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An isolated growth cone-enriched fraction from developing rat brain has substance P binding sites
DevelopmentalBrain Research, 40 (1988) 1-9 Elsevier
1
BRD 50712
Research Reports
An isolated growth cone-enriched fraction from developing rat brain has substance P binding sites R. Owen Lockerbie, Jean-Claude Beaujouan, Monique Saffroy and Jacques Glowinski College de France-INSERM U 114, Chairede Neuropharmacologie, Paris (France) (Accepted 13 October 1987)
Key words: Isolated growth cone; Rat; Substance P receptor; Pharmacology; Development
A fraction enriched in neuronal growth cones isolated from developing rat forebrain was shown to possess binding sites for the substance P analog, Bolton-Hunter substance P ([125I]BHSP). Specific binding of this ligand reached an equilibrium after 10 min at 2(I °C, and was reversible and temperature-dependent. Removal of extracellular Na ÷ did not block but rather augmented [125I]BHSP binding suggesting that the labeled analog was not transported into the growth cone fraction. Scatchard analysis of the binding indicated a single class of non-interacting binding sites in the growth cone fraction (Kd: 257 pM; Bmax:56 fmol/mg protein). From competition studies using substance P and other tachykinins, their rank order of potency for inhibiting [125I]BHSP binding was SP > physalaemin > > eledoisin > kassinin > NKB/> NKA. Such order is consistent with the presence of an SP receptor (Neurokinin-1) in the growth cone fraction. The N-terminal fragments of substance P, SPI_7 and SPl_ll free acids, and the C-terminal fragment, SP7_i1, were devoid of affinity for the [125I]BHSP binding site. However SP6_I1and SPl_ll methyl esters showed more potency.
INTRODUCTION Binding sites for the tachykinin substance P (SP) have been characterized, by using a labeled derivative of SP, the [125I]Bolton-Hunter SP (BHSP), with various m a m m a l i a n central nervous system (CNS) preparations including rodent embryonic neurons 1'32, synaptosomes from various regions of the adult rat CNS 3&36"37 and adult rat brain cortex membranes 4. The same findings have also been made with other ligands such as [3H]SP and [3H]physalaemin on adult rat brain m e m b r a n e s 2'24'28-3°. Although SP has been d e m o n s t r a t e d in the brain of several species at its early stages of d e v e l o p m e n t 9:3'21, very little is presently known on the d e v e l o p m e n t of SP receptors. A few recent studies, however, including those with primary cultures of mesencephalic embryonic neurons from the mouse I and with embryonic and young postnatal rat brain 3~, have d e m o n s t r a t e d the
early a p p e a r a n c e of SP receptors. To further substantiate the early acquisition of the SP r e c e p t o r during brain ontogeny we report here on the pharmacological characterisation of the receptor, by using the [125I]BHSP ligand, in an isolated fraction enriched in neuronal growth cones from neonatal rat brain 11. MATERIALS AND METHODS
Preparation of growth cone-enriched fractions Growth cone-enriched fractions were isolated from the forebrain or brainstem of 5-day-old (day of birth is day 1) rats (Iffa Credo, France) as previously described 11. F r o m electron microscopical analysis, more than 90% of m e m b r a n e - b o u n d , identifiable objects in these fractions fulfil the morphological criteria for the identification of growth cones. G r o w t h cones are enriched in the fraction by a factor of 7 over the starting h o m o g e n a t e . Using morphological con-
Correspondence: R.O. Lockerbie, Coll~ge de France-INSERM U 114, Chaire de Neuropharmacologie, 11, place Marcelin Berthelot, 75231 Paris, France. 0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
trois along with biochemical and immunological controls, the growth cone-enriched fraction has been shown not to be contaminated by synaptosomes, glial or neuronal cell soma elements, and contains only small amounts of isolated rough endoplasmic reticulure and mitochondria. The major contaminants of the growth cone fraction are large membrane sacs many of which have a similar diameter to that of growth cones and vesicles of unidentified origin! 1. The isolation method involves Ficoll-sucrose gradients, and for this reason the growth cone fraction is returned to isotonic conditions by incubation in Krebs' buffer 7. The growth cone fraction was kept at 4 °C until the binding assays, when the fraction was pelleted (10,000 g for 30 s) and resuspended and incubated at 37 °C for 30 min in a buffer used for the binding assays (see below).
Binding assays ]-'he composition of the Krebs-Ringer phosphate buffer (pH 7.0) used for the binding assays was the following (in mM); NaCI 120; KCI 4.8; CaC12 1.2; MgSO4 1.2; NaH2PO 4 15.6. To this buffer was added glucose (1 mg/ml) bovine serum albumin (BSA; 0.4 mg/ml) and bacitracin (60 ~g/ml). Binding assays were performed in Eppendorf tubes (vol. 1.5 ml) which were precoated with the binding assay buffer in which the BSA concentration was 5 mg/ml. Routinely, 20/~1 of the growth cone fraction (approx. 60 ktg of protein) was incubated in a final volume of 200 /A of the binding assay buffer for 10 min at 20 °C with [J25I]BHSP (approx. 25,000 cpm; 40 pM; see ref. 23 for synthesis). At the end of this incubation, the tubes were centrifuged for 30 s at 10,000 g and the pellets washed once with 1 ml of the 'cold' medium used in incubation. Radioactivity bound to tissues was estimated using a Packard gamma counter. All assays were performed in triplicate, and included blanks in which the growth cone fraction was omitted from the incubation mix. Non-specific binding (NSB) was determined by co-incubating the growth cone-fraction with the radioactive ligand and 1 ~M SP. Specific binding (SB) was calculated by subtracting NSB from total binding (TB). To determine reversibility of binding, the growth cone fraction was incubated with radioactive ligand as described above, and then SP (1/~M) was added ~md the concentration of [t25I]BHSP specifically
bound with time was followed. For analyzing saturation kinetics of [125I]BHSP binding by the Scatchard method 33, the growth cone fraction was incubated as described above with different concentrations of radioactive iigand and specific binding measured at each concentration. For studies of the competitive inhibition of [I25I]BHSP binding, the radioactive ligand was mixed with increasing concentrations of non-labeled SP and structurally related peptides or derivatives, and incubated with the growth cone fraction under the conditions described above with specific binding measured at each concentration.
Effect of Na + on [125I]BHSP binding In experiments designed to ensure that [125I]BHSP is not transported into the growth cone fraction, the effect of Na+-free media was investigated by performing binding assays as described above in Krebs' buffer from which all Na + was removed. In Na+-free media, isotonicity was maintained by removing NaCI and NaH2PO 4 and adding sucrose (0.28 M), KHzPO4 (15.6 mM) and Tris (1.2 mM). The pH of this media was corrected to pH 7.0 by the addition of K O H . In parallel, control experiments involved pre-incubating the growth cone fraction for 30 rain at 37 °C in Krebs' buffer and then incubating 60/~g protein for 15 min at 20 °C with [3H]GABA (1.0/~Ci) diluted with unlabeled amino-acid to a final ligand concentration of 5/~M in Krebs' buffer or in Na+-free Krebs' buffer (1 ml) in the presence (blank) or absence of nipecotic acid (400/~M), an inhibitor of G A B A uptake into the growth cone fraction 19. Amino-oxyacetic acid (100/~M) was included to inhibit G A B A transaminase ~. Incubations were terminated by rapid centrifugation (30 s, 10,000 g) and pellets washed once with cold incubating medium (1 ml). Radioactivity was determined by counting in a Packard 3255 spectrometer using Aquasol-2 (NEN) as scintillant. Other methods Liquid chromatographic (HPLC) analysis involving C18 ktBondapak columns (0.4 x 30 cm; Waters Instruments, Rochester, MN) and methanol-ammonium acetate (50 mM, pH 4; 540-460) as solvent (flow rate: 2 ml/min) was used to identify the radioactive material recovered in the supernatant at the end of the 10-rain incubation period in order to assess the
extent of [12SI]BHSP degradation by peptidases. Protein content was estimated by the method of Bradford 3 with the suggested modification of Gogstad and Krutnes m. Regression analysis was used to draw lines through data points for Scatchard and Hill plots.
