Identification of the motoneuron-depolarizing peptide in bovine dorsal root as hypothalamic substance P

Brain Research, 73 (1974) 59-69 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

59

I D E N T I F I C A T I O N OF T H E M O T O N E U R O N - D E P O L A R I Z I N G PEPTIDE IN BOVINE D O R S A L ROOT AS H Y P O T H A L A M I C SUBSTANCE P

TOMOYUKI TAKAHASHI, SHIRO KONISHI, DAVID POWELL, SUSAN E. LEEMAN AND MASANORI OTSUKA Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo (Japan) and Laboratory of Human Reproductionand Reproductive Biology, Harvard Medical School, Boston, Mass. 02115 (U.S.A.)

(Accepted December 15th, 1973)

SUMMARY

Previous studies have shown that bovine dorsal root contains a peptide which depolarizes the frog's spinal motoneurons. In the present study, pharmacological, chemical, enzymatic and immunological properties of this motoneuron-depolarizing peptide were investigated, and the results indicated that the peptide is identical with substance P recently isolated from hypothalamus. The amount of substance P in the dorsal root was 24-130 pmoles/g wet weight, while that in the ventral root was 9-27 times less. These results support the hypothesis that substance P might be an excitatory transmitter released from primary sensory neurons.

I NTRODUCTION It has recently been shown z3 that a fraction obtained by chromatography of an extract of bovine spinal dorsal root tissue exerts a depolarizing action on the spinal motoneurons of a frog. The same fraction also produced a fall of blood pressure in a rat and a contraction o f the guinea pig ileum. Since all these activities were abolished by incubation with chymotrypsin, they were tentatively ascribed to a peptide which we called 'dorsal root peptide'. The corresponding fraction o f the ventral root extract, by contrast, did not display such activities to any appreciable extent. Based on these findings a hypothesis was proposed that the dorsal root peptide might be an excitatory transmitter released from the primary sensory neurons. A series of peptides having a common C-terminal sequence is known to have the biological activities similar to those o f the dorsal root peptide. Among these peptides, physalaemin, eledoisin and hypothalamic substance P are naturally-occurring undecapeptides and exert a remarkably strong depolarizing action on frog's spinal

60

T. TAKAHASHIet al.

motoneuronsa,9,~a, t4. It therefore seemed possible that the dorsal root peptide is identical with one o f these motoneuron-depolarizing peptides. This possibility was explored in the present study. A preliminary report of this work has already been published 14. MATERIALS AND METHODS

Preparation of crude extracts Lumbar and sacral parts o f bovine spinal cords together with nerve roots were obtained within 30 min after slaughter, and were kept in ice or frozen. Dorsal and ventral roots were dissected within several hours and stored in a deep freezer. A crude extract of the nerve roots was prepared according to the procedures o f Chang and Leeman 1. Virtis 45 homogenizer (40,000 rev./min for 10 min) was used for homogenizing the nerve roots (for details of preparation seee also Otsuka et al.a3). Cation exchange chromatography on Amberlite CG-50 column The crude extract originating from 30 to 45 g dorsal or ventral roots dissolved in 6-9 ml water was applied to an Amberlite CG-50 column (0.7 cm × 14 cm). After the column was washed with 100 ml of 0.1 ~ acetic acid, stepwise elution was carried out with 20 ml each of 1.9 M and then 3.8 M pyridine-acetate buffers (30:4, v/v, pH 5.9). Eluates were collected and then lyophilized. Gel chromatography on Sephadex LH-20 column The tissue extract or several known peptides were applied to a Sephadex LH-20 column (1.4 cm × 80 cm) equilibrated with 3.8 M pyridine-acetate buffer (pH 5.9). The column was eluted with the same buffer and the eluates were collected in 3-ml fractions and lyophilized. The amounts of dorsal root peptide and other gut-contracting substances in the fractions were bioassayed on guinea pig ileum. The amounts o f synthetic peptides and glucagon were determined by the ninhydrin reaction 16. Paper chromatography Solutions of 10 #1 containing synthetic peptides or partially purified tissue extract were applied to an ascending paper chromatogram. The buffer system was n-butanol-pyridine-water (1 : 1 : 1, pH 8.0) and the paper was Toyo No. 51 (2 cm × 38 cm). After being chromatographed for 24 h at 4 °C, the paper was dried at room temperature, and cut into 1-cm strips. Each piece of paper was eluted with 1 ml Tyrode solution for bioassay on guinea pig ileum. High voltage electrophoresis Synthetic peptides or partially purified tissue extract were submitted to a high voltage electrophoresis of the enclosed strip type 19. The buffer system was either formic acid-acetic acid-water (20:80:900, pH 2.0) or pyridine-acetic acid-water (80:3.2:720, pH 6.2). Acetone ( 1 0 ~ v/v) was added to buffers to reduce the vapor pressure. Samples in 5/21 buffer were spotted on the starting line of Whatman 3 MM

