ucn-1028-c and adenosine-3--5--cyclic-phosphorothioate

Parathyroid hormone (PTH), the major systemic calcium-regulating hormone, has been linked to uremic vascular changes. Considering the possible deleterious action of PTH on vascular structures, it seemed logical to evaluate the impact of PTH on the receptor of advanced glycation end products (RAGE) and interleukin 6 (IL-6) mRNA and protein expression, taking into account that such parameters might be involved in the pathogenesis of vascular calcification, atherosclerosis, and/or arteriolosclerosis. Human umbilical vein cord endothelial cells (HUVEC) were stimulated for 24 h with 10(-12)-10(-10) mol/l PTH. The mRNA expression of RAGE and IL-6 was established by reverse transcriptase/PCR techniques. RAGE protein levels were determined by Western blot and IL-6 secretion was measured by ELISA. The pathways by which PTH may have an effect on HUVEC functions were evaluated. PTH (10(-11)-10(-10)mol/l) significantly increased RAGE mRNA and protein expression. PTH also significantly increased IL-6 mRNA expression without changes at protein levels. The addition of protein kinase (PKC or PKA) inhibitors or nitric oxide (NO) synthase inhibitors significantly reduced the RAGE and IL-6 mRNA expression and the RAGE protein expression. PTH stimulates the mRNA expressions of RAGE and IL-6 and the protein expression of RAGE. These stimulatory effects are probably through PKC and PKA pathways and are also NO dependent. Such data may explain the possible impact of PTH on the atherosclerotic and arteriosclerotic progression.

Topics: Atherosclerosis; Cyclic AMP; Endothelium, Vascular; Female; Gene Expression; Humans; Interleukin-6; Naphthalenes; NG-Nitroarginine Methyl Ester; Parathyroid Hormone; Pregnancy; Protein Kinases; Receptor for Advanced Glycation End Products; Receptors, Immunologic; RNA, Messenger; Thionucleotides

CEACAM1 (also known as biliary glycoprotein, C-CAM or CD66a) is a cell adhesion molecule of the immunoglobulin family behaving as a tumor inhibitory protein in colon, prostate, liver, endometrial and breast cancers. Inhibition of tumor development is dependent upon the presence of the long 71-73 amino acid cytoplasmic domain of the CEACAM1 protein (CEACAM1-L). We have recently defined a number of cis-acting motifs within the long cytoplasmic domain participating in tumor cell growth inhibition. These are Tyr488, corresponding to an Immunoreceptor Tyrosine-based Inhibition Motif, as well as the three terminal lysine residues of the protein. In this study, we provide evidence that treatment with phorbol esters leads to increased phosphorylation of in vivo (32)P-labeled CEACAM1-L in mouse CT51 carcinoma cells, in the mouse 1MEA 7R.1 liver carcinoma cells and in 293 human embryonic kidney cells transfected with the Ceacam1-L cDNA. Basal level Ser phosphorylation was abrogated by treatment with the staurosporine inhibitor, but not by the protein kinase C-specific inhibitor calphostin C or other inhibitors such as H7 or sphingosine. Specific inhibitors of protein kinase A or calmodulin kinase had only minimal effects on the levels of basal or PMA-induced Ser phosphorylation. Furthermore, PMA treatment of the CT51 cells induced cell spreading and cellular relocalization of the CEACAM1-L protein. Since Ser503 has been described as a PMA-induced phosphorylation site in other cell systems, we investigated whether Ser503 was involved in these responses in mouse intestinal cells. No differences were noticed in the basal or the PMA-induced phosphorylation levels, kinase inhibitor sensitivity or the PMA-induced relocalization of the protein between the wild-type and the Ser503Ala mutant CEACAM1-L. However, we provide evidence that Ser503 participates in CEACAM1-L-mediated tumor inhibition as its mutation to an Ala led to in vivo tumor development, contrary to the tumor inhibitory phenotype observed with the wild-type CEACAM1-L protein.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amino Acid Sequence; Animals; Antigens, CD; Antigens, Differentiation; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoembryonic Antigen; Carcinogenicity Tests; Carcinoma; Cell Adhesion Molecules; Colonic Neoplasms; Cyclic AMP; Enzyme Inhibitors; Epithelial Cells; Female; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Naphthalenes; Point Mutation; Protein Kinase C; Serine; Staurosporine; Tetradecanoylphorbol Acetate; Thionucleotides; Tumor Cells, Cultured

