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Characterization of kininases in testicular cells
Immunopharmacology ELSEVIER
Immunopharmacology32 (1996) 169-171
Characterization of kininases in testicular cells Thomas K. Monsees *, Werner Miska, Wolf-Bernhard Schill Dept. qf Dermatology and Andrology, Justus Liebig Unicersitv, Gaflkystr. 14, 35385 Giessen. Germany
Abstract Kininases are an important part of the kallikrein-kinin system. We investigated the pattern of kininases in rat Sertoli cells. Sertoli cells are found in the seminiferous tubule of the testis and play a key role in spermatogenesis. Bradykinin was actively cleaved by cultivated Sertoli cells at the ProV-Phe 8, PheS-Ser 6 and Gly4-Phe 5 bonds as demonstrated by high performance liquid chromatography analysis. Addition of phosphoramidon and thiorphan, which are specific inbibitors of neutral metalloendopeptidase 3.4.24.11 (NEP), strongly inhibited the degradation of bradykinin. In contrast, the kininase type II-specific inhibitors captopril and enalapril were only partially effective in preventing peptidolysis. NEP and kininase type II were shown to be located on Sertoli cell membranes. The action of kininase type I leads to the formation of the metabolite bradykinin(1-8) which could be detected in small amounts by HPLC analysis. Cleavage of the PheS-Ser 6 bond might be caused by the action of the endopeptidases 24.15 and 24.16, which are phosphoramidon-insensitive. Our results indicate that neutral metalloendopeptidase 24.11 is the main kininase responsible for rapid bradykinin inactivation in Sertoli cells. Further kininases with minor activities are the kininases type I and II and probably the metalloendopeptidases 24.15 and 24.16. Keywords: Kininase type I and II: Neutral metalloendopeptidase; Kallikrein-kinin system; Peptide degradation; Sertoli cell
1. Introduction Kinins are potent local tissue hormones involved in many physiological processes. The biological effects are mediated by specific, membrane-bound receptors. These and peptidolytic enzymes called kininases are located on the cell surface and compete for the kinins (Regoli and Barabe, 1980). In the rat, kallikrein activates Sertoli cell function (Rohen and Buschhiiter, 1975) and increases the relative number of spermatocytes (Rohen and Stuttman, 1977). Kallikrein also increases the concentration of the
Abbreviations: NEP, neutral metalloendopeptidase; HPLC, high performance liquid chromatography; Bk, bradykinin • Corresponding author.
testicular androgen binding component (Saito et al., 1989). H o w e v e r , the effective substance is bradykinin, which is liberated from kininogen by the proteinase kallikrein (Schill et al., 1989). Sertoli cells are found in the seminiferous tubules of the testis and form an epithelium that is in close contact with germ cells. Because of the close morphological and biochemical relationships of Sertoli cells and germ cells, Sertoli cells are regarded as the 'nurse cells' of spermatogenesis (Bardin et a l , 1988). Consequently, a modification of Sertoli cell function by a potential involvement of the kallikrein-kinin system could lead to altered spermatozoa development. Here we report the kininase pattern in cultured rat Sertoli cells. Using high performance liquid chro-
0162-3109/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 I 62-3 I 09(95)()0084-4
170
T.K. Monsees et al. / lmmunopharmacology 32 (1996) 169-171
matography (HPLC) we analyzed the degradation of bradykinin and the appearance of resulting cleaving products. The kininases responsible for bradykinin inactivation were identified by means of specific proteinase inhibitors.
• Bk(1-4) -- *--Bk(6-9) = Bk(1-5)
Bk(8-9) -- ~---Bk(1-7) = Bk
g. ~ 2. M a t e r i a l s a n d m e t h o d s
Cell culture. Sertoli cells from 18-day-old Sprague-Dawley rats were prepared according to published procedures (Galdieri et al., 1981; Onoda et al., 1990). High performance liquid chromatography. HPLC analyses were performed using a system containing a L-6200A pump, a L-3000 Multi channel photo detector (Merck-Hitachi), and a C-R5A Chromatopac integrator (Shimadzu). Peptides were applied to a LiChrosorb RP-8 column, 250 × 4 mm, 5 /zm particle size (Merck). The separation was carried out isocratically with 24% acetonitrile and 76% 75 mM NaC104, 5 mM NaH2PO 4, pH 2.2 at a flow rate of 1.3 m l / m i n . The degradation products detected at 214 nm were identified by co-elution with peptide standards. Degradation of bradykinin by Sertoli cells. On day 6 of culture, cells were washed twice with phosphate buffered saline (PBS). For degradation experiments, cells were incubated at 34°C in 10 mM PBS, pH 7.3, supplemented with 1.42 mM CaC12. Bradykinin (100 /zM) was added at time = 0. In some experiments proteinase inhibitors were added at final concentrations as shown in Fig. 2. At the indicated time intervals, aliquots were removed, boiled for 5 min and stored at - 2 0 ° C until HPLC analysis.
