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Metalloendopeptidase (EC 3.4.24.11) but not angiotensin converting enzyme is involved in the inactivation of substance P by synaptic membranes of the rat substantia nigra
Life Sciences, Vol. 44, p p . Printed in the U.S.A.
1467-1474
P e r g a m o n Press
NETAT.TJ~ERDOPEPTIDASE (EC 3.4.24.11) BUT NOT AI~IOTENSIN CONVERTII~ IS INVOLVED IN THE INACTIVATION OF SUBSTANCE P BY SYNAPTIC NENBRARES OF THE RAT SUBSTANTIA RIGRA.
OBLIN A., DANSE M.J. and ZIVKOVIC B.
Laboratoires d'Etudes et de Recherches Synthdlabo (L.E.R.S.) 31 Ave P.V. Couturier 92220 - Bagneux, France (Received in final form March 13, 1989) S-mmary Substance P is a neuropeptide released in vlvo from the substantia nigra, the principal substance P nerve terminal region in the rat brain. Its inactivation was investigated in a purified nlgral synoptic membrane preparation. The membrane-bound enzyme shares many features with the endopeptidase 24-11 (EC 3.4.24.11): i) hydrolysis of peptlde bonds Gln6-Phe 7 , Phe7-Phe 8 and Gly9-Leu I0 , 2) sensitivity to the inhibition by phosphoramidon and 3) relative affinity for substance P. Bestatlne and captopril inhibit only the hydrolysls of the metabolites. These results suggest that substance P is inactivated in substantla nigra by endopeptidase 24-11 and that a bestatin-sensltive aminopeptidase and anglotensln converting enzyme may play a role in subsequent degradation of the substance P metabolites.
In the central nervous system, the highest density of substance P containing nerve terminals is found in the substantia nigra. It is believed that the decrease in nigral substance P levels after chronic impairment of the D 2 dopamine receptor function [1] results from an increased release of this neuropeptide in the synaptlc cleft followed by an enzymatic hydrolysis [2]. However, the enzymes involved in substance P inactivation in the substantia nigra are not yet elucidated. The a m i n o a c i d s e q u e n c e o f s u b s t a n c e P ( A r g - P r o - L y s - P r o - G l n - G l n - P h e Phe-Gly-Leu-MetNH2) suggests that the presence of proline residue and methionine amide in the N-terminal and C-terminal parts, respectively confers a resistance to the actions of exopeptidases. Thus, only membrane endopeptidases may be involved in the initial metabolisation of substance P : a post proline dipeptidylamlnopeptidase (EC 3.4.14.5) [3,4] ; a membrane metalloendopeptidase ("enkephalinase", EC 3.4.24.11) [5,6] ; a "substance P-degradlng enzyme" [7], and a peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1) [6,8]. However their physiological function, partlcularly in the substance Pergic nerve terminals, has remained obscure.
0024-3205/89 $3.O0 + .00 Copyright (c) 1989 Pergamon Press plc
1468
Endopeptldase
24-11 Inactivates
Substance P
Vol. 44, No. 20, 1989
We have recently suggested that a phosphoramldon-sensltive endopeptidase is involved in substance P degradation in the nerve terminals of the striatonigral pathway [9]. Here we report that this enzyme is a metalloendopeptidase (EC 3.4.24.11) which is essential for this neuropeptlde degradation in the substantia nlgra and that angiotensin converting enzyme contributes only to the subsequent degradation of some substance P metabolites.
