Regulation of Substance P Receptor System in Rat Striatum by Chronic Naltrexone Treatment

BRESM 70640

Regulation of substance P receptor system in rat striatum by chronic naltrexone treatment

Orisa J. Igwe

Dit'ision of Pharmacology, School of Pharmacy, Unit,ersity of Missouri-Kansas City Kansas City, MO 64108-2792 (USA)

(Accepted 2 March 1993)

Key words: Substance P receptor regulation: Naltrexone; Opioid receptor antagonism; Inositol 1,4,5-trisphosphate; Inositol 1,4,5-trisphosphate receptor; Striatum

Chronic blockade of opioid receptors by naltrexone increases opioid peptides in the striatum, and up-regulates brain opioid receptors resulting in functional supersensitivity. Striatal SP content was increased 3.5-fold after 8 days of naltrexone treatment relative to control animals. The present study was undertaken to determine whether SP receptors in the striatum and SP receptor-coupled second messenger system are modulated by increased striatal SP content induced by chronic opioid receptor blockade. The binding affinity and capacity of SP receptors, determined using [E251]Bolton_Hunter SP ([1251]BHSP) labeled at Lys3, in striatal synaptosomal membranes were not significantly altered by chronic blockade of opioid receptors. Although the concentrations of [Sar,Met (O2)II]SP, a NK-1 receptor-specific agonist, and SP(1-7), an aminoterminal major metabolite of SP, required to inhibit half of [1251]BHSP binding (ICs0) in striatal synaptosomal membranes were significantly decreased, the ICsos for SP and an NK-2 receptor-specific agonist, [Nlen~]NK A(4-10), remained unchanged by chronic naltrexone treatment. The data suggest that naltrexone which has no SP receptor antagonistic action, not only indirectly acts on SP-ergic neurons but also causes a change in the apparent affinity of NK-I receptor (as reflected by changes in IC50 values) in the striatum. Cellular inositol-l,4,5-trisphosphate [lns(l,4,5)P3], quantified by a highly sensitive and selective radioreceptor mass assay, was increased in the striatum by 28%, relative to control levels. With [~H]Ins(I,4,5)P 3 as a ligand, Scatchard analyses of the concentration-dependent saturation curves showed that the density of striatal intracellular Ins(1,4,5)P 3 receptors was increased by 53%. The levels of SP and cellular lns(1,4,5)P 3, and the density of Ins(1,4,5)P 3 receptors in the cerebellum, used as a positive control, were unchanged by chronic naltrexone treatment. The findings of opiate antagonist-induced increases in SP striatal content and Ins(l,45)P3 receptor densities, appear to support the concept of a role of endogenous opioids in the regulation of SP receptor activity. The data also suggest that inter-regulatory mechanisms exist between phospholipase C/phosphoinositide-coupled receptors such as SP receptors, and adenylate cyclase-coupled inhibitory receptors, such as opioid receptors.

INTRODUCTION same striatonigral terminal l''. As a substantial part of the striatal efferent system, SP-ergic neurons play an Substance P (SP), a member of the tachykinin family important role in the functioning of the basal of neuropeptides, is synthesized by neurons in the ganglia 1s,34. striatum 55'x3 and found in high concentrations in two of The subcellular localization of SP in striatal synap- the three primary striatal target nuclei, the entopedun- tosomes and vesicle fractions 24, are consistent with the cular nucleus and the substantia nigra pars reticu- role of SP as neurotransmitter in the striatum. Accord- lata 6'2~. Striatal medium spiny neurons are the primary ing to this role, SP has been reported to increase the source of striatal projections 38'8~'~2 and account for firing rate of some striatal neurons 5s, to evoke the 90-95% of the striatal cell population 52. Striatal neu- release of endogenous dopamine vT, and acetylchol- rons consist of two major neurotransmitter-specific ine 3'76 in the striatum. Neurotransmitter receptors in types, those containing SP, dynorphin and y-aminobu- the striatum include tachykinin receptors 34'6~ and opi- tyric acid (GABA), and those containing enkephalin old receptors of various subtypes 39, both linked to and GABA 1'9"1°'16"21'29'5l. GABA, SP and dynorphin dopaminergic and GABA-ergic neurons 54. Striatal neu- might coexist together, and with enkephalin in the rons that contain SP synapse on the somatodendritic

Correspondence. O.J. Igwe, Division of Pharmacology, School of Pharmacy, University of Missouri-Kansas City, Medical School Building; M3-104, 2411 Holmes, Kansas City, MO 64108-2792, USA. Fax: (1) (816) 235-5194. 41