Dopamine D4 receptors (D4 receptors) mediate dopamine-stimulated, folate-dependent phospholipid methylation. To investigate possible regulation of this multi-step D4 receptor-mediated phospholipid methylation cycle by protein kinases, specific kinase activators and inhibitors were studied in SK-N-MC human neuroblastoma cells, using [14C] formate to label folate-derived single-carbon groups. Phorbol dibutyrate (PDB), an activator of protein kinase C, stimulated basal phospholipid methylation and also shifted the dose-response curve for dopamine-stimulated phospholipid methylation to the right by more than an order of magnitude. Calphostin C, an inhibitor of protein kinase C, had little effect on basal phospholipid methylation but significantly inhibited dopamine-stimulated phospholipid methylation and also blocked the stimulatory response to PDB. Chelerythrine, which inhibits protein kinase C and other kinases, strongly inhibited both basal and dopamine-stimulated phospholipid methylation. Forskolin, an activator of protein kinase A, inhibited basal and dopamine-stimulated phospholipid methylation, but only at high concentrations while Rp-cAMP, an inhibitor of protein kinase A, did not block this effect. Inhibition of protein kinase G produced a modest decrease in dopamine-stimulated phospholipid methylation, but neither sodium nitroprusside, which increases nitric oxide (NO) production and activates protein kinase G, nor the NO synthase inhibitor N-nitro-L-arginine had any effect on basal or dopamine-stimulated phospholipid methylation. These observations indicate that protein kinase C is an important regulator of basal and D4 receptor-mediated folate-dependent phospholipid methylation, whereas protein kinase A and protein kinase G have a lesser or minimal role.

Topics: Alkaloids; Benzophenanthridines; Carbazoles; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP-Dependent Protein Kinases; Dopamine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Indoles; Methylation; Naphthalenes; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Phenanthridines; Phorbol 12,13-Dibutyrate; Phospholipids; Protein Kinase C; Receptors, Dopamine D2; Receptors, Dopamine D4; Thionucleotides; Tumor Cells, Cultured

The modulation of a transient T-type calcium current by the five muscarinic receptor subtypes, stably expressed in NIH 3T3 cells, was studied with the whole-cell patch-clamp technique. Voltage-step depolarizations applied to the NIH 3T3 cells revealed a low-voltage-activated (LVA) T-type calcium current that was inhibited by Ni2+ and unaffected by omega-conotoxin GVIA. In cells transfected with the m3 and m5 muscarinic receptors, application of acetylcholine (ACh) resulted in a pertussis-toxin-insensitive increase in peak T-type calcium current amplitude. The m3-induced atropine-sensitive increase in current amplitude was accompanied by a shift in the voltage dependence of activation to more hyperpolarized potentials. The increase in peak T-type calcium current amplitude and the shift in voltage dependence was mimicked by incubation with 500 microM 8-bromo-cAMP. Conversely, T-type calcium current amplitudes were reduced by incubation with 10 microM RpcAMPS, an inhibitor of cAMP-dependent protein kinase (PKA). Preincubation with 500 microM 8-bromo-cAMP or with 10 microM RpcAMPS abolished the increase in T-type calcium current amplitude previously noted on stimulation of the m3 muscarinic receptor by ACh. Application of ACh to NIH 3T3 cells stably transformed with the m1 muscarinic receptor resulted in no discernable change in T-type calcium current amplitude. However, on pre-incubation of the cells with calphostin C, an inhibitor of protein kinase C (PKC), application of ACh to the cells now resulted in a robust increase in T-type calcium current amplitude. Application of 500 nM PDBu, an activator of PKC, reduced the T-type calcium current amplitude. No significant changes in T-type calcium currents were observed on application of ACh to cells stably transfected with the m2 or m4 muscarinic receptors. However, after pre-incubation with forskolin, the m2 muscarinic receptor induced a decrease in T-type calcium current amplitude. Stimulation of the ml, m3 and m5 muscarinic receptors in the NIH 3T3 cell resulted in dose-dependent increases in the concentration of intracellular cAMP in comparison to control as determined by cAMP immunoassay. Conversely, stimulation of the m2 and m4 muscarinic receptors by carbachol resulted in a dose-dependent reduction in intracellular concentrations of cAMP, as compared with control basal levels. It is concluded that the m3 and m5 muscarinic receptors enhance T-type calcium channel activity. At least in the case of the m3 mu

Topics: 3T3 Cells; 8-Bromo Cyclic Adenosine Monophosphate; Acetylcholine; Animals; Calcium Channel Blockers; Calcium Channels, T-Type; Colforsin; Cyclic AMP; Enzyme Inhibitors; Gene Expression; Membrane Potentials; Mice; Naphthalenes; Nickel; Nifedipine; Patch-Clamp Techniques; Receptor, Muscarinic M1; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Receptor, Muscarinic M5; Receptors, Muscarinic; Thionucleotides; Transfection; Vasodilator Agents

