The Experts below are selected from a list of 15 Experts worldwide ranked by ideXlab platform
Alexandros Makriyannis - One of the best experts on this subject based on the ideXlab platform.
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Endocannabinoid Enhancement Protects against Kainic Acid-Induced Seizures and Associated Brain Damage
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: David A. Karanian, Alexandros Makriyannis, Sanjida L. Karim, Jodianne T. Wood, John S. Williams, Ben A. BahrAbstract:Endocannabinoids are released in response to pathogenic insults, and inhibitors of endocannabinoid inactivation enhance such on-demand responses that promote cellular protection. Here, AM374 (Palmitylsulfonyl Fluoride), an irreversible inhibitor of fatty acid amide hydrolase (FAAH), was injected i.p. into rats to test for endocannabinoid enhancement. AM374 caused a prolonged elevation of anandamide levels in several brain regions, including the hippocampus, and resulted in rapid activation of the extracellular signal regulated-kinase/mitogen-activated protein kinase pathway that has been linked to survival. To evaluate the neuroprotective nature of the FAAH inhibitor, we tested AM374 in a seizure model involving rats insulted with kainic acid (KA). AM374 was injected immediately after KA administration, and seizure scores were significantly reduced throughout a 4-h observation period. The KA-induced seizures were associated with calpain-mediated cytoskeletal breakdown, reductions in synaptic markers, and loss of CA1 hippocampal neurons. FAAH inhibition protected against the excitotoxic damage and neuronal loss assessed 48 h postinsult. AM374 also preserved pre- and postsynaptic markers to levels comparable with those found in noninsulted animals, and the synaptic marker preservation strongly correlated with reduced seizure scores. With regard to behavioral deficits in the excitotoxic rats, AM374 produced nearly complete functional protection, significantly improving balance and coordination across different behavioral paradigms. These data indicate that AM374 crosses the blood-brain barrier, enhances endocannabinoid responses in key neuronal circuitries, and protects the brain against excitotoxic damage.
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Dual Modulation of Endocannabinoid Transport and Fatty Acid Amide Hydrolase Protects against Excitotoxicity
The Journal of Neuroscience, 2005Co-Authors: David A. Karanian, Alexandros Makriyannis, Queenie B. Brown, Therese A. Kosten, Ben A. BahrAbstract:The endocannabinoid system has been suggested to elicit signals that defend against several disease states including excitotoxic brain damage. Besides direct activation with CB1 receptor agonists, cannabinergic signaling can be modulated through inhibition of endocannabinoid transport and fatty acid amide hydrolase (FAAH), two mechanisms of endocannabinoid inactivation. To test whether the transporter and FAAH can be targeted pharmacologically to modulate survival/repair responses, the transport inhibitor N -(4-hydroxyphenyl)-arachidonamide (AM404) and the FAAH inhibitor Palmitylsulfonyl Fluoride (AM374) were assessed for protection against excitotoxicity in vitro and in vivo . AM374 and AM404 both enhanced mitogen-activated protein kinase (MAPK) activation in cultured hippocampal slices. Interestingly, combining the distinct inhibitors produced additive effects on CB1 signaling and associated neuroprotection. After an excitotoxic insult in the slices, infusing the AM374/AM404 combination protected against cytoskeletal damage and synaptic decline, and the protection was similar to that produced by the stable CB1 agonist AM356 ( R -methanandamide). AM374/AM404 and the agonist also elicited cytoskeletal and synaptic protection in vivo when coinjected with excitotoxin into the dorsal hippocampus. Correspondingly, potentiating endocannabinoid responses with the AM374/AM404 combination prevented behavioral alterations and memory impairment that are characteristic of excitotoxic damage. The protective effects mediated by AM374/AM404 were (1) evident 7 d after insult, (2) correlated with the preservation of CB1-linked MAPK signaling, and (3) were blocked by a selective CB1 antagonist. These results indicate that dual modulation of the endocannabinoid system with AM374/AM404 elicits neuroprotection through the CB1 receptor. The transporter and FAAH are modulatory sites that may be exploited to enhance cannabinergic signaling for therapeutic purposes.