SUBSTANCE

P

IN BOVINE DORSAL ROOT

61

paper (12 cm × 50 cm). As reference, L-glutamic acid (electrophoresis at pH 2.0) or L-lysine (at pH 6.2) in 1 #1 buffer was spotted on both sides of the samples. After electrophoresis for 60-90 min at 80 V/cm, the paper was dried at room temperature. Reference amino acids and certain peptides were detected by staining the paper with 0.2~o ninhydrin acetone 17. For surveying the gut-contracting activities in electropherograms, divided bands of 2-4 cm width were cut into 1-cm strips, which were eluted with 1 ml Tyrode solution and applied to a guinea pig ileum.

Bioassays Distal portion of the ileum was isolated from an overnight fasted guinea pig and suspended in a 0.4 ml bath of Tyrode solution at 37 °C oxygenated with 95 ~o 02-5 ~ CO2. The Tyrode solution contained 10-7 g/ml atropine, 10-6 g/ml mepyramine and 10-6 g/ml tryptamine in order to keep the ileum insensitive to acetylcholine, histamine and serotonin. Hypotensive action was tested on the carotid blood pressure of a rabbit anesthetized with urethan. The motoneuron-depolarizing activities were determined using isolated spinal cord of bullfrog as described in the previous papersS,L Radioimmunoassay of substance P The lyophilized tissue extracts were taken up in sodium barbital buffer and assayed for substance P by radioimmunoassay as described by Powell et al. 1~. The only modifications of the method were that sample, antibody, label and buffer were combined without any preincubation and incubated for a total time of 18 h. Peptides Hypothalamic substance P was synthesized as described in the previous paper 18. Synthetic substance P was also kindly supplied by Dr. H. Yajima, Faculty of Pharmaceutical Sciences, Kyoto University. Physalaemin was a gift of Dr. Y. Nobuhara, Dainippon Pharmaceutical Company, Osaka. Eledoisin and glucagon were obtained from Sigma Chemical Company; angiotensin I, bradykinin, and a shorter homolog of eledoisin were from Protein Research Foundation, Osaka. RESULTS

Pharmacological properties of the dorsal root peptide, substance P, physalaemin and eledoisin The crude extract of bovine dorsal roots was fractionated on an Amberlite CG-50 column. As reported in the previous papers 13,14, most of the gut-contracting activity, which could be inactivated by incubation with chymotrypsin, occurred in the fraction eluted by 3.8 M pyridine-acetate buffer (pH 5.9). This fraction was further submitted to gel chromatography on a Sephadex LH-20 column. As shown in Fig. 2, the main peak of gut-contracting activity was found at the elution volume (Ve) of about 1.4 times the void volume (Vo). The active fraction was collected, lyophilized and tested for the pharmacological activities. As illustrated in Fig. 1,

62

T. TAKAHASHI et al. a

b

c

L ~ i ira,.L

A

11

II

II

I

I

I i

0

I

L

~

I

70mmH g

i

,

1

i

2min

'

Fig. 1. Comparison of the biological activities of the dorsal root peptide and synthetic substance P. Dorsal root peptide was partially purified from the crude extract of dorsal root through Amberlite CG-50 and Sephadex LH-20 columns. A: contraction of guinea pig ileum treated with atropine, mepyramine and tryptamine. B: blood pressure of the rabbit. C: changes of membrane potential of spinal motoneurons were recorded from the 8th ventral root with Ag-AgCI electrode in an isolated and hemisected spinal cord of the frog soaked in 0.6 mM Ca Ringer's solution. Corresponding dorsal root was stimulated maximally at 0.1 Hz. a: activities of the dorsal root peptide. Active fraction derived from 320 mg wet wt. of dorsal root was added to 0.4-ml bath between arrows in A; that from 200 mg tissue injected intravenously at arrow in B; and that from 6 g tissue added to 0.3-ml bath between arrows in C. b: activities of the dorsal root peptide after incubating with chymotrypsin (20 yg/ml, 37 °C for 30 min). The amounts of the dorsal root extract were the same as in a. c: activities of synthetic substance P; 16 pmoles./0.4-ml bath in A, 10 pmoles injected in B, and 390 pmoles/ 0.3-ml bath in C.

