Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Labelling of lipids by D-[1-14C]glucose, D-[6-14C]glucose and D-[3-3H]glucose in pancreatic islets from normal and GK rats

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-¹⁴C]glucose than D-[6-¹⁴C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired ¹⁴C/³H ratio found in islets exposed to both D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose and D-[3-³H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-¹⁴C]glucose. The labelling of islet lipids by D-[6-¹⁴C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C₁-C₂-C₃ moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C₄-C₅-C₆ moiety of the hexose, (iii) that only a limited amount of [3-³H]glycerone 3-phosphate generated from D-[3-³H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose rather than D-[3-³H]glucose.     

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.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: isotopic discrimination between hydrogen and deuterium

Summary The discrimination between the isotopes of hydrogen in the reaction catalyzed by yeast phosphoglucoisomerase is examined by NMR, as well as by spectrofluorometric or radioisotopic methods. The monodirectional conversion of D-glucose 6-phosphate to D-fructose 6-phosphate displays a lower maximal velocity with D-[2-²H]glucose 6-phosphate than unlabelled D-glucose 6-phosphate, with little difference in the affinity of the enzyme for these two substrates. About 72% of the deuterium located on the C₂ of D-[1-¹³C,2-²H]glucose 6-phosphate is transferred intramolecularly to the C₁ of D-[1-¹³C,1-²H]fructose 6-phosphate. The velocity of the monodirectional conversion of D-[U-¹⁴C]glucose 6-phosphate (or D-[2-³H]glucose 6-phosphate) to D-fructose 6-phosphate is virtually identical in H₂O and D₂O, respectively, but is four times lower with the tritiated than ¹⁴C-labelled ester. In the monodirectional reaction, the intramolecular transfer from the C₂ of D-[2-³H]glucose 6-phosphate is higher in the presence of D₂O than H₂O. Whereas prolonged exposure of D-[1-¹³C]glucose 6-phosphate to D₂O, in the presence of phosphoglucoisomerase, leads to the formation of both D-[1-¹³C,2-²H]glucose 6-phosphate and D-[1-¹³C,1-²H]fructose 6-phosphate, no sizeable incorporation of deuterium from D₂O on the C₁ of D-[1-¹³C]fructose 1,6-bisphosphate is observed when the monodirectional conversion of D-[1-¹³C]glucose 6-phosphate occurs in the concomitant presence of phosphoglucoisomerase and phosphofructokinase. The latter finding contrasts with the incorporation of hydrogen from ¹H₂O or tritium from ³H₂O in the monodirectional conversion of D-[2-³H]glucose 6-phosphate and unlabelled D-glucose 6-phosphate, respectively, to their corresponding ketohexose esters.     

Modulation of GABA binding sites by CNS depressants and CNS convulsants

[³H]Muscimol binding at 23°C and muscimol stimulated [³H]flunitrazepam binding at 37°C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [³H]muscimol binding sites can be observed. At 23°C [³H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0°C. The CNS depressants etazolate and pentobarbital reversibly enhance [³H]muscimol binding and they increase the affinity of muscimol as a stimulator of [³H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [³H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [³H]flunitrazepam binding. Binding of [³H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag⁺, Hg⁺ and Cu²⁺ in μM concentrations, Ag⁺ being most potent. The effects of 100 μM AgNO₃ persist after repeated washing of the membranes. When membranes are pretreated with AgNO₃ only one apparent population of [³H]muscimol binding sites with high affinity (K_d: 6–8 nM) is found. In AgNO₃ pretreated membranes, the affinity of muscimol as stimulator of [³H]flunitrazepam binding is increased 18 times (EC₅₀ 14 nM) when compared to control membranes, (EC₅₀ 253 nM). In AgNO₃ pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [³H]muscimol binding or baseline and muscimol stimulated [³H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [³H]dihydropicrotoxinin binding.     

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.     

In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase

[³H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [³H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [³H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [³H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [³H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K₁) and dissociation (K₂) rate constants of 2.21 · M^? · min^?1 and 3.21 · 10^?1, respectively, were obtained. The dissociation constant (K_d) of 15 nM for [³H]dihydroalprenolol obtained from saturation binding data closely agreed with the (K_d) derived from the ratio of dissociation and association rate constants (K₂/K₁).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [³H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [³H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [³H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [³H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (K_i) values for the antagonists determined from inhibition of [³H]dihydroalprenolol binding agreed closely with the (K_i) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [³H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor.     