3. R e s u l t s a n d d i s c u s s i o n
HPLC analysis clearly demonstrated the degradation of bradykinin (Bk) in Sertoli cell culture supernatants. The main products of bradykinin peptidolysis were identified as Bk(1-7), Bk(1-5), Bk(6-9), Bk(1-4), and Bk(8-9). These results indicate that primary cleavages of bradykinin occur at the Pro 7Phe 8, PheS-Ser 6, and G l y 4 - P h e 5 bonds. The relative amount of bradykinin degradation and resulting
40 2o 0 0
30
60
90
120
180
240
Incubation time (min)
Fig. I. Time course of bradykinin degradation by cultivated Sertoli cells and appearance of metabolites. 100% peptide corresponds to the maximum HPLC peak area of the respective peptide during the incubation time. Each point is the mean of three to four individual experiments. metabolites plotted against incubation time are shown in Fig. 1. The metabolites Bk(1-7) and Bk(6-9) show the typical profile of intermediate cleavage products. Bk(1-4), Bk(1-5), and Bk(8-9) were identified as stable final products after 5 h of incubation. The kininase pattern of Sertoli cells was further investigated by means of specific proteinase inhibitors. The results are shown in Fig. 2. Thiorphan and phosphoramidon are specific inhibitors of NEP - " @" - • no inhibitor ,0. Phosphoramidon •,~ Thiorphan - - -I- - - Phosp+Capto 100 . . . .
_
_~___
-~ ¢ . . . . .__.~ ~
Captopril Enalapril E64 o-Phenanthroline . . . . . . .
!° 0
30
60 90 120 180 In©ulmtion time |rain)
240
Fig. 2. Effect of several protease inhibitors on bradykinin degradation by Sertoli cells. Specificityand concentrations used: captopril (4.6 /xM; kininase II), enalapril (500 /xM; kininase II), phosphoramidon (1.8 k~M; NEP 24.11), thiorphan (1 txM; NEP 24.11), E64 (5 /xg/ml; cysteine proteases), phosphoramidon+captopril (1.8 /~M+4.6 /xM), and o-phenanthroline (1 raM; metalloenzymes). 100% bradykinin corresponds to the initial bradykinin concentration (100 /xM). Each point is the mean of three to four individual experiments.
T.K. Monsees et al. / lmmunopharmacology 32 (1996) 169-171
24.11. Both substances strongly inhibit the degradation of bradykinin. The half-life values of bradykinin were prolonged to about 260 min (phosphoramidon) and 300 min (thiorphan), respectively, compared to 50 rain for peptidolysis without inhibitor. In contrast, only weak inhibition was observed by the action of the kininase type II-specific inhibitors captopril or enalapril. The corresponding bradykinin half-life values are 55 rain (captopril) and 60 rain (enalapril), respectively. A combination of NEP and kininase type II specific inhibitors caused a marked, but not absolute, reduction in bradykinin degradation. The action of kininase type I leads to the formation of the metabolite bradykinin(l-8) which was detected in small amounts by HPLC analysis (data not shown). Cleavage of the PheS-Ser 6 bond leads to the release of Bk(6-9). This might result from the action of the endopeptidases 24.15 and 24. 16 which are phosphoramidon-insensitive and were recently described in rat testes (Orlowski et al., 1989; Mentlein and Dahms, 1994). The bradykinin degrading activity of Sertoli cells is mainly attributed to metalloenzymes, as was clearly demonstrated by use of the metal chelator o-phenanthroline. Both kininase type II and neutral metalloendopeptidase are zinc-containing enzymes. Moreover, both enzymes are reported to be membrane-bound. This is in good agreement with our observation that cell-free, Sertoli cell-conditioned media shows no bradykinin degrading activity. The cysteine protease-specific inhibitor E-64 had no effect on the peptidolysis of bradykinin. We have demonstrated the involvement of different kininases in isolated Sertoli cells. However, the putative function of bradykinin in regulating Sertoli
171
cell activity and of the kallikrein-kinin system in the testis remains to be investigated.
