Effect of repeated administration of manganese on the striatal cholinergic and dopaminergic receptors in the rat

Previous studies suggest that chronic administration of manganese produces symptoms akin to Parkinson's disease, which is believed to be due to derangement in the central cholinergic and dopaminergic system. In the present study the effect of repeated administration of manganese chloride to male Sprague-Dawley rats on the binding of [3H]spiroperidol and [3H]quinuclidinyl benzilate to striatal dopaminergic and cholinergic receptors, respectively, was determined. Daily intraperitoneal injections of manganese chloride (15 mg/kg) for 15 days followed by a 24 h drug-free period did not alter the receptor density (Bmax) or the apparent dissociation constant (Kd) of either [3H]spiroperidol or [3H]quinuclidinyl benzilate to the rat striatal membranes. It is concluded that chronic treatment with manganese does not change the plasticity of post-synaptic dopaminergic or cholinergic receptors in the rat striatum.     

Triethyllead-induced peroxidative damage in various regions of the rat brain

Adult male Fisher 344 rats (8-10 wk old) were dosed ip with 1.75 mg/kg body weight of triethyllead chloride (TEL) for 5 consecutive days. Rats were sacrificed 1, 7, or 21 d after the last injection. The rate of lipid peroxidation was significantly elevated in frontal cortex at all three time points assayed (1, 7, or 21 d). However, hippocampal and cerebellar membranes showed no changes in peroxidative capacity at these time points. In order to determine whether cortical membrane damage was reflected in alteration of a restricted protein population, a series of high-affinity receptor binding sites was determined in cortical membranes derived from treated rats 7 d after the last injection of triethyllead. The rate of lipid peroxidation was significantly increased in the frontal cortex of triethyllead treated rats; however, no changes in the binding of [3H]spiroperidol, [3H]quinuclidinyl benzilate, and [3H]benzodiazepine were seen in animals exposed to triethyllead. The cortical wet weight, protein content, and cell number were also unchanged by TEL treatment, reflecting an absence of gross damage.     

Muscarinic receptor subsensitivity in the longitudinal muscle of the rat ileum following chronic anticholinesterase treatment with diisopropylfluorophosphate

Chronic administration of diisopropylfluorophosphate to rats at a dose regimen producing 70 per cent inhibition of ileal cholinesterase was accompanied by a decrease in the sensitivity of the isolated ileum to oxotremorine and carbachol. The same treatment produced an increase in the sensitivity of the ileum to acetylcholine during the first ten days of anticholinesterase treatment, but after ten days of treatment the sensitivity of the ileum to acetylcholine was reduced. When [³H]quinuclidinyl benzilate binding was measured in ileal longitudinal muscle homogenates from anticholinesterase-treated rats, a decrease in binding was observed which was due primarily to an increase in the dissociation constant of [³H]quinuclidinyl benzilate. Chronic diisopropylfluorophosphate treatment also increased the K_i of various cholinergic ligands as determined by competitive displacement of [³H]quinuclidinyl benzilate binding. Scatchard analysis of agonist displacement ol [³H]quinuclidinyl benzilate binding revealed two agonist binding sites which were present in approximately equal concentrations. Chronic diisopropylfluorophosphate treatment caused an increase in the dissociation constant of the high affinity site but produced only small effects on the dissociation constant of the low affinity site and on the relative concentration of the two sites. These results indicate that the tolerance to chronic anticholinesterase treatment is, in part, a receptor-mediated phenomenon.     

Alteration of cerebral neurotransmitter receptor function by exposure of rats to manganese

Adult male rats were treated on 15 successive days with i.p. injection of manganese chloride at 10 or 15 mg/kg body weight. 24 h after the last dose, the binding of the dopaminergic antagonist [3H]spiroperidol to striatal membranes was elevated significantly. At the lower dose, no other high-affinity binding site measured was altered. However, at the 15 mg/kg body weight dose, cerebellar GABA, frontal cortical serotonin, and striatal muscarinic binding were all depressed. Neither dose level altered striatal levels of enkephalin, substance P, dopamine, serotonin, or dihydroxyphenylacetic acid (DOPAC). Receptor binding measurements may be a sensitive means of detecting disturbances of specific neural circuits.     