Hexose metabolism in pancreatic islets

Summary In rat pancreatic islets, a rise in extracellular D-glucose concentration is known to cause a greater increase in the oxidation of D-[6-¹⁴C]glucose than utilization of D-[5-³H]glucose. In the present study, such a preferential stimulation of acetyl residue oxidation relative to glycolytic flux was mimicked by nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate, 3-phenylpyruvate, L-leucine, 2-ketoisocaproate, D-fructose and ketone bodies. The preferential stimulation of D-[6-¹⁴C]glucose oxidation by these nutrients was observed at all hexose concentrations (0.5, 6.0 and 16.7 mM), coincided with an unaltered rate of D-[3,4-¹⁴C]glucose oxidation, was impaired in the absence of extracellular Ca²⁺, and failed to be affected by NH₄ ⁺. Although the ratio between D-[6-¹⁴C]glucose oxidation and, D-[5-³H]glucose utilization in islets exposed to other nutrient secretagogues could be affected by factors such as isotopic dilution and mitochondrial redox state, the present data afford strong support to the view that the preferential stimulation of oxidative events in the Krebs cycle of nutrient-stimulated islets is linked to the activation of key mitochondrial dehydrogenases, e.g. 2-ketoglutarate dehydrogenase. The latter activation might result from the mitochondrial accumulation of Ca²⁺, as attributable not solely to stimulation of Ca²⁺ inflow into the islet cells but also to an increase in ATP availability.     

Characterization of adenosine receptors in Mullus surmuletus

The intent of the present study was to investigate adenosine receptor sites in brain membranes of the saltwater teleost fish, Mullus surmuletus, using the A₁ receptor selective agonist, [³H]CHA, and A_2a receptor selective agonist [³H]CGS 21680. The A₁ selective agonist, [³H]CHA, bound saturably, reversibly and with high affinity to a single-class of binding sites (K_d 1.47 nM; B_max 100–190 fmol/mg protein, dependent on fish length). The A_2a selective agonist, [³H]CGS 21680, also bound saturably, reversibly and with relative high affinity to a single-class of binding sites (K_d 44.2 nM; B_max 150–300 fmol/mg protein dependent on fish length). In equilibrium competition experiments, adenosine analogous, NECA, CGS 21680, CHA, CPA, S-PIA, R-PIA, CPCA, DPMA, and xanthine antagonists, DPCPX, XAC, and THEO all displaced [³H]CHA and [³H]CGS 21680 specifically bound to brain membranes from Mullus surmuletus. Specific binding of both [³H]CHA and [³H]CGS 21680 was inhibited by GDPβS. For [³H]CHA the IC₅₀ value was 2.5 ± 0.1 μM, while for [³H]CGS 21680 the IC₅₀ value was 7.7 ± 0.3 μM. Our results indicate that the high affinity binding sites for [³H]CHA have some pharmacological characteristics of mammalian A₁ adenosine receptors, while the binding sites for [³H]CGS 21680 appear to be virtually identical to the binding sites for [³H]CHA.     

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.     

Synthesis and binding characteristics of tritiated tipp[Φ], a highly specific and stable delta opioid antagonist

The pseudopeptide H-Tyr-TicΦ[CH₂-NH]Phe-Phe-OH (TIPP[Φ]) is a δ opioid antagonist with high δ receptor affinity and unprecedented δ selectivity. TIPP[Φ] was radiolabelled by catalytic tritiation of its precursor [Tyr(3′,5′-I₂)¹]TIPP[Φ]. The resulting radioligand, [³H]TIPP[Φ], had a specific activity of 1.77 TBq/mmol (47.9 Ci/mmol) and showed high stability against enzymatic degradation. [³H]TIPP[Φ] binding to rat brain membranes was saturable and Scatchard analysis indicated a single binding site with a K_d of 0.98 nM and a B_max of 105.4 fmol/mg. A study of [³H]TIPP[Φ] binding displacement by various receptor-selective opioids showed the expected rank order of potency (δ ⪢ μ > κ). [³H]TIPP[Φ] represents an excellent new radioligand for δ receptor labelling studies in vitro and in vivo.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates

Summary The exchange of protons and deuterons by phosphoglucoisomerase during the single passage conversion of D-[2-¹³C,1-²H]fructose 6-phosphate in H₂O or D-[2-¹³C]fructose 6-phosphate in D₂O to D-[2-¹³C]glucose 6-phosphate, as coupled with the further generation of 6-phospho-D-[2-¹³C]gluconate in the presence of excess glucose-6-phosphate dehydrogenase was investigated by ¹³C NMR spectroscopy of the latter metabolite. In H₂O, the intramolecular deuteron transfer from the C₁ of D-fructose 6-phosphate to the C₂ of D-glucose 6-phosphate amounted to 65%, a value only slightly lower than the 72% intramolecular proton transfer in D₂O. Both percentages, especially the latter one, were lower than those previously recorded during the single passage conversion of D-[1-¹³C,2-²H]glucose 6-phosphate in H₂O or D-[1-¹³C]glucose 6-phosphate in D₂O to D-fructose 6-phosphate and then to D-fructose 1,6-bisphosphate. These differences indicate that the sequence of interactions between the hexose esters and the binding sites of phosphoglucoisomerase is not strictly in mirror image during, respectively, the conversion of the aldose phosphate to ketose phosphate and the opposite process.     

