okadaic-acid and 1-2-dioctanoylglycerol

Arginine vasopressin (AVP) stimulates adrenocorticotropin (ACTH) secretion from corticotroph cells of the anterior pituitary via activation of the V1b vasopressin receptor, a member of the G protein-coupled receptor (GPCR) family. Recently, we have shown that treatment of ovine anterior pituitary cells with AVP for short periods results in reduced responsiveness to subsequent stimulation with AVP. The aim of this study was to investigate mechanisms involved in this desensitization process. Among the GPCR family, rapid desensitization is commonly mediated by receptor phosphorylation, with resensitization being mediated by internalization and subsequent dephosphorylation of the receptors by protein phosphatases. Since desensitization of V1a vasopressin receptors is mediated by protein kinase C-mediated receptor phosphorylation, we investigated the involvement of this enzyme in desensitization of the ACTH response to AVP. Treatment of perifused ovine anterior pituitary cells with the specific protein kinase C (PKC) activator 1,2-dioctanoyl-sn-glycerol (300 microM) did not induce any reduction in response to a subsequent 5-min stimulation with 100 nM AVP, despite potently stimulating ACTH secretion. Likewise, the results obtained using the PKC inhibitor Ro 31-8220 were not consistent with involvement of PKC in AVP desensitization: 2 microM Ro 31-8220 did not reduce the ability of a 10 nM AVP pretreatment to induce desensitization to a subsequent stimulation with 100 nM AVP. Pharmacologic blockade of receptor internalization by treatment with 0.25 mg/ml concanavalin A significantly impaired the ability of a 15-min pretreatment with 10 nM AVP to induce desensitization, rather than affecting resensitization. Treatment with 10 nM okadaic acid, an inhibitor of protein phosphatase 1 and 2A, had no effect on either resensitization or desensitization. In contrast, inhibition of protein phosphatase 2B (PP2B) with 1 microM FK506 decreased the rate of resensitization: complete recovery from desensitization took 40 min, whereas in controls recovery was complete 20 min after termination of the pretreatment. These results indicate that desensitization of the ACTH response to AVP is not mediated by PKC-catalyzed phosphorylation, suggesting subtype-specific differences in the regulation of V1a and V1b vasopressin receptors. The data demonstrate that desensitization was dependent, at least in part, upon receptor internalization and that resensitization was dependent upon PP2B

Topics: Adrenocorticotropic Hormone; Animals; Arginine Vasopressin; Calcineurin Inhibitors; Casein Kinase I; Cells, Cultured; Concanavalin A; Diglycerides; Dose-Response Relationship, Drug; Indoles; Okadaic Acid; Phosphoprotein Phosphatases; Pituitary Gland, Anterior; Protein Kinase C; Protein Phosphatase 1; Protein Phosphatase 2; Receptors, Vasopressin; Sheep; Stimulation, Chemical; Tacrolimus; Time Factors

The eggs of Urechis unicinctus Von Drasche, an echiuroid, are arrested at P-I stage in meiosis. The meiosis is reinitiated by fertilization. Immunoblotting analysis using anti-ERK2 and anti-phospho-MAPK antibodies revealed a 44 kDa MAP kinase species that was constantly expressed in U. unicinctus eggs, quickly phosphorylated after fertilization, and dephosphorylated slowly before the completion of meiosis I. Phosphorylation of the protein was not depressed by protein synthesis inhibitor Cycloheximide (CHX), but was depressed by the MEK1 inhibitor PD98059. Under PD98059 treatment, polar body extrusion was suppressed and the function of centrosome and spindle was abnormal though GVBD was not affected, indicating that MAP kinase cascade was important for meiotic division of U. unicinctus eggs. Other discovery includes: A23187 and OA could parthenogenetically activate U. unicinctus eggs and phosphorylated 44 kDa MAP kinase species, indicating that the effect of fertilization on reinitiating meiosis and phosphorylation of 44 kDa MAP kinase specie is mediated by raising intracellular free calcium and by phosphorylation of some proteins, and that phosphotase(s) sensitive to OA is responsible for arresting U. unicinctus eggs in prophase I. diC8, an activator of PKC, accelerated the process of U. unicinctus egg meiotic division after fertilization and accelerated the dephosphorylation of 44 kDa MAP kinase specie, which implied that the acceleration effect of PKC on meiotic division was mediated by inactivation of MAP kinase cascade. Elevating cAMP/PKA level in U. unicinctus eggs had no effect on meiotic division of the eggs.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Annelida; Calcimycin; Chromosomes; Colforsin; Cycloheximide; Diglycerides; Extracellular Signal-Regulated MAP Kinases; Fertilization; Flavonoids; MAP Kinase Signaling System; Meiosis; Okadaic Acid; Ovum; Parthenogenesis; Phosphorylation

