Comparative 99mTc-sestamibi and 3H-daunomycin uptake in human carcinoma cells: relation to the MDR phenotype and effects of reversing agents.
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Because 99mTc-sestamibi (MIBI) appears to be a potent candidate for multidrug resistance (MDR) evaluation in tumors, its cellular uptake should be similar to that of 3H-daunomycin in a variety of conditions of expression and inhibition of MDR activity. METHODS: We used a human rhinopharyngeal carcinoma cell line (KB-3-1) and its MDR variant (KB-A1). Cells were incubated 2 h with 99mTc-MIBI and 3H-daunomycin under control conditions or in the presence of a reversing agent such as verapamil (10 pmol/L), PSC833 (1 micromol/L) or S9788 (5 micromol/L). RESULTS: Relative to the KB-3-1-sensitive cells, accumulations of 99mTc-MIBI and 3H-daunomycin were reduced to 31% +/- 5% and 36% +/- 11% (P < 0.001 for both) in KB-A1-resistant cells. In sensitive cells, accumulation of both agents was increased by verapamil and PSC833 (range 115%-140%; P < 0.05) but not by S9788. In KB-A1 cells, only S9788 significantly increased the cellular uptake of 99mTc-MIBI (138% +/- 25%; P < 0.01), whereas the intracellular uptake of 3H-daunomycin was markedly increased with the three reversing agents (up to 311% +/- 37% with S9788; P < 0.001). With this last treatment, uptake of 3H-daunomycin in KB-A1 cells nearly returned to its basal level in sensitive cells. CONCLUSION: 99mTc-MIBI monitors the MDR phenotype of tumor cells effectively but responds to reversing agents differently than 3H-daunomycin. (+info)
Development and organization of ocular dominance bands in primary visual cortex of the sable ferret.
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Thalamocortical afferents in the visual cortex of the adult sable ferret are segregated into eye-specific ocular dominance bands. The development of ocular dominance bands was studied by transneuronal labeling of the visual cortices of ferret kits between the ages of postnatal day 28 (P28) and P81 after intravitreous injections of either tritiated proline or wheat germ agglutinin-horseradish peroxidase. Laminar specificity was evident in the youngest animals studied and was similar to that in the adult by P50. In P28 and P30 ferret kits, no modulation reminiscent of ocular dominance bands was detectable in the pattern of labeling along layer IV. By P37 a slight fluctuation in the density of labeling in layer IV was evident in serial reconstructions. By P50, the amplitude of modulation had increased considerably but the pattern of ocular dominance bands did not yet appear mature. The pattern and degree of modulation of the ocular dominance bands resembled that in adult animals by P63. Flat mounts of cortex and serial reconstructions of layer IV revealed an unusual arrangement of inputs serving the two eyes in the region rostral to the periodic ocular dominance bands. In this region, inputs serving the contralateral eye were commonly fused along a mediolateral axis, rostral to which were large and sometimes fused patches of ipsilateral input. (+info)
Implication of proteasome in estrogen receptor degradation.
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In MCF-7 breast cancer cells, estradiol (E2) and pure antiestrogen RU 58668 down-regulate the estrogen receptor (ER). Interestingly, the protein synthesis inhibitor cycloheximide (CHX) abrogated solely the effect of E2 suggesting a selective difference in the degradation of the receptor induced by estrogenic and antiestrogenic stimulations. A panel of lysosome inhibitors (i.e. bafilomycin, chloroquine, NH4Cl, and monensin), calpain inhibitors (calpastatin and PD 150606) and proteasome inhibitors (lactacystin and proteasome inhibitor I) were tested to assess this hypothesis. Among all inhibitors tested, lactacystin and proteasome inhibitor I were the sole inhibitors to abrogate the elimination of the receptor induced by both E2 and RU 58668; this selective effect was also recorded in cells prelabeled with [3H]tamoxifen aziridine before exposure to these ligands. Hence, differential sensitivity to CHX seems to be linked to the different mechanisms which target proteins for proteasome-mediated destruction. Moreover, the two tested proteasome inhibitors produced a slight increase of ER concentration in cells not exposed to any ligand, suggesting also the involvement of proteasome in receptor turnover. (+info)
Interleukin-1alpha and tumour necrosis factor-alpha modulate airway smooth muscle DNA synthesis by induction of cyclo-oxygenase-2: inhibition by dexamethasone and fluticasone propionate.
