Butanol extracts from myelin fragments. VI. Lack of specificity of 5-hydroxytryptamine binding to recombinate fraction with sulphatides and phosphatidylinositol

To clarify the participation of sulphatides and PI for the binding of 5-HT to myelin butanol extracts, binding experiments of ¹⁴C·5-HT to the recombinate fraction of these two acidic lipids were performed. The recombinate fraction was incubated with 5 × 10^?7 M of ¹⁴C·5-HT, and elution profile of ¹⁴C·5-HT, sulphatides and PI was examined by Sephadex LH₂₀ column chromatography. Three components were eluted with CM 4:1 and elution areas of those relatively corresponded to each other. On the other hand, when 3 fold excess volumes of solvents were used ¹⁴C·5-HT was also eluted with CM 4:1 but the clear difference was observed in the elution pattern of sulphatides and PI. In the myelin butanol extracts, we have reported that the 5-HT binding macromolecules present in the myelin extracts would be composed of saturable and non-saturable components, and ACh, DA and tryptamine specifically inhibited the saturable 5-HT binding. Therefore, on the recombinate system of two acidic lipids the specificity of ¹⁴C·5-HT binding was investigated by displacement experiments. The results indicated that only ACh slightly inhibited the ¹⁴C·5-HT binding but 5-HT, DA and tryptamine had no effect. All these observations suggest that other component(s) besides sulphatides and PI may be implicated in the binding of 5-HT to myelin butanol extracts.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

3H-imipramine binding in membrane preparations from brains of insects (Periplaneta americana,Locusta migratoria) and mollusc (Helix pomatia)

1. The binding of [³H]-imipramine to brain membrane preparations of the insects, Periplaneta americana, Locusta migratoria and the mollusc, Helix pomatia was investigated.2. [³H]-Imipramine binding is similar in invertebrates and vertebrates although, in insects, the binding is independent of sodium.3. In the three invertebrate systems studied, [³H]-imipramine binding was inhibited by 5-hydroxytryptamine (5-HT) only at high concentrations (100 μM 5-HT caused 20–30% inhibition in insects and 10% inhibition in snail).4. Studies on binding and incorporation of [³H]-imipramine and [³H]-5-hydroxytryptamine indicate that the imipramine binding site differs from the 5-hydroxytryptamine recognition site of uptake.5. The [³H]-imipramine binding sites are not specific for 5-hydroxytryptamine and have similar affinity for dopamine.     

Characterization of Multiple [3H]5–Hydroxytryptamine Binding Sites in Rat Spinal Cord Tissue

Abstract: High-affinity [³H]5-hydroxytryptamine ([³H]5-HT) binding in the rat spinal cord is similar to that demonstrated in the frontal cortex. [³H]5-HT binds with nearly the same affinity to sites in both tissues. Furthermore, similar patterns of displacement of [³H]5–HT were seen in both tissues, with either spiperone or LSD as the unlabeled ligand. This high-affinity binding appears to be to multiple sites, since displacement studies using 2 nM [³H]5–HT result in Hill coefficients less than unity for spiperone, LSD, and quipazine [Hill coefficients (n_H): 0.44, 0.39, 0.40, respectively]. These sites apparently have an equal affinity for [³H]5-HT, since unlabeled 5-HT did not discriminate between them. Thus, the high-affinity [³H]5-HT binding in the spinal cord may be analogous to that observed in the frontal cortex, where two populations of sites have previously been described (5-HT_IA, 5-HT_IB). In addition to the multiple high-affinity spinal cord binding sites, a low-affinity [³H]5-HT binding component was also identified. A curvilinear Scatchard plot results from saturation studies using [³H]5-HT (0.5–100 nM) in the spinal cord. The plot can be resolved into sites having apparent dissociation constants of 1.4 nM and 57.8 nM for the high-and low-affinity components, respectively. Additional support for a change in affinity characteristics at higher radioligand concentrations comes from the displacement of 30 nM [³H]5-HT by the unlabeled ligand. A nonparallel shift in the dissociation curve was seen, resulting in a Hill coefficient less than unity (0.32). None of the specifically bound [³H]5-HT in the spinal cord is associated with the 5-HT uptake carrier, since fluoxetine, an inhibitor of 5-HT uptake, does not alter binding characteristics. In addition, a 5-HT binding site analogous to the site designated 5-HT, was not apparent in the spinal cord. Ketanse-rin and cyproheptadine, drugs that are highly selective for 5-HT, sites, did not displace [³H]5-HT from spinal tissue, and [³H]spiperone, a radioligand that binds with high affinity to 5-HT₂ sites, did not exhibit saturable binding in the tissue. Thus, the 5-HT₂ binding site reported in other regions of the central nervous system, and the serotonin uptake carrier do not appear to contribute to the multiple binding sites demonstrated in the spinal cord.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS. STEREOSELECTIVITY OF THE BINDING OF L-[14C]GLUTAMIC ACID IN CEREBRAL CORTEX

