Anandamide Inactivation Uptake and Enzymatic Hydrolysis

When added to the medium of neurons or astrocytes in culture, radioactive anandamide is rapidly taken up by the cells this process occurs with the rapid time-course, high affinity, temperature-dependence, saturability and selectivity typical of a carrier-mediated uptake (Fig. 6.11). Further experiments will have to address the precise mechanism and kinetic properties of this uptake system in neurons and astrocytes, in order to determine which of these cell types takes up anandamide more effectively. Also, it will be essential to establish whether anandamide uptake is present in the adult central nervous system and whether it is localized to synaptic fields where anandamide release is expected to take place (e.g., where cannabinoid receptors are localized). 

In addition to uptake, anandamide may be disposed of by a process of enzymatic hydrolysis. This reaction yields arachidonate and ethanolamine (Fig. 6.11), and it is carried out by a membrane-associated amidohydrolase activity which has been characterized kinetically in some detail and has been partially purified from porcine brain. 

In rat brain microsomes, anandamide amidohydrolase activity is optimal at pH 6 and 8, is independent of divalent cations, has an apparent Km for PH] anandamide of about 13 pM and has a Vmax of about 5600 pmol/minutes/mg protein. Its activity is blocked by both serine protease inhibitors (e.g., phenyl methyl 

Small changes in the chemical structure of anandamide are important for the catalytic activity of anandamide amidohydrolase (Fig. 6.12). Structural modifications that result in reduced hydrolysis include (1) elongating the fatty acyl chain above 20 carbon atoms (2) replacing cis double bonds with trans double bonds (3) decreasing the number of double bounds to one, or eliminating them.  

It should be emphasized that, while rat brain microsome amidohydrolase shows little or no activity towards N-palmitoyl-ethanolamine and other saturated NAEs, another enzyme activity 

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