Peptides, Vol. 17, No. 4, pp. 635-639, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/96 $15.00 + .OO ELSEVIER zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA PI1 SO196-9781(96)00067-S Vasorela.xation by Casomokinin L, a Derivative of ,O-Casobmorphin and Casoxin D, is Mediated by NK1 Receptor HIROYUKI FUJITA, * HIROYUKI RYUSUKE NAKAGIRI,? SUGANUMA, t’ HACHIRO USUI, $ KAZUYOSHI KURAHASHI, 3 RYUZO SASAKIT AND MASAAKI YOSHIKAWAt** zyxwvutsrqponmlkjihgfedcbaZYXW *Nippon Synthetic Chemical Industry Co., Ltd., Ibaraki-shi, Osaka 567, Japan ?Department of Food Science and Technology, Faculty of Agriculture and $Pharmacology Division, Radioisotope Research Center, Kyoto University, Sakyo-ku, Kyoto 60601, Japan Received 7 September 1995 R. SASAKI AND M. YOSHIKAWA. zyxwvutsrqponmlkjihgfedcbaZYX Vasorelaxarion zyxwvutsrqponmlk PEPIIDES 17(4) 635-639, 1996.-Casomokinin L (Tyr-Pro-Phe-Pro-Pro-Leu), a derivative of P-casomorphin and casoxin D, showed endothelium-dependent vasorelaxing a.ctivity on the canine mesenteric artery (EC& = 7 X lo-* M). This relaxing activity was partly blocked by lo-’ M NAME (nitric oxide synthase inhibitor), and the inhibition by NAME was restored by addition of 10m4 M arginine, suggesting the involvement of nitric oxide as an endothelium-dependent relaxing factor. The relaxing activity was blocked by lo-* M CP-99994 and 1O-7 M FK888 (NK, antagonists), but not by 1O-6 M SR-48968 (NK2 antagonist). Casomokinin L binds to neurokinin NK, receptors (K, = 5.8 X 10-s M ), and lowered blood pressure in SHR (0.1 mglkg, IV and 3 mg/kg, PO). Thus, despite its only three residues in common with substance P, casomokinin L binds to NK, receptors, relaxes the artery, and exerts an antihypertensive effect. FUIITA, H., H. SUGANUMA, H. USUI, K. KURAHASHI, by casomokinin L, a derivative of P-casomorphin Casomokinin L Mesenteric artery Neurokinin NKI receptor Vasorelaxing peptide R. NAKAGIRI, and casoxin D, is mediated by NK, receptor. Nitric oxide Blood pressure Endothelium-dependent relaxing Substance P Casoxin D peptide casomokinin laxation mechanism nin L. MANY kinds of biologically active peptides are released from food proteins by enzymatic digestion ( l,lO, 14). Casoxin D (TyrVal-Pro-Phe-Pro-Pro-Phe I), which was isolated as an antiopioid peptide from pepsin-chymotrypsin digest of human casein, shows vasorelaxing activity that i.s mediated by bradykinin Br receptors (24,25). [ des-Val’] Casoxin D shows opioid agonist activity and vasorelaxing activity, which is also mediated by B, receptors (24) ; we named this pept ide casomokinin F. It is known that [de+Arg9]bradykinin, an endogenous Br agonist peptide ( 18), is converted to an antagonist by substitution of Phe at the carboxy-terminus to Leu ([des-Arg’][ Leu*] bradykinin) ( 17). We substituted Phe at the carboxy-terminus of casomokinin F to Leu, with the aim of obtaining an opioid agonist/B1 antagonist peptide that might have potent analgesic activity. The peptide obtained, Tyr-Pro-Phe-Pro-Pro-Leu, showed opioid activity in the guinea pig ileum assay system with an I& value of 10 ,uM (Z!4). However, this peptide was not an antagonist for Br receptors. Furthermore, this peptide showed potent vasorelaxing activity that was not antagonized by [desArg9] [Leu8]bradykinin, a B1 antagonist (24). We named this factor Opioid P-Casomorphin L. In this study, we examined the vasoreand antihypertensive effect of casomoki- METHOD Chemicals and Reagents Aspirin, indomethacin, nitroarginine methyl ester (NAME), L-arginine, prostaglandin F*u, chymostatin, leupeptin, bacitratin, BSA (radioimmunoassay grade), and polyethyleneimine were obtained from Sigma Chemical Co. Acetylcholine was obtained from Daiichi Seiyaku Co., Ltd. Papaverine hydrochloride was obtained from Dainihon Seiyaku Co., Ltd. Substance P and [des-Arg’] [ Leu’] bradykinin ( 17) were obtained from The Peptide Institute, Inc. ( + ) - (2 S,3 S) -3- (2-methoxybenzyl)amino-2-phenylpiperidine (CP-99994) was a gift from Pfizer Inc. N*- [ (4 R) -4-hydroxy- 1- ( 1-methyl-lH-indol3-yl)carbonyl-~-prolyl]-N-methyl-N-phenylmethyl-3-(2-naphthyl)-L-alanineamide (FK888) was a gift from Fujisawa Pharmaceutical Co., Ltd. (S) -N-methyl-N[ 4- (4-acetylamino- 1 Present address: Kagome Co., Ltd. Nishinasuno-cho, Nasu-gun, Tochigi 329-27, Japan. * Requests for reprints should be addressed to Masaaki Yoshikawa, Department of Food Science University, Sakyo-ku, Kyoto 606-01, Japan. 