piperidines and adenosine-3--5--cyclic-phosphorothioate

Acute ethanol overdose can induce dysfunction of cerebellar motor regulation and cerebellar ataxia. In this study, we investigated the effect of ethanol on facial stimulation-evoked inhibitory synaptic responses in cerebellar Purkinje cells (PCs) in urethane-anesthetized mice, using in vivo patch-clamp recordings. Under voltage-clamp conditions, ethanol (300 mM) decreased the amplitude, half-width, rise time and decay time of facial stimulation-evoked outward currents in PCs. The ethanol-induced inhibition of facial stimulation-evoked outward currents was dose-dependent, with an IC50 of 148.5 mM. Notably, the ethanol-induced inhibition of facial stimulation-evoked outward currents were significantly abrogated by cannabinoid receptor 1 (CB1) antagonists, AM251 and O-2050, as well as by the CB1 agonist WIN55212-2. Moreover, the ethanol-induced inhibition of facial stimulation-evoked outward currents was prevented by cerebellar surface perfusion of the PKA inhibitors H-89 and Rp-cAMP, but not by intracellular administration of the PKA inhibitor PKI. Our present results indicate that ethanol inhibits the facial stimulation-evoked outward currents by activating presynaptic CB1 receptors via the PKA signaling pathway. These findings suggest that ethanol overdose impairs sensory information processing, at least in part, by inhibiting GABA release from molecular layer interneurons onto PCs.

Topics: Action Potentials; Animals; Benzoxazines; Central Nervous System Depressants; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dronabinol; Ethanol; gamma-Aminobutyric Acid; Isoquinolines; Membrane Potentials; Mice; Mice, Inbred ICR; Morpholines; Naphthalenes; Patch-Clamp Techniques; Physical Stimulation; Piperidines; Purkinje Cells; Pyrans; Pyrazoles; Receptor, Cannabinoid, CB1; Reflex; Signal Transduction; Sulfonamides; Thionucleotides

Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 microM). WIN 55,212-2 was also shown to inhibit the NMDA-induced increase in intracellular calcium concentration ([Ca2+](i)) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca2+](i) occurred with similar concentrations (25-100 nM) and in the same time-dependent manner (pre-exposure 1-15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in [Ca2+](i) was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositol-trisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing [Ca2+](i) via a cAMP/PKA-dependent process during the neurotoxic event.

Topics: Animals; Benzoxazines; Calcium; Cell Culture Techniques; Cell Death; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclohexanols; Dantrolene; Drug Interactions; Estrenes; Fetus; Hippocampus; Macrocyclic Compounds; Morpholines; N-Methylaspartate; Naphthalenes; Neurons; Neuroprotective Agents; Okadaic Acid; Oxazoles; Piperidines; Pyrazoles; Pyrrolidinones; Rats; Rats, Inbred Strains; Receptor, Cannabinoid, CB1; Rimonabant; Ryanodine; Thionucleotides

The cloned 5-HT3 receptor from NCB-20 neuroblastoma cells was expressed in Xenopus oocytes and the effect of the endogenous cannabinoid ligand, anandamide, was investigated on the function of this receptor. The oocytes expressing the cloned 5-HT3 receptors were voltage-clamped at -70 mV. Anandamide, at the concentration range of 0.1-100 microM, reversibly inhibited 1 microM 5-HT induced currents. The inhibition of 5-HT induced currents by anandamide was concentration-dependent with an EC50 of 3.7 microM and slope value of 0.94. This inhibitory effect was not dependent on the membrane potential and anandamide did not have an effect on the reversal potential of 5-HT-induced currents. In the presence of 10 microM anandamide, the maximum 5-HT-induced response was also inhibited and the respective EC50 values were 3.4 microM and 3.1 microM in the absence and presence of anandamide, indicating that anandamide acts as a noncompetitive antagonist on 5-HT3 receptors. CB1 receptor antagonist SR-141716A (1 microM) and pertussis toxin (5 microg/ml) did not cause a significant change on the inhibition of 5-HT responses by anandamide. The effect of anandamide was not changed by preincubating the oocytes with 0.2 mM 8-Br-cAMP, a membrane-permeable analog of cAMP, or Sp-cAMPS (0.1 mM), a membrane-permeable protein kinase A activator. These results suggest that the effect of anandamide is independent of the activation of cAMP pathway and not mediated by the activation of PTX sensitive G-proteins. In conclusion, we demonstrated that the endogenous cannabinoid anandamide inhibits the function of 5-HT3 receptors expressed in Xenopus oocytes in a cannabinoid-receptor independent and noncompetitive manner.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Analysis of Variance; Animals; Arachidonic Acids; Biguanides; Cannabinoids; Chelating Agents; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Egtazic Acid; Electrophysiology; Endocannabinoids; Enzyme Inhibitors; Female; Indazoles; Membrane Potentials; Oocytes; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Serotonin, 5-HT3; Rimonabant; RNA, Complementary; Serotonin; Serotonin 5-HT3 Receptor Antagonists; Serotonin Receptor Agonists; Thionucleotides; Tropanes; Xenopus laevis