the active fraction elicited a c o n t r a c t i o n o f guinea pig's ileum, caused a fall in r a b b i t ' s b l o o d pressure, a n d d e p o l a r i z e d the frog's spinal m o t o n e u r o n s . Since all 3 activities were a b o l i s h e d by i n c u b a t i n g the fraction with c h y m o t r y p s i n (Fig. lb), they were ascribed to the dorsal r o o t peptide. A t present, 3 n a t u r a l l y - o c c u r r i n g peptides, i.e., h y p o t h a l a m i c substance P, p h y s a l a e m i n a n d eledoisin, are k n o w n to have such p h a r m a c o l o g i c a l activities as described above. In Fig. lc, the activities o f authentic substance P are illustrated f o r c o m p a r i s o n , a n d clearly they are very similar to those o f the d o r s a l r o o t peptide. I f one assumes that the d o r s a l r o o t p e p t i d e is either substance P, p h y s a l a e m i n o r eledoisin, for each case it is possible to calculate the a m o u n t s o f the p e p t i d e in the d o r s a l r o o t extract, by c o m p a r i n g the relative potencies o f gut-contracting, b l o o d pressure-lowering a n d m o t o n e u r o n - d e p o l a r i z i n g activities o f the d o r s a l r o o t p e p t i d e with those o f the undecapeptides. T a b l e I s u m m a r i z e s the result o f such calculation. It can be seen t h a t o n l y the a s s u m p t i o n t h a t the d o r s a l r o o t p e p t i d e is substance P gives the values which are consistent with each o t h e r in the 3 b i o a s s a y systems.

SUBSTANCE P IN BOVINE DORSAL ROOT

63

TABLE I AMOUNT OF THE DORSAL ROOT PEPTIDE ESTIMATED ON AN ASSUMPTION THAT 11 IS SUBSTANCE

P, PHYSAL-

AEMIN OR ELEDOISIN

Bioassays

Peptide (pmoles/g wet wt. of dorsal root)

Contraction of ileum Fall of blood pressure Depolarization of motoneurons

Substance P

Physalaemin

Eledoisin

38 41 50

27 150 7

48 500 5

Chemical properties of the dorsal root peptide Estimation of the molecular weight by gel chromatography The active fraction of the dorsal root extract eluted from an Amberlite CG-50 column was submitted to gel chromatography using a Sephadex LH-20 column. As illustrated in Fig. 2, the peak of gut-contracting activity occurred at Ve/Vo -- 1.34 ± 0.03 (mean ± S.E.M. of 3 experiments). Three peptides of known molecular weights were also run on the same column and the relationship between the logarithm of the molecular weight and the ratio Ve/Vo was plotted in Fig. 3. F r o m these data, the molecular weight o f the dorsal root peptide was estimated at 1250 ± 150 which coincides with that o f substance P as well as physalaemin and eledoisin (Table II). High voltage paper electrophoresis The active fraction of the dorsal root extract eluted from an Amberlite CG-50 Peak of hypofhQlamic subsfence P Blue dex~ron

1

NoCI

1

1

E

6

5~9

o

-

-

I ]5

2, 30

_ 45 Elution

60

75

90

volurne ( m l )

Fig. 2. Gel chromatography of dorsal root extract. Crude extract was fractionated by Amberlite CG-50 column and the active fraction was applied to Sephadex LH-20 column 0 . 4 c m x 80 cm) which was eluted with 3.8 M pyridine-acetate buffer (pH 5.9). Each 3-ml fraction was bioassayed on guinea pig ileum. Ordinate: gut-contracting activity of 1-g tissue expressed as an amount o f substance P that causes the same effect. Peak elution volumes of blue dextran, synthetic substance P and NaCI are shown by arrows.

