Agonist Stimulated [35SGTPyS Binding to Cell Membrane Homogenates

When performing these assays in a new tissue for the first time, it is helpful to first perform an assay to optimize assay conditions by determining nonspecific, basal, and agonist-stimulated (using a maximally effective concentration of agonist) with different amounts of membrane protein (see Fig. 2). Typically, using brain homogenates, 3-20 ^g of membrane protein per test tube yields good results. With cultured cells, the amount of protein required depends on the receptor expression level and, perhaps more importantly, the G protein expression level, which is often not as high as in brain, where the amount of Go-type G protein is estimated to make up nearly 1% of total brain protein. Experience has shown that even in a cell line in which cannabinoid inhibition of adenylyl cyclase has been observed, there may be no significant activity of cannabinoids for [35S]GTPyS binding. In tissues in which cannabinoid-stimu-lated [35S]GTPyS is observed (including brain tissue), the percent stimulation by agonist increases with decreasing protein (up to a point), but specific binding and total disintegrations per minute (DPM) decreases (see Fig. 2). A protein concentration that yields approx 1000-2000 DPM of basal [35S]GTPyS binding has been found to work well (using 0.05-0.1 nM [35S]GTPyS).

Next, using the previously determined protein concentration, the optimal concentration of GDP is determined by assaying the amount of basal and agonist-stimulated [35S]GTPyS binding using a maximally effective concentration of agonist (see Fig. 3). In this assay, nonspecific binding need only be determined once, including the highest concentration of GDP and 30 ^M unlabeled GTPyS. Concentrations of GDP from 0.1 to 300 ^M typically give a full range of effects. Generally, total and specific DPM decrease with increasing GDP, but percent stimulation increases up to approx 100 ^M, where specific DPM are quite low. Ten to fifty ^M GDP appears to give the best results (see Fig. 3).

Finally, using the optimal concentrations of membrane protein and GDP, a time-course could be performed to determine optimal incubation time. At each time point, nonspecific, basal, and agonist-stimulated binding should be determined.

Fig. 2. Effect of protein content on [35S]GTPyS binding to brain membranes.The figure shows an assay typical of varying amounts of membrane protein. In particular, nonspecific, basal, anandamide-stimulated, and WIN55,212-2-stimulated [35S]GTPyS binding to whole mouse brain membranes is depicted. Membranes were incubated in assay buffer with 0.1% BSA for 1 h at 30°C. Values shown are the mean of three values measured in consecutive assay tubes in the same rack. Nonspecific binding was determined in the presence of 25 |M unlabeled GTPyS, basal in the absence of GTPyS or agonists, and anandamide and WIN55,212-2 with 30 |M and 10 |M of these agonists, respectively (A). Net binding (A, inset) was calculated by subtracting mean basal values from mean values obtained in the presence of anandamide or WIN55,212-2 at each amount of protein. Percent stimulation (B) was determined by dividing net binding values by basal binding values obtained at each amount of protein. Notice that though overall and net-agonist-stimulated binding increase with increasing protein, due to the concurrent increase in basal binding, percent stimulation (i.e., the signal-to-noise ratio) is optimal at lower amounts of membrane protein.

Incubations are typically 1 or 2 h. Using brain tissue and cannabinoid agonist, it was found that increasing incubation time from 1 to 2 h increased both basal and total binding somewhat, decreasing percent stimulation to a degree, but at 2 h the assay was at a steady state, where there was little further increase in binding beyond that time (14). Times between 0.5 and 4 h give a full range of effects in brain tissue (from very little specific binding to a steady-state condition).

3.3.1. [35S]GTPyS Membrane Binding Procedure

1. Assay buffer: 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl with 0.1% (w/v) BSA is added to test tubes on ice.

2. GDP is added to all test tubes (see Note 4).

3. GTPyS is added to triplicate(s) of nonspecific binding (see Note 4).

4. Cannabinoid ligands are added to the assay tubes (see Note 2)

5. Membrane homogenate is added to the test tubes: from CNS tissues (5-10 |g/tube) or cultured cells expressing cannabinoid receptors (3-50 |g/tube, depending on the level of expression).

6. Add 0.05-0.2 nM [35S]GTPyS (see Note 4) for a final volume of 0.2- 1.0 mL.

7. Initiate the assay by transferring to a temperature-controlled, shaking water bath at 30°C; incubate for 1-2 h.

8. Assays are terminated by rapid filtration onto Whatmann GF/B glass fiber filters, followed by three rinses with 50 mM cold Tris-HCl, pH 7.4.

9. Filters are placed in scintillation vials with several milliliters of scintillation fluid and are allowed to remain so overnight (or for 1 h of vigorous shaking); either will allow extraction of the sample into the scintillation fluid.

10. Determine the amount of binding by liquid scintillation spectrophotometry at 95% efficiency for 35S.

Fig. 3. Effect of GDP concentration on [35S]GTPyS binding to brain membranes.The figure shows an assay typical of varying amounts of GDP. In particular, basal and WIN55,212-2-stimulated [35S]GTPyS binding to rat cerebellar membranes is depicted. Membranes were incubated in assay buffer with 0.1% BSA for 1 h at 30°C. Values shown are the mean of three values measured in consecutive assay tubes in the same rack. Binding was determined in the absence (basal) and presence of 30 |M WIN55,212-2 (A). Net binding (A, inset) was calculated by subtracting mean basal values from mean values obtained in the presence of WIN55,212-2 at each concentration of GDP. Percent stimulation (B) was determined by dividing net binding values by basal binding values obtained at each concentration of GDP. While basal and agonist-stimulated binding decrease with increasing GDP (A), net agonist-stimulated binding (A, inset) and percent stimulation (B) do not change much at GDP concentrations below 10-6 M (1 |M). Above 1 |M GDP, net agonist-stimulated binding decreases sharply, but is accompanied by a more rapid decrease in basal binding, leading to optimal percent stimulation values. Above 10-4 M (100 |M), binding values are too low and the difference between agonist-stimulated and basal values are too small to yield usable results.

3.3.2. Data Analysis

Each condition in the assay is repeated in triplicate. Basal [35S]GTPyS binding is determined in the absence of receptor ligands, and nonspecific binding is determined in the absence of receptor ligands and the presence of 10 |M unla-beled GTPyS. Mean values ± SD are calculated for each triplicate, and triplicates for which the SD is greater than 10% of the mean are inspected for values that are outliers. The mean value obtained for nonspecific binding in each assay is subtracted from every other mean value to obtain specific binding values, which are used for all further calculations. Net-agonist-stimulated binding is calculated by subtracting basal values from each value obtained in the presence of ligand. Percent stimulation by agonist is calculated by dividing each net-agonist-stimulated value by the basal value. Concentration-effect is determined by nonlinear regression using an appropriate model in Prism (GraphPad Software, San Diego, CA) to determine EC50 (the concentration producing halfmaximal effect) and Emax (the theoretical maximal value obtained with each lig-and) values for each agonist in each assay.

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