calyculin-a and 1-3-dipropyl-8-cyclopentylxanthine

We have previously reported that varying stimulus intensity produces qualitatively different types of synaptic plasticity in area CA1 of hippocampal slices: brief low-intensity (LI) theta-burst (TB) stimuli induce long-term potentiation (LTP), but if the stimulus intensity is increased (to mimic conditions that may exist during seizures), LTP is not induced; instead, high-intensity (HI) TB stimuli erase previously induced LTP ("TB depotentiation"). We now have explored the mechanisms underlying TB depotentiation using extracellular field recordings with pharmacological manipulations. We found that TB depotentiation was blocked by okadaic acid and calyculin A (inhibitors of serine/threonine protein phosphatases PP1 and PP2A), FK506 (a specific blocker of calcineurin, a Ca(2+)/calmodulin (CaM) protein phosphatase), and 8-Br-cAMP (an activator of protein kinase A) with 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor). These results suggest that protein phosphatase pathways are involved in the TB depotentiation similar to other type of down-regulating synaptic plasticity such as low-frequency stimulation (LFS)-induced long-term depression (LTD) and depotentiation in the rat hippocampus. However, TB depotentiation and LFS depotentiation could have differential functional significance.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcineurin; Calcineurin Inhibitors; Cyclic AMP-Dependent Protein Kinases; Electrophysiology; Enzyme Inhibitors; Hippocampus; Male; Marine Toxins; Membrane Potentials; Okadaic Acid; Organ Culture Techniques; Oxazoles; Phosphodiesterase Inhibitors; Phosphoprotein Phosphatases; Rats; Rats, Sprague-Dawley; Theta Rhythm; Xanthines

Recent studies have demonstrated that several compounds with diverse structures can activate wild-type cystic fibrosis transmembrane conductance regulator (CFTR) by non-receptor-mediated mechanisms. Some of these compounds have been shown to enhance cAMP-dependent activation of DeltaF508-CFTR. This study was undertaken to compare the mechanisms by which genistein, IBMX, milrinone, 8-cyclopentyl-1, 3-dipropylxanthine (CPX), the benzimidazolone NS004, and calyculin A increase CFTR activity. Our studies demonstrate that, in transfected NIH-3T3 cells, maximal enhancements of forskolin-dependent DeltaF508-CFTR activity are greatest with genistein, IBMX, and NS004. Milrinone, genistein, CPX, NS004, and calyculin A do not increase cellular cAMP. Because forskolin and calyculin A increase in vivo phosphorylation of cAMP binding response element (CREB), the inability of milrinone, genistein, CPX, and NS004 to increase CREB phosphorylation suggests that they do not stimulate protein kinase A or inhibit phosphatase activity. Our data suggest that the mechanisms by which genistein and NS004 activate CFTR differ. We also demonstrate that, in NIH-3T3 cells, IBMX-dependent enhancement of cAMP-dependent CFTR activity is not due to an increase in cellular cAMP and may involve a mechanism like that of genistein.

Topics: 1-Methyl-3-isobutylxanthine; 3T3 Cells; Animals; Benzimidazoles; Chlorophenols; Colforsin; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Inhibitors; Genistein; Humans; Kinetics; Marine Toxins; Mice; Oxazoles; Phosphopeptides; Phosphorylation; Recombinant Proteins; Sequence Deletion; Transfection; Xanthines