MATERIALS ARD METHODS
Peptides and inhibitors. Substance P and substance P fragments were purchased from Cambridge Research Biochemlcals (England) and other reagents from Sigma. Trltlated substance P was obtained from HEN Research Products (2-L-prolyl-S-4-SH substance P ; specific activity 39.9 Ci/mmol). S ynaptlc membranes preparations. Sprague-Dawley rats (150 - 200 g) were killed by decapitation and substantiae nigrae were dissected out of brains on a cold plate, Purified synaptlc membranes were prepared by a previously described procedure of Jones and Matus [10]. The membranes were washed in 5 mM Tris-HCl (pH 7.8) and the resulting pellet was resuspended in 25 raM Trls-HCl (pH 7.8) containing 0.2 M NaCI at a protein concentration of 0.75 mg/ml. Hydrolysis of substance P. Samples of nlgral synaptlc membranes (40-60 ~g of protein) were incubated with substance P (600 pM) in 25 mM Trls-HCl (pH 7.8) containing 0.2 M NaCI in the presence or in the absence of irthibltors (final volume = i00 pl). The reaction was stopped by addition to the samples 50 pl of mobile HPLC phase, before sample analysis on reversed-phase column. Inhibitory effect of phosphoramidon was evaluated as the difference between the area of the substance P peak observed after incubation performed in the absence and in the presence of the inhibitor. In this study, the initial concentration of substance P was only 75 ~M. Kinetic study. Km value for hydrolysis of substance P was determined by incubating various concentrations of substance P (20 - 250 ~M) and approximately 15 pmoles of [3H] peptide with synaptic membranes (30 pg of protein) for 1 hour at 37"C. The [3H] metabolites were separated from intact [3H] peptide by HPLC. The [3H] substance P peak was collected directly into scintillation vials and radioactivity was estimated by liquid scintillation counting. Enzymatic activity was estimated from the decrease of substance P concentration. HPLC analysis of substance P cleavage products. Reaction products were separated by analytical HPLC on an 0DS Spherisorb C18 , 5 pm, column (0.4 mm x 25 cm) using a Sprectra Physics chromatograph. For the kinetic study and the concentratlon-dependent effect by phosphoramidon, the separation of substance P from its degradation products was performed using an isocratlc elutlon as described for substance P fragments chromatography [9]. For the metabollte identification, the breakdown products were separated with a 15 min linear acetonltrile gradient established between 20 % in a 10 mM potassium phosphate pH 2.5 and 50 % in the same buffer pH 2.2, followed by 20 min elutlon at the final conditions. The elutlon was performed at flow rate of 1 ml/mln at room temperature with detection at 214 nm. Products were identified either by using marker peptides or by amino acid analysis of individual peaks using a Kontron amino acid analyzer fitted with o-phthalaldehyde detection.
Vol. 44, No. 20, 1989
Endopeptidase
24-11 Inactivates
Substance P
1469
RESULTS
Hydrolysis of substance P by endopeptidase-containing synaptic membranes from substantla nigra. The HPLC profile of the degradation products obtained when substance P was incubated in the presence of bestatlne (in order to avoid subsequent hydrolysis of metabolites) is shown in Figure IA. Seven major peptide-containing peaks that absorb at 214 nm could be separated. The products shown in Table 1 were identified by amino acid analysis and by comparison of their retention times with those of synthetic peptlde fragments [9]. The metabolites formed are consistent with the hydrolysis at three sites :
Arg-Pr o-Lys-Pr o-Gln-Gln-Phe-Phe-G ly-Leu-Me t NIt2 1 2 3 4 5 6 7 8 9 i0 ii
®
5
10
15
20
9
® u~ z
4(
u
30
25
4
O
7 HE~
~H ==
.:"
, 5
iiil
10
15
2~0
25
TIME (mln)
Figure 1 HPLC elution profiles of unlabelled substance P (0.6 mM) (A) and tritlated substance P (30 pmoles) (B) incubated at 37°C with nlgral synaptic membranes (60 pg of protein). The peptlde products were separated with a linear gradient of acetonitrile and pH.
1470
Endopeptldase
24-11 Inactivates
Substance P
Vol. 44, No. 20, 1989
T h r e e 3H-labelled peptldes were formed (Figure IB). Based upon their retention times, the peaks at 9.08 min and 16.05 mln correspond to the 3H-N terminal peptide fragments 1-7 and 1-9. The early peak at 3.37 mln was assigned to the N-terminal product of the Gln-Phe peptide bond cleavage. Thus, the major N-termlnal radioactive fragment (59 % of the total 3H-substance P degraded) suggests a predominant cleavage of the PheT-Phe 8 peptlde bond. The rate of degradation was found to increase with increasing concentration of substance P. A Lineweaver-Burk plot of these results indicates an apparent K m of 38.8 pM (Figure 2). TABLE 1 Peptides Formed During Incubation of Substance P With Synaptic Membranes from Substantia Nigra
Peak n°
Retention time (mln)
Peptide Identified
Radioactivity Content (~ of Total Radioactivity Recovered)
10.7
Fragments of Substance P
1
3.37
n.d
2
5.05
F-G
*
8-9
3
5.29
L-Ms
*
i0-ii
4
9.08
5
ii. 38
6
15.03
7
16.05
8
20.55
F-F-G-L-Ma
9
24.31
R-P-K-P-Q-Q-F-F-G-L-Ma
R-P-K-P-Q-Q-F F-F-G
33.9
i-6 (?)
