TABLE 3

Comparison of filtration and centrifugation assays for determination of bound and free radioligand The binding of two radioligands ([3H]spiperone and [3H]nemonapride) at the concentrations indicated in the presence of 100 mM Na+ or NMDG) to membranes of Sf9 cells expressing D2 dopamine receptors was determined in parallel centrifugation and filtration assays as described under Materials and Methods. In the filtration assays, 50 μg of membrane was used, and total, nonspecific, and specific radioligand bindings were determined. In the centrifugation assays, 100 μg of membrane was used, and the total, nonspecific, and specific radioligand bindings in the pellet were determined as well as the free radioligand in the supernatant after the centrifugation. Bound radioligand is expressed as a concentration (picomolar) in the 1-ml assay volume and for filtration assays has been normalized to 100 μg of membranes to allow for comparison with the centrifugation assays. For the free radioligand, the concentration measured (picomolar) is given along with the concentration calculated derived from the amount bound in the pellet in the centrifugation assays. The data are values from one experiment performed in triplicate (data are shown as mean ± S.E.M.), which has been replicated three times with similar results. From the three experiments, it is possible to analyze the effects of different conditions. Specific [3H]spiperone binding in filtration assays was 109.8 ± 3.8% of that in centrifugation assays (P < 0.05, paired t test). Total [3H]spiperone binding in filtration assays was 102.0 ± 3.8% of that in centrifugation assays (P > 0.05). Specific [3H]spiperone binding in the presence of Na+ ions was 106.8 ± 2.4% of that in the presence of NMDG (P < 0.05), and total [3H]spiperone binding in the presence of Na+ ions was 104.0 ± 3.0% of that in the presence of NMDG (P > 0.05). Calculated free [3H]spiperone as a percentage of the measured free [3H]spiperone was 102.5 ± 5.4 (P > 0.05) and 109.0 ± 8.1% (P > 0.05) in the nonspecific and total tubes, respectively. Specific [3H]nemonapride binding in filtration assays was 102.2 ± 5.3% of that in centrifugation assays (P > 0.05, paired t test). Total [3H]nemonapride binding infiltration assays was 92.0 ± 2.4% of that in centrifugation assays (P > 0.05). Calculated free [3H]nemonapride as a percentage of the measured free [3H]nemonapride was 114.3 ± 2.3 (P < 0.05) and 114.5 ± 4.3% (P < 0.05) in the nonspecific and total tubes, respectively.

Radioligand Added Radioligand Filtration Bound Radioligand Centrifugation
Bound Radioligand Free Radioligand
Total Nonspecific Specific Total Nonspecific Specific Total Tubes Nonspecific Tubes
Calculated Actual Calculated Actual
[3H]Spiperone + Na+ 193 ± 2 64 ± 2 4 ± 1 60 70 ± 2 9 ± 1 61 123 130 ± 1 184 210 ± 1
[3H]Spiperone – Na+ 219 ± 4 66 ± 2 6 ± 1 60 66 ± 3 10 ± 1 56 153 140 ± 1 209 200 ± 2
[3H]Nemonapride + Na+ 304 ± 8 86 ± 1 4 ± 1 82 101 ± 1 11 ± 1 90 203 190 ± 1 293 270 ± 10
[3H]Nemonapride – Na+ 317 ± 4 38 ± 2 4 ± 1 34 40 ± 1 12 ± 1 28 277 250 ± 1 305 270 ± 10