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1. Previous studies have established that glucocorticoids inhibit airway smooth muscle DNA synthesis. The effects of a combination of the pro-inflammatory cytokines, interleukin-1alpha (IL-1alpha) and tumour necrosis factor-alpha (TNF-alpha) on the inhibition of DNA synthesis by glucocorticoids in human cultured airway smooth muscle have now been investigated, since these cytokines are chronically expressed in asthmatic airways. 2. Thrombin (0.3 u ml(-1)) and basic fibroblast growth factor (bFGF, 300 pM) stimulated increases in DNA synthesis which were concentration-dependently inhibited by dexamethasone (1-1000 nM). 3. The cytokine mixture, comprising IL-1alpha (0.01 and 0.1 pM) and TNF-alpha (3 and 30 pM), directly evoked increases in DNA synthesis which were attenuated by dexamethasone. However, the cytokine mixture prevented responses to bFGF or thrombin. 4. Paradoxically, in the presence of the cytokine mixture and bFGF, dexamethasone (1-1000 nM) concentration-dependently increased DNA synthesis. Furthermore, neither dexamethasone (100 nM) nor fluticasone propionate (1 nM) inhibited DNA synthesized in response to bFGF/cytokine mixture combination and dexamethasone was similarly inactive against the thrombin/cytokine mixture. 5. The levels of prostaglandin E2 (PGE2), an established inhibitor of airway smooth muscle DNA synthesis, remained below the limits of assay detection (0.05 nM) under basal conditions or following stimulation with either thrombin or bFGF. In contrast, the cytokine mixture alone, and in the presence of thrombin or bFGF, induced biologically active levels of PGE2. Dexamethasone (100 nM), the non-selective cyclo-oxygenase (COX) inhibitor indomethacin (3 microM) or the selective COX-2 inhibitor L-745,337 (0.3 microM) completely inhibited synthesis of PGE2. 6. Neither indomethacin (3 microM) nor L-745,337 (0.3 microM) influenced thrombin- or bFGF-induced DNA synthesis. However, each COX inhibitor enhanced DNA synthesis in cytokine-treated cells. 7. In unstimulated airway smooth muscle cells, COX-1, but not COX-2 protein was detectable by Western blotting. The induction of COX-2 protein by the cytokine mixture was attenuated by dexamethasone (100 nM), whereas the level of COX-1 protein was unaffected by either the cytokines or by dexamethasone. 8. Cytokine-induced, COX-2-dependent eicosanoid production inhibits DNA synthesis. The paradoxical increase in DNA synthesis observed in glucocorticoid treated airway smooth muscle stimulated by cytokine/bFGF combinations may be explained by the ability of glucocorticoids to repress COX-2 induction and prevent cytokine-induction of the DNA synthesis inhibitor, PGE2. (+info)
A non-pungent triprenyl phenol of fungal origin, scutigeral, stimulates rat dorsal root ganglion neurons via interaction at vanilloid receptors.
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1. A [3H]-resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl-scutigeral, ovinal, neogrifolin, and methyl-neogrifolin), scutigeral (Ki=19 microM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization. 2. Scutigeral induced a dose-dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 microM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 microM). 3. [3H]-RTX binding isotherms were shifted by scutigeral (10-80 microM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 microM. 4. Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements in the rat (up to 100 microM) upon intraocular instillation. 5. In accord with being non-pungent, scutigeral (5 microM) did not elicit a measurable inward current in isolated rat dorsal root ganglion neurons under voltage-clamp conditions. It did, however, reduce the proportion of neurons (from 61 to 15%) that responded to a subsequent capsaicin (1 microM) challenge. In these neurons, scutigeral both delayed (from 27 to 72 s) and diminished (from 5.0 to 1.9 nA) the maximal current evoked by capsaicin. 6. In conclusion, scutigeral and its congeners form a new chemical class of vanilloids, the triprenyl phenols. Scutigeral promises to be a novel chemical lead for the development of orally active, non-pungent vanilloids. (+info)
GABA(B) receptor isoforms GBR1a and GBR1b, appear to be associated with pre- and post-synaptic elements respectively in rat and human cerebellum.
