h-89 and staurosporine-aglycone

The excitatory action of brain-derived neurotrophic factor (BDNF) on synaptic transmission is triggered by adenosine A2A receptor activation. Since high-frequency neuronal firing, such as that inducing long-term potentiation (LTP), favours both A2A receptor activation and BDNF effects on transmission, we now evaluated the influence of adenosine on the facilitatory action of BDNF upon CA1 hippocampal LTP. theta-Burst stimulation of the pyramidal inputs induced a significant and persistent increase in field EPSP slopes, and this potentiation was augmented in the presence of BDNF (20 ng/ml), an action prevented by the inhibitor of Trk receptor autophosphorylation, K252a (200 nM). Removal of endogenous extracellular adenosine with adenosine deaminase (ADA, 1 U/ml), as well as the antagonism of adenosine A2A receptors with SCH58261 (100 nM), prevented the excitatory action of BDNF upon LTP. In an adenosine depleted background (with ADA), activation of adenosine A2A receptors (with 10nM CGS21680) restored the facilitatory effect of BDNF on LTP; this was fully prevented by the protein kinase A inhibitor, H-89 (1 microM) and mimicked by the adenylate cyclase activator, forskolin (10 microM). In similar experiments, activation of adenosine inhibitory A1 receptors (with 5 nM CPA) did not affect the facilitatory effect of BDNF. In conclusion, the facilitatory action of BDNF upon hippocampal LTP is critically dependent on the presence of extracellular adenosine and A2A receptor activation through a cAMP/PKA-dependent mechanism. Since extracellular adenosine accumulates upon high-frequency neuronal firing, the present results reveal a key process to allow the influence of BDNF upon synaptic plasticity.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Animals; Brain-Derived Neurotrophic Factor; Carbazoles; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data Interpretation, Statistical; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Indole Alkaloids; Isoquinolines; Long-Term Potentiation; Male; Neuroprotective Agents; Phenethylamines; Phosphorylation; Pyrimidines; Rats; Rats, Wistar; Receptor, Adenosine A2A; Sulfonamides; Triazoles

Ligusticum chuanxiong (LC) is a traditional Chinese herbal medicine used to treat various cardiovascular diseases. In this study, the butanol extract of LC was found to protect neuronal-like pheochromocytoma cells from serum deprivation-induced apoptosis. Both a serine/threonine kinase inhibitor and a specific protein kinase A (PKA) inhibitor blocked the protective effect of LC. A transcription inhibitor (actinomycin D) and a protein synthesis inhibitor (cyclohexamide) also attenuated the protective effect of LC, suggesting the requirement of gene expression for the protection of LC. On the other hand, LC increased both the formation of cyclic-AMP and the phosphorylation of the cyclic-AMP response element-binding protein (CREB), a downstream target of PKA and a nuclear transcription factor known for neuroprotective mechanism. Furthermore, LC-induced CREB phosphorylation and protective effect could be blocked by a PKA inhibitor and overexpression of the dominant negative CREB, respectively. Taken together, the protective mechanism of LC in antagonizing serum deprivation-induced PC12 cell apoptosis might be mediated through a PKA/CREB-dependent pathway.

Topics: Animals; Apoptosis; Carbazoles; CREB-Binding Protein; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Indole Alkaloids; Isoquinolines; Ligusticum; PC12 Cells; Plant Extracts; Protein Kinase Inhibitors; Rats; Rhizome; Serum; Sulfonamides; Transfection

Cyclic AMP-dependent proteolysis of GATA-6(Delta50) was characterized using inhibitors for intracellular signaling pathways. Among these kinase inhibitors, only H-89 and K252a inhibited the proteolysis induced by dbcAMP, a membrane permeable cAMP analogue, others such as PD98059, SB203580, calphostine C, PP1, and KN-93 did not do so. These results suggest that A-kinase, but not C-kinase, MEK, P38 MAP-kinases or Src kinase, could participate in the observed phenomenon. We further demonstrated that an inhibitor for ubiquitin isopeptidase (Delta12-PGJ2) inhibited the degradation of GATA-6(Delta50) in the presence of dbcAMP, suggesting that the cAMP-dependent proteolysis could be mediated through the ubiquitin-proteasome pathway, although proteasome activity did not change significantly during dbcAMP treatment. The full-length GATA-6 was also responsive to the induced degradation. Furthermore, mutation of a potential phosphorylation site (Ser-290-->Ala) for A- and C-kinases, and deletion of the PEST sequence of GATA-6 did not abolish the degradation. All these results suggest that cellular factor(s) may play a crucial role in mediating the activation of the cAMP-dependent process.