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FATTY ACID SULFONYL FluorideS INHIBIT ANANDAMIDE METABOLISM AND BIND TO THE CANNABINOID RECEPTOR
Biochemical and Biophysical Research Communications, 1997Co-Authors: Dale G. Deutsch, W.a.g. Hill, K.l. Morse, David Salehani, Gladys Arreaza, Romelda L. Omeir, Alexandros MakriyannisAbstract:Arachidonoyl ethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors (CB1, CB2) and a putative neurotransmitter. Phenylmethylsulfonyl Fluoride (PMSF) is an inhibitor of the enzyme (an amidase) which hydrolyzes anandamide to arachidonic acid and ethanolamine. We report here that fatty acid sulfonyl Fluorides are potent inhibitors of anandamide metabolism. In order to investigate the SAR of these anandamide amidase inhibitors we tested a series of fatty acid (C12 to C20) sulfonyl Fluorides both as inhibitors of anandamide degradation and as ligands for the central cannabinoid receptor (CB1). AM374 (Palmitylsulfonyl Fluoride, C16) was approximately 20 times more potent than PMSF and 50 times more potent than arachidonyltrifluoromethyl ketone in preventing the hydrolysis of anandamide in brain homogenates. AM374 was over a thousand-fold more effective than PMSF in inhibiting the amidase in cultured cells. The C12 to C18 sulfonyl Fluoride analogs were equipotent as inhibitors of the amidase and the reverse reaction (the synthase) with nanomolar IC50values. These compounds generally showed decreasing affinity for the CB1 receptor as the chain length increased; thus, C12 sulfonylFluoride had an IC50of 18 nM and C20 sulfonylFluoride had an IC50of 78 μM. The C14, C16, and C18 sulfonyl Fluorides showed high selectivity for the amidase over the CB1 receptor and thus are potentially useful selective anandamide amidase inhibitors.
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High-performance liquid chromatographic determination of anandamide amidase activity in rat brain microsomes.
Analytical Biochemistry, 1996Co-Authors: Wensheng Lang, W. Adam Hill, Atmaram D. Khanolkar, Alexandros MakriyannisAbstract:A rapid, sensitive, and reliable method for measuring anandamide amidase activity in rat brain microsomes by reversed-phase high-performance liquid chromatography (RP-HPLC) and its applications are described. Enzymatic activity was assayed by the determination of the rates of hydrolysis of anandamide or its analogs at 37°C. The reaction products were separated using an ODS guard column eluted with aqueous phosphoric acid–acetonitrile and quantitated with uv detection at 204 nm and an external standard method. Baseline separation of the acid products from their substrates was completed in less than 2 min. The detection limits were 1.4 pmol for arachidonic acid and 0.22 pmol for anandamide at a signal to noise ratio of 4:1. The stability of anandamide in the acidic mobile phase was tested, and no significant decomposition was observed up to 1 h. The method was successfully applied to the examination of substrate specificity as well as for testing the ability of amidase inhibitors to block its hydrolysis. Kinetic constants obtained for (S)-methanandamide were an apparentKmof 8.6 ± 1.3 μMand aVmaxof 362 ± 16 pmol/min/mg of protein. A highly potent inhibitor, Palmitylsulfonyl Fluoride (PSF), was found to have an IC50of 50 nM. PSF is 210 times as potent as phenylmethylsulfonyl Fluoride. The method offers several advantages over existing methodology using radioisotopes or a solvent extraction procedure.
Ben A. Bahr - One of the best experts on this subject based on the ideXlab platform.
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Endocannabinoid Enhancement Protects against Kainic Acid-Induced Seizures and Associated Brain Damage
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: David A. Karanian, Alexandros Makriyannis, Sanjida L. Karim, Jodianne T. Wood, John S. Williams, Ben A. BahrAbstract:Endocannabinoids are released in response to pathogenic insults, and inhibitors of endocannabinoid inactivation enhance such on-demand responses that promote cellular protection. Here, AM374 (Palmitylsulfonyl Fluoride), an irreversible inhibitor of fatty acid amide hydrolase (FAAH), was injected i.p. into rats to test for endocannabinoid enhancement. AM374 caused a prolonged elevation of anandamide levels in several brain regions, including the hippocampus, and resulted in rapid activation of the extracellular signal regulated-kinase/mitogen-activated protein kinase pathway that has been linked to survival. To evaluate the neuroprotective nature of the FAAH inhibitor, we tested AM374 in a seizure model involving rats insulted with kainic acid (KA). AM374 was injected immediately after KA administration, and seizure scores were significantly reduced throughout a 4-h observation period. The KA-induced seizures were associated with calpain-mediated cytoskeletal breakdown, reductions in synaptic markers, and loss of CA1 hippocampal neurons. FAAH inhibition protected against the excitotoxic damage and neuronal loss assessed 48 h postinsult. AM374 also preserved pre- and postsynaptic markers to levels comparable with those found in noninsulted animals, and the synaptic marker preservation strongly correlated with reduced seizure scores. With regard to behavioral deficits in the excitotoxic rats, AM374 produced nearly complete functional protection, significantly improving balance and coordination across different behavioral paradigms. These data indicate that AM374 crosses the blood-brain barrier, enhances endocannabinoid responses in key neuronal circuitries, and protects the brain against excitotoxic damage.
