h-89 and 1-9-dideoxyforskolin

In a previous study we showed that activation of a presynaptically located metabotropic glutamate receptor (mGluR) with pharmacological properties of mGluR4a causes a facilitation of glutamate release in layer V of the rat entorhinal cortex (EC) in vitro. In the present study we have begun to investigate the intracellular coupling linking the receptor to transmitter release. We recorded spontaneous alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated excitatory postsynaptic currents (EPSCs) in the whole cell configuration of the patch-clamp technique, from visually identified neurons in layer V. Bath application of the protein kinase A (PKA) activator, forskolin, resulted in a marked facilitation of EPSC frequency, similar to that seen with the mGluR4a specific agonist, ACPT-1. Preincubation of slices with the PKA inhibitor H-89 abolished the effect of ACPT-1, as did preincubation with the adenylate cyclase inhibitor, SQ22536. Activation of protein kinase C (PKC) using phorbol 12 myristate 13-acetate (PMA) did not affect sEPSC frequency; however, it did abolish the facilitatory effect of ACPT-1 on glutamate release. A robust enhancement of EPSC frequency was seen in response to bath application of the specific PKC inhibitor, GF 109203X. Both H-89 and the group III mGluR antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) abolished the effects of GF 109203X. These data suggest that in layer V of the EC, presynaptic group III mGluRs facilitate release via a positive coupling to adenylate cyclase and subsequent activation of PKA. We have also demonstrated that the PKC system tonically depresses transmitter release onto layer V cells of the EC and that an interaction between mGluR4a, PKA, and PKC may exist at these synapses.

Topics: Adenine; Animals; Colforsin; Cyclic AMP-Dependent Protein Kinases; Cyclopentanes; Entorhinal Cortex; Enzyme Activation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Glycine; In Vitro Techniques; Indoles; Isoquinolines; Male; Maleimides; Presynaptic Terminals; Protein Kinase C; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Sulfonamides; Tricarboxylic Acids

X-adrenoleukodystrophy (X-ALD) is an inherited fatty acid metabolic disorder with secondary manifestation of neuroinflammatory disease process. We report that compounds (forskolin, 8-bromo cAMP, and rolipram) that increase cAMP and activate protein kinase A (PKA) were found to stimulate the peroxisomal beta-oxidation of lignoceric acid (C24:0) whereas compounds (H-89 and myristoylated PKI) that decrease cAMP and PKA activity inhibited the peroxisomal beta-oxidation of lignoceric acid in cultured skin fibroblasts from X-ALD patients. Consistent with the stimulation of beta-oxidation of lignoceric acid, activators of PKA normalized the level of very long chain fatty acids (VLCFA) in X-ALD cultured skin fibroblasts. This normalization of VLCFA in X-ALD cells with forskolin, 8-Br cAMP or with rolipram, an inhibitor of cAMP phosphodiesterase, was realized independent of expression of mRNA or protein of the ALD gene, suggesting that cAMP derivatives can correct the metabolic defect in X-ALD fibroblasts without involving the candidate gene for the disease. Because astrocytes and microglia in demyelinating lesions of X-ALD brain express proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), we examined the effect of cAMP derivatives or rolipram on lipopolysaccharide-stimulated rat primary astrocytes and microglia and found that cAMP derivatives and rolipram inhibited the induction of TNF-alpha and IL-1beta in both astrocytes and microglia. The ability of cAMP derivatives and rolipram to block the induction of TNF-alpha and IL-1beta in astrocytes and microglia and to normalize the fatty acid pathogen in skin fibroblasts of x-adrenoleukodystrophy (X-ALD) clearly identify cAMP analogs or rolipram as candidates for potential therapy for X-ALD patients.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenoleukodystrophy; Animals; Astrocytes; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytokines; Enzyme Inhibitors; Fatty Acids; Genetic Linkage; Interleukin-1; Isoquinolines; Neuroglia; Oxidation-Reduction; Protein Kinase Inhibitors; Pyrrolidinones; Rats; Rolipram; Sulfonamides; Tumor Necrosis Factor-alpha; X Chromosome

Prolonged activation of an A2A adenosine receptor significantly inhibits the cellular response to subsequent stimulation (A2A desensitization). We have reported previously that activation of phosphodiesterase (PDE) contributes to A2A desensitization in PC12 cells. In the present study, we show that a type IV PDE (PDE4)-selective inhibitor (Ro 20-1724) effectively blocks the increase in PDE activity in desensitized cells. Thus, PDE4 appears to be the PDE specifically activated during A2A desensitization in PC12 cells. Prolonged treatment of PC12 cells with an A2A-selective agonist (CGS21680) leads to increased PDE4 activity in a dose-dependent manner, which can be blocked by an A2A-selective antagonist [8-(3-chlorostyryl)caffeine]. Using two PDE4 antibodies, we were able to demonstrate that the levels of two PDE4-immunoreactive bands (72 and 79 kDa) were increased significantly during A2A desensitization. Prolonged treatment with forskolin to elevate intracellular cyclic AMP contents also resulted in increased PDE4 activity. In addition, activation of PDE4 activity during A2A desensitization could be blocked by a protein kinase A (PKA)-selective inhibitor (H89) and was not observed in a PKA-deficient PC12 cell line (A123). Taken together, activation of PDE4 via a cyclic AMP/PKA-dependent pathway plays a critical role in dampening the signal of the A2A receptor.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenosine; Adrenal Gland Neoplasms; Animals; Blotting, Western; Caffeine; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 4; Enzyme Activation; Enzyme Inhibitors; Isoquinolines; Kinetics; PC12 Cells; Phenethylamines; Pheochromocytoma; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Polymerase Chain Reaction; Purinergic P1 Receptor Agonists; Rats; Receptor, Adenosine A2A; Receptors, Purinergic P1; Recombinant Proteins; Sulfonamides

