultured rat mesenteric VSMC with udabelled iso-rANP,, -,5,, rBNP, and rANP(,-,2,,. As evident from this figure, the concentrations of rBNP and iso-rANPf,-,,, required to displace labelled rANP were about fourfold higher than that of rANP(wNo detectable differEach value on ence was present between rBNP and iss-rANP(, the curve represents the mean of two experiments, each performed in duplicate. NSB, nonspecific binding.
,,.
-,,.
%isof the gene sequence for iso-rANP, however, also revealed coding for Leu at position 44 (Roy and Flynm 1990), and there exists a possibility that the Gln/Eeu substitution at this position may represent a polymorphic form of this peptide in rats. To investigate the biological activity o f rBNP and iso-rANP, we have compared the two peptides on the basis of h o w n biochemical, pharmacological, and physioBogical activities for natriaretic peptides. In bioassay experiments in anesthetized intact rats, cardiovascular and urine flow measurements were comparable during the control periods. We chose to administer a Q.8-pmol dose sf peptide since this dose of rANP produced maximal urinary responses Vennings and Flynn 1989). The effects of the three peptides on mean arterial pressure, heart rate, and urine flow of bioassay rats in relation to control (vehicle) are illustrated in Fig. 1. At a dose of 0.8 prnol, eardiovascula~and urine responses for the three peptides were similar; there were no significant differences
8
7
6
FIG.4.Extracellular (upper panel) and intracelluliar (lower panel) accumulation of cGMP in rat n~esentericVSMC in response to porcine BNP (pBNP), rANP rBNP, and iso-rANP,, (Upper panel) The extracellular accumulation of cGMP as induced by atrial peptides (lO-y M) was similar with rBNP and iso-rANP,,-,,,, but was significantly lower than that induced by rANP(,-,,,, and pBNP (one-way ANOVA).Each value on the curve represents: the mean f SD of three experiments, each performed in duplicate. (Lower panel) At 10-' M, the intracellular cGMP accumulation induced by pBNP and rANP,,,was significantly higher than that induced by rBNP Similar levels of intracellular cGMP were induced and iso-rANP(, by rBNP and ~ S O - ~ A N-45). P(~
,,- ,,,
control
9
-,,.
,, -,,.
between responses at any of the time periods. While injection of iso-rANP and rBNP had no effect on hematocrit, injection of rANP significantly lowered hematocrit compared with the other three groups ( p < 0.05, data not shown). This latter finding was consistent with previous results (Jennings and Flynn 1989). In isolated rat aortae, all three peptides elicited csncentratisn-dependent relaxation respnses (Fig. 2). The potency for relaxation by the three peptides was of similar magnitude, with no statistically significant differences in the Ee5()values for relaxation. The maximal relaxation response at the highest dose tested (180 nM) was the same for all three natriuretic peptides . Receptor binding studies using cultured sat mesenteric VSMC indicated that rBNP and ~ S O - ~ A N Pcompete ( ~ - ~ ~ with ) equd affinity for the same receptor population in this cell type. As shown in Fig. 3, rANP displaces labelled rANP with an ICSOvalue sf 2.1 x 1W9 M, while rBNP and iso-rANP do the same at an identical but higher ICso value of 7.9 x 164-%E%%. The BIBo ratios for rANP were significantly differfrom both iso-rANP and rBNP at ent (one-way ANOVA) and M unlabelled peptide, but n o significant difference was detectable between rBNP and iso-rANP. As illustrated in
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CAN. J. PHYSIOL. PWARMACOL. VOL. 70, 1992
Fig. 4, the extracellular and intracellular accumuBation sf cGMP in cultured rat mesenteric VSMC induced by natriuretic peptides showed no significant difference between rBNP and iso-rANP. These data indicate that both rBNP and iso-rANP are capable sf activating the guanylate cyclase linked receptor system at high doses (1V4M), and do so with similar efficacy. However, the concentration s f iss-rANP and rBNP required to elicit a cGMP response was significantly higher than that sf porcine BNP @BNP), a related natriuretic peptide with different amino acid sequence. This would suggest that rBNP and iss-rANP are not likely to be the endogenous Bigands for the guanylate cyclase linked B-receptor subtype prevalent in cultured rat mesenteric VSMC. These results demonstrate that despite the single amino acid residue substitution in rBNP and iso-rANP, the two peptides are virtually identical within the limits sf experimental variation in their ability to induce the characteristic bischemicd, pharmacological, and physiological responses of atrial watriuretic pepaides. The original amino acid sequence s f iso-rANP may represent a polymorphic form of the rat B-type natriuretic peptide, but our results indicate that both iso-rANP and rBNP are in essence the same peptide.
