okadaic-acid and adenosine-3--5--cyclic-phosphorothioate

Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 microM). WIN 55,212-2 was also shown to inhibit the NMDA-induced increase in intracellular calcium concentration ([Ca2+](i)) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca2+](i) occurred with similar concentrations (25-100 nM) and in the same time-dependent manner (pre-exposure 1-15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in [Ca2+](i) was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositol-trisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing [Ca2+](i) via a cAMP/PKA-dependent process during the neurotoxic event.

Topics: Animals; Benzoxazines; Calcium; Cell Culture Techniques; Cell Death; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclohexanols; Dantrolene; Drug Interactions; Estrenes; Fetus; Hippocampus; Macrocyclic Compounds; Morpholines; N-Methylaspartate; Naphthalenes; Neurons; Neuroprotective Agents; Okadaic Acid; Oxazoles; Piperidines; Pyrazoles; Pyrrolidinones; Rats; Rats, Inbred Strains; Receptor, Cannabinoid, CB1; Rimonabant; Ryanodine; Thionucleotides

Studies have suggested that integration of kinase and phosphatase activities maintains the steady-state L-type Ca(2+) current in ventricular myocytes, a balance disrupted in failing hearts. As we have recently reported that the PP1/PP2A inhibitor calyculin A evokes pronounced increases in L-type I(Ca), the goal of this study was to identify the counteracting kinase and phosphatase that determine 'basal'I(Ca) in isolated mouse ventricular myocytes. Whole-cell voltage-clamp studies, with filling solutions containing 10 mm EGTA, revealed that calyculin A (100 nm) increased I(Ca) at test potentials between -42 and +49 mV (44% at 0 mV) from a holding potential of -80 mV. It also shifted the V(0.5) (membrane potential at half-maximal) of both activation (from -17 to -25 mV) and steady-state inactivation (from -32 to -37 mV) in the hyperpolarizing direction. The broad-spectrum protein kinase inhibitor, staurosporine (300 nm), was without effect on I(Ca) when added after calyculin A. However, by itself, staurosporine decreased I(Ca) throughout the voltage range examined (50% at 0 mV) and blocked the response to calyculin A, indicating that the phosphatase inhibitor's effects depend upon an opposing kinase activity. The PKA inhibitors Rp-cAMPs (100 microm in the pipette) and H89 (1 microm) failed to reduce basal I(Ca) or to block the calyculin A-evoked increase in I(Ca). Likewise, calyculin A was still active with 10 mm intracellular BAPTA or when Ba(2+) was used as the charge carrier. These data eliminate roles for protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMKII) as counteracting kinases. However, the protein kinase C (PKC) inhibitors Ro 31-8220 (1 microm) and Gö 6976 (200 nm) decreased steady-state I(Ca) and blunted the effect of calyculin A. PP2A is not involved in this regulation as intracellular applications of 10-100 nm okadaic acid or 500 nm fostriecin failed to increase I(Ca). However, PP1 is important, as dialysis with 2 microm okadaic acid or 500 nm inhibitor-2 mimicked the increases in I(Ca) seen with calyculin A. These in situ studies identify constitutive activity of PP1 and the counteracting activity of certain isoforms of PKC, in pathways distinct from receptor-mediated signalling cascades, as regulatory components that determine the steady-state level of cardiac L-type I(Ca).

Topics: Animals; Biophysical Phenomena; Biophysics; Buffers; Calcium Channels, L-Type; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Electric Conductivity; Enzyme Inhibitors; Homeostasis; Isoenzymes; Male; Marine Toxins; Mice; Mice, Inbred Strains; Myocytes, Cardiac; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Kinase C; Protein Phosphatase 1; Staurosporine; Thionucleotides

