Substance P fragments and striatal endogenous dopamine outflow: interaction with substance P
S. Khan, J. Sandhu, R. Whelpton, A.T. Michael-Titus Department of Pharmacology, Division of Biomedical Sciences, Queen Mary and Westfield College, Mile End Road, London E1 4NS, UK
Summary Accumulating evidence shows that N- and C-terminal substance P fragments have significant biological activity. Substance P(1-9) and substance P(6-11) have been reported to be major substance P metabolites in rat striatum. We investigated the effects of these fragments on endogenous dopamine outflow in rat striatal slices. Substance P-(1-9) and substance P-(6-11) induced a significant increase in dopamine outflow at 0.1 and 1 nM. The effects of substance P-(6-11) (1 nM) were reversed by the tachykinin NK 1 antagonist WIN 51,708 (17~-hydroxy-17c~- ethynyl-5c~-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) (2.5 nM), whereas the effects of substance P-(1-9) were not modified by the antagonist. Substance P-(1-9) and substance P-(6-11) (1 nM) did not increase the dopamine overflow induced by 25 mM KCI. The effects of the two fragments were reversed by the muscarinic antagonist atropine (1 gM) but not by nicotinic antagonists dihydro-~-erythroidine (0.5 gM) and pempidine (10 gM). The co-incubation of tissue with substance P and each fragment in a 1/1 or 10/1 ratio of substance P to metabolite revealed a negative interaction between parent and fragments. A similar pattern was observed when substance P was co-administered with the active fragments substance P(1-4), substance P(1-7), substance P(5-11) and substance P(8-11). The data show that substance P-(1-9) and substance P-(6-11) have modulatory effects similar to substance P. However, the presence of active substance P metabolites does not appear to amplify the signal mediated by the parent peptide.
INTRODUCTION
Substance P is an undecapetide which can be a substrate for a variety of peptidases, such as 'enkephalinase' (neu- tral metalloendopeptidase, EC 3.4.24.11) ~ angiotensin- converting enzyme (EC 3.14.5.1) 2 and a post-proline cleaving enzyme (EC 3.4.21.26). 3 The metabolism of the peptide results in a complex mixture of fragments, as shown after incubation of substance P with synaptoso- real membranes. 1,4 Certain species, such as substance P-(1-7) or C-terminal fragments, have been detected in significant concentrations in several brain structures and in the spinal cord? Studies in various experimental mod- els have shown that such fragments have intrinsic activ-
Received 9 June 1998 Accepted 13 August 1998
Correspondence to: Dr A.T. Michael-Titus, Department of Pharmacology, Division of Medical Sciences, Queen Mary and Westfield College, Mile End Road, London E1 4NS, UK. Tel. 0171-982-6361; Fax 0181-983-0470; e-mail A.T. [email protected]
ity, which is similar or opposite to that induced by the parent peptide. 6-1°
Of particular interest is the role of substance P and its metabolites in the basal ganglia, which contain high con- centrations of the peptide. Substance P is localized in two types of striatal neurons. 1~ The most numerous are medinm-size spiny neurons, which represent a major stri- atal output. 12,~3 The peptide can be released from nerve terminals in the substantia nigra or from axon collaterals in the striatum) 4,~s The latter also contains a significant density of binding sites for substance P, which can be detected either using the peptide or selective agonists for NK~ receptors, 16-~8 at which substance P has been pro- posed as a preferential endogenous ligand. 19
One of the functional models used to study the effects of substance P in the basal ganglia is the outflow of dopamine from nigrostriatal neurones. After nigral administration the peptide modulates dopamine release from striatal termi-
2021 nals and from nigral dendrites. , Furthermore, local stri- atal modulation of radiolabelled dopamine outflow has been shown with substance P or NK1 agonists, 22-z4 and has
519
520 Khan et al
been confmned in studies measuring endogenous dopamine outflow? ~ Thus, the data suggest that the pep- tide may exert a dual control, i.e. on the cell bodies and the nerve terminals of nigrostriatal dopaminergic neurones. Our observations with striatal slices have shown that N- and C-terminal substance P fragments have intrinsic mod- ulatory effects, which are very similar to those induced by the parent pepfide. 25,2~ The effects are mediated by striatal cholinergic interneurones, and involve the activation of muscarinic receptors. 27 The pattern of the effects of flag- ments in the sttiatum is significantly different from that reported previously after intranigral administration of N- and C-terminal fragments. 2s
Therefore, not only do substance P fragments have intrinsic activity, but the neuromodulatory patterns for the same peptide differ in the terminal area (i.e. substan- tia nigra) vs. the axon collateral area (i.e. striatum). The simultaneous presence in the extracellular fluid of sub- stance P and several active metabolites may lead to a complex synaptic signal. Its nature will depend on the number and characteristics of the metabolites and their relative abundance. Interestingly, the analysis of sub- stance P metabolism in vivo has confirmed the presence of substance P-(1-7) and substance P-(5-1 1), and has shown that substance P-(1-9) and substance P-(6-1 1) are major species produced in the striatum. 29 Our study on dopamine outflow in striatal slices analyzed the effects of these fragments. In parallel, we studied the effects of the shortest N- and C-terminal substance P sequences, sub- stance P-(1-2) and substance P-(10-1 1). Finally, we inves- tigated the interaction between substance P and the various active N- and C-terminal fragments of the pep- tide that have been documented to date.
