Journal of .\'eurochernistry,
1975. Vol. 25, pp. 305-307. Pergamon Press. Printed in Great Britain.
SUBCELLULAR DISTRIBUTION OF SUBSTANCE P IN BOVINE HYPOTHALAMUS AND SUBSTANTIA NIGRA M. J. D L ~ - YD. , *MULHALL and D. POWELL Department ot Biochemistry, Trinity College, Dublin 2, Ireland (Received 23 October 1974. Accepted 6 March 1975)
Abstract-Subcellular distribution of substance P as measured by radioimmunoassay was studied in fractions and subfractions from bovine hypothalamus and substantia nigra. Most of the substance P was found in the crude mitochondrial fraction. Subfractionation of the crude mitochondrial fraction by density gradient centrifugation showed most of substance P was present in nerve ending particles. Subcellular particle integrity was assessed using enzyme markers.
SUBSTANCE P is found in the brain and intestine of most vertebrate species (LEMBECK & ZETLER,1972). As isolated from bovine hypothalami substance P is an undempeptide (CHANG& LEEMAN,1970). In human brain highest concentrations of the substance as measured both by bioassay (ZETLER, 1970) and by et al., 1974) are found in radioimmunoassay (DUFFY the substantia nigra and hypothalamus with lowest amounts in areas containing white matter. This uneven distribution of substance P in brain suggested that the molecule might be a neurotransmitter (LEMBECK, 1953). Recently it has been shown that the pure peptide causes depolarization of motor neurons (OTSUKAet al., 1972) and increases the concentration of glycine in mouse brain (STERNet al., 1974). In this investigation we show that in bovine hypothalamus and substantia nigra highest concentrations of the substance are found in subcellular organnelles containing the nerve ending particles.
sucrose and centrifuged for 10min at 9009 at 0-4"C. The precipitate was washed twice in the sucrose medium and centrifuged again as above to obtain the nuclear fraction. The supernatant fluid was pooled and centrifuged at 11,500g for 20min a t (t4"C. The precipitate was washed once in the same medium and centrifuged again. The precipitate was denoted the mitochondrial fraction. The supernatant fraction was centrifuged at 100,ooOg for 60min at 0-4°C. The precipitate was designated the microsomal fraction and the supernatant fluid as the supernatant fraction. The submitochondrial fractions were prepared from the crude mitochondrial fraction prepared as above. It was resuspended in 032 M-sucrose at 33% of the original tissue weight and was layered on top of a discontinuous gradient density of sucrose at 0.8, 1.0, 1.2 and 1.4 M. After centrifugation at 50,000 g for 2 h at WIT the layered subfractions was separated by aspiration, diluted with 0.t 5 M-SUCIOS~ and precipitates were collected by centrifugation at 150,000g for 30 min. Radioimmunoassay of substance P
Substance P was assayed by the method of POWELL et al. (1973). To permit labelling with '''I a substance P dcrivative was used with a tyrosyl residue substituted for the MATERIALS AND METHODS phenylalanine at position 8. Iodination was carried out Materials at room temperature (about 20°C) in small glass vials. The Substance P was purchased from Beckman Instruments reaction mixture contained 1 mCi of sodium '''1 iodide, S.A. Geneva, Switzerland. Tyr-8 substance P was supplied Tyr-8 substance P (5 pg), 0.5 M-sodium phosphate buffer by Drs. Niall, Tregear and Potts, Endocrine