Naunyn-Schmiedeberg's
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 301, 1 7 - 2 2 (1977)
Pharmacology
9 by Springer-Verlag 1977
Substance P in Rat Brain Synaptosomes F. LEMBECK, N. MAYER, and G. SCHINDLER Institut ffir experimentelle und klinische Pharmakologie der Universitfit Graz, Universitfitsplatz 4, A-8010 Graz, Austria
Summary. 1. Rat brain synaptosomes were incubated under different conditions to study the release of substance P (SP). 2. Potassium ions and electrical field stimulation induced a loss of SP from synaptosomes. The release of SP by potassium in high concentrations (23.8 mM) was shown to be calcium dependent. 3. Substance P was retained in synaptosomes during incubation in 0.32 M sucrose at + 4 ~ up to 120 min. During incubation at 30~ the SP content fell initially (30 min) but was gradually restored (120 min). 4. If these pre-incubated synaptosomes were reincubated for 45 min at 30 ~C in potassium free KrebsRinger-phosphate buffer a further rise in their SP content occurred which was taken as indication that SP is being synthesized in synaptosomes. 5. The newly synthesized SP is presumably stored by binding to phosphatidyl serine until a sudden release is initiated by depolarization. Key words." Substance P - Synaptosomes.
INTRODUCTION All known putative transmitter substances were found to be present in synaptosomes. This was demonstrated for acetylcholine, noradrenaline, dopamine, 5-hydroxytryptamine, GABA, glycine, glutamate (Ryall, 1962; Ryall, 1964; Whittaker, 1970) as well as for histamine (Kuhar et al., 1971) and taurine (Schmied et al., 1975). A role of substance P as a central neurotransmitter has first been suggested by Lembeck (1953). Further evidence for this function of substance P was recently Send offprint requests to F. Lembeck at the above address
obtained in electrophysiological and histochemical studies (Konishi and Otsuka, 1974; Otsuka et al., 1975; H6kfelt et al., 1975; Cuello et al., 1976). Substance P has also been found in synaptosomal preparations (Lembeck and Holasek, 1960; Inouye and Kataoka, 1962; Schenker et al., 1976). The release of a neurotransmitter can be caused by depolarisation of the cell membrane. Thus incubation of synaptosomes in media containing depolarizing concentrations (50 raM) of potassium enhanced the release of acetylcholine (Haga, 1971) and GABA (De Belleroche and Bradford, 1972a). The release of noradrenaline and GABA from rat brain synaptosomes incubated in the presence of potassium (56 raM) was reported by Osborne et al. (1973). Transmitter release depends also on the calcium concentration in the medium as shown for acetylcholine by De Belleroche and Bradford (1972b) and Cooke et al. (1973), for noradrenaline by Hukovi6 and Muscholl (1962), Baldessarini and Kopin (1967) and Hfiusler et al. (1968) and for GABA by De Belleroche and Bradford (1972 a). The present experiments were performed to investigate (a) under which conditions synaptosomes are able to retain a constant amount of substance P, (b) if SP is released from synaptosomes by depolarisation (increase of the potassium concentration in the incubation medium or electrical field stimulation) and (c) if the release of SP is influenced by the calcium concentration in the medium. METHODS Preparation of Synaptosomes. Sprague Dawley rats (strain OFA, SD, SPF, both sexes, 150-250 g) were killed by a blow on the neck. The brains were quickly removed and weighed. All further manipulations were carried out at 4~ The brains were homogenized in 0.32 M sucrose (10 ml/g tissue) containing aprotinin (20 units/ml) to inhibit proteolytic enzymes and Cialit ' [(2-ethyl-mercury-mer-
18 capto)-benzoxazol-5-Na-carbonic acid] (2.5 rag/l) to inhibit bacterial growth. Synaptosomes were prepared according to the method of Gray and Whittaker (/962) by centrifuging of the resuspended (in 0.32 M sucrose) P2 fraction into a discontinuous density gradient (0.8 and 1.2 M sucrose) at 100000 g for 60 rain in a Beckman L-2 ultra centrifuge (SW 27 rotor). The mainly synaptosomes containing "Bfraction" (Gray and Whittaker, 1962) was removed by gentle suction with a motor driven syringe. In the foliowing text the term "synaptosomes" refers to this fraction. Protein was measured in the brain tissue to be 94.2 i 3.2 mg/g, in fraction Pz 39.8 _+ 2.4 and in fraction B 4.1 ,+ 0.2 mg/ml. The SP content per mg protein was found to be in brain tissue to be 0.8 ,+ 0.1 ng/mg in fraction P2 2.6 ,+ 0.4 ng/mg and in the fraction B 5.4 _+ 0.3 ng/mg.
Incubation. Five ml "synaptosomes" (containing on average 20.5 mg protein) were added to 10 ml of the incubation media. The final concentrations of ions in the incubation media were therefore 2/3 of the following solutions : 1. Sucrose, 0.32 M; 2. Krebs-Ringer-phosphate buffer (KR) pH 7.4 (118 mM NaC1, 4.75 mM KC1, 0.88 mM CaCI2, 1.18 mM MgSO~, 3.18ram NaHzPO4, 13.02mM NazHPO4); 3. KrebsRinger-phosphate buffer without KCI; 4. Krebs-Ringer-phosphate buffer with KC1 23.8 mM ; 5. Krebs-Ringer-phosphate buffer without CaCIz; 6. Krebs-Ringer-phosphate buffer wifh CaCI2 4.4 raM; 7. Krebs-Ringer-phosphate buffer with CaCI~ 8.8 mM. Inconsistencies in the amount of SP present in synaptosomal preparations which occurred in preliminary experiments could be prevented by inhibiting proteolytic enzymes with aprotinin (20 units/ ml). It was added to all incubation media. Aprotinin in the concentration of 0.5 units/ml has, however, recently be found to cause little inhibition of the in vitro degradation of SP (Skrabanek et al., 1977). More recently bacitracin was found to be an even better inhibitor of proteolysis (Cuello et al., 1977). It was added in a 20 gM concentration in addition to aprotinin in the experiments shown in Figure 4. As the incubation of synaptosomes lasted sometimes up to 165 min breakdown of SP by bacterial enzymes could take place. Thus a bactericidal substance (Cialit | 25 gg/ml) was added to all incubation media throughout the present experiments. The incubations were carried out at + 4 ~C or + 30~C in a shaking water bath and terminated by centrifugation at +4~ (Christ Zeta 20 refrigerated centrifuge, 20000 g, 10 min). SP was extracted from the resulting pellet. Depolarisation of synaptosomal membranes by electrical field stimulation was carried out in a 8 ml containing bath using 2 ring electrodes with a distance of 4 cm (square wave pulses 3 ms, 10 Hz, constant current of 0.5 mA). Extraction of Substance P from Synaptosomes. SP was extracted by the method of Chang and Leeman (1970) with slight modifications. The pellet obtained after centrifugation of the incubate was resuspended in a 0 . 9 ~ NaC1 solution; 0.3 ml 0.1 N HC1 and 10 ml acetone were added and the samples shaken for 18 h in a water bath at 4 ~C. The insoluble precipitate formed was removed by centrifugation and the supernatant freed from lipids by shaking with 25 ml petroleum ether (b.p. 4 0 - 60 ~C). The aqueous phase was evaporated in vacuo and the dry residue redissolved in 0.5 ml of an 0.9 ~ NaC1 solution which contained per 100 ml i mg bovine serum albumine and 0.05 mg Cialit | The pH was adjusted to 6 - 7 by addition of 5 ~o NaHCO3 solution shortly before bioassay. Bioassay. The bioassay of SP was performed on the isolated guinea pig ileum suspended in an oxygenated Tyrode solution at 37 ~C which contained per ml 0.1 gg atropine, 1.0 gg mepyramine and 0.1 lag methysergide. Synthetic SP was used as standard. Prostaglandins
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977) would have been removed by petroleum ether during the extraction (see above). Several samples of the fraction B preparation were tested on the rat duodenum which is relaxed by bradykinin like peptides; no relaxation was observed. It seems to be justified to regard the bioassay used as being reliable for the estimation of SP.