MATERIALS AND METHODS
Preparation of slices
The housing of the animals and their sacrifice were car- tied out in accordance with current UK regulations on animal experimentation. Male Wistar rats (200-250 g) were housed in standard cages, under alternating 12 h light/dark periods, with food and water ad libitum. The animals were killed by stunning and decapitation. The brain was rinsed in ice-cold Krebs-bicarbonate buffer and coronal striatal slices (approximate thickness 400-500 ~tm) were cut over ice and washed in buffer (2x 20 ml for 4 hemistriata).
Monitoring dopamine outflow
The outflow of endogenous doparnine was monitored in striatal slices using a sequential incubation method described previously. B° Striatal slices were incubated in
Krebs-bicarbonate buffer in the absence or presence of tested substances, introduced after collection of the first two samples. Slices were exposed to substance P and its fragments for 10 rain. 25 mM KC1 was applied as a 1 rain pulse. WIN 51,708 (17-[3-hydroxy-17-a-ethynyl-5-(z- andr o s tano [3,2- b]pyr imido [ 1,2-a]b enz imidazole) , atropine, pempidine or dihydro-[3-erythroidine was intro- duced after the first two samples and was maintained throughout the experiment. Basal outflow represented the average dopamine concentration measured in the first two samples. At the end of the experiment the slices were homogenized and the dopamine and protein con- tent determined. Dopamine concentrations were mea- sured by high performance liquid chromatography with electrochemical detection as previously described. B° The protein content of the slices was determined using Lowry's method and bovine serum albumin as a stan- dard. Dopamine outflow was expressed as pmol/ml/mg protein. Changes in dopamine outflow were expressed as a percentage of the controls, calculated by extrapolating the basal outflow for the duration of the exposure to the drugs. The results were expressed as means + S.E.M. and were analyzed by one way or two way analysis of vari- ance and Bonferroni's test for post-hoc comparisons.
Reagents
Dopamine, substance P, substance P-(1-7), substance P- (1-9), substance P-(5-11), substance P-(6-11), substance P-(8-11) and pempidine were from Sigma Chemicals Ltd. (Poole, Dorset). Substance P-(1-4) was from Calbiochem- Novabiochem (Beeston, Nottingham). Substance P-(1-2) and substance P-(10-11) were from Bachem (Saffron Walden, Essex). WIN 51,708, atropine and dihydro- [3-erythroidine were from Research Biochemicals Inter- national (Semat, St. Albans, Hertfordshire). All other reagents were from Fisons Scientific Ltd. (Loughborough, Leicestershire) and BDH Laboratory Supplies (Lutter- worth, Leicestershire).
RESULTS
Effect of substance P-(1-9), substance P-(6-11), substance P-(1-2) and substance P-(10-11) on dopamine outflow
As shown in Figure 1, at the concentrations of 0.1 and 1 nM substance P-(1-9) and substance P-(6-11) signifi- cantly modified the outflow of endogenous dopamine from rat striatal slices (F(6,65) = 16.1 and 11.4, P < 0.05, respectively). The maximum increases were seen at 0.1 nM for substance P-(1-9) and 1 nM for substance P- (6-11). Lower or higher concentrations of both fragments failed to modify endogenous dopamine outflow.
Substance P fragments and striatal endogenous dopamine outflow 521
500 -
oo
1
400
300 -
200-
100
-*-sP0-9) --II-SP(6-11) o Sl'0-2) A SP(IO=II)
I I [ [ [ I -11 -10 -9 -8 -7 -6
Log pcpfide concentration (M)
Fig. 1 Striatal slices were exposed to substance P-(1-9) (SP(1-9), and substance P-(6-11) (SP(6-11)) (0.01 nM-1 #M), or substance P-(1-2) (SP(1-2)) and substance P-(10-11) (SP(10-11)) (0.1 and 1 nM). Typical basal dopamine outflow was 5.4 _+ 0.3 pmol/ml/mg protein. Results are the mean + S.E.M of 6 determinations. *P < 0.05 vs. respective controls.
At the concentrations of 0.1 and 1 nM, the fragments substance P-(1-2) and substance P-(10-11) were devoid of activity (Fig. 1).