Extracellular Zn2+ modulates current passage through voltage- and neurotransmitter-gated ion channels, at concentrations less than, or near, those produced by release at certain synapses. Electrophysiological effects of cytoplasmic Zn2+ are less well understood, and effects have been observed at concentrations that are orders of magnitude greater than those found in resting and stimulated neurons. To examine whether and how neurons are affected by lower levels of cytoplasmic Zn2+, we tested the effect of Zn2+-selective chelators, Zn2+-preferring ionophores, and exogenous Zn2+ on neuronal somata during whole-cell patch-clamp recordings. We report here that cytoplasmic zinc facilitates the downward regulation of a background Cl- conductance by an endogenous protein kinase C (PKC) in fish retinal ganglion cell somata and that this regulation is maintained if nanomolar levels of free Zn2+ are available. This regulation has not been described previously in any tissue, as other Cl- currents have been described as reduced by PKC alone, reduced by Zn2+ alone, or reduced by both independently. Moreover, control of cation currents by a zinc-dependent PKC has not been reported previously. The regulation we have observed thus provides the first electrophysiological measurements consistent with biochemical measurements of zinc-dependent PKC activity in other systems. These results suggest that contributions of background Cl- conductances to electrical properties of neurons are susceptible to modulation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Calcium; Cations; Chelating Agents; Chlorides; Cyclic AMP; Cytoplasm; Enzyme Activation; Goldfish; Indoles; Ion Channel Gating; Ion Channels; Ionophores; Maleimides; Membrane Potentials; Naphthalenes; Patch-Clamp Techniques; Protein Kinase C; Retinal Ganglion Cells; Thionucleotides; Zinc

Promoter sequences responsive to cyclic AMP (cAMP) are found in a number of cellular genes, and bind transcription factors of the cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1) family. We have used a human T-lymphotropic virus type 1 (HTLV-1) model of cAMP response element (CRE) transcription to investigate the influence of lymphocyte activation on transcription from homologous regions in the viral promoter. We previously demonstrated increased HTLV-1 transcription following CD2 but not CD3 receptor cross-linking. We hypothesized that this increased viral transcription was mediated, in part, through the phosphorylation of CREB. Therefore, we investigated CD2 and CD3 receptor-mediated signalling in primary human peripheral blood mononuclear cells (PBMC). CD2, but not CD3, cross-linking increased cAMP detected by competitive enzyme-linked immunosorbent assay (ELISA) approximately fourfold. CD2 cross-linking concurrently increased phosphorylation of CREB detected by immunoblot assay eightfold. Consistent with post-translational regulation, no change in total level of CREB protein was observed. Phosphorylation of CREB occurred through a herbimycin A and Rp-cAMP-sensitive pathway, suggesting phosphorylation required antecedent activation of both protein tyrosine kinases (PTK) and protein kinase A (PKA). Both CD2 and CD3 cross-linking increased binding of nuclear proteins to a radiolabelled CRE oligonucleotide probe in electrophoretic mobility shift assays suggesting that lymphocyte activation enhances binding independently of phosphorylation of CREB at serine 133. These data indicate specific modulation of the CREB/ATF-1 family of transcription factors by the CD2 signalling pathway and suggest CD2 receptor modulation of CRE-mediated transcription following ligand engagement (e.g. cell-to-cell contact).

Topics: Activating Transcription Factor 2; Benzoquinones; Blotting, Western; CD2 Antigens; CD3 Complex; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Electrophoresis; Enzyme Inhibitors; Human T-lymphotropic virus 1; Humans; Lactams, Macrocyclic; Lymphocyte Activation; Monocytes; Naphthalenes; Oligonucleotide Probes; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Receptor Cross-Talk; Rifabutin; Signal Transduction; Thionucleotides; Transcription Factors; Transcription, Genetic