Protein Estimation. The amounts of protein contained in the tissue homogenates, the P2-fractions and the synaptosomal preparations were measured by the method of Lowry et al. (1951) using bovine serum albumine as standard. Materials. Substance P (Beckman Instruments, Palo Alto, California), Aprotinin (Bayer AG Leverkusen), Bacitracin (Biochemie G.m.b.H., Wien), Bovine serum albumine (Miles Inc., Kankakee, Ill.), Cialit | (2-ethyl-mercury-mercapto)-benzoxazol-5-Na-carbonic acid (Asid G.m.b.H., Miinchen), Methysergid bimaleate (Sandoz S.A., Basel), Mepyramine hydrochloride (Specia S.A., Paris), Atropine sulphate (Merck A.G., Darmstadt). All other chemicals used were of analytical grade and obtained from Merck A.G., Darmstadt. When a drug was used in form of a salt the dosen given refers to the "free substance".
RESULTS
Loss of Substance P from Synaptosomes in the Presence of High Potassium Concentrations (23.8mM). A Krebs-Ringer-phosphate buffer (23.8 mM KC1) was used for the depolarisation of the synaptosomal membranes. Synaptosomes incubated at 30~ in this K + rich buffer contained after 15 rain significantly (P < 0.001) less SP than synaptosomes incubated in normal KR (see Fig. 1A). Loss of Substance P from Synaptosomes by Electrical Field Stimulation. Synaptosomes suspended in normal KR and subjected to electrical field stimulation lost within 15 rain twice as much SP as unstimulated synaptosomes (P < 0.005, see Fig. 1 B). When expressed in per cent of the amount of SP originally present the loss after field stimulation ( - 5 0 ~o) was smaller than the loss which occurred in the presence of 23.8 mM K + ( - 63 ~, see Fig. 1A). Influence of the Calcium Concentration in the Incubation Medium on the Loss of Substance P from Synaptosomes. Synaptosomes were incubated for 15 rain at 30~ either in normal KR (controls) or in K + rich (23.8 raM) KR containing different concentrations of calcium (Fig. 2). 23.8 mM K + caused no change of SP in the absence of Ca 2+, when compared with normal Krebs-Ringer solution. Ca 2+ in concentrations between 0.88 and 8.8 mM enhanced the release of SP from the synaptosomes elicited by potassium. The magnitude of the SP loss was proportional to the Ca z + concentration in the incubation medium. Comparison between the Loss of Substance P from Synaptosomes Incubated at 4~ and 30~ At 4~ the concentration of SP in synaptosomes remained
F. Lembeck et al. : Substance P in Synaptosomes
19
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Fig. 1. Effect of high K + (A) or electrical field stimulation (B) on the substance P (SP) content of rat brain synaptosomes (Mean values • S.E.M., n = 6). (A) Not incubated controls: 12I;incubated for 15 or 45 min at 30~ in Krebs-Ringer phosphate buffer (KR): IN; in K + free KR: []: in K + rich (23.8 raM) KR:N. (B) D and Nlas in A; 9 Incubated in KR for 15 min at 30~ and subjected to electrical field stimulation
0
15
I i
30
/,5
60
120 rain
Fig.3. SubstanceP (SP) content of rat brai~ synaptosomes after incubation for different periods in 0.32 M sucrose at 4~ [3, in 0.32 M sucrose at 30~ []: in Krebs-Ringer phosphate buffer (KR) at 4~ []; in KR at 30:C: ~. (Mean values • S.E.M,;
n = 4-7)
it was n e a r l y as high as t h a t o f s y n a p t o s o m e s i n c u b a t e d at 4 ~ C for 120 m i n (see Fig. 3). +
5--
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23,80
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23,80
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Fig.2. Effect of Ca 2+ on the release of substance P (SP) from rat brain synaptosomes by high K +. Not incubated controls: 13. Incubated for 15 rain at 30~C in Krebs-Ringer phosphate buffer (KR) : IN: in K + rich KR in the absence or presence of Ca2+ in different concentrations : ~ : K + and Ca2+ : mM. (Mean values +_ S,E.M. ; n = 6)
c o n s t a n t for 45 m i n w h e n i n c u b a t e d in K R a n d for 120 rain when i n c u b a t e d in 0.32 M sucrose. A t 30~ a loss o f SP f r o m s y n a p t o s o m e s by m o r e t h a n 50 % c o u l d be d e t e c t e d after 15 m i n w h e t h e r i n c u b a t e d in 0.32 m M sucrose o r in K R . I n K R no f u r t h e r loss o f SP was seen after 45 inin. The SP c o n t e n t o f s y n a p t o s o m e s in 0.32 M sucrose at 30~ was higher after 60 m i n i n c u b a t i o n t h a n after 30 min. A f t e r 120 rain
The Effect of Increased Potassium Concentrations on the Substance P Content of Synaptosomes "Pre-Incubated" in Sucrose for t20 min. T h e r e s t o r a t i o n after 120 rain o f the t r a n s i e n t l y d e c r e a s e d SP c o n t e n t o f s y n a p t o s o m e s i n c u b a t e d in 0.32 M sucrose at 30~ p r o m p t e d a n i n v e s t i g a t i o n into the b e h a v i o u r o f this p o s s i b l y newly synthesized SP t o w a r d s d e p o l a r i s a t i o n o f the s y n a p t o s o m a l m e m b r a n e by K +. Thus, after 120 rain o f the initial i n c u b a t i o n the s y n a p t o s o m e s were i s o l a t e d by the c e n t r i f u g a t i o n a n d r e s u s p e n d e d in v a r i o u s fresh i n c u b a t i o n m e d i a (see Fig. 4). W h e n r e - i n c u b a t e d for 45 rain at 30~ in 0.32 M sucrose a further rise in the SP c o n t e n t f r o m 4.5 4- 0.8 n g / m g p r o t e i n to 6.1 _+ 1.0 n g / m g p r o t e i n occurred. In the e x p e r i m e n t s illustrated in F i g u r e 4 the m e a n concent r a t i o n o f SP in the s y n a p t o s o m e s after 120 rain inc u b a t i o n in 0.32 M sucrose was 5.6 n g / m g protein. W h e n these s y n a p t o s o m e s were r e - i n c u b a t e d for 45 rain at 30~ in K +-free K R a further rise in the s y n a p t o s o m a l SP c o n c e n t r a t i o n o c c u r r e d . T h e presence o f p h y s i o l o g i c a l K + c o n c e n t r a t i o n s in the K R inh i b i t e d this rise, elevated K + c o n c e n t r a t i o n s caused a loss o f SP. A d d i t i o n o f b a c i t r a c i n (20 r a M ) to the K +free s o l u t i o n resulted in a 4-fold increase o f the SP c o n t e n t o f the r e - i n c u b a t e d s y n a p t o s o m e s a n d led also to h i g h e r values o f the SP c o n t e n t o f the s y n a p t o somes i n c u b a t e d in m e d i a c o n t a i n i n g p o t a s s i u m (see Fig. 4). It is n o t e d t h a t t h e r e is n o difference b e t w e e n