The effects of increases in cellular adenosine 3'5'-cyclic monophosphate (cAMP) on 5-hydroxytryptamine-(5-HT-) induced generation of inositol phosphates (IPs) and increases in intracellular Ca2+ ([Ca2+]i) were investigated using canine cultured tracheal smooth muscle cells (TSMCs). Cholera toxin and forskolin induced concentration- and time-dependent cAMP formation with half-maximal effects (-logEC50) produced at concentrations of 7.0 +/- 0.5 and 4.9 +/- 0.4 respectively. Pretreatment of TSMCs with either forskolin or dibutyryl cAMP inhibited 5-HT-stimulated responses. Even after treatment for 24h, these agents still inhibited the 5-HT-induced Ca2+ mobilization. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration response curves of 5-HT. The water-soluble forskolin analogue L-858051 [7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated accumulation of IPs. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on this response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-isoquinolinesulphonamide] and HA-1004[N-(2-guanidinoethyl)-5-isoquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IPs. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The AlF4--induced accumulation of IPs was inhibited by forskolin, suggesting that G protein(s) are directly activated by AlF4-- and uncoupled from phospholipase C by forskolin treatment. These results suggest that activation of cAMP/PKA might inhibit the 5-HT-stimulated phosphoinositide breakdown and consequently reduce the [Ca2+]i increase or inhibit both responses independently.

Topics: Adenine; Aluminum Compounds; Animals; Bucladesine; Calcium; Cholera Toxin; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diterpenes; Dogs; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Fluorides; GTP-Binding Proteins; Isoquinolines; Male; Muscle, Smooth; PC12 Cells; Rats; Serotonin; Serotonin Antagonists; Sulfonamides; Trachea

The brain creatine kinase (CKB) gene is expressed in a variety of tissues with highest expression seen in the brain. We have previously shown in primary rat brain cell cultures that CKB mRNA levels are high in oligodendrocytes and astrocytes and low in neurons (Molloy et al.: J Neurochem 59:1925-1932, 1992). In this report we show that treatment of human U87 glioblastoma cells with forskolin and IBMX, to elevate intracellular cAMP, induces expression of CKB mRNA from the transiently transfected rat CKB gene by 14-fold and also increases expression from the endogenous human CKB gene. This induction of CKB mRNA i) is due to increased transcription; ii) occurs rapidly (with maximal induction after 6 hr; iii) requires the activity of protein kinase A (PKA), but iv) does not require de novo protein synthesis and, in fact, is superinduced in the presence of cycloheximide. Given the role of oligodendrocytes in the energy-demanding process of myelination and of astrocytes in ion transport, these results have physiological significance, since they suggest that changes in cellular energy requirements in the brain during events, such as glial cell differentiation and increased neuronal activity, may in part be met by a cAMP-mediated modulation of CKB gene expression. Of particular importance is the possible modulation of CKB gene expression during myelinogenesis, since oligodendrocyte differentiation has been shown previously to be stimulated by increases in cAMP.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Animals; Brain; Brain Neoplasms; Cloning, Molecular; Colforsin; Creatine Kinase; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Energy Metabolism; Enzyme Activation; Enzyme Induction; Glioblastoma; Glioma; HeLa Cells; Humans; Isoenzymes; Isoquinolines; Myelin Sheath; Nerve Tissue Proteins; Neuroglia; Protein Synthesis Inhibitors; Rats; Recombinant Fusion Proteins; RNA, Antisense; Sulfonamides; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The effect of intracellular cyclic AMP (cAMP) on N-methyl-D-aspartate (NMDA) receptor-mediated stimulation of nitric oxide (NO) formation was investigated in rat cerebellar slices. Forskolin (30-120 microM), while lacking any direct effect on NO production, elicited a concentration-dependent enhancement of the response to 10 microM NMDA. Dideoxyforskolin, which does not activate adenylyl cyclase did not influence the NMDA response. Increasing intracellular cAMP directly by incubation with the membrane-permeant analogue of cAMP, 2'-o-dibutyryladenosine 3'5'-cyclic monophosphate (dibutyryl cAMP) (1 mM), similarly enhanced NO formation, as did prevention of cAMP degradation with the phosphodiesterase inhibitor theophylline. The enhancement of NMDA activity appeared to involve protein phosphorylation (possibly of the receptor itself) since the protein kinase A inhibitor H-89, abolished the enhancements with both forskolin and dibutyryl cAMP. Thus cAMP may have a physiological role in the modulation of NMDA receptor-stimulated synthesis of NO.

Topics: Animals; Cerebellum; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Isoquinolines; Male; N-Methylaspartate; Nitric Oxide; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Sulfonamides