Effect of Allopurinol on Hypoxia-Induced Modification of the NMDA Receptor in Newborn Piglets

The present study tests the hypothesis that pretreatment with allopurinol, a xanthine oxidase inhibitor, will prevent modification of the NMDA receptor during cerebral hypoxia in newborn piglets. Eighteen newborn piglets were studied. Six normoxic control animals were compared to six untreated hypoxic and six allopurinol (20 mg/kg i.v.) pretreated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 hour and tissue hypoxia was confirmed biochemically by the measurement of ATP and phosphocreatine. Brain cell membrane Na⁺,K⁺-ATPase activity was determined to assess membrane function. Na⁺,K⁺-ATPase activity was decreased from control in both the untreated and treated hypoxic animals (46.0 ± 1.0 vs 37.9 ± 2.5 and 37.3 ± 1.4 mol Pi/mg protein/hr, respectively, p < 0.05). [³H]MK-801 binding was determined as an index of NMDA receptor modification. The receptor density (Bmax) in the untreated hypoxic group was decreased compared to normoxic control (1.09 ± 0.17 vs 0.68 ± 0.22 pmol/mg protein, p < 0.01). The dissociation constant (Kd) was also decreased in the untreated group (10.0 ± 2.0 vs 4.9 ± 1.4 nM, p < 0.01), indicating an increase in receptor affinity. However, in the allopurinol treated hypoxic group, the Bmax (1.27 ± 0.09 pmol/mg protein) was similar to normoxic control and the Kd (8.1 ± 1.2 nM, p < 0.05) was significantly higher than in the untreated hypoxic group. The data show that the administration of allopurinol prior to hypoxia prevents hypoxia-induced modification of the NMDA receptor-ion channel binding characteristics, despite neuronal membrane dysfunction. By preventing NMDA receptor-ion channel modification, allopurinol may produce a neuromodulatory effect during hypoxia and attenuate NMDA receptor mediated excitotoxicity.     

Pre- and Postsynaptic Localization of 3H-Imipramine Binding Sites in Rat Cerebral Cortex

Abstract

The subcellular localization of ³H-imipramine binding sites in brain was investigated with the aim of learning about the possible mechanism of action of this antidepressant. The rat cerebral cortex was submitted to a systematic fractionation and both the nuclear and the synaptosomal fractions were purified by gradient centrifugation. Using a centrifugation assay for the binding, we found that the synaptosomal membranes had the highest specific activity and showed two binding sites, one of high affinity with a K_D of 14 nM and a Bmax of 3.1 pmol per mg protein, and another of lower affinity with a K_D of 99 nM and a Bmax of 14.2 pmol per mg protein. Purified nuclei have a lower specific activity than the synaptosomal membrane, specially when expressed per g tissue. On the other hand, myelin and capillaries have few binding sites. Synaptosomal membranes were treated with 0.1, 0.2 and 0.5% Triton X-100 to dissolve the pre- and post-synaptic membrane and submitted to ³H-imipramine binding in the presence of the detergent or after washing of the residue. The results obtained suggest that although most ³H-imipramine binding sites are localized pre-synaptically, a certain proportion are post-synaptic. These findings are discussed in relation to previous studies from this laboratory on the localization of central receptors with reference to the synaptic region and to the antidepressant action of imipramine.     

Identification and Characterization of Specific Binding Sites for Somatostatin in Cytosol of Bovine Cystic Duct Mucosa

Abstract

Specific binding sites for somatostatin have been detected in cytosolic fraction of bovine cystic duct mucosa. At 37°C, the interaction of ¹²⁵I-Tyr¹¹-somatostatin with cytosolic fraction was rapid, reversible, specific and saturable. At equilibrium, the binding of tracer was competitively inhibited by native peptide in the 1 nM to 2 µ M range of concentrations. Scatchard analysis of binding data suggested the presence of two distinct classes of somatostatin binding sites: a class with a high affinity (K_d = 7.8 ± 0.3 nM) and a low capacity (1.3 ± 0.3 pmol somatostatin/mg protein) and a class with a low affinity (K_d = 129.1 ± 2.0 nM) and a high capacity (43.5 ± 6.7 pmol somatostatin/mg protein). The binding sites were shown to be highly specific for somatostatin since neuropeptides present in cystic duct such as Leu-enkephalin, neurotensin, substance P and vasoactive intestinal peptide did practically not show competition. These findings suggest that somatostatin could contribute to the regulation of the functions of the cystic duct mucosa in physiological and pathological conditions.     

