herbimycin and Pituitary-Neoplasms

The high basal level of prolactin (PRL) gene expression in rat pituitary GH3 cells is maintained through the spontaneous activity of voltage-sensitive calcium channels (VSCCs). This can be observed experimentally by addition of 0.5 mM CaCl2 to GH3 cells cultured in a low calcium, serum-free medium. CaCl2 specifically induces PRL gene expression and this induction is inhibited by VSCC blockers. PRL gene expression is also stimulated by several hormones and growth factors. In the present study, we examined the effects of tyrosine kinase inhibitors on the ability of CaCl2, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and thryrotropin-releasing hormone (TRH) to increase PRL mRNA levels. Of several PTK inhibitors used, one PTK inhibitor, herbimycin A, specifically inhibited the CaCl2-induced increase in cytoplasmic and nuclear prolactin (PRL) mRNA without affecting cell viability, cell-cell and cell-matrix adhesion, or the expression of several other genes. The effects of herbimycin A were reversible. In cells pretreated with herbimycin A, PRL mRNA levels were reduced by 69 +/- 12% (P < 0.001; n = 4). Western blot analysis using anti-phosphotyrosine antibody revealed a decrease of 91 +/- 1% (P < 0.001; n = 4) in the phosphotyrosine content of proteins in the molecular weight range of 130-160 kDa. After changing the medium back to SFM plus 0.5 mM CaCl2, levels of PRL mRNA increased over a period of several hours, and this increase was accompanied by the tyrosine phosphorylation of two or more proteins in the approximate size range of 130-160 kDa. Herbimycin A also inhibited PRL gene expression in the independently-derived 235-1 lactotrope cell line and lowered the tyrosine specific phosphorylation of protein(s) in a similar size range. Herbimycin A inhibited the ability of bFGF, EGF and TRH to stimulate PRL gene expression in GH3 cells. Again, in cells pretreated with herbimycin A, bFGF induced a reappearance of tyrosine-specific phosphorylation, followed by a reappearance of PRL mRNA. These findings provide evidence for a role for at least one PTK which is necessary for basal and stimulated PRL gene expression.

Topics: Animals; Benzoquinones; Calcium Chloride; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression; Lactams, Macrocyclic; Phosphorylation; Pituitary Neoplasms; Prolactin; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; RNA, Messenger; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured

The aim of this study was to evaluate the effect of protein-tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) inhibitors on Ca2+ channels in GH3 cells. The activity of Ca2+ channels was monitored either by single-cell microfluorometry or by the whole-cell configuration of the patch-clamp technique. Genistein (20-200 micron) and herbimycin A (1-15 micron) inhibited [Ca2+]i rise induced either by 55 mM K+ or 10 micron Bay K 8644. In addition, genistein and lavendustin A inhibited whole-cell Ba2+ currents. By contrast, daidzein, a genistein analogue devoid of PTK inhibitory properties, did not modify Ca2+ channel activity. The inhibitory action of genistein on the [Ca2+]i increase was completely counteracted by the PTP inhibitor vanadate (100 micron). Furthermore, vanadate alone potentiated -Ca2+-i response to both 55 mM K+ and 10 micron Bay K 8644. The possibility that genistein could decrease the [Ca2+]i elevation by enhancing Ca2+ removal from the cytosol seems unlikely since genistein also reduced the increase in fura-2 fluorescence ratio induced by Ba2+, a cation that enters into the cells through Ca2+ channels but cannot be pumped out by Ca2+ extrusion mechanisms. Finally, in unstimulated GH3 cells, genistein caused a decline of [Ca2+]i and the disappearance of [Ca2+]i oscillations, whereas vanadate induced an increase of [Ca2+]i and the appearance of [Ca2+]i oscillations in otherwise non-oscillating cells. The present results suggest that in GH3 cells PTK activation causes an increase of L-type Ca2+ channel function, whereas PTPs exert an inhibitory role.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Barium; Benzoquinones; Calcium; Calcium Channels; Calcium Channels, L-Type; Cell Line; Enzyme Inhibitors; Genistein; Isoflavones; Kinetics; Lactams, Macrocyclic; Membrane Potentials; Patch-Clamp Techniques; Phenols; Pituitary Gland; Pituitary Neoplasms; Potassium; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Quinones; Rifabutin; Time Factors