Acknowledgements This project was supported by grants from the Medical Research Council of Canada and the Faculty of Medicine and the Principal's Development Fund, Queen's University. The technical assistance of Maureen St. James and Barbara Lawson and the secretarial work of Dianne Snider were much appreciated. The authors also thank Dr. Seon Shin of the Department of Physiology for the use s f his gamma counter. Aburaya, M., Hino, J., Minamino, N., Kangawa, K., and Matsuo, H. 1989. Isolation and identification of rat brain natriuretie peptides in cardiac atrium. Biochem. Biophys. Res. Cornmun. 163: 226-232. Cantin, M., and Genest, J. 1985. The heart and the atrial natriuretic factor. Endocr. Rev. 6 : 107- 127. de Bold, A. J., Borenstein, H. B., Veress, A. T., and Sonnenberg, PI. 1981. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rdts. Life Sci. 28: 89-94. FBynn, T. G., Brar, A., Trernblay, L., Sarda, I., Lyons, $I., and Jennings, D. B. 1989. Isolation and characterization of iso-rANP, a new natriuretic peptide from rat atria. Biockem. Biophys. Res. Commun. 161: 8369-837. Fumya, M., Takehisa, M . , Minarnitake, Y., Kitajima,Y.,
Hayashi,Y., Ohnuma, N., Hshihara, T., Minamino, N., Kangawa, K., and Matsuo, H. 1990. Novel natriuretic pegtide, CNP, potently stimulates cyclic GMP production in rat cultured vascular smooth muscle cel8s. Biochem. Biophys. Res. Comrnun. 170: 2631 -208. Hamet, P., Tremblay, J., Pang, So C., Shherska, R., Schiffrin, E. L., Garcia, R., Cantin, M., Genest, J., Balmour, R., Ervin, F. R., Martin, S., and Goldwater, R. 1986. Cyclic GMP as mediator and biological marker of atrial natriuretic factor. S . Hypertens. 4(Suppl.): S49 - S56. Hamet, P., Pang, S. C., and Tremblay, S. 1989. Atrial natriuretic factor-induced egression of cyclic guanosine 3':s'-monophosphate in cultured vascular smooth muscle and endothelial cells. J . Biol. Chem. 264: 12 364 - 12 369. Jennings, D. B., and Flynn, T. G. 1989. Cardiovascular and renal effects of iso-rANP, a second natriuretic peptide from rat atria. Can. J. Physiol. Pharmacol. 6 7 11372- 1379. Kojima, M., Minamino, N., Kangawa, K., and Matsuo, H. 1989. Cloning and sequence analysis of cDNA encoding a precursor for rat brain natriuretic peptide. Biwhem. Biophys. Res. Commun. 158: 1420- 1426. Eeitman, D. C., Andresen, J. W., Catalano, R. M., Waldman, S. A . , Tuan, J. J., and Murad, F. 1988. Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cycrase activity in cultured cells. J. Biol. Chern. 263: 3720 -3728. Rapport, R. M.,Winquist, R. S., Baskin, E. P., Faison, E. P., Waldrnan, S. A., and Murad, F. 1986. Effects of atriopeptins on relaxation and cyclic GMP levels in rat and rabbit aortas. Eur. J. Pharrnacol. 120: 123- 126. Roy, R. N., and FIynn, T. G. 1990. Organization of the gene for isorANP, a rat B-type natriuretic peptide. Biochem. Biophys. Res. Commun. 871: 416 -423. Sarda, I. W., de Bold, M. L., and de Bold. A. S. 1889. Optimization of atrial natriuretic factor radioimmamsassay. Clin. Biochem. 22: 11- 15. Steiner, A. L.,Parker, C. W., and Kipnis, D. M. 1972. bdiesimmunoassay for cyclic nucleotides. 61. Adenosine 3 ' ,5 '-monophessphate and guanosine 3',5'-monophosphate in mammalian tissues and body fluids. J. Biol. Chem. 249: 1106-1113. Stewart, D. H., Hayward, L. D., and Bennett, B. M. 1989. Differential bistransfomation of the enantiomers of isoidide dinitrate in isolated rat aorta. Can. J. PhysioB. Pharrnacol. 67: I403 - 1408. Sredoh, T., Kangawa, K., Minarnino, N., and Matsuo, H. 1988. A new natriuretic peptide in porcine brain. Nature (London): 332: 78-81. Waldman, S . A., Rapoport, R. M . , and Maarad, F. 1984. Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues. J. Biol. Chem. 259: 14 332 14 334.
