kn-62 and adenosine-3--5--cyclic-phosphorothioate

After naturalistic odor preference training, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was rapidly phosphorylated in the olfactory bulb, specifically in the odor encoding regions of the glomerular layer and external plexiform layer. Intrabulbar CaMKII antagonist experiments revealed that CaMKII supports short- and long-term preference memory formation. With bulbar PKA activation as the unconditioned stimulus odor preferences could be induced despite CaMKII blockade, but now odor specificity was lost, with odor preference generalizing to an untrained odor. Odor-specific learning was associated with increased membrane-associated AMPA receptors, while nonspecific odor preference was not. Thus CaMKII activation provides a tag to confer stimulus specificity as well as supporting natural odor preference learning.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Analysis of Variance; Animals; Animals, Newborn; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cyclic AMP; Enzyme Inhibitors; Female; Learning; Male; Memory; Odorants; Olfactory Bulb; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Thionucleotides; Time Factors

The cardiac ankyrin repeat protein (CARP), a nuclear transcription co-factor that negatively regulates cardiac gene expression, is increased in human heart failure and in animal models of cardiac hypertrophy. The mechanism by which CARP expression is regulated and the consequences of CARP overexpression on cardiac contractility are unknown.. Compared to vehicle treated controls, 4-day treatment of male Wistar rats with the beta-adrenoceptor agonist isoprenaline (2.4 mg/kg per day) induced hypertrophy and significantly increased CARP mRNA and CARP protein levels in left ventricles. The signalling pathways were investigated in more detail in isolated neonatal rat cardiomyocytes. Treatment of cells with isoprenaline (1 micromol/l) caused a significant increase in CARP mRNA and protein by approximately 50%. Combined beta(1)- and beta(2)-adrenoceptor blockade, inhibition of protein kinase A (PKA; Rp-cAMPS, 100 micromol/l), and inhibition of calmodulin-dependent protein kinases (CaMK; KN-62, 10 micromol/l) completely reversed the effects of isoprenaline. To examine the consequences of CARP overexpression on contractile function, an adenovirus encoding human CARP as well as a control virus were constructed. Although the basal force of contraction was not different, contractile response to Ca(2+) and isoprenaline was significantly diminished in engineered heart tissue infected with the recombinant adenovirus that carries the CARP gene (Ad.CARP).. Our study provides the first evidence that overexpression of CARP, which is thought to act as a transcriptional co-repressor, may deteriorate contractile function of the heart tissue. Furthermore, beta-adrenoceptor stimulation and activation of PKA and CaMK have been identified as mechanisms that induce expression of CARP in cardiomyocytes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenergic beta-Agonists; Animals; Blotting, Northern; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression; Hypertrophy, Left Ventricular; Infusion Pumps, Implantable; Isoproterenol; Male; Muscle Proteins; Myocardial Contraction; Myocardium; Nuclear Proteins; Protein Kinase Inhibitors; Protein Kinases; Rats; Rats, Wistar; Receptors, Adrenergic, beta; Repressor Proteins; RNA, Messenger; Thionucleotides

In the auditory system, inhibitory transmission from the medial nucleus of the trapezoid body (MNTB) to neurons of the lateral superior olivary nucleus (LSO) undergoes activity-dependent long-term depression, and may be associated with developmental elimination of these synapses [Sanes DH, Friauf E (2000).. development and influence of inhibition in the laterial superior olivary nucleus. Hear Res 147:46-58]. Although GABA(B) receptor activation and postsynaptic free calcium are implicated in this depression, little is known about intracellular signaling mechanisms in this or other forms of inhibitory plasticity. In this study, we asked whether the calcium dependency of inhibitory depression was associated with the activation of calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and/or cAMP-dependent protein kinase A (PKA). Whole-cell voltage-clamp recordings were obtained from LSO neurons in a brain slice preparation, permitting for the selective pharmacologic manipulation of individual postsynaptic LSO neurons. Inclusion of a CaMKII antagonist (KN-62) in the internal pipet solution blocked inhibitory synaptic depression. A second CaMKII inhibitor (autocamtide peptide fragment) significantly decreased inhibitory depression. Inclusion of a specific antagonist of protein kinase C (PKC fragment 19-36) in the internal recording solution also blocked inhibitory depression. To test involvement of a cAMP-dependent intracellular cascade, two different manipulations were performed. Inclusion of PKA antagonists (Rp-cAMPS or a cAMP dependent protein kinase inhibitor peptide) prevented inhibitory depression. In contrast, when a nonhydrolyzable cAMP analog (Sp-cAMPS) was permitted to enter the postsynaptic cell, the MNTB-evoked IPSCs became depressed in the absence of low-frequency stimulation. Thus, three key postsynaptic kinases, CaMKII, PKC, and PKA, participate in the activity-dependent depression of inhibitory MNTB-LSO synapses during postnatal development.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Animals, Newborn; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Gerbillinae; Long-Term Synaptic Depression; Neuronal Plasticity; Olivary Nucleus; Organ Culture Techniques; Patch-Clamp Techniques; Peptide Fragments; Peptides; Protein Kinase C; Protein Kinases; Protein Serine-Threonine Kinases; 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

We have previously demonstrated that neuropeptide Y (NPY) inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma (PC12) cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF). The present study uses multiple selective Ca2+ channel and protein kinase agonists and antagonists to elucidate the mechanisms by which NPY modulates catecholamine synthesis as determined by in situ measurement of DOPA production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The L-type Ca2+ channel blocker nifedipine inhibited the depolarization-induced stimulation of DOPA production by approximately 90% and attenuated the inhibitory effect of NPY. In contrast, the N-type Ca2+ channel blocker omega-conotoxin GVIA inhibited neither the stimulation of DOPA production nor the effect of NPY. Antagonism of Ca2+/calmodulin-dependent protein kinase (CaM kinase) greatly inhibited the stimulation of DOPA production by depolarization and prevented the inhibitory effect of NPY, whereas alterations in the cyclic AMP-dependent protein kinase pathway modulated DOPA production but did not prevent the effect of NPY. Stimulation of Ca2+/phospholipid-dependent protein kinase (PKC) with phorbol 12-myristate 13-acetate (PMA) did not affect the basal rate of DOPA production in NGF-differentiated PC12 cells but did produce a concentration-dependent inhibition of depolarization-stimulated DOPA production. In addition, NPY did not produce further inhibition of DOPA production in the presence of PMA, and the inhibition by both PMA and NPY was attenuated by the specific PKC inhibitor chelerythrine. These results indicate that NPY inhibits Ca2+ influx through L-type voltage-gated Ca2+ channels, possibly through a PKC-mediated pathway, resulting in attenuation of the activation of CaM kinase and inhibition of depolarization-stimulated catecholamine synthesis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenine; Animals; Calcium Channel Blockers; Calcium Channels; Calcium-Calmodulin-Dependent Protein Kinases; Carcinogens; Catecholamines; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Enzyme Inhibitors; Imidazoles; Ion Channel Gating; Isoquinolines; Neuropeptide Y; Nifedipine; omega-Conotoxin GVIA; PC12 Cells; Peptides; Piperazines; Protein Kinase C; Protein Kinases; Protein Synthesis Inhibitors; Rats; Tetradecanoylphorbol Acetate; Thionucleotides