α-Bungarotoxin Binding in House Fly Heads and Torpedo Electroplax

Abstract: House fly heads contain a site that binds α-bungarotoxin with high affinity. It is present at about 23 pmol/g of heads and binds α-bungarotoxin (labeled with [³H]pyridoxamine phosphate) reversibly with a K _d of 6 nM. The effects of 48 drugs have been compared on the α-bungarotoxin binding sites of house fly and Torpedo. The pharmacology of the house fly site is similar to that previously reported for neuronal α-bungarotoxin binding sites in both vertebrates and invertebrates and is distinguishable from that of the classic nicotinic neuromuscular acetylcholine receptor, as exemplified by that of Torpedo electroplax. Differences between the house fly site and Torpedo include higher affinities of the Torpedo receptor for decamethonium, hexamethonium, carbamylcholine, and acetyl-β-methylcholine, but lower affinities for nicotine, atropine, and dihydro-β-erythroidine.     

Muscarinic Binding of Pial Vessels and Arachnoid Membrane

Abstract: The muscarinic sites in arachnoid and pial vessels were compared by analysis of the binding of quinuclidinyl benzilate (QNB) to membrane preparations. Saturation analysis indicated that the process was saturable, high affinity, and related to protein concentration in both structures. Although the affinities in the two structures [ K _D= 0.039 (arachnoid) and 0.097 n M (pial vessels)] were similar, the arachnoid had ∼ 10-fold more binding sites ( B _max= 2,100 fmol/mg of protein) than the pial vessels ( B _max= 250 fmol/ mg of protein). This difference was found in both bovine and porcine fractions. Pharmacological analysis of [³H]-QNB displacement by muscarinic and nonmuscarinic ligands gave the typical pattern of muscarinic receptors in both structures. Inhibition of binding to pial vessels by the M, antagonist pirenzepine revealed only one low-affinity site ( K _i= 7.8 × 10⁻⁷ M ), whereas, the arachnoid had a small proportion (21%) of high-affinity sites ( K _i= 2.2 × 10⁻⁹ M ) associated with low-affinity sites ( K _i= 5.50 × 10⁻⁷ M ). It is concluded that muscarinic-mediated effects that do not involve the M, subtype are induced in bovine pial vessels by a relatively low concentration of binding sites. The high content of muscarinic binding sites and their diversity in the arachnoid suggest a functional role for muscarinic cholinergic receptors in this structure.     

Identification of Strychnine Binding Sites in the Rat Retina

Abstract: [³H]Strychnine specifically binds to membrane fractions isolated from rat retinae. The binding is saturable, with an apparent dissociation constant, K _D, of 14.3 × 10⁻⁹ M and 205 fmol bound/mg protein. Specific binding is time-dependent and proportional to protein concentration. Glycine and taurine are equally potent inhibitors of [³H]strychnine binding ( K _i= 4 × 10⁻⁵ M); no other amino acids endogenously present in the retina inhibited [³H]strychnine binding.     

Effect of Electroconvulsive Shock on Muscarinic Cholinergic Receptors in Rat Cerebral Cortex and Hippocampus

Abstract: Single electroconvulsive shock (ECS) induced no change in [³H]quinuclidinyl benzilate ([³H]QNB) binding to muscarinic cholinergic receptors in rat cortex and hippocampus. ECS administered once daily for 7 days induced a significant reduction in [³H]QNB binding in both brain areas. Concurrent ECS reversed the significant increase in cortical [³H]QNB binding induced by chronic atropine administration. These findings may have relevance to the antidepressant or amnestic effects of electroconvulsive therapy.     

Specificity of Muscarinic Acetylcholine Receptor Regulation by Receptor Activity

Abstract— Regulation of muscarinic acetylcholine receptor concentration by receptor activity in neuron-like NG108-15 hybrid cells is a highly specific process. Receptor levels, monitored by binding of [³H]quinuclidinyl benzilate ([³H]QNB), decreased 50-75% following 24-h incubation of cells with muscarinic agonists, but none of the following cellular processes was altered by this chronic receptor stimulation: (1) glycolytic energy metabolism, measured by [³H]deoxy- d -glucose ([³H]DG) uptake and retention; (2) rate of cell division; (3) transport, measured by [³H]valine and [³H]uridine uptake; (4) RNA biosynthesis, measured by [³H]uridine incorporation; (5) protein biosynthesis, measured by [³H]valine and [³⁵S]methionine incorporation into total protein and into protein fractions obtained by polyacrylamide gel electrophoresis. In contrast, chronic stimulation did cause a threefold decrease in the capacity of carbachol to stimulate phosphatidylinositol (PI) turnover, a receptor-mediated response. In addition to cholinomimetics, the neuroeffector adenosine (1 m m for 24 h) also caused a decrease in [³H]QNB binding levels, but chronic stimulation of α -adrenergic, opiate, prostaglandin E₁, and prostaglandin F_2α receptors found on NG108-15 cells caused no changes. The data indicate that loss of muscarinic receptors caused by receptor stimulation is not a consequence of fundamental changes evoked in overall cellular physiology but reflects a specific regulation of cholinoceptive cell responsiveness.     