635 and Technology, Faculty of Agriculture, Kyoto FUJITA 636 in the presence of inhibitors or antagonists applied prior to prostaglandin zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH FzIr To determine the complete relaxation point, 1 @f papaverine was added at the end of the examination. Binding assay. Neurokinin NK, receptor assay was performed (A) Intact Casomokinin L (nM) 10s M Prostaglandin F,, 1 min (B) Endotheliumremoved Casomokinin L (nM) : A l@ M Prostaglandin FPa ET AL. H 1 min FIG. 1. Effect of removing endothelium upon vasorelaxation. Cumulative addition of casomokinin L was performed at the times indicated by closed circles. Relaxation of the intact (A) or the endothelium-removed canine mesenteric artery (B) by casomokinin L. Papav.: 10m4 M papaverine. 4-phenyl piperidino) -2- (3,4-dichlorophenyl) butyl] benzamide (SR-48968) was a gift from Sanofi Recherche. D-Arg[ Hyp3, Thi’,D-Tic’,Oic’] bradykinin (Hoe 140) ( 11) was a gift from Hoechist Company. [ 3H] [ Sar’-Met( 0,) “1 Substance P (38.3 Ci/mmol) was obtained from Du Pont NEN Research Products. Experimental Procedures Amino acid sequence analysis andpeptide sy nthesis. The peptide was synthesized by the t-Boc method with a Sam Two peptide synthesizer (Biosearch Inc.). The peptide was deprotected using the anisolelhydrogen fluoride method and purified by reverse-phase HPLC on an ODS column. The amino acid sequence of the purified peptide was analyzed using a 477A protein sequencer (Applied Biosystems Inc.). Vasorelaxation assay. Beagle dogs of either sex weighing 812 kg were exsanguinated from the common carotid artery under sodium pentobarbital anesthesia (25 mg/kg, IV). The mesenteric arteries were then removed and cut into ring strips. The endothelium was removed mechanically by gently rubbing the intimal surface. The absence of functional endothelium was confirmed by the inability of acetylcholine to induce relaxation. Intact and endothelium-denuded arteries were mounted between two stainless steel hooks in an organ bath containing Krebs-Henseleit solution ( 120 mM NaCl, 4.7 mM KCl, 1.2 mM MgS04, 1.2 mM KH2P04, 2.5 mM NaHC03, and 10 mM glucose) at 37°C and bubbled with a mixture of 95% 02/.5% C02. The end of the hook was attached to the lever of a force-displacement transducer (T78-240, Orientec Co.), which was connected to a Recti-Horiz-8K recorder (NEC San-ei Instrument Co. Ltd.,) on which the isometric tension changes were recorded. Applied tension was adjusted to 1.5 g. The artery was then constricted by prostaglandin Fz, at 1O-6 M. The relaxing activity of samples was assayed by measuring the binding of [ 3H] [ Sar’-Met( 02) “1 substance P (5) to rat submaxillary gland membranes (12). Submaxillary glands from adult male Wister rats were homogenized in 10 volumes (w/v) of ice-cold buffer A (50 mM Tris-HCl, pH 7.4, containing 120 mM NaCl and 5 mM KCl) with a Brinkmann Polytron PT-10 at a setting of 7 for lo- 15 s. The homogenate was incubated on ice in the presence of 300 mM KC1 and 10 mM EDTA for 30 min with intermittent mixing and then centrifuged at 50,000 x g for 10 min. The pellet was resuspended in 20 volumes of ice-cold buffer B (50 mM Tris-HCl) by a Polytron at a setting of 7 for 5- 10 s and centrifuged again. This washing procedure was repeated twice, and the final pellet was resuspended in 60 volumes of buffer B. For binding experiments, the reaction was initiated by adding 400 ~1 of freshly prepared membrane preparation in triplicate to 0.2-10 nM [3H] [Sar’Met( 0,) ‘I] substance P at 20°C for 90 min in a final volume of 500 ~1 containing 50 mM Tris-HCl (pH 7.4)) 0.02% BSA, 1 pg of chymostatin, 2 pg of leupeptin, 20 pg of bacitracin, and various test drugs. At the end of the incubation period, 5 ml of icecold buffer A was added to each tube and contents were filtered immediately under reduced pressure through Whatman GF/F glass-fiber filters (pretreated with 0.1% polyethyleneimine in water for 3 h prior to use). Each of the filters was then washed twice with 5 ml of ice-cold buffer A and radioactivity was determined by liquid scintillation