Units, Massa- (pH 7.4) and 'Chloramine T (supplied by Kodak Ltd., chussets General Hospital Boston, U.S.A. [8-3H]Adeno- Kirkby, Livcrpool) (50 pg) in a final volume of 60 pl. The sine 3'3' cyclic monophosphate, ammonium salt (20,000- reaction was terminated after 20 s by addition of sodium 30,000 mCi/mmol) and 1251(1MS.30)were supplied by The meta-bisulphite (150 pg in 0 1 ml) followed by 1 ml of blood Radiochemical Centre, Amersham, Bucks, U.K. bank plasma. After incubation of antiserum and synthetic substance Methods P standard (or unknown sample) at 4°C for 24 h, iodinated The primary subcellular fractions were prepared as fol- peptide was added and incubation continued for another lows. Bovine hypothalami and substantia nigra were 48 h. Antibody-bound label was then separated from label obtained from International Meat Packers Ltd., Dublin. free in the incubate by adding 2 0 0 4 of 1.5% charcoal immediately after death of the animal. Primary subcellular coated with 0.15% dextran T 20. fractions were prepared as previously described by DE Brain extracts were purified to the step before chromatROBERTIS et al. (1962). The brain areas were cut into small ography (CHANG& LEAMAN,1970) before assay for subpieces and homogenised in 0 3 2 M-sucrose for 2 min using stance P. a glass homogeniser with a loose Teflon pestle (0.25mm clearance). The homogenate was diluted to 10% in 0.32 M- Enzyme assays Lactate dehydrogenase activity (LDH) was assayed by * Present address: Department of Radioisotopes, St. the method of BERGMEYER et al. (1963) succinate dehydroVincent's Hospital, Dublin 4, Ireland. genase (SDH) activity as described by DE ROBERTISet al. N.C
2513-H
305
M. J. DUFFY, D. MULHALL and D. POWELL
306
TABLE1. DISTRIBUTION OF PROTEIN, SUCCINATE DEHYDRWENASE ACTIVITY (SDH.), LACTATE DEHYDROGENASE ACTIVITY (LDH.), ADENYL CYCLASE ACTIVITY AND SUBSTANCE P IN SUBCELLULAR ORGANELLES FROM BOVINE HYPOTHALAMI Protein
Subcellular organelle
SDH. activity
LDH. activity (%)
(%I
(%)
Adenyl cyclase activity
Substance P
(%)
(%)
Substance P (ngimg protein) 2-43
-~ ~~~
Nuclear fraction Mitochondria1 fraction Microsomd fraction Supernatant fraction Total recovery (%)
12.6 f 0 3 2
6.91 f 0 4 0
3.65 f 0 4 5
8.25 f 1.6
9.44 f 1.1
50.1 f 2.9
83.0 f 3.5
37.8 f 2 6
468 f 3.4
59.0 f 3.5
524
41.3 f 4.0
I56
202
+ 1.7
2.4
*
0.21
87.9 f 5.6
+ 1.9 16.0 + 0 8 3
3.6 f 051
53.3 f 4 7
7.7 f 0 3 8
17.1 f 0.8 94.3 f 4.3
0-73
86.2 f 51
905 f 4.8
377 238 2.99
857 f 3.9
Procedure as described in section on Methodology. Values are means of 3 determinations k S.E.M.
TABLE 2: DISTRIBUTION OF PROTEM,
SUCCINATE DEHYDROCENASE ACTIVITY, LACTATE DEHYDRWENASE ACTIVITY, ADENYL CYCLASE ACTIVITY AND SUBSTANCE P IN SUBCELLULAR ORGANELLES FROM BOVINE SUBSTANTIA NIGRA Protein
Subcellular organelle
SDH. activity
(%I
(%I
Nuclear lraction Mitochondria1 fraction Microsomai fraction Supernatant fraction Total recovery (%)
8.0
13.1 f 0 5 6 477
+ 4.3
*
LDH. activity
Adenyl cyclase activity
Substance P
(%)
(%)
(%)
1.2
7.7 f 0 7 4
85.3 f 5 4
208 +_ 2.2
1.3 +_ 0 3 3
18.4 f 1.7
5 4 f076
409
4.2 +_ 0 2 9
839 f 4 4
8.7 f 0.82 556
k 56
5.90
39.5 +_ 3 8
18.4 f 2 7
4.46
17.3 f 3.8
4.73
92.1 f 5 7
89.2 f 4 7
Procedure as described in section on Methodology. Values are means of 3 determinations
TABLE 3.
3-35
45.9 f 3.6
3 3 f 025
47.1 f 3 8
85.2 f 3.9
920 f 3 2
11.3 +_ 0.89
k 2.3
Substance P (ng/mg pratein)
S.E.M.