20
Naunyn-Schmiedeberg'sArch. Pharmacol. 301 (1977) 25-
20
c"
15 0 O_ Cn
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0
4,8 23,8
mM [K*1
Fig. 4. Substance P (SP) in pre-incubated (120 min in 0.32 M sucrose at 30~ synaptosomes. Controls SP in synaptosomes preincubated for 120 rain and sedimented without further incubation: ffl; SP in pre-incubated synaptosomes separated from first incubation medium and re-incubated for 45 min at 30~C in KrebsRinger phosphate buffer without (IN) or with bacitracin (20 raM) (N) in the absence of presence of 4.8 or 23.8 mM K + (see abscissa). (Mean values _+ S.E.M.; n = 6)
the SP content of synaptosomes in 4.8 m M K + and 23.8 m M K + ; this remains unexplained and an effect of bacitracin besides the inhibition of proteolytic enzymes cannot be excluded. DISCUSSION The observations made in the present experiments are in good agreement with recent reports from other laboratories on the release of SP from nervous tissues. Thus Otsuka and Konishi (1976) found a release of SP from the isolated spinal cord of newborn rats with a potassium rich (55 m M ) physiological salt solution. The superfusion of hypothalamus slices with a potassium rich solution (47 m M ) for 4 min led to a calcium dependent release of SP (Iversen et al., 1976). Similar results were obtained by Cuello et al. (1977) using slices of substantia nigra. Schenker et al. (1976) superfused synaptosomal preparations with a potassium rich
(60 m M ) medium and measured the efflux of SP at 3 min intervals. They found that immediate release of SP due to the increased K § concentration occurred only in the presence of calcium ions. The results quoted above were obtained using the radioimmunoassay for SP. In contrast, in the present experiments SP was measured by bioassay. This method is less sensitive than the radioimmunoassay and does not allow to measure the amount of SP released into the incubation medium. Concentrating the SP present in the incubation medium after the synaptosomes have been removed would lead to an increase in the concentration of inorganic ions which would interfere with the bioassay. In the experiments described in this paper it was observed that the SP content of synaptosomes remained constant for 120min when incubated in 0.32 M sucrose at 4 ~C. When incubated at 30~ there was an initial loss. This is in agreement with observations on the release of other transmitter substances from synaptosomes (De Belleroche and Bradford, 1972a; Raiteri et al., 1974). The rate of the decrease of the SP content caused by high potassium concentrations (23.8 m M ) agrees well with results of experiments in which the efflux of SP from synaptosomes was measured under similar conditions (Schenker et al., 1976). The decrease of the synaptosomal SP content after electrical field stimulation provides further evidence for a release of SP by depolarisation. The calcium sensitivity of the potassium induced SP release (see Fig. 3) confirmed and extended the findings of Otsuka and Konishi (1976) and Schenker et al. (1976) on the efflux of SP into the incubation media of synaptosomes. Our results are also in agreement with the results of Inouye and K a t a o k a (1962). Several observations made in the present experiments allow us to speculate about the storage mechanism and the synthesis of SP in synaptosomes. The fact that the concentration of SP in synaptosomes incubated in 0.32 M sucrose at 4~ is maintained constant up to 120 min suggests that under these conditions no efflux of SP occurs, as it is unlikely that considerable quantities of SP are synthesized at 4~ The fall of the SP content of synaptosomes incubated in K R or 0.32 M sucrose at 30 ~C over 30 min indicates that the efflux greatly exceeds any possible synthesis or reuptake. When the incubation of the synaptosomes in 0.32 M sucrose was continued for 60 or 120 min at 30~ a gradual increase in synaptosomal SP content occurred. The concentrations found at 120 min were similar to those of synaptosomes incubated in 0.32 M sucrose at 4~ for the same length of time. This could either be due to a re-uptake of released SP or to the synthesis of new SP in synaptosomes. No
F. Lembeck et al. : Substance P in Synaptosomes
observations on the uptake of SP by tissues have so far been reported. There is also little known about its biosynthesis. However, the observation that the SP content of pre-incubated (120 rain in 0.32 M sucrose at 30~ synaptosomes increased after 45 rain reincubation in potassium free KR by about 50 ~ (see Fig. 4) can at present best be explained by synthesis of new SP in the synaptosomes and not by re-uptake. Re-incubation of pre-incubated synaptosomes in sucrose resulted only in a small further rise of their SP content. Re-incubation of pre-incubated synaptosomes was also carried out in potassium free KR to which in addition to aprotinin (20 units/ml) a second inhibitor of proteolytic enzymes, bacitracin (20 raM) has been added. This increased the SP content of the synaptosomes four fold when compared with synaptosomes reincubated in potassium free KR containing aprotinin only and confirms that aprotinin does not inhibit proteolytic enzymes completely. Assuming that in the presence of bacitracin the catabolism of SP is completely prevented, then it can be calculated that approximately 300 pg of SP are synthesized per mg synaptosomal protein per rain in potassium free KR. As the initial amount of SP in pre-incubated synaptosomes was 5.6 ng/mg protein the rate of synthesis of SP per rain in a potassium free and bacitracin containing medium was calculated to be as high as 5 ~ of the initial amount (compare Fig.4). According to Schenker et al. (1976) the release of SP by depolarisation is a sudden event. The amount of SP released could be replaced by a rapid de novo synthesis or from a storage site at which SP is continously, but slowly synthesized. Recent findings (Lembeck et al., 1977) suggest that SP can be stored by binding to phosphatidyl serine. REFERENCES Baldessarini, R. J., Kopin, L : The effect of drugs on the release of norepinephrin-3-H from central nervous system tissues by electrical stimulation in vitro. J. Pharmacol. exp. Ther. 157, 3 1 - 38 (1967) Chang, M. M., Leeman, S. E.: Isolation of a sialogocic peptide from bovine hypothalamic tissue and its characterisation as substance P. J. Biol. Chem. 245, 4784-4790 (1970) Cooke, J. D., Okamoto, K., Quastel, D. M. J. : The role of calcium in depolarisation-secretion coupling of the motor nerv-terminal. J. Physiol. 