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dc.contributor.authorPan, Xianghua
dc.date.accessioned2015-05-18T19:53:48Zen
dc.date.available2015-05-18T19:53:48Zen
dc.date.issued1996-09en
dc.identifier.urihttp://hdl.handle.net/10675.2/553074
dc.description.abstractThe rat brain cannabinoid receptor (CB1) is a member of the G protein coupled receptor family. The present study directly tested the functional coupling of CB1 receptor with neuronal ion channels. The rat CB1 receptor was heterologously expressed by microinjection o f cRNA into the enzymatically dissociated adult rat superior cervical ganglion (SCG) neurons. The cannabinoid receptor agonists WIN 55,212-2 and CP 55940 produced a voltagedependent inhibition of whole cell Ca2+ currents. The maximal inhibitions o f the Ca2+ current by WIN 55,212-2 and CP 55,940 were 73% and 38%, respectively. The E C 50 of WIN 55,212-2 was 47 nM and the EC50 of CP 55940 was 7 nM. The inhibition was mediated by pertussis toxin (PTX) sensitive G protein and the N-type Ca2+ channels are the targets. The L-type Ca2+ channels, M type K+ current and the A current were not modulated by WIN 55,212-2. An inwardly rectifying current was activated by WIN 55,212-2. The selective CB1 receptor antagonist SR 141716A and LY 320135 abolished the inhibition of the Ca2+ currents by WIN 55,212-2. However, when given alone, SR 141716A and LY 320135 increased the Ca2+ current in SCG neurons expressing the CB1 receptor. SR 141716A increased Ca2+ current with an EC50 of 32 nM and a maximal current increase of 41% at 1 p.M. This increase of Ca2+ current by SR 141716A was a reversal o f a tonic inhibition of Ca2+ current in neurons expressing CB 1 receptors. A K192A mutant of the human CB1 receptor was tested to determine whether the tonic activation o f the cannabinoid receptor is due to endogenous arachidonyl ethanolam ide (anandam ide). The K192A m utant receptor was expressed by microinjection of receptor cDNA into nucleus of the SCG neurons. WIN 55,212-2 inhibited the Ca2+ current in these SCG neurons, but SR 141716A did not increase the Ca2+ current. However, SR 141716A inhibited the effect of WIN 55,212-2. Ca2+ currents from male rat major pelvic ganglion neurons were examined for modulation by native cannabinoid receptors. WIN 55,212-2 inhibited and SR 141716A increased the \ '>ltage-dependent Ca2+ currents in a subpopulation of the rat major pelvic ganglion neurons. 1 (J.M WIN 55,212-2 inhibited current by 26.1±1.8% and 1 pM SR 141716A increased current by 27.4±6.9%. These findings indicate that both heterologously expressed CB1 cannabinoid receptors and the native neuronal cannabinoid receptors can inhibit voltage-dependent Ca2+ channels. There is a tonic activation of both the heterologously expressed rat and human CB1 receptor and the native rat cannabinoid receptor. Two possible mechanisms may be responsible for the tonic receptor activation: an endogenous agonist may exist or the cannabinoid receptor can adopt an active conformational state such that SR 141716A may act as an inverse agonist.
dc.relation.urlhttp://ezproxy.augusta.edu/login?url=http://search.proquest.com/docview/304329725?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectCannabinoid Receptoren
dc.subjectCa2+ Channelsen
dc.subjectSympathetic Neuronen
dc.subjectHeterologous Expressionen
dc.subjectWIN 55,23 12-2en
dc.subjectAnandamideen
dc.subjectInverse Agonisten
dc.titleModulation of Ion Channels by Cannabinoid Receptors in Rat Sympathetic Neuronsen
dc.typeDissertationen
dc.contributor.departmentDepartment of Pharmacology and Toxicologyen
dc.description.advisorLewis, Deborah L.en
dc.description.degreeDoctor of Philosophy (Ph.D.)en
dc.description.committeeIkeda, Stephen R.; Caldwell, Robert W.; Carrier, Gerald O.; Creazzo, Tony L.; Wei, Xiangyangen
html.description.abstractThe rat brain cannabinoid receptor (CB1) is a member of the G protein coupled receptor family. The present study directly tested the functional coupling of CB1 receptor with neuronal ion channels. The rat CB1 receptor was heterologously expressed by microinjection o f cRNA into the enzymatically dissociated adult rat superior cervical ganglion (SCG) neurons. The cannabinoid receptor agonists WIN 55,212-2 and CP 55940 produced a voltagedependent inhibition of whole cell Ca2+ currents. The maximal inhibitions o f the Ca2+ current by WIN 55,212-2 and CP 55,940 were 73% and 38%, respectively. The E C 50 of WIN 55,212-2 was 47 nM and the EC50 of CP 55940 was 7 nM. The inhibition was mediated by pertussis toxin (PTX) sensitive G protein and the N-type Ca2+ channels are the targets. The L-type Ca2+ channels, M type K+ current and the A current were not modulated by WIN 55,212-2. An inwardly rectifying current was activated by WIN 55,212-2. The selective CB1 receptor antagonist SR 141716A and LY 320135 abolished the inhibition of the Ca2+ currents by WIN 55,212-2. However, when given alone, SR 141716A and LY 320135 increased the Ca2+ current in SCG neurons expressing the CB1 receptor. SR 141716A increased Ca2+ current with an EC50 of 32 nM and a maximal current increase of 41% at 1 p.M. This increase of Ca2+ current by SR 141716A was a reversal o f a tonic inhibition of Ca2+ current in neurons expressing CB 1 receptors. A K192A mutant of the human CB1 receptor was tested to determine whether the tonic activation o f the cannabinoid receptor is due to endogenous arachidonyl ethanolam ide (anandam ide). The K192A m utant receptor was expressed by microinjection of receptor cDNA into nucleus of the SCG neurons. WIN 55,212-2 inhibited the Ca2+ current in these SCG neurons, but SR 141716A did not increase the Ca2+ current. However, SR 141716A inhibited the effect of WIN 55,212-2. Ca2+ currents from male rat major pelvic ganglion neurons were examined for modulation by native cannabinoid receptors. WIN 55,212-2 inhibited and SR 141716A increased the \ '>ltage-dependent Ca2+ currents in a subpopulation of the rat major pelvic ganglion neurons. 1 (J.M WIN 55,212-2 inhibited current by 26.1±1.8% and 1 pM SR 141716A increased current by 27.4±6.9%. These findings indicate that both heterologously expressed CB1 cannabinoid receptors and the native neuronal cannabinoid receptors can inhibit voltage-dependent Ca2+ channels. There is a tonic activation of both the heterologously expressed rat and human CB1 receptor and the native rat cannabinoid receptor. Two possible mechanisms may be responsible for the tonic receptor activation: an endogenous agonist may exist or the cannabinoid receptor can adopt an active conformational state such that SR 141716A may act as an inverse agonist.


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