Agents and Actions, vol. 29, 3/4 (1990)
0065-4299/90/040172-09 $1.50+0.20/0 9 1990 Birkh/iuser Verlag, Basel
Degradation pathway of kinins in tumor ascites and inhibition by kininase inhibitors: Analysis by HPLC Y. Matsumura, H. Maeda 1 and H. Kato 2 Department of Microbiology, Kumamoto University Medical School, Kumamoto 860, and 2 National Cardiovascular Center Research institute, Suita, Osaka 565, Japan
Abstract
We have recently found presence of a high concentration of a novel type of kinin, hydroxyprolyl 3 bradykinin (Hyp3-BK) in human tumor ascites in addition to conventional bradykinin (BK). Because of their potential physiological activity, it is of interest to know how these bradykinins can be degraded in ascites. Degradation of two synthetic kinins, BK and Hyp3-BK, added to the ascitic fluid from patients with ovarian carcinoma and hepatoma, were analyzed by reversed phase HPLC. Both kinins were degraded into their desArg9-BK or -Hyp3-BK and desPheS-Arg-9-BK or -Hyp3-BK products following incubation with the ascitic fluid. The rate of the degradation of BK and Hyp3-BK was the same. The formation of desArg9-BK was completely inhibited by kininase I inhibitor, while the formation of desPheS-Arg9-BK was not completely inhibited by a kininase II inhibitor. The degradation of both kinins was inhibited completely by EDTA. The results indicate the presence of other metalloprotease(s) which cleaves kinins in the ascitic fluid, in addition to kininase I and kininase IX. The carboxypeptidase A and carboxypeptidase B inhibitor, benzyl malic acid, failed to block degradation of both kinins. A rapid cleave of Phe-Arg into Phe and Arg was also found in the ascitic fluid. Thus, the major degradation products of kinins in the ascitic fluid were demonstrated to be either desArgg-BK or Hyp3-BK, desPhe 8Arg9-BK or -Hyp3-BK, phenylalanine and arginine. Lysyl-BK and lysylhydroxyprolyl 3-BK were rapidly converted into BK and hydroxyprolyl 3-BK by the ascitic fluid.
Introduction
It has been well documented recently that extravascular permeability in tumor tissue is enhanced [1-4] and it is sustained by at least two factors, one being protein in nature [5] and the other being kinin [6]. However, the mechanism of the tumor vascular permeability is still not clear. We have demonstrated the presence of two kinins, Abbreviations: BK, Bradykinin; Hyp3-BK, Hydroxyprolyl 3-
bradykinin; CPNI, carboxypeptidase N inhibitor; APMSF, paraamidinophenylmethylsulfonyl fluoride. i Author for correspondence.
bradykinin (BK) and hydroxyprolyl 3-bradykinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg), (Hyp 3BK), in human tumor ascites and concluded that they are generated from high molecular weight kininogen [7, 8]. We and other workers have also identified Hyp3-BK from normal human plasma or urine [9-12]. Because of the large capacity of generation of kinins and the potent activity of these peptides, they may be important in various pathological conditions. With regard to the extravascular permeability in the tumor tissue, the concentration and degradation of kinins in the ascitic fluid from cancer patients need to be eluci-
Agents and Actions, vol. 29, 3/4 (1990) dated. During a recent study, we found the content of Hyp3-BK to be much higher than that of BK [7, 8]. The" question arose as to which of these kinins was most potent and long lasting in vivo, while their activity in vitro was found to be about the same. It has been established that kinins are rapidly inactivated by kininase I and kininase II (angiotensin converting enzyme) in plasma. The mechanism of the degradation of kinins has been examined in detail by using each enzyme [13-15]. However, it seems that the degradation pathway of kinins in plasma or in extravascular fluid is a complex phenomenon because the degradation is caused by two kininases and other