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Dopamine receptors mediate alterations in striato-nigral dynorphin and substance p pathways
~~ro~~T~acoia~y Vol. 26, No. 9, pp. 1295-1302,1987 Printed in Great Britain. All rights reserved
~28-3~8/87 $3.00+ 0.00 Copyright Q 1987Pergamon Journals Ltd
DOPAMINE RECEPTORS MEDIATE ALTERATIONS STRIATO-NICRAL DYNORPHIN AND SUBSTANCE P PATHWAYS
IN
INGRID NYLANDER and L. H. TERENIUS Department of Pharmacology, Uppsala University, Box 591, S-751 24 Uppsala, Sweden (Accepted 9 March 1987)
Summary-Peptides derived from prodynorphin, dynorphin A and B, (mu)-enkephalin and (~u~nkephaiyl-Arg, as well as substance P, were measured in substantia nigra, striatum and globus pallidus, after subacute (5 doses at 6 hr intervals) treatment of rats with a number of dopamine receptor agonists and antagonists. Drugs selective for the dopamine D, and D, receptors, respectively, as well as unselective drugs were used. In the substantia nigra, levels of immunoreactive dynorphin A and dynorphin B were increased after treatment with a D,-antagonist (sulpiride) and a D,-agonist (SKF 38393), while a D,-antagonist (SCH 23390) reduced levels. The mixed D, and D, antagonist cis-fluwnthixol reduced only the level of dynorphin A. A corresponding increase of the levels-of (Leu)enkephalin in the nigra was found after treatment with sulpiride. In contrast to dynorphin peptides, the levels of (Leu)enkephalyi-Arg were markedly increased after both D,- and Dz (LY 17155.5)-stimulation. Substance P tended to be reduced after D,-stimulation and treatment with all the dopamine antagonists; the reduction was significant with sulpiride and c~-flu~nthixol. Levels of peptides in striatum and globus pallidus were generally affected in the same direction as levels in the nigra. The results in this study present further evidence that dopamine receptor agents affect dynorphin peptides and substance P, differentially. Effects on (Leu)enkephalin and (Leu)enkephalyl-Arg only partly paralleled the effects on levels of dynorphin. Thus, the D, and D, receptors differentially affect levels of different products of prodynorphin, that is, seem to affect certain steps of the processing of prodynorphin selectively. Key words: prodynorphin peptides, substance P, striate-nigh antagonists.
The opioid peptides deriving from prodynorphin, i.e. the dynorphin peptides (Goldstein, Fischli, Lowney, Hunkapiller and Hood, 1981; Kakidani, Furutani, Takahashi, Noda, Morimoto, Hirose, Asai, Inayama, Nakanishi and Numa, 1982), as well as substance P
and other protachykinin peptides, have been shown to be present in high levels in the substantia nigra and the basal ganglia (Brownstein, Mroz, Tappaz and Leeman, 1977; Christensson-Nylander and Terenius, 1985; Dores, Lewis, Khachaturian, Watson and Akil, 1985; Gale, Hong and Guidotti, 1977; Haber and Watson, 1985; Hong, Yang and Costa, 1978; Kanazawa, Emson and Cuello, 1977; Maysinger, Hollt, Seizinger, Mehraein, Pasi and Herz, 1982; Weber, Roth and Barchas, 1982). These two peptide systems are expressed in separate but anatomically overlapping striato-nigral neurones (Brownstein et al., 1977; Christensson-Nylander, Herrera-Marschitz, Staines, Hdkfelt, Terenius, Ungerstedt, Cuello, Oertel and Goldstein, 1986; Gale, et al., 1977; McLean, Bannon, Zamir and Pert, 1985; Vincent, HBkfelt, Christensson and Terenius, 1982a, b) and possibly also in st~ato-pallidal (Staines, Nagy, Vincent and 1980) and/or pallidonigral pathways Fibiger, (Brownstein et al., 1977; Kanazawa et al., 1977). The N.P. 2619-D
pathways, dopamine agonists, dopamine
interactions between these peptidergic pathways and the nigro-striatal dopamine pathway are at present being examined. For instance, several investigators have reported alterations in levels of substance P when different dopamine agonists and antagonists were administered to rats. In general, reduced levels of substance P in the nigra were found after repeated administration of different dopamine antagonists, which was thought to reflect an increased activity (increased release) in striato-nigral substance P neurones as a consequence of the blockade of dopamine receptors (Hanson, Alphis, Wolf, Levine and Lovenberg, 1981; Hong, Yang, Racagni and Costa, 1977). With the agonists, a selective D, receptor agonist (SKF 38393) also reduced substance P in the nigra, whereas a selective D, agonist (RU 24926; Nn-propyl-di-beta-(3-hydroxyphenyl)ethylamine hy drochloride) gave increased levels, indicating that the D, and Dz receptors, respectively, are differently coupled, functionally (Sonsalla, Gibb and Hanson, 1984). The indirect dopamine receptor agonist, methamphet~ine also enhanced levels of substance P in the nigra (Ritter, Schmidt, Gibb and Hanson, 1984). Much less information is available concerning the interaction between the dopamine and dynorphin
INGRID NYLANDER and
1296
L. H.
TERENIUS
DYN PROENKEPHALIN
DYN
6
Q NEOENDORPHIN
DYNORPHIN
[._NEO
29 I
I
A
DYNORPHIN
B
I
pr-pq
pq
I
l-8)
A(
LA
LA
I
32
I LE
LE
I
I
I
LA
LE 0
Fig. 1. Possible known opioid peptide fragments of prodynorphin. u-NE0 = alpha-neoendorphin, /?-NE0 = beta-neoendorphin, A(l-8) = dynorphin A&8), LA = (Leu)enkephalyl-Arg, LE = (Leu) enkephalin.
systems. Discordant results have been obtained after chronic treatment with haloperidol; unchanged levels of dynorphin in the nigra were reported after 10 days of treatment (Nylander and Terenius, 1986), while
treatment for 21 days increased levels (Quirion, Gaudreau, Martel, St-Pierre and Zamir, 1985). Previous studies have revealed evidence for an interaction between dynorphin and dopamine. Dynorphin peptides, injected unilaterally into the substantia nigra, (1) inhibit release of dopamine in the striatum (Christensson-Nylander et al., 1986; Herrera-Marschitz, Christensson-Nylander, Sharp, Staines, Hokfelt, Terenius and Ungerstedt, 1986) and (2) produce contralateral turning (HerreraMarschitz, Hiikfelt, Ungerstedt, Terenius and Goldstein, 1984; Herrera-Marschitz et al., 1986). Since this latter effect is also recorded in animals lesioned with 6-hydroxydopamine (6-OHDA) it may involve other pathways than the nigro-striatal dopamine (DA) pathway. In this study, a more thorough examination has been performed to analyze the interaction between dynorphin and dopamine systems. For comparison, substance P was also measured. Both dopamine agonists and antagonists have been used, some with selective actions on the D, and D, dopamine receptor, respectively, some more unselective. The drugs were given in 5 doses every 6 hr. Dynorphin peptides (dynorphin A and dynorphin B) and substance P were measured with radioimmunoassays in the substantia nigra, globus pallidus and striatum. In addition, (Leu)enkephalyl-Arg and (Leu)-enkephalin were measured in substantia nigra and the levels compared to levels of dynorphin. These enkephalins are likely to be processing products of prodynorphin in this area of the CNS (Christensson-Nylander et al., 1986; Zamir, Palkovitz, Weber, Mezey and Brownstein, 1984) (Fig. 1). METHODS
Drug treatment
Male Sprague-Dawley
rats (Anticimex,
Sweden)
weighing 170-190 g were used. The drugs were injected intraperitoneally every 6 hr on 5 occasions. Six hours after the last dose the animals were decapitated. Control animals received equivalent volume of saline. Since the purpose of this study was to test possible effects on levels of peptides of different kinds of substances acting on dopamine receptors, only one dose of each drug was used, chosen according to information in the literature. Seven groups of rats were used, with 8-10 rat in each group, receiving one of the following drugs: apomorphine (2 mg/kg), cis-flupenthixol (0.1 mg/kg) (Nielsen, Pedersen, Nymark, Franck, Boeck, Fjalland and Christensen, 1973), LY 171555 (0.5 mg/kg) (Fuller, Hemrick-Luecke, Wong, Pearson, Threlkeld and Hynes, 1983; Titus, Kornfeld, Jones, Clemens, Smalstig, Fuller, Hahn, Hynes, Mason, Wong and ‘Foreman, 1983; Tsuruta, Frey, Grewe, Cote, Eskay and Kebabian, 1981), SKF 38393 (15 mg/kg) (Setler, Sarau, Zirkle and Saunders, 1978; Sonsalla et al., 1984), SCH 23390 (0.1 mg/kg) (Hyttel, 1983; Iorio, Barnett, Leitz, Houser and Korduba, 1983), sulpiride (100 mg/kg) (Oblin, Zivkovic and Bartholini, 1984) or saline. Extraction
of tissue and separation
of extract
After decapitation, the substantia nigra, globus pallidus and striatum were rapidly dissected out on an ice-cold plate, frozen and kept at -90°C until processed further. Extraction was performed with 1 M acetic acid, containing 0.01% mercaptoethanol, at 95°C for 5 min. The tissues were homogenized by sonication, heated another 5 min at 95”C, again cooled before centrifugation for 15 min in a Beckman Microfuge@. The supematants were collected and transferred to ion exchange columns (SP Sephadex C-25, Pharmacia, Sweden) for separation and purification of the peptides. The procedure is described in detail elsewhere (Bergstrom, Christensson, Folkesson, Stenstrom and Terenius, 1983). In brief, the columns were eluted stepwise with a series of buffers containing pyridine and formic acid of increasing ion strength. (Leu)Enkephalin and
Dopaminergic drugs affect dynorphin and substance P (Leu)enkephalyl-Arg were eluted in separate fractions, while dynorphin A, B and substance P eluted together in one fraction. The fractions were dried in a vacuum centrifuge and redissolved in an appropriate volume of methanol/O. 1 M HCl (1: 1) before measurement of each peptide with radioimmunoassay. Radioimmunoassays
0.9% NaCI. Sulpiride France) was dissolved acetic acid. The solution with NaOH. All drugs volumes.
