Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.     

Inhibition of Dopamine Agonist-Induced Phosphoinositide Hydrolysis by Concomitant Stimulation of Cyclic AMP Formation in Brain Slices

Abstract: We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp -cyclic adenosine monophosphothioate ( Sp -cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D₁ receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp -cyclic adenosine monophosphothioate ( Rp -cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 µ M Rp -cAMPS, the dose-response curves of the dopamine D₁ receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC₅₀ values, however, were not significantly altered by protein kinase A inhibition. Neither Sp -cAMPS nor Rp -cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D₁ receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.     

Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N-and L-type calcium channels.     

Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin     

Hormone- and growth factor-stimulated NADH oxidase

An NADH oxidase activity of animal and plant plasma membrane is described that is stimulated by hormones and growth factors. In plasma membranes of cancer cells and tissues, the activity appears to be constitutively activated and no longer hormone responsive. With drugs that inhibit the activity, cells are unable to grow although growth inhibition may be more related to a failure of the cells to enlarge than to a direct inhibition of mitosis. The hormone-stimulated activity in plasma membranes of plants and the constitutively activated NADH oxidase in tumor cell plasma membranes is inhibited by thiol reagents whereas the basal activity is not. These findings point to a thiol involvement in the action of the activated form of the oxidase. NADH oxidase oxidation by Golgi apparatus of rat liver is inhibited by brefeldin A plus GDP. Brefeldin A is a macrolide antibiotic inhibitor of membrane trafficking. A model is presented where the NADH oxidase functions as a thiol-disulfide oxidoreductase activity involved in the formation and breakage of disulfide bonds. The thiol-disulfide interchange is postulated as being associated with physical membrane displacement as encountered in cell enlargement or in vesicle budding. The model, although speculative, does provide a basis for further experimentation to probe a potential function for this enzyme system which, under certain conditions, exhibits a hormone- and growth factor-stimulated oxidation of NADH.     

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.