peptidases and proteases in the fluids. Recently, Majima et al. developed an enzyme immunoassay for the degradation product of BK and studied the degradation of BK in the inflammatory exudate [16]. In this study, the degradation pathway of both BK and Hyp 3BK in the ascitic fluids was analyzed using reversed phase HPLC in the presence or absence of various kininases and peptidase inhibitors. Materials and methods Chemicals: Captopril, D-2-methyl-3-mercaptopropionyl-L-proline (kininase II inhibitor), was a gift from Sankyo Co., Tokyo [17]. CPNI, carboxypeptidase N inhibitor, DL-2-mercaptomethyl3-guanidino-ethylthio-propanoic acid (kininase I inhibitor), was obtained from Calbiochem-Behring, San Diego, CA, USA [18]. APMSF, p-amidinophenylmethylsulfonyl fluoride was from Wako Pure Chemical Industry Co., Ltd. Osaka. BK, Lys-BK, Hyp3-BK, Lys-Hyp3-BK and desArg9-BK were from Peptide Institute Inc., Minoh, Osaka. Des-Phe8-Arg9-BK and a pentapeptide, Arg-Pro-Pro-Gly-Phe, were generous gifts from Prof. M. Katori, Kitasato University School of Medicine. Phe-Arg was from Bachem Feinchemikalien AG, Bubendorf, Switzerland. (S)-c~Benzylmalic acid [19] was generous gift from Dr. T. Aoyagi, Institute for Microbial Chemistry, Tokyo. Ser-Pro-Phe was synthesized from Bocamino acids by an Applied Biosystems Peptide Synthesizer (Model 430A). Its amino acid sequence was confirmed by amino acid analysis and sequence analysis. All other reagents were from local commercial sources. Human ascitic exudates were obtained from two patients with carcinomatosis of ovarian cancer and a patient with hep-
173 atoma and centrifuged to remove precipitates and residues, and their supernatants were used. Their protein concentrations were 45 mg per ml, 66 mg per ml and 20 mg per ml which were determined by Lowry's method. Amino acid composition of peptides was determined by a Pico-Tag HPLC System (Waters Associates, Bedford, MA, USA), as reported previously [8]. The amino acid sequence was determined by an Applied Biosystems GasPhase Protein Sequencer (Model 470A) equipped with an on-line Model 120A phenylisothiohydantoin (PTH) analyzer, as reported previously [8]. Degradation of Synthetic kinins in Tumor Ascitic Fluid: Each supernatant of tumor ascites was diluted three fold with the following incubation buffer and then a 10 gl aliquot and 100gl of 0.02 M Tris-HC1 buffer, pH 7.4, containing 0.15 M NaC1 were mixed with 10 lal of I m M synthetic kinin solution in distilled water. In order to examine the effect of kininase in the ascites the following reaction mixture was also tested. A 10 gl aliquot of 3 fold diluted ascitic fluid was mixed with 10 gl of following specific inhibitors in 90 ~tl of the same incubation buffer: 1 m M CPNI to inhibit kininase I, 1 m M captopril to inhibit kininase II, 2 mg/ml (S)~-benzylmalic acid to inhibit carboxypeptidase A and carboxypeptidase B and 40 m M EDTA to inhibit metalloenzymes. After 5 rain preincubation, i0 t~1 of 1 m M synthetic BK solution was added into the incubation mixture. The mixture was incubated for the various periods of time at 37~ and 100 ~1 aliquot removed and subjected to reversed-phase HPLC on column (4.6 m m x 25 cm) of ODS-120A from Toyo Soda Manufacturing Co., Ltd., Tokyo, with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of I ml per rain. Peptide peaks were detected at 210 nm and the peak area was calculated using an integrator, Chromatocorder 11, System Instruments, Tokyo and converted to the amounts of the peptide by the standard curve of synthetic peptide. Results and discussion
Figure 1 shows the elution patterns on reversed phase HPLC of the 6 h digests of synthetic BK and Hyp3-BK with three fold diluted ascitic fluid from a patient with ovarian carcinoma. Three main peaks (peaks I - t h r o u g h III) were found in addition to undigested BK or Hyp3-BK in each digest.
