Characterization of Neurotransmitter Receptors in the Rat Hippocampal Formation

The hippocampal formation has been extensively research in terms of its putative neurotransmitters, anatomical connections, and behavioral relevance. An aspect of importance is the assessment of apparent neurotransmitter receptors by using receptor binding assays. In the present study, such assays were done in vitro to investigate alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic, muscarinic cholinergic, benzodiazepine, and opiate receptors in the rat hippocampal formation. The corresponding radioligands for these receptors were [3H]prazosin, [3H]p-aminoclonidine, [3H]dihydroalprenolol, [3H]quinuclidinyl benzilate, [3H]flunitrazepam, and [3H]naloxone. An analysis of the binding parameters for the ligands indicated saturable binding of a high affinity and the following rank order of maximal binding capacities: [3H]flunitrazepam greater than [3H]quinuclidinyl benzilate greater than [3H]naloxone greater than [3H]p-aminoclonidine greater than [3H]prazosin greater than [3H]dihydroalprenolol. Competition experiments with pharmacologic agonists and antagonists confirmed the specificity of each ligand. The results are integrated with information on other types of receptors and with neurotransmitter concentrations, and discussed in terms of hippocampal function.     

Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.     

Ligand-binding propeties of the muscarinic acetylcholine receptor in mouse neuroblastoma cells.

1. Muscarinic acetylcholine receptors in a plasma-membrane fraction derived from mouse neuroblastoma clone NIE-115 bind [3-3H]quinuclidinyl benzilate according to the Law of Mass Action (Kdissociation 40 pM, h0.96). 2. Antagonist and agonist binding to the receptor was studied by displacement of [3-3H]quinuclidinyl benzilate with non-radioactive ligands. The data show good agreement with similar data obtained on rat brain and ideal smooth muscle [Birdsall & Hulme (1976) J. Neurochem. 27, 7-16] indicating that the receptor is very similar in these three tissues.     

Decreased Number of Benzodiazepine Receptors in Frontal Cortex of Rat Brain Following Long-Term Lithium Treatment

Chronic administration of lithium led to a decreased number of benzodiazepine receptors (ca. 20%) in frontal cortex of rat brain, whereas no change was observed in the binding characteristics in the remaining part of the cortex and in the hippocampus and the cerebellum. Long-term lithium treatment did not change the binding of [3H]lysergic acid diethylamide and [3H]quinuclidinyl benzilate to membranes of various brain regions in the rat. We concluded that the effect of lithium on the benzodiazepine receptor is brain region specific and cannot be explained as a consequence of a reduced gamma-aminobutyric acid-ergic stimulation of benzodiazepine receptor, as the change in receptor binding was due to a change in the number of receptors rather than in the affinity constant.     

Increased muscarinic receptor binding in heart membranes by an inhibitor of protein kinase C

The number of muscarinic acetylcholine receptors (MAChRs), as detected by binding of [3H]quinuclidinyl benzilate (QNB), was investigated under conditions which promote protein phosphorylation. Incubation of a crude heart membrane preparation in the presence of ATP/Mg2+ reduced MAChR number by 50%. Incubation with polymyxin B, an inhibitor of protein kinase C, blocked the effect of ATP/Mg2+ and increased MAChR number by 74%.     

Dopaminergic and cholinergic muscarinic receptor effects of L-alphacetylmethadol (LAAM) and its metabolites

In isolated rat hearts L-alphacetylmethadol (LAAM) produced a concentration-dependent decrease in the spontaneous beating rate. This effect was completely prevented by 1.0 microM atropine. Chronic treatment of rats with LAAM increased the number of striatal dopamine receptors measured by [3H]spiroperidol binding. The affinity of these binding sites for [3H]spiroperidol was unchanged by LAAM treatment. There were no significant changes in the number or affinity of binding sites for the labeled muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) with chronic LAAM treatment. The ability of LAAM, nor-LAAM, or dinor-LAAM to antagonize the binding of [3H]spiroperidol (40 pM) or [3H]QNB (125 pM) to striatal membrane fragments was tested. The measured affinity constants for LAAM and metabolites were 100-3000 times higher than the affinity constants of unlabeled spiroperidol at [3H]spiroperidol binding sites. The affinity constants of LAAM and metabolites at muscarinic binding sites were 10-20 times higher than pilocarpine and 5000-8000 times higher than atropine. These results suggest that LAAM can produce some of its effects by acting as a weak agonist at muscarinic receptor sites.     