tree of large intrastriatal aspiny neurons l~, shown to activity of functional striatal SP receptors coupled to selectively express SP receptors 33 and mRNA encoding phosphatidylinositol second messenger system through choline acetyltransferase32. Other neurons on which modulation of striatal Ins (1,4,5)P 3 level and Ins SP-containing neurons synapse in the striatum and (1,4,5)P 3 receptor. substantia nigra, express GABA receptors, but no SP receptors 33. Although cholinergic interneurons repre- MATERIALS AND METHODS sent a small population in the striatum 37, they exert a profound influence on striatal function89. Chemicals Mammalian tachykinin receptors are classified as Synthetic SP, [Sar 9, Met(O2)ll]SP, [p-Glu6pro9]SP(6-11) (Sep- tide), [Pro7]NKB, [Nlel°]NKA(4-10), SP(1-7), phosphoramidon, NK-1, NK-2 and NK-3 receptor subtypes, based on chyinostatin and leupeptin were purchased from Peninsular Labora- whether their putative endogenous ligand is SP, neu- tories, Inc. (Belmont, CA). Stock solutions (10 -3 M) of these com- rokinin A (NK A), or neurokinin B (NK B), respec- pounds were made in double distilled water and stored frozen in aliquots until use. Thawed aliquots were used once and discarded. tively 4°'7°. The mRNA for NK-1 receptor is abundant Tris-HC1, bovine serum albumin (BSA), disodium EDTA, bacitracin, in the striatum, consistent with the known distribution sucrose, and polyethylenimine were obtained from Sigma Chemical Co. (St. Louis, MO). d-Myo-inositol 1,4,5-trisphosphate hexasodium of SP binding sites 43'44. Although SP receptor (NK-1) salt, naltrexone HCI and hydroxypropyl-/3-cyclodextrin (cyclodextrin) expression is affected by post-transcriptional mecha- were purchased from Research Biochemical, Inc. (Nattick, MA). nisms, the molecular mechanisms underlying the regulation of its activity are unknown4°. In both neuronal Animals, drug delivery and tissue collection Male Sprague-Dawley rats (250-300 g body wt.; Sasco, Inc., and non-neuronal tissues, activation of NK-1, NK-2 or Omaha, NE) were used in all experiments. Animals were maintained NK-3 receptor-coupled to a specific phospholipase C 2 per cage, had free access to food and water, and were acclimatized (PLC), leads to the hydrolysis of phosphatidyl inositol to the holding areas for at least 24 h before use in experiments. For binding and biochemical studies involving chronic drug treatment, 4,5-bisphosphate (PIPE)4°'8s with the formation of two each animal, under ether anesthesia, was implanted with Model 2001 putative second messengers: inositol 1,4,5-trisphos- Alzet mini-osmotic pump (delivery rate = 1 /zl/h for 7 days) (Alza phate [Ins(1,4,5)P 3] which mobilizes intracellular Ca 2+8 Corp., Palo Alto, CA) subcutaneously in the dorsal subscapular space. Pumps were filled with either naltrexone HCl (70 mg/ml) or through Ins(1,4,5)P 3 receptor s4 and diacylglycerol vehicle (5% cyclodextrin in double distilled water), and maintained (DAG) which activates protein kinase C(PKC) 72. An in the animals for 7 days. Animals were sacrificed on day 8. For the excellent correlation has been observed between the study of time course alterations in striatal SP content in which longer delivery times were required, animals were implanted with Model density/distribution of [3H]SP binding sites and the 2002 mini-osmotic pumps (delivery rate = 0.5/.d/h for 14 days) filled ability of SP to stimulate PIP2 hydrolysis62, further with either naltrexone HC1 (140 mg/ml) or vehicle (control). Ani- mals were sacrificed by decapitation on days 1, 4, 6, 8, 12 and 14. providing a functional link between SP receptors and Brains were rapidly removed. Dissections to obtain the brain regions this second messenger system. Since receptor-mediated of interest were carried out immediately on an ice-chilled glass plate PIP2 hydrolysis is largely a neuronal and post-synaptic using coordinates determined from the reference atlas of Paxinos and Watson 73. membrane response 36, this intracellular second mes-

senger system may play important roles in regulating Preparation of striatal synaptosomal membranes neurotransmitter receptor signaling and functionally Striatum from individual rats was homogenized in 10 vol of interacting with other receptor-coupled effectors. ice-cold 10% (w/w) sucrose with a motor driven Teflon glass-homo- genizer (15-20 stokes, 800 rpm). The homogenate was centrifuged at SP and other tachykinins have not been shown to 1,500 × g for 5 rain, and the resulting supernatant was centrifuged at activate or inhibit adenylate cyclase activity 47. The only 20,000× g for 10 min. The crude synaptosomal pellet was lysed by second messenger system so far linked to /x, ~ and K resuspension in ice-cold 10 vols. of 5 mM Tris-HC1, pH 8.0, followed by incubation on ice for 10 min. This resuspension was centrifuged at opioid receptor subtypes is adenylate cyclase 15'59 which 39,000× g for 10 min, and the resulting pellet was resuspended once activity is inhibited by opioid receptor stimulation in more in the same buffer and recentrifuged. The final pellet was contrast to other G protein-liked receptors ~2. Further- resuspended in 50 mM Tris HCI, pH 7.4 at a dilution of 1 mg protein/ml, and aliquots were stored at - 80°C, or used fresh for SP more, in striatal neurons, neuropeptide and receptor receptor binding assay. mRNAs are under chronic synaptic control TM. These Before use in a binding assay, frozen membranes were thawed, diluted 3-fold with binding buffer (see below), and left to stand at observations have raised questions as to how SP recep- room temperature for 30 min. The suspension was then centrifuged tors are modulated in vivo by increased tissue levels of at 39,000x g for 10 min, and the resulting pellet was resuspended in SP, induced by chronic naltrexone treatment, since the binding buffer for use in binding experiments. Aliquots were removed for protein analysis performed according to the bicin- neurotransmitter receptors can be regulated by their choninic acid methods°, using BSA as a standard. own ligands (homologous regulation), as well as by other regulatory molecules (heterologous regulation) 79. Binding assays for [1251]Bolton-Hunter-labeled Lys 3 substance P The principal objective of this study is to determine ([125I]BHSP) One striatum was used per binding assay (n = 5 experiments whether chronic blockade of opioid receptors, which conducted in triplicate). Saturation experiments at equilibrium, were significantly elevates striatal SP level, also regulates the performed in conical polypropylene culture tubes, containing binding 42