Effect of substance P-(1-9) and substance P-(6-11) on the dopamine overflow induced by KCI 25 mM
A 1 min exposure of slices to KC1 (25 mM) induced a sig- nificant increase in dopamine outflow (Table 1A). Administration of KC1 (25 mM) with each of the peptides (1 riM) led to increases in dopamine outflow compared to controls. The analysis of the effects of KC1 in the pres- ence of each of the two peptides showed a negative inter- action (F(1,32) = 5.16 and 5.67, P < 0.05, respectively), the effect of the combination on dopamine outflow being sig- nificantly less than the sum of the two intrinsic effects.
Table 1 Interaction of substance P fragments with potassium and an NK~ antagonist
Slices were incubated with A) KC125 mM or B) WIN 51,708 2.5 nM, in the absence or presence of substance P-(1-9) (SP(1-9)) or substance P-(6-11) (SP(6-11)) at a concentration of 1 nM, as described in Methods. Mean + S.E.M. of 6 determinations. *P < 0.05 vs. non-treated control.
Interaction of substance P-(1-9) and substance P-(6-11) with a tachykinin NK 1 receptor antagonist
The tachykinin NK 1 receptor antagonist WIN 51,708 (2.5 nM) had no intrinsic effect on the spontaneous outflow of endogenous dopamine (Table 1B). Exposure of slices to WIN 51,708 (2.5 nM) and the peptides (1 nM) produced different effects for the two fragments. The effect of sub- stance P-(6-11) ( lnM) on dopamine outflow was reversed by WIN 51,708, whereas the NK1 receptor antagonist had no effect on substance P-(1-9) induced dopamine overflow (F(1,44) = 31, P < 0.05, and 0.02, n.s, respectively).
Effect of substance P-(1-9) and substance P-(6-11) on dopamine outflow in the presence of muscarinic and nicotinic antagonists
The effects of 1 nM substance P-(1-9) and substance P- (6-11) were completely reversed by the muscarinic antagonist atropine (1 ~M) (F(1,28) = 30.5 for substance P-(1-9) and 13.3 for substance P-(6-11), P < 0.05), but were unaffected by the nicotinic antagonists dihydro-[~- erythroidine (0.5 glVI) (F(1,28) = 0.816, for substance P- (1-9) and 0.89, n.s, for substance P-(6-11) or pempidine (10 pM)(F(1,28) = 0.03 and 1.97, n.s, for substance P-(1-9) and substance P-(6-11) respectively) (Fig. 2). The cholin- ergic antagonists were devoid of intrinsic effects on dopamine outflow.
Interaction of substance P and its N- and C- terminal fragments
Figure 3 shows the effects of co-incubating slices with substance P (1 nM) and N- or C- terminal fragments at
Fig. 2 Striatal slices were exposed to 1 nM substance P-(1-9) (SP(1-9)) or substance P-(6-11) (SP(6-11)), in the presence of atropine (1 I~M), dihydro-13-erythroidine(DHE) (0.5 ~LM) or pempidine (Pemp) (10 ~LM). Results represent the means + S.E.M of 6 determinations. *P < 0.05 vs. non-treated controls.
300 -
~ 200
1o0
0
• no SP [] with SP
** ** T ** # T * *
i" ! i
I
J I I
& '7, '7, '7,
Fig. 3 Striatal slices were co-incubated with substance P(SP) (1 nM) and each of the substance P fragments, (1-9), (1-7), (1-4), (5-11), (6-11) and (8-11) (0.1 nM). Means + S.E.M of 6 determinations. *P < 0.05 vs. respective controls.
the concentration of 0.1 nM, i.e. a 10:1 ratio parent to metabolite. Substance P and the fragments separately elicited significant increases in dopamine outflow, but their combination revealed no potentiation or additivity. The interaction between the parent peptide and its frag- ments was negative in each case (F(1,28) = 53.9 and 23.6, P < 0.05 for substance P-(1-9) and substance P-(6-11) respectively; F(1,20) = 78.9, 9.27 and 28.3, P < 0.05 for SP(1-4), SP(5-11) and SP(8-11) respectively; F(1,20) = 11.53, P < 0.05, for SP(1-7)).
Striatal slices were also co-incubated with substance P (0.02 nM) and substance P-(1-7) (0.02 nM) or substance P-(6-11)(0.02nM), i.e. a 1:1 ratio parent to metabolite. Each peptide induced a small but significant increase in dopamine outflow (% controls: 127 _+ 9, 130 _+ 11 and 128 _+ 15, P < 0.05, for substance P, substance P-(1-7) and substance P-(6-11) respectively). When combined, a neg- ative interaction was observed again, the outflow from tissue exposed concomittantly to parent peptide and fragments not differing from control values (% controls): 98 + 6, F = 19.8, P < 0.05, for substance P and the N-ter- minal fragment, or 110 _+ 8, F = 8.5, P < 0.05 for substance P and its C-terminal fragment.
