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Biomedical subjects

C Hertel

Publications and source records attributed to C Hertel.

At least 55 records · Page 3Linked to original sources
PubMed1986

The involvement of cellular ATP in receptor-mediated internalization of epidermal growth factor and hormone-induced internalization of beta-adrenergic receptors.

Beta-Adrenergic receptors and epidermal growth factor receptors are both expressed on the cell surface of human astrocytoma cells. Incubation with a catecholamine or epidermal growth factor results in rapid internalization of the respective receptor. The internalized receptors co-migrate in light fractions on sucrose gradients. Astrocytoma cells maintain a constant ATP concentration by either glycolytic or mitochondrial ATP production. When cells are incubated in a medium depleted of substrates for glycolysis and gluconeogenesis, addition of inhibitors of mitochondrial ATP synthesis causes a rapid reduction in cellular ATP content. An immediate return to control ATP levels occurs upon addition of an appropriate nutrient, such as glucose. Decreasing the cellular ATP content to less than 10% of control markedly inhibits internalization of beta-adrenergic receptors and epidermal growth factor. The inhibition of endocytosis is reversed as soon as the intracellular ATP content is restored. Previous work by others (Clarke, B.L., and Weigel, P.H. (1985) J. Biol. Chem. 260, 128-133) suggested that ATP is not required for internalization (per se) of asialoglycoprotein in hepatocytes but was required for recycling of the asialoglycoprotein receptor. In contrast, our results indicate that in astrocytoma cells the process of internalization of epidermal growth factor and beta-adrenergic receptors, per se, is highly ATP dependent.

Adenosine Triphosphate↗
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

A comparison of catecholamine-induced internalization of beta-adrenergic receptors and receptor-mediated endocytosis of epidermal growth factor in human astrocytoma cells. Inhibition by phenylarsine oxide.

The ligand-induced internalization of beta-adrenergic receptors and the receptor-mediated internalization of epidermal growth factor were blocked, under similar conditions, by phenylarsine oxide (PAO) in human astrocytoma cells (1321N1). The inhibition was not prevented or reversed by monofunctional sulfhydryl agents such as 2-mercaptoethanol or glutathione; however, the inhibitory action of PAO was blocked and reversed by bifunctional thiols such as 2,3-dimercaptoethanol or dithiothreitol. The results are consistent with the interaction of PAO with vicinal sulfhydryl groups to form a stabile ring structure. PAO did not prevent isoproterenol-induced uncoupling (desensitization) of beta-adrenergic receptors even though receptor internalization was completely blocked. The effects of PAO on receptor internalization could not be explained by any action of the trivalent arsenical to lower ATP levels. Ligand binding to both receptors was not detectably altered by PAO under conditions selective for inhibition for endocytosis. The results suggest a common mechanism for internalization of beta-adrenergic receptors and epidermal growth factor by a process that involves vicinal sulfhydryl groups.

Adenosine Triphosphate↗
PubMed1985

Cellular redistribution of beta-adrenergic receptors in a human astrocytoma cell line: a comparison with the epidermal growth factor receptor in murine fibroblasts.

The redistribution of beta-adrenergic receptors (beta-AR) during agonist-induced desensitization has been compared to the process of receptor-mediated endocytosis of epidermal growth factor (EGF) in human astrocytoma cells (1321N1). [125I]EGF exhibited saturable binding to high affinity (KD = 1-2 nM) receptor sites on intact 1321N1 cells. [125I]EGF was found to internalize rapidly using an acid wash technique to remove surface bound hormone. Sucrose density gradient fractionation following exposure to EGF revealed a redistribution of EGF binding sites from high density (heavy peak) to low density (light peak) regions of the gradient. The light peak binding probably represents EGF in internalized vesicles formed during endocytosis. Low temperature (4 degrees C) or the presence of the lectin concanavalin A (con A) inhibited this ligand-induced movement of EGF receptors. When cells were incubated simultaneously with EGF and the beta-AR agonist isoproterenol, both receptors were found to co-migrate in the low density regions of sucrose gradients. No evidence of heterologous ligand-induced receptor endocytosis was found. These results suggest that the EGF receptors and beta-AR are processed in parallel by 1321N1 cells.

Animals↗
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↗
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

Receptor-specific mechanisms of desensitization of beta-adrenergic receptor function.

beta-Adrenergic receptor (beta AR)-specific, agonist-induced desensitization of adenylate cyclase can be shown in most mammalian cells examined to involve at least three reactions. An initial 'uncoupling' reaction leads to a 40-60% loss of catecholamine-stimulated adenylate cyclase activity at a time when no detectable loss of beta AR has occurred. This process precedes by 45-90 sec the appearance of beta AR in cytoplasmic vesicles. Such beta AR exhibit ligand binding properties consistent with their existence on the inside of membrane vesicles; thus, they appear to be formed by a process of agonist-induced beta AR internalization (endocytosis). A third process results in the loss of beta AR, at least in some cases due to receptor degradation. In general, agonist-induced desensitization or down-regulation reactions do not require protein synthesis. Recovery from the desensitized states does not require protein synthesis, whereas recovery from beta AR down-regulation (degraded receptors) requires new receptor synthesis. Agonist-induced beta AR desensitization and down-regulation reactions appear to have much in common with the process of polypeptide hormone-induced receptor down-regulation. The availability of a large number of ligands (agonists, partial agonists and antagonists) for the beta AR should allow the use of this receptor system to gain unique insights into the general processes of ligand-induced, cell surface receptor endocytosis.

Adenosine Triphosphate↗
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

Effects of amiprophosmethyl on massive and limited calcium loading of maize mitochondria.

In maize mitochondria the effect of the herbicide amiprophosmethyl was studied on massive and limited Ca2+-loading and on mitochondrial energy transduction. Under massive Ca2+-loading conditions amiprophosmethyl inhibited the Ca2+ transport system directly without a significant effect on the respiratory chain, the membrane potential or Ca2+ efflux. Under limited Ca2+-loading conditions an increased Ca2+ efflux was noted, which could be partially responsible for the inhibition of the net Ca2+ accumulation.

Calcium↗
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↗