buffer (composition in mM: NaCI, 120; KC1, 4.8; CaCI 2, 1.0; MgSO4, temperature, and the pellet was used to determine tissue protein 1.0; Tris HC1, 50, pH 7.4) with 3 mM MnCI 2, 1 pM to 1 nM content. [1251]BHSP (spec. act. 2200 Ci/mmol; New England Nuclear, Wil- Water soluble components in the supernatant were extracted mington, DE), and protease inhibitor cocktail prepared in the bind- with a 3:1 (v/v) mixture of Freon (1,1,2-trichlorotrifluoroethane or ing buffer and containing 0.01% BSA, 2/zg/ml chymostatin, 4/.tg/ml TCTFE) and tri-n-octylamine (2 ml of mixture/ml of TCA extract) leupeptin, 40 /zg/ml bacitracin, and 4 /~g/ml phosphoramidon. In by vortexing for 20 s. The layers were separated by centrifugation at competition studies, [125I]BHSP (50 pM) was incubated in the same 1,000× g for 2 min. The clear aqueous upper phase, containing assay buffer but with varying concentrations (0.1 pM to 10/.tM) and lns(1,4,5)P 3, was carefully transferred to clean tubes, stored at 4°C, using a concentration spacing of one log unit for each unlabeled and within 7 days of sample preparation, was assayed for Ins(1,4,5)P 3 competitor peptide. In both 'hot' saturation and competition studies, as described in the Dupont radioreceptor assay (RRA) kit (NEN, the incubation was started by adding 100 /zl of the membrane Wilmington, DE). The slightly cloudy organic bottom layer was suspension (50-100/zg protein) to the pre-equilibrated mixture. The appropriately discarded. The assay combines the use of a high final incubation volume was 500 ~1 in all experiments. specific activity tracer [3H]Ins(1,4,5)P3 with a specific binding protein After incubation at room temperature for 60 min, bound for Ins(1,4,5)P3. [125I]BHSP was separated from free ligand by rapid filtration under Briefly, sample aliquots were incubated on ice in a Tris-HCl vacuum through Whatman GF/C filters using a Brandel harvester buffer (pH 8.5) containing EDTA in the presence of a specific with a precise volume delivery timer. To minimize non-specific binding protein and tracer amounts (~ 10,000 cpm) of [3H]Ins binding, the filters were presoaked overnight at 4°C in 50 mM (1,4,5)P 3. The protein-bound fractions were then separated by cen- Tris-HCl (pH 7.4) buffer containing 0.01% polyethylenimine and trifugation, the pellets dissolved in 0.5 M NaOH, neutralized with 0.5 0.01% BSA, followed by oven drying at 70°C for 10-12 h. Before use, M HCI, and counted for radioactivity in a liquid scintillation counter filters were wetted on the filtration stage with ice-cold washing after addition of a biodegradable cocktail, Ultima Gold ® (Packard buffer (50 mM Tris-HCl (pH 7.4), 0.12 M NaCI) at 4°C). The Instruments Co., Meriden, CT). Assays with known amounts of incubation tubes were rapidly washed 4 × with 3 ml of the washing Ins(1,4,5)P 3 were determined from the calibration curve. The buffer per wash. The filter discs were carefully removed with forceps, lns(1,4,5)P 3 content of each sample was determined from the calibra- placed in polystyrene test tubes, and counted in an auto-gamma tion curve after appropriate correction for non-specific binding de- counter (Packard Instruments) with 75% efficiency. Specific binding termined in the same assay protocol. Appropriate buffer samples was defined as the difference between total binding and non-specific were taken through the TCA/TCTFE-tri-n-octylamine extraction binding. Non-specific binding was determined as the amount of protocol to provide diluent for the RRA standard curves. The cross ligand bound in the presence of 2/~M unlabeled SP. reactivity of the specific binding protein was less than 10% for Ins(2,4,5)P 3 and Ins(1,3,4,5)P4, and less than i% for other inositol phosphates. Membrane preparation for the binding of D-myo[3H]-inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) Extraction and radioimmunoassay (RL4) for SP Striatum, whole brain, or cerebellum were homogenized (Brink- SP was extracted from the striatum and cerebellum of chronic man Polytron setting 9 for 4 s) in 10 vols. of ice-cold buffer A (50 naltrexone-treated and control rats as was previously described as. SP mM Tris-HCl, pH 8.3 at 25°C, 2.5 mM EDTA, 1 mM dithiothreitol), was quantified by RIA using a commercial kit (Penninsular Labs, pelleted by centrifugation (39,000× g for 10 min), and resuspended Belmont, CA). The antiserum used for RIA has less than 0.01% in the same buffer. The particulate preparations were either used in cross-reactivity with any of the tested SP-related peptides. binding assays on the same day or the pellet was covered with buffer A and stored at - 20°C for use within 7 days. In all tissues examined, frozen membranes gave higher specific binding than fresh mem- Protein determination branes. Before use in binding experiments, frozen membranes were The pellets resulting from the extraction of Ins(1,4,5)P 3 and SP thawed, diluted 2-fold with buffer A and centrifuged at 39,000× g were washed with isotonic saline, dissolved in 1M NaOH by incubat- for 10 min at 4°C. Pelleted membranes were washed with buffer A, ing at 37°C for 3 h, and used for the determination of protein and finally resuspended in the same buffer for use in binding assays. concentration 8° to allow values to be normalized to the total protein Aliquots were used for protein determination by the bicinchoninic content of each sample. acid method 8°. In 'cold' saturation experiments, membranes were incubated with Data analysis a constant amount of [3H]Ins(1,4,5)P3 and increasing concentrations For each radiolabeled ligand, binding assays of tissue ho- of the unlabeled ligand. Binding assays were carried out in microcen- mogenates from control and naltrexone treated rats were carried out trifuge tubes and contained 500-600 /zg protein of the particulate in parallel on the same day. The data from competition studies with preparations per tube for whole brain and striatum (or 200-400 ~g [tzSI]BHSP were analyzed by the computer program EBDAe4 to protein/tube for cerebellum), 0.2 nM (~ 10,000 dpm) of [3H]Ins determine the inhibitory concentration (IC50) and the dissociation (1,4,5)P 3 (spec. act. = 20-60 Ci/mmol, Amersham Corp., Arlington, constant for each competitor (K,). On the basis of the raw data from IL), and different concentrations (0.1-100 nM) of unlabeled the ligand-binding studies, the EBDA program was used to process Ins(1,4,5)P3. The reaction was started by adding tissue suspension the data from both [12SI]BHSP and [3H]Ins(1,4,5)P3 binding assays (250 pA) to the mixture to give a final incubation volume of 0.5 ml, into a form that could be further analyzed by the non-linear curve- and vortexing the tubes. Incubations were carried out for 20 min on fitting program LIGAND 68. ice, and stopped by centrifugation at 13,600× g for 5 min at room Statistical analyses of binding data were carried out by 1-way temperature followed by aspiration of the supernatant. Pellets were analysis of variance (ANOVA), followed by Scheffe's F-test of multi- resuspended in 5 ml of Ready Protein + scintillation cocktail (Beck- ple comparisons, using a commercial computer software (Statview). man) and the radioactivity determined in a Beckman LS3801 counter Statistical significance between SP and Ins(1,4,5)P 3 tissue contents in with a counting efficiency for 3H of 45%. Non-specific binding was control and naltrexone-treated rats were determined using a 2-tailed determined in the presence of 1/~M Ins(1,4,5)P 3, and was subtracted unpaired Student's t-test with a probability of < 0.05 taken as from total binding to yield specifically bound [ 3H]Ins(1,4,5)P 3. significant. All values are reported as mean _+ S.E.M.