1297 (Delagrange Laboratories, by adding a few drops of was diluted and neutralized were injected in equivalent
Statistics
Analysis of differences between means was by the Student’s two-tailed t-test. In addition, analysis of variance was tested using the General Linear Models procedure, GLM (SAS Institute, Cary, North Carolina). Differences were considered significant at
Cross-reactivity studies were performed with each antiserum, using a variety of peptides with sequence homologies to the peptide to be measured. Only P < 0.05. cross-reacting peptides (> 0.1%) are listed below. (Leu)enkephalyl-Arg and substance P. [‘251]labelled (Leu)enkephalyl-Arg and Tyr*-substance P reRESULTS spectively, were used as tracer. Iodination was performed using chloramine T and the labelled peptide The effects of subacute treatment with different was purified with high pressure liquid chrodrugs on levels of neuropeptides are presented for matography (HPLC) (on reversed phase column, each area of the CNS studied separately. The followlinear gradient of acetonitrile and H,O in 0.04% ing drugs were used: SKF 38393 (15 mg/kg), a selectrifluoroacetic acid). Incubation with antibody and tive D, agonist; LY 171555 (0.5 mg/kg) a selective Dz sample or standard peptide was performed overnight. agonist; apomorphine (2 mg/kg) an unselective dopaFree and antibody-bound peptides were separated by minergic agonist, although mainly acting on D, reincubation with dextrane-coated charcoal for 10 min. ceptors; SCH 23390 (0.1 mg/kg) a D, antagonist; The (Leu)enkephalyl-Arg antiserum showed 10% sulpiride (100 mg/kg), a D, antagonist and ciscross-reactivity with (Met)enkephalyl-Arg, 1% with flupenthixol (0.1 mg/kg) an antagonist with activity (Met)enkephalyl-Arg-Phe, 0.5% with dynorphin A on both D,- and D,-receptors. (l-8) and alpha-neoendorphin. Fragments of substance P (3-11) and (4-11) cross-reacted 100% with Substantia nigra (Figs 2 and 3) the antiserum for substance P. (Leu)Enkephalin (Fig. 2). The only drug that (Leu)Enkephalin. [3H]labelled peptide (Amersham, affected levels of this peptide was sulpiride, which England) was used as the tracer. Otherwise the gave an elevation. procedure was the same as for (Leu)enkephalyl-Arg Dynorphin A and B (Fig. 3). Increased levels were and substance P. The antiserum recognized Argmeasured after treatment with SKF 38393, whereas (Leu)enkephahn to 100% and (Met)enkephalin to no change was observed after LY 171555 or apo3%. morphine. The D, antagonist, sulpiride caused a Dynorphin A and B. [‘251]labelled peptides were marked increase in the dynorphins, contrasting the prepared according to the procedure used for subdecrease observed after treatment with the stance P and (Leu)enkephalyl-Arg. Dynorphin A D,-antagonist SCH 23390. Also, cis-flupenthixol antiserum showed 100% cross-reactivity with dynorgave a marked reduction in levels of dynorphin. phin A (9-17), while no cross-reacting peptides were (Leu)Enkephafyl-Arg (Fig. 3). Levels of this pepfound with dynorphin B antiserum. Radiotide significantly increased after treatment with both immunoassay was performed as for the other pepSKF 38393 and LY 171555. No other drug caused tides with the exception that separation of free and significant changes. antibody-bound peptide occurred by addition of preSubstance P (Fig. 3). The D, agonist SKF 38393 cipitate of normal rabbit serum and sheep anti-rabbit caused a decrease, as did all the antagonists; the serum. The procedure has previously been described decrease caused by sulpiride and cis-flupenthixol in detail (Christensson-Nylander, Nyberg, Ragreached statistical significance. narsson and Terenius, 1985). Drugs
Globus pallidus (Fig. 4)
Apomorphine HCI (Apoteksbolaget, Sweden), cisflupenthixol (Lundbeck, Denmark) LY 171555 [(rrans-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-lH) (or 2H)-pyrazolo(3,4-g)quinoline)] (Lilly, U.S.A.), SKF 38393 HBr [(2,3,4,5,-tetrahydro-7,8-dihydroxy-lphenyl-lH-3-benzazepine) Smith, Kline and French, U.S.A.] and SCH 23390 [(R)-( +)-8-chloro-2,3,4,5tetrahydro-3-methyl-5-phenyl-lH-3-benzazepine-7-ol hemimaleate) Schering, U.S.A.] were dissolved in
In general, levels of prodynorphin-derived peptides were affected in a similar way to those in the substantia, nigra. Dynorphin A and B. The dopaminergic agonists had fairly small effects. With sulpiride, marked increases in both dynorphins were observed. However, they were significantly reduced by cis-flupenthixol, an effect which is in contrast to that observed in substantia nigra.
INGRID NYLANDER
1298 pm01
/g
and L. H. TERENWS
Antagonists
Agonists
’ DI ’ D, ’ D ’
Ic
T
T
T
’ ‘JI I Dz I IJ I
a
_
Leu - Enk
Fig. 2. Immunoreactive levels of (Leu)enkephalin (mean values SEM) in rat substantia nigra expressed = saline, as pmol/g tissue, after tsatment with substances acting on dopamine receptors. D, agonist =SKF 38393, D, [7 = dopamine agonists, w = dopamine antagonists. C =control; antagonist = SCH 23390; D, agonist = LY 171555, D, antagonist = sulpiride; D agonist = apomorphine, unselective action on dopamine receptors. Leuwith D antagonist = cis-flupenthixol enk = (Leu)enkephalin. *P < 0.05, (Students’s two-tailed t-test).
(Leu)Enkephafyl-Arg. Levels of this peptide were markedly increased by the two agonists, SKF 38393 and LY 171555. No other significant effects were recorded. Substance P. Levels were not significantly affected.
pmoledg
AGONISTS
ANTAGONISTS
’ 4’ 0; D ’ Lcu -Arg
Striatum (Fig. 5) Dynorphin A and B. Levels were affected by the various treatments; SKF 38393 gave an increase whereas LY 171555 gave a significant decrease. The D, antagonist, sulpiride markedly increased levels of dynorphin. (Leu)Enkephalyl-Arg. As in the other structures investigated, SKF 38393 and LY 171555 markedly increased levels. No other treatment produced significant effects. Substance P. Only sulpiride produced significant effects, a marked reduction. Other treatments left levels essentially unchanged.
Dyn 6
200
150
Dyn A
DISCUSSION
Probes for dopamine receptor types
The dopamine receptors can be subdivided into D, and D2 types, using biochemical and pharmacological criteria (Gower and Marriott, 1982; Kebabian and Calne, 1979; Seeman, 1981). For instance, the D, receptor activates adenylate cyclase, while the D, receptor is either not coupled or inactivates the enzyme. A number of substances with selective actions on the D, and DZ receptor, respectively, have been synthesized. Based on information in the literature, the probes used in this investigation act mostly on one type of receptor and have negligible actions on the other: SKF 38393 has D, agonistic actions (Setler et al., 1978; Sonsalla et al., 1984), and SCH 23390 inhibits this receptor (Hyttel, 1983; Iorio et al., 1983). The drug LY 171555 stimulates the D, receptor (Fuller et al., 1983; Titus et al., 1983; Tsuruta et al.,
100 I
.h iu
SP
600 ZU-
Fig. 3. Immunoreactive levels of (Leu)enkephalyl-Arg, dynorphin A, dynorphin B and substance P (mean values SEM) in the substantia nigra of the rat, expressed as pmol/g tissue, after treatment with substances acting on dopamine receptors. Leu-Arg = (Leu)enkephalyl-Arg, Dyn A = dynorphin A, Dyn B = dynorphin B, SP = substance P. See also legend to Fig. 2. *P ~0.05, **P ~0.01, ***P < 0.001 (Student’s two-tailed f-test). Analysis of variance confirmed the effects of treatment; P < 0.001 for dynorphins A and B and (Leu)enkephalyl-Arg, P < 0.05 for substance P.