spectrometry using a Tri-carb scintillation spectrometer D3225 (Hewlett-Packard). Nonspecific binding was defined as binding in the presence of 1 ph4 substance P. Specific binding was calculated by subtracting the nonspecific from total binding. M easurement of blood pressure. Male spontaneously hypertensive rats (SHR) weighing 260-290 g were used. To estimate antihypertensive activity of the peptide after IV administration, SHRs were anesthetized with a-D-chloralose at a dose of 45 mg/ kg (IV). A catheter for drug injection was inserted into the femoral vein. The carotid arterial blood pressure and heart rate were measured with a pressure transducer (Spectramed Medical Products Pte., Ltd.). For estimation of antihypertensive activity of the 10.8 1o-7 Casomokinin L (M) FIG. 2. Effect of nitric oxide synthase 1o-s 10-s inhibitor on vasorelaxation. Concentration-response curve for casomokinin L-induced relaxation of canine mesenteric artery in the absence of the inhibitor (O), and in the presence of 10m5 M NAME (A)or 10e5 M NAME and 10e4 M arginine (0). Casomokinin L was added 30 min after the inhibitors. Each value is expressed as the mean k SE of four experiments. Significant differences from control: *p < 0.05, **p < 0.01. CASOMOKININ L IS AN NK, AGONIST 637 zyxwvutsrqp PEPTIDE c c CP99994 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA SR 48968 10-7 Casomokinin L (M) 10-6 10-e 10-7 10-6 1 5 Casomokinin L(M) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA FIG. 3. Effects of neurokinin antagonists on vasorelaxation. Neurokinin NK, antagonists (CP-99994, FK888) and NK2 antagonist (SR 48968) were added 30 min before casomokinin L. Concentrations of antagonists are shown in the figure. Values are expressed as means -C SE of four experiments. Significant differences from control: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON *p < 0.05, **p < 0.01. peptide after oral administration, by the tail cuff method using blood pressure was measured a UR-5000 (Ueda Seisakusho). SHR were used in groups of five animals each. The peptide was dissolved in saline, then emulsified with egg yolk. Test compounds were administered orally in SHR using a metal zonde in a volume of 0.5 ml. Data Analyses All results are expressed as means and SE. The differences between the presence and absence of inhibitors or antagonists were analyzed statistically using Student’s t-test for paired observations (p < 0.05 and 0.01). RESULTS Relaxation Mechanism of Casomokinin activity. Thus, the relaxation caused by casomokinin L was endothelium dependent [Fig. zyxwvutsrqponmlkjihgfedcbaZYXWVUTS 1( B zyxwvutsrqponmlkjihgfedcbaZYXW )] . Effects of NO synthase and cyclooxygenase inhibitors. It is known that vasorelaxation caused by endogenous peptides is mediated by endothelium-dependent relaxing factors, such as prostaglandin IZ (22), or nitric oxide (NO) ( 15) released from endothelium. The vasorelaxation was partly blocked by the NO synthase inhibitor NAME at a 10m5 M. Moreover, inhibition by NAME was restored by addition of 10 m4M arginine (Fig. 2). On the other hand, the relaxation caused by casomokinin L was not blocked by 5 X 10m5 M aspirin or 3 X lo-’ M indomethacin, both of which are cyclooxygenase inhibitors (data not shown). These results suggest that the relaxation caused by casomokinin L is mediated at least partly by endothelium-dependent relaxing factor, NO. L Endothelium dependence. As shown in Fig. 1, casomokinin L showed vasorelaxing activity in canine mesenteric artery that had been preconstricted b:y prostaglandin Fti. Cumulative addition of casomokinin L showed dose-dependent vasorelaxation with an ECso value of 70 nM [Fig. 1 (A)]. To determine the mechanism of vasorelaxation of casomokinin L, we examined whether the relaxation was endothelium dependent. In endothelium-denuded arteries, casomokinin L showed no vasorelaxing Determination of the Receptor Neurokinins are vasodilators of vascular beds and relaxants of many isolated arteries. Vasorelaxation by neurokinins is mediated by NK, or NK, receptors in the arteries ( 19). The effects of NKI and NK2 antagonists on the vasorelaxation elicited by casomokinin L were examined. As shown in Fig. 3, the relaxation was blocked by NK, antagonists, lo-‘M CP-99994 (3) and lo-’ M FK888 (7 ). On the other hand, the relaxation was not blocked by NK, antagonist, 10 m6M SR-48968 (6). These results suggest that the vasorelaxing activity of casomokinin L is mediated by NK, receptors. Substance P showed vasorelaxing activity in the 80 Saline 6 70 E g60 ; I Casomokinin L (0.1mcv%) 50 E40 i3 $ 30 p 20 10 . ...