Extraction of and radioreceptor assay for [3H]Ins(1,4.5)P3 RESULTS The striatum or cerebellum (80-100 mg wet wt.) were homogenized in 2 ml of ice-cold 1 M trichloroacetic acid (TCA) (Brinkman Naltrexone-induced alterations in striatal SP let,el Polytron setting 8 for 8 s). The homogenate was incubated on ice for 15 rain, and centrifuged at 1,000× g for 10 min at 4°C. The super- The time course of alterations in SP content in the natant was removed and further incubated for 15 min at room striatum following chronic blockade of opioid receptors 43

300 significantly different from the values on day 6 or day 12. Thus, striatal SP content remained elevated iiiiiiiiii i!iiiiiiiii throughout naltrexone treatment. 200 ,,-,,,,,,. ,{~,~,~,~ ,,,,,,,,,, ~,~,~,~,~,: 3;,;,;,;' ;,;,:,;,~- .;,;-~,;,;, ,;,;,;,;-; Number and affinity of [125I]BHSP binding sites The concentration dependence of SP binding at equilibrium was determined in the presence of increas- 100 .';,';.';,';," ing concentrations of [125I]BHSP. To determine the number and affinity of SP receptors, synaptosomal membranes from the striatum of control and naltrexone-treated rats were incubated with [125I]BHSP in the :?;?;?;?;? ;?;?;';';' ;?;?;?:?:i ;?;:;?;?;? ";?;?;:;?;: 0 ..... presence or absence of 2 /zM unlabeled SP. Non- 1 4 6 8 12 14 specific binding represented 5-20% of the total bind- # of Day• Treated wAh NaRrexone ing throughout the study, with the higher values occur- Fig. 1. Time course of naltrexone-induced changes in striatal SP ring with ~25I]BHSP older than 10 days from date of level. SP was extracted and quantified by RIA as described under Materials and Methods. Histograms represent the mean+S.E.M. manufacture. Specific binding was saturable with in- from 3-4 independent experiments (each experiment = 1 animal creasing ligand concentration (Fig. 2A). Scatchard examined independently) * P < 0.05, significantly different from con- analyses of the saturation curves from naltrexone- trol (unpaired Student's t-test). treated and control rats yielded straight lines (Fig. 2B), indicating that SP binds to a single population of sites was investigated (Figure 1) to determine the point at in striatal synaptosomal membranes even following which optimal effects occur. SP was extracted from the chronic blockade of opioid receptors. The dissociation striatum of rats treated with naltrexone for 1, 4, 6, 8, 12 constant (K d) and binding capacity (Bmax) determined or 14 days and quantified by RIA. At all the time following naltrexone treatment were not significantly points examined except for the first day, striatal SP was different relative to control membranes (Fig. 2B). Hill significantly elevated above control values. A 1.7- and a plots generated from [125I]BHSP binding data in both 2.8-fold increase were observed on days 4 and 8, re- control and naltrexone-treated membranes were spectively, following naltrexone treatment. Although monophasic and showed no indication of cooperativity the highest increase in SP content in absolute amount for striatal SP receptors (Hill coefficient = 1.02 ___ 0.2; appears to be on day 8, the value on day 8 was not n = 12). The data indicate that the density of SP recep-

e- 20 A • Control lS .... -o.--- Nalt-Treated B • Control o o Nan-Treated Q.