DISTRIBUTION OF PROTEIN, SUCCINAE DEHYDRWENASE ACTIVITY, ADENYL CYCLASE ACTIVITY AND SUBSTANCE P IN SUE-MI'NKHONDRIAL FRACTIONS FROM BOVINE HYPOTHALAMI
Protein Fraction
A. Myelin B. Nerve endings C. Nerve endings D. Nerve endings E. Free. mitochondria Total recovery %
(%I
32.1 f 2.8 8-73f 0.92 9-92 k 0.83 24.7 f 1.7 24.5 f 1.5 95.6 f 5.0
SDH. activity
(%I
Adenyl cyclase
(%I
(%)
5.43 0.79 17.8 & 1.23 34.2 f 3.0 39.9 f 4.5 2.7 f 042 87.8 3.6
6.28 f 1.3 33.0 f 3.7 607 f 5.8 83.0 f 4.2
Substance P
2.97 f 0.80 20.4 f 1.5 35-7 f 2.6 362 2.3 4.7 f 0.48 86.2 4.0
+
Procedure as described in section on Methodology. Values are means of 3 determinations
Substance P (ng/mg protein)
0.09 8.83 13.6 5.52 0.70
S.E.M.
TABLE 4. DISTRIBUTION OF PROTEIN,
SUCCINATE DEHYDROGENASE ACTIVITY, ADENYL CYCLASE ACTIVITY AND SUBSTANCE IN SUB-MITOCHONDRIAL FRACTIONS FROM BOVINE SUBSTANTIA NIGRA
Fraction A. Myelin
B. C. D. E.
Nerve endings Nerve endings Nerve endings Free mitochondria Total recovery %
Protein
SDH. activity
Adenyl cyclase activity
Substance P
29.6 f 3.1 10.9 f 0.96 16.3 f 1.8 23Q f 2.7 202 f 3.0 89.9 f 5.5
2.8 f 0.34 4.9 f 038 305 f 2.6 614 5.3 84.6 f 3.7
8.4 f 0.73 142 f 1.5 35.3 f 2.9 38.8 f 2.6 3.3 f 0.47 91.7 f 4-9
4 7 f 0.50 23.0 f 1.6 35.1 & 2.6 336 +_ 4.9 3-6 f 1.4 901 f 4.6
(%I
(%I
~~
(%I
~
(%I
P
Substance P (ng/mg protein)
0930 12.5 12-6 8.6 1.05
~
Procedure as described in section on Methodology. Values are means of 3 determinations +_ S.E.M.
(1962) and adenyl cyclase activity by the method of chondrial fraction. Small amounts were detected in ALBANOet al. (1973). the supernatant, microsomal and nuclear fractions. d. Protein was ~-~~easured as described by LOWRY Gradient centrifugation of the crude mitochondria1 (1951) using bovine Serum albumin as standard. fractions showed most of the substance P was located
RESULTS The results in Table 1 and 2 show that both in
the bovine hypothalamus and substantia nigra highest amounts of substance P were found in the crude mito-
in fractions B, C and D (nerve ending particles) (Tables 3 and 4). Little substance P was found in fraction A (myelin) and E (mitochondria). No electron microscopic studies on the various subcellular fractions were carried out in the present
Subcellular distribution of substance P
307
investigation. However the results using enzyme The results suggest a close relationship of substance markers are in general agreement with previous P to neurotransmission in brain. However whether reports (DEROBERTIS et al., 1962, 1967). Most of the the peptide is a neurotransmitter or a modulator of succinate dehydrogenase activity was found in the transmission remains to be established. crude mitochondria1 fraction (83435%) especially in subfraction E. Although about half the lactate dehydRESULTS rogenase activity was located in the supernatant frac- ALBANOJ., MAUDSLEY D., BROWNB. & BARNESG. (1973) tion, 37-40:4, also appeared in the crude mitochonBiochem. Soc. Trans. 1, 477-479. drial fraction. After separation of the crude mitochon- BERGMEYER H., BERNTE. & HESS B. (1963) in Methods of Enzymatic Analysis, (BERGMEYER H., ed.) pp, 73C741. drial fraction by density gradient centrifugation most Academic Press, New York. of the adenyl cyclase activity was found in fractions, CHANG M. & LEEMAN S. (1971) J. biol. Chem. 245, 4784containing the nerve ending particles.