1-7
*
7-9
Bestatine
*
-
R-P-K-P-Q-Q-F-F-G
9.8
1-9
* 44.4
7 - 11 Substance P
D a t a a r e f r o m i n c u b a t i o n a c h i e v i n g 55-65 % d e g r a d a t i o n o f s u b s t a n c e P. V a l u e s represent the radioactivity contents of each peak expressed as the percentage of total radioactivity after HPLC (* ( 0.5 Z) (n.d. : not determined). Effect of vepttdase inhibttors. The e f f e c t s of various peptidase inhtbitors on t h e d e g r a d a t i o n o f s u b s t a n c e P and i t s m e t a b o l i t e s a r e shown i n T a b l e 2. Captopril (100 pM), a s e l e c t i v e inhibitor of angiotensin converting e n z y m e , h a d no i n h i b i t o r y a c t i o n on t h e s u b s t a n c e P d e g r a d a t i o n . The p a t t e r n of metabolites f o r m e d was s i m i l a r t o t h a t o b s e r v e d when t h e n e u r o p e p t i d e was incubated in the absence of the inhibitor. However, as compared t o t h e control, captopril inhibited the degradation of the tripeptide (Phe-Phe-Gly) but failed to increase t h e p e a k a r e a of t h e m e t a b o l i t e s with unblocked C - t e r m i n a l s p a r t s 1-7 and 1-9 ( T a b l e 2 ) . S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h
Vol. 44, No. 20, 1989
Endopeptidase
I
V 5.C
24-11 Inactivates Substance P
1471
//
2.5
5
Io
25
50
v
I
(Substance P)[raM] Figure
2
Lineweaver-Burk plot of substance P hydrolysis by nigral endopeptidase. Approximately 1 pmole of labeled peptlde was incubated with unlabelled peptide (20 - 250 mM) in the presence of 30 pg of synaptlc membranes. Breakdown products were determined from the radioactivity content of the substance P peak. The enzymatic rate was expressed in pmoles peptlde hydrolysed per hour per mg proteins. TABLE 2 Effect of Peptldase Inhlbltors on the Degradation of Substance P and on the Formation of Substance P Metabolltes
Relative Activity
Bestatine
(100 pM) Substance
P
(% of Control)
Phosphoramidon
(10 ]~'~I)
Captopril
(IOO).LM)
107
262
104
1-9
104
25
126
7-9
245
7
196
1-7
106
13
103
In the absence of any inhibitors, 55 - 65 % of substance P (0.6 mM) was hydrolysed under the conditions used (3 h at 37°C). Metabolltes were quantified by integration of the absorbance of the peak at 214 run.
1472
Endopeptidase 24-11 Inactivates Substance P
Vol. 44, No. 20, 1989
the bestatine (i00 pM), an amlnopeptldase inhibitor. Bestatin enhanced the absorbance of the peak eluted at 5.29 min (Leu - Met NH2) , 20.55 min (fragment 7-11) (not shown) and 11.38 mln (Phe-Phe-Gly) by preventing their degradation by aminopeptidase. When the incubation was carried out in the presence of phosphoramidon (I0 pM), the enzymatic activity was strongly inhibited. This potent inhibitor of endopeptidase 24-11 produced a concentration-dependent effect on the substance P hydrolysis with an IC50 of i00 nM (Figure 3). 100
o~
o
50
llO -log
I 9
h 8
[phos ph . . . .
Figure
I 7
I 6
idon]( M )
3
E f f e c t o f p h o s p h o r a m i d o n on t h e h y d r o l y s i s by n i g r a l s y n a p t i c m e m b r a n e s .
of substance
P
DISCUSSION
The present study showed that metalloendopeptidase (EC 3.4.24.11), peptldyl dipeptidase A and aminopeptidase are present in purified synaptic membranes from the rat substantla nigra. Identity of the substance P hydrolysinK niKral endopeptidase. The results show that a membrane-bound endopeptidase hydrolyses substance P by cleaving the Gln6-Phe 7 , Phe7-Phe 8 and Gly9-Leu I0 peptide bonds and suggest that this enzymatic activity is similar to that of endopeptidase 24-11. In support of this are the following findings : i) Endopeptidase 24-11 purified from the pig kidney hydrolyses the peptide bonds involving the amino-groups of hydrophoblc residues [5]; 2) the apparent Km for the degradation of substance P by nigral synaptic membranes (38.8 ~M) agrees well with the value (31.9 ~M) obtained with purified endopeptidase 24-11 [ii]; 3) the formation of substance P metabolites was inhibited in a dose-dependent manner by phosphoramidon (IC50 = i00 nM), a specific inhibitor of endopeptidase 24-11. However, the most abundent hydrolysis appeared at the board Phe7-Phe 8 and not Gly9-Leud I0 as observed previously [5] after substance p hydrolysis by purified kidney endopeptidase 24-11.