From the total lipid extract of ncrve-ending membranes or the homogenate of cerebral cortex a hydrophobic protein fraction binding L-[¹⁴C]glutamic acid was separated by chromatography on Sephadex LH20. This protein could only be partially separated from the [¹⁴C]GABA-binding protein and from the lipids that are present in the fraction; however, it was demonstrated that both amino acids bind to different sites. The saturation of the binding showed a high (Kd₁= 0.3μM), a medium (Kd, = 5 μM) and a low (Kd, = 55 μM) affinity binding site. The high affinity binding site had a binding capacity of 0.53 nmol/mg of protein and was highly stereoselective for the L-enantiomer. The binding of L-[¹⁴C]glutamic acid was not inhibited by GABA, was slightly inhibited by glycine and glutamine and was strongly inhibited in a progressive order by DL-a-methylglutamic acid, L-nuciferine, L-aspartic acid and L-glutamic acid diethyl ester. These results are compared with those previously obtained with the L-glutamic acid-binding protein isolated from crustacean muscle. The stereoselectivity of the binding and the possible role of this protein in synaptic transmission are discussed.     

Subcellular distribution and chemical nature of the receptor for 5-hydroxytryptamine in the central nervous system

Abstract— In vitro binding experiments with 5-hydroxy[¹⁴C]tryptamine (3.3 × 10^?6 M) were carried out on subcellular fractions of the cat brain. The highest specific activity was observed in some fractions of nerve-ending membranes isolated from the hypothalamus, basal ganglia, and gray areas of the mesencephalon. The specificity of this high affinity binding was demonstrated by competition with reserpine, butanolamide of lysergic acid, and desmethylimipramine. With butanol-water extraction the [¹⁴C]5-HT was found in the butanol while the gangliosides were separated in the water phase. Several experiments with thin layer and column chromatography suggest that in the organic phase the [¹⁴C]5-HT is not bound to the lipids but to a special proteolipid. This proteolipid is different from that found in myelin and has similar chromatographic properties to that previously observed in the proteolipid which binds d-[¹⁴C]tubocurarine in nerve-ending membranes of the cerebral cortex.     

Identification of 5-hydroxytryptamine binding substances from rat brain stem

Abstract— Godwin & Sneddon (1975) reported the binding of 5-hydroxy-[³H]tryptamine (5-HT) on a Sephadex LH-20 column to‘proteolipid material’extracted with n-butanol from rat brain stem. An examination of this‘proteolipid material’with TLC showed the main constituents to be cerebroside sulfate (CS), monophosphoinositide (PI), and diphosphoinositide. The elution profiles of [³H]5-HT incubated with purified CS or with a mixture of CS and PI were similar to that of the brain extract on the same column. Because the elution profile of the mixture of CS and PI was more similar to that of the brain extract, it was concluded that what was suggested to be a possible proteolipid‘5-HT receptor’was mainly two acidic lipids. The elution profile of [³H]5-HT incubated with purified PI, however, was similar to [³H]5-HT eluted alone. This suggested that either PI did not bind to 5-HT or that the PI-5-HT complex possesses different Chromatographie behavior than PI. To test this latter possibility, [¹⁴C]5-HT and [³H]PI were incubated then eluted on a Sephadex LH-20 column with a continuous gradient of increasing polarity. The gradient first eluted PI, then an apparent PI-5-HT complex, and finally 5-HT. This demonstrated that PI will bind to 5-HT on a Sephadex LH-20 column and that the PI-5-HT complex is probably more polar than PI.     

