0022-3042/78/06001-I321 802.00/0

Jourrrol of N~wroclwrriivtr~ VOI 30. pp. 1 3 2 1 ~13?h Pergamon Press Lld. 1978 Printed in Great Britain 0 International Society for Neurochernirlry Lld.

STIMULATORY EFFECTS O F SUBSTANCE P O N NEURITE EXTENSION AND CYCLIC AMP LEVELS I N CULTURED NEUROBLASTOMA CELLS SHIGEHIKO NARUMI and YOSHITAKA MAKI Biological Research Laboratories, Central Research Divisions Takeda Chemical Industries, Ltd., Yodogawa-ku, Osaka, Japan (Received 19 April 1977. Revised 30 September 1977. Accepted 19 October 1977)

Abstract-Synthetic substance P initially increased cyclic AMP levels and subsequently induced neurite extension in cultured neuroblastoma N 18 cells. The magnitude of these effects depended on the concentration of fetal calf serum (FCS) in the culture medium, being more evident in the presence of a lower (0.1%) concentration of FCS. In Eagle’s medium containing 0.1% FCS, low concentrations of substance P (10-’-10-5 M) increased cyclic AMP levels and stimulated neurite extension. In Eagle’s medium containing 5% FCS, both substance P at concentrations of 10-5-10-3 M and dibutyryl cyclic AMP at concentrations of 10-4-10-2 M increased cyclic AMP levels and stimulated neurite extension. The activities of acetylcholinesterase, (Na* K+)-, HCO; and Mg’+-stimulatedATPase were also increased. Cell growth was inhibited. Substance P at concentrations of 10-7-10-5 M also stimulated the adenylate cyclase activity of a particulate fraction of N 18 in a concentration-dependent manner.

+

SUBSTANCE P is an undecapeptide found in high con- including neurite extension (SEEDSet al., 1970), acetylcentrations in intestine and brain. In the brain it is cholinesterase activity (BLUMEet al., 1970) and et al., 1975). especially high in the substantia nigra and hypothala- ATPase activity (LEDIG In the present communication we report the effects mus, as shown by radioimmunoassay (DUFFYet al., 1974). In uiuo the peptide stimulates salivary secretion of substance P on the morphological and enzymatic (CHANG & LEEMAN, 1970), Nai excretion (MILLSet differentiation and cyclic A M P contents of N 18 a{., I974), causes hypotension (VON EULER & GAD- neuroblastoma cells. These results are compared with the effects of dibutyryl cyclic AMP. The possible role DUM, 1931) and increases the concentration of glycine in brain (STERNet al., 1974). Its effect in uitro include of substance P as a modulator or local hormone in stimulation of gut contraction (VON EULER & GAD- the nervous system is also discussed. DUM, 1931) and depolarization of motor neurons MATERIALS AND METHODS (OTSUKA et al., 1972). OTSUKA e t al. (1972); OTSUKA & KONISHI(1974) and KONISHI& OTSUKA(1974) Cell culture found that substance P is 200 times more potent than Neuroblastoma cells, clone N 18 derived from the L-glutamate in depolarizing motor neurons in the iso- C-1300 tumor cell line (AMANO et al., 1972), were grown lated frog and newborn rat spinal cord. These findings routinely in Falcon lOOmm tissue culture Petri dishes in & FREEsuggest that substance P may be a transmitter Dulbecco’s modified Eagle’s medium (DULBECCO released from sensory nerve terminals in the spinal MAN, 1959), which was supplemented with 10% fetal calf serum (FCS) and Kanamycin (100pg/ml) in a humidified cord (OTSUKA et al., 1975). atmosphere of 10% co2-90%air at 37°C. Recently, DUFFY& POWELL (1975) reported that substance P also stimulates adenylate cyclase activity Determination of neurite extension in particulate fractions from rat and human brain. To determine the percentage of cells undergoing morCyclic AMP is found in high concentrations in ner- phological differentiation (neurite extension), lo5 t r y p vous tissue where it is thought to function as a post- sinized neuroblastoma N 18 cells were plated per 60mm synaptic ‘second messenger’. The complexity of the petri dish. After 24 h of incubation in Eagle’s medium supstructure of nervous tissue, however, makes the inter- plemented with 5%FCS, the medium was replaced with pretation of data obtained from biochemical and medium containing the substance to be tested and pharmacological experiments on brain tissue difficult. 0.1-5.0% FCS. At various times after this, all cells with least three ranWe therefore considered that studies on cultured and without neurites were counted in at domly selected areas of each culture plate. Of the total neuroblastoma cells (N 18 clone) derived from ner- cells counted per dish, cells having at least two neurites vous tissue might serve as a simple model system for longer than the diameter of the cell body were scored as the elucidation of the physiological role@) of sub- differentiated cells. The degree of neurite extension represtance P. The N 18 clone retains several functions as- sented by these differentiated cells was then expressed as sociated with neurons in the intact nervous system the percentage of the total cells. 1321

