Binding of leukotriene B4 and its analogs to human polymorphonuclear leukocyte membrane receptors

LTB₄-induced proinflammatory responses in PMN including chemotaxis, chemokinesis, aggregation and degranulation are thought to be initiated through the binding of LTB₄ to membrane receptors. To explore further the nature of this binding, we have established a receptor binding assay to investigate the structural specificity requirements for agonist binding. Human PMN plasma membrane was enriched by homogenization and discontinuous sucrose density gradient purification. [³H]-LTB₄ binding to the purified membrane was dependent on the concentration of membrane protein and the time of incubation. At 20°C, binding of [³H]-LTB₄ to the membrane receptor was rapid, required 8 to 10 min to reach a steady-state and remained stable for up to 50 min. Equilibrium saturation binding studies showed that [³H]-LTB₄ bound to high affinity (dissociation constant, K_d = 1.5 nM), and low capacity (density, B_max=40 pmol/mg protein) receptor sites. Competition binding studies showed that LTB₄, LTB₄-epimers, 20-OH-LTB₄, 2-nor-LTB₄, 6-trans-epi-LTB₄ and 6-trans-LTB₄, in decreasing order of affinity, bound to the [³H]-LTB₄ receptors. The mean binding affinities (K₁) of these analogs were 2, 34, 58, 80, 1075 and 1275 nM, respectively. Thus, optimal binding to the receptors requires stereospecific 5(S), 12(R) hydroxyl groups, a cis-double bond at C-6, and a full length eicosanoid backbone. The binding affinity and rank-order potency of these analogs correlated with their intrinsic agonistic activities in inducing PMN chemotaxis. These studies have demonstrated the existence of high affinity, stereoselective and specific receptors for LTB₄ in human PMN plasma membrane.     

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions

A leukotriene B₄ (LTB₄) analog, 20-trifluoromethyl LTB₄ (20CF₃−LTB₄), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF₃−LTB₄ was equipotent to LTB₄ as a chemoattractant (EC₅₀, 3 nM), produced 50% of maximal activity of LTB₄, and competed with [H] LTB₄ for binding to intact human neutrophil LTB₄ receptors. In contrast to chemotactic activity, 20CF₃−LTB₄ in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 μM on LTB₄-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB₄, 20CF₃−LTB₄ induced neutrophil desensitization to degranulation by LTB₄. The results indicate that hydrogen atoms at C-20 of LTB₄ are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF₃−LTB₄ is a partial agonist for chemotaxis and an antagonist for degranulation syggests that different LTB₄ receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB₄ can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.     

Binding of a thromboxane A2/prostaglandin H2 agonist [H]U46619 to washed human platelets

The binding characteristics of [³H]U46619 to washed human platelets were studied. [³H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a K_d of 11 ± 4 nM (n=4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a K_d of 20 ± 7nM and a B_max of 9.1 ± 2.3 fmole/10⁷ platelets (550 ± 141 binding sites per platelet) (n=4). A number of compounds that act as either agonists or antagonists of the TXA₂/PGH₂ receptor were tested for their ability to inhibit the binding of [³H]U46619 to washed human platelets. The K_ds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [³H]U46619 binds to the putative human platelet TXA₂/PGH₂ receptor.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [³H]-leukotriene D₄ ([³H]-LTD₄), in the presence or absence of unlabeled LTD₄, the diastereoisomer of LTD₄ (5R,6S-LTD₄), leukotriene E₄ (LTE₄) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [³H]-LTD₄ in the crude membrane fraction isolated from guinea pig lung. The time required for [³H]-LTD₄ binding to reach equilibrium was approximately 20 to 25 min at 37°C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [³H]-LTD₄ to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (B_max), derived from Scatchard analysis, was approximately 320±200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5±4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD₄, FPL 55712 and 5R,6S-LTD₄ competed with [³H]-LTD₄. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD₄, did not significantly compete with [³H]-LTD₄ at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.     