174
Agents and Actions,vol. 29, 3/4 (1990) |
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0065-4299/90/040172-09 $1.50+0.20/0 9 1990 Birkh/iuser Verlag, Basel
Degradation pathway of kinins in tumor ascites and inhibition by kininase inhibitors: Analysis by HPLC Y. Matsumura, H. Maeda 1 and H. Kato 2 Department of Microbiology, Kumamoto University Medical School, Kumamoto 860, and 2 National Cardiovascular Center Research institute, Suita, Osaka 565, Japan
Abstract
We have recently found presence of a high concentration of a novel type of kinin, hydroxyprolyl 3 bradykinin (Hyp3-BK) in human tumor ascites in addition to conventional bradykinin (BK). Because of their potential physiological activity, it is of interest to know how these bradykinins can be degraded in ascites. Degradation of two synthetic kinins, BK and Hyp3-BK, added to the ascitic fluid from patients with ovarian carcinoma and hepatoma, were analyzed by reversed phase HPLC. Both kinins were degraded into their desArg9-BK or -Hyp3-BK and desPheS-Arg-9-BK or -Hyp3-BK products following incubation with the ascitic fluid. The rate of the degradation of BK and Hyp3-BK was the same. The formation of desArg9-BK was completely inhibited by kininase I inhibitor, while the formation of desPheS-Arg9-BK was not completely inhibited by a kininase II inhibitor. The degradation of both kinins was inhibited completely by EDTA. The results indicate the presence of other metalloprotease(s) which cleaves kinins in the ascitic fluid, in addition to kininase I and kininase IX. The carboxypeptidase A and carboxypeptidase B inhibitor, benzyl malic acid, failed to block degradation of both kinins. A rapid cleave of Phe-Arg into Phe and Arg was also found in the ascitic fluid. Thus, the major degradation products of kinins in the ascitic fluid were demonstrated to be either desArgg-BK or Hyp3-BK, desPhe 8Arg9-BK or -Hyp3-BK, phenylalanine and arginine. Lysyl-BK and lysylhydroxyprolyl 3-BK were rapidly converted into BK and hydroxyprolyl 3-BK by the ascitic fluid.
Introduction
It has been well documented recently that extravascular permeability in tumor tissue is enhanced [1-4] and it is sustained by at least two factors, one being protein in nature [5] and the other being kinin [6]. However, the mechanism of the tumor vascular permeability is still not clear. We have demonstrated the presence of two kinins, Abbreviations: BK, Bradykinin; Hyp3-BK, Hydroxyprolyl 3-
bradykinin; CPNI, carboxypeptidase N inhibitor; APMSF, paraamidinophenylmethylsulfonyl fluoride. i Author for correspondence.
bradykinin (BK) and hydroxyprolyl 3-bradykinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg), (Hyp 3BK), in human tumor ascites and concluded that they are generated from high molecular weight kininogen [7, 8]. We and other workers have also identified Hyp3-BK from normal human plasma or urine [9-12]. Because of the large capacity of generation of kinins and the potent activity of these peptides, they may be important in various pathological conditions. With regard to the extravascular permeability in the tumor tissue, the concentration and degradation of kinins in the ascitic fluid from cancer patients need to be eluci-
Agents and Actions, vol. 29, 3/4 (1990) dated. During a recent study, we found the content of Hyp3-BK to be much higher than that of BK [7, 8]. The" question arose as to which of these kinins was most potent and long lasting in vivo, while their activity in vitro was found to be about the same. It has been established that kinins are rapidly inactivated by kininase I and kininase II (angiotensin converting enzyme) in plasma. The mechanism of the degradation of kinins has been examined in detail by using each enzyme [13-15]. However, it seems that the degradation pathway of kinins in plasma or in extravascular fluid is a complex phenomenon because the degradation is caused by two kininases and other peptidases and proteases