cytochalasin-b and Pituitary-Neoplasms

Using digitonin-permeabilized GH3 cells, we investigated both the release of prolactin (PRL) and changes in the cytoskeleton. We determined that permeabilized GH3 cells released PRL in a dose-dependent manner upon addition of micromolar Ca(2+). Phalloidin, a filamentous actin (F-actin) stabilizing agent, inhibited both Ca(2+)-dependent and -independent PRL release, whereas cytochalasin B, a destabilizing agent, had almost no effect on the release. Observation with a confocal laser scanning microscope revealed that F-actin existed mainly in the cortical region in the quiescent state. Increased cytosolic Ca(2+) induced a change in F-actin distribution: F-actin in the cortical region decreased, whereas F-actin inside the cells increased. This change in F-actin distribution was not observed when phalloidin was added. Addition of cytochalasin B induced patchy F-actin spots, but the pattern of the changes of F-actin distribution did not change. The time course of change in F-actin distribution showed that the F-actin network in the cortical region was reduced within 1 min, and Ca(2+)-dependent release of PRL continued for up to 20 min. These results suggest that the F-actin network near the membrane acts as a barrier to exocytosis and that Ca(2+) directly controls the cytoskeletal changes.

Topics: Actin Cytoskeleton; Actins; Adenoma; Animals; Calcium; Cytochalasin B; Cytoskeleton; Exocytosis; Microscopy, Confocal; Phalloidine; Pituitary Neoplasms; Prolactin; Rats; Stimulation, Chemical; Tumor Cells, Cultured

The goal of these studies was to define the rate-limiting steps in the inactivation of type 2 iodothyronine deiodinase (D2). We examined the effects of ATP depletion, a lysosomal protease inhibitor, and an inhibitor of actin polymerization on D2 activity in the presence or absence of cycloheximide or 3,3', 5'-triiodothyronine (reverse T3, rT3) in rat pituitary tumor cells (GH4C1). We also analyzed the effects of the proteasomal proteolysis inhibitor carbobenzoxy- L-leucyl-L-leucyl-L-leucinal (MG132). The half-life of D2 activity in hypothyroid cells was 47 min after cycloheximide and 60 min with rT3 (3 nM). rT3 and cycloheximide were additive, reducing D2 half-life to 20 min. D2 degradation was partially inhibited by ATP depletion, but not by cytochalasin B or chloroquine. Incubation with MG132 alone increased D2 activity by 30-40% for several hours, and completely blocked the cycloheximide- or rT3-induced decrease in D2 activity. These results suggest that D2 is inactivated by proteasomal uptake and that substrate reduces D2 activity by accelerating degradation through this pathway. This is the first demonstration of a critical role for proteasomes in the post-translational regulation of D2 activity.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chloroquine; Cycloheximide; Cysteine Endopeptidases; Cytochalasin B; Iodide Peroxidase; Iodothyronine Deiodinase Type II; Isoenzymes; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Pituitary Neoplasms; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Rats; Thyroxine; Triiodothyronine, Reverse; Tumor Cells, Cultured; Ubiquitins

Cultured cells in vitro from estrogen-induced rat prolactin-secreting adenomas (prolactinomas) were examined by indirect immunofluorescence microscopy for the distribution of cytoskeletal proteins and alterations of cytoskeleton after treatment with bromocriptine, colchicine and cytochalasin B (CB). After 8 days in culture, prolactinoma cells were well expanded and developed cytoplasmic processes were seen. The cytoplasmic microtubules were observed as fine reticular networks radiating from perinuclear portions toward the cell periphery when decorated with an antibody against tubulin. On the other hand, the actin filaments showed diffuse and spotty distribution when detected with an anti-actin antibody. Contaminated fibroblasts showed a reticular distribution of microtubules and a parallel array of actin cables which corresponds to "stress fibers" throughout the cytoplasm. After treatment with bromocriptine, the reticular distribution of microtubules in prolactinoma cells changed into a coarse and sparse pattern, which was identical with the changes in the distribution of tubulin after treatment with colchicine. On the other hand, distribution of actin was not affected by bromocriptine. Bromocriptine treatment did not alter the distribution of microtubules and actin filaments in fibroblasts, whereas colchicine changed the distribution of microtubules in both prolactinoma cells and fibroblasts. CB treatment changed the localization of actin filaments in both kinds of cells. These in vitro studies indicated bromocriptine would selectively affect the cytoplasmic microtubular system of prolactinoma cells.

Topics: Actins; Adenoma; Animals; Bromocriptine; Cells, Cultured; Colchicine; Cytochalasin B; Estradiol; Female; Fluorescent Antibody Technique; Microtubule Proteins; Neoplasm Proteins; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred Strains; Tubulin

When added to intact GH4Cl or GH3 cells, cytochalasin b, dansylcadaverine, and chloroquine inhibited the binding of L-125I-triiodothyronine (T3) to nuclear receptors within 30 min. These drugs also reduced the amount of 125I-T3 in the post-nuclear supernatant fraction of cells lysed with Triton X-100. If 125I-T3 was added before the drugs, 125I-T3 that was already bound did not dissociate, but further binding was blocked. Inhibition due to 10 microM cytochalasin b or 150 microM dansylcadaverine was reversible within 2 h of drug removal, and inhibition due to 150 microM chloroquine was partially reversible. Drug inhibition was overcome as the T3 concentration was raised. These drugs did not significantly inhibit 125I-T3 binding to isolated nuclei, and nuclei isolated from drug-treated cultures bound as much 125I-T3 as control nuclei. The results suggest that thyroid hormones reach their nuclear binding sites by an active transport process which is inhibited by the drugs studied.

Topics: Animals; Antineoplastic Agents; Biological Transport, Active; Cadaverine; Cell Line; Cell Nucleus; Chloroquine; Cytochalasin B; Kinetics; Neoplasms, Experimental; Pituitary Neoplasms; Rats; Receptors, Cell Surface; Receptors, Thyroid Hormone; Triiodothyronine

Topics: Animals; Colchicine; Cytochalasin B; Depression, Chemical; Female; Growth Hormone; In Vitro Techniques; Male; Microtubules; Neoplasm Transplantation; Neoplasms, Experimental; Perphenazine; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Tritium; Vinblastine; Vinca Alkaloids; Vincristine