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Biomedical subjects

M Staehelin

Publications and source records attributed to M Staehelin.

At least 19 recordsLinked to original sources
PubMed1988

Changes in beta-adrenoceptors and leucocyte subpopulations after physical exercise in normal subjects.

Seven healthy volunteers were subjected to standardized bicycle ergometry. Before and at the end of exercise, leucocyte and lymphocyte subset distribution was assessed by immunofluorescence labelling with monoclonal antibodies and the cytofluorograf. Competition binding studies were performed on mononuclear leucocytes with a fixed amount of the radioligand 125I-(-)-cyanopindolol (125I-CYP) and increasing concentrations of the hydrophilic ligand (-)4-(3-tertiary-butylamino-2-hydroxypropoxy)-benzimidazol-2-one hydrochloride (CGP-12177). Total numbers of beta-adrenoceptors per cell and the receptor-ligand affinities were then derived by computer analysis. In separate experiments with blood obtained from resting subjects, beta-adrenoceptor numbers of lymphocyte subsets, which had been sorted by the fluorescence activated cell sorter, were determined by saturation binding of 125I-CYP. During exercise there was a twofold increase in total leucocyte numbers. The ratio between monocytes (4%) and lymphocytes (55%) remained constant, but the composition of lymphocyte subsets had changed. A twofold increase was observed for the lymphocyte population carrying the Leu-7 and the CD8 antigens or only the Leu-7 antigen, whereas the number of B cells and CD4 positive T cells increased only slightly. The lymphocyte phenotype changes appeared after 3 min, reached a maximum at the end of ergometry and had disappeared 30 min after exercise. Exercise led to a doubling of the number of beta-adrenoceptors in unseparated mononuclear leucocytes from 240 +/- 46 to 535 +/- 190 sites per cell (SD, n = 6). beta-Adrenoceptor numbers were higher on sorted Leu-7 positive cells (KD 14 +/- 6 pmol/l, Bmax 1174 +/- 233 sites/cell) and on CD8 positive cells (KD 145 +/- 79 pmol/l, Bmax 1577 +/- 670 sites/cell) than on monocytes (KD 39 +/- 31 pmol/l, 647 +/- 91 sites/cell). Very low specific 125I-CYP binding was found on Leu-7 and CD8 negative cells (less than 0.2 pmol/l). Before exercise the displacement curves with CGP-12177 fitted best a single site model, whereas after exercise 12% of the receptors were in a low affinity state for CGP-12177 indicating internalized receptors. (ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Bromoacetylalprenololmenthane binding to beta-receptors modulates the rate of hormone-induced internalization but not desensitization in S49 cells.

Bromoacetylalprenololmenthane was found to inhibit hormone-induced beta-adrenergic receptor internalization in a dose-dependent fashion in S49 lymphoma cells, besides its known ability to bind to beta-receptors irreversibly. This new found property of BAAM+ was taken advantage of in studying whether receptor internalization is a necessary step in the desensitization of adenylate cyclase. BAAM-treated cells showed functional desensitization even when receptor internalization had been blocked substantially by 50-65%. This finding suggests that receptor internalization is not directly involved in desensitization.

Alprenolol↗
PubMed1987

Similarities in cAMP responses between murine lymphoid cell lines and subsets of mouse lymphocytes.

Intracellular cAMP responses to forskolin (which stimulates independently of hormone receptors) and to several hormones were studied in mouse lymphoid cell lines of B- and T-cell origin and in subpopulations of mouse lymphocytes. In the T-cell lines, isoproterenol caused an increase in cAMP amounting at most to 28% of that caused by 100 microM forskolin. In contrast, in the B-cell lines the increase ranged from 50% to over 100%. In 11 of 13 T-cell lines, forskolin at 1 microM concentration potentiated the isoproterenol response five- to tenfold. Forskolin potentiation was also found in thymocytes and peripheral T-cells but not in B-cell lines. The relative increases in intracellular cAMP evoked by isoproterenol, prostaglandin E2 (PGE2) and histamine varied markedly from cell line to cell line. For instance, three lines carrying the T-helper cell marker L3T4 responded only to PGE2 and not to isoproterenol, whereas two cell lines carrying the Ly2 marker gave a higher response to isoproterenol than to PGE2. The results indicate that subsets of lymphocytes respond differently to various hormones and that these differences are maintained in continuous cell lines.