228, 459-497 (1973) Cuello, A.C., Polak, J. M., Pearse, A. G . E . : Substance P: A naturally occurring transmitter in human spinal cord. The Lancet 1976 II, 1054-1056 Cuello, A. C., Emson, P., del Eiacco, M., Gale, J., Iversen, L. L., Jessel, T. M., Kanazawa, I., Paxinos, G., Quik, M. : Distribution and release of substance P in the central nervous system. Centrally acting peptides (Hughes, J., ed.). London: Macmillan 1977 (in press)
21 De Belleroche, J. S., Bradford, H . F . : Metabolism of beds of mammalian cortical synaptosomes: response to depolarising influences. J. Neurochem. 19, 585-602 (1972a) De Belleroche, J. S., Bradford, H. F. : The stimulus-induced release of acetylcholine from synaptosome beds and its calcium dependence. J. Neurochem. 19, 1817-1819 (1972b) Gray, E. G., Whittaker, V. P.: The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenisation and centrifugation. J. Anat. (Lond.) 96, 7 9 - 8 7 (1962) Haga, T. J. : Synthesis and release of 14C acetylcholine in synaptosomes. J. Neurochem. 18, 781-798 (1971) Hfiusler, G., Thoenen, H., Haefely, W., Huerlimann, A. : Electrical events in cardiac adrenergic nerves and noradrenaline release from the heart induced by acetylcholine and KCI. NaunynSchmiedeberg's Arch. Pharmakol. exp. Pathol. 261, 389-411 (1968) H6kfett, T., Kelterth, J., Nillson, G., Pernow, B.: Experimental immunhistochemical studies on localisation and distribution of substance P in cat primary sensory neurons. Brain Res. 100, 235-252 (1975) Hukovi6, S., Muscholl, E. : Die Noradrenalin-Freisetzung aus dem isolierten Herzen durch sympathische Nervenreizung. NaunynSchmiedebergs Arch. exp. Path. Pharmakol. 243, 348 (1962) Inouye, A., Kataoka, K.: Sub-cellular distribution of the substance P in the nervous tissues. Nature (Lond.) 193, 585 (1962) Iversen, L. L., Jessel, J., Kanazawa, I. : Release and metabolism of substance P in rat hypothalamus. Nature (Lond.) 264, 8 1 - 8 3 (1976) Konishi, S., Otsuka, M. : Excitatory action of hypothalamic substance P on spinal motoneurons of new born rats. Nature (Lond.) 252, 7 3 4 - 735 (1974) Kuhar, M. J., Taylor, K. M., Snyder, S. M. : The sub-cellular localisation &histamine and histamine methyltransferase in rat brain. J. Neurochem. 18, 1515-1527 (1971) Lembeck, F. : Zur Frage der zentralen Ubertragung afferenter Impulse. III. Mitteilung: Das Vorkommen und die Bedeutung der Substanz P in den dorsalen Wurzeln des Rfickenmarks. NaunynSchmiedeberg's Arch. exp. Path. Pharmacol. 219, 197-213 (1953) Lembeck, F., Holasek, A.: Die intracellulS, re Lokalisation der Substanz P. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmakol. 238, 542-545 (1960) Lembeck, F., Mayer, N., Schindler, G. : Lipid bound substance P in central nervous system. Naunyn-Schmiedeberg's Arch. Pharmakoi. 297, R55 (1977) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Osborne, R. H., Bradford, H. F., Jones, D. G. : Pattern of amino acid release from nerve endings isolated from spinal cord and medulla. J. Neurochem. 21, 407-419 (1973) Otsuka, M., Konishi, S.: Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat. Nature (Lond.) 264, 83 - 84 (1976) Otsuka, M., Konishi, S., Takahashi, T. : Hypothalamic substance P as a candidate for transmitter of primary afferent neurons. Fed. Proc. 34, I 9 2 2 - 1928 (1975) Raiteri, M., Angelini, F., Levi, G. : A simple apparatus for studying the release of neurotransmitters from synaptosomes. Europ. J. Pharmacol. 25, 411-414 (1974) RyaI1, R. W. : Sub-cellular distribution of pharmacologically active substances in guinea pig brain. Nature (Lond.) 196, 680-681 (1962) RyaI1, R. W. : The sub-celluiar distribution of acetylchoiine, substance P, 5-hydroxytryptamine, 7-amino-butyric acid and glu-
22 tamic acid in brain homogenates. J. Neurochem. 11, 131-145 (1964) Schenker, C., Mroz, E. A., Leeman, S. E. : Release of substance P from isolated nerve endings. Nature (Lond.) 264, 790-792 (1976) Schmied, R., Sieghart, W., Karobath, M.: Taurine uptake in synaptosomal fractions of the rat cerebral cortex. J. Neurochem. 25, 5 - 9 (1975)
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977) Skrabanek, P., Cannon, D., Kirrana, J., Legge, D., Powe11, D. : Circulating immunoreactive substance P in man. Irish. J. Med. Sci. 145, 399-468 (1977) Whittaker, V. P. : The synaptosome. Handbook of Neurochemistry, II (A. Lajtha, ed.). New York-London: Plenum 1970
Received July 15~Accepted September 8, 1977
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 301, 1 7 - 2 2 (1977)
Pharmacology
9 by Springer-Verlag 1977
Substance P in Rat Brain Synaptosomes F. LEMBECK, N. MAYER, and G. SCHINDLER Institut ffir experimentelle und klinische Pharmakologie der Universitfit Graz, Universitfitsplatz 4, A-8010 Graz, Austria
Summary. 1. Rat brain synaptosomes were incubated under different conditions to study the release of substance P (SP). 2. Potassium ions and electrical field stimulation induced a loss of SP from synaptosomes. The release of SP by potassium in high concentrations (23.8 mM) was shown to be calcium dependent. 3. Substance P was retained in synaptosomes during incubation in 0.32 M sucrose at + 4 ~ up to 120 min. During incubation at 30~ the SP content fell initially (30 min) but was gradually restored (120 min). 4. If these pre-incubated synaptosomes were reincubated for 45 min at 30 ~C in potassium free KrebsRinger-phosphate buffer a further rise in their SP content occurred which was taken as indication that SP is being synthesized in synaptosomes. 5. The newly synthesized SP is presumably stored by binding to phosphatidyl serine until a sudden release is initiated by depolarization. Key words." Substance P - Synaptosomes.