I322

SHIGEHIKO NARUMI and YOSHITAKA MAKI

Assay for cyclic AMP content

Materials

The cells were harvested by removing the medium and scraping the cells with 0.01% EDTA-phosphate buffered saline at room temperature. The cells were then washed quickly with cold Dulbecco's phosphate buffered saline without calcium and magnesium (PBS) and then homogenized with 5 0 0 ~ 1of 5% trichloracetic acid (TCA). The homogenate was then allowed to stand for 30min at 4°C. Each TCA precipitate was solubilized in 0.5 N-NaOH for protein determination. The TCA soluble supernatants, after removal of TCA by extraction with water-saturated ether 3 times, were then neutralized with 1 0 0 ~ 1of 0.5N-Tris-HCI buffer (pH 7.4) and treated with Sop1 of 0.3 M-ZnSO, and 50 p1 of 0.3 M-BaCI,. The solution was clarified by centrifugation at 5009 for 5 min and cyclic AMP was determined by the method of GILMAN(1970) using 2.5pg of binding protein from bovine muscle and 1 pmol of [3H-8]cyclic AMP (specific activity 30 Ci/mmol) in 2 0 0 4 of reaction mixture.

The following materials were used: Fetal calf serum (Microbiological Associates, Inc.); kanamycin (Takeda Chemical Ind., Ltd.); substance P (synthesized by Dr. Fujino's group in Takeda Chemical Ind. Ltd. in conventional method and checked purity). Its structure was

Assay of enzyme activities ( I ) Adenylate cyclase (EC 4.6.1.1.). The standard assay 1975) for adenylate cyclase consystem (DUFFY& POWELL, 0.2 mM-ATP, 0.3 mM-MgCI,, 1.0mM-NaC1, tained 1.0 mM-KCI, 0.6 mwtheophylline, 100 mM-Tris-HCI buffer (pH 7.4) in a total volume of 50 pl. The reaction was started by the addition of the enzyme preparation (5-1Opg protein), which was the pellet obtained after homogenization of the cells in 0.32 M-sucrose followed by centrifugation at 50,OOOg for 30min. Incubation was carried out with shaking--60 strokes/min-at 37°C for 30 min. The enzyme activity was terminated by addition of Sop1 of 0.3 M-Z~SO,and 0.3 M-BaCI,. The test tubes were then allowed to stand for 1 or 2 h at 4"C, and subsequently centrifuged at 500 g for 5 min to remove insoluble materials. Portions of the supernatant were then assayed for cyclic AMP using the method described above. (2) Acetylcholinesterase (EC 3.1.1.7.). Acetylcholinesterase was measured by a modification of the method of BLUME er af. (1970). The reaction mixture contained in a final volume of 0.05 ml, 0.05 M-pOtaSSiUm phosphate buffer @H 6.8), 0.2 M-NaCI, 1 mM-EDTA, 0.5% Triton X-100, 3.3 mM-acetylcholine choloride containing 0.01 pCi [l-'4C]acetylcholine (3.84 mCi/mmol) and 100-300 p g protein of N 18 homogenate. The reaction mixtures were incubated for 30min at 37°C and activity was terminated by the addition of 500 p1 of 10pM-physostigmine salicylate solution. The diluted reaction mixtures plus 1.0 ml water were then passed through a 0.6 x 3 cm column of Dowex 50 x 8 (Na+ form). The eluates were collected in scintallation vials and 10ml scintillation fluid (toluene scintillator containing 33% Nonion) was added and the radioactivity was determined. (3) ATPase (EC 3.6.1.3.). ATPase activity was assayed l~ in a medium containing 5 mwATP-Na2, 5 m ~ - M g C and 100mM-Tris-acetate buffer (PH7.5) with or without 20 mM-NaHCO, or 20 mM-KCI and 100 mM-NaC1. Enzyme preparation (50-100 pg protein) was the homogenate of N 18 cells in 0.32~-sucrose.The mixtures, in a final volume of 1 ml, were incubated for 20min at 37°C and the enzymatic reaction was then terminated by adding 0.5ml of ice-cold 5% TCA. The inorganic phosphate liberated during the incubation was estimated according to the method of TAKAHASHI, 1966. Protein was measured as described by LOWRYet al. (1951) using bovine serum albumin as a standard.

