Release of arachidonic acid by 2-arachidonoyl glycerol and HU210 in PC12 cells; roles of Src, phospholipase C and cytosolic phospholipase A(2)alpha.
The phospholipase A(2) (PLA(2))-prostanoid cascade is involved in cannabinoid receptor-mediated neuronal functions. We investigated the signaling mechanism for the release of arachidonic acid by cannabinoids, 2-arachidonoyl glycerol (2-AG) and HU210, in rat PC12 cells and in primary cultured cells from the mouse cerebellum. The effect of selective inhibitors for signaling pathways and/or enzymes (alpha type cytosolic PLA(2) (cPLA(2)alpha), G protein, Src kinases, phospholipase C, protein kinase C) was assessed. Methods included translocation of the chimeric protein GFP-cPLA(2)alpha, the activities of Src family kinases, Ca(2+)-dependent fluorescence and cyclic AMP accumulation. Treatment with 2-AG and HU210 at greater concentrations than 3 muM caused the release of arachidonic acid, and the response was inhibited by AM251 (an antagonist of cannabinoid CB(1) receptor) and by pyrrophenone (a selective inhibitor of cPLA(2)alpha) in PC12 cells. The cannabinoid treatment caused the intracellular translocation of cPLA(2)alpha and an increase in the intracellular Ca(2+) level. Treatment with HU210 caused tyrosine phosphorylation of Src and Fyn, and increased their kinase activities. Pretreatment with inhibitors of tyrosine kinases or phospholipase C abolished the cannabinoids-induced release of arachidonic acid and Ca(2+) response, and protein kinase C inhibitor reduced the release of arachidonic acid. 2-AG caused the release of arachidonic acid from cultured cells of the mouse cerebellum via similar mechanisms. These data reveal that cannabinoids activated cPLA(2)alpha in a Src-phospholipase C-protein kinase C-dependent manner probably via cannabinoid CB(1) receptor and/or CB(1)-like receptor in neuronal cells.
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8675, Japan.
Maiko
M
Nabemoto
Masato
M
Mashimo
Akiyoshi
A
Someya
Hiroyuki
H
Nakamura
Tetsuya
T
Hirabayashi
Hiromichi
H
Fujino
Masayuki
M
Kaneko
Yasunobu
Y
Okuma
Takeshi
T
Saito
Naoto
N
Yamaguchi
Toshihiko
T
Murayama
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001095:Arachidonic Acids / Q000494:pharmacology*; D000242:Cyclic AMP / Q000096:biosynthesis; D003600:Cytosol / Q000201:enzymology; D013759:Dronabinol / Q000031:analogs & derivatives* / Q000494:pharmacology; D063388:Endocannabinoids; D005989:Glycerides / Q000494:pharmacology*; D054513:Group IV Phospholipases A2 / Q000502:physiology*; D051379:Mice; D008813:Mice, Inbred ICR; D016716:PC12 Cells; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D051076:Proto-Oncogene Proteins c-fyn / Q000502:physiology; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D010738:Type C Phospholipases / Q000502:physiology*; D019061:src-Family Kinases / Q000502:physiology*
D001095:Arachidonic Acids; D063388:Endocannabinoids; D005989:Glycerides; D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D016718:Arachidonic Acid; C103505:AM 251; D013759:Dronabinol; C094503:glyceryl 2-arachidonate; D000242:Cyclic AMP; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D011493:Protein Kinase C; D054513:Group IV Phospholipases A2; D010738:Type C Phospholipases; C062018:HU 211