Mitogen-activated protein (MAP) kinase, protein kinase C (PKC), cAMP, and okadaic acid (OA)-sensitive protein phosphatases (PPs) have been suggested to be involved in oocyte meiotic resumption. However, whether these protein kinases and phosphatases act by independent pathways or interact with each other in regulating meiosis resumption is unknown. In the present study, we aimed to determine the regulation of meiosis resumption and MAP kinase phosphorylation by PKC, cAMP, and OA-sensitive PPs in rat oocytes using an in vitro oocyte maturation system and Western blot analysis. We found that ERK1 and ERK2 isoforms of MAP kinases existed in a dephosphorylated (inactive) form in germinal vesicle breakdown (GVBD)-incompetent and GVBD-competent germinal vesicle intact (GVI) oocytes as well as GVBD oocytes at equivalent levels. These results indicate that MAP kinases are not responsible for the initiation of normal meiotic resumption in rat oocytes. However, when GVBD-incompetent and GVBD-competent oocytes were incubated in vitro for 5 h, MAP kinases were phosphorylated (activated) in GVBD-competent oocytes, but not in meiotic-incompetent oocytes, suggesting that oocytes acquire the ability to phosphorylate MAP kinase during acquisition of meiotic competence. We also found that both meiosis resumption and MAP kinase phosphorylation were inhibited by PKC activation or cAMP elevation. Moreover, these inhibitory effects were overcome by OA, which inhibited PP1/PP2A activities. These results suggest that both cAMP elevation and PKC activation inhibit meiosis resumption and MAP kinase phosphorylation at a step prior to OA-sensitive protein phosphatases. In addition, inhibitory effects of cAMP elevation on meiotic resumption and MAP kinase phosphorylation were not reversed by calphostin C-induced PKC inactivation, indicating that cAMP inhibits both meiotic resumption and MAP kinase activation in a PKC-independent manner.

Topics: Animals; Blotting, Western; Cyclic AMP; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Female; Kinetics; Meiosis; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Naphthalenes; Okadaic Acid; Oocytes; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate

Neutrophils contain a 21-kDa phosphoprotein that undergoes rapid dephosphorylation upon stimulation of these cells with the chemoattractant N-fMet-Leu-Phe (fMLP), activators of protein kinase C [e.g., 4 beta-phorbol 12-myristate 13-acetate (PMA)] or the calcium ionophore A23187. This phosphoprotein was identified as the non-muscle form of cofilin by peptide sequencing and immunoblotting with specific antibodies. Evidence is presented that in neutrophils cofilin is regulated by a continual cycle of phosphorylation and dephosphorylation, and that the phosphatase undergoes activation during cell stimulation. Experiments with a wide variety of antagonists further suggested that the protein kinase that participates in these reactions may be a novel enzyme. The kinetics of cofilin dephosphorylation in neutrophils stimulated with fMLP or PMA were very similar to those observed for superoxide (O2-) release. Immunofluorescent studies revealed that cofilin was present throughout the cytosol of resting neutrophils and underwent rapid translocation to the F-actin-rich, ruffled membranes of stimulated cells. Cytochemical analysis further revealed that the ruffled membranes also contained large amounts of hydrogen peroxide (H2O2), a product of the O2-/H2O2-generating activity of stimulated neutrophils (NADPH oxidase). Cofilin is therefore well placed to participate in the continual polymerization and depolymerization of F-actin that is thought to give rise to the oscillatory pattern of H2O2 production observed under certain conditions.

