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High-affinity neurotensin receptor is involved in phosphoinositide hydrolysis stimulation by carbachol in neonatal rat brain
Developmental Brain Research 154 (2005) 247 – 254 www.elsevier.com/locate/devbrainres
Research report
High-affinity neurotensin receptor is involved in phosphoinositide hydrolysis stimulation by carbachol in neonatal rat brain S. Pereyra-Alfonsoa, M.G. Lo´pez Ordieresa,b, M. del V. Armaninoa, G. Rodrı´guez de Lores Arnaiza,b,* a
Instituto de Biologı´a Celular y Neurociencias bProf. E. De RobertisQ, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1121) Buenos Aires, Argentina b Ca´tedra de Farmacologı´a, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, (1113) Buenos Aires, Argentina Accepted 17 November 2004 Available online 11 January 2005
Abstract Ontogenetic studies indicate that inositol phosphate accumulation in rodent brain tissue by cholinergic muscarinic agonists as well as expression of high-affinity neurotensin receptor (NTS1) peak at 7 days after birth. Herein, potential participation of this receptor in such effect was investigated. Cerebral cortex prisms of 7-day-old rats were preloaded with [3H]myoinositol and later incubated during 60 or 20 min in the presence of muscarinic agonist carbachol plus neurotensin and SR 48692, a non-peptide NTS1 antagonist. In 60-min incubation experiments, inositol phosphate accumulation by 10 3 M carbachol was roughly 320%, an effect which remained unaltered plus 10 6 M to 10 4 M neurotensin but partially decreased with equimolar SR 48692 concentration. In 20-min incubation experiments, inositol phosphate accumulation by 10 3 M carbachol was circa 240%, a value which attained 320–360% plus 10 7 M neurotensin; this effect was totally blocked by 10 7 M SR 48692. It was concluded that in inositol phosphate accumulation by carbachol, besides the cholinergic muscarinic receptor, the NTS1 receptor is likewise involved; findings at 60 min are attributable to the effect of endogenous neurotensin whereas those at 20 min most likely involve both endogenous and exogenously added peptide. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Peptide receptor structure and function Keywords: Neurotensin; Neurotensin receptors; Carbachol; Phosphoinositide hydrolysis; Neonatal brain cortex
1. Introduction Phosphoinositide (PI) metabolism is enhanced by activation of several brain neurotransmitter receptors, an effect which takes place through G proteins [14,15]. It has been described that muscarinic agonists acetylcholine and carba-
Abbreviations: IP3, inositol 1,4,5-triphosphate; IPs, inositol phosphates * Corresponding author. Instituto de Biologı´a Celular y Neurociencias bProf. E. De RobertisQ, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1121) Buenos Aires, Argentina. Fax: +54 11 4508 3645 or 4964 8274. E-mail address: [email protected] (G. Rodrı´guez de Lores Arnaiz). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.11.003
chol, a nonhydrolyzable analog of acetylcholine, stimulate inositol phosphate (IP) accumulation in rat brain cortex slices, an effect recorded on day three after birth with a peak at day seven and a progressive decrease thereafter [2,4]. Neurotensin is a tridecapeptide present in mammalian central nervous system (CNS) and peripheral tissues, whose widespread distribution in cell bodies and nerve terminals in the brain suggests that it may play a major role in neurotransmission or neuromodulation, subserving diverse physiological CNS functions [35,52]. In adult rat brain, neurotensin can bind to two distinct sites, one of high and the other of low affinity, corresponding to NTS1 and NTS2 receptor, respectively [53]. Structurally unrelated to these
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two receptor types, a third one (NTS3) has been described [28]. Physiological neurotensin effects seem due to its interaction with high-affinity sites, which can be blocked by a selective non-peptide neurotensin receptor antagonist termed SR 48692 [20]; therefore, this substance is a valuable tool for investigating neurotensin action in numerous experimental models [40]. The ability of neurotensin to stimulate PI hydrolysis has been documented [18,31]. NTS1 receptor has been linked to a variety of transductional effects including Ca2+ mobilization as well as stimulation of cGMP and inositol phosphate production [53]. Anatomical, physiological, and pharmacological evidence point to a relationship between muscarinic cholinergic and neurotensinergic systems. Immunoreactive neurotensin has been detected in nerve endings innervating Meynert basal nucleus [23,55]. Neurotensin is localized throughout the cytoplasm and along the plasma membrane of cholinergic magnocellular neurons, where neurotensin receptors are present on the perikaryia and proximal dendrites [46,47]. Monoiodo-neurotensin-labeled binding sites are selectively associated with cholinergic nerve cell bodies in the diagonal band and substantia innominata, suggesting that endogenous neurotensin may directly influence forebrain cholinergic function in the CNS [45]. The association of high-affinity neurotensin binding sites with basal forebrain cholinergic neurons has been reported [13]. Autoradiography of classical and neuropeptide receptor distribution disclosed that neurotensin and muscarinic receptors follow a similar distribution in human cortex, with the highest densities occurring in the most superficial gray matter areas [22]. Patch-clamp studies in nucleus basalis neurons indicate that neurotensin excites cholinergic neurons by reducing inwardly rectifying K+ conductance [16]. When added in vitro, neurotensin is able to modify ligand binding to muscarinic receptor in CNS membranes [33,43]. Interestingly, neurotensin administration into rat hippocampus enhances acetylcholine and GABA extracellular levels. These changes were prevented with SR 48692 and bicuculline, suggesting that neuromodulatory action of neurotensin on release of such neurotransmitters is due to the interaction with high-affinity neurotensin and GABAA receptors present in cholinergic nerve endings [36]. The coupling between neurotensin receptors, PI turnover and intracellular Ca2+ mobilization in HT-29 colonic epithelial cells has been demonstrated [9,50,54]. However, potential involvement of neurotensin receptors in brain PI hydrolysis stimulation by carbachol has not yet been explored so far. Ontogenic studies have disclosed that rodent mRNA NTS2 is detected only from postnatal day fourteen [41] whereas mRNA NTS1 is already present at birth and peaks at day seven [21,27,42] and maximal brain IP accumulation by carbachol also peaks at day seven [2]. Based on these facts, NTS1 antagonist SR 48692 was used to test the participation of high-affinity neurotensin receptor
on carbachol-induced PI hydrolysis in cerebral cortex of neonatal rats.
