anandamide and adenosine-3--5--cyclic-phosphorothioate

Rats were bilaterally implanted with indwelling cannulae in the CA1 region of the dorsal hippocampus. After recovery from surgery, they were trained in a one-trial, step-down inhibitory avoidance task using a 0.5 mA foot shock. The animals received intrahippocampal infusions of either vehicle or anandamide (100 microM, 0.5 microl/side) 30 min before training. Then, either immediately post-training or 3 h later, they received infusions of saline, noradrenaline (0.5 microg/side), SKF 38393 (1.5 microg/side), oxotremorine (0.6 microg/side) or Sp-cAMPs (0.5 microg/side) also in the hippocampus. All animals were tested for retention 24-h post-training. Anandamide produced anterograde amnesia. Immediate, but not delayed, post-training treatment with Sp-cAMPs and noradrenaline reversed this effect. SKF 38393 and oxotremorine had no influence on the amnesia caused by anandamide either when given immediately or 3 h after training. The results suggest that the amnesic effect of anandamide is related to the known noradrenergic regulation of cAMP-dependent protein kinase (PKA) activity previously described in the hippocampus immediately after avoidance training, which is crucial to long-term memory (LTM) formation.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Amnesia, Anterograde; Animals; Arachidonic Acids; Avoidance Learning; Behavior, Animal; Calcium Channel Blockers; Chi-Square Distribution; Cyclic AMP; Dopamine Agonists; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Hippocampus; Memory; Morphine; Muscarinic Agonists; Narcotics; Norepinephrine; Oxotremorine; Polyunsaturated Alkamides; Rats; Rats, Wistar; Reaction Time; 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

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