Supersensitivity of the cholinergic muscarinic system in the rat’s brain is induced by high concentrations of Cu⁺².

Elsa Gutiérrez-Reyes, Darwin Castañeda-Perozo, Jhan Papale-Centofanti, Carlota Nello-Pérez, Carmine Pascuzzo-Lima, José Moreno-Yanez and Rafael Bonfante-Cabarcas.

Unidad de Investigaciones Bioquímicas “José Antonio Moreno Yánez”.
Centro de Investigaciones Biomédicas, Decanato de Medicina.
Universidad Centroccidental “Lisandro Alvarado”. Barquisimeto, Venezuela. Correo electrónico: rafaelabc@hotmail.com

Abstract. 

    Transition metals have been described as regulators of receptor’s function. Here, we studied the effects of chronic administration of Cu²⁺ or the Cu²⁺ chelator penicillamine (PA) on the functional and binding properties of the muscarinic receptors (MR) on selected areas of rat’s brain. Groups of 10 Sprague-Dawley rats were treated daily, for 45 days with either 1) 1mg/Kg CuSO₄ (Cu²⁺), 2) 100 mg/Kg PA, or 3) saline solution. Double T-maze and motility cages were used for behavioral testing and the binding assays were performed using [³H]-QNB or [³H]-N-MSCP as MR’s ligands. Cu²⁺ brain levels were measured in the cerebral cortex by atomic absorption spectrophotometer. Results showed that PA treated rats displayed a significant decrease of locomotor’s activity (LA) and rearing behavior (RB), but a significant increases in memory efficiency (ME). Cu²⁺ treated rats displayed diminished RB with no significant changes in LA. Cu²⁺ treated rats displayed higher MR’s density (Bmax) in cortex (C), striatum (S), and hippocampus (H). An increase in Bmax was also observed in PA treated rats, but only in C and S. Finally, Cu²⁺ tissue concentration was significantly higher in C of both Cu²⁺ and with PA treated animals. In conclusion, 45 days of Cu²⁺ or PA treatment induced brain hypercuprosis, which was associated with MR binding supersensitivity; however, change in ME was only observed in PA treated rats suggesting that might be still another factor in these experiments besides Cu²⁺ (i.e., Zn²⁺ or PA itself) involved in memory modulation.

Key words:Muscarinic receptor, Cu²⁺, locomotor activity, memory,super-sensitivity, [³H]-QNB binding.

Resumen. 

Palabras claves:Receptor muscarínico, Cu²⁺, actividad locomotora, memoria, supersensibilidad, unión de [³H]-QNB.

Received: 13-11-2001. Accepted: 14-05-2002

INTRODUCTION

    Muscarinic receptors (MRs) belong to the super family of seven transmembrane helix G-proteins-coupled receptors (1-3). Five MR subtypes: m1, m2, m3, m4, and m5 have been described based on molecular cloning studies; however, only four of these cloned subtypes have been pharmacologically and functionally defined, in primary tissues, corresponding to the described receptors M1, M2, M3, and M4 (2).

    Cholinergic neurotransmission, besides its classical role in peripheral neurotransmission, is also involved in a variety of cerebral-controlled functions such as learning, memory, and cognition (4-6). Loss of cholinergic integrity, including low levels of brain’s cholineacetyltransferase, loss of cortical cholinergic projections on the forebrain, and reduced number of MRs have been demonstrated in Alzheimer’s disease, which is the most prevalent type of memory disorders (7). Furthermore, MR antagonists can disrupt acquisition and performance of learned behaviors by interfering with the interaction between acetylcholine and MRs (8).

    Several endogenous factors have been reported to be able to modify functions of MR, among then, endogenous putative regulatory ligands (9,10), guanine nucleotides (11), and ions such as Cu²⁺ and Zn²⁺ (12).

    Copper (Cu²⁺) is an essential trace metal, which plays an important role in the biochemistry of human nervous system. Several studies have indicated that alterations in Cu²⁺ homeostasis is implicated in the pathogenesis of certain diseases characterized by neurodegenerative processes such as Menkes’ and Wilson’s diseases (both are inherited disorders of Cu²⁺ metabolism) (13) as well as in Alzheimer’ disease, which is characterized by the imbalance of the Cu²⁺, Fe, and Zn²⁺ homeostasis among other features (13, 14).