Insulin Receptor Sub-Types in a Human Lymphoid-Derived Cell Line (IM-9): Differential Regulation by Insulin,Dexamethasone and Monensin

Abstract

The cells of the human IM-9 lymphocyte-derived line contain a sub-population of insulin binding sites which differ from classical insulin binding sites in their higher binding affinity for insulin-like growth factor II (IGF-II) and insulin-like growth factor I (IGF-I). These atypical insulin binding sites are identified on IM-9 cells by [¹²⁵I]IGF-II binding.

To determine whether the atypical and classical insulin receptors of IM-9 cells were subject to different modes of in vivo regulation, we treated IM-9 cells with agents known to alter the surface expression of insulin receptors - insulin, dexamethasone and monensin. We then measured insulin and IGF-II binding to the surface of the washed cells.

Pretreatment of IM-9 cells with 1 μM insulin for 20 h at 37°C induced a 44–48% decrease in the number of high affinity insulin binding sites, but no change in the number of IGF-II binding sites. In contrast, the surface expression of both insulin and IGF-II binding sites (classical and atypical insulin receptors) increased 1.3 to 1.7-fold after treatment with dexamethasone (200 nM) and decreased 30 to 45% after monensin (1 μM). These results suggest that atypical and classical insulin receptors are differentially susceptible to down-regulation by insulin.     

Binding of DL-[3H]-alpha-Amino-3-Hydroxy-5-Methyl-Isoxazole-4-Propionic Acid (AMPA) to Rat Cortex Membranes Reveals Two Sites or Affinity States

Abstract

A method for measuring [³H]-AMPA binding in rat cortex membranes is described. Specific binding was saturable and accounted for 95% of total binding at 5 nM of [³H]-AMPA. Non linear curve fitting of [³H]-AMPA saturation isotherms suggested the presence of two binding sites: the high affinity site showed a pKd of 8.26 ± 0.07 (Kd = 5.49 nM) and a Bmax of 0.19 ± 0.03 pmol/mg protein, whereas the low affinity site indicated a pKd of 7.28 ± 0.05 (Kd = 52 nM) and a Bmax of 1.30 ± 0.23 pmol/mg protein. The pharmacological profile of [³H]-AMPA binding has been determined by studying a series of compounds in binding displacement experiments: Quisqualate was the most potent inhibitor of [³H]-AMPA binding (IC₅₀ = 9.7 nM), followed by AMPA (19 nM), CNQX, DNQX and L-Glutamate (272–373 nM). Kainate was a moderate displacer (6.2 μM); Ibotenic acid and glycine were very weak inhibitors (74 and 92 μM, respectively). CPP, GAMS and L-Aspartic acid showed IC₅₀-values of over 400 μM and MK-801, DL-AP5 and NMDA were almost inactive at the maximal concentration used in our experiments.     

Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

Effect of salicylic acid on glucucorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma

The binding activity of [³H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [³H]dexamethasone of 3.64 · 10^?8 M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 · 10⁵.Total binding activity to [³H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid at 37°C. The maximum enhancement was seen at the concentration of 10^?3 M and in 3 h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicylic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin, also enhanced the total binding activity to [³H]dexamethasone of the cytoplasmic fraction at the concentration of 2 · 10^?5 M and 2 · 10^?7 M, respectively.     