Vol. 44, No. 20, 1989
Endopeptidase 24-11 Inactivates Substance P
1473
PhysioloKical implications. Several enzymes appear as candidates for the hydrolysis of substance P. Previous reports suggest that angiotensln converting enzyme is involved in the substance P degradation. This is based on the finding that intraventricular injections of captoprll increased cerebral levels of this peptide in particular in zonal recitulata of subtantia nigra [12]. Moreover, striatal lesions carried out using Ibotenlc or kainic acid showed that angiotensin converting enzyme in the substantia nigra is present on presynaptlc terminals of axons originating the caudate putamen [ 1 3 , 14]. Although, it was reported that purified angiotensln converting enzyme (free enzyme form) can also hydrolyse substance P in vitro [6, 8], in the present experiments, captopril failed to produce any significant influence on the substance P cleavage by nigral synaptic membranes (bound enzyme form). This result may be interpreted that angiotensin converting enzyme does not play an essential role in physiological degradation of substance P in nerve terminals of striato-nigral pathway. On the other hand, this non-specific dipeptldyl carboxypeptidase releases mainly dipeptlde fragments from the unblocked-C terminal peptides [15]. This mechanism is confirmed in the present study by prevention of the substance P metabollte (Phe-Phe-Gly) degradation by captopril. However, the degradation of metabolites (SP 1-7) and (SP 1-9) were not or only little affected by captopril. It cannot be excluded that the N-termlnal part of substance P (Arg-Pro-Lys-Pro) plays an important role in determining the substrate specificity for the brain peptidyl dipeptidase A. Morevoer, a recent study has reported that purified angiotensin-converting enzyme fails to hydrolyse at the significant rate neurokinin A a tachykinin that shares an identical C terminal sequence as substance P but a different amino acids composition in N terminal sequence [16, 17]. Thus the physiological importance of angiotensin converting enzyme in substantia nigra remains to be established. In conclusion, These in vitro studies clearly imply that the metalloendopeptidase (EC 3.4.24.11) is the enzyme responsible for the first peptlde bond(s) hydrolyzed when substance P is incubated with the synaptlc membranes. Amlnopeptldase and anglotensln converting enzyme may play only se secondary role in catabolims of substance P metabolltes. However, whether substance P is degraded in substantla nigra by endopeptldase 3.4.24.11 remains to be established in vivo. Further studies including the immunohistochemical determination of the exact enzymatic localization to substance P pathway and the prolongation of the biological actions of substance P in vivo by preventing its degradation witth selective metalloendopeptidase (EC 3.4.24.11) inhibltors are needed.
AcknowledKements. We thank Dr. fragments.
K.G.
Lloyd
and Mrs Voltz
for amlno-acld analysis of peptlde
REFEP~ENCES
I. 2.
0BLIN, A., ZIVKOVIC, B. and G. BARTHOLINI, Brain Res. 421 387-390 (1987). ZIVKOVIC, B., OBLIN, A. and G. BARTHOLINI, Eur. J. Pharmacol.ll2 253-255 (1985).
1474
3. 4. 5. 6.
7. 8. 9. I0. 11. 12. 13. 14.
Endopeptidase 24-11 Inactivates Substance P
Vol. 44, No. 20, 1989
KATO, T . , NAGATSU, T . , FUKASAWA,K, HARADA, M., NAGATSU, I . and S. SAKAKIBARA, Biochem. Biophys. A c t s . 525 417-422 (1978). HEI~IANN, E. and R. NENTLEIN, FEBS L e t t . 91 360-364 (1978) MATSAS, R., FULCHER, I.S., KENNY, A.J. and A.J. TURNER, Proc. Natl. Acad. Sci. USA, 80 3111-3115 (1983). SKIDGEL, R., ENGELBRECHT, S., JOHNSON, A.R. and E. ERDOS, Peptldes ~, 769-776 (1984). LEE, C.M., SANDBERG, B.E.B., HANLEY, M.R. and L.L. IVERSEN, Eur. J. Biochem. 114 315-327 (1981). YOKASAWA, H., ENDO, S., OGURA, Y. and S.I. ISHII, Biochem. Biophys. Res. Commun. 11~, 735-742 (1983). OBLIN, A., DANSE, M.J. and B. ZIVKOVIC, Neuroscience Lett. 84, 91-96. (1988). JONES, D.H. and A.I. MATUS, Blochem. Biophys. Acta. ~5~ 276-287 (1974). MATSAS, R., KENNY, A.J. and A.J. TURNER, Biochem. J. 223 433-440 (1984). HANSON, G.R. and W. LOVENBERG, J. Neurochem. 35 1370-1374 (1980). STRITTMATTER, M.M.S., LO, M.M.S., JAVITCH, J.A. and S.H. SNYDER, Proc. Natl. Acad. Sci. U.S.A. 81 1599-1603 (1984). KOSHIYA, K., KAT0, T., TANAKA, R. and T. KATO. Brain Res. 324 261-270
(1984). 15. ERDOS, E.G. Handbook of gxvertmental Pharmacology, pp. 427-484, Springer, Heidelberg (1979). 16. HOOPER, N.M., KENNY, A.J. and A.J. TURNER, Blochem. J. 231 357-361 (1985) 17. HOOPER, N.M. and A.J. TURNER, Biochem J. 241 625-633 (1987).