2. Material and methods 2.1. Materials Reagents were analytical grade. Carbachol, myoinositol, and neurotensin acetate were purchased from Sigma (St. Louis, MO, USA). Peptide solutions in bidistilled water were freshly prepared for each experiment. Dimethyl sulfoxide (DMSO) was purchased from J.T. Baker Chemical Co.(Phillipburg, N.J.). A Dowex anion exchange resin (AG 1-X8, 100–200 mesh, formate form) was from Bio-Rad Laboratories (Richmond, CA, USA). SR 48692 {2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxy phenyl) pyrazol 3-yl) carbonylamino]tricyclo (3.3.1.13.7) decan-2carboxylic acid} was kindly provided by Dr. Danielle Gully, SANOFI Recherche, France. OptiPhase bHisafeQ 3 was purchased from Wallac Oy (Turku, Finland). [3H]myoinositol (20 Ci/mmol) was from New England Nuclear (Boston, MA, USA). 2.2. Animals Wistar neonatal rats (6–7 days old) of either sex were used, considering bday 0Q the day of birth. All studies described were conducted in accordance with the Guide for Care and Use of Laboratory Animals provided by the National Institutes of Health, USA. 2.3. Determination of [3H]-inositol phosphates (IPs) A procedure based on described methods [7,10], with modifications, was performed. The cerebral cortex of three neonatal rats were processed for each experimental procedure. Pooled tissue was placed on ice on a Petri dish with gassed Krebs–Henseleit buffer containing (mM): NaCl, 120; KCl, 4.7; CaCl2, 1.3; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; and glucose, 11.7, equilibrated to pH 7.4 with O2/CO2 (95:5); tissue was lightly minced [51] and prisms dispersed at 10% (w/v) in the same buffer. Samples were incubated in bulk at 37 8C for 1 h under gentle shaking with an intermediate change of buffer, followed by 60 min incubation with [3H]myoinositol (6 ACi/ml; final concentration 3 10 7 M) and four washes with fresh buffer replaced every 5 min under O2/CO2 (95:5). Fifty microliters of packed prisms containing 1.1 F 0.10 mg protein (mean values F SEM, n = 16) were transferred to tubes with 0.24 ml of the same buffer which contained LiCl (7.5 mM final concentration, with NaCl iso-osmotically reduced), and the indicated additions to 0.3 ml final volume. To test drug effect, prisms were incubated for 15 min in the presence or absence of SR 48692 dissolved in DMSO 10% (v/v), then carbachol alone or neurotensin plus carbachol were added
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and incubation proceeded under O2/CO2 (95:5) with shaking at 37 8C for 20 or 60 min, as indicated in the figure legends. Incubation was carried out for 0 to 60 min to investigate time course of IPs accumulation. Prism samples preloaded with [3H]myoinositol were processed throughout without additions (in duplicate) to determine basal IPs accumulation. Incubations were stopped by the addition of 940 Al chloroform:methanol (1:2), followed by chloroform (310 Al) and bidistilled water (310 Al) to separate phases. Tubes were vortex-mixed for 15 s, then centrifuged at 1000 g for 10 min to facilitate phase separation. Radiolabeled IPs were separated from inositol by ion-exchange chromatography using small columns containing 0.5 ml of a 50% slurry AG 1-X8 resin in the formate form. Upper phase aliquots (750 Al) diluted to 3 ml with bidistilled water were added to the resin suspension, centrifuged, and washed 4 times with 3 ml of myoinositol (5 mM). [3H]-IPs were eluted with 1 ml of 1 M ammonium formate/0.1 M formic acid and 800 Al of this eluate added to 10 ml of OptiPhase bHisafeQ 3 and counted in a Tracor Analytic scintillation spectrometer with 30% efficiency. The relatively long time of incubation (10–60 min) did not allow to measure the accumulation of the more polar hydrolysis products inositol-1,4-diphosphate and inositol1,4,5-triphosphate, since changes in these compounds are detectable at very short times after addition of receptor agonists to the incubation buffer [6]. However, since the concentration of ammonium formate employed allows the recovery of all IPs, the term IPs instead of IP1 (inositol monophosphate) was used. Radioactivity in the lipid fraction was monitored by counting aliquots from the lower organic phase (200 Al) after drying overnight at room temperature and adding OptiPhase bHisafeQ 3 solution with 45% efficiency. In all experiments, aliquots containing the same amount of brain tissue (that is, similar intraexperimental protein content) were processed, which allowed the comparison between basal condition and treatment in the same sample. Accumulation of [3H]-IPs was thus expressed as the percentage of basal value (without additions).
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After 60-min prism incubation, basal [3H]-IPs accumulation was 5092 F 270 dpm per mg protein and total [3H]inositol incorporation into phospholipids was 70,462 F 1700 dpm per mg protein (mean values F SEM from 10 experiments). In the presence of 10 3 M carbachol, IPs accumulation was roughly 260% of basal value; such IPs accumulation was decreased 26% by 10 4 M SR 48692 but it was almost unaffected by the antagonist at 10 6 M concentration (Fig. 1A). To serve as controls, experiments in the presence of SR 48692 either at 10 6 or 10 4 M
2.4. Protein measurement Protein was determined by the method of Lowry et al. [24] using bovine serum albumin as standard.
3. Results Labeling of inositol phosphates (IPs) and inositol lipids in brain prisms of neonatal rats was studied after incubation with [3H]myoinositol in the presence or absence of carbachol with or without neurotensin and/or NTS1 receptor antagonist SR 48692 dissolved in DMSO 10% (v/v).
Fig. 1. IPs formation in cerebral cortex prisms obtained from neonatal rats in the presence of carbachol and NTS1 receptor antagonist SR 48692. Prisms preloaded with [3H]myoinositol were incubated in duplicate for 15 min in the absence or presence of 10 6 M or 10 4 M SR 48692 in DMSO 10% (v/v), then 10 3 M carbachol was added and incubation continued for 60 min, when samples were processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously (in duplicate) without additions to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of n experiments. (A) carbachol (CCh) alone or plus SR 48692 (n = 5). (B) 10 6 M SR 48692 (n = 7) or 10 4 M SR 48692 (n = 13). ***P b 0.005; ns, nonsignificant difference, by Student’s t test.