INTRODUCTION All known putative transmitter substances were found to be present in synaptosomes. This was demonstrated for acetylcholine, noradrenaline, dopamine, 5-hydroxytryptamine, GABA, glycine, glutamate (Ryall, 1962; Ryall, 1964; Whittaker, 1970) as well as for histamine (Kuhar et al., 1971) and taurine (Schmied et al., 1975). A role of substance P as a central neurotransmitter has first been suggested by Lembeck (1953). Further evidence for this function of substance P was recently Send offprint requests to F. Lembeck at the above address
obtained in electrophysiological and histochemical studies (Konishi and Otsuka, 1974; Otsuka et al., 1975; H6kfelt et al., 1975; Cuello et al., 1976). Substance P has also been found in synaptosomal preparations (Lembeck and Holasek, 1960; Inouye and Kataoka, 1962; Schenker et al., 1976). The release of a neurotransmitter can be caused by depolarisation of the cell membrane. Thus incubation of synaptosomes in media containing depolarizing concentrations (50 raM) of potassium enhanced the release of acetylcholine (Haga, 1971) and GABA (De Belleroche and Bradford, 1972a). The release of noradrenaline and GABA from rat brain synaptosomes incubated in the presence of potassium (56 raM) was reported by Osborne et al. (1973). Transmitter release depends also on the calcium concentration in the medium as shown for acetylcholine by De Belleroche and Bradford (1972b) and Cooke et al. (1973), for noradrenaline by Hukovi6 and Muscholl (1962), Baldessarini and Kopin (1967) and Hfiusler et al. (1968) and for GABA by De Belleroche and Bradford (1972 a). The present experiments were performed to investigate (a) under which conditions synaptosomes are able to retain a constant amount of substance P, (b) if SP is released from synaptosomes by depolarisation (increase of the potassium concentration in the incubation medium or electrical field stimulation) and (c) if the release of SP is influenced by the calcium concentration in the medium. METHODS Preparation of Synaptosomes. Sprague Dawley rats (strain OFA, SD, SPF, both sexes, 150-250 g) were killed by a blow on the neck. The brains were quickly removed and weighed. All further manipulations were carried out at 4~ The brains were homogenized in 0.32 M sucrose (10 ml/g tissue) containing aprotinin (20 units/ml) to inhibit proteolytic enzymes and Cialit ' [(2-ethyl-mercury-mer-
18 capto)-benzoxazol-5-Na-carbonic acid] (2.5 rag/l) to inhibit bacterial growth. Synaptosomes were prepared according to the method of Gray and Whittaker (/962) by centrifuging of the resuspended (in 0.32 M sucrose) P2 fraction into a discontinuous density gradient (0.8 and 1.2 M sucrose) at 100000 g for 60 rain in a Beckman L-2 ultra centrifuge (SW 27 rotor). The mainly synaptosomes containing "Bfraction" (Gray and Whittaker, 1962) was removed by gentle suction with a motor driven syringe. In the foliowing text the term "synaptosomes" refers to this fraction. Protein was measured in the brain tissue to be 94.2 i 3.2 mg/g, in fraction Pz 39.8 _+ 2.4 and in fraction B 4.1 ,+ 0.2 mg/ml. The SP content per mg protein was found to be in brain tissue to be 0.8 ,+ 0.1 ng/mg in fraction P2 2.6 ,+ 0.4 ng/mg and in the fraction B 5.4 _+ 0.3 ng/mg.
Incubation. Five ml "synaptosomes" (containing on average 20.5 mg protein) were added to 10 ml of the incubation media. The final concentrations of ions in the incubation media were therefore 2/3 of the following solutions : 1. Sucrose, 0.32 M; 2. Krebs-Ringer-phosphate buffer (KR) pH 7.4 (118 mM NaC1, 4.75 mM KC1, 0.88 mM CaCI2, 1.18 mM MgSO~, 3.18ram NaHzPO4, 13.02mM NazHPO4); 3. KrebsRinger-phosphate buffer without KCI; 4. Krebs-Ringer-phosphate buffer with KC1 23.8 mM ; 5. Krebs-Ringer-phosphate buffer without CaCIz; 6. Krebs-Ringer-phosphate buffer wifh CaCI2 4.4 raM; 7. Krebs-Ringer-phosphate buffer with CaCI~ 8.8 mM. Inconsistencies in the amount of SP present in synaptosomal preparations which occurred in preliminary experiments could be prevented by inhibiting proteolytic enzymes with aprotinin (20 units/ ml). It was added to all incubation media. Aprotinin in the concentration of 0.5 units/ml has, however, recently be found to cause little inhibition of the in vitro degradation of SP (Skrabanek et al., 1977). More recently bacitracin was found to be an even better inhibitor of proteolysis (Cuello et al., 1977). It was added in a 20 gM concentration in addition to aprotinin in the experiments shown in Figure 4. As the incubation of synaptosomes lasted sometimes up to 165 min breakdown of SP by bacterial enzymes could take place. Thus a bactericidal substance (Cialit | 25 gg/ml) was added to all incubation media throughout the present experiments. The incubations were carried out at + 4 ~C or + 30~C in a shaking water bath and terminated by centrifugation at +4~ (Christ Zeta 20 refrigerated centrifuge, 20000 g, 10 min). SP was extracted from the resulting pellet. Depolarisation of synaptosomal membranes by electrical field stimulation was carried out in a 8 ml containing bath using 2 ring electrodes with a distance of 4 cm (square wave pulses 3 ms, 10 Hz, constant current of 0.5 mA). Extraction of Substance P from Synaptosomes. SP was extracted by the method of Chang and Leeman (1970) with slight modifications. The pellet obtained after centrifugation of the incubate was resuspended in a 0 . 9 ~ NaC1 solution; 0.3 ml 0.1 N HC1 and 10 ml acetone were added and the samples shaken for 18 h in a water bath at 4 ~C. The insoluble precipitate formed was removed by centrifugation and the supernatant freed from lipids by shaking with 25 ml petroleum ether (b.p. 4 0 - 60 ~C). The aqueous phase was evaporated in vacuo and the dry residue redissolved in 0.5 ml of an 0.9 ~ NaC1 solution which contained per 100 ml i mg bovine serum albumine and 0.05 mg Cialit | The pH was adjusted to 6 - 7 by addition of 5 ~o NaHCO3 solution shortly before bioassay. Bioassay. The bioassay of SP was performed on the isolated guinea pig ileum suspended in an oxygenated Tyrode solution at 37 ~C which contained per ml 0.1 gg atropine, 1.0 gg mepyramine and 0.1 lag methysergide. Synthetic SP was used as standard. Prostaglandins
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977) would have been removed by petroleum ether during the extraction (see above). Several samples of the fraction B preparation were tested on the rat duodenum which is relaxed by bradykinin like peptides; no relaxation was observed. It seems to be justified to regard the bioassay used as being reliable for the estimation of SP.