References Bardin C, Yan Cheng C, Musto NA, Gunsalus GL. The Ser~oli cell. In: Knobil E, Neill J, Eds. The Physiology of Reproduction. New York: Raven Press, 1988: 933-974. Galdieri M, Ziparo E, Palombi F, Russo MA, Stefani M. Pure Sertoli cell cultures: a new model for the study of somatic-germ cell interactions. J Androl 1981; 5: 249-254. Mentlein R, Dahms P. Endopeptidases 24.16 and 24.15 are responsible for the degradation of somastatin, neurotensin, and other neuropeptides by cultivated astrocytes. J Neurochem 1994; 62: 27-36. Onoda M, Suarez-Quian CA, Dym M. Characterization of Sertoli cells cultured in the bicameral chamber system: relationship between formation of permeability barriers and polarized secretion of transferrin. Biol Reprod, 1990; 43: 672-683. Orlowski M, Reznik S, Ayala J, Pierotti AR. Endopeptidase 24.15 from rat testis. Biochem J 1989; 261:951 958. Regoli D, Barabe J. Pharmacology of bradykinin and related kinins. Pharmacol Rev 1980: 32: 1-46. Rohen JW, Buschhiiter H. Karyometric measurements on the Sertoli cell nuclei in kallikrein-treated albino rats. In: Haberland GL, Rohen JW, Schirren C, Huber P, Eds. Kininngenases. Kallikrein. Stuttgart-New York: Schattauer. 1975: 85 97. Rohen JW, Stuttman R. The early postnatal development of the germinanative epithelium of the testis in the albino rat under the influence of kallikrein. In: Haberland GL, Rohen, JW, Suzuki T, Eds. Kininogenases. Kallikrein 4. Stuttgart-New York: Schattauer, 1977: 217-223. Saito S, Mikuma N, Kumamoto Y. Eftect of kallikrein on the Sertoli cell function. Urol lnt 1989: 44: 87-89. Schill WB, Miska W, Parsch EM, Fink E. Significance of the kallikrein-kinin-system in andrology. In: Schmidt I, Dietze G, Eds. The Kallikrein-Kinin System in Health and Disease. Braunschweig: Limbach, 1989:171-203.
Immunopharmacology32 (1996) 169-171
Characterization of kininases in testicular cells Thomas K. Monsees *, Werner Miska, Wolf-Bernhard Schill Dept. qf Dermatology and Andrology, Justus Liebig Unicersitv, Gaflkystr. 14, 35385 Giessen. Germany
Abstract Kininases are an important part of the kallikrein-kinin system. We investigated the pattern of kininases in rat Sertoli cells. Sertoli cells are found in the seminiferous tubule of the testis and play a key role in spermatogenesis. Bradykinin was actively cleaved by cultivated Sertoli cells at the ProV-Phe 8, PheS-Ser 6 and Gly4-Phe 5 bonds as demonstrated by high performance liquid chromatography analysis. Addition of phosphoramidon and thiorphan, which are specific inbibitors of neutral metalloendopeptidase 3.4.24.11 (NEP), strongly inhibited the degradation of bradykinin. In contrast, the kininase type II-specific inhibitors captopril and enalapril were only partially effective in preventing peptidolysis. NEP and kininase type II were shown to be located on Sertoli cell membranes. The action of kininase type I leads to the formation of the metabolite bradykinin(1-8) which could be detected in small amounts by HPLC analysis. Cleavage of the PheS-Ser 6 bond might be caused by the action of the endopeptidases 24.15 and 24.16, which are phosphoramidon-insensitive. Our results indicate that neutral metalloendopeptidase 24.11 is the main kininase responsible for rapid bradykinin inactivation in Sertoli cells. Further kininases with minor activities are the kininases type I and II and probably the metalloendopeptidases 24.15 and 24.16. Keywords: Kininase type I and II: Neutral metalloendopeptidase; Kallikrein-kinin system; Peptide degradation; Sertoli cell
1. Introduction Kinins are potent local tissue hormones involved in many physiological processes. The biological effects are mediated by specific, membrane-bound receptors. These and peptidolytic enzymes called kininases are located on the cell surface and compete for the kinins (Regoli and Barabe, 1980). In the rat, kallikrein activates Sertoli cell function (Rohen and Buschhiiter, 1975) and increases the relative number of spermatocytes (Rohen and Stuttman, 1977). Kallikrein also increases the concentration of the
Abbreviations: NEP, neutral metalloendopeptidase; HPLC, high performance liquid chromatography; Bk, bradykinin • Corresponding author.