DISCUSSION
The results show that in bovine hypothalamus and substantia nigra most of substance P is located in nerve ending particles. This finding is in general agreement with previous reports using the relatively non specific bioassay (RYALL,1964) and preliminary et al., 1973) to results using immunoassay (POWELL detect the substance. The occurrence of relatively high concentrations of the peptide in the nerve ending particles suggests that substance P is closely involved in neurotransmission. Adenyl cyclase activity was also highest in the nerve ending particles. It has previously been shown that synthetic substance P stimulates adenyl cyclase et al., activity in both human and rat brain (DUFFY 1974). A common subcellular location suggests that stimulation of the cyclase by the peptide may be of physiological signficance.
4790. DE ROBERTISE., DE NALDIA,, DE LORESARNAIZG. & SALGANICOFF L. (1962) J. Neuroohem. 9, 23-35. DE ROBERTISE., DE LORESARXAIZ G. & ALBERICI M. (1967) J. biol. Chem. 242, 3487-3493. DUFFYM. J., WONGJ. & POWELL D. (1974) Biochem. SOC. Trans. 2. 1262-1264. LEMBECK F. (1953) Naunyn-Schmiedebergs Archs. exp. Path. Pharmak. 219, 197-213. LEMBECK F. & ZETLERG. (1972) Int. Encycl. Pharmacol. Ther. Section 72, 1, 29-71. LOWRY0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) J . biol. Chem. 193, 265-275. OTSUKAM., KONISHIS. & TAKAHASHI T. (1972) Proc. Jap. Acad. 48, 747-751. POWELL D., LEEMAN S., TKEGEAR G., NIALLH. & POTTS J., JR. (1973) Nature, New Bid. 241, 252-254. RYALLR. W. (1964) J. Neurochem. 11, 131-145. STERNP., CATOVICS. & STERNM. (1974) Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmk. 281, 233-239. ZETLERG. (1970) in Aspects of Neuroendocrinology (BARGMANN W. & SCHANER B., eds) pp. 287-295. Springer, Berlin.
1975. Vol. 25, pp. 305-307. Pergamon Press. Printed in Great Britain.
SUBCELLULAR DISTRIBUTION OF SUBSTANCE P IN BOVINE HYPOTHALAMUS AND SUBSTANTIA NIGRA M. J. D L ~ - YD. , *MULHALL and D. POWELL Department ot Biochemistry, Trinity College, Dublin 2, Ireland (Received 23 October 1974. Accepted 6 March 1975)
Abstract-Subcellular distribution of substance P as measured by radioimmunoassay was studied in fractions and subfractions from bovine hypothalamus and substantia nigra. Most of the substance P was found in the crude mitochondrial fraction. Subfractionation of the crude mitochondrial fraction by density gradient centrifugation showed most of substance P was present in nerve ending particles. Subcellular particle integrity was assessed using enzyme markers.
SUBSTANCE P is found in the brain and intestine of most vertebrate species (LEMBECK & ZETLER,1972). As isolated from bovine hypothalami substance P is an undempeptide (CHANG& LEEMAN,1970). In human brain highest concentrations of the substance as measured both by bioassay (ZETLER, 1970) and by et al., 1974) are found in radioimmunoassay (DUFFY the substantia nigra and hypothalamus with lowest amounts in areas containing white matter. This uneven distribution of substance P in brain suggested that the molecule might be a neurotransmitter (LEMBECK, 1953). Recently it has been shown that the pure peptide causes depolarization of motor neurons (OTSUKAet al., 1972) and increases the concentration of glycine in mouse brain (STERNet al., 1974). In this investigation we show that in bovine hypothalamus and substantia nigra highest concentrations of the substance are found in subcellular organnelles containing the nerve ending particles.