1467-1474
P e r g a m o n Press
NETAT.TJ~ERDOPEPTIDASE (EC 3.4.24.11) BUT NOT AI~IOTENSIN CONVERTII~ IS INVOLVED IN THE INACTIVATION OF SUBSTANCE P BY SYNAPTIC NENBRARES OF THE RAT SUBSTANTIA RIGRA.
OBLIN A., DANSE M.J. and ZIVKOVIC B.
Laboratoires d'Etudes et de Recherches Synthdlabo (L.E.R.S.) 31 Ave P.V. Couturier 92220 - Bagneux, France (Received in final form March 13, 1989) S-mmary Substance P is a neuropeptide released in vlvo from the substantia nigra, the principal substance P nerve terminal region in the rat brain. Its inactivation was investigated in a purified nlgral synoptic membrane preparation. The membrane-bound enzyme shares many features with the endopeptidase 24-11 (EC 3.4.24.11): i) hydrolysis of peptlde bonds Gln6-Phe 7 , Phe7-Phe 8 and Gly9-Leu I0 , 2) sensitivity to the inhibition by phosphoramidon and 3) relative affinity for substance P. Bestatlne and captopril inhibit only the hydrolysls of the metabolites. These results suggest that substance P is inactivated in substantla nigra by endopeptidase 24-11 and that a bestatin-sensltive aminopeptidase and anglotensln converting enzyme may play a role in subsequent degradation of the substance P metabolites.
In the central nervous system, the highest density of substance P containing nerve terminals is found in the substantia nigra. It is believed that the decrease in nigral substance P levels after chronic impairment of the D 2 dopamine receptor function [1] results from an increased release of this neuropeptide in the synaptlc cleft followed by an enzymatic hydrolysis [2]. However, the enzymes involved in substance P inactivation in the substantia nigra are not yet elucidated. The a m i n o a c i d s e q u e n c e o f s u b s t a n c e P ( A r g - P r o - L y s - P r o - G l n - G l n - P h e Phe-Gly-Leu-MetNH2) suggests that the presence of proline residue and methionine amide in the N-terminal and C-terminal parts, respectively confers a resistance to the actions of exopeptidases. Thus, only membrane endopeptidases may be involved in the initial metabolisation of substance P : a post proline dipeptidylamlnopeptidase (EC 3.4.14.5) [3,4] ; a membrane metalloendopeptidase ("enkephalinase", EC 3.4.24.11) [5,6] ; a "substance P-degradlng enzyme" [7], and a peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1) [6,8]. However their physiological function, partlcularly in the substance Pergic nerve terminals, has remained obscure.
0024-3205/89 $3.O0 + .00 Copyright (c) 1989 Pergamon Press plc
1468
Endopeptldase
24-11 Inactivates
Substance P
Vol. 44, No. 20, 1989
We have recently suggested that a phosphoramldon-sensltive endopeptidase is involved in substance P degradation in the nerve terminals of the striatonigral pathway [9]. Here we report that this enzyme is a metalloendopeptidase (EC 3.4.24.11) which is essential for this neuropeptlde degradation in the substantia nlgra and that angiotensin converting enzyme contributes only to the subsequent degradation of some substance P metabolites.