    In vitro studies have shown that Cu²⁺ is able to affect MRs by inhibiting the muscarinic’s antagonists binding to MR in hippocampus, forebrain, cortex, and adrenal medulla, but not to the brainstem’s MRs (12, 15, 16). On the other hand, it has been reported that Cu²⁺ facilitates the binding of agonists by either decreasing the agonist’s Ki (i.e., in displacement curves) or by increasing [³H]-acetylcholine binding capacity in rat cortex (12, 16-19). The in vivo effect of Cu²⁺ has been explored only in rats with induced Cu²⁺-deficincy, but the results are controversial. Farrar and Hoss (20) reported that homogenates prepared from forebrains of Cu²⁺-deficient animals displayed a significant decrease in MR occupancy and affinity. Feller et al. (21) found that Cu²⁺ decreased MR occupancy only in corpus striatum and cerebral cortex however, Geiger et al. (22) reported the opposite phenomenon, with Cu²⁺ promoting increased [³H]-QNB binding in striatum and cerebellum. Still, there are not studies addressing the in vivo effect of an excess of Cu²⁺ in the MRs’ binding on experimental animals.

    Regardless the cumulated knowledge related with the in vitro effect of Cu²⁺ on antagonist and agonist binding and the effect of Cu²⁺ deficiency on the expression of MR in rats, the role of Cu²⁺ on the MR-mediated aspects of behavior has not been clearly defined. For this reason, we decided to assess the effects of chronic administration of Cu²⁺ or the Cu²⁺ chelator penicillamine (PA) on learning, memory, and locomotor activities of young Sprague Dawley rats, followed by determination of MR binding affinity and occupancy in brain’s homogenates, in order to correlate binding with behavioral findings. Our results showed that Cu²⁺ and/or PA both induced brain hypercuprosis and MR up-regulation. These molecular findings were translated in behavior changes (i.e., hypomotility and learning facilitation) suggestive of a functional supersensitivity of these receptors.

Animals and treatments

    3 groups, each composed of ten (n=10) 21-days old male Sprague-Dawley rats, received a daily intraperitoneal injection of: 1) 1 mg/kg CuSO₄ (Cu²⁺), 2) 100 mg/kg PA, or 3) equal volume of 0.85% NaCl (control group) for 45 days. To avoid the PA’s collateral effects, 2 mg/kg/day ZnSO₄ and 4 mg/kg/day pyridoxine were added to the drinking water. After treatment, each animal from every group was evaluated for its learning capacity and locomotor activity. Then, the animals were sacrificed by decapitation and their brains rapidly removed. The striatum, brainstem, frontal cortex, and hippocampus were dissected. Frontal cortex and hipocampus were chosen because their involvement in memory processes and striatum because their involvement in motor funtions respectively; furthermore, frontal cortex and hippocampus, as well as striatum, are characteristically described to be rich in MR of the M1 subtype (6). The brainstem region was chosen because its high content of M2-subtypes MRs (2). The dissected regions from each group were cut in small pieces and homogenates prepared 1: 50 w/v in 25 mM Hepes Buffer pH 7.3, aliquoted in small volumes, and stored at –70°C until used. Protein concentration was determined by bicinchioninic acid colorimetric (BCA) assay (23).

Binding assays

    [³H]-QNB (an hydrophobic muscarinic ligand non subtype specific) and [³H]-N-MSCP (an hydrophilic muscarinic ligand non subtype specific) were used to perform binding assays, according to protocols already published (12), in order to estimate the MR number in the dissected brain’s regions. Briefly, 100 µg of total protein were incubated with 5-1000 pM [³H]-QNB or 20-4000 pM of [³H]-N-MSCP in absence (total binding) or presence (nonspecific binding) of 2 µM atropine in 2 mL final volume completed with 25mM Hepes buffer, at 37°C for 60 min. Binding reaction was terminated by vacuum filtration through GF/B glass-fiber filters (Whatman Inc. Clifton, NJ). Filters were washed three times with 5 mL of ice-cold phosphate buffer, dried (60°C for 12 hours) and placed into scintillation vials with 5 mL of scintillation cocktail (PPO, POPOP, Triton X-100, and Toluene). Radioactivity retained in the filters was measured in a liquid scintillation counter (57% counting efficiency; Wallac 1410, Pharmacia, Inc., Finland). Non-labeled atropine used in saturating concentrations blocks all muscarinic sites therefore, only non-muscarinic sites (nonspecific binding) are then available to the radioligands. In absence of atropine, all sites became accessible to the radioligand (total binding); therefore, specific (muscarinic) binding is obtained by subtracting nonspecific binding from the total binding values. Also, Bmax was determined at saturating concentrations of the ligand (500 pM of [³H]-QNB).