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concentration were run, to observe that the antagonist failed to modify basal IPs accumulation (Fig. 1B). DMSO 10% (v/ v) did not alter either IPs accumulation by 10 3 M carbachol or basal IP accumulation (data not shown). Since these findings suggested the participation of NTS1 receptor in carbachol effect, and due to the ability of neurotensin to enhance IPs accumulation by carbachol [33], experiments were carried out with 10 3 M carbachol plus 10 7 to 10 4 M neurotensin. In the presence of the peptide, carbachol effect was not increased whereas a trend to diminish PI turnover was recorded (Fig. 2). In the simultaneous presence of 10 3 M carbachol plus 10 6 M neurotensin, 10 6 M SR 48692 reduced roughly 30% IPs accumulation (Fig. 3A). Similar results were recorded if neurotensin and the antagonist were increased to 10 4 M concentration (Fig. 3B). Neurotensin alone either at 10 6 or 10 4 M concentration failed to alter IPs accumulation (Figs. 3A and B). DMSO 10% (v/v) alone did not change basal IP accumulation (data not shown). Since experiments summarized so far showed that IPs accumulation by 10 3 M carbachol decreased by blocking high-affinity neurotensin receptor with SR 48692, negative findings with neurotensin alone were unexpected. To explore further this issue, the time course of IPs accumulation by 10 3 M carbachol was studied in the absence or presence of 10 7 M neurotensin. Without neurotensin, 10 3
Fig. 2. IPs formation in cerebral cortex prisms obtained from neonatal rats in the presence of 10 3 M carbachol (CCh) and neurotensin (NT). Prisms preloaded with [3H]myoinositol were incubated in duplicate for 60 min in the presence of 10 3 M carbachol with or without neurotensin at 10 7 M, 10 6 M, or 10 4 M concentration and processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously without additions (in duplicate) to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of ten experiments.
Fig. 3. IPs formation in cerebral cortex prisms obtained from neonatal rats in the presence of 10 3 M carbachol (CCh), neurotensin (NT), and SR 48692. Prisms preloaded with [3H]myoinositol were incubated in duplicate for 15 min in the presence of 10 6 M or 10 4 M SR 48692, then 10 6 M or 10 4 M neurotensin plus 10 3 M carbachol were added and incubation continued for 60 min, when samples were processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously without additions (in duplicate) to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of n experiments. (A) SR 48692 and neurotensin concentration was 10 6 M (n = 6). (B) SR 48692 and neurotensin concentration was 10 4 M (n = 8). *P b 0.05; **P b 0.02; ***P b 0.005, by Student’s t test.
M carbachol increased IP accumulation 160–200% when incubation lasted up to 30 min and 350% at 60 min incubation; addition of 10 7 M neurotensin only enhanced IP accumulation at 20 min incubation (Fig. 4). Therefore, further experiments were performed with 20 min incubation time.
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Fig. 4. Time course of IPs formation in cerebral cortex prisms obtained from neonatal rats in the presence of 10 3 M carbachol (CCh) and 10 7 M neurotensin (NT). Prisms preloaded with [3H]myoinositol were incubated in duplicate up to 60 min in the presence of 10 3 M carbachol (CCh) with or without 10 7 M neurotensin (NT) and processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously without additions (in duplicate) to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of 2–4 experiments. *P b 0.05, by Student’s t test.
In this condition, basal [3H]-IPs accumulation was 3061 F 236 dpm per mg protein and total [3H]-inositol incorporation into phospholipids was 48,761 F 1493 dpm per mg protein (mean values F SEM from 11 experiments). IPs accumulation by 10 3 M carbachol was 270%, but it remained unaltered by addition of neurotensin at 10 7 M concentration; when added together, 365% accumulation was recorded, which was totally blocked by 10 7 M SR 48692 (Fig. 5). Antagonist SR 48692 at 10 7 M concentration or DMSO 10% (v/v) did not change basal IP accumulation (data not included). Neurotensin at 10 6 M and 10 4 M concentration failed to alter basal or IPs accumulation produced by 10 3 M carbachol (data not shown). Since carbachol failed to alter total [3H]-inositol incorporation, carbachol-induced IPs accumulation was calculated as the percentage of basal accumulation without additions, as an expression of phosphoinositide hydrolysis [7].
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SR 48692, suggesting the involvement of high-affinity neurotensin receptor in such effect. In previous studies, PI hydrolysis in neonatal and adult rat brain was analyzed in the presence of several additions, including carbachol and inhibitors of Na+, K+-ATPase activity; results showing increases in IPs accumulation after 60 min incubation of cerebral cortex prisms with [3H]myoinositol were recorded [11]. To investigate whether neurotensinergic system was involved in these effects, experiments following a similar experimental schedule were performed in the presence of antagonist SR 48692, where preliminary results indicated a reduction of IPs accumulation, suggesting the involvement of NTS1 receptor [39]. To analyze further neurotensinergic system participation in carbachol effect, assays were carried out and it was observed that PI hydrolysis stimulation by carbachol, known to be totally blocked by cholinergic antagonist atropine [3], was partially reduced with SR 48692. The same pattern of results was recorded if neurotensin was exogenously added, suggesting that endogenous neurotensin may be at least partially involved in IPs accumulation by carbachol, and that an interaction between muscarinic and NTS1 receptors could occur. The ability of neurotensin to enhance PI turnover stimulation by carbachol has been reported [33]; unexpectedly, present results showed that exogenously added neuro-
4. Discussion PI hydrolysis is stimulated in rat brain by several agents, including carbachol, a muscarinic agonist (see Introduction). In the present study, potential involvement of neurotensin and/or its high-affinity receptor in carbachol effect was tested by assaying IPs accumulation in neonatal rat brain prisms. Experiments were performed in the presence of carbachol, neurotensin, with or without SR 48692, a nonpeptidic antagonist for NTS1 receptor. Results obtained indicate that PI hydrolysis induced by carbachol, either alone or plus neurotensin, is partially or totally blocked by
Fig. 5. Effect of 20 min incubation with 10 3 M carbachol (CCh) plus 10 7 M neurotensin (NT) and SR 48692 on IPs formation in cerebral cortex prisms obtained from neonatal rats. Prisms preloaded with [3H]myoinositol were incubated in duplicate for 15 min in the absence or presence of 10 7 M SR 48692, then 10 3 M carbachol or 10 7 M neurotensin or both were added and incubation continued for 20 min, when samples were processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously without additions (in duplicate) to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of 3–11 experiments. ***P b 0.005 by Student’s t test.