sucrose and centrifuged for 10min at 9009 at 0-4"C. The precipitate was washed twice in the sucrose medium and centrifuged again as above to obtain the nuclear fraction. The supernatant fluid was pooled and centrifuged at 11,500g for 20min a t (t4"C. The precipitate was washed once in the same medium and centrifuged again. The precipitate was denoted the mitochondrial fraction. The supernatant fraction was centrifuged at 100,ooOg for 60min at 0-4°C. The precipitate was designated the microsomal fraction and the supernatant fluid as the supernatant fraction. The submitochondrial fractions were prepared from the crude mitochondrial fraction prepared as above. It was resuspended in 032 M-sucrose at 33% of the original tissue weight and was layered on top of a discontinuous gradient density of sucrose at 0.8, 1.0, 1.2 and 1.4 M. After centrifugation at 50,000 g for 2 h at WIT the layered subfractions was separated by aspiration, diluted with 0.t 5 M-SUCIOS~ and precipitates were collected by centrifugation at 150,000g for 30 min. Radioimmunoassay of substance P
Substance P was assayed by the method of POWELL et al. (1973). To permit labelling with '''I a substance P dcrivative was used with a tyrosyl residue substituted for the MATERIALS AND METHODS phenylalanine at position 8. Iodination was carried out Materials at room temperature (about 20°C) in small glass vials. The Substance P was purchased from Beckman Instruments reaction mixture contained 1 mCi of sodium '''1 iodide, S.A. Geneva, Switzerland. Tyr-8 substance P was supplied Tyr-8 substance P (5 pg), 0.5 M-sodium phosphate buffer by Drs. Niall, Tregear and Potts, Endocrine Units, Massa- (pH 7.4) and 'Chloramine T (supplied by Kodak Ltd., chussets General Hospital Boston, U.S.A. [8-3H]Adeno- Kirkby, Livcrpool) (50 pg) in a final volume of 60 pl. The sine 3'3' cyclic monophosphate, ammonium salt (20,000- reaction was terminated after 20 s by addition of sodium 30,000 mCi/mmol) and 1251(1MS.30)were supplied by The meta-bisulphite (150 pg in 0 1 ml) followed by 1 ml of blood Radiochemical Centre, Amersham, Bucks, U.K. bank plasma. After incubation of antiserum and synthetic substance Methods P standard (or unknown sample) at 4°C for 24 h, iodinated The primary subcellular fractions were prepared as fol- peptide was added and incubation continued for another lows. Bovine hypothalami and substantia nigra were 48 h. Antibody-bound label was then separated from label obtained from International Meat Packers Ltd., Dublin. free in the incubate by adding 2 0 0 4 of 1.5% charcoal immediately after death of the animal. Primary subcellular coated with 0.15% dextran T 20. fractions were prepared as previously described by DE Brain extracts were purified to the step before chromatROBERTIS et al. (1962). The brain areas were cut into small ography (CHANG& LEAMAN,1970) before assay for subpieces and homogenised in 0 3 2 M-sucrose for 2 min using stance P. a glass homogeniser with a loose Teflon pestle (0.25mm clearance). The homogenate was diluted to 10% in 0.32 M- Enzyme assays Lactate dehydrogenase activity (LDH) was assayed by * Present address: Department of Radioisotopes, St. the method of BERGMEYER et al. (1963) succinate dehydroVincent's Hospital, Dublin 4, Ireland. genase (SDH) activity as described by DE ROBERTISet al. N.C
2513-H
305
M. J. DUFFY, D. MULHALL and D. POWELL
306
TABLE1. DISTRIBUTION OF PROTEIN, SUCCINATE DEHYDRWENASE ACTIVITY (SDH.), LACTATE DEHYDROGENASE ACTIVITY (LDH.), ADENYL CYCLASE ACTIVITY AND SUBSTANCE P IN SUBCELLULAR ORGANELLES FROM BOVINE HYPOTHALAMI Protein
Subcellular organelle
SDH. activity
LDH. activity (%)
(%I
(%)
Adenyl cyclase activity
Substance P
(%)
(%)
Substance P (ngimg protein) 2-43
-~ ~~~
Nuclear fraction Mitochondria1 fraction Microsomd fraction Supernatant fraction Total recovery (%)
12.6 f 0 3 2
6.91 f 0 4 0
3.65 f 0 4 5
8.25 f 1.6
9.44 f 1.1
50.1 f 2.9
83.0 f 3.5
37.8 f 2 6
468 f 3.4
59.0 f 3.5
524
41.3 f 4.0
I56
202
+ 1.7
2.4
*
0.21
87.9 f 5.6
+ 1.9 16.0 + 0 8 3
3.6 f 051
53.3 f 4 7
7.7 f 0 3 8
17.1 f 0.8 94.3 f 4.3
0-73
86.2 f 51
905 f 4.8
377 238 2.99
857 f 3.9
Procedure as described in section on Methodology. Values are means of 3 determinations k S.E.M.
