kn-93 and decamethrin

When capsaicin is applied repeatedly to dorsal root ganglion (DRG) neurons for brief periods (10-15 s) at short intervals (5-10 min), the evoked responses rapidly decline, a phenomenon termed tachyphylaxis. In addition to this phenomenon, the present study using Ca(2+) imaging revealed that repeated application of capsaicin to rat dissociated DRG neurons at longer intervals (20-40 min) or during multiple applications at short intervals elicited an enhancement of the responses, termed potentiation. The potentiation occurred in 50-60% of the capsaicin-responsive cells, on average representing a 20- to 30% increase in the peak amplitude of the Ca(2+) signal, and was maximal at a 40-min application interval. An analysis of the mechanisms underlying potentiation revealed that it was suppressed by block of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) with 5 μM KN-93 or block of the activation of extracellular signal-regulated kinase (ERK) 1/2 with 2 μM U-0126. Lowering the extracellular Ca(2+) concentration from 2 to 1 mM or pretreatment with deltamethrin (1 μM), which blocks calcineurin and tachyphylaxis, enhanced potentiation. Potentiation was not affected by: 1) inhibition of protein kinase C or protein kinase A, 2) block of the three subtypes of neurokinin receptors, or 3) block of the trafficking of transient receptor potential V1 channel to the membrane. These results indicate that the potentiation is a slowly developing Ca(2+)-modulated process that is mediated by a complex intracellular signaling pathway involving activation of CaMKII and ERK1/2. Potentiation may be an important peripheral autosensitization mechanism that occurs independently of the pronociceptive effects of inflammatory mediators and neurotrophic factors.

Topics: Animals; Benzylamines; Butadienes; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Capsaicin; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Ganglia, Spinal; Male; Membrane Potentials; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuronal Plasticity; Neurons; Nitriles; Patch-Clamp Techniques; Protein Kinase C; Protein Kinase Inhibitors; Pyrethrins; Rats; Rats, Sprague-Dawley; Recovery of Function; Sensory System Agents; Substance P; Sulfonamides; Tachyphylaxis; Time Factors; TRPV Cation Channels

Axon pathfinding depends on attractive and repulsive turning of growth cones to extracellular cues. Localized cytosolic Ca2+ signals are known to mediate the bidirectional responses, but downstream mechanisms remain elusive. Here, we report that calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase provide a switch-like mechanism to control the direction of Ca(2+)-dependent growth cone turning. A relatively large local Ca2+ elevation preferentially activates CaMKII to induce attraction, while a modest local Ca2+ signal predominantly acts through CaN and phosphatase-1 (PP1) to produce repulsion. The resting level of intracellular Ca2+ concentrations also affects CaMKII/CaN operation: a normal baseline allows distinct turning responses to different local Ca2+ signals, while a low baseline favors CaN-PP1 activation for repulsion. Moreover, the cAMP pathway negatively regulates CaN-PP1 signaling to inhibit repulsion. Finally, CaMKII/CaN-PP1 also mediates netrin-1 guidance. Together, these findings establish a complex Ca2+ mechanism that targets the balance of CaMKII/CaN-PP1 activation to control distinct growth cone responses.

Topics: Animals; Benzylamines; Calcineurin; Calcium; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Chlorocebus aethiops; COS Cells; Cyclic AMP; Cyclic GMP; Cyclosporine; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Embryo, Mammalian; Embryo, Nonmammalian; Enzyme Inhibitors; Growth Cones; Humans; Models, Neurological; Nerve Growth Factors; Netrin-1; Neurons; Nitriles; Okadaic Acid; Phosphoprotein Phosphatases; Photolysis; Protein Phosphatase 1; Pyrans; Pyrethrins; Semaphorin-3A; Spinal Cord; Spiro Compounds; Sulfonamides; Time Factors; Tumor Suppressor Proteins; Xenopus

Depolarization has been known to play an important role in the neuronal damage that occurs following cerebral ischemia. In the present study, we investigated the roles of calmodulin (CaM) and CaM-dependent enzymes in depolarization-induced neuronal cell death. Treatment of primary cortical neurons with 10 microM veratridine, a voltage sensitive Na(+) channel activator, induced cell death as indicated by lactate dehydrogenase leakage from neurons. CaM antagonists (calmidazolium, trifluoperazine, W-7, and W-5) inhibited cell death induced by veratridine in a concentration-dependent manner. CaM kinase II (CaMKII) inhibitors (KN-62, KN-93, and myristoylated autocamtide-2 related inhibitory peptide), but not inhibitors of nitric oxide synthase or calcineurin, prevented veratridine-induced neuronal cell death. Veratridine rapidly activated CaMKII in neurons, and CaM antagonists and a CaMKII inhibitor suppressed the CaMKII activation. These results suggest that the CaM-CaMKII pathway contributes to depolarization-evoked cell death in neurons.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Death; Cells, Cultured; Cerebral Cortex; Egtazic Acid; Enzyme Inhibitors; Fetus; Kinetics; Membrane Potentials; Neurons; NG-Nitroarginine Methyl Ester; Nifedipine; Nitriles; Pyrethrins; Rats; Sulfonamides; Tacrolimus; Trifluoperazine; Veratridine