Receptors for vasoactive intestinal peptide on isolated epithelial cells of rat ventral prostate

Receptors for porcine vasoactive intestinal peptide have been characterized in isolated epithelial cells of rat ventral prostate. The interaction of ¹²⁵I-labelled VIP with cells was rapid, reversible, specific, saturable and dependent on temperature. Degradation of peptide and receptors was minimized at 15°C. At apparent equilibrium, the binding of ¹²⁵I-labelled peptide was competitively inhibited by native VIP in the 1·10⁻¹⁰−10⁻⁷ M range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a K_d = 4.0 nM and a low binding capacity (0.12 pmol VIP/mg cell protein), and a low-affinity class with a K_d = 17.8 nM and a high binding capacity (1.6 pmol VIP/mg cell protein). Chicken VIP and porcine secretin exhibited a 7-fold higher and a 7-fold lower affinity than porcine VIP for binding sites, respectively. Glucagon, Leu-enkephalin, Met-enkephalin and somatostatin were ineffective. The presence of high-affinity receptors for VIP together with previous reports on the occurrence of VIP-containing neurones innervating the male genitourinary tract strongly suggest that this peptide may be important in the physiological regulation of the functions of prostatic epithelium.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

Studies on glucocorticoid-induced cytolysis in cultured mouse lymphoma cells, L5178Y : I. Glucocorticoid specificity and cytoplasmic receptors in sensitive and resistant cells

1. 1. The cytolytic action of glucocorticoids such as dexamethasone was studied in a murine cultured lymphoma L5178Y cell.

2. 2. Dexamethasone-resistant cells, which could grow even in a culture medium containing 2 × 10⁻⁵ M dexamethasone, were selected from hormone-sensitive L5178Y lymphoma cells by stepwise increasing concentrations of dexamethasone in culture medium.

3. 3. The synthetic activities of macromolecules corresponded closely with the viabilities and with the resistibilities at various concentrations of dexamethasone in both cells.

4. 4. Studies of binding of dexamethasone to lymphoma L5178Y cells in vivo demonstrated that (1) equilibrium of binding at 37 °C was established within 5 min in both cells; (2) the specific binding to either whole cell or the subcellular fractions was, in sensitive cells, about double that in resistant cells.

5. 5. The cytoplasm of L5178Y lymphoma cells contains specific binding sites for dexamethasone and its binding sites (5.7 × 10⁻¹⁰ mmol/mg protein) were about 2.5-fold more than that of dexamethasone-resistant cells (2.3 × 10⁻¹⁰ mmol/mg protein).

6. 6. Studies on the dissociation constant, competition of various steroids for the specific binding with dexamethasone, and the sedimentation constant of steroid-receptor complex, suggested that the nature of cytoplasmic binding in resistant cells might be the same as that in sensitive cells.

7. 7. The nuclear binding of dexamethasone was extremely dependent on the affinity of cytoplasm to steroid. Studies suggested an equal level of nuclear association sites for cytoplasmic steroidreceptor complex in the two cell types.

     

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells.

The protein neurotoxin II from the venom of the scorpion $\text{Androctonus}\text{australis}$ Hector was labeled with ¹²⁵I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (K_D = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na⁺ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone $\text{Anemona sulcata}$ also affects the closing of the action potential Na⁺ ionophore in nerve axons. The unlabelled sea anemone toxin modifies ¹²⁵I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent K_D for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both $\text{Androctonus}$ scorpion toxin II and $\text{Anemona}$ sea anemone toxin II interact competitively with a regulatory component of the action potential Na⁺ channel.     

High affinity binding sites on plasma membrane obtained from the lymphoblastoid cultured 1301 cell line for highly radioactive serum thymic factor

The interaction of the synthetic serum thymic factor (FTS, facteur thymique sérique) with a plasma membrane preparation of human T lymphocytes from the lymphoblastoid T cell line 1301 was studied using ³H-labelled FTS (specific activity 120 Ci/mmol). The binding is temperature dependent and function of the concentration of both ³H-labelled FTS and membrane proteins. At 37°C, using 1 nM of ³H-labelled FTS a steady state is observed within 80 min. The binding is reversible, specific and saturable. Scatchard analysis reveals the existence of at least two binding sites with respective K_d of the order of 0.516±0.2 nM and 110±27.8 nM with concentrations of 0.186±0.045 pmol and 2.026±0.367 pmol per mg of membrane protein.     