Dopaminergic drugs affect dynorphin and substance P
pmoL?s/g rc’
AGONISTS
ANTAGONISTS
‘D&‘D
‘D,‘D,‘D’
1299
pmolcs /g AGONtSTS ‘Dl’y2-D’
rvw
Lw -Arg
Lcu -Arg
Dyn B
Dyn B
Dyn A
Dyn A 15
flh 10 = ka
Fig. .4. Immunoreactive levels of (Leu)cnkephalyl-Arg, dynorphin A, dynorphin B. and substance P in globus palbdus, see further legend to Figs 2 and 3. Analysis of variance confirmed a treatment effect for dynorphins A and
B and (Leu)enkephalyl-Acg (P c 0.00I ).
1981) and sulpiride is a D, antagonist (Hyttel, Larsen, Christensen and Arnt, 1985; Oblin et al., 1984)
Apomorphine and cis-flupenthixol are an unselective dopamine agonist and antagonist, respectively (Arnt and Hyttel, 1985; Herrera-Marschitz and Ungerstedt, 1984b; Hyttel ef al., 1985 and Nielsen ef al., 1973). The purpose of this study was to test the effect of such agents on several peptides in three areas of the brain. To limit the chemical analyses to reasonable proportions, only one dose of each substance was chosen, instead of the ideal experimental protocol with several doses. Functional studies, performed by measuring the rotational response of dopamine agonists, suggest that the two dopamine receptors are associated with different striatal efferents; apomorphine induces rotation by an action mainly on D, receptors through striatal efferents running through the globus pallidus to the substantia nigra, while pergolide induces rotation through D2 receptors acting through other striatal efferent pathways (Arnt and Hyttel, 1985; Herrera-Marschitz and Ungerstedt, 1984a; Herrera-
ANTAGONISTS
.‘:zz .~:~ ..;i.. ..ii .: : ,; ,_: : ;: ::: :, j:. .::
T
l
-
1
Fig. 5. Immunoreactive levels of (Leu)enkephalyl-Arg, dynorphin A, dynorphin B and substance P in the striatum (see further legend to Figs 2 and 3). Analysis of variance confirmed a treatment effect for dynorphins A and B (P -CO.OOl), (Leu)enkephalytArg and substance P (P < 0.01).
Marschitz and Ungerstedt, 1984b). Consequently, the striatum, globus pallidus and substantia nigra were included in this study. Prodynorphin-related peptides in the substmtia nigra
Dynorphin peptides are present in cell bodies in the striatum and in nerve terminals in the zona rcticulata of substantia nigra (Vincent et al., 1982b). The levels of these peptides in the substantia nigra are high compared to other brain areas. In addition, high levels of (Leu)enkephalin are found in this area: whereas other areas of the brain have a 4: 1 ratio of (Met)enkephalin: (Leu)enkephalin, this ratio is 1: 2 in substantia nigra. (Christensson-Nylander et al., 1986). (Met)-Enkephalin is mainly present in neurones within the zona compacta. Cross-reaction of (Met)enkephalin with the (Leu)enkephalin antiserum is only 3% and HPLC has confirmed that most of the immunoreactivity relates to (Leu)enkephalin (Christensson-Nylander et al., 1986). (Lcu)Enkephalyl-Arg is present as the N-terminus in all peptides derived from prodynorphin and is
INGRID NYLANDER
1300
unique to this precursor. (Met)-Enkephalyl-Arg, a peptide present in proenkephalin, cross-reacted 10% in the radioimmunoassay for (Leu)enkephalyl-Arg. However, HPLC experiments have shown that the immunoreactivity measured is indeed (Leu)enkephalyl-Arg (Christensson-Nylander et al., 1985). Dopamine-peptide
interactions
Levels of dynorphin in the substantia nigra were increased by a selective D, agonist and decreased by a D, antagonist. An increase in levels of dynorphin in the nigra were also detected after D, inhibition. Unfortunately, tissue levels of peptides are very imperfect measures of turn-over; for example, increased levels may be due to increased biosynthesis, but may also depend on reduced release. Therefore the present data is hard to evaluate in terms of activation/ deactivation of the peptide pathways. An interaction between the two dopamine receptors has been proposed (Onali, Olianas and Gessa, 1984; Rosengarten, Schweitzer and Friedhoff, 1983; Sailer and Salama, 1985) and inhibition of the D, receptor might cause an imbalance of D,/D, responses, resulting in a similar effect as D, stimulation, as was indeed found in this study. Substance P, also present in striato-nigral neurones, tended to be affected in the direction opposite to that of dynorphin peptides, indicating that the two peptide systems are not co-regulated. This agrees with experiments where these peptides were injected into the substantia nigra (Herrera-Marschitz et al., 1984). Substance P-containing neurones may act as part of a positive feed-back loop increasing the activity in the nigro-striatal dopamine pathway and causing release of dopamine, while the dynorphin pathway might serve as a negative feed-back loop, causing the opposite effects and, in addition, control efferent nondopaminergic neurones in the substantia nigra et al., 1986; Herrera(Christensson-Nylander Marschitz et al., 1986). Efsects of treatment with drugs on processing prodynorphin
of
Figure 1 shows the possible cleavage products from prodynorphin. Since (Leu)enkephalin is mainly derived from prodynorphin, rather than proenkephalin (Christensson-Nylander et al., 1986; Zamir et al., 1984), it could be expected that the levels of this peptide would be affected in the same way as dynorphin peptides. Indeed, levels of (Leu)enkephalin in the nigra increased after Dr inhibition. However, the substances acting on the D, receptor were without effect on levels of (Leu)enkephalin. (Leu)Enkephalyl-Arg was markedly increased in all structures after D, activation, thus following the dynorphin peptides. In contrast to the dynorphins however, levels of (Leu)enkephalyl-Arg were also markedly increased after D2 activation. Therefore, it could be suggested that the processing of prodynorphin is dependent on the type of dopamine
and L. H.
TERENKJS
receptor stimulated, and possibly secondary to the activation of different processing enzymes in the prodynorphin neurone. Studies on cerebrospinal fluid (Nyberg, Nordstrom and Terenius, 1985) and spinal cord (Nyberg et al., unpublished observation) have shown that an enzyme exists which may be responsible for the generation of (Leu)enkephalyl-Arg from dynorphin A, dynorphin B and alpha-neoendorphin. The partly different effects of D, and D, receptor agents on levels of dynorphin compared to those of the shorter prodynorphin-derived peptides cannot be fully explained by this study. At present it is necessary to consider at least two possibilities: (a) the different dopamine receptor types are present on the same neurone, but different postsynaptic effects are elicited after D, or D, stimulation, respectively; (b) the dopamine receptors are present on different striatal efferents. The second alternative agrees well with a model proposed by Ungerstedt, Herrera-Marschitz, Stable, Tossman and Zetterstrljm (1985), through analysis of rotation behaviour, elicited by different dopamine agonists (Herrera-Marschitz and Ungerstedt, 1984a and b; Ungerstedt et al., 1985). In conclusion, this study has presented evidence for alterations in the biosynthesis and processing of dynorphin peptides after the administration of different drugs acting on dopamine receptors. Dynorphin peptides were affected differently to substance P, in agreement with the presence of these peptides in separate striatonigral pathways, as well as different connections with the motor system. Other prodynorphin-derived peptides, (Leu)enkephalin and (Leu)enkephalyl-Arg, were found to be affected only partly as dynorphin peptides, suggesting a possible difference in enzymatic processing after activation of different types of dopamine receptors. Acknowledgements-The authors are grateful to Mrs lngrid
Eriksson for skilful technical assistance. The work was SUDDOrtedbv the Swedish Medical Research Council (No. 37bb and 5696) and the Bank of Sweden Tercentennary Fund (83/208) to L. Terenius.
REFERENCES
Amt J. and Hyttel J. (1985) Differential involvement of dopamine D, and D, receptors in the circling behaviour induced by apomorphine, SKF 38393, pergolide and LY 171555 in 6-hydroxydopamine-lesioned rats. Psychopharmacology 85: 346352.
Bergstrom L., Christensson I., Folkesson R.. Stenstrom B. and Terenius L. (1983) An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P. Life Sci. 33: 1613~1619. Brownstein M. J., Mroz E. A., Tappaz M. L. and Leeman S. E. (1977) On the origin of substance P and glutamic decarboxylase (GAD) in the substantia nigra. Brain Res. 135: 315-323.
Christensson-Nylander I., Nyberg F., Ragnarsson U. and Terenius L. (1985) A general procedure for analysis of proenkephalin B derived opioid peptides. Reg. Peptides 11: 65-76.
Dopaminergic drugs affect dynorphin and substance P Christensson-Nylander I. and Terenius L. (1985) Dynorphin peptides in human substantia nigra. Neuropeptides 6: 391-396.