Histamine H3 receptors modulate histamine synthesis, although little is known about the transduction mechanisms involved. To investigate this issue, we have used a preparation of rat brain cortical miniprisms in which histamine synthesis can be modulated by depolarization and by H3 receptor ligands. When the miniprisms were incubated in presence of forskolin, dibutyryl-cAMP, or 3-isobutyl-1-methylxanthine (IBMX), histamine synthesis was stimulated in 34, 29, and 47%, respectively. These stimulations could be prevented by the selective cAMP protein kinase blocker Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPs). Preincubation with the H3 receptor agonist imetit prevented IBMX- (100% blockade) and forskolin- (70% blockade) induced stimulation of histamine synthesis. The H3 inverse agonist thioperamide enhanced histamine synthesis in the presence of 1 mM IBMX or 30 mM potassium (+47 and +45%, respectively). Similarly, the H3 antagonist clobenpropit enhanced histamine synthesis in the presence of 30 mM potassium (+ 59%). The cAMP-dependent protein kinase blockers Rp-cAMPs and PKI14-22 could impair the effects of thioperamide and clobenpropit, respectively. These results indicate that the adenylate cyclase-protein kinase A pathway is involved in the modulation of histamine synthesis by H3 autoreceptors present in histaminergic nerve terminals.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Brain; Bucladesine; Colforsin; Cyclic AMP; Drug Interactions; Histamine; Imidazoles; In Vitro Techniques; Male; Piperidines; Potassium; Rats; Rats, Sprague-Dawley; Receptors, Histamine H3; Thionucleotides; Thiourea

Long-term potentiation (LTP) and long-term depression (LTD) are two main forms of activity-dependent synaptic plasticity that have been extensively studied as the putative mechanisms underlying learning and memory. Current studies have demonstrated that prior synaptic activity can influence the subsequent induction of LTP and LTD at Schaffer collateral-CA1 synapses. Here, we show that prior short-term synaptic disinhibition induced by type A gamma-aminobutyric acid (GABA) receptor antagonist picrotoxin exhibited a facilitation of LTP induction and an inhibition of LTD induction. This effect lasted between 10 and 30 min after washout of picrotoxin and was specifically inhibited by the L-type voltage-operated Ca2+ channel (VOCC) blocker nimodipine, but not by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphopentanoic acid (D-APV). Moreover, this picrotoxin-induced priming effect was mimicked by forskolin, an activator of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), and was blocked by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536) and the PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). It was also found that following picrotoxin application, CA1 neurons have a higher probability of synchronous discharge in response to a population of excitatory postsynaptic potential (EPSP) of fixed slope (EPSP/spike potentiation). However, picrotoxin treatment did not significantly affect paired-pulse facilitation (PPF). These findings suggest that a brief of GABAergic disinhibition can act as a priming stimulus for the subsequent induction of LTP and LTD at Schaffer collateral-CA1 synapses. The increase in Ca2+ influx through L-type VOCCs in turn triggering a cAMP/PKA signalling pathway is a possible molecular mechanism underlying this priming effect.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Colforsin; Cyclic AMP; Electric Stimulation; Excitatory Postsynaptic Potentials; GABA-A Receptor Antagonists; Hippocampus; In Vitro Techniques; Long-Term Potentiation; Male; Picrotoxin; Piperidines; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Synapses; Thionucleotides

1. Calcitonin gene-related peptide (CGRP) is found in dense-cored vesicles in the motor nerve terminal. 2. Exogenous CGRP increased the size of the quanta. The increase in size reached a maximum after about 40 min. The lowest effective concentration of human CGRP (hCGRP) was 0.8 nM. The action of hCGRP was antagonized by (-)-vesamicol, a drug that blocks active acetylcholine (ACh) uptake into synaptic vesicles, so it appears that hCGRP increases size by adding more ACh to the quanta. The action of hCGRP was antagonized by drugs that block the activation of protein kinase A (PKA). (In other preparations CGRP also activates PKA.) 3. The hCGRP effect was not blocked by fragment 8-37, an antagonist of one class of CGRP receptor. 4. hCGRP increases evoked quantal output and miniature endplate potential (MEPP) frequency, again by activating PKA. 5. CGRP release was measured by radioimmunoassay. Release was increased by depolarization with elevated K+, but the amounts released appear to be below those needed to affect quantal size or output. Moreover, although elevated K+ can increase quantal size it acts by a pathway that does not involve PKA. We suggest that the most likely target of endogenously released CGRP is the regulation of circulation of the muscle.

Topics: Acetylcholine; Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endocannabinoids; Enzyme Inhibitors; Evoked Potentials; Humans; In Vitro Techniques; Kinetics; Motor Neurons; Muscle, Skeletal; Neuromuscular Junction; Patch-Clamp Techniques; Peptide Fragments; Piperidines; Polyunsaturated Alkamides; Quantum Theory; Rana pipiens; Thionucleotides

The aim of this study was to study the possible intracellular mechanisms underlying the anoxia-induced long-term potentiation (anoxic LTP) in the CA1 neurons of rat hippocampal slices using extra- and intracellular recording techniques. Superfusion of the hippocampal slices with the protein kinase C (PKC) inhibitors NPC-15437 (20 microM) or H-7 (20 microM) specifically prevented the induction of anoxic LTP. Moreover, the anoxic LTP was completely abolished in neurons intracellularly recorded with the selective PKC inhibitor PKCI 19-36 (50 microM). The specific cAMP-dependent protein kinase (PKA) inhibitor Rp-cyclic adenosine 3',5'-monophosphate (Rp-cAMPS, 25 microM) had no effect on the anoxic LTP. It is concluded that induction of anoxic LTP requires the activation of postsynaptic PKC.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cell Hypoxia; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Hippocampus; Long-Term Potentiation; Male; Neurons; Organ Culture Techniques; Piperidines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Thionucleotides