h-89 and 5-7-dichlorokynurenic-acid

Ischemic episodes lead to profound functional and structural alterations of the gastrointestinal tract which may contribute to disorders of intestinal motility. Enhancement of glutamate overflow and the consequent activation of NMDA (N-methyl-D-aspartate) receptors may participate to such changes by modulating different enteric neurotransmitter systems, including cholinergic motor pathways.. The molecular mechanism/s underlying activation of NMDA receptors in the guinea pig ileum were investigated after glucose/oxygen deprivation (in vitro ischemia) and during reperfusion.. The number of ileal myenteric neurons positive for NR1, the functional subunit of NMDA receptors, and its mRNA levels were unchanged after in vitro ischemia/reperfusion. In these conditions, the protein levels of NR1, and of its phosphorylated form by protein kinase C (PKC), significantly increased in myenteric neurons, whereas, the levels of NR1 phosphorylated by protein kinase A (PKA) did not change, with respect to control values. Spontaneous glutamate overflow increased during in vitro ischemia/reperfusion. In these conditions, the NMDA receptor antagonists, D(-)-2-amino-5-phosphonopentanoic acid [(D)-AP5] (10 μmol L(-1)) and 5,7-dichlorokynurenic acid (5,7-diClKyn acid) (10 μmol L(-1)) and the PKC antagonist, chelerythrine (1 μmol L(-1)), but not the PKA antagonist, H-89 (1 μmol L(-1)), were able to significantly depress the increased glutamate efflux.. The present data suggest that in the guinea pig ileum during in vitro ischemia/reperfusion, NR1 protein levels increase. Such event may rely upon posttranscriptional events involving NR1 phosphorylation by PKC. Increased NR1 levels may, at least in part, explain the ability of NMDA receptors to modulate a positive feedback on ischemia/reperfusion-induced glutamate overflow.

Topics: 2-Amino-5-phosphonovalerate; Animals; Benzophenanthridines; Guinea Pigs; Ileum; In Vitro Techniques; Isoquinolines; Kynurenic Acid; Male; Models, Animal; Myenteric Plexus; Phosphorylation; Protein Kinase C; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Sulfonamides

1. Protein kinase modulation of glycine-activated currents was examined in acutely dissociated ganglion cells from tiger salamander retina using whole-cell voltage-clamp techniques. 2. Glycine (100 microM) induced an outward chloride current in cells clamped at 0 mV. Co-application of 50 microM forskolin made the glycine-induced current more transient. The combination of forskolin and glycine reduced the later portion of current response without changing the initial peak amplitude. 3. 3-Isobutyl-1-methylxanthine (IBMX) or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) produced effects similar to those of forskolin. H-89, a protein kinase A (PKA) inhibitor, blocked the effect of forskolin. 4. A protein kinase C (PKC) activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), also made the glycine response more transient. Unlike PKA analogues, OAG enhanced the glycine peak response without changing the glycine late response. OAG effects were blocked by 1 microM GF-109203X, a PKC inhibitor. 5. Nanomolar concentrations of strychnine selectively blocked the fast phase of the glycine current and reversed the effect of OAG, but not that of forskolin. Conversely, forskolin occluded the effect of 5,7-dichlorokynurenic acid, which selectively suppresses the late phase of the glycine current. The action of OAG was not blocked by 5,7-dichlorokynurenic acid. 6. Thus, through a differential modulation, both protein kinase A and C shorten the decay time of the glycine current. PKA suppresses the slow component, while PKC potentiates the fast component.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Ambystoma; Animals; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glycine; Glycine Agents; Isoquinolines; Kinetics; Kynurenic Acid; Membrane Potentials; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Protein Kinase C; Retinal Ganglion Cells; Strychnine; Sulfonamides