The relationship between cytotoxic effect and binding to mammalian cultured cells of Clostridium perfringens enterotoxin

Abstract The relationship between the cytotoxic effect and binding to different cell lines of Clostridium perfringens enterotoxin was investigated. The enterotoxin released ⁵¹Cr from Vero and MDCK cells labeled with Na₂-⁵¹CrO₄. The effect varied depending upon the dose of enterotoxin and the duration and temperature of the interaction. The enterotoxin gave no effect on FL, KB, or L-929 cells. [¹²⁵I]Enterotoxin bound specifically to Vero and MDCK cells via a binding site of distinct nature, but not to FL, KB, or L-929 cells. The number of the binding sites located on one MDCK cell (1.98 × 10⁶ sites/cell) was three times that on one Vero cell (5.64 × 10⁵ sites/cell), although the binding affinity of MDCK cell ( K _a/ 3.76 × 10⁷ M⁻¹) was 0.1 that of Vero cells ( K _a/ 3.23 × 10⁸ M⁻¹). Binding of the enterotoxin to susceptible cells was temperature-independent.     

[1251]2α-BUNGAROTOXIN AND [3H]QUINUCLIDINYLBENZILATE BINDING IN CENTRAL NERVOUS SYSTEMS OF DIFFERENT SPECIES

Abstract— [¹²⁵I]Diiodo α-bungarotoxin ([¹²⁵I]₂BuTx) and [³H]quinuclidinylbenzilate ([³H]QNB) binding sites were measured in post-nuclear membrane fractions prepared from whole brains or brain regions of several species. Species studied included Drosophila melanogaster (fruit fly), Torpedo californiea (electric ray), Carassius auratus (goldfish), Ram pipiens (grass frog), Kana cutesheiana (bullfrog), Rattus norvegicus (rat, Sprague-Dawley), Mus muscalus (mouse, Swiss random, C58/J, LG/J), Oryctolagus cuniculus (rabbit, New Zealand Whitc), and Bos (cow). Acetyl-CoA: choline O -acetyltransferase (EC 2.3.1.6) levels were also determined in the post nuclear supernatants and correlated with the number of binding sites.
All species and regions except Drosophila had 16–150 fold more [³H]QNB binding sites than [¹²⁵I]₂BuTx binding sites. Brain regions with the highest levels of [¹²⁵I]₂BuTx binding were Drosophila heads (300 fmol/mg), goldfish optic tectum (80fmol/mg), and rat and mouse hippocampus (3040 fmol/mg). The highest levels of [³H]QNB binding were seen in rat and mouse caudate (1.3–1.6 pmol/mg). Lowest levels of [³H]QNB and [¹²⁵I]₂BuTx binding were seen in cerebellum. The utility of [¹²⁵I]₂BuTx and [³H]QNB binding as quantitative measures of nicotinic and muscarinic acetylcholine receptors in CNS is discussed.     

Concanavalin A-Sepharose Affinity Chromatography of Axon Plasma Membrane Proteins. Heterogeneity of Cholinergic Binding Sites

Abstract: Lysolecithin-solubilized proteins from axon plasma membranes of lobster walking leg nerve bundles were chromatographed on concanavalin A (Con A)-sepharose. Bound glycoproteins were eluted with α-methyl-D- mannoside. Near quantitative recovery of total protein was observed, 20–30% of the total protein being eluted in the Con A-binding glycoprotein fraction. A 5-fold enrichment of acetylcholinesterase (AChE) activity was achieved, demonstrating the glycoprotein nature of the axonal enzyme. The chromatographed fractions were characterized for binding of [³H]quinuclidinyl benzilate (QNB), [³nicotine (Nic), and [1251]α-bung arotoxin (BgTx) in an attempt to distinguish possible "muscarinic" and "nicotinic" binding sites in axonal membranes. All of the high-affinity "muscarinic" [³H]QNB binding activity appeared in the non-Con A-binding protein fractions, while binding of the two "nicotinic" ligands, [³Nic and ¹²⁵I-BgTx, was found in both the glycoprotein and non-Con A-binding protein fractions. BgTx interaction with the Con A-binding glycoproteins could be blocked with dtubocurarine, but BgTx binding in the non-Con A-binding proteins was not inhibited by curare. The significance of multiple cholinergic binding sites in axonal membranes is discussed. These data suggest a closer similarity between the cholinergic ligand binding proteins of peripheral nerve membrane and ganglia than between the axonal cholinergic binding sites and the ACh receptor of the neuromuscular junction.     

