Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long-term subculturing. In order to study in vitro age-related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast-specific genes in early-passage (< 50% lifespan completed) with those of late-passage cells (> 90% lifespan completed). We found a significant reduction in mRNA levels of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late-passage cells compared to early-passage cells, suggesting an in vitro age-related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic cells inhibited their proliferation by 57 +/- 1% and stimulated steady state mRNA levels of AP (1.7 +/- 0.1-fold) and OC (1.8 +/- 0.2-fold). Similarly, calcitriol treatment increased mRNA levels of AP (1.7 +/- 0.2-fold) and OC (3.0 +/- 0.3-fold) in late-passage osteoblastic cells. Thus, in vitro senescent osteoblastic cells maintain their responsiveness to calcitriol and some of the observed in vitro age-related decreases in biological markers of osteoblast functions can be reverted by calcitriol treatment.     

Characterization of human interleukin-3 receptors on a multi-factor-dependent cell line

Recombinant human interleukin-3 (hIL-3) was radioiodinated by Bolton-Hunter method with maintenance of biological activity. Using 125I-hIL-3, hIL-3 receptors were characterized on a multi-factor-dependent cell line TF-1. Equilibrium binding studies revealed the existence of a single class of binding sites (667 +/- 306 sites/cell) with a Kd of 173 +/- 25 pM. Affinity labeling of TF-1 cells with 125I-IL-3 yielded two bands of 150 kDa and 85 kDa, implying molecular weights of 135 kDa and 70 kDa for the hIL-3 receptors.     

Solubilisation of the 5-Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested (1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside), deoxycholate (0.5%) yielded the best solubilisation (54.6 +/- 6% of receptor, 70.5 +/- 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 +/- 6 fmol/mg of protein; KD = 0.33 +/- 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4 degrees C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) greater than quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) greater than ICS 205-930 (0.93 nM) greater than GR 38032F (2.2 nM) greater than BRL 24924 (4.1 nM) greater than MDL 72222 (23.4 nM) greater than ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.     

Solubilization of Peripheral-Type Benzodiazepine Binding Sites from Cat Cerebral Cortex

The present study demonstrates for the first time the solubilization of peripheral-type benzodiazepine binding sites (PBS) from cat cerebral cortex. Of all detergents tested [digitonin, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholate, and Triton X-100] in the presence of NaCl, the best solubilization (15% of initial activity) was obtained using 0.5% of the zwitterionic detergent CHAPS plus 2 M NaCl. Specific binding of [3H]PK 11195 to membrane-bound and solubilized PBS was saturable, yielding equilibrium dissociation constants (KD) of 1.3 +/- 0.2 and 1.9 +/- 0.3 nM, respectively, and maximal numbers of binding sites of 1,435 +/- 150 and 980 +/- 126 fmol/mg protein, respectively. The KD value of PK 11195 binding to solubilized PBS obtained from experimental kinetic analysis was 0.95 +/- 0.09 nM. The relative potencies of various compounds (PK 11195, Ro 5-4864, diazepam, flunitrazepam, clonazepam, methyl-beta-carboline-3-carboxylate, and Ro 15-1788) in displacing [3H]PK 11195 specific binding from membrane-bound and solubilized PBS were similar. Most of the solubilized binding activity was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein-binding site in the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4 degrees C, which will enable future application of purification procedures without major concern for stability of the material.     