01 o.o°°°° ~ i.i 15 0 . """" I I x E O v I e 10 "O .c_ c- | O m 10 n

m E o 0 E 4) O. (n Q D. u) L "I- CD ! ,-,1 i 0 m 0 I I I I I I 0 0 200 400 600 800 1000 1200 0 5 10 15 20

1251-BHSP Concentration (pM) 1251-BHSP Bound (fmol/mg protein) Fig. 2. Saturation binding isotherms of [125I]BHSP (A) in control and chronic naltrexone-treated (NALT-treated) rat striatal synaptosomal membranes. Binding assays were performed with increasing concentrations of [IzSI]BHSP (1-1000 pM) in the absence (total binding) and presence (non-specific binding) of 2/zM unlabeled SP. Each data point represents the mean 5: S.E.M. of 5 independent experiments performed in triplicate. B is a Scatehard plot transformation of the data in A with K d = 0.185:0.04 nM and Bmax = 18.45:1.9 fmol/mg protein (for control) and K a = 0.21 5:0.02 nM and Bmax = 22.9 5:1.5 fmol/mg protein following chronic NALT treatment. 44

TABLE I Substance P-like immunoreactiL,ity (SP-Li) and Inositol 1.4,5-trisphosphate[Ins(1,4,5)P~] contents in rat brain regions following chronic naltrexone treatment Rats were treated with either naltrexone or vehicle for 8 days. Brains from control and naltrexone-treated animals were dissected into striatum and cerebellum. Radioimmunoassay (SP) and radioreceptor assay Ins(1,4,5)P3 were performed as described in Materials and Methods. Values are mean± S.E.M. from 4-6 independent experiments (i.e., one animal = one experiment) performed in duplicate.

Brain region SP-Li (pmol / mg protein) % Change Ins(1,4,5)P3 (pmol / mg protein) % Change Control NAL T-treated Control NAL T-treated Striatum 0.72 _+ 0.10 2.49 ± 0.48 * + 246 127 _+ 37 162 ± 38 + 28 Cerebellum 0.07_+ 0.01 0.06 ± 0.01 - 14 205 _+ 5 246 ± 73 + 20

Statistically significant difference, *P < 0.05 (unpaired 2-tailed Student's t-test). tors in the striatum was not significantly altered follow- specific binding of [1251]BHSP to striatal synaptosomal ing naltrexone treatment, although striatal SP content membranes following chronic blockade of opioid re- was significantly elevated (Table I). ceptors (Table III). The overall rank order of potency of competitors was SP = [Sar '~, Met(Oz)II]sp >>> SP(1- Competitive inhibition of [ 125I]BHSP binding 7) = [p-Glu ~, Pro 9] SP(6-11) >> [NleI°]NK A(4-10) Different SP-related peptides (0.001 nM to 10/zM) >>>>[Pro7]NK B using striatal control membranes, were tested for their ability to competitively inhibit the whereas that using striatal membranes derived from naltrexone-treated rats was [Sar ~, Met(O2)tq SP >> SP >> SP(1-7)> [p-Glu ~, Pro~]SP(6-11) >> [Nlem]NK TABLE II A(4-10) >>>> [Pro 7] NK B. Chronic naltrexone treat- lnositol 1, 4,5-trisphosphate {Ins(1, 4,5) P3] receptor characteristics in rat brain membrane preparations ment did not significantly change the inhibitory po- Particulate membrane preparations (500-700 ~g protein) from whole tency of SP, the endogenous ligand for NK-1 receptor. brain, cerebelum and striatum in control and naltrexone-treated rats However, the concentration of [Sar 9, Met(O2)ll]SP, an were incubated with a single concentration of [3H]Ins(1,4,5)P3 and NK-1 receptor specific agonist 25 needed to half maxi- eight (8) concentrations of unlabeled Ins(1,4,5)P3 under conditions described in the Materials and Methods section. " K,~ and Bm~~ mally inhibit [125I]BHSP binding (ICs0), was decreased values are mean _+ S.E.M. of 5-7 independent experiments con- by about 5-fold in striatal membranes derived from ducted in triplicate. naltrexone-treated rats relative to control. The (ICs0) Brain region Control NAL T-treated for [p-Glu 6, Pro~]SP(6-11) or septide, was unaffected by chronic naltrexone treatment, but the inhibitory K a (nM) Bm,.~(pmol/ K a (nM) Bin, ~. (pmol/ mg protein ) mg protein) potency of SP(1-7), a primary N-terminal metabolite Whole brain 14.3_+4.0 a 3.0_+0.3 ~ 15.5±2.1 4.5±0.01 * of SP in the CNS TM, was increased 2-fold relative to Cerebellum 14.9±2.2 15.3_+2.8 13.6_+ 1.2 16.6_+3.1 control. With increased striatal synthesis/release (and Striatum 15.9± 1.9 0.9±0.2 20.2±4.0 1.8±0.2 * possibly catabolism) of SP, the present data suggest For comparisons of values between and within control and naltrex- that the putative binding sites for SP(1-7) 49 might be one-treated groups. Ft. w = 7.9 (P = 0.018), Fi. s = 28.4 (P _< 0.001), increased in the striatum following chronic blockade of *P _< 0.05 (ANOVA followed by Scheffe's F-test of multiple comparisons). opioid receptors. The inhibitory potency of [NleW]NK

TABLE IIl Inhibition parameters of [1eSl]BHSP specific binding to synaptosomal membranes from rat striatum Binding of IzsI-BHSP (50pM) was determined in the presence or the absence of different concentrations (10 ~q to 10 5 M) of competing peptides. A concentration spacing of one log unit was used for each inhibitor. The concentration of the competing peptide needed to cause 50% inhibition of specific binding (IC50) and the apparent inhibition constant, K i, were calculated using the computer program EBDA64. Values for IC5o and K i are the mean ± S.E.M. of 3-4 independent experiments conducted in triplicate. Ratio is control ICso/naltrexone-treated IC50.