Muscarinic antagonist binding site heterogeneity as evidenced by autoradiography after direct labeling with [3H]-QNB and [3H]-pirenzepine

Saturable, high affinity binding of tritiated pirenzepine [( 3H]-PZ) was obtained in slide mounted tissue sections prior to performing autoradiographic localization of these binding sites. The binding in tissue sections of rostral rat forebrain gave a KD of 18nM and a Bmax of 51 fmoles/mg tissue. These binding characteristics are similar to those previously obtained in homogenate membrane preparations and indicate the binding is taking place in a similar manner. The distribution of the binding sites labeled with [3H]-PZ represented a subpopulation of those which could be labeled with tritiated quinuclidinyl benzilate [( 3H]-QNB). Thus, [3H]-PZ and [3H]-QNB both label regions of the cerebral cortex, hippocampus, striatum and dorsal horn of the spinal cord, while sites in the cerebellum, nucleus tractus solitarius, facial nucleus and ventral horn of the spinal cord are labeled with [3H]-QNB and not by [3H]-PZ. These observations indicate separate regions of the brain where antagonists bind to subtypes of muscarinic receptors.     

Aging and rat brain muscarinic receptors as measured by quinuclidinyl benzilate binding

Measurement of cholinergic muscarinic receptor binding in various rat brain areas using the ligand [³H]quinuclidinyl benzilate indicates that receptor binding is decreased in striatum and cerebellum of aged female rats (22 months old) as compared to younger rats (4 months old). Decreases were not observed in cortex, hippocampus, hypothalamus, or amygdala areas. Further examination of [³H]quinuclidinyl benzilate binding in subcellular fractions of aged and young rat cerebellum and striatum indicated a decrease in binding in the crude nuclear and crude synaptosomal fractions. Binding data indicate the observed decrease in specific ligand binding is due to a decrease in number of binding sites while receptor affinity does not appear to change.Supported by the Research Service of the Veterans Administration and by Research Grant NS 13227 from NINCDS.     

Myocardial muscarinic acetylcholine receptor: Choline and tris unmask heterogeneity of antagonist binding sites

The binding properties of myocardial muscarinic acetylcholine receptors are altered in the presence of choline or Tris. The binding of the antagonist [³H]quinuclidinyl benzilate is reduced in the presence of choline or Tris buffer, when compared to parallel determinations in a physiologic salt solution or phosphate buffer. Scatchard analysis indicates the reduced binding is due to a decrease in the apparent number of receptor sites. Experiments with other organic buffers exclude the possibility that the reduced binding in Tris is due to the absence of sodium ions. In the presence of choline or Tris up to 45% of the receptors are not accessible to [³H]quinuclidinyl benzilate. The remaining sites maintain their high affinity for the antagonist. A heterogeneity of antagonist sites is evident.     

Sodium Nitroprusside Induces Internalization of Muscarinic Receptors Stably Expressed in Chinese Hamster Ovary Cell Lines

Abstract: We have characterized the internalization of muscarinic acetylcholine receptors induced by the nitric oxide (NO)-generating compound sodium nitroprusside. When Chinese hamster ovary cells, stably transfected with the human m4 muscarinic receptor subtype, were incubated for 1 h in the presence of 700 µ M sodium nitroprusside, the number of receptors measured in intact cells with the hydrophilic ligand N -[³H]methylscopolamine was reduced by 30%. The effect was dose dependent, beginning with a concentration of sodium nitroprusside as low as 45 µ M . Removal of sodium nitroprusside from the incubation medium did not result in a recovery of the binding sites. The phenomenon was temperature dependent and was blocked by the muscarinic antagonist atropine. No receptor diminution was detected when the number of binding sites was evaluated with the lipophilic antagonist [³H]quinuclidinyl benzilate. This indicates that sodium nitroprusside induces a redistribution of the muscarinic receptors between the plasma membrane and an internal compartment of the cell. Receptor loss was readily reversed by treatment with the sulfhydryl reducing agent diethyldithiocarbamate. Our data provide evidence that muscarinic receptors are internalized by sodium nitroprusside through the oxidation of sulfhydryl groups; they also suggest that NO could play a role in muscarinic receptor desensitization.     