Characterization of [3H]Vesamicol binding in rat brain preparations

The binding of (1)-[³H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P₂ fraction from the rat cerebral cortex reached equilibrium within 4 min at 37°C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P₂ fractions from the cerebral cortex (K_d:21 nM and 980 nM), striatum (K_d:28 nM and 690 nM), and cerebellum (K_d:22 nM and 833 nM). High affinity B_max values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity B_max values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC₅₀:17 nM) and efficaciously (over 90%). Both high affinity and low affinity B_max values for [³H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by puriffied synaptosomes, crude membranes and P₂ fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [³H]vesamicol binding. Isoosmotic replaement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [³H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin.     

Cell-free synthesis of opiate binding sites

A procedure is described for the detection of opiate binding sites synthesized during in vitro translation of various mRNA preparations. RNA were isolated from membrane bound polysomes which were prepared from NG 108-15 hybridoma, C6BU1 glioma cells, as well as from N18TG2, NB2aAg and NB41A3 neuroblastoma cells. Polyadenylated [poly(A)⁺] RNA were purified, translated in vitro in a rabbit reticulocyte lysate and the translation products assayed for their ability to bind [³H] bremazocine. Bound and free ligands were separated by column chromatography. After translation of poly(A)⁺ RNA obtained from NG 108-15 cells we demonstrated a stereospecific, saturable binding of [³H]bremazocine (displaced by levorphanol and not by dextrorphan) with a K_d of 2.4 ± 1.0 nM. The total amount of opiate binding sites synthesized was 6.2 ± 0.5 fmol per μg of poly(A)⁺ RNA. Opiate binding sites were undetectable at zero time and a plateau was reached after translation had proceeded for 20 min. Five time less opiate binding sites were synthesized when the poly(A)⁺ RNA purified from N18TG2 neuroblastoma cells were used under the same experimental conditions. There was no detectable binding of opiate ligands with poly(A)⁺ RNA obtained from C6BU1 glioma cells, NB2aAg or NB41A3 neuroblastoma cells.     

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [³H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [³H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a K_d of 113 nM and B_max of 0.65 pmol/10⁶ cells in freshly isolated cell suspension and K_d of 1.65 μM and B_max of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [³H]PAF itself was found in the same cell preparations. The binding of [³H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K_i of 3.8 nM and B_max of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam also competed for the [³H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [³H]WEB 2086 with a rank order BN 50739⪢Ro 5-4864≥clonazepam. Interestingly, bicuculline, a specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [³H]WEB 2086. The binding of [³H]flunitrazepam was inhibited with a rank potency BN 50739⪢WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

Binding of thyroid hormone to mouse granulosa cell nuclei and its biological relevance

Thyroid hormone showed specific binding ability to mouse granulosa cells from immature mice, primed with post menopausal gonadotropin. Saturation of specific binding sites was reached by 2 nM concentration of the hormone. A Scatchard analysis of thyroid hormone binding exhibited a K_d of 42 x l0_-9M/mg nuclear DNA and a maximum binding capacity of 1 pmol/mg nuclear DNA. Competitive inhibition studies showed thyroid hormone binding to be analogue specific. Addition of 100 ng of thyroid hormone to granulosa cell incubations (1 x 10⁶ cells/well) resulted in a three-fold increase in cellular protein synthesis. Thyroid hormone resulted in a dose dependant increase in progesterone release from granulosa cell. It also stimulated the formation of pregnenolone (83%) and progesterone (81%) from radiolabeled cholesterol as compared to control. This stimulation by thyroid hormone was completely inhibited by cycloheximide. Results indicate a direct effect of thyroid hormone on granulosa cells, its binding to nuclei causing an increase in steroidogenesis through the mediation of protein(s).     