Christensson-Nylander I., Herrera-Marschitz M., Staines W., Hiikfelt T., Terenius L., Ungerstedt U., Cuello C., Gertel W. H. and Goldstein M. (1986) Striato-nigral dynorphin and substance P. I: Biochemical and immunohistochemical evidence. Exp. Bruin Res. 64: 169-192. Dores R. M., Lewis M. E., Khachaturian H., Watson S. J. and Akil H. (1985) Analysis of opioid and non-opioid end products of prodynorphin in the substantia nigra of the rat. Neuropeptides 5: 501-504. Fuller R. W., Hemrick-Luecke D., Wong D. T., Pearson D., Threlkeld P. G. and Hynes M. D. (1983) Altered behavioural response to a D,-agonist LY 141865 in spontaneously hypertensive rats exhibiting biochemical and endocrine responses similar to those in normal rats. J. Pharmac. exp. Ther. 227: 354359.
Gale K., Hong J. S. and Guidotti A. (1977) Presence of substance P and GABA in separate striato-nigral neurons. Brain Res. 136: 371-375. Goldstein A., Fischli W., Lowney L. I., Hunkapiller M. and Hood L. (1981) Porcine pituitary dynorphin: Complete amino-acid sequence of the biologically active heptadecapeptide. Proc. natn. Acad. Sci. U.S.A. 78: 7219-7223. Gower A. J. and Marriott A. S. (1982) Pharmacological evidence for the subclassification of central dopamine receptors in the rat. Br. J. Pharmac. 77: 185-194. Haber S. N. and Watson S. J. (1985) The comparative distribution of enkephalin, dynorphin and substance P in the human globus pallidus and basal forebrain. Neuroscience 14: 101l-1024. Hanson G. R., Alphis L., Wolf W., Levine R. and Lovenberg W. (1981) Haloperidol-induced reduction of nigral substance P-like immunoreactivity: A probe for the interactions between dopamine and substance P neuronal systems. J. Pharmac. exp. Ther. 218: 568-574. Hererra-Marschitz M. and Ungerstedt U. (1984a) Evidence that striatal efferents relate to different dopamine receptors. Brain Res. 323: 269-278. Herrera-Marschitz M. and Ungerstedt U. (1984b) Evidence that apomorphine and pergolide induce rotation in rats by different actions on D, and D, receptor sites. Eur. J. Pharmac. 98: 165-176.
Herrera-Marschitz M., Hiikfelt T., Ungerstedt U., Terenius L. and Goldstein M. (1984) Effect of intranigral injections of dynorphin, dynorphin fragments and a-neoendorphin on rotational behaviour in the rat. Eur. J. Pharmac. 102: 213-227.
Herrera-Marschitz M., Christensson-Nylander I., Sharp T., Staines W., Hijkfelt T., Terenius L. and Ungerstedt U. (1986) Striato-nigral dynorphin and substance P pathways in the rat. II: Functional evidence. Exp. Brain Res. 64: 193-207. Hong J. S., Yang H-Y. T. and Costa E. (1978) Substance P content of substantia nigra after chronic treatment with antischizophrenic drugs. Neuropharmacology 17: 83-85. Hong J. S., Yang H-Y. T., Racagni G. and Costa E. (1977) Projections of substance P containing neurons from neostriatum to substantia nigra. Brain Res. 122: 541-544. Hyttel J. (1983) SCH 23390-the first selective dopamine D, antagonist. Eur. J. Pharmac. 91: 153-154. Hyttel J., Larsen J-J., Christensen A. V. and Amt J. (1985) Receptor binding profiles of neuroleptics. In: Dyskinesiu -Research and Treatment (Casey D. E., Chase T. N., Christensen A. V. and Gerlach J., Eds), Psychopharmacology suppl. 2, pp. 9-17. Springer, Berlin. Iorio L. C., Bamett A., Leitz F. H., Houser V. P. and Korduba C. A. (1983) SCH 23390, a potent benzazepine antipsychotic with unique interactions on dopaminergic systems. J. Pharmac. exp. Ther. 226: 462-468. Kakidani H., Furutani Y., Takahashi H., Noda M., Morimoto Y., Hirose T., Asai M., Inayama S., Nakanishi S.
1301
and Numa S. (1982) Cloning and sequence analysis of cDNA for porcine beta-neoendorphin/dynorphin precursor. Nature 298: 245249. Kanazawa I., Emson P. C. and Cue110A. C. (1977) Evidence for the existence of substance P containing fibers in striato-nigral and pallido-nigral pathways in rat brain. Brain Res. 119: 447453.
Kebabian J. W. and Calne D. B. (1979) Multiple receptors for dopamine. Nature 277: 93-96. Maysinger D., Hollt V., Seizinger B. R., Mehraein P., Pasi A. and Herz A. (1982) Parallel distribution of immunoreactive a-neo-endorphin and dynorphin in rat and human tissue. Neuropeptides 2: 21 l-225. McLean S., Bannon M. J., Zamir N. and Pert C. B. (1985) Comparison of the substance P- and dynorphincontaining projections to the substantia nigra: A radioimmunocytochemical and biochemical study. Brain Res. 361: 185-192. Nielsen I. M., Pedersen V., Nymark M., Franck K. F., Boeck V., Fjalland B. and Christensen A. V. (1973) The comparative pharmacology of flupenthixol and some reference neuroleptics. Acta pharmac. tox. 33: 353-362.
Nyberg F., Nordstrom K. and Terenius L. (1985) Endopeptidase in human cerebrospinal fluid which cleaves proenkephalin B opioid peptides at consecutive basic amino acids. Biochem. biophys. Res. Commun. 131: 1069-1074. Nylander I. and Terenius L. (1986) Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat. Brain Res. 380: 3441.
Oblin A., Zivkovic B. and Bartholini G. (1984) Involvement of the D-2 dopamine receptor in the neuroleptic induced decrease in nigral substance P. Eur. J. Pharmac. 105: 175-177. Onali P., Olianas M. C. and Gessa G. L. (1984) Selective blockade of dopamine D, receptors by SCH 23390 discloses striatal dopamine D, receptors mediating the inhibition of adenylate cyclase in rats. Eur. J. Pharmac. 99: 127-128.
Quirion R., Gaudreau P., Martel J.-C., St-Pierre S. and Zamir N. (1985) Possible interactions between dynorphin and dopaminergic systems in rat basal ganglia and substantia nigra. Brain Res. 331: 358-362. Ritter J. K., Schmidt C. J., Gibb J. W. and Hanson G. R. (1984) Increases of substance P-like immunoreactivity within striatal-nigral structures after subacute methamphetamine treatment. J. Pharmac. exp. Ther. 229: 487492.
Rosengarten H., Schweitzer J. W. and Friedhoff A. J. (1983) Induction of oral dyskinesias in naive rats by D, stimulation. Life Sci. 33: 24792482. Sailer C. F. and Salama A. I. (1985) Dopamine receptor subtypes: in viuo biochemical evidence for functional interaction. Eur. J. Pharmac. 109: 297-300. Seeman P. (1981) Brain dopamine receptors. Pharmac. Rev. 32: 229-313. Setler P. E., Sarau H. M., Zirkle C. L. and Saunders H. L. (1978) The central effects of a novel dopamine agonist. Eur. J. Pharmac. SO: 419430. Sonsalla P. K., Gibb J. W. and Hanson G. R. (1984) Opposite responses in the striato-nigral substance P system to D, and D, receptor activation. Eur. J. Pharmac. 105, 1855187. Staines W. A., Nagy J. I., Vincent S. R. and Fibiger H. C. (1980) Neurotransmitters contained in the efferents of striatum. Brain Res 194: 391-402. Titus R. D., Komfeld E. C., Jones N. D., Clemens J. A., Smalstia E. B.. Fuller R. W.. Hahn R. A.. Hvnes M. D.. Mason\. R.,‘Wong D. T. and Foreman M. N. (1983) Resolution and absolute configuration of an ergolinerelated dopamine agonist, trans-4,4a,5,6,7,8,8a,g_Octa-
INGRID NYLANDER
1302 hydro-5-propyl-IH
(or 2H)-pyrazolo(3,4-g)quinoline.
J.
Med. Chern. M: 1112-1116.
Tsuruta K., Frey E. A., Grewe C. W., Cote T. E., Eskay R. L. and Kebabian J. W. (1981) Evidence that LY 141865 specifically stimulates the Dz receptor. Nature 292: 463-465. Ungerstedt U., Herrera-Marschitz M., Stlhle L., Tossman U. and Zetterstriim Y. (1985) Functional classification of different dopamine receptors. In: Dyskinesia-Research and Treatment (Casey D. E., Chase T. N., Christensen A. V. and Gerlach J., Eds), Psychopharmacology suppl. 2, pp. 1930. Springer, Berlin. Weber E., Roth K. A. and Barchas J. D. (1982) Immunohistochemical distribution of or-neoendorphin/dynorphin
and L. H. TERENIUS neuronal systems in rat brain: Evidence for co-localization. Proc. natn. Acad. Sci. U.S.A. 79: 3062-3066. Vincent S. R., Hokfelt T., Christensson I. and Terenius L. (1982a) Dynorphin immunoreactive neurons in the central nervous system of the rat. Neurosci. .%zr. 33: 185190. Vincent S., Hokfelt T., Christensson I. and Terenius L. (1982b) Immunohistochemical evidence for a dynorphin immunoreactive striato-nigral pathway. Eur. J. Pharmnc. 85: 251-252.
