Biochemistry
-
Comparative Study
Comparison of anandamide transport in FAAH wild-type and knockout neurons: evidence for contributions by both FAAH and the CB1 receptor to anandamide uptake.
The cellular inactivation of the endogenous cannabinoid (endocannabinoid) anandamide (AEA) represents a controversial and intensely investigated subject. This process has been proposed to involve two proteins, a transporter that promotes the cellular uptake of AEA and fatty acid amide hydrolase (FAAH), which hydrolyzes AEA to arachidonic acid. However, whereas the role of FAAH in AEA metabolism is well-characterized, the identity of the putative AEA transporter remains enigmatic. ⋯ The results of these studies reveal that (i) care should be taken to avoid the presence of artifacts when studying the cellular uptake of lipophilic molecules like AEA, (ii) FAAH significantly contributes to AEA uptake, especially with longer incubation times, and (iii) a UCM707-sensitive protein(s) distinct from FAAH also participates in AEA uptake. Interestingly, the FAAH-independent component of AEA transport was significantly reduced by pretreatment of neurons with the cannabinoid receptor 1 (CB1) antagonist SR141716A. Collectively, these results indicate that the protein-dependent uptake of AEA is largely mediated by known constituents of the endocannabinoid system (FAAH and the CB1 receptor), although a partial contribution of an additional UCM707-sensitive protein is also suggested.
-
The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. ⋯ The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.