Topics: Actin Depolymerizing Factors; Actins; Amino Acid Sequence; Animals; Calcimycin; Cell Membrane; Diglycerides; Diterpenes; Guinea Pigs; Humans; Hydrogen Peroxide; Immunoblotting; Marine Toxins; Microfilament Proteins; Molecular Sequence Data; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Neutrophil Activation; Neutrophils; Okadaic Acid; Oxazoles; Oxides; Phosphorylation; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate

Protein phosphorylation and subsequent dephosphorylation was studied in digitonin-permeabilized neuroblastoma SH-SY5Y cells by measuring the incorporation of [32P]phosphate into myelin basic protein (MBP). 1,2-Dioctanoyl-sn-glycerol (DOG) and calcium synergistically induced phosphorylation of MBP, which was inhibited by the protein kinase C (PKC) pseudosubstrate peptide (PKC19-36). The phosphorylation increased for 10 min when a net dephosphorylation started to appear. The dephosphorylation was inhibited by okadaic acid. Regardless of calcium concentration, the presence of DOG was necessary for significant effects of okadaic acid on MBP phosphorylation. H7 and staurosporine dose-dependently inhibited the phosphorylation of MBP, induced by DOG and calcium in the presence of okadaic acid. Different PKC pseudosubstrate peptides were applied and all showed an inhibitory effect on the phosphorylation of MBP under these conditions. These results demonstrate the presence, in SH-SY5Y cells, of a protein phosphatase, possibly protein phosphatase 2A, with a high basal activity that counteracts PKC-induced phosphorylation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Amino Acid Sequence; Calcium; Cell Membrane Permeability; Diglycerides; Ethers, Cyclic; Humans; Isoenzymes; Isoquinolines; Molecular Sequence Data; Myelin Basic Protein; Neoplasm Proteins; Neuroblastoma; Okadaic Acid; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorylation; Piperazines; Polymyxin B; Protein Kinase C; Protein Phosphatase 2; Protein Processing, Post-Translational; Signal Transduction; Staurosporine; Substrate Specificity; Tumor Cells, Cultured

Recently, the ligand-receptor signal transduction mechanism has been implicated in mediating the zona pellucida (ZP)-induced acrosome reaction. Little is known about the role of protein phosphorylation in this specific event. We examine whether modification of protein phosphorylation and dephosphorylation affects the kinetics of the acid-solubilized ZP-induced acrosome reaction of mouse sperm. Mouse epididymal sperm were incubated in modified Krebs-Ringer bicarbonate medium for a period of 90 to 120 min and then treated with 2 acid-solubilized ZP/microliters for an additional 60 min. The chlortetracycline fluorescence assay was used to monitor the acrosome reaction. Capacitated sperm were inhibited from undergoing acid-solubilized ZP-induced acrosome reaction in the presence of an inhibitor of cyclic nucleotide-dependent protein kinase, H8; activators of the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C); an inhibitor of phosphatases 1 and 2A, okadaic acid; or an inhibitor of protein tyrosine kinases, genistein. The addition of inhibitors of protein kinase C, such as staurosporine, H7, and protein kinase C [19-36] pseudosubstrate, inhibited the phorbol ester-dependent inhibition of the acid-solubilized ZP-induced acrosome reaction. The present study suggests that protein phosphorylation and dephosphorylation play a regulatory role in the process of the ZP-induced acrosome reaction.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acrosome; Alkaloids; Animals; Diglycerides; Ethers, Cyclic; Exocytosis; Female; Genistein; Isoflavones; Isoquinolines; Male; Membrane Fusion; Mice; Okadaic Acid; Phorbol Esters; Phosphoprotein Phosphatases; Phosphorylation; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Protein Processing, Post-Translational; Signal Transduction; Sperm-Ovum Interactions; Staurosporine; Tetradecanoylphorbol Acetate; Zona Pellucida

