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Degradation of substance P by neuronal and glial cells cultured from rat fetal brain and their membranes
Neuropeptides (1989) 14,31-37 0 Longman Group UK Ltd 1989
0143-4179/89/0014-dO31/$10.00
Degradation of Substance P by Neuronal and GlIial Cells Cultured from Rat Fetal Brain and their Membranes S. ENDO. H. YOKOSAWA and S. ISHIt Department of Biochemistry, Faculty of Pharmaceutical Japan. (Reprint requests to H. Y.)
Sciences, Hokkaido University, Sapporo 060,
Abstract-By HPLC analysis, neuronal and glial cells cultured from rat fetal brain and their membrane preparations were shown to degrade substance P (SP) added exogenously. ~The degradation by neuronal cells and their membranes resulted in marked accumulation dr SP fragments (1-4) and (l-6), and the accumulation, as well as the initial cleavage of SP, was strongly inhibited by metal chelators but not by phosphoramidon and captopril. Prop0 ed cleavage sites by neuronal cells were almost identical to those by a substance P-degra ing endopeptidase previously purified from rat brain by us (J. Biochem. 104: 999-1006 (19 )). On the other hand, the action of glial cells and their membranes on SP produced the fragm i nts (l-6), (l-4), (10-l’)) and (8-9) in high amounts. The production, as well as the initial cleavage of SP, was inhibited not only by metal chelators and p-chloromercuribenzenesulfonic acid1but also by phosphoramidon. Proposed cleavage sites by glial cells were almost identical to those attacked by endopeptidase-24.11. Thus, the proteases that degrade SP in neuronal cellsland glial cells seem different.
Introduction
degradation in the synapse, the enzymes involved have not been well elucidated. Membrane-bound proteases seem to ‘be involved in the degradation of SP just as lacetylcholinesterase degrades acetylcholine !n the synapse. Several membrane-bound proteases in brain have been reported to be involved1 in the degradation of SP. These enzymes include a substance P-degrading enzyme (EC 3.4.24.;) from human brain (3), endopeptidase-24.11, (EC 3.4.24.11, enkephalinase) (4-6), angiotensm-converting enzyme (ACE, EC 3.4.15.1) (6-9); post-
Substance P (SP) is an undecapeptide with the sequence Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-GlyLeu-MetNHz. This peptide exists in the central and peripheral nervous systems as well as in the gastrointestinal tract, and is thought to function as a neurotransmitter after secretion into the synapse (1, 2). Although the function of SP as a neurotransmitter is probably terminated by enzymatic Date received 31 January 1989 Date accepted 7 February 1989
31
32 proline dipeptidylaminopeptidase (EC 3.4.14.-) from guinea pig brain synaptic membranes (10) and a substance P-degrading endopeptidase from rat brain (11). In order to determine which proteases among all those above would principally function in initiating the cleavage of SP in the synapse, we used neuroblastoma N-18 cells (12) and glioma C6 cells (Endo et al., unpublished) as models of the neurons and glia, respectively, and investigated the degradation of SP by these cells and their plasma membranes. On the basis of our experimental results with the former cells, we have proposed that metalloendopeptidase(s) distinct from either endopeptidase-24.11 or ACE may play a key role in initiating the degradation of SP by neurons (12). We succeeded in purifying this key enzyme from the rat brain. It is a substance P-degrading endopeptidase, distinct from all the proteases that had been reported to cleave SP (11). Results obtained with glioma cells suggested that endopeptidase-24.11 initiates the cleavage of SP in glia (Endo et al., unpublished), but it does not seem to be very active in brain synaptic membranes (11). In this paper, we examined the degradation of SP by primary cultures enriched either in the neuronal cells or glial cells and by membranes from these cells. We expected that these cell cultures were more typical of the brain than the established cell lines used in our previous studies. Materials SP and phosphoramidon were purchased from the Peptide Institute Inc., Osaka, Japan. SP fragp-chloromercuribenzenesulfonic acid ments, (PCMBS) , diisopropylfluorophosphate (DFP) and poly-D-Lys HBr (M.W. 30000-70000) were obtained from Sigma Chemical Co., St. Louis, MO, USA. Methanesulfonic acid (4M) containing 0.2% 3-(2-aminoethyl)indole was purchased from Pierce Chemical Co., Rockford, IL, USA. Captopril was kindly donated by Dr A. Awaya of Mitsui Pharmaceuticals, Inc., Tokyo, Japan. Methods Cell culture Cell suspensions were prepared from forebrains of
NEUROPEPTIDES