Arg-Pro-Lys-Pro-Glu-Glu-Phe-Phe-Gly-Leu-Met-NH2 ; dibutyryl cyclic AMP monosodium salt (Boehringer Mannheim Co. Ltd); ATP disodium salt (Boehringer Mannheim Co. Ltd.); physostigmine salicylate (Merck) and Nonion (Nippon Yushi Co). RESULTS

Effects of substance P on neurite extension and cyclic A M P levels of N 18 neuroblastoma cells in Eagle's medium containing 0.1% FCS

Substance P showed a stimulating effect on neurite extension as well as increasing cyclic AMP levels (Fig. 1). These effects depended on the concentration of FCS in the culture medium, that is, the effects were more evident at a lower concentration of FCS, when the cells were retarded in growth and tended to differentiate. The addition of substance P (10-7-10-5 M) to the medium containing 0.1% FCS, significantly increased cyclic AMP levels by about 1.5 times compared to control levels at 20min after addition (Fig. 1 and Table 1). A later increase was seen in both control and experimental cells in medium containing O.l%FCS. The same treatment in O.l%FCS stimulated the neurite extension of N 18 which reached a maximum between 60 and 90min. This effect was dependent on the concentration of added substance P (Table 1). Effects of substance P and dibutyryl cyclic A M P on the growth, neurite extension and cyclic A M P levels of N 18 neuroblastoma cells in Eagle's medium containing 5% FCS

Substance P and dibutyryl cyclic A M P at higher concentrations of M and l o T 2M respectively sharply increased cyclic A M P levels 10-30 min after addition, in media containing a higher concentration of FCS (5%). Substance P and dibutyryl cyclic A M P both stimulated neurite extension but maximum effects were observed later than the increase in cyclic A M P levels, at 90min after addition (Fig. 2). The effect of substance P (10-5-10-3 M) and dibutyryl cyclic A M P (10-3-10-2~) added in medium containing 5% FCS on neurite extension and inhibition of cell growth was concentration-dependent. The greatest effects were seen at the highest concentration tested of each drug, 2 4 4 8 h after their addition (Table

2). Effects of substance P on the activity of adenylate cyclase in particulate fractions

Substance P (10-7-10-5 M) significantly stimulated adenylate cyclase activity in a particulate preparation from N 18 in a concentration-dependent manner (Table 3).

Effects of substance P on neuroblastoma cells

1323

Effects of substance P and dibutyryl cyclic A M P on the activities of acetylcholinesterase, Mg2+-,Na+-K and HCOi-stimulated ATPase of cultured cells + -

The addition of substance P (W4 or M) or dibutyryl cyclic AMP or lo-* M) to the medium 1401-

.