    In average, binding experiments were carried out in six independent points and repeated at least twice.

Behavioral testing

    After 2 days of habituation trials, which consisted in let the animals freely explore the testing maze for 30 minutes (one trial per day), learning and memory were measured using a 20 seconds latency foot-shock double T Maze, based on a protocol described by Farr et al. (24). Memory was scored as the percent of successes and time that each rat expended in solving the maze in the trial (10 trials per test). Failures were defined as incapacity to solve the maze, enter in a wrong arm, or when receiving a foot-shock. Memory efficiency was defined as the percentage of success per unit of time. A motility cage, which is divided in four chambers, was used to score locomotor activity as well as the rearing behavior. Motility activity was recorded in 10 min periods preceded by a 5 min latency.

    Behavior experiments were carried on each rat independently (n = 10 for each group).

Tissue’s Cu²⁺ content determination

    Cortex Cu²⁺ content were determined on each rat independently (n = 10 for each group).

Data analysis

    The maximum number of receptors (Bmax) and dissociation constant (Kd) were calculated based on the Hill’s equation using a sigmoid curve from Graphpad Inplot Software (San Diego Ca.); adjustment was done according to the least square method. Data are expressed as the mean ± standard error. Statistical analysis of the differences between groups was performed using ANOVA test, followed by the Bonferroni post-test, accepting as significant a p<0.05.

Materials

    [³H]-QNB (l-quinuclidinyl[phenyl-4-³H]-benzilate) with a specific activity of 48 Ci/mmol was purchased from Amersham Pharmacia Biotech UK and [³H]-N-MSCP (scopolamine [N-methyl-³H]-methyl chloride) with a specific activity of 80.4 Ci/mmol was obtained from New England Nuclear USA. All other reagents were purchased from Sigma Chemical Company (St. Louis, MO, USA).

RESULTS

Binding characteristics of rat brain MRs

    Brain’s homogenates from Cu²⁺-treated rats displayed a significant increase in MR density (Bmax) in cortex (2535 ± 41fmol/mg), striatum (1195 ± 30 fmol/mg), and hippocampus (2456 ± 26 fmol/mg) than control groups (2331 ± 39 fmol/mg in cortex, 1076 ± 9 fmol/mg is striatum, and 2309 ± 35 fmol/mg in hippocampus) as measured using [³H]-QNB as radioligand. No significant changes in the number of binding sites in the brainstem between treated and control were observed. Unexpectedly, brain’s homogenates of PA treated rats also shown a significant increase in the number of MRs in cortex (2555 ± 57 fmol/mg) and striatum (1248 fmol/mg); however, we did not detected significant changes in the number of MRs in hippocampus and brainstem (Table I). Similar results were observed using [³H]-N-MSCP as radioligand (Fig.1)

                                                                                              TABLE I ]]>                                    MUSCARINIC RECEPTOR DENSITIES IN DIFFERENT BRAIN AREAS

Data are the mean ± standard error (n = 6 for each group).

Units are fentomols of [³H]-QNB bound per mg of protein.

]]> *p < 0.05 by ANOVA followed by Bonferroni post-test.

Fig.1. Hyperbolic isotherm of [3H]-N-MSCP binding in crebral cortex. 100 µg of protein from rat brain cortex were incubated with 20-4000 pM of  [³H] -N-MSCP for 1 hour at 37°C. [³H] -N-MSCP binding to MRs from brain cortex is greater (p<0.05) in animals treated with Cu²⁺ (gray diamond) and PA (black square) as compared with control group (open circle). Data are the mean ± standard error calculated from 6 independent data points.    

