h-89 and Seizures

Leptomycin B (LMB), originally developed as an anti-fungal agent, has potent neuroprotective properties against status epilepticus (SE, a prolonged seizure activity). However, the pharmacological profiles and mechanisms of LMB for neuroprotection remain elusive. In the present study, we found that LMB increased phosphorylation levels of protein kinase A (PKA) catalytic subunits, protein phosphatase 2B (PP2B, calcineurin) and extracellular signal-regulated kinase 1/2 (ERK1/2) under normal condition, and abolished SE-induced neuronal death. Co-treatment of H-89 (a PKA inhibitor) with LMB could not affect the seizure latency and its severity in response to pilocarpine. However, H-89 co-treatment abrogated the protective effect of LMB on SE-induced neuronal damage. Cyclosporin A (CsA, a PP2B inhibitor) co-treatment effectively prevented SE-induced neuronal death without altered seizure susceptibility in response to pilocarpine more than LMB alone. H-89 co-treatment inhibited LMB-mediated ERK1/2 phosphorylation, but CsA enhanced it. U0126 (an ERK1/2 inhibitor) co-treatment abolished the protective effect of LMB on SE-induced neuronal death without alterations in PKA and PP2B phosphorylations. To the best of our knowledge, the present data demonstrate a previously unreported potential neuroprotective role of LMB against SE via PKA- and PP2B-mediated ERK1/2 activation.

Topics: Animals; Calcineurin; Cell Death; Cyclic AMP-Dependent Protein Kinases; Cyclosporine; Disease Models, Animal; Fatty Acids, Unsaturated; Hippocampus; Isoquinolines; Male; MAP Kinase Signaling System; Neurons; Neuroprotective Agents; Phosphorylation; Rats; Rats, Sprague-Dawley; Seizures; Status Epilepticus; Sulfonamides; Temporal Lobe

Preconditioning by N-methyl-d-aspartate (NMDA) may be promoted in vivo by the administration of a sub-convulsing dose of NMDA, with a neuroprotective effect against seizures and neuronal death induced by the infusion of quinolinic acid (QA) in mice. This study aimed to evaluate the participation of protein kinase C (PKC), cyclic AMP-dependent protein kinase (PKA), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), Ca(2+)/calmodulin dependent protein kinase II (CaMKII) and phosphatidilinositol-3 kinase (PI3K) signaling pathways in this neuroprotection model. Adult Swiss male mice were preconditioned with NMDA 24 h before the infusion of QA, and were treated with inhibitors of the aforementioned signaling pathways either 15 min before the preconditioning or infusion of QA. Inhibition of the PKA and PI3K pathways abolished the protection evoked by NMDA, and inhibition of the MEK pathway significantly diminished this protection. Treatment with PKC and CaMKII inhibitors did not alter the protection rate. Inhibition of the MEK and PKC pathways resulted in an increased mortality rate when followed by the infusion of QA, or NMDA preconditioning and QA infusion, respectively. These results suggest that the PKA, PI3K and MEK pathways have a crucial role in the achievement of a neuroprotective state following preconditioning.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Androstadienes; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Conditioning, Psychological; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Injections, Intraventricular; Isoquinolines; Male; Mice; Mitogen-Activated Protein Kinases; N-Methylaspartate; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolinic Acid; Receptors, N-Methyl-D-Aspartate; Seizures; Signal Transduction; Sulfonamides; Wortmannin

The present study shows interactive effects of pentoxifylline (PTX) as a phosphodiesterase (PDE) inhibitor, H-89 as a protein kinase A (PKA) inhibitor and bucladesine (db-cAMP) as a cAMP agonist on pentylenetetrazol (PTZ)-induced seizure in mice. Different doses of pentoxifylline (25, 50, 100 mg/kg), bucladesine (50, 100, 300 nM/mouse), and H-89 (0.05, 0.1, 0.2 mg/100g) were administered intraperitoneally (i.p.), 30 min before intravenous (i.v.) infusion of PTZ (0.5% w/v). In combination groups, the first and second components were injected 45 and 30 min before PTZ infusion. In all groups, the control animals received an appropriate volume of vehicle. Single administration of PTX had no significant effect on both seizure latency and threshold. Bucladesine significantly decreased seizure latency and threshold only at a high concentration (300 nM/mouse). Intraperitoneal administration of H-89 (0.2 mg/100g) significantly increased seizure latency and threshold in PTZ-treated animals. All applied doses of bucladesine in combination with PTX (50 mg/kg) caused a significant reduction in seizure latency. Pretreatment of animals with PTX (50 and 100 mg/kg) attenuated the anticonvulsant effect of H-89 (0.2 mg/100g) in PTZ-exposed animals. H-89 (0.05, 0.2 mg/100g) prevented the epileptogenic activity of bucladesine (300 nM) with significant increase of seizure latency and seizure threshold. In conclusion, we showed that seizure activities were affected by pentoxifylline, H-89 and bucladesine via interactions with intracellular cAMP and cGMP signaling pathways, cyclic nucleotide-dependent protein kinases, and related neurotransmitters.

Topics: Animals; Bucladesine; Dose-Response Relationship, Drug; Drug Interactions; Isoquinolines; Male; Mice; Pentoxifylline; Pentylenetetrazole; Phosphodiesterase Inhibitors; Protein Kinase Inhibitors; Seizures; Sulfonamides

Mitogen-activated protein kinase-activated protein kinase (MK)2 is one of several downstream targets of p38 mitogen-activated protein kinase and has a well documented role in inflammation. Here, we describe a possible new function of MK2. We show that triggering depolarization by potassium chloride or increasing the cellular cAMP by forskolin treatment led to elevated levels of expression and activity of mouse MK2. In both treatments, the kinase inhibitor H89 completely prevented the up-regulation of MK2 at the transcript level. By the use of different cell lines we demonstrated that the induction of MK2 expression is characteristic of neuronal cells and is absent in fibroblasts, macrophages and kidney cells. In vivo, induction of a status epilepticus by systemic administration of the chemoconvulsant kainic acid resulted in markedly reduced neurodegeneration in the pyramidal layer of the hippocampus, dentate gyrus and hilus of MK2-deficient mice compared with wild-type mice. Together, our data suggest a possible role of MK2 in the cellular response after neuronal depolarization, in particular in excitotoxicity.

Topics: Animals; Cells, Cultured; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Stability; Excitatory Amino Acid Agonists; Hippocampus; Humans; Intracellular Signaling Peptides and Proteins; Isoquinolines; Kainic Acid; Membrane Potentials; Mice; Mice, Knockout; Neurons; p38 Mitogen-Activated Protein Kinases; PC12 Cells; Potassium Chloride; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Rats; RNA, Messenger; Seizures; Signal Transduction; Sulfonamides

This study assessed the effects of drugs which manipulate the cAMP system on afterdischarges (ADs) induced in the CA1 region of rat hippocampal slices. The adenylate cyclase activator forskolin (50 microM) and the phosphodiesterase inhibitor rolipram (0.1 and 1 microM) enhanced AD generation. These effects were reversed by the cAMP-dependent protein kinase inhibitors H-89 (5 microM) and Rp-cAMPS (100 microM). These findings suggest that AD generation can be modulated through cAMP generation and the subsequent activation of the cAMP-dependent protein kinase.

Topics: Action Potentials; Adenylyl Cyclases; Animals; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Enzyme Activators; Enzyme Inhibitors; Epilepsy; Hippocampus; Isoquinolines; Male; Phosphodiesterase Inhibitors; Rats; Rats, Sprague-Dawley; Rolipram; Seizures; Sulfonamides; Thionucleotides