Endogenous molecules stimulating N-acylethanolamine-hydrolyzing acid amidase (NAAA)

Abstract

Fatty acid amide hydrolase (FAAH) plays the central role in the degradation of bioactive N-acylethanolamines such as the endocannabinoid arachidonoylethanolamide (anandamide) in brain and peripheral tissues. A lysosomal enzyme referred to as N-acylethanolamine-hydrolyzing acid amidase (NAAA) catalyzes the same reaction with preference to palmitoylethanolamide, an endogenous analgesic and neuroprotective substance, and is therefore expected as a potential target of therapeutic drugs. In the in vitro assays thus far performed, the maximal activity of NAAA was achieved in the presence of both nonionic detergent (Triton X-100 or Nonidet P-40) and the SH reagent dithiothreitol. However, endogenous molecules that might substitute for these synthetic compounds remain poorly understood. Here, we examined stimulatory effects of endogenous phospholipids and thiol compounds on recombinant NAAA. Among different phospholipids tested, choline- or ethanolamine-containing phospholipids showed potent effects, and 1 mM phosphatidylcholine increased NAAA activity by 6.6-fold. Concerning endogenous thiol compounds, dihydrolipoic acid at 0.1-1 mM was the most active, causing 8.5-9.0-fold stimulation. These results suggest that endogenous phospholipids and dihydrolipoic acid may contribute in keeping NAAA active in lysosomes. Even in the presence of phosphatidylcholine and dihydrolipoic acid, however, the preferential hydrolysis of palmitoylethanolamide was unaltered. We also investigated a possible compensatory induction of NAAA mRNA in brain and other tissues of FAAH-deficient mice. However, NAAA expression levels in all the tissues examined were not significantly altered from those in wild-type mice.

Keywords: N-Acylethanolamine-hydrolyzing acid amidase; NAAA; dihydrolipoic acid; fatty acid amide hydrolase; palmitoylethanolamide; phospholipid.

Figure 1

Effects of various detergents on NAAA activity. Recombinant NAAA (300 ng of protein) was allowed to react with 100 μM [¹⁴C]PEA in the presence of 3 mM DTT and the indicated detergent at 0.1%. The enzyme activity in the absence of detergent (39.0 nmol/min/mg of protein) is expressed as 100%, and relative activities are shown (means ± SD, n = 3).

Figure 2

Effects of various phospholipid classes on NAAA activity. Recombinant NAAA (300 ng of protein) was allowed to react with 100 μM [¹⁴C]PEA. (A) The reactions were carried out in the presence of 3 mM DTT and either Nonidet P-40 at 0.1% (w/v) or the indicated phospholipid at 100 μM. (B) Varying concentrations of PC in the presence of 3 mM DTT. The enzyme activity in the absence of phospholipid or detergent (44.5 nmol/min/mg of protein) was expressed as 100%, and relative activities are shown (means ± SD, n = 3).

Figure 3

Effects of various thiol compounds on NAAA activity. Recombinant NAAA (300 ng of protein) was allowed to react with 100 μM [¹⁴C]PEA. (A) The reactions were carried out in the presence of 0.1% Nonidet P-40 and the indicated compound at 3 mM. (B) Varying concentrations of DTT (○) and dihydrolipoic acid (●) both with 0.1% Nonidet P-40. The enzyme activities in the absence of thiol compound (29.0 (A) and 28.5 (B) nmol/min/mg of protein) are expressed as 100%, and relative activities are shown (means ± SD, n = 3).

Figure 4

Substrate specificity of NAAA in the presence of different stimulators. Recombinant NAAA (0.3–3 μg of protein) was allowed to react with the indicated ¹⁴C-labeled NAEs at 100 μM in the presence or absence of 0.1% Nonidet P-40, 100 μM PC, 3 mM DTT, and 0.1 mM dihydrolipoic acid. Means ± SD of the enzyme activities are shown (n = 3).

Figure 5

Effects of various buffers on NAAA activity. Recombinant NAAA (300 ng of protein) was allowed to react with 100 μM [¹⁴C]PEA in the presence of 3 mM DTT and 0.1% Nonidet P-40 at pH 4.5. The pH was adjusted with the following buffers (100 mM): citrate-Na₂HPO₄ (a), sodium citrate (b), 3,3-dimethylglutaric acid-NaOH (c), sodium acetate (d), sodium succinate (e), sodium phthalate (f), sodium tartrate (g), and sodium formate (h). The enzyme activity in citrate-Na₂HPO₄ buffer (219 nmol/min/mg of protein) is expressed as 100%, and relative activities are shown (means ± SD, n = 3).

Figure 6

Expression levels of NAAA mRNA in various tissues of wild-type and FAAH^–/– mice. Total RNAs were isolated from the indicated tissues of wild-type (open columns) and FAAH^–/– (closed columns) mice and were reverse-transcribed. The resultant cDNAs were analyzed by real-time quantitative PCR. The obtained NAAA mRNA levels are normalized to GAPDH mRNA levels, and the NAAA/GAPDH ratios are shown (means ± SE, n = 3).