Topics: Animals; Base Sequence; Benzylamines; Binding Sites; Bucladesine; Carbazoles; Cell Nucleus; CHO Cells; Cricetinae; Cyclic AMP; DNA-Binding Proteins; Enzyme Inhibitors; Flavonoids; GATA6 Transcription Factor; Humans; Imidazoles; Indole Alkaloids; Isoquinolines; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Molecular Sequence Data; Mutation; Naphthalenes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plasmids; Prostaglandin D2; Proteasome Endopeptidase Complex; Protein Binding; Pyridines; src-Family Kinases; Sulfonamides; Transcription Factors

The hyperpolarization-activated Ih channel is modulated by neurotransmitters acting through the cAMP messenger system. In rat olfactory receptor neurons (ORNs), dopamine, by inhibition of adenylyl cyclase, shifts the voltage of half-maximal activation (V1/2) of Ih to more negative potentials and decreases Ih maximal relative conductance. Whether these effects result from a phosphorylation-dependent mechanism is unclear. Therefore, we used whole-cell patch-clamp recording techniques to study cAMP-dependent phosphorylation via PKA on Ih in rat ORNs. General protein kinase inhibition (50 nM K252a) produced a hyperpolarizing shift in Ih V1/2 and decreased Ih maximal conductance. Specific inhibition of PKA with H-89 (500 nM) also shifted the V1/2 of Ih to more negative potentials, and, in some cells, decreased Ih maximal conductance. PKA-mediated phosphorylation (cBIMPS, 50 mM) shifted Ih V1/2 more positive, modulated the kinetics of Ih channel activation and increased Ih peak current amplitude. Internal perfusion of the catalytic subunit of PKA (84 nM) also shifted Ih V1/2 positive and this shift was blocked by co-perfusion with PKI (50 nM). These results show that in rat ORNs, the voltage dependence of Ih activation can be modulated by PKA-dependent phosphorylation. We also show that PKA and other protein kinases may be involved in the regulation of Ih maximal conductance. Our findings suggest that changes in the phosphorylation state of ORNs may affect resting properties as well as modulate odor sensitivity.

Topics: Animals; Carbazoles; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Dichlororibofuranosylbenzimidazole; Electric Conductivity; In Vitro Techniques; Indole Alkaloids; Ion Channels; Isoquinolines; Male; Membrane Potentials; Olfactory Receptor Neurons; Patch-Clamp Techniques; Rats; Reference Values; Sulfonamides; Thionucleotides

We studied the phosphorylation of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid acinar cells. We have previously shown that NKCC1 activity in these cells is dramatically upregulated in response to beta-adrenergic stimulation and that this upregulation correlates with NKCC1 phosphorylation, possibly due to protein kinase A (PKA). We show here that when ATP is added to purified acinar basolateral membranes (BLM), NKCC1 is phosphorylated as a result of membrane-associated protein kinase activity. Additional NKCC1 phosphorylation is seen when PKA is added to BLMs, but our data indicate that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities, rather than its direct phosphorylation of NKCC1. Also, phosphopeptide mapping demonstrates that these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by beta-adrenergic stimulation. However, this upregulatory phosphorylation can be mimicked by the addition of cAMP to permeabilized acini, and this effect can be blocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatory phosphorylation of NKCC1 and suggest that an additional factor present in the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.

Topics: Adenosine Triphosphate; Animals; Carbazoles; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Indole Alkaloids; Indoles; Isoproterenol; Isoquinolines; Maleimides; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Parotid Gland; Peptides, Cyclic; Phenols; Phosphorus Radioisotopes; Phosphorylation; Rats; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2; Staurosporine; Sulfonamides; Sympathomimetics; Vanadates