/--* 20 C

0

0

+

Times after addition of substance P,

min

80

0

CJI

6 60 m

FIG. 1. Effect of substance P on cyclic AMP levels (upper figure) and neurite extension (lower figure) of N 18 neuroblastoma cells in Eagle’s medium containing 0.1, 1.0 and 5% FCS. To determine the percent of neurite extension, los trypsinized N 18 cells were plated per 60 mm dish. Each point used 2 dishes. After 24 h incubation in Eagle’s medium suppleaented with 5% FCS, the medium was replaced by the medium to be tested. At various times later, all cells with and without neurites were counted in at least three randomly selected areas of each culture plate. (Total counted cells were 200.) To assay cyclic AMP levels of N 18, the cells were homogenized with 5% TCA. Cyclic AMP contents in TCA soluble fraction were determined according to the methods of GILMAN. See details in Materials and Methods. 0.1% 1.0% 5.0% FCS 0 A 0 Control 0 A Substance P ( W 5M)

0

Ea

40

C

..

a-“

20 0 0 1020 30 60 90 Times a f t e r addition of drugs,

FIG.2. Effects of substance P and dibutyryl cyclic AMP on neurite extension and cyclic AMP levels of neuroblastoma N 18 cells in Eagle’s medium containing 5% FCS. In each 60mm dish lo5 cells were plated and each point used 2 dishes and a total of 200 cells from at least 6 different areas were counted. Substance P ( 1 0 - 3 ~ ) ,dibutyryl cyclic AMP lo-'^) or drug vehicle alone were tested. Protein, cyclic AMP and neurite extension were determined as described in Materials and Methods. o Control, cl Substance P A Dibutyryl cyclic AMP M).

TABLE1. EFFECTS OF SUBSTANCE P ON THE NEURITE EXTENSION AND CYCLIC AMP LEVELS TOMA N 18 CELLS IN EAGLE’S MEDIUM CONTAINING 0.1% FCS

% Neurite Concentration Treatment

(M)

120

rnin

extension 90 min

OF NEUROBLAS-

Cyclic AMP levels (pmol/mg protein) 20 min

% Control Substance P

10-7

lo-6 10- 5

26 f 6 46 f 5* 47 3** 15 1***

*

33 f 3 46 k 10 50 k 11* 53 f 2***

(100) (139) (151) (161)

Values are means f S.D., N = 3. * p < 0.05, ** p < 0.01, *** P c 0.001 compared with control. lo5 cells of N 18 were plated per 60 mm dish. ARer 24 h, the medium was replaced with the medium containing O.l%FCS and substance P, or drug vehicle alone. The effect on neurite extension and cyclic AMP levels were tested as described in Materials and Methods.

SHIGEHIKO NARUMI and YOSHITAKAMAKI

1324

OF SUBSTANCE P TABLE2. EFFECTS

A N D DIBUTYRYL CYCLIC AMP ON THE NEURITE EXTENSION AND CELL GROWTH OF NEUROBLASTOMA N 18 IN EAGLE'SMEDIUM CONTAINING 5% FCS

% Neurite extension 24 h 48 h

Treatment (MI

Cell growth 48 h

24 h x 105

Control

21 f 5

35 f 3

3.2

5.7

15 f 2 43 f 5 87 f 10

50 f 10 41 f 3 91 f 2

3.9 4.0 3.0

5.7 5.7 3.4

30 f 5 33 4 81 & 15

66 f 1 5 71 f 20 94 3

3.2 3.0 2.6

4.5 4.2 3.8

Substance P 10- 3

Dibutyryl cyclic AMP 10-4

*

*

Immediately after 5 x 10 cells of N 18 were plated per 60mm dish, substance P, dibutyryl cyclic AMP or drug vehicle alone was added to the medium and the effect on cell growth was determined using a Coulter counter. Each value was the mean of 2 dishes. For neurite extension n = 6 and the means f S.D.are shown. TABLE3. STIMULATION OF

ADENYLATE CYCLASE ACTIVITY BY SUBSTANCE P IN THE PARTICULATE PREPARATION FROM NEUROBLASTOMA N 18 CELLS

Concentration Agent added

(MI

Adenylate cyclase activity (pmol cyclic AMP/mg prot./min) 23.8 f 3.2

Control Substance P

10-7

42.9 2.7** 62.8 9.8.. 95.0 f 31.0***

(180) (263) (399)

Values are means f S.D. and N = 4. ** P < 0.01. *** P < 0.001 compared with control. The assay system contained 0.3 mM-ATP, 0.3 rnM-MgCl,, 1.0 mM-NaC1, 1.0 mM-KCl, 0.6 mM-theophylline, 100 mM-Tris-HC1 buffer @H 7.4) with or without substance P (10-7-10-5 M) and 5-10pg protein of enzyme preparation from N 18. The procedure is described in detail in Materials and Methods.

