cyclic-gmp and Pituitary-Neoplasms

To investigate the effects of IL-2 on the proliferation, cell cycle, protein kinase C(PKC) and cAMP/cGMP of cultured human pituitary adenoma cells.. MTT colorimetry, (3)H-TdR and flow cytometry were used to detect the proliferation and radioimmunoassay was used to observe the effect of IL-2 on PKC activity and cAMP/cGMP levels of cultured human pituitary adenoma cells.. (1) IL-2 (1 x 10(4), 1 x 10(5) and 5 x 10(5) U/L) stimulated the proliferation and DNA synthesis of cultured human pituitary adenoma cells in a dose-dependent manner. (2) IL-2 (1 x 10(4), 1 x 10(5) and 5 x 10(5) U/L) decreased the ratio of pituitary adenoma cells in G(1) phase and increased the ratio in S and G(2) phase markedly. (3) Compared with control, PMA, a PKC activator, increased the activity of membrane and total PKC in human pituitary adenoma cells. However, after treatment with IL-2 (1 x 10(5) U/L), a significant increase of the activity of cytoplasmic, membrane and total PKC in human pituitary adenoma cells was observed. (4) IL-2 (5 x 10(4), 1 x 10(5) U/L) decreased the amount of cAMP in the cytoplasm of human pituitary adenoma cells, but had no effect on that of cGMP.. IL-2 can stimulate the proliferation of pituitary adenoma cells through PKC and cAMP/cGMP signaling pathways.

Topics: Antineoplastic Agents; Cell Cycle; Cell Proliferation; Cyclic AMP; Cyclic GMP; Enzyme Activation; Humans; Interleukin-2; Pituitary Neoplasms; Protein Kinase C; Signal Transduction; Tumor Cells, Cultured

We found previously that ACh can significantly inhibit the proliferation of cultured human pituitary adenoma cells. In order to make a further investigation of the mechanism of the inhibitory effect of ACh on the proliferation of pituitary adenoma cells, we observed the levels of protein kinase C (PKC), [Ca(2+)](i) and cAMP/cGMP in cultured pituitary adenoma cells after treatment with ACh. The results demonstrate that (1) compared with control, PMA, a PKC activator, increased the activity of cytoplasm, membrane and total PKC in human pituitary adenoma cells. However, after a 15-min treatment with ACh (10 micromol/L), a significant reduction of the activity of cytoplasm, membrane and total PKC in human pituitary adenoma cells was observed, and the reduction effect could be blocked by atropine. (2) The level of [Ca(2+)](i) of single adenoma cells was found to decrease immediately on the addition of ACh (10 micromol/L), which could also be blocked by atropine. (3) ACh increased the amount of cAMP in the cytoplasm of human pituitary adenoma cells, but had no effect on that of cGMP. These data provide an important clue to explore the molecular mechanisms of the inhibitory effect of ACh on the proliferation of pituitary adenoma cells, and suggest that the modulating effect of ACh on the proliferation of pituitary adenoma cells results from the interactions of several cellular signaling pathways.

Topics: Acetylcholine; Adenoma; Calcium; Cyclic AMP; Cyclic GMP; Humans; Pituitary Neoplasms; Protein Kinase C; Signal Transduction; Tumor Cells, Cultured

Methemoglobin (metHb) has been reported to be present in areas surrounding solid tumors. The effects of human metHb on the growth of one human hepatocellular carcinoma cell line and one human glioma cell line that simply replicate in Ham's nutrient mixture F12 (F12) were investigated. MetHb, depending on its concentration, stimulated or inhibited the in vitro growth of both cancer cell lines. The stimulatory or inhibitory effect was due to the release of hemin from metHb, which was recognized by its characteristic light absorption spectrum. The possibility of metHb or hemin acting initially through a 3', 5'-cyclic guanosine monophosphate- (cGMP-) or prostaglandin E2- (PGE2-) mediated pathway to enhance cell growth was excluded. Ferric iron derived from the catabolic degradation of hemin increased cell growth, whereas biliverdin (Bv) and its reduction product, bilirubin (Br), decreased cell growth. Hemoglobin oxidized to metHb in conditions found in tumors showing neovascularization and hemorrhage may contribute significantly to increased proliferation of cancerous cells.

Topics: Adult; Aged; Bilirubin; Biliverdine; Carcinoma, Hepatocellular; Cell Division; Cyclic GMP; Dinoprostone; Dose-Response Relationship, Drug; Female; Glioma; Globins; Hemin; Humans; Liver Neoplasms; Methemoglobin; Pituitary Neoplasms; Tumor Cells, Cultured

Topics: Adenoma; Animals; Cyclic GMP; Female; Humans; Leptin; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Pituitary Hormones; Pituitary Neoplasms; Rats

Familial hypothalamic diabetes insipidus is an autosomal dominant disorder characterized by deficient vasopressin synthesis. Different point mutations in the vasopressin-neurophysin (VP-NP) precursor gene have been found in affected families. In a Dutch kindred, a single G to T transversion in the NP-encoding exon B of one allele converts the highly conserved glycine 17 to a valine residue. In order to examine whether this point mutation affects the processing and transport of the VP-NP precursor, the normal (HV2) and mutant (MT6) vasopressin cDNAs were stably expressed in the mouse pituitary cell line AtT20. The normal precursor was correctly glycosylated and processed, and NP was detected in the culture medium. Secretion of NP was stimulated by 8-bromo-cAMP, indicating that the normal precursor was targeted to the regulated secretory pathway. In contrast, the mutant precursor was synthesized, but processing and secretion were dramatically reduced. The mutant precursor was core-glycosylated but remained endoglycosidase H-sensitive, suggesting that the protein did not reach the trans-Golgi network. These results were supported by immunocytochemical studies. In HV2 cells, NP derived from the precursor was concentrated in the tips of the cell processes where secretory granules accumulate. In MT6 cells, NP staining was restricted to the endoplasmic reticulum (ER) as determined by colocalization with an ER-resident protein, BiP. These results suggest that the mutation within the conserved part of NP alters the conformation of the precursor and thus triggers its retention in the ER.

Topics: Amino Acid Sequence; Animals; Arginine Vasopressin; Cell Line; Conserved Sequence; Cyclic GMP; Diabetes Insipidus; Fluorescent Antibody Technique; Glycine; Glycosylation; Humans; Mice; Netherlands; Neurophysins; Oxytocin; Pituitary Gland; Pituitary Neoplasms; Point Mutation; Protein Precursors; Recombinant Proteins; Transfection; Valine; Vasopressins

Preincubation of AtT-20 mouse pituitary tumour cells with the phorbol ester PMA resulted in a concentration-dependent inhibition of CNP-stimulated cyclic GMP production. The phorbol ester analogue 4 alpha phorbol had no inhibitory effect and 24 h preincubations with PMA resulted in a characteristic down-regulation of the response indicating that the inhibitory actions were mediated via the activation of protein kinase C. Forskolin in the presence of the phosphodiesterase inhibitor IBMX stimulated intracellular cyclic AMP concentrations by up to eight fold, but did not alter basal nor CNP-stimulated cyclic GMP production. These results indicate that CNP-stimulated guanylate cyclase activity associated with the GC-B natriuretic peptide receptor expressed in AtT-20 cells is inhibited by protein kinase C.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Atrial Natriuretic Factor; Cell Line; Colforsin; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Kinetics; Mice; Natriuretic Peptide, C-Type; Pituitary Neoplasms; Protein Kinase C; Proteins; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

Although C-type natriuretic peptide (CNP) has been shown to exist at the highest concentration in the anterior pituitary in rat tissues, its physiological role(s) there is (are) not clear. In this study, we report a novel function of CNP examined with anterior pituitary-derived cell lines, GH3 and AtT20/D16v-F2. Both CNP and atrial natriuretic peptide (ANP) increased cellular cGMP levels in both cell lines in dose-dependent manners. CNP, but not ANP, stimulated growth hormone (GH) release from GH3 cells. In contrast, neither ANP nor CNP had any significant effect on the corticotropin release from AtT20/D16v-F2 cells. An activator for cGMP-dependent protein kinase (cGK), dibutyryl cGMP, mimicked the stimulation of GH release from GH3 cells by CNP. Constitutive GH release from GH3 cells was greatly diminished in the presence of inhibitors for cAMP-dependent protein kinase, while stimulative GH release by CNP was not affected. However, inhibitors which can block cGK almost completely diminished the stimulative effect of CNP. An inhibitor for protein kinase C did not show any effect on either constitutive or CNP-stimulative GH release. Our observations indicate that the stimulation of GH release from GH3 cells by CNP is mediated mainly by the cGK signal-transduction pathway, not by cAMP-dependent protein kinase or protein kinase C, through a CNP-specific receptor (possibly ANP-B receptor). Thus, CNP may act as a local modulator in the anterior pituitary.