TABLE 2: DISTRIBUTION OF PROTEM,
SUCCINATE DEHYDROCENASE ACTIVITY, LACTATE DEHYDRWENASE ACTIVITY, ADENYL CYCLASE ACTIVITY AND SUBSTANCE P IN SUBCELLULAR ORGANELLES FROM BOVINE SUBSTANTIA NIGRA Protein
Subcellular organelle
SDH. activity
(%I
(%I
Nuclear lraction Mitochondria1 fraction Microsomai fraction Supernatant fraction Total recovery (%)
8.0
13.1 f 0 5 6 477
+ 4.3
*
LDH. activity
Adenyl cyclase activity
Substance P
(%)
(%)
(%)
1.2
7.7 f 0 7 4
85.3 f 5 4
208 +_ 2.2
1.3 +_ 0 3 3
18.4 f 1.7
5 4 f076
409
4.2 +_ 0 2 9
839 f 4 4
8.7 f 0.82 556
k 56
5.90
39.5 +_ 3 8
18.4 f 2 7
4.46
17.3 f 3.8
4.73
92.1 f 5 7
89.2 f 4 7
Procedure as described in section on Methodology. Values are means of 3 determinations
TABLE 3.
3-35
45.9 f 3.6
3 3 f 025
47.1 f 3 8
85.2 f 3.9
920 f 3 2
11.3 +_ 0.89
k 2.3
Substance P (ng/mg pratein)
S.E.M.
DISTRIBUTION OF PROTEIN, SUCCINAE DEHYDRWENASE ACTIVITY, ADENYL CYCLASE ACTIVITY AND SUBSTANCE P IN SUE-MI'NKHONDRIAL FRACTIONS FROM BOVINE HYPOTHALAMI
Protein Fraction
A. Myelin B. Nerve endings C. Nerve endings D. Nerve endings E. Free. mitochondria Total recovery %
(%I
32.1 f 2.8 8-73f 0.92 9-92 k 0.83 24.7 f 1.7 24.5 f 1.5 95.6 f 5.0
SDH. activity
(%I
Adenyl cyclase
(%I
(%)
5.43 0.79 17.8 & 1.23 34.2 f 3.0 39.9 f 4.5 2.7 f 042 87.8 3.6
6.28 f 1.3 33.0 f 3.7 607 f 5.8 83.0 f 4.2
Substance P
2.97 f 0.80 20.4 f 1.5 35-7 f 2.6 362 2.3 4.7 f 0.48 86.2 4.0
+
Procedure as described in section on Methodology. Values are means of 3 determinations
Substance P (ng/mg protein)
0.09 8.83 13.6 5.52 0.70
S.E.M.
TABLE 4. DISTRIBUTION OF PROTEIN,
SUCCINATE DEHYDROGENASE ACTIVITY, ADENYL CYCLASE ACTIVITY AND SUBSTANCE IN SUB-MITOCHONDRIAL FRACTIONS FROM BOVINE SUBSTANTIA NIGRA
Fraction A. Myelin
B. C. D. E.
Nerve endings Nerve endings Nerve endings Free mitochondria Total recovery %
Protein
SDH. activity
Adenyl cyclase activity
Substance P
29.6 f 3.1 10.9 f 0.96 16.3 f 1.8 23Q f 2.7 202 f 3.0 89.9 f 5.5
2.8 f 0.34 4.9 f 038 305 f 2.6 614 5.3 84.6 f 3.7
8.4 f 0.73 142 f 1.5 35.3 f 2.9 38.8 f 2.6 3.3 f 0.47 91.7 f 4-9
4 7 f 0.50 23.0 f 1.6 35.1 & 2.6 336 +_ 4.9 3-6 f 1.4 901 f 4.6
(%I
(%I
~~
(%I
~
(%I
P
Substance P (ng/mg protein)
0930 12.5 12-6 8.6 1.05
~
Procedure as described in section on Methodology. Values are means of 3 determinations +_ S.E.M.