The effects of a diet rich in fish oil on human neutrophils: Identification of leukotriene B5 as a metabolite

Diets that are enriched with fish oil have been shown to alter arachidonic acid metabolism via the cyclooxygenase pathway. Recently it has been shown that one of the major component fatty acids of fish oil, eicosapentaenoate (EPA), is a substrate for the leukotriene B (LTB) pathway when added exogenously to human neutrophils . We fed a diet that contained 8–10 gm/day of EPA to four human subjects for three weeks and compared the arachidonate metabolism of their neutrophils to the same functions while the subjects were on their usual diet. The fish oil-supplementation increased neutrophil EPA content from undetectable levels to 7.4 ± 2.4% (p<0.01, expressed as % of total fatty acid), and decreased arachidonate from 15.4 ± 2.3% to 12.8 ± 2.3% (p<0.05). Leukotriene B₅ was identified as a metabolite during the fish oil-diet by its chromatographic profile and mass spectrum. During the experimental diet LTB₄ decreased from 160 ± 37 ng/10⁷ neutrophils to 120 ± 12 (p<0.05), and LTB₅ increased from 0 to 39 ± 9 ng/10⁷ neutrophils (p<0.005). The diet had no effect on neutrophil aggregation or adherence to nylon fibers.     

Influences of cell size and aging on l-[H]norepinephrine binding sites in rat epididymal fat cells

[³H]norepinephrine binding to isolated rat fat cells was studied as a function of adipose cell age and size. Rats aged from 4 to 78 weeks were used.Scatchard analysis of norepinephrine binding revealed in old fat cells like in young ones the existence of two orders of binding sites with respectively high and low affinity for norepinephrine. The apparent association constants Ka₁ and Ka₂ associated with these binding sites did not differ consistently in the different groups of fat cells studied (Ka₁ = 1.7 to 2.2 × 10⁶ × M⁻¹; Ka₂ = 1.9 to 2.5 × 10⁴ × M⁻¹), suggesting that age and cell size do not modify the apparent affinity of norepinephrine-binding sites in rat fat cells.On the contrary, the total amount of norepinephrine bound to each of these sites was dependent upon cell age and size. In fact, maximum binding of norepinephrine to the high affinity sites was 0.9 and 9 pmol/10⁵ cells in small (diameter: 35 μ) and large (diameter: 105 μ) adipocytes, respectively, the values found for the low affinity sites being 13 and 135 pmol/10⁵ cells. When expressed per unit of fat cell area, however, the total binding capacity for these sites appeared practically constant (2.4 — 2.8 pmol × 10⁻³/mm² and 34.2 — 38.2 pmol × 10⁻³/mm² for the high and low affinity sites respectively). These data suggest that the total norepinephrine binding capacity of the fat cell is directly proportional to its surface.     

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

Evidence for a dual pathway in platelet activating factor-induced aggregation of rat polymorphonuclear leucocytes

The purpose of this study was to determine the role, if any, of Leukotriene B₄ (LTB₄) in Platelet Activating Factor (PAF)-induced aggregation of rat polymorphonuclear leucocytes (PMNs). Exposure of rat PMNs to 10⁻⁷ M PAF resulted in the release of 4.5 ± 0.7 ng/10⁷ cells of LTB₄ measured by radioimmunoassay. However, the maximum aggregation of PMNs achieved by exposure to LTB₄ (10⁻⁷M) was only 50% of that produced by maximally aggregating concentrations of PAF (10⁻⁷M). 5-Lipoxygenase inhibitors, BW755c and Nafazatrom at concentrations that completely abolished LTB₄ synthesis inhibited the aggregation induced by PAF only by 40% and 50% respectively. Furthermore, desensitisation experiments revealed that the aggregatory response of PMNs to PAF was only partially refractory to prior treatment with LTB₄ whereas the aggregatory response to LTB₄ was completely refractory to prior treatment with PAF. These results suggest that PAF-induced aggregation of rat PMNs is in part mediated by LTB₄ and in part directly by an as yet unidentified mechanism.     

