3H] prostaglandins binding to dispersed bovine luteal cells: evidence for discrete prostaglandin receptors.

Suspensions of dispersed bovine luteal cells prepared by collagenase digestion of luteal tissue specifically bound [³H]Prostaglandin (PG) E₁ and [³H]PGF_2α. While the number of sites per cell (～ 1.8 × 10⁵) were about the same for both [³H]PGs, the apparent Kds were different: [³H]PGE₁ ? 2.4 nM; [³H]PGF_2α ? 11 nM. The [³H]PGs binding was inhibited in a dose-dependent manner in the presence of increasing concentrations of unlabeled PGs. Potency order for inhibition of [³H]PGE₁ binding was: PGE₂ > PGE₁ > PGF_2α > PGF_1α. The corresponding data for [³H]PGF_2α was: PGF_2α > PGF_1α > PGE₂ > PGE₁. While [³H]PGE₁ and [³H]PGF_2α bind to their own receptors with high affinity, their affinities for each other's binding were extremely low. Thus, these results demonstrate that luteal cells, like plasma membranes isolated from luteal tissue, contain receptors for PGEs and PGF_2α which are discrete with respect to specificity and affinity.     

Properties of [3H] prostaglandin F2α binding to dispersed bovine luteal cells

Intact viable bovine luteal cell suspensions prepared by collagenase digestion of luteal tissue were used in studying the selected properties of [³H] prostaglandin (PG) F_2α binding and compared with those observed in plasma membranes. [³H]PGF_2α specific binding to luteal cells was a rapid (K₁ = 8.4 × 10⁴M^?12αS^?1), reversible (K_?1 = 1.8 × 10^?4S^?1) and saturable process at 24°. There was a single class of receptors with an apparent dissociation constant of 10.6 nM and 1.8 × 10⁵ receptors per cell. The presence of increasing amounts of unlabeled PGs inhibited [³H]PGF_2α binding in a dose-dependent manner. The potency order for this inhibition was: (15S) 15-methyl-PGF_2α methyl ester > ICI-80,996 > PGF_2α > ICI-81,008 > PGF_1α > PGE₂, (15S) 15-methyl-PGE₂ methyl ester > PGF_2α metabolites > other PGs, PGF_1α metabolites and PGE metabolites. Other than the homegeneous nature of binding and a greater association rate in cells, the rest of the [³H]PGF_2α binding properties in cells were in good agreement with those observed in plasma membranes.     

Prostaglandin F2α antagonizes thromboxane A2-induced human platelet aggregation

The effects of prostaglandin F_2α on human blood platelet function were investigated. PGF_2α at 15 μM completely blocked platelet aggregation induced by 500 μM arachidonic acid or 3 μM U46619 but had no effect on aggregatin induced by 7.5 μM ADP. A similar specificity of action was not obtained with either PGI₂ or PGE₂. Thus concentrations of PGI₂ (3 nM) or PGE₂ (20 μ M) which inhibited U46619-induced aggregation by 100% also blocked ADP-stimulated aggregation.The inhibitory properties of PGF_2α were not related to increases in platelet cAMP, since direct measurement of intracellular cAMP revealed that 15 μ M PGF_2α produced no substantial change in cAMP levels. This finding was in direct contrast to results obtained using either PGI₂ or PGE₂. Both PGI₂ (3 nM) and PGE₂ (20 μ M) induced significant increases in platelet cAMP levels.The possibility that PGF_2α directly interacts at the platelet TXA₂/PGH₂ receptor was investigated by measuring [³H]PGF_2α binding to isolated platelet membranes. It was found that [³H] PGF_2α binding reached equilibrium within 30 min at room temperature and could be 90% displaced by addition of 1000 fold excess of unlabelled PGF_2α. Furthermore, when 1000 fold excess of either the TXA₂/PGH₂ “mimetic” U46619 or the TXA₂/PGH₂ antagonist 13-azaprostanoic acid was added, specific [³H] PGF_2α binding was displaced by 95% and 85% respectively. In contrast, the same molar excess of 6-keto-PGF_1α, azo analog 1, or TXB₂, caused displacement of only 15%, 20% or 25% of the [³H] PGF_2α binding. Scatchard analysis indicated that [³H] PGF_2α has two binding sites; i.e., a high affinity binding site with an apparent Kd of 50 nM and a low affinity binding site with apparent Kd of 320 nM. These results suggest that the selective inhibition by PGF_2α of AA or U46619-induced aggregation may be mediated through interaction at the platelet TXA₂/PGH₂ receptor.     