Chemica& BSA was purchased from Calbiochem. Bacitracin, nipecotic acid and amino-oxyacetic acid were from Sigma. [3H]GABA (spec. act. 57 Ci/mmol) was from Amersham, and Aquasol-2 from NEN. Physalaemin, kassinin, eledoisin, SP6-11, SPT-ll, SPl-7 and SPl_ll free acids, SPl_ll methyl ester and spantide were from Peninsula. Neurokinin-A and -B (NKA and NKB) were synthesized by S. Lavielle (University of Paris VI). RESULTS
Binding of [1251]BHSP to growth cone-enriched fractions Preliminary studies were performed in which binding assays were carried out in the presence of different combinations of BSA and peptidase inhibitors at various concentrations. The presence of BSA (0.4 mg/ml) and bacitracin (60 ~g/ml) resulted in the best compromise between a good specific binding of [125I]BHSP and a low percentage value for degradation of the ligand. HPLC analysis showed that in the presence of these two compounds, approximately 76% of the radioactivity recovered at the end of a 10min incubation period migrated as authentic [125I]BHSP. Fig. 1 shows that when a growth cone-enriched fraction isolated from 5-day-old rat forebrain was incubated at 20 °C with a low concentration of [12Sl]BHSP (48 pM) for 10 min, specific binding increased linearly over the range from 30 to 120/,g of protein. Specific binding of [12SI]BHSP to the growth cone fraction after 10 min at 20 °C was approximately 76% of the total binding and represented about 3% of the total number of counts per minute added to the incubation medium. When binding assays were performed for 10 min at 4 °C, approximately 80% of specific binding was lost. Under identical assay conditions, specific [125I]BHSP binding to a growth cone fraction from
the brainstem of 5-day-old rats showed the same linearity with increasing protein concentration as the forebrain growth cone fraction (data not shown). However, in absolute terms (fmol/mg protein) specific binding to the brainstem growth cone fraction was greater than specific binding to the forebrain growth cone fraction with the difference being 61% (mean of 3 experiments). A period of 10 min was used in all experiments since, as Fig. 2A shows, an equilibrium of specific [12SI]BHSP binding was reached around this time. Specific binding of [125I]BHSP was reversible, since rapid dissociation of the labeled iigand was observed when 1/xM unlabeled SP was added to the incubation medium for various times after the initial 10 min incubation (Fig. 2B). Dependence of Na + is a characteristic of neurotransmitter high-affinity uptake systems 6. Fig. 3 shows that under similar assay conditions, the absence of extracellular Na + reduced [3H]GABA uptake into the growth cone fraction by >80% as previously described 12J9 while enhancing [125I]BHSP binding to the growth cone fraction by > 100%.
Equilibrium binding studies Under equilibrium conditions of [125I]BHSP bindI
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20 "c. B: dissociation: growth cone,enriched fractions (60 ag of protein) were incubated with the ligand (39 pM) for 10 min
at 20 o C. Substance P (1 pM) was then added and the concentration of [ 125 I]BHSP specifically bound was followed with time. Each point represents the mean of data obtained in 3 separate experiments each performed in triplicate.
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ing to the growth cone fraction, non-specific binding of the ligand was a linear function of the labeled ligand concentration. In contrast, specific binding of [tasI]BHSP was saturable (Fig. 4A). Scatchard analysis of the specific binding data gave linear plots indicating the presence of a single class of binding sites (Fig. 4B). F r o m 4 i n d e p e n d e n t experiments, average
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cone-enriched fractions (60/~g protein) were incubated for 10 min at 20 °C with []~SI]BHSP(39 pM) in Krebs' medium with or without Na ÷ and the specific binding determined. Right: [3H]GABA uptake into growth cone-enriched fractions was evaluated under the same conditions as for [12SI]BHSPbinding (see Materials and Methods section). Blank values were determined by incubating growth cone-enriched fractions in the presence of nipecotic acid (400/~M). Each value represents the mean ± S.E.M. of 3 experiments each performed in triplicate or quadriplicatc.
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Fig. 4. [I251]BHSP binding to growth cone-enriched fraction from 5-day-old rat forebrain as a function of radiolabeled peptide concentration. A: growth cones (60pg of protein) were incubated with different concentrations of radioactive ligand for 10 min at 20 °C. Total (0), specific (©) and non-specific (&) binding. B: Scatchard plot of specific [125I]BHSP binding data. Each point is the mean of 3 determinations obtained in a single experiment.
values for the dissociation constant (Kd) and the density of sites (Bmax) were 257 pM +51 (mean + S.E.M.) and 56 fmol/mg protein +11 (mean _+ S.E.M.), respectively. Hill plots of the data (nil = 0.93 _+ 0.15) revealed that []25I]BHSP was bound to a single population of non-interacting sites.
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Competitive inhibition of [~251]BHSP binding When the efficacies of SP and other tachykinins were tested for inhibition of [125I]BHSP-specific binding to the growth cone fraction, they were found to do so in a competitive manner (Fig. 5, Table I). The rank order of potency was SP > physalaemin > > eledoisin > kassinin > NKB/> NKA with the inhibitory effect of physalaemin, eledoisin, kassinin and NKA being about 61,2, 0.7 and 0.3% that of SP, respectively (Table I). Two SP C-terminal fragments were also tested for their ability to inhibit specific [125I]BHSP binding and were found to be much less potent than SP in attenuating binding. Thus, SP6_I] and SP7_II reduced spe-
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log [TK](M) Fig. 5. Competition inhibition of [1251]BHSP binding to growth cone-enriched fractions from 5-day-old forebrain by SP and other tachykinins (TK). Growth cone-enriched fractions (60 pg of protein) were incubated with radioactive ligand (62 pM) and increasing concentrations of non-labeled tachykinin for 10 min at 20 °C. Results are expressed as percentage of the specific binding of [ 125I]BHSP alone. Each value is the mean of 4 separate experiments each performed in triplicate.