One prominent manifestation of mechanical activity in hair cells is spontaneous otoacoustic emission, the unprovoked emanation of sound by an internal ear. Because active hair bundle motility probably constitutes the active process of nonmammalian hair cells, we investigated the ability of hair bundles in the bullfrog's sacculus to produce oscillations that might underlie spontaneous otoacoustic emissions. When maintained in the normal ionic milieu of the ear, many bundles oscillated spontaneously through distances as great as 80 nm at frequencies of 5-50 Hz. Whole-cell recording disclosed that the positive phase of movement was associated with the opening of transduction channels. Gentamicin, which blocks transduction channels, reversibly arrested oscillation; drugs that affect the cAMP phosphorylation pathway and might influence the activity of myosin altered the rate of oscillation. Increasing the Ca 2+ concentration rendered oscillations faster and smaller until they were suppressed; lowering the Ca 2+ concentration moderately with chelators had the opposite effect. When a bundle was offset with a stimulus fiber, oscillations were transiently suppressed but gradually resumed. Loading a bundle by partial displacement clamping, which simulated the presence of the accessory structures to which a bundle is ordinarily attached, increased the frequency and diminished the magnitude of oscillation. These observations accord with a model in which oscillations arise from the interplay of the hair bundle's negative stiffness with the activity of adaptation motors and with Ca 2+-dependent relaxation of gating springs.

Topics: Animals; Biological Clocks; Calcium; Cilia; Colforsin; Cyclic AMP; Diacetyl; Diffusion Chambers, Culture; Enzyme Inhibitors; Gentamicins; Hair Cells, Auditory; Iontophoresis; Microscopy, Video; Models, Biological; Molecular Motor Proteins; Okadaic Acid; Patch-Clamp Techniques; Physical Stimulation; Rana catesbeiana; Saccule and Utricle; Stress, Mechanical; Thionucleotides; Xanthines

Using the whole-cell patch-clamp technique in freshly isolated rat osteoclasts we examined the effects of estrogen on ionic channels. The predominant current was an inward rectifier K+ current (IKir). In the absence of non-osteoclastic cells, extracellularly applied 17beta-estradiol (>0.1 microM) inhibited IKir, indicating that estrogen acts directly on osteoclasts. Application of 17beta-estradiol (10 microM) for 10 min reduced IKir at the membrane potential of -120 mV to 70 +/- 15% of control. Removal of 17beta-estradiol partially restored the inhibition. The inhibition of IKir was dependent on concentration and application time. Intracellularly applied 17beta-estradiol had no effect on IKir. 17alpha-estradiol also inhibited the IKir, whereas progesterone and testosterone had no effect. The inhibitory action of 17beta-estradiol was not affected by guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), adenosine 3',5'-cyclic monophosphothioate Rp diastereomer (Rp-cAMPS), okadaic acid, staurosporine and phorbol ester, and was independent of intracellular Ca2+ concentration ([Ca2+]i). With no influence from soluble factors secreted from non-osteoclastic cells, preincubation of the osteoclasts for more than 60 min with much lower concentrations of 17beta-estradiol (1 and 10 nM) caused a reduction of IKir. In current-clamp configuration, application of 17beta-estradiol (10 microM) depolarized the membrane associated with a decrease in a membrane conductance, indicating that 17beta-estradiol inhibits IKir and depolarizes the membrane of osteoclasts. These results suggest that the 17beta-estradiol-induced inhibition of IKir might be mediated via non-genomic mechanisms. This direct action of 17beta-estradiol on osteoclasts may contribute to the regulation of [Ca2+]i and partially account for the protective effects of estrogen against bone loss.

Topics: Animals; Barium; Calcium; Cells, Cultured; Chelating Agents; Cyclic AMP; Dose-Response Relationship, Drug; Estradiol; Guanosine Diphosphate; Membrane Potentials; Okadaic Acid; Osteoclasts; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Inwardly Rectifying; Progesterone; Rats; Rats, Wistar; Staurosporine; Testosterone; Tetradecanoylphorbol Acetate; Thionucleotides; Time Factors

Neurotrophins have been implicated in activity-dependent synaptic plasticity, but the underlying intracellular mechanisms remain largely unknown. Synaptic potentiation induced by brain-derived neurotrophic factor (BDNF), but not neurotrophin 3, was prevented by blockers of adenosine 3',5'-monophosphate (cAMP) signaling. Activators of cAMP signaling alone were ineffective in modifying synaptic efficacy but greatly enhanced the potentiation effect of BDNF. Blocking cAMP signaling abolished the facilitation of BDNF-induced potentiation by presynaptic activity. Thus synaptic actions of BDNF are gated by cAMP. Activity and other coincident signals that modulate cAMP concentrations may specify the action of secreted neurotrophins on developing nerve terminals.