    In all regions examined, Hill’s coefficient (n_H) values were greater than 1, which suggests positive homotropic cooperativity in the binding of [³H]-QNB to the MRs; however, no differences were observed among groups. Likewise, there were not significant differences in the dissociation constant (Kd) for all tissues studied, suggesting that both Cu²⁺ and PA did not affect receptor affinity (Tabla II).

Locomotor activity, rearing behaviour and memory efficiency

    After 24 hours from the last doses of Cu²⁺ or PA, we tested locomotor activity, rearing behaviour, and memory. Results indicated that chronic administration of PA induced a significant decrease of locomotor’s activity (control rats: 14.7 ± 1.23 events/10 min, PA rats: 7.5 ± 1.12 events/10 min) and rearing behaviour (control rats: 25.5 ± 2.26 events/10 min, PA rats: 10.1 ± 2.12 events/10 min). On the other hand, PA increases significantly memory efficiency (control rats: 17.85 ± 6.7% of success/sec, PA rats: 23.59 ± 5.07% of success/sec). Cu²⁺ decreased significantly the rearing behaviour (16.3 ± 0.91 events/10 min), but no significant difference was observed on locomotor’s activity and memory efficiency (Tabla III).

Cu²⁺ concentrations measured in brain tissue

    We only determined Cu²⁺ levels in cortex because of the tissue sample’s size required for this type of analysis in our experimental conditions. As expected, Cu²⁺ concentration was significantly higher (p<0.05) in the cortex of rats treated with Cu²⁺ (3.24 ± 0.17 µg/g) as compared with the control groups (2.54 ± 0.54 µg/g); however, PA treated rats, also displayed higher concentrations of copper as compared with control animals (3.01 ± 0.26 µg/g) (Tabla IV).

                                                                                    TABLE II ]]>               [³H]-QNB dissociation constant and Hill’s coefficient in homogenates from cortex and brainstem

Data are the mean ± standard error (n = 6 for each group). Dissociation constant (Kd) are expressed in picomolar and n_H was calculated based on Hill’s equation using Graphpad Inplot software.

                                                                                         TABLE III
                                                                           BEHAVIOUR PARAMETERS

Locomotor activity and rearing behaviour are expressed as the mean of the number of events in 10 minutes ± standard error (n = 10 for each group). Memory efficiency is expressed as a percentage of success by unit time in seconds. *p < 0.05 by ANOVA followed by Bonferroni’ post test.

                                                                                         TABLE IV
                                                                TISSUE LEVELS OF Cu²⁺ IN CORTEX

Determined by AAS, expressed in g/g of tissue. Values are the mean ± S.E.M (n = 10 for each group. * Different from control group, values p < 0.05 (ANOVA followed by Bonferroni’ post test).

    Copper is a transition metal of broad distribution in mammalian tissues including the central nervous system (26, 27). Brain’s areas of high Cu²⁺ content (i.e., hippocampus and striatum) are described also have a high content of muscarinic synapses, which are highly sensitive to alterations in the Cu²⁺ homeostasis (16).

    There are few reports on the neurological effects of Cu²⁺ administration in experimental animals. Oral administration of Cu²⁺ affected neither locomotor activity nor learning and/or memory abilities; however, Cu²⁺ in combination with divalent manganesum (Mn²⁺) impaired learning ability and memory (28). Analysis of bioorganic amines, in the brains of animals receiving these cations, revealed significant increment in the levels of dopamine and noradrenaline levels (28, 29). These findings suggested that Cu²⁺ increased the release of dopamine, noradrenaline, and 5-hydroxytryptamine and inhibited the reuptake of dopamine and 5-hydroxytriptamine in striatum and cortex synapses (30). Similarly, Cu²⁺ deficiency decreased both dopamine and noradrenaline levels (22).

    In this report, we showed for the first time, that intraperitoneal administration of high doses of Cu²⁺ results in the induction of up-regulation of the MR number in striatum, cerebral cortex, and hippocampus, but it did not affect the MR population in brainstem. On the other hand, administration of the Cu²⁺-chelator PA induced MR up-regulation only in the striatum and cerebral cortex. Both, PA and Cu²⁺ treatment increased the Cu²⁺ levels in cerebral cortex, suggesting that the MR up-regulation might be related to the increased copper levels in the selected brain’s areas.