Interaction of the D1 Receptor Antagonist Sch 23390 with the Central 5-HT System: Radioligang Binding Studies,Measurements of Biochemical Parameters and Effects on L-5-HTP Syndrome

Abstract

The interaction of SCH 23390 with dopamine (DA) and serotonin (5-HT) systems has been examined in vivo and in vitro. Like selective 5-HT₂ blockers, SCH 23390 inhibited in vivo [³H]spiperone binding in the rat frontal cortex (ID₅₀: 1.5 mg/kg) without interacting at D₂ sites. SCH 23390 was equipotent to cinanserin and methysergide. In vitro, SCH 23390 inhibited [³H]ketanserin binding to 5-HT₂ sites (IC₅₀ = 30 nM). Biochemical parameters linked to DA and 5-HT were not changed excepted in striatum where SCH 23390 increased HVA and DOPAC. In the L-5-HTP syndrome model, SCH 23390 clearly showed antagonism of 5-HT₂ receptors. SCH 23390 had weak affinity for 5-HT_1B (IC₅₀ = 0.5 μM), 5-HT_1A (IC₅₀ = 2.6 μM) and α;₁-adenergic receptors (IC₅₀ = 4.4 μM).     

Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei,and chromatin from rat hepatocytes

Histamine and polyamines have been implicated in the mediation of cell proliferation. Our previous work linked the growth-modulatory effects of histamine with its binding to intracellular sites in microsomes and nuclei of various tissues. In this study, we identify cytochrome P450 enzymes as a major component of microsomal intracellular sites in hepatocytes and demonstrate that polyamines compete with high affinity for histamine binding to them. Spectral measurement of histamine binding to P450 in liver microsomes resolved high and intermediate affinity binding sites (K_s1 = 2.4 ± 1.6 μM; K_s2 = 90 ± 17 μM) that corresponded to microsomal binding sites (K_d1 = 1.0 ± 0.9 μM; K_d2 = 57 ± 13 μM) resolved by ³H-histamine binding; additional low affinity (K_d3 ∼ 3 mM), and probably physiologically irrelevant, sites were resolved only by ³H-histamine radioligand studies. As determined spectrally, treatment of microsomes with NADPH/carbon monoxide decreased histamine binding to P450 by about 90% and, as determined by ³H-histamine binding, abolished the high affinity sites and reduced by 85% the number of intermediate sites. Spermine competed potently for ³H-histamine binding: in microsomes, K_i = 9.8 ± 5.8 μM; in nuclei, K_i = 13.7 ± 3.1 μM; in chromatin, K_i = 46 ± 33 nM. Polyamines inhibited the P450/histamine absorbance complex with the rank order of potency: spermine > spermidine ≫ putrescine. In contrast, histamine did not compete for ³H- spermidine binding in nuclei or microsomes, suggesting that polyamines modulate histamine binding allosterically. We propose that certain P450 isozymes that modulate gene function by controlling the level of oxygenated lipids, represent at least one common intracellular target of growth-regulatory endogenous bioamines and, as shown previously, of exogenous growth-modulatory drugs including antiestrogens, antiandrogens, and certain antidepressants and antihistamines. J. Cell. Biochem. 69:233–243, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of erythrocyte membrane (Ca2+ + Mg2+)-ATPase by the organophosphorus insecticides parathion and methylparathion

Organophosphorus insecticides parathion and methylparathion non-competitively inhibited the activity of (Ca²⁺ + Mg²⁺)-ATPase bound to and solubilized from pig erythrocyte membrane. Both enzyme preparations exhibited biphasic substrate curves displaying the existence of two functional active sites with low and high affinity to ATP. Also, the relationship between the activity of bound enzyme and Ca²⁺ concentration was biphasic. The activity reached maximum at 20 μM then dropped progressively as the Ca²⁺ concentration was raised. The inhibition of the activity was more pronounced for parathion than for methylparathion and the solubilized enzyme preparation was more affected than the bound one. The inhibition constants (K_i) for parathion for bound enzyme were 55 and 158 μM for high- and low-affinity active sites, respectively; for methylparathion these values equalled 74 and 263 μM, respectively. K_i values for parathion were 36 and 118 μM for solubilized enzyme (high- and low-affinity sites, respectively), for methylparathion −62 and 166 μM, respectively. The magnitude of the effect was greater for a low Ca²⁺ concentration, which could arise from different conformational states of the enzyme at different calcium concentrations. The results of the experiment suggest that the insecticides inhibited the ATPase by binding to a site on the enzyme rather than by the interaction with associated lipids, although lipids could weaken the action of the compounds due to the strong affinity of organophosphorus insecticides to lipids.     

