h-89 and Pituitary-Neoplasms

The L-type Ca(2+) channel is the primary voltage-dependent Ca(2+)-influx pathway in many excitable and secretory cells, and direct phosphorylation by different kinases is one of the mechanisms involved in the regulation of its activity. The aim of this study was to evaluate the participation of Ser/Thr kinases and tyrosine kinases (TKs) in depolarization-induced Ca(2+) influx in the endocrine somatomammotrope cell line GH3. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using a spectrofluorometric method with fura 2-AM, and 12.5 mM KCl (K(+)) was used as a depolarization stimulus. K(+) induced an abrupt spike (peak) in [Ca(2+)](i) that was abolished in the presence of nifedipine, showing that K(+) enhances [Ca(2+)](i), preferably activating L-type Ca(2+) channels. H89, a selective PKA inhibitor, significantly reduced depolarization-induced Ca(2+) mobilization in a concentration-related manner when it was applied before or after K(+), and okadaic acid, an inhibitor of Ser/Thr phosphatases, which has been shown to regulate PKA-stimulated L-type Ca(2+) channels, increased K(+)-induced Ca(2+) entry. When PKC was activated by PMA, the K(+)-evoked peak in [Ca(2+)](i), as well as the plateau phase, was significantly reduced, and chelerythrine (a PKC inhibitor) potentiated the K(+)-induced increase in [Ca(2+)](i), indicating an inhibitory role of PKC in voltage-dependent Ca(2+) channel (VDCC) activity. Genistein, a TK inhibitor, reduced the K(+)-evoked increase in [Ca(2+)](i), but, unexpectedly, the tyrosine phosphatase inhibitor orthovanadate reduced not only basal Ca(2+) levels but, also, Ca(2+) influx during the plateau phase. Both results suggest that different TKs may act differentially on VDCC activation. Activation of receptor TKs with epidermal growth factor (EGF) or vascular endothelial growth factor potentiated K(+)-induced Ca(2+) influx, and AG-1478 (an EGF receptor inhibitor) decreased it. However, inhibition of the non-receptor TK pp60 c-Src enhanced K(+)-induced Ca(2+) influx. The present study strongly demonstrates that a complex equilibrium among different kinases and phosphatases regulates VDCC activity in the pituitary cell line GH3: PKA and receptor TKs, such as vascular endothelial growth factor receptor and EGF receptor, enhance depolarization-induced Ca(2+) influx, whereas PKC and c-Src have an inhibitory effect. These kinases modulate membrane depolarization and may therefore participate in the regulation of a plethora of int

Topics: Animals; Calcium; Calcium Channels, L-Type; Carcinogens; Cell Line; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; ErbB Receptors; Female; Fluorescent Dyes; Fura-2; Isoquinolines; Membrane Potentials; Pituitary Gland; Pituitary Neoplasms; Potassium Chloride; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins pp60(c-src); Rats; Rats, Inbred WF; Sulfonamides; Tetradecanoylphorbol Acetate

The mechanisms mediating cAMP effects to stimulate transcription of the PRL gene have been examined. Treatments that elevate intracellular cAMP concentrations were found to stimulate the mitogen-activated protein kinase (MAPK) in GH(3) cells. Elevated cAMP was also found to stimulate activation of the GTP-binding protein, Rap1. Rap1GAP1 reduced cAMP-induced phosphorylation of MAPK, offering evidence that Rap1 may play a role in mediating activation of MAPK. Treatment of GH(3) cells with PD98059, an inhibitor of the MAPK pathway, reduced the ability of forskolin to activate a PRL reporter gene, providing evidence that MAPK contributes to cAMP-mediated effects on the PRL promoter. As previous studies have implicated Ets factor binding sites within the PRL promoter in mediating responses to MAPK, we expected that the Ets sites would also play a role in cAMP responsiveness. Surprisingly, mutation of all of the consensus Ets factor binding sites in the proximal PRL promoter greatly reduced responsiveness to epidermal growth factor (EGF) and TRH but did not reduce cAMP responsiveness. Experiments using an expression vector for adenovirus 12S E1a provided evidence that the coactivators, CREB binding protein and/or p300, probably play a role in cAMP responsiveness of the PRL promoter. Interestingly, the ability of a GAL4-p300 fusion protein to enhance reporter gene activity was stimulated by cAMP in a MAPK-dependent manner. These findings provide evidence for a model for cAMP-induced PRL transcription involving Rap1-induced MAPK activity leading to stimulation of the transcriptional coactivators, CBP and p300.

Topics: Animals; Binding Sites; Colforsin; CREB-Binding Protein; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; E1A-Associated p300 Protein; Enzyme Inhibitors; Epidermal Growth Factor; ets-Domain Protein Elk-1; Flavonoids; Isoquinolines; Mitogen-Activated Protein Kinases; Nuclear Proteins; Pituitary Neoplasms; Potassium Channels; Prolactin; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; rap1 GTP-Binding Proteins; Rats; Sulfonamides; Thyrotropin-Releasing Hormone; Trans-Activators; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Rat pituitary malignant tumor cells; mGH3, show hypervascularization in in vivo xenografts and overexpress parathyroid hormone-related peptide (PTHrP) compared to original GH3 cells. To elucidate whether PTHrP is involved in tumor-derived angiogenesis, we examined the effect of PTHrP on vascular endothelial cells both in vitro and in vivo. Results of in vivo diffusion chamber assay showed a clear hypervascularization on the outer surface of diffusion chambers containing mGH3 tumor cell implants but not in those containing GH3 cells. Co-incubation with antisense PTHrP oligonucleotide (10 microM), but not sense or mismatched PTHrP oligonucleotide, suppressed hypervascularization in diffusion chambers. To further examine the role of PTHrP on endothelial cell function, PTHrP(1-34) was added at various concentrations to cultured bovine endothelial cells (BAECs) harvested from the aorta. PTHrP(1-34) did not alter the proliferation or migration of endothelial cells, but rather dose-dependently increased capillary formation by endothelial cells on the collagen gel matrix. Furthermore, 0.1 mM of 8-bromo-cAMP caused a similar increase in tube formation, which was dose-dependently inhibited by H89, a protein kinase A inhibitor. Our results indicate for the first time that PTHrP is a potential paracrine factor acting via the PKA pathway to enhance angiogenesis through capillary tube formation by endothelial cells in malignant pituitary tumors.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cell Line; Cyclic AMP-Dependent Protein Kinases; Endothelium, Vascular; Enzyme Inhibitors; Growth Hormone; Isoquinolines; Neovascularization, Pathologic; Oligonucleotides, Antisense; Parathyroid Hormone-Related Protein; Pituitary Gland; Pituitary Neoplasms; Proteins; Rats; Sulfonamides; Tumor Cells, Cultured