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Dual Modulation of Endocannabinoid Transport and Fatty Acid Amide Hydrolase Protects against Excitotoxicity
The Journal of Neuroscience, 2005Co-Authors: David A. Karanian, Alexandros Makriyannis, Queenie B. Brown, Therese A. Kosten, Ben A. BahrAbstract:The endocannabinoid system has been suggested to elicit signals that defend against several disease states including excitotoxic brain damage. Besides direct activation with CB1 receptor agonists, cannabinergic signaling can be modulated through inhibition of endocannabinoid transport and fatty acid amide hydrolase (FAAH), two mechanisms of endocannabinoid inactivation. To test whether the transporter and FAAH can be targeted pharmacologically to modulate survival/repair responses, the transport inhibitor N -(4-hydroxyphenyl)-arachidonamide (AM404) and the FAAH inhibitor Palmitylsulfonyl Fluoride (AM374) were assessed for protection against excitotoxicity in vitro and in vivo . AM374 and AM404 both enhanced mitogen-activated protein kinase (MAPK) activation in cultured hippocampal slices. Interestingly, combining the distinct inhibitors produced additive effects on CB1 signaling and associated neuroprotection. After an excitotoxic insult in the slices, infusing the AM374/AM404 combination protected against cytoskeletal damage and synaptic decline, and the protection was similar to that produced by the stable CB1 agonist AM356 ( R -methanandamide). AM374/AM404 and the agonist also elicited cytoskeletal and synaptic protection in vivo when coinjected with excitotoxin into the dorsal hippocampus. Correspondingly, potentiating endocannabinoid responses with the AM374/AM404 combination prevented behavioral alterations and memory impairment that are characteristic of excitotoxic damage. The protective effects mediated by AM374/AM404 were (1) evident 7 d after insult, (2) correlated with the preservation of CB1-linked MAPK signaling, and (3) were blocked by a selective CB1 antagonist. These results indicate that dual modulation of the endocannabinoid system with AM374/AM404 elicits neuroprotection through the CB1 receptor. The transporter and FAAH are modulatory sites that may be exploited to enhance cannabinergic signaling for therapeutic purposes.
David A. Karanian - One of the best experts on this subject based on the ideXlab platform.
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Endocannabinoid Enhancement Protects against Kainic Acid-Induced Seizures and Associated Brain Damage
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: David A. Karanian, Alexandros Makriyannis, Sanjida L. Karim, Jodianne T. Wood, John S. Williams, Ben A. BahrAbstract:Endocannabinoids are released in response to pathogenic insults, and inhibitors of endocannabinoid inactivation enhance such on-demand responses that promote cellular protection. Here, AM374 (Palmitylsulfonyl Fluoride), an irreversible inhibitor of fatty acid amide hydrolase (FAAH), was injected i.p. into rats to test for endocannabinoid enhancement. AM374 caused a prolonged elevation of anandamide levels in several brain regions, including the hippocampus, and resulted in rapid activation of the extracellular signal regulated-kinase/mitogen-activated protein kinase pathway that has been linked to survival. To evaluate the neuroprotective nature of the FAAH inhibitor, we tested AM374 in a seizure model involving rats insulted with kainic acid (KA). AM374 was injected immediately after KA administration, and seizure scores were significantly reduced throughout a 4-h observation period. The KA-induced seizures were associated with calpain-mediated cytoskeletal breakdown, reductions in synaptic markers, and loss of CA1 hippocampal neurons. FAAH inhibition protected against the excitotoxic damage and neuronal loss assessed 48 h postinsult. AM374 also preserved pre- and postsynaptic markers to levels comparable with those found in noninsulted animals, and the synaptic marker preservation strongly correlated with reduced seizure scores. With regard to behavioral deficits in the excitotoxic rats, AM374 produced nearly complete functional protection, significantly improving balance and coordination across different behavioral paradigms. These data indicate that AM374 crosses the blood-brain barrier, enhances endocannabinoid responses in key neuronal circuitries, and protects the brain against excitotoxic damage.