TABLE
I
Inhibition qf [1251]BHSP binding to growth cones and adult rat brain synaptosomes by substance P, analogs of SP, and other endogenous tachykinins
The concentrations of the peptides required to inhibit the specific binding of [~25I]BHSPto growth cones by 50% (ICs0) were estimated from the HiU plots of the titration curves exampled in Fig. 5. (a) Each value represents the mean of the number of experiments given in parentheses. The S~D. of each value is less than 75%. (b) Data are from Viger et al. (1983) and Lavielle et al. (1986). Hill coefficients for these compounds were close to unity (0.79-1.16). Peptide
Growth cones
Rat brain synaptosomes
(a) ICsr~(M) SP Physalaemin Eledoisin
8.5 × 10 "~ 1.4 × 10 ~ 4.4 x 10 -s
Kassinin
1.2 × 10 -7
Neurokinin A Neurokinin B SP,,_ll SP7 ii SP~ 7 free acid SPI.II free acid
3.0 x 10 7 2.9 × 10 -7 7.6 × 10 -s >10 5 > 1 0 -5 5.1 x 10 .6
(4) (4) (2) (2) (3) (3) (3) (2) (2) (4)
SPI-it methyl ester Spantide
2,1 x 10-s 9.5 x Ill 7
(3) (5)
Relative affinity
(b) 1C5o (M)
Relative affinity
1 0.61 0.02
6.4 x 10 l0 2.4 x 10 -9 1.1 x 10 -7
1 0.3 0.006
0.007
2.0 x 10 -7
0.003
0.003 0.003 0.01
0.005 0.0003 0.001
0.0002
1.4 x 10 -7 2.2 × 10 -6 7.0 X 10 -7 >10 5 n.d. 6.7 × 10 -6
0.04 0.0009
9. l x Ill ~ 2.6 x 10 7
0.007 0.002
cific [~251]BHSP binding with about 1/100 and more than 1/100,000, respectively, the potency of SP (Table 1). N-terminal fragments of SP such a s S P I _ 7 and SP 1_1~ free acids were without appreciable effect on specific [125I]BHSP binding. Hence, S P I _ l l free acid reduced [1251]BHSP binding with about 1/5000 the potency of SP while S P I _ 7 free acid did not significantly modify binding even at 10 -5 M concentrations. However, SPl_ll methyl ester had a considerably more potent effect on p251]BHSP binding, its affinity being 1/25 that of SP (Table I). Hill coefficients for all these compounds were close to unity. Spantide, a proposed SP receptor antagonist, proved to be a weak inhibitor of [tzSI]BHSP binding with 1/1000 the potency of SP (Table I). Dimethylsulphoxide which was used to solubilize NKB and SP6_li was without effect on [125I]BHSP binding to the growth cone fraction (data not shown). DISCUSSION
We have shown in this paper that a fraction enriched in growth cones isolated from developing rat forebrain possesses binding sites for substance P. Using a labeled analog of SP, [125I]BHSP, specific binding to the growth cone-fraction was linearly dependent on tissue concentration and was saturable.
0.0001
of high affinity, reversible and temperature-dependent. The binding assay conditions adopted in the present conditions were similar to those using this ligand in other receptor binding studies (for review see ref. 17). Under these conditions, H P L C analysis showed that after 10 min of incubation at 20 °C when binding had reached equilibrium, only slight peptidasic cleavage of the ligand had occurred. Thus, most of the binding observed in this study was attributable to authentic [125I]BHSP. [125I]BHSP-specific binding sites have been demonstrated in rat brain synaptosome fractions 3~ and recently on intact glial cells from several brain structures of the mouse 35. However, the binding reported in the present study is unlikely to have a contribution from these sources nor from neuronal cell soma elements since they do not contaminate the growth cone preparation 11. Although the growth cone fraction contains only small amounts of isolated rough endoplasmic reticulum and mitochondria 11, these organelles too would not be expected to contribute to [125I]BHSP binding in the fraction since binding sites so far have only been located on plasma membrane. However, the growth cone fraction does contain large membrane sacs, many of which have a similar diameter to that of growth cones, and vesicles of unidentified origin 11. We do not know yet what is the
distribution of [125I]BHSP binding sites between the growth cones and these elements in the fraction. For several reasons, it is unlikely that all the growth cones in the fraction possess binding sites. Thus, autoradiographical studies have shown that only a fixed proportion of embryonic mouse mesencephalic neurons in primary culture have SP receptors (Beaujouan eta!., unpublished observations). Further, autoradiographic studies of SP receptor binding sites in rat brain have demonstrated differential distributions of SP receptors between and within various regions, and that such distributions can undergo major changes during ontogeny 3~. Under assay conditions in which all Na + was removed, [3H]GABA uptake into the growth cone fraction was blocked by more than 80%, while [125I]BHSP binding was not reduced but rather increased. These findings suggest that the labeled SP analog was not transported into the growth cone fraction. The increase in [125I]BHSP binding in Na+-free media probably reflects the ion substitutions that were made for such media since it is known that binding to the SP receptor can be modulated by both monovalent and divalent ions 2~. The saturability of the specific binding of [125I]BHSP to the growth cone fraction was demonstrated from equilibrium studies. Scatchard analysis of the specific binding with increasing concentrations of the ligand revealed a single population of non-interacting binding sites with an apparent affinity (Kd) of 257 pM and a density (Bmax) of 56 fmol/mg protein. The Kj of [125I]BHSP binding to the growth cone fraction is in good agreement with the binding of this ligand under similar conditions to mouse embryonic mesencephalic neurons in culture (Kd: 169 pM) 1. However, slight differences are apparent when comparisons are made with [125I]BHSP binding to adult preparations. Thus, a K a of 470 pM was derived from equilibrium studies of [125I]BHSP binding to a crude synaptosome fraction of adult rat brain 38, and this value shows little variation for synaptosomes from different regions of the rat central nervous system 36. These differences may in part be due to differences in the assay conditions for the SP binding. Alternatively, as previously suggested 3s, perhaps the lower affinity of [125I]BHSP binding sites observed in the adult is related to a decrease in receptor sensitivity when SP receptor-containing neurons are fully developed
and functionally in contact with their target cells. A similar decrease in receptor affinity during development has also been found for instance with/~-adrenergic receptors 22. However, Quirion and D a m ~l did not find any significant change in the affinity of [1251]BHSP binding sites in rat brain membranes during ontogeny. The potential of SP and other endogenous tachykinins, and of N- and C-terminal fragments of SP to inhibit specific [125I]BHSP binding to the growth cone fraction was also investigated. For the tachykinins, their rank order of potency for inhibiting binding was SP > physalaemin > > eledoisin > kassinin > NKB /> NKA. Such order is similar to that found in previous studies of [1251]BHSP binding to various CNS preparations 5'3°'37. However, NKB in the present study shows more potency than in studies with the other preparations. As with other preparations 1'4'38of the two C-terminal fragments tested in the present study, SP6_ll showed a marked reduction compared to SP in inhibiting [125I]BHSP binding. The non-biologically active SP7_II was devoid of affinity for the [1251]BHSP binding site. Of the N-terminal fragments tested, SPI_ 7 and SPl_ll free acids, they were either with little or no effect on binding. However, SP1 ~ methyl ester, which has been shown to have a selective affinity for the SP receptor in the peripheral nervous system 39 and CNS 5"34, showed more potency although its inhibitory effect on [125I]BHSP binding to the growth cone-fraction was still only 1/25 that of SP. Hence, the pharmacology of the [1251]BHSP binding site in the growth cone fraction is consistent with the presence of a SP receptor 17 (Neurokinin-1 ; nomenclature designated at the Montreal meeting, July 1986). It is interesting to note that the relative affinities (comparison with SP) of some of the ligands tested on the growth cone fraction are higher than those reported for adult rat brain synaptosomes ls'3s. This is true for NKB and SP6_ll whose relative potencies on the [125I]BHSP binding site in the growth cone fraction were 10 times more than in the adult (see Table I in Results). Furthermore, in comparison with studies using adult rat brain cortex membranes 4. the relative potency of SP6_ll on the [125I]BHSP binding site is 25 times more in the growth cone fraction. However, our results with the growth cone fraction compare well with those obtained for mouse embryonic mes-