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A_2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A_2A receptors ([³H]CGS 21680) or A₁ receptors ( N ⁶-[³H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A_2A versus A₁ receptors ( K _i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [³H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K _D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [³H]SCH 58261 with the following estimated K _i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N ⁶-cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg²⁺ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [³H]CGS 21680.     

Stimulation of Guanosine 5'-O-(3-[35S]Thiotriphosphate) Binding by Cholinergic Muscarinic Receptors in Membranes of Rat Olfactory Bulb

Abstract: In membranes of rat olfactory bulb, a brain region in which muscarinic agonists increase cyclic AMP formation, the muscarinic stimulation of guanosine 5'- O -(3-[³⁵S]thiotriphosphate) ([³⁵S]GTPγS) binding was used as a tool to investigate the receptor interaction with the guanine nucleotide-binding regulatory proteins (G proteins). The stimulation of the radioligand binding by carbachol (CCh) was optimal (threefold increase) in the presence of micromolar concentrations of GDP and 100 m M NaCl. Exposure to N -ethylmaleimide and pertussis toxin markedly inhibited the CCh effect, whereas it increased the relative stimulation of [³⁵S]GTPγS binding elicited by pituitary adenylate cyclase-activating polypeptide (PACAP). On the other hand, membrane treatment with cholera toxin curtailed the PACAP stimulation of [³⁵S]GTPγS binding but did not affect the response to CCh. Like CCh, a number of cholinergic agonists stimulated [³⁵S]GTPγS binding in a concentration-dependent and saturable manner. The antagonist profile of the muscarinic stimulation of [³⁵S]GTPγS binding was highly correlated with that displayed by the muscarinic stimulation of adenylyl cyclase. These data indicate that the olfactory bulb muscarinic receptors couple to G_i/G_o, but not to G_s, and support the possibility that activation of G_i/G_o mediates the stimulatory effect on adenylyl cyclase activity.     

Rapid Communication: cis-Methyldioxolane Specifically Recognizes the m2 Muscarinic Receptor

Abstract: cis -Methyldioxolane (CD) is a muscarinic receptor agonist. [³H] CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [³H] CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [³H] CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [³H] CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty-eight percent of Hm2 receptors bound [³H] CD with a K _D of 3.5 ± 0.5 nM. Binding was eliminated in the presence of guanosine 5'- O -(3-thiotriphosphate), indicating that the Hm2 receptors labeled by [³H] CD are those that are associated with GDP-bound G protein. Binding of [³H] CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [³H] CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [³H] CD should aid research in these areas.     

Pharmacological and Regional Characterization of [3H]LY278584 Binding Sites in Human Brain

Abstract: Binding of [³H]LY278584, which has been previously shown to label 5-hydroxytryptamine₃ (5-HT₃) receptors in rat cortex, was studied in human brain. Saturation experiments revealed a homogeneous population of saturable binding sites in amygdala ( K _D= 3.08 ± 0.67 n M, B _max= 11.86 ± 1.87 fmol/mg of protein) as well as in hippocampus, caudate, and putamen. Specific binding was also high in nucleus accumbens and entorhinal cortex. Specific binding was negligible in neocortical areas. Kinetic studies conducted in human hippocampus revealed a K _on of 0.025 ± 0.009 n M ⁻¹ min⁻¹ and a K _off of 0.010 ± 0.002 min⁻¹. The kinetics of [³H]LY278584 binding were similar in the caudate. Pharmacological characterization of [³H]LY278584 specific binding in caudate and amygdala indicated the compound was binding to 5-HT₃ receptors. We conclude that 5-HT₃ receptors labeled by [³H]LY278584 are present in both limbic and striatal areas in human brain, suggesting that 5-HT₃ receptor antagonists may be able to influence the dopamine system in humans, similarly to their effects in rodent studies.     