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Dual Modulation of Endocannabinoid Transport and Fatty Acid Amide Hydrolase Protects against Excitotoxicity
The Journal of Neuroscience, 2005Co-Authors: David A. Karanian, Alexandros Makriyannis, Queenie B. Brown, Therese A. Kosten, Ben A. BahrAbstract:The endocannabinoid system has been suggested to elicit signals that defend against several disease states including excitotoxic brain damage. Besides direct activation with CB1 receptor agonists, cannabinergic signaling can be modulated through inhibition of endocannabinoid transport and fatty acid amide hydrolase (FAAH), two mechanisms of endocannabinoid inactivation. To test whether the transporter and FAAH can be targeted pharmacologically to modulate survival/repair responses, the transport inhibitor N -(4-hydroxyphenyl)-arachidonamide (AM404) and the FAAH inhibitor Palmitylsulfonyl Fluoride (AM374) were assessed for protection against excitotoxicity in vitro and in vivo . AM374 and AM404 both enhanced mitogen-activated protein kinase (MAPK) activation in cultured hippocampal slices. Interestingly, combining the distinct inhibitors produced additive effects on CB1 signaling and associated neuroprotection. After an excitotoxic insult in the slices, infusing the AM374/AM404 combination protected against cytoskeletal damage and synaptic decline, and the protection was similar to that produced by the stable CB1 agonist AM356 ( R -methanandamide). AM374/AM404 and the agonist also elicited cytoskeletal and synaptic protection in vivo when coinjected with excitotoxin into the dorsal hippocampus. Correspondingly, potentiating endocannabinoid responses with the AM374/AM404 combination prevented behavioral alterations and memory impairment that are characteristic of excitotoxic damage. The protective effects mediated by AM374/AM404 were (1) evident 7 d after insult, (2) correlated with the preservation of CB1-linked MAPK signaling, and (3) were blocked by a selective CB1 antagonist. These results indicate that dual modulation of the endocannabinoid system with AM374/AM404 elicits neuroprotection through the CB1 receptor. The transporter and FAAH are modulatory sites that may be exploited to enhance cannabinergic signaling for therapeutic purposes.
Dale G. Deutsch - One of the best experts on this subject based on the ideXlab platform.
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The Cellular Uptake of Anandamide Is Coupled to Its Breakdown by Fatty-acid Amide Hydrolase
Journal of Biological Chemistry, 2000Co-Authors: Dale G. Deutsch, Sherrye T. Glaser, Judy M. Howell, Jeffrey S. Kunz, Robyn A. Puffenbarger, Cecilia J. Hillard, Nada A. AbumradAbstract:Abstract Anandamide is an endogenous compound that acts as an agonist at cannabinoid receptors. It is inactivated via intracellular degradation after its uptake into cells by a carrier-mediated process that depends upon a concentration gradient. The fate of anandamide in those cells containing an amidase called fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and ethanolamine. The active site nucleophilic serine of FAAH is inactivated by a variety of inhibitors including methylarachidonylfluorophosphonate (MAFP) and Palmitylsulfonyl Fluoride. In the current report, the net uptake of anandamide in cultured neuroblastoma (N18) and glioma (C6) cells, which contain FAAH, was decreased by nearly 50% after 6 min of incubation in the presence of MAFP. Uptake in laryngeal carcinoma (Hep2) cells, which lack FAAH, is not inhibited by MAFP. Free anandamide was found in all MAFP-treated cells and in control Hep2 cells, whereas phospholipid was the main product in N18 and C6 control cells when analyzed by TLC. The intracellular concentration of anandamide in N18, C6, and Hep2 cells was up to 18-fold greater than the extracellular concentration of 100 nm, which strongly suggests that it is sequestered within the cell by binding to membranes or proteins. The accumulation of anandamide and/or its breakdown products was found to vary among the different cell types, and this correlated approximately with the amount of FAAH activity, suggesting that the breakdown of anandamide is in part a driving force for uptake. This was shown most clearly in Hep2 cells transfected with FAAH. The uptake in these cells was 2-fold greater than in vector-transfected or untransfected Hep2 cells. Therefore, it appears that FAAH inhibitors reduce anandamide uptake by cells by shifting the anandamide concentration gradient in a direction that favors equilibrium. Because inhibition of FAAH increases the levels of extracellular anandamide, it may be a useful target for the design of therapeutic agents.