Characterization of a dopamine-sensitive [3H]LSD binding site in honeybee (Apis mellifera) brain

[³H]Lysergic acid diethylamide ([³H]LSD) binds on membrane homogenate of honeybee brain to both a dopamine-sensitive site (D-site) and a serotonin-sensitive site (S-site). Under suitable conditions the properties of the two sites can be studied separately. Specific binding of [³H]LSD to both the D-site and the S-site has high affinity and is saturable. The mean equilibrium dissociation constants (K_D) were 3.8 nM for the D- and 0.89 nM for the S-site. The densities (B_max values) of both binding sites were 1.7 pmol/mg protein for the D-site and 0.79 pmol/mg protein for the S-site. [³H]LSD binding to the D-site was reversible and reached equilibrium in about 30 min. Pharmacological displacement studies display a high binding affinity of the putative natural agonist dopamine to the D-site (K_i = 22 nM). The most potent displacers of D-site binding were lisuride, (+)-bromocriptine, chlorpromazine, S(+)-butaclamol, and 6,7-ADTN. The [³H]LSD labelled D-site seems to be G-protein coupled, since addition of the stable GTP analogue GTPγS or NaCl to the incubation medium evoked a decrease of specific [³H]LSD binding to the D-site.     

D-glucose metabolism in normal dispersed islet cells and tumoral INS-1 cells

The metabolism of D-glucose was characterized in both normal dispersed rat islet cells and the 2-mercaptoethanol-dependent insulin-secreting cells of the INS-1 line. The normal and tumoral islet cells differed from one another by the relative magnitude, concentration dependency and hierarchy of the increase in the production of ³HOH from D-[5-³H]glucose and ¹⁴C-labelled CO₂, acidic metabolites and amino acids from D-[U-¹⁴C]glucose at increasing concentrations of the hexose. For instance, whilst the paired ratio between D-[U-¹⁴C]glucose oxidation and D-[5-³H]glucose utilization augmented in a typical sigmoidal manner in normal islet cells exposed to increasing concentrations of D-glucose, it progressively decreased under the same experimental conditions in INS-1 cells. Nevertheless, the absolute values and concentration-response relationship for the increase in ATP generation rate attributable to the catabolism of D-glucose were virtually identical in normal and tumoral cells. These findings indicate that the analogy in the secretory response to D-glucose of normal and INS-1 islet cells, although coinciding with a comparable response to the hexose in terms of ATP generation, contrasts with a vastly different pattern of D-glucose metabolism in these two cell types.     

Calcium alters the properties of [3H]diazepam binding sites in rat brain membranes

• 1.1. [³H]diazepam ([³H]DZ) was used as a ligand to study the effects of Ca²⁺ on benzodiazepine binding to rat brain membranes.
• 2.2. [³H]DZ bound at a single class of binding sites showing K_D and B_max values of 5.4 nM and 852 fmol/mg protein respectively. These values are consistent with previous reports.
• 3.3. Amongst the various divalent cations tested Mg²⁺, Fe²⁺, Cd²⁺, and Sr²⁺ had no significant effect on [³H]DZ binding. Mn²⁺, Ba²⁺, Co²⁺, Ni²⁺ and La²⁺ enhanced radioligand binding, whereas Ca²⁺, Zn²⁺ and Pb²⁺ inhibited [³H]DZ binding to brain freeze-thawed membranes.
• 4.4. The inhibition of [³H]DZ binding by Ca²⁺ was concentration-dependent. 50% inhibition occurred at a Ca²⁺ concentration of 5.6mM. The Hill coefficient for the inhibition was 1.03, displaying noncoperativity. The effect of Ca²⁺ on [³H]DZ binding could be prevented by La³⁺ but was not reversed by EGTA.
• 5.5. A kinetic analysis of Ca²⁺ inhibition of [³H]DZ binding indicates that Ca²⁺ inhibited competitively. Analysis of binding isotherms indicates that both K_D and B_max were altered at the [³H]DZ binding sites. The marked increase in K_D value in the presence of Ca²⁺ (5 mM) can be explained by a drastic increase in the dissociation rate constant.
• 6.6. It was suggested that Ca²⁺ may induce a conformational change in the diazepam binding sites on rat brain membranes. The unchanged Hill coefficient in the presence or absence of Ca²⁺ indicates that a single population of binding sites was labeled by [³H]DZ.
• 7.7. The calmodulin antagonists, trifluoperazine and W-7 were weak inhibitors of [³h]dz binding.