# 0 10-5 10-4 Casomokinin 10-3 J 10.2 L(M) 4. Displacement of [3H][Sar9~Met(Oz)“]substance P by casomokinin L. Values are expressed as means 2 SE percentage inhibition of [3H][Sar9,Met(OZ)“]substance P binding of three experiments (0). FIG. FIG. 5. Antihypertensive activity of casomokinin L after intravenous injection in SHR. Casomokinin L was administered at a dose of 0.1 mgl kg. Injection of saline or casomokinin L is indicated by arrow. FUJITA 638 ET AL. zyxwvutsrq 10 2 5- (A) Eo z? $ -5 I f m IT -10 iv $ -15 g -20: ii : C% -25a -30 " 9 0 8 ” 2 ” 4 8' a 6 ” Time after Administration (hr) aot,6,""""'.""""""' 2 4 6 6 10 12 Time after Administration (hr) FIG. 6. Antihypertensive activity of casomokinin L after oral administration in SHR. SHRs with systolic blood pressure of about 220-250 mmHg were used. Casomokinin L was administered as an emulsion in 30% egg yolk at doses of 3 mglkg (A) or 10 mg/ kg (B). Control groups (0) received the same amounts of egg yolk as experimental groups (0). Changes in systolic blood pressure from zero time are expressed as means s SE(n = 5). - - ( EC50 = 3.4 X 10e6 M) mediated by bradykinin B, receptors canine mesenteric artery with an EC5,, value of 5.0 X lOmy M (23,25). The vasorelaxation was blocked by aspirin and indo(data not shown), whereas that of casomokinin L was 7.0 X methacin, cyclooxygenase inhibitors, and mediated by prostalo-EM. We further tested the effects of casomokinin L on the binding glandin I?. Casomokinin F, a derivative peptide of casoxin D, of 1.2 x 10e9 M [3H] [Sar’-Met(02)“]substance P, a selective also showed vasorelaxation mediated by bradykinin B, receptors ligand for NKI receptors, to submaxillary glands of Wister rats. (24 ) ( ECsO = 8.6 X 10 -7 M) . On the other hand, the vasorelaxAs shown in Fig. 4, the inhibition of specific binding to the NKIing activity of casomokinin L is mediated by neither bradykinin selective radioligand by casomokinin L occurred with a zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA K, value B, nor B2 receptors but by NK, receptors. The endothelium-deof 5.8 X 10m5 h4. The Ki value of substance P was 8.6 X lo-’ pendent relaxation of the artery was partly blocked by an inhibM under the same conditions. itor for NO synthase, but not by inhibitors for cyclooxygenase. Substance P binds to NK, receptors, stimulating the release of nitric oxide from the endothelium and relaxing artery smooth Antihypertensive Effect of Casomokinin L muscle (4). We have shown that casomokinin L evokes vasoThe endogenous NK1 receptor-selective peptide substance P relaxation by stimulating NKI receptors and nitric oxide release. has antihypertensive activity (2,16). Thus, we examined the efCasomokinin L showed vasorelaxing activity in the canine fects of casomokinin L after IV administration in SHR. As shown mesenteric artery with an E& value of 7.0 x 10m8 M, whereas in Fig. 5, casomokinin L administered IV to SHR lowered blood that of substance P was 5.0 X 10m9 M. Casomokinin L showed pressure by 25 mmHg at a dose of 0.1 mg/kg. similar activity in the porcine coronary artery (data not shown). We further tested the effects of casomokinin L after oral adMoreover, casomokinin L was not bound to bradykinin B, or B2 ministration in SHR. Antihypertensive activities of relatively receptors (data not shown). large angiotensin-converting enzyme inhibitory peptides after The K, value of casomokinin L in the NK, binding assay was oral administration were reported previously to be potentiated by 5.8 X lo-’ M whereas that of substance P was 8.6 X 10m8 M. emulsification in egg yolk (21) . We also found that the antihyThe E& value of the former in the vasorelaxation assay was 7.0 pertensive activity of ovokinin, a bradykinin B, agonist octax 10m8 M whereas that of the latter was 5. 