Peptide Control NAL T-treated ICso ratio ICso (nM) K, (nM) IC5o (nM) K, (nM) SP 1.15_+0.85 0.81_+0.57 0.65_+0.17 0.54_+0.15 1.8 [Sar q, Met(O2)l 1]SP 0.42 _+ 0.06 0.33 _+ 0.05 0.09 _+ 0.05 * 0.08 _+ 0.02 * 4.7 [Nle IO]NK A(4-10) 2 302 + 385 1876 ± 298 2 212 ± 675 1821 ± 546 1 [ProV]NK b > 100,000 > 100,000 > 100,000 > 100,000 N.D. [p-Glu 6, Prog]SP(6 - 11 ) 744 ± 202 578 _+ 146 660 _+ 184 516 ± 138 1.1 SP(1-7) 562±232 454_+ 198 264±79 * 207±61 * 2

For comparisons between and within control and naltrexone-treated groups, FI.4 = 24 (P < 0.01), F1, 4 = 25.8 ( P _< 0.01); F3,8 = 28.3 (P _< 0.001), F3. s = 36 (P _< 0.001), *P _< 0.05 (ANOVA followed by Scheffe's F-test of multiple comparisons). N.D., not determined. 45

A(4-10), an NK-2 receptor-specific agonist 25, was un- Number and affinity of Ins(1,4,5)P3 binding sites affected by chronic naltrexone treatment. [Pro7]NKB, a In 'cold' saturation studies, specific binding of specific NK-3 receptor agonist 57, was virtually inactive Ins(1,4,5)P 3 to striatal membranes was saturable at as a competitor for striatal 125I]BHSP binding sites about 45 nM (Fig. 3A) in both control and chronic with several orders of magnitude (> 100,000) lower naltrexone-treated rats. Scatchard plots of saturation than the homologous ligand for SP. This data further data were linear, showing specific binding to a single confirms the low density of NK-3 receptors in the class of sites (Fig. 3B). Under the conditions of the striatum 4°. binding assay, two saturation experiments (out of six) showed evidence of two sites in the striatum, but Coordinate alteration in SP and Ins(1,4,5)P 3 tissue levels LIGAND analysis failed to give statistically significant To determine whether SP and Ins(1,4,5)P 3 contents two-site fits. Table II shows the binding constants for were altered in a coordinate fashion following chron- Ins(1,4,5)P 3 obtained in whole brain, cerebellum and ic-naltrexone treatment, SP and Ins(1,4,5)P 3 were ex- striatum. Naltrexone-treatment did not significantly al- tracted from the striatum and cerebellum of control ter the binding affinities in the tissues examined, but and naltrexone-treated rats. Striatal SP and Ins(1,4,5)P 3 the mean binding capacity (Bmax) was increased by contents, quantified by RIA and RRA, respectively, 33% and 53% in whole brain and striatum respectively, were normalized to the total protein content of each following chronic opioid receptor blockade. Hill coeffi- tissue, as detailed in Materials and Methods. Striatal cients were close to unity in both whole brain and the SP content, following opioid receptor blockade for 8 d, brain regions examined, showing that no cooperativity increased by 3.5-fold relative to control (Table I). No was involved in the binding. In preliminary experi- significant changes in SP levels were apparent in the ments (3 × ), a single i.p. injection of naltrexone (10 cerebellum, further supporting the specificity of nal- mg/kg body wt.) 24 h before sacrifice, produced no trexone treatment in inducing increased SP in specific change in the density of striatal Ins(1,4,5)P 3 receptors brain regions as was reported earlier 87. Ins(1,4,5)P 3 (data not shown) compared to vehicle-treated controls. contents of the striatum and cerebellum were not sig- Thus, it is unlikely that the increased Ins(1,4,5)P 3 den- nificantly altered by chronic naltrexone treatment rela- sity observed in the striatum in response to chronic tive to control values (Table I). naltrexone treatment is due to an increased capacity of

1.84 A 12 ~ B s Control Control .... •o---- Nalt-Treated k- o Nalt-Treated A ooooO°° 10 1.39 ,°o,ooo°oOO°°°°'°°°° ,

0.93

4 0.48

0.02 i I I I I I I 0 i ~ I i ~, 0 15 30 45 60 75 90 105 1

Ins(1,4,5)P3 Concentration (nM) [3H]lns(1,4,5)P3 Bound (pmol/mg protein) Fig. 3. Saturation binding isotherms of [3H]InsP3 (A) in striatal membranes derived from control and naltrexone-treated (NALT-teated) rats. Binding assays were performed with increasing concentrations of unlabeled Ins(1,4,5)P 3 (0.1-100 nM) and a single concentration of [3H]Ins(1,4,5)P3. Non-specific binding was determined in the presence of 1 /zM unlabeled Ins(1,4,5)P 3. Each data point represents the mean+S.E.M, of 6 independent experiments performed in triplicate. B shows a Scatchard plot transformation of the data in A with a K d = 15.9 + 1.9 nM and Bma~ = 0.9 + 0.2 pmol/mg (for control) and a K a = 20.2 + 4 nM and Bmax = 1.8 5:0.2 pmol/mg protein following chronic NALT treatment. 46