64

T. TAKAHASHI

et

al.

2.0

~Gly-Phe (222) v,/vo 1.5

Dorsa[~Hypothalarnic

substanceP(1340)

"~Glucagon ( 3485)

1.0 I 100

I I I 500 I000 5000 Log molecularweight

Fig. 3. Estimation of molecular weight of the dorsal root peptide. Peptides of known molecular weights and the dorsal root peptide were applied to Sephadex LH-20 column. Procedures of gel chromatography were as in Fig. 2. Ordinate: ratio of elution volume to the void volume. Abscissa: molecular weight in logarithmic scale. Numbers in parentheses indicate the molecular weights of peptides. Arrow indicates the peak of gut-contracting activity of the dorsal root peptide. column was submitted to high voltage paper electrophoresis as described in Methods. Electrophoretic mobilities o f the dorsal r o o t peptide at p H 2.0 and 6.2 were c o m p a r e d with those o f several k n o w n peptides, and the results are summarized in Fig. 4. This combination o f 2 systems o f electrophoresis provided a satisfactory separation o f the peptides. It can be seen from Fig. 4, that the electrophoretic mobilities o f the dorsal root peptide are identical with those o f substance P (see also Table II).

Paper chromatography and enzymatic degradation The results presented in the preceding sections suggest that the dorsal root peptide is identical with substance P. This was further supported by the following experiments. Dorsal r o o t peptide was partially purified from the crude dorsal root extract using an Amberlite CG-50 column and then Sephadex LH-20 chromatography. This fraction as well as synthetic peptides was submitted to paper chromatography or enzymatic degradation. R f values o f the dorsal root peptide, substance P TABLE II COMPARISON OF CHEMICAL PROPERTIES OF THE DORSAL ROOT PEPTIDE AND HYPOTHALAMIC SUBSTANCE P

Values represent mean ± S.E.M. and number of determinations in parentheses.

Dorsal root peptide Hypothalamic substance P Molecular weight estimated by gel chromatography Electrophoretic mobilities relative to L-glutamic acid at pH 2.0 Electrophoretic mobilities relative to L-lysine at pH 6.2 Rfin paper chromatography (n-butanol-pyridine-water)

1250 ~ 150 1.17 ± 0.02 0.61 ± 0.01 0.62

(3) (3) (3) (1)

1340 1.19 ± 0.01 (6) 0.59 ~ 0.01 (4) 0.62 ± 0.02 (3)

SUBSTANCE

P

IN BOVINE

DORSAL

65

ROOT -e- Dorsal root peptide --o-- Hypothalomic substance p

I.O pH2.0 Eledoisin reloted peptide

08 (,j ,6

Oorsol root pegtide @

o6

~

o

Hyoot halomic substance P i Bradykinin

*6

0

0.2

0

EledolSln i 0

pHo2

/!

Angiotensin I

Physoloemin

o

o!2 o~ o'o o18 ,'o

,!2 ,~

o

o12 o'.4 06t h 08i

Mobilify at pH 2.0

Fig. 4. Electrophoretic mobilities of the dorsal root peptide and synthetic peptides having similar biological activities. In A, mobilities of the peptides at pH 2.0 relative to L-glutamic acid were plotted

against those at pH 6.2 relative to L-lysine.Eledoisinrelated peptide: Lys-Phe-Ile-Gly-Leu-Met-NH2. In B and C, electrophoretic mobilities of the dorsal root peptide were compared with those of hypothalamic substance P. Ordinate: gut-contracting activity expressed as an amount of substance P. Abscissa: electrophoretic mobility relative to L-glutamate (in B) and to L-lysine (in C). Migrations of L-glutamate and L-lysinewere 17 cm and 34 cm respectively.

and physalaemin were 0.62, whereas that of eledoisin was 0.73 (Table II). The gutcontracting activities o f both the dorsal root peptide and substance P were inactivated by incubating with chymotrypsin (Boehringer, 20 #g/ml, 37 °C for 30 min), but insensitive to TPCK-treated trypsin (Worthington, 100/~g/ml, 37 °C, 40 min) or carboxypeptidase A (Boehringer, 10 #g/ml, 37 °C, 60 min). Physalaemin was inactivated by chymotrypsin and trypsin but not by carboxypeptidase A.