Differential Regulation of Transferrin Receptor Gene Expression in Human Hemopoietic Cells: Molecular and Cellular Aspects

Abstract

As we have shown earlier (-)¹²⁵lodocyanopindolol (¹²⁵ICYP) binding to β-adrenoceptors (β-AR) in human mononuclear leucocytes (MNL) yields evidence for the existence of high affinity (B_hiaff) and low affinity (B_loaff) binding sites. We studied the regulation of these 2 classes of binding sites during 240 min of (-)-epinephrine (EPI) infusion (0.1 μg/kg/min) (n=8) in male healthy volunteers. Saturation experiments were performed on MNL membranes with ¹²⁵ICYP over a large concentration range (1–550 pmol/l). Binding parameters were calculated by computer analysis assuming 2 classes of binding sites. We found a preinfusion value of 830±50 [sites/cell] (K_D=1.5±0.2 pmol/l) of B_hiaff binding sites and 5210±510 [sites/cell] (K_D=420±80 pmol/l) of B_loaff. During EPI infusion we observed biphasic modulation of the B_hiaff and an inverse modulation of the B_loaff. After 40 min of EPI B_hiaff increased to 1970±280 [sites/cell] (K_D=4.2±0.8 pmol/l), whereas B_loaff decreased to 2720±280 [sites/cell] (K_D=140±70 pmol/l); despite constant plasma epinephrine concentration (PEC) after 240 min of EPI B_hiaff changed to 1310±240 [sites/cell] (K_D=2.8±1.0 pmol/l) vs. 4370±760 [sites/cell] (K_D=190±100 pmol/l) B_loaff. These results suggest an interdependent inverse modulation of the 2 classes of binding sites for ¹²⁵ICYP on MNL during EPI infusion.     

L-Glutamate Receptor Heterogeneity: Labeling of Distinct Receptor Sub-Types Using Radioligand Binding Techniques

Abstract

This study demonstrates (1) that L-[³H]glutamate labels 3 distinct binding sites (types A1, A2 and A4) in isolated rat brain membranes and (2) that only the N-methyl-aspartate (A1) and quisqualate (A2) receptor classes are associated with the postsynaptic density (PSD). L-[³H]glutamate bound to PSDs with K_d 339 nM and B_max 6.1 pmol/mg protein. These sites were resolved into 2 distinct sub-types on the basis of inhibition studies. N-Methyl-aspartate maximally inhibited 57% of PSD-located L-glutamate binding sites (the A1 site) and quisqualate 43% (the A2 site); the effects of both substances were additive. The ligand selectivities of these 2 sites indicated their identity with the N-methyl-D-aspartate and quisqualate receptor classes defined electrophysiologically. The Cl^?-dependent population of L-glutamate binding sites (the A4 site) which predominates in synaptic membranes was absent from PSDs.     

Leukotriene C4 ([3H] - LTC4) binding to membranes isolated from a hamster smooth muscle cell line (DDTIMF2)

Leukotriene C₄ (LTC₄) has been demonstrated to induce contraction of the smooth muscle cell line DDTIMF2. A partially purified membrane fraction obtained from these cells exhibited a high affinity binding site for LTC₄. Binding of [³H]-LTC₄ was saturable, specific and reversible with a dissociation constant (K_d) of 21 ± 4 nM. The maximum number of binding sites (B_max) was 55 ± 5 pmol/mg of protein. Specificity was demonstrated in competition studies in which the Ki of LTC₄ against specifically bound [³H] - LTC₄ was 12 nM whereas Leukotriene D₄ (LTD₄) and Leukotriene E₄ (LTE₄) had a Ki of 38 ± 4 and 4.7 ± 0.5 nM respectively. A previously described antagonist of leukotriene-induced smooth muscle contraction PFL 55712 had a K_i of 23 ± 2 nM as determined by competition binding experiments.