1518-day rat fetuses according to the method of Arimatsu and Hatanaka (13). The cells were cultured at 37°C under 10% COG-90% air in poly-D-Lys coated plastic dishes (Falcon) with Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 5% (v/v) heat-inactivated horse serum (GIBCO), 5% (v/v) fetal calf serum (GIBCO), SOIU/ml penicillin G (Meiji Seika, Japan) and 0.1 mg/ml streptomycin sulfate (Meihi Seika, Japan). The cultures enriched in neurons, which we called neurons in this paper, were prepared by treatment of primary cultured cells with 10l.r.M arabinosylcytosine (Sigma) for 24hr (14). Cultures enriched in glia, glial cells, were obtained by subculturing the confluent primary cultures without arabinosylcytosine treatment. Isolation of membranes from primary cultures All procedures were done at 4°C. Cells cultured in petri dishes were washed with 5mM HEPESNaOH buffer (pH 7.4) containing 155NaC1, 5.4mM KCI, 1.8mM CaCl2 and 0.8mM MgCl2 (HEPES Buffer) without detaching the cells from the dishes. Then, the cells were detached with a cell scraper, suspended in the above solution, and centrifuged at 100 x g for 3 min. Ten volumes of 1OmM Tris-HCl buffer (pH 7.5) containing 10% (w/v) sucrose were added to the resulting pellet, and the cells were homogenized with a Teflon homogenizer. The homogenate was centrifuged at 800 x g for 20 min and then at 9 000 x g for 20 min. The resulting pellet was suspended in 20mM Tris-HCl buffer containing 10% (w/v) sucrose and again centrifuged at 9000 x g for 20 min. The pellet was suspended in HEPES Buffer. Degradation of SP by cultured cells and their membranes The cells were washed five times with HEPES Buffer without detaching them from their 16mm petri dishes. Four hundred ~1 of HEPES Buffer and 50 ~1 of inhibitor solution or of HEPES Buffer were then added to the dishes. After the mixture (0.45ml) was preincubated at 37°C for 30 min, 501.~1of 0.5mM SP dissolved in HEPES Buffer were added to the dishes, and the reaction was allowed to proceed at 37°C under 10% CO&Xl% air (final pH of the solution was 7.4). Aliquots of
DEGRADATION
OF SUBSTANCE P BY NEURONAL
33
AND GLIAL CELLS
the reaction mixture (lOOt.~l)were withdrawn at various times and heated at 100°C for 5 min to stop the reaction. Then the sample was centrifuged, filtered through a membrane filter (Millipore Millex-HV, pore size 0.45pm) and subjected to high-performance liquid chromatography (HPLC) for analysis of the cleavage products. The HPLC was carried out under the conditions described in the legend to Figure 1. The degradation of SP by membranes prepared from cultured cells was carried out at 37°C in a reaction mixture (0.1 ml) consisting of 70~1 of HEPES Buffer, 10~1 of 0.5mM SP and 60-12Opg of protein from the membrane preparation. The reaction was terminated by heating at 100°C for 5 min. The reaction mixture was treated and subjected to HPLC analysis as described above. Protein determination
Protein was determined by the method of Bradford (15) using bovine serum albumin (Sigma) as a standard.
Retention
time (min)
Pig 1 Degradation of SP by neurons. The neuronal IIs were incubated at 37°C for 6hr in the absence of inhibitor a) and in t the presence of 1mM EDTA (b), 1mM o-phenanth line (c) and 1Oa phosphoramidon (d). Aliquots in a volum of SOJ were subjected to HPLC using a reversed-phase lumn of Nucleosil 5Crs (Marchery, Nagel and Co.) with a 16 ’ linear concentration gradient of l-65% acetonitrile in .l% trifluoroacetic acid at a flow rate of 1 mUmin. The abso1 bance at 210~1 was monitored. The peaks A to K in (a) rep nt the fragments identified. The peaks i and n represe t those originated from inhibitors and cells, respectively. Th9 arrows indicated the peaks whose appearance was inhibited in the presence of inhibitors.
Results
The similarity between the experimental ~results obtained with neurons and their membran s sugincubation with neurons or their membranes in the gested that the degradation of SP by th cells presence or absence of protease inhibitors, and the would probably occur at the plasma me i rane. reaction products were analyzed by HPLC (Figs 1 Observation of the same metal-chelator-suscepand 2). In the absence of inhibitors (Figs la and tible cleavage products in both of the experfments 2a), the area of peak P (SP) decreased as a function (indicated by arrows in Figures 1 and 2 and also by of time, whereas those of other peaks (A to K in thick lines in Figures 3a and 3b) also suggested that Figure 1 and A to L in Figure 2, except for peaks the cells cleaved SP at the bonds between Pro4originating from cells) increased. The analytical Gin’, Gln5-Gln6 and Gln6-Phe’ probably by the results on ammo acid compositions of the cleavage action of a protease present in the plasma 1memproducts isolated by HPLC, together with the brane. These cleavage sites were identical to the comparison of their chromatographic retention target of the substance P-degrading endope tidase times with those for authentic SP fragments, that we previously purified from rat brain and was allowed the assignment of these products as shown shown to be susceptible to metal chelators but not in Figures 3a and 3b. Fragments (l-4) and (l-6), to phosphoramidon (11). The effects of various and free phenylzlanine were the major products inhibitors on the initial cleavage of SP by neuronal produced by the cells and their membranes. The membranes - determined by the decrease of peak appearance of these products was markedly P in HPLC - are shown in the Table. The ‘initial depressed by the addition of metal-chelators, cleavage of SP by neurons was strongly ’ ‘bited EDTA or o-phenanthroline, to the reaction mix- by metal chelators, as well as PCMBS, but‘4 eakly ture (Figs lb, lc and 2c), whereas the addition of by captopril and phosphoramidon. Thus, a etalphosphoramidon, an effective inhibitor of endo- chelator-sensitive but phosphoramidon- ‘“,senpeptidase-24.11, had little effect (Figs Id and 2b). sitive metalloprotease, probably, the subdtance The digestion of SP was allowed to proceed by