the same number of active receptors, i.e., the data ized to two different striatal neuronal subpopula- suggest that new Ins(1,4,5)P 3 receptors were synthe- tions 32'74 suggesting that naltrexone acts on multiple sized by chronic naltrexone treatment. The data also types of striatal neurons. The increased striatal SP suggest that naltrexone-induced up-regulation of content following opioid receptor blockade also sug- Ins(1,4,5)P 3 receptors is specific for striatum as was the gests that opioids are required for normal control of increase in SP content. The cerebellum, which exhibits processes which govern striatal neuropeptide levels. the highest density of Ins(1,4,5)P 3 receptors in the rat Indeed, motor deficits in certain pathological states brain 9° and low SP tissue level and SP receptor den- (e.g., Parkinson's disease) may be due, in part, to sity 75, was unaffected by naltrexone treatment. The functional alterations in striatal peptidergic neurons high density of cerebellar Ins(1,4,5)P 3 receptor and the since striatal dopaminergic system is involved in the low level of cerebellar SP content were confirmed in regulation of the activity of neurons producing SP and this study. opioid peptides 34. Available evidence also show that dopaminergic terminals make direct synaptic contacts DISCUSSION with SP-positive neurons in the striatum 56, and opioid receptors on dopaminergic terminals participate in the The present study examined the effects of chronic regulation of striatal SP metabolism 2°. Interestingly, opioid blockade by naltrexone on several components intranigral SP enhances dopamine release in the stria- of the substance P system. The data show that chronic tum 41, but the metabolically generated SP(1-7) TM an- naltrexone a) increased striatal substance P levels, b) tagonizes SP-induced dopamine release 42. In the pre- had little effect on binding sites for [125I]BHSP, and c) sent study, the IC50 value for SP(1-7) was decreased did not significantly increase the levels of striatal 2-fold following chronic blockade of opioid receptors Ins(1,4,5)P3, but increased the number of binding sites (Table III). Conceivably, the metabolic regulation of for Ins(1,4,5)P 3. In addition, data on inhibition of sub- striatal SP could result in positive feedback modulation stance P binding indicated that several analogs of sub- of SP functions and of plasticity in striatal circuits. The stance P inhibited binding of [125I]BHSP at lower con- coordinate increases in enkaphalin and SP biosynthe- centrations in naltrexone-treated tissue than in control ses 87 cannot result exclusively from modulation of dop- tissue. Since opioid antagonists do not directly interact amine release, and complex interactions among neu- with tachykinin receptors, the data could not have ronal subpopulations must be proposed. Taken to- arisen by direct antagonist blockade of SP receptors. gether with the localization of enkephalin in striatopal- Opioid agonists have not been shown to have any lidal neurons and SP (and dynorphin) in striatonigral effect on PIP e hydrolysis 6°. Despite a considerable neurons, the present data suggest that naltrexone mod- effort, the only second messenger system so far clearly ulates neurons contributory to both the striatopallidal established for all opioid receptors is inhibition of and striatonigral pathways. adenylate cyclase 15"59. Therefore, the results in the A myriad of functional links exists in the striatum present study cannot be due to direct effect of endoge- between SP and endogenous opioid systems in the nous opioids on PIP, hydrolysis. modulation of sensory input. The inhibition of SP Neurotrophic and neuroprotective effects of SP have release in the striatum by enkaphalin 67 has been postu- recently been demonstrated in 6-hydroxydopamine-in- lated to occur either postsynaptically or 'parasynapti- duced lesion of the substantia nigra 63. With a relatively cally', via diffusion of SP z° as there is no ultrastruc- high density of SP receptor in the striatum 43'44 as rural evidence for axo-axonic synapses between en- confirmed in this study, and the close anatomical and kephalin and SP elements 46. Conceivably, the increase functional interactions between dopamine and SP in in striatal SP contents observed in this study might the striato-nigral system 56, it appears that an increase result from enhanced tonicity of opioid peptides 86, thus in SP and the density of its second messenger-coupled disinhibiting SP neurons. This disinhibition would cause receptor in the striatum brought about by chronic increased SP release and catabolism, necessitating an opioid blockade, would have value in future therapeu- increase in PPT mRNA 87, and SP in the striatum. tic strategies related to recovery from brain damage or Components of second messenger system involved in its prevention. modulation of neurotransmitter release are present in The results in the present study confirm the increase the striatum, e.g., CaZ+-dependent protein kinase C in striatal SP levels (Table I) following chronic opioid (PKC), Ins(1,4,5)P 3 receptors 9°, cAMP-dependent pro- receptor blockade, as found by other investigators s7. tein kinase (PKA) 31, and L and/or N Ca 2÷ Thus, chronic naltrexone treatment regulates SP and channeIst9'53. enkephalin, two neuropeptides that are largely local- Curiously, chronic naltrexone treatment did not sig- 47