Animals↗
PubMed1986

Evidence for the appearance of an uncoupled form of the beta-adrenergic receptor distinct from the internalized receptor.

Agonist treatment of C6-glioma cells induces two altered states in beta-adrenergic receptors, a low affinity for the hydrophilic antagonist CGP-12177 and a low affinity for agonists like isoproterenol. We present evidence that, in cells not treated to inhibit receptor internalization, the two properties occur with a different time course, the low affinity for isoproterenol preceding that for CGP-12177. In that the low affinity for CGP-12177 is due to the internalization of the receptor, the results indicate that uncoupling of the receptor, indicated by the low affinity for isoproterenol, occurs while the receptor is still located on the cell surface. Removal of the agonist leads to reappearance of the receptor to the plasma membrane followed by loss of the uncoupled state.

Adrenergic beta-Agonists↗
PubMed1985

(-)-S-[3H]CGP-12177 and its use to determine the rate constants of unlabeled beta-adrenergic antagonists.

The enantiomers of the hydrophilic beta-adrenergic blocker CGP-12177 have been synthesized and the S-enantiomer radiolabeled with tritium. The dissociation constant (Kd) of the S-enantiomer for binding to the beta-adrenergic receptor is one-half of that of the racemic mixture and at least 2 orders of magnitude lower than that of the R-enantiomer. The kinetic parameters of the latter were determined by analyzing its effect on the association kinetics of (-)-S-[3H]CGP-12177. A computer program was developed that allows the association and dissociation rate constants of unlabeled ligands to be calculated. This method was validated using Monte Carlo simulations. In addition, the rate constants of unlabeled S-CGP-12177 and S-alprenolol calculated using this method were in good agreement with those of S-[3H]CGP-12177 and S-[3H]dihydroalprenolol, respectively, determined independently. The method was also used to measure the rate constants of the enantiomers of pindolol. These antagonists as well as S- and R-CGP-12177 form their receptor complexes with similar association rate constants. In contrast, the dissociation of the R-enantiomers from receptor-ligand complexes were found to be at least 100 times faster than those of the corresponding S-enantiomers.

Adrenergic beta-Antagonists↗
PubMed1985

beta-Adrenergic receptors and adenylate cyclase activity in murine lymphoid cell lines.

beta-Adrenergic receptors were determined in sixteen cultured tumor cell lines of T- and B-lymphoid derivation by the binding of the hydrophilic ligand [3H]CGP-12177. The number of receptors per cell varied from less than a hundred to over three thousand. Intracellular cAMP responses to isoproterenol and forskolin were measured for seven cell lines possessing a range of receptor numbers. No direct relationship was found between the isoproterenol-induced elevation of cAMP and the receptor number, and forskolin-induced increases in cAMP varied by more than 100-fold between cell lines. Adenylate cyclase activity was also measured in membrane preparations from the seven cell lines. The T-lymphoma line WEHI-22, the line with the highest receptor number (3700 per cell), showed very little response to any stimulatory agent. With the other four cell lines possessing receptors, the membrane-bound adenylate cyclase activity evoked by isoproterenol relative to that evoked by forskolin was found to be proportional to the number of receptors per cell. The number of receptors thus seems to determine the proportion of the inherent adenylate cyclase that can be activated by a beta-adrenergic agent. The levels of adenylate cyclase induced in membranes by GppNHp and by fluoride, relative to those induced by forskolin, were substantially higher with some cell lines than with others. These data and the results from WEHI-22 cells indicate that lymphoid cell lines differ in their content of functional C and N proteins.

Adenylyl Cyclase Inhibitors↗
PubMed1984

Desensitized beta-adrenoceptors of C6-glioma cells have distinct binding properties.