encephalic neurons (see ref. 1 and unpublished results) The early acquisition of an SP r e c e p t o r is implic a t e d from the present e x p e r i m e n t s with the growth cone p r e p a r a t i o n , as well as from studies with primary cultures of mouse e m b r y o n i c mesencephalic neurons ~ and with e m b r y o m c and early postnatal rat brain 3t It is conceivable that the early a p p e a r a n c e of a receptor for SP implies a role for the r e c e p t o r in one or more aspects of synaptogenesis. In this respect it has been r e p o r t e d that SP can stimulate neurite outgrowth both from embryonic chick dorsal root ganglia and n e u r o b l a s t o m a cells 26'27. H o w e v e r , it should be noted that in these studies SP p r o d u c e d its effects only at high concentrations ( > 1 0 -5 M). In addition, the N-terminal t e t r a p e p t i d e of SP was found in the experiments with n e u r o b l a s t o m a cells 27 to have almost the same effect as SP, while the C-terminal heptapeptide h a d little effect. This physiological profile is different from the binding profile in which N-terminal fragments are devoid o f affinity for the SP receptor as found in the present study and in others (see ref. 17 for review). M o r e specifically, a growth-stimulatory effect has been p r o p o s e d for SP on rat brain catecholaminergic neurons. Thus. Jonsson and Hallman ~4 showed that SP can counteract 6-hydroxydopamine-induced denervation of distant nerve terminal projections of n o r a d r e n a l i n e neurons of the locus coeruleus and also p r o m o t e fiber growth of these neurons when n e o n a t a i l y axotomized 16. SP has also been shown to have similar actions on d a m a g e d central serotonergic neurons during o n t o g e n y 15. M o r e over. SP has been r e p o r t e d to accelerate the regener-
ation of central catecholaminergic fibers in cat occipital cortex 25. H o w e v e r , with respect to the studies demonstrating counteracting effects of SP on neurotoxin-induced alteration of the d e v e l o p m e n t of catecholaminergic neurons, o t h e r interpretations can be envisaged. These include interference by SP of the neurotoxin's action at the level of the catecholamine uptake mechanism and/or nerve terminal and axonal degeneration. As a possible corollary to these effects of SP on brainstem neurons it has recently been demonstrated that there is a very high density of SP binding sites in this brain region over the first postnatal week in the rat relative to o t h e r a g e - m a t c h e d regions and to adult levels 3a. O u r results in the p r e s e n t study support this finding in that the growth cone fraction from brainstem exhibits a rich binding of [125I]BHSP. SP receptors m a y play o t h e r roles during brain ontogeny. It has b e e n d e m o n s t r a t e d that the growth cone-enriched fraction from developing rat forebrain can accumulate and release G A B A and n o r a d r e n aline 19. F u r t h e r , the fraction has b e e n shown to possess an a u t o r e c e p t o r - m e d i a t e d mechanism for modulating G A B A release 2°. Perhaps, therefore, SP receptors could contribute to a m o d u l a t i o n of transmitter release from the growth cone fraction.
ACKNOWLEDGEMENTS R . O . L . is a recipient of an E M B O long term fellowship (ALT-133). This study was s u p p o r t e d by grants from I N S E R M , D R E T (85.078), and RhOne Poulenc SantO.
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7 8
hibition of radioligand binding by guanine nucleotides, J. Biol. Chem., 258 (1983) 5158-5164. Cascieri, M.A., Chicchi, G.G. and Liang, T., Demonstration of two distinct tachykinin receptors in rat brain cortex, J. Biol. Chem., 260 (1985) 1501-1507. Fonnum, F., Lund Karlsen, R., Malthe-Sorenseen, D., Stern, S. and Walaas, I., High-affinity transport systems and their role in transmitter action. In C.W. Cotman, G. Poste and G.L. Nicolson (Eds.), The Cell Surface and Neuronal Function, Elsevier, Amsterdam, 1980, pp. 455-504. Fried, R.C. and Blaustein, M.P., Retrieval and recycling of synaptic vesicle membrane in pinched-off nerve terminals (synaptosomes), J. Cell Biol.. 78 (1978) 685-700. Gardner. C.R. and Richards, M.H., Presence of radiolabelled metabolites in release studies using 3H-GABA, J. Neurochem.. 36 (1981) 1590-1593.
9 Gilbert, R.F.T. and Emson, P.C., Substance P in rat CNS and duodenum during development, Brain Res., 171 (1979) 166-170. 10 Gogstad, G.A. and Krutnes, M.B., Measurement of protein in cell suspensions using the Coomassie brilliant blue dye-binding assay, Anal. Biochem., 126 (1982) 355-359. 11 Gordon-Weeks, P.R. and Lockerbie, R.O., Isolation and partial characterisation of neuronal growth cones from neonatal rat forebrain, Neuroscience, 13 (1984) 119-136. 12 Gordon-Weeks, P.R., Lockerbie, R.O. and Pearce, B.R., Uptake and release of 3H-GABA by growth cones isolated from neonatal rat brain, Neurosci. Lett., 52 (1984) 205-210. 13 lnagaki, S., Sakanaka, M., Shiosaka, S., Senba, E., Takatsuki, K., Takagi, H., Kawai, Y., Minagawa, H. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis. I. Forebrain and upper brainstem, Neuroscience, 7 (1982) 251-277. 14 Jonsson, G. and Hallman, H., Substance P modifies the 6hydroxydopamine induced alteration of postnatal development of central noradrenaline neurons, Neuroscience, 7 (1982) 2909-2918. 15 Jonsson. G. and Hallman, H., Effect of substance P on the 5,7-dihydroxytryptamine induced alteration of postnatal development of central serotonin neurons, Med. Biol.. 61 (1983) 105-112. 16 Jonsson. G. and Hallman, H., Effect of substance P on neonatally axotomized noradrenaline neurons in rat brain, Med. Biol., 61 (1983) 179-185. 17 Lavielle, S., Beaujouan, J.C., Torrens, Y., Saffroy, M., Viger, A., Chassaing, G., Marquet, A. and Glowinski, J., Central nervous system receptors for tachykinins; structure activity relationships and biochemical studies. In C.C. Jordan and P. Oehme (Eds.), Substance P Metabolism and Biological Actions, Taylor and Francis, London, 1985, pp. 83-93. 18 Lavielle, S., Chassaing, G., Julien, S., Besseyre, J. and Marquet, A., Influence of the aminoacids of substance P in the recognition of its receptor: affinities of synthesized SP analogues for the specific 125I-BHSP binding site on rat brain synaptosomes, Neuropeptides, 7 (1986) 191-200. 19 Lockerbie, R.O., Gordon-Weeks, P.R. and Pearce, P.R., Growth cones isolated from developing rat forebrain: uptake and release of GABA and noradrenaline, Dev. Brain Res., 21 (1985) 265-275. 20 Lockerbie. R.O. and Gordon-Weeks, P.R., y-Aminobutyric acid-A (GABA-A) receptors modulate (3H)GABA release from isolated neuronal growth cones in the rat, Neurosci. Lett., 55 (1985) 273-277. 21 McGregor, G.P., Woodhams, P.L., O'Shaughnessy, D.J., Ghatei, M.A., Polak, J.M. and Bloom, S.R., Developmental changes in bombesim substance P, somatostatin and vasoactive intestinal polypeptide in the rat brain, Neurosci. Lett., 28 (1982) 21-27. 22 Maderspach, K. and Fajszi, C., Development offl-adrenergic receptors and their function in glia-neuron communication in cultured chick brain, Dev. Brain Res., 6 (1983) 252-257.