Topics: Animals; Brain-Derived Neurotrophic Factor; Carbazoles; Cells, Cultured; Cyclic AMP; Cycloleucine; Excitatory Postsynaptic Potentials; Indoles; Nerve Growth Factors; Neuronal Plasticity; Neurons; Neurotrophin 3; Okadaic Acid; Patch-Clamp Techniques; Pyrroles; Signal Transduction; Synapses; Synaptic Transmission; Thionucleotides; Xenopus

Bovine aortic endothelial cells produce prostacyclin as their major arachidonic acid metabolite. cAMP, in turn, is the second messenger for prostacyclin. In the present study, we investigated the effects of cAMP-elevating agents on prostacyclin production by bovine aortic endothelial cells. Treatment of resting bovine aortic endothelial cells with cAMP-elevating agents inhibited prostacyclin production and cyclooxygenase activity, without affecting arachidonic acid release. No change was detected in cyclooxygenase-1 protein expression. The specific inhibitor of protein kinase A, Rp-cAMPS (adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt), and the phosphatase inhibitor, okadaic acid, both suppressed cAMP-induced inhibition, suggesting that this inhibition is mediated by a phosphorylation-dephosphorylation cascade, which is possibly protein kinase A-dependent. In lipopolysaccharide-treated cyclooxygenase-2 expressing bovine aortic endothelial cells, where cyclooxygenase-1 activity was selectively inhibited, dibutyryl cAMP failed to inhibit cyclooxygenase-2 activity. Cyclooxygenase-2 protein was induced upon treatment with dibutyryl cAMP and further induction of cyclooxygenase-2 protein was effected by IBMX (3-isobutyl-1-methyl-xanthine) and dibutyryl cAMP in bacterial lipopolysaccharide-stimulated cells. These results suggest that increased cellular cAMP selectively inhibits cyclooxygenase-1 activity without altering cyclooxygenase-1 protein expression, and at the same time, up-regulates cyclooxygenase-2 protein. This complex regulation of cyclooxygenase activity and protein expression by cAMP may represent a prostacyclin-induced autoregulatory mechanism in bovine aortic endothelial cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Aorta; Arachidonic Acid; Blotting, Western; Bucladesine; Cattle; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Drug Interactions; Endothelium, Vascular; Enzyme Inhibitors; Epoprostenol; In Vitro Techniques; Isoenzymes; Lipopolysaccharides; Okadaic Acid; Prostaglandin-Endoperoxide Synthases; Proteins; Thionucleotides

Inhibition of Na+,K+-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na+,K+-ATPase activity in response to dopamine is associated with endocytosis of its alpha- and beta-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na+,K+-ATPase and inhibition of its activity were accompanied by phosphorylation of the alpha-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the alpha-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent alpha-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the alpha-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent alpha-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the alpha-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.

Topics: Animals; Cell Membrane; Cells, Cultured; Cyclic AMP; Endocytosis; Endosomes; Genes, myc; Kidney Tubules, Proximal; Macromolecular Substances; Male; Mutagenesis, Site-Directed; Okadaic Acid; Opossums; Phosphorylation; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Serine; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Thionucleotides; Transfection