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1. Metabotropic gamma-aminobutyric acid (GABA) receptors, GABA(B), are coupled through G-proteins to K+ and Ca2+ channels in neuronal membranes. Cloning of the GABAB receptor has not uncovered receptor subtypes, but demonstrated two isoforms, designated GBR1a and GBR1b, which differ in their N terminal regions. In the rodent cerebellum GABA(B) receptors are localized to a greater extent in the molecular layer, and are reported to exist on granule cell parallel fibre terminals and Purkinje cell (PC) dendrites, which may represent pre- and post-synaptic receptors. 2. The objective of this study was to localize the mRNA splice variants, GBR1a and GBR1b for GABA(B) receptors in rat cerebellum, for comparison with the localization in human cerebellum using in situ hybridization. 3. Receptor autoradiography was performed utilizing [3H]-CGP62349 to localize GABA(B) receptors in rat and human cerebellum. Radioactively labelled oligonucleotide probes were used to localize GBR1a and GBR1b, and by dipping slides in photographic emulsion, silver grain images were obtained for quantification at the cellular level. 4. Binding of 0.5 nM [3H]-CGP62349 demonstrated significantly higher binding to GABA(B) receptors in the molecular layer than the granule cell (GC) layer of rat cerebellum (molecular layer binding 200+/-11% of GC layer; P<0.0001). GBR1a mRNA expression was found to be predominantly in the GC layer (PC layer grains 6+/-6% of GC layer grains; P<0.05), and GBR1b expression predominantly in PCs (PC layer grains 818+/-14% of GC layer grains; P<0.0001). 5. The differential distribution of GBR1a and GBR1b mRNA splice variants for GABA(B) receptors suggests a possible association of GBR1a and GBR1b with pre- and post-synaptic elements respectively. (+info)
Analysis of the behaviour of selected CCKB/gastrin receptor antagonists in radioligand binding assays performed in mouse and rat cerebral cortex.
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1. The previously described complex behaviour of the CCKB/gastrin receptor antagonist, L-365,260, in radioligand binding assays could be explained by a variable population of two binding sites. We have investigated whether other CCKB/gastrin receptor ligands (PD134,308, PD140,376, YM022 and JB93182) can distinguish between these sites. 2. In the mouse cortex assay, Hill slopes were not different from unity and the ligand pKI values did not differ when either [125I]-BH-CCK-8S or [3H]-PD140,376 was used as label as expected for a single site (G2). 3. In the rat cortex, where previous analysis of replicate (n=48) L-365,260 data indicated the presence of two CCKB/gastrin sites (G1 and G2), the competition data for PD134,308, PD140,376, YM022 and JB93182 could be explained by a homogeneous population of CCKB/gastrin sites because the Hill slope estimates were not significantly different from unity. However, the estimated affinity values for JB93182 and YM022 were significantly higher and that for PD134,308 was significantly lower than those obtained in the mouse cortex when the same radioligand was used. In view of our previous data obtained with L-365,260, the rat cortex data were also interpreted using a two-site model. In this analysis, SR27897 expressed approximately 9 fold, PD134,308 approximately 13 fold and PD140,376 approximately 11 fold selectivity for the G2 site. In contrast, JB93182 expressed approximately 23 fold and YM022 approximately 4 fold selectivity for the G1 site. If the two-site interpretation of the data is valid then, because of its reverse selectivity to L-365,260, JB93182 has been identified as a compound which if radiolabelled could provide a test of this receptor subdivision. (+info)
Characterization of the binding of a novel radioligand to CCKB/gastrin receptors in membranes from rat cerebral cortex.
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1. We have investigated the binding of a novel radiolabelled CCKB/gastrin receptor ligand, [3H]-JB93182 (5[[[(1S)-[[(3,5-dicarboxyphenyl)amino]carbonyl]-2-phenylethyla mino]-carbonyl]-6-[[(1-adamantylmethyl) amino]carbonyl]-indole), to sites in rat cortex membranes. 2. The [3H]-JB93182 was 97% radiochemically pure as assessed by reverse-phase HPLC (RP-HPLC) and was not degraded by incubation (150 min) with rat cortex membranes. 3. Saturation analysis indicated that [3H]-JB93182 labelled a homogeneous population of receptors in rat cortex membranes (pKD=9.48+/-0.08, Bmax=3.61+/-0.65 pmol g(-1) tissue, nH=0.97+/-0.02, n=5). The pKD was not significantly different when estimated by association-dissociation analysis (pKD=9.73+/-0.11; n=10). 4. In competition studies, the low affinity of the CCKA receptor antagonists, L-364,718; SR27897 and 2-NAP, suggest that, under the assay conditions employed, [3H]-JB93182 (0.3 nM) does not label CCKA receptors in the rat cortex. 5. The affinity estimates obtained for reference CCKB/gastrin receptor antagonists were indistinguishable from one of the affinity values obtained when a two site model was used to interpret [125I]-BH-CCK8S competition curves obtained in the same tissue (Harper et al., 1999). 6. This study provides further evidence for the existence of two CCKB/gastrin sites in rat cortex. [3H]-JB93182 appears to label selectively sites previously designated as gastrin-G1 and therefore it may be a useful compound for the further discrimination and characterization of these putative receptor subtypes. (+info)