Covalent affinity labeling, detergent solubilization, and fluid-phase characterization of the rabbit neutrophil formyl peptide chemotaxis receptor

The formyl peptide chemotaxis receptor of rabbit neutrophils and purified rabbit neutrophil plasma membranes has been identified by several affinity labeling techniques: covalent affinity cross-linking of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys (125I-hexapeptide) to the membrane-bound receptor with either dimethyl suberimidate or ethylene glycol bis(succinimidyl succinate) and photoactivation of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-N epsilon-[6-[(4-azido-2-nitrophenyl)amino]hexanoyl]Lys(125I-PAL). These techniques specifically identify the receptor as a polypeptide that migrates as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with Mr 50 000-65 000. The receptor has been solubilized in active form from rabbit neutrophil membranes with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and from whole cells with CHAPS. Chemotaxis receptor activity was measured by the ability of the solubilized membrane material to bind 125I-hexapeptide or fMet-Leu-[3H]Phe with gel filtration or rapid filtration through poly(ethylenimine)- (PEI) treated filters as assay systems. 125I-PAL was specifically cross-linked to the same molecular weight material in the CHAPS and digitonin solubilized extract, but no specific labeling of the receptor was seen when membranes were extracted with Nonidet P-40 and Triton X-100. Therefore, although a large number of detergents are able to solubilize the receptor, it appears that some release the receptor in an inactive form. The ligand binding characteristics of fMet-Leu-[3H]Phe to the CHAPS-solubilized receptor shared properties with the membrane-bound formyl peptide receptor, both of which showed curvilinear, concave-upward Scatchard plots. Computer curve fitting with NONLIN and statistical analyses of the binding data indicated that for both the membrane-bound and solubilized receptors a two saturable sites model fitted the data significantly better (p less than 0.01) than did a one saturable site model. The characteristics of the two saturable sites model for the soluble receptor were a high-affinity site with a KD value of 1.25 +/- 0.45 nM and a low-affinity site with a KD value of 19.77 +/- 3.28 nM. A total of 35% of the two sites detected was of the higher affinity. In addition, a Hill coefficient of 0.61 +/- 0.12 was observed.     

Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3

Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3.     

Bombesin receptor in membranes from Swiss 3T3 cells. Binding characteristics, affinity labelling and modulation by guanine nucleotides.

Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.     

6-Fluoro-vitamin D3: a new antagonist of the biological actions of vitamin D3 and its metabolites which interacts with the intestinal receptor for 1 alpha,25(OH)2-vitamin D3

The biological activity and the binding affinity for the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] intestinal receptor of a new fluorine-containing vitamin D compound, namely 6-fluoro-vitamin D3 (6-F-D3), is reported. A significant interaction of 6-F-D3 with the 1,25(OH)2D3 receptor was found, with a relative competitive index (RCI) of 0.26 +/- 0.04, which is intermediate between 25-hydroxyvitamin D3 (0.14 +/- 0.01) and 1 alpha-hydroxyvitamin D3 (0.46 +/- 0.08), where the RCI of 1,25(OH)2D3 is defined to be 100. In contrast, vitamin D3 was unable to interact with the 1,25(OH)2D3 receptor. Also, the biological activity of 6-F-D3 was assessed in vivo in the vitamin D-deficient chick. 6-F-D3 at doses up to 130 nmol displayed no biological action on either intestinal calcium absorption (ICA) or bone calcium mobilization (BCM) over the time interval of 14-48 h after dosing. However, when 130 nmol 6-F-D3 was given 2 h before and 6 h after vitamin D3 (1.62 nmol), a significant inhibition of vitamin D-mediated ICA was noted. Also, a dose of 130 nmol 6-F-D3 given 2 h before and 6 h after 1,25(OH)2D3 (0.26 nmol) significantly inhibited ICA, as measured at 12 h. 6-F-D3 is the first vitamin D analog found which has an ability to both bind to the 1,25(OH)2D3 receptor and to antagonize the production of biological responses by 1,25(OH)2D3.     

Interaction of detergents with cytochrome c oxidase.

The binding of ionic and nonionic, nondenaturing detergents to cytochrome c oxidase has been examined. All bind and displace part but not all of the phospholipid that is associated with the enzyme after isolation. From 6 to 10 phospholipid molecules, depending on the detergent used, do not exchange and these are mostly diphosphatidylglycerol molecules as first shown by Awasthi et al. ((1971) Biochim. Biophys. Acta 226, 42). The binding of Triton X-100 and deoxycholate to the cytochrome c oxidase complex has been studied in detail. Both bind to the enzyme above their critical micelle concentrations: Triton X-100 in the amount of 180 +/- 10 molecules per complex and deoxycholate in the amount of 80 +/- 4 molecules per complex. In nonionic detergents, cytochrome c oxidase exists as a dimer (4 heme complex). The enzyme is dissociated into the monomer or heme aa3 complex by delipidation in bile salts. Activity measurements in different detergents suggest that cytochrome c oxidase requires a flexible, hydrophobic environment for maximal activity and that the dimer or 4 heme complex may be the active species.     

Expression of ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta, Diptera) in E. coli and purification in a functional state.

Full length clones of ecdysteroid receptor (EcR) and Ultraspiracle (USP) from Chironomus tentans were expressed as GST fusion proteins in E. coli and purified by affinity chromatography. The absence of detergents during the purification procedure is essential for retaining receptor function, especially ligand binding. Presence of USP is mandatory for ligand binding to EcR, but no other cofactors or posttranslational modifications seem to be important, since Scatchard plots revealed the same characteristics (two high affinity binding sites for Ponasterone A with K(D1)=0.24+/-0.1nM and K(D2)=3.9+/-1.3.nM) as found in 0.4 M NaCl extracts of Chironomus cells. Gel mobility shift assays showed binding of the heterodimer to PAL and DR5 even after removal of the GST-tag, whereas EcR binding to PAL1 is GST-dependent. USP binds preferentially to DR5. Addition of unprogrammed reticulocyte lysate improves ligand binding only slightly. Removal of GST has no effect on (3)H-ponasterone A binding, but alters DNA binding characteristics. Calculation of specific binding (5.3+3.0 nmol/mg GST EcR) revealed that 47+/-26% of purified receptor protein was able to bind ligand. The addition of purified EcR to cell extracts of hormone resistant subclones of the epithelial cell line from C. tentans, which have lost their ability to bind ligand, restores specific binding of (3)H-ponasterone A.     

Age-related changes in adrenergic alpha 1, alpha 2, and beta receptors of rat white fat cell membranes: an analysis using [3H]bunazosin as a novel ligand for the alpha 1 adrenoceptor.

Age-related changes in alpha 1-, alpha 2-, and beta-catecholamine receptors on membrane of rat epididymal fat cells were investigated. Both young (6 weeks old, weight about 190 g) and aged (20 weeks old, weight about 490 g) Sprague-Dawley male rats were used. For the alpha 1-adrenoceptor binding experiment, we developed a novel analytical method using the hydrophilic alpha 1-receptor selective antagonist, [3H]bunazosin. The binding of [3H]bunazosin to its binding sites was rapid, reversible, saturable, and stereospecific. Scatchard binding analysis showed a single class of binding site. The sites were characterized as alpha 1-adrenoceptors by inhibition experiments using various agonists and antagonists. The number of maximum binding sites (Bmax) of alpha 1-receptor binding was 37.0 +/- 6.5 (young) versus 24.0 +/- 3.2 (aged) fmol/mg protein (P less than 0.01). [3H]Rauwolscine and [3H]CGP-12177 were used for alpha 2- and beta-receptor binding, respectively. In alpha 2-receptor detection using [3H]rauwolscine as a ligand, Bmax increased markedly from 19.8 +/- 4.9 to 86.2 +/- 19.5 fmol/mg protein (P less than 0.01). In contrast, Bmax for beta-receptor decreased from 69.7 +/- 9.7 to 45.4 +/- 13.9 fmol/mg protein with increasing rat age (P less than 0.05). Kd showed no change in each of the binding experiments between young and aged rats. The cell volume increased from 0.07 +/- 0.02 to 0.15 +/- 0.06 nl. It is implied that anti-lipolytic activity strengthened on the whole mainly with the marked increase of alpha 2-receptor number and decrease of beta-receptor number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of N-methylation of selected peptide bonds on the biological activity of insulin. [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin

Hydrogen bonding involving peptide bonds of the backbone of the insulin molecule may play an important role in insulin-receptor interaction. Our previous work suggested that the A2-A8 helical segment of the hormone molecule participates in this interaction. To investigate the possible involvement of peptide bonds of this segment in insulin-receptor interaction the [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin ([MeIle2-A]- and [MeVal3-A]insulins) were synthesized. The circular dichroic spectra of the analogues were obtained and their properties were examined in several biological assays. The circular dichroic spectra suggested that the analogues remained monomeric at concentrations at which insulin is predominantly dimeric, and that their A2-A8 helical segments are distorted. The in vitro biological activity and the receptor binding affinity of these analogues were compared with that of natural insulin. Both analogues are weak full agonists. [MeIle2-A]insulin displayed a potency of 5.4 +/- 0.3% in stimulating lipogenesis and 4.6 +/- 2.3% in receptor binding affinity in rat fat cells and rat liver plasma membranes respectively. [MeVal3-A]insulin displayed a potency of 2.1 +/- 0.2% in lipogenesis and 1.0 +/- 0.3% in receptor binding assays. In radioimmunoassays [MeIle2-A]- and [MeVal3-A]insulins exhibited potencies of 13% and 11% respectively relative to the natural hormone. The substantially decreased biological activity and receptor binding affinity of these analogues may be attributed partly to the change of conformation and partly to the loss of hydrogen bonding capacity of the A2-A8 segment brought about by N-methylation of the A1-A2 or A2-A3 peptide bonds.     

A cellular 3,3',5-triiodo-L-thyronine binding protein from a human carcinoma cell line. Purification and characterization

A cellular binding protein for 3,3',5-triiodo-L-thyronine (T3) was solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) from A431 human epidermoid carcinoma cells. The binding activity is T3 specific. Analysis of the equilibrium binding data indicated that the binding protein has one class of binding sites for T3 with a Kd of (17 +/- 3) nM and Bmax of (1.8 +/- 0.6) pmol/50 micrograms of protein. The pH optimum for binding is 6.8. The T3 binding protein elutes from Sephadex G-200 in an included peak which has a Stokes radius of 40 A and sediments on glycerol gradients at 3.7 S. By affinity labeling with [3,5-125I]thyroxine a protein with a molecular weight of 58,000 was specifically labeled. Its isoelectric point was determined to be 7.1, which is different from the reported pIs of other thyroid hormone binding proteins. p58 was successively purified to apparent homogeneity by chromatography on Sephadex G-200, QAE-Sephadex, SP-Sephadex, and hydroxylapatite. Approximately 50 micrograms of purified protein was obtained from 2.5 X 10(9) cells with a yield of 1.1%. The purified protein retains its binding activity. The specific binding activity is enriched by approximately 1000-fold. With the availability of a purified protein with T3 binding activity, it becomes possible to study its cellular function.     

A major ozonation product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, induces apoptosis in H9c2 cardiomyoblasts

Cholesterol, a major neutral lipid component of biological membranes and the lung epithelial lining fluids, is susceptible to oxidation by reactive oxygen and nitrogen species including ozone. The oxidation by ozone in biological environments results in the formation of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (cholesterol secoaldehyde or CSeco, major product) along with some other minor products. Recently, CSeco has been implicated in the pathogenesis of atherosclerosis and Alzheimer's disease. In this communication, we report that CSeco induces cytotoxicity in H9c2 cardiomyoblasts with an IC(50) of 8.9+/-1.29 microM (n=6). The observed effect of CSeco at low micromolar concentrations retained several key features of apoptosis, such as changes in nuclear morphology, phosphatidylserine externalization, DNA fragmentation, and caspase 3/7 activity. Treatment of cardiomyocytes with 5 microM CSeco for 24h, for instance, resulted in 30.8+/-3.28% apoptotic and 1.8+/-1.11% of necrotic cells as against DMSO controls that only showed 1.3+/-0.33% of apoptosis and 1.6+/-0.67% of necrosis. In general, the loss of cellular viability paralleled the increased occurrence of apoptotic cells in various CSeco treatments. This study, for the first time, demonstrates the induction of apoptotic cell death in cardiomyocytes by a cholesterol ozonation product, implying a role for ozone in myocardial injury.     