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tensin to brain prisms for 60 min was ineffective. To test whether this discrepancy was due to the extent of prism incubation, time course of IP accumulation by carbachol in the absence or presence of neurotensin was investigated. It was observed that IP accumulation by 10 3 M carbachol was enhanced by 10 7 M neurotensin only at 20 min incubation. In this condition, 10 7 M SR 48692 completely abolished IP accumulation, again indicating the involvement of NTS1 receptor. Since SR 48692 selectively blocks high-affinity neurotensin receptor [20], present results on PI hydrolysis may indicate that this drug interferes with a potential cross talk between NTS1 and muscarinic receptors, impairing muscarinic receptor activation which leads to phospholipase C stimulation. In the light of results recorded at various times of prism incubation, it could be speculated that different time courses occur for sequestration of both receptor types: at 20 min incubation, neurotensin binding to NTS1 receptor favors interaction with muscarinic receptor, leading to carbachol effect enhancement; at longer incubation periods, differential internalization pattern for neurotensin and muscarinic receptors may be responsible for disappearance of neurotensin effect. In support, incubation of whole SN17 cells with 125I-NT results in a time- and temperature-dependent internalization of the specifically bound peptide [5]. Most interestingly, the t 1/2 of this internalization is 13 min [17], a value nearly identical for neurons in culture [12], whereas longer t 1/2 for muscarinic receptor internalization of roughly 1.6 h has been found [44]. Present experiments carried out at 20 min incubation showed that concentrations of neurotensin higher than 10 7 M (10 6 M to 10 4 M) failed to enhance PI hydrolysis stimulation by carbachol. A suitable explanation may be based on the observation that 125I-NT internalization is dependent on the concentration of extracellular neurotensin and that peptide internalization process is receptor-dependent [5]. At low neurotensin concentration (10 7 M), the peptide may be effective whereas at higher concentrations, a greater proportion of peptide is most likely internalized, impairing neurotensin effect on PI hydrolysis. Within the cell, peptide–receptor complexes will be part of lysosomes and endosomes. Endocytosis mediates downregulation of G-protein-coupled receptors [48,49]. Confocal microscopy disclosed that ligand–receptor complex is sequestered within a granular compartment both in lysosomal and endosomal bodies. Enzyme catabolism and receptor internalization seem to be involved in removal and/or inactivation of neurotensin [5]. Therefore, our negative results at 60 min may well be explained by endocytosis. It may be hypothesized that differences recorded at 20 min versus 60 min are due to different time courses for internalization of muscarinic and neurotensin receptors. To explain different incubation time results, dimerization of receptors may offer a plausible explanation. Many Gprotein-coupled receptors are able to constitute dimers of
identical or distinct monomeric subunits, an important process for receptor function, including agonist affinity, potency, and efficacy and G protein specificity. Heterodimerization may modify ligand binding or coupling properties and trafficking characteristics of the receptors [1]. Examples from the literature indicated heterodimerization between muscarinic receptor subtypes [25] or between a classical neurotransmitter receptor and a peptide receptor [38]. Global effect seems to depend on incubation time: exogenously added neurotensin is able to enhance PI stimulation by carbachol at 20 min incubation, a transient effect, since at longer incubation time, results did not differ from those recorded with carbachol alone. Most interestingly, in this condition, neurotensin effect was abolished by SR 48692. The presence of SR 48692 decreases cross talk, diminishing heterodimerization and further phospholipase C stimulation. The relationship between cholinergic muscarinic and neurotensinergic systems has been demonstrated in other physiological responses. To illustrate, the ability of neurotensin [8,26,32] and carbachol [30,34] to induce hypothermia has been described. Results showing that hypothermic action of carbachol is mediated via endogenous neurotensin have been reported [19]. In another line of research, a functional interaction between muscarinic antagonists and neurotensin on in vivo dopamine metabolism has been shown in rat nucleus accumbens and striatum [37]. Although in some models there is a relationship between muscarinic and neurotensin system regulation, it does not seem to be a general phenomenon. It is known that IP accumulation by carbachol is totally blocked by atropine, confirming the involvement of muscarinic receptor [3]. When added in vitro, neurotensin is able to modify ligand binding to muscarinic receptor in membranes of the central nervous system, an effect not altering the number or affinity of [3H]-QNB binding sites nor the agonist conformation states of the muscarinic receptor [33]. This is attributable to a functional agonist-induced regulation of muscarinic receptor, as suggested for carbachol effect in developing oligodendrocytes [29]. [3H]-QNB binding studies performed in membranes of several CNS areas showed a decrease in ligand binding by neurotensin, an effect most likely independent of NTS1 receptor [43]. Results suggest that in 60-min incubation experiments, SR 48692 most likely impairs the effect of endogenous neurotensin, whereas in 20-min incubation experiments, the antagonist may well block the effect of both endogenous and exogenously added neurotensin. Since SR 48692 decreased carbachol effect, NTS1 receptor involvement is suggested. However, the possibility that presynaptic mechanisms or local–neuronal circuits are operative cannot be disregarded. To summarize, present results suggest that high-affinity neurotensin receptor is involved in IP accumulation
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enhancement by carbachol, besides cholinergic muscarinic receptor, supporting the idea that agonist-stimulated PI turnover is regulated at multiple steps in the signal transduction pathway.
Acknowledgments G. R. de L. A. is chief investigator from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET). The authors are indebted to Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica, CONICET, Universidad de Buenos Aires and Fundacio´n Antorchas, Argentina, as well as to Committee for Aid and Education, International Society for Neurochemistry (CAEN, ISN), for financial support.
