Pitfalls and solutions in assaying anandamide transport in cells

Abstract

Nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fluorescence microscopy.

Fig. 1.

Interaction of AEA with plastic and glass supports. Plastic culture wells without cells were incubated for 10 min with increasing concentrations of [³H]AEA, alone [plastic, at 37°C (closed circle), or at 4°C (open circle)], or in the presence of coverslips [glass, at 37°C (closed square) or at 4°C (open square)]. The accumulation was stopped by buffer removal, and the amount of tritium adsorbed nonspecifically to the different supports was measured after washing. To compare AEA adsorption on coverslips and plastic wells, the total amount of AEA was normalized to the total wet surface of the two types of support and was expressed as pmol/min/cm². The temperature-sensitive component of the nonspecific uptake associated to the plastic wells was obtained by subtracting the uptake at 37°C from that at 4°C, and is shown as dotted line. The inset (on the right) shows an enlargement of the graph relative to [³H]AEA retained by the coverslips. Values are means ± SEM of at least three independent experiments performed in triplicate. The points were generated using GraphPad Prism, and the curves were fitted using the one site binding hyperbola.

Fig. 2.

Binding of AEA to plastic or glass supports in the presence or absence of cells. A: Cells or supports alone were incubated for 10 min with 400 nM [³H]AEA in cell culture media. The uptakes (means ± SEM, n = 9) at 37°C (black columns), 4°C (gray columns), and 37°C − 4°C (unfilled columns) are shown, each measured in the presence (+) or absence (−) of HaCaT cells. The rate of uptake was related to the total surface in contact with AEA solution, thus allowing the comparison of uptake values among different conditions. B: Concentration dependence of [³H]AEA uptake by HaCaT cells. These cells were grown onto plastic wells (closed circle) or coverslips (closed square) and were incubated with varying concentrations of AEA for 15 min at 37°C. C:Time-dependent uptake of [³H]AEA in HaCaT cells. Cells were grown onto plastic wells (closed circle) or coverslips (closed square) and were incubated with 400 nM AEA at 37°C for the indicated periods of time, then they were washed and radioactivity was evaluated as described in “Materials and Methods.” Inhibitory effects of 50 μM OMDM-1 (×) on both concentration- and time-dependent [³H]AEA uptake were evaluated in cells grown onto coverslips. Nonspecific adsorption of AEA to cell membranes or cell supports was estimated by running the identical experiments at 4°C and was subtracted from each data point. The lines drawn are the best fit of the data to the one site binding hyperbola. Values are means ± SEM of at least three independent experiments, each performed in triplicate.

Fig. 3.

Time- and temperature-dependent release of [³H]AEA from HaCaT cells grown on coverslips. HaCaT cells were preloaded with 400 nM [³H]AEA for 10 min at 37°C. Fresh buffer was added and the cells were incubated at either 4°C (open circle) or 37°C (closed circle) for the indicated time periods. Release was measured by extracting [³H]AEA retained onto coverslips with NaOH. The temperature-dependent efflux (closed square) was calculated by subtracting the fraction of [³H]AEA remaining within the cells at 37°C from the fraction remaining at 4°C. The line drawn through the filled squares is the best fit of the data to the one phase exponential association using least squares, nonlinear regression analysis (GraphPad Prism). Values are means ± SEM of at least three independent experiments performed in triplicate.

Fig. 4.

Kinetics of accumulation and release of b-AEA in HaCaT cells grown onto coverslips. A: Concentration-dependent accumulation of b-AEA. HaCaT cells were incubated with the indicated concentrations of b-AEA for 10 min at 37°C, then cells were washed and subjected to microscopy detection. B: Time-dependent accumulation of b-AEA. HaCaT cells were incubated with 10 μM b-AEA at 37°C for the indicated time periods, then cells were washed and subjected to immunodetection. C: Time-dependent release of b-AEA. HaCaT cells were preloaded with 10 μM b-AEA at 37°C for 30 min, then were placed in fresh incubation medium (containing 0.15% BSA) at 37°C (closed square) or at 4°C (open square). At the indicated time points, cells were washed, fixed, and analyzed by immunofluorescence. The temperature-dependent release (closed circle) was calculated by subtracting the fraction of [³H]AEA remaining within the cells at 37°C from the fraction remaining at 4°C. Inhibitory effects of 50 μM OMDM-1 (×) on both import and export of b-AEA were also evaluated. Plotted data represent the mean ± SEM fluorescence intensity measured in five fields of three independent experiments. Representative images coming from b-AEA immunostainings are shown on the right side of each graphs. Scale bars, 50 μm. Fluorescence intensity was quantified by the ImageJ software. The points were generated using GraphPad Prism, and the curves were fitted using the one site binding hyperbola for accumulation data and the one phase exponential association for release data.