Zamir N., Palkovitz M., Weber E., Mezey E. and Brownstein M. J. (1984) A dyno~hiner~c pathway of Leuenkephalin production in substantia nigra. Nature 307r 643-645.
~28-3~8/87 $3.00+ 0.00 Copyright Q 1987Pergamon Journals Ltd
DOPAMINE RECEPTORS MEDIATE ALTERATIONS STRIATO-NICRAL DYNORPHIN AND SUBSTANCE P PATHWAYS
IN
INGRID NYLANDER and L. H. TERENIUS Department of Pharmacology, Uppsala University, Box 591, S-751 24 Uppsala, Sweden (Accepted 9 March 1987)
Summary-Peptides derived from prodynorphin, dynorphin A and B, (mu)-enkephalin and (~u~nkephaiyl-Arg, as well as substance P, were measured in substantia nigra, striatum and globus pallidus, after subacute (5 doses at 6 hr intervals) treatment of rats with a number of dopamine receptor agonists and antagonists. Drugs selective for the dopamine D, and D, receptors, respectively, as well as unselective drugs were used. In the substantia nigra, levels of immunoreactive dynorphin A and dynorphin B were increased after treatment with a D,-antagonist (sulpiride) and a D,-agonist (SKF 38393), while a D,-antagonist (SCH 23390) reduced levels. The mixed D, and D, antagonist cis-fluwnthixol reduced only the level of dynorphin A. A corresponding increase of the levels-of (Leu)enkephalin in the nigra was found after treatment with sulpiride. In contrast to dynorphin peptides, the levels of (Leu)enkephalyi-Arg were markedly increased after both D,- and Dz (LY 17155.5)-stimulation. Substance P tended to be reduced after D,-stimulation and treatment with all the dopamine antagonists; the reduction was significant with sulpiride and c~-flu~nthixol. Levels of peptides in striatum and globus pallidus were generally affected in the same direction as levels in the nigra. The results in this study present further evidence that dopamine receptor agents affect dynorphin peptides and substance P, differentially. Effects on (Leu)enkephalin and (Leu)enkephalyl-Arg only partly paralleled the effects on levels of dynorphin. Thus, the D, and D, receptors differentially affect levels of different products of prodynorphin, that is, seem to affect certain steps of the processing of prodynorphin selectively. Key words: prodynorphin peptides, substance P, striate-nigh antagonists.
The opioid peptides deriving from prodynorphin, i.e. the dynorphin peptides (Goldstein, Fischli, Lowney, Hunkapiller and Hood, 1981; Kakidani, Furutani, Takahashi, Noda, Morimoto, Hirose, Asai, Inayama, Nakanishi and Numa, 1982), as well as substance P
and other protachykinin peptides, have been shown to be present in high levels in the substantia nigra and the basal ganglia (Brownstein, Mroz, Tappaz and Leeman, 1977; Christensson-Nylander and Terenius, 1985; Dores, Lewis, Khachaturian, Watson and Akil, 1985; Gale, Hong and Guidotti, 1977; Haber and Watson, 1985; Hong, Yang and Costa, 1978; Kanazawa, Emson and Cuello, 1977; Maysinger, Hollt, Seizinger, Mehraein, Pasi and Herz, 1982; Weber, Roth and Barchas, 1982). These two peptide systems are expressed in separate but anatomically overlapping striato-nigral neurones (Brownstein et al., 1977; Christensson-Nylander, Herrera-Marschitz, Staines, Hdkfelt, Terenius, Ungerstedt, Cuello, Oertel and Goldstein, 1986; Gale, et al., 1977; McLean, Bannon, Zamir and Pert, 1985; Vincent, HBkfelt, Christensson and Terenius, 1982a, b) and possibly also in st~ato-pallidal (Staines, Nagy, Vincent and 1980) and/or pallidonigral pathways Fibiger, (Brownstein et al., 1977; Kanazawa et al., 1977). The N.P. 2619-D
pathways, dopamine agonists, dopamine
interactions between these peptidergic pathways and the nigro-striatal dopamine pathway are at present being examined. For instance, several investigators have reported alterations in levels of substance P when different dopamine agonists and antagonists were administered to rats. In general, reduced levels of substance P in the nigra were found after repeated administration of different dopamine antagonists, which was thought to reflect an increased activity (increased release) in striato-nigral substance P neurones as a consequence of the blockade of dopamine receptors (Hanson, Alphis, Wolf, Levine and Lovenberg, 1981; Hong, Yang, Racagni and Costa, 1977). With the agonists, a selective D, receptor agonist (SKF 38393) also reduced substance P in the nigra, whereas a selective D, agonist (RU 24926; Nn-propyl-di-beta-(3-hydroxyphenyl)ethylamine hy drochloride) gave increased levels, indicating that the D, and Dz receptors, respectively, are differently coupled, functionally (Sonsalla, Gibb and Hanson, 1984). The indirect dopamine receptor agonist, methamphet~ine also enhanced levels of substance P in the nigra (Ritter, Schmidt, Gibb and Hanson, 1984). Much less information is available concerning the interaction between the dopamine and dynorphin
INGRID NYLANDER and
1296
L. H.
TERENIUS
DYN PROENKEPHALIN
DYN
6
Q NEOENDORPHIN
DYNORPHIN
[._NEO
29 I
I
A
DYNORPHIN
B
I
pr-pq
pq
I
l-8)
A(
LA
LA
I
32
I LE
LE
I
I
I
LA
LE 0
Fig. 1. Possible known opioid peptide fragments of prodynorphin. u-NE0 = alpha-neoendorphin, /?-NE0 = beta-neoendorphin, A(l-8) = dynorphin A&8), LA = (Leu)enkephalyl-Arg, LE = (Leu) enkephalin.
systems. Discordant results have been obtained after chronic treatment with haloperidol; unchanged levels of dynorphin in the nigra were reported after 10 days of treatment (Nylander and Terenius, 1986), while
treatment for 21 days increased levels (Quirion, Gaudreau, Martel, St-Pierre and Zamir, 1985). Previous studies have revealed evidence for an interaction between dynorphin and dopamine. Dynorphin peptides, injected unilaterally into the substantia nigra, (1) inhibit release of dopamine in the striatum (Christensson-Nylander et al., 1986; Herrera-Marschitz, Christensson-Nylander, Sharp, Staines, Hokfelt, Terenius and Ungerstedt, 1986) and (2) produce contralateral turning (HerreraMarschitz, Hiikfelt, Ungerstedt, Terenius and Goldstein, 1984; Herrera-Marschitz et al., 1986). Since this latter effect is also recorded in animals lesioned with 6-hydroxydopamine (6-OHDA) it may involve other pathways than the nigro-striatal dopamine (DA) pathway. In this study, a more thorough examination has been performed to analyze the interaction between dynorphin and dopamine systems. For comparison, substance P was also measured. Both dopamine agonists and antagonists have been used, some with selective actions on the D, and D, dopamine receptor, respectively, some more unselective. The drugs were given in 5 doses every 6 hr. Dynorphin peptides (dynorphin A and dynorphin B) and substance P were measured with radioimmunoassays in the substantia nigra, globus pallidus and striatum. In addition, (Leu)enkephalyl-Arg and (Leu)-enkephalin were measured in substantia nigra and the levels compared to levels of dynorphin. These enkephalins are likely to be processing products of prodynorphin in this area of the CNS (Christensson-Nylander et al., 1986; Zamir, Palkovitz, Weber, Mezey and Brownstein, 1984) (Fig. 1). METHODS
Drug treatment
Male Sprague-Dawley
rats (Anticimex,
Sweden)
weighing 170-190 g were used. The drugs were injected intraperitoneally every 6 hr on 5 occasions. Six hours after the last dose the animals were decapitated. Control animals received equivalent volume of saline. Since the purpose of this study was to test possible effects on levels of peptides of different kinds of substances acting on dopamine receptors, only one dose of each drug was used, chosen according to information in the literature. Seven groups of rats were used, with 8-10 rat in each group, receiving one of the following drugs: apomorphine (2 mg/kg), cis-flupenthixol (0.1 mg/kg) (Nielsen, Pedersen, Nymark, Franck, Boeck, Fjalland and Christensen, 1973), LY 171555 (0.5 mg/kg) (Fuller, Hemrick-Luecke, Wong, Pearson, Threlkeld and Hynes, 1983; Titus, Kornfeld, Jones, Clemens, Smalstig, Fuller, Hahn, Hynes, Mason, Wong and ‘Foreman, 1983; Tsuruta, Frey, Grewe, Cote, Eskay and Kebabian, 1981), SKF 38393 (15 mg/kg) (Setler, Sarau, Zirkle and Saunders, 1978; Sonsalla et al., 1984), SCH 23390 (0.1 mg/kg) (Hyttel, 1983; Iorio, Barnett, Leitz, Houser and Korduba, 1983), sulpiride (100 mg/kg) (Oblin, Zivkovic and Bartholini, 1984) or saline. Extraction
of tissue and separation
of extract
After decapitation, the substantia nigra, globus pallidus and striatum were rapidly dissected out on an ice-cold plate, frozen and kept at -90°C until processed further. Extraction was performed with 1 M acetic acid, containing 0.01% mercaptoethanol, at 95°C for 5 min. The tissues were homogenized by sonication, heated another 5 min at 95”C, again cooled before centrifugation for 15 min in a Beckman Microfuge@. The supematants were collected and transferred to ion exchange columns (SP Sephadex C-25, Pharmacia, Sweden) for separation and purification of the peptides. The procedure is described in detail elsewhere (Bergstrom, Christensson, Folkesson, Stenstrom and Terenius, 1983). In brief, the columns were eluted stepwise with a series of buffers containing pyridine and formic acid of increasing ion strength. (Leu)Enkephalin and
Dopaminergic drugs affect dynorphin and substance P (Leu)enkephalyl-Arg were eluted in separate fractions, while dynorphin A, B and substance P eluted together in one fraction. The fractions were dried in a vacuum centrifuge and redissolved in an appropriate volume of methanol/O. 1 M HCl (1: 1) before measurement of each peptide with radioimmunoassay. Radioimmunoassays
0.9% NaCI. Sulpiride France) was dissolved acetic acid. The solution with NaOH. All drugs volumes.