Are 5-HT receptors involved in the sprouting of serotoninergic terminals following neonatal 5,7-dihydroxytryptamine treatment in the rat?

The neonatal administration of 5,7-dihydroxytryptamine to rats (100 mg kg^?1 s.c. on the 1st and 2nd day after birth) resulted in marked reductions in serotoninergic presynaptic markers ([³H]-5-HT synaptosomal uptake, tryptophan hydroxylase activity and endogenous 5-HT content) in various forebrain areas, particularly the cerebral cortex and the hippocampus. In contrast, this treatment produced an increased outgrowth of serotoninergic terminals in the brain stem as judged by the significant increments of these presynaptic markers in this region. Both in the hippocampus and the brain stem, these 5,7-dihydroxytryptamine-induced changes in serotoninergic innervation were associated with a transient increase in 5-HT-sensitive adenylate cyclase activity. No significant alteration of the specific high affinity binding of [³H]-5-HT to synaptosomal membranes from various brain regions was detected in 5,7-dihydroxytryptamine-treated rats for at least the first postnatal month.The chronic blockade of 5-HT receptors by metergoline (5 mg kg^?1 day^?1 from day 3 to day 22 after birth) altered neither the changes in presynaptic markers nor the evolution of [³H]-5-HT high affinity binding in 5,7-dihydroxytryptamine-treated rats.These findings further illustrate that the high affinity binding sites for [³H]-5-HT do not correspond to postsynaptic 5-HT receptors coupled to adenylate cyclase in the rat brain. Apparently, 5-HT receptors play no role in the increased outgrowth of serotoninergic systems in the brain stem following neonatal 5,7-dihydroxy-tryptamine treatment.     

Diazepam-Sensitive Specific Binding of Phenytoin in Rat Brain

Abstract

[³H]Phenytoin binding to rat cortical membrane was significantly enhanced in the presence of diazepam. This binding is saturable, reversible and displacable by unlabelled phenytoin. Analyses of the binding data either by the Scatchard plot or by the displacement curve revealed a high and a low affinity sites with K_d values of 32 ± 5 nM and 8.5 ± 1.1 μM, respectively. Similar enhancement of [³H]phenytoin binding was observed when diazepam was replaced by Ro 5–4864 (4″-chlorodiazepam) which is selective for the ‘peripheral’ type benzodiazepine binding sites. In contrast, neither the ‘central’ type receptor selective agonist clonazepam nor the antagonist Ro 15–1788 enhanced [³H]phenytoin binding. Therefore, it seems that these phenytoin binding sites in rat cerebral cortex are associated with a benzodiazepine site similar to the ‘peripheral’ type binding site for its selective affinity for Ro 5–4864. However, judging from the micromolar concentrations required for the enhancement of [³H]phenytoin binding, they appear unlikely to be the same ‘peripheral’ type binding sites as measured by [³H]Ro 5–4864 binding (K_d approx. 1 nM). The micromolar affinity benzodiazepine recognition sites are a possibility, if they indeed exist.     

Development of cross-tolerance to 5-hydroxytryptamine in organotypic cultures of mouse spinal cord-ganglia during chronic exposure to morphine