Topics: Animals; Atrial Natriuretic Factor; Bucladesine; Cell Line; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; Growth Hormone; Kinetics; Natriuretic Peptide, C-Type; Pituitary Gland, Anterior; Pituitary Neoplasms; Protein Kinase Inhibitors; Proteins; Rats

Receptors for the natriuretic peptide family have been characterized in the adrenocorticotrophic hormone (ACTH)-secreting AtT-20 pituitary tumour cell line. Northern blot analysis detected mRNA transcripts for the guanylate cyclase-linked GC-B receptor subtype. There was no evidence for the expression of either guanylate cyclase-linked GC-A receptor or atrial natriuretic peptide (ANP)-C (clearance) receptor mRNAs. Cyclic GMP production in AtT-20 cells was stimulated up to 200-fold by C-type natriuretic peptide (CNP), which was 10- and 20 times as effective as equivalent concentrations of brain natriuretic peptide and ANP respectively. Cyclic GMP dose-response curves to CNP failed to show any signs of saturation even at concentrations up to 30 microM, indicating a relatively low affinity of CNP for the GC-B receptor. Although CNP induced large stimulations in cyclic GMP production, specific binding of [125I-Tyr0]CNP could not be demonstrated in AtT-20 cells. The absence of specific binding with this radiolabelled analogue is possibly due to a reduced affinity for the GC-B receptor, as CNP analogues with N-terminal modifications such as [Tyr0]CNP and [127I-Tyr0]CNP exhibited reduced abilities to stimulate cyclic GMP production in these cells. Despite elevating cyclic GMP levels, CNP had no effect on basal or corticotrophin-releasing factor-stimulating ACTH release from the cells. These results show that the guanylate cyclase-coupled GC-B receptor is the only natriuretic peptide receptor subtype expressed in AtT-20 cells. Although CNP can markedly stimulate cyclic GMP production in these cells, there is incomplete expression of the normal natriuretic peptide-induced inhibitory pathway of ACTH secretion at some point distal to the production of cyclic GMP.

Topics: Adrenocorticotropic Hormone; Animals; Atrial Natriuretic Factor; Cell Line; Corticotropin-Releasing Hormone; Cyclic GMP; Kinetics; Natriuretic Peptide, C-Type; Nerve Tissue Proteins; Pituitary Neoplasms; Receptors, Atrial Natriuretic Factor; RNA, Messenger; Tumor Cells, Cultured

Natriuretic peptides inhibit the release and action of many hormones through cyclic guanosine monophosphate (cGMP), but the mechanism of cGMP action is unclear. In frog ventricular muscle and guinea-pig hippocampal neurons, cGMP inhibits voltage-activated Ca2+ currents by stimulating phosphodiesterase activity and reducing intracellular cyclic AMP; however, this mechanism is not involved in the action of cGMP on other channels or on Ca2+ channels in other cells. Natriuretic peptide receptors in the rat pituitary also stimulate guanylyl cyclase activity but inhibit secretion by increasing membrane conductance to potassium. In an electrophysiological study on rat pituitary tumour cells, we identified the large-conductance, calcium- and voltage-activated potassium channels (BK) as the primary target of another inhibitory neuropeptide, somatostatin. Here we report that atrial natriuretic peptide also stimulates BK channel activity in GH4C1 cells through protein dephosphorylation. Unlike somatostatin, however, the effect of atrial natriuretic peptide on BK channel activity is preceded by a rapid and potent stimulation of cGMP production and requires cGMP-dependent protein kinase activity. Protein phosphatase activation by cGMP-dependent kinase could explain the inhibitory effects of natriuretic peptides on electrical excitability and the antagonism of cGMP and cAMP in many systems.