MATERIALS ARD METHODS
Peptides and inhibitors. Substance P and substance P fragments were purchased from Cambridge Research Biochemlcals (England) and other reagents from Sigma. Trltlated substance P was obtained from HEN Research Products (2-L-prolyl-S-4-SH substance P ; specific activity 39.9 Ci/mmol). S ynaptlc membranes preparations. Sprague-Dawley rats (150 - 200 g) were killed by decapitation and substantiae nigrae were dissected out of brains on a cold plate, Purified synaptlc membranes were prepared by a previously described procedure of Jones and Matus [10]. The membranes were washed in 5 mM Tris-HCl (pH 7.8) and the resulting pellet was resuspended in 25 raM Trls-HCl (pH 7.8) containing 0.2 M NaCI at a protein concentration of 0.75 mg/ml. Hydrolysis of substance P. Samples of nlgral synaptlc membranes (40-60 ~g of protein) were incubated with substance P (600 pM) in 25 mM Trls-HCl (pH 7.8) containing 0.2 M NaCI in the presence or in the absence of irthibltors (final volume = i00 pl). The reaction was stopped by addition to the samples 50 pl of mobile HPLC phase, before sample analysis on reversed-phase column. Inhibitory effect of phosphoramidon was evaluated as the difference between the area of the substance P peak observed after incubation performed in the absence and in the presence of the inhibitor. In this study, the initial concentration of substance P was only 75 ~M. Kinetic study. Km value for hydrolysis of substance P was determined by incubating various concentrations of substance P (20 - 250 ~M) and approximately 15 pmoles of [3H] peptide with synaptic membranes (30 pg of protein) for 1 hour at 37"C. The [3H] metabolites were separated from intact [3H] peptide by HPLC. The [3H] substance P peak was collected directly into scintillation vials and radioactivity was estimated by liquid scintillation counting. Enzymatic activity was estimated from the decrease of substance P concentration. HPLC analysis of substance P cleavage products. Reaction products were separated by analytical HPLC on an 0DS Spherisorb C18 , 5 pm, column (0.4 mm x 25 cm) using a Sprectra Physics chromatograph. For the kinetic study and the concentratlon-dependent effect by phosphoramidon, the separation of substance P from its degradation products was performed using an isocratlc elutlon as described for substance P fragments chromatography [9]. For the metabollte identification, the breakdown products were separated with a 15 min linear acetonltrile gradient established between 20 % in a 10 mM potassium phosphate pH 2.5 and 50 % in the same buffer pH 2.2, followed by 20 min elutlon at the final conditions. The elutlon was performed at flow rate of 1 ml/mln at room temperature with detection at 214 nm. Products were identified either by using marker peptides or by amino acid analysis of individual peaks using a Kontron amino acid analyzer fitted with o-phthalaldehyde detection.
Vol. 44, No. 20, 1989
Endopeptidase
24-11 Inactivates
Substance P
1469
RESULTS
Hydrolysis of substance P by endopeptidase-containing synaptic membranes from substantla nigra. The HPLC profile of the degradation products obtained when substance P was incubated in the presence of bestatlne (in order to avoid subsequent hydrolysis of metabolites) is shown in Figure IA. Seven major peptide-containing peaks that absorb at 214 nm could be separated. The products shown in Table 1 were identified by amino acid analysis and by comparison of their retention times with those of synthetic peptlde fragments [9]. The metabolites formed are consistent with the hydrolysis at three sites :
Arg-Pr o-Lys-Pr o-Gln-Gln-Phe-Phe-G ly-Leu-Me t NIt2 1 2 3 4 5 6 7 8 9 i0 ii
®
5
10
15
20
9
® u~ z
4(
u
30
25
4
O
7 HE~
~H ==
.:"
, 5
iiil
10
15
2~0
25
TIME (mln)
Figure 1 HPLC elution profiles of unlabelled substance P (0.6 mM) (A) and tritlated substance P (30 pmoles) (B) incubated at 37°C with nlgral synaptic membranes (60 pg of protein). The peptlde products were separated with a linear gradient of acetonitrile and pH.
1470
Endopeptldase
24-11 Inactivates
Substance P
Vol. 44, No. 20, 1989
T h r e e 3H-labelled peptldes were formed (Figure IB). Based upon their retention times, the peaks at 9.08 min and 16.05 mln correspond to the 3H-N terminal peptide fragments 1-7 and 1-9. The early peak at 3.37 mln was assigned to the N-terminal product of the Gln-Phe peptide bond cleavage. Thus, the major N-termlnal radioactive fragment (59 % of the total 3H-substance P degraded) suggests a predominant cleavage of the PheT-Phe 8 peptlde bond. The rate of degradation was found to increase with increasing concentration of substance P. A Lineweaver-Burk plot of these results indicates an apparent K m of 38.8 pM (Figure 2). TABLE 1 Peptides Formed During Incubation of Substance P With Synaptic Membranes from Substantia Nigra
Peak n°
Retention time (mln)
Peptide Identified
Radioactivity Content (~ of Total Radioactivity Recovered)
10.7
Fragments of Substance P
1
3.37
n.d
2
5.05
F-G
*
8-9
3
5.29
L-Ms
*
i0-ii
4
9.08
5
ii. 38
6
15.03
7
16.05
8
20.55
F-F-G-L-Ma
9
24.31
R-P-K-P-Q-Q-F-F-G-L-Ma
R-P-K-P-Q-Q-F F-F-G
33.9
i-6 (?)