Protein Estimation. The amounts of protein contained in the tissue homogenates, the P2-fractions and the synaptosomal preparations were measured by the method of Lowry et al. (1951) using bovine serum albumine as standard. Materials. Substance P (Beckman Instruments, Palo Alto, California), Aprotinin (Bayer AG Leverkusen), Bacitracin (Biochemie G.m.b.H., Wien), Bovine serum albumine (Miles Inc., Kankakee, Ill.), Cialit | (2-ethyl-mercury-mercapto)-benzoxazol-5-Na-carbonic acid (Asid G.m.b.H., Miinchen), Methysergid bimaleate (Sandoz S.A., Basel), Mepyramine hydrochloride (Specia S.A., Paris), Atropine sulphate (Merck A.G., Darmstadt). All other chemicals used were of analytical grade and obtained from Merck A.G., Darmstadt. When a drug was used in form of a salt the dosen given refers to the "free substance".
RESULTS
Loss of Substance P from Synaptosomes in the Presence of High Potassium Concentrations (23.8mM). A Krebs-Ringer-phosphate buffer (23.8 mM KC1) was used for the depolarisation of the synaptosomal membranes. Synaptosomes incubated at 30~ in this K + rich buffer contained after 15 rain significantly (P < 0.001) less SP than synaptosomes incubated in normal KR (see Fig. 1A). Loss of Substance P from Synaptosomes by Electrical Field Stimulation. Synaptosomes suspended in normal KR and subjected to electrical field stimulation lost within 15 rain twice as much SP as unstimulated synaptosomes (P < 0.005, see Fig. 1 B). When expressed in per cent of the amount of SP originally present the loss after field stimulation ( - 5 0 ~o) was smaller than the loss which occurred in the presence of 23.8 mM K + ( - 63 ~, see Fig. 1A). Influence of the Calcium Concentration in the Incubation Medium on the Loss of Substance P from Synaptosomes. Synaptosomes were incubated for 15 rain at 30~ either in normal KR (controls) or in K + rich (23.8 raM) KR containing different concentrations of calcium (Fig. 2). 23.8 mM K + caused no change of SP in the absence of Ca 2+, when compared with normal Krebs-Ringer solution. Ca 2+ in concentrations between 0.88 and 8.8 mM enhanced the release of SP from the synaptosomes elicited by potassium. The magnitude of the SP loss was proportional to the Ca z + concentration in the incubation medium. Comparison between the Loss of Substance P from Synaptosomes Incubated at 4~ and 30~ At 4~ the concentration of SP in synaptosomes remained
F. Lembeck et al. : Substance P in Synaptosomes
19
A
B G ll)
/
C)-
5--
5--
c, 5 E 2
12_ CO
C 0)
03 G
O
& -
O_
E _ IX_ CO r
-O
15
/*Stain
O
1--Stain
Fig. 1. Effect of high K + (A) or electrical field stimulation (B) on the substance P (SP) content of rat brain synaptosomes (Mean values • S.E.M., n = 6). (A) Not incubated controls: 12I;incubated for 15 or 45 min at 30~ in Krebs-Ringer phosphate buffer (KR): IN; in K + free KR: []: in K + rich (23.8 raM) KR:N. (B) D and Nlas in A; 9 Incubated in KR for 15 min at 30~ and subjected to electrical field stimulation
0
15
I i
30
/,5
60
120 rain
Fig.3. SubstanceP (SP) content of rat brai~ synaptosomes after incubation for different periods in 0.32 M sucrose at 4~ [3, in 0.32 M sucrose at 30~ []: in Krebs-Ringer phosphate buffer (KR) at 4~ []; in KR at 30:C: ~. (Mean values • S.E.M,;
n = 4-7)
it was n e a r l y as high as t h a t o f s y n a p t o s o m e s i n c u b a t e d at 4 ~ C for 120 m i n (see Fig. 3). +
5--
K / - - ( K R ) - - /,,75
c-
2+
Ca 0,88
23,80
0
E
23,80
0,88
0_ CO
23.80
L,40
23.80
8.80
o L. o,_
c
II 15 min
Fig.2. Effect of Ca 2+ on the release of substance P (SP) from rat brain synaptosomes by high K +. Not incubated controls: 13. Incubated for 15 rain at 30~C in Krebs-Ringer phosphate buffer (KR) : IN: in K + rich KR in the absence or presence of Ca2+ in different concentrations : ~ : K + and Ca2+ : mM. (Mean values +_ S,E.M. ; n = 6)
c o n s t a n t for 45 m i n w h e n i n c u b a t e d in K R a n d for 120 rain when i n c u b a t e d in 0.32 M sucrose. A t 30~ a loss o f SP f r o m s y n a p t o s o m e s by m o r e t h a n 50 % c o u l d be d e t e c t e d after 15 m i n w h e t h e r i n c u b a t e d in 0.32 m M sucrose o r in K R . I n K R no f u r t h e r loss o f SP was seen after 45 inin. The SP c o n t e n t o f s y n a p t o s o m e s in 0.32 M sucrose at 30~ was higher after 60 m i n i n c u b a t i o n t h a n after 30 min. A f t e r 120 rain
The Effect of Increased Potassium Concentrations on the Substance P Content of Synaptosomes "Pre-Incubated" in Sucrose for t20 min. T h e r e s t o r a t i o n after 120 rain o f the t r a n s i e n t l y d e c r e a s e d SP c o n t e n t o f s y n a p t o s o m e s i n c u b a t e d in 0.32 M sucrose at 30~ p r o m p t e d a n i n v e s t i g a t i o n into the b e h a v i o u r o f this p o s s i b l y newly synthesized SP t o w a r d s d e p o l a r i s a t i o n o f the s y n a p t o s o m a l m e m b r a n e by K +. Thus, after 120 rain o f the initial i n c u b a t i o n the s y n a p t o s o m e s were i s o l a t e d by the c e n t r i f u g a t i o n a n d r e s u s p e n d e d in v a r i o u s fresh i n c u b a t i o n m e d i a (see Fig. 4). W h e n r e - i n c u b a t e d for 45 rain at 30~ in 0.32 M sucrose a further rise in the SP c o n t e n t f r o m 4.5 4- 0.8 n g / m g p r o t e i n to 6.1 _+ 1.0 n g / m g p r o t e i n occurred. In the e x p e r i m e n t s illustrated in F i g u r e 4 the m e a n concent r a t i o n o f SP in the s y n a p t o s o m e s after 120 rain inc u b a t i o n in 0.32 M sucrose was 5.6 n g / m g protein. W h e n these s y n a p t o s o m e s were r e - i n c u b a t e d for 45 rain at 30~ in K +-free K R a further rise in the s y n a p t o s o m a l SP c o n c e n t r a t i o n o c c u r r e d . T h e presence o f p h y s i o l o g i c a l K + c o n c e n t r a t i o n s in the K R inh i b i t e d this rise, elevated K + c o n c e n t r a t i o n s caused a loss o f SP. A d d i t i o n o f b a c i t r a c i n (20 r a M ) to the K +free s o l u t i o n resulted in a 4-fold increase o f the SP c o n t e n t o f the r e - i n c u b a t e d s y n a p t o s o m e s a n d led also to h i g h e r values o f the SP c o n t e n t o f the s y n a p t o somes i n c u b a t e d in m e d i a c o n t a i n i n g p o t a s s i u m (see Fig. 4). It is n o t e d t h a t t h e r e is n o difference b e t w e e n
