The Effect of Methyl-ß-Cyclodextrin treatment upon the viability and anandamide uptake capability of C6 glioma and P19 embryonic carcinoma cells

Lina Thors, Christopher J. Fowler. Department of Pharmacology and Clinical Neuroscience, Umeå, Sweden.

There is conflicting data in the literature as to whether cholesterol depletion of membranes affects the rate of uptake of anandamide (AEA). Thus, for example, Bari et al. (2005) reported that the cholesterol-depleting agent methyl-ß-cyclodextrin (MCD) greatly reduced the rate of uptake of AEA into C6 glioma cells. On the other hand, AEA uptake by P19 cells is not affected by mevinolin treatment, which disrupts cholesterol synthesis (Sandberg & Fowler, 2005). The lack of effect of mevinolin may indicate that the cholesterol dependency of AEA uptake into P19 cells differs from that for C6 cells.

In order to explore this possibility, uptake of AEA by P19 and C6 cells was investigated using a standard method (Sandberg & Fowler, 2005). MCD was preincubated with the cells for 30 min at 37°C prior to the addition of AEA (assay concentration 100 nM) and incubation for 1 or 4 min at 37 °C. Initially, MCD (concentration range 2-50 mM) was applied and uptake measured without a wash prior to addition of AEA. A clear concentration-dependent reduction in uptake was observed for both cells. The IC50 values obtained at 1 and 4 min incubation times, respectively, were 9.4 and 9.9 mM for C6 cells, and 6.5 and 7.3 mM for P19 cells. However, a similar pattern of inhibition of [3H]AEA retention by wells alone was also seen, with IC50 values of 3.7 and 5.1 mM being found for 1 and 4 min incubation times, respectively. The most likely explanation for this effect is that MCD forms complexes with AEA (Jarho et al., 1996) and thereby reduces the concentration available for uptake. In order to avoid this problem, a wash phase was used. Under these conditions, retention of AEA by the wells was not affected by the MCD treatment, but the sensitivity of the cells was right shifted. In the case of P19 cells, the IC50 values were 16 and 12 mM for AEA incubation times of 1 and 4 min, respectively. For the C6 cells, concentrations of MCD ≤20 mM did not produce any effect on the uptake, whilst total uptake was 31 and 38% of control for 1 and 4 min incubation times, respectively, following pretreatment with 50 mM MCD.

A reduction in measured total uptake can be due to an effect on the uptake process itself, but also be secondary to an effect on cell viability, since non-viable cells will detach and thereby be lost during the washing phase of the uptake assay. In consequence, in a separate experiment the cellular densities of C6 and P19 cells were measured after the treatment with MCD followed by a wash phase. For C6 cells, 10 mM MCD was without effect upon the observed cell density, whereas this concentration greatly reduced the number of P19 cells remaining. A higher MCD concentration (50 mM) also dramatically reduced the number of C6 cells remaining after the wash phase. This would suggest that in our hands, the ability of MCD to affect cell viability is the major determinant of its effects on uptake.

Bari et al. (2005) J Biol Chem 280: 12212-12220

Jarho et al. (1996) Life Sci 58: 181-185

Sandberg & Fowler (2005) Chem Phys Lipids 134: 131-139