Membrane Permeability Characteristics of Metaphase II Mouse Oocytes at Various Temperatures in the Presence of Me2SO

In this study, the hydraulic conductivity (L_p), Me₂SO permeability ( _Me2SO), and the reflection coefficients (ς) and their activation energies were determined for Metaphase II (MII) mouse oocytes by exposing them to 1.5 M Me₂SO at temperatures of 30, 20, 10, 3, 0, and −3°C. These data were then used to calculate the intracellular concentration of Me₂SO at given temperatures. Individual oocytes were immobilized using a holding pipette in 5 μl of an isosmotic PBS solution and perfused with precooled or prewarmed 1.5 M Me₂SO solutions. Oocyte images were video recorded. The cell volume changes were calculated from the measurement of the diameter of the oocytes, assuming a spherical shape. The initial volume of the oocytes in the isoosmotic solution was considered 100%, and relative changes in the volume of the oocytes after exposure to the Me₂SO were plotted against time. Mean (means ± SEM) L_pvalues in the presence of Me₂SO ( ^Me₂SO_p) at 30, 20, 10, 3, 0, and −3°C were determined to be 1.07 ± 0.03, 0.40 ± 0.02, 0.18 ± 0.01, 7.60 × 10⁻²± 0.60 × 10⁻², 5.29 × 10⁻²± 0.40 × 10⁻², and 3.69 × 10⁻²± 0.30 × 10⁻²μm/min/atm, respectively. The _Me2SOvalues were 3.69 × 10⁻³± 0.3 × 10⁻³, 1.07 × 10⁻³± 0.1 × 10⁻³, 2.75 × 10⁻⁴± 0.15 × 10⁻⁴, 7.83 × 10⁻⁵± 0.50 × 10⁻⁵, 5.24 × 10⁻⁵± 0.50 × 10⁻⁵, and 3.69 × 10⁻⁵± 0.40 × 10⁻⁵cm/min, respectively. The ς values were 0.70 ± 0.03, 0.77 ± 0.04, 0.81 ± 0.06, 0.91 ± 0.05, 0.97 ± 0.03, and 1 ± 0.04, respectively. The estimated activation energies (E_a) for ^Me₂SO_p, _Me2SO, and ς were 16.39, 23.24, and −1.75 Kcal/mol, respectively. These data may provide the fundamental basis for the development of more optimal cryopreservation protocols for MII mouse oocytes.     

Specific binding of [H]heparin to human carcinoma SW-13 and other mammalian cells

This study reports on the specific binding of [³H]heparin to human adrenocortical carcinoma cell line SW-13. Heparin binding to SW-13 cells is specific, saturable, and time- and temperature-dependent with maximum binding occurring between 90 and 120 min at 22 °C. Scatchard analysis revealed two classes of binding sites. The apparent K_d for high-affinity receptors is 2.14 × 10⁻⁸ M with 1.48 × 10⁶ sites per cells. Six other tested mammalian cell lines also have specific binding sites for heparin.     

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.     

Leukotriene B4, polymorphonuclear leukocytes and inflammatory exudates in the rat

Leukocyte numbers and Leukotriene B₄- (LTB₄-) and LTC₄-immunoreactivity were measured in inflammatory exudates obtained from sponges impregnated with several irritants implanted subcutaneously in the rat. Sponges containig 1% uric acid, carragennan or zymosan were implanted for 5h and compared to saline sponges. Increases in leukocyte numbers and LTB₄-immunoreactivity were found in the presence of irritants, the highest concentrations being observed in the presence of zymosan. The presence of LTB₄ was confirmed by liquid chromatographic (HPLC) analysis. A time course study was carried out with zymosan-impregnated sponges and the maximal rate of leukocyte infiltrations was found to coincide with the maximal levels of LTB₄-immunoreactivity. The LTC₄-immunoreactivity was low and following analysis by HPLC was concluded to be unrelated to leukotrienes. The levels of LTB₄-immunoreactivity, but not the numbers of leukocytes, were elevated compared to corresponding controls in sponges containing 0.01% ionphore A23187 (untreated rats) or in sponges containing zymosan (rats pretreated with indomethacin; 3 and 10 mg/kg p.o.). Impregnation of sponges with 3 × 10⁻⁶M LTB₄ but not 3 × 10⁻⁷M LTB₄ induced a significant leukocyte migration. It was concluded that LTB₄ can induced leukocyte migration into sponge exudates in the rat but that measurements of LTB₄ in such exudates can not be correlated with the degree of leukocyte infiltration.     