20
Naunyn-Schmiedeberg'sArch. Pharmacol. 301 (1977) 25-
20
c"
15 0 O_ Cn
E 12_ CO r
0
4,8 23,8
mM [K*1
Fig. 4. Substance P (SP) in pre-incubated (120 min in 0.32 M sucrose at 30~ synaptosomes. Controls SP in synaptosomes preincubated for 120 rain and sedimented without further incubation: ffl; SP in pre-incubated synaptosomes separated from first incubation medium and re-incubated for 45 min at 30~C in KrebsRinger phosphate buffer without (IN) or with bacitracin (20 raM) (N) in the absence of presence of 4.8 or 23.8 mM K + (see abscissa). (Mean values _+ S.E.M.; n = 6)
the SP content of synaptosomes in 4.8 m M K + and 23.8 m M K + ; this remains unexplained and an effect of bacitracin besides the inhibition of proteolytic enzymes cannot be excluded. DISCUSSION The observations made in the present experiments are in good agreement with recent reports from other laboratories on the release of SP from nervous tissues. Thus Otsuka and Konishi (1976) found a release of SP from the isolated spinal cord of newborn rats with a potassium rich (55 m M ) physiological salt solution. The superfusion of hypothalamus slices with a potassium rich solution (47 m M ) for 4 min led to a calcium dependent release of SP (Iversen et al., 1976). Similar results were obtained by Cuello et al. (1977) using slices of substantia nigra. Schenker et al. (1976) superfused synaptosomal preparations with a potassium rich
(60 m M ) medium and measured the efflux of SP at 3 min intervals. They found that immediate release of SP due to the increased K § concentration occurred only in the presence of calcium ions. The results quoted above were obtained using the radioimmunoassay for SP. In contrast, in the present experiments SP was measured by bioassay. This method is less sensitive than the radioimmunoassay and does not allow to measure the amount of SP released into the incubation medium. Concentrating the SP present in the incubation medium after the synaptosomes have been removed would lead to an increase in the concentration of inorganic ions which would interfere with the bioassay. In the experiments described in this paper it was observed that the SP content of synaptosomes remained constant for 120min when incubated in 0.32 M sucrose at 4 ~C. When incubated at 30~ there was an initial loss. This is in agreement with observations on the release of other transmitter substances from synaptosomes (De Belleroche and Bradford, 1972a; Raiteri et al., 1974). The rate of the decrease of the SP content caused by high potassium concentrations (23.8 m M ) agrees well with results of experiments in which the efflux of SP from synaptosomes was measured under similar conditions (Schenker et al., 1976). The decrease of the synaptosomal SP content after electrical field stimulation provides further evidence for a release of SP by depolarisation. The calcium sensitivity of the potassium induced SP release (see Fig. 3) confirmed and extended the findings of Otsuka and Konishi (1976) and Schenker et al. (1976) on the efflux of SP into the incubation media of synaptosomes. Our results are also in agreement with the results of Inouye and K a t a o k a (1962). Several observations made in the present experiments allow us to speculate about the storage mechanism and the synthesis of SP in synaptosomes. The fact that the concentration of SP in synaptosomes incubated in 0.32 M sucrose at 4~ is maintained constant up to 120 min suggests that under these conditions no efflux of SP occurs, as it is unlikely that considerable quantities of SP are synthesized at 4~ The fall of the SP content of synaptosomes incubated in K R or 0.32 M sucrose at 30 ~C over 30 min indicates that the efflux greatly exceeds any possible synthesis or reuptake. When the incubation of the synaptosomes in 0.32 M sucrose was continued for 60 or 120 min at 30~ a gradual increase in synaptosomal SP content occurred. The concentrations found at 120 min were similar to those of synaptosomes incubated in 0.32 M sucrose at 4~ for the same length of time. This could either be due to a re-uptake of released SP or to the synthesis of new SP in synaptosomes. No
F. Lembeck et al. : Substance P in Synaptosomes
observations on the uptake of SP by tissues have so far been reported. There is also little known about its biosynthesis. However, the observation that the SP content of pre-incubated (120 rain in 0.32 M sucrose at 30~ synaptosomes increased after 45 rain reincubation in potassium free KR by about 50 ~ (see Fig. 4) can at present best be explained by synthesis of new SP in the synaptosomes and not by re-uptake. Re-incubation of pre-incubated synaptosomes in sucrose resulted only in a small further rise of their SP content. Re-incubation of pre-incubated synaptosomes was also carried out in potassium free KR to which in addition to aprotinin (20 units/ml) a second inhibitor of proteolytic enzymes, bacitracin (20 raM) has been added. This increased the SP content of the synaptosomes four fold when compared with synaptosomes reincubated in potassium free KR containing aprotinin only and confirms that aprotinin does not inhibit proteolytic enzymes completely. Assuming that in the presence