(1962) and adenyl cyclase activity by the method of chondrial fraction. Small amounts were detected in ALBANOet al. (1973). the supernatant, microsomal and nuclear fractions. d. Protein was ~-~~easured as described by LOWRY Gradient centrifugation of the crude mitochondria1 (1951) using bovine Serum albumin as standard. fractions showed most of the substance P was located
RESULTS The results in Table 1 and 2 show that both in
the bovine hypothalamus and substantia nigra highest amounts of substance P were found in the crude mito-
in fractions B, C and D (nerve ending particles) (Tables 3 and 4). Little substance P was found in fraction A (myelin) and E (mitochondria). No electron microscopic studies on the various subcellular fractions were carried out in the present
Subcellular distribution of substance P
307
investigation. However the results using enzyme The results suggest a close relationship of substance markers are in general agreement with previous P to neurotransmission in brain. However whether reports (DEROBERTIS et al., 1962, 1967). Most of the the peptide is a neurotransmitter or a modulator of succinate dehydrogenase activity was found in the transmission remains to be established. crude mitochondria1 fraction (83435%) especially in subfraction E. Although about half the lactate dehydRESULTS rogenase activity was located in the supernatant frac- ALBANOJ., MAUDSLEY D., BROWNB. & BARNESG. (1973) tion, 37-40:4, also appeared in the crude mitochonBiochem. Soc. Trans. 1, 477-479. drial fraction. After separation of the crude mitochon- BERGMEYER H., BERNTE. & HESS B. (1963) in Methods of Enzymatic Analysis, (BERGMEYER H., ed.) pp, 73C741. drial fraction by density gradient centrifugation most Academic Press, New York. of the adenyl cyclase activity was found in fractions, CHANG M. & LEEMAN S. (1971) J. biol. Chem. 245, 4784containing the nerve ending particles.
DISCUSSION
The results show that in bovine hypothalamus and substantia nigra most of substance P is located in nerve ending particles. This finding is in general agreement with previous reports using the relatively non specific bioassay (RYALL,1964) and preliminary et al., 1973) to results using immunoassay (POWELL detect the substance. The occurrence of relatively high concentrations of the peptide in the nerve ending particles suggests that substance P is closely involved in neurotransmission. Adenyl cyclase activity was also highest in the nerve ending particles. It has previously been shown that synthetic substance P stimulates adenyl cyclase et al., activity in both human and rat brain (DUFFY 1974). A common subcellular location suggests that stimulation of the cyclase by the peptide may be of physiological signficance.
4790. DE ROBERTISE., DE NALDIA,, DE LORESARNAIZG. & SALGANICOFF L. (1962) J. Neuroohem. 9, 23-35. DE ROBERTISE., DE LORESARXAIZ G. & ALBERICI M. (1967) J. biol. Chem. 242, 3487-3493. DUFFYM. J., WONGJ. & POWELL D. (1974) Biochem. SOC. Trans. 2. 1262-1264. LEMBECK F. (1953) Naunyn-Schmiedebergs Archs. exp. Path. Pharmak. 219, 197-213. LEMBECK F. & ZETLERG. (1972) Int. Encycl. Pharmacol. Ther. Section 72, 1, 29-71. LOWRY0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) J . biol. Chem. 193, 265-275. OTSUKAM., KONISHIS. & TAKAHASHI T. (1972) Proc. Jap. Acad. 48, 747-751. POWELL D., LEEMAN S., TKEGEAR G., NIALLH. & POTTS J., JR. (1973) Nature, New Bid. 241, 252-254. RYALLR. W. (1964) J. Neurochem. 11, 131-145. STERNP., CATOVICS. & STERNM. (1974) Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmk. 281, 233-239. ZETLERG. (1970) in Aspects of Neuroendocrinology (BARGMANN W. & SCHANER B., eds) pp. 287-295. Springer, Berlin.