SUBSTANCE p AND DIBUTYRYL CYCLIC AMP ON THE ACETYLCHOLINFSTERASE ACTIVITY OF NEUROBLASTOMA N 18 CELLS

TABLE4. EFFECTSOF

Concentration Treatment

(M)

Control Substance P

1010-4

w3 Dibutyryl cyclic AMP

10-4

Acetylcholinesterase activity (nmol/mg protein/min) 24 h 48 h 22 f 3

43 f 3

21 8 28 I** 39 f 6***

53 f 5 46 f 9 41 f 1

+

*

21 4 31 f 6*** 29 f 1***

52 & 10 54 5 29 f 4

*

Values are means f S.D.with N = 4. ** P i0.01. *** P c 0.001 compared with Control. Immediately after N 18 cells were plated per 60 mm dish in Eagle's medium containing 5%FCS, substance P, dibutyryl cyclic AMP, or drug vehicle alone was added to the medium. After 24 or 48 h, the activity of acetylcholinesterase was determined as follows: The assay system contained 3.3 mM-acetylcholine chloride containing 0.01 pCi [1-'4C]acetylcholine, 0.2 M-NaCI, 1 mM-EDTA, 0.5% Triton X-100 0.05 M-potassium phosphate buffer @H 6.8) and 100-300 pg N 18 homogenate protein. The detailed procedure is described in Materials and Methods.

Effects of substance P on neuroblastoma cells

1325

TABLE 5. EFFECTS OF SUBSTANCE P AND DIBUTYRYL CYCLIC AMP ON THE ACTIVITIES Mg’+, (Na’ + K+)-, HCO;-STIMULATED ATPASEIN NEUROBLASTOMA N 18 CELLS

OF

Treatment (MI

Mg2+

Control

5.6

Activities of ATPase (pmol Pi/mg proteinfi) % Na+-K+ % HCO;

%

100

2.4

100

1.8

100

5.9 7.3 13.7

245 304 570

1.2 4.2 5.8

67 233 322

2.9

120 325 675

1.4 3.0 6.8

78 166 377

Substance P 10-5 10-4

5.1

91

9.5 10.5

169 187

6.2 6.0 9.5

110 107 169

Dibutyryl cyclic AMP 10-4 10-3

7.8

16.2

For culture conditions see the legend to Table 4. The activity of ATPase in N 18 cells homogenates was assayed in the medium containing 5 mM-ATP-Na,, 5 mM-MgCI,, 100 mM-Tris-acetate buffer (pH 7.5) with or without 20 mM-NaHCO,, or 20mM-KC1 plus 100mM-NaC1. For further details see Materials and Methods. Activities were determined 24 h after addition of drugs. All values were mean of 2 experiments. containing 5% FCS, significantly increased the specific activity of acetylcholinesterase of neuroblastoma N 18 cell 24 h after addition and the effect on acetylcholinesterase activity was transient and disappeared at 48 h (Table 4). The addition of substance P (10-5-10-3~) and dibutyryl cyclic AMP (10-4-10-2 M) to the medium containing 5% FCS, also markedly increased the specific activity of Na+-K+-stimulated ATPase. This effect was concentration-dependent increasing to 5o(r600% at M-substance P or lo-’ M-dibutyryl cyclic AMP (Table 5). Similar but less marked effects were also seen for Mg2+-ATPase or HC0;-stimulated ATPase (Table 5).