D I S C U S S I O N
The activity of substance P fragments has been docu- mented in a range of experimental models, from macrophage activation to antinociception, modulation of blood pressure or locomotor activity, z1°,3~-33 Various N- and C-terminal fragments have been studied, over a
range of concentrations, using various modes of adminis- tration. This heterogeneity of experimental conditions may, at least in part, be the reason why it is not clear yet which fragments are likely to play physiological roles in various processes, and how their presence may interfere with the signal mediated by the parent peptide.
The basal ganglia are rich in substance P, which is pre- sent in one of the main striatal efferent pathways, that projects to the substantia nigra) 3 Tissue concentrations of substance P metabolites such as substance P-(1-7) and substance P-(5-11) have been reported in the mes- encephalon and the striatum, where they represent 2-4% and 1-2% of the parent peptide levels, respec- tively. ~ Substance P-(1-7) is also produced after incuba- tion of the peptide with synaptosomal striatal or nigral membranes, 1,4 or after injection of substance P intrani- grally? 4 The other N-terminal species which appears to be a major form relative to other fragments is substance P-(1-9), detected in rat striatum during infusion of sub- stance p.29 In the same study, substance P-(6-11) was shown to be a very abundant C-terminal fragment. Internal fragments, e.g. substance P-(4-8), or substance P-(3-11) were minor species. Interestingly, a recent study by Kostel and Lunte 35 suggests that the latter is a major fragment.
We have shown significant modulatory effects with the substance P fragments (1-4), (1-7), (5-11) and (8-11) ill striatal slices, 25,26 and in the present study we extended our analysis to the major species recently reported. 29 Substance P-(1-9) and substance P-(6-11) showed a sim- ilar profile to that described previously with the other
Substance P fragments and striatal endogenous dopamine outflow 523
fragments, inasmuch as the dose-response curve for dopamine outflow was bell-shaped. Concentrations of 0.1 and 1 nM induced a significant increase. The effects of C- terminal fragments were sensitive to an NK~ antagonist, whereas the effects of N-terminal fragments were not. The maximum effect on dopamine outflow was compara- ble in amplitude to that induced by substance P-(1-7) and substance P-(5-1 1). 25 Neither substance P-(1-9) nor substance P-(6-1 1) potentiated potassium-induced over- flow. As shown by our studies in this preparation, this is unlikely to be due to the inability of dopamine tissue stores to respond to intense stimulation. 3° As with previ- ous observations, the effect on dopamine outflow involved a cholinergic link and activation of muscarinic receptors, 2z which ultimately modulate dopamine out- flOW,
It has been suggested that the effect of acetylcholine on striatal dopamine outflow reflects the balance between the excitatory effects mediated through mus- carinic M 1 receptors and the inhibitory effects due to M R receptor stimulation. 3~ Tachykinin-induced acetylcholine release in striatal slices appears to be linear. 37,3s Therefore, it could be suggested that concentrations of peptides higher than 1 nM lead to a large acetylcholine outflow and a predominant activation of M 2 receptors. This would abolish the increased dopamine release due to stimula- tion of M 1 receptors. Preliminary data with substance P and selective M 1 and M 2 antagonists confirm this hypoth- esis. The M1 antagonist pirenzepine reverses the increased dopamine outflow elicited by substance P (1 nM), whereas a concentration of 10 nM of substance P can induce an increased dopamine outflow in the pres- ence of the M 2 antagonist methoctramine (unpublished data). Therefore, the modulation by peptides of striatal dopamine outflow appears to be under the indirect per- missive influence of muscarinic receptors.
The previous observations and the present study lead somewhat unexpectedly to the conclusion that a series of fragments of substance P, representing the N- and C-ter- minal domains of the peptide, are intrinsically active at concentrations similar to those of the parent pepfide. This prompted us to investigate the activity of the two shortest N- and C-terminal sequences, substance P-(1-2) and substance P-(10-1 1), which could be generated for example by a dipeptidyl aminopeptidase 39 or neutral met- allo-endopeptidase. ],4° These fragments were inactive, thus confirming the specificity of the effects seen with the other fragments. As substance P-(1-4) and substance P-(8-1 1) were found to be active, 26 the implication is that the! loss of Lys3-Pro 4 and Phe s- Gly 9 or the reduced size of the peptides leads to loss of activity.
N- and C-terminal fragments appear to activate the same target, i.e. the striatal cholinergic cell, with compa- rable potencies, but the effects of the N-terminal flag-
ments could not be blocked with an NK~ antagonist. In our previous studies we showed that even very high con- centrations of NK~ antagonist fail to reverse the effects of N-terminal peptidesY However, the interpretation of the data obtained at high concentrations of antagonist is problematic, because of the intrinsic decrease in dopamine outflow induced by the antagonist. Should a reversal be observed, it could merely reflect an additivity of opposite effects and not true antagonism.
