Accumulation of N-arachidonoylethanolamine (anandamide) into cerebellar granule cells occurs via facilitated diffusion.
N-Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [3H]AEA accumulation by cerebellar granule cells is dependent on incubation time (t(1/2) of 2.6 +/- 0.8 min at 37 degrees C) and temperature. The accumulation of AEA is saturable and has an apparent Km of 41 +/- 15 microM and a Vmax of 0.61 +/- 0.04 nmol/min/10(6) cells. [3H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 microM phloretin (57.4 +/- 4% of control) in a noncompetitive manner. [3H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [3H]AEA accumulation is fairly selective for AEA among other naturally occurring N-acylethanolamines; only N-oleoylethanolamine significantly inhibited [3H]AEA accumulation at a concentration of 10 microM. The ethanolamides of palmitic acid and linolenic acid were inactive at 10 microM. N-Arachidonoylbenzylamine and N-arachidonoylpropylamine, but not arachidonic acid, 15-hydroxy-AEA, or 12-hydroxy-AEA, compete for AEA accumulation. When cells are preloaded with [3H]AEA, temperature-dependent efflux occurs with a half-life of 1.9 +/- 1.0 min. Phloretin does not inhibit [3H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein-mediated transport process that has the characteristics of facilitated diffusion.
Hillard
C J
CJ
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee 53226, USA.
Edgemond
W S
WS
Jarrahian
A
A
Campbell
W B
WB
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D001667:Binding, Competitive; D001692:Biological Transport; D002186:Cannabinoids; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D004058:Diffusion; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D008297:Male; D010042:Ouabain; D010693:Phloretin / Q000494:pharmacology; D053284:Polyunsaturated Alkamides; D051381:Rats; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors; D013696:Temperature
D001095:Arachidonic Acids; D002186:Cannabinoids; D002352:Carrier Proteins; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D053284:Polyunsaturated Alkamides; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D010693:Phloretin; C078814:anandamide