cyclic-gmp and tautomycin

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

In the present study, the role of phosphoprotein phosphatase in the regulation of L-type Ca2+ channel currents in rat pinealocytes was investigated using the whole-cell version of the patch-clamp technique. The effects of three phosphatase inhibitors, calyculin A, tautomycin, and okadaic acid, were compared. Although all three inhibitors were effective in inhibiting the L-type Ca2+ channel current, calyculin A was more potent than either tautomycin or okadaic acid, suggesting the involvement of phosphoprotein phosphatase-1. To determine the kinase involved in the regulation of these channels, cells were pretreated with H7 (a nonspecific kinase inhibitor), H89 (a specific inhibitor of cyclic AMP-dependent kinase), KT5823 (a specific inhibitor of cyclic GMP-dependent kinase), or calphostin C (a specific inhibitor of protein kinase C). Pretreatment with either H7 or calphostin C decreased the inhibitory effect of calyculin A on the L-type Ca2+ channel current. In contrast, pretreatment with H89 or KT5823 had no effect on the inhibition caused by calyculin A. Based on these observations, we conclude that basal phosphatase activity, probably phosphoprotein phosphatase-1, plays an important role in the regulation of L-type Ca2+ channel currents in rat pinealocytes by counteracting protein kinase C-mediated phosphorylation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenergic beta-Agonists; Alkaloids; Animals; Antifungal Agents; Calcium Channels; Calcium Channels, L-Type; Carbazoles; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Indoles; Isoproterenol; Isoquinolines; Male; Marine Toxins; Membrane Potentials; Nerve Tissue Proteins; Norepinephrine; Okadaic Acid; Oxazoles; Patch-Clamp Techniques; Phosphoric Monoester Hydrolases; Phosphorylation; Pineal Gland; Protein Kinase C; Pyrans; Rats; Rats, Sprague-Dawley; Spiro Compounds; Sulfonamides; Sympathomimetics

The role of phosphoprotein phosphatase in the regulation of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rat pinealocytes was investigated using the three phosphatase inhibitors calyculin A, tautomycin, and okadaic acid. Calyculin A (0.1 microM) was found to enhance the isoproterenol- and norepinephrine-stimulated cAMP accumulation six- and threefold, respectively, whereas tautomycin and okadaic acid were less effective. The effect of calyculin A was rapid (within 5 min) and persisted in the presence of phosphodiesterase inhibition. However, in contrast to protein kinase C activation or intracellular calcium elevation, the phosphatase inhibitors were less effective in potentiating the cAMP response stimulated by forskolin or cholera toxin, and their effects were not blocked by calphostin C or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. The adrenergic-stimulated cGMP response was also less sensitive to the phosphatase inhibition. Therefore, our results suggest that 1) the adrenergic-stimulated cAMP signal is subjected to the tonic inhibition by phosphoprotein phosphatase; 2) phosphatase inhibitors enhance cAMP synthesis through their actions at the receptor level; and 3) the cAMP signal is more sensitive to the regulation by phosphorylation than cGMP in rat pinealocytes.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Antifungal Agents; Cyclic AMP; Cyclic GMP; Drug Synergism; Ethers, Cyclic; Isoproterenol; Kinetics; Male; Marine Toxins; Norepinephrine; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Pineal Gland; Protein Phosphatase 2; Pyrans; Rats; Rats, Sprague-Dawley; Spiro Compounds