23 Michelot, R., Gozlan, H., Beaujouan, J.-C., Besson, M.J., Torrens, Y. and Glowinski, J., Synthesis and biological activities of substance P iodinated derivatives, Biochem. Biophys. Res. Commun., 95 (1980) 491-498. 24 Mohini, P., Bahouth, S.N., Brundish, D.E. and Musacchio, J.M., Specific labelling of rat brain substance P receptor with ~H-physalaemin, J. Neurosci., 5 (1985) 2078-2085. 25 Nakai, K. and Kasamatsu, T., Accelerated regeneration of central catecholamine fibers in cat occipital cortex: effects of substance P, Brain Res., 323 (1984) 374-379. 26 Narumi, S., and Fujita, T., Stimulatory effects of substance P and nerve growth factor (NGF) on neurite outgrowth in embryonic chick dorsal root ganglia, Neuropharmacology, 17 (1978) 73-76. 27 Narumi, S. and Maki, Y., Stimulatory effects of substance P on neurite extension and cyclic AMP levels in cultured neuroblastoma cells, J. Neurochem.. 30 (1978) 1321-1326. 28 Park, C.H., Massari, V.J., Quirion, R., Tizaki, Y., Shults, V.W. and O'Donohue, T.L., Characteristics of 3H-substance P binding sites in rat brain membranes, Peptides, 5 (1984) 833-836. 29 Perrone, M.H., Diehl, R.E. and Hanbrich, D.R., Binding of (3H)substance P to putative substance P receptors in rat brain membranes, Eur. J. Pharmacol., 95 (1983) 131-133. 30 Quirion, R. and Pilapil, C., Comparative potencies of substance P, substance K and neuromedin K on brain substance P receptors, Neuropeptides, 4 (1984) 325-329. 31 Quirion, R. and Dam, V.-T., Ontogeny of substance P receptor binding sites in rat brain, J. Neurosci., 6 (1986) 2187-2199. 32 Rfiegg, V.T., A binding site for substance P on dissociated cells from cultures of rat CNS. In P. Skrabanek and D. Powell (Eds.), Substance P, Boole, Dublin, 1983, pp. 65-66. 33 Segal, I.H., Biochemical Calculations, Wiley, New York, 1976, pp. 241-244. 34 Torrens, Y., Beaujouan, J.C. and Glowinski, J., Pharmacological characterisation of two tachykinin binding sites in the rat cerebral cortex, Neuropeptides. 6 (1985) 59-70. 35 Torrens, Y., Beaujouan, J.C., Saffroy, M., Daguet de Montety, M.C., Bergstr0m, L. and Glowinski, J., Substance P receptors in primary cultures of cortical astrocytes from the mouse, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9216-9220. 36 Torrens, Y., Beaujouan, J.C., Viger, A. and Glowinski, J., Properties of a 125I-substance P derivative binding to synapfosomes from various brain structures and the spinal cord of the rat, Naunyn-Schmiedeberg's Arch. Pharmacol., 324 (1983) 134-139. 37 Torrens, Y., Lavielle, S., Chassaing, G., Marquet, A., Glowinski, J. and Beaujouan, J.C., Neuromedin K, a tool to further distinguish two central tachykinin binding sites, Eur. J. Pharmacol., 102 (1984) 381-382. 38 Viger, A., Beaujouan, J.C., Torrens, Y. and Glowinski, J., Specific binding of a 125I-substance P derivative to rat brain synaptosomes, J. Neurochem., 40 (1983) 1030-1039. 39 Watson S.P., Sandberg, B.E.B., Hanley, M.R. and Iversen, L.L., Tissue selectivity of substance P alkyl esters suggesting multiple receptors, Eur. J. Pharmucol., 87 (1983) 77-84.
1
BRD 50712
Research Reports
An isolated growth cone-enriched fraction from developing rat brain has substance P binding sites R. Owen Lockerbie, Jean-Claude Beaujouan, Monique Saffroy and Jacques Glowinski College de France-INSERM U 114, Chairede Neuropharmacologie, Paris (France) (Accepted 13 October 1987)
Key words: Isolated growth cone; Rat; Substance P receptor; Pharmacology; Development
A fraction enriched in neuronal growth cones isolated from developing rat forebrain was shown to possess binding sites for the substance P analog, Bolton-Hunter substance P ([125I]BHSP). Specific binding of this ligand reached an equilibrium after 10 min at 2(I °C, and was reversible and temperature-dependent. Removal of extracellular Na ÷ did not block but rather augmented [125I]BHSP binding suggesting that the labeled analog was not transported into the growth cone fraction. Scatchard analysis of the binding indicated a single class of non-interacting binding sites in the growth cone fraction (Kd: 257 pM; Bmax:56 fmol/mg protein). From competition studies using substance P and other tachykinins, their rank order of potency for inhibiting [125I]BHSP binding was SP > physalaemin > > eledoisin > kassinin > NKB/> NKA. Such order is consistent with the presence of an SP receptor (Neurokinin-1) in the growth cone fraction. The N-terminal fragments of substance P, SPI_7 and SPl_ll free acids, and the C-terminal fragment, SP7_i1, were devoid of affinity for the [125I]BHSP binding site. However SP6_I1and SPl_ll methyl esters showed more potency.
INTRODUCTION Binding sites for the tachykinin substance P (SP) have been characterized, by using a labeled derivative of SP, the [125I]Bolton-Hunter SP (BHSP), with various m a m m a l i a n central nervous system (CNS) preparations including rodent embryonic neurons 1'32, synaptosomes from various regions of the adult rat CNS 3&36"37 and adult rat brain cortex membranes 4. The same findings have also been made with other ligands such as [3H]SP and [3H]physalaemin on adult rat brain m e m b r a n e s 2'24'28-3°. Although SP has been d e m o n s t r a t e d in the brain of several species at its early stages of d e v e l o p m e n t 9:3'21, very little is presently known on the d e v e l o p m e n t of SP receptors. A few recent studies, however, including those with primary cultures of mesencephalic embryonic neurons from the mouse I and with embryonic and young postnatal rat brain 3~, have d e m o n s t r a t e d the
early a p p e a r a n c e of SP receptors. To further substantiate the early acquisition of the SP r e c e p t o r during brain ontogeny we report here on the pharmacological characterisation of the receptor, by using the [125I]BHSP ligand, in an isolated fraction enriched in neuronal growth cones from neonatal rat brain 11. MATERIALS AND METHODS
Preparation of growth cone-enriched fractions Growth cone-enriched fractions were isolated from the forebrain or brainstem of 5-day-old (day of birth is day 1) rats (Iffa Credo, France) as previously described 11. F r o m electron microscopical analysis, more than 90% of m e m b r a n e - b o u n d , identifiable objects in these fractions fulfil the morphological criteria for the identification of growth cones. G r o w t h cones are enriched in the fraction by a factor of 7 over the starting h o m o g e n a t e . Using morphological con-
Correspondence: R.O. Lockerbie, Coll~ge de France-INSERM U 114, Chaire de Neuropharmacologie, 11, place Marcelin Berthelot, 75231 Paris, France. 0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
trois along with biochemical and immunological controls, the growth cone-enriched fraction has been shown not to be contaminated by synaptosomes, glial or neuronal cell soma elements, and contains only small amounts of isolated rough endoplasmic reticulure and mitochondria. The major contaminants of the growth cone fraction are large membrane sacs many of which have a similar diameter to that of growth cones and vesicles of unidentified origin! 1. The isolation method involves Ficoll-sucrose gradients, and for this reason the growth cone fraction is returned to isotonic conditions by incubation in Krebs' buffer 7. The growth cone fraction was kept at 4 °C until the binding assays, when the fraction was pelleted (10,000 g for 30 s) and resuspended and incubated at 37 °C for 30 min in a buffer used for the binding assays (see below).