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Research report
High-affinity neurotensin receptor is involved in phosphoinositide hydrolysis stimulation by carbachol in neonatal rat brain S. Pereyra-Alfonsoa, M.G. Lo´pez Ordieresa,b, M. del V. Armaninoa, G. Rodrı´guez de Lores Arnaiza,b,* a
Instituto de Biologı´a Celular y Neurociencias bProf. E. De RobertisQ, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1121) Buenos Aires, Argentina b Ca´tedra de Farmacologı´a, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, (1113) Buenos Aires, Argentina Accepted 17 November 2004 Available online 11 January 2005
Abstract Ontogenetic studies indicate that inositol phosphate accumulation in rodent brain tissue by cholinergic muscarinic agonists as well as expression of high-affinity neurotensin receptor (NTS1) peak at 7 days after birth. Herein, potential participation of this receptor in such effect was investigated. Cerebral cortex prisms of 7-day-old rats were preloaded with [3H]myoinositol and later incubated during 60 or 20 min in the presence of muscarinic agonist carbachol plus neurotensin and SR 48692, a non-peptide NTS1 antagonist. In 60-min incubation experiments, inositol phosphate accumulation by 10 3 M carbachol was roughly 320%, an effect which remained unaltered plus 10 6 M to 10 4 M neurotensin but partially decreased with equimolar SR 48692 concentration. In 20-min incubation experiments, inositol phosphate accumulation by 10 3 M carbachol was circa 240%, a value which attained 320–360% plus 10 7 M neurotensin; this effect was totally blocked by 10 7 M SR 48692. It was concluded that in inositol phosphate accumulation by carbachol, besides the cholinergic muscarinic receptor, the NTS1 receptor is likewise involved; findings at 60 min are attributable to the effect of endogenous neurotensin whereas those at 20 min most likely involve both endogenous and exogenously added peptide. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Peptide receptor structure and function Keywords: Neurotensin; Neurotensin receptors; Carbachol; Phosphoinositide hydrolysis; Neonatal brain cortex
1. Introduction Phosphoinositide (PI) metabolism is enhanced by activation of several brain neurotransmitter receptors, an effect which takes place through G proteins [14,15]. It has been described that muscarinic agonists acetylcholine and carba-
Abbreviations: IP3, inositol 1,4,5-triphosphate; IPs, inositol phosphates * Corresponding author. Instituto de Biologı´a Celular y Neurociencias bProf. E. De RobertisQ, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1121) Buenos Aires, Argentina. Fax: +54 11 4508 3645 or 4964 8274. E-mail address: [email protected] (G. Rodrı´guez de Lores Arnaiz). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2004.11.003
chol, a nonhydrolyzable analog of acetylcholine, stimulate inositol phosphate (IP) accumulation in rat brain cortex slices, an effect recorded on day three after birth with a peak at day seven and a progressive decrease thereafter [2,4]. Neurotensin is a tridecapeptide present in mammalian central nervous system (CNS) and peripheral tissues, whose widespread distribution in cell bodies and nerve terminals in the brain suggests that it may play a major role in neurotransmission or neuromodulation, subserving diverse physiological CNS functions [35,52]. In adult rat brain, neurotensin can bind to two distinct sites, one of high and the other of low affinity, corresponding to NTS1 and NTS2 receptor, respectively [53]. Structurally unrelated to these
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two receptor types, a third one (NTS3) has been described [28]. Physiological neurotensin effects seem due to its interaction with high-affinity sites, which can be blocked by a selective non-peptide neurotensin receptor antagonist termed SR 48692 [20]; therefore, this substance is a valuable tool for investigating neurotensin action in numerous experimental models [40]. The ability of neurotensin to stimulate PI hydrolysis has been documented [18,31]. NTS1 receptor has been linked to a variety of transductional effects including Ca2+ mobilization as well as stimulation of cGMP and inositol phosphate production [53]. Anatomical, physiological, and pharmacological evidence point to a relationship between muscarinic cholinergic and neurotensinergic systems. Immunoreactive neurotensin has been detected in nerve endings innervating Meynert basal nucleus [23,55]. Neurotensin is localized throughout the cytoplasm and along the plasma membrane of cholinergic magnocellular neurons, where neurotensin receptors are present on the perikaryia and proximal dendrites [46,47]. Monoiodo-neurotensin-labeled binding sites are selectively associated with cholinergic nerve cell bodies in the diagonal band and substantia innominata, suggesting that endogenous neurotensin may directly influence forebrain cholinergic function in the CNS [45]. The association of high-affinity neurotensin binding sites with basal forebrain cholinergic neurons has been reported [13]. Autoradiography of classical and neuropeptide receptor distribution disclosed that neurotensin and muscarinic receptors follow a similar distribution in human cortex, with the highest densities occurring in the most superficial gray matter areas [22]. Patch-clamp studies in nucleus basalis neurons indicate that neurotensin excites cholinergic neurons by reducing inwardly rectifying K+ conductance [16]. When added in vitro, neurotensin is able to modify ligand binding to muscarinic receptor in CNS membranes [33,43]. Interestingly, neurotensin administration into rat hippocampus enhances acetylcholine and GABA extracellular levels. These changes were prevented with SR 48692 and bicuculline, suggesting that neuromodulatory action of neurotensin on release of such neurotransmitters is due to the interaction with high-affinity neurotensin and GABAA receptors present in cholinergic nerve endings [36]. The coupling between neurotensin receptors, PI turnover and intracellular Ca2+ mobilization in HT-29 colonic epithelial cells has been demonstrated [9,50,54]. However, potential involvement of neurotensin receptors in brain PI hydrolysis stimulation by carbachol has not yet been explored so far. Ontogenic studies have disclosed that rodent mRNA NTS2 is detected only from postnatal day fourteen [41] whereas mRNA NTS1 is already present at birth and peaks at day seven [21,27,42] and maximal brain IP accumulation by carbachol also peaks at day seven [2]. Based on these facts, NTS1 antagonist SR 48692 was used to test the participation of high-affinity neurotensin receptor
on carbachol-induced PI hydrolysis in cerebral cortex of neonatal rats.