The role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases in the process of secretin stimulation of fluid and bicarbonate secretion from biliary epithelium was examined using a novel isolated bile duct unit (IBDU) model from rat liver. Sp-adenosine 3',5'-cyclic monophosphothiolate (Sp-cAMPS), 100 microM, a PKA-specific agonist, significantly increased secretion during a 30-min perfusion (+61%, P < 0.01). In contrast, preincubation and perfusion of Rp-cAMPS, 100 microM, a specific PKA inhibitor, reduced the ability of secretin to stimulate both fluid secretion (111 vs. 25%; P < 0.01) and Cl-/HCO3- exchanger activity (80 vs. 28%). Neither the PKC agonist phorbol 12-myristate 13-acetate, 10 microM, nor the PKC antagonist staurosporine showed any effect on either basal or secretin-stimulated fluid secretion or Cl-/HCO3- exchange activity in IBDU. Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, also had no effect on basal fluid secretion or on the basal activity of the Cl-/HCO3- exchanger. However, okadaic acid resulted in persistence of secretion after removal of secretin, in contrast to the reduction in secretion observed in controls. These findings indicate that PKA but not PKC is involved in the signal transduction of secretin-stimulated fluid secretion and Cl-/HCO3- exchange activity in rat bile duct epithelium, a process inactivated by dephosphorylation by protein phosphatases 1 and/or 2A.

Topics: Animals; Antiporters; Bile Ducts; Body Fluids; Chloride-Bicarbonate Antiporters; Culture Techniques; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Male; Okadaic Acid; Phosphoric Monoester Hydrolases; Protein Kinase C; Rats; Rats, Sprague-Dawley; Secretin; Thionucleotides

(1) Glucagon activates hepatic glutaminase in vivo. Mitochondria from glucagon-injected rats retain an enhanced capacity to catabolize glutamine and this is more sensitive to activation by inorganic phosphate. The glucagon-elicited stimulation of glutaminase is not evident in broken mitochondria. A similar activation of glutaminase occurs in a number of situations which are associated with elevated glucagon levels in vivo, i.e., after a high-protein meal, after injection of bacterial endotoxin and in diabetes mellitus. (2) Studies in isolated hepatocytes revealed that glutaminase could be activated, not only by glucagon, but also by a cell-permeable protein kinase A activator (Sp-cAMPS) and by a cell-permeable protein phosphatase 1 and 2A inhibitor (okadaic acid). However, the activation of glutaminase by glucagon was not inhibited by a cell-permeable protein kinase A inhibitor (Rp-8-Br-cAMPS). We suggest that the signalling pathway, for glutaminase activation by glucagon, is complex and possibly contains redundant elements.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus, Experimental; Dietary Proteins; Endotoxins; Enzyme Activation; Enzyme Inhibitors; Ethers, Cyclic; Glucagon; Glutamic Acid; Glutaminase; Glutamine; Mitochondria, Liver; Okadaic Acid; Phosphates; Rats; Signal Transduction; Thionucleotides

The objective of this study was to investigate the importance of cAMP during capacitation of bovine sperm. The competitive antagonist of cAMP, Rp-adenosine-3'5'-cyclic monophosphorothioate (Rp-cAMP), blocked heparin-induced capacitation (p < 0.05). The effect of Rp-cAMP on heparin-induced capacitation was reversed by 8-bromo-cAMP. The maximal inhibitory effect on capacitation occuroff when Rp-cAMP was added at the start of sperm incubation. These results support an important role for cAMP-dependent protein kinases during heparin-induced capacitation of bovine sperm. Further support for a role for protein phosphorylation during capacitation came from the use of the protein phosphatase inhibitor, okadaic acid. Okadaic acid had no affect on heparin-induced capacitation of bovine sperm (p > 0.05); however, bovine sperm were capacitated by okadaic acid in the absence of heparin (p < 0.05). The relationship of cAMP to capacitation-associated changes in sperm intracellular pH (pHi) was investigated using image analysis of bovine sperm. The pHi of sperm increased during capacitation, and Rp-cAMP did not affect the change in pHi. We conclude that heparin-induced capacitation of bovine sperm involves an increase in cAMP and a protein phosphorylation event but that these do not induce the increase in pHi associated with capacitation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cattle; Cyclic AMP; Drug Interactions; Ethers, Cyclic; Heparin; Hydrogen-Ion Concentration; Male; Okadaic Acid; Phosphorylation; Protein Kinase Inhibitors; Sperm Capacitation; Spermatozoa; Thionucleotides