Binding assays ]-'he composition of the Krebs-Ringer phosphate buffer (pH 7.0) used for the binding assays was the following (in mM); NaCI 120; KCI 4.8; CaC12 1.2; MgSO4 1.2; NaH2PO 4 15.6. To this buffer was added glucose (1 mg/ml) bovine serum albumin (BSA; 0.4 mg/ml) and bacitracin (60 ~g/ml). Binding assays were performed in Eppendorf tubes (vol. 1.5 ml) which were precoated with the binding assay buffer in which the BSA concentration was 5 mg/ml. Routinely, 20/~1 of the growth cone fraction (approx. 60 ktg of protein) was incubated in a final volume of 200 /A of the binding assay buffer for 10 min at 20 °C with [J25I]BHSP (approx. 25,000 cpm; 40 pM; see ref. 23 for synthesis). At the end of this incubation, the tubes were centrifuged for 30 s at 10,000 g and the pellets washed once with 1 ml of the 'cold' medium used in incubation. Radioactivity bound to tissues was estimated using a Packard gamma counter. All assays were performed in triplicate, and included blanks in which the growth cone fraction was omitted from the incubation mix. Non-specific binding (NSB) was determined by co-incubating the growth cone-fraction with the radioactive ligand and 1 ~M SP. Specific binding (SB) was calculated by subtracting NSB from total binding (TB). To determine reversibility of binding, the growth cone fraction was incubated with radioactive ligand as described above, and then SP (1/~M) was added ~md the concentration of [t25I]BHSP specifically
bound with time was followed. For analyzing saturation kinetics of [125I]BHSP binding by the Scatchard method 33, the growth cone fraction was incubated as described above with different concentrations of radioactive iigand and specific binding measured at each concentration. For studies of the competitive inhibition of [I25I]BHSP binding, the radioactive ligand was mixed with increasing concentrations of non-labeled SP and structurally related peptides or derivatives, and incubated with the growth cone fraction under the conditions described above with specific binding measured at each concentration.
Effect of Na + on [125I]BHSP binding In experiments designed to ensure that [125I]BHSP is not transported into the growth cone fraction, the effect of Na+-free media was investigated by performing binding assays as described above in Krebs' buffer from which all Na + was removed. In Na+-free media, isotonicity was maintained by removing NaCI and NaH2PO 4 and adding sucrose (0.28 M), KHzPO4 (15.6 mM) and Tris (1.2 mM). The pH of this media was corrected to pH 7.0 by the addition of K O H . In parallel, control experiments involved pre-incubating the growth cone fraction for 30 rain at 37 °C in Krebs' buffer and then incubating 60/~g protein for 15 min at 20 °C with [3H]GABA (1.0/~Ci) diluted with unlabeled amino-acid to a final ligand concentration of 5/~M in Krebs' buffer or in Na+-free Krebs' buffer (1 ml) in the presence (blank) or absence of nipecotic acid (400/~M), an inhibitor of G A B A uptake into the growth cone fraction 19. Amino-oxyacetic acid (100/~M) was included to inhibit G A B A transaminase ~. Incubations were terminated by rapid centrifugation (30 s, 10,000 g) and pellets washed once with cold incubating medium (1 ml). Radioactivity was determined by counting in a Packard 3255 spectrometer using Aquasol-2 (NEN) as scintillant. Other methods Liquid chromatographic (HPLC) analysis involving C18 ktBondapak columns (0.4 x 30 cm; Waters Instruments, Rochester, MN) and methanol-ammonium acetate (50 mM, pH 4; 540-460) as solvent (flow rate: 2 ml/min) was used to identify the radioactive material recovered in the supernatant at the end of the 10-rain incubation period in order to assess the
extent of [12SI]BHSP degradation by peptidases. Protein content was estimated by the method of Bradford 3 with the suggested modification of Gogstad and Krutnes m. Regression analysis was used to draw lines through data points for Scatchard and Hill plots.
Chemica& BSA was purchased from Calbiochem. Bacitracin, nipecotic acid and amino-oxyacetic acid were from Sigma. [3H]GABA (spec. act. 57 Ci/mmol) was from Amersham, and Aquasol-2 from NEN. Physalaemin, kassinin, eledoisin, SP6-11, SPT-ll, SPl-7 and SPl_ll free acids, SPl_ll methyl ester and spantide were from Peninsula. Neurokinin-A and -B (NKA and NKB) were synthesized by S. Lavielle (University of Paris VI). RESULTS
Binding of [1251]BHSP to growth cone-enriched fractions Preliminary studies were performed in which binding assays were carried out in the presence of different combinations of BSA and peptidase inhibitors at various concentrations. The presence of BSA (0.4 mg/ml) and bacitracin (60 ~g/ml) resulted in the best compromise between a good specific binding of [125I]BHSP and a low percentage value for degradation of the ligand. HPLC analysis showed that in the presence of these two compounds, approximately 76% of the radioactivity recovered at the end of a 10min incubation period migrated as authentic [125I]BHSP. Fig. 1 shows that when a growth cone-enriched fraction isolated from 5-day-old rat forebrain was incubated at 20 °C with a low concentration of [12Sl]BHSP (48 pM) for 10 min, specific binding increased linearly over the range from 30 to 120/,g of protein. Specific binding of [12SI]BHSP to the growth cone fraction after 10 min at 20 °C was approximately 76% of the total binding and represented about 3% of the total number of counts per minute added to the incubation medium. When binding assays were performed for 10 min at 4 °C, approximately 80% of specific binding was lost. Under identical assay conditions, specific [125I]BHSP binding to a growth cone fraction from
the brainstem of 5-day-old rats showed the same linearity with increasing protein concentration as the forebrain growth cone fraction (data not shown). However, in absolute terms (fmol/mg protein) specific binding to the brainstem growth cone fraction was greater than specific binding to the forebrain growth cone fraction with the difference being 61% (mean of 3 experiments). A period of 10 min was used in all experiments since, as Fig. 2A shows, an equilibrium of specific [12SI]BHSP binding was reached around this time. Specific binding of [125I]BHSP was reversible, since rapid dissociation of the labeled iigand was observed when 1/xM unlabeled SP was added to the incubation medium for various times after the initial 10 min incubation (Fig. 2B). Dependence of Na + is a characteristic of neurotransmitter high-affinity uptake systems 6. Fig. 3 shows that under similar assay conditions, the absence of extracellular Na + reduced [3H]GABA uptake into the growth cone fraction by >80% as previously described 12J9 while enhancing [125I]BHSP binding to the growth cone fraction by > 100%.
Equilibrium binding studies Under equilibrium conditions of [125I]BHSP bindI
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y.g protein /assay Fig. 1. [12SI]BHSP binding at various concentrations of a growth cone-enriched fraction from 5-day-old rat forebrain. Increasing concentrations of the growth cone fraction were incubated with the radioactive ligand (48 pM) for 10 min at 20 °C as described in Materials and Methods. Specific binding was calculated by subtracting non-specific binding from total binding. All values are the mean +_S.E.M. of 3 determinations.