of bacitracin the catabolism of SP is completely prevented, then it can be calculated that approximately 300 pg of SP are synthesized per mg synaptosomal protein per rain in potassium free KR. As the initial amount of SP in pre-incubated synaptosomes was 5.6 ng/mg protein the rate of synthesis of SP per rain in a potassium free and bacitracin containing medium was calculated to be as high as 5 ~ of the initial amount (compare Fig.4). According to Schenker et al. (1976) the release of SP by depolarisation is a sudden event. The amount of SP released could be replaced by a rapid de novo synthesis or from a storage site at which SP is continously, but slowly synthesized. Recent findings (Lembeck et al., 1977) suggest that SP can be stored by binding to phosphatidyl serine. REFERENCES Baldessarini, R. J., Kopin, L : The effect of drugs on the release of norepinephrin-3-H from central nervous system tissues by electrical stimulation in vitro. J. Pharmacol. exp. Ther. 157, 3 1 - 38 (1967) Chang, M. M., Leeman, S. E.: Isolation of a sialogocic peptide from bovine hypothalamic tissue and its characterisation as substance P. J. Biol. Chem. 245, 4784-4790 (1970) Cooke, J. D., Okamoto, K., Quastel, D. M. J. : The role of calcium in depolarisation-secretion coupling of the motor nerv-terminal. J. Physiol. 228, 459-497 (1973) Cuello, A.C., Polak, J. M., Pearse, A. G . E . : Substance P: A naturally occurring transmitter in human spinal cord. The Lancet 1976 II, 1054-1056 Cuello, A. C., Emson, P., del Eiacco, M., Gale, J., Iversen, L. L., Jessel, T. M., Kanazawa, I., Paxinos, G., Quik, M. : Distribution and release of substance P in the central nervous system. Centrally acting peptides (Hughes, J., ed.). London: Macmillan 1977 (in press)
21 De Belleroche, J. S., Bradford, H . F . : Metabolism of beds of mammalian cortical synaptosomes: response to depolarising influences. J. Neurochem. 19, 585-602 (1972a) De Belleroche, J. S., Bradford, H. F. : The stimulus-induced release of acetylcholine from synaptosome beds and its calcium dependence. J. Neurochem. 19, 1817-1819 (1972b) Gray, E. G., Whittaker, V. P.: The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenisation and centrifugation. J. Anat. (Lond.) 96, 7 9 - 8 7 (1962) Haga, T. J. : Synthesis and release of 14C acetylcholine in synaptosomes. J. Neurochem. 18, 781-798 (1971) Hfiusler, G., Thoenen, H., Haefely, W., Huerlimann, A. : Electrical events in cardiac adrenergic nerves and noradrenaline release from the heart induced by acetylcholine and KCI. NaunynSchmiedeberg's Arch. Pharmakol. exp. Pathol. 261, 389-411 (1968) H6kfett, T., Kelterth, J., Nillson, G., Pernow, B.: Experimental immunhistochemical studies on localisation and distribution of substance P in cat primary sensory neurons. Brain Res. 100, 235-252 (1975) Hukovi6, S., Muscholl, E. : Die Noradrenalin-Freisetzung aus dem isolierten Herzen durch sympathische Nervenreizung. NaunynSchmiedebergs Arch. exp. Path. Pharmakol. 243, 348 (1962) Inouye, A., Kataoka, K.: Sub-cellular distribution of the substance P in the nervous tissues. Nature (Lond.) 193, 585 (1962) Iversen, L. L., Jessel, J., Kanazawa, I. : Release and metabolism of substance P in rat hypothalamus. Nature (Lond.) 264, 8 1 - 8 3 (1976) Konishi, S., Otsuka, M. : Excitatory action of hypothalamic substance P on spinal motoneurons of new born rats. Nature (Lond.) 252, 7 3 4 - 735 (1974) Kuhar, M. J., Taylor, K. M., Snyder, S. M. : The sub-cellular localisation &histamine and histamine methyltransferase in rat brain. J. Neurochem. 18, 1515-1527 (1971) Lembeck, F. : Zur Frage der zentralen Ubertragung afferenter Impulse. III. Mitteilung: Das Vorkommen und die Bedeutung der Substanz P in den dorsalen Wurzeln des Rfickenmarks. NaunynSchmiedeberg's Arch. exp. Path. Pharmacol. 219, 197-213 (1953) Lembeck, F., Holasek, A.: Die intracellulS, re Lokalisation der Substanz P. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmakol. 238, 542-545 (1960) Lembeck, F., Mayer, N., Schindler, G. : Lipid bound substance P in central nervous system. Naunyn-Schmiedeberg's Arch. Pharmakoi. 297, R55 (1977) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Osborne, R. H., Bradford, H. F., Jones, D. G. : Pattern of amino acid release from nerve endings isolated from spinal cord and medulla. J. Neurochem. 21, 407-419 (1973) Otsuka, M., Konishi, S.: Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat. Nature (Lond.) 264, 83 - 84 (1976) Otsuka, M., Konishi, S., Takahashi, T. : Hypothalamic substance P as a candidate for transmitter of primary afferent neurons. Fed. Proc. 34, I 9 2 2 - 1928 (1975) Raiteri, M., Angelini, F., Levi, G. : A simple apparatus for studying the release of neurotransmitters from synaptosomes. Europ. J. Pharmacol. 25, 411-414 (1974) RyaI1, R. W. : Sub-cellular distribution of pharmacologically active substances in guinea pig brain. Nature (Lond.) 196, 680-681 (1962) RyaI1, R. W. : The sub-celluiar distribution of acetylchoiine, substance P, 5-hydroxytryptamine, 7-amino-butyric acid and glu-
22 tamic acid in brain homogenates. J. Neurochem. 11, 131-145 (1964) Schenker, C., Mroz, E. A., Leeman, S. E. : Release of substance P from isolated nerve endings. Nature (Lond.) 264, 790-792 (1976) Schmied, R., Sieghart, W., Karobath, M.: Taurine uptake in synaptosomal fractions of the rat cerebral cortex. J. Neurochem. 25, 5 - 9 (1975)
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977) Skrabanek, P., Cannon, D., Kirrana, J., Legge, D., Powe11, D. : Circulating immunoreactive substance P in man. Irish. J. Med. Sci. 145, 399-468 (1977) Whittaker, V. P. : The synaptosome. Handbook of Neurochemistry, II (A. Lajtha, ed.). New York-London: Plenum 1970
Received July 15~Accepted September 8, 1977