Characterization of the progesterone receptor of rabbit uterus with the synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione

The synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione (Org 2058) was used to characterize the progesterone receptor in the uterine cytosol of the rabbit. [³H] Org 2058 binds to a homogeneous population of protin binding sites with an apparent association equilibrium constant of 7.7· 10⁸ M⁻¹ at 0°C. The concentration of protein-bound steroid at saturation is 2.3 pmol per mg of cytosol protein. [³H] Progesterone binds to the same set of binding sites but exhibits a 4–5 fold lower apparent association constant. The difference in affinity is mainly due to a 13-fold slower rate of dissociation of the synthetic progestin compared with progesterone. Org 2058 competes very efficiently for the binding of [³H] progesterone to the uterine cytosol, and progesterone also competes, although less efficiently, for the binding of [³H]-Org 2058. There is a good correlation between the progestational activity of various steroids and their ability to compete with [³H] Org 2058 binding to the cytosol. At 0°C, there is no metabolic transformation of either Org 2058 or progesterone in the uterine cytosol.When filled with the steroid, the progesterone receptor is stable, but in the absence of the steroid the receptor binding sites are thermolabile and show a rapid decay at 20°C . Org 2058 is more effective than progesterone in protecting the receptor against thermal inactivation. The rate constant of association and dissociation of [³H] Org 2058 and the cytosol receptor are strongly dependent on temperature and the activation energy of the dissociation reaction is 17.8 kcal/mol. The equilibrium association constant is less dependent on temperature and exhibits ΔH° of −4.7 kcal/mol. The binding reaction shows a positive entropy change of 23 cal · K⁻¹ · mol⁻¹.At low ionic strength the complex of Org 2058 and the progesterone receptor tends ot aggregate. It sediments as a broad peak on sucrose gradients (4–6 S), and is excluded from columns of Sephadex G-100 and G-200. At concentrations of NaCl above 0.15 M, the receptor sediments in sucrose gradients as an homogeneous peak at 3.6 S, but upon gel filtration it aggregates and a complex elution pattern is observed, that prevents a precise estimation of the molecular weight.     

Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: Possible pitfall of using the calcium ionophore A23187 to stimulate 5′ lipoxygenase

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5′ lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5′ lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B₄ by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC₅₀ 1.6 × 10⁻⁴M) was approximately 100 times less potent than BW755C (IC₅₀ 1.7 × 10⁻⁶M) at inhibiting leukotriene B₄ synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 × 10⁻⁶M). The data obtained using STZ as stimulus are consistent with previous studies and indicate that benoxaprofen is a relatively selective inhibitor of cylco-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5′ lipoxygenase for evaluating inhibitors of eicosanoid synthesis.     

Nuclear benzodiazepine binding: Possible interaction with thyroid hormone receptors

The biochemical and pharmacological properties of nuclear [³H]flunitrazepam in brain tissues were studied. Nuclear [³Hflunitrazepam binding is saturable for both central and peripheral binding sites. Inosine and hypoxanthine displace nuclear [³H]flunitrazepam binding with greater potency than the membrane [³H]flunitrazepam binding. Triiodothyronine (T₃) increases the maximum number of binding sites (B_max) of nuclear [³H]flunitrazepam binding in vitro while thyroxine (T₄) does not have any effect. Diazepam reduces the affinity of nuclear¹²⁵I-T₃ binding in vitro, while the B_max is not affected significantly. Mild digestion of chromatin, using micrococcal nuclease, reveals that a major portion of nuclear [³H]flunitrazepam binding sites are located on chromatin. These data suggest a functional role for nuclear benzodiazepine binding and a possible modulatory effect of benzodiazepines on T₃ binding with its nuclear receptors.     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.