in the fluids. Recently, Majima et al. developed an enzyme immunoassay for the degradation product of BK and studied the degradation of BK in the inflammatory exudate [16]. In this study, the degradation pathway of both BK and Hyp 3BK in the ascitic fluids was analyzed using reversed phase HPLC in the presence or absence of various kininases and peptidase inhibitors. Materials and methods Chemicals: Captopril, D-2-methyl-3-mercaptopropionyl-L-proline (kininase II inhibitor), was a gift from Sankyo Co., Tokyo [17]. CPNI, carboxypeptidase N inhibitor, DL-2-mercaptomethyl3-guanidino-ethylthio-propanoic acid (kininase I inhibitor), was obtained from Calbiochem-Behring, San Diego, CA, USA [18]. APMSF, p-amidinophenylmethylsulfonyl fluoride was from Wako Pure Chemical Industry Co., Ltd. Osaka. BK, Lys-BK, Hyp3-BK, Lys-Hyp3-BK and desArg9-BK were from Peptide Institute Inc., Minoh, Osaka. Des-Phe8-Arg9-BK and a pentapeptide, Arg-Pro-Pro-Gly-Phe, were generous gifts from Prof. M. Katori, Kitasato University School of Medicine. Phe-Arg was from Bachem Feinchemikalien AG, Bubendorf, Switzerland. (S)-c~Benzylmalic acid [19] was generous gift from Dr. T. Aoyagi, Institute for Microbial Chemistry, Tokyo. Ser-Pro-Phe was synthesized from Bocamino acids by an Applied Biosystems Peptide Synthesizer (Model 430A). Its amino acid sequence was confirmed by amino acid analysis and sequence analysis. All other reagents were from local commercial sources. Human ascitic exudates were obtained from two patients with carcinomatosis of ovarian cancer and a patient with hep-
173 atoma and centrifuged to remove precipitates and residues, and their supernatants were used. Their protein concentrations were 45 mg per ml, 66 mg per ml and 20 mg per ml which were determined by Lowry's method. Amino acid composition of peptides was determined by a Pico-Tag HPLC System (Waters Associates, Bedford, MA, USA), as reported previously [8]. The amino acid sequence was determined by an Applied Biosystems GasPhase Protein Sequencer (Model 470A) equipped with an on-line Model 120A phenylisothiohydantoin (PTH) analyzer, as reported previously [8]. Degradation of Synthetic kinins in Tumor Ascitic Fluid: Each supernatant of tumor ascites was diluted three fold with the following incubation buffer and then a 10 gl aliquot and 100gl of 0.02 M Tris-HC1 buffer, pH 7.4, containing 0.15 M NaC1 were mixed with 10 lal of I m M synthetic kinin solution in distilled water. In order to examine the effect of kininase in the ascites the following reaction mixture was also tested. A 10 gl aliquot of 3 fold diluted ascitic fluid was mixed with 10 gl of following specific inhibitors in 90 ~tl of the same incubation buffer: 1 m M CPNI to inhibit kininase I, 1 m M captopril to inhibit kininase II, 2 mg/ml (S)~-benzylmalic acid to inhibit carboxypeptidase A and carboxypeptidase B and 40 m M EDTA to inhibit metalloenzymes. After 5 rain preincubation, i0 t~1 of 1 m M synthetic BK solution was added into the incubation mixture. The mixture was incubated for the various periods of time at 37~ and 100 ~1 aliquot removed and subjected to reversed-phase HPLC on column (4.6 m m x 25 cm) of ODS-120A from Toyo Soda Manufacturing Co., Ltd., Tokyo, with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of I ml per rain. Peptide peaks were detected at 210 nm and the peak area was calculated using an integrator, Chromatocorder 11, System Instruments, Tokyo and converted to the amounts of the peptide by the standard curve of synthetic peptide. Results and discussion
Figure 1 shows the elution patterns on reversed phase HPLC of the 6 h digests of synthetic BK and Hyp3-BK with three fold diluted ascitic fluid from a patient with ovarian carcinoma. Three main peaks (peaks I - t h r o u g h III) were found in addition to undigested BK or Hyp3-BK in each digest.
174
Agents and Actions,vol. 29, 3/4 (1990) |
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