2. Material and methods 2.1. Materials Reagents were analytical grade. Carbachol, myoinositol, and neurotensin acetate were purchased from Sigma (St. Louis, MO, USA). Peptide solutions in bidistilled water were freshly prepared for each experiment. Dimethyl sulfoxide (DMSO) was purchased from J.T. Baker Chemical Co.(Phillipburg, N.J.). A Dowex anion exchange resin (AG 1-X8, 100–200 mesh, formate form) was from Bio-Rad Laboratories (Richmond, CA, USA). SR 48692 {2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxy phenyl) pyrazol 3-yl) carbonylamino]tricyclo (3.3.1.13.7) decan-2carboxylic acid} was kindly provided by Dr. Danielle Gully, SANOFI Recherche, France. OptiPhase bHisafeQ 3 was purchased from Wallac Oy (Turku, Finland). [3H]myoinositol (20 Ci/mmol) was from New England Nuclear (Boston, MA, USA). 2.2. Animals Wistar neonatal rats (6–7 days old) of either sex were used, considering bday 0Q the day of birth. All studies described were conducted in accordance with the Guide for Care and Use of Laboratory Animals provided by the National Institutes of Health, USA. 2.3. Determination of [3H]-inositol phosphates (IPs) A procedure based on described methods [7,10], with modifications, was performed. The cerebral cortex of three neonatal rats were processed for each experimental procedure. Pooled tissue was placed on ice on a Petri dish with gassed Krebs–Henseleit buffer containing (mM): NaCl, 120; KCl, 4.7; CaCl2, 1.3; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; and glucose, 11.7, equilibrated to pH 7.4 with O2/CO2 (95:5); tissue was lightly minced [51] and prisms dispersed at 10% (w/v) in the same buffer. Samples were incubated in bulk at 37 8C for 1 h under gentle shaking with an intermediate change of buffer, followed by 60 min incubation with [3H]myoinositol (6 ACi/ml; final concentration 3 10 7 M) and four washes with fresh buffer replaced every 5 min under O2/CO2 (95:5). Fifty microliters of packed prisms containing 1.1 F 0.10 mg protein (mean values F SEM, n = 16) were transferred to tubes with 0.24 ml of the same buffer which contained LiCl (7.5 mM final concentration, with NaCl iso-osmotically reduced), and the indicated additions to 0.3 ml final volume. To test drug effect, prisms were incubated for 15 min in the presence or absence of SR 48692 dissolved in DMSO 10% (v/v), then carbachol alone or neurotensin plus carbachol were added
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and incubation proceeded under O2/CO2 (95:5) with shaking at 37 8C for 20 or 60 min, as indicated in the figure legends. Incubation was carried out for 0 to 60 min to investigate time course of IPs accumulation. Prism samples preloaded with [3H]myoinositol were processed throughout without additions (in duplicate) to determine basal IPs accumulation. Incubations were stopped by the addition of 940 Al chloroform:methanol (1:2), followed by chloroform (310 Al) and bidistilled water (310 Al) to separate phases. Tubes were vortex-mixed for 15 s, then centrifuged at 1000 g for 10 min to facilitate phase separation. Radiolabeled IPs were separated from inositol by ion-exchange chromatography using small columns containing 0.5 ml of a 50% slurry AG 1-X8 resin in the formate form. Upper phase aliquots (750 Al) diluted to 3 ml with bidistilled water were added to the resin suspension, centrifuged, and washed 4 times with 3 ml of myoinositol (5 mM). [3H]-IPs were eluted with 1 ml of 1 M ammonium formate/0.1 M formic acid and 800 Al of this eluate added to 10 ml of OptiPhase bHisafeQ 3 and counted in a Tracor Analytic scintillation spectrometer with 30% efficiency. The relatively long time of incubation (10–60 min) did not allow to measure the accumulation of the more polar hydrolysis products inositol-1,4-diphosphate and inositol1,4,5-triphosphate, since changes in these compounds are detectable at very short times after addition of receptor agonists to the incubation buffer [6]. However, since the concentration of ammonium formate employed allows the recovery of all IPs, the term IPs instead of IP1 (inositol monophosphate) was used. Radioactivity in the lipid fraction was monitored by counting aliquots from the lower organic phase (200 Al) after drying overnight at room temperature and adding OptiPhase bHisafeQ 3 solution with 45% efficiency. In all experiments, aliquots containing the same amount of brain tissue (that is, similar intraexperimental protein content) were processed, which allowed the comparison between basal condition and treatment in the same sample. Accumulation of [3H]-IPs was thus expressed as the percentage of basal value (without additions).
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After 60-min prism incubation, basal [3H]-IPs accumulation was 5092 F 270 dpm per mg protein and total [3H]inositol incorporation into phospholipids was 70,462 F 1700 dpm per mg protein (mean values F SEM from 10 experiments). In the presence of 10 3 M carbachol, IPs accumulation was roughly 260% of basal value; such IPs accumulation was decreased 26% by 10 4 M SR 48692 but it was almost unaffected by the antagonist at 10 6 M concentration (Fig. 1A). To serve as controls, experiments in the presence of SR 48692 either at 10 6 or 10 4 M
2.4. Protein measurement Protein was determined by the method of Lowry et al. [24] using bovine serum albumin as standard.
3. Results Labeling of inositol phosphates (IPs) and inositol lipids in brain prisms of neonatal rats was studied after incubation with [3H]myoinositol in the presence or absence of carbachol with or without neurotensin and/or NTS1 receptor antagonist SR 48692 dissolved in DMSO 10% (v/v).
Fig. 1. IPs formation in cerebral cortex prisms obtained from neonatal rats in the presence of carbachol and NTS1 receptor antagonist SR 48692. Prisms preloaded with [3H]myoinositol were incubated in duplicate for 15 min in the absence or presence of 10 6 M or 10 4 M SR 48692 in DMSO 10% (v/v), then 10 3 M carbachol was added and incubation continued for 60 min, when samples were processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously (in duplicate) without additions to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of n experiments. (A) carbachol (CCh) alone or plus SR 48692 (n = 5). (B) 10 6 M SR 48692 (n = 7) or 10 4 M SR 48692 (n = 13). ***P b 0.005; ns, nonsignificant difference, by Student’s t test.