    It has been reported, that Cu²⁺ inhibits the binding of antagonists to MRs in vitro experiments (12, 15, 16), but increases the binding of agonists (12, 16-19). In agreement with reports of agonist-induced MR down-regulation (31), the administration of Cu²⁺ might induce MR down-regulation by facilitating endogenous agonist binding however, this phenomenon was not observed in our experiments. On the contrary, we found that administration of Cu²⁺ promotes up-regulation of the MR population.

    A possible mechanism for the observed Cu²⁺ effect on the MR population could involve its known affinity for cystein moieties and its reducing capacity. Once bound, Cu²⁺ is able to modify the cysteine’s sulfhydryl groups’ redox state (18). Modifications of these groups could result in marked changes in muscarinic agonist’s binding affinity (17, 18).

    On the other hand, Cu²⁺-reduction of sulfhydryl groups may protect the receptors to be desensitized and degraded as is seen when agonists bind the MR inducing receptor phosphorylation and subsequent receptor degradation (31). Supporting this concept, site-direct mutagenesis studies have demonstrated the role of the cysteine residues at the carboxyl-terminus of several transmembrane receptors. These residues are described to be critical for functional desensitization and/or for internalization of alpha adrenergic and glucose-dependent insulinotropic receptors (32-34). For this reason, the up-regulation of MR density on the described Cu²⁺-responsive brain’s areas could be explained by a decreased in the degradation rate of the MRs. This observation is sustained by former observations that rats fed with Cu²⁺-deficient diets displayed a decrease in the number of receptors at those brain regions (20, 21). These results suggest that Cu²⁺ regulates MRs in situ by altering the receptor’s redox state and therefore, increasing the half-life of the receptor.

    On the other hand, Cu²⁺ could modulate the MR gene expression. Cu²⁺-mediated activation of yeast’s gene expression occurs through Cu²⁺-regulation of DNA binding regions. Cu²⁺ binding stabilizes a DNA-specific conformation, favoring high affinity interaction between the receptor’s specific DNA promoter region and transcriptions elements (35).

    Similarly, we observed that PA treatment induced up-regulation of MRs in most of the brain’s areas studied, which is correlated with an increased Cu²⁺ tissue level. PA is used in the treatment of Wilson’s disease, which is characterized by a generalized hypercuprosis (36). In Wilson’s disease patients receiving PA, the half-life to decrease Cu²⁺ concentrations in the cerebro-spinal fluid is about 23.5 months (37). During the first months of treatment, PA seems to worsen the neurological manifestations of the disease, this phenomenon has been explained in relationship to a disturbance of blood-brain barrier (37).

    In our protocol, experimental animals received PA for 1.5 months, which might be not enough time to decrease the total brain’s copper levels, although sufficient to mimic the PA-induced worsening of neurological conditions. Systemic PA treatment may induce Cu²⁺ redistribution from peripheral tissues to the brain aided by a disruption on the blood-brain barrier integrity allowing for easier access of Cu²⁺ to the central nervous system and inducing a transient hypercuprosis. We do not have a plausible explanation for the effect observed in hippocampus, where Cu²⁺ failed to induce an up-regulation of the MR population; although this observation will be object of further studies, we speculate that hippocampus might have a higher sensitivity to PA treatment that might be promoting a reduction in the hippocampus’s Cu²⁺ concentrations earlier.

    Finally, our data showed that Cu²⁺ up-regulates muscarinic cholinergic receptors in selected brain areas. Although Cu²⁺ by itself does not seem to affect animal’s behavior, the impact of Cu²⁺-induced MR up-regulation on the behavior responses might be related to the levels of Zn²⁺ in the central nervous system.

ACKNOWLEDGMENTS

    We thank Dr Carla Lankford for the proof reading of the manuscript. This work was supported by grants 02-23M-99 and 005-ME-2000 from CDCHT (Consejo de Desarrollo Científico y Tecnológico, Barquisimeto, Venezuela).

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