d
34
NEUROPEI’TIDES
P-degrading endopeptidase (ll), may function in initiating cleavage of SP by neurons cultured from 5 rat fetal brain. p 0.021 1 iu I I Next, degradation of SP by glia and their membranes was analyzed by HPLC (Figs 4 and 5), and the cleavage products were isolated (A to I in Figure 4 and A to K in Figure 5) and characterized (Figs 6a and 6b). The area of peak P of SP decreased as a function of time in the absence of inhibitors (Fig 4a and 5a). Among the products identified when cells or membranes were incuRetention time (min) bated with SP, fragments (l-6), (l-4), (10-11) and Fig 2 Degradation of SP by membrane preparation of (8-9) were found to accumulate in high amounts, neurons. SP was degraded by membrane preparation (6Ot.~g together with free phenylalanine. In the presence protein) for 3 hr in the absence of inhibitor (a) and in the of either metal chelators or phosphoramidon, the presence of 10 PM phosphoramidon (b), 1 mM EDTA (c) and formation of major fragments from SP was 1 mM PCMBS (d). Aliquots in a volume of SO+1were analyzed strongly inhibited (Figs 4b, 4c, 4d, 5c and 5d). The by HPLC as in Fig 1. The peaks A to L in (a) represent the fragments identified. The peak i represents that originated from cleavage sites, the bonds between Gln6-Phe’, the inhibitor. The arrows indicated the peaks whose appearPhe’-Phe’ and Gly’-Leu”, proposed on the basis ance was inhibited in the presence of inhibitors. of finding on the phosphoramidon-susceptible peaks (indicated by arrows in Figures 4 and 5 and also by thick lines in Figures 6a and 6b) were identical to those known as the targets of the action of endopeptidase-24.11(4-6,16). The initial cleavage of SP by glial membranes was also susceptible to metal chelators and phosphoramidon (see 2 3 4 5 6 7 6 9 10 11 Table). Other inhibitors such as thiol-blocking (a) reagents and DFP showed substantial inhibitory effects too, whereas catopril and Z-Gly-ProCH$l did not (Table). These results suggested (a)
(b)
I J(6)
t
21
I W
I
I I
A(12)
I
B(27) CW
II *
Table Effects of Various Protease Inhibitors on The Degradation of SP by The Membranes Prepared from Neurons or Glia
I
Inhibitor
lap;),-
Ft4 ,
I I
F(6) GO
/
~__f!!)-_;
Fig3 Summary of cleavage products of SP by neurons (a) and ,.. ~_ their membranes (b). Numbers in parentheses represent yields of cleavage products determined on the basis of SP degraded (extents of degradation of SP were 63% (a) and 53% (b)). Fragments indicated by thick lines represent those whose appearance was inhibited by EDTA.
o-Phenanthroline EDTA Phosphoramidon PCMBS Iodoacetic acid DFP Chymostatin Leupeptin Captopril Z-Gly-Pro-CH&l
Concentration WW 1.0 1.0 0.01 1.0 1.0 0.1 0.01 0.01 0.01 0.01
Inhibition (%) Neurons Gfia 50 59 26 80 18 31 18 6 23 26
56 57 48 12 39 52 26 31 0 5
The extent of degradation of SP was determined by measuring the decrease of SP detected by HPLC.
DEGRADATION
OF SUBSTANCE P BY NEURONAL
35
AND GLIAL CELLS
that a metal-chelator-sensitive and phosphoramidon-sensitive protease, probably endopeptidase24.11, present in the plasma membrane would play a principal role in the initial stage of cleavage of SP in the glia cultured from rat fetal brains. Discussion Though the architectural and functional interaction between the neurons and glia is not conserved in dissociated cultures enriched in one cell1or the
I
0
I
0
5
IO Retenlion
I
0
time
5
1
10
fmin)
Fig 4 Degradation of SP by gha. The glial cells were incubated at 37°C for 5hr in the absence of inhibitor (a) and in the presence of 1mM EDTA (b), 1OuM phosphoramidon (c) and 1 mM o-phenanthroline (d). Aliquots in a volume of 50~1 were subjected to HPLC as in Fig 1. The peaks A to I in (a) represent the fragments identified. The peaks i and g represent those originated from inhibitors and cells, respectively. The arrows indicate the peaks whose appearance was inhibited in the presence of inhibitors.
c
FM\ C_P(2or5)__4 i _ _H(2 --- or 4)
I ;
Summary of cleavage products of SP by glia l(a) and their membranes (b). Numbers in parentheses represent yields of cleavage products determined on the basis of SP d graded (extents of degradation of SP were 44% (a) and 83/o & (b)). Fragments indicated by thick lines represent phosphoramidonsensitive ones.
Fig 6
h-b4
00
I
5
IO Retentv3nOtime
5
10
(min)
Fig 5 Degradation of SP by membrane preparation of gha. SP was degraded by membrane preparation (1OO~g protein) for 2 hr in the absence (a) and in the presence of 1 mM PCMBS (b), 10uM phosphoramidon (c) and 1mM o-phenanthroline (d). Aliquots in a volume of 50~1 were subjected to HPLC as in Fig 1. The peaks A to K in (a) represent the fragments identified. The peak i represents that originated from the inhibitor. The arrows indicated the peaks whose appearance was inhibited in the presence of inhibitors.