1297 (Delagrange Laboratories, by adding a few drops of was diluted and neutralized were injected in equivalent
Statistics
Analysis of differences between means was by the Student’s two-tailed t-test. In addition, analysis of variance was tested using the General Linear Models procedure, GLM (SAS Institute, Cary, North Carolina). Differences were considered significant at
Cross-reactivity studies were performed with each antiserum, using a variety of peptides with sequence homologies to the peptide to be measured. Only P < 0.05. cross-reacting peptides (> 0.1%) are listed below. (Leu)enkephalyl-Arg and substance P. [‘251]labelled (Leu)enkephalyl-Arg and Tyr*-substance P reRESULTS spectively, were used as tracer. Iodination was performed using chloramine T and the labelled peptide The effects of subacute treatment with different was purified with high pressure liquid chrodrugs on levels of neuropeptides are presented for matography (HPLC) (on reversed phase column, each area of the CNS studied separately. The followlinear gradient of acetonitrile and H,O in 0.04% ing drugs were used: SKF 38393 (15 mg/kg), a selectrifluoroacetic acid). Incubation with antibody and tive D, agonist; LY 171555 (0.5 mg/kg) a selective Dz sample or standard peptide was performed overnight. agonist; apomorphine (2 mg/kg) an unselective dopaFree and antibody-bound peptides were separated by minergic agonist, although mainly acting on D, reincubation with dextrane-coated charcoal for 10 min. ceptors; SCH 23390 (0.1 mg/kg) a D, antagonist; The (Leu)enkephalyl-Arg antiserum showed 10% sulpiride (100 mg/kg), a D, antagonist and ciscross-reactivity with (Met)enkephalyl-Arg, 1% with flupenthixol (0.1 mg/kg) an antagonist with activity (Met)enkephalyl-Arg-Phe, 0.5% with dynorphin A on both D,- and D,-receptors. (l-8) and alpha-neoendorphin. Fragments of substance P (3-11) and (4-11) cross-reacted 100% with Substantia nigra (Figs 2 and 3) the antiserum for substance P. (Leu)Enkephalin (Fig. 2). The only drug that (Leu)Enkephalin. [3H]labelled peptide (Amersham, affected levels of this peptide was sulpiride, which England) was used as the tracer. Otherwise the gave an elevation. procedure was the same as for (Leu)enkephalyl-Arg Dynorphin A and B (Fig. 3). Increased levels were and substance P. The antiserum recognized Argmeasured after treatment with SKF 38393, whereas (Leu)enkephahn to 100% and (Met)enkephalin to no change was observed after LY 171555 or apo3%. morphine. The D, antagonist, sulpiride caused a Dynorphin A and B. [‘251]labelled peptides were marked increase in the dynorphins, contrasting the prepared according to the procedure used for subdecrease observed after treatment with the stance P and (Leu)enkephalyl-Arg. Dynorphin A D,-antagonist SCH 23390. Also, cis-flupenthixol antiserum showed 100% cross-reactivity with dynorgave a marked reduction in levels of dynorphin. phin A (9-17), while no cross-reacting peptides were (Leu)Enkephafyl-Arg (Fig. 3). Levels of this pepfound with dynorphin B antiserum. Radiotide significantly increased after treatment with both immunoassay was performed as for the other pepSKF 38393 and LY 171555. No other drug caused tides with the exception that separation of free and significant changes. antibody-bound peptide occurred by addition of preSubstance P (Fig. 3). The D, agonist SKF 38393 cipitate of normal rabbit serum and sheep anti-rabbit caused a decrease, as did all the antagonists; the serum. The procedure has previously been described decrease caused by sulpiride and cis-flupenthixol in detail (Christensson-Nylander, Nyberg, Ragreached statistical significance. narsson and Terenius, 1985). Drugs
Globus pallidus (Fig. 4)
Apomorphine HCI (Apoteksbolaget, Sweden), cisflupenthixol (Lundbeck, Denmark) LY 171555 [(rrans-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-lH) (or 2H)-pyrazolo(3,4-g)quinoline)] (Lilly, U.S.A.), SKF 38393 HBr [(2,3,4,5,-tetrahydro-7,8-dihydroxy-lphenyl-lH-3-benzazepine) Smith, Kline and French, U.S.A.] and SCH 23390 [(R)-( +)-8-chloro-2,3,4,5tetrahydro-3-methyl-5-phenyl-lH-3-benzazepine-7-ol hemimaleate) Schering, U.S.A.] were dissolved in
In general, levels of prodynorphin-derived peptides were affected in a similar way to those in the substantia, nigra. Dynorphin A and B. The dopaminergic agonists had fairly small effects. With sulpiride, marked increases in both dynorphins were observed. However, they were significantly reduced by cis-flupenthixol, an effect which is in contrast to that observed in substantia nigra.
INGRID NYLANDER
1298 pm01
/g
and L. H. TERENWS
Antagonists
Agonists
’ DI ’ D, ’ D ’
Ic
T
T
T
’ ‘JI I Dz I IJ I
a
_
Leu - Enk
Fig. 2. Immunoreactive levels of (Leu)enkephalin (mean values SEM) in rat substantia nigra expressed = saline, as pmol/g tissue, after tsatment with substances acting on dopamine receptors. D, agonist =SKF 38393, D, [7 = dopamine agonists, w = dopamine antagonists. C =control; antagonist = SCH 23390; D, agonist = LY 171555, D, antagonist = sulpiride; D agonist = apomorphine, unselective action on dopamine receptors. Leuwith D antagonist = cis-flupenthixol enk = (Leu)enkephalin. *P < 0.05, (Students’s two-tailed t-test).
(Leu)Enkephafyl-Arg. Levels of this peptide were markedly increased by the two agonists, SKF 38393 and LY 171555. No other significant effects were recorded. Substance P. Levels were not significantly affected.
pmoledg
AGONISTS
ANTAGONISTS
’ 4’ 0; D ’ Lcu -Arg
Striatum (Fig. 5) Dynorphin A and B. Levels were affected by the various treatments; SKF 38393 gave an increase whereas LY 171555 gave a significant decrease. The D, antagonist, sulpiride markedly increased levels of dynorphin. (Leu)Enkephalyl-Arg. As in the other structures investigated, SKF 38393 and LY 171555 markedly increased levels. No other treatment produced significant effects. Substance P. Only sulpiride produced significant effects, a marked reduction. Other treatments left levels essentially unchanged.
Dyn 6
200
150
Dyn A
DISCUSSION
Probes for dopamine receptor types
The dopamine receptors can be subdivided into D, and D2 types, using biochemical and pharmacological criteria (Gower and Marriott, 1982; Kebabian and Calne, 1979; Seeman, 1981). For instance, the D, receptor activates adenylate cyclase, while the D, receptor is either not coupled or inactivates the enzyme. A number of substances with selective actions on the D, and DZ receptor, respectively, have been synthesized. Based on information in the literature, the probes used in this investigation act mostly on one type of receptor and have negligible actions on the other: SKF 38393 has D, agonistic actions (Setler et al., 1978; Sonsalla et al., 1984), and SCH 23390 inhibits this receptor (Hyttel, 1983; Iorio et al., 1983). The drug LY 171555 stimulates the D, receptor (Fuller et al., 1983; Titus et al., 1983; Tsuruta et al.,
100 I
.h iu
SP
600 ZU-
Fig. 3. Immunoreactive levels of (Leu)enkephalyl-Arg, dynorphin A, dynorphin B and substance P (mean values SEM) in the substantia nigra of the rat, expressed as pmol/g tissue, after treatment with substances acting on dopamine receptors. Leu-Arg = (Leu)enkephalyl-Arg, Dyn A = dynorphin A, Dyn B = dynorphin B, SP = substance P. See also legend to Fig. 2. *P ~0.05, **P ~0.01, ***P < 0.001 (Student’s two-tailed f-test). Analysis of variance confirmed the effects of treatment; P < 0.001 for dynorphins A and B and (Leu)enkephalyl-Arg, P < 0.05 for substance P.