similar to physiological concentrations of substance P. For example substantia nigra contains 528 & 67, and hypothalamus contains 295 f 14ng substance P/g wet weight (DUFFYet al., 1974). In order to show that the effects of substance P may be mediated via changes in intracellular cyclic A M P levels it is important to show that cyclic A M P itself has similar effects. However, cyclic AMP itself penetrates into cells with difficulty, and so we used dibutyryl cyclic AMP. As can be seen from Fig. 2 the effects of dibutyryl cyclic AMP and substance P in increasing neurite extension were very similar. The treatment of dibutyryl cyclic AMP increased intracellular cyclic AMP levels in neuroblastoma N 18 cells. In the case of the highest concentration of dibutyryl cyclic AMP (10 mM), most of intracellular cyclic AMP levels might be due to DISCUSSION dibutyryl cyclic AMP itself or monobutyryl cyclic In the present studies, we have shown that addition AMP. In the binding protein assay of cyclic AMP, of a synthetic preparation of substance P can lead dibutyryl cyclic AMP and monobutyryl cyclic AMP to increased morphological and enzymatic differenti- are also bound to the binding protein 100 and 10 ation and the elevation of cyclic AMP levels in neuro- times less than cyclic AMP. Though we have no data blastoma N 18 cells. The results show that substance that dibutyryl cyclic AMP is degraded to cyclic AMP P stimulated the activity of adenylate cyclase in uitro, and transferred into the cells as cyclic AMP, most and increased cyclic AMP levels 10-20min after addi- of dibutyryl cyclic AMP is degraded to monobutyryl tion, and subsequently induced neurite extension cyclic AMP in the cells, and monobutyryl cyclic AMP which reached a maximum 60-90 min after addition. has been shown to act similarly to cyclic AMP in The specific activities of acetylcholinesterase and intracellular physiological functions. (BLECHER& ATPase were increased 24 h after the addition of sub- HUNT,1972; KAUKEL& HILZ,1972). stance P. The morphological alterations and the inNeurite extension in cultured neuroblastoma N 18 crease in cyclic AMP levels thus showed a positive cells has been termed morphological differentiation, correlation. In Eagle’s medium containing 5% FCS, and its importance as a model for neural differentiwe needed high concentrations M) of substance ation has been pointed out by many authors (FURP because FCS counteracted the effect of substance MANSKI et a/., 1971; PRASAD & HSIE, 1971; SCHUBERT P. However, in the medium containing O.l%FCS or et al., 1969; SEEDS et al., 1970). X-irradiation (PRASAD, in the in uitro adenylate cyclase assay system, sub- 1971), removal of serum (SEEDSet al., 1970) and addistance P was effective a t ~ O - ’ M and its effects in- tion of dibutyryl cyclic AMP (GILMAN& NIRENBERG, ; & HSIE, 1971), 5-bromodeoxyuridine creased in a concentration-dependent manner. Con- 1 9 7 1 ~ PRASAD centrations of 10-~-10-~ (SCHUBERT M (16&1600ng/mI) are & JACOB,1970), or other drugs which in-