other, the cultured cells retain their characteristic features. These include a morphological pattern, synaptogenesis, receptor distribution and 60 on (17). Furthermore, it has been reported that they are useful models for the analysis of the degradation of neuropeptides (M-20). Horsthemke et al. (20) have previously examined the degradation of SP by neuronal and glial cells of rat brain in primary cultures in the presence of various inhibitors and reported that endo eptidase-24.11, which had been shown to be g, eterogeneously distributed in neuronal and glial cells
36 (19), was involved in the degradation of SP only in the glia cell, whereas post-proline dipeptidylaminopeptidase (EC 3.4.14.5) and a bacitracin-sensitive protease catalyzed the degradation of SP in both of the neuronal and glial cells. In this paper, we also investigated the degradation of SP by cultures enriched in neurons and glia from rat fetal brain and obtained further evidence that membrane-bound proteases playing key roles in the degradation of SP are diierent in neuronal and glial cells. While Horsthemke et al. did not analyze the cleavage products of SP in their study (20), we separated the major products by HPLC and identified them in order to define the protease involved in the SP degradation by each of the cells. SP added exogenously was found to be degraded by both neurons and glia in our study (Figs 1 and 4), as observed by Horsthemke et al. (19). Metal chelating reagents inhibited SP degradation in either of the cells markedly, but phosphoramidon was a strong inhibitor only of the glial enzyme. The fragments, whose formation was inhibited by corresponded well to those phosphoramidon, reported as the products from SP by the action of endopeptidase-24.11 (4-6,16). These results confirm the previous suggestion (N-20) that endopeptidase-24.11 is not evenly distributed between glia and neurons. We further analyzed the mode of degradation by membranes of neurons and glia in the presence and absence of various protease inhibitors (Fig 2 and 5), in order to determine the subcellular location of proteases measured in intact cells. Almost the same results were obtained with the cells and their respective membranes with respect to the products of SP degradation and inhibitor-sensitivities. Thus, the heterogeneous distribution of endopeptidase-24.11 between neurons and glia was reflected in the properties of their respective membranes. Recently, we have purified a novel substance P-degrading endopeptidase from rat brain and proposed that this enzyme plays an important role in degrading SP in the synapse (11). The inhibitorsusceptivities and the sites of cleavage by the neurons were quite similar to those observed when the substance P-degrading endopeptidase acts on SP. Therefore, this substance P-degrading endopeptidase would play a key role in degrading SP released into neuronal cultures from rat fetal
NEUROPEPTIDES
brain. Although further studies using specific inhibitors of this enzyme are necessary to clarify its precise role in the degradation of SP, we would like to propose a principal role of the substance P-degrading endopeptidase, abundant in the neurons, in initiating degradation of SP in the synapse and a role of endopeptidase-24.11 present abundantly in glia as a “scavenger” in the SP degradation.
Acknowledgements We wish to thank Dr. I. Matsuoka and Professor K. Kurihara of Hokkaido University for their help for preparing primary cultures and for their helpful discussion. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (H.Y., SE. and S.I.), from the Research Foundation for Pharmaceutical Sciences (H.Y .) and from the Japan Foundation for Applied Enzymolozy (H.Y.). S. Endo is a recipient of a fellowship of Japan Society for the Promotion of Sciences for Japanese Junior Scientists.
References 1. Nicoll, R. A., Schenker, C. and Leeman, S. E. (1980). Substance P as a transmitter candidate. Ann. Rev. Neurosci. 3: 227-268. 2. Pemow, B. (1980). Substance P. Pharmacol. Rev. 35: 85-141. 3. Lee, C.-M., Sandberg, B. E. B., Hanley, M. R. and Iversen, L. L. (1980). Purification and characterisation of a membrane-bound substance-P-degrading enzyme from human brain. Eur. J. Biochem. 114: 315-327. 4. Matsus, R., Fulcher, I. S., Kenny, A. J. and Turner, A. J. (1983). Substance P and [Leulenkephalin are hydrolyzed by an enzyme in pig caudate synaptic membranes that is identical with the endopeptidase of kidney microviUi. Proc. Natl. Acad. Sci. USA 80: 3111-3115. 5. Matsus, R., Kenny, A. J. and Turner, A. J. (1984). The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11. Biochem. J. 223: 433-440. 6. Skidgel, R. A., Engelbrecht, S., Johnson, A. R. and Erdos, E. G. (1984). Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase. Peptides 5: 769-776. 7. Yokosawa, H., Endo, S., Ogura, Y. and Ishii, S. (1983). A new feature of angiotensin-converting enzyme: hydrolysis of substance P. B&hem. Biophys. Res. Commun. 116: 735-742. 8. Strittmatter, S. M., Thiele, E. A., Kapiloff, M. S. and Snyder, S. H. (1985). A rat brain isozyme of angiotensinconverting enzyme. J. Biol. Chem. 260: 9825-9832.
DEGRADATION
OF SUBSTANCE P BY NEURONAL AND GLIAL CELLS
9. Hooper, N. M. and Turner, A. J. (1987). Isolation of two differentially gfycosylated forms of peptidyl-dipeptidase A (angiotensin-converting enzyme) from pig brain: a reevaluation of their role in neuropeptide metabolism. Biochem. J. 241: 625633. 10. O’Connor, B. and O’Cuinn, G. (1986). Post-proline didpeptidyl-aminopeptidase from synaptosomal membranes of guinea-pig brain. Eur. J. B&hem. 154: 329-335. 11. Endo, S., Yokosawa, H. and Ishii, S. (1988). Purification and characterization of substance P-degrading endopeptidase from rat brain. J. Biochem. (Tokyo) 104: 999-1006. 12. Endo, S., Yokosawa, H. and Ishii, S. (1985). Degradation of substance P by the neuroblastoma cells and their membrane. B&hem. Biophys. Res. Commun. 129: 684700. 13. Arimatsu, Y. and Hatanaka, H. (1986). Estrogen treatment enhances survival of cultured fetal rat amygdala neurons in a defined medium. Dev. Brain Res. 26: 151-159. 14. Dichter, M. A. (1978). Rat cortical neurons in cell culture: culture methods, cell morphology, electrophysiology, and synapse formation. Brain Res. 149: 279-293.