Effects of hypoxia on atrial muscarinic cholinergic receptors and cardiac parasympathetic responsiveness

Chronic exposure of rats to hypoxia resulted in a lower resting heart rate and a supranormal increase in heart rate in response to parasympathetic blockade by atropine. The density of muscarinic cholinergic receptors labeled by the antagonist [³H]quinuclidinyl benzilate was elevated significantly in the atria of animals kept hypoxic for 2–4 weeks. Chronic hypoxia did not change the affinity of the receptor for [³H]quinuclidinyl benzilate, the weight of the atria, or the amount of protein per atrial pair. Thus, the decrease in resting heart rate may be explained by the increase in the density of atrial muscarinic cholinergic receptors.     

Muscarinic receptor alterations as a mechanism of anticholinesterase tolerance

Tolerance to the toxic signs of the organophosphorus ester acetylcholinesterase inhibitor, O,O-diethyl S-[2-(ethylthio)ethyl] phosphorodithioate (disulfoton), was induced in rats by giving 10 doses of 2.0 mg/kg/day. Concurrent with the induction of tolerance, decreased sensitivity to the cholinergic agonists carbachol and oxotremorine could be demonstrated in studies of heart rate in vivo and in isolated preparations of ileum and atria. A significant decrease in the binding of the muscarinic antagonist [³H]quinuclidinyl benzilate could be demonstrated in ileum from disulfoton-tolerant animals. However, no alterations in the binding of [³H]quinuclidinyl benzilate, [³H]oxotremorine-M, or oxotremorine were evident in atria from tolerant animals. The results suggest that, in addition to receptor loss, other mechanisms distal to ligand recognition sites or removed from the receptor complex may contribute to the subsensitivity of tissues to muscarinic cholinergic agonists.     

Muscarinic cholinergic binding in striatal and mesolimbic areas of the rat: reduction by 6-hydroxydopa.

The pilocarpine-induced catalepsy and the specific binding of [³H]quinuclidinyl benzilate ([³H]QNB) in synaptic membranes were investigated in the adult rat neonatally treated with 6-hydroxydopa (6-OHDOPA). An i.p. administration of pilocarpine (75–150 mg/kg) elicited less catalepsy in the 6-OHDOPA-treated rats than in the controls. The specific binding of [³H]QNB in the striatal or mesolimbic homogenates proved to be saturable. In addition, treatment with 6-OHDOPA caused a significant reduction in B_max values of [³H]QNB binding in both regions but did not alter the K_D values as compared with those of the controls. These results suggest that neonatal treatment with 6-OHDOPA induces a muscarinic-cholinergic hyposensitivity in the rat brain which is probably due to the decrease in the number of muscarinic-cholinergic receptors in the striatal and/or mesolimbic areas.     

Tricyclic antidepressant drug treatment decreases alpha 2-adrenoreceptors on human platelet membranes

The rate of thermal inactivation of [³H]5-HT, [³H]spiroperidol and [³H]LSD binding sites in a membrane preparation from the rat frontal cortex has been studied. The binding sites were inactivated as different rates and the results are interpreted as supporting the claim that [³H]5-HT and [³H]spiroperidol bind to different sites within the frontal cortex of the rat.     

3H]spiperone and [3H]quinuclidinyl benzilate binding in striatal membranes from rats chronically treated with manganese chloride throughout development and for over two years

Chronic treatment of rats with manganese (1 mg MnCl2 X 4H2O per ml of drinking water) from conception till adulthood only slightly decreased [3H]spiperone binding in striatal membranes whereas [3H]quinuclidinyl benzilate binding was not affected. The age-related decreases in dopaminergic and muscarinic cholinergic receptor binding in striatal membranes were not affected by the life-span manganese treatment.     

Long-Term Alterations in Benzodiazepine,Muscarinic and α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) Receptor Density Following Continuous Cocaine Administration

Abstract: Animal models of clinical phenomena, such as stimulant-induced psychosis have focused primarily on persisting alterations that develop in brain after chronic stimulant administration. The present study utilized autoradiographic measures to examine changes in the density of benzodiazepine ([³H] flunitrazepam), muscarinic ([³H] quinuclidinyl benzilate), and non-NMDA glutamatergic (³H α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid: AMPA) receptor binding in rats 21 days following two exposures to cocaine administered continuously for 5 days via subcutaneous pellets. A marked, selective increase in [³H] flunitrazepam binding in both the lateral and medial habenula nucleus was observed. Reduced [³H] quinuclidinyl benzilate binding was observed in various brain areas, including large decreases in the anterior cingulate cortex and ventral thalamus. A reduction in [³H]AMPA binding was observed in the ventral striatum and was suggested in the nucleus accumbens. [³H] Flunitrazepam binding was also examined 12 hr following a single 5 day cocaine exposure to determine if the long-term habenular changes were evident at acute withdrawal. No alterations in [³H] flunitrazepam binding were observed in the habenula or any other structure analyzed at this time point. The relation of these results to persisting alterations in mesocorticolimbic pathways and previous findings of cocaine-induced degeneration in lateral habenula circuitry is discussed.     