When C6-rat glioma cells were incubated for 20 min with beta-adrenoceptor agonists, a part of the beta-adrenoceptors was localized in light density vesicles. These receptors have the same affinity for dihydroalprenolol as plasma membrane receptors but have a lowered affinity for agonists as well as for the hydrophilic beta-adrenoceptor antagonist CGP-12177. The affinity of these vesicular receptors for a variety of hydrophobic beta-blockers as well as for the hydrophilic beta-blocker timolol is compared with that of plasma membrane receptors. None of the ligands investigated showed any difference in affinity but CGP-12177. Both, introducing a lipophilic side chain into CGP-12177 or altering the benzimidazol-2-one ring system of CGP-12177 led to an increase in the affinity of the vesicular receptors. Since in the presence of the pore-forming agent alamethicin the same affinity was determined for vesicular receptors as for those located on the plasma membrane, it is concluded that the apparent low affinity of the vesicular receptors in the absence of alamethicin is caused by a membrane barrier. The lower affinity of the vesicular receptors for agonists was only slightly increased by alamethicin.

Animals↗
PubMed1984

Some unique properties of CGP-12177.

CGP-12177, like isoproterenol, has a lower affinity for desensitized receptors. Experiments were performed to study whether this property of CGP-12177 is due to its partial agonist activity or its impermeability for membranes. Reduced binding of [3H]DHA to desensitized receptors at 0 degrees C as well as a reversal of the reduced binding in the presence of digitonin indicate a permeability barrier. A partial agonist effect of CGP-12177 was only found in intact C6 cells and neither in C6 membranes nor in S49 cells. This peculiar effect in intact C6 cells is stereospecific and not due to an inhibition of phosphodiesterases.

Adenylyl Cyclases↗
PubMed1983

Determination of the desensitization of beta-adrenergic receptors by [3H]CGP-12177.

Isoprenaline treatment of C6-glioma cells induced a fast decrease in the number of beta-adrenergic receptors as determined by binding of [3H]CGP-12177, which paralleled the decrease in the hormonally stimulated adenylate cyclase activity. The total number of receptors, as determined by binding of (-)-[3H]dihydroalprenolol, did not decrease. Separation of the beta-adrenergic receptors on a sucrose density gradient showed that the decrease in the number of receptors detectable with CGP-12177 was due to a movement of the receptors from the plasma membrane to a vesicular cell compartment. By using both (-)-[3H]dihydroalprenolol and [3H]CGP-12177 it is thus possible to differentiate between the total number of receptors and those present at the plasma membrane in an unfractionated cell lysate.

Adenylyl Cyclases↗
PubMed1983

CGP-12177. A hydrophilic beta-adrenergic receptor radioligand reveals high affinity binding of agonists to intact cells.

A new, hydrophilic beta-adrenergic receptor radioligand, (+/-)-[3H]CGP-12177 (4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride), was synthesized and radiolabeled to 40 Ci/mmol. The nonspecific binding of this compound to turkey erythrocyte ghosts and C6 glioma cell membranes was less than 5% of the total binding at five times the appropriate KD. Binding assays of intact C6 glioma cells also showed low nonspecific binding, less than 20% of the total binding at five times the appropriate KD. The affinities of the antagonists (-)- and (+)-propranolol as well as of the agonist (-)-isoproterenol were examined by their potency to displace various radioligands from intact C6 glioma cell and membrane preparations. With membrane preparations, both [3H] CGP-12177 and [3H]dihydroalprenolol (DHA) were displaced stereospecifically by the antagonists (-)- and (+)-propranolol and the agonist (-)-isoproterenol. With whole cells, [3H]CGP-12177 and [3H]DHA behaved differently. [3H]DHA and [3H]carazolol could be stereospecifically displaced by antagonists but only partially displaced by agonists, while [3H]CGP-12177 could be completely displaced by both antagonists and agonists as in membranes. In contrast to [3H]CGP-12177, the lipophilic ligand [3H]DHA is actually taken up by cells. The inability of agonists to displace lipophilic radioligands from receptors on intact cells may not be due to a low affinity of agonists for receptors on cells, but to an agonist-induced change in the receptors which renders them inaccessible to hydrophilic agonists and antagonists. This change is likely to be their internalization into the cell.