Sphingosine inhibits muscarinic cholinergic receptor binding in rat parotid acinar cells

1. Sphingosine inhibited the binding of [³H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells.2. The inhibition of [³H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites.3. The effect of Sphingosine on the binding was not affected by the chelation of extracellular Ca²⁺.4. H-7, an inhibitor of protein kinase C, failed to decrease [³H]QNB binding.5. Stearylamine, an analogue of Sphingosine, was as effective as Sphingosine in inhibiting [³H]QNB binding.6. These results suggest that Sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca²⁺ and protein kinase C.     

Quantitative Relationships of Five Putative Neurotransmitter Receptor Sites in Rat Hippocampal Formation

The hippocampus is well suited for studies of the interrelationships of various neurotransmitter systems in the CNS by reason of its simple laminated organization, defined connections, and variety of identified neurotransmitters. We have studied the biochemical and pharmacological properties of five radiolabeled ligand binding sites in a membrane fraction prepared from rat hippocampal formation. These binding sites are thought to identify recognition sites for neurotransmitter receptors. The rank order of ligand binding sites is [³H]muscimol > [³H]quinuclidinyl benzilate > [³Hdihydroergocryptine > [³H]dihydroalprenolol > ¹²⁵I-labeled α-bungarotoxin. All ligands have a single, saturable, high-affinity binding site. Pharmacological characterization of the ligand binding sites indicates properties consistent with the identification of these sites as neurotransmitter receptors.     

Mechanism of Agonist-Induced Down-Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.     

Binding of N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H] quinuclidinyl benzilate to CNS extracts of the cockroach Periplaneta americana

The nerve cord of the cockroach (Periplaneta americana) contains distinct saturable components of specific binding for the ligands N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H]quinuclidinyl benzilate. N-[Propionyl-3H]propionylated alpha-bungarotoxin bound reversibly to homogenates with a Kd of 4.8 nM and Bmax of 910 fmol mg-1. The association rate constant (1.9 X 10(5) M-1 s-1) and dissociation rate constant (1.2 X 10(-4) s-1) yielded a Kd of 0.6 nM. Nicotinic ligands were found to displace toxin binding most effectively. The binding sites characterized in this way showed many similarities with the properties of the vertebrate neuronal alpha-bungarotoxin binding site. For a range of cholinergic ligands, inhibition constants calculated from toxin binding studies closely corresponded to their effectiveness in blocking the depolarizing response to acetylcholine recorded by electrophysiological methods from an identified cockroach motoneurone. The N-[propionyl-3H]propionylated alpha-bungarotoxin binding component therefore appears to be a constituent of a functional CNS acetylcholine receptor. Binding of L-[benzilic-4,4'-3H]quinuclidinyl benzilate was reversible with a Kd of 8 nM and Bmax of 138 fmol mg-1, determined from equilibrium binding experiments. The Kd calculated from the association rate constant (2.4 X 10(5) M-1 s-1) and dissociation rate constant (1.3 X 10(-4) s-1) was 1.9 nM. Muscarinic ligands were the most potent inhibitors of quinuclidinyl benzilate binding. The characteristics of this binding site resembled those of vertebrate CNS muscarinic cholinergic receptors. In contrast with vertebrate CNS, the nerve cord of Periplaneta americana contains more (approximately X 7) alpha-bungarotoxin binding sites than quinuclidinyl benzilate binding sites.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

Isolation of rat brain subcellular fraction enriched in putative neurotransmitter receptors and synaptic junctions

A simple reliable method was developed for the rapid isolation of a synaptic plasma membrane-enriched fraction from rat brain. The procedure involves the direct lysis of a crude mitochondrial fraction followed by a combined flotation-sedimentation density gradient centrifugation in a fixed-angle centrifuge rotor. All fractions have been characterized with respect to relative enrichment of (Na⁺–K⁺) ATPase activity as well as putative cholinergic neurotransmitter receptors determined by [¹²⁵I]-bungarotoxin and [³H]quinuclidinyl benzilate binding. The 2-to 4-fold relative enrichment of putative receptor binding sites correlated well with the 4-fold enrichment of morphologically identifiable synaptic junctions in the synaptic plasma membrane enriched fraction.     