Solubilization and characterization of [3H]spiroperidol binding sites from subcellular fractions of the calf striatum

Specific [³H]spiroperidol binding sites were obtained from subcellular fractions of the calf striatum in a solubilized form using salt extraction with potassium chloride. Binding activity was assayed by adsorption on Norit SGX to separate unbound radioactive ligand from the macromolecular ligand complex. The solubilized membrane preparation exhibited stereoselectivity for butaclamol with the (+)-enantiomer being the active agent. Neuroleptic drugs were found to have an affinity for the soluble preparation which was identical to that of the native membrane-bound receptor. The effects of various parameters on the binding of [³H]spiroperidol to the soluble extract, including temperature, salt concentration, ionic strength and stabilizing agents, suggest that the binding site is biochemically stable. Electron micrographs of the soluble material reveal the absence of any recognizable membrane structures. Overall, these data demonstrate that a macromolecular component with high affinity and stereospecificity for [³H]spiroperidol can be isolated from calf striatal synaptosomes and microsomes, and suggest that at least one of the receptors for dopamine may be an extrinsic protein.     

Effect of acute and chronic morphine administration on brain cholinergic muscarinic receptors

The binding of [3H]quinuclidinyl benzilate (QNB) to various brain regions was determined in rats after acute or chronic treatment with morphine. Morphine and naloxone, in vitro, inhibited the binding of [3H]QNB to striatal membranes only at high concentrations. Thirty minutes after a single injection, morphine (5 or 40 mg/kg s.c.) did not alter the Bmax or Kd values for [3H]QNB binding to striatal receptors. The binding of [3H]QNB to membranes of different brain regions was not changed in morphine tolerant-dependent rats or rats undergoing abrupt or naloxone precipitated withdrawal. The results suggest that central cholinergic muscarinic receptors are unaffected by acute or chronic treatment with morphine, or during abstinence.     

Ontogeny of dopamine D2 receptor mRNA in rat brain

The postnatal development of rat brain dopamine D₂ receptor gene expression was investigated in animals 1 day to 1 year old. The level of expression of the striatal D₂ mRNA was appreciable at birth (day 1), steadily increased to a maximum at day 28, and showed declines at ages 6 months and one year. The mRNA development profile was similar to that of [³H]spiroperidol binding in striatal membranes except that there was a lack of correlation between mRNA levels and [³H]spiroperidol binding during the early developmental periods. For example, although the mRNA expression at day 1 is about 75% of the 28-day value, the corresponding level of [³H]spiroperidol binding is only 15% of the value observed at day 28. Polymerase chain reaction (PCR) analysis of alternatively spliced forms of D₂ receptor mRNA showed that the developmental expression of the two isoforms proceeded in parallel as the ratio of D₂₁ and D₂₅ mRNAs remained more or less constant in different age group of rats. In situ hybridization revealed a differential developmental profile of D₂ mRNA for major dopaminergic regions of rat brain such as caudate putamen, nucleus accumbens, olfactory tubercle and substantia nigra.     

Reduced [H]quinuclidinyl benzilate binding to muscarinic receptors in disulfoton-tolerant mice

The organophosphate, acetylcholinesterase inhibitor, disulfoton, O,O-diethyl S-[2-(ethylthio)ethyl]phosphorodithioate, was given daily for 2 weeks to male mice at two different dosages. Clinical signs of poisoning disappeared in 5 days after the beginning of the treatment, i.e., the animals developed apparent tolerance to disulfoton toxicity. Tolerant mice were less sensitive to a lethal dose of carbachol and exhibited a decrease of [³H]quinuclidinyl benzilate ([³H]QNB) binding in forebrain, hindbrain, and ileum. Scatchard analysis of saturation experiments revealed a decrease in the density of receptors (B_max) in the disulfoton-treated mice, as compared with controls. No significant changes in affinity were found, except in the ileum. A time-course study showed a good parallelism between the decrease of [³H]QNB binding and the development of tolerance. Twenty-one days after the end of the disulfoton treatment AChE activity was still inhibited, but [³H]QNB binding had returned to normal levels. The recovery of [³H]QNB binding appears to be faster in ileum than in forebrain and hindbrain. These findings indicate that the development of tolerance to chronic organophosphate treatment is, at least partially, due to a reduction in the number of cholinergic receptors.     