Topics: Animals; Atrial Natriuretic Factor; Charybdotoxin; Cyclic AMP; Cyclic GMP; Ethers, Cyclic; Membrane Potentials; Okadaic Acid; Phosphoprotein Phosphatases; Pituitary Neoplasms; Potassium Channels; Protein Kinases; Scorpion Venoms; Tetraethylammonium; Tetraethylammonium Compounds; Thionucleotides; Tumor Cells, Cultured

Whether atrial natriuretic peptide (ANP)-evoked inhibition of corticotrophin-releasing factor (CRF)-stimulated ACTH secretion was also manifest in ACTH secreting AtT-20 pituitary tumour cells was investigated. ANP stimulated increases in cGMP accumulation at concentrations of the peptide above 10(-8) M which indicates the presence of the ANP receptors on these cells. CRF stimulated a concentration-dependent increase in ACTH secretion from AtT-20 cells which was unaffected by ANP, 8-bromo-cGMP, or sodium nitroprusside (SNP). Calcium stimulated a concentration-dependent increase in ACTH secretion from electrically permeabilised cells which was unaffected by co-incubation with cGMP but potentiated by cAMP. These results reveal the presence of ANP receptors on AtT-20 cells but suggest that an incomplete expression of the stimulus-secretion coupling mechanisms for ANP, at some point after cGMP production, prevents the effects of natriuretic peptides upon ACTH secretion being manifest in these cells.

Topics: 1-Methyl-3-isobutylxanthine; Adrenocorticotropic Hormone; Animals; Atrial Natriuretic Factor; Calcium; Cell Membrane Permeability; Corticotropin-Releasing Hormone; Cyclic AMP; Cyclic GMP; Hypothalamo-Hypophyseal System; Mice; Nitroprusside; Pituitary Neoplasms; Receptors, Atrial Natriuretic Factor; Tumor Cells, Cultured

The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 microM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 microM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 microM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.

Topics: Animals; Bucladesine; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP; Cyclic GMP; Dibutyryl Cyclic GMP; Gene Expression Regulation, Enzymologic; Kinetics; Macromolecular Substances; Pituitary Neoplasms; Protein Kinases; Protein Processing, Post-Translational; RNA, Messenger

The 1,4-dihydropyridine BAY-K-8644 [methyl-1,4-dihydro-2, 6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate] acts as both a calcium channel agonist and antagonist by stimulating or inhibiting inward calcium current. In AtT-20 mouse pituitary tumor cells, BAY-K-8644 both stimulates and blocks adrenocorticotropin (ACTH) secretion. Because in several cell systems the cytoplasmic enzyme guanylate cyclase is activated, presumably by calcium entry, the effect of BAY-K-8644 on cyclic GMP (cGMP) synthesis in AtT-20 cells was assessed. BAY-K-8644 increased cGMP accumulation in a time-dependent manner. The concentrations of BAY-K-8644, however, required to increase cGMP formation were not associated with its stimulatory effects on secretion but rather with its ability to antagonize basal and (-)-isoproterenol-induced ACTH secretion. The inhibitory effect of BAY-K-8644 on ACTH secretion was not mimicked by 8-Br-cGMP. The cGMP response to BAY-K-8644 was not mimicked by the cationophore, A-23187, or depolarizing concentrations of K+. Other calcium channel antagonists such as nifedipine or verapamil had markedly smaller effects on cGMP formation compared to BAY-K-8644. Sodium nitroprusside and sodium azide both increased cGMP synthesis in AtT-20 cells and both inhibited, to a lesser extent than BAY-K-8644, both basal- and (-)-isoproterenol-stimulated ACTH release. The data suggest that BAY-K-8644 stimulates cGMP synthesis by binding to sites less accessible or poorly activated by other dihydropyridines, and that stimulation of guanylate cyclase is independent of inward calcium current.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenocorticotropic Hormone; Animals; Azides; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Isoproterenol; Mice; Nifedipine; Nitroprusside; Pituitary Neoplasms; Sodium Azide; Stimulation, Chemical; Verapamil

The dihydropyridine calcium channel activator, BAY-K-8644, stimulates cGMP formation in ACTH-secreting mouse AtT-20 clonal corticotrophs. The recent report that calmodulin antagonists could inhibit dihydropyridine binding in several tissues suggested that these agents might also affect the cyclic nucleotide response to BAY-K-8644. In fact, TMB-8, trifluoperazine, and melittin, described as in vitro antagonists of calmodulin-dependent enzyme activities, all inhibited BAY-K-8644 induced cGMP synthesis in a concentration-dependent manner. The antagonists had no effect on cGMP formation stimulated by sodium nitroprusside or sodium azide. The calcium channel antagonist, nifedipine, did not stimulate cGMP formation nor did it alter the effect of BAY-K-8644 on accumulation of the nucleotide; one explanation thus is that the cyclase involved in cGMP formation is coupled to a low affinity binding site for BAY-K-8644, which is less accessible to other dihydropyridines. The relation of cyclic GMP formation to the function of the calcium channel in AtT-20 cells remains unknown.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Calcium Channel Blockers; Calmodulin; Cells, Cultured; Cyclic GMP; Gallic Acid; Humans; Ion Channels; Melitten; Pituitary Neoplasms; Trifluoperazine

The regulation of T4 5'-deiodinase activity was studied in cultured GH4C1 cells. Enzyme activity was measured in cell sonicates as the release of radioiodide from [125I]T4. Enzyme activity was stimulated 2- to 3-fold by hypothyroid serum and activators of protein kinase C, such as TRH and phorbol esters. The hypothyroid serum effect was maximal by 3 h, whereas TRH and phorbol esters required 6 h to achieve a maximal effect. The hypothyroid serum effect was gone within 4 h of returning the cells to control medium. In contrast, the TRH and phorbol ester effects persisted 24-48 h after removal of those agents. Both T4 and rT3 were at least as potent as T3 in blocking the effect of hypothyroid serum. The stimulation of 5'-deiodinase induced by hypothyroid serum was additive with that induced by kinase C activators. Trifluoperazine blocked the effect of TRH and phorbol esters, but not that of hypothyroid serum. It is concluded that stimulation of 5'-deiodinase activity can occur by at least two independent mechanisms: one involving hypothyroidism and another involving activation of protein kinase C. The relative potencies of various iodothyronines for abolishing the hypothyroid effect differ markedly from the relative binding affinities of these agents for the nuclear T3 receptor, suggesting that this thyroid hormone effect may not be mediated by the classical nuclear thyroid hormone receptor.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcimycin; Cell Line; Colforsin; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Hypothyroidism; Iodide Peroxidase; Phorbol Esters; Pituitary Neoplasms; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Thyroid Hormones; Thyrotropin-Releasing Hormone; Thyroxine; Trifluoperazine; Triiodothyronine; Triiodothyronine, Reverse

Cyclo(Histidyl-Proline) is a metabolite of thyrotropin-releasing hormone. It has been suggested that this peptide plays a role in regulating prolactin secretion in GH cells. An investigation of the effect of cyclo(His-Pro) on GH cells indicated that it does not affect basal prolactin release or accumulation or the levels stimulated by TRH. cAMP levels in GH cells are elevated by TRH or VIP, but not influenced by cyclo(His-Pro). cGMP levels in GH cells are not affected by either TRH or cyclo(His-Pro). While there is specific binding of TRH to receptors in GH cells, no such receptors for cyclo(His-Pro) are detectable. It is suggested that GH cells are unresponsive to cyclo(His-Pro).

Topics: Animals; Clone Cells; Cyclic AMP; Cyclic GMP; Haplorhini; Humans; Peptides, Cyclic; Piperazines; Pituitary Neoplasms; Prolactin; Rats; Receptors, Cell Surface; Receptors, Thyrotropin-Releasing Hormone; Thyrotropin-Releasing Hormone

Topics: Animals; Calcium; Cells, Cultured; Cobalt; Cyclic AMP; Cyclic GMP; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Theophylline; Thyrotropin-Releasing Hormone

Topics: Adipose Tissue; Adrenocorticotropic Hormone; Animals; Bucladesine; Clofibrate; Cyclic AMP; Cyclic GMP; Growth Hormone; Lipolysis; Male; Neoplasms, Experimental; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred F344