M1 Muscarinic Acetylcholine Receptor in Cultured Rat Neostriatum Regulates Phosphoinositide Hydrolysis

Abstract: : Muscarinic acetylcholine receptor expression and function in cultured rat neostriatal neurons were examined. All experiments were performed on intact neurons grown in vitro for 12-14 days. The muscarinic antagonist N-[³H]methylscopolamine ([³H]NMS) binds to a single site in cultures with a K_D of 89 pM and a B_max of 187 fmol/mg of protein, or 32,000 sites/neuron. Competition studies using [³H]NMS were performed to determine what receptor sur > types were present. Nonlinear analysis of competition curves was best described with a single binding site for atropine, pirenzepine, and AF-DX 116 {11-[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one}, with K_i values of 0.6, 62, and 758 nM, respectively. These results indicate that the muscarinic receptors present in neostriatal cultures are of the M₁subtype, having high affinity for pirenzepine and low affinity for AF-DX 116. In contrast with antagonists, carbachol displaced [³H]NMS from two sites with K_i values of 6.5 and 147 μM, with the higher-affinity form predominant (83% of sites). The M₁ receptor subtype was linked to phosphoinositide turnover. Carbachol stimulated the formation of phosphoinositides with an EC₅₀ of 37 μM and was antagonized by atropine. At equimolar doses, pirenzepine was more potent than AF-DX 116 at antagonizing the response.     

Characterization of the transferrin-iron uptake system in Neisseria meningitidis

Abstract The transferrin-iron uptake system of six Neisseria meningitidis strains was characterized using ¹²⁵I-transferrin in receptor assays and ⁵⁵Fe-loaded transferrin in uptake assays. Receptors for transferrin varied among the strains both in number (from 700 to 4700 receptors per cell) and in their affinity constants for the protein ( K _a ranged from 0.7×10⁷ to 4.0×10⁷ 1 mol⁻¹). Neither receptor numbers nor affinity constants were significantly different in carrier and invasive strains, although the K_a seem to be somewhat higher in the latter. Iron uptake from transferrin was also variable among the strains, but showed the same lack of correlation with their origin.     

Differential Effects of Hypoxia on Ligand Binding Properties of Nicotinic and Muscarinic Acetylcholine Receptors on Cultured Bovine Adrenal Chromaffin Cells

Abstract: Hypoxia is known to disturb neuronal signal transmission at the synapse. Presynaptically, hypoxia is reported to suppress the release of neurotransmitters, but its postsynaptic effects, especially on the function of neurotransmitter receptors, have not yet been elucidated. To clarify the postsynaptic effects, we used cultured bovine adrenal chromaffin cells as a model of postsynaptic neurons and examined specific binding of l -[³H]nicotine (an agonist for nicotinic acetylcholine receptors: nAChRs) and ²²Na⁺ flux under control and hypoxic conditions. Experiments were performed in media preequilibrated with a gas mixture of either 21% O₂/79% N₂ (control) or 100% N₂ (hypoxia). Scatchard analysis of the specific binding to the cells revealed that the K_D under hypoxic conditions was twice as large as that under control conditions, whereas the B _max was unchanged. When the specific [³H]nicotine binding was kinetically analyzed, the association constant ( k ₁) but not the dissociation constant ( k ₋₁) was decreased to 40% of the control value by hypoxia. When the binding assay was performed using the membrane fraction, these changes were not observed. Nicotine-evoked ²²Na⁺ flux into the cells was suppressed by hypoxia. In contrast, specific [³H]quinuclidinyl benzilate binding to the intact cells was unaffected by hypoxia. These results demonstrate that hypoxia specifically suppresses the function of nAChRs (and hence, neuronal signal transmission through nAChRs), primarily by acting intracellularly.     

The binding of hepatitis A virus to cell surfaces shows evidence of positive co-operativity

Abstract Radio-iodinated hepatitis A virus binds to cultured mammalian cells in a saturable manner, with about 1.4 × 10³ sites/cell and a S _0.5 of about 1.4 × 10⁻¹¹ M for FRhK-4 cells. This binding to FRhK-4 cells shows evidence of positive co-operativity, with a Hill coefficient of 2.1 (±0.1). This implies that the cellular receptor for the virus may have multiple binding sites and that the affinity of HAV for its receptor is increased if one of the binding sites is occupied by virus. Binding is completely blocked by two neutralising monoclonal antibodies, which also inhibit viral haemagglutination. A non-neutralising monoclonal antibody partially inhibits binding to FRhK-4 cells, but has no effect on haemagglutination.     