Effects of reserpine and adenosine agonist on α2-adrenergic receptor of rat vas deferens examined with [3H]yohimbine and [3H]clonidine

The changes of [³H]yohimbine and [³H]clonidine binding sites in rat vas deferens on treatments with adenosine receptor agonists (2-chloroadenosine, adenosine) or reserpine were examined. Treatment with adenosine agonist in vitro increased [³H]clonidine binding sites but had no influence on affinity and number of binding sites of α₂-antagonist, [³H]yohimbine. Amount of [³H]yohimbine binding sites was found to be higher than that of [³H]clonidine with or without the treatment. Inhibition curves of α₂-agonists, clonidine and norepinephrine, on [³H]yohimbine binding were less than unity though α₂-antagonist inhibited with about 1.0 of n_H. The treatment with adenosine agonist reduced the IC₅₀ value of agonists on the [³H]yohimbine binding but had no influence on the inhibitory effect of antagonist. These effect of adenosine agonists was completely blocked by theophylline. Accordingly it was considered that activation of adenosine receptor caused configurational change in α₂-adrenergic receptor from low affinity state for agonist to the high affinity state, though both states had same affinity for antagonist.On the other hand, treatment with reserpine in vivo increased the affinity of clonidine for α₂-adrenergic receptors and also increased the amount of the α₂-receptors.     

Prostaglandin F2α specific binding in equine corpora lutea

Preliminary studies indicate the presence of PGF_2α specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 × 10^?9M and the concentration of binding sites of ～0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF_2α specific binding sites indicates specificity for the 9α-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF_2α analogs with a phenyl ring introduced at carbons 16 or 17. Specific PGF_2α binding was demonstrated in corpora lutea collected at known stages of the estrous cycle. There was no pattern in these values based on the stage of the cycle. While specific ³H-PGE₁ binding could be demonstrated, no high affinity sites could be quantitated. ³H-PGE₁ binding appeared unaffected by changes in temperature or time of incubation, whereas PGF_2α specific binding was significantly modified by both these factors.     

Catecholamine binding to the α-adrenergic receptors of hamster adipocytes evidence that guanine nucleotides regulate this binding to the α2-receptor subtype

The binding characteristics of the α-component of (?)-[³H]norepinephrine to hamster adipocyte membranes were studied. Binding was rapid, reaching equilibrium in 20 min at 25°C. Dissociation of specific binding by 10 μM phentolamine suggested dissociation from two different sites. The time course of dissociation induced by a 50-fold dilution was unchanged by the addition of norepinephrine, suggesting the absence of cooperative binding sites. [³H]norepinephrine binding was saturable, yielding curvilinear Scatchard plots. Computer modeling of these data further supported the existence of two classes of binding sites, one with high affinity (_D = 23 nM) but low binding capacity (96 fmol/mg protein) and one with low affinity (K_D = 400 nM) but high binding capacity (1000 fmol/mg protein). Adrenergic ligands of competed with [³H]norepinephrine binding in the following order of potency: (?)-norepinephrine>(?)-epinephrine>>(+)-norepinephrine>(?)-isoproterenol. Displacement by the selective α-adrenergic drugs prazosin, clonidine and yohimbine yielded biphasic curves consistent with binding of [³H]norepinephrine to both α₁- (14–22%) and α₂- (78–86%) receptor subtypes. Although Gpp(NH)p failed to alter the binding of [³H]dihydroergocryptine, it severely reduced the binding affinity of (?)-epinephrine, (?)-norepinephrine and the selective α₂-agonist, clonidine. The inhibitory effects of clonidine and of the α-component of (?)-epinephrine on the adrenocorticotropin-stimulated cyclic AMP production in the intact adipocyte were closely correlated with their effects on the binding of both [³H]norepinephrine and [³H]dihydroergocryptine. Conversely, yohimbine but not prazosin markedly antagonised the α-inhibitory effect of norepinephrine on cyclic AMP production. These data led to concluded that [³H]norepinephrine can be successfully used to study the entire α-adrenergic receptor population of hamster fat cells and that the predominant α₂ -receptor subtype exists in two different affinity states for agonists, the proportions of which are modulated by guanine nucleotides.     