Exposure of organotypic explants of mouse spinal cord with attached dorsal root ganglia (DRGs) to low concentrations (～10nM) of 5-hydroxytryptamine (5-HT) markedly depressed sensory-evoked dorsal-horn network responses, resembling the acute effects of opioids in these cultures. Attenuation of cord responses by 5-HT was not prevented by exposure to the 5-HT antagonists, methysergide and cyproheptadiene, nor to the opiate antagonist, naloxone. Explants that had become tolerant to morphine after chronic exposure (1 μM) for > 2 days often developed cross-tolerance to 5-HT. Acute exposure of morphine-tolerant explants to naloxone (1 μM) further attenuated the effects of 5-HT so that the minimum depressant levels of 5-HT were often increased up to 30-fold. Increasing the extra-cellular Ca⁺⁺ concentration (to 5 mM) and/or introduction of 4-aminopyridine markedly antagonized the depressant effects of 5-HT on DRG-evoked cord responses, so that 5-HT levels comparable to those used on morphine-tolerant explants were required to depress naive explants. These depressant effects of 5-HT on cord-DRG explants are consonant with antinociceptive actions of 5-HT administered to dorsal cord $\text{in situ}$. Our data suggest that 5-HT may block neuronal components of dorsal horn networks at similar regions to those that are depressed by opiates, e.g. presynaptic DRG nerve terminals where abundant opiate receptors are located. The marked attenuation of the depressant effects of both 5-HT and opiates on cord-DRG explants by high Ca⁺⁺ raises the possibility that cross-tolerance to 5-HT in morphine-tolerant explants may result from the same neuronal alterations that render dorsal-horn networks tolerant to opiates. Furthermore, the increased degree of cross-tolerance to 5-HT after acute introduction of naloxone in morphine-tolerant cultures may be an expression of opiate dependence.     

The binding of phloridzin to the isolated luminal membrane of the renal proximal tubule

The binding of [³H]ploridzin by isolated luminal membranes of the rabbit proximal tubule and by slices of rabbit kidney cortex was studied.Kinetic analyses of the relationship between the concentration of phloridizin in the incubation medium and the binding of phloridzin to the membrane indicated two distinct classes of receptors sites. One class, comprising high affinity sites, reached saturation at 20–25 μM phloridzin, had a K(phloridzin) of 8 μM, and 8·10⁺² nmoles interacted with 1 mg of brush border protein. The other class, comprising low affinity sites, had a K(phloridzin) of 2.5 mM, and the number of binding sites was 1.25 nmoles/mg Na⁺ was required for the binding of phloridzin at the high affinity sites. Na⁺ decreased the apparent K_i for phloridzin; the apparent V of binding was not altered. Binding at the low affinity sites was independent of Na⁺. Ca²⁺ was necessary for maximal binding at the high affinity sites. Binding of phloridzin at high affinity sites was more sensitive to N-ethylmalcimide and mersalyl than was binding at low affinity sites. Binding at high affinity sites, but not at low affinity sites, was temperature dependent.d-Glucose was a competitive inhibitor of the high affinity binding of phloridzin. The apparent K₁ was 1 mM. D-Glucoe inhibited non-competitively at the low affinity sites. l-Glucose had no influence on phloridzin binding. Phloretin was a competitive inhibitor of high affinity phloridzin binding with an apparent K_i of 16 μM. Phloretin inhibited low affinity bindings of phloridizin non-competitively. Binding of phloridzin at high affinity sites was completely reversible. Binding at low affinity sites was only partially reversed. Phloridzin bound at high affinity sites on the brush border was displaced by phloridzin and phloretin but not by d-glucose.The mechanism of the high affinity binding of phloridzin was distinguished from that of the initial interaction of d-glucose with the membrane. Binding of phloridzin required Na⁺, whereas the interaction of d-glucose with the membranes had a prominent Na⁺-independent component.Intact renal cells in cortical slices accumulated phloridzin. The uptake did not saturate, was Na⁺ independent, and was not competitively inhibited by sugars. These characteristics resemble those for the low affinity binding of phloridzin by isolated membranes. It is suggested that low affinity binding may represent an initial binding followed by uptake of the glycoside into membrane vesicles.     

Butanol extracts from myelin fragments: identification of 5-hydroxytryptamine binding components

To investigate the nature of 5-HT binding components originated from myelin butanol extracts (i.e. proteolipids), the recombinate experiments with acidic lipids were planned. Binding assay of 14C . 5-Ht to recombinates was carried out by Sephadex lh20 column chromatography. Among several recombinate fractions, recombinate of sulphatides, phosphatidylserine and phosphatidylinositol showed the same elution profile and binding capacity for 14C . 5-HT with those of the myelin extracts. The displacement studies with other neurotransmitters revealed that the recombinate of these three acidic lipids completely regenerated the specificity of the original myelin proteolipids. Namely, tryptamine, DA and ACh inhibited the 5-HT binding but NA had no effect. A double reciprocal plot of 5-HT binding represented a multiple binding mode, not a straight line. This observation suggested that, at least, three binding components (or sites) are implicated in the binding of 14C . 5-HT to this recombinate system having 3 Kds of 2.2 x 10(-7), 4.1 x 10(-7) and 4.2 x 10(-6) m. All these observations infer that the 5-HT binding components of myelin proteolipids are mainly three acidic lipids in nature.     

In vitro effect of phencyclidine and other psychomotor stimulants on serotonin uptake in human platelets

Phencylidine, ketamine and fluoxetine inhibited serotonin (5-HT) uptake in a non-competitive manner in human blood platelets whereas d- and 1-amphetamine produced a competitive inhibition of 5-HT uptake. Phencyclidine (IC₅₀, 2.5 μM) was one-hundredth as potent as fluoxetine (IC₅₀, 22 νM) but ten times more potent than ketamine (IC₅₀, 25 μM) and d-amphetamine (IC₅₀, 24 μM) and three times more potent than 1-amphetamine (IC₅₀, 80 μM) in inhibition of 5-HT uptake by human blood platelets. The possibility that inhibition of 5-HT may contribute to some of the proposed serotonergic effects of psychomotor stimulants is discussed.     

[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [³H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K_d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, K_ds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, K_d 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A₂ subtype. It is concluded that [³H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Effects of thiol modification and Ca++ on agonist-specific state transitions of nicotinic acetylcholine receptor from Torpedo californica electroplax.

The agonist binding affinity of nicotinic acetylcholine receptor (nAChR) from $\text{Torpedo}$$\text{californica}$ electroplax, as inferred from ability of agonist to inhibit specific curaremimetic neurotoxin binding to nAChR, is sensitive to the duration of exposure to agonist. The concentration of carbachol necessary to prevent one-half of toxin binding over a 30 min incubation with nAChR (K₃₀) is 10 μM when toxin and carbachol are simultaneously added to membrane-bound nAChR, and 3 μM when nAChR are pretreated with carbachol for 30 min prior to the addition of toxin. These alterations in agonist affinity may be mimicked by modification of nAChR thiol groups. Affinity of nAChR for carbachol is decreased following treatment with dithiothreitol (DTT). Dithio-bis-nitrobenzoic acid treatment of DTT-reduced membranes yields K₃₀ values of 5 μM for carbachol, while N-ethylmaleimide treatment of DTT-reduced nAChR produces nAChR with reduced affinity for carbachol, reflected in K₃₀ values of about 400 μM. In the absence of Ca⁺⁺, K₃₀ values for carbachol binding to native and DTT-reduced nAChR are diminished 3–6 fold. These affinity alterations are not observed with d-tubocurarine (antagonist) binding to nAChR. Thus, Ca⁺⁺ and the oxidation state of nAChR thiols appear to affect the affinity of nAChR for agonists (but not antagonists), and may therefore be related to agonist-mediated events in receptor activation and/or desensitization.     

Characterization of an Adenylate Cyclase-Linked Serotonin (5-HT1) Receptor in a Neuroblastoma × Brain Explant Hybrid Cell Line (NCB-20)

Clonal cell line NCB-20 (a hybrid of mouse neuroblastoma N18TG2 and Chinese hamster 18-day embryonic brain expiant) expressed both high- (K_D 180 nM) and low-affinity (>3000 nM) binding sites for [³H]serotonin (5-HT) which were absent from the parent neuroblastoma. The low-affinity binding site was eliminated by 1 μM spiperone. The order of drug potency for inhibition of high-affinity [³H]5-HT binding was consistent with a 5-HT₁ receptor (5,6 - dihydroxytryptamine = 5-HT = methysergide = 5-methoxytryptamine > cyproheptadine = clozapine = mianserin > spiperone > dopamine = morphine = ketanserin = norepinephrine). [³H]5-HT binding was inhibited by guanine nucleotides (e.g., GTP and Gpp(NH)p), whereas antagonist binding was not; as-corbate was also inhibitory. A 30-min exposure of cells to 1—2 μM 5-HT or other agonists produced a three- to fivefold stimulation of cyclic AMP levels. The order of potency for 5-HT agonist stimulation of basal cyclic AMP levels and 5-HT antagonist reversal of agonist-stimulated levels was the same as the order of drug potency for inhibition of high-affinity [³H]5-HT binding, suggesting linkage of the 5-HT₁ receptor to adenylate cyclase in NCB-20 cells.