Several hypotheses could be put forward to explain the resistance of N-terminal fragments to the NK 1 antagonist. The N- and C-terminal fragments may activate two differ- ent receptors, one of which is not the NK] receptor. Although receptors for N-terminal fragments such as substance P-(1-7) have been described, 4~ their character- istics appear to be different from the receptors which mediate the effects of N-terminal fragments in our model. Alternatively, the N-terminal fragments could stimulate an isoform of the NK1 receptor which is insen- sitive to the non-peptide antagonist. Isoforms of the receptor have been reported. 42 However, a study of these isoforms in the striatum suggests that this structure con- tains only one population of sites, which correspond to the long isoform of the receptor. 43 Finally, it could be sug- gested that all fragments activate the same receptor, most probably the NK] type. This explanation would appear at first very unlikely, considering the low affinity of N-ter- minal fragments for NK~ sites reported in conventional binding assays. 44,45 The determination of the affinities of peptides in such studies is based on their ability to dis- place the radiolabelled ligand (often the parent peptide). In the case of septide, a C-terminal analog of substance P, differences of several orders of magnitude have been reported between the potency of the peptide in func- tional assays for NK2 receptors and the affinities calcu- lated in displacement studies. 4~,47 Septide has been shown to modulate striatal acetylcholine release with higher efficacy than substance p.4s
In a study on striatal radiolabelled dopamine release, septide and an NK 1 agonist displayed different sensitivity to an NK ~ antagonist. 22 Furthermore, NK] agonists can abolish the effects of septideY ,49 Whether septide-sensi- tive receptors exist is an open question, but the pharma- cology of this peptide highlights some of the unresolved questions concerning the interaction of tachykinins with their receptors. Recent analyses of.data generated using several tachykinin ligands, challenge the established views concerning the methodology of binding. 5° In par- ticular, as agonists may select different receptor confor- mations, it is suggested that affinities should be measured in homologous binding assays (i.e. using the radiolabelled form of the tested peptide) and :not using a displacement paradigm in heterologous binding assays. 5~ The flexible conformation of peptides may lead to modes
of activation of receptors which differ from the interac- tion described for more rigid ligands, such as cate- cholamines. Analysis of the NK 1 receptor by mutagenesis has shown that peptides bind to residues in both the extracellular and the t ransmembrane domains, whereas the binding of non-peptide antagonists is mainly to intra- helical residues. 52,53 Furthermore, it cannot be ruled out that changes in receptor conformation when the peptide agonist is bound and which lead to G-protein activation, resuk in poor or non-recognition by a non-peptide antag- onist. 54-56 However, our experimental model is unique, in that the activities of C- and N-terminal substance P frag- ments are comparable both in pattern and amplitude. The ability of substance P-(1-7) to down-regulate the NK 1 receptor ~z leaves open the question of the interac- tion of N-terminal fragments with this receptor. Preliminary data in our laboratory show that substance P-(1-7) can trigger internalization of NK 1 receptors.
Data on the half-life of substance P after release from nerve terminals are not available and the clearance kinet- ics of active substance P fragments remains to be deter- mined. It is likely that the parent peptide and its metabolites may transiently co-exist in the vicinity of the receptors. Therefore, we tested the effect of a concomi- tant exposure of the tissue to substance P and its frag- ments, at equimolar concentrations or using a ratio parent/metabolite which may be closer to physiological conditions, z A negative interaction was observed in all the combinations tested, which suggests that the frag- ments do not amplify the signal mediated by the parent peptide. In particular, for short fragments such as sub- stance P-(1-4) and substance P-(8-1 1), which induced a smaller increase in dopamine outflow than the parent peptide, the effect of substance P was reduced in ampli- tude during coincubation. Interactions between sub- stance P and its fragments could follow several patterns. It has been suggested that a fragment such as substance P-(1-7) may act as an endogenous antagonist for the par- ent peptide or C-terminal fragments. 28 However, in some models, such as the modulation of blood pressure, the N- terminal heptapeptide appears to be responsible for the effect of the parent peptide, as the activity of substance P is abolished if formation of substance P-(1-7) is pre- vented. 6 The negative interaction that we observed is unlikely to be due to a 'ceiling' effect which may be a consequence of the biphasic response, because similar results were obtained by co-infusion of near-threshold concentrations of peptides. The in vivo ratios of parent to metabolites after peptide release are unknown, but our data suggest that in spite of their intrinsic effects, the fragments may not amplify the effects of substance P.
The results obtained so far show that in the striatum a multitude of substance P fragments are potential neuro- modulators. Although some fragments differ only by one
aminoacid, e.g. substance P-(5-11) and substance P-(6-11), their interaction with receptors may be significantly differ- ent, for example in terms of desensitization. 5s The intrastri- atal infusion of substance P, or the observations we have reported after striatal injection of substance p,59 give some indication about the fragments formed in vivo. However, studies using high concentrations of exogenous peptide may lead to metabolic patterns which are not identical to those observed during physiological release of substance P. In the basal ganglia, as well as in other structures, the pres- ence and fate in the extracellular space of endogenous pep- tides and their active metabolites awaits elucidation with techniques such as liquid chromatography coupled to mass-spectrometry. 6° Finally, it must be borne in mind that in the central nervous system there may be regional differ- ences between patterns of metabolism for the same pep- tide? 1 The numerous observations showing that peptide metabolites are active also raise the question whether the real neurotransmitter is the peptide or its metabolite(s). We have shown that the intrastriatal disappearance of sub- stance P is extremely rapid. 59 Furthermore, ongoing experi- ments suggest that the N-terminal species have a longer half-life in vivo in the striatum than C-terminal fragments. It cannot be ruled out that in some circumstances active peptide metabolites with longer half-lives may have impor- tant biological effects.
REFERENCES
1. Matsas R, Fulcher IS, Kenny AJ, Turner AJ. Substance P and [Leu]enkephalin are hydrolyzed by an enzyme in pig caudate synaptic membranes that is identical with the endopeptidase of kidney microvilli. Proc Natl Acad Sci USA 1983; 80:3111-3115.
2. Skidgel R, Engelbrecht S, Johnson AR, Erd6s ED. Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase. Peptides 1982; 3: 851-85Z
3. Blumberg S, Teichberg VI, Charli JL et al. Cleavage of substance P to an N-terminal tetrapeptide and a C-terminal heptapeptide by a post-proline cleaving enzyme from bovine brain. Brain Res 1980; 192: 477-486.
4. Oblin & Danse MJ, Zivkovic B. Metalloendopeptidase (EC 3.4.24.11) but not angiotensin converting enzyme is involved in the inactivation of substance P by synaptic membranes of the rat substantia nigra. Life Sci 1989; 44: 1467-1474.
5. Sakurada T, Le Gr~ves P, Stewart J, Terenius L Measurement of substance P metabolites in rat CNS. J Neurochem 1985; 44: 718-722.
6. Hall ME, Miley E Stewart JM. The role of enzymatic processing in the biological actions of substance R Peptides 1989; 10: 895-901.
Z Herrera-Marschitz M, Terenius L, Sakurada T et al. The substance P(l-7) fragment is a potent modulator of substance P actions in the brain. Brain Res 1990; 521:316-320.
8. Sakurada T, Tan-No K, Yamada T et al. N-terminal substance P fragments inhibit the spinally induced, NK 1 receptor mediated behavioural responses in mice. Life Sci 1990; 47:109-113.
9. Skilling SR, Smullin DH, Larson AA. Differential effects of C- and N-terminal substance P metabolites on the release of amino acid neurotransmitters from the spinal cord:potential role in nociception. J Neurosci 1990; 10: 1309-1318.
Substance P fragments and striatal endogenous dopamine outflow 525
10. Hall ME, Stewart JM. The substance P fragment SP(1-7) stimulates motor behavior and nigral dopamine release. Pharmac Biochem Behav 1991; 41 : 75-78.
11. Bolam JP, Somogyi P, Takagi H e t al. Localization of substance P-like immunoreactivity in neurons and nerve terminals in the neostriatum of the rat: a correlated light and electron microscopic study. J Neurocytol 1983; 12: 325-344.
12. Gerfen CR, Young WS3rd. Distribution of striatonigral and striatopallidal peptidergic neurons in both patch and matrix compartments: an in situ hybridization histochemistry and fluorescent retrograde tracing study. Brain Res 1988; 460: 161-16Z
13. Gerfen CR. The neostriatal mosaic: multiple levels of compartmental organization in the basal ganglia. Ann Rev Neurosci 1992; 15: 285-320.
14. Humpel C, Saria A. Effects of GABA and L-glutamic acid on the potassium-evoked in vitro release of substance P and neurokinin A-like immunoreactivities are different in the rat striatum and substantia nigra. Neurosci Lett 1989; 105: 159-163.
15. Lindefors N, Brodin E, Tossman U et al. Tissue levels and in vivo release of tachyMnins and GABA in striatum and substantia nigra of rat brain after unilateral striatal dopamine denervation. Exp Brain Res 1989; 74: 527-534.
16. Dam TV, Martinelli B, Quirion R. Autoradiographic distribution of brain neurokinin-1/substance P receptors using a highly selective ligand [3H]-[Sarg,Met(O2)n]-substance P. Brain Res 1990; 531: 333-33Z
17. Quirion R, Shults CW, Moody TW et al. Autoradiographic distribution of substance P receptors in rat central nervous system. Nat, 1983; 303: 714-716.