Beta-adrenergic receptors: evidence for negative cooperativity.

The specific binding of [³H] (?)alprenolol to sites in frog erythrocyte membranes provides a tool for directly assessing ligand binding to adenylatecyclase coupled β-adrenergic receptors. Hill Plots of such binding data yield slopes (n_H=“Hill Coefficients”) less than 1.0, suggesting that negatively cooperative interactions among the β-adrenergic receptors may occur. The existence of such negative cooperativity was confirmed by a direct kinetic method. The dissociation of receptor bound [³H] (?)alprenolol was studied under two conditions: 1) with dilution of the ligand-receptor complex sufficient to prevent rebinding of the dissociated tracer and 2) with this same dilution in the presence of excess unlabeled (?)alprenolol. If the sites are independent, the dissociation rates must be the same in both cases. However, the presence of (?)alprenolol increases the rate of [³H] (?)alprenolol dissociation, indicating that negatively cooperative interactions among the β-adrenergic receptor binding sites do occur.     

Binding of [3H]batrachotoxinin A-20-α-benzoate to a high affinity site associated with house fly head membranes

1. [³H]Batrachotoxinin A-20-α-benzoate (BTX-B), a radioligand that labels the alkaloid activator recognition site of the voltage-sensitive sodium channel, was bound specifically to high affinity, saturable sites in a subcellular preparation from house fly (Musca domestica L.) heads that was shown previously to contain binding sites for other sodium channel-directed ligands.2. Specific binding of [³H]BTX-B was observed in the presence of 140 mM sodium or potassium and was inhibited by choline ion.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom stimulated the specific binding of [³H]BTX-B four-fold, increasing the proportion of specific binding of 10 nM [³H]BTX-B from less than 15% to 40%. Equilibrium dissociation studies in the presence of scorpion venom gave an equilibrium dissociation constant (K_D) for [³H]BTX-B of 80 nM and a maximal binding capacity (B_max) of 1.5 pmol/mg protein.4. Parallel experiments in the absence of venom gave a K_D value of 140 nM and a B_max of 1.3 pmol/mg protein, indicating that scorpion venom stimulated [³H]BTX-B binding by increasing the affinity of this site approximately two-fold.5. The specific binding of [³H]BTX-B was inhibited by the sodium channel activators aconitine and batrachotoxin and, to a lesser extent, by the anticonvulsant diphenylhydantoin. However, several other sodium channel-directed neurotoxins known to exert allosteric effects on the binding of [³H]BTX-B to mammalian brain preparations did not affect the binding of [³H]BTX-B to house fly head membranes.6. These studies provide evidence for a high affinity binding site in house fly head membrane preparations that exhibits properties expected of the activator recognition site of the voltage-sensitive sodium channel but does not respond to several compounds known to modify allosterically the binding of [³H]BTX-B to sodium channels in mammalian brain.     

The oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity states

The binding of the chemoattractant N-formyl-methionylleucyl-[³H]phenylalanine to intact polymorphonuclear leukocytes and membrane preparations was analyzed by computer methods. Whole viable cells bind the chemoattractant with a single dissociation constant (K_D) of 22.3 ± 2.4 nM and contain an average of 55,000 receptors percell. In contrast, the binding data using membrane preparations were consistent with the presence of two classes of binding sites with average K_Ds of 0.53 ± 0.01 nM and 24.4 ± 1.2 nM. The high affinity receptors accounted for ca. 25% of the binding sites. Increasing the receptor occupancy did not affect the rate of dissociation of the ligand-receptor complex thus negative cooperativity is not a likely explanation for the complex binding isotherms. On the other hand, the dissociation kinetics did agree with the two affinity receptor model.     