     

Characterisation of [3H]MK-801 Binding and its Cooperative Modulation by PIG Brain Membranes

Abstract

Cooperative modulation of [³H]MK-801 binding to extensively washed pig cortical brain membranes in the presence of various concentrations of L-glutamate, glycine, spermine, CPP and DCKA was evaluated in association experiments. In saturation experiments [³H]MK-801 labelled a homogeneous population of binding sites with a K_d-value of 1.26 ± 0.18 nmol 1^?1 and a B_max-value of 2130 ± 200 fmol/mg protein. The pharmacological profile of this site was further evaluated in competition experiments with known NMDA receptor channel blockers. In nonequilibrium binding experiments EC₅₀-values of reference compounds acting at the L-glutamate, at the glycine, and at the polyamine site, were determined by increasing or decreasing [³H]MK-801 binding. Ifenprodil reduced [³H]MK-801 binding in a biphasic manner. All the data obtained are in agreement with results from [³H]MK-801 binding to rodent as well as human brain membranes. This study therefore strongly suggests, that pig cortical membranes are a suitable alternative to rodent brain membranes, and an acceptable substitute for human brain membranes in [³H]MK-801 binding experiments.     

Evaluation of the negative cooperativity model for fat cell beta-adrenergic receptors

Cooperative site-to-site interactions among beta-adrenergic receptors of fat cell membranes are probed with the potent beta-adrenergic antagonist (?)-[³H]-dihydroalprenolol according to the kinetic method of De Meyts et al. (De Meyts, P., Roth, J., Neville, Jr., D.M., Gavin, III, J.R. and Lesniak, M.A. (1973) Biochem. Biophys. Res. Commun. 55, 154–161). Dissociation of specific (?)-[³H]dihydroalprenolol binding from fat cell membranes following a 100-fold dilution was rapid at 37°C; only 40% of the initial equilibrium binding remained 30 s after dilution. Dissociation of (?)-[³H]dihydroalprenolol bound under conditions yielding approximately 20% initial occupancy was performed in the absence and in the presence of a large molar excess of beta-adrenergic agonist ((?)-isoproterenol) or beta-adrenergic antagonist ((?)-alprenolol or(?)-propanalol). Neither agonists nor antagonists influenced the rate of (?)-[³H]dihydroalprenolol dissociation from fat cell membranes performed at 4, 22 or 37°C. Although analysis of the steady-state binding of (?)-[³H]-dihydroalprenolol to fat cell membranes yields Hill coefficients, n_H, less than 1.0, the present study indicates that these fat cell beta-adrenergic receptors display no cooperative site-to-site interactions.     

Two Classes of [3H]Spiperone Binding Sites in Bovine Neurohypophysis: D-2 Receptors and Putative 5-Ht2 Receptors

Abstract

Binding of [³H]spiperone was studied in membranes obtained from bovine neurohypophyses devoid of intermediate lobe tissue. Non-linear Scatchard plot suggested the presence of more than a single class of binding sites. Competition experiments using ketanserin, a ligand selective for 5-HT₂ receptors, were carried out to ascertain whether serotonergic, in addition to dopaminergic receptors, might be responsible for the heterogeneity of [³H]spiperone binding. Computer-assisted modeling suggested the presence of two classes of binding sites for ketanserin (K_a = 1.6 ± 0.2 and 366.7 ± 20.5 nM, respectively). The K_a value for ketanserin binding to the high-affinity sites, as well as the K_a of [³H]spiperone for these sites suggested by the 2 sites model indicate that they represent serotonin 5-HT₂ receptors. The [³H]spiperone K_a at the ketanserin low-affinity sites (65 ± 7 pM) and the rank order of inhibitory potencies for several antagonists show that the lowaffinity sites represent dopamine D-2 receptors.     

Effects of chronic ethanol exposure on the gaba-benzodiazepine receptor complex in rat brain

Chronic treatment of male Wistar rats with ethanol by inhalation did not affect the binding of [³H]flunitrazepam, [³H]GABA or [³H]muscimol to extensively washed synaptic membranes. Neither the affinity (K_d) nor the number of binding sites (Bmax) for these ligands was changed. However, GABA enhancement of [³H]flunitrazepam binding was significantly decreased by approx. 40% in ethanol-treated animals (172% compared to 215%). Acute treatment with ethanol did not produce changes in the binding of [³H]flunitrazepam or [³H]muscimol. These findings suggest that chronic ethanol treatment leads to uncoupling of the various receptor sites on the GABA—benzodiazepine receptor ionophore-complex in the brain.