Adrenergic beta-Antagonists↗
PubMed1983

Reappearance of beta-adrenergic receptors after isoproterenol treatment in intact C6-cells.

The reappearance of beta-adrenergic receptors in C6-glioma cells after desensitization with isoproterenol was studied using the antagonist [3H]CGP-12177. Reappearance had the following properties: (a) it occurred in intact cells only, (b) it was temperature dependent, (c) it required an Na+/H+ gradient, low intracellular Ca2+ activity, and (d) it required ATP, and (e) intact lysosomes. The results suggest endocytosis and recycling of the beta-adrenergic receptor after agonist treatment.

Adrenergic beta-Antagonists↗
PubMed1983

[3H]CGP-12177, a beta-adrenergic ligand suitable for measuring cell surface receptors.

[3H]CGP-12177, in contrast to lipophilic ligands like [3H]Dihydroalprenolol, shows very little non-specific binding to intact cells. It also differs from lipophilic ligands in that it can be completely displaced from the receptors on intact cells by isoproterenol. Furthermore, binding sites for this ligand disappear rapidly upon exposure of cells to agonists. These binding sites reappear rapidly upon removal of the agonist, if any possible interaction with residual traces of the agonist are prevented. These results indicate that [3H]CGP-12177 binds only to cell surface receptors and that agonist causes a rapid and reversible shift of receptors from the cell surface to the interior of the cell.

Adrenergic beta-Agonists↗
PubMed1983

Evidence for intravesicular beta-adrenergic receptors in membrane fractions from desensitized cells: binding of the hydrophilic ligand CGP-12177 only in the presence of alamethicin.

Two fractions of beta-adrenergic receptors have been separated from desensitized C62B cells by centrifugation on a sucrose gradient. Using two beta-adrenergic receptor (beta AR) antagonists, 125I-iodopindolol and (3H)CGP-12177, the different binding characteristics of these fractions have been investigated. While iodopindolol binds to beta AR both in a light (30% sucrose) and a heavy (45-50% sucrose) peak, CGP-12177 binds only to those in the heavy peak. Incubation of these two gradient fractions with the antibiotic alamethicin selectively increases the binding of CGP-12177 only in the light peak. This result suggests that the desensitization-induced light peak beta AR exist on the inside of vesicles. The hydrophilic ligand CGP-12177 is capable of reaching these receptors only through alamethicin-formed pores.

Alamethicin↗
PubMed1982

Rapid and reversible disappearance of beta-adrenergic cell surface receptors.

The water soluble beta-adrenergic ligand [3H]CGP-12177 was used to measure the cell surface receptors in intact cells. In two cell lines, C6 glioma and WEHI 7 lymphoma cells, -50% of the cell surface receptors disappear within minutes of incubation of the cells with isoproterenol. The receptors can still be detected in homogenates and reappear on the cell surface when cells are washed and reincubated at 37 degrees C. The data agree with a disappearance of the receptors from the cell surface by an agonist-mediated endocytosis.

Animals↗
PubMed1977

Chemical basis for brain-specific serine transfer RNAs.

Serine tRNA from rat brain can be resolved into six isoaccepting species. Three of these species show the same chromatographic behavior as the seryl tRNAs from other rat organs, whereas the remaining species appear to be specific for brain. The isoacceptor tRNAs were purified to homogeneity by chromatography on benzoylated DEAE-cellulose followed by reversed-phase chromatography. We found that the additional species of serine tRNA in brain differ from their counterparts derived from other rat organs by a lack of a specific guanosine ribose-methylation in the dihydrouridine loop. In addition, when total liver tRNA was compared with total brain tRNA, the same degree of undermethylation with respect to 2'-O-methylguanosine was found as a general phenomenon.

Animals↗