Competition for in vitro [h]gibberellin a(4) binding in cucumber by substituted phthalimides: comparison with in vivo gibberellin-like activity

Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA-binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity [³H]GA₄ binding sites (K_d ~ 30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-[3-chlorophthalimido]-cyclohexanecarboximide (AC-94,377), the K_d for [³H]GA₄ increased, whereas the maximum number of saturable [³H]GA₄ binding sites did not change significantly. The dissociation of [³H]GA₄ from its binding sites was complex and was best described by a bi-exponential equation. AC-94,377 did not affect the rates of [³H]GA₄ dissociation from its binding sites. These results implied that AC-94,377 and [³H]GA₄ compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs.     

Prostaglandin E2 receptor in the myometrium: distribution in subcellular fractions

[³H]Prostaglandin (PG) E₂ bound specifically to several subcellular fractions from bovine myometrium. The binding was temperature dependent, rapid, and reversible. PGE₂ and PGE₁ competed for the [³H]PGE₂ binding site. The PGs inhibited in the following decreasing order: PGE₂ = PGE₁ ? PGF_2α > PGA₂ > PGF_1β > PGB₂. No competitive effect could be found for oxytocin. Scatchard analysis of the binding data were interpreted as showing a single high-affinity binding constant. There was no difference in the binding constant between the various fractions. The average molar dissociation constant was 2.74 ± 0.14 × 10^?9. Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 ± 2.3 × 10^?11 and the sarcoplasmic reticulum contained 11.2 ± 0.8 × 10^?11 mol binding sites/g. The results suggest that there is a high-affinity PGE₂ receptor present in both plasma membrane and sarcoplasmic reticulum.     

Characterization of α2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[³H]Yohimbine, a potent α₂-adrenergic antagonist, was used to label the α₂-adrenergic receptors in membranes isolated from human platelets. Binding of [³H]yohimbine to platelet membranes appears to have all the characteristics of binding to α₂-adrenergic receptors. Binding reached a steady state in 2–3 min at 37°C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10^?5 M;$\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.37}\text{min}$). [³H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [³H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (?) isomer was 11-times more potent than the (+) isomer. Cathecholamine agonists competed for the occupancy of the [³H]yohimbine-binding sites with an order of potency: clonidine > (?)-epinephrine > (?)-norepinephrine >> (?)-isoproterenol. The potent α₂-adrenergic antagonist, phentolamine, competed for the sites whereas the β-antagonist, (±)-propanolol, was a very weak inhibitor. 0.1 mM GTP reduced the bindng affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonine competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [³H]yohimbine binding. These results suggest tht [³H]yohimbine binding to human platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label α₂-adrenergic receptors.     

Binding of [3H]batrachotoxinin A-20-α-benzoate and [3h]saxitoxin to receptor sites associated with sodium channels in trout brain synaptoneurosomes