15. Badford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. B&hem. 72: 248-254. 16. Matsus, R., Kenny, A. J. and Turner, A. J. (1985). Endopeptidase-24.11 in human synaptic membrane preparation hydrolyzes substance P. B&hem. J. 228: 487-492. 17. Thomas, W. E. (1986). Studies of neurotratrsmitter chemistry of central nervous system neurons in primary tissue culture. Life Sci. 38: 297-308. 18. Lentzen, H. and Palenker, J. (1983). Localization of the thiorphan-sensitive endopeptidase, termed enkephalinase A, on gha cells. PEBS Lett. 153: 93-97. 19. Horsthemke, B., Hamprecht, B. and Bauer, K. ,(1983). Heterogeneous distribution of enkephalin-degradmg peptidases between neuronal and ghal cells. Biochem. Biophys. Res. Commun. 115: 423-429. 20. Horsthemke, B., Schulz, M. and Bauer, K. (1984).lDegradation of substance P by neurons and ghal cells. Bhxhem. Biophys. Res. Commun. 125: 728-733.
0143-4179/89/0014-dO31/$10.00
Degradation of Substance P by Neuronal and GlIial Cells Cultured from Rat Fetal Brain and their Membranes S. ENDO. H. YOKOSAWA and S. ISHIt Department of Biochemistry, Faculty of Pharmaceutical Japan. (Reprint requests to H. Y.)
Sciences, Hokkaido University, Sapporo 060,
Abstract-By HPLC analysis, neuronal and glial cells cultured from rat fetal brain and their membrane preparations were shown to degrade substance P (SP) added exogenously. ~The degradation by neuronal cells and their membranes resulted in marked accumulation dr SP fragments (1-4) and (l-6), and the accumulation, as well as the initial cleavage of SP, was strongly inhibited by metal chelators but not by phosphoramidon and captopril. Prop0 ed cleavage sites by neuronal cells were almost identical to those by a substance P-degra ing endopeptidase previously purified from rat brain by us (J. Biochem. 104: 999-1006 (19 )). On the other hand, the action of glial cells and their membranes on SP produced the fragm i nts (l-6), (l-4), (10-l’)) and (8-9) in high amounts. The production, as well as the initial cleavage of SP, was inhibited not only by metal chelators and p-chloromercuribenzenesulfonic acid1but also by phosphoramidon. Proposed cleavage sites by glial cells were almost identical to those attacked by endopeptidase-24.11. Thus, the proteases that degrade SP in neuronal cellsland glial cells seem different.
Introduction
degradation in the synapse, the enzymes involved have not been well elucidated. Membrane-bound proteases seem to ‘be involved in the degradation of SP just as lacetylcholinesterase degrades acetylcholine !n the synapse. Several membrane-bound proteases in brain have been reported to be involved1 in the degradation of SP. These enzymes include a substance P-degrading enzyme (EC 3.4.24.;) from human brain (3), endopeptidase-24.11, (EC 3.4.24.11, enkephalinase) (4-6), angiotensm-converting enzyme (ACE, EC 3.4.15.1) (6-9); post-
Substance P (SP) is an undecapeptide with the sequence Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-GlyLeu-MetNHz. This peptide exists in the central and peripheral nervous systems as well as in the gastrointestinal tract, and is thought to function as a neurotransmitter after secretion into the synapse (1, 2). Although the function of SP as a neurotransmitter is probably terminated by enzymatic Date received 31 January 1989 Date accepted 7 February 1989
31
32 proline dipeptidylaminopeptidase (EC 3.4.14.-) from guinea pig brain synaptic membranes (10) and a substance P-degrading endopeptidase from rat brain (11). In order to determine which proteases among all those above would principally function in initiating the cleavage of SP in the synapse, we used neuroblastoma N-18 cells (12) and glioma C6 cells (Endo et al., unpublished) as models of the neurons and glia, respectively, and investigated the degradation of SP by these cells and their plasma membranes. On the basis of our experimental results with the former cells, we have proposed that metalloendopeptidase(s) distinct from either endopeptidase-24.11 or ACE may play a key role in initiating the degradation of SP by neurons (12). We succeeded in purifying this key enzyme from the rat brain. It is a substance P-degrading endopeptidase, distinct from all the proteases that had been reported to cleave SP (11). Results obtained with glioma cells suggested that endopeptidase-24.11 initiates the cleavage of SP in glia (Endo et al., unpublished), but it does not seem to be very active in brain synaptic membranes (11). In this paper, we examined the degradation of SP by primary cultures enriched either in the neuronal cells or glial cells and by membranes from these cells. We expected that these cell cultures were more typical of the brain than the established cell lines used in our previous studies. Materials SP and phosphoramidon were purchased from the Peptide Institute Inc., Osaka, Japan. SP fragp-chloromercuribenzenesulfonic acid ments, (PCMBS) , diisopropylfluorophosphate (DFP) and poly-D-Lys HBr (M.W. 30000-70000) were obtained from Sigma Chemical Co., St. Louis, MO, USA. Methanesulfonic acid (4M) containing 0.2% 3-(2-aminoethyl)indole was purchased from Pierce Chemical Co., Rockford, IL, USA. Captopril was kindly donated by Dr A. Awaya of Mitsui Pharmaceuticals, Inc., Tokyo, Japan. Methods Cell culture Cell suspensions were prepared from forebrains of
NEUROPEPTIDES
1518-day rat fetuses according to the method of Arimatsu and Hatanaka (13). The cells were cultured at 37°C under 10% COG-90% air in poly-D-Lys coated plastic dishes (Falcon) with Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 5% (v/v) heat-inactivated horse serum (GIBCO), 5% (v/v) fetal calf serum (GIBCO), SOIU/ml penicillin G (Meiji Seika, Japan) and 0.1 mg/ml streptomycin sulfate (Meihi Seika, Japan). The cultures enriched in neurons, which we called neurons in this paper, were prepared by treatment of primary cultured cells with 10l.r.M arabinosylcytosine (Sigma) for 24hr (14). Cultures enriched in glia, glial cells, were obtained by subculturing the confluent primary cultures without arabinosylcytosine treatment. Isolation of membranes from primary cultures All procedures were done at 4°C. Cells cultured in petri dishes were washed with 5mM HEPESNaOH buffer (pH 7.4) containing 155NaC1, 5.4mM KCI, 1.8mM CaCl2 and 0.8mM MgCl2 (HEPES Buffer) without detaching the cells from the dishes. Then, the cells were detached with a cell scraper, suspended in the above solution, and centrifuged at 100 x g for 3 min. Ten volumes of 1OmM Tris-HCl buffer (pH 7.5) containing 10% (w/v) sucrose were added to the resulting pellet, and the cells were homogenized with a Teflon homogenizer. The homogenate was centrifuged at 800 x g for 20 min and then at 9 000 x g for 20 min. The resulting pellet was suspended in 20mM Tris-HCl buffer containing 10% (w/v) sucrose and again centrifuged at 9000 x g for 20 min. The pellet was suspended in HEPES Buffer. Degradation of SP by cultured cells and their membranes The cells were washed five times with HEPES Buffer without detaching them from their 16mm petri dishes. Four hundred ~1 of HEPES Buffer and 50 ~1 of inhibitor solution or of HEPES Buffer were then added to the dishes. After the mixture (0.45ml) was preincubated at 37°C for 30 min, 501.~1of 0.5mM SP dissolved in HEPES Buffer were added to the dishes, and the reaction was allowed to proceed at 37°C under 10% CO&Xl% air (final pH of the solution was 7.4). Aliquots of
DEGRADATION
OF SUBSTANCE P BY NEURONAL
33
AND GLIAL CELLS
the reaction mixture (lOOt.~l)were withdrawn at various times and heated at 100°C for 5 min to stop the reaction. Then the sample was centrifuged, filtered through a membrane filter (Millipore Millex-HV, pore size 0.45pm) and subjected to high-performance liquid chromatography (HPLC) for analysis of the cleavage products. The HPLC was carried out under the conditions described in the legend to Figure 1. The degradation of SP by membranes prepared from cultured cells was carried out at 37°C in a reaction mixture (0.1 ml) consisting of 70~1 of HEPES Buffer, 10~1 of 0.5mM SP and 60-12Opg of protein from the membrane preparation. The reaction was terminated by heating at 100°C for 5 min. The reaction mixture was treated and subjected to HPLC analysis as described above. Protein determination
Protein was determined by the method of Bradford (15) using bovine serum albumin (Sigma) as a standard.
Retention
time (min)
Pig 1 Degradation of SP by neurons. The neuronal IIs were incubated at 37°C for 6hr in the absence of inhibitor a) and in t the presence of 1mM EDTA (b), 1mM o-phenanth line (c) and 1Oa phosphoramidon (d). Aliquots in a volum of SOJ were subjected to HPLC using a reversed-phase lumn of Nucleosil 5Crs (Marchery, Nagel and Co.) with a 16 ’ linear concentration gradient of l-65% acetonitrile in .l% trifluoroacetic acid at a flow rate of 1 mUmin. The abso1 bance at 210~1 was monitored. The peaks A to K in (a) rep nt the fragments identified. The peaks i and n represe t those originated from inhibitors and cells, respectively. Th9 arrows indicated the peaks whose appearance was inhibited in the presence of inhibitors.
Results
The similarity between the experimental ~results obtained with neurons and their membran s sugincubation with neurons or their membranes in the gested that the degradation of SP by th cells presence or absence of protease inhibitors, and the would probably occur at the plasma me i rane. reaction products were analyzed by HPLC (Figs 1 Observation of the same metal-chelator-suscepand 2). In the absence of inhibitors (Figs la and tible cleavage products in both of the experfments 2a), the area of peak P (SP) decreased as a function (indicated by arrows in Figures 1 and 2 and also by of time, whereas those of other peaks (A to K in thick lines in Figures 3a and 3b) also suggested that Figure 1 and A to L in Figure 2, except for peaks the cells cleaved SP at the bonds between Pro4originating from cells) increased. The analytical Gin’, Gln5-Gln6 and Gln6-Phe’ probably by the results on ammo acid compositions of the cleavage action of a protease present in the plasma 1memproducts isolated by HPLC, together with the brane. These cleavage sites were identical to the comparison of their chromatographic retention target of the substance P-degrading endope tidase times with those for authentic SP fragments, that we previously purified from rat brain and was allowed the assignment of these products as shown shown to be susceptible to metal chelators but not in Figures 3a and 3b. Fragments (l-4) and (l-6), to phosphoramidon (11). The effects of various and free phenylzlanine were the major products inhibitors on the initial cleavage of SP by neuronal produced by the cells and their membranes. The membranes - determined by the decrease of peak appearance of these products was markedly P in HPLC - are shown in the Table. The ‘initial depressed by the addition of metal-chelators, cleavage of SP by neurons was strongly ’ ‘bited EDTA or o-phenanthroline, to the reaction mix- by metal chelators, as well as PCMBS, but‘4 eakly ture (Figs lb, lc and 2c), whereas the addition of by captopril and phosphoramidon. Thus, a etalphosphoramidon, an effective inhibitor of endo- chelator-sensitive but phosphoramidon- ‘“,senpeptidase-24.11, had little effect (Figs Id and 2b). sitive metalloprotease, probably, the subdtance The digestion of SP was allowed to proceed by
d
34
NEUROPEI’TIDES
P-degrading endopeptidase (ll), may function in initiating cleavage of SP by neurons cultured from 5 rat fetal brain. p 0.021 1 iu I I Next, degradation of SP by glia and their membranes was analyzed by HPLC (Figs 4 and 5), and the cleavage products were isolated (A to I in Figure 4 and A to K in Figure 5) and characterized (Figs 6a and 6b). The area of peak P of SP decreased as a function of time in the absence of inhibitors (Fig 4a and 5a). Among the products identified when cells or membranes were incuRetention time (min) bated with SP, fragments (l-6), (l-4), (10-11) and Fig 2 Degradation of SP by membrane preparation of (8-9) were found to accumulate in high amounts, neurons. SP was degraded by membrane preparation (6Ot.~g together with free phenylalanine. In the presence protein) for 3 hr in the absence of inhibitor (a) and in the of either metal chelators or phosphoramidon, the presence of 10 PM phosphoramidon (b), 1 mM EDTA (c) and formation of major fragments from SP was 1 mM PCMBS (d). Aliquots in a volume of SO+1were analyzed strongly inhibited (Figs 4b, 4c, 4d, 5c and 5d). The by HPLC as in Fig 1. The peaks A to L in (a) represent the fragments identified. The peak i represents that originated from cleavage sites, the bonds between Gln6-Phe’, the inhibitor. The arrows indicated the peaks whose appearPhe’-Phe’ and Gly’-Leu”, proposed on the basis ance was inhibited in the presence of inhibitors. of finding on the phosphoramidon-susceptible peaks (indicated by arrows in Figures 4 and 5 and also by thick lines in Figures 6a and 6b) were identical to those known as the targets of the action of endopeptidase-24.11(4-6,16). The initial cleavage of SP by glial membranes was also susceptible to metal chelators and phosphoramidon (see 2 3 4 5 6 7 6 9 10 11 Table). Other inhibitors such as thiol-blocking (a) reagents and DFP showed substantial inhibitory effects too, whereas catopril and Z-Gly-ProCH$l did not (Table). These results suggested (a)
(b)
I J(6)
t
21
I W
I
I I
A(12)
I
B(27) CW
II *
Table Effects of Various Protease Inhibitors on The Degradation of SP by The Membranes Prepared from Neurons or Glia
I
Inhibitor
lap;),-
Ft4 ,
I I
F(6) GO
/
~__f!!)-_;
Fig3 Summary of cleavage products of SP by neurons (a) and ,.. ~_ their membranes (b). Numbers in parentheses represent yields of cleavage products determined on the basis of SP degraded (extents of degradation of SP were 63% (a) and 53% (b)). Fragments indicated by thick lines represent those whose appearance was inhibited by EDTA.
o-Phenanthroline EDTA Phosphoramidon PCMBS Iodoacetic acid DFP Chymostatin Leupeptin Captopril Z-Gly-Pro-CH&l
Concentration WW 1.0 1.0 0.01 1.0 1.0 0.1 0.01 0.01 0.01 0.01
Inhibition (%) Neurons Gfia 50 59 26 80 18 31 18 6 23 26
56 57 48 12 39 52 26 31 0 5
The extent of degradation of SP was determined by measuring the decrease of SP detected by HPLC.
DEGRADATION
OF SUBSTANCE P BY NEURONAL
35
AND GLIAL CELLS
that a metal-chelator-sensitive and phosphoramidon-sensitive protease, probably endopeptidase24.11, present in the plasma membrane would play a principal role in the initial stage of cleavage of SP in the glia cultured from rat fetal brains. Discussion Though the architectural and functional interaction between the neurons and glia is not conserved in dissociated cultures enriched in one cell1or the
I
0
I
0
5
IO Retenlion
I
0
time
5
1
10
fmin)
Fig 4 Degradation of SP by gha. The glial cells were incubated at 37°C for 5hr in the absence of inhibitor (a) and in the presence of 1mM EDTA (b), 1OuM phosphoramidon (c) and 1 mM o-phenanthroline (d). Aliquots in a volume of 50~1 were subjected to HPLC as in Fig 1. The peaks A to I in (a) represent the fragments identified. The peaks i and g represent those originated from inhibitors and cells, respectively. The arrows indicate the peaks whose appearance was inhibited in the presence of inhibitors.
c
FM\ C_P(2or5)__4 i _ _H(2 --- or 4)
I ;
Summary of cleavage products of SP by glia l(a) and their membranes (b). Numbers in parentheses represent yields of cleavage products determined on the basis of SP d graded (extents of degradation of SP were 44% (a) and 83/o & (b)). Fragments indicated by thick lines represent phosphoramidonsensitive ones.