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FATTY ACID SULFONYL FluorideS INHIBIT ANANDAMIDE METABOLISM AND BIND TO THE CANNABINOID RECEPTOR
Biochemical and Biophysical Research Communications, 1997Co-Authors: Dale G. Deutsch, W.a.g. Hill, K.l. Morse, David Salehani, Gladys Arreaza, Romelda L. Omeir, Alexandros MakriyannisAbstract:Arachidonoyl ethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors (CB1, CB2) and a putative neurotransmitter. Phenylmethylsulfonyl Fluoride (PMSF) is an inhibitor of the enzyme (an amidase) which hydrolyzes anandamide to arachidonic acid and ethanolamine. We report here that fatty acid sulfonyl Fluorides are potent inhibitors of anandamide metabolism. In order to investigate the SAR of these anandamide amidase inhibitors we tested a series of fatty acid (C12 to C20) sulfonyl Fluorides both as inhibitors of anandamide degradation and as ligands for the central cannabinoid receptor (CB1). AM374 (Palmitylsulfonyl Fluoride, C16) was approximately 20 times more potent than PMSF and 50 times more potent than arachidonyltrifluoromethyl ketone in preventing the hydrolysis of anandamide in brain homogenates. AM374 was over a thousand-fold more effective than PMSF in inhibiting the amidase in cultured cells. The C12 to C18 sulfonyl Fluoride analogs were equipotent as inhibitors of the amidase and the reverse reaction (the synthase) with nanomolar IC50values. These compounds generally showed decreasing affinity for the CB1 receptor as the chain length increased; thus, C12 sulfonylFluoride had an IC50of 18 nM and C20 sulfonylFluoride had an IC50of 78 μM. The C14, C16, and C18 sulfonyl Fluorides showed high selectivity for the amidase over the CB1 receptor and thus are potentially useful selective anandamide amidase inhibitors.
Nada A. Abumrad - One of the best experts on this subject based on the ideXlab platform.
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The Cellular Uptake of Anandamide Is Coupled to Its Breakdown by Fatty-acid Amide Hydrolase
Journal of Biological Chemistry, 2000Co-Authors: Dale G. Deutsch, Sherrye T. Glaser, Judy M. Howell, Jeffrey S. Kunz, Robyn A. Puffenbarger, Cecilia J. Hillard, Nada A. AbumradAbstract:Abstract Anandamide is an endogenous compound that acts as an agonist at cannabinoid receptors. It is inactivated via intracellular degradation after its uptake into cells by a carrier-mediated process that depends upon a concentration gradient. The fate of anandamide in those cells containing an amidase called fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and ethanolamine. The active site nucleophilic serine of FAAH is inactivated by a variety of inhibitors including methylarachidonylfluorophosphonate (MAFP) and Palmitylsulfonyl Fluoride. In the current report, the net uptake of anandamide in cultured neuroblastoma (N18) and glioma (C6) cells, which contain FAAH, was decreased by nearly 50% after 6 min of incubation in the presence of MAFP. Uptake in laryngeal carcinoma (Hep2) cells, which lack FAAH, is not inhibited by MAFP. Free anandamide was found in all MAFP-treated cells and in control Hep2 cells, whereas phospholipid was the main product in N18 and C6 control cells when analyzed by TLC. The intracellular concentration of anandamide in N18, C6, and Hep2 cells was up to 18-fold greater than the extracellular concentration of 100 nm, which strongly suggests that it is sequestered within the cell by binding to membranes or proteins. The accumulation of anandamide and/or its breakdown products was found to vary among the different cell types, and this correlated approximately with the amount of FAAH activity, suggesting that the breakdown of anandamide is in part a driving force for uptake. This was shown most clearly in Hep2 cells transfected with FAAH. The uptake in these cells was 2-fold greater than in vector-transfected or untransfected Hep2 cells. Therefore, it appears that FAAH inhibitors reduce anandamide uptake by cells by shifting the anandamide concentration gradient in a direction that favors equilibrium. Because inhibition of FAAH increases the levels of extracellular anandamide, it may be a useful target for the design of therapeutic agents.