1. [³H]Batrachotoxinin A-20-α-benzoate ([³H]BTX-b) and [³H]saxitoxin ([³H]STX), radioligands that bind to distinct sites on the voltage-sensitive sodium channel, were bound specifically to saturable sites in rainbow trout (Oncorhynchus mykiss) brain synaptoneurosomes.2. Specific [³H]BTX-B binding was temperature dependent with highest levels of specific [³H]BTX-B binding observed at 7°C. Specific binding was inversely correlated with assay temperature at temperatures above 7°C.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom (ScV) stimulated specific [³H]BTX-B binding at 27°C, but not at 7°C. The dihydropyrazole insecticide RH 3421 inhibited specific [³H]BTX-B binding at 7°C but had no effect on specific binding at 27°C. The sodium channel activators veratridine and aconitine and the local anesthetic dibucaine inhibited specific [³H]BTX-B binding at both 7°C and 27°C.4. Displacement experiments in the presence of ScV at 27°C gave an equilibrium dissociation constant (K_d) for [³H]BTX-B of 710 nM and a maximal binding capacity (B_max) of 11.3 pmol/mg protein. Kinetic experiments established the rates of association (1.17 × 10⁵min⁻¹ nM⁻¹) and dissociation (0.0514min⁻¹) of the ligand-receptor complex.5. The binding of [³H]STX reached apparent saturation at 7.5 nM. Scatchard analysis of the saturation data indicated a K_d of 3.8nM and a B_max of 1.9 pmol/mg protein.6. These studies provide evidence for high affinity, saturable binding sites for [³H]BTX-B and [³H]STX in trout brain preparations. Whereas certain neurotoxins modified the specific binding of [³H]BTX-B in trout brain synaptoneurosomes in a predictable fashion, other compounds known to affect specific [³H]BTX-B binding in mammalian brain preparations had no effect on specific [³H]BTX-B binding in the trout.     

Characterization of the Binding of the GABA Agonist [3H]Piperidine-4-Sulphonic Acid to Bovine Brain Synaptic Membranes

Abstract: The binding of radioactive piperidine-4-sulphonic acid ([³H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: K_D= 17 ± 7 nM (B_max= 0.15 ± 0.07 pmol/mg protein) and K_D= 237 ± 100 nM (B_max= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [³H]P4S. The slow binding component (k_on= 5.6 × 10⁷ or 8.8 × 10⁷ M^-1 min^-1, k_Off= 0.83 min^-1, and K_D= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (K_D= 11 nM, B_max= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [³H]P4S appears to involve the same two populations of sites with B_max values similar to those for [³H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [³H]P4S and [³H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.     

Specific Binding of L-[3h]-Glutamic Acid to Rat Substantia Nigra Synaptic Membranes

Abstract

The specific binding of L-[³H] -glutamic acid (GLU) was investigated in synaptic membranes from rat substantia nigra. L-[³H]-GLU binding to the membrane preparations occurred in a reversible and saturable way. The specific binding was stimulated by the presence of CaCl₂ and was reduced by freezing and thawing the membranes. Scatchard analysis of the saturation isotherms yielded a non-linear plot suggesting that the binding reaction does not occur through a simpla bimolecular association. Assuming non-interacting binding sites, a high (K_D1, 139 nM; Bmax1, 3.5 pmoles/mg protein) and a low (K_D2, 667 nM; Bmax2, 15.1 pmoles/mg protein) affinity L-[³H]-GLU binding site were obtained. The kinetics of dissociation of bound L-[³H]-GLU was biphasic; the respective dissociation rate constant (k-1) being 0.20 min^?1 and 0.013 min^?1. A series of amino acid receptor agonists and antagonists were tested as inhibitors of L-[³H]-GLU specific binding. Quisqualic acid, L-GLU and D-α-aminoadipate (D-α-AA) were the most potent inhibitors. DL-2-amino-4-phosphonobutyrate (APB), N-Methy1-D-aspartate (NMDA) and D-GLU were moderate inhibitors, whereas diamino-pimelic acid (DAPA) and glutamate diethyl ester (GDEE) exhibited the lowest relative potency. Kainic acid (KA), γ-aminobutyric acid (GABA) and bicuculline were not able to modify at any concentration used the specific binding of L-[³H]-GLU. These data demonstrate the presence of specific GLU binding sites in synaptic structures at substantia nigra level and support the idea that excitatory amino acids may play a role in synaptic transmission in this brain region.     

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that (±)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist $\text{(±)-[}{}^3\text{H]hydroxybenzylisoproterenol}$ to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k₁=1.37·10⁷·M^?1·min^?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k₂ of 0.106·min^?1 and 0.011·min^?1.[³H]Hydroxybenzylisoproterenol binding was saturable (B_max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [³H]hydroxybenzylisoproterenol binding sites, one having high affinity (K_D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity (K_D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [³H]hydroxybenzylisoproterenol binding with the following order of potency=(?)-propranolol>(?)-isoproterenol>(?)-norepinephrine≈ (?)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β₁-adrenergic receptors. Competition curves of [³H]hydroxybenzylisoproterenol binding by the β-agonist (?)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (?)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [³H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (?)-isoproterenol, then number of [³H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (?)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (?)-isoproterenol to displace [³H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [³H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [³H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [³H]dihydroalprenolol in these cells.     

Multiple benzodiazepine receptors and their regulation by gamma-aminobutyric acid

The interaction of propyl β-carboline-3-carboxylate (PCC) with benzodiazepine receptors in the cerebral cortex of the rat was investigated by direct measurements of [³H]PCC binding and by competitive inhibition of [³H]flunitrazepam (FLU) binding. Initial experiments showed that [³H]PCC binding exhibited characteristics of saturability, stereospecificity and a pharmacological specificity remarkably similar to that of [³H]FLU binding. Analysis of [³H]PCC binding isotherms and PCC/[³H]PCC competition curves revealed the presence of a small population of super high affinity PCC binding sites (K_SH = 30–100 pM) which represents approximately 3–6% of the total sites. When measured by competitive inhibition of [³H]FLU binding, receptor occupancy by PCC was generally consistent with that determined by direct measurements of [³H]PCC binding. Analysis of the PCC/[³H]FLU competition curve revealed the presence of two major populations of high and low affinity PCC binding sites with dissociation constants of 0.54 and 10 nM and relative abundances of 52 and 45%, respectively. Collectively, the results of the [³H]PCC binding isotherm, PCC/[³H]PCC competition curve and PCC/[³H]FLU competition curve are internally consistent when rationalized in terms of three populations of benzodiazepine receptors - super high, high, and low affinity - each having different affinities for PCC and equal affinity for FLU. The effects of γ-aminobutyric acid (GABA) on PCC and FLU binding were investigated, and it was observed that GABA enhanced the binding of FLU to the various receptor subtypes whereas no significant effect of GABA on the binding of PCC was detected.     

[3H]-BIBP3226 and [3H]-NPY Binding to Intact SK-N-MC Cells and CHO Cells Expressing the Human Y1 Receptor

Abstract

We have studied the binding of [³H]-NPY and the newly developed non-peptide Y₁ receptor antagonist [³H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y₁ receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [³H]-NPY binding was slow, the binding kinetics of [³H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y₁ agonists NPY, PYY and [Leu³¹-Pro³⁴]-NPY completely and potently displaced [³H]-NPY binding, they could only displace 70 to 80 % of the [³H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [³H]-NPY binding sites in CHO-Y1 cells whereas [³H]-BIBP3226 binding parameters remained unchanged.     

Autoantibodies Enhance Agonist Action and Binding to Cardiac Muscarinic Receptors in Chronic Chagas' Disease

Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M₂-muscarinic acetylcholine receptors (M₂AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M₂AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M₂AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [³H]-N-methyl scopolamine ([³H]-NMS) in allosterism binding assays. A peptide corresponding to the M₂AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [³H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [³H]-NMS dissociation right shifted from an IC₅₀ of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 × 10^{? 8}, 1.33 × 10^{? 7}, and 2.0 × 10^{? 7} mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M₂AChRs as a positive cooperativity effect on acetylcholine action.     

Characteristics of Binding of [3H]WAY100635 to Rat Hippocampal Membranes

Kinetic analysis of binding of [³H][N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide ([³H]WAY100635) to 5-HT_1A receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding experiments revealed that [³H]WAY100635 binds to a single class of receptors with very high apparent affinity (K _D = 87 ± 4 pM, B _max = 15.1 ± 0.2 fmol/mg protein). The binding was almost irreversible, as the dissociation rate constant obtained k _off = (7.8 ± 1.1) × 10⁻³ min⁻¹, means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25°C. Systematic association kinetic studies of [³H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive cooperativity. The affinities of antagonists determined from competition with [³H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of [³⁵S]GTPγS binding probably due to the ligand’s kinetic peculiarities. Thus, [³H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies is strongly limited.