Binding of Agonists and Antagonists to Muscarinic Acetylcholine Receptors on Intact Cultured Heart Cells

The binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured cardiac cells has been compared with the binding observed in homogenized membrane preparations. The antagonists [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine bind to a single class of receptor sites on intact cells with affinities similar to those seen in membrane preparations. In contrast with the heterogeneity of agonist binding sites observed in membrane preparations, the agonist carbachol binds to a homogeneous class of low-affinity sites on intact cells with an affinity identical to that found for the low-affinity agonist site in membrane preparations in the presence of guanyl nucleotides. Kinetic studies of antagonist binding to receptors in the absence and presence of agonist did not provide evidence for the existence of a transient (greater than 30 s) high-affinity agonist site that was subsequently converted to a site of lower affinity. Nathanson N. M. Binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured heart cells.     

Possible Allosteric Interaction of 4-Aminopyridine with Rat Brain Muscarinic Acetylcholine Receptors

The interaction of the potassium channel blocker 4-aminopyridine (4-AP) and its analogs with muscarinic acetylcholine receptors was studied in rat brain homogenate. 4-AP displaced specific [3H]quinuclidinyl benzilate [( 3H]QNB) binding in a concentration-dependent fashion. Hill coefficient values decreased with increasing the concentration of [3H]QNB and different analogs of 4-AP demonstrated varying potencies. Scatchard analysis of saturation isotherms of specific [3H]QNB binding showed that low concentrations of 4-AP slightly reduced maximum binding without affecting the equilibrium dissociation constant, whereas higher concentrations reduced maximum binding further and significantly increased the equilibrium dissociation constant. Schild plots of these data resulted in curvilinear functions. The results are discussed in terms of possible allosteric interactions between potassium channels and muscarinic receptor binding sites.     

Quaternary forms of classical muscarinic antagonists distinguish subpopulations of muscarinic receptors: correlation with gallamine-defined subpopulations

Atropine and scopolamine inhibit the binding of [3H] quinuclidinyl benzilate (QNB) to muscarinic receptors of rat forebrain in a manner that suggests homogeneity of the binding sites. Under the same conditions, the inhibition by N-methylatropine (NMA) and N-methylscopolamine (NMS) of the binding of [3H]QNB is consistent with the presence of subpopulations of receptors that differ greatly in affinities toward these quaternary ligands. The subpopulations that are defined according to the affinities of NMA and NMS correlate very well with those that are defined by the use of gallamine. It is suggested that the heterogeneity in the binding of NMS explains some of the anomalous interactions between NMS and gallamine that have been reported previously.     

Ganglion cells of chicken retina possess nicotinic rather than muscarinic acetylcholine receptors

Chicken retinas were exposed to intravitreal kainic acid to destroy amacrine and bipolar cells at low concentrations, and horizontal cells at high concentrations in addition. Ganglion cells were destroyed by intravitreal injections of colchicine. Low doses of kainic acid reduced the number of binding sites for both [³H]quinuclidinyl benzilate (muscarinic acetylcholine receptors) and N-[propionyl ³H]-bungarotoxin (nicotinic acetylcholine receptors), with little additional loss at higher doses. In contrast, colchicine reduced the number of binding sites for N-[propionyl-³H]-bungarotoxin, but had little or no effect on the number of binding sites for [³H]quinuclidinyl benzilate. These results are consistent with the idea that, in chicken retina, cholinergic amacrine cells make contact with ganglion cell dendrites at sites which possess mainly nicotinic acetylcholine receptors, while both types of receptor are involved in interactions between amacrine cells and perhaps bipolar cells.