Detection of the response regulator AgrA in the cytosolic fraction of Staphylococcus aureus by monoclonal antibodies

Abstract The interaction of salivary lysozyme with the surface protein antigen (PAc) of Streptococcus mutans and the interaction of lysozyme with the pathogen were examined by ELISA using S. mutans MT8148 (PAc⁺) and the PAc-defective mutant EM-2 (PAc⁻). The lysozyme clearly bound to the S. mutans wild type but not to the S. mutans mutant. Furthermore, lysozyme bound directly in the fluid phase to the rPAc, of which the binding kinetics were determined ( K _on= 3.63 ± 0.04 × 10³M⁻¹ s ⁻¹, K _off= 1.72 ± 0.04 × 10⁻⁵s⁻¹ and K_on / K_off= 2.11 × 10⁸M⁻¹) using surface plasmon resonance. The kinetics of both association and dissociation were relatively slow. In addition, anti-lysozyme antibody significantly inhibited the binding of salivary components to the rPAc. The present findings indicate that lysozyme is one of the major salivary components interacting with S. mutans PAc.     

Neurosteroids Modulate Nicotinic Receptor Function in Mouse Striatal and Thalamic Synaptosomes

Abstract: Progesterone and its A-ring reduced metabolites are allosteric activators of GABA_A receptors. The studies reported here examined the effects of these steroids on brain nicotinic receptors using an ⁸⁶Rb⁺ efflux assay that likely measures the function of α4β2-type nicotinic receptors and [³H]dopamine release, which may be modulated by an α3-containing nicotinic receptor. Both of the A-ring reduced metabolites of progesterone were noncompetitive inhibitors of both assays, whereas progesterone inhibited only the ⁸⁶Rb⁺ efflux assay. The ⁸⁶Rb⁺ efflux assay was slightly more sensitive than was the dopamine release assay to steroid inhibition. Inhibition developed slowly for both assays ( t _1/2 = 0.4 min) and was reversed even more slowly ( t _1/2 = 10–15 min). Steroid addition did not alter either the rate of association of [³H]nicotine binding to brain membranes, nor was equilibrium binding changed. These findings argue that neurosteroids are allosteric inhibitors of brain nicotinic receptors.     

Characterization of the pColV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein of Escherichia coli; isolation and purification of the protein and analysis of its nucleotide sequence and primary structure

Abstract The cloacin DF13/aerobactin receptor protein from Escherichia coli (pFS8) and from Klebsiella edwardsii were isolated by repeated Triton X-100 extractions and purified by affinity chromatography. Both receptor proteins ran as a single protein band on SDS-PAGE. Their apparent M_r values were 74 000 and 76 000, respectively. The binding constants of the purified receptor proteins from E. coli (pFS8) and K. edwardsii and cloacin DF13 were determined. Values of 2.0 × 10⁸ M⁻¹ and 1.0 × 10⁹ M⁻¹, respectively, were found.
The nucleotide sequence of the pColV-K30 gene, contained on pFS8 and encoding the cloacin DF13/aerobactin receptor protein, was determined and the primary structure of the protein as well as its secondary structure were deduced. The results revealed that the pColV-K30-specified receptor protein might be synthesized as a precursor, with a signal sequence of 25 amino acid residues. The mature protein has an M_r of 77 345.     

Sequestration of 125I-Labeled p Nerve Growth Factor by Embryonic Sensory Neurons

Abstract: Nerve growth factor (NGF) binds to two specific receptors on sensory nerve cells. These two receptors are characterized by different equilibrium dissociation constants. The higher affinity (type I) receptors have an equilibrium dissociation constant of 3.3 × 10^-11 M. The lower affinity (type II) receptors have an equilibrium dissociation constant of 1.7 × 10^-9M. These two receptors are not a result of negative cooperatively, but apparently are different receptors. At 22°C the rate of association is 1 × 10⁷ M^-1 S^-1 and the rates of dissociation are 6.5 × 10^-4 s^-1 (type I) and 3.2 × 10^-2 s^-1'(type II). After binding, a time-dependent process occurs that makes the NGF inaccessible to the external milieu (sequestered). The sequestration process is energy-dependent, but apparently temperature-independent. The data suggest that only the type I receptors are involved in the sequestration process. This process is similar to that observed on sympathetic neurons and may be the first step in the internalization of NGF by responsive cells.