guanosine-diphosphate and edelfosine

Adenosine 5'-triphosphate (ATP) mediates a variety of biological functions following nerve-evoked release, via activation of either G-protein-coupled P2Y- or ligand-gated P2X receptors. In smooth muscle, ATP, acting via P2Y receptors (P2YR), may act as an inhibitory neurotransmitter. The underlying mechanism(s) remain unclear, but have been proposed to involve the production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] by phospholipase C (PLC), to evoke Ca(2+) release from the internal store and stimulation of Ca(2+)-activated potassium (K(Ca)) channels to cause membrane hyperpolarization. This mechanism requires Ca(2+) release from the store. However, in the present study, ATP evoked transient Ca(2+) increases in only ∼10% of voltage-clamped single smooth muscle cells. These results do not support activation of K(Ca) as the major mechanism underlying inhibition of smooth muscle activity. Interestingly, ATP inhibited Ins(1,4,5)P(3)-evoked Ca(2+) release in cells that did not show a Ca(2+) rise in response to purinergic activation. The reduction in Ins(1,4,5)P(3)-evoked Ca(2+) release was not mimicked by adenosine and therefore, cannot be explained by hydrolysis of ATP to adenosine. The reduction in Ins(1,4,5)P(3)-evoked Ca(2+) release was, however, also observed with its primary metabolite, ADP, and blocked by the P2Y(1)R antagonist, MRS2179, and the G protein inhibitor, GDPβS, but not by PLC inhibition. The present study demonstrates a novel inhibitory effect of P2Y(1)R activation on Ins(1,4,5)P(3)-evoked Ca(2+) release, such that purinergic stimulation acts to prevent Ins(1,4,5)P(3)-mediated increases in excitability in smooth muscle and promote relaxation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium Channel Agonists; Calcium Signaling; Carbachol; Colon; Guanosine Diphosphate; Guinea Pigs; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Myocytes, Smooth Muscle; Patch-Clamp Techniques; Phospholipid Ethers; Purinergic P2Y Receptor Agonists; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2Y1; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Thionucleotides; Type C Phospholipases

Whereas the entorhinal cortex (EC) receives profuse serotonergic innervations from the raphe nuclei in the brain stem and is critically involved in the generation of temporal lobe epilepsy, the function of serotonin (5-hydroxytryptamine, 5-HT) in the EC and particularly its roles in temporal lobe epilepsy are still elusive. Here we explored the cellular and molecular mechanisms underlying 5-HT-mediated facilitation of GABAergic transmission and depression of epileptic activity in the superficial layers of the EC. Application of 5-HT increased sIPSC frequency and amplitude recorded from the principal neurons in the EC with no effects on mIPSCs recorded in the presence of TTX. However, 5-HT reduced the amplitude of IPSCs evoked by extracellular field stimulation and in synaptically connected interneuron and pyramidal neuron pairs. Application of 5-HT generated membrane depolarization and increased action potential firing frequency but reduced the amplitude of action potentials in presynaptic interneurons suggesting that 5-HT still increases GABA release whereas the depressant effects of 5-HT on evoked IPSCs could be explained by 5-HT-induced reduction in action potential amplitude. The depolarizing effect of 5-HT was mediated by inhibition of TASK-3 K(+) channels in interneurons and required the functions of 5-HT(2A) receptors and Galpha(q/11) but was independent of phospholipase C activity. Application of 5-HT inhibited low-Mg(2+)-induced seizure activity in slices via 5-HT(1A) and 5-HT(2A) receptors suggesting that 5-HT-mediated depression of neuronal excitability and increase in GABA release contribute to its anti-epileptic effects in the EC.

Topics: Action Potentials; Animals; Animals, Newborn; Antibodies; Arachidonic Acids; Dose-Response Relationship, Drug; Entorhinal Cortex; Estrenes; gamma-Aminobutyric Acid; Guanosine Diphosphate; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Interneurons; Magnesium; Neural Inhibition; Patch-Clamp Techniques; Phospholipid Ethers; Platelet Aggregation Inhibitors; Potassium Channel Blockers; Potassium Channels, Tandem Pore Domain; Pyramidal Cells; Pyrrolidines; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Ruthenium Red; Serotonin; Serotonin Agents; Suramin; Thionucleotides

Nitric oxide (NO) is a ubiquitous multifunctional free radical produced during sepsis, shock, reperfusion injury, and allograft rejection. Many studies are presently evaluating the functional roles of NO production in these settings. However, the signal transduction mechanisms underlying initiation of NO production are largely unknown. This study defines the cell surface receptor proteins that mediate endotoxin-induced NO synthesis in ANA-1 murine macrophages.. Endotoxin (LPS, 10 micrograms/ml) was added to ANA-1 macrophages to induce NO synthesis. In selected instances guanosine 5'-O-(2-thiodiphosphate)-trilithium salt (GOTP), pertussis toxin, cholera toxin, or suramin were added as inhibitors of specific subclasses of heterotrimeric G proteins. Calphostin was added as a protein kinase C inhibitor, and ET-OCH3 was added as a phospholipase C-beta inhibitor. NO release was quantified by measurement of the NO metabolite, nitrite. Membrane guanosine triphosphatase (GTPase) activity was also analyzed. Steady-state levels of inducible nitric oxide synthase (iNOS) mRNA were determined by using reverse transcription-polymerase chain reaction analysis.. Inhibition of G protein function by suramin or GOTP significantly decreased synthesis of NO and expression of iNOS mRNA. Pertussis and cholera toxin did not alter NO synthesis, suggesting that the Gi and Gs classes are not involved. Inhibition of protein kinase C or upstream phospholipase C-beta activity decreased NO synthesis, implicating the Gq class of heterotrimeric G proteins.. In ANA-1 macrophages, endotoxin-mediated NO synthesis is dependent on heterotrimeric Gq protein-phospholipase C-beta-protein kinase C signal transduction.

Topics: Animals; Cell Line; Cholera Toxin; Endotoxins; Enzyme Inhibitors; Escherichia coli; Genistein; GTP-Binding Proteins; Guanosine Diphosphate; Isoflavones; Kinetics; Lipopolysaccharides; Macrophages; Mice; Naphthalenes; Nitric Oxide; Nitric Oxide Synthase; Pertussis Toxin; Phospholipid Ethers; Polymerase Chain Reaction; Protein Kinase C; Protein-Tyrosine Kinases; RNA, Messenger; Signal Transduction; Suramin; Tetradecanoylphorbol Acetate; Thionucleotides; Transcription, Genetic; Type C Phospholipases; Virulence Factors, Bordetella