We examined effects of modulators of protein kinases and phosphatases on the kinetics of mouse sperm capacitation. The chlortetracycline fluorescence assay was used to monitor the process of capacitation (in terms of the appearance of the B pattern). The treatment of sperm with dibutyryl cyclic AMP (cAMP) or dibutyryl cGMP resulted in a higher percentage B pattern at various times during capacitation compared with the control. The addition of 100 microM H8 inhibited the cyclic nucleotide-dependent stimulation of capacitation. Tumor promotors, 12-O-tetradecanoyl phorbol 13-acetate (TPA; a stimulator of protein kinase C) and okadaic acid (an inhibitor of protein phosphatases 1 and 2A), induced a rapid appearance of the B pattern (15 min after addition) and maintained a percentage B pattern similar to that of the control in the later period of capacitation. An inhibitor of protein kinase C, staurosporine, inhibited the TPA-dependent acceleration of capacitation. Furthermore, the addition of genistein, an inhibitor of protein tyrosine kinases, resulted in a strong inhibition of capacitation. All agents tested did not affect sperm motility. These data suggest that protein phosphorylation and dephosphorylation may play regulatory roles in mediating mouse sperm capacitation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Bucladesine; Chlortetracycline; Dibutyryl Cyclic GMP; Diglycerides; Ethers, Cyclic; Fluorescence; Genistein; Isoflavones; Isoquinolines; Male; Mice; Okadaic Acid; Peptide Fragments; Phorbol Esters; Phosphoprotein Phosphatases; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Sperm Capacitation; Staurosporine; Tetradecanoylphorbol Acetate

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine; Adrenal Medulla; Animals; Biological Transport; Calcimycin; Caprylates; Cells, Cultured; Diglycerides; Dinucleoside Phosphates; Enzyme Activation; Ethers, Cyclic; Isoquinolines; Kinetics; Okadaic Acid; Phorbol 12,13-Dibutyrate; Piperazines; Protein Kinase C; Tetradecanoylphorbol Acetate; Triglycerides

Laminin mediates neural adhesion and process formation. A possible signal transduction pathway for laminin was investigated in both NG108-15 and PC12 neuronal cells using radiolabeling studies as well as various stimulators and inhibitors of phosphatases and kinases. Using [32P]-ortho-phosphate, laminin caused a decrease in the TCA-precipitable counts. Further, laminin stimulated dephosphorylation of laminin binding proteins of 110 kDa, 67 kDa, and 45 kDa and this dephosphorylation was blocked by the phosphatase inhibitor, okadaic acid, and the protein kinase C stimulator, TPA. The phosphatase inhibitors okadaic acid and vanadate, as well as the protein kinase C stimulators, TPA and DAG, blocked laminin-mediated process formation. Inhibitors of kinase activity such as H-7, H-8, and H-9 increased laminin-mediated neural process formation. Since phosphate incorporation into laminin-binding proteins is decreased by laminin and because both phosphatase inhibitors and kinase stimulators inhibit laminin-mediated process formation, we conclude that dephosphorylation events promote the neural cell response to laminin.

Topics: Adrenal Gland Neoplasms; Animals; Cell Adhesion; Diglycerides; Ethers, Cyclic; Glioma; Laminin; Neoplasm Proteins; Neuroblastoma; Neurons; Okadaic Acid; Pheochromocytoma; Phorbol Esters; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Rats; Signal Transduction; Tumor Cells, Cultured; Vanadates