Radioimmunoassay for substance P A specific radioimmunoassay for substance P has recently been devisedlL The antibody for hypothalamic substance P was shown to react with substance P but not with physalaemin and eledoisin. Dorsal root extracts containing the dorsal root peptide before and after purification with Amberlite CG-50 chromatography were submitted to this test and shown to contain a material reacting with the antibody (for quantitative aspect, see below). Distribution of hypothalamic substance P in bovine dorsal and ventral roots The variety of examinations described above altogether indicate that the dorsal root peptide is identical with hypothalamic substance P. It is of interest to compare the amount of substance P in the dorsal and ventral roots, because, if the dorsal root peptide, i.e. substance P, is a neurotransmitter of the primary sensory neurons, one would expect its selective distribution in the dorsal root. So far acetylcholine, norepinephrine and GABA have been shown to be concentrated specifically in the neurons which release these transmitters. Application of the crude extract o f bovine dorsal root elicited a contraction of

T. TAKAHASHIet al.

66 C r u d e e x t r a c t s of th e ~:

o~

+

d o r s a l root

-o-

v e n t r a l root

eo

-6

£

IE o-

tq L

Z

-

O

I

1.0

1.5

2.0

Ve/Vo

Fig. 5. Comparison of dorsal and ventral root extracts by gel chromatography. Crude extracts of dorsal and ventral roots (original tissue 4 and I0 g respectively) were applied to Sepbadex LH-20 column (1.4 cm × 80 cm), which was eluted with 3.8 M pyridine-acetate buffer (pH 5.9). Each 3-ml fraction was bioassayed on guinea pig ileum. Ordinate: same as in Fig. 2.

guinea pig ileum treated with atropine, mepyramine and tryptamine. The gut-contracting activity of the crude extract of dorsal root was about 2.5 times more potent than that of the ventral root (Table III, cf Lembeck t°, and Hellauer6). Not all the activity of the crude extracts, however, can be ascribed to substance P, because the activity of the dorsal root extract was only partially, and that of the ventral root extract was not at all inactivated by chymotrypsin. The ratio of the chymotrypsinsensitive activity of crude dorsal root extract to that o f the ventral root extract was more than 11 (Table III). Crude extracts of both dorsal and ventral roots were, therefore, fractionated on a Sephadex LH-20 column. As shown in Fig. 5, the chromatographic pattern of the dorsal root extract displayed 3 peaks of gut-contracting activity at Ve/Vo = 1.0, 1.34 and 1.6. Gut-contracting activities of the first 2 fractions were completely abolished by incubation with chymotrypsin. The second fraction is very probably substance P because the peak elution volume was the same as that of substance P. This prediction was further confirmed by submitting the second fraction to a high voltage paper electrophoresis. Electropherograms at both p H 2.0 and 6.2 displayed single peaks with the same mobilities as those of substance P. The first small fraction at void volume may also represent substance P in a bound form because, when electrophoresed at p H 2.0 and 6.2 the fraction gave the peaks of gut-contracting activity at the same distances of migration as those of substance P. The third fraction with the peak at Ve/Vo = 1.6 occurred in both dorsal and ventral root extracts and was not inactivated by chymotrypsin. Fractions corresponding to the second peak were collected for both dorsal and ventral root extracts, lyophilized and bioassayed on guinea pig ileum. The amount of substance P thus estimated was 43 pmoles/g in the dorsal and 1.6 pmoles/g in the ventral root extracts. The ratio of the amount in dorsal to ventral root extracts was 27.

SUBSTANCE

P

67

IN BOVINE DORSAL ROOT

T A B L E III ESTIMATED AMOUNTSOF SUBSTANCEP IN THE EXTRACTSOF DORSAL AND VENTRAL ROOTS Values represent m e a n i

Purification step

S.E.M. a n d n u m b e r of determinations in parentheses.

Amount of substance P (pmoles/g wet wt.) Dorsalroot

C r u d e extract C r u d e extract C r u d e extract Amberlite C G - 5 0 c o l u m n Sephadex L H - 2 0 c o l u m n

160 :~ 5.5 130 ± 8 30 24 43 ± 2.1

D~ V*

Method of determination

65 4- 8.5 (3) < 12 (3)

2.5 > 11

Bioassay o n i l e u m * * Bioassay on i l e u m * * *

3.5 (1) 2.5 (1) 1.6 4- 0.1 (3)

9 10 27

Radioimmunoassay Radioimmunoassay Bioassayonileum**

Ventralroot (3) (3) (1) (1) (3)

* R a t i o o f a m o u n t s o f s u b s t a n c e P in dorsal to ventral root extracts. ** Estimated f r o m the total gut-contracting activity. *** Estimated f r o m the gut-contracting activity w h i c h was inactivated by c h y m o t r y p s i n .

Crude extracts of dorsal and ventral roots, as well as the active fraction eluted from the Amberlite CG-50 column, were also submitted to radioimmunoassay of substance P (see ref. 15). The obtained values were similar to those of the bioassay (Table III). DISCUSSION

The results described in the present paper provide convincing evidence that the motoneuron-depolarizing peptide in bovine dorsal root, i.e., dorsal root peptide is identical with substance P recently isolated from the hypothalamus by Chang et al.l,2. Pharmacological, chemical, enzymatic and immunological properties of both peptides were shown to be identical. Quantitative data concerning the pharmacological and chemical properties o f both peptides are given in Tables I and II. Since Euler and Gaddum 4 reported the presence o f substance P in brain and intestine, much interest has been focused on its physiological function, particularly its possible transmitter role (for review see Lembeck and Zetlerll,12). Studies o f substance P, however, have been greatly hampered by the absence of a pure substance P preparation. This obstacle has been removed by recent studies o f Chang et al. 1,2 and Tregear et ak 18, and as a result, several findings now support the hypothesis that substance P is an excitatory transmitter in the spinal cord released from the primary sensory neurons. Namely, the amount of substance P in bovine dorsal root is about 9-27 times larger than that in the ventral root a4. Substance P exerts a remarkably strong excitatory action on frog's spinal motoneurons T M as well as on the spinal neurons o f a newborn rat (Konishi and Otsuka, unpublished observation). When the subcellular fractions of rat hypothalamic tissue were analysed for substance P, the highest concentration was found in the nerve ending particles 15. The amount o f

68

T. TAKAHASHI et al.

substance P in cat's spinal cord, p a r t i c u l a r l y in the dorsal horn, was decreased to below o n e - h a l f after the section o f the d o r s a l r o o t s ( T a k a h a s h i a n d Otsuka, u n p u b lished observation). Hellauer a n d U m r a t h 7 have a l r e a d y m a d e an a t t e m p t to extract a sensory transmitter substance f r o m bovine dorsal roots, a n d f o u n d t h a t the d o r s a l r o o t extract contains an agent p r o d u c i n g a v a s o d i l a t a t i o n o f d e n e r v a t e d r a b b i t ear. The a m o u n t o f this v a s o d i l a t o r agent was f o u n d to be much larger in the d o r s a l r o o t t h a n in the ventral r o o t 7. Based on these findings they p r o p o s e d a hypothesis t h a t the active agent might be a t r a n s m i t t e r o f p r i m a r y sensory n e u r o n s which induces axon reflex v a s o d i l a t a t i o n at the p e r i p h e r a l synapses a n d excitation o f spinal n e u r o n s at the central synapses ( c f Dalea). L a t e r studies o f L e m b e c k x° a n d H e l l a u e r 6 showed that the v a s o d i l a t o r agent has also a p o t e n t a c t i o n on a t r o p i n e - t r e a t e d intestine to induce a c o n t r a c t i o n . L e m b e c k assumed that the active agent is substance P, which he p r o posed as a sensory transmitter. Since then, however, neither the chemical identity o f this v a s o d i l a t o r a n d gut-contracting agent n o r its a c t i o n on central n e u r o n s has been elucidated until o u r recent study. The results o b t a i n e d on h y p o t h a l a m i c substance P, as discussed above, are in a r e m a r k a b l e parallel with the hypothesis o f L e m b e c k a n d Hellauer. It should be n o t e d t h a t the a m o u n t o f substance P in the d o r s a l r o o t is m u c h s m a l l e r than, e.g., t h a t o f acetylcholine in the ventral r o o t 5. This m a y m e a n t h a t substance P is a t r a n s m i t t e r o f only a p a r t o f whole p r i m a r y sensory neurons. It is also conceivable t h a t substance P is c o n c e n t r a t e d o n l y in their nerve terminals. A l t h o u g h further studies, such as, for example, the e x a m i n a t i o n o f release o f substance P f r o m sensory neurons, w o u l d be needed before it could be accepted as a n e u r o t r a n s m i t t e r , available d a t a at present m a k e substance P one o f the p r o m i s i n g c a n d i d a t e s for the t r a n s m i t t e r o f p r i m a r y sensory neurons.

REFERENCES 1 CHANG, M. M., AND LEEMAN, S. E., Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P, J. biol. Chem., 245 (1970) 4784-4790. 2 CHANG, M. M., LEEMAN,S. E., AND NIALL, H. D., Amino-acid sequence of substance P, Nature (Lond.)., 232 (1971) 86-87. 3 DALE, H. H., Pharmacology and nerve-endings, Proc. roy. Soc. Med., 28 (1935) 319-332. 4 EULER,U. S. v., AND GADDUM,J. H., An unidentified depressor substance in certain tissue extracts, J. Physiol. (Lond.), 72 (1931) 74-87. 5 EVANS,C. A. N., AND SAUNDERS,N. R., The distribution of acetylcholine in normal and in regenerating nerve~, J. Physiol. (L~n.t.), 192 (1967) 79-92. 6 HELLAUER,H., Zur Charakterisierung der Erregungssubstanz sensibler Nerven, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 219 (1953) 234-241. 7 HELLAUER,H., UND UMRATH,K., Uber die Aktionssubstanz der sensiblen Nerven, Pfliigers Arch. ges. Physiol., 249 (1948) 619-630. 8 KONISHI, S., AND OTSUKA, M,, Actions of certain polypeptides on frog spinal neurons, Jap. J. Pharmaeol., 21 (1971) 685-687. 9 KONISHI,S., AND OTSUKA, M., The effects of substance P and other peptides on spinal neurons of the frog, Brain Research, 65 (1974) 397-410. 10 LEMBECK,F., Zur Frage der zentralen Obertragung afferenter Impulse. III. Mitteilung, Naunyn Sehmiedeberg's Arch. exp. Path. Pharmak., 219 (1953) 197-213.

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11 LEMBECK,F., AND ZETLER, G., Substance P: A polypeptide of possible physiological significance, especially within the nervous system. In C. C. PFEIFFER AND J. R. SMYTmES(Eds.), International Review ofNeurobiology, Vol. 4, Academic Press, New York, 1962, pp. 159-215. 12 LEMBECK,F., AND ZETLER, G., Substance P. In J. M. WALKER(Ed.), lnternationalEncyclopedia of Pharmacology and Therapeutics, Section 72, Vol. I, Pergamon Press, Oxford, 1971, pp. 29-71. 13 OTSUKA,M., KONISm, S., AND TAKAHASHI,Z., The presence of a motoneuron-depolarizing peptide in bovine dorsal roots of spinal nerves, Proc. Jap. Acad., 48 (1972) 342-346. 14 OTSUKA, M., KONISHt, S., AND TAKAHASHI,T., A further study of the motoneuron-depolarizing peptide extracted from dorsal roots of bovine spinal nerves, Proc. Jap. Acad., 48 (1972) 747-752. 15 POWELL,D., LEEMAN,S. E., TREGEAR,G. W., NIALL, H. D., AND POTTS, J. T., JR., Radioimmunoassay for substance P, Nature (Lond.), 241 (1973) 252-254. 16 ROSEN, H., A modified ninhydrin colorimetric analysis for amino acids, Arch. Biochem., 67 (1957) 10-15. 17 TOENNIS,G., AND KoLa, J. J., Techniques and reagents for paper chromatography, dnalyt. Chem., 23 (1951) 823-826. 18 TREGEAR,G. W., NIALL, H. D., POTTS,J. T., JR., LEEMAN,S. E., AND CHANG, M. M., Synthesis of substance P, Nature (Lond.), 232 (1971) 87-89. 19 WrtITAKER,J. R., Electrophoresis in stabilizing media. In G. ZWEIG AND J. R. WHITAKER(Eds.), Paper chromatography and electrophoresis, Vol. 1, Academic Press, New York, 1967, pp. 25-27