0 X 10m9 M. The peptide derived from ovalbumin (8)) was potentiated by emulreason for the large discrepancy between the Ki value in the bindsification in egg yolk (9). Therefore, we examined the ing assay and the EC5o value in the relaxation assay for casoantihypertensive activity of orally administered casomokinin L mokinin L is not clear. Casomokinin L might be resistant to pepafter emulsification in 30% egg yolk. As shown in Fig. 6, casotidases because it is rich in Pro residues. This might contribute mokinin L dose-dependently lowered systolic blood pressure afto its potent vasorelaxing activity. ter administration in SHR; at a dose of 3 mg/kg, blood pressure It is generally agreed that there are three types of neurokinin was lowered after 2 h by 11 mmHg [Fig. 6(A)]. Furthermore, receptors, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI NK, , NK2, and NK3, whose preferred endogenous at a dose of 10 mg/kg, blood pressure was significantly lowered substrates are substance P, neurokinin A, and neurokinin B, re4 h after administration, and this activity was observed even after spectively. These peptides have the characteristic arrangement of 8 h [Fig. 6(B)]. These results suggest that casomokinin L, a their C-termini Phe-X-Gly-Leu-Met-NH,. However, casomokivasorelaxing peptide, has potent antihypertensive activity. nin L, of which the C-terminal arrangement is Pro-Pro-Leu, shows vasorelaxing activity mediated by NK, receptors, because DISCUSSION the vasorelaxation elicited by casomokinin L was blocked only by NK, antagonists. Casomokinin L, a derivative of casoxin D, showed potent Figure 7 shows primary structures of casoxin D, casomovasorelaxing activity for canine mesenteric artery ( ECso = 7.0 X kinin F ([des-Val’] casoxin D), casomokinin L, and [des10m8 M). Casoxin D, the original peptide from which casomoArg’] bradykinin. Casomokinin F is a derivative of casoxin D. kinin F and casomokinin L are derived, showed vasorelaxation CASOMOKININ L IS AN NKi AGONIST 639 zyxwvutsrqp PEPTIDE mokinin L will be useful for studying the mode of binding to neurokinin receptors. Substance P lowers blood pressure in various species (2,16), and therefore we examined whether casomokinin L also shows Casomokinin F : Bl this effect. We used SHR in this study, as it is a good animal Casoxin D Receptor Ides-Argg]BK : zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA model of essential hypertension. It was reported that substance P lowered blood pressure after IV injection at a dose of 1.25-2.5 FIG. 7. Homology among substance P, casomokinin L, casomokinin F, ,ug/kg in SHR ( 16). Casomokinin L showed antihypertensive casoxin D, and [des-Arg’lbradykinin. Common residues are enclosed. activity after IV administration in SHR at a dose of 100 pg/kg. Casoxin D is a bradykinin B1 agonist peptide derived from a pepsin/ Moreover, Casomokinin L lowered systolic blood pressure after chymotrypsin digest of human cu,,-casein (23,25). oral administration in SHR. Casomokinin L bound to NK, receptors, stimulating the release of nitric oxide and exerting a potent antihypertensive effect. The vasorelaxing activities of casoxin D and casomokinin F Substance P also act as a neuro- and immunomodulator (20). Casomokinins F and L also can be regarded as derivatives of pare mediated by bradykinin B, receptors. It should be noted that the Bi agonist casomokinin F is converted to the NK, casomorphin (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) , which is an opioid agonist casomokinin L by substitution of Phe at the carboxypeptide derived from p-casein. In fact, casomokinins F and L terminus to Leu. This figure also shows primary structure of have opioid activity (24). Sizheng et al. reported that Tyr-D-Alasubstance P. It should also be noted that casomokinin L has Gly-Phe-Phe-Gly-Met-Leu-NH,, a hybrid between opioid peponly three homologous residues to substance P, even if gaps tide and substance P, showed different neuromodulatory propare inserted in its primary structure to fit maximally. Casoerties from substance P ( 13). Substance P Casomokinin L : : REFERENCES 1. Brantl, V.; Teschemacher, H.; Henschen, A.; Lottspeich, F. Novel opioid peptides derived from casein (P-casomorphins) I. Isolation from bovine casein peptone. Hoppe Seylers Z. Physiol. Chem. 360:1211-1216; 1979. 2. Constantine, .I. 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