Fig 6
h-b4
00
I
5
IO Retentv3nOtime
5
10
(min)
Fig 5 Degradation of SP by membrane preparation of gha. SP was degraded by membrane preparation (1OO~g protein) for 2 hr in the absence (a) and in the presence of 1 mM PCMBS (b), 10uM phosphoramidon (c) and 1mM o-phenanthroline (d). Aliquots in a volume of 50~1 were subjected to HPLC as in Fig 1. The peaks A to K in (a) represent the fragments identified. The peak i represents that originated from the inhibitor. The arrows indicated the peaks whose appearance was inhibited in the presence of inhibitors.
other, the cultured cells retain their characteristic features. These include a morphological pattern, synaptogenesis, receptor distribution and 60 on (17). Furthermore, it has been reported that they are useful models for the analysis of the degradation of neuropeptides (M-20). Horsthemke et al. (20) have previously examined the degradation of SP by neuronal and glial cells of rat brain in primary cultures in the presence of various inhibitors and reported that endo eptidase-24.11, which had been shown to be g, eterogeneously distributed in neuronal and glial cells
36 (19), was involved in the degradation of SP only in the glia cell, whereas post-proline dipeptidylaminopeptidase (EC 3.4.14.5) and a bacitracin-sensitive protease catalyzed the degradation of SP in both of the neuronal and glial cells. In this paper, we also investigated the degradation of SP by cultures enriched in neurons and glia from rat fetal brain and obtained further evidence that membrane-bound proteases playing key roles in the degradation of SP are diierent in neuronal and glial cells. While Horsthemke et al. did not analyze the cleavage products of SP in their study (20), we separated the major products by HPLC and identified them in order to define the protease involved in the SP degradation by each of the cells. SP added exogenously was found to be degraded by both neurons and glia in our study (Figs 1 and 4), as observed by Horsthemke et al. (19). Metal chelating reagents inhibited SP degradation in either of the cells markedly, but phosphoramidon was a strong inhibitor only of the glial enzyme. The fragments, whose formation was inhibited by corresponded well to those phosphoramidon, reported as the products from SP by the action of endopeptidase-24.11 (4-6,16). These results confirm the previous suggestion (N-20) that endopeptidase-24.11 is not evenly distributed between glia and neurons. We further analyzed the mode of degradation by membranes of neurons and glia in the presence and absence of various protease inhibitors (Fig 2 and 5), in order to determine the subcellular location of proteases measured in intact cells. Almost the same results were obtained with the cells and their respective membranes with respect to the products of SP degradation and inhibitor-sensitivities. Thus, the heterogeneous distribution of endopeptidase-24.11 between neurons and glia was reflected in the properties of their respective membranes. Recently, we have purified a novel substance P-degrading endopeptidase from rat brain and proposed that this enzyme plays an important role in degrading SP in the synapse (11). The inhibitorsusceptivities and the sites of cleavage by the neurons were quite similar to those observed when the substance P-degrading endopeptidase acts on SP. Therefore, this substance P-degrading endopeptidase would play a key role in degrading SP released into neuronal cultures from rat fetal
NEUROPEPTIDES
brain. Although further studies using specific inhibitors of this enzyme are necessary to clarify its precise role in the degradation of SP, we would like to propose a principal role of the substance P-degrading endopeptidase, abundant in the neurons, in initiating degradation of SP in the synapse and a role of endopeptidase-24.11 present abundantly in glia as a “scavenger” in the SP degradation.
Acknowledgements We wish to thank Dr. I. Matsuoka and Professor K. Kurihara of Hokkaido University for their help for preparing primary cultures and for their helpful discussion. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (H.Y., SE. and S.I.), from the Research Foundation for Pharmaceutical Sciences (H.Y .) and from the Japan Foundation for Applied Enzymolozy (H.Y.). S. Endo is a recipient of a fellowship of Japan Society for the Promotion of Sciences for Japanese Junior Scientists.
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DEGRADATION
OF SUBSTANCE P BY NEURONAL AND GLIAL CELLS
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15. Badford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. B&hem. 72: 248-254. 16. Matsus, R., Kenny, A. J. and Turner, A. J. (1985). Endopeptidase-24.11 in human synaptic membrane preparation hydrolyzes substance P. B&hem. J. 228: 487-492. 17. Thomas, W. E. (1986). Studies of neurotratrsmitter chemistry of central nervous system neurons in primary tissue culture. Life Sci. 38: 297-308. 18. Lentzen, H. and Palenker, J. (1983). Localization of the thiorphan-sensitive endopeptidase, termed enkephalinase A, on gha cells. PEBS Lett. 153: 93-97. 19. Horsthemke, B., Hamprecht, B. and Bauer, K. ,(1983). Heterogeneous distribution of enkephalin-degradmg peptidases between neuronal and ghal cells. Biochem. Biophys. Res. Commun. 115: 423-429. 20. Horsthemke, B., Schulz, M. and Bauer, K. (1984).lDegradation of substance P by neurons and ghal cells. Bhxhem. Biophys. Res. Commun. 125: 728-733.