3H]Pirenzepine and [3H]quinuclidinyl benzilate binding to brain muscarinic cholinergic receptors. Differences in measured receptor density are not explained by differences in receptor isomerization

Muscarinic receptor densities were measured in membranes prepared from rat cerebral cortex using [3H]pirenzepine and [3H]quinuclidinyl benzilate. Isotherms of equilibrium binding data modeled to a single apparent binding site for both ligands. However, as has been reported previously, [3H]pirenzepine labeled only a small fraction of the binding sites that were labeled by [3H]quinuclidinyl benzilate. This observation has been used to support the hypothesis that subtypes of muscarinic receptors exist. Several investigators have previously suggested that antagonist binding to muscarinic receptors involves an isomerization of the receptor-antagonist complex, and it is only the isomerized form of the receptor that is identified by radioligand binding studies. To examine the possibility that the difference in the density of binding sites identified by [3H]pirenzepine and [3H]quinuclidinyl benzilate is due to differences in the degree of isomerization of the receptor associated with the binding of each ligand, the kinetics of the binding of [3H]pirenzepine and [3H]quinuclidinyl benzilate to membranes prepared from rat cerebral cortex were examined. The pseudo-first-order rate constant of association for both ligands showed a nonlinear (hyperbolic) dependence on ligand concentration. These results suggested that a rapidly equilibrating initial binding step was followed by a more slowly equilibrating isomerization of the initially formed ligand-receptor complex. The kinetic data were computer-modeled to obtain estimates of the equilibrium constants for both reaction steps. The equilibrium constants for the isomerization step were 0.1 and 0.004 for [3H]pirenzepine and [3H]quinuclidinyl benzilate, respectively. Our measurements, in agreement with others, suggested that only the fraction of receptors which isomerized were measurable using filtration binding assays. Although essentially all (99.6%) of the [3H]quinuclidinyl benzilate binding sites appeared to isomerize, only 90% of the [3H]pirenzepine binding sites isomerized, and thus only 90% were measured in our assay. It therefore appears that differences in receptor isomerization can partially, but not wholly, account for the differences between [3H]pirenzepine and [3H]quinuclidinyl benzilate binding in rat cerebral cortex.     

Binding characteristics of [3H]quinupramine to rat brain membrane fractions

The binding characteristics of [3H]quinupramine to rat brain membrane fractions were studied. The specific binding of [3H]quinupramine to rat brain membrane fractions was stable, reversible and saturable. Scatchard analysis of the data from saturation experiments indicated that the specific binding was a single population with an affinity (KD) of 3.04 nM, a maximal binding site number (Bmax) of 714 fmol/mg protein, and a Hill coefficient (nH) of 1.08. Compounds known to inhibit muscarinic cholinergic receptors such as atropine and quinuclidinyl benzilate were the most potent competitors of [3H]quinupramine binding. When the drug potencies in inhibiting [3H]quinupramine binding were tested in the presence of 10 nM atropine, mianserin was the most potent competitor. Studies of the subcellular fractions showed that there was an enrichment of [3H]quinupramine binding sites in the synaptosome fraction. The regional distribution study revealed the highest densities of binding sites in the cerebral cortex and the lowest in the cerebellum. Thus, the specific binding of [3H]quinupramine observed here can be accounted for by both muscarinic cholinergic and serotonin S2 receptors.     

Neurotransmitter receptors in brain regions of acrylamide- treated rats. I: Effects of a single exposure to acrylamide

A single oral dose of acrylamide (25 or 50 mg/kg) increased the level of striatal 3H-spiroperidol binding in six week old male rats. This enhanced dopamine receptor activity was specific since treatment caused no significant changes in glycine, serotonin, and muscarinic cholinergic binding. At the highest acrylamide dose tested (100 mg/kg), elevations of the medullary glycine and frontal cortical serotonin receptors were also found. Pretreatment of animals with a blocker of hepatic mixed function oxidase (SKF 525a) or a thiol blocker (methylmercuric chloride) prevented the acrylamide-induced elevation of striatal spiroperidol binding, indicating that the causative agent was a secondary metabolite of acrylamide. Apomorphine-induced motility was significantly attenuated by 24 hr predosing with acrylamide, suggesting a change in the sensitivity of the dopamine receptor. The behavioral relevance of observed biochemical changes was thus shown by the altered response of treated animals to apomorphine.     