1-7
*
7-9
Bestatine
*
-
R-P-K-P-Q-Q-F-F-G
9.8
1-9
* 44.4
7 - 11 Substance P
D a t a a r e f r o m i n c u b a t i o n a c h i e v i n g 55-65 % d e g r a d a t i o n o f s u b s t a n c e P. V a l u e s represent the radioactivity contents of each peak expressed as the percentage of total radioactivity after HPLC (* ( 0.5 Z) (n.d. : not determined). Effect of vepttdase inhibttors. The e f f e c t s of various peptidase inhtbitors on t h e d e g r a d a t i o n o f s u b s t a n c e P and i t s m e t a b o l i t e s a r e shown i n T a b l e 2. Captopril (100 pM), a s e l e c t i v e inhibitor of angiotensin converting e n z y m e , h a d no i n h i b i t o r y a c t i o n on t h e s u b s t a n c e P d e g r a d a t i o n . The p a t t e r n of metabolites f o r m e d was s i m i l a r t o t h a t o b s e r v e d when t h e n e u r o p e p t i d e was incubated in the absence of the inhibitor. However, as compared t o t h e control, captopril inhibited the degradation of the tripeptide (Phe-Phe-Gly) but failed to increase t h e p e a k a r e a of t h e m e t a b o l i t e s with unblocked C - t e r m i n a l s p a r t s 1-7 and 1-9 ( T a b l e 2 ) . S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h
Vol. 44, No. 20, 1989
Endopeptidase
I
V 5.C
24-11 Inactivates Substance P
1471
//
2.5
5
Io
25
50
v
I
(Substance P)[raM] Figure
2
Lineweaver-Burk plot of substance P hydrolysis by nigral endopeptidase. Approximately 1 pmole of labeled peptlde was incubated with unlabelled peptide (20 - 250 mM) in the presence of 30 pg of synaptlc membranes. Breakdown products were determined from the radioactivity content of the substance P peak. The enzymatic rate was expressed in pmoles peptlde hydrolysed per hour per mg proteins. TABLE 2 Effect of Peptldase Inhlbltors on the Degradation of Substance P and on the Formation of Substance P Metabolltes
Relative Activity
Bestatine
(100 pM) Substance
P
(% of Control)
Phosphoramidon
(10 ]~'~I)
Captopril
(IOO).LM)
107
262
104
1-9
104
25
126
7-9
245
7
196
1-7
106
13
103
In the absence of any inhibitors, 55 - 65 % of substance P (0.6 mM) was hydrolysed under the conditions used (3 h at 37°C). Metabolltes were quantified by integration of the absorbance of the peak at 214 run.
1472
Endopeptidase 24-11 Inactivates Substance P
Vol. 44, No. 20, 1989
the bestatine (i00 pM), an amlnopeptldase inhibitor. Bestatin enhanced the absorbance of the peak eluted at 5.29 min (Leu - Met NH2) , 20.55 min (fragment 7-11) (not shown) and 11.38 mln (Phe-Phe-Gly) by preventing their degradation by aminopeptidase. When the incubation was carried out in the presence of phosphoramidon (I0 pM), the enzymatic activity was strongly inhibited. This potent inhibitor of endopeptidase 24-11 produced a concentration-dependent effect on the substance P hydrolysis with an IC50 of i00 nM (Figure 3). 100
o~
o
50
llO -log
I 9
h 8
[phos ph . . . .