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concentration were run, to observe that the antagonist failed to modify basal IPs accumulation (Fig. 1B). DMSO 10% (v/ v) did not alter either IPs accumulation by 10 3 M carbachol or basal IP accumulation (data not shown). Since these findings suggested the participation of NTS1 receptor in carbachol effect, and due to the ability of neurotensin to enhance IPs accumulation by carbachol [33], experiments were carried out with 10 3 M carbachol plus 10 7 to 10 4 M neurotensin. In the presence of the peptide, carbachol effect was not increased whereas a trend to diminish PI turnover was recorded (Fig. 2). In the simultaneous presence of 10 3 M carbachol plus 10 6 M neurotensin, 10 6 M SR 48692 reduced roughly 30% IPs accumulation (Fig. 3A). Similar results were recorded if neurotensin and the antagonist were increased to 10 4 M concentration (Fig. 3B). Neurotensin alone either at 10 6 or 10 4 M concentration failed to alter IPs accumulation (Figs. 3A and B). DMSO 10% (v/v) alone did not change basal IP accumulation (data not shown). Since experiments summarized so far showed that IPs accumulation by 10 3 M carbachol decreased by blocking high-affinity neurotensin receptor with SR 48692, negative findings with neurotensin alone were unexpected. To explore further this issue, the time course of IPs accumulation by 10 3 M carbachol was studied in the absence or presence of 10 7 M neurotensin. Without neurotensin, 10 3
Fig. 2. IPs formation in cerebral cortex prisms obtained from neonatal rats in the presence of 10 3 M carbachol (CCh) and neurotensin (NT). Prisms preloaded with [3H]myoinositol were incubated in duplicate for 60 min in the presence of 10 3 M carbachol with or without neurotensin at 10 7 M, 10 6 M, or 10 4 M concentration and processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously without additions (in duplicate) to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of ten experiments.
Fig. 3. IPs formation in cerebral cortex prisms obtained from neonatal rats in the presence of 10 3 M carbachol (CCh), neurotensin (NT), and SR 48692. Prisms preloaded with [3H]myoinositol were incubated in duplicate for 15 min in the presence of 10 6 M or 10 4 M SR 48692, then 10 6 M or 10 4 M neurotensin plus 10 3 M carbachol were added and incubation continued for 60 min, when samples were processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously without additions (in duplicate) to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of n experiments. (A) SR 48692 and neurotensin concentration was 10 6 M (n = 6). (B) SR 48692 and neurotensin concentration was 10 4 M (n = 8). *P b 0.05; **P b 0.02; ***P b 0.005, by Student’s t test.
M carbachol increased IP accumulation 160–200% when incubation lasted up to 30 min and 350% at 60 min incubation; addition of 10 7 M neurotensin only enhanced IP accumulation at 20 min incubation (Fig. 4). Therefore, further experiments were performed with 20 min incubation time.
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Fig. 4. Time course of IPs formation in cerebral cortex prisms obtained from neonatal rats in the presence of 10 3 M carbachol (CCh) and 10 7 M neurotensin (NT). Prisms preloaded with [3H]myoinositol were incubated in duplicate up to 60 min in the presence of 10 3 M carbachol (CCh) with or without 10 7 M neurotensin (NT) and processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously without additions (in duplicate) to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of 2–4 experiments. *P b 0.05, by Student’s t test.
In this condition, basal [3H]-IPs accumulation was 3061 F 236 dpm per mg protein and total [3H]-inositol incorporation into phospholipids was 48,761 F 1493 dpm per mg protein (mean values F SEM from 11 experiments). IPs accumulation by 10 3 M carbachol was 270%, but it remained unaltered by addition of neurotensin at 10 7 M concentration; when added together, 365% accumulation was recorded, which was totally blocked by 10 7 M SR 48692 (Fig. 5). Antagonist SR 48692 at 10 7 M concentration or DMSO 10% (v/v) did not change basal IP accumulation (data not included). Neurotensin at 10 6 M and 10 4 M concentration failed to alter basal or IPs accumulation produced by 10 3 M carbachol (data not shown). Since carbachol failed to alter total [3H]-inositol incorporation, carbachol-induced IPs accumulation was calculated as the percentage of basal accumulation without additions, as an expression of phosphoinositide hydrolysis [7].
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SR 48692, suggesting the involvement of high-affinity neurotensin receptor in such effect. In previous studies, PI hydrolysis in neonatal and adult rat brain was analyzed in the presence of several additions, including carbachol and inhibitors of Na+, K+-ATPase activity; results showing increases in IPs accumulation after 60 min incubation of cerebral cortex prisms with [3H]myoinositol were recorded [11]. To investigate whether neurotensinergic system was involved in these effects, experiments following a similar experimental schedule were performed in the presence of antagonist SR 48692, where preliminary results indicated a reduction of IPs accumulation, suggesting the involvement of NTS1 receptor [39]. To analyze further neurotensinergic system participation in carbachol effect, assays were carried out and it was observed that PI hydrolysis stimulation by carbachol, known to be totally blocked by cholinergic antagonist atropine [3], was partially reduced with SR 48692. The same pattern of results was recorded if neurotensin was exogenously added, suggesting that endogenous neurotensin may be at least partially involved in IPs accumulation by carbachol, and that an interaction between muscarinic and NTS1 receptors could occur. The ability of neurotensin to enhance PI turnover stimulation by carbachol has been reported [33]; unexpectedly, present results showed that exogenously added neuro-
4. Discussion PI hydrolysis is stimulated in rat brain by several agents, including carbachol, a muscarinic agonist (see Introduction). In the present study, potential involvement of neurotensin and/or its high-affinity receptor in carbachol effect was tested by assaying IPs accumulation in neonatal rat brain prisms. Experiments were performed in the presence of carbachol, neurotensin, with or without SR 48692, a nonpeptidic antagonist for NTS1 receptor. Results obtained indicate that PI hydrolysis induced by carbachol, either alone or plus neurotensin, is partially or totally blocked by
Fig. 5. Effect of 20 min incubation with 10 3 M carbachol (CCh) plus 10 7 M neurotensin (NT) and SR 48692 on IPs formation in cerebral cortex prisms obtained from neonatal rats. Prisms preloaded with [3H]myoinositol were incubated in duplicate for 15 min in the absence or presence of 10 7 M SR 48692, then 10 3 M carbachol or 10 7 M neurotensin or both were added and incubation continued for 20 min, when samples were processed for the assay of IPs. Prism samples preloaded with [3H]myoinositol were processed simultaneously without additions (in duplicate) to determine basal IPs accumulation. Results are expressed as percentage of labeling taking as 100% values of basal IPs accumulation and are mean values (FSEM) of 3–11 experiments. ***P b 0.005 by Student’s t test.