Biological activity of 24,24-difluoro-1 alpha, 25-dihydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone in inducing differentiation of human myeloid leukemia cells

Vitamin D compounds added to the culture medium induce differentiation of human myeloid leukemia cells (HL-60 cells) by binding to a specific cytosol receptor protein. This system provides a biologically relevant and technically simple assay to examine the relationship between molecular structure and biological activity of vitamin D compounds. Using this culture system, the biological activity of 24,24-F2-1 alpha,25(OH)2D3 and 1 alpha,25(OH)2D3-26,23-lactone was assayed. 24,24-F2-1 alpha,25(OH)2D3 was four to seven times more potent than 1 alpha,25(OH)2D3 in inducing phagocytosis and C3 rosette formation of HL-60 cells, though both compounds bound equally well to the cytosol receptor, suggesting that the defuorination at the 24-carbon position may stimulate membrane permeability of the compound. 1 alpha,25(OH)2D3-26,23-lactone, on the other hand, was only 1/200th as active as 1 alpha,25(OH)2D3. The binding affinity of the lactone for the cytosol receptor was identical with that of 1 alpha (OH)D3, suggesting that the lactone formation between the 26 and 23 positions masks the function of the 25-hydroxyl group. The binding affinity of vitamin D3 derivatives to the specific cytosol receptor of HL-60 cells was well correlated with that of intestinal cytosol protein specifically bound to 1 alpha,25(OH)2D3.     

Characterization of Two Cerebellar Binding Sites of[3H]Ro 15-4513

Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H- imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate), a partial inverse agonist of central benzodiazepine receptors, binds to two distinct sites in the cerebellum. The binding to diazepam-sensitive (DZ-S) sites is displaced by different benzodiazepine receptor ligands, whereas the other site is insensitive to benzodiazepine agonists [diazepam-insensitive (DZ-IS)]. The binding of [3H]Ro 15-4513 was studied in pig cerebellar membranes and in receptors solubilized and purified from these. Micromolar concentrations of gamma-aminobutyric acid (GABA) decreased DZ-S binding at both 0 and 37 degrees C, whereas it had no effect on DZ-IS binding at 0 degrees C and was stimulatory at 37 degrees C. The pH profiles of [3H]Ro 15-4513 binding were quite similar in both binding sites in the pH range of 5.5-10.5 but differed at acidic pH values from those reported for flunitrazepam and Ro 15-1788 (flumazenil; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H- imidazol[1,5-a][1,4]benzodiazepine-3-carboxylate) binding in DZ-S sites, suggesting that [3H]Ro 15-4513 does not interact with a histidine residue apparently present in the binding site. Zn2+, Cu2+, Co2+, and Ni2+ enhanced the binding to DZ-S sites, and the first three mentioned also enhanced the binding to DZ-IS sites. [3H]Ro 15-4513 binding activity was solubilized by various detergents. All detergents tested were more efficient in solubilizing DZ-S binding activity. High ionic strength improved especially the solubility of DZ-IS binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A reliable assay for beta-adrenoceptors in intact isolated human fat cells with a hydrophilic radioligand, [3H]CGP-12177