The incubation of rat peritoneal macrophages in the presence of staurosporine, a non-specific protein kinase inhibitor, induced interleukin-6 (IL-6) production in a time- and concentration-dependent manner at 6.3-63 nM, but at 210 nM, the stimulant effect on IL-6 production was reduced. The levels of IL-6 mRNA as determined by a reverse transcription-polymerase chain reaction were also increased by staurosporine in parallel with the ability to induce IL-6 production. Compounds structurally related to staurosporine including K-252a (non-specific protein kinase inhibitor) and KT-5720 (inhibitor of cyclic AMP-dependent protein kinase, PKA), did not increase IL-6 production by peritoneal macrophages. Staurosporine-induced increases in IL-6 production and expression of IL-6 mRNA were decreased by the PKC inhibitors, H-7 (2.7-27 microM), Ro 31-8425 (1-10 microM) and calphostin C (0.3-3 microM) and by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 (30-100 microM), but were further increased by the protein tyrosine kinase (PTK) inhibitor, genistein (12-37 microM). The staurosporine-induced increase in IL-6 production was not affected by the PKA inhibitor, H-89 (0.1-3 microM). These findings suggest that the induction of IL-6 production by staurosporine is secondary to elevation of IL-6 mRNA level, which, in turn, is positively regulated by the activation of PKC and PI 3-kinase and negatively regulated by the activation of PTK. PKA does not appear to play a significant role.

Topics: Animals; Carbazoles; Chromones; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genistein; Indole Alkaloids; Indoles; Interleukin-6; Isoquinolines; Macrophages, Peritoneal; Male; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Pyrroles; Rats; Rats, Sprague-Dawley; RNA, Messenger; Staurosporine; Sulfonamides; Time Factors

A high-throughput screening (HTS) assay for inhibitors of nitric oxide (NO) production by activated microglia was developed and used to compare the relative activities of various anti-inflammatory compounds and cell-permeable protein kinase inhibitors. BV-2 cells, an immortalized line that retains phenotypic features of microglia and produces NO in response to lipopolysaccharide (LPS), were used in the activation paradigm for the HTS assay. A characteristic feature of the compounds that were the most potent dose-dependent inhibitors of NO production is their ability to modulate serine/threonine protein kinases. The anti-inflammatory compound K252a, an inhibitor of calmodulin (CaM)-regulated protein kinases, had one of the highest potencies in the assay. Other classes of kinase inhibitors, including the protein kinase A inhibitor H-89, the mitogen activated protein kinase inhibitors PD98059 and SB203580, and the tyrosine kinase inhibitor genistein, were less potent and efficacious than K252a or the general serine/threonine/tyrosine kinase inhibitor staurosporine. K252a suppresses production of the inducible nitric-oxide synthase (iNOS). The inhibitory effect of K252a is not due to cell toxicity and does not correlate with inhibition of NFkappaB nuclear translocation. The mechanism of action appears to involve inhibition of phosphorylation of the transcription factor CREB, a protein whose activity is modulated by phosphorylation by CaM-dependent protein kinases. These data suggest that signal transduction pathways mediated by CaM-dependent protein kinases warrant future study as potential drug discovery targets.

Topics: Amidines; Animals; Benzylamines; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cell Line; Cell Nucleus; Cyclic AMP Response Element-Binding Protein; Drug Design; Enzyme Inhibitors; Flavonoids; Imidazoles; Indole Alkaloids; Isoquinolines; Lipopolysaccharides; Mice; Microglia; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Phosphorylation; Pyridines; Staurosporine; Sulfonamides

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

Topics: Adenoviruses, Human; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Humans; Indole Alkaloids; Indoles; Isoflavones; Isoquinolines; Lactoferrin; Low Density Lipoprotein Receptor-Related Protein-1; Maleimides; Melanoma; Naphthalenes; Phenols; Protein Kinase C; Receptors, Immunologic; Signal Transduction; Staurosporine; Sulfonamides; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase, time- and dose-dependently induced the differentiation of M1 cells, a murine myeloid leukemic cell line, into macrophage-like cells as revealed by the phagocytosis of polystyrene latex particles. This differentiation was inhibited not only by actinomycin D and cycloheximide but also by ST-638 (an inhibitor of tyrosine kinase). However, it was affected neither by K-252a (an inhibitor of C-kinase) nor by H-89 (an inhibitor of A-kinase), in contrast to the M1 cell differentiation induced by leukemia inhibitory factor (LIF). Okadaic acid inhibited both the bafilomycin A1-induced and LIF-induced differentiation of M1 cells.