Dopaminergic drugs affect dynorphin and substance P
pmoL?s/g rc’
AGONISTS
ANTAGONISTS
‘D&‘D
‘D,‘D,‘D’
1299
pmolcs /g AGONtSTS ‘Dl’y2-D’
rvw
Lw -Arg
Lcu -Arg
Dyn B
Dyn B
Dyn A
Dyn A 15
flh 10 = ka
Fig. .4. Immunoreactive levels of (Leu)cnkephalyl-Arg, dynorphin A, dynorphin B. and substance P in globus palbdus, see further legend to Figs 2 and 3. Analysis of variance confirmed a treatment effect for dynorphins A and
B and (Leu)enkephalyl-Acg (P c 0.00I ).
1981) and sulpiride is a D, antagonist (Hyttel, Larsen, Christensen and Arnt, 1985; Oblin et al., 1984)
Apomorphine and cis-flupenthixol are an unselective dopamine agonist and antagonist, respectively (Arnt and Hyttel, 1985; Herrera-Marschitz and Ungerstedt, 1984b; Hyttel ef al., 1985 and Nielsen ef al., 1973). The purpose of this study was to test the effect of such agents on several peptides in three areas of the brain. To limit the chemical analyses to reasonable proportions, only one dose of each substance was chosen, instead of the ideal experimental protocol with several doses. Functional studies, performed by measuring the rotational response of dopamine agonists, suggest that the two dopamine receptors are associated with different striatal efferents; apomorphine induces rotation by an action mainly on D, receptors through striatal efferents running through the globus pallidus to the substantia nigra, while pergolide induces rotation through D2 receptors acting through other striatal efferent pathways (Arnt and Hyttel, 1985; Herrera-Marschitz and Ungerstedt, 1984a; Herrera-
ANTAGONISTS
.‘:zz .~:~ ..;i.. ..ii .: : ,; ,_: : ;: ::: :, j:. .::
T
l
-
1
Fig. 5. Immunoreactive levels of (Leu)enkephalyl-Arg, dynorphin A, dynorphin B and substance P in the striatum (see further legend to Figs 2 and 3). Analysis of variance confirmed a treatment effect for dynorphins A and B (P -CO.OOl), (Leu)enkephalytArg and substance P (P < 0.01).
Marschitz and Ungerstedt, 1984b). Consequently, the striatum, globus pallidus and substantia nigra were included in this study. Prodynorphin-related peptides in the substmtia nigra
Dynorphin peptides are present in cell bodies in the striatum and in nerve terminals in the zona rcticulata of substantia nigra (Vincent et al., 1982b). The levels of these peptides in the substantia nigra are high compared to other brain areas. In addition, high levels of (Leu)enkephalin are found in this area: whereas other areas of the brain have a 4: 1 ratio of (Met)enkephalin: (Leu)enkephalin, this ratio is 1: 2 in substantia nigra. (Christensson-Nylander et al., 1986). (Met)-Enkephalin is mainly present in neurones within the zona compacta. Cross-reaction of (Met)enkephalin with the (Leu)enkephalin antiserum is only 3% and HPLC has confirmed that most of the immunoreactivity relates to (Leu)enkephalin (Christensson-Nylander et al., 1986). (Lcu)Enkephalyl-Arg is present as the N-terminus in all peptides derived from prodynorphin and is
INGRID NYLANDER
1300
unique to this precursor. (Met)-Enkephalyl-Arg, a peptide present in proenkephalin, cross-reacted 10% in the radioimmunoassay for (Leu)enkephalyl-Arg. However, HPLC experiments have shown that the immunoreactivity measured is indeed (Leu)enkephalyl-Arg (Christensson-Nylander et al., 1985). Dopamine-peptide
interactions
Levels of dynorphin in the substantia nigra were increased by a selective D, agonist and decreased by a D, antagonist. An increase in levels of dynorphin in the nigra were also detected after D, inhibition. Unfortunately, tissue levels of peptides are very imperfect measures of turn-over; for example, increased levels may be due to increased biosynthesis, but may also depend on reduced release. Therefore the present data is hard to evaluate in terms of activation/ deactivation of the peptide pathways. An interaction between the two dopamine receptors has been proposed (Onali, Olianas and Gessa, 1984; Rosengarten, Schweitzer and Friedhoff, 1983; Sailer and Salama, 1985) and inhibition of the D, receptor might cause an imbalance of D,/D, responses, resulting in a similar effect as D, stimulation, as was indeed found in this study. Substance P, also present in striato-nigral neurones, tended to be affected in the direction opposite to that of dynorphin peptides, indicating that the two peptide systems are not co-regulated. This agrees with experiments where these peptides were injected into the substantia nigra (Herrera-Marschitz et al., 1984). Substance P-containing neurones may act as part of a positive feed-back loop increasing the activity in the nigro-striatal dopamine pathway and causing release of dopamine, while the dynorphin pathway might serve as a negative feed-back loop, causing the opposite effects and, in addition, control efferent nondopaminergic neurones in the substantia nigra et al., 1986; Herrera(Christensson-Nylander Marschitz et al., 1986). Efsects of treatment with drugs on processing prodynorphin
of
Figure 1 shows the possible cleavage products from prodynorphin. Since (Leu)enkephalin is mainly derived from prodynorphin, rather than proenkephalin (Christensson-Nylander et al., 1986; Zamir et al., 1984), it could be expected that the levels of this peptide would be affected in the same way as dynorphin peptides. Indeed, levels of (Leu)enkephalin in the nigra increased after Dr inhibition. However, the substances acting on the D, receptor were without effect on levels of (Leu)enkephalin. (Leu)Enkephalyl-Arg was markedly increased in all structures after D, activation, thus following the dynorphin peptides. In contrast to the dynorphins however, levels of (Leu)enkephalyl-Arg were also markedly increased after D2 activation. Therefore, it could be suggested that the processing of prodynorphin is dependent on the type of dopamine
and L. H.
TERENKJS
receptor stimulated, and possibly secondary to the activation of different processing enzymes in the prodynorphin neurone. Studies on cerebrospinal fluid (Nyberg, Nordstrom and Terenius, 1985) and spinal cord (Nyberg et al., unpublished observation) have shown that an enzyme exists which may be responsible for the generation of (Leu)enkephalyl-Arg from dynorphin A, dynorphin B and alpha-neoendorphin. The partly different effects of D, and D, receptor agents on levels of dynorphin compared to those of the shorter prodynorphin-derived peptides cannot be fully explained by this study. At present it is necessary to consider at least two possibilities: (a) the different dopamine receptor types are present on the same neurone, but different postsynaptic effects are elicited after D, or D, stimulation, respectively; (b) the dopamine receptors are present on different striatal efferents. The second alternative agrees well with a model proposed by Ungerstedt, Herrera-Marschitz, Stable, Tossman and Zetterstrljm (1985), through analysis of rotation behaviour, elicited by different dopamine agonists (Herrera-Marschitz and Ungerstedt, 1984a and b; Ungerstedt et al., 1985). In conclusion, this study has presented evidence for alterations in the biosynthesis and processing of dynorphin peptides after the administration of different drugs acting on dopamine receptors. Dynorphin peptides were affected differently to substance P, in agreement with the presence of these peptides in separate striatonigral pathways, as well as different connections with the motor system. Other prodynorphin-derived peptides, (Leu)enkephalin and (Leu)enkephalyl-Arg, were found to be affected only partly as dynorphin peptides, suggesting a possible difference in enzymatic processing after activation of different types of dopamine receptors. Acknowledgements-The authors are grateful to Mrs lngrid
Eriksson for skilful technical assistance. The work was SUDDOrtedbv the Swedish Medical Research Council (No. 37bb and 5696) and the Bank of Sweden Tercentennary Fund (83/208) to L. Terenius.
REFERENCES
Amt J. and Hyttel J. (1985) Differential involvement of dopamine D, and D, receptors in the circling behaviour induced by apomorphine, SKF 38393, pergolide and LY 171555 in 6-hydroxydopamine-lesioned rats. Psychopharmacology 85: 346352.
Bergstrom L., Christensson I., Folkesson R.. Stenstrom B. and Terenius L. (1983) An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P. Life Sci. 33: 1613~1619. Brownstein M. J., Mroz E. A., Tappaz M. L. and Leeman S. E. (1977) On the origin of substance P and glutamic decarboxylase (GAD) in the substantia nigra. Brain Res. 135: 315-323.
Christensson-Nylander I., Nyberg F., Ragnarsson U. and Terenius L. (1985) A general procedure for analysis of proenkephalin B derived opioid peptides. Reg. Peptides 11: 65-76.
Dopaminergic drugs affect dynorphin and substance P Christensson-Nylander I. and Terenius L. (1985) Dynorphin peptides in human substantia nigra. Neuropeptides 6: 391-396.
Christensson-Nylander I., Herrera-Marschitz M., Staines W., Hiikfelt T., Terenius L., Ungerstedt U., Cuello C., Gertel W. H. and Goldstein M. (1986) Striato-nigral dynorphin and substance P. I: Biochemical and immunohistochemical evidence. Exp. Bruin Res. 64: 169-192. Dores R. M., Lewis M. E., Khachaturian H., Watson S. J. and Akil H. (1985) Analysis of opioid and non-opioid end products of prodynorphin in the substantia nigra of the rat. Neuropeptides 5: 501-504. Fuller R. W., Hemrick-Luecke D., Wong D. T., Pearson D., Threlkeld P. G. and Hynes M. D. (1983) Altered behavioural response to a D,-agonist LY 141865 in spontaneously hypertensive rats exhibiting biochemical and endocrine responses similar to those in normal rats. J. Pharmac. exp. Ther. 227: 354359.
