Isotope dilution mass spectrometric measurements indicate that arachidonylethanolamide, the proposed endogenous ligand of the cannabinoid receptor, accumulates in rat brain tissue post mortem but is contained at low levels in or is absent from fresh tissue

被引:96
作者
Kempe, K
Hsu, FF
Bohrer, A
Turk, J
机构
[1] WASHINGTON UNIV,SCH MED,DEPT MED,DIV ENDOCRINOL,ST LOUIS,MO 63110
[2] WASHINGTON UNIV,SCH MED,DEPT MED,DIV DIABET,ST LOUIS,MO 63110
[3] WASHINGTON UNIV,SCH MED,DEPT MED,DIV METAB & LAB MED,ST LOUIS,MO 63110
[4] WASHINGTON UNIV,SCH MED,DEPT PATHOL,DIV METAB & LAB MED,ST LOUIS,MO 63110
[5] WASHINGTON UNIV,SCH MED,DEPT PATHOL,DIV DIABET,ST LOUIS,MO 63110
[6] WASHINGTON UNIV,SCH MED,DEPT PATHOL,DIV ENDOCRINOL,ST LOUIS,MO 63110
关键词
D O I
10.1074/jbc.271.29.17287
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Arachidonylethanolamide (AEA) isolated from porcine brain binds to cannabinoid receptors, mimics nabinoid pharmacologic effects, and has been proposed as an endogenous cannabinoid receptor ligand, Demonstration of co-distribution of AEA and cannabinoid receptors in various brain regions could provide supportive evidence for this role, are have performed isotope dilution mass spectrometric measurements of AEA and have demonstrated AEA production by rat tissue homogenates in vitro from exogenous arachidonate and ethanolamine, No detectable endogenous AEA (<3.5 pmol/g of tissue) was observed in fresh rat brain, whether or not inhibitors of AEA hydrolysis were present during tissue processing, AEA (>1 nmol/g) was produced during saponification of brain phospholipid extracts, This appears not to reflect hydrolysis of N-arachidonylethanolamine phospholipid precursors of AEA, because Streptomyces chromfucsis phospholipase D, which is active against NAPE, failed to generate AEA from brain phospholipids despite substantial conversion of phospholipids to phosphatidic acid. Such experiments suggested that the abundance of N-arachidonylethanolamine phospholipid in fresh rat brain may be less than 1 in 10(6) phospholipid molecules, AEA generated during saponification of tissue phospholipids appears to arise from base-catalyzed aminolysis of arachidonate containing glycerolipids, because AEA was produced from synthetic (1-stearoyl, 2-arachidonoyl)-phosphatidylethanolamine under saponification conditions, and the amount produced increased 300-fold when free ethanolamine was included in the hydrolysis solution, Although AEA was not detectable (<0.17 pmol/mg of protein) in fresh rat brain, AEA accumulated post mortem to levels of 126 pmol/mg of brain protein. These findings do not exclude the possibility that AEA is rapidly synthesized and degraded locally in vivo, but they indicate that the AEA content of fresh rat brain and of NAPE precursors from which AEA might be derived are exceedingly low and that AEA can be produced artifactually from biological materials.
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页码:17287 / 17295
页数:9
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