nificantly change the binding capacity (Bmax) of Cellular Ins(1,4,5)P 3 is increased by a number of ago- [125I]BHSP to striatal synaptosomal membranes, al- nists interacting with cell-surface receptors coupled to though it elevated striatal SP. The inhibition data PLC 7A7. Changes in the concentration of this second (Table III) suggest that the density of NK-1 receptor messenger have been causally linked to the mobiliza- might be increased. Taken together, the data (Table tion of non-mitochondrial Ca 2÷ stores 69 by binding to III) provides an indirect evidence that [125I]BHSP may an Ins(1,4,5)P 3 receptor protein 84, shown by functional be binding to multiple NK-1 sites within the same reconstitution studies to contain Ins(1,4,5)P 3 recogni- receptor complex, suggesting heterogeneity of striatal tion site and its associated calcium channel 28. NK-1 receptors and their possible differential regula- In the present study, a novel RRA method was used tion following chronic opioid receptor blockade. The to monitor changes in mass accumulation of Ins differences in IC50 values are unlikely to arise from (1,4,5)P3, thus circumventing problems that can arise in differential affinities of [125I]BHSP in striatal mem- [3H]-inositol prelabeling protocols 4. The advantage of branes since the binding affinities are similar in control the RRA for Ins(1,4,5)P 3 over the more conventional and following chronic naltrexone treatment (Table II). technique, which determines changes in the incorpora- It also appears unlikely that SP (at the concentrations tion of radiolabeled myo-inositol into inositol phos- used in this study) is binding to either NK-2 or NK-3 phates, is that it allows absolute quantification of the receptors since [Nlel°]NK A(4-10) and [Pro7]NK B are amount of the inositol phosphate present, and avoids poor inhibitors of SP binding. Two isoforms of human possible complications arising from changes in specific NK-1 receptors with differential activation of intracel- radioactivity of different inositol phosphate pools 14. lular effectors have been cloned and characterized Furthermore, the assessment of Ins(1,4,5)P 3 rather than suggesting > 92% sequence homology between rat and its degradation products removed the complication of human gene 3°. Furthermore, functional in vitro studies inositol phospholipids other than PIP 2 undergoing hy- using early peptide antagonists have suggested the exis- drolysis in the striatum at the time animals were sacri- tence of subtypes of NK-1 receptors 13 and more recent ficed. The results of the present study indicates that studies using NK-1 specific non-peptide antagonists the basal Ins(1,4,5)P 3 level is relatively high in the rat appear to confirm this heterogeneity2,91. striatum and was 28% higher following chronic naltrex- SP receptor binding studies have shown dense bind- one treatment. Interestingly, the magnitude of striatal ing in the striatum, but with a clear absence of binding SP increase did not correspond to that of the increase within the substantia nigra 23. Also, chronic naltrexone in striatal Ins(1,4,5)P 3. This difference suggests that treatment increased SP level in the striatum with no newly synthesized SP is not available for release, change in SP level in the substantia nigra87. Within the and/or that tissue PIP 2 hydrolysis is under other physi- striatum, SP has been shown to induce acetylcholine ological regulation5°. An important question raised by release 3'76, suggesting a functional role for striatal SP. this data then, is the functional significance of the Intrastriatal cholinergic neurons selectively express the change in SP content, assuming that some of the in- SP receptor which is not expressed by other striatal crease in striatal Ins(1,4,5)P 3 resulted from increased neurons 33. Speculatively, increased striatal SP follow- SP synthesis, release and receptor activation. Would ing chronic blockade of opioid receptors would in- this magnitude of change reflect, on the part of SP- crease SP release (and catabolism), activating SP re- containing neurons, an adaptive response, or an in- ceptors on cholinergic neurons and causing release of crease in the activity of cholinergic SP receptor33? acetylcholine from these neurons 34. This mechanism Since the cerebellum has very low SP and endogenous may account for interaction between enkaphalin-con- opioid contents but high Ins(1,4,5)P 3 content and taining striatopallidal neurons and SP/dynorphin-con- Ins(1,4,5)P 3 receptor density, cerebellum was used as a taining striatonigral neurons as they also express mus- positive control in this study. Neither cerebellar SP and carinic receptors. Conceivably, complex interactions Ins(1,4,5)P 3 or Ins(1,4,5)P 3 receptor density was af- occur between subpopulations of striatal output neu- fected by chronic naltrexone treatment (Tables I and rons and cholinergic interneurons to maintain a bal- II). These findings suggest that changes observed in ance in the two output pathways. striatal Ins(1,4,5)P 3 content might be due to increased Functional SP receptors in both neuronal and non- activity of SP neurons in the striatum. neuronal tissues are associated with increased hydroly- Although chronic blockade of opioid receptors pro- sis of PIP62"88 yielding Ins(1,4,5)P 3 and DAG. Hydroly- duced a significant increase in striatal SP with no sis of PIP 2 might serve a number of functions in nerve elevation in SP receptor density, it markedly increased cells, such as, excitability, secretion of neurotransmit- striatal Ins(1,4,5)P 3 receptor density with a modest ters, post-tetanic potentiation and differentiation2s. increase in cellular Ins(1,4,5)P 3. These findings provide 48 preliminary evidence that the relationship between SP The data suggests that an interregulation exists in the occupancy of its receptor and functional response in- striatum between SP receptors coupled to PLC/phos- duced by PIP 2 hydrolysis might be altered by chronic phoinositide system and opioid receptors coupled to naltrexone treatment. The possible mechanism for such adenylate cyclase, although these receptors are local- an alteration in 'cell surface receptor' - 'second mes- ized in different neuronal subpopulations. senger receptor' coupling is not understood. The mechanism underlying the increase in the density of striatal Acknowledgments. I thank Dr. Cynthia Nyquist-Battie for com- ments and critical reading of the manuscript and Ms. Sue Khan for Ins(1,4,5)P 3 receptor following chronic blockade of opi- secretarial support. This work was supported by a UMKC Faculty oid receptors is not clear. 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