18. Rothman RB, Herkenham M, Pert C et al. Visualization of rat brain receptors for the neuropeptide substance R Brain Res 1984; 309: 47-54.
19. Mussap CJ, Geraghty DP, Burcher E. Tachykinin receptors: a radioligand binding perspective. J Neurochem 1993; 60: 1987-2009.
20. Baruch P, Artaud E Godeheu G e t al. Substance P and neurokinin A regulate by different mechanisms dopamine release from dendrites and nerve terminals of the nigrostriatal dopamine neurons. Neuroscience 1988; 25: 889-898.
21. Reid MS, Herrera-Marschitz M, H6kfelt T et al. Effects of intranigral substance P and neurokinin A on striatal dopamine release - I. interactions with substance P antagonists. Neuroscience 1990; 36: 643-658.
22. Gauchy C, Desban M, Glowinski J, Kernel ML. Distinct regulations by septide and the neurokinin-1 tachykinin receptor agonist [Prog]substance P of the N-methyl-D- aspartate-evoked release of dopamine in striosome- and matrix- enriched areas of the rat striatum. Neuroscience 1996; 73: 929-939.
23. Petit F, Glowinski J. Stirnulatory effect of substance P on the spontaneous release of newly synthesized [3H]dopamine from rat striatal slices: a tetrodotoxin-sensitive process. Neuropharmacology 1986; 25:1015-1021.
24. Tremblay L, Kernel ML, Desban Me t al. Distinct presynaptic control of dopamine release in striosomal- and matrix-enriched areas of the rat striatum by selective agonists of NK1, NK2 and NK3 tachykinin receptors. Proc Natl Acad Sci USA 1992; 89: 11214-11218.
25. Khan S, Brooks N, Whelpton R, Michael-Titus AT. Substance P- (1-7) and substance P-(5-11) locally modulate dopamine release in rat striatum. Eur J Pharmacol 1995; 282: 229-233.
26. Khan S, Whelpton R, Michael-Titus AT. Evidence for modulatory effects of substance P fragments (1-4) and (8-11) on
endogenous dopamine outflow in rat striatal slices. Neurosci Lett 1996; 205: 33-36.
2Z Khan S, Grogan E, Whelpton R, Michael-Titus AT. N- and C- terminal substance P fragments modulate striatal dopamine outflow through a cholinergic link mediated by muscarinic receptors. Neuroscience 1996; 73: 919-92Z
28. Reid MS, Herrera-Marschitz M, Terenius L, Ungerstedt U. Intranigral substance P modulation of striatal dopamine: interaction with N-terminal and C-terminal substance P fragments. Brain Res 1990; 526: 228-234.
29. kndr6n PE, Caprioli RM. In vivo metabolism of substance P in rat striatum utilizing microdialysis/liquid chromatography/micro-electrospray mass spectrometry. J Mass Spect 1995; 30: 817-824.
30. Khan S, Whelpton R, Michael-Titus AT. Endogenous dopmnine outflow from rat striatal slices in static and dynamic conditions. Neurosci Res Commun 1995; 16: 145-153.
31. Bar-Shavit Z, Goldman R, Stabinsky Y, et al. EnhamLcement of phagocytosis - a newly found activity of substance P residing in its N-terminal tetrapeptide sequence. Biochem Biophys Res Commun 1980; 94: 1445-1451.
32. Hall ME, Miley FB, Stewart JM. Modulation of blood pressure by substance P: opposite effects of N-terminal and C-terminal fragments on anaesthetized rats. Life Sci 1987; 40: 1909-1914.
33. Cridland RA, Henry JL. N- and C-terminal SP-fragments of substance P: spinal effects in the rat tail flick test. Brain Res Bull 1988; 20: 429-432.
34. Humpel C, Saria A, Regoli D. Injection of tachykinins and selective neurokinin receptor ligands into the substantia nigra reticulata increases striatal dopamine and 5-hydroxytryptamine metabolism. Eur J Pharm 1991; 195:107-114.
35. Kostel KL, Lunte SM. Evaluation of capillary electrophoresis with post-column derivatization and laser-induced fluorescence detection for the determination of substance P and its metabolites. J Chromatogr 1997; 695: 27-38.
36. Xu M, Mizobe F, Yamamoto T, Kato T. Differential effect of M1- and M2-muscarinic drugs on striatal dopamine release and metabolism in freely moving rats. Brain Res 1989; 495: 232-242.
3Z Arenas E, Alberch J, Perez-Navarro E et al. Neurokinin receptors differentially mediate endogenous acetylcholine release evoked by tachyldnins in the neostriatum. J Neurosci 1991; 11 : 2332-2338.
38. Emonds-Alt X, Doutremepuich J-D, Heaulme Me t al. In vitro and in vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK 1 receptor antagonist. Eur J Pharmacol 1993; 250: 403-413.