Our purpose was to determine the role of protein kinases in the mediation of the stimulatory effects of lead on catecholamine secretion. Pheochromocytoma cells were incubated for 90 minutes with W-7 (calmodulin antagonist), calphostin C (protein kinase C inhibitor), Sp-cAMPS (cAMP agonist), Rp-cAMPS (cAMP antagonist), forskolin (activator of adenylyl cyclase), or lead nitrate. Catecholamines were measured by liquid chromatography. Lead had a stimulatory effect on catecholamine secretion, whereas W-7 was inhibitory. In the presence of both lead and W-7, the response was markedly decreased compared to that seen with lead alone. Calphostin C suppressed the secretion of catecholamines; however, in the presence of lead and calphostin C, the secretion was similar to that seen with lead alone. Compared to control, Sp-cAMPS was stimulatory. Co-incubation of Sp-cAMPS and lead had a slight synergistic effect. Rp-cAMPS decreased catecholamine secretion, but co-incubation of Rp-cAMPS and lead resulted in a slight reduction compared to lead alone. Forskolin markedly increased the secretion of catecholamines, and co-incubation of lead and forskolin resulted in a synergistic increase. In the absence of calcium, lead had no effect. We conclude that lead stimulates catecholamine secretion by acting through the calcium/calmodulin-dependent protein kinase II system and not through the protein kinase C or protein kinase A system, and requires the presence of calcium for its action.

Topics: Adenylyl Cyclases; Animals; Calmodulin; Catecholamines; Colforsin; Cyclic AMP; Enzyme Activation; Enzyme Inhibitors; Lead; Models, Biological; Naphthalenes; Nitrates; Pheochromocytoma; Protein Kinase C; Rats; Sulfonamides; Thionucleotides; Tumor Cells, Cultured

The mechanism of endothelin-1 (ET-1)-induced atrial natriuretic peptide (ANP) release was studied in neonatal rat ventricular cardiomyocytes. These cells expressed a single high-affinity class of ETA receptor (dissociation constant = 54 +/- 18 pM, n = 3), but no ETB receptors. Incubation of cardiomyocytes with ET-1 led to concentration-dependent ANP release and prostacyclin production. ET-1-induced ANP release was affected by neither protein kinase C (PKC) inhibition or downregulation nor by cyclooxygenase inhibition, indicating that ET-1-stimulated ANP secretion is not a PKC-mediated, prostaglandin-dependent process. Furthermore, ET-1 significantly stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production and increased cytosolic calcium concentration in these preparations. Both ET-1-induced calcium influx and ANP release were decreased by the cAMP antagonist Rp-cAMPS, the Rp diastereoisomer of cAMP. Moreover, ET-1-induced ANP secretion was strongly inhibited in the presence of nifedipine as well as in the absence of extracellular calcium. Thus our results suggest that ET-1 stimulates ANP release in ventricular cardiomyocytes via an ETA receptor-mediated pathway involving cAMP formation and activation of a nifedipine-sensitive calcium channel.

Topics: Alkaloids; Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzophenanthridines; Calcium; Cells, Cultured; Cyclic AMP; Cyclooxygenase Inhibitors; Egtazic Acid; Endothelin-1; Enzyme Inhibitors; Epoprostenol; Heart; Heart Ventricles; Kinetics; Myocardium; Naphthalenes; Nifedipine; Phenanthridines; Protein Kinase C; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Staurosporine; Thionucleotides

1. The effects of the vasoconstrictor angiotensin II (Ang II) on whole-cell ATP-sensitive K+ currents (IK,ATP) of smooth muscle cells isolated enzymatically from rat mesenteric arteries were investigated using the patch clamp technique. 2. Ang II, at a physiological concentration (100 nM), reduced IK,ATP activated by 0.1 mM internal ATP and 10 microM levcromakalim by 36.4 +/- 2.3%. 3. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-sn-glycerol (OAG, 1 microM) reduced IK,ATP by 44.1 +/- 2.7%. GDP beta S (1 mM), included in the pipette solution, abolished the inhibition by Ang II, while that by OAG was unaffected. 4. Pretreatment with the PKC inhibitors staurosporine (100 nM) or calphostin C (500 nM) prevented the Ang II-induced inhibition of IK,ATP. 5. Ang II inhibition was unaffected by cell dialysis with PKA inhibitor peptide (5 microM), and the PKA inhibitor Rp-cAMPS (100 microM) did not reduce IK,ATP. 6. Our results suggest that Ang II modulates KATP channels through activation of PKC but not through inhibition of PKA.

Topics: Adenosine Triphosphate; Angiotensin II; Animals; Cromakalim; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diglycerides; Enzyme Inhibitors; Glyburide; Guanosine Diphosphate; Hypoglycemic Agents; Male; Membrane Potentials; Mesenteric Arteries; Muscle, Smooth, Vascular; Naphthalenes; Patch-Clamp Techniques; Potassium; Potassium Channel Blockers; Potassium Channels; Protein Kinase C; Rats; Rats, Wistar; Staurosporine; Thionucleotides; Vasoconstrictor Agents; Vasodilator Agents