1326

SHIGEHIKO NARUMI and YOSHITAKA MAKI

crease intracellular cyclic AMP levels, can all induce their valuable advice and discussion. We should also like GRAHAM for her invaluable help in mophological differentiation in neuroblastoma and to thank Mrs ELVIRA glioma cells. Prostaglandin E, and catecholamines typing the manuscript. cause a rapid increase in cyclic AMP levels of neuroREFERENCES & NIRENBERG, blastoma and glioma cells (GILMAN 1971a ) and subsequent morphological alterations M. (1972) Proc. & NIRENBERG,AMANO I., RICHELSONE. & NIRENBERG have also been reported (GILMAN natn. Acad. Sci. U.S.A. 69, 25&263. I971 h). BLECHERM. & HUNTN. H. (1972) J. biol. Chem. 247, Recently, DUFFY & POWELL (1975) reported that 7479-7484. synthetic substance P stimulated adenylate cyclase ac- BLUMEA,, GILBERT S., WILSONS., FARBER J., ROSENBERG tivity in particulate preparations from rat and human R. & NIRENBERG M. (1970) Proc. nafn. Acad. Sci., U.S.A. brain. This may be one of the first reports describing 67, 786-792. stimulation of brain adenylate cyclase activity by a CHANCM. & LEEMAN S. (1970) J. biol. Chem. 245, 478M790. naturally occurring peptide, and suggested that subD. (1975) Biochim. biophys. Acta stance P, like the other drugs mentioned above, might DUFFYM. J. & POWELL 385, 275-280. cause morphological differentiation in neuroblastoma DUFFYM. J., WONGJ. R. & POWELL D. (1974) Biochem. cells. SOC. Trans. 2, 1262-1264. In order to obtain such information, we have carDULBECCO R. & FREEMAN G. (1959) Virology 8, 396397. ried out the present experiments to elucidate a poss- FERMANSKI P., SILVERMAN D. J. & LUBINM. (1971) Nature, ible relationship between substance P and the concenLond. 233, 413-415. tration of the intracellular ‘second messenger’, cyclic GILMANA. G . (1970) Proc. natn. Acad. Sci., U.S.A. 67, AMP. The results show that substance P stimulated 305-3 12. adenylate cyclase activity and increased cyclic AMP GILMAN A. G. & NIRENBERG M. (1971a) Nature, Lond. 234, 356357. levels, and subsequently promoted neurite extension. A. G. & NIRENBERG M. (1971b) Proc. natn. Acad. The increased cyclic AMP formation should not be GILMAN Sci., U.S.A. 68, 2165-2168. due to the inhibition of phosphodiesterase as subE. & HILZH. (1972) Biochem. biophys. Res. Comstance P had no effect on phosphodiesterase activity KAUKEL mun. 46, 1011-1018. & POWELL,1975; in rat brain homogenate (DUFFY KONISHIS . & OTSUKA M. (1974) Brain Res. 65, 397-410. our unpublished observation). LEDIGM., CIESIELSKI-TRESKA J., CAMY., MONTAGNON D. Recently, we have also obtained stimulation of fibre & MANDEL P. (1975) J . Neurochem. 25, 635-640. outgrowth by substance P in chick dorsal ganglia LOWRY 0. H., ROSEBROUGHN. J., FARRA. L. & RANDALL & FUJITA,1978). From these results, we (NARUMI R. J. (1951) J. biol. Chem. 193, 265-274. speculate that substance P may be exerting an MILLS1. H., MACFARLANE N. A. A. & WARDP. E. (1974) Nature, Lond. 247, 108-109. homonal influence on neurite extension in nerve cells S. & FUJITAT. (1978) Neuropharmacology, 17, in both central and spinal nervous systems. It will NARUMI 73-76. be of interest to determine whether the initial increase of cyclic AMP levels due to substance P is directly OTSUKAM. & KONISHIS. (1974) Nature, Lond. 252, 733-735. responsible for the subsequent morphological and/or OTSUKA M., KONISHI S. & TAKAHASHI T. (1972) Proc. Jap. the enzymatic differentiation and how these effects are Acad. 48, 342-346. achieved. OTSUKA M., KONISHI S. & TAKAHASHI T. (1975) Fedn Proc. The N-terminal tetrapeptide of substance P (ArgFedn Am. SOCSexp. Biol. 34, 1922-1928. Pro-Lys-Pro-OH) had almost the same effect of sub- PRASAD K. N. (1971) Nature, Lond. 254, 471-473. stance P and C-terminal heptapeptide amide (Gln- PRASAD K. N. & HSIEA. W. (1971) Nature, N e w Biol. 233, 141-142. Gln-Phe-Gly-Leu-Met-NH,) had a little effect on the D. & JACOBF. (1970) Proc. natn. Acad. Sci., neurite extension of neuroblastoma N 18 cells (unpub- SCHUBERT U.S.A. 67, 247-254. lished observation). From these results, we speculate D., HUMPHRIES S., BARONIC. & COHNM. (1969) that the active site of substance P in these effects may SCHUBERT Proc. rtafn. Acad. Sci., U.S.A. 64, 316-323. be Arg-Pro-Lys-Pro-NH,. SEEDSN. W., GILMAN A. G., AMANOI. & NIRENBERG M. wish to thank Drs H. K. KIMELBERG of the Division of Neurosurgery, Albany Medical College of Union University and Z. SUZUOKI and T. Acknowlcdgmients-We

FUJITAof the Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., for

(1970) Proc. natn. Acad. Sci., U.S.A. 66, 16Cb167. STERNP., GATOVIC S. & STERN M. (1974) Naunyn-Schmiedbergs Arch. exp. Path. Pharmak. 281, 233-239.

TAKAHASHI Y.(1955) Seikagaku (in Japanese), 26, 69e698. VON EULFR V. S. & GADDUM J. H. (1931) J. Physiol., Lond. 12, 7487.