39. Kato T, Nagatsu T, Fukasawa K et al. Successive cleavage of N- terminal ArgO-Pro 2 and Lys3-Pro 4 from substance P but no release of Argl-Pro 2 from bradykinin, by X-Pro dipeptidyl- aminopeptidase. Biochim Biophys Acta I978; 525:417-422.
40. Matsas R, Kenny AJ, Turner AJ. The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachyldnins and their analogues, by endopeptidase 24.11. Biochem J 1984; 223: 433-440.
41. Igwe OJ, Kim DC, Seybold VS, Larson AA. Specific binding of substance P aminoterminal heptapeptide [SP(I-7)] to mouse brain and spinal cord membranes. J Neurosci 1990; 10: 3653-3663.
42. Kage R, Leeman SE, Boyd ND. Biochemical characterization of two different forms of the substance P receptor in rat submaxillary gland. J Neurochem 1993; 60: 347-351.
43. Mantyh PW, Rogers SD, Ghilardi JR et al. Differential expression of two isoforms of the neurokinin-1 (substance P) receptor in vivo. Brain Res 1996; 719: 8-13.
44. Ingi T, Kita]ima Y, Minamitake Y, Nakanishi S. Characterization of ligand-binding properties and selectivities of three rat tachykinin receptors by transfection and functional expression of their cloned cDNAs in mammalian cells. J Pharm exp Ther 1991; 238: 968-975.
45. Cascieri MA, Huang R-R C, Fong TM et al. Determination of the amino acid residues in substance P conferring selectivity and specificity for the rat neurokinin receptors. Mol Pharmacol 1992; 41: 1096-1099.
46. Pradier L, Menager J, Le Guern Je t al. Sepfide: an agonist for the NK1 receptor acting at a site distinct from substance P. Mol Pharmacol 1994; 45: 287-293.
47. Sagan S, Chassaing G, Pradier L, Lavielle S. Tachykinin peptides affect differently the second messenger pathways after binding to CHO-expressed human NK-1 receptors. J Pharm exp Ther 1996; 276: 1039-1048.
48. Steinberg R, Rodier D, Souilhac Je t al. Pharmacological characterization of tachykinin receptors controlling acetylcholine release from rat striatum: an in vivo microdialysis study. J Neurochem 1995; 65: 2543-2548.
49. Torrens Y, Saffroy M, GlowinskiJ, Beaujouan JC. Substance P(6-11) and natural tachykinins interact with septide-sensitive tachykinin receptors coupled to a phospholipase C in the rat urinary bladder. Neuropeptides 1997; 31:243-251.
50. Maggi CA, Schwartz TW. The dual nature of the tachykinin NK1 receptor. TIPS 1997; 18: 351-355.
51. Hastrup H, Schwartz TIN. Septide and neurokinin A are high- affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis. FEBS Lett 1996; 399: 264-266.
52. Fong T, Huang CR, Yu H et al. Mutational analysis of neurokinin receptor function. CanJ Physiol Pharmacol 1995; 73: 860-865.
53. Turcatti G, Zoffmann S, Lowe JA 3rd et al. Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands. J Biol Chem 1997; 272: 21167-21175.
54. Rosenkilde MM, Cahir M, Gether U et al. Mutations along transmembrane segments II of the NK-1 receptor affect substance P competition with non-peptide antagonists but not substance P binding. J Biol Chem 1994; 269: 28160-28164.
55. Schwartz TW. Locating ligand-binding sites in 7TM receptors by protein engineering. Curr Biol 1994; 5: 434-444.
56. Schwartz TW, Gether U, Schambye HT, Hjorth SA. Molecular mechanism of action of non-peptide ligands for peptide receptors. Curr Pharm Design 1995; 1: 325-342.
57. Yukhananov RY, Larson AA. An N-terminal fragment of substance P, substance P(1-7), down-regulates neurokinin-1 binding in the mouse spinal cord. Neurosci Lett 1994; 178: 163-166.
58. Tsch6pe C, Jost N, Unger T, Culman J. Central cardiovascular and behavioral effects of carboxy- and amino-terminal fragments of substance P in conscious rats. Brain Res 1995; 690: 15-24.
59. Michael-Titus AT, Whelpton R, Stephens SM, Yau KW. Metabolism of substance P in rat striatum studied using a combination of high performance liquid chromatography (HPLC) and capillary electrophoresis. Br J Pharmacol 1997; 122: 79P.
60. Emmett MR, Andrdn PE, Caprioli RM. Specific molecular mass detection of endogenously released neuropeptides using in vivo microdialysis/mass spectrometry. J Neurosci Meth 1995; 62: 141-147.
61. Davis TP, Gillespie TJ, Konings PNM. Specificity of neurotensin metabolism by regional rat brain slices. J Neurochem 1992; 58: 608-61Z