Figure
I 7
I 6
idon]( M )
3
E f f e c t o f p h o s p h o r a m i d o n on t h e h y d r o l y s i s by n i g r a l s y n a p t i c m e m b r a n e s .
of substance
P
DISCUSSION
The present study showed that metalloendopeptidase (EC 3.4.24.11), peptldyl dipeptidase A and aminopeptidase are present in purified synaptic membranes from the rat substantla nigra. Identity of the substance P hydrolysinK niKral endopeptidase. The results show that a membrane-bound endopeptidase hydrolyses substance P by cleaving the Gln6-Phe 7 , Phe7-Phe 8 and Gly9-Leu I0 peptide bonds and suggest that this enzymatic activity is similar to that of endopeptidase 24-11. In support of this are the following findings : i) Endopeptidase 24-11 purified from the pig kidney hydrolyses the peptide bonds involving the amino-groups of hydrophoblc residues [5]; 2) the apparent Km for the degradation of substance P by nigral synaptic membranes (38.8 ~M) agrees well with the value (31.9 ~M) obtained with purified endopeptidase 24-11 [ii]; 3) the formation of substance P metabolites was inhibited in a dose-dependent manner by phosphoramidon (IC50 = i00 nM), a specific inhibitor of endopeptidase 24-11. However, the most abundent hydrolysis appeared at the board Phe7-Phe 8 and not Gly9-Leud I0 as observed previously [5] after substance p hydrolysis by purified kidney endopeptidase 24-11.
Vol. 44, No. 20, 1989
Endopeptidase 24-11 Inactivates Substance P
1473
PhysioloKical implications. Several enzymes appear as candidates for the hydrolysis of substance P. Previous reports suggest that angiotensln converting enzyme is involved in the substance P degradation. This is based on the finding that intraventricular injections of captoprll increased cerebral levels of this peptide in particular in zonal recitulata of subtantia nigra [12]. Moreover, striatal lesions carried out using Ibotenlc or kainic acid showed that angiotensin converting enzyme in the substantia nigra is present on presynaptlc terminals of axons originating the caudate putamen [ 1 3 , 14]. Although, it was reported that purified angiotensln converting enzyme (free enzyme form) can also hydrolyse substance P in vitro [6, 8], in the present experiments, captopril failed to produce any significant influence on the substance P cleavage by nigral synaptic membranes (bound enzyme form). This result may be interpreted that angiotensin converting enzyme does not play an essential role in physiological degradation of substance P in nerve terminals of striato-nigral pathway. On the other hand, this non-specific dipeptldyl carboxypeptidase releases mainly dipeptlde fragments from the unblocked-C terminal peptides [15]. This mechanism is confirmed in the present study by prevention of the substance P metabollte (Phe-Phe-Gly) degradation by captopril. However, the degradation of metabolites (SP 1-7) and (SP 1-9) were not or only little affected by captopril. It cannot be excluded that the N-termlnal part of substance P (Arg-Pro-Lys-Pro) plays an important role in determining the substrate specificity for the brain peptidyl dipeptidase A. Morevoer, a recent study has reported that purified angiotensin-converting enzyme fails to hydrolyse at the significant rate neurokinin A a tachykinin that shares an identical C terminal sequence as substance P but a different amino acids composition in N terminal sequence [16, 17]. Thus the physiological importance of angiotensin converting enzyme in substantia nigra remains to be established. In conclusion, These in vitro studies clearly imply that the metalloendopeptidase (EC 3.4.24.11) is the enzyme responsible for the first peptlde bond(s) hydrolyzed when substance P is incubated with the synaptlc membranes. Amlnopeptldase and anglotensln converting enzyme may play only se secondary role in catabolims of substance P metabolltes. However, whether substance P is degraded in substantla nigra by endopeptldase 3.4.24.11 remains to be established in vivo. Further studies including the immunohistochemical determination of the exact enzymatic localization to substance P pathway and the prolongation of the biological actions of substance P in vivo by preventing its degradation witth selective metalloendopeptidase (EC 3.4.24.11) inhibltors are needed.
AcknowledKements. We thank Dr. fragments.
K.G.
Lloyd
and Mrs Voltz
for amlno-acld analysis of peptlde
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I. 2.
0BLIN, A., ZIVKOVIC, B. and G. BARTHOLINI, Brain Res. 421 387-390 (1987). ZIVKOVIC, B., OBLIN, A. and G. BARTHOLINI, Eur. J. Pharmacol.ll2 253-255 (1985).
1474
3. 4. 5. 6.
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Endopeptidase 24-11 Inactivates Substance P
Vol. 44, No. 20, 1989
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(1984). 15. ERDOS, E.G. Handbook of gxvertmental Pharmacology, pp. 427-484, Springer, Heidelberg (1979). 16. HOOPER, N.M., KENNY, A.J. and A.J. TURNER, Blochem. J. 231 357-361 (1985) 17. HOOPER, N.M. and A.J. TURNER, Biochem J. 241 625-633 (1987).