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tensin to brain prisms for 60 min was ineffective. To test whether this discrepancy was due to the extent of prism incubation, time course of IP accumulation by carbachol in the absence or presence of neurotensin was investigated. It was observed that IP accumulation by 10 3 M carbachol was enhanced by 10 7 M neurotensin only at 20 min incubation. In this condition, 10 7 M SR 48692 completely abolished IP accumulation, again indicating the involvement of NTS1 receptor. Since SR 48692 selectively blocks high-affinity neurotensin receptor [20], present results on PI hydrolysis may indicate that this drug interferes with a potential cross talk between NTS1 and muscarinic receptors, impairing muscarinic receptor activation which leads to phospholipase C stimulation. In the light of results recorded at various times of prism incubation, it could be speculated that different time courses occur for sequestration of both receptor types: at 20 min incubation, neurotensin binding to NTS1 receptor favors interaction with muscarinic receptor, leading to carbachol effect enhancement; at longer incubation periods, differential internalization pattern for neurotensin and muscarinic receptors may be responsible for disappearance of neurotensin effect. In support, incubation of whole SN17 cells with 125I-NT results in a time- and temperature-dependent internalization of the specifically bound peptide [5]. Most interestingly, the t 1/2 of this internalization is 13 min [17], a value nearly identical for neurons in culture [12], whereas longer t 1/2 for muscarinic receptor internalization of roughly 1.6 h has been found [44]. Present experiments carried out at 20 min incubation showed that concentrations of neurotensin higher than 10 7 M (10 6 M to 10 4 M) failed to enhance PI hydrolysis stimulation by carbachol. A suitable explanation may be based on the observation that 125I-NT internalization is dependent on the concentration of extracellular neurotensin and that peptide internalization process is receptor-dependent [5]. At low neurotensin concentration (10 7 M), the peptide may be effective whereas at higher concentrations, a greater proportion of peptide is most likely internalized, impairing neurotensin effect on PI hydrolysis. Within the cell, peptide–receptor complexes will be part of lysosomes and endosomes. Endocytosis mediates downregulation of G-protein-coupled receptors [48,49]. Confocal microscopy disclosed that ligand–receptor complex is sequestered within a granular compartment both in lysosomal and endosomal bodies. Enzyme catabolism and receptor internalization seem to be involved in removal and/or inactivation of neurotensin [5]. Therefore, our negative results at 60 min may well be explained by endocytosis. It may be hypothesized that differences recorded at 20 min versus 60 min are due to different time courses for internalization of muscarinic and neurotensin receptors. To explain different incubation time results, dimerization of receptors may offer a plausible explanation. Many Gprotein-coupled receptors are able to constitute dimers of
identical or distinct monomeric subunits, an important process for receptor function, including agonist affinity, potency, and efficacy and G protein specificity. Heterodimerization may modify ligand binding or coupling properties and trafficking characteristics of the receptors [1]. Examples from the literature indicated heterodimerization between muscarinic receptor subtypes [25] or between a classical neurotransmitter receptor and a peptide receptor [38]. Global effect seems to depend on incubation time: exogenously added neurotensin is able to enhance PI stimulation by carbachol at 20 min incubation, a transient effect, since at longer incubation time, results did not differ from those recorded with carbachol alone. Most interestingly, in this condition, neurotensin effect was abolished by SR 48692. The presence of SR 48692 decreases cross talk, diminishing heterodimerization and further phospholipase C stimulation. The relationship between cholinergic muscarinic and neurotensinergic systems has been demonstrated in other physiological responses. To illustrate, the ability of neurotensin [8,26,32] and carbachol [30,34] to induce hypothermia has been described. Results showing that hypothermic action of carbachol is mediated via endogenous neurotensin have been reported [19]. In another line of research, a functional interaction between muscarinic antagonists and neurotensin on in vivo dopamine metabolism has been shown in rat nucleus accumbens and striatum [37]. Although in some models there is a relationship between muscarinic and neurotensin system regulation, it does not seem to be a general phenomenon. It is known that IP accumulation by carbachol is totally blocked by atropine, confirming the involvement of muscarinic receptor [3]. When added in vitro, neurotensin is able to modify ligand binding to muscarinic receptor in membranes of the central nervous system, an effect not altering the number or affinity of [3H]-QNB binding sites nor the agonist conformation states of the muscarinic receptor [33]. This is attributable to a functional agonist-induced regulation of muscarinic receptor, as suggested for carbachol effect in developing oligodendrocytes [29]. [3H]-QNB binding studies performed in membranes of several CNS areas showed a decrease in ligand binding by neurotensin, an effect most likely independent of NTS1 receptor [43]. Results suggest that in 60-min incubation experiments, SR 48692 most likely impairs the effect of endogenous neurotensin, whereas in 20-min incubation experiments, the antagonist may well block the effect of both endogenous and exogenously added neurotensin. Since SR 48692 decreased carbachol effect, NTS1 receptor involvement is suggested. However, the possibility that presynaptic mechanisms or local–neuronal circuits are operative cannot be disregarded. To summarize, present results suggest that high-affinity neurotensin receptor is involved in IP accumulation
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enhancement by carbachol, besides cholinergic muscarinic receptor, supporting the idea that agonist-stimulated PI turnover is regulated at multiple steps in the signal transduction pathway.
Acknowledgments G. R. de L. A. is chief investigator from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET). The authors are indebted to Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica, CONICET, Universidad de Buenos Aires and Fundacio´n Antorchas, Argentina, as well as to Committee for Aid and Education, International Society for Neurochemistry (CAEN, ISN), for financial support.
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