testicular androgen binding component (Saito et al., 1989). H o w e v e r , the effective substance is bradykinin, which is liberated from kininogen by the proteinase kallikrein (Schill et al., 1989). Sertoli cells are found in the seminiferous tubules of the testis and form an epithelium that is in close contact with germ cells. Because of the close morphological and biochemical relationships of Sertoli cells and germ cells, Sertoli cells are regarded as the 'nurse cells' of spermatogenesis (Bardin et a l , 1988). Consequently, a modification of Sertoli cell function by a potential involvement of the kallikrein-kinin system could lead to altered spermatozoa development. Here we report the kininase pattern in cultured rat Sertoli cells. Using high performance liquid chro-
0162-3109/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 I 62-3 I 09(95)()0084-4
170
T.K. Monsees et al. / lmmunopharmacology 32 (1996) 169-171
matography (HPLC) we analyzed the degradation of bradykinin and the appearance of resulting cleaving products. The kininases responsible for bradykinin inactivation were identified by means of specific proteinase inhibitors.
• Bk(1-4) -- *--Bk(6-9) = Bk(1-5)
Bk(8-9) -- ~---Bk(1-7) = Bk
g. ~ 2. M a t e r i a l s a n d m e t h o d s
Cell culture. Sertoli cells from 18-day-old Sprague-Dawley rats were prepared according to published procedures (Galdieri et al., 1981; Onoda et al., 1990). High performance liquid chromatography. HPLC analyses were performed using a system containing a L-6200A pump, a L-3000 Multi channel photo detector (Merck-Hitachi), and a C-R5A Chromatopac integrator (Shimadzu). Peptides were applied to a LiChrosorb RP-8 column, 250 × 4 mm, 5 /zm particle size (Merck). The separation was carried out isocratically with 24% acetonitrile and 76% 75 mM NaC104, 5 mM NaH2PO 4, pH 2.2 at a flow rate of 1.3 m l / m i n . The degradation products detected at 214 nm were identified by co-elution with peptide standards. Degradation of bradykinin by Sertoli cells. On day 6 of culture, cells were washed twice with phosphate buffered saline (PBS). For degradation experiments, cells were incubated at 34°C in 10 mM PBS, pH 7.3, supplemented with 1.42 mM CaC12. Bradykinin (100 /zM) was added at time = 0. In some experiments proteinase inhibitors were added at final concentrations as shown in Fig. 2. At the indicated time intervals, aliquots were removed, boiled for 5 min and stored at - 2 0 ° C until HPLC analysis.
3. R e s u l t s a n d d i s c u s s i o n
HPLC analysis clearly demonstrated the degradation of bradykinin (Bk) in Sertoli cell culture supernatants. The main products of bradykinin peptidolysis were identified as Bk(1-7), Bk(1-5), Bk(6-9), Bk(1-4), and Bk(8-9). These results indicate that primary cleavages of bradykinin occur at the Pro 7Phe 8, PheS-Ser 6, and G l y 4 - P h e 5 bonds. The relative amount of bradykinin degradation and resulting
40 2o 0 0
30
60
90
120
180
240
Incubation time (min)
Fig. I. Time course of bradykinin degradation by cultivated Sertoli cells and appearance of metabolites. 100% peptide corresponds to the maximum HPLC peak area of the respective peptide during the incubation time. Each point is the mean of three to four individual experiments. metabolites plotted against incubation time are shown in Fig. 1. The metabolites Bk(1-7) and Bk(6-9) show the typical profile of intermediate cleavage products. Bk(1-4), Bk(1-5), and Bk(8-9) were identified as stable final products after 5 h of incubation. The kininase pattern of Sertoli cells was further investigated by means of specific proteinase inhibitors. The results are shown in Fig. 2. Thiorphan and phosphoramidon are specific inhibitors of NEP - " @" - • no inhibitor ,0. Phosphoramidon •,~ Thiorphan - - -I- - - Phosp+Capto 100 . . . .
_
_~___
-~ ¢ . . . . .__.~ ~
Captopril Enalapril E64 o-Phenanthroline . . . . . . .