Topics: Animals; Anti-Bacterial Agents; Carbazoles; Cinnamates; Ethers, Cyclic; Growth Inhibitors; Indole Alkaloids; Interleukin-6; Isoquinolines; Leukemia Inhibitory Factor; Leukemia, Myeloid; Lymphokines; Macrolides; Macrophages; Mice; Okadaic Acid; Phagocytosis; Phosphoprotein Phosphatases; Phosphorylation; Protein Biosynthesis; Protein Kinase C; Protein Kinase Inhibitors; Proton-Translocating ATPases; RNA; Sulfides; Sulfonamides; Tumor Cells, Cultured; Vacuoles

1. In an air pouch-type allergic inflammation model in rats, leucocytes that had infiltrated into the pouch fluid collected 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils when they were incubated in the medium. 2. To clarify the mechanism of activation of the infiltrated leucocytes in producing these factors, the effects of protein kinase inhibitors on neutrophil chemotactic factor production were examined. 3. When the infiltrated leucocytes were incubated for 4 h in medium containing the non-selective protein kinase inhibitor K-252a (1-100 ng ml-1, 2.14-214 nM), the tyrosine kinase inhibitor genistein (1-50 micrograms ml-1, 3.7-185 microM), and the more selective protein kinase C inhibitor H-7 (5-100 micrograms ml-1, 13.7-274 microM); neutrophil chemotactic activity in the conditioned medium was decreased in a concentration-dependent manner, but the adenosine 3':5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor H-89 (1-1000 ng ml-1, 2.24-2240 nM) showed no effect. 4. Isoelectric focusing of the conditioned medium revealed that the leucocytes produced two neutrophil chemotactic factors, leucocyte-derived neutrophil chemotactic factor (LDNCF) 1 and LDNCF-2. Treatment of the leucocytes with K-252a, genistein, and H-7, but not H-89, inhibited production of both LDNCF-1 and LDNCF-2. 5. These results suggest that activation of tyrosine kinase and protein kinase C, but not cAMP-dependent protein kinase, is responsible for the production of LDNCF-1 and LDNCF-2. 6. The steroidal anti-inflammatory drug dexamethasone and the protein synthesis inhibitor cycloheximide inhibited neutrophil chemotactic factor production in a concentration-dependent manner. Time-course experiments showed that the inhibitory effect by dexamethasone was apparent even 30 min after the incubation.7. Mechanism for inhibiting the production of LDNCF-1 and LDNCF-2 by dexamethasone is also discussed.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Carbazoles; Cells, Cultured; Chemotactic Factors; Culture Media, Conditioned; Cycloheximide; Depression, Chemical; Dexamethasone; Genistein; Hypersensitivity; Indole Alkaloids; Inflammation; Isoelectric Focusing; Isoflavones; Isoquinolines; Leukocytes; Male; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Sulfonamides

Rapid changes in morphology of PC12D cells, a subline of PC12 cells, in response to various agents were studied in relation to the subsequent outgrowth of neurites. A few minutes after addition of NGF or of dbcAMP, staining of F-actin with rhodamine phalloidin revealed the formation of ruffles around the periphery of cells. Simultaneous relocalization of F-actin to the area of ruffles occurred in response to NGF. A moderate relocalization of F-actin occurred in dbcAMP-treated cells. Other neurite-promoting agents on PC12D cells, such as bFGF, EGF and PMA, also caused ruffling and an identical redistribution of F-actin. The actin bundles then condensed into several dot-like aggregates that subsequently became the growth cones of neurites. When an inhibitor of protein kinase, K-252a, was added, only the NGF-induced morphological change was selectively decreased. By contrast, an inhibitor of protein kinase A, H-89, selectively blocked the dbcAMP-induced change. These are analogous to the effects of those inhibitors on the outgrowth of neurites. These observations indicate that the formation of ruffles with the redistribution of F-actin might be one of the earliest steps in the neurite outgrowth and that the morphological changes might be triggered by the activation of specific protein kinases. Neither cytochalasin B nor colchicine prevented the series of morphological changes. However, processes formed in the presence of cytochalasin B had no filopodium and protrusions formed in the presence of colchicine were shaped like large filopodia. It appears that microtubules and microfilaments may not be absolutely required for the initiation of the rapid morphological changes, but that complete neurites might be formed with contribution by microtubules and by microfilaments.

Topics: Actins; Animals; Bucladesine; Carbazoles; Colchicine; Cytochalasin B; Cytoskeleton; Epidermal Growth Factor; Fibroblast Growth Factor 2; Indole Alkaloids; Isoquinolines; Nerve Growth Factors; Neurons; Protein Kinases; Rats; Sulfonamides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured