calcimycin and adenosine-3--5--cyclic-phosphorothioate

Antipsychotic drugs can modulate transcription factors and also nuclear receptors, but their action on glucocorticoid receptors (GR)-members of the steroid/thyroid hormone receptor family has not been studied so far. In the present study we investigated effects of various antipsychotics on the glucocorticoid-mediated gene transcription in fibroblast cells, stably transfected with a mouse mammary tumor virus promoter (LMCAT cells). Chlorpromazine (3-100 microM) inhibited the corticosterone-induced gene transcription in a concentration- and time-dependent manner. Clozapine showed a similar, but less potent effect, while haloperidol acted only in high concentrations, and other antipsychotic drugs (sulpiride, raclopride, remoxipride) were without any effect. It was also found that a phorbol ester (an activator of protein kinase C (PKC)) and A-23187 (Ca(2+)-ionophore) attenuated the inhibitory effect of chlorpromazine on the GR-induced gene transcription. An antagonist of the L-type Ca(2+) channel, as well as an inhibitor of phospholipase C (PLC) inhibited the corticosterone-induced gene transcription, but had no effect on the chlorpromazine-induced changes. The involvement of a PKC/PLC pathway in the chlorpromazine action was confirmed by Western blot analysis which showed that the drug in question decreased the PLC-beta(1) protein level, and to a lesser extent that of the PKC-alpha protein in LMCAT cells. The aforementioned data suggest that inhibition of the glucocorticosteroid-induced gene transcription by chlorpromazine and clozapine may be a mechanism by which these drugs block some effects induced by glucocorticoids. The inhibitory effect of chlorpromazine on the corticosterone-induced gene transcription seems to depend on the inhibition of Ca(2+) influx and/or the inhibition of some calcium-dependent enzymes, e.g. phospholipase beta(1).

Topics: Animals; Antipsychotic Agents; Calcimycin; Calcium; Calcium Channel Blockers; Cell Line; Cell Line, Transformed; Chloramphenicol O-Acetyltransferase; Chlorpromazine; Clozapine; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Estrenes; Fibroblasts; Gene Expression Regulation; Genes, Reporter; Haloperidol; Histamine; Histamine H1 Antagonists; Ionophores; Mammary Tumor Virus, Mouse; Mice; Nifedipine; Phorbol Esters; Phosphodiesterase Inhibitors; Protein Kinase C; Protein Kinase C-alpha; Pyrilamine; Pyrrolidinones; Raclopride; Receptors, Glucocorticoid; Remoxipride; Sulfonamides; Sulpiride; Tetradecanoylphorbol Acetate; Thionucleotides; Transcription, Genetic; Type C Phospholipases

Corpora lutea (CL) from Days 5, 10, and 15 after superovulation were enzymatically dispersed, and a portion of the cells were elutriated to obtain fractions enriched with small or large luteal cells. Mixed, small, and large luteal cell fractions were incubated with no treatment or with agonists or antagonists of cAMP (dbcAMP or Rp-cAMPS), protein kinase C (PKC; TPA or H-7), or calcium (A23187, EGTA, or A23187 + EGTA). The rate of contact-dependent gap junctional intercellular communication (GJIC) was evaluated by laser cytometry. Media were collected for progesterone (P(4)) radioimmunoassay, and luteal cells cultured with no treatment were fixed for immunocytochemistry or frozen for Western blot analysis. Luteal cells from each stage of the estrous cycle exhibited GJIC. The dbcAMP increased (P < 0.05) GJIC for all cell types across the estrous cycle. The Rp-cAMPS decreased (P < 0.05) GJIC for small luteal cells on Day 5 and for all cell types on Days 10 and 15. The TPA inhibited (P < 0.01), but H-7 did not affect, GJIC for all cell types across the estrous cycle. The A23187 decreased (P < 0.05) GJIC for large luteal cells touching only small or only large luteal cells, whereas A23187 + EGTA decreased (P < 0.05) GJIC for all cell types across the estrous cycle. For the mixed and large luteal cell fractions, dbcAMP increased (P < 0.05), but TPA and A23187 + EGTA decreased (P < 0.05), P(4) secretion. The A23187 alone decreased (P < 0.05) P(4) secretion by large, but not by mixed, luteal cells. For all days and cell types, the rate of GJIC and P(4) secretion were correlated (r = 0.113-0.249; P < 0.01). Connexin 43 was detected in cultured luteal cells by immunofluorescence and Western immunoblotting. Thus, intracellular regulators like cAMP, PKC, or calcium appear to regulate GJIC, which probably is an important mechanism for coordinating function of the ovine CL.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Blotting, Western; Bucladesine; Calcimycin; Calcium; Cells, Cultured; Chelating Agents; Culture Media, Conditioned; Cyclic AMP; Egtazic Acid; Enzyme Inhibitors; Estrous Cycle; Female; Fluorescent Antibody Technique; Gap Junctions; Immunohistochemistry; Ionophores; Luteal Cells; Progesterone; Protein Kinase C; Second Messenger Systems; Sheep; Tetradecanoylphorbol Acetate; Thionucleotides

The interaction between Ca2+ and cAMP in the mediation of ecdysteroid secretion from prothoracic glands (PGs) of Bombyx mori was investigated in vitro. Omission of Ca2+ from the PGs' incubation medium decreased basal ecdysteroid secretion from day 3 until day 6. On day 6, the ability of forskolin or 3-isobutyl-1-methylxanthine (IBMX) to stimulate ecdysteroid secretion was affected by the omission of Ca2+ from the medium. The cAMP agonist Sp-adenosine 3',5'-cyclic monophosphothioate (Sp-cAMPS) and the cAMP analogue dibutyryl cyclic AMP (dbcAMP) stimulated ecdysteroid secretion even in the absence of Ca2+ from the medium. The Sp-cAMPS-stimulated ecdysteroid secretion was inhibited by the cAMP antagonist Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) and the L-type Ca2+ channel blocker verapamil. Both the Ca2+ ionophore A23187 and the L-type Ca2+ channel agonist S(-) Bay K 8644 could stimulate ecdysteroid secretion. The A23187-induced ecdysteroid secretion was partially inhibited by Rp-cAMPS. The combined results indicate that Ca2+ and cAMP signaling pathways can cooperatively, as well as independently, stimulate ecdysteroid secretion from the PGs.

Topics: 1-Methyl-3-isobutylxanthine; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Bombyx; Bucladesine; Calcimycin; Calcium; Colforsin; Cyclic AMP; Drug Interactions; Ecdysterone; Enzyme Inhibitors; Phosphodiesterase Inhibitors; Second Messenger Systems; Signal Transduction; Thionucleotides; Verapamil

Effects of YT-146 [2-(1-octynyl) adenosine], an adenosine A2 receptor agonist, on cAMP production and noradrenaline (NA) release were investigated in PC12 cells. YT-146 caused a concentration-dependent cAMP accumulation (EC50: 1.2+/-0.9 nM). In [3H]NA-prelabeled cells, YT-146 increased the basal NA release and enhanced ATP-evoked NA release in a concentration-dependent manner (EC50: 0.23+/-0.15 nM). YT-146 augmented the maximal response to ATP without affecting the EC50 value of ATP. These effects of YT-146 were inhibited by several adenosine receptor antagonists with a characteristic of adenosine A2A receptor subtype. The effects of YT-146 were mimicked by forskolin, dibutylyl cAMP and Sp-cAMPS, and inhibited by H-89, a cAMP-dependent protein kinase inhibitor. YT-146 had little effect on ATP-induced increase in intracellular Ca2+ concentration. YT-146 enhanced the NA release induced by several different stimuli including Ca2+ ionophore A23187. The present results suggest that YT-146 is a potent agonist on adenosine A2A receptors in PC12 cells and causes a cAMP-dependent enhancement of NA release by affecting the exocytosis process at a point downstream of the intracellular Ca2+ increase.

Topics: Adenosine; Adenosine Triphosphate; Alkynes; Animals; Bucladesine; Calcimycin; Calcium; Carbachol; Cholinergic Agonists; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Ionophores; Isoquinolines; Membrane Potentials; Norepinephrine; PC12 Cells; Potassium Chloride; Purinergic P1 Receptor Agonists; Rats; Receptor, Adenosine A2A; Sulfonamides; Thionucleotides; Tritium; Vasodilator Agents

The present study was performed to characterize the direct involvement of cAMP-dependent protein kinase (PKA) in the regulation of collagen synthesis by parathyroid hormone (PTH) and PTH-related peptide (PTHrP) in osteoblastic osteosarcoma cells, UMR-106. Sp-cAMPS (10(-4)M), a direct activator of PKA, as well as dibutyryl cAMP (dbcAMP, 10(-4)M) significantly inhibited collagen synthesis. Human (h) PTH-(1-34) (10(-7)M) and hPTHrP (10(-7) M) inhibited collagen synthesis to the same degree. Although Rp-cAMPS, which acted directly as an antagonist in the activation of PKA, did not affect collagen synthesis by itself, it significantly antagonized dbcAMP- and Sp-cAMPS-induced inhibition of collagen synthesis. Moreover, Rp-cAMPS antagonized PTH- and PTHrP-induced inhibition of collagen synthesis to the same degree. The present study first indicated that the activation of PKA was directly linked to the regulation of collagen synthesis by PTH in osteoblast and that PTHrP had the same effect on collagen synthesis presumably through the same mechanism as PTH.

Topics: Animals; Bucladesine; Calcimycin; Cell Line; Collagen; Cyclic AMP; Ionomycin; Kinetics; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proline; Protein Kinases; Proteins; Rats; Tetradecanoylphorbol Acetate; Thionucleotides; Tritium