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20 "c. B: dissociation: growth cone,enriched fractions (60 ag of protein) were incubated with the ligand (39 pM) for 10 min
at 20 o C. Substance P (1 pM) was then added and the concentration of [ 125 I]BHSP specifically bound was followed with time. Each point represents the mean of data obtained in 3 separate experiments each performed in triplicate.
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ing to the growth cone fraction, non-specific binding of the ligand was a linear function of the labeled ligand concentration. In contrast, specific binding of [tasI]BHSP was saturable (Fig. 4A). Scatchard analysis of the specific binding data gave linear plots indicating the presence of a single class of binding sites (Fig. 4B). F r o m 4 i n d e p e n d e n t experiments, average
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cone-enriched fractions (60/~g protein) were incubated for 10 min at 20 °C with []~SI]BHSP(39 pM) in Krebs' medium with or without Na ÷ and the specific binding determined. Right: [3H]GABA uptake into growth cone-enriched fractions was evaluated under the same conditions as for [12SI]BHSPbinding (see Materials and Methods section). Blank values were determined by incubating growth cone-enriched fractions in the presence of nipecotic acid (400/~M). Each value represents the mean ± S.E.M. of 3 experiments each performed in triplicate or quadriplicatc.
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Fig. 4. [I251]BHSP binding to growth cone-enriched fraction from 5-day-old rat forebrain as a function of radiolabeled peptide concentration. A: growth cones (60pg of protein) were incubated with different concentrations of radioactive ligand for 10 min at 20 °C. Total (0), specific (©) and non-specific (&) binding. B: Scatchard plot of specific [125I]BHSP binding data. Each point is the mean of 3 determinations obtained in a single experiment.
values for the dissociation constant (Kd) and the density of sites (Bmax) were 257 pM +51 (mean + S.E.M.) and 56 fmol/mg protein +11 (mean _+ S.E.M.), respectively. Hill plots of the data (nil = 0.93 _+ 0.15) revealed that []25I]BHSP was bound to a single population of non-interacting sites.
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Competitive inhibition of [~251]BHSP binding When the efficacies of SP and other tachykinins were tested for inhibition of [125I]BHSP-specific binding to the growth cone fraction, they were found to do so in a competitive manner (Fig. 5, Table I). The rank order of potency was SP > physalaemin > > eledoisin > kassinin > NKB/> NKA with the inhibitory effect of physalaemin, eledoisin, kassinin and NKA being about 61,2, 0.7 and 0.3% that of SP, respectively (Table I). Two SP C-terminal fragments were also tested for their ability to inhibit specific [125I]BHSP binding and were found to be much less potent than SP in attenuating binding. Thus, SP6_I] and SP7_II reduced spe-
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log [TK](M) Fig. 5. Competition inhibition of [1251]BHSP binding to growth cone-enriched fractions from 5-day-old forebrain by SP and other tachykinins (TK). Growth cone-enriched fractions (60 pg of protein) were incubated with radioactive ligand (62 pM) and increasing concentrations of non-labeled tachykinin for 10 min at 20 °C. Results are expressed as percentage of the specific binding of [ 125I]BHSP alone. Each value is the mean of 4 separate experiments each performed in triplicate.
TABLE
I
Inhibition qf [1251]BHSP binding to growth cones and adult rat brain synaptosomes by substance P, analogs of SP, and other endogenous tachykinins
The concentrations of the peptides required to inhibit the specific binding of [~25I]BHSPto growth cones by 50% (ICs0) were estimated from the HiU plots of the titration curves exampled in Fig. 5. (a) Each value represents the mean of the number of experiments given in parentheses. The S~D. of each value is less than 75%. (b) Data are from Viger et al. (1983) and Lavielle et al. (1986). Hill coefficients for these compounds were close to unity (0.79-1.16). Peptide
Growth cones
Rat brain synaptosomes
(a) ICsr~(M) SP Physalaemin Eledoisin
8.5 × 10 "~ 1.4 × 10 ~ 4.4 x 10 -s
Kassinin
1.2 × 10 -7
Neurokinin A Neurokinin B SP,,_ll SP7 ii SP~ 7 free acid SPI.II free acid
3.0 x 10 7 2.9 × 10 -7 7.6 × 10 -s >10 5 > 1 0 -5 5.1 x 10 .6
(4) (4) (2) (2) (3) (3) (3) (2) (2) (4)
SPI-it methyl ester Spantide
2,1 x 10-s 9.5 x Ill 7
(3) (5)
Relative affinity
(b) 1C5o (M)
Relative affinity
1 0.61 0.02
6.4 x 10 l0 2.4 x 10 -9 1.1 x 10 -7
1 0.3 0.006
0.007
2.0 x 10 -7
0.003
0.003 0.003 0.01
0.005 0.0003 0.001
0.0002
1.4 x 10 -7 2.2 × 10 -6 7.0 X 10 -7 >10 5 n.d. 6.7 × 10 -6
0.04 0.0009
9. l x Ill ~ 2.6 x 10 7
0.007 0.002
cific [~251]BHSP binding with about 1/100 and more than 1/100,000, respectively, the potency of SP (Table 1). N-terminal fragments of SP such a s S P I _ 7 and SP 1_1~ free acids were without appreciable effect on specific [125I]BHSP binding. Hence, S P I _ l l free acid reduced [1251]BHSP binding with about 1/5000 the potency of SP while S P I _ 7 free acid did not significantly modify binding even at 10 -5 M concentrations. However, SPl_ll methyl ester had a considerably more potent effect on p251]BHSP binding, its affinity being 1/25 that of SP (Table I). Hill coefficients for all these compounds were close to unity. Spantide, a proposed SP receptor antagonist, proved to be a weak inhibitor of [tzSI]BHSP binding with 1/1000 the potency of SP (Table I). Dimethylsulphoxide which was used to solubilize NKB and SP6_li was without effect on [125I]BHSP binding to the growth cone fraction (data not shown). DISCUSSION
We have shown in this paper that a fraction enriched in growth cones isolated from developing rat forebrain possesses binding sites for substance P. Using a labeled analog of SP, [125I]BHSP, specific binding to the growth cone-fraction was linearly dependent on tissue concentration and was saturable.