Corticotropin-releasing hormone (CRH) plays an important role in regulating the development and function of hypothalamic-pituitary-adrenal axis. The mechanisms by which CRH regulates tissue-specific growth, differentiation and gene expression remain to be established. In the present study, we show that CRH differentially regulates MAP kinase activity in normal ovine anterior pituitary cells and mouse corticotrope AtT20 cells. Incubation of ovine normal anterior pituitary cells with CRH increased MAP kinase activity, an effect mimicked by cAMP and inhibited by the protein kinase A inhibitor H89. In contrast, incubation of mouse pituitary tumor AtT20 cells with CRH inhibited MAP kinase activity, an effect also mimicked by forskolin and inhibited by H89. This decrease in MAP kinase activity occurred with a time course similar to the increase seen in normal anterior pituitary cells. Furthermore, both effects of CRH on MAP kinase activity were inhibited by atrial natriuretic peptide (ANP). ANP also reversed the inhibition of DNA synthesis induced by CRH in AtT20 cells. Thus, CRH may differentially regulate cell growth in sheep normal anterior pituitary and mouse tumor corticotropes by modulating MAP kinase activity through a mechanism dependent on cAMP production and subject to regulation by ANP.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Colforsin; Corticotropin-Releasing Hormone; Cyclic AMP; DNA Replication; Enzyme Inhibitors; Flavonoids; Isoquinolines; Mice; Pituitary Gland, Anterior; Pituitary Neoplasms; Sheep; Sulfonamides; Thymidine

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), members of the glucagon-secretin family, have recently been suggested to be involved in the regulation of corticotropin (ACTH) secretion. In this study, we examined the effects of both peptides on POMC gene expression. Using AtT20PL, a clone of the AtT20 mouse corticotroph tumor cells stably transfected with 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene, the effects of both peptides on the POMC promoter activity were estimated by a luciferase assay. PACAP stimulated POMC 5' promoter activity as well as cAMP generation and ACTH secretion in a dose- and time-dependent manner, with the maximal effect being observed 3 h after the start of incubation. A similar effect was observed with VIP. Although the combined effects of PACAP/CRH or VIP/CRH were greater than that of either hormone alone, no such effect was observed between PACAP and VIP. Furthermore, RT-PCR analysis showed the presence of only the PVR3 receptor subtype in this cell line, which is known to have a similar affinity to PACAP and VIP, indicating that both peptides exert their effects through the same receptor. In contrast to the effect of CRH, which was completely abolished by a protein kinase A inhibitor H89, the effects of PACAP/VIP on POMC expression persisted during H89 treatment, suggesting the involvement of alternative intracellular signaling pathway(s) distinct from the protein kinase A system. Our results suggest that PACAP and VIP have positive effects on POMC gene expression and that multiple signaling pathways are involved in the transcriptional event.

Topics: Adenylyl Cyclases; Adrenocorticotropic Hormone; Animals; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Isoquinolines; Mice; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Pituitary Neoplasms; Polymerase Chain Reaction; Pro-Opiomelanocortin; Rats; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Sulfonamides; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The effects of human growth hormone-releasing hormone (hGHRH) on Ca2+ channels were examined in human growth hormone-producing adenoma cells using the perforated whole cell clamp technique. These cells exhibited T- and L-type Ca2+ channel currents, and application of 10(-8) M hGHRH increased the amplitude of both currents. Application of 10(-5) M 8-bromoadenosine 3',5'-cyclic monophosphate also increased T- and L-type currents. Additional application of 10(-8) M hGHRH did not further increase the current amplitudes. Treatment with the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (10(-5) M) or H-89 (10(-5) M) inhibited the enhancement of Ca2+ channel currents by hGHRH, as did intracellular injection of protein kinase A (PKA) inhibitor peptide [PKI-(5-24)], indicating that hGHRH increased the amplitude of Ca2+ channel currents through the activation of the adenosine 3',5'-cyclic monophosphate (cAMP)-PKA system. When intracellular Ca2+ concentration ([Ca2+]i) was chelated to < 30 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTAAM), hGHRH failed to increase the Ca2+ channel currents. In this condition, hGHRH activated nonselective cation channels, which revealed that the cAMP-PKA system operated after treatment with BAPTA-AM and that the site of low [Ca2+]i-induced inhibition of hGHRH effects on Ca2+ channels was at a step after PKA activation.

Topics: Adenoma; Barium; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Enzyme Inhibitors; Growth Hormone-Releasing Hormone; Human Growth Hormone; Humans; Isoquinolines; Membrane Potentials; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptide Fragments; Peptides; Pituitary Neoplasms; Stereoisomerism; Sulfonamides; Thionucleotides; Tumor Cells, Cultured