Protein malnourishment: a predisposing factor in acrylamide toxicity in pregnant rats.

Exposure to acrylamide (3-10 mg/kg body weight) was found to be lethal for protein-deficient pregnant rats as evidenced by their increased mortality. It had no such effect on the normal protein diet fed pregnant and nonpregnant rats and the protein-malnourished nonpregnant rats. Protein deficiency during pregnancy caused a significant decrease in the activity of brain monoamine oxidase and acetylcholinesterase and striatal [3H]spiperone binding, known to label dopamine receptors; had no significant effect on the binding of 3H-QNB (quinuclidinyl benzilate) to cerebellar and [3H]diazepam to frontocortical membranes, known to label muscarinic and benzodiazepine receptors, respectively; and had no significant effect on brain glutathione (GSH) levels in comparison with pregnant rats fed normal protein diet. Exposure to acrylamide (2 mg/kg body weight) in protein-malnourished pregnant rats caused a marked decrease in the activity of monoamine oxidase and acetylcholinesterase and also in the binding of [3H]spiperone, [3H]QNB, and [3H]diazepam to striatal, cerebellar, and frontocortical membranes, respectively. Kinetic studies revealed that decreased binding of these ligands in the specific brain regions were due to decreased receptor sites (Bmax). A reduction in the brain glutathione content was also observed in these animals in comparison with those fed a low-protein diet during pregnancy. Pregnant rats fed a normal-protein diet on acrylamide exposure, however, showed no such biochemical changes in comparison with the pregnant rats fed normal protein diet. Also, no effect on any of the parameters studied was observed in the adult nonpregnant rats fed a low-protein diet (for 18 d) and those exposed to the monomer (d 6-17) fed either a normal- or low-protein diet in comparison with respective controls. The results indicate that pregnancy under conditions of malnutrition modifies the susceptibility of pregnant rats toward acrylamide.     

EFFECT OF THYROID STATUS ON β-ADRENORECEPTORS AND MUSCARINIC RECEPTORS IN THE RAT LUNG

• 1 The effects of thyroid status on the specific binding of the muscarinic ligand (–)-[³H] quinuclidinyl benzilate (QNB) and of the β-adrenoreceptor ligand (–)-[³H] dihydroalprenolol (DHA) in the adult rat lung were investigated.
• 2 The specific binding of (–)-[³H] quinuclidinyl benzilate (QNB) to lung membranes was saturable and the equilibrium dissociation constant (K_D) determined from Scatchard analysis was 54 pM. Kinetic analysis of the binding of [³H] QNB yielded a K_D of 42 pM. [³H] QNB binding was inhibited by muscarinic agonists and antagonists, the order of their potency was l-hyoscyamine>atropine>scopolamine>oxotremorine>carbachol. These data were consistent with [³H] QNB binding to the muscarinic receptor.
• 3 Adult male rats treated for 2 weeks with the antithyroid agent 3-amino-1,2,4-triazole (ATZ) showed a 52% and 80% reduction in the serum concentration of triiodothyronine (T₃) and thyroxine (T₄) respectively. These hypothyroid rats also had a 39% decrease in the concentration of lung β-adrenoreceptors and a 37% decrease in the concentration of lung muscarinic receptors as compared to euthyroid controls. Concurrent treatment of rats with ATZ and T₄ for 2 weeks resulted in a reduction of 15% and 20% in the concentration of lung β-adrenoreceptors and muscarinic receptors respectively. The K_D values for [³H] DHA and [³H] QNB binding did not change with the ATZ or ATZ + T₄ treated groups.
• 4 Administration of T₄ (500 μg/kg/day) to male rats for 12 days did not result in any significant change in the concentration of either β-adrenoreceptors or muscarinic receptors compared to euthyroid controls. No change in the K_K values for [³H] DHA or [³H] QNB binding were detected.
• 5 The results show that hypothyroid rats have a reduced lung concentration of both β-adrenoreceptors and muscarinic receptors whereas in hyperthyroid rats these receptors do not significantly change from euthyroid controls.

     

Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site

Experimental probing of the model of the muscarinic M₂ receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8–15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [³H] N-methylscopolamine and [³H] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [³H] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [³H] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (<10-fold) to the binding site of the M₂ receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M₂ receptor binding site but W155 is important for proper ligand binding on the muscarinic M₂ receptor, as predicted by the proposed model.