The beta-adrenergic receptors of isolated human fat cells were identified using a new hydrophilic beta-adrenergic radioligand (+/-)[3H]CGP-12177. The results were compared with those from [3H]dihydroalprenolol binding to fat cells and membranes. [3H]CGP-12177 binding to isolated fat cells showed lower nonspecific binding (less than 15% of total binding) than the lipophilic [3H]dihydroalprenolol (40-60%) at 3 times the KD. At 37 degrees C, [3H]CGP-12177 binding was rapid, reversible, of high affinity (1.2 +/- 0.3 nM) and saturable. The total number of binding sites per cell in subcutaneous adipocytes was 25,000 +/- 6,000 and was equivalent to that found using membrane fractions. Displacement of [3H]CGP-12177 bound to adipocytes by propranolol was stereoselective, consistent with competition at a single site, and had the same characteristics as in membranes. The displacement curves of the beta 1-selective antagonists (atenolol and betaxolol) were biphasic, the high affinity displacement accounting for 70% of the total binding sites. Beta-adrenergic agonists also competed with [3H]CGP-12177 binding in the order of potency: (-) isoproterenol greater than (-) norepinephrine greater than (-) epinephrine, similar to that found in membranes and in in vitro studies on the lipolytic activity of isolated fat cells. This study demonstrates that the sites specifically labeled by [3H]CGP-12177 are the physiological beta-adrenoceptors and also shows that the ligand is better than [3H]dihydroalprenolol for the accurate identification of these receptors in intact human adipocytes. The methodology, which requires biopsies of less than 1 gram of adipose tissue, can be of potential interest for clinical studies investigating the status of fat cell beta-adrenoceptors in various pathophysiological situations.     

Identification of specific [3H]adenine-binding sites in rat brain membranes

We recently reported that adenine acts as a neurotrophic factor independent of adenosine or P2 receptors in cultured Purkinje cells [Watanabe S. et al. (2003) J. Neurosci. Res. 74, 754-759], suggesting the presence of specific receptors for adenine in the brain. In this study, the characterization of adenine-binding activity in the rat brain was performed to further characterize the receptor-like adenine-binding sites. Specific binding sites for [(3)H]adenine were detected in membrane fractions prepared from rat brains. The kinetics of [(3)H]adenine binding to membranes was described by the association and dissociation rate constants, 8.6 x 10(5) M(-1) min(-1) and 0.118 +/- 0.045 min(-1), respectively. A single binding site for [(3)H]adenine with a K (D) of 157.1 +/- 20.8 nM and a B (max) of 16.3 +/- 1.1 pmol/mg protein (n = 6) was demonstrated in saturation experiments. A displacement study involving various related compounds showed that the [(3)H]adenine binding was highly specific for adenine. It was also found that [(3)H]adenine-binding activity was inhibited by adenosine, although other adenosine receptor ligands were ineffective as to [(3)H]adenine binding. The brain, especially the cerebellum and spinal cord, showed the highest [(3)H]adenine-binding activity of the tissues examined. These results are consistent with the presence of a novel adenine receptor in rat brain membranes.     

High-affinity [3H]octopamine-binding sites in Drosophila melanogaster: interaction with ligands and relationship to octopamine receptors

[3H]Octopamine binds to a particulate preparation from heads of Drosophila melanogaster at a level of 0.5 +/- 0.1 pmol/mg protein, with an apparent dissociation constant of 6.0 +/- 0.9 x 10(-9) M at 26 degrees C. The binding is reduced or abolished by heat, trypsin, detergents, sulfhydryl reagents and EDTA. Low concentrations of MgCl2 or CaCl2 increase binding but high ionic strength is inhibitory. Low concentrations of dihydroergotamine, phentolamine, clonidine, chlorimipramine and chlorpromazine, but not of serotonin and propranolol, displace the labeled biogenic amine from its binding sites. The stable GTP analogue, guanosine-5'-(beta-gamma-imido)triphosphate (Gpp(NH)p), at the microM range, decreases the maximal number of the high-affinity [3H]octopamine-binding sites. The properties of the [3H]octopamine-binding sites are compared to the properties of octopamine receptors as revealed by stimulation of adenylate cyclase in insects, including Drosophila.