Gale K., Hong J. S. and Guidotti A. (1977) Presence of substance P and GABA in separate striato-nigral neurons. Brain Res. 136: 371-375. Goldstein A., Fischli W., Lowney L. I., Hunkapiller M. and Hood L. (1981) Porcine pituitary dynorphin: Complete amino-acid sequence of the biologically active heptadecapeptide. Proc. natn. Acad. Sci. U.S.A. 78: 7219-7223. Gower A. J. and Marriott A. S. (1982) Pharmacological evidence for the subclassification of central dopamine receptors in the rat. Br. J. Pharmac. 77: 185-194. Haber S. N. and Watson S. J. (1985) The comparative distribution of enkephalin, dynorphin and substance P in the human globus pallidus and basal forebrain. Neuroscience 14: 101l-1024. Hanson G. R., Alphis L., Wolf W., Levine R. and Lovenberg W. (1981) Haloperidol-induced reduction of nigral substance P-like immunoreactivity: A probe for the interactions between dopamine and substance P neuronal systems. J. Pharmac. exp. Ther. 218: 568-574. Hererra-Marschitz M. and Ungerstedt U. (1984a) Evidence that striatal efferents relate to different dopamine receptors. Brain Res. 323: 269-278. Herrera-Marschitz M. and Ungerstedt U. (1984b) Evidence that apomorphine and pergolide induce rotation in rats by different actions on D, and D, receptor sites. Eur. J. Pharmac. 98: 165-176.
Herrera-Marschitz M., Hiikfelt T., Ungerstedt U., Terenius L. and Goldstein M. (1984) Effect of intranigral injections of dynorphin, dynorphin fragments and a-neoendorphin on rotational behaviour in the rat. Eur. J. Pharmac. 102: 213-227.
Herrera-Marschitz M., Christensson-Nylander I., Sharp T., Staines W., Hijkfelt T., Terenius L. and Ungerstedt U. (1986) Striato-nigral dynorphin and substance P pathways in the rat. II: Functional evidence. Exp. Brain Res. 64: 193-207. Hong J. S., Yang H-Y. T. and Costa E. (1978) Substance P content of substantia nigra after chronic treatment with antischizophrenic drugs. Neuropharmacology 17: 83-85. Hong J. S., Yang H-Y. T., Racagni G. and Costa E. (1977) Projections of substance P containing neurons from neostriatum to substantia nigra. Brain Res. 122: 541-544. Hyttel J. (1983) SCH 23390-the first selective dopamine D, antagonist. Eur. J. Pharmac. 91: 153-154. Hyttel J., Larsen J-J., Christensen A. V. and Amt J. (1985) Receptor binding profiles of neuroleptics. In: Dyskinesiu -Research and Treatment (Casey D. E., Chase T. N., Christensen A. V. and Gerlach J., Eds), Psychopharmacology suppl. 2, pp. 9-17. Springer, Berlin. Iorio L. C., Bamett A., Leitz F. H., Houser V. P. and Korduba C. A. (1983) SCH 23390, a potent benzazepine antipsychotic with unique interactions on dopaminergic systems. J. Pharmac. exp. Ther. 226: 462-468. Kakidani H., Furutani Y., Takahashi H., Noda M., Morimoto Y., Hirose T., Asai M., Inayama S., Nakanishi S.
1301
and Numa S. (1982) Cloning and sequence analysis of cDNA for porcine beta-neoendorphin/dynorphin precursor. Nature 298: 245249. Kanazawa I., Emson P. C. and Cue110A. C. (1977) Evidence for the existence of substance P containing fibers in striato-nigral and pallido-nigral pathways in rat brain. Brain Res. 119: 447453.
Kebabian J. W. and Calne D. B. (1979) Multiple receptors for dopamine. Nature 277: 93-96. Maysinger D., Hollt V., Seizinger B. R., Mehraein P., Pasi A. and Herz A. (1982) Parallel distribution of immunoreactive a-neo-endorphin and dynorphin in rat and human tissue. Neuropeptides 2: 21 l-225. McLean S., Bannon M. J., Zamir N. and Pert C. B. (1985) Comparison of the substance P- and dynorphincontaining projections to the substantia nigra: A radioimmunocytochemical and biochemical study. Brain Res. 361: 185-192. Nielsen I. M., Pedersen V., Nymark M., Franck K. F., Boeck V., Fjalland B. and Christensen A. V. (1973) The comparative pharmacology of flupenthixol and some reference neuroleptics. Acta pharmac. tox. 33: 353-362.
Nyberg F., Nordstrom K. and Terenius L. (1985) Endopeptidase in human cerebrospinal fluid which cleaves proenkephalin B opioid peptides at consecutive basic amino acids. Biochem. biophys. Res. Commun. 131: 1069-1074. Nylander I. and Terenius L. (1986) Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat. Brain Res. 380: 3441.
Oblin A., Zivkovic B. and Bartholini G. (1984) Involvement of the D-2 dopamine receptor in the neuroleptic induced decrease in nigral substance P. Eur. J. Pharmac. 105: 175-177. Onali P., Olianas M. C. and Gessa G. L. (1984) Selective blockade of dopamine D, receptors by SCH 23390 discloses striatal dopamine D, receptors mediating the inhibition of adenylate cyclase in rats. Eur. J. Pharmac. 99: 127-128.
Quirion R., Gaudreau P., Martel J.-C., St-Pierre S. and Zamir N. (1985) Possible interactions between dynorphin and dopaminergic systems in rat basal ganglia and substantia nigra. Brain Res. 331: 358-362. Ritter J. K., Schmidt C. J., Gibb J. W. and Hanson G. R. (1984) Increases of substance P-like immunoreactivity within striatal-nigral structures after subacute methamphetamine treatment. J. Pharmac. exp. Ther. 229: 487492.
Rosengarten H., Schweitzer J. W. and Friedhoff A. J. (1983) Induction of oral dyskinesias in naive rats by D, stimulation. Life Sci. 33: 24792482. Sailer C. F. and Salama A. I. (1985) Dopamine receptor subtypes: in viuo biochemical evidence for functional interaction. Eur. J. Pharmac. 109: 297-300. Seeman P. (1981) Brain dopamine receptors. Pharmac. Rev. 32: 229-313. Setler P. E., Sarau H. M., Zirkle C. L. and Saunders H. L. (1978) The central effects of a novel dopamine agonist. Eur. J. Pharmac. SO: 419430. Sonsalla P. K., Gibb J. W. and Hanson G. R. (1984) Opposite responses in the striato-nigral substance P system to D, and D, receptor activation. Eur. J. Pharmac. 105, 1855187. Staines W. A., Nagy J. I., Vincent S. R. and Fibiger H. C. (1980) Neurotransmitters contained in the efferents of striatum. Brain Res 194: 391-402. Titus R. D., Komfeld E. C., Jones N. D., Clemens J. A., Smalstia E. B.. Fuller R. W.. Hahn R. A.. Hvnes M. D.. Mason\. R.,‘Wong D. T. and Foreman M. N. (1983) Resolution and absolute configuration of an ergolinerelated dopamine agonist, trans-4,4a,5,6,7,8,8a,g_Octa-
INGRID NYLANDER
1302 hydro-5-propyl-IH
(or 2H)-pyrazolo(3,4-g)quinoline.
J.
Med. Chern. M: 1112-1116.
Tsuruta K., Frey E. A., Grewe C. W., Cote T. E., Eskay R. L. and Kebabian J. W. (1981) Evidence that LY 141865 specifically stimulates the Dz receptor. Nature 292: 463-465. Ungerstedt U., Herrera-Marschitz M., Stlhle L., Tossman U. and Zetterstriim Y. (1985) Functional classification of different dopamine receptors. In: Dyskinesia-Research and Treatment (Casey D. E., Chase T. N., Christensen A. V. and Gerlach J., Eds), Psychopharmacology suppl. 2, pp. 1930. Springer, Berlin. Weber E., Roth K. A. and Barchas J. D. (1982) Immunohistochemical distribution of or-neoendorphin/dynorphin
and L. H. TERENIUS neuronal systems in rat brain: Evidence for co-localization. Proc. natn. Acad. Sci. U.S.A. 79: 3062-3066. Vincent S. R., Hokfelt T., Christensson I. and Terenius L. (1982a) Dynorphin immunoreactive neurons in the central nervous system of the rat. Neurosci. .%zr. 33: 185190. Vincent S., Hokfelt T., Christensson I. and Terenius L. (1982b) Immunohistochemical evidence for a dynorphin immunoreactive striato-nigral pathway. Eur. J. Pharmnc. 85: 251-252.
Zamir N., Palkovitz M., Weber E., Mezey E. and Brownstein M. J. (1984) A dyno~hiner~c pathway of Leuenkephalin production in substantia nigra. Nature 307r 643-645.