0.0001
of high affinity, reversible and temperature-dependent. The binding assay conditions adopted in the present conditions were similar to those using this ligand in other receptor binding studies (for review see ref. 17). Under these conditions, H P L C analysis showed that after 10 min of incubation at 20 °C when binding had reached equilibrium, only slight peptidasic cleavage of the ligand had occurred. Thus, most of the binding observed in this study was attributable to authentic [125I]BHSP. [125I]BHSP-specific binding sites have been demonstrated in rat brain synaptosome fractions 3~ and recently on intact glial cells from several brain structures of the mouse 35. However, the binding reported in the present study is unlikely to have a contribution from these sources nor from neuronal cell soma elements since they do not contaminate the growth cone preparation 11. Although the growth cone fraction contains only small amounts of isolated rough endoplasmic reticulum and mitochondria 11, these organelles too would not be expected to contribute to [125I]BHSP binding in the fraction since binding sites so far have only been located on plasma membrane. However, the growth cone fraction does contain large membrane sacs, many of which have a similar diameter to that of growth cones, and vesicles of unidentified origin 11. We do not know yet what is the
distribution of [125I]BHSP binding sites between the growth cones and these elements in the fraction. For several reasons, it is unlikely that all the growth cones in the fraction possess binding sites. Thus, autoradiographical studies have shown that only a fixed proportion of embryonic mouse mesencephalic neurons in primary culture have SP receptors (Beaujouan eta!., unpublished observations). Further, autoradiographic studies of SP receptor binding sites in rat brain have demonstrated differential distributions of SP receptors between and within various regions, and that such distributions can undergo major changes during ontogeny 3~. Under assay conditions in which all Na + was removed, [3H]GABA uptake into the growth cone fraction was blocked by more than 80%, while [125I]BHSP binding was not reduced but rather increased. These findings suggest that the labeled SP analog was not transported into the growth cone fraction. The increase in [125I]BHSP binding in Na+-free media probably reflects the ion substitutions that were made for such media since it is known that binding to the SP receptor can be modulated by both monovalent and divalent ions 2~. The saturability of the specific binding of [125I]BHSP to the growth cone fraction was demonstrated from equilibrium studies. Scatchard analysis of the specific binding with increasing concentrations of the ligand revealed a single population of non-interacting binding sites with an apparent affinity (Kd) of 257 pM and a density (Bmax) of 56 fmol/mg protein. The Kj of [125I]BHSP binding to the growth cone fraction is in good agreement with the binding of this ligand under similar conditions to mouse embryonic mesencephalic neurons in culture (Kd: 169 pM) 1. However, slight differences are apparent when comparisons are made with [125I]BHSP binding to adult preparations. Thus, a K a of 470 pM was derived from equilibrium studies of [125I]BHSP binding to a crude synaptosome fraction of adult rat brain 38, and this value shows little variation for synaptosomes from different regions of the rat central nervous system 36. These differences may in part be due to differences in the assay conditions for the SP binding. Alternatively, as previously suggested 3s, perhaps the lower affinity of [125I]BHSP binding sites observed in the adult is related to a decrease in receptor sensitivity when SP receptor-containing neurons are fully developed
and functionally in contact with their target cells. A similar decrease in receptor affinity during development has also been found for instance with/~-adrenergic receptors 22. However, Quirion and D a m ~l did not find any significant change in the affinity of [1251]BHSP binding sites in rat brain membranes during ontogeny. The potential of SP and other endogenous tachykinins, and of N- and C-terminal fragments of SP to inhibit specific [125I]BHSP binding to the growth cone fraction was also investigated. For the tachykinins, their rank order of potency for inhibiting binding was SP > physalaemin > > eledoisin > kassinin > NKB /> NKA. Such order is similar to that found in previous studies of [1251]BHSP binding to various CNS preparations 5'3°'37. However, NKB in the present study shows more potency than in studies with the other preparations. As with other preparations 1'4'38of the two C-terminal fragments tested in the present study, SP6_ll showed a marked reduction compared to SP in inhibiting [125I]BHSP binding. The non-biologically active SP7_II was devoid of affinity for the [1251]BHSP binding site. Of the N-terminal fragments tested, SPI_ 7 and SPl_ll free acids, they were either with little or no effect on binding. However, SP1 ~ methyl ester, which has been shown to have a selective affinity for the SP receptor in the peripheral nervous system 39 and CNS 5"34, showed more potency although its inhibitory effect on [125I]BHSP binding to the growth cone-fraction was still only 1/25 that of SP. Hence, the pharmacology of the [1251]BHSP binding site in the growth cone fraction is consistent with the presence of a SP receptor 17 (Neurokinin-1 ; nomenclature designated at the Montreal meeting, July 1986). It is interesting to note that the relative affinities (comparison with SP) of some of the ligands tested on the growth cone fraction are higher than those reported for adult rat brain synaptosomes ls'3s. This is true for NKB and SP6_ll whose relative potencies on the [125I]BHSP binding site in the growth cone fraction were 10 times more than in the adult (see Table I in Results). Furthermore, in comparison with studies using adult rat brain cortex membranes 4. the relative potency of SP6_ll on the [125I]BHSP binding site is 25 times more in the growth cone fraction. However, our results with the growth cone fraction compare well with those obtained for mouse embryonic mes-
encephalic neurons (see ref. 1 and unpublished results) The early acquisition of an SP r e c e p t o r is implic a t e d from the present e x p e r i m e n t s with the growth cone p r e p a r a t i o n , as well as from studies with primary cultures of mouse e m b r y o n i c mesencephalic neurons ~ and with e m b r y o m c and early postnatal rat brain 3t It is conceivable that the early a p p e a r a n c e of a receptor for SP implies a role for the r e c e p t o r in one or more aspects of synaptogenesis. In this respect it has been r e p o r t e d that SP can stimulate neurite outgrowth both from embryonic chick dorsal root ganglia and n e u r o b l a s t o m a cells 26'27. H o w e v e r , it should be noted that in these studies SP p r o d u c e d its effects only at high concentrations ( > 1 0 -5 M). In addition, the N-terminal t e t r a p e p t i d e of SP was found in the experiments with n e u r o b l a s t o m a cells 27 to have almost the same effect as SP, while the C-terminal heptapeptide h a d little effect. This physiological profile is different from the binding profile in which N-terminal fragments are devoid o f affinity for the SP receptor as found in the present study and in others (see ref. 17 for review). M o r e specifically, a growth-stimulatory effect has been p r o p o s e d for SP on rat brain catecholaminergic neurons. Thus. Jonsson and Hallman ~4 showed that SP can counteract 6-hydroxydopamine-induced denervation of distant nerve terminal projections of n o r a d r e n a l i n e neurons of the locus coeruleus and also p r o m o t e fiber growth of these neurons when n e o n a t a i l y axotomized 16. SP has also been shown to have similar actions on d a m a g e d central serotonergic neurons during o n t o g e n y 15. M o r e over. SP has been r e p o r t e d to accelerate the regener-
ation of central catecholaminergic fibers in cat occipital cortex 25. H o w e v e r , with respect to the studies demonstrating counteracting effects of SP on neurotoxin-induced alteration of the d e v e l o p m e n t of catecholaminergic neurons, o t h e r interpretations can be envisaged. These include interference by SP of the neurotoxin's action at the level of the catecholamine uptake mechanism and/or nerve terminal and axonal degeneration. As a possible corollary to these effects of SP on brainstem neurons it has recently been demonstrated that there is a very high density of SP binding sites in this brain region over the first postnatal week in the rat relative to o t h e r a g e - m a t c h e d regions and to adult levels 3a. O u r results in the p r e s e n t study support this finding in that the growth cone fraction from brainstem exhibits a rich binding of [125I]BHSP. SP receptors m a y play o t h e r roles during brain ontogeny. It has b e e n d e m o n s t r a t e d that the growth cone-enriched fraction from developing rat forebrain can accumulate and release G A B A and n o r a d r e n aline 19. F u r t h e r , the fraction has b e e n shown to possess an a u t o r e c e p t o r - m e d i a t e d mechanism for modulating G A B A release 2°. Perhaps, therefore, SP receptors could contribute to a m o d u l a t i o n of transmitter release from the growth cone fraction.
ACKNOWLEDGEMENTS R . O . L . is a recipient of an E M B O long term fellowship (ALT-133). This study was s u p p o r t e d by grants from I N S E R M , D R E T (85.078), and RhOne Poulenc SantO.
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