!° 0
30
60 90 120 180 In©ulmtion time |rain)
240
Fig. 2. Effect of several protease inhibitors on bradykinin degradation by Sertoli cells. Specificityand concentrations used: captopril (4.6 /xM; kininase II), enalapril (500 /xM; kininase II), phosphoramidon (1.8 k~M; NEP 24.11), thiorphan (1 txM; NEP 24.11), E64 (5 /xg/ml; cysteine proteases), phosphoramidon+captopril (1.8 /~M+4.6 /xM), and o-phenanthroline (1 raM; metalloenzymes). 100% bradykinin corresponds to the initial bradykinin concentration (100 /xM). Each point is the mean of three to four individual experiments.
T.K. Monsees et al. / lmmunopharmacology 32 (1996) 169-171
24.11. Both substances strongly inhibit the degradation of bradykinin. The half-life values of bradykinin were prolonged to about 260 min (phosphoramidon) and 300 min (thiorphan), respectively, compared to 50 rain for peptidolysis without inhibitor. In contrast, only weak inhibition was observed by the action of the kininase type II-specific inhibitors captopril or enalapril. The corresponding bradykinin half-life values are 55 rain (captopril) and 60 rain (enalapril), respectively. A combination of NEP and kininase type II specific inhibitors caused a marked, but not absolute, reduction in bradykinin degradation. The action of kininase type I leads to the formation of the metabolite bradykinin(l-8) which was detected in small amounts by HPLC analysis (data not shown). Cleavage of the PheS-Ser 6 bond leads to the release of Bk(6-9). This might result from the action of the endopeptidases 24.15 and 24. 16 which are phosphoramidon-insensitive and were recently described in rat testes (Orlowski et al., 1989; Mentlein and Dahms, 1994). The bradykinin degrading activity of Sertoli cells is mainly attributed to metalloenzymes, as was clearly demonstrated by use of the metal chelator o-phenanthroline. Both kininase type II and neutral metalloendopeptidase are zinc-containing enzymes. Moreover, both enzymes are reported to be membrane-bound. This is in good agreement with our observation that cell-free, Sertoli cell-conditioned media shows no bradykinin degrading activity. The cysteine protease-specific inhibitor E-64 had no effect on the peptidolysis of bradykinin. We have demonstrated the involvement of different kininases in isolated Sertoli cells. However, the putative function of bradykinin in regulating Sertoli
171
cell activity and of the kallikrein-kinin system in the testis remains to be investigated.
References Bardin C, Yan Cheng C, Musto NA, Gunsalus GL. The Ser~oli cell. In: Knobil E, Neill J, Eds. The Physiology of Reproduction. New York: Raven Press, 1988: 933-974. Galdieri M, Ziparo E, Palombi F, Russo MA, Stefani M. Pure Sertoli cell cultures: a new model for the study of somatic-germ cell interactions. J Androl 1981; 5: 249-254. Mentlein R, Dahms P. Endopeptidases 24.16 and 24.15 are responsible for the degradation of somastatin, neurotensin, and other neuropeptides by cultivated astrocytes. J Neurochem 1994; 62: 27-36. Onoda M, Suarez-Quian CA, Dym M. Characterization of Sertoli cells cultured in the bicameral chamber system: relationship between formation of permeability barriers and polarized secretion of transferrin. Biol Reprod, 1990; 43: 672-683. Orlowski M, Reznik S, Ayala J, Pierotti AR. Endopeptidase 24.15 from rat testis. Biochem J 1989; 261:951 958. Regoli D, Barabe J. Pharmacology of bradykinin and related kinins. Pharmacol Rev 1980: 32: 1-46. Rohen JW, Buschhiiter H. Karyometric measurements on the Sertoli cell nuclei in kallikrein-treated albino rats. In: Haberland GL, Rohen JW, Schirren C, Huber P, Eds. Kininngenases. Kallikrein. Stuttgart-New York: Schattauer. 1975: 85 97. Rohen JW, Stuttman R. The early postnatal development of the germinanative epithelium of the testis in the albino rat under the influence of kallikrein. In: Haberland GL, Rohen, JW, Suzuki T, Eds. Kininogenases. Kallikrein 4. Stuttgart-New York: Schattauer, 1977: 217-223. Saito S, Mikuma N, Kumamoto Y. Eftect of kallikrein on the Sertoli cell function. Urol lnt 1989: 44: 87-89. Schill WB, Miska W, Parsch EM, Fink E. Significance of the kallikrein-kinin-system in andrology. In: Schmidt I, Dietze G, Eds. The Kallikrein-Kinin System in Health and Disease. Braunschweig: Limbach, 1989:171-203.