Nocodazole and taxol affect subcellular compartments but not secretory activity of GH3B6 prolactin cells.

The effects of two drugs known to affect microtubules (nocodazole, a depolymerizing agent, and taxol, a polymerizing and stabilizing agent) have been tested in GH3B6 prolactin (PRL) cells, a rat pituitary cell line. Under basal condition, GH3B6 cells displayed a dense and entangled microtubule (MT) network, and a tight perinuclear cage of cytokeratin fibers with branching bundles in the cytoplasm. Nocodazole induced a disappearance of MT in the cytoplasm accompanied by the formation of tubulin blebs at the cell periphery, and a slackening of the perinuclear cage of cytokeratin. Taxol induced the formation of straight MT bundles in the cytoplasm, and a tightening of the cytokeratin cage. In parallel, nocodazole induced a fragmentation of the Golgi apparatus which appeared, after staining with antibodies against PRL or against mannosidase II, a Golgi membrane antigen, as small subunits dispersed in the cytoplasm. Taxol induced a perturbation of the Golgi apparatus which, however, remained located near the nucleus. Surprisingly, despite their obvious effects on the subcellular organization, the two MT drugs did not perturb the basal and thyroliberin (TRH)-stimulated PRL release. Moreover, they do not seem to affect the intracellular transport and release of neosynthesized PRL as appreciated by "pulse-chase" experiments. These observations demonstrate that, although MT assume an important role in the spatial compartmentalization of GH3B6 cells, they are not directly involved in the different steps of the intracellular PRL transport from its synthesis site to its release site, as well as in the associated membrane traffic.

Rapid and transient reorganization of the cytoskeleton in GH3B6 cells during short-term exposure to thyroliberin.

The cytoskeletal organization of the rat pituitary tumor cell line GH3B6 was analysed using immunofluorescence, in basal conditions and after stimulation by thyroliberin (TRH). Under basal conditions, a dense and entangled cytoplasmic microtubule network, a perinuclear cage of cytokeratin fibers, and a diffuse distribution of F-actin were revealed. Short-term stimulation of these cells by TRH induces a first early phase of PRL release (0-2 min), concomitant with a rarefaction of cytoplasmic PRL-containing granules, followed by a second plateau phase (5-30 min), concomitant with modifications of the Golgi zone. We show that TRH induced early and transient modifications in the cytoskeletal distribution during these short periods of stimulation. First, after 2 min of stimulation, small fluorescent tubulin blebs appeared under the plasma membrane. Then, after 5 min they disappeared, and a thin actin network, accentuated by thicker fibers, organized transiently in the cytoplasm. After 30 min, the microtubules and cytokeratin networks had extended throughout the cytoplasm and the actin distribution was diffuse again. So, in this study, we have shown the existence of a parallelism between the redistribution of intracellular PRL compartments and the reorganization of cytoskeletal elements, during exposure to TRH. We could not clearly correlate these modifications with transduction mechanisms involved in TRH action.

Laminin induces formation of neurite-like processes and potentiates prolactin secretion by GH3 rat pituitary cells.

Tumor-derived GH3 rat pituitary cell lines are widely utilized to study mechanisms of prolactin secretion and responsiveness to secretagogues. These cells served here as a model with which to study relationships between shape and function. When GH3 cells were routinely grown in serum-supplemented medium, they exhibited the polygonal phenotype of epithelial cells, with scarce secretory granules. In contrast, when seeded in a serum-free medium, they attached loosely and contained more secretory granules. In both cases, they released prolactin in a nonpolarized manner. We show in the present work that laminin extracted from Englebreth-Holm-Swarm (EHS) tumors was a potent attachment and spreading factor for GH3/B6 cells seeded in serum-free medium. Moreover, it induced the formation of neurite-like processes, which were increased in number and length by chronic treatment with a specific secretagogue, thyroliberin (TRH). These changes in cell shape were correlated with a potentiation of prolactin secretion, both basal and TRH-stimulated. Furthermore, using immunocytochemistry and electron microscopy, we revealed--at the dilated tip of processes--an accumulation not only of prolactin, but also of synaptophysin, a vesicle membrane marker, and of several organelles, such as secretory granules, smooth vesicles, dense bodies and mitochondria. The cytoplasmic processes contained long parallel bundles of microtubules and showed a strong immunoreactivity for beta 2-tubulin. In addition, we found immunocyto-chemical evidence for the presence of 200-k Da neurofilament protein in GH3/B6 cell processes as well as in neurites of cultured hypothalamic neurons. We conclude that, in GH3/B6 cells